No title

ADVANCES IN GENETICS VOLUME 19 Edited by E. W. CASPARI Department of Biology University o f Rochester Rochester, New Yor...

Author: Author Unknown

4 downloads 289 Views 33MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!

Report copyright / DMCA form

DOWNLOAD PDF

ADVANCES IN GENETICS VOLUME 19 Edited by E. W. CASPARI Department of Biology University o f Rochester Rochester, New York

1977 ACADEMIC PRESS

NEW YORK SAN FRANCISCO LONDON

A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT 0 1977, nY ACADEMIC PRESS, INC.

ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR nY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

Unifed Kingdom Edifion published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London N W l

LIBRARY OF CONGRESS CATALOG CARD NUMBER:47-30313 ISBN 0-12-017619-X PRINTED IN THE UNITED STATES OF AMERICA

CONTRIBUTORS TO VOLUME 19 Numbers in parentheses indicate the pages on which the authors’ contributions begin.

EDWARD G. BARRY(133), Department of Botany, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina ETTAKAFER(33), Department of Biology, McGill University, Montreal, Canada CALVIN F. KONZAK (407), Department of Agronomy and Soils, and Program in Genetics, Washington State University, Pullman, Washington N. C. MISHRA(341), Department of Biology, University of South Carolina, Columbia, South Carolina P. A. PARSONS ( l ) ,Department of Genetics and Human Variation, L a Trobe University, Bundoora, Victoria, Australia DAVIDD. PERKINS (133) , Department of Biological Sciences, Stanford University, Stanford, California RUTHSAGER(287), Department of Microbiology and Molecular Genetics, Harvard Medical School, and Sidney Farber Cancer Institute, Boston, Massachusetts

vii

MEIOTIC AND MITOTIC RECOMBINATION IN Aspergillus AND ITS CHROMOSOMAL ABERRATIONS Etta Kafer Department of Biology, McGill University, Montreal, Canada

I. Introduction . . . . . . . . . . . . . . . . . . 11. Meiotic Crossing-over and Nondisjunction: Effects of Chromosomal Aberrations, Especially Translocations . . . . . . . . . . . A. Meiotic Crossing-over in Standard Crosses, . . . . . . . . B. Reduction of Crossing-over by Chromosomal Aberrations, Especially Reciprocal Translocations . . . . . . . . . . . . . C. Aneuploids from Controls, and from Crosses Heteroeygous for Reciprocal Translocations . . . . . . . . . . . . . . . . . D. Meiotic Nondisjunction Frequencies in Crosses Heteroeygous for Reciprocal Translocations . . . . . . . . . . . . . E. Interchromosomal Effects of Translocations in Meiosis . . . . . 111. Mitotic Recombination . . . . . . . . . . . . . . . A. Mitotic Crossing-over and Nondisjunction in Standard Diploids . . B. Crossing-over in the Centromere Area of Group I and Genetic Differences between Strains . . . . . . . . . . . . . . . . C. Mitotic Recombination in Triploids . . . . . . . . . . I). Induced Mitotic Recombination in Standard Diploids . . . . . E. Mitotic Recombination in Translocation Heterozygotes . . . . . F. Mitotic Crossing-over in Disomics from Translocation Crosses, Especially in “Stable” Disomics . . . . . . . . . . . IV. Genetic Mapping and the Use of Translocations . . . . . . . . A. Genetic Mapping of Mutants in Aspergilhs nidulans and Effects of Translocations . . . . . . . , . . . . . . . . . B. Frequencies, Detection, and Mapping of Translocations . . . . . C. Mapping of Centromeres and the Use of Translocations for Sequencing of Meiotic Fragments. . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

33 35 35 40

43 50 56 58 58

67 74 81 85 97 100 101 107 116 124

I. Introduction

When mitotic recombination and the parasexual cycle were first demonstrated in Aspergillus nidulans, it was expected and hoped that these processes would permit mapping and breeding in commercially used asexual fungi, like Penicillium chrysogenum, Aspergillus oryzae, etc. 33

34

ETTA KAFER

(Pontecorvo et al., 1953 ; Pontecorvo, 1956). Indeed the feasibility of such work, and the existence of somatic fusion of cells and nuclei followed by mitotic segregation, have been demonstrated in a large number of species (Roper, 1966). These include not only important parasitic fungi, like Ascochyta imperfecta (Sanderson and Srb, 1965) or Ustilago hordei (Megginson and Person, 1974), but also mammalian cells and recently even those of higher plants (Gamborg et al., 1974). However, most of these investigations have not yet progressed to any practical stage, since various unexpected problems were encountered [for example, scarcity of spontaneous recombinants in Penicillium (Macdonald, 1971 ; Ball. 1973) 1. From the experience with Aspergillus nidulans (Kafer, 1965), it seems likely that chromosomal aberrations, especially translocations, which are induced simultaneously with desired mutations and recombinants, produce a major problem for genetic analysis by mitotic recombination, even though these do not explain all unexpected features of mitotic segregation encountered with asexual species. Such aberrations are frequently induced in fungi by practically all commonly used mutagens, especially the relatively high and multiple doses of irradiation which have been applied in many asexual species (even low doses of UV induce a considerable frequency of translocations in Neurospora, as reported by Perkins, 1974). Since heterozygous translocations prevent random recovery of chromosomes in mitotic haploids from diploids, it is obvious that mitotic analysis of asexual species becomes frustratingly complicated by induced translocations, especially if chromosome numbers are small [e.g., possibly only three in Penicillium (Ball, 1973) 1. Detailed genetic analysis can, therefore, be successful if mutants are obtained spontaneously (e.g., selected by resistance to analogs and inhibitors) or with mutagens that cause no or few aberrations (e.g., chemicals that induce mainly base-pair substitutions). So far few mutagens of this type are known that are effective in fungi [except possibly nitrous acid (Abbondandolo and Bonatti, 1970) ] ; however, low doses of highly mutagenic chemicals, like nitrosoguanidine (NG) , also appear to produce relatively fewer chromosomal aberrations than point mutations [ e.g., permitting fast progress of mitotic mapping in the slime mold Dictyostelium, where mainly NG-induced mutants are being used (Katz and Kao, 1974; Kessin et al., 1974) 1. Although unrecognized aberrations complicate genetic analysis, translocations, once identified by their effects on meiotic and mitotic nondisjunction, can be used to advantage in mitotic mapping of markers and centromeres. This certainly is the case for Aspergillus nidulans, where meiotic recombination also occurs, so that translocations can be combined with suitable markers. In this species, meiotic recombination frequencies are so high that markers of the same chromosome arm often are com-

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

35

pletely unlinked in crosses; such markers can be sequenced either by mitotic crossing-over, or by means of overlapping translocations (Pontecorvo and Kafer, 1958; Kafer, 1958, 1975). I n asexual species, such use of translocations is bound to be very difficult and will probably be restricted to a few favorable cases. One major problem is that in most fungi, mitotic, and even meiotic, chromosomes are too small for recognition of translocations cytologically. However, new techniques might solve this problem, as has recently been the case for mammalian chromosbmes, which has led to fast progress in the mapping of translocations and deletions and to their use for mitotic mapping of genes in human cells (Jacobs et al., 1974; Ruddle, 1973; Shows and Brown, 1975). One great advantage of techniques that use chromosomal aberrations for genetic mapping is the fact that the obtained results are qualitative, and often comlementary to evidence from meiotic mapping [as has been demonstrated in many species of higher plants, e.g., in maize by Kasha and Burnham (1965) and in Drosophila, as in phages and bacteria, even at the microlevel within genes, e.g., by Welshons and Keppy (1975) 1. In addition, chromosomal aberrations may give information on basic processes, e.g., those of pairing and disjunction [as demonstrated in Drosophila by Grell (1962, 1967) ; or in maize by Burnham et al. (1972) 1. This investigation, therefore, had three purposes : 1. T o analyze the effects of one, or two, overlapping, reciprocal translocations on meiotic crossing-over and nondisjunction ; 2. To identify the processes of mitotic recombination in diploids with and without translocations, in triploids, and also in disomics from single and double translocation crosses, as well as the effects of inducing agents on these ; 3. T o assess the various methods of genetic mapping, the uses of translocations for mapping, and the various problems arising from chromosomal aberrations for mapping by the parasexual cycle. All the techniques and information on mutants used in these investigations have been written up in detail and are available from the author on request (origins of most of the translocations are shown in Table 1 ; for other strains, see Barratt et al., 1975). II. Meiotic Crossing-over and Nondisjunction: Effects of Chromosomal Aberrations, Especially Translocations

A. MEIOTIC CROSSING-OVER IN STANDARD CROSSES 1. Homogeneity of Linkage Values

Meiotic recombination in standard strains of Aspergillus nidulans produces typical values between linked markers, which differ no more

TABLE 1 Mitotic and Meiotic Tests for Translocations (T) in Original or Progeny Strains of UV-Induced Mutants'

UVinduced mutant

Residual genotype

FGSC No.*

Linkage group of mutant andarm

ActAl

riboA yA; nicB

23 1

I11 L

adE2O~

biA pabaA biA

50 429

I

adG14 anAl

biA; p A biA

choAla

biA (AcrA; lysB; choA+)

lacAl luAl lysB6

YA; P w A biA biA; smA

lysD2O

biA; sB pabaA y A

35 31d 1 354 58d 5.5 66 395 418

"1

I L I L

R1 VI R I L V L

Mitotic test in diploidsc Recombination between groups Deduced translocation None

T1(ZZ;VZZ) None None

No. of Lowest hap% loids found -

-

-

67

3

51 81

Groups with

Meiotic test in crossesC Frequencies of disomics for T-groups No./Total

%

0/1126

<0.1

1I;VII

17/ 958 -

1.8 -

36 34

None None

0/2660

-

-

T1(Z;VZZ)

48 -

0

1;VII

-

-

29/1187

None None None

48 39 31

31 36 26

None None None

0/1603 -

27/1437 60/1499

1.9 4.0

TI (ZZZ;VIZ)

31 125

1II;VII

-

-

-

biA biA

219 34

IV L I11 L

~ h e n A 2 ~ riboA adG y A proA1 pabaA y A

304 332

III

phenAP proAl

biA; nirA biA

260 32

111 It I L

pr0A2~

biA riboA g A ; AclA

111 414

I")

pyroA4 riboAl riboB2

biA biA biA; ActA w A 3

33 158

sAl'

biA anA y A ; w A 2 ; pyroA adG; pyroA; chaA

370 40

biA; chaA2 pabaA; chaA2

372 4 13

biA b i A ; lysB

398

methGl methH2

sAaa sD6O

3d

"I

IV It I L VIII It

None None

80 41

T1(I;VZZZ) T1(Z;ZV)e

None

T1 (ZZ;ZZZj None None None

32 -

"1

1;IV -

-

-

22

0

1I;III

50

2.5 22

34 40 18

None None None

-

-

-

29

T2(I;VZZZ)

For translocation-free progeny, see Barratt et al. (197,5), Section I. FGSC, Fungal Genetics Stock Center. c A few of these results are included in Kafer (196.5) or in Upshall and Kafer (1974). d Poor strain, containing additional morphological mutants. c Independently identified earlier by Sinha (1967). a

3 -

-

86

TI (V;VZZZ)

None None

1;VIII

T1 (V;VZ)

II1

37 34

V ;\? Y;VIII 1;VIII

7/ 8.57

0.8

19/1648 0/1603

1.2 <1.0

34/ 867

3.9

71 310

-

14/ 398

2.6 2.4

18/1786 23/ 347

1.0 4.2

-

1.8

-

14/ 799

m

2

0

g

E z

5U g+I

5p 0 0

9

2

u2

2

k

x

-iz 3

3.

38

ETTA KAFER

from cross to cross than expected from random sampling. This is exemplified by Table 2, where values from different laboratories are compared and approximate averages are calculated [only one of our 13 values deviates enough to be significant a t the 57%level, while 1/20 are expected to do so by chance; standard errors are calculated as in Kafer (1958) using angular transformation and weighting with the total number of tested progeny (Snedecor and Cochran, 1967); since for important crosses usually 400 progeny are tested rather than the routine sample of 200, and in some of the most informative crosses even 600-1000, it did not seem suitable to use the computer program of Dorn (1972), which produces nonweighted averages]. This uniformity of recombination values in Aspergillus is almost unique, which in part must be due to the fact that in this homothallic species all strains could be started from a single haploid nucleus (Pontecorvo et al., 1953). Except for induced mutations, all strains are therefore naturally isogenic. Aspergillus also shows extremely long genetic maps-e.g., compared to Neurospora-with three to four meiotically unlinked regions in several chromosomes or even arms. This relatively high rate of meiotic recombination may also, a t least in part, be a consequence of the high level of isogenicity. But it probably is also species specific, just like the differences observed in various mammalian species [ e.g., man showing higher chiasma frequencies than mouse, even though mice used for such investigations are usually more inbred (Ford and Clegg, 1969) ; or the extremely high frequency of chiasmata in amphibians correlated with relatively low chromosome numbers (e.g., Wallace, 1974) 1. 2. Lack of Chiasrna Interference

Aspergillus also differs from many other genetically well analyzed organisms by its complete lack of (positive) chiasma interference (Strickland, 1958), or even production of slight negative interference in certain crosses (Kafer, 1958) or under certain conditions (Elliott, 1960b). These results agree with the computer-analyzed data of Dorn (1972), which show no interference for crosses between markers in three of the linkage groups, and slight negative interference (i.e,, values of > 1 for coincidence = observed double crossovers/expected) in the two other groups (contrary to a statement of Dorn, referred to by Clutterbuck, 1974). I n one of these latter cases, namely in the crosses involving group IV, apparent negative interference might possibly be due to a reciprocal translocation Tl (IV;VIII) associated with frA 1 (Kiifer, 1975) as pointed out by Clutterbuck (1974). However, such effects are more likely caused by inversions than by translocations, since the latter reduce double as well as single crossing-over.

TABLE 2 Combined Recombination Frequencies ( %) in Controls and Heterozygous Translocation Crosses for Intervals in the Right Half of Linkage Group Io Markers and intervals Crosses

riboA

Control crosses

-

anA

19.2 f 1 . 3 (960)

19.4 & 1 . 0 (1446) 17.4 & 1 . 5 (3121) Combined values (Total)

18.3 (5.527)

-

adG

6.4 f 0 . 8 (960)

6 . 7 f 0.5 (1022) 8.2 f 0 . 5 (1284)

-

proA

25.3 k 1.4 (960)

29.8 & 1.2 (2747) 33.9 f 1.05 (2591)

7.2

30.6 (6298)

(3266)

- .-

pabaA

-

yA

-

biA

Reference

6.9 k 1.4 (960) 8.8 (1119)

13.6 f 1 . 1 (960) 16.5 (623)

3 . 1 f 0.6 (960) 5.5 (623)

Elliott (1960b)

11.1 f 1 . 3 (593) 7.9 f 1.2 (3938) 8.7 k 0.3 (3854)

16.8 f 1.2 (938) 15.7 f 1.0 (4228) 16.7 k 0.5 (3126)

6.9 f 0 . 5 (2962) Tj.7 5 0.4 (4098) 5.8 k 0 . 3 (2549)

Dorn (1972)

8.4 (10,464)

16.0 (987.5)

6.8 (11,192)

5.5b (901)

11.4b (1611)

5.4 (812)

Elliott (1960b)

Kafer (1958) Kiifer, Unpublished

33.1 Crosses heterozygous

for TI(Z;VZZ) or TZ(1;VIZZ)

12. I* (921)

20. 4b (543)

Totals tested are given in parentheses. Difference from control average significant at 1 % level. c 5% level.

-

a

b

w

CD

40

ETTA KAFER

Additional evidence for lack of interference is also contained in the values of Table 3 (Section 11, B, 2), where control recombination frequencies for all pairwise combinations of five markers of group I are shown; these can be analyzed for coincidence in six groups of three markers each. The obtained coincidence values are very close to 1 in five of the six cases (two of them slightly less than 1, namely, 0.91 and 0.85, and three slightly larger than 1, namely 1.06, 1.17, 1.25), and only one indicates possible negative interference (coincidence value of 1.75, for the two adjacent intervals suA - 12.2% - gnlD - 18.7% - fpaB, compared t o 24.3% for suA - f p a B ) . The published meiotic data from different laboratories are therefore in excellent agreement and indicate that, in standard crosses under standard conditions, distribution of multiple crossing-over in Aspergillus nidulans is usually random. As a consequence, the values of meiotic linkage maps are approximately additive only for small intervals, ca. up to 15 centiMorgans (cMo), while for three adjacent but more distant markers, the frequency of recombinants between the outside markers is considerably less than the sum for the other two pairs (see Section IV, on mapping).

B. REDUCTION OF CROSSING-OVER BY CHROMOSOMAL ABERRATIONS, ESPECIALLY RECIPROCAL TRANSLOCATIONS The effects of chromosomal aberrations on meiotic recombination are well known and are of two general types: ( 1 ) if single (or equivalent) crossing-over leads to the formation of inviable products, then most recovered types are noncrossovers or double crossovers; or (2) if aberrations interfere with meiotic pairing and chiasma formation, all crossovers, single as well as multiple ones, are reduced in frequency. 1. Inversions

The best-known crossover suppressors are inversions, which are commonly used in Drosophila and also are now being developed for mutagenicity tests in mice (Evans and Phillips, 1975). They act mainly, but not exclusively, by the first mechanism (Roberts, 1967). In most other organisms inversions have been isolated only rarely, probably mainly because of their relatively cryptic cytological and genetic properties (as demonstrated, e.g., in mice by Roderick and Hawes, 1974). I n fungi so far only three pericentric inversions have been identified in Neurospora; these inversions all produce inviable crossover progeny in heterozygous crosses, as is evident from the occurrence of asci with two white abortive spores (Newmeyer and Taylor, 1967; Turner et al., 1969). I n Aspergillus, only a single case of a likely inversion has been encountered, which

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

41

seems to be associated with lysA1. This inversion appears to overlap with T1 (VZ;VIZ) (“Ab VI” in Pollard et nl., 1968) but probably not T1 (V;VZ),as indicated by the finding that it produces many stable and partially stable aneuploids in crosses only with the former. No other methods for detection of inversions are available in this species which produces neither ascospore patterns nor cytologically identifiable chromosomes, and where reduced recombination frequencies would be detectable only in relatively well-mapped segments. 2. Translocations

I n Drosophila many translocations with one break fairly distal on a chromosome arm have been shown to reduce meiotic recombination even more effectively. In such cases single as well as double crossovers are absent, probably owing to synaptic disturbances (Roberts, 1970, 1972). I n Aspergillus, reduction of meiotic recombination has become evident around the break points of all translocations that have been mapped so far. For markers that are fairly closely linked to the breaks, the map distances in heterozygous crosses are often reduced to about half (see Fig. 15). To check whether such reductions were also observed in more distant intervals, recombination was analyzed in detail in linkage group I, in which the breaks of two translocations were mapped, namely that of T2(Z;VZZZ), close but proximal to suA (Ma and Kafer, 1974), and that of T1 (Z;VZZ), very close and probably distal to fpnB (Kafer, 1975). A special set of crosses was analyzed in which the same multiply marked standard strain was crossed to a standard strain, and to both translocations [namely: fpaB galD suAadE SulA riboA anA pabaA y A adE biA; FGSC No. 483 (Barratt et al., 1975) 1. Homozygous translocation crosses heterozygous for these same markers were also carried out (except for fpaR in the Tl(Z;VZZ) cross, because no recombinant containing this marker in coupling with this translocation has been obtained so far). The results for markers distal on I L and also for the most proximal ones on VII and VIII, combined with similar data that were homogeneous, are presented in Table 3 (and for intervals to the right of riboA in Table 2; data in both tables are based on totals of 1000-2000 tested in most cases, and always on totals of over 500). Since all values from homozygous crosses were indistinguishable from controls, these have been omitted, except for the intervals between the markers riboA - SulA - fwA - facC, which are on the same chromosome in homozygous T2(Z;V1ZI) crosses but not in controls (sD50 cannot be used as a marker in T x T crosses because these are homozygous for sD) . From the data in Table 3 it can be seen that recombination in intervals

42

.

ETTA KAFER

TABLE 3 Interchromosomal Linkages, and Reduced Recombination Frequencies ( %) for Intrachromosomal Intervals, in Crosses Heterozygous for Tf (Z;VZZ)or Ti2(Z;VZZZ)a Crosses heterozygous for Tf (Z;VZZ) Linkage group I riboA

group

SulA

I

suA galD fpaB

1

fpaB1- ,*'"I

SulA

suA

galD

16.0 (19.2)'

27.1 (42.0)

28.9 (41.6)

29.3 (44.0)

22.1 (35.1)

25.3 (37.3)

26.4 (42.7)

(12.2)

(24.3)

(19.2) 15.4 (35.1)

28.1 (41.6)

21.8 (37.3)

(12.2)

SD

27.3 (44.0)

21.8 (42.7)

16.5 (24.3)'

(18.7)'

2.8

6.1

VIII

fwA

facC

2(6;

:;:1

(4

(#I

13.4 (74

33.7 20.2 [45.31* [48.31

16.8 (2)

16.2

33.0 21.8 [42.8]* [50.2]

13.8

17.6 (2)

22.4

(#)

suA

galD

fpaB

riboA

SulA

(2)

32.4 (#)

t

(2)

(18.7)

I

Group

34.8

19.2

25.8 (42.0)

L

Group VII

(#)

22.3

(#I

(Z)

\ 10.5

-$"

Linkage group I Crosses heterozygous for T2(Z;VZZZ)

13.7

6.6 (8.8)"

SD

fwA

Group VIII

Values for controls in parentheses (19.2)*; for homozygous T in brackets [45.3]. Symbols for degree of significance: a = difference not significant; * = significantly different a t 5% level; all other values significantly different at 1% level. # No linkage; on different chromosomes. -+ = positions of T-breaks. a

b

close to the breaks shows highly significant differences between controls and heterozygous translocation crosses ; for the more distant intervals recombination values are also somewhat smaller in translocation crosses, but not significantly so. I n Table 2, the values from crosses heterozygous for either translocation are compared to the average standard values for

RECOMBINATION

AND TRANSLOCATIONS IN

43

Aspergillus

intervals to the right of riboA, and in such tests significant differences show up for all except the most distant intervals. However, in longer chromosomes these effects do not spread beyond intervals that show free recombination; e.g., no effects of these translocations are found for markers distal on VII R, or VIII R, respectively (data not shown). 3. Double Translocation Crosses

Even in crosses between two overlapping translocations, recombination is normal in distant, long intervals of the involved groups. On the other hand, for shorter intervals, closer to the breaks, the obtained values are usually even more reduced than in single translocation crosses [as shown also for Drosophila by Robinson and Curtis (1972), who in a double translocation cross, found very much reduced recombination in one differential segment and none at all in the other]. For example, in a cross of T1 ( V ; V I ) X T l (V1;VIl)(cross 2141, genotypes in Table 6) meiotic recombination between the four markers of linkage group VI (lacA - b w A - s B - s b A ) appears completely normal, i.e., no significant linkage was found for any pair except s B - sbA, and the value for the latter did not differ from controls. On the other hand, in cross 2146, linkages of markers around the breaks, e.g., galD and phenB of T l ( I ; V I I ) or palF - lysD - malA of T1 (ZI1;Vll) became very tight. FROM CONTROLS, AND FROM CROSSES C. ANEUPLOIDS HETEROZYGOUS FOR RECIPROCAL TRANSLOCATIONS

1. Typical Disomics from Standard Crosses

I n Aspergillus, ascospores from crosses between haploid strains are usually haploid, but rare aneuploids or diploids are also found (Pritchard, 1954), and in Neurospora ascospores are the best source of diploids (Smith, 1974). Most aneuploids from standard crosses are n 1 types, but occasionally n 2 or 2n 1 colonies are encountered. The frequency of aneuploids of all these types from standard crosses or selfed cleistothecia is about 0.1-0.2% [Upshall and Kafer, 1974; the lower values of Pollard et al. (1968), are now known t o be due to storage of the ascospore suspension a t 4OC, which leads to fairly rapid loss of all disomics]. Eight different disomic types corresponding to eight mitotic linkage groups can be distinguished visually (Kafer, 1961 ; Kafer and Upshall, 1973). These different types show very different rates of abnormal growth, with n 1 for group I V (“n IV”) often so normal that it is hard to detect, especially when morphological mutants segregate, and n 1 for group V I I I ( [ [ n VIII”) growing so poorly that often i t can hardly be seen and is easily overgrown by normal colonies. These differences are expected to

+

+

+

+

+

+

+

44

ETTA KAFER

influence the frequencies of their recovery, especially in ascospore platings of high density. 2. Disomics from Crosses Heterozygous for One Translocation

In meiosis, heterozygous reciprocal translocations produce unbalanced products even with 2 : 2 disjunction of centromeres, but these all have deficiencies and none are likely to survive in haploid organisms (also in the mouse, but not in man, these are invariably lethal; Ford and Clegg, 1969). I n addition, 3:l and 4:O disjunctions occur with increased frequencies, so that additional specific aneuploids are produced. Recovery of such aneuploids depends on two main factors: (a) they have t o survive long enough to be classified; (b) for genetic analysis they also have to produce identifiable phenotypes. a. Viability. In haploid organisms hypoploids are usually not viable whereas hyperploids show varying degrees of viability. For example, in yeast, viability is excellent up to about four extra chromosomes (Parry and Cox, 1970; James and Inhaber, 1975) and in Neurospora disomics are very unstable, but these and large duplications show very good viability and cannot be detected visually except when incompatibility alleles become heterozygous (e.g., Perkins, 1975;Smith, 1975). I n higher organisms, hypoploidy for small elements may be tolerated in some cases [as in certain tertiary monosomics of the tomato of Khush and Rick (1967)1, especially in species with a few disproportionately small chromosomes [e.g., in Drosophila, where the large autosomes are not recovered in trisomics, but haplo and triplo types can be obtained for chromosomes X and IV from translocation crosses (e.g. Grell, 1959)1. b. Identification. Aneuploid products may be recognizable by a system that uses genetic markers plus phenotypic criteria [such as patroclinous males and matroclinous females after X-nondisjunction, produced for example, by elevated temperature in Drosophila (Grell, 1971a)1. Or identification may be possible from special features of the general appearance alone, presumably resulting from imbalance a t many loci; as for the famous trisomic series of Datura (Blakeslee and Belling, 1924) or similarly recognizable types in tomato [Rick and Khush (1968),but not in the related potato (Kessel and Rowe, 1974)l. I n fungi other than Aspergillus, such phenotypic specificity is either absent or hard to recognize (as in yeast ; James et al., 1974). c. Recovery. I n Aspergillus only four of the potential 34 types of aneuploids from any T X cross (Ford and Clegg, 1969) are expected to be fairly viable, namely the hyperploid products from 3:1 disjunctions. There are four n 1 types, disomic either for one of two normal chromosomes or for one of the two translocation chromosomes [as shown for TI (VZ;VII) and T l ( I I I ; V I I ) (Kafer, 1975)1. Such n 1 always result

+

+

+

RECOMBINATION AND TRANSLOCATIONS IN

45

Aspergillus

from primary meiotic nondisjunction so that the “typical looking” disomics with a normal chromosome extra contain both translocation chromosomes in the balanced haploid set, while the two types of translocation disomics contain both standard chromosomes (mitotic crossingover occasionally produces exceptions to this rule). The viability of the “typical looking” types does not differ from that of standard n 1, while the translocation disomics have specific phenotypes and different but specific viabilities for every translocation. They differ much more than the standard n 1, since many translocations produce chromosomes that are either much longer or much shorter than the standard ones. Some of these translocation disomics are therefore even less viable than n V I I I and only rarely recovered [e.g., one type from crosses with T1 (Z;VZZ) or T,?(Z;VlII),namely the n 1 which is disomic for the longer translocation chromosome, in which most of the long arms of V I I or V I I I are attached to almost the whole chromosome I (see Fig. 15)]. Generally, both types of translocation disomics are expected t o survive for translocations between linkage groups for which viable n 2 have repeatedly been obtained [like I11 or I V with each other, or with 11, V, VI, or V I I (Pollard et al., 1968; E. Kafer, unpublished)]. On the other hand, phenotypic recognition of highly viable types like disomics for very short translocation chromosomes may become very difficult or impossible, since these may not show differences distinct enough to be identified in single colonies. This is postulated to be the case for the other, short, translocation chromosomes of both T1 (Z;VZZ) and TZ(I;VZZZ) (above), as well as for the missing type of T1 ( V ; V I ) (Fig. 1 and Table 4 ; genotypes in Table 5 ) . The progressive reduction of phenotypic effects of all or part of a chromosome is best exemplified by comparison of disomics with duplications, as generated by nonreciprocal translocations (e.g., for group 111 ; Bainbridge, 1970; Clutterbuck, 1970). Even though some correlation between the severity of effects on viability, i.e., growth rate and conidiation, and the size of the duplicated segments is generally found, it is not expected to be consistent or accurate, since the genes responsible are not likely to be distributed at random in a chromosome. Indeed it is obvious that such correlation does not provide accurate information on chromosome sizes: for example, group VII is longer than many others, but has the least phenotypic effect as judged by the difficulties of identifying 2 n + V I I . More precise information, like that for Drosophila produced by Lindsley et al. (1972) is, however, not available, and will be difficult to obtain except for fairly large segments.

+

+

+

+

+

3. Disomics from Crosses between Overlapping Translocatwns

From crosses between two reciprocal translocations, new disomic types are obtained when one of the breaks of each translocation is located in

46

ETTA KAFER

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

47

the same chromosome (the extreme case where both breaks are in the same chromosome will be discussed in the next section). For example, one new disomic type is expected as a consequence of meiotic crossing-over between the breaks, which produces a new “double-translocation” chromosome as the complementary product t o a normal homolog for the linkage group involved in both translocations [called “tripartite” by Tuleen (1973)working with barley translocations]. I n addition, and much more striking, new types of almost stable disomics are formed. These arise frequently, and three types are expected. One type, the most frequent of these, is genetically and phenotypically a segregant that contains the equivalent of two normal homologs of the common linkage group, each being split up into two segments, so that the extra chromosome cannot be lost (Figs. 2a and 11). Such “stable” n 1, disomic for a standard chromosome, are produced, for example, in the absence of meiotic crossingover by nondisjunction in the “common” group when the four translocation chromosomes segregate to one pole and the two normal homologs to the other [very frequent trisomics, 2n 1 for the “common” chromosome, from double-T crosses have been demonstrated in the mouse (White et al., 1974)1. The other two types can be formed after meiotic crossing-over between the breaks, which produces the new “tripartite” chromosome plus a standard one (Fig. 3).If both these crossover products segregate to the same pole, which may occur frequently if they have centromeres of different groups, no third element exists that would produce a viable haploid type; however, if they segregate with the two residual chromosomes from either parent (namly, one normal and one translocation chromosome in either case) stable disomics with the equivalent of one extra translocation chromosome are produced (Figs. 2b and 3 show one of these, which has been recovered repeatedly). Such disomics have been analyzed in detail from the relatively wellmarked crosses shown in Table 6. For the three crosses of T1 (Z;VZZ) to either T1 (VZ;VIZ) or T1 (ZZZ;VZZ) detailed results are shown in Table 7. All the listed types could be identified visually, and their heterozygous markers confirmed the phenotypic classification. For TI (Z;VZZ), which has one break extremely distal in group I and the other proximal in V I I R (see Fig. 14), one type of translocation disomic is usually inviable and the other probably not identifiable (as mentioned above). Since T1 (Z;VZI)

+

+

FIG. 1. Platings of disomics from a cross heteroeygous for TI(V;VI).(a and b) Typical-looking disomics: (a) n V; (b) n VI. (c) Only type of recovered translocation disomic, n V-VI, disomic for centromere of V. (d) Haploid. FIG.2. Transfers of “stable” disomics from a cross of TI(Z;VZI)x TI(ZIZ;VZI) showing rare haploid sectors. (a) Stable n + VII, p a l F / + , producing dark paZF and lighter pal’ haploid sectors. (b) “Stable” translocation disomic, n VII-111.

+

+

+

+

TABLE 4 Frequencies of Disomics in Crosses Heterozygous for Reciprocal Translocations

Cross No. T1(V;V I ) 9 Crosses. 19.53 2130 2131 21.52 2132 All Tl(V;VZ)

Total number of colonies

Total disomics, all types

Relative frequencies of specific n T-disomics n V-VI

+

~

(%)

(No.)

n+V

4557 2750 1628 1872 3120 3695

2.5% 2.3% 2.7% 8.9%. 3.9% 4.1%

(112) (64) (44) (166) (121) (153)

48% 19% 26% 57%. 24% 27%

(49) (13) (11) (90) (29) (41)

17622

3.7%

(660)

37% (233)

26% 62% 62% 37%. 72% 56%

n

(3ga) (30) (26) (58) (87) (84)

+VI

14% 19% 12% 6% 4% 17%

51 % (324)

+ 1types

Typical disomics (absolute %)

T-disomics n +VI-V

(15) (13) (5) (10) (5) (26)

n+V

?a

-

-

-

12% (74)

1

n +\'I

1.1 0.5 0.7 4.W 0.9 1.1

0.33 0.47 0.31 0.53 0.23 0.70

1.3

0.42

Total specific disomics

T1(VI;VZZ)

(%)

(No.)

n

+ VI

n

+ VI-VII

~~

n ~

+ VII

n

+ VII-VI

12

+ VI

n

+ VII

~~~

4 Crossesa 5 Crosses* 5 recent crosses

3643 12740 6090

2.7% 5.0% 4.8%

(100) (643) (290)

35% (35) 42% (270) 42% (121)

13% (13) 16% (102) 11% (31)

30% (30) 23% (145) 27% (79)

22% (22) 20% (126) 16% (45)

1.0 2.1 2.0

0.8 1.1 1.3

All Tl(VZ;VZZ)

22473

4.6%

(1033)

41 % (426)

14% (146)

25% (254)

19% (193)

1.9

1.1

z* r3

w %-

cj

I

RECOMBINATION AND TRANSLOCATIONS IN

49

Aspergillus

TABLE 5 Genotypes of Crosses Heterozygous for T1 (V;VZ) (Markers on Groups I’ and 1’1 Only)

Cross of origin for T-strain

Linkage groups

Cross No.

T

(noT)

1953

T

2152

(noT)

T 2130 2131

VI

V

(noT)

x

T (noT)

T 2132

(noT)

2136

(noT)

T

+ + + + + ; + + + + facA + + ; lacA + + + facA + + lacA + lysB p A + + riboD ; + + + + facA + + lacA + + + + + + lacA bwA + + facA + + lacA + + + lysB p A + hxA riboD ;

; ;

+ + facA + + + + hxA riboD + + facA + + ZysB p A + hxA riboD

ZysB

Homozygous T1( V ; V I ) cross T p A facA 2153 T lysB

+

+

+ + +

; ;

; ;

: ;

ribOD ;

+

;

lacA bwA

+ +

sB sB

+ +

+ + sB + + sbA sB sbA + 4+ sbA

(990, not shown)

1953 1953 2133 2130

+ + 4lacA bwA + sbA + + + + + + 4- sbA lacA bwA + +

2130

2131 2132

is one of the parents in all these crosses, this considerably reduced the numbers of aneuploid types and facilitated analysis (even though the other two translocations each produced two recognizable types of translocation disomics). For example, no types, stable or sectoring, disomic for the tripartite crossover chromosome I-VII-VI or I-VII-111 (Le., with centromere of group I) are expected to be viable; and only two, rather than three, types of stable disomics were likely to be obtained (as was found, Table 7).One of these stable types is a typical-looking n VII in all crosses (Fig. 2a), and the other is “disomic” for the translocation chromosome with the centromere of VII [namely, in the two crosses with T1 (ZZ1;VZZ) disomic for the VII-I11 chromosome as shown in Figs. 2b and 31. As can be seen in Fig. 2, both types of “stable” disomics have extremely large disomic centers, and these show the phenotypes of the much smaller centers of the corresponding sectoring disomics (published recently, Kafer, 1975); but they only produce very rare haploid sectors, since haploids can be formed only after mitotic crossing-over between the breaks in VII has occurred (Fig. 3 and, below, Fig. 12 and Table 17).

+

50

ETTA KAFER

PARENTS

I-VII-III

---- + Act'

lllN 8-

VII tcrossover)

t IL

Mitotic Crossing Over

I

I-v11

111

VII-l

F I ~ .3. Meiotic crossing-over between the breaks in a cross of Tf(1;VII)x Tl(IIZ;VZI) (cross 2146) and formation of stable translocation disomic n + VII-I11

by meiotic nondisjunction ; mitotic crossing-over in one of the intervals between T-breaks (see Table 17) leading to formation of a normally unstable disomic, able to produce haploid sectors of a crossover type.

D. MEIOTICNONDISJUNCTION FREQUENCIES IN CROSSES HETEROZYGOUS FOR RECIPROCAL TRANSLOCATIONS 1. Single Translocation Crosses

Since reciprocal translocations cause disturbances in pairing a t meiosis, it is not surprising that nondisjunction for the two involved chromosomes is

++

+I

u

a

+% - '",+

H

Aspergillus

?+ +; ++

++

d

+.f 1

+ I ;+ $+

._.-

R

& + +?j + +

rq .c

X

._..

z+ ++

.- .-

++

+%

s + % E ++ ++ -

3.

+ $ g + +? +; + % + $1 +%

++ %+

+g

%+

+;

++ +; EL

rq % +

&+

4

L

1

$+

H

rq

jt

j + I1 + Y

RECOMBINATION AND TRANSLOCATIONS IN

Y

F

H

$L +

JY +

J+ Y

51

TABLE 7 Frequencies and Types of Disomics from Crosses between Partially Overlapping Translocations: TI (ZL;VZZR)Crossed to Either TI (VZR;VZZR)or TI (ZZZL;VZZR) Normally unstable disomics with frequent haploid sectors Cross (No.) and 2nd T involved

Total Total prog- abnoreny mals

Mainly n 1. n

+

+ VI

n+ VI-VII

+

n VII-VI

Cross- All unover' stable n V I I types n

+

"Stable" disomics

+ VII

n

+

VII-VI

Both types"

Other abnormals

Ef:

x TI (VZ;VZZ) (1944) Nos. Frequencies

331

154 46.5%

x TI (ZIZ;VZZ) (1946) (2146)

413 236 -

Total Nos. 649 Frequencies

n

+ I11

7

29

n+ 111-VII

n+ VII-111

21

67 20.2%

50

29

79 23.9%

8 2.4%

n+ VII-I11

145

5

8

131 -

5 -

1 -

20 10 -

35 28 -

35 35 -

103 79 -

16 34 -

19 18 -

276 42.5%

10

9

30

63

70

182 28.0%

50

37

35

7

52 -

-0

87 13.4%

7 1.1%

The very inviable n + I-VII type from TI (Z;VZZ), the corresponding stable disomic, and the crossover types reciprocal to + V I I with the resulting "tripartite" chromosome extra, have been obtained only rarely, and could not be identified with certainty. a

n

7

3

2 w

B

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

53

greatly increased. Aspergillus presents an especially suitable system for the genetic analysis of these effects, since all eight types of standard disomics can be recognized visually and, provided ascospores are plated a t low densities, such aneuploids can be recovered fairly reliably. For all known translocations it was found that the frequencies of disomy for the heteromorphic groups increased 50- to 100-fold in heterozygous crosses, the highest values being found in those cases where all of the four expected disomics are recovered (Upshall and Kafer, 1974). When one or both types of translocation disomics are not recoverable or identifiable (see above), these obtained values for aneuploid frequencies are minimal estimates of the nondisjunction frequencies. On the other hand, typical looking disomics that contain an extra standard chromosome have known viabilities, and their absolute and relative frequencies show characteristic values typical for each translocation (Table 4).Such frequencies seem to show a fairly good inverse correlation with the distance of the T-break from the centromere; that is, the closer the break to the centromere the higher the resulting nondisjunction in meiosis (Kafer, 1975). This relationship may make i t possible to predict the approximate position of the breaks of new translocations from their aneuploid frequencies in heterozygous crosses, especially if similar information is available from crosses heterozygous for mapped cases that have one break in the same chromosome arm [this has been attempted for T1 (V ;V I),especially the break in group V I , and its n+VI frequencies are compared to those of T1 (VZ;VII) in Table 4; T1 ( V ; V l ) has subsequently been mapped, and details of the relevant crosses are given in Table 5 and in Section TV on mapping]. 2. Aneuploid Frequencies in Crosses between Translocations

When two translocations are intercrossed, the types and frequencies of aneuploids depend very much on the relative positions of the four breaks. There are three possibilities (as outlined in detail by Curtis and Robinson, 1971): (a) they may all be located in different chromosomes; (b) in the opposite extreme, the same two chromosomes are involved in both translocations; or (c) the breaks map in three chromosomes. Results from these three types of crosses in Aspergillus are shown in Table 8. a. Breaks in Four Groups. As expected, when all four breaks map in different groups, no interaction is detectable and the observed frequencies of disomics are not significantly different from the sum of the values obtained for each translocation alone (in Table 8, part a ) : three of the four cases fall well within the expected range, while in one cross, 2159, aneuploids are slightly but not significantly higher than expected (namely 10.5%, compared to 4% plus 3.5%, giving a x2 of about 2.8, P = 0.10).

54

ETTA KAFER

TABLE 8 Aneuploid Frequencies ( %) in Crosses between Two Translocations and Variation with Plating Densities5 Type of cross and translocations involved (a) Breaks in four different chromosomes Tl(I1;III)X Tl(V;VI) T2(I;VIII) X Tl(V1;VII) T2(I;VIII) X Tl(II1;VII) Tl(II1;VII) X Tl(1V;VIII) (b) Two breaks each in two chromosomes Tl(1L;VIIR) X TB(1L;VIIR) Tl(1R;VIII) X TB(1L;VIIIR) (c) Breaks in three chromosome arms, two breaks in same one Mapped translocations

Tl(1;VII) X T1(V1;VII) Tl(1;VII) X Tl(II1;VII) Tl(V1;VII) X Tl(II1;VII) T2(I;VIII) X Tl(1;VII) TP(1;VIII) X Tl(1V;VIII)

Arm of overlap, distance

Cross

-

2159 1822 2156 2158

-

1790 18% (131/725) 1656c 2 % (23/1038)

No.

Plating densities High

-

VII It Fairly close

Low

10.5 % (20/ 189) 7 . 1 % (29/407) 8 . 5 % (18/2ll) 7 . 5 % (33/430) 16% (68/420)“ -

1788 29 % (258/902) 1944 Fairly distant 1946 35 % (145/413) 2146 29 % (306/1054) Fairly close 2145 I L, very close 1645” 11 % (171/1555) 2142 25% (165/711) VIII R, very 2143 13% (112/844) distant 2157 1943b 10% (35/35@)

47 % (154/331) 55 % (131/236) 46 % (165/362) 20% (76/390) 14 % (46/320) -

VIII VIII

14 % (30/220) -

Crosses of unmapped T1 (V;VZZZ) (distances unknown) and

TI ( V;VZ)

Tl(V;VIII) Tl(V;VIII) Tl(V;VIII) Tl(V1;VII)

X X X X

TB(1;VIII) Tl(1V;VIII) Tl(V;VI) Tl(V;VI)

V

VI R, close?

213Sb 22% 2137b 11% 213gb 13 % 2141b 27%

(160/75@) (57/500b) (41/300b) (145/54@)

Observed numbers are given in parentheses. Totals estimated from number of colonies with “recombinant” color, since partly selfed, twin cleistothecia. Results of A. Upshall (unpublished).

b. Breaks in Two Groups. The situation is quite different when both breaks map in the same two groups or even in the same arm, so that types and frequencies of the aneuploids depend on the relative positions of breaks within a chromosome or arm. Such crosses have been used in vari-

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

55

ous organisms, either to identify the chromosome arms in which breaks are located (as in barley; Kasha and Burnham, 1965) or to generate specific duplications [ e.g., in Neurospora using ascospore patterns (Perkins, 1974) ; or in Drosophila for gene dosage studies (O’Brien and Gethmann, 1973) 1. Only two pairs of translocations of this type are available in Aspergillus. One of these, T l ( I ; V I I I ) x T2(Z;VIIZ), showed no detectable interaction (cross 1656, Table 8,b). This agrees with the postulated positions of the breaks in group I, namely a t opposite ends of both arms, and indicates a similar relationship in VIII [one break of Tl(1;VIZI) maps distal in I R (Klfer, 1965), but the other break has not yet been mapped, because of extremely low aneuploid frequencies obtained in certain crosses to markers of group VIII (Upshall and Kafer, 1974) 1. I n the other cross (1790 in Table 8,b) an unexpectedly found 1;VII translocation which showed breaks in the same chromosome regions as T1 ( I ; V l l ), was crossed to the latter to check for identity. Since 16-18% aneuploids were observed, it is evident that a new TZ(I;VZI) was present [ T 2 ( I ; V I I )could have originated in an ancestral UV-treated multitranslocation strain, or in one of the parents which is a reverted duplication strain; the latter produced another derivative, which contained a T (1;V;VII;VIIZ) complex, after a few years of occasional subculturing]. c. Crosses between Translocations with Breaks in Three Groups. When one break of each of the two translocations that are crossed together is in the same chromosome, it is expected that all three chromosome pairs involved will form joint pairing figures in meiosis. These can be used to determine breaks cytologically in some species. Such pairing results in increased disturbance of normal disjunction for the various centromeres, especially those of the chromosome pair involved in both translocations. A high frequency of nondisjunction is therefore expected, leading to the formation of various aneuploid types (as explained in detail in Section 11, C, 3 ) . Three main variables must influence the overall frequencies: (i) the distance of the more proximal break from the centromere, since proximity seems t o increase nondisjunction ; (ii)the distance between the breaks, especially their relative position to the centromere, i.e., whether they are located on the same or opposite arms; (iii) the viability of the various aneuploid products. A large number of such crosses have been analyzed, because it was hoped that these relationships could be used to get approximate estimates of the positions of the breaks for unmapped cases, as is possible by cytological techniques in higher plants, like barley (Tuleen and Gardenhire, 1974). The crosses between translocations with mapped breaks used for this investigation are grouped according to the arm of overlap (which corresponds either to VII R, I L or VIII R, Table 8,c). Some of the pairs of

56

ETTA KAFER

translocations were crossed more than once, using various suitably marked strains (genotypes in Table 6, for crosses that were analyzed in detail). Since rather large differences were found in such pairs, repeated platings from the same crosses were also carried out. From the obtained results, it became evident that a major variable was the plating density. This high variability obscured all minor differences between pairs of similar crosses. I n addition, since three variables interact, similar values can result from quite different situations. This is demonstrated in Table 8,c, where no distinction is possible between the first two listed cases, both involving T1 (Z;VZZ),even though the distance between the breaks in VII R must be about twice as long in the second [cross to T1 (ZIZ;VZZ)]as in the first [to T1 (V1;VZZ)1, and no meiotic linkage is detectable in standard crosses for any of these intervals. And for the crosses with Ti?(Z;VIZI)no significant difference in aneuploid frequencies is detectable for crosses with overlap in I L compared to those in VIII R, even though the two situations are quite different: in the first case the two breaks are quite close, but both distal in I L, while in the second the breaks are exceedingly far apart, but one is quite proximal in VIII R (Fig. 14, and Table 8,c, crosses 1645 and 2142, compared to crosses 2143 and 2157). Such results, therefore, have no predictive value, unless they are quite extensive, and only if several similar cases are well analyzed and can be compared and used as standards. And even well established results may fit several situations [as do those of the unmapped translocations TI (V;VZ)ahd T1 (V;VIZZ),quite apart from the fact that the latter can hardly be used when they differ no more from each other and from the crosses between mapped translocations than repeats of the same cross in several cases]. I n conclusion, tentative mapping based on results from crosses with overlapping translocations is probably not worth while in most cases. The effort needed to get consistent values in several such pairs is hardly greater than that needed for mapping by other methods (especially since partly selfed cleistothecia are frequent, which unpredictably changes the survival rate of ascospore samples). On the other hand, very extreme cases can be recognized even in small samples: breaks are probably distal in opposite arms when barely additive values of disomic frequencies are found; or fairly close and in proximal areas, when 4 0 4 0 % abnormals are produced in double-?' crosses. E. INTERCHROMOSOMAL EFFECTS OF TRANSLOCATIONS IN MEIOSIS Since the heterozygous translocation crosses of Aspergillus show many of the features investigated in detail in Drosophila, it is of interest to

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

57

consider whether the general mechanism postulated in the latter organism also may apply to Aspergillus. Based on an extensive analysis of effects by recombination-reducing aberrations on the disjunction of other chromosomes, two phases of pairing have been postulated for the meiosis of Drosophila: one early one, leading to and reinforced by chiasma formation, and a second one, “distributive pairing,” which involves all noncrossover chromosomes and may result in pairing of heterologous types (Grell, 1962, 1967). According to this hypothesis, reciprocal translocations may not only produce increased nondisjunction of the involved chromosomes, but, indirectly, also of others, especially if these have affinities and pair with chromosomes of the rearranged type. This is expected to lead to increased recovery of n 1 for other linkage groups, as well as to increased frequencies of n+ 2 among the T-specific types [observations in support of a general occurrence of these kinds of effects have also been reported in man (Grell, 1971b) 1. I n Aspergillus, occasionally some translocation crosses seemed to give higher than expected aneuploid frequencies for noninvolved chromosomes. For example, the early crosses of T1 ( V ; V I ) gave an unusually high frequency of “other” n 1 types, mainly disomics for group 111, some of them originating from n V I11 types. Similarly, a well-analyzed cross heterozygous for Tl(1;VZZ) gave unusually many n 2 types (namely 11 out of 4400 progeny, or almost 10% of all 129 recovered aneuploids; and in this case also over half of the n 2 were disomic for group 111). However, such observations have remained entirely sporadic and their overall frequencies do not conform to the expectations based on Grell’s hypothesis. They may, on the other hand, be comparable to results by Rick and Gill (1973), who obtained various aneuploids, usually trisomic for shorter chromosomes, from crosses with primary trisomics of tomato. But if it is taken into account that in various situations n I11 types are the most frequent [e.g., among aneuploids isolated from diploids (Pollard et al., 1968), or among n 2 types from any source, see above], the inconsistent recovery of n+ 2 types may be attributable to the very poor viability of most types. Consequently, double disomics would be viable and expected only in certain favorable cases, and their low frequencies could not be used as an argument for or against any hypothesis. As a further check, crossing-over in n and n+ 1 progeny has been compared in a random sample of values from crosses heterozygous for T1 (ZZZ;VZZ) or T l (V;VZ). It was found that in the 27 randomly chosen comparisons from these crosses, recombination is as high in disomics as it is in haploids (namely, values were larger in n 1 than in haploids for 14 of the 27 analyzed pairs of markers, and smaller in 13). This agrees with the general similarities of values between n and n 1 obtained from

+

+ + +

+

+

+

+

+

+

58

ETTA KAFER

all other crosses and may be analogous to results with Y-autosome translocations obtained by Novitski (1975). From all the combined evidence, it seems possible that there is no regular occurrence of distributive pairing in Aspergillus; since noncrossover chromosomes hardly ever seem to exist, this is not really surprising. I n extreme situations, with very high or no crossing-over, distributive pairing may not be needed [as indicated by the lack of it in Drosophila males; or in the cases of “uncoupled” mutants which lack distributive pairing but do not affect exchange, or the disjunction of bivalents that have undergone exchanges (Carpenter, 1973) 1. Ill. Mitotic Recombination

A. MITOTICCROSSING-OVER AND NONDISJUNCTION IN STANDARD DIPLOIDS Several types of mitotic segregation which lead to spotting or variegation have been demonstrated in various organisms. I n Aspergillus, two main types occur spontaneously: (1) Mitotic crossing-over, which seems to occur in all fungi, and possibly all Diptera, generalizing from cases in Drosophila (Stern, 1936) and the house fly (Nothiger and Dubendorfer, 1971). So far it has been reported only in very few higher plants and animals (e.g., recently in Tradescantia by Christianson, 1975). (2) Mitotic nondisjunction, which seems to be a universal process, the reported frequencies of which vary mainly with the ease of identification. Absolute Frequencies. I n higher organisms it is extremely difficult t o estimate the frequencies of these processes, since the somatic cells involved show various rates and extents of division during development, and clone size may also be affected by the mutants used for detection. I n fungi, where vegetative growth is fairly uniform and mononucleate units can be sampled (e.g., in conidia) approximate estimates can be made, provided proper correction for clonal distribution is applied [ Luria and Delbriick (1943), as shown for crossing-over in the right arm of group I1 (Kafer, 1958) 1 . The incidence of mitotic crossing-over using color markers has been estimated in two similar sets of experiments. It varies from about 0.1 to 0.3% per chromosome arm, probably correlated with arm length (Kafer, 1961), giving a total estimate of over 3% [since a t least 6 of the established 13 arms are very long, and probably only 3 relatively short (Clutterbuck, 1974) ; see Fig. 151 . Nondisjunction, based mainly on aneuploid frequencies, probably is also a t least 2 3 % , considering the difficulties of identification and lower survival [ as evident

IN

RECOMBINATION AND TRANSLOCATIONS

59

Aspergillus

from the fact that none of these types were recovered in similar experiments of Pontecorvo et al. (1954) 1. Such information is valuable mainly for estimates of coincidence of the various types of segregation, e.g., double crossing-over vs. nondisjunction (see below). For all other purposes, mitotic recombination frequencies between genes in diploids are so low, and such determinations are so laborious, that more convenient measures of relative frequencies are used by most investigators, and also in all other experiments discussed here. 1. Mitotic Crossing-over

Mitotic crossing-over occurs a t the four-strand stage of mitosis, SO that there are two possibilities for the segregation of centromeres (Stern, 1936; Fig. 4). Since mitotic recombination is rare, products are usually selected. Namely, intergenic crossovers are obtained as segregants with increased resistance or striking phenotypes, when fully or partially recessive markers become homozygous (type I segregation) ; and interallelic crossovers are selected as wild-type recombinants between different alleles which cause a requirement or sensitivity to an inhibitor (type I1 segregation). The latter type of selection can lead to recovery of both strands

I

B ;'

+

su

+

+

SUI

Ps;

+

+ 4

Y

+ +

independent exchanges at 4-strand stage of mitosts

bx-" cha 4

Two possible types of segregation of centromeres

I

Twin-spots. homozygous distal to CO .1

30 pius

PIUS

2.

-

40

a Parental

phenotypes Heterozygous segregants. unchanged

1.

4o

Reciprocal 2.

cr0-m30

sy

:

+

+ 8YI

+BuI

:

u:

rib

i

+

: :

i

;,bO

pro 1

- .

IYS

-

I

4

+

ly.

7.t

I

i5 i

/

Cha

fw

i7 i Pro 2

\

I

+

+ +

cha C b

-v

FIG. 4. Reciprocal mitotic crossing-over and genotypes of various segregants resulting from coincidence of exchanges in I L and VIII R, considering the two possible types of segregation of centromeres in mitosis.

60

ETTA KAFER

involved in the exchange [in about half of the cases, Fig. 4 (Roper and Pritchard, 1955) 1 . However, such recombinants frequently result not from reciprocal crossing-over, but from gene conversion [as shown even in Drosophila (Kelly, 1974)l. Because of the ease of selection, allelic recombination is used extensively as a tool for measuring recombination frequencies [e.g., in various uvs mutants of Ustilago (Holliday, 1971) ] or for the mapping of noncomplementing alleles [induced in yeast, e.g., by sunlamp irradiation (Lawrence and Christensen, 1974) 1. Also, its frequencies are relatively high, often barely ten times rarer than meiotic ones. However, because mitotic conversion occurs with relatively high frequencies and does not seem to be coupled to outside marker exchange, sequencing of outside markers or centromeres is not easily possible from segregation of markers in selected allelic recombinants (Davies et al., 1975). This method has therefore been tried only once (for group IV; Kafer 1958), but not used further, and only intergenic crossing over is discussed here. In selected intergenic crossovers, only one of the two strands involved in an exchange is recovered. I n Aspergillus, these have been identified first by color (Pontecorvo et al., 1953, 1954) and later by resistance (Roper and Kiifer, 1957), and selection of the latter type is so far the only method available in Dictyostelium (Williams et al., 1974; Gingold and Ashworth, 1974; Katz and Kao, 1974). However, in systems that select by visual criteria it is possible, with suitable markers in repulsion, to recover all four strands: namely, one crossover and one parental each in two “reciprocal” diploid segregants, as “twin spots” (Stern, 1936 ; Wood and Kiifer, 1967) ; (Fig. 4 ) . Indeed, since no other process is known that would produce two fully viable segregants with this type of complementarity, such twin spots are the one conclusive criterion for somatic crossing-over in some species of higher organisms [e.g., in soybeans (Vig and Paddock, 1968) 1, Relative Frequencies, Distribution. As in Drosophila, mi totic crossingover in Aspergillus is concentrated in the centromere areas and shows characteristic distribution in each arm which differs from that of meiotic crossing-over when large enough samples are compared (e.g., Table 10). However, even small samples can be used in a qualitative way to determine the sequence of markers on a chromosome arm, provided a distal marker is available for selection. One basic problem is to identify crossover segregants and distinguish them from those produced by other processes. This is especially difficult in the first stages of analysis, as exemplified by the problem of interpretation encountered by Pontecorvo and Roper before haploids were distinguished (in Pontecorvo et al., 1953). And even a t later stages, nondisjunctional segregants are often

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

61

impossible to identify with certainty ; for example, after definite mapping of the centromere to the “left” of sD and coA ( M a and Klfer, 1974; Clutterbuck and Cove, 1974), it is now known that some of the co ribo cha “nondisjunctional” segregants of Kafer (1961) must have been crossover types. This latter distinction is not crucial for the sequencing of markers. But it is essential for the mapping of centromeres, as is good recovery of diploid vs. haploid segregants and uniform viability of all segregant types (discussed in detail in the section on genetic mapping). Specially marked diploids have now been prepared that facilitate visual distinction of the various types of segregants and therefore make it easier to observe and measure the relative frequencies of the various types of segregation (Table 9 ) . All of them contain two color mutants in repulsion on the right arm of linkage group VIII, fawn (fwA) and chartreuse (chaA) ; in addition they have two or three selective markers in coupling on the two arms of group I (including the suppressor suAadE on I L, and yA = yellow on I R in all cases). Therefore, if markers on I L are selected (e.g., suA), all haploids are either fawn (yA;fwA) or yellow chartreuse (yA;chaA) and can easily be distinguished from diploid segregants, which may be green crossovers (wild-type color) or yellow (yA/yA) nondisjunctionals. Very rarely, resulting from two events of recombination, diploids may be chartreuse (chaA/chaA) or fawn (fwA/ fwA) ; the latter can be visually distinguished from lighter fawn, yA;fwA, haploids only under optimal conditions. The distribution of crossing-over in both arms of group I is shown in Table 10 (results of diploids 2196 and 2200, genotypes in Table 9, and of diploid 859, genotype in Table 11). These results demonstrate the general agreement, but also the discrepancy in the prod-centromere area, compared to the similar published results of Pontecorvo and Kafer (1958; diploids Y and Z, Table 10; extensive analysis to explain these discrepancies is discussed below). Diploid 2196 is heterozygous for fpaB distal on I L, which permits selection of resistant segregants on complete medium (CM) supplemented with p-fluorophenylalanine (pfp). This extends the mitotic map of I L and was intended to provide a simple method for teaching of mitotic recombination, since, on media containing pfp, all sectors, even haploid ones, are usually large enough to be tested without prior purification. However, selection on CM + p f p produces such a predominance of haploids that in two large samples analyzed here, only the few diploid segregants shown in Table 10 were obtained. These results are, therefore, very approximate and, judging from larger samples published previously ( M a and Klfer, 1974), the value for interval I1 may be underestimated. Selection for f p a B is therefore very suitable when haploids

62

ETTA KAFER

TABLE Genotypes of Diploids for Teaching Diploid

No.

475 2196

2198

2201 2200

Linkage groups

Haploids FGSCNo.

473 475 474

fpu-sensitive

1 f p a B galD

suA

+

riboA

a n A pabaA

+ galD s u A + riboA a n A + + + SulA + + + galD s u A + riboA a n A (s+) + + SulA + + 479 -

yA

+ + + SulA + + + + f p a B galD s u A + riboA a n A pabaA y A + + + SulA + + + + diploids

513 466

yA

+

+

pro-94

biA

+

+

+

are wanted, but not economical for quantitative analysis of crossing-over (the reverse is true for selection using sulfite-requiring mutants, like sB or sC on selenate media ; see Tables 19 and 20). 2. Nondisjunction

Haploid and diploid segregants resulting from misdistribution of whole chromosomes may arise from several processes, especially when inducing agents are used (see below). I n Aspergillus, spontaneous nondisjunction usually involves missegregation of single or few chromosomes, producing trisomics as primary products (see Fig. 5 ) . Such trisomics have reduced growth rate, but produce diploid nondisjunctional segregants repeatedly by chromosome loss or %econdary nondisjunction.” The determination of absolute frequencies of nondisjunction is therefore very approximate (see above), and it is not surprising that even their relative frequencies (e.g., compared to diploid crossovers) may differ under different conditions. Haploid segregants are likely to be the result of several steps of chromosomal loss, presumably starting with the 2 n - 1 products which only survive in heterokaryotic condition (Kafer, 1961) (for viability and phenotypes of aneuploids see above, “meiotic” aneuploids) . Cytological evidence for these postulated steps has recently been produced by Brody and Williams ( 1974) in Dictyostelium, where diploids produced aneuploid nuclei of all classes, from 2 n 2 down to n 1. However, no recognizable aneuploid clones could be maintained even from spores with sizes falling between the usual diploid or hapoid ones. Observed Belative Frequencies of (Diploid Crossover a n d ) Nondisjunctional Segregants. For the left arm of group I , using suA as a selective

+

+

+ +

biA

adE biA adE lysF pabaA y A

+

pabaA

adE adE adE adE

+

RECOMBINATION AND TRANSLOCATIONS IN

63

Aspergillus

9 and Induction of Segregation Linkage groups

I

I1

I11

v

IV

+ + ++ + + + AcrA w A ActA pyroA facA

+

+

+ --

+

+

+

+ - -+ -

+

AcrA

ActA

+

+

+

+

Selection of

su

+

+

suI

+ lacA

+ sB

+

+

+ +

VIII

s D f OliA choA

+ +

+

choA

w

A

+

+

+ + riboB chaA s D f w A + + + + + chaA

+ + + + + + + + + + + choA + + + chaA lacA sB + + + + + + + + + +

AcrA ActA pyroA facA a d E+ --adE AcrA pyroA facA

+

VII

VI

lacA

sB

+

OliA

+

+

+

chaA

z: Nondisjunction of single chromosomes ( 2 steps)

0

I

Double Nondisjunct ion

I

Yad

+

+ ad

bi

su

+

su

+

-

Yad + Y d +

+ ad

bi

trisom

/i

loss of extra homolog

+

su 2n

-

-

Y

d

+

ad

+

nondislunctional ~y

su

+

+

SUI

parental 2n

y

A

v

+ a d bi

FIG.5. Mitotic nondisjunction in group I producing sulsu, y/y segregants: either double nondisjunction, or two steps of single nondisjunction with an “unstable” trisomic 2n I intermediate.

+

TABLE 10: Relative Frequencies of Mitotic Crossing-over in Various Intervals of Both Arms in Group 10 DIploid and soled

Selective marker

I

I I I U su

fpaB gal

+

m B gal

or

&

S

U

S u l +

+

+

S d +

+

+

+

+

+

+

+

gal

sUA

1pMB ad

sUA

+

suA

fPaB gal

+

gal

su

m B gal +

rib0 rib0

an

ribo

+

--

pro

an

+

+

+

fiaB gal

s

S su

t

+

+

-

u

l

+

+

+

d

+

-

-

+

rib0

+

+

an

pro

--

+

I

z

o

s

7%

5%

Y

II

1 6

+

+

m

38%

+

+ +

N

20%

5%

(12

+

+

v

18%

5%

(6

+

Y

+ V

+

+

+

+

+

+

Y

x

J y s Paba Y Paba Y

paaa Y

+

Y

+

+

Y

I Y Pnba ~ Y

Observed numbers are given in parentheses.

10% (24

+ 48)

17%

+ 44)

14%

I

21%

Y

1

+

%

+ +

vmIx

-

lntervab

X

1 marker 859 Y a n d Z SuA SUA

Paba Y

+

~ Y S

I

+

su +

+

---

po

ribo an

+

=

+

+

su

fpaB gal +

+

+

~YS

-

+ + s u l + +

JpaB gal su + + + & YA

+

+

SUA

an pro

+

su i

A

--

V V I V I I V I I I M

ribo

S d +

+ +

JpaB

YY

+

rn W

+

Meiotic

1 VI

+

29%

68%

(127)

(73)

(169

20)

6!%

(102)

51%

(30)

+ 244)

16

I11% (998)

yA (1) <0.5%

YA 3%

vm

“4%

(164)

49%

48%

Ix

3%

(62)

18%

10%

X

16%

(123)

33%

33%

y/y double Co

23%

7%

nondisjunctionals

(3)

Totals:

(374)

1%

2

p: 6s96

>28 (407)

YA 5%

68%

(1) <0.5%

M

22%

-

1%

6%

5%

(181)

(310)

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

65

marker, yellow ( y A / y A ) nondisjunctional diploids showed frequencies of 6 1 0 % (not shown in Table 10, these are “yellow suppressed” sectors, among all LLsuppressed’’ sectors from diploids of Table 9 ; totals inspected, over 2000). These values vary slightly according to the method used for the choosing of “random” samples of sectors: yellow nondisjunctional sectors are conspicuous and easier to identify than green crossover sectors, so that the former are isolated preferentially when “all” sectors are counted rather than the “first” large one from each colony. However, from seven other diploids, which did not show such color differences, a truly random and similar average of 6.5% nondisjunctionals was obtained among suA/suA diploid segregants (120/1883 sectors tested). For the right arm of group I , nondisjunctional types among yellow segregants, selected as single heads from colonies of various diploids on CM, seem to show a lower frequency, which generally ranges from 1 to 6% (see y A / y A segregants in Tables 10 and 12). The overall average was found to be 3.7% (239 nondisjunctionals among 6367 yellow segregants isolated from 30 diploids) ; that is, only slightly more than half that found among suA/suA segregants. This is contrary to expectation, since the distance from the centromere to suA on the left arm of I is much longer than that on the right arm to y A , so that nondisjunctional types should be a relatively higher fraction among the y A / y A than among suA/suA. The discrepancy may, however, be the result of different conditions of selection which are expected to affect growth of unbalanced 2n 1 types and, consequently frequencies of secondary nondisjunction. However, it is hard to imagine more favorable conditions than exist in the heteroknryotic situation on CM when yellow heads are picked ; but i t has been found that growth conditions of heterozygous suA/+ diploids on medium lacking adenine seem to be unexpectedly favorable for aneuploids [especially suA/suA/+ types, but others as well (Kafer, 1976) 1. Because of such variables which influence recovery, i t therefore does not seem possible to use the incidence of nondisjunctionals as a common base line for comparisons of crossing-over frequencies in both arms of a chromosome, as theoretically should be the case.

+

3. Double Crossing-over us. Nondisjunction, and Coincidence

Considering the relative frequencies of mitotic crossing-over and nondisjunction, i t was concluded (and assumed above) that segregants homozygous for all markers of a chromosome are much more likely to be of nondisjunctional origin. Crossovers, resulting from double or consecutive crossing-over in both arms, are expected to be much rarer, in spite of the relatively high concentration of crossing-over close to the centromere. Evidence for this assumption has been obtained recently, using

66

ETTA KAFER

diploids of Table 9, which contain markers facilitating rough estimates of double crossing-over or coincident segregation. I n these diploids, 2-strand double crossovers with exchanges in both arms of group I or nondisjunctionals will be yellow diploid segregants, if suAadE is used as a selective marker; but, in addition, four-strand doubles are also recognizable since y+/y+ are darker green and such diploids are easily identified (because in these diploids all haploids are lighter colored). Assuming no chromatid interference, these two types, y A / y A or should be equally frequent (and so far we have no evidence against this assumption). The values obtained for four-strand double crossing-over ranged from zero to about 1% with an approximate average of 0.245% (in over 20 samples of about 300 each). Double crossing-over is, therefore, no more frequent than expected from random coincidence of single exchanges (unless two-strand doubles across the centromere are disproportionately frequent). To obtain a further estimate of the relative frequencies of nondisjunction and two-strand double crossing-over (other than across the centromere) “suppressed” yellow segregants (suA/suA y A / y A ) from diploid 2200 were tested. Among these it was found that a minimum of 6.5% (8/127) were definitely double crossovers (not homozygous for pro) ; this value leads to a minimum estimate of 0.4% double crossovers among all suA/suA segregants, well within the range indicated above ; therefore, nondisjunction is about ten times more frequent than double crossingover in this chromosome. Coincidence of mitotic crossing over in different chromosomes is also found with frequencies no greater than expected from random occurrence of single exchanges (but in most cases nondisjunction was distinguishable from crossing-over only for group I ) . For example, diploids of Table 9 permit a rough estimate of coincidence in I L and VIII R, when suA/suA sectors are inspected: all “suppressed” chartreuse (suA/suA; chaA/chaA) sectors are the result of crossing over in I L plus crossing over in VIII R (or nondisjunction, but presumably no more than in ca. 10% of the cases). Their frequency was found to be roughly 0.5% (3/413 in one tested sample with presumably slightly preferential isolation). Coinfor AcrA) among cidence of segregation for AcrA (producing segregants selected for a marker of group I has been observed with similarly low frequencies (5/1476 segregants from three different diploids = 0.3%). These results therefore indicate that multiple events of mitotic recombination occur a t random in Aspergillus. This was found not only for spontaneous segregants, but also for induced ones (Shanfield and Kafer,

+/+,

+/+

RECOMBINATION AND TRANSLOCATIONS IN

67

Aspergillus

1971). In Drosophila, on the other hand, results by Garcia-Bellido (1972) suggest that after induction with X-rays double-crossover types are too frequent to be due to independent events in all cases.

B. CROSSING-OVER IN THE CENTROMERE AREAOF GROUPI GENETICDIFFERENCES BETWEEN STRAINS

AND

When mitotic analysis of T1 (ZL;VIIR) was started (Kafer, 1962), i t soon became obvious that the mitotic mapping of proA and of the centromere of group I presented special problems. From the control (diploid 600, which is p r o A l / + , Table 1 1 ) results similar t o those shown above for diploid 859 (Table 10) were obtained: namely, significantly more of the “suppressed” (suA/suA) crossovers were homozygous for proA1 than obtained previously, and considerably fewer proA/proA were found among the “yellow” crossovers than observed previously in diploids Y and Z (Pontecorvo and Kafer, 1958). To trace the possible mutation and to check whether genetic or technical differences caused these discrepancies, twelve proA1 strains from the two proA1 pedigrees were combined with suitable tester strains and checked for segregation of proA1, by selecting “suppressed” as well as “yellow” crossovers. As a first finding, i t could be shown that when high levels of proline were used in the selective medium lacking adenine (rather than normal levels of arginine, as used previously) all diploids heterozygous for proA1 in coupling with suAadE and y A , produced significant frequencies of “suppressed” pro/pro crossovers in the left arm, which always were considerably higher than those of yellow pro/pro crossovers on the right. It was concluded, that the centromere must, therefore be located t o the right of proA, rather than to the left as deduced from earlier results [and also concluded by Strickland (1958) on the basis of results from unordered tetrads]. It became clear that only poor viability under the conditions used previously, had failed to show up the relatively high amount of mitotic crossing-over on the left arm between proA and the centromere which occurred in all diploids. However, if this was the case, an explanation would have to be found for the “yellow” pro/pro “crossovers” (still heterozygous for all other markers on the left arm, e.g., suAadE) . These diploid pro-segregants could possibly have arisen by “double” events (like crossing-over between proA and suAadE, followed by nondisjunction ; as previously assumed for the corresponding “suppressed” pro segregants of diploid Z ) ; but, since they were consistently found with frequencies about half that of normal nondisjunctional yellow segregants, they are much more frequent than expected from chance

TABLE 11 Genotypes of proA/+ Diploids (Groups I and I1 Only) Diploid Nos.

Available testers: FGSC Nos.

1408" 600 348 854 1388" 863 348 85 1

+

+

anA

luA

suA riboA

+

+

+

850

+ proA1

+

anA

+

+

suA

+

+

+

proAl

anA

adG

+

+

+

proA11

+ +

+

+ suA

12760

proA1

+

suA 1276"

864

+

s u A riboA

859

I1

Linkage groups

+ suA

riboA fpaA91

+ + + + + +

anA

+

+

+

anA

luA

+

+

anA

luA

+

+

+ proA1

+ proA2

+ pro-94

-

0

0

-

m

rn

0

lysF P a b a A

+ + + +

+

yA

adE

+

+

adE

biA

pabaA

yA

adE

+

+

+ +

adE

biA

adE

biA

YA

adE

pabaA

AcrA

+

+

wA

AcrA

+

. + +

0

wA

wA

M

e e

P

m

+

pabaA

YA

+

+ +

+

+

+

adE

biA

+

wA

+

+

yA

adE

+

Acra

wA

+

+

0

AcrA

wA

+

+

-

AmA

wA

+

+

+

+

+

adE

biA

+

+

yA

adE

+

+

+

+

adE

biA

+

+

yA

adE

+

AcrA

+

+

+

P

4

M

s 0

0

867

866

((

suA 50

+ suA

871

suA

+ +

+

+

+

+

+ +

+ +

+

pro-94

+

proA2

+

+

868

suA

+

+

869

suA

+ +

+ + + +

55

+ 865

suA

896

suA

+ +

+

riboA

897

{{

509

suA

+

Tester strain lost, Montreal number.

+

+ +

ZuA

+ + adG

+

+ + +

proA2

+

+

-

1

-

pro-94 proA2

+ pro-94 proA2

+ pro-94

-

+

+

YA

+ +

+

+

adE

yA

adE

YA

adE

+

+

yA

adE

yA

adE

+ +

+

+

+

yA

adE

yA

adE

+

+ +

adE

+

yA

adE

+ + + + + + + + +

+ + + + + + + +

+

+

+ biA

+ + biA

+

AcrA

wA

+

+

Acra

wA

AcrA

wA

+

+

AcrA

wA

AcrA

wA

+

+

AcrA

wA

AcrA

wA

+

+

AcrA

wA

-

A

70

ETTA KAFER

coincidence of crossing-over and nondisjunction. I n addition, however many other markers were present (e.g., the close markers EysF in I R and luA in I L) in no case was there evidence for any type of segregation in these yellow or suppressed pro-types other than “single crossing-over” on either side of proA (the genotypes of all, or a t least ten, pro segregants from both selections from every test diploid were analyzed by haploidization) . Since such ambiguous results might possibly be produced by diploids heterozygous for an intrachromosomal aberration, it was provisionally assumed that a small pericentric inversion had been induced together with proA1 and closely linked to it. To explain the ambiguous results, all test diploids would have to be heterozygous and the aberration would not be present in any of the pro+ tester strains used. This actually might be expected if the inversion was small enough and meiotic recombination reduced enough, that it remained virtually associated with proA1. As a first check of this hypothesis two other available proA mutants, proA2 and proA11, were analyzed for mitotic segregation. The results were very similar to those obtained with proAl (Table 1 2 ) . Therefore, if a chromosomal aberration was the cause of the ambiguous results with proA1, this same aberration would also have to be present in proA2 (and proA11) and presumably originate in biAl in which both proA1 and 2 had been induced. The only strain definitely without i t would then be the original wild type, mutants induced in it like yA2, and possibly progeny from backcrosses to the former. To test this prediction, a new pro mutant was induced in the original wild-type strain. In several tests this mutant, pro-94, gave consistent results, namely a considerable frequency of ‘Lsuppressed’l pro/pro mitotic crossovers and practically no “yellow” ones (e.g., diploid 2200, Tables 9 and 10; pro-94 is closely linked to proA2, but probably an allele of p r o R ) . To demonstrate clearly that a pericentric inversion was present in the biAl strain it was necessary to construct a diploid homozygous for the aberration and heterozygous for pro. I n such a “biA” diploid proA should map consistently to the right of the centromere, just as it mapped to the left in homozygous “wild type”; only structurally heterozygous diploids should produce ambiguous results. Pairs of diploids were therefore constructed using the most suitable pro+ tester strains, each against both the original proA2 and the pro-94 strains (in which suAadE, y A adE, had been introduced by mitotic crossing-over in distal segments, Fig. 6). These pairs of diploids are shown in Table 11. The results, obtained when crossovers were selected on both arms of group I, are given in Table 12. They do not show the pattern expected from a small inversion in the centromere area of the original birl-strain. Only the diploid homozygous for “wild type” centromere area (diploid 865)

TABLE 12 Frequencies of pro Crossovers among Segregants Selected on I L (suA or fpaA) or I R ( y A ) in Diploids Containing Different pro Strains Combined with Various Tester Strains ~

Centromere type -+ type 1 Selection: Tester strain FGSC No. “Wild type” Mixed

17

Suppressed suA/suA Diploid No. 865

% -15%

864

“biA”

55

868 -13%

“biA”

50

866

Mixed (Fd

(Fraction)

897 2 3 . 5 % (71/302)

Centromere type +

1

863 “Mixed” 348 or (Fs) M 1276

(Nondisjunctionals %)

Diploid No.

(3 %)

869

1. I % (8/715)

(2%)

1 . 0 % (3/292)

(1.3%)

%

(Fraction)

(46 incl. N)4 221 1 . 5 % (3/196)

13% (190/1483)

Suppressed

Yellow ( y A / y A )

(67 incl. N). 283 1 . 0 % (2/207)

18% (187/992)

(M 1276 lost) 509

(Fs)

“biA-type” (proA2)

“Wild-type” (pro-94)

9 % (10/109)

%

(Fraction)

850

(4.2%)

896

12% (48/339)

4 . 3 % (16/368)

(3.5%)

867

”Mixed” (proAll) Selected fpaA/fpaA

(Fraction)

(Nondisjunctionals %)

(19 incl. N). 2 . 1 % (6/281) 136 11 % (63/597) 2 . 6 % (13/478)

871

1 . 8 % (12/653) (3.5%)

%

Yellow

-’%

-10%

(43 incl. N). 238 ~

.

10% (119/1228)

-

(4.6%)

_

5% (21/426)

(5.4%)

“biA-type” (proAl) Yellow

2 % (6/251)

(3.1%)

851

8 % (18/217)

5 % (7/141)

(7.5%)

a suA pro/++ diploids homozygous for yA and adE, were used t o select suA/suA types, among which pro crossovers could not be distinguished from nondisjunctionals (N).

72

ETTA KAFER

+ proA- strain -

-

Marker strain suA yA adE

-

-

3) selected white

C>

proA

+

bjA

AUA

WA

haplold:

FIG.6. Stepwise insertion of selective markers .yA (and simultaneously adE) distal on I R, and suAadE distal on I L, into pro strains by mitotic crossing-over, followed by haploidisation. gives unambiguous results: these again place pro consistently onto the left arm, provided 1% exceptional pro-yellow types are disregarded as possible coincidence of two events of segregation. All other diploids give significant frequencies of “yellow” pro/pro “crossovers,” even though considerably less than “suppressed” pro/pro. These results, therefore, rule out the existence of a pericentric inversion in the original biAl strain but, on the other hand, definitely implicate genetic differences between biA1 and the original wild type ~ b sthe basis of the different results obtained with proA1 (or proA2) compared to pro-94. Since only diploids with “wild type” centromeres give results in agreement with current models of mitotic crossing-over, it looks as if the original biA 1 strain contains a mutation that affects mitotic segregation close to the prod-centromere area. Because no testable hypothesis comes to mind, details of these results are given here as accurately as possible for future consideration: Twelve different derived proAl strains were checked for mitotic crossing over in 18 different diploids. These produced on the average 9.3 k 1.1% of %uppressed” pro/pro crossovers (among 4913 selected ; range 5-15%). From 13 of these diploids, “yellow” crossovers could also be selected (total tested 1880) ; these were homozygous for the proA allele in coupling with yA with an average frequency of 3.4 f 0.5%.However, there appeared to be differences between different proA1-strains ; e.g., the “early” proAy+strain from the published diploid Z gave the highest values of apparent

RECOMBINATION AND TRANSLOCATIONS IN

Aspergil lus

73

crossing over to the left of proA among yA/yA (when tested in three different diploids, including diploid 854, genotype in Table 11): i t showed a range of 5-7% among a total of 452 yA/yA (which all had t o be analyzed for heteroxygosis of proA1 by selection of suA-wA haploids, since in these diploids proA was in replusion to yA). On the other hand, the derived proAl-“control” strain for the translocation work, when tested in four different diploids (one of them being diploid 600, Table 11) showed significantly lower values, ranging from 1.1 to 3.6% (average 1.8%, 539 yA/yA tested; x2 = 10.5, P < 0.01). Most other strains of the pedigree between these two proAl-strains gave, however, intermediate values (average 4.0%, 689 yA/yA tested from 6 diploids). The following procedure was used to check the original proA11 (pabaAl yA2; pyroA4) strain for proA-segregation. T o avoid all crossing, a new fpaA-allele (fpaA91) was induced in this strain by nitrosoguanidine treatment, so that fpaA/fpaA crossover segregants could be isolated on the left arm of group I, as well as yellow ones on I R (diploid 863, Table 12; comparable to the selection in both arms after introduction of suAadE on the left arm, and yA ad23 on the right arm, into proA2 and pro-94 strains, Fig. 6, and Table 11). The general trends emerging from a closer inspection of the data of Table 12 are the following: All diploids which contain suA in coupling with pro-94 of “wild type” origin, give higher frequencies of “suppressed” pro mitotic crossovers on I L (ranging from 13 to 23%, average 15.8%), than the corresponding diploids with proA2 of “biAl” origin (which show a range of 9-1276, average 10.8%). In addition, all the former diploids show lower frequencies of “yellow” pro/pro crossovers on I R (1.0-1.8%) than the latter (range from 2 to 570, average 3.4%). While for any one pair of diploids these differences are barely significant a t the 5% level, the combined values show highly significant differences (x2 > 10, P < 0.01, as expected from the fact that there is no overlap of values). I n addition, the centromere type of the pro+ ‘V.ester” strain also seems to influence results, since less yellow pro exceptional types are produced when pro-94 is combined with a tester having a “wild type” centromere (diploid 865) than when the tester is a “biA”-type strain (diploids 866 and 868). When all data are considered, it seems clear that only diploids with “wild type” centromeres give unambiguous results as expected from reciprocal mitotic crossing-over, while none of the diploids with “biA”-type centromeres conform to these patterns, presumably due to some type of mutation (that may also cause the correlated higher nondisjunction). To speculate on the kind of mutation which would cause the observed results one could postulate that i t creates a weak alternate centromere position (“spindle fiber” attached to the left of proA) which would result

74

ETTA KAFER

in about 1.5-20/0 single crossovers on the “right” arm when the mutation is heteroaygous (diploids 969, 866, 868), and twice that much when it is homoaygous (diploids 854,Z and 867,896, etc.) . At this time it is difficult to visualize how this or any other alternate hypothesis could be tested, since the possibilities of mitotic analysis have been pretty well exhausted, and the cytology of mitosis is obviously too imprecise to be a suitable tool (Robinow and Caten, 1969). The only remaining possibility, namely investigation of the genetic differences between pro-94 and various proA mutants and the anomalous centromere behavior in the latter, by means of meiotic tetrad analysis might, however, be worthwhile. While previously no markers close to any other centromeres were known so that the centromere distances computed from unordered tetrads were, by necessity, very approximate (Strickland, 1958) markers very close to their centromeres have now been identified in both arms of groups 11, 111, and IV, and new potentially linked ones have become known also for all other groups (Fig. 15; Clutterbuck, 1974). Mutations causing changes, e.g., in heterochromatic content, that affect meiotic crossing over as well as nondisjunction, have been observed in several organisms (in maize, by Ward, 1973; or in Drosophila, by Carpenter and Sandler, 1974). I n the above case in Aspergillus, however, extensive crossing of the described strains with “wild type” or “biA” centromere areas to the same tester strains has not revealed any effect of the postulated mutation on meiotic recombination (results are included in Table 2 ) . Whether any other of the reported cases that show reduction of repetitive sequences in the centromere areas correlated with increases of nondisjunction, or diffuse centromeres, are relevant here is hard to guess, even though at least in one case differences between meiosis and mitosis were observed (Comings and Okada, 1972; Laird, 1973). C. MITOTICRECOMBINATION IN TRIPLOIDS Triploids of Aspergillus nidulans have been obtained previously. Provided they were heteroaygous for conidial color markers, it could be seen (Fig. 7) that they are considerably less stable than diploids, and that they produce a high frequency of color segregants, probably by chromosomal segregation (Elliott, 1956; E. Kafer, unpublished). However, i t turned out to be impossible to obtain quantitative information, as long as mainly recessive markers were available, even when almost every homolog contained a marker (as shown for one of these early triploids, triploid 407, Table 13). These triploids were constructed with the aim of recogniaing as many segregants as possible by having two mutant alleles of most

TABLE 13 Genotypes of Triploids Triploid HomoNo. logs

I

Linkage groups :

m

Il

a 407

b C

a

2300

b C

Groups :

Iv a

407

b C

a

2300

b C

+ +

V

GPYYrOA

QymA

---. ---. methG

+

+

methG

+ +

+

QymA

M

VI

==-L- + +

+

nicA

+

nicA

+

VIII

sB

+-

+

+

+

+

+

+

+ +

+

+ + +

+

m'cB

choA

+

-++

choA

+

+

0

+ + +

+

+

nicA

+

+

facA

+ + + + + sbA lacA sB

+

+ +

+ malA

+ +

OIiA

+

choA

+ +

+

-+

+ + i

riboB

+ +

chd ~~

s D f w A +

+ + +

+

nboB

~

chaA

+ +

nboB chaA

76

ETTA KAFER

recessive markers present in the triploid. Crossing-over or nondisjunction would then produce segregants differing in their requirements or color from the original triploid, so that such segregants could fairly easily be recognized. However, it turned out that segregation frequencies are so high, that the problem is not to obtain spontaneous segregants, but to distinguish the various types obtained. To assess the relative frequencies of crossing over vs. chromosomal loss or nondisjunction, crossovers should be distinguishable from nondisjunctionals, and triploids from diploids and haploids. Since it is impossible to prove whether a segregant is homoor hemizygous for all recessive markers of a homolog, such distinctions were difficult or impossible for most segregants from triploid 407. The situation is quite different for the recently constructed triploid 2300 (Table 13). It contains not only more recessive color mutants than triploid 407 (five rather than three), but also a relatively large number of mutants that can be recognized in the heterozygous condition ( f p a B , SulA, suAadE if adE is present, IodA, AcrA, ActA, sB, and OZiA). These can be used to distinguish diploid or triploid segregants from haploid ones, since in the former usually a t least one of them remains heterozygous; also loss of homologs containing these mutants can easily be followed. In addition, mutants are generally present only as a single allele which makes identification of all homologs in haploids possible (except for some white haploids, Table 15; the haploids selected on CM+pfp were isolated to check the genotype of the triploid). Only the mutant rib& of group VIII was present in two homologs, since it served as a balancing marker to obtain the heterokaryon between a diploid containing two of the homologs (b/c) and the haploid strain (a) from which the triploid was selected. I n addition, yA2 and yA91 (a recently obtained olive mutant) are noncomplementing alleles, which, however, can easily be distinguished in haploids (but not in the various diploid or triploid combinations) . Haploids and diploids were also distinguished visually from triploids (Fig. 8 ) . The results obtained with this triploid (2300) demonstrate very clearly that chromosome loss is occurring extremely frequently in triploids of A. nidulans. When conidia from the newly synthesized triploid colonies were plated onto complete medium, very high frequencies of unstable aneuploid colonies of many different types were obtained (30-3576 in two platings with totals of about 200 each). I n addition, most triploid colonies produced one or two color patches or segments large enough to be seen by the naked eye (see Fig, 7b) compared to 1-2 per 100 diploid colonies (Wood and Kafer, 1969) (Fig. 7a), and practically all of these resulted from chromosome loss. To obtain a random sample of segregants, all easily visible color segments from the triploid colonies of the first plating were purified by

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

77

FIG.7. Low-density platings of conidia on complete medium: (a) diploid; (b) triploid. FIG.8. Shape and arrangement of conidial heads permitting visual classification of (a) diploids, (b) triploids, as well as (c) haploid segregants.

78

ETTA KAFER

streaking, and the resulting stable types were tested for markers and for ploidy (285 segregants from 145 triploid-looking colonies, Table 14). Since few segregants remained triploid, chromosome loss is obviously the major process a t work, while nondisjunction is quite rare and mitotic crossing-over even more so. This is also evident from the large frequency of aneuploids found, not only among the plated conidia which resulted in sectoring usually hyperdiploid colonies, but also as precursors of many of the isolated color segregants. Triploids of A . nidulans therefore constitute an excellent source of aneuploids (comparable to triploid meiosis in yeast, Parry and Cox, 1970). a. Random Loss of Chromosomes. That the frequent loss of chromosomes was basically a random process was demonstrated in two ways: (i) All haploids, which constituted about one-third of all color segregants, were classified for loss of homologs for every linkage group. The resulting frequencies then were compared with that expected on the basis of random loss (Table 15). As can be seen from these results, very few cases are found where the observed frequencies differ significantly from the expected ones, and in three of these cases the known reduced viability of the corresponding mutants is likely to be the main cause (homologs Ia, Ic, and I I I b ; only for one, sbA on VIb, does the extremely low recovery remain unexplained). (ii) All diploid yellow segregants (mainly yA2/yA91) were checked for loss of any of the mutants conferring resistance as an indication of loss of one of the homologs of groups 11,111,VI, TABLE 14 Relative Frequencies of Various Types of Spontaneous Color Segregants from Triploid Colonies of 2300

Crossovers Color

Haploids

Yellow-olive

23

Darker or paler green White Fawn Chartreuse Yellow-cha

3 33 16 2 9

Total Nos. and frequencies

86 30 %

Chromosomaltype segregants

Total

2n

3n

2n

3n

No.

%

7

3

151

16

200

12%

25 33

9% 12%

i2

> 10 > 4%

(max. 9%)

> 167 > 60%

285

TABLE 15 Relative Frequencies of Recovery of the Three Homologs a, b, or c in Haploids from Triploid No. 2300

a

F!

Bis

m

Linkage groups and homologs

I a

b

I1

I11

IV

-c

a

b

c

a

b

c

a

b

V

VI

VII

VIII

--c

a

b

c

a

b

c

a

b

c

2el

(a, b a o r c ) b c

(b orc)

Totals (No.)

8 Z

.+ r3

No. from CM So 64 14b 33 19 34 30 22 34 41 lga 26 36 26 24 46 6" 34 33 26 27 29 (1) 23 llb (22) No. from 68c 1 2 24 24 23 48 Oc 23 27 15. 29 29 22 20 37 10 33 28 18 25 21 (8) 21 9' (12) C M pfp Total No. 76c 65 16 57 43 57 78 22c 57 68 34a 55 65 48 44 83 7a 67 61 44 52 50 (9) 44 20 (34)

+

Combined % x2

P

a

4gC 41 10 45.1' <.005

36 28 36 50 14c 36 43 22 35 41 31 28 53 4a 43 39 28 33 32 2.4 0.9 11.3e 4.7 61.P 2.8 >0.25 >.5 <0.01 -0.1 <0.005 >0.25

Homolog with reduced recovery.

63 <0.1 >0.99

* Small chartreuse or haploid green color segments are difficult to detect in the light green heterozygous triploid.

+

Ratios distorted in sample from CM pfp, which selects for fpaB on Ia, and against phenA on IIIb. d x2 refers to haploids from C M only. c Significant deviations from expected random recovery.

86 71 157

p r

0

P

z 3

0

2

P P '1

3. c1

80

ETTA KAFER

and VII. It was found that also in this case, the pattern of marker loss agrees well with that expected from random loss of one of the three homologs: The various combinations of loss of the same number of markers ( 1 or 2 or 3) occurred with similar frequencies, while th*eaverage number of cases for loss of any specific combination of markers decreased as the number of lost markers increased (from 0 to 4 ) . b. Mitotic Crossing-over. To check for mitotic crossing over vs. nondisjunction, all yellow nonhaploid segregants were analyzed for markers on both arms of group I. It was found that 10% of these (17/167) were still triploid, but only very few of them (3/17) were 3n crossovers (still heterozygous for fpaB as well as SulA). In addition, seven of the 151 diploid yellow segregants were crossovers (namely six fpaB+,SulA+ but not proA, and one f p a B SulA+ but not anA or p a b a i l ) . For these it is difficult to be sure whether crossing over occurred in the 3n, 2n or aneuploid stage. But there is no doubt that mitotic crossover segregants from triploids are exceedingly rare. c. Nondisjunction. The majority of the few obtained yellow triploid segregants (13/16) apparently are of a nondisjunctional type, in which loss of homolog Ic usually was compensated for by nondisjunction of I b (the most viable homolog, which was also the one most frequently recovered in haploids). As expected, few (1/144) of the yellow diploid segregants resulted from nondisjunction (i.e., were homozygous for all markers of I a or I b ) , since in triploid 2300 simple loss of homolog I a (carrying the only y+ allele) produced yellow segregants. This indicated that the observed process is not one of random distribution of chromosomes, but more likely one of preferential division of nuclei of lower ploidy. Therefore, all results agree, and they indicate that triploids of Aspergillus show relatively low frequencies of mitotic crossing over but randomly lose chromosomes a t a high rate. Triploids cannot be transferred since even in freshly synthesized 3n colonies up to a third of the conidia are already aneuploid. Such aneuploids are mainly hyperdiploid, and diploid segregants represent a relatively stable intermediate stage, similar to relatively stable clonal variants in mammalian cell lines (e.g., Terzi and Hawkins, 1975). I n addition, haploid segregants are also more frequent than in diploids. Such triploids appear, therefore, to be even less competitive than triploid cells in man (Mittwoch and Delhanty, 1972). The whole segregation pattern rather resembles that found in hybrid mammalian cells used for mapping of human genes, and is quite different from the situation in yeast, for example, where polyploids and even aneuploids are relatively stable [as analyzed in detail recently by Campbell et al. (1975) ].

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

81

D. INDUCED MITOTICRECOMBINATION IN STANDARD DIPLOIDS Since spontaneous somatic segregation is very rare, induction of such recombination is often resorted to, especially when it is used as a tool for analysis of various problems. The main question in these cases is whether the inducing agents actually increase the frequency of spontaneous recombination, or produce segregants hy entirely different mechanisms, or both. In either of the latter two cases the problem arises how to distinguish segregants that look identical but are the end products of different types of processes. This is all the more difficult because the end result depends on the relative viabilities of the intermediate products, like aneuploids or heterozygous deletions, and these differ greatly from species to species. Generalizations from one organism or system t o another are, therefore, difficult even if these are relatively closely related (as shown, for example, by the entirely different aneuploid tolerance in yeast, Aspergillus, and Ustilago). In addition, the results depend on the developmental stage of the treated nuclei and cells, especially in higher organisms like Drosophila, but also in fungi, (e.g., in Aspergillus) where quiescent or germinating conidia, or growing hyphae may be exposed t o inducing agents. The final results also depend on relative frequencies of induced and spontaneous recombination, and the abilities of various segregants to conipete with normal cells. For example, in Drosophila, X-rays are routinely used to increase mitotic crossing-over not only in developmental, hut also in behavioral studies (Hotta and Benzer, 1972) ; and evidence for reciprocal crossingover is based mainly on the production of “twin spots” when markers in repulsion on thc same chromosome arm are used (Stern, 1936). It is well known that X-rays also produce chromosomal aberrations and breakage which can be demonstrated by cytological techniques (e.g., Gatti et al., 1974), but survival of such nuclei is poor and no visible spots are expected from most of these (Stern and Tokunaga, 1971). Therefore, only secondary and very subtle differences between spontaneous and induced crossing-over show u p ; in this system these differences are quite impossible to trace directly to any primary effects of ionizing radiation [e.g.) differential effects of “Minutes” (Ferrus, 1975) ; or differences in relative distances in mitotic recombination maps (Garcia-Bellido, 1972) 1. Indirect deductions from relative frequencies of stable products, however, are possible. This is demonstrated by Vig (1973a,b) working with chlorophyll mutants in soybean, which so far is the best-analyzed system in higher plants. Several types of primary effects are deduced froin results in hornozygous controls compared to heterozygotes, which give spontaneous twin spots after treatment with various inhibitors of DNA

82

ETTA KAFER

or protein synthesis. Taking into account the known cytological effects of these chemicals on plant chromosomes, the following effects were distinguished: (1) induced mitotic crossing-over, indicated by proportional increases in twin and single spots (e.g., mitomycin C) ; (2) induced nondisjunction, recognized by lesser increases of twin spots than single spots (e.g., by sodium azide) ; and (3) segmental aneuploidy or terminal deletions by increases of certain single spots only [e.g., by fluorodeoxyuridine (FUdR) 1. a. Selective Methods. In fungi, induced segregation is being used in commercially important asexual species (e.g., in Penicillium, Ball 1973). I n such situations it is more important to obtain segregants with desired properties than to understand their mode of induction. Fungal systems, however, have the advantage that induced segregants can be investigated further, since subculturing [recently also achieved in higher plants (Dulieu, 1975) ] and analysis of their genotypes is possible by haploidization. Also, segregants can be enriched by selective techniques, which, on the other hand, have the disadvantage of restricting recovery to certain stable types. Much interesting information is lost in this way, not only about reciprocal types but also about unstable intermediate products, which could be recovered with appropriate methods. But when very rare events are being investigated, as in the case of recombination between alleles, selective methods may permit isolation of informative types not obtainable in any other way [ e.g., the long-stretch conversions obtained by Bandiera et al. (1973) 1. Also for fast tests (e.g., of possible environmental mutagens) , labor-saving selective techniques are very valuable [e.g., the efficient one of Bignami et al. (1974) 1. Or visual selection for color sectors can be used (Kappas et al., 1974) in a system comparable to that of Vig in soybean, provided well-marked diploids are used [like those in the top part of Table 9 (KBfer et al., 1976) 1. b. Nonselective Detailed Analysis. With nonselective techniques, analysis of the actual effects of inducing agents is possible in Aspergillus nidulans, since these permit to isolate unstable precursor products of induced segregation. In addition, their genotypes and their likely mode of origin can be deduced from the types of secondary segregants that any one primary type is able to produce. Using this information, and comparing the stable segregants obtained from different selective situations, it is then possible to deduce the interplay between induced primary effects and spontaneous secondary ones, as modified by competition between differentially viable types (induced poorly viable and spontaneously recovered segregants) . Such analysis is very time consuming and, therefore, can be carried out only on a small scale. To avoid competition between types with different growth rates, single

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

83

nuclei should ideally be treated and grown under optimal conditions. I n

A . nidulans conidia are uninucleate and suitable on that account, but

many agents act only on dividing nuclei. In most cases, therefore, quiescent and germinating conidia should be treated. The latter are only partly synchronized even in controls, and understandably less so when treatment delays germination (Scott and Alderson, 1974). The approximate time of the ,first mitosis can be recognized by the first signs of bulging in conidia which start producing germ tubes. For short treatments, effective chemicals can be added briefly before that time, during presumptive periods of DNA replication; less reactive ones may have to be present during the whole “preincubation” period, and either type can be removed by diluting and plating onto solid media at a time when many nuclei are in the first or second division. I n low-density platings normal diploid colonies can be distinguished from modified types, which represent aneuploid segregants or induced mutations, and the level of killing can be determined. If well-marked diploids are used, the following frequently encountered types can be distinguished: 1. Colonies of mutant color or with large mutant segments (often “twin” halves or quarters) that result from induced mitotic crossing-over. Testing of a few well-spaced spots around the periphery of normal colonies permits detection of segregation for biochemical markers and may reveal events at the half-chromatid level (Wood and Kafer, 1969). Such twin segments are induced, e.g., by UV, FUdR, or mitomycin C. But, as expected from work with other organisms (e.g., Ustilago; Esposito and Holliday, 1964), great differences are observed in relative rates compared to killing, or to induction of other segregant types (Shanfield and Kafer, 1971). 2. Visually identifiable trisomics as a result of mitotic nondisjunction. These show mainly a chromosomal type of segregation of markers, and give 1 : 2 ratios for recessive :dominant alleles, if the recessive one is present on two homologs [no specifically nondisjunction inducing agent has been identified; nitrosoguanidine (NG) produced a fair number of trisomics ; however, Na-azide successfully used by Vig (1973a) has not yet been checked]. 3. Abnormally growing types which, from single nuclei, produce a variety of haploid and multiply nondisjunctional diploid patches or sectors (but few and relatively small areas of hyperploid conidia). These seem to result from malfunction of spindles and show a general misdistribution of chromosomes. Such “breakdown” types are exceedingly rare among spontaneous segregants, but very frequent after treatment with agents that induce haploids [ e.g., p-fluorophenylalanine (pfp) (Morpurgo, 1963; Shanfield and Kafer, 1971)l. Evidence for effects on spindles has

84

ETTA KAFER

been obtained by Crackower (1972) using cytological methods for the fungicide griseofulvin, which also induces haploid segregants (Kappas and Georgopoulos, 1974). 4. Other abnormally growing types that frequently produce segregant sectors and often contain semidoininant lethals, e.g., terminal deletions. These are the most difficult to recognize since many resemble aneuploids, and produce stable segregants of the usual three euploid types [which often is interpreted as induced segregation (e.g., Kafer, 1960) 1. Only detailed analysis of a sufficient number of cases shows that they produce a wide variety of phenotypes, each one resulting from specific reduction in growth rate and conidiation (specific for the monosomic segment) and each with a characteristic pattern of subsequent spontaneous segregation [that is, ratios of crossovers and nondisjunctionals of a few types, and of haploids, are found to differ in each case (Kafer, 1963, 1969) 1. 5. I n normally growing survivors completely recessive lethals and chromosomal translocations are further detectable by haploidization, provided markers are present in all linkage groups (Kafer and Chen, 1964; Clutterbuck, 1974). That different techniques can produce different information in the same organism and different results in different species, is best illustrated by the effects of pfp in several Aspergillus species and Ustilago. While in A. nidulans pfp-treated conidia on CM produce abnormal colonies with a wealth of haploid patches in which aneuploids are relatively inconspicuous, diploid colonies grown on Chi containing pfp often form large bulges that are aneuploid and secondarily show conidiating haploid patches or sectors. A few of the most frequent and viable aneuploids, like 2n I1 or 2n V are even regularly obtained among purification streaks, as are also disomics for the same groups (Kafer and Upshall, 1973). Such specific and quite stable dieomics have also been observed in Ustilago violacea by Day and Jones (1971). They find that in this spccies resistant mutants are being selected for a t a fairly high rate, possibly in line with the observation of Talmud and Lewis (1974) that in some eukaryotes pfp is highly mutagenic. However, in A. nidulans, haploids induced by pfp do not show increased resistance (McCully and Forbes, 1965). If also in other Aspergillus species aneuploids are a relatively long-lasting stage in the production of haploids when segregants are selected from CM containing pfp, this could explain partly the observed, relatively high, rates of crossing-over in A. niger (Lhoas, 1967) or in A. flavus (Papa, 1973) : recently it has been found that disomics of A . nidulans show much higher rates than diploids for mitotic crossingover between markers of opposite arms (Section 111, F ; Upshall et al., 1977).

+

+

RECOMBINATION

AND TRANSLOCATIONS IN

Aspergillus

85

E. hlITOTIC RECOMBINATION I N TRANSLOCATION HETEROZYGOTES Diploids heterozygous for reciprocal translocations are expected to produce unbalanced products when crossovers are selected in one of the structurally heterozygous chromosome arms (see Fig. 9 ) . Such a crossover will be trisomic for one of the two translocated segments and monosomic for the other. Provided this unbalanced segregant is able to grow and produce further mitotic segregation, balanced diploid types, which form better growing sectors, will be produced. These result from an occasional event of mitotic crossing-over (or nondisjunction) in the other of the two involved chromosome arms. Theoretically such selected 2-step crossovers should therefore give information on the positions of the translocation breaks (Kafer, 1962). However, it has been found that results are usually more complex than expected and rarely useful for the mapping of T-breaks for the following reasons: Monosomy reduces viability drastically; standard 2n - 1 types, for example, are known to exist only as transient nuclear stages in heterokaryotic condition in the mycelium, but never to form viable conidia. The selected unbalanced crossovers, therefore, show reduced growth rates to various degrees, and recovery of these primary types becomes difficult or impossible except when the monosomic segments are small. In addition, the phenotypes of any viable crossovers are unknown, and their genotypes are usually indistinguishable from those of their second-order nondisjunctional segregants, so that the latter may be mistaken for primary types (Kafer, 1976). Therefore, it usually is impossible to identify the various segregant types before a translocation is well mapped, so that suitable markers can be placed onto the various chromosome segments. 1. Types of Recovered Crossovers and Steps of Segregation

a. Recovery and Viability of Primary Crossover Types. Since mitotic crossovers from translocation heterozygotes are monosomic for one translocated segment and trisomic for the other, it depends very much on the size and genetic content of these segments, which primary crossovers are detectable and/or grow enough to produce further stable segregants. Reciprocal crossing-over in either of the two heteromorphic chromosome arms is expected to produce a twin pair of unbalanced crossovers. Such twins are reciprocal for their imbalance, each one being trisomic for that translocation segment for which the other is monosomic. It was found, however, that, in both of the two cases chosen for detailed analysis, only some of the expected types could be selected. Half of them (one type of imbalance) was probably far too unbalanced to produce conidia on the selective media. This, most likely, is due to the fact that in both cases

86

ETTA KAFER

, * n v I

+

fpaA anA

A

VII

+

\ : - -

%A

+ +

,

Balanced TO VIO f

+

bA .

A

+

’

YA

+

phene

Selected croswer

+ -momK)somic W l t r i s o m r : segment malA

tho.

pink fluffy-aconial unbalanced type

+ SU*

biA

fpaA anA

7

slightly fluffy green. lrisomlc (tho*)

su’

malA

cho*

fpaA anA YA

+

darker green diploid

FIG.9. Genotypes of segregants resulting from three steps of segregation in diploids heterozygous for Tl(Z;VZZ) with jpaA in coupling (and suAadE = su in repulsion), selected on CM pfp: step 1. Mitotic crossing over in I L producing “fluffy’’ unbalanced types, fpaA/jpaA. Steps 2 and 3. Spontaneous steps of nondisjunction in group VII, resulting in well-growing trisomic 2n VII sectors that produce stable diploid segments, segregant for markers in VII R.

+

+

RECOMBINATION AND TRANSLOCATIONS

IN

Aspergillus

87

one break is fairly distal, and the second one is quite proximal in a long chromosome arm. Therefore, only crossovers monosomic for the smaller distal segment and trisomic for the very long one, have been recovered, but never the reciprocal types monosomic for the latter segments. Two translocations were chosen for analysis, both with one break in I L in which three convenient selective markers are located (suAadE, f p a A , and fpaB, see Fig. 15). I n the case of T2(Z;VIZI), the distal two of these markers are translocated (Ma and Kafer, 1974) so that selection becomes possible in both involved arms and all the viable crossovers could be selected (two from each diploid). But such primary crossovers, selected from T2(I;VIZZ)/+ diploids, were difficult to recover and to identify, since they grew very poorly and barely produced conidia; in addition, they always were outgrown by their spontaneous second- and third-order diploid segregants (Kafer, 1976). I n the case of Tl(1;VII)on the other hand, the break in I L is distal to all three selective markers (Kafer, 1975), and only one expected type of primary crossovers could be isolated: Selection was possible only in I L, and the selective marker had to be in coupling with the translocation (shown for fpaA in Fig. 9 ) . I n this case, the monosomic segment of I L is sufficiently smaller so that the primary crossovers grow quite well and show up as distinct sectors on selective media. Their conidiation is, however, reduced and they look “pink” and “fluffy,” so that any second-order diploid sectors show up clearly. On the other hand, when selective markers are used in repulsion to T1 (I;VII),prolonged incubation produces patches or small sectors of two unstable types that show unexpected genotypes and segregation patterns. Evidence has now been obtained (presented in the next section) that these unexpected types originate as translocation monosomics, which apparently are viable in this case, because the VII-I T-chromosome is so very small. b. Steps of Segregation in Primary Crossovers. Details of the sequence of steps of segregation, following selection of primary crossovers from T/+ diploids, are shown in Fig. 9 for Tl(I;VII).I n the first selected step, segregation usually results from mitotic crossing-over, nondisjunction being relatively rare (see Table 16). These primary crossovers are selected because they lose the dominant wild type allele, and, in the case of T1 (I;VII), they become homozygous for the selective mutant. They are unbalanced, because they are trisomic for the long segment of VII R attached distal on I L, and monosomic for the tip of I L. They grow quite well (as mentioned above), and their “pink-fluffy” appearance is very similar to that of the standard disomics, n 1 for group VII. Like the n VII types, they conidiate almost normally on thin medium especially if grown a t room temperature rather than a t 37OC. When

+

+

88

ETTA KAFER

transferred or replated, such unbalanced crossovers regularly produce green sectors (looking like the colonies in Fig. l l e ) . Most of these turn out to be unstable and are slightly abnormal 2 n + V I I types, arising presumably by nondisjunction in VII (step 2, Fig. 9 ) . Occasionally diploid dark green sectors are produced by compensating crossing-over in VII R (not shown in Fig. 9 ) . The 2n VII intermediates are mainly identified by their phenotypes [in comparison to standard 2n VII (Kafer and Upshall, 1973)l ; in addition, marker segregation in their stable diploid dark green sectors confirms the postulated genotypes (step 3, Fig. 9 ) . Segregants of this type have been analyzed in large numbers from diploids heterozygous for various markers and for T1 (1;T711)in coupling with the selective marker suAadE [a recessive suppressor used for selection from diploids homozygous for adE, on media lacking adenine; details are shown in Kafer (1976) 1. Recently, diploids with suAadE in repulsion, but fpaA in coupling to T1 ( I ; V I I ) , have also been successfully used to obtain unbalanced fpaA/fpaA crossovers. Such a diploid (as shown in Fig. 9, but heterozygous for several additional markers) was grown on media containing pfp, and conidia were “needle-plated” from 50 apparent or real sectors. Among these, 34 fpA-resistant, near-diploid segregants were obtained. They all showed one of the two expected phenotypes: (i) 20 of them were phenotypically indistinguishable from the pink, poorly conidiating but well growing, primary crossovers obtained previously when suAadE in coupling with T l ( 1 ; V I I ) was used for selection. These segregants produced the familiar two- or three-step patterns, with light green 2n VII intermediates and dark green diploid sectors; (ii) the other 14 represented such light-green secondary segregants, namely 2n VII trisomics, with the occasional darker diploid sectors. As expected, segregation for markers on I L was found in the first step, and primary crossovers were either anA/anA or ansi/+ depending on the position of the exchange (and nondisjunction was rare; see Fig. 9 and Table 16). Subsequently markers of VII R segregated in the last step, when, e.g., cho+ on the trisomic segment in VII was lost, so that thc two choA alleles on the segments attached to I L become expressed. In conclusion, it has been possible to select primary unbalanced crossovers from all diploids heterozygous for T l ( 1 ; V I I ) and Td(l;VIII),but in all cases only half of the possible types were viable enough to be recoverable. This is not unexpected, since in both translocations one break is located quite distal, the other close, to a centromere, creating translocation segments of very different lengths. I n general, it is expected that translocations with two distal breaks will produce viable crossovers of all expected types, while those with two proximal breaks may well pro-

+

+

+

+

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

89

duce none. The former is probably the case for T1 (VI;VII), since four reciprocally segregating unbalanced types were recovered from a translocation heterozygote, among large samples of nonselected conidia (Kafer, 1961). I n addition, as expected, these four likely crossover types formed pairs of identical phenotypes (however, with markers available a t the time genetic evidence was very incomplete), In all cases, viable unbalanced crossovers show distinct phenotypes and reduced growth rate. Therefore, even very rare types of compensating crossing-over between the break and the centromere show up as large diploid sectors. I n addition, nondisjunction is unusually frequent in the primary unbalanced types, especially when breaks are proximal; and trisomics are regularly found. These grow sufficiently better to produce conspicuous unstable sectors that form stablediploid sectors by chromosome loss in a third step. Formation of trisomics can only be inferred from the changes in growth rate and phenotype, since nondisjunction rarely produces a genetically detectable change. However, it is confirmed by the exclusively nondisjunctional segregation in the next step. For each translocation the situation is expected to be different. The relative growth rates and conidiation of the four chromosome segments produced by the two breaks, when mono- or trisomic, determine the chances for recovery and recognition of the various types. In Aspergillus, inviability seems to be the real problem, while the various phenotypes are unusually well distinguishable, so that cytological identification can be dispensed with. 2. Translocation Monosornics

From both, diploids heterozygous for T l ( 1 ; V I I ) and for T1 (VI;VIZ), unexpected unbalanced segregants have been obtained which mainly produce stable haploid sectors. These are now believed to be translocation monosomics. Usually these types are rare, but in one case, when the selective marker suAadE is used in repulsion to T1 (I;V II),they can be selected-even if only indirectly-on media lacking adenine : they grow slightly better because they produce stable suAadE segregants and are detected because no selected suAadE crossovers are viable and competing in this case. For detailed analysis, therefore, a diploid of the type shown a t the top of Figs. 9 and 10 was used (but with many additional markers). Such diploids previously have been found to produce unexpected unstable segregants of two types, both of them with stable suAadE sectors: (i) very slow growing segregants with a new phenotype which produce mainly haploid sectors ; and (ii) segregants of the unbalanced “pink-fluffy” type which are still heterozygous for suAadE, but form diploid suAadE sectors either by crossing over in I L or nondisjunction of I (Kafer, 1976). During the most recent study of these types, it has

90

ETTA KAFER

finally been possible to demonstrate conclusively that the first of these types originates by loss of the small translocation chromosome, and the second is usually produced by nondisjunction of chromosome VII in the fikt one (steps 1 and 2, Fig. 10). While the first type produces mainly conspicuous stable haploid sectors, the second forms mainly diploid sectors, either by one step of mitotic crossing-over in I L, or by two Balanced

VII

-,

1 ) Loss of ViI-l T- chromosome

2n:

TI ( 1 . v /suAadE ~

I

\

indirect 8election

on media

lacking adenine

\

J. Ts a

Crosslng Over in vIIR

2 ) Nondisjunction of normal VII

I

VII

Irisam I C A

J*

manosom~e segments el

"pink-fluffy"

(relatively

SU

su

Phen

unbalanced types

3 ) Nondisjunction

frequent)

green

2n

yellow 2n

FIG.10. Genotypes of segregants produced in four steps of segregation from repulsion diploids TI(I;VZI)/suAadE on media lacking adenine: (1) Loss of the very small VII-I translocation chromosome producing slow-growing translocation monosomics, mainly with normal haploid sectors (not shown) ; (2) nondisjunction of the normal VII in T-monosomics resulting in much faster growing, but fluffy, unbalanced types (crossing-over proximal in VII R occasionally produces this type in one step) ; (3) crossing-over in I L of the unbalanced type produces normal suA/suA diploids. while nondisjunction of I results in a light green translocation trisomic (yA/yA/+) ; (4) loss of the long I-VII T-chromosome from the latter results in balanced yellow 272 sectors.

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

91

steps of nondisjunction of I (steps 3 and 4 of Fig. 10). Identification of these four steps is possible by changes in phenotypes (Fig. l l ) ,and at the same time genetic markers segregate in some of them. The finding that one of the regularly obtained unexpected types can produce the other (step 2) explains the recurring problems of classification of the two types, since every so often, some cases seemed to change overnight from one to the other, especially on further incubation. a. Phenotypic Evidence. The relationship between the unexpected unstable types first became evident, when some of the postulated translocation monosomics were plated a t very low densities on unusually thick medium to improve photographic recording. Under these conditions “abnormal” centers of all types conidiate less and show up much better, and unstable sectors have a chance to produce second- and third-order sectors. Therefore, it suddenly was easy to see that T-monosomics not only produce stable haploid (and rare diploid) sectors, but quite frequently show sectors or large areas of the unstable “pink-fluffy” type (Figs. llb-d) . It also became evident, that any diploid sectors always arise from these unstable intermediates, in one or two further steps of segregation (Figs. l l c and l l d ) . On thin media, such “pink-fluffy” areas, which may surround the centers, often become indistinguishable from it (except on transillumination) so that only euploid segregants show up as sectors (Fig. l l a ) ; in such cases monosomics look quite different from those shown in Fig. l l b but are almost indistinguishable from stable disomics (Fig. 2a). To confirm that the unstable intermediates can always be found proximal to any diploid sectors, conidia from such areas were replated: the expected “pink-fluffy” unstable type was obtained in all cases (Fig. l l e ) . These colonies produced diploid sectors much more readily than the original T-monosomic (Fig. l l d ) , and such 2n sectors always were either green, or yellow originating from a light-green unstable intermediate. The latter must be a translocation trisomic type which produces only yellow 2n sectors, nondisjunctional for group I (replated in Fig. l l f ) . b. Genetic Evidence. The original Tl ( I ; V I I )/suAadE diploid was plated at low densities on media lacking adenine and incubated for over a week. When sectorlike areas of improved growth were formed by most colonies, abnormal conidia from their proximal parts were plated on complete media (as described recently in detail; Kafer, 1976). The result was that in the majority of cases (18/23 platings) slow-growing types with haploid sectors were recovered, in a few cases simultaneously with faster growing “pink-fluffy” types. When tested for genetic segregation all these segregants were found to be only mutant for phenB, as expected from loss of phenB+ due to loss of the small T-chromosome (step 1, Fig.

92

ETTA KAFER

RECOMBINATION A N D TRANSLOCATIOKS IN

Aspergillus

93

10). The other platings (5/23) showed only colonies of the pink-fluffy type; of these, four had segregated for phenB, and one had not. The latter presumably remained heterozygous for phenB as a consequence of mitotic crossing-over between phenB and the break proximal in VII R (alternative to steps 1 and 2, Fig. 10; tests for the leaky mutant phenB are quite difficult but are clear-cut in the resulting stable sectors; with nicB used in coupling to nzaZA, these are liomozygous for nicB and therefore completely negative on media lacking nicotinic acid if they are phenB+, but partially growing if they are phenB) . The postulated translocation monosomics always produced stable haploid sectors, under crowded conditions exclusively so (450 colonies showed 390 haploid sectors, 140 of which were tested). Such haploids were mutant for all markers of I and V I I in coupling with suAadE and segregated randomly for markers on all other chromosomes, as expected from translocation monosomics. In addition, in low-density platings, sectors of the unstable pink-fluffy type were frequent (about half as many as haploid ones were identified). No marker segregation could be demonstrated for this step (step 2, Fig, 10). This agrees with the hypothesis that the pink-fluffy types result from nondisjunction of the normal VII-chromosome, hemizygosis for phenB not being distinguishable from homozygosis. From the pink-fluffy unstable sectors (total about 200 from the 450 colonies) over 100 green diploid sectors were obtained in a final step of segregation. When tested, these all showed segregation for suAadE (loss of suA+) and other markers in coupling on I L, dependent on the position of the exchange (results are combined with earlier ones from the same diploid in Table 16) ; in addition all recessive markers distal to the break on VII R in coupling to suA (e.g., maZA) simultaneously showed up, owing to the loss of the trisomic segment of VII R which contained their wildtype alleles (Fig. 10, alternative to steps 3 and 4 ) . These segregants therefore arose by mitotic crossing-over in I L. Alternatively, yellow diploid sectors usually grew from segments Gf lightish-green areas. ForFIG. 11. Phenotypes and patterns of sectoring in segregants from diploids

T1(I;VZl)/suAndE selected on media lacking adenine and replated on complete

medium (genotypes in Fig. 10). (a-c) Replated T-monosomics in different growth conditions: (a) on thin medium, 2n - 1 center surrounded by unbalanced type, both conidiating, with white n sectors; (b) on thick medium, incubated 6 days, with haploid and unbalanced fluffy sectors; (c) incubated 9 days, large “fluffy” areas, one with third-order green crossover sector. (d) Unbalanced, “fluffy” types, one with 2n - 1 center, the other with 2n green crossover sectors and two unstable Ttrisomic segments with yellow 2n sectors. (e) Replated “pink-fluffy” unbalanced type with green trisomic and yellow 2n sectors. ( f ) Replated translocation trisomic with yellow 2n sectors (step 4, Fig. 10).

94

ETTA KAFER

mation of the latter showed no marker segregation, but a pronounced change in phenotype, as expected from nondisjunction of the normal I-chromosome (step 3, Fig. 10). The light-green types are therefore translocation trisomics, and as expected, on replating they produced only yellow, mainly diploid suA yA, sectors (Fig. llf). Like the green crossovers, these stable yellow sectors show simultaneous segregation for markers on I (in this case all markers proximal to the break on I L as well as those on I R) and on VII R (distal to the break), as expected from loss of the long I-VII translocation chromosome (39 tested; step 4, Fig. 10). In conclusion, all evidence is consistent and indicates that from diploids T1 (Z;VZZ)/suAadE, translocation monosomics are obtained on media lacking adenine, even though these segregants are not homo- or hemizygous for suAadE. It appears likely that their growth is favored by the following three factors: (i) their stable sectors all are hemi- or homozygous for suAadE and nuclei of this type in the mycelium would confer a selective advantage and be responsible for the improved growth; (ii) loss of the VII-I translocation chromosome would be relatively frequent and the resulting monosomic relatively viable, because this chromosome is extremely small; (iii) inviability of the selected crossover types from this diploid prevents competition. No such translocation monosomics have ever been obtained from diploids heterozygous for Td(Z;VIZZ), since in this case suAadE is translocated and point iii would not apply. On the other hand, the diploid heterozygous for TI (VZ;VZZ), which was analyzed in detail previously (Kafer, 1961), indeed produced almost identical looking types, among random samples of conidia. These presumed translocation monosomics always were heterozygous for markers on six normal chromosomes and for choA of VII R which is translocated to VI R, but not for the mutants on the other VIV I I translocation chromosome. This indicates that in this case, it is also the rearranged chromosome with centromere area of VII, which is lacking in the monosomic. This explains the similarity of the phenotypes of the monosomics; but since in this translocation the break in VII R is further distal (see Fig. 15), it is somewhat surprising that the viability also appears to be quite similar. 3. Frequencies of Different Types of Mitotic Segregation in Translocation Heterox ygotes

So far, no accurate data bearing on effects of translocations on mitotic recombination are available. All measurements of absolute frequencies of the primary products of crossing-over vs. nondisjunction are unreliable, and in addition they are valid only for the specific translocations used,

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

95

mainly for the following two reasons: (a) viabilities of primary segregants resulting from these two processes differ tremendously, crossovers being much more unbalanced than the primary trisomics from nondisjunction; (b) these absolute viabilities as well as their relative abilities to compete with normal colonies depend very much on the position of the breaks, i t . , the lengths and types of mono- or trisomic segments. Attempted measurements of such frequencies, by analysis of the frequencies of various disomics from translocation heterozygotes (Pollard e t al., 1968), or the frequencies of primary unbalanced segregants in random samples of plated conidia from T6(I;VZZZ)/+ diploids (unpublished data), are therefore, of very limited use. The only somewhat indicative data are relative frequencies of crossovers and nondisjunctional types, especially if translocation heterozygotes are used, in which both types of segregants show relatively good viabilities in comparison with controls. The disadvantage is, that such relatively viable segregants are only obtained when brcaks arc distal, and therefore when effects are minimal. This was the case for diploids heterozygous for Ti!(Z;VIIZ) (Kafer, 1976), and for diploids with T1 (Z;VlZ) in coupling to the selective marker (Table 16). The obtained results suggest that, as expected, nondisjunction frequencies as well as the distribution of crossing over, are influenced by the translocation, even though for I L the effects are quite small. That is, in heterozygous translocation diploids where pairing and crossing-over in the regions of the breaks is presumably reduced, crossingover tends to become relatively more frequent in the intervals closer to the centromere, and mitotic nondisjunction appears to be slightly increased (but samples are too small to demonstrate significant differences). The situation is much more abnormal, however, in the unbalanced firstorder segregants, where nondisjunction may be relatively frequent. However, in such cases the frequencies of diploid crossover sectors are compared with those of diploid nondisjunctional ones resulting from two steps of nondisjunction (see Table 16, SUA selection). The latter vary somewhat with plating densities and may represent a maximum value in cases where one event of primary nondisjunction (one segment of the T-trisomic type) produces more than one distinct yellow secondary nondisjunctional sector. The alternative is to compare the frequencies of dark green crossover sectors with those of translocation trisomic type (in replated unstable types by visual classification). I n this way an attempt was made to compare nondisjunction in the two different heteromorphic chromosomes of T l (Z;VIZ) a t two different temperatures ( 2 5 O and 37OC). While it was found that crossover sectors showed no significant differences at the two temperatures, nondisjunctional sectors were in-

TABLE 16 Nondisjunction and Crossing-over in TI (Z;VZZ) Diploids and Controls

Type of diploid

NondisSelective No. of junction tested in group I marker on I crossovers (%) su

Relative frequencies of crossing-over in various intervals of I L

33 % 34 % -

76

6% 5-9 % 8%

TIT

suA

580

8-12 %

21 %

T/

suA fPaA

57 98

10-15% 11 %

22 % -

15-25 %

22 % 23 %

Controls

+

TI +

2nd-step segregants

suA fpaA

{5:;

riboA

-57

.

-

anA

14 %

*

14 %

-

%

28 %

I

-50%

%

-

centromere

* 9%

@%

* 77 %

5%

proA 53 % *

64%

I

-61

-

c

15 % P

*

95 % +

16 %

*

M

3*

w $

B

RECOMBINATION

AND TRANSLOCATIONS

IN

Aspergillus

97

creased 3-5 times in numbers and 2-3 times in relative frequencies. This applied equally for both chromosomes I and VII. However, nondisjunction was relatively much higher in group VII, where the break is proximal ( 6 0 4 5 % ; total of 218 colonies inspected) than in group I where the break is distal ( 1 6 3 5 % ; total of 419 colonies). This agrees with results in higher plants where especially very small translocation chromosomes show relatively high nondisjunction frequencies. These results show that, while the effect of the translocation in the original balanced heterozygote may be quite small, exceedingly high frequencies of nondisjunctional segregants may be isolated, since primary segregants usually show much higher nondisjunction, especially if breaks are proximal. Unfortunately no suitable genetic markers were available in the above cases on the small translocation chromosomes VII-I of T1 (Z;VZZ), or VZZZ-I of T2(Z;VZZZ), to check on their nondisjunction frequencies in the original heterozygous diploids, and, translocation trisomics with these chromosomes extra are not likely to be phenotypically recognizable. On the other hand, the translocation monosomics found regularly as segregants from some of these diploids indicate that, also in Aspergillus, loss of the small translocation chromosome is relatively frequent.

F. MITOTICCROSSING-OVER IN DISOMICS FROM TRANSLOCATION CROSSES,ESPECIALLY IN “STABLE”DISOMICS 1. Normally Unstable Disomics

In Aspergillus nidulans mitotic crossing-over in disomics seems to be much more frequent than that observed in diploids. The highest values obtained in disomics from translocation crosses were those observed as sectors of unusual color in disomics for group I which were heterozygous for y A and T1 (Z;VZZ). These showed about 8% recombination between the translocation break near fpaB in I L and yA in I R (Kafer, 1975; see also Fig. 15). Even higher values were observed for standard n 1 for the same group (between suAadE and yA) and somewhat lower values between markers on both arms of other groups (Upshall et al., 1977). This would suggest an average level of 3-570 mitotic crossing-over per arm in disomics, compared to 0.1-0.3% in diploids. These differences are in agreement with the inverse correlation between stability and frequency of mitotic recombination (in diploids and disomics of Neurosporu, Aspergillus, and yeast) as postulated by Smith (1974), even though no logical reason for such a relationship has been proposed. Such differences, therefore, have to be taken into account when comparisons are made between different species, i.e., crossover frequencies in diploids should be

+

98

ETTA KAFER

compared separately from those in aneuploids or derived haploids [which probably would reduce the differences between A . nidulans and A . niger (Lhoas, 1967) ; as mentioned above]. Selected Crossovers, Distribution, and Coincidence of Recombination. Unstable disomics from translocation crosses usually produce only one type of haploid sector, even if many markers are heterozygous, because such disomics are structurally heteromorphic (see Section 11, C on translocation disomics). Heterozygosis, therefore, only becomes evident when the common balanced haploids contain the recessive allele, so that the growth responses of the disomic center differs from that of the sector. From such heterozygous n 1 types, crossovers can be selected by transferring them to a medium that selects against one recessive allele present in the haploid noncrossover sectors. The frequencies of such selected crossover sectors depend on the distance of the “selective” marker to the translocation break. They are quite rare, except when selective marker and break map on opposite arms. That is, as in diploids and standard disomics, crossing over occurs mainly close to the centromeres. Indeed, among the selected rare types, double crossovers with an additional exchange in the centromere area have been observed fairly frequently (the relatively small samples analyzed showed no significant difference from frequencies expected on the basis of random coincidence).

+

2. “Stable” Disomics

Three types of unusually stable disomics are expected from crosses between translocations that have one break each in the same chromosome (Section 11, C ) . The recovery of such types depends on their viabilities and phenotypic distinctness [and only two such types were obtained from crosses of Tl(I;VIZ) to other translocations with a break in VII R, Table 711. Very rarely the extra chromosome material of such “stable” disomics is lost and normal haploid sectors are produced (Fig. 2 ) . This is expected to be possible after the occurrence of a reciprocal mitotic crossing-over in the segment between the two breaks (as diagrammed in Figs. 3 and 12). Such a mitotic exchange in a “stable” n 1 which is disomic, e.g., for the two segments of a standard chromosome, produces as one of the two complementary products a new type of “tripartite” chromosome, and as the other a normal (or standard) chromosome; the latter can then be lost to form a balanced haploid containing both translocations (Fig. 12 and Table 17). These “stable disomics” are almost indistinguishable phenotypically from unstable duplication types in which the duplicated material involves a large segment of the same linkage group (Bainbridge and Roper, 1966). Such duplications also form rare haploid sectors, but apparently lose the extra chromosome segment by some un-

+

/-\ 1

RECOMBINATION AND TRANSLOCATIONS IN

99

Aspergillus

malA*

OitA’

I-VII

A,-~A

---+*--

- +.

wl-lll

Ill-VII

. eF’.

OioA

.

pIF

lY.0

,, ,,’

“stable“n+VII

Pairing and mitotic crossing over

I-VII

YA

+

normally unstable n + V I I lost

. 7 I-VII-Ill

._ly.0

\2y+

regularly

VII-l

III-vII

double T/ haploid

FIG.12. Stable n + V I I from cross of Tl(I;VII)x Tf(ZZI;VII)(2146) resulting irom meiotic nondisjunction, and mitotic crossing-over in the disomic segment between the two breaks in VII R, leading to formation of normally unstable n VII able t o form haploid “crossover” sectors.

+

orthodox mechanism [termed mitotic nonconformity in Aspergillus by Roper and Nga, (1969)l. This process appears to be either the consequence of nonrandom chromosome breaks in some cases [e.g., in Neurospora (Perkins, 1972, 1975) ] or of transposition of some segments causing loss of others (Azevedo and Roper, 1970). It was therefore of interest to analyze in detail the genetic changes associated with the loss of the disomic material in the “stable disomics” obtained here. For a detailed analysis the best-marked disomics with n VII phenotype (Fig. 2a) were chosen from a cross between 7’1 (1;VZI) and T l ( I I I ; V I I ) (cross 2146, Table 6 ) . It was found that six (of the 34, Table 7) cases with this phenotype were still heterozygous for all markers of VII R in the parental arrangement (since meiotic recombination was only partially reduced in the segment between the two breaks which must be over 100 cMo units long). These six disomics were used to inoculate the centers of a large number of CM plates, and all obvious haploid sectors were isolated (1-2 per plate). Tests of these haploids produced the results shown in Table 17. The segregation of markers in these haploids from stable disomics clearly supports the postulated mechanism of single mitotic crossing-over,

+

100

ETTA KAFER

TABLE 17 Types and Relative Frequencies of Mitotic Crossover Haploids from Stable n VII Disomics from Cross 2146 (Table 6): TI (ZL;VZZR) X TI (ZZZL;VZZR)

+

T-break niR mddk p w e Intervals 1 - 4 in T-overlap

MIA

+

palF T-break

Crossing-over in intervals: Genotypes of sectors:

+

Stable n VII isolate No. 273 222 244 211 252 256

1

2

3

Oli + pal

++ pal

+sF pal

3 0 10 6 6 4 29 23 %

16 0 6 3 2 0 27 21 %

5 5 7 2 3 -6 28 22 %

4 +sF+

Number of haploid sectors 30 7 37 18 14 21 127

Relative frequencies :

6 2 14 7 3 11 43 34 %

followed by loss of the normal VII crossover product. I n addition, the data show that mitotic exchanges occurred with similar frequencies in the four marked intervals between the breaks, which are all on the same arm and of roughly similar size (the slightly more frequent type is the most viable one, since it is the only one which is p a l F + ) . IV. Genetic Mapping and the Use of Translocations

A large variety of methods combining genetic, cytological, and biochemical techniques are used in various organisms for the mapping of genes to specific chromosome segments. Since the methods based on mitotic recombination have been new and especially useful in Aspergillus nidulans, emphasis will be mainly on these: in addition, techniques making use of translocations for genetic mapping, in conjunction with meiotic and mitotic recombination, are considered in detail. These latter techniques logically are the same as the ones employed for the mapping of translocations ; similarly, mapping of markers to chromosome arms leads to mapping of centromeres.

RECOMBINATION

AND TRANSLOCATIONS IN

Aspergillus

101

A. GENETICMAPPINGOF MUTANTSIN Aspergillus nidulans A N D EFFECTS OF TRANSLOCATIONS The procedure for genetic mapping in A . nidulans takes into account that meiotic distances are relatively large, so that markers on the same chromosome or even on the Same arm frequently are unlinked in crosses (as discussed in Section 11, A ) . Mapping therefore generally proceeds in two or three stages: (1) Identification of the mitotic linkage group or syntenic group, i.e., mapping to one of the eight chromosomes; (2) meiotic mapping in crosses to a selection of widely spaced markers of that, chromosome; and (3) if this second step does not identify the precise location within the linkage group and exact location is of special interest, additional mapping to chromosome arms or segments by one or more further methods, which are less generally applicable: (a) mitotic crossingover in standard diploids heterozygous for suitable markers ; (b) mitotic recombination in homozygous translocations diploids ; or (c) mapping by means of disomics from heterozygous translocation crosses. 1. Mapping of Mutants to One of the Eight Chromosomes

(Which Detects Translocutions) The first step of mapping of any new marker consists of a check for mitotic linkage to markers on all eight chromosomes, in haploids which are selected from heterozygous test diploids. This mapping technique was a logical extension of the work of Pontecorvo et al. (1954), who demonstrated the usefulness of haploid segregants for identification of all markers which are located on the same chromosome. It became feasible after, and was developed simultaneously with, the mapping of the first pairs of markers on all eight chromosomes (Kafer, 1958). This method is relatively easy and much more efficient than meiotic mapping, so that in addition to the almost 200 markers actually placed on the current Aspergillus map, almost half as many are mapped just to one of the eight groups (Clutterbuck, 1974). Similar mapping of markers to whole chromosomes is also possible by means of disomics, especially in organisms where these are stable (such as yeast, Mortimer and Hawthorne, 1973). a. Mapping Procedure. The mitotic mapping technique involves the following three steps: (i) The mutant strain has to be combined into a heterokaryon with a suitably marked “mitotic mapping strain” (Barratt et al., 1965, 1975; or “master strain,” McCully and Forbes, 1965). From the heterokaryon, the test diploid can readily be isolated (Roper, 1952). Since simple standard strains are usually used to obtain new mutants (e.g. biA in various colors, to obtain nutritional mutants by the “bi-

102

ETTA KAFER

starvation” technique; Pontecorvo et al., 1953), we find it worthwhile to first cross all induced mutants to a “meiotic tester” strain. I n such crosses, we can not only introduce markers that facilitate heterokaryon formation, but, in addition, eliminate any of the frequently induced morphological mutants, and check for gross aberrations (which increase aneuploid frequencies, Upshall and Kafer, 1974). (ii) HapZoids are selected by one or two of the methods for which the test diploid is suitable: either by “double selection” using markers on two different chromosomes to obtain some of the rare spontaneous haploids resulting from mitotic nondisjunction (Kafer, 1958) ; or by induced haploidization on media containing chemicals that interfere with mitosis and favor the growth of haploid types (Lhoas, 1961; Hastie, 1970). (iii) The segregation of markers in the haploids is analyzed, and any cases of complete linkage rather than free recombination are identified. These procedures generally map new mutants to one of the eight mitotic linkage groups provided they are not cytoplasmic ones [like some of the oligomycinresistant mutants of Rowlands and Turner (1973)l. If any unusual linkages are found, selection of haploids by more than one method, or analysis of differently marked diploids distinguishes cases due to translocations from those caused by special conditions, like poor viabilities or interaction of mutants [e.g., suppression of f p a A or fpaB by Zys mutants, as found here, and similarly in Neurospora (Kinsey and Stadler, 1969)l. b. Identification of Mitotic Linkage Groups with Chromosomes. Many methods are known that permit assignment of linkage groups to chromosomes. For example, mono-, di-, or trisomics can be used, usually in somatic cells (since they are often sterile). This is possible only if the mitotic chromosomes are sufficiently distinct (such identification has recently been achieved with biochemical techniques in yeast, for chromosome and group I, which contain many rRNA genes; Finkelstein et al., 1972). Or meiotic pairing figures of zygotes heterozygous for translocations can permit identification, if two translocations are used that involve one common chromosome that is identifiable in meiotic prophase {as has become feasible in mice with the recently improved staining methods for mammalian chromosomes (Miller and Miller, 1972) ; or in cytologically more favorable material like the mosquito (Bhalla et al., 1974) 1. In Aspergillus nidulans cytological identification of chromosomes is obviously difficult, since problems were encountered even in counting the total number of meiotic chromosomes: only with the help and experience gained with the larger chromosomes of Neurospora (McClintock, 1945) was the haploid chromosome number recognized as eight (Elliott, 1960a) rather than four (Pontecorvo et al., 1953). Mitotic chromosomes are even harder to count, and in addition they are somatically paired, so that diploids can be recognized only by the increased amount of total

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

103

DNA (Robinow and Caten, 1969). This rules out the use of aneuploids, which in Aspergillus are unstable and never go through meiosis. The meiotic method using translocations, which has turned out to be feasible, if difficult, in Neurospora (Barry and Perkins, 1969) would seem to be the most promising approach, especially if rings of translocations could be used that would tie up all but a few chromosomes. However, even though translocation pairing figures can in some cases be identified clearly, determination of the relative sizes of all eight chromosomes and identification of specific ones in translocation crosses, is probably not feasible with current cytological techniques (E. R. Boothroyd, personal communication). It seems, therefore, unprofitable to attempt numbering of chromosomes by size, and the unconventional practice of referring to chromosomes with the number of the corresponding linkage groups (which is done by some investigators using Aspergillus, and regularly for yeast) probably is the most sensible procedure under the circumstances. 2. Meiotic Mapping of Mutants

For linkage groups with many mapped markers and few stretches of 50% crossing-over, meiotic linkages can usually be found. This is especially true for relatively short ones, like IV or V, but also applies to the well-sequenced groups I, 11, and VIII (Clutterbuck, 1974; see also Fig. 15). On the other hand, meiotic linkages are often not found in the remaining groups, and in these cases the alternative techniques, described below for sequencing of markers and centromeres, are especially useful. Furthermore, meiotic mapping of new markers may be unsuccessful not only because of the meiotically long distances, but also because generally no positive, or cven slightly negative, interference is observed (Section 11, A ) . Three-point crosses of loosely linked markers often do not clearly establish their sequence, nor do crosses of two closely linked ones to more distant markers [e.g., the position of dilA closely linked to argB and proximal of methH (Fig. 14) is only tentatively established, even though 400 progeny were tested from a cross heterozygous for these mutants]. To map loosely linked markers, certain translocations can be very useful, because recombination is reduced in heterozygous crosses, and a t least markers linked to the breaks can often be sequenced more easily (Section 11, B ) ; in addition, the orientation of distal segments may become apparent. One such case is illustrated in Fig. 13, which shows the values for intra- and interchromosomal recombination in crosses heterozygous for T1 (ZV;VZZZ). These results clearly map sE between chaA and nirA and place nirA distal to all other markers, confirming mitotic crossing-over data of A. Niklewicz (Gravel et al., 1970; details shown in M a and Kafer, 1974). On the other hand, when translocations are present that are not recog-

104

ETTA KAFER

pabaB 29%,+

(680

12% 11063)

FIG.13. Meiotic recombination frequencies (‘36) between markers of groups IV and VIII in crosses heterozygous for !l’l(ZV;VZZZ) (values based on 1-5 samples of about 200 each; total tested number in parentheses).

nized or not sufficiently taken into account, sequencing based on results from heterozygous crosses may lead to erroneous maps. A case in point is the map of group 111, where reduction of recombination by unidirec+ VZZZ) by Clutterbuck, tional translocations (observed, e.g., for T,%?(ZII 1970) was more extreme than originally assumed (Bainbridge, 1970; Kafer, 1975). In this regard it is interesting to note that, even though it is stated that in the recent map “translocations have been taken into account where necessary” (Clutterbuck, 1974), all the quoted discrepancies are in areas where markers tightly linked to translocations are known to have been used for mapping [at least in some of the crosses: namely, suCpro and T1 (ZZZ + VZZZ) in 111; frAl and TI (ZV;VZZZ) in IV R ; sD50 and T,%?(Z;VIZI)proximal in VIII R, evident also in the data of Dorn (1972) ; and one additional case in VII R, namely markers around the break of T1 (ZZZ;VZZ) associated with ZysD20; see below, subsection 3 , ~ .

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

105

3. Mapping of Markers to Specific Chromosome Arms or Segments

Of the three methods that permit identification of the chromosome arm or segment in which a mutant is located, two make use of mitotic recombination, one ( a ) of mitotic crossing-over in standard diploids; the other (b) of mitotic haploidization and crossing-over in homozygous translocation diploids ; the third (c) uses disomics from heterozygous translocation crosses. This last type of analysis in addition identifies suitable strains containing markers in coupling with translocations, which then can be used for mitotic mapping in homozygous translocation diploids. Such diploids are useful also for centromere mapping when selective markers are translocated to new groups (as in diploids of Table 19). Of the three techniques, only the first has been in general use for some time. However, since it is efficient mainly in linkage groups in which selective markers are located distally on both arms, as in groups I and 11, it has only rarely been used for mapping in other groups (for example, for the mapping of centromeres in groups I11 and I V ) . The other two techniques are even more restricted and can be applied only when suitable, mapped translocations are available ; these, therefore, have only recently been used in special cases. a. Sequencing b y Mitotic Crossing-over in Standard Diploids. Mapping by mitotic crossing-over is most efficient, when recessive mutants are used in coupling (or dominant ones in repulsion) to the selective marker and to a marker on the opposite arm. A small sample of crossover segregants will then identify the relative position of any meiotically unlinked mutants, since results are basically qualitative: the new mutant is located either distal to the selective marker, if all crossovers are homozygous for i t ; or on the other arm, if the new mutant is homozygous only simultaneously with the marker on the opposite arm (i.e., in nondisjunctional segregants) ; or proximal, if it is homozygous in a fraction of crossover segregants. However, if centromeres have not yet been mapped with certainty, or when segregants are so rare that they approach mutation frequencies, results are much less easy to interpret (see below, Section IV, C on centromere mapping). b. Mapping to Chromosome Segments b y Mitotic Analysis of Homozygous Translocation Diploids. When mapped translocations are available with a break in the chromosome to which a marker has been assigned, these can be used for mapping to scgrnents in several ways; the simplest is mapping by mitotic analysis of homozygous translocation diploids (Ma and Kafer, 1974). This is relatively easy once strains with the new mutant and suitable markers in coupling with the translocation have been

106

ETTA KAFER

constructed, or when a translocation is tightly linked to a marker. From diploids heterozygous for markers and homozygous for the translocation, haploids can then be isolated that map any new mutant to one of the two segments defined by the translocation break. Selection of mitotic crossovers may, in addition, define the sequence of markers in the distal translocated segment or in the residual part of that arm, if a suitable selective system is available. Such mapping has recently been carried out in many cases, usually either preceding or confirming results obtained by other methods. For example, the sequence palF - lysD - malA, choA was firmly established in this way, when over 100 haploids from three diploids homozygous for Tl(III;VII)with the break a t lysD were analyzed: in all cases palF was completely linked to the proximal markers phenB and sF, but not to the translocated ones, malA, choA, or nicB. These results agree with those obtained from translocation disomics, not only from crosses with T1 (III;VII)but also with Tl (VI;VII).[In addition, in the latter palF is the only one of these markers that shows definite, if slight, meiotic linkage to sF or pantoB close to the break, and similar linkage was also found for O l d , on the other side of the break (Fig. 15) (Klfer, 1975).] I n Table 19 below, results from diploids homozygous for T 1 (VI;VII) confirm that, while palF in this translocation is on the same distal segment as maZA, choA and nicB, the mutant OliA maps proximal (since it segregates with phenB, the only nontranslocated marker in Tl(I;VII); for results based on mitotic crossovers from these diploids, see Section IV, C ) . c. Mapping by Means of Disomics from Heterozygous Translocation Crosses. This method also depends on the availability of a translocation that has one mapped break in the relevant linkage group and, in addition, produces a reasonable frequency of n 1, especially translocation disomics. From heterozygous translocation crosses, four types of disomics are expected and found for some translocations (see Section 11, C, 2, c) : two types of “typical looking” n+ 1 types, which contain one of the two involved standard chromosomes in addition to a haploid set including both translocation chromosomes ; and two types of translocation disomics, each disomic for one of the two rearranged chromosomes and containing in addition a balanced standard haploid set. Since haploid sectors from typical looking disomics usually contain the translocation, these are very useful for construction of homozygous diploids. However, because of the relatively high rate of mitotic crossing-over that may occur a t the fourstrand stage in disomics, cases sometimes are found that do not agree with this general rule, but seem to be homomorphic as well as homozygous [e.g., apparent double crossovers around the breaks are often of this type (Kafer, 1975) 1.

+

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

107

If one, or preferably two, recognizable types of translocation disomics are found, these can be checked for heterozygous markers (especially in cases with recessive alleles in haploid sectors). Since any unmapped mutant will be found to be heterozygous in only one of the two types, such results usually assign the marker to one of the two segments defined by the translocation break (exactly like those obtained with haploids from homozygous T-diploids) . This type of mapping corresponds to methods in various higher plants that show visually identifiable aneuploids, e.g., translocation trisomics in tomato (Khush and Rick, 1967) or secondary trisomics in Datura (Carlson, 1972). I n addition to this qualitative mapping, which depends on the recovery of translocation disomics, it is possible to check for meiotic linkages to the translocation breaks, using both types of disomics, namely, translocation disomics and/or typical looking disomics (as explained in detail in the next section). Also meiotic linkages of the new mutant to normally unlinked markers may show up in such crosses because of the resulting reduction of crossing-over (Section 11,B). Such mapping was, for example, first obtained for ad1 and s A on 111L, which both showed very close meiotic linkages to the break of Tl(II1;VII)in disomics (and to each other in haploids) ; since only adI was heterozygous simultaneously with the proximal marker A c t A in one type of disomic, and sA was heterozygous in the other, these results mapped sA close, but distal t o adI on I11 L (see Fig. 14; as confirmed subsequently by the haploids from the homozygous T-diploids mentioned above). Similarly, the first mapping of OliA to a proximal segment of VII R has been obtained with translocation disomics (40 isolated) from two crosses heterozygous for Tl (VI;VII). Of the 17 that. produced haploid OZi+sectors, five were heterozygous for OliA simultaneously with phenB, but not for pantoB or any of the other markers of VII R ; and all these five disomics showed the phenotypes typical for disomics heterozygous for the proximal part of VII R and the distal segment of VI R, thus mapping OZiA proximal to the break on VII [as shown also in T/T diploids, Table 19; OZiA has recently been renamed O K (A. J. Clutterbuck, personal communication) ].

B. FREQUENCIES, DETECTION, AND MAPPING OF TRANSLOCATIONS The various steps for mapping of translocation breaks are basically the same as those outlined in the preceding section for mapping of mutants: ( 1 ) Translocations are detected, and the linkage groups involved can be determined by isolation of haploids from heterozygous diploids, Table 19; OZiA has recently been renamed OliC (A. J. Clutterin the standard mapping methods, (Kafer, 1958; McCully and Forbes,

108

ETTA KAFER

1965) , but not the previous modification used by Forbes (1959) 1. (2) I n favorable cases, meiotic mapping may be possible, especially when small linkage groups or well-mapped arms are involved. (3) Mitotic recombination in diploids and analysis of disomics from T-crosses can be used for further analysis, and combined results may permit to map the breaks.

1. Zdentification and Frequencies of Translocatwns a. Detection. The detection of translocations in well-marked diploids is easy (Kafer, 1962) except when unexpected interactions occur, either between certain types of mutants and selective media, or between markers under certain conditions [e.g., inviability of morphological mutants on CM with pfp (Kafer, 1965; Bainbridge and Roper, 1966) ; or inhibition of f p a mutants by 2ys mutants (Section IV, A, p. 102) ; or inviability of paba on CM containing sulfonilamide (Bignami et al., 1974) ; etc.]. As mentioned above, these can be recognized if more than one system of selection or more than one type of mapping strain are used: consistent linkage of all markers of two groups, provided both reciprocal types of haploids can be recovered, clearly identifies the chromosomes containing the breaks. Crosses to standard strains also reveal most translocations, since they cause increased nondisj unction, which produces disomics for the involved groups (demonstrated in Upshall and Kafer, 1974). Results from tests using both these methods, which identified nine UV-induced translocations, are shown in Table 1. It should, therefore, be relatively easy to keep all strains translocation-free, except those being used specifically for translocation work. However, while this has been achieved to a considerable extent for most of the retained Montreal strains, it also has become obvious that such a procedure is much too time-consuming when translocations are not of primary interest. This is the case because unexpected or undesired translocations remain in many strains for the following reasons: widespread distribution of early translocations in the strains of all laboratories ; high rate of induction of translocations with most mutagenic treatments ; spontaneous occurrence of new translocations even in tested strains, especially those with “reverted” duplications ; mix-up of strains of origin with indistinguishable phenotypes ; tight linkage of desired mutants to induced translocations; etc. It seems, therefore, that as a general rule aberrations have to be expected in many strains, and that the best procedure is probably to check strains immediately before they are needed, a t least for those genetic investigations where translocations could interfere. b. Association of Mutants with Breaks. I n 5 of the 16 analyzed cases it has not been possible to separate an induced mutant from a chromosomal aberration present in the same strain, even though analysis was quite extensive. Some of these may represent cases of position effects of

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

109

the breaks [as shown for brZA12 and T 2 ( N I + V I I I ) by Clutterbuck, (1970) 1, while others are known to be due to close linkage; the latter must be the case for pantorll, induced in a strain containing T I (ZII + V I I I ) and not yet separated from it. Three other cases are more likely of the former type, since mutant and translocation were induced simultaneously and remained inseparable when over 100 haploid or disomic progeny from crosses were tested (namely, lysD20 and sD50, Table 1 ; and frA1, Fig. 13) ; this, however, does not constitute conclusive proof (as, e.g., available in Drosophila; Lefevre, 1973). c. Frequencies of Induced Translocations. Induction of balanced reciprocal translocations without any phenotypic effects is much more frequent than was originally assumed, not only after irradiation with Xor 7-rays, but also after UV treatment (Kafer, 1965). It appears now that ultraviolet light at a level of killing used in Aspergillus work ( 2 4 % survival) induces translocations with frequencies of 15% to 25% (9/21 in Table 1, which are all well analyzed cases, plus 1/21 further UVinduced mutants for which the original strain was not analyzed). Analysis of random samples of UV-treated strains produced slightly lower values (A. Upshall, personal communication). These results are in line with similar observations in Neurospma [somewhat lower UV dose, much less killing because conidia are multinucleate and 8% aberrations (Perkins, 1974) 1. Lower frequencies of UV-induced translocations have been found after treatment of diploids (Kiifer and Chen, 1964), but this could be due to somatic pairing which, a t least in Drosophila, leads mainly to fusion of homologs rather than to interchromosomal translocations when breaks are induced (Gatti et al., 1974). Whether some of the powerful chemical mutagens currently in use also frequently induce chromosomal aberrations is not yet known. So far only a few mutants induced by nitrosoguanidine (NG) have been checked, and no translocations have been found, in line with results by Malling and DeSerres (1970) in Neurospora. Also the recent progress in mitotic mapping in slime molds using mainly NG-induced mutants, indicates that translocations may be relatively rare in this case (Rothman and Alexander, 1975). However, NG is known to cause breaks in higher organisms (in human cells found by Kelly and Legator, 1970) and possibly also in diploids of Aspergillus (Shanfield and Kafer, 1971). Certainly EMS has been shown to induce translocations in mice, where such aberrations are recognized as the main cause of sterility (Cacheiro et aZ., 1974).

2. Meiotic Mapping As expected, meiotic mapping of translocation breaks is usually difficult in Aspergillus, even though the reduction of recombination in heterozy-

110

ETTA KAFER

gous crosses is most helpful. Homozygous crosses, furthermore, have barely been usable, except in cases where a self-fertile marker is associated with the break. Such markers also greatly enhance meiotic mapping in heterozygous crosses (as shown for Tl(1V;VIII) associated with jrAl in Fig. 13).

3. Mapping of Translocation Breaks to Specific A m s or Segments a. Mapping of Breaks to Specific Chromosome Arms. This can be relatively easy in heterozygous diploids when a selective marker is available in the same arm as one break. In such diploids, mitotic crossovers are either abnormal because unbalanced, or entirely absent if inviable (for problems with this method, see Section 111, E). b. Haploids and Mitotic Crossovers from Homozygous T / T Diploids. Such segregants easily map the breaks, but accurate mapping is possible only if the closest markers can be used, which therefore has to be in coupling with the translocation. This is most readily obtained when a marker is either induced closely linked to or associated with one of the breaks; or, on the other hand, when even the closest markers are barely meiotically linked. In other cases, the closest markers will be recognized more likely by the difficulties encountered in getting them into coupling (Le., by recognizable linkage in disomics, see below). However, results in haploids from T/T diploids are always clear-cut and useful for confirmation; and they are especially useful when only one marker is found linked to the breaks, and/or when no translocation disomics are obtained. c. Mapping of T-Breaks in Translocation Disomics. Such mapping of translocation breaks is no different from mapping of new mutants (discussed above) ; all translocation disomics are checked for heterozygous markers, and if two types have been obtained, these show complementary patterns: one type is heterozygous (with characteristic frequencies) for all the markers of the distal translocated segment of one chromosome and also for the nontranslocated markers and the centromere area of the other; the second type is found heterozygous for all other markers of the two involved chromosomes. These results, therefore, qualitatively map the translocation breaks if the sequence of all mapped markers is known [as shown for T l ( V ; V l ) and markers of Group V, Table 18, discussed below]. d. Unidirectional Translocations. Mapping these translocations by identification of heterozygous markers in phenotypically recognizable duplication types is basically the same technique as the previous one (Bainbridge, 1970, Clutterbuck, 1970 ; Perkins, 1972). The details differ because duplication types are much more frequent (one-third of the viable progeny); their haploid sectors, on the other hand, are much rarer-i.e., their heterozygous centers are more stable and much easier

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

111

to test [like the stable disomics from crosses between overlapping translocations (Fig. 2) ]. I n duplication types all heterozygous markers belong to the same linkage group and they indicate the extent of the translocated segment. e. Meiotic Linkage of Markers to Breaks in All Types of Disomics. Since the majority of haploid sectors from T-disomics obtained in heterozygous T-crosses contain the standard chromosome set, and those from typical-looking n 1 contain the rearranged set, their sectors show up meiotic linkages of markers to the transolcation breaks in the form of complementary allele ratios. Testing one sector from each disomic and comparing marker segregation in the sectors of the two groups of disomics, therefore, identifies meiotic linkages of markers to the translocation breaks (Kafer, 1975).

+

4. Example, Mapping

of T1 (V ;V I)

Genetic mapping of translocations by a combination of all the described methods is illustrated here, using the case of the reciprocal translocation T1 (V;VZ). This translocation was mapped in detail to check on predictions of approximate break positions, based on disomic frequencies in crosses heterozygous for translocations (Kafer, 1975). Details are given here to demonstrate not only how a combination of methods is used to advantage, but also to show some of the problems encountered in this type of work. The translocation T1 (V ;V I) was discovered in an F, progeny of a cross with the UV-induced sAl mutant of group 111, and subsequently also demonstrated in the original W-treated strain (Table 1 ) . Eleven other sAl descendants were checked for the translocation, and seven were found to show increased frequencies of disomics, especially of n V and n VI (range 1-5% in relatively small platings), while four others showed less than 0.5% and therefore were translocation free (total of 9 heterozygous translocation crosses in Table 4 ) . Larger platings were made from five well marked crosses heterozygous for T1 (V ;V I) (genotypes in Table 5 ) . These definitely produced only one type of translocation disomic, which grows almost normally and produces large conidiating aneuploid centers (Fig. l c ) , rather than the two variants postulated earlier on the basis of the nine small samples (Upshall and Kafer, 1974). Tests for heterozygous markers confirmed that all translocation disomics were of the same type; they were found to be heterozygous for lysB, but not facA, hxA, riboD of group V (bottom of Table 18). They also were heterozygous for the distal markers of VI, namely sB and sbA, but not for the proximal marker lacA (Fig. 15). These results place the breaks of TI (V;VI) between lysB and facA on V (where the sequence has been

+

+

CL CI

XI

TABLE 18 Frequencies of Recombination between Breaks of TI (V;VI)and Relevant Markers in All Types of Disomics, and Heterozygous Markers in Translocation Disomics, from Crosses Heterozygous for T1(V;VZ)

Cross No.

+

Total TZ 1 (Typical disomics Translocation disomics)

1953 2130 2152 2132 2131

Total disomics:

+5630) (16 + 26) 42 (35 + 87) 122 (67 + 84) (26

151 (100 58) 158

+

+

VL ZysB

pA

-

-

- proxima1 - V R facA

+ 7) 29 % (7 + 6)

- distal hxA ribOD -

lacA

+

-

proximal - VI R - distal bwA sB sbA

(14 18) 57 % (4 7) 26% 31 % ( 5 25) (3 10) (9 22) (17 38) (15 44) 25% 11% 25% 45 % 48 % 13) (18 26) (22 24) (24 34) (32 33) (35 44) (15 29 % 31% 38% 43% 52 % 18 % (23 20) (4 11) (16 22) (23 24) (45 26) (45 35) 9% 24% 30% 45% 27 % 51 %

+ +

+

+

+

(9

+

+ +

+

+ +

+ +

+

+

+

+

+

+ +

(15 15) 54 % (9 14) (9 16) 55% 60% (15 +48) 52 % (33 38) 47 % (49 30) (37 34) 50% 45%

+

+

+

+

+

(244 285) 529 Crossovers/total : Combined percent:

117/431 27%

T-disomics: heterozygous/tested 140/127

28/280 10%

144/529 27%

01 /124a 0/73

105/309 34%

0/57

191/431 44% 0/96

1

250/487 51%

39/193

0/91

O?/llOa

90%

Conidial color of disomic centers is difficult to recognize in T-disomics, especially in the case of bwA.

195/378 52%

167/351 48%

47/67

36/56

M

3P

2

$

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

113

established by meiotic recombination) and between lacA and sB (sequenced by mitotic crossing over; see Section C, p. 120). Heterozygosis for the color mutants p A and bwA could not be determined visually in these brownish disomic types, and mapping with respect to these two markers was carried out subsequently by other methods. Only in one cross were contradictory results obtained, namely many similar looking unusual disomics often heterozygous for lysB and f a c A ; however, since in this cross a progeny strain from a double translocation cross had been used, it probably contained a differently modified chromosome V (and since all other crosses gave consistent results in large Samples, this cross was excluded from the final analysis and Table 18). To obtain meiotic mapping of markers to the breaks, all types of disomics were analyzed for marker frequencies in their haploid sectors. Table 18 gives the numbers of observed recombinants between the breaks and the markers of V and VI, listed separately for “typical looking” disomics (namely n + V and n + V I combined) and for translocation disomics. The pooled frequencies clearly demonstrate meiotic linkage of the translocation breaks to bwA, but not to any other marker on VI. On the other hand, all markers on V show meiotic linkage to the break on V. The obtained values map this break in the middle between 1ysB and f a c A , close to p A but probably proximal to it, since p A maps closer to f a c A (27%) than to 2ysB (40%; Fig. 15). These results agree with the above approximate mapping indicated by the heterozygous markers in the T-disomics. They are also confirmed by meiotic intergroup linkages obtained in these crosses : namely, bwA becomes meiotically linked to some of the markers on V, closest to p A and less close, but equidistant, to lysB and facA (31% recombination in each case). Such intergroup linkages between markers located close to the breaks usually show smaller values than expected from the distance estimated for each marker to its break, because disomics show a relatively high level of mitotic crossingover (as discussed in Section 111, F) . To map the breaks of T1 (V;VZ) with respect to the linked color markers, p A in V, and bwA on VI, hoinozygous translocation diploids were constructed (Table 19, diploids 2208 PA/+, 2182 and 2184 bwA/+). The haploids isolated from these diploids clearly mapped p A on the translocated piece, i.e., the T-break to “left” and proximal to p A in V, in agreement with the meiotic results. On the other hand, b w A was mapped on the proximal part of VI R , since it segregated in haploids together with lacA and was now unlinked to the translocated markers sB and sbA. This must mean, that bzcA and p A are located on the same translocation chromosome in T1 (V;VZ). To confirm this hypothesis, and to measure the meiotic distance between them, cross 2153, homozygous

TABLE 19 Haploid Segregants from Diploids Homozygous for TI (V;VZ)or TI (VZ;VZZ) v - VI VI - v lysB - sB sbA lacA bwA - p A facA hxA riboD VII - VI VI - VII phenB OliA - sB sbA IacA bwA - pantoB palF malA choA nicB

Markers

2184 2208 2211

sB

a

+

a

+

6 + 6 a

+

6 a 6 +

TI (VI;VZZ)

1

- -

+- +++ -+ -+ - ++

I" - +

a

1996 1997

a I; a - + - -

1998

a - + - -

a

6

+ ++ +- -+ - -

- + ++ -+ +- +- - + + - -

6 - -

45

6 + + + + + +

6 - + + + + + a - + +--+ 6 + + + + + +

a

a + + - - + -

+- +

++ + +

VI a

Parental

Recombinants

V-VI a VI-V a VI-V b

Total numbers of haploids of four types:

1992

6

a

V I I - VI

++

Total

VI - v

v - VI

TI ( V ;VI) 2182

Numbers and types of haploids

Arrangement of markers on homologs a/b

Diploid No.

7

10

44"

16

16

54

18

44" -

16 57

187

- VII

-+ + ----

Parental

V-VI b VI-V a VI-V b 12

16

5

7

12

14

10

8 46

11 42

42

VII-VI a VI-VII a VI-VII b

9

VII-VI b VI-VII a VI-VII b

6 + - + + + + +

42

7

22

3

lo

b

64

24

11

13

16

39

18

6

11

4

36

2 51

5 44

3 30

26

a t

b a i;

++ +- +- ++ +- +- -+ ++ ++ ++ ++ +- -+- + _ _ + - - - + ++ +++++

Totals:

Two rare crossover types from selenate selection not included.

181

%

H

2 E 1

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

115

for T1 (V;VZ), but heterozygous pA X bwA was carried out (genotypes in Table 5 ) . It was possible to distinguish all four color combinations visually, and slight but significant linkage was found between PA and bwA (314 recombinants, 4476, among a total of 703 classified). As expected, this value is considerably higher than the corresponding one in heteroxygous T-crosses [estimated a t less than 35% in cross 2136, in which double-mutant types pA;bwA were difficult to classify (Table 5 ) 1. 5. Approximate Mapping of T-Breaks, Based on Disomic Frequencies in Crosses By comparing the absolute frequencies of “typical looking” disomics in crosses heterozygous for all mapped translocations, an inverse correlation of the distances from breaks to centromeres was clearly apparent (Kafer, 1975) ; that is, meiotic nondisjunction was high when breaks were proximal and low when they were distal. As mentioned in Section 11, D, 1, it should be feasible to use this relationship and to predict the approximate position of the breaks for an unmapped translocation from aneuploid frequencies in heteroxygous crosses (especially if these could be compared t o other, mapped, cases with breaks in the same arm). Such predictions were attempted for T1 ( V ; V I ) ,which now has been mapped. The absolute frequencies of typical-looking disomics from all TI (V;VZ) crosses are shown in Table 4. On the average, these values are fairly high for n V, and about half as much for n VI (ca. 1% and 0.50/0, respectively, after correction for abnormal viabilities in cross 2131). These results place one of the breaks proximal in group V, which, considering the above mapping results, may well be the case; on the other hand, the break in VI R appears to be located quite distal in VI, and certainly much further distal than that of T1 (VI;VZZ),since the latter gives many more n VI in heterozygous crosses (Table 4). This contradicts the meiotic mapping results, which place both breaks close to each other in VI, distal but linked to bwA. The difference in the n VI frequencies is, therefore, not explained and the postulated correlation is not found; rather, breaks in close proximity on the same arm show quite different frequencies of typical disomics. This finding is rather disappointing, even though not entirely unexpected. It indicates that additional factors may influence nondisjunction frequencies of centromeres of heteromorphic pairs. One such factor could well be the length of the attached translocated segment, which is very much longer in the case of T1 (V1;VII) than T1 (V;V1).However, if the situation is indeed more complicated and based on a t least two unknown variables, predictions are likely to be very inaccurate, possibly even misleading.

+

+

+

+

116

ETTA KAFER

C. MAPPINGOF CENTROMERES AND THE USE OF TRANSLOCATIONS FOR SEQUENCING OF MEIOTIC FRAGMENTS While centromeres probably now could be mapped meiotically by analysis of unordered tetrads because quite a lot of markers close to their centromeres have become available, no such analysis has been attempted in recent years. In Aspergillus, early results from standard crosses between markers of groups I-IV indicated loose linkage of markers to three of the centromeres (Strickland, 1958). All of these have been confirmed by mitotic mapping, even though the actual values calculated for the meiotic distances do not agree exactly with current estimates (three of them being smaller, and one considerably larger). All recent centromere mapping is, therefore, based on mitotic crossing-over or on special techniques that make use of translocations. These latter are of two types: The centromere position can be postulated either on the basis of mapped translocation breaks if breaks are very close to the centromeres; or centromeres can be mapped by mitotic crossing-over in diploids homozygous for translocations ‘in cases where the latter transfer suitable selective markers to new linkage groups. 1. Mapping of Centromeres by Mitotic Crossing-over in Standard

Diploids For reliable mapping of centromeres by mitotic crossing-over, selection should ideally be practicable in both chromosome arms. When all types of selection are considered (including, for example, selection for auxotrophy), this probably is the case in Aspergillus for all groups in which markers on both arms have been isolated. However, in the case of acrocentric chromosomes, or when all available markers are located on the same arm, centromere mapping presents more of a problem (as is the case for markers in groups VI, VII, and VIII used here). Also, if selection is possible in only one arm, it is practically impossible to distinguish between crossover segregants and nondisjunctional ones, unless more than one marker is available on the other arm. Fortunately, crossingover is relatively frequent close to the centromere. Therefore, two markers, which only rarely, and always simultaneously, become homozygous (especially if they are not closely linked in meiosis) can usually be identified as markers on the opposite arm. Hence the rare homozygous segregants are probably of nondisjunctional origin. In other cases, where selected crossovers apparently show recombination between all “proximal” markers, the centromere position cannot definitely be identified. It can, however, be tentatively assigned if values for the most ‘Lproximal” interval are either extremely large (over 30%) or very small (less than

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

117

570). Such guesses are somewhat easier if the same selective system has been used in less ambiguous situations, and the relative viabilities of all types can be judged. No expected percent value for nondisjunctionals can be given because the relative frequencies of nondisjunctionals among selected diploid segregants vary not only with the relative distance of the selective marker from the centromere, but also with the different conditions of selection (values of less than 3% and over 25% have been obtained). It seems that under different conditions, the absolute frequencies of nondisjunction vary considerably and in unpredictable ways (as evident for chromosome I : Section 111, A, 2 ) . This is understandable if different conditions permit growth and further segregation of trisomics, which always are the primary product, to different degrees. Also it is possible that certain selective media actually induce “double” nondisjunction (e.g., p-fluorophenylalanine when used for selection of fpaA or fpaB, since it causes general missegregation of chromosomes). Additional problems also arise when selective or other markers are used that become homoaygous by recombination not much more frequently than by mutation [as, e.g., ActA, see below; or as found for two of the markers recently used in Dictyostelium (Mosses et al., 1975)]. Problems naturally also may be caused by intrachromosomal aberrations, e.g., inversions (or minor ones like the one postulated for the centromere area of group I in the biA strain: Section 111,B). Any other aspects of centromere mapping will be discussed by presenting as examples the details for the centromers of groups I to I V and VI, which all were mapped by mitotic crossing-over in standard diploids. a. Linkage Group I . In Aspergillus nidulans good selective markers are available distal in both arms only in two groups: groups I and 11. On I R, selection for yellow conidial heads (yA) has been used extensively even though it is rather laborious ; however, relatively efficient selection is possible on the other arm (using suAadE, and fpaA or fpaB). The centromere position between lysF - yA on the one hand and suAadE or fpaA - 1uA on the other, is therefore easy to demonstrate; unexpectedly, however, the mapping between proA and lysF in certain strains does not give completely unambiguous results (see Section 111,B). b. Group 11. This centromere was first mapped between wA and thiA by selecting for AcrA on I1 L, and acrB on I1 R (Kafer, 1958), but it has more recently been mapped between two closer markers by Clutterbuck (1974), namely palcA and riboE, the color mutant ygA being used as a selective marker on I1 R. c. Group IZI. The best selective marker in this group is sC, which has recently been mapped close and distal to ad1 on I11 L (Kafer, 1975). On selenate media, homozygous sC segregants can easily be obtained (Jan-

118

ETTA KAFER

sen, 1975; Arst, 1968). Jansen’s results show the sequence of markers on the SC arm (clearly the left arm, see Fig. 14) as cnzH sC - dilA - gaZA, and the centromere between galA and phenA was identified by obtaining a few phenA crossovers after selection for auxotrophy (among homozygous sC, 16% of nondisjunctionals were obtained). We have used selection for SC in several diploids heterozygous for a variety of markers. The two most informative ones are shown in Table 20 (diploids 1873 and 1877). The obtained results confirm and extend those of Jansen; they map the centromere between ActA and phenA (as postulated by Bainbridge, 1970) and place SuBpro on the same arm as phenA, that is to the right of p h e d (no recombination between phenA and SuBpro was obtained in sC/sC segregants, so that the types that are homozygous for all markers are recognized as nondisjunctionals ; their frequencies, 15-20y0, agree with those obtained by Jansen). In addition galE, a d l , and methH are confirmed as markers of the left arm (as placed by Dorn, 1967) ; however, galE is shown to be definitely located distal to sC, and methH, galA, and ActA proximal to it [as shown in Figs. 14 and 15 and in the most recent map of Clutterbuck (1974) ; confirming evidence for the position of suBpro has recently also been published by Klimczuk and Wegledski (1974) 1. Selection of crossovers using ActA (Bainbridge, 1970) on very high levels of actidione, was found to be very difficult, since most of the rare sectors turned out to be haploids, or heterokaryons between haploids and the parental diploid. From one diploid, with a d l , ActA, and phenA in coupling, no diploid actidione-resistant crossovers could be obtained. From a second one, with sC, galA, ActA, and phenA in coupling, a few

-

I

I

edE

meaB 28

14

I I

.-I

I 39'

-

1 m H

I I

27-1

I

i 3.8 ;d' sA

I 145

I galA

methH dilA 9

1 !I

28

I

I ActA

phenA

1

I--

I

6'

11.

1

-

suBpro 24.

I

1

11. -*

FIG. 14. Linkage map of group 111: Meiotic recombination (%) from standard crosses, except for distances adjacent to break of Tl(ZZZ;VZZ); * = well established values, standard errors
TABLE 20 Diploid Mitotic Recombinants from Diploids 1873 and 1877, Heteroaygous for sC and Other Markers of Group 111 Diploid 1873

Diploid 1877

cross-

Meiotic overs in units interval

Intervakl 2 galE SC No. of egregants

+

40

1

'0

5

2

0

50

3 s28}2,

10

SO

4

2

1'

5 - } g

10

8

25

6

(1 9

Nondisjunctionals

z 1'

15 0

0

+

+

3

+

d methU

5

4

dilA

+

+

8

7

8

+ - p .h - , -

gola ActA

9

d

+

i

-

I

+

SuBpro

1

2

3

4

5

SC f

+

+ -

- -

t

6

7 8 9

-

- - -+ " + + + +

:Gr:!

ApprOX.

combined eettmate

gpnts

Distal

+ -

sc+

+ + +

sc+

+

- sc+

+

-

-

-

-

-

s c + s c +

s c i

s c +

+

s c +

+

s c +

+

s c i

+

s c +

+

i

+ + + -

-

+

-

+

-

+ + +

+ - - + i-

= , +-

-

+

+ - $ ++ - +

-- +

-

I + s c + - -

35-45%

1-3%

-

+ I*sc+++--li-

+ s c + + + + +

-

i

+

d

+

+

+

I + s c + + + - -

-

+

10-15%

+ +

-

2-5%

+- +

+ - )

+ * c + + + - - + I

cated type is dentified in this way.

+ + + +

+I :- + l+sc+++--g+-

+

+

-

0%

+ a + + + - +- -

+

I

154

15-208

1 I I

Other arm 15-2096

120

ETTA KAFER

crossovers homozygous for ActA, as well as sC and gaZA but not phenA, were isolated. Prototrophic sectors were also isolated, but after extensive purification only two of these were genuine diploid segregants; one of the two was haploidized, and it turned out to be still heterozygous, containing the Act+ allele as well as a mutation to a much more resistant allele in the other homolog. d. Group IV. The centromere of group I V has originally been mapped between methG and pyroA in a diploid homozygous for biA, using “bistarvation” for selection (Kafer, 1958) ; and the data with T l (1V;VIII) (Fig. 13) showed that jrAl and all markers to the right (paZC - pabaB pyrod) are on the same arm. To map the centromere more accurately, diploids heterozygous for the new suppressor of adE20, w C l ladE20, were constructed with all these markers (except j r A l ) in coupling to suCadE. Since suCadE is very closely linked to jrAl in crosses heterozygous for TI (1V;VlII), it was expected t o map proximal on the right arm. However, the following results (of K. Schafer) map the centromere between suCadE and jrA 1 : all (ca. 300) diploid suCadE segregants were found to be also homozygous for methG, placing this marker distal to suCadE; on the other hand, only a fraction (27%) were homozygous for markers of the right arm (paZC - pabaB - pyroA) and, as expected, always simultaneously homozygous for all of them. These latter segregants must therefore be nondisjunctional diploids and their high frequency a result of the very close position of suCadE to the centromere. These results confirm the mapping of the markers paZC to pyroA on the right arm of IV, and map the centromere to the right of suCadE, hence between suCadE and jrA; but suCadE, like methG‘, must be located on the left arm. e. Group VI. The centromere of this group was mapped to the left of ZacA, using sB (on selenate media) for selection, in diploids heterozygous for sB in coupling with ZacA, b.wA, and sbA (Fig. 15). All the obtained diploid segregants were homozygous for sbA as well as sB, placing sbA distal to sB. In addition, many segregants (ca. 65%) were also homozygous for bwA and over half of these lacA/ZacA. This confirms the sequence lacA - bwA - sB,sbA deduced from the mapping of T l ( V I ; V I I ) (Kafer, 1975) and of TI ( V ; V I )(above) and places the centromere to the “left” of lacA and of all other markers used here (unless nondisjunction is as high as 30% in this case).

-

2. The Use of Mapped Translocations for Positioning of Centromeres and Sequencing of Meiotic Fragments

The mapping of reciprocal translocations can produce two types of information if incompletely sequenced linkage groups are involved: (a)

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

121

Markers that are translocated together map on the same chromosome tip or arm, while all other markers of that group are located on the residual part of that chromosome, which must contain the centromere. (b) Results from several translocations with breaks in the same arm identify overlaps and may sequence meiotically unlinked markers or meiotic fragments of that arm. a. The centromere of VIII was identified to the left of sD50 and all other markers used here (Fig. 15), when the break of T2(I;VIZZ) was mapped a t sD and all other markers of VIII were found to be translocated to linkage group I ( M a and Kafer, 1974) ; if diploids homozygous for this translocation are used, mapping of the centromere of VIII in relation to any markers proximal to sD will be easy, since in such diploids two selective markers, suAadE and fpaB, are translocated to VIII R ) . Similarly the first evidence for the position of the centromere of group V to the “left” of pA - f a c A - hxA -riboD was obtained from the mapping of the translocation T1 (V;VZ), since all these markers are translocated t o the centromere-ZacA segment of group VI (Tables 18 and 19 and Fig. 15). b. The mapping of the breaks of T1 (Z;VIZ), T1 (VI;VII), and T1 (ZII;VZZ) lead to the following sequencing of meiotic fragments of two groups: on VI, lacA - bwA - sB, sbA; and on VII, probably all on the same arm, phenB - OliA - sF,pantoB - palF - lysD - malA - choA - nicB (Kafer, 1975).

5. Mapping of the Centromeres by Mitotic Crossing-over in T / T Diploids To map the centromeres of V and VII, the two translocations that transfer the selective marker sB to these chromosomes have been used in T/T diploids, heterozygous for sB and a variety of markers. Segregants homo- or hemizygous for sB were selected on selenate media. Table 19 gives the genotypes of these diploids and of the mitotic haploids isolated from them, which confirmed the positions of the breaks with respect to markers on the two segments. Three diploids homozygous for T l (V;VZ) were used to map the centromere of group V (2182, 2184, and 2211) and these gave the following types of diploid segregants. All sB/sB scgregants (total ca. 400) were simultaneously homozygous for sbA, placing sbA distal to sB in the translocated segment from group VI (as expected from the corresponding results in standard diploids mentioned above). None of these sB/sB segregants was homozygous for any other marker but lysB [even though about 90 could have shown homozygosis for lacA or bwA, two markers proximal on VI R ; and 100-200, homozygosis for either facA, hxA, or

122

ETTA KAFER

111

14

hiA

*

;

-

so0 47

bwA

I

38

.8

-500

nbd)

38

17

k A

VI

frA

27

V

sbA

41'

20

35

VII

fscA

COA

Vlll

@ 21'

T2(1;VIII)

CnlB

5

fr0

0

4

r,ka

50

pal0 chaA

.E

I

I

FIG.15. Sequence of markers and mapped translocation breaks in all eight chromosomes and meiotic recombination frequencies between adjacent markers in percent. Values above the lines are from recent standard crosses; below, from heterozygous translocation crosses; * = well established values, based on totals >low, standard errors less than 10%; "(4", etc.) = tentative values based on totals <5W. For linkage group 11, no new mapping data were available, and those of Kafer (1958) are shown.

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

123

riboD of group V which presumably are translocated to VI, judging from their mapping on the “other” translocation chromosome of T1 (V;VZ)1. The mutant, lysB was found to be homozygous only rarely ( 1 4 % in three samples of 50-100 each from diploid 2211, the only diploid with sB and lysB in coupling). These results indicate that the centromere of V is probably located between lysB and the break of T1 ( V ; V I ) ,proximal to P A : such a low frequency of lysB/lysB types is more likely to be due to nondisjunction than to crossing-over (provided the viability of ZysB types was normal on selenate media; diploids with sB in coupling to nicA, which maps very close to lysB, could be used to check this; or, even more conclusive, haploidization of sB/sB segregants from diploids 2184 and 2208, which carry sB and l y s B in repulsion; the latter might be rather time consuming, but would avoid construction of new strains with nicA, as well as sB and the translocation in coupling). The centrornere of group VIZ, on the other hand, was mapped by the break of TI (1;VIZ) as to the left of all markers used here, except possibly phenB which is located proximal, but fairly closely linked, t o the break (Fig. 15). To identify the position of the centromere either t o the left of phenB, or between phenB and the translocation break, two diploids homozygous for Tl(VZ;VZZ) and heterozygous for phenB and sB in coupling were constructed (diploids 1997 and 1998, Table 19). When homozygous sB segregants were selected on selenate, over 25% of these were found to be also homozygous for phenB. This most likely maps the ccntromere to the left of phenB (as in the case of lacA on VI above). I n addition the sB/sB mitotic crossovers (from diploid 1997) segregated for OZiA: namely, about 70% of these segregants were still heterozygous for OliA as well as phenB, a few of them were homozygous for OliA+but not for phenB, while all homozygous phenB also were homozygous for OliA’. This confirms the mapping of OZiA proximal to the break of T1 (VZ;VZZ) and definitely places it distal to phenB, that is, between phenB and sF or pantoB, on the standard map. All markers of VII used so far, therefore, map on one long “right” arm of this group, which may possibly represent an acrocentric chromosome. In conclusion, these recent results show that the use of translocations, and the various methods of mapping that they make possible, has led to a considerable change in the overall maps of Aspergillus (some of these results are already taken into account in the recent map of Clutterbuck, 1974). Not only have all centromeres now been placed, even if somewhat tentatively, but in addition most of the unlinked meiotic fragments have been sequenced. However, several of these fragments contain groups of meiotically linked markers that still need to be oriented. This applies specifically t o fragments on which only one marker was used in the translocation mapping; a second one a t the other end might possibly

124

ETTA KAFER

have shown up additional meiotic linkages in heterozygous translocation crosses. This, for example, is the case for group V I ; however in early attempts to use the markers lysA, nicC, and pacC, difficulties of various types were encountered (aberrations, excessive selfing, etc.). It seems quite likely that crosses of the latter two and e.g., molA (Clutterbuck, 1974) to the sequenced markers used here, and heterozygous for one of the translocations with breaks in VI, would produce some new meiotic linkages. Or, failing this, diploids heterozygous for sB and the above markers in coupling could sequence a t least some of them, even if-at the worstall of them were located on the unidentified “left” arm. The only other nonoriented fragment a t this time is that containing nicB (as well as palD and f l A ) distal on VII R. Since early crosses heterozygous for palD, choA, and T1 (V1;VIZ) did not produce any detectable linkage between these mutants, the only other useful experiment might be to use flA in a similar cross (such morphological mutants were not used here, because they make classification of disomics impossible). It is clear, however, that even complete sequencing of markers that would orient these known fragments would not in any sense finish or establish the total AspergiZlus map, since it is still expanding a t a considerable rate. New, loosely linked markers are being found not only a t the ends of mapped groups, but not infrequently have to be placed onto completely unlinked meiotic fragments. And it seems that the overall rate of increase in size has barely changed from the first map (Kafer, 1958) to the second one (Dorn, 1967; increase >600 units in 9 years) compared to the next one (Clutterbuck, 1974; increase >450 units in 7 years). It is, therefore, quite clear that the genetic map of Aspergillus nidulans is still far from being “saturated,” and considerable increases in the near future will presumably result just from the meiotic mapping of the over 75 genes, which a t this time are mapped only to one of the eight linkage groups (Clutterbuck, 1974). ACKNOWLEDGMENTS The excellent technical assistance of Mrs. P. Marshall and the expert photography of Mr. R. Lamarche are greatfully acknowledged. Thanks are due also to Drs. E. R. Boothroyd, A. J. Clutterbuck, and A. Upshall for help in the preparation of this publication. This work was supported by operating grant No. 2564 from the National Research Council of Canada.

REFERENCES Abbondandolo, A., and Bonatti, S. 1970. The production by nitrous acid of complete and mosaic mutations during defined nuclear stages in cells of Schizosaccharomyces pombe. Mutat. Res. 9, 59-69.

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

125

Arst, H. N. 1968. Genetic analysis of the first steps of sulphate metabolism of Aspergillus nidulans. Nature (London) 219, 265-270. Azevedo, J. L., and Roper, J. A. 1970. Mitotic non-conformity in Aspergillus: Successive and transposable genetic changes. Genet. Res. 16, 79-93. Bainbridge, B. W. 1970. Genetic analysis of an unequal chromosomal translocation in Aspergillus nidulans. Genet. Res. 15, 317-326. Bainbridge, B. W., and Roper, J. A. 1966. Observations on the effects of a chromosome duplication in Aspergillus nidulans. J. Gen. Microbiol. 42, 417-424. Ball, C. 1973. Improvement of penicillin productivity in Penicillium chrysogenum by recombination. In “Genetics of Industrial Microorganisms” (Z. VanEk, Z. HOE k l e k , and J. Cudlin, eds.), Vol. 2, pp. 227-237. Elsevier, Amsterdam. Bandiera, M., Armaleo, D., and Morpurgo, G. 1973. Mitotic intragenic recombination as a consequence of heteroduplex formation in Aspergillus nidulans. Mol. Gen. Genet. 122, 137-148. Barratt, R. W., Johnson, G. B., and Ogata, W. N. 1965.Wild-type and mutant stocks of Aspergillus nidulans. Genetics 52, 233-246. Barratt, R. W., Ogata, W. N., and Kiifer, E. 1975. Fungal Genetics Stock Center: Aspergillus Stocklist (1st revision, July 1975). Aspergillus Newsl. 13, 23-58. Barry. E. G., and Perkins, D. D. 1969. Position of linkage group V markers in chromosome 2 of Neurospora crassa. J. Hered. 60, 12&125. Bhalla, S. C., Cajaiba, A. C. I., Carvalho, W. M. P., and Santos, J. M. 1971. Translocations, inversions and correlation of linkage groups to chromosomes in the mosquito Culex pipiens fatignns. Can. J . Genet. Cytol. 18, 837-350. Bignami, M., Morpurgo, G., Pagliano, R., Carere, A., Conti, G., and Di Giuseppe, G. 1974. Nondisjunction and crossing over induced by pharmaceutical drugs in Aspergillus nidulans. Mutat. Res. 26, 159-170. Blakeslee, A . F., and Belling, J. 1924. Chromosomal mutations in the Jimson weed (Datura stramonium).J . Hered. 15, 194-206. Brody, T., and Williams, K. L. 1974. Cytological analysis of the parasexual cycle in Dictyostelium discoideum. J. Gen. Microbiol. 82, 371-383. Burnham, C. R.,Stout, J. T., Weinheimer, W. H., Kowles, R. V., and Phillips, R. L. 1972.Chromosome pairing in maize. Genetics 71, 111-126. Cacheiro, N. L., Russell, L. B., and Swartout, M. S.1974. Translocations, the predominant cause of total sterility in sons of mice treated with mutagens. Genetics 76,7%91. Campbell, D. A., Fogel, S., and Lusnak, K. 1975. Mitotic chromosome loss in a disomic haploid of Saccharomyces cerevisiae. Genetics 79, 383-396. Carlson, P. S. 1972. Locating genetic loci with aneuploids. MoZ. Gen. Genet. 114, 273-280. Carpenter, A. T. C. 1973. A meiotic mutant defective in distributive disjunction in Drosophila melanognster. Genetics 73, 39W28. Carpenter, A. T. C., and Sandler, L. 1974. On recombination-defective meiotic mutants in Drosophila melanogaster. Genetics 76, 45g475. Christianson, M. L. 1975. Mitotic crossing-over as an important mechanism of floral sectoring in Tradescantia. Mutat. Res. 28, 389-395. Clutterbuck, A. J. 1970. A variegated position effect in Aspergillus nidulans. Genet. Res. 16, 303-316. Clutterbuck, A. J. 1974. Aspergillus nidulans. In “Handbook of Genetics” (R. C. King, ed.), Vol. 1, pp. 447-510. Plenum, New York. Clutterbuck, A . J., and Cove, D. J. 1974. The genetic loci of Aspergillus nidulans. In “Handbook of Microbiology” (H. Lechevalier, ed.), pp. 665476. Chem. Rubber Publ. Co., Cleveland, Ohio.

126

ETTA KAFER

Comings, D. E., and Okada, T. A. 1972. Holocentric chromosomes in Oncopeltus: Kinetochore plates are present in mitosis but absent in meiosis. Chromosoma 37, 177-192. Crackower, S. H. B. 1972. The effects of griseofulvin on mitosis in Aspergillus nidulans. Can. J . Microbiol. 18, 683-687. Curtis, C. F., and Robinson, A. S. 1971. Computer simulation of the use of double translocations for pest control. Genetics 69,97-113. Davies, P. J., Evans, W. E., and Parry, J. M. 1975. Mitotic recombination induced by chemical and physical agents in the yeast Saccharomyces cerevisiae. Mutat. Res. 29, 301-314. Day, A. W., and Jones, J. K. 1971. p-Fluorophenylalanine-inducedmitotic haploidization in Ustilago violacea. Genet. Res. 18, 299-309. Dorn, G. L. 1967. A revised map of the eight linkage groups of Aspergillus nidulans. Genetics 56, 619-631. Dorn, G. L. 1972. Computerized meiotic mapping in Aspergillus nidulans. Genetics 72, 595-605.

Dulieu, H. L. 1975. Somatic variants on a yellow mutant in Nicotiana tabacum L. (al+/al az+/az).11. Reciprocal genetic events occurring in leaf cells. Mutat. Res. 28, 69-77.

Elliot, C. G. 1956. Triploid Aspergillus nidulans. Microb. Genet. Bull. 13, 7. Elliot, C. G. 1960a. The cytology of Aspergillus nidulans. Genet. Res. 1, 462-476. Elliot, C. G. 1960b. Non-localized negative interference in Aspergillus nidulans. Heredity 15, 247-262. Esposito, R. E., and Holliday, R. 1964. The effect of 5-fluorodeoxyuridine on genetic replication and mitotic crossing over in synchronized cultures of Ustilago maydis. Genetics 50, 1009-1017. Evans, E. P., and Phillips, J. S. 1975. Inversion heterozygosity and the origin of XO daughters of Bpa/+ female mice. Nature (London) 256, 40-41. Ferrus, A. 1975. Parameters of mitotic recombination in minute mutants of Drosophila melanogaster. Genetics 79, 589-599. Finkelstein, D. B., Blamire, J., and Marmur, J. 1972. Location of ribosomal RNA cistrons in yeast. Nature ( L o n d o n ) , N e w Biol. 240, 279-281. Forbes, E. C. 1959. Use of mitotic segregation for assigning genes to linkage groups in Aspergillus nidulans. Heredity 13, 67-80. Ford, C. E., and Clegg, H. M. 1969. Reciprocal translocations. Br. M e d . Bull. 25, 110-1 14.

Gamborg, 0. L., Constabel, F., Fowke, L., Kao, K. N., Oyama, K., Kartha, K., and Pelcher, L. 1974. Protoplast and cell culture methods in somatic hybridisation in higher plants. Can. J. Genet. Cytol. 16, 737-750. Garcia-Bellido, A. 1972. Some parameters of mitotic recombination in Drosophiln melanogaster. Mol. Gen. Genet. 115, 54-72. Gatti, M., Tonzarella, C., and Oliveri, G. 1974. Analysis of the chromosome aberrations induced by X-rays in somatic cells of Drosophila melanogaster. Genetics 77, 701-719. Gingold, E. B., and Ashworth, J. M. 1974. Evidence for mitotic crossing-over during the parasexual cycle of the cellular slime mould Dictyostelium diwoideum. J. Gen. Microbiol. 84, 70-78. Gravel, R. A,, Kiifer, E., Niklewicz-Borkenhagen, A., and Zambryski, P. 1970. Genetic and accumulation studies in sulfite-requiring mutants of Aspergillus nidulans. Can. J . Genet. Cytol. 12,831-840.

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

127

Grell, R. F. 1959. Non random assortment of non-homologous chromosomes in Drosophila melanogaster. Genetics 44, 421-435. Grell, R. F. 1962. A new hypothesis on the nature and sequence of meiotic events in the female of Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S.A. 48, 165-1 72. Grell, R. F. 1967. Pairing a t the chromosomal level. J. Cell. Physiol. 70, Suppl. 1, 119-146. Grell, R. F. 1971a. Induction of sex chromosome nondisjunction by elevated temperature. Mutat. Res. 11,347-349. Grell, R. F. 1971b. Distributive pairing in man? Ann. Genet. 14, 165-171. Hastie, A. C. 1970. Benlate-induced instability of Aspergillus diploids. Nature (London) 226, 771. Holliday, R. 1971. Biochemical measure of the time and frequency of radiation-induced allelic recombination in Ustilago. Nature ( L o n d o n ) , N e w Biol. 232, 233236. Hotta, Y., and Benzer, S. 1972. Mapping behaviour in Drosophila. Nature (London) 240,527-535. Jacobs, P. A,, Buckton, K. E., Cunningham, C., and Newton, M. 1974. An analysis of the break points of structural rearrangements in man. J . M e d . Genet. 11, 50-61. James, A. P., and Inhaber, E. R. 1975. Evidence of preferential pairing of chromosomes at meiosis in aneuploid yeast. Genetics 79, 561-571. James, A. P., Inhaber, E. R., and Prkfontaine, G. J. 1974. Lethal sectoring and the delayed induction of aneuploidy in yeast. Genetics 77, 1-9. Jansen, G. J. 0. 1975. The order of some loci and the position of the centromere on chromosome I11 of Aspergillus nidulans. Aspergillus Newsl. 12, 20-21. Kafer, E. 1958. An 8-chromosome map of Aspergillus nidulans. A d v . Genet. 9, 105145. KLfer, E. 19W. High frequency of spontaneous and induced somatic segregation in Aspergillus nidulans. Nature (London) 186,619-620. Kiifer, E. 1961. The processes of spontaneous recombination in vegetative nuclei of Aspergillus nidulans. Genetics 46, 1581-1609. Kafer, E. 1962. Translocations in stock strains of Aspergillus nidulans. Genetica ( T h e Hague) 33, 59-68. Kiifer, E. 1963. Radiation effects and mitotic rerombination in diploids of Aspergillus nidulans. Genetics 48, 2745. Kafer, E. 1965. Origins of translocations in Aspergillus nidulans. Genetics 52, 217-232. Kafer, E. 1969. Effects of ultraviolet irradiation on heterozygous diploids of Aspergillus nidulnns. 11. Recovery from W-induced mutation in mitotic recombinant sectors. Genetics 63, 821-841. Kafer, E. 1975. Reciprocal translocations and translocation disomics of Aspergillus and their use for genetic mapping. Genetics 79,7-30. Kiifer, E. 1976. Mitotic crossing over and nondisjunction in translocation heterozygotes of Aspergillus. Genetics 82, 605-627. Kafer, E., and Chen, T. L. 1964. Translocations and recessive lethals induced in Aspergillus nidulans by ultraviolet light and gamma-rays. Can. J . Genet. Cytol. 6, 249-254. Kafer, E., and Upshall, A. 1973. The phenotypes of the eight disomics and trisomics of Aspergillus niddans. J. Hered. 64, 35-38. Kafer, E., Marshall, P., and Cohen, G. 1976. Well-marked diploids of Aspergillus for tests of environmental mutagens : Identification of induced mitotic recombination and mutation. Mutat. Res. 38, 141-146.

128

ETTA KAFER

Kappas, A., and Georgopoulos, S. G. 1974. Interference of griseofulvin with the segregation of chromosomes a t mitosis in diploid Aspergillus nidulans. J. Bacteriol. 119,334-335. Kappas, A., Georgopoulos, S. G., and Hastie, A. C. 1974. On the genetic activity of benzimidazole and thiophanate fungicides on diploid Aspergillus nidulans. Mutat. Res. 28, 17-27. Kasha, K. J., and Burnham, C. R. 1965. The location of interchange break points in barley. 11. Chromosome pairing and the intercross method. Can. J. Genet. Cytol. 7, 620-632. Katz, E. R., and Kao, V. 1974. Evidence for mitotic recombination in the cellular slime mold Dictyostelium discoideum. Proc. Natl. Acad. Sci. U S A . 71, 40254026. Kelley, F. I., and Legator, M. 1970. Effect of N-methyl-N'-nitro-N-nitrosoguanidine on the cell cycle and chromosomes of human embryonic lung cells. Mutat. Res. 10, 237-246. Kelly, P. T. 1974. Non-reciprocal intragenic mitotic recombination in Drosophila melanogaster. Genet. Res. 23, 1-12. Kessel, R., and Rowe, P. R. 1974. Interspecific aneuploids in the genus Solanum. Can. J. Genet. Cytol. 18, 515-528. Kessin, R. H., Williams, K. L., and Newell, P. C. 1974. Linkage analysis in Dictyostelium discoideum using temperature-sensitive growth mutants selected with bromodeoxyuridine. J. Bacteriol. 119, 776-783. Khush, G.S.,and Rick, C. M. 1967.Tomato tertiary trisomics: Origin, identification, morphology and use in determining position of centromeres and arm location of markers. Can. J. Genet. Cytol. 9,610-631. Kinsey, J. A., and Stadler, D. R. 1969. Interaction between analogue resistance and amino acid auxotrophy in Neurospora. J. Bacteriol. 97, lllP1117. Klimczuk, J., and Weglefiski, P. 1974. New proline suppressor mutants in Aspergillus nidulans. Acta Microbiol. Pol. 8,93-100. Laird, C. D. 1973. DNA of Drosophila chromosomes. Annu. Rev. Genet. 7, 177-204. Lawrence, C. W.,and Christensen, R. 1974. Fine structure mapping in yeast with sunlamp radiation. Genetics 78, 723-733. Lefevre, G., Jr. 1973. Mutant vs nonmutant chromosome breakpoints in the X chromosome of Drosophila melanogaster. Genetics 74, Suppl. 2, Part 2, s. 154. Lhoas, P. 1961. Mitotic haploidization by treatment of Aspergillus niger diploids with para-fluorophenylalanine. Nature (London) 190, 744. Lhoas, P. 1967. Genetic analysis by means of the parasexual cycle in Aspergillus niger. Genet. Res. 10, 45-61. Lindsley, D.L., Sandler, L.et al. 1972.Segmental aneuploidy and the genetic gross structure of the Drosophila genome. Genetics 71, 157-184. Luria, S. E.,and Delbruck, M. 1943. Mutations from virus sensitivity to virus resistance. Genetics 28, 491-511. Ma, G. L. C., and Kafer, E. 1974. Genetic analysis of the reciprocal translocation T 2 U ; V I I I ) of Aspergillus using the technique of mitotic mapping in homozygous diploids. Genetics 77, 11-23. McClintock, B. 1945. Neurospora. I. Preliminary observations of the chromosomes of Neurospora crassa. Am. J. Bot. 32, 671478. McCully, K.S.,and Forbes, E. 1965. The use of p-fluorophenylalanine with "master strains" of Aspergillus nidulans for assigning genes to linkage groups. Genet. Res. 8, 352-359.

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

129

Macdonald, K. D. 1971. Segrcgants from a heterozygous diploid of Penicillium chrysogenum following different physical and chemical treatment. J . Gen. Microbiol. 67, 247-2.50. Malling, H. V., and DeSerres, F. J. 1970. Genetic effects of N-methyl-N’-nitro-Nnitrosoguanidine in Neurospora crnssa. Mol. Gen. Genet. 106, 195-207. Megginson, F. G. A., and Person, C. 0. 1974. Somatic recombination in Ustilago hordei during the parasitic phase on barley. Can. J . Genet. Cytol. 16, 851-855. Miller, D. A., and Miller, 0. J. 1972. Chromosome mapping in the mouse. Science 178, 949-955. Mittwoch, U., and Delhanty, J. D. A. 1972. Inhibition of mitosis in human triploid cells. Nature (London), New Biol. 283, 11-13. Morpurgo, G. 1963. Somatic segregation induced by p-fluorophenylalanine. Aspergillus N e d . 4, 8. Mortimer, R. K., and Hawthorne, D. C. 1973. Genetic mapping in Saccharomyces. IV. Mapping of temperature-sensitive genes and use of disomic strains in localizing genes. Genetics 74, 33-54. Mosses, D., Williams, K. L., and Newell, P. C. 1975. The use of mitotic crossing-over for genetic analysis in Dictyostelium discoideum: Mapping of linkage group 11. J . Gen. Microbiol. 90, 247-259. Newmeyer, D., and Taylor, C. W. 1967. A pericentric inversion in Neurospora, with unstable duplication progeny. Genetics 56, 771-791. Nothiger, R., and Diibendorfer, A., 1971. Somatic crossing-over in the housefly. Mol. Gen. Genet. 112, 9-13. Novitski, E . 1975. Evidence for the single phase pairing theory of meiosis. Genetics 79, 63-71. O’Brien, S. J., and Gethmann, R. C. 1973. Segmental aneuploidy as a probe for structural genes in Drosophiln: Mitochondria1 membrane enzymes. Genetics 75, 155-167. Papa, K. E . 1973. The parasexual cycle in Aspergillus pavus. Mycologin 65, 1201-1205. Parry, E. M., and Cox, B. S. 1970. The tolerance of aneuploidy in yeast. Genet. Res. 16, 333-340. Perkins, D. D. 1972. An insertional translocation in Neurospora that generates duplications heterozygous for mating type. Genelics 71, 25-51. Perkins, D. D. 1974. The manifestation of chromosome rearrangements in unordered asci of Neurosporn. Genetics 77, 459-489. Perkins, D. D. 1975. The use of duplication-generaiing rearrangements for studying heterokaryon incompatibility genes in Neurosporn. Genetics 80, 87-105. Pollard, R., Kafer, E., and Johnston, M. 1968. Influence of chromosomal aberrations on meiotic and mitotic nondisjunction in Aspergillus nidulans. Genetics 60, 743-757. Pontecorvo, G. 1956. The parasexual cycle in fungi. Annu. Rev. Microbiol. 10, 393-100. Pontecorvo, G., and Kafer, E. 1958. Genetic analysis based on mitotic recombination. A d v . Genet. 9, 71-104. Pontccorvo, G., Roper, J. A,, Hemmons, L. M., Macdonald, K. D., and Bufton. A. W. J. 1953. The genetics of Aspergillus nidulnns. Adv. Genet. 5, 141-238. Pontecorvo, G., TRrr Gloor, E., and Forbes, E. 1954. Analysis of mitotic recombination in Aspergillus nitlidnns. J . Ge)iet. 52, 226-237. Pritchard, R . H. 1954. Ascospores with diploid nuclei in Aspergillus nidulans. Caryologin 6, Suppl. 1, 1117.

130

ETTA KAFER

Rick, C. M., and Gill, B. S. 1973. Reproductive errors in aneuploids: Generation of variant extra-chromosomal types by tomato primary trisomics. Can. J. Genet. Cytol. 15, 299-308. Rick, C. M., and Khush, G. S. 1968. Cytogenetic explorations in the tomato genome. I n “Seminars in Genetics” (R. Bogart, ed.), pp. 45-68. Oregon State Univ. Press, Corvallis. Roberts, P. A. 1967. A positive correlation between crossing over within heterozygous pericentric inversions and reduced egg hatch of Drosophila females. Genetics 58, 179-187.

Roberts, P. A. 1970. Screening for X-ray-induced crossover suppressors in Drosophila melanogaster: Prevalance and effectiveness of translocations. Genetics 85, 429448.

Roberts, P. A. 1972. Differences in synaptic affinity of chromosome arms of Drosophila melanogaster revealed by differential sensitivity to translocation heteroaygosity. Genetics 71, 401415. Robinow, C. F., and Caten, C. E. 1969. Mitosis in Aspergillus nidulans. J. Cell Sci. 5, 403-431. Robinson, A. S., and Curtis, C. F. 1972. Crossing over in a double translocation in Drosophila. Can. J. Genet. Cytol. 14, 129-137. Roderick, T. H., and Hawes, N. L. 1974. Nineteen paracentric chromosomal inversions in mice. Genetics 78, 109-117. Roper, J. A. 1952. Production of heterozygous diploids in filamentous fungi. Experientiu 8, 14-15. Roper, J. A. 1966. Mechanisms of inheritance: The parasexual cycle. I n “The Fungi” (G. C. Ainsworth and S. S. Sussman, eds.), Vol. 2, pp. 589-617. Academic Press, New York. Roper, J. A., and Kafer, E. 1957. Acriflavin-resistant mutants of Aspergillus nidulans. J. Gen. Microbiol. 18, 660-667. Roper, J. A., and Nga, B. H. 1969. Mitotic non-conformity in Aspergillus nidulans: The production of hypodiploid and hypohaploid nuclei. Genet. Res. 14, 127-136. Roper, J. A., and Pritchard, R. H. 1955. The recovery of the complementary products of mitotic crossing over. Nature (London) 175,639. Rothman, F. G., and Alexander, E. T. 1975. Parasexual genetic analysis of the cellular slime mold Dictyostelium discoideum A3. Genetics 80, 715-731. Rowlands, R. T., and Turner, G. 1973. Nuclear and extranuclear inheritance of oligomycin resistance in Aspergillus nidulans. Mol. Gen. Genet. 128, 201-216. Ruddle, F. H. 1973. Linkage analysis in man by somatic cell genetics. Nature (London) 242, 165-169. Sanderson, K. E., and Srb, A. M. 1965. Heterokaryosis and parasexuality in the fungus Ascochyta imperfecta. Am. J. Bot. 52, 72-81.. Scott, B. R., and Alderson, T. 1974. A rapid method for preparing single-cell suspensions of Aspergillus conidia depleted in germination inhibitor. J. Gen. Microbiol. 85, 173-176.

Shanfield, B., and Kafer, E. 1971. Chemical induction of mitotic recombination in Aspergillus nidulans. Genetics 87, 209-219. Shows, T. B., and Brown, J. A. 1975. Human X-linked genes regionally mapped utilizing X-autosome translocations and somatic cell hybrids. Proc. Natl. Acad. Sci. U S A . 72, 212.52129. Sinha, U. 1967. Aromatic amino acid biosynthesis and para-fluorophenylalanine resistance in Aspergillus nidulans. Genet. Res. 10, 261-272.

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

131

Smith, D. A. 1974. Unstable diploids of Neurospora and a model for their somatic behavior. Genetics 76, 1-17. Smith, D. A. 1975. A mutant affecting meiosis in Neurospora. Genetics 80, 125-133. Snedecor, G. W., and Cochran, W. G. 1967. “Statistical Methods,” 6th ed. Iowa State Univ. Press, Ames. Stern, C. 1936. Somatic crossing-over and segregation in Drosophila melanogaster. Genetics 21, 625-730. Stern, C., and Tokanuga, C. 1971. On cell lethals in Drosophih. Proc. Natl. Acad. Sci. U S A . 68, 329-331. Strickland, W. N. 1958. An analysis of interference in Aspergillus nidulans. Proc. R . SOC.London, Ser. B 149,82-101. Talmud, P. J., and Lewis, D. 1974. The mutagenicity of amino acid analogues in Coprinus lagopus. Genet. Res. 23, 47-61. Terzi, M., and Hawkins, T. S. C. 1975. Chromosomal variation and the establishment of somatic cell lines in vitro. Nature (London) 253, 361-362. Tuleen, N. A. 1973. Karyotype analysis of multiple translocation stocks of barley. Can. J. Genet. Cytol. 15, 267-273. Tuleen, N. A,, and Gardenhire, J. H. 1974. Determination of break point positions in barley translocations by karyotype analysis of permanent rings of six chromosomes. Can. J. Genet. Cytol. 16, 539-548. Turner, B. C., Taylor, C. W., Perkins, D. D., and Newmeyer, D. 1969. New duplication-generating inversions in Neurospora. Can. J. Genet. Cytol. 11, 622-638. Upshall, A., and Kafer, E. 1974. Detection and identification of translocations by increased specific nondisjunction in Aspergillus nidulans. Genetics 76, 19-31. Upshall, U., Croft, J. H., Sargent, J., and Dales, D. 1977. Mitotic crossing over in disomic isolates of Aspergillus nidulans. (Submitted for publication.) Vig, B. K. 1973a. Somatic crossing over in Glycine maz (L.) Merrill: Effect of some inhibitors of DNA synthesis on the induction of somatic crossing over and point mutations. Genetics 73, 583-596. Vig, B.K.197313.Somatic crossing over in Glycine max (L.)Merrill: Mutagenicity of sodium azide and lack of synergistic effect with caffeine and mitomycin C. Genetics 75, 265-277. Vig, B. K., and Paddock, E. F. 1968. Alteration by mitomycin C of spot frequencies in soybean leaves. J. Hered. 59, 225-229. Wallace, H.1974.The chiasmata of axolotls. Arch. Genet. (Zurich) 47,8695. Ward, E. J. 1973. Nondisjunction: Localization of the controlling site in the maize B chromosome. Genetics 73, 387-391. Welshons, W. J., and Keppy, D. 0. 1975. Intragenic deletions and salivary band relationships in Drosophila. Genetics 80, 143-155. White, B. J., Tjio, J.-H., Van De Water, L. C., and Crandall, C. 1974. Trisomy 19 in the laboratory mouse. I. Frequency in different crosses at specific developmental stages and relationship of trisomy to cleft palate. Cytogenet. Cell Genet. 13, 217-231. Williams, K. L., Kessin, R. H., and Newell, P. C. 1974. Parasexual genetics in Dictyostelium discoideum: Mitotic analysis of acriflavin resistance and growth in axenic medium. J. Gen. Microbiol. 84,59-69. Wood, S., and Kafer, E. 1967. Twin spots as evidence for mitotic crossing-over in Aspergillus induced by ultra-violet light. Nature (London) 216, 63-64. Wood, S., and Kafer, E. 1969. Effects of ultraviolet irradiation on heterozygous diploids of Aspergillus nidulans. I. UV-induced mitotic crossing over. Genetics 62, 507-518.

THE CYTOGENETICS

OF

Neurospora

.

David D Perkins Department of Biological Sciences. Stanford University. Stanford. California

and

.

Edward G Barry Department of Botany. University of North Carolina at Chapel Hill. Chapel Hill. North Carolina

I . Introduction . . . . . . . . . . I1. The Life Cycle. Incompatibility. and Ploidy . I11. Meiosis and Mitosis . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . in the Ascus . . .

. . . .

A . Description of Normal Meiosis and Mitosis B . Somatic Divisions . . . . . . . . . . . . . . . . C Genetic Variants Affecting Meiosis or Related Stages (Meiotic Mutants. Aneuploids. Spore Killers. Bubble Asci) . . . . . . . . . IV The Wild-Type Genome . . . . . . . . . . . . . . . A . The Chromosomes . . . . . . . . . . . . . . . . 1 . Chromosome Size . . . . . . . . . . . . . . . 2. Chromosome Composition . . . . . . . . . . . . 3. Techniques for Chromosome Cytology . . . . . . . . . 4. Chromosome Identification and Morphology . . . . . . . B . Marker Distribution and Genetic Mapping . . . . . . . . 1 . The Markers . . . . . . . . . . . . . . . . 2. Genetic Maps and Marker Distribution . . . . . . . . 3. Crossing-over Variability and Its Basis . . . . . . . . 4 . Mapping Methodology . . . . . . . . . . . . . C . Linkage Group-Chromosome Correlations . . . . . . . . D . Cytoplasmic Genes . . . . . . . . . . . . . . . V . Chromosome Rearrangements . . . . . . . . . . . . . A . Methods of Identifying and Mapping Rearrangements . . . . . B . The Identified Rearrangements: Types. Frequencies. Origins. and Phenotypes. . . . . . . . . . . . . . . . . . C . Rearrangements That Do Not Make Viable Duplications . . . . 1 . Reciprocal Translocations . . . . . . . . . . . . . 2 . Mutual Insertions . . . . . . . . . . . . . . . 3. Inversions . . . . . . . . . . . . . . . . . D . Rearrangements That Generate Viable Nontandem Duplications via Meiotic Recombination . . . . . . . . . . . . . . 1 . Insertions (Insertional Translocations. Transpositions) . . . .

.

.

133

134 138 142 142 148 148 152 152 152 152 155 156 162 162 163 168 170 172 177 177 178 183 186 187 191 191 193 197

134

DAVID D. PERKINS AND EDWARD G . BARRY

2. Quasiterminal Rearrangements (Translocations, Pericentric

Inversions) .

. . . . . . . . . . . . . . . . . . . . . . . . .

3. Partially Overlapping Rearrangements

E. Nontandem Duplications and Their Characteristics (Mapped Duplications and Their Extent; Source and Mode of Origin; Vegetative Phenotype and Fertility; Instability) . . . . . . . . . . F. Rearrangements in Other Eukaryotic Microorganisms (Recognized Rearrangements; Errors froni Failure to Detect Rearrangements) . . VI. Summary . . . . . . . . . . . . . . . . . . . Appendix: Chromosome Aberrations of Neurospora crassa. . . . . . Explanatory Foreword . . . . . . . . . . . . . . . Index Tables . . . . . . . . . . . . . . . . . . Appendix Table 1: Rearrangements That Do Not Produce Viable Duplications, Listed by Linkage Groups . . . . . . . . . Appendix Table 2: Duplication-Generating Rearrangements, Listed by Type and Linkage Group . . . . . . . . . . . . . Descriptions of Individual Identified Rearrangements . . . . . . References . . . . . . . . . . . . . . . . . .

204 208 210 220 223 226 226 228 228 230 230 266

1. Introduction

Neurospora was chosen by Beadle and Tatum (1941) for obtaining the first nutritional mutants, thus opening a new era of biochemical genetics and molecular biology. Since then, 5000 papers concerned with Neurospora have been published, and several fundamental genetic contributions have been made using Neurospora, for example, the first proof of gene conversion (Mitchell, 1955), the first demonstration of complementation between alleles (Fincham and Pateman, 1957; Giles e t al., 1957), and the discovery of genes that regulate recombination precisely in specific chromosome regions (Jessup and Catcheside, 1965). Despite this activity, and despite Neurospora’s proved advantages for genetic research, cytogenetic knowledge has until recently progressed rather slowly. Neurospora is still far behind Drosophila (see Lindsley and Grell, 1968) and maize (Rhoades, 1950, 1955; Neuffer and Coe, 1974) in this respect, and is perhaps more nearly comparable t o tomato (Rick and Butler, 1956; Rick, 1971, 1974), the mouse (Miller and Miller, 1975), or barley (Nilan, 1974). The slow progress in Neurospora cytogenetics is partly because of the small size of the chromosomes. The DNA content per haploid genome is only one-third that in Drosophila and less than 1/115 that in maize. Reliable, detailed observations of meiotic chromosome structure and behavior can be made using conventional light microscopy, as was first shown by Barbara McClintock (1945), but the process is necessarily slower and more difficult than with maize pachytene chromosomes or Drosophila salivaries.

THE CYTOGENETICS OF

Neurospora

135

However, Neurospora cytogenetics entered a new phase when i t was realized that chromosome rearrangements could be identified and partially characterized simply by inspecting the ascospores that are shot out of fruiting bodies in groups that represent individual meiotic tetrads (Perkins, 1966a, 1967). At about the same time, simplified mapping methods were developed that have made it easier to locate point mutants and aberration break points. Since then, progress has been rapid. Over 160 rearrangements have been identified and mapped, and the number of known gene loci has been brought t o over 400. Many of the rearrangements produce viable duplications covering specific chromosome segments, and these are being used extensively for cytogenetic experiments. Scope. This review concerns genetically significant aspects of Neurospora cytology, and the relation between genes and chromosomes, with special emphasis on chromosome rearrangements. In addition to reviewing the published literature, numerous results are presented that have not been published previously, Many of these are cytological, appearing under various headings ; one section concerns the morphology and identification of individual chromosomes. New genetic results mainly concern chromosome rearrangements ; general characteristics of aberrations are given in the text, and a brief description of each known rearrangement is given in the Appendix. If not otherwise identified, cytological contributions are by Barry; genetic contributions are primarily by Perkins. The Neurospora results are related briefly to findings in other organisms. Some sections devoted to accessory topics are set in smaller print. These are not essential for understanding the sections that follow them. Some topics are omitted because of space limitations. N o attempt is made to deal with crossing over and interference (see Emerson, 1963, 1966, 1969; Perkins, 1962a,b; Bole-Gowda et al., 1962; Fincham and Day, 1971), intragenic recombination (see Emerson, 1969; Fincham and Day, 1971 ; Stadler, 1973), fine-structure mapping (see Catcheside, 1966; Fincham, 1974), molecular models of recombination (see Holliday, 1974; Hotchkiss, 1974; Sobell, 1974; Meselson and Radding, 1975 ; Wildenberg and Meselson, 1975; Wagner and Radman, 1975), or genetic aspects of DNA degradation, repair and synthesis (see Schroeder, 1975; Worthy and Epler, 1972, 1973). Other topics are dealt with only very briefly, such as the genetic control of recombination, and the cytoplasmic genome. Literature citations do not include publications that appeared after December 1975, when this review was completed. Advantages of Neurospora for Cytogenetics. Neurospora possesses several favorable features compared to the more conventional organisms that are used in cytogenetic research, and these in part compensate for the small size of its chromosomes. (1) All four products of individual

136

DAVID D. PERKINS AND EDWARD G. BARRY

meioses survive, (2) Viable meiotic products are black-pigmented spores, allowing white inviable deficiency spores to be seen in stark contrast. A chromosome rearrangement is signaled by the presence of white spores in characteristic frequencies and patterns. Major rearrangement types can be distinguished simply from a visual inspection of the numbers of white spores in individual asci. (3) Progeny can be obtained either as random meiotic products, as unordered tetrads, or as ordered tetrads whose linear spore arrangement reflects the events of meiosis. The spores show high viability and germination. (4) The vegetative (somatic) part of the life cycle is haploid. (5) Duplications are more readily identified as partial diploids against a haploid background than as partial triploids against a diploid background. (6) Somatic variants can be obtained in pure culture. Any somatic cell can serve as a germ cell. (7) The multinucleate nature of vegetative stages and the absence of cross walls in the ascus allow internuclear interaction and complementation to be studied. Intranuclear interactions can be studied in duplications. (8) The life cycle is short. Precise environmental control is possible. (9) Stocks can be maintained indefinitely in suspended animation. (10) Culture requirements and growth habit are such that stable mutants are readily obtained. Selective enrichment is often possible. Smallness of the genome may itself be a potential advantage for molecular cytogenetics (see Petes et ul., 1973). Neurosporu also shares advantageous features with the organisms more conventionally used in cytogenetic research, such as a low chromosome number, meiosis that is amenable to cytological examination, and distinctive chromomere patterns that enable individual chromosomes to be identified a t pachynema. Finally, a vast store of information already exists on Neurosporu, the exchange of research information is well organized, and stocks are readily available for research. A stock center (FGSC) maintains about 3000 Neurospora cultures, including most of the described mutant genes, rearrangements, wild strains, special-purpose stocks, and cytoplasmic variants (Fungal Genetics Stock Center, California State University, Humboldt, Arcata, California 95521 ; founded 1960 under R . W. Barratt and W. M. Ogata). Research methods are well developed (Davis and de Serres, 1970; Ryan, 1950 ; Emerson, 1955 ; Stanford Neurospora Methods, 1963; Perkins et al., 1969; Perkins, 1972b,c, 1975). A newsletter publishes research notes, stock lists, bibliographies, a directory of Neurospora workers listing their special interests, and summaries of the biennial Neurospora Research Conferences (Neurosporu Newsletter, founded 1962 and edited by B. J. Bachmann). Bibliographies list all research publications on Neurospora (Bachmann and Strickland, 1965 ; Bachmann, 1970, 1971-1976).

THE CYTOGENETICS OF

Neurospora

137

The genus Neurospora. Neurospora is a filamentous ascomycete belonging to the Class Pyrenomycetes and the Order Sphaeriales. This order includes other fungi well known to geneticists-Bombardia, Gelasinospora, Podospora, Sordaria, Ophiostoma, Glomerella, and Venturia. The genus was described by Shear and Dodge (1927) soon after the sexual stages were first recognized. Several conidiating Neurospora species are known, of which three are heterothallic with 8-spored asci (N. crassa, N. sitophila, and AT. intermedia) , and a t least one is pseudohomothallic with 4-spored asci (N. tetrasperma) . Crosses between species are sufficiently fertile that genes can be transferred from one species to another (see, for example, Fincham, 1951 ; Howe and Haysman, 1966; Metzenberg and Ahlgren, 1973). In addition, five true-homothallic species have been described-N. terricola, N. africana, N. dodgei, N. galapagosensis, and N. lineolata; these are all 8-spored and without conidia. For a conventional key to Neurospora species, based largely on morphology, see Frederick et al. (1969). An unconventional key, based on behavior in crosses with species-reference strains, is given by Perkins et al. (1976). The most used species hss been Neurospora crassa. This resulted largely from its choice for genetic work by C. C. Lindegren in the 1930s and by Beadle and Tatum in 1940. (B. 0. Dodge, who did the first genetic work on Neurospora, favored the species N. sitophila and N . tetrasperma in his own research.) If statements in this review do not name another species, they refer to N. crassn. Early History. Details of most investigations before the 1950s will not be covered in this review. Only highlights are sketched in the following paragraphs. Informal accounts of the early Neurospora work, by Lindegren (1973)) Catcheside (1973), Horowitz (1973), and others, will be found in Neurospora Newsletter 20. Shear and Dodge (1927) showed the mating-type system to be bipolar, with 4: 4 segregation of the two mating-type alleles among unordered asci that were shot from the perithecium. Wilcox (1928) examined ascus development cytologically in N. sitophila, and showed that spindles do not overlap and that the linear array of ascospores reflects the events of meiosis. Wilcox (1928) and Dodge (1929) dissected out ascospores of N. sitophila in linear order, and showed that the mating-type alleles segregated either a t first division (prereduction) or a t second division (postreduction). Dodge (1931) also described the segregation of spontaneously arising morphological differences in N. sitophila. The cytological basis of pseudohomothallism in N. tetrasperma was shown by Dodge (1927) and Colson (1934) to involve overlapping spindles in the ascus and inclusion of two nonsister nuclei in one spore. Lindegren studied the segregation in asci of mating type and of some morphological traits. He proposed that postreduction frequencies, as

138

DAVID D. PERKINS AND EDWARD G. BARRY

reflected in ascospore order, measured the frequency of crossing over in the segment between a gene locus and its centromere. He discovered linked genes and showed (1933) that crossing over in Neurospora occurred reciprocally between chromatids a t a 4-strand stage, just as in Drosophila. Lindegren (1936a,b) and Lindegren and Lindegren (1937, 1939, 1942) went on to construct the first linkage maps (eight genes in two linkage groups) and to study interference. The mutants obtained by Beadle, Tatum, and their associates, beginning in 1940, provided a basis for constructing more complete maps. By 1949 six linkage groups had been established (Houlahan et al., 1949; Barratt and Garnjobst, 1949), and the seventh group was soon added (see Barratt et at., 1954). McClintock (1945) first showed that chromosome morphology and behavior in the Neurospora ascus are similar to those in higher organisms, and that they can be studied by methods used in plant cytogenetics. She and Singleton (1948, 1953) described normal chromosome morphology and behavior in the ascus, identified and numbered the seven chromosomes, and noted that meiosis was similar in its main features to that in higher eukaryotes, though it differed in some details of chromosome contraction, pairing, and coiling. The presence of chromosome rearrangements was suggested as an explanation for aborted ascospores (“partial sterility”) in crosses involving an irradiated parent (Lindegren and Lindegren, 1941; Sansome et al., 1945). The first genetic evidence of chromosome rearrangements, pseudolinkage, was obtained in the 1940s and reported by Houlahan et al. (1949). Several translocations were confirmed cytologically and aspects of their meiotic behavior were described by McClintock (1945, 1947, 1955), Singleton (1948), and St. Lawrence (1953). II. The life Cycle, Incompatibility, and Ploidy

The Life Cycle Figure 1 shows the life cycle of a heterothallic species. The stages are described in some detail in the figure legend. For other diagrams and descriptions of the life cycle and of events in the ascus, see Fincham and Day (1971) ,Beadle (1945), and Emerson (1955, 1963, 1966). There are two major phases-vegetative and sexual. The latter begins with fertilization and concludes with ascospore differentiation ; it coincides with perithecial development and occurs within the perithecium. The vegetative phase begins with ascospore germination. Under ordinary laboratory conditions, generation time is 3 4 weeks.

THE CYTOGENETICS OF

Neurospora

139

N . tetrasperma is pseudohomothallic (secondarily homothallic) and differs from the heterothallic species in producing asci with four large ascospores, each of which contains nuclei representing two of the meiotic products-usually A and a. On germination, single ascospores thus give rise to heterokaryotic strains capable of self-fertilization (see Dodge et al., 1950; Fincham and Day, 1971). Mating T y p e Sexual compatibility is governed by two alleles, A (earlier called A or +) and a (earlier called B or -). Extensive attempts t o produce a new mating-type allele by recombination in N . crassa have failed (Newmeyer et al., 1973), nor has any different mating-type allele been found among a large number of isolates collected from nature (Perkins et al., 1976). Mating-type alleles in the three heterothallic species and in the pseudohomothallic species N . tetrasperma are all indistinguishable from A and a of N . crassa. This has been shown by interspecific matings and by introgression. There have been reports in the Sphaeriales of complete asci being produced in a normally heterothallic species by a single mating type. Lewis (1969) observed that ascospores from a cross between two Sordaria species were predominantly unpigmented. Rare asci that contained all black ascospores apparently originated by fusion between nuclei of only one of the parental types. I n Neurospora crassa, a preliminary observation of Vigfussen and Can0 (1974) is potentially important. They report that crude protein extracts of fertilized cultures induced the sexual phase in single-mating-type cultures, producing complete asci from which all eight progeny are of the original making type. Heterokaryosis and Vegetative (Heterokaryon) Incompatibility The pseudohomothallic species N . tetrasperma is normally a heterokaryon containing two genetically different nuclei in the same cytoplasm. Heterokaryons may be produced also in heterothallic species by fusion of somatic cells, by breakdown of rare disomics, or by mutation. Formation of stable heterokaryons in N . crassa requires that the participating nuclei be genetically identical a t all of the numerous heterokaryon-incompatibility ( h e t ) loci. At least ten different het loci have been identified (Mylyk, 1975). The presence of different alleles a t even one of the het loci precludes formation of stable heterokaryons (Garnjobst, 1953, 1955 ; Holloway, 1953, 1955). Anastomosis occurs but is followed by a vegetative incompatibility reaction that kills any cell in which unlike alleles are present (Garnjobst and Wilson, 1956). I n some combinations the reaction is

140

DAVID D. PERKINS AND EDWARD G . BARRY

/

IA mature OSCUS

macroconidiurn

2

bronched multinucleate mycelium

L a - ! $rnicroconidium !

1

8 perithecium

\

meiosis

7

5 'Qiscus 6

rnicroconidiurn rnacroconidium

nuclear \ fusion

protoperithecium ascogenous hypha

FIQ.1. Life cycle of Neurospora crassa, a heterothallic species (adapted from Fincham and Day, 1971). Stages 1 to 4, from ascospore germination to fertilization, constitute the vegetative phase, and stages 4 to 8, from fertilization through ascospore maturation, the sexual phase. Nuclear fusion and meiotic prophase occur 4-5 days after fertilization at 25"C, and ascospores are shot from the perithecium from the ninth day. 1. The black ascospores, approximately 17 x 26 pm, can be isolated manually from an agar surface without use of micromanipulation apparatus. Ascospore dormancy is broken by heat or chemicals. The diagram shows a germinating ascospore, with hyphae growing from both ends. 2. The mycelium consists of branched, threadlike hyphae, made up of multinucleate cells separated by perforate cross-walls through which nuclei and cytoplasm can readily pass. Vegetative growth is loose and rapidly spreading (over 5 mm per hour at 37°C. 3. Vegetative spores are of two types: powdery airborne orange macroconidia (68 pm in diameter, mostly multinucleate) and smaller uninucleate microconidia. 4. Protoperithecia are formed by coiling of hyphae around the ascogonial cells. Specialized hyphae, the trichogynes, pick up nuclei of opposite mating type and transport them to the ascogonium within the protoperithecium. Fertilizing nuclei may originate from macroconidia, microconidia, or mycelium. Usually only one fertilizing nuclear type contributes to the

THE CYTOGENETICS OF

Neurospora

141

milder, and incompatible nuclei are eliminated without killing the cells (Pittenger and Brawner, 1961). A similar but nonlethal vegetative incompatibility reaction also occurs when unlike het alleles are present in heterozygous condition in the same nucleus. This behavior has been used extensively in studying duplications. In Neurospora crassa the mating-type genes function not only in determining sexual compatibility, but also vegetatively as unlike het alleles (Beadle and Coonradt, 1944; Sansome, 1946; Gross, 1952; see Newmeyer et al., 1973). T h a t is, both mt" and mt" ( A and a ) must be present to make functional perithecia in the sexual phase, but if they are brought together in the vegetative phase they result in an incompatibility reaction similar to that shown by other het genes, both in heterokaryons (Garnjobst and Wilson, 1956) and in heteroaygous duplications (Newmeyer, 1965; Newmeyer and Taylor, 1967; Perkins, 1972a, 1975). The vegetative incompatibility function has never been separated from the mating-type locus in extensive tests (Newmeyer et al., 1973). In N . sitophila (Mishra, 1971) and in N . tetrasperma, opposite mating types do not show any heterokaryon-incompatibility reaction. However, Metzenberg and Ahlgren (1973) have shown that when the mating-type alleles of N . tetrasperma are introgressed into N . crassa they are vegetatively incompatible with each other and with the oposite mating types of N . crassa. The same is true for the N . sitophila mating-type genes introgressed into N . crassa (Perkins, 1976). It therefore appears that contents of a perithecium, but exceptions are revealed when the fertilizing parent is heterokaryotic (see, for example, Nakamura and Egashira, 1961). Selective fertilization has also been detected when a heterokaryon is used a s male parent (Egashira and Nakamura, 1972). 5. Upon fertilization, the haploid A and a nuclei do not fuse, but proliferate within the perithecium, forming heterokaryotic ascogenous hyphae. Emerson (1966) estimates that seven to ten divisions occur before nuclear fusion. 6 . The final, conjugatc division before nscus formation occurs in a binucleate hookshaped structure, the crozier. A and a fuse, and the diploid zygote nucleus immediately enters meiosis. Although the fusion nucleus is the only diploid nucleus in the life cycle, the entire sexual phase until ascospore formation is functionally similar to a diploid, in that the two gametic parental genomes are present in a 1:l ratio in a common cytoplasm. 7. Meiosis is described in Section 111, A. For details of meiosis and ascus development, see drawings in Fincham and Day (1971, Fig. 1) and photographs in Barry (1969) and Singleton (1953). Asci do not develop synchronously; numerous asci are initiated successively from the same ascogenous hypha. Mature, 8-spored asci mcasure about 20 x 200 pm. 8. The perithecial wall is maternal in origin. Derivation of perithecial tissues has been analyzed using genetic mosaics obtained when maternal cultures are heterokaryotic for a maternally expressed gene per, governing perithecial color (Johnson, 1975a,b). Mature perithecia (400-600 pm in diameter) develop a beak terminating in an ostiole through which ascospores are forcibly extruded in groups of eight, each comprising the contents of a n ascus.

142

DAVID D. PERKINS AND EDWARD G. BARRY

the mating-type alleles of N . tetrasperma and N . sitophila possess an innate vegetative incompatibility similar to N . crassa, but that this is suppressed in their normal genetic background. An unlinked gene, to1 (tolerant), has been found in N . crassa that suppresses the vegetative incompatibility of the crassa mating-type alleles without affecting crossing behavior (Newmeyer, 1970). The vegetative incompatibility associated with mating type or with other het genes disappears completely during the sexual stages, from fertilization through ascospore maturation. For a review of heterokaryosis and its genetic control see Davis (1966). More recent references will be found in Perkins (1975) and in Section V, E, 6, b. Diploidy and Numerical Aneuploids in Normal Sequence. Numerous attempts have been made to obtain stable diploids in Neurospora, but without success (e.g., by Smith, 1974; Sansome, 1956; Jean M. Foley, personal communication 1963). Case and Giles (1962) tested the possibility that somatic fusion of haploid nuclei would occur in a heterokaryon, followed by chromosome reassortment, and they showed that it did not occur. However, “pseudowild-type” progeny (from crosses with closely linked complementing markers in repulsion phase) originate as rare disomic ascospores which break down rapidly to form heterokaryons made up of haploid nuclei (first described by Mitchell et al., 1952). Multiply disomic ascospores and presumably completely diploid ascospores are readily produced meiotically in crosses of appropriate genotype, for example those involving meiotic mutants (see Section 111, C, 1 and Smith, 1975), but the disomic or diploid condition is rapidly reduced to haploid. Haploidization is frequently accompanied by mitotic recombination (Coyle and Pittenger, 1965; Threlkeld and Stoltz, 1970; Smith, 1974). Unstable disomics also originate in Neurospora by 3 :1 segregation from heterozygous translocations (Section V, C, 1, c). Disomics are known in other fungi that are relatively much more stable (see, for example, Kafer and Upshall, 1973; Shaffer et al., 1971). Stable diploids can be obtained readily in Aspergillus (Roper, 1952), and they are a normal feature of the life cycle in Saccharomyces cerevisiae. Ill. Meiosis and Mitosis

A. DESCRIPTION OF NORMAL MEIOSISAND MITOSISIN

THE

Ascus

Encouraging progress has been made in the cytological study of meiosis in fungi since the subject was reviewed by Olive (1965). Electron

THE CYTOGENETICS OF

Neurospora

143

microscopy has been used with increasing effectiveness (e.g., by Westergaard and von Wettstein, 1966; Lu, 1967a; Zickler, 1970; Schrantz, 1970; Gillies, 1972; Byers and Goetsch, 1975), effective variations of staining techniques have been applied in light microscopy (e.g., Lu, 196713; Zickler, 1967; Lu and Raju, 1970), quantitative spectrophotometry has been used to study DNA replication (Rossen and Westergaard, 19661, and meiotic mutants have been examined (e.g., Simonet and Zickler, 1972). However, the basic description of Neurospora meiosis by McClintock (1945) and Singleton (1953) still stands as a model for filamentous fungi, though it has been augmented. 1 . N . crassa and Other Heterothallic Neurospora Species

Four nuclear divisions occur in the ascus-two meiotic, followed by two mitotic. The latter are sometimes referred to as the third and fourth divisions in the ascus. All the divisions are conventional, although there are interesting minor differences from higher organisms. The sequence of events in the ascus was first described by McClintock (1945) for N . crassa, then in greater detail with photographic documentation by Singleton (1948, 1953). A further description of meiotic prophase relating to diplonema was added by Barry (1969). Gillies (1972) found synaptonemal complexes a t pachynema, and described their characteristics. Meiosis in other ascomycetes has been studied with the light and electron microscopes, and the contributions of Rossen and Westergaard (1966) on Neottiella, and Zickler (1970, 1973) on species of Ascobolus, Podospora, and Sordaria are of particular interest in interpreting similar or contrasting features in Neurospora. A detailed study by Lu (196713) of the very closely related fungus Gelasinospora caZospora shows that events in the ascus are very similar to those in Neurospora. A summary description of divisions in the ascus will be given here. Chromosome structure and morphology will be described in Section IV, A. a. Stages through Pachynema. In Neurospora crassa the ascus is generated from the penultimate cell of a crozier. The two haploid nuclei, derived from a conjugate nuclear division in the crozier, fuse in the young budding ascus. Then the two nucleoli fuse as the chromosomes condense into very short univalents, which begin to pair. Meiosis in Neurospora and other ascomycetes differs from that in higher organisms in this respect (see McClintock, 1945; Westergaard, 1964) : the chromosomes begin to pair when they are very short, but as pairing continues the chromosomes elongate through pachynema and into a diffuse diplonema, rather than contracting from leptonema to diplonema as in higher eukaryotes, such as maize and lily. At pachynema in Neurospora the paired homologous chromosomes lie parallel t o one another 2000-5000 A apart and show

144

DAVID D. PERKINS AND EDWARD G. BARRY

little relational coiling (see Fig. 4). The varied spacing may be an artifact of fixation and staining because the characteristic “railroad track” appearance varies according to the fixative used. Alternatively, it could be due to actual changes as pachynema progresses, with the widest separation a t late pachynema. Pachytene chromosomes appear to be much more closely paired in maize than in Neurospora, yet the synapsed chromosomes are separated by roughly comparable distances in each. Synaptonemal complexes are the same width in both organisms. Probably the distance seems to be greater in Neurospora became the amount of chromatin is so much smaller relative to the width of the synaptonemal complex. b. Synaptonemal Complex. From serial sections of pachytene nuclei of N . crassa, Gillies (1972) was able to reconstruct the entire set of seven complexes, determining their size and other characteristics. Each complex of a bivalent is attached to the nuclear envelope a t each end, except for the nucleolus organizer end of chromosome 2, which is free. Width of the complex is 2000 A, including the 1200 A central region plus two 400 A lateral elements. The lateral elements are banded, which seems characteristic of the ascomycetes (Westergaard and von Wettstein, 1972). The synaptonemal complexes are one-third shorter than pachytene chromosomes prepared with acetic-lactic-ethanol fixative (Table 1 of Gillies, 1972). They may differ in length because the complexes were measured before the chromosomes had extended to the full length characteristic of late pachynema, when the chromosome measurements were made. Alternatively, if the measurements were from cells of comparable stages, differences in processing must have resulted in shrinking or stretching (Gillies, 1972). Gillies observed thickenings of the central component (“nodes”) in varying numbers in the complexes in most nuclei. Schrantz (1970), Zickler (1973), and Radu et al. (1974) have found similar structures in other fungi. These apparently correspond to the “recombination nodules” observed by Carpenter (197513) in female Drosophila. c. Postpachytene Stages. After pachynema the chromosomes become indistinct during a diffuse diplonema (Barry, 1969). The stage recognized as conventional diplonema follows, but it is rarely observed, indicating that it must be of very brief duration. Chiasmata are visible then and in the following stage, diakinesis (see Singleton, 1953, Figs. 34, 35). Singleton (1953) made general chiasma counts a t diakinesis, observing that most bivalents usually have two chiasmata, with sometimes three in the longer chromosomes or only one in the shorter. This would indicate that the total genetic map should be more than 700 units, which agrees with other estimates of its minimum length (Section IV, B, 2, c ) .

THE CYTOGENETICS OF

Neurospora

145

The chromosomes are very short a t metaphase I; the longest does not exceed 2 pm. Anaphase is asynchronous so that often some dyad chromosomes reach the poles before other bivalents separate on the metaphase plate (see Fig. 2). The anaphase-I (and 11) chromosomes are rounded rather than linear (V or J shaped). The spindle is intranuclear. The nuclear membrane is present throughout the division, disintegrating only near the spindle equator, and closing by constriction and new synthesis around the daughter nuclei a t telophase (Wells, 1970; Westergaard and von Wettstein, 1970; Zickler, 1970; Mu’Azu, 1973). Divisions at all other stages are intranuclear, both in the ascus and in vegetative cells. After the first telophase the chromosomes become extended again during a brief interphase, when a small nucleolus is formed. Soon the chromosomes contract to enter the second meiotic division, which is conventional in appearance. During both meiotic divisions the spindle is usually parallel or slightly oblique to the long axis of the ascus. At the second division the spindles are tandem to one another without overlapping. The two meiotic divisions are followed by a mitotic division. No cross walls in the ascus separate any of the daughter nuclei. The nuclei retain their sequential placement in the ascus such that the top and bottom four nuclei are the separate products of the first meiotic division, and the top and bottom two nuclei in each half of the ascus are the products of

FIG.2. Anaphase I in Neurospora crassa. The division is asynchronous, with five chromosomes near each pole while two bivalents are still disjoining in the center. Note the generally spherical shape of the anaphase chromosomes. (Orcein staining, X3000.)

146

DAVID D. PERKINS AND EDWARD

a.

BARRY

the second division. Adjacent nuclei by pairs (1-2, 34, 5-6, 7-8) are identical sister nuclei. An ascospore wall is then formed around each of the eight nuclei, including adjacent cytoplasm. A second mitotic division occurs in each immature ascopore. I n both mitotic divisions in the ascus, the aggregation of chromosomes on a metaphase plate and subsequent disjunction of chromatids to the poles on a spindle is very clear. These divisions are typical mitoses. At late anaphase and telophase of the mitotic division, a ring of chromosomes is formed (Weijer and McDonald, 1965) which is suggestive of the appearance of some division figures seen in somatic nuclei. The centrosomes become progressively more conspicuous a t the second and third divisions. [The centrosome is also called centriole, centrosomal plaque, spindle-pole body, polar organelle, and other names (see Lu, 1967b; Zickler, 1970; Wells, 1970; Mu’Azu, 1973). Centrosome is used here, for brevity.] Zickler (1970) observed variations in centrosome shape, structure, and degree of attachment to the nuclear membrane in species of Ascobolus and Podospora. The centrosome may not be primarily involved in delimiting the ascospore (see Zickler, 1970), contrary to earlier interpretations (Singleton, 1953), but i t is the site to which the spindle fibers (microtubules) converge, and it seems also to be involved in the spacing and orientation of the nuclei within the ascus. d. The D N A Cycle. Meiosis and mitosis in the ascus of Neottiella rutilans are similar to those in Neurospora. Rossen and Westergaard (1966) have measured the comparative DNA content of Neottiella nuclei spectrophotometrically, using Feulgen staining. They found that the amounts of DNA indeed correspond to what might be expected for each division stage: nuclei of vegetative hyphae, paraphyses, and ascogenous hyphae have the haploid amount of DNA; nuclei in the croziers before the conjugate division have up to the diploid amount; conjugating nuclei just prior to fusion in the ascus each have the diploid amount; the fusion nucleus has a tetraploid level; each nucleus of the tetrad a t the completion of meiosis has the haploid amount. There is a round of DNA replication preceding each of the third and fourth divisions in the ascus. The critical finding is that replication of the bulk of the DNA for the meiotic divisions has been completed before the haploid gametic nuclei fuse in Neottiella. This observation precludes a direct relation between crossing over and total DNA replication. A similar study has not been made in Neurospora because of inability to obtain Feulgen staining or to label chromosomal DNA specifically with a radioisotope (Baer and St. Lawrence, 1964; Fink and Fink, 1962a,b). It is reasonable to assume, however, that a similar pattern of DNA

THE CYTOGENETICS OF

147

Neurospora

replication would be found, with the bulk of premeiotic DNA replication completed prior to karyogamy. Meiosis and ascus development in the other heterothallic Neurospora species N . sitophila and N . intermedia follow the pattern of N . crassa (E. G. Barry, unpublished; Wilcox, 1928, on N . sitophila). 2. Ascus Development in Homothallic Species

N. B. Raju (1976) has examined meiosis and related stages in the five homothallic Neurospora species. Two nuclei fuse in the crozier, just as in the heterothallic and pseudohomothallic species, and subsequent stages are essentially similar to those described for N . crassa. 3. Ascus Development in Pseudohomothallic Species The 4-spored species N . tetrasperma exhibits a precise genetic control of cellular events coordinated with meiosis. The divisions in the ascus are similar to those in N . crassa (Colson, 1934; Cutter, 1946). However, spindles overlap a t the second nuclear division in the ascus, and ascospore walls are formed in such a way that each of the four spores contains daughter nuclei derived from two different first-division products. The mating-type locus is close to centromere, and alleles A and a usually segregate a t division I, so that 98% of ascospores are heterokaryotic A a, and each resulting culture is thus capable of entering the sexual cycle and forming perithecia without fertilization from an external source. The rare asci where crossing over has occurred in the short interval between m t and centromere produce homokaryotic A A and a a spores (see Dodge, 1927; Sansome, 1946; Howe, 1963, 1964; Hung, 1972). The tetrasperma system undoubtedly evolved from ancestors that were heterothallic with 8-spored asci. Reversion from the 4-spored to the 8-spored condition can be accomplished by a single dominant mutant gene E (Dodge et al., 1950). The opposite change from 8- to 4-spored asci is not readily accomplished in a normally 8-spored species, as might be expected. Pateman (1959) selected over several generations for large spores in N . crassa and obtained strains that produced occasional 4spored asci, but the basis of this condition was multigenic and recessive, and many of the asci had defective spores.

+

+

+

Pseudohomothallic species having asci with four heterokaryotic spores are known in other genera. In some (for example Gelasinospora tetraspema-Dodge, 1937) heterokaryosis for the mating-type alleles is achieved as in N . tetrasperma. I n Podospora anserina, however, the same end result is achieved in a completely different way. The mating-type locus is far from the centromere, and a nearly invariable exchange occurs so that the mating-type alleles segregate at the second division in 98% of asci (Rizet and Engelmann, 1949). Spindles are aligned and

148

DAVID D. PERKINS AND EDWARD G. BARRY

ascospore walls are delimited so that each of the four spores includes daughter nuclei derived from two different second-division products (for diagrams and references, see Fincham and Day, 1971, p. 302). Pseudohomothallism must therefore have evolved independently in different ascomycetes.

B. SOMATIC DIVISIONS

Mitosis in the Hyphae. Coordinated studies with both light and electron microscopes now show the somatic nuclear divisions in fungi to be mitotic in nature, complete with spindles, separate anaphase chromosomes, and centrosomes. The clearest demonstration of the mitotic divisions in ascomycetes, both in somatic hyphae and in the ascus, is by Zickler (1970, 1971). There has been a long history of controversy (see Robinow and Bakerspigel, 1965), some investigators interpreting the somatic nuclear divisions as classical mitoses, others maintaining that they were not because spindles and chromosomes could not be resolved at metaphase and anaphase in the tiny nuclei. Zickler employed both light-microscope and phase-contrast observations of living material as well as of hematoxylin-stained material, and electron microscopy of double-stained preparations. She found that the divisions are extremely rapid, taking only 6-7 minutes, with the spindle and metaphase-anaphase stages visible for only 2-3 minutes, whereas interphase lasts 3 4 hours. Her observations were made with numerous species of Ascobolus, Sordaria, and Podospora. These are so closely related that there is no hesitation in extrapolating the results to Neurospora, but it would be gratifying, even so, to have electron-microscope studies showing the fine specific detail of: somatic mitosis in Neurospma, which has been a t the center of the controversy.

c. GENETICVARIANTS AFFECTINGMEIOSISOR

RELATED STAGES

1. Meiotic Mutants

Several mutant genes have been identified in Neurospora crassa that block the normal course of development during the sexual phase, between fertilieation and ascospore maturation. Of special interest are those that affect meiosis itself, or the stages immediately before meiosis. Mutants of this type have also been used for the genetic dissection of meiosis in higher organisms (e.g., Sandler et al., 1968; Rhoades and Dempsey, 1966; Gottschalk, 1973) and in other fungi (Bresch et al., 1968; Esposito et al., 1970; Simonet and Zickler, 1972; Wheeler and Driver, 1953; Heslot, 1958; Esser and Straub, 1958; El Ani and Olive, 1962). Of the genes that affect chromosome pairing and disjunction in Neurospora, the recessive mei-1 was the first to be thoroughly studied (Smith,

THE CYTOGENETICS OF

Neurospora

149

1975). mei-1 apparently originated from a wild-collected strain, and was long known as the Abbott factor, responsible for ascospore abortion. There is little or no crossing over in mei-1 x mei-1, nondisjunction is high, and the ascospores are mostly white and inviable. The few viable ascospores are always heterozygous for markers in one or more linkage groups, and they probably originate as unstable diploids. Cytological observations of mei-1 have been made by B. C. Lu and D. R. Galeazzi (personal communication). I n homozygous condition, mei-1 results in failure of chromosome pairing during prophase I. Chromosomes may be associated occasionally a t points or regions. The components of the synaptonemal complex are present, but normal complexes are formed only rarely. Daughter chromosomes fail to separate cleanly a t the second and third divisions, and may then be enclosed in the same nuclear envelope. When the centrosomes divide, they may form an abnormal spindle with four poles. Thus, when the division is completed some centrommes may have no associated chromosomes, or else a subnormal number. The recessive gene mei-3 (Newmeyer and Galeazzi, 1974) completely blocks early stages of meiotic development, so that perithecia are barren. Karyogamy occurs, but chromosomes fail to pair normally. Few asci are seen a t later stages; these are abnormal with few or no spores (N. B. Raju, unpublished). mei-3 also causes loss of duplicated chromosome segments (see Section V, E, 4). Another recessive gene, mei-4 varies in its expression from highly barren to almost normally fertile, depending on the genetic backgound (Newmeyer and Galeazzi, 1974). I n the moderately barren crosses studied by B. C. Lu and D. Galeazzi (personal communication), the first meiotic division is normal, but in the second and third divisions daughter chromosomes fall to separate cleanly in some asci, much as in crosses homozygous for mei-1 . Consequently, irregular disjunctions occur. Some centrosomes appear to separate from the nucleus a t the third division. Abnormal spores are delimited that may lack nuclei, or they may already possess two nuclei before the fourth division. The dominant Mei-2 results in extensive nondisjunction (Smith, 1975). The defect is a t aygonema when chromosome pairing fails, although some segments may show partial pairing. At metaphase I some bivalents are found, but most chromosomes are univalent; disjunction is abnormal. Thereafter the divisions are normal (B. C. Lu, unpublished). Several other strains have been tentatively identified as dominant meiotic mutants ; characteristically most of the ascospores from heterozygous crosses that involve thcm are white and inviable. Except for mei-3, the meiotic mutants described so far have all been

150

DAVID D. PERKINS AND EDWARD G. BARRY

recognized initially by their effects on fertility or on ascospore viability, resulting from defects of pairing, disjunction, or spore formation. Several radiation-sensitive mutants are also homozygous-barren, with development blocked usually but not always before karyogamy (uvs-3, -5, and -6; Schroeder, 1970; N. B. Raju, unpublished). More subtle effects are characteristic of the rec and cog genes which regulate meiotic recombination in specific local regions (see D. G. Catcheside, 1974, 1975, and Section IV, B, 3 ) . Mutant genes acting at stages of the sexual cycle other than meiosis may be of no less interest for other aspects of cytology and development, but they can hardly be called meiotic mutants. This category includes mutants with altered ascospore morphology (Barry et al., 1972; Novak and Srb, 1973; Srb et al., 1973, 1974), notably the dominant gene R : Round spore (Mitchell, 1966) ; mutants with abnormal ascus shape such as p k : peak ( = bis:biscuit) (see Srb et al., 1974) ; and female-sterile mutants which are unable to form perithecia (see Mylyk and Threlkeld, 1974). Mutant strains have been described by Vigfusson et al. (1971) and Weijer and Vigfusson (1972) that are male-sterile and female-fertile. 2. Effects of Aneuploidy

Most or all of the segmental duplications known in Neurospora result in barren perithecia when they are crossed to either euploid or duplication strains of the opposite mating type. The block in perithecial development varies, but is most commonly at the crozier stage (N. B. Raju, unpublished). Further details are given in Section V, E, 3. Numerical aneuploids such as disomics are apparently too unstable to have recognizable effects on perithecial development or fertility. 3. Spore Killers

Three factors are known in Neurospora that resemble Segregation Distorter (SD)in Drosophila (for SD, see Hart1 and Hiraizumi, 1976; Peacock and Miklos, 1973). The Neurospora factors, called Spore Killer ( S K ) , were first recognized because asci from SK/SK+ contained exclusively four black and four shrunken white spores. No chromosome rearrangement would be expected to have such a result; instead, the observation suggested an ascospore-color mutant that was expressed autonomously in each spore. However, the surviving, black ascospores proved to be those carrying the factor responsible for spore death, S K , rather than its sensitive allele, SKg. Ascospores that did not contain S K deteriorated, a behavior exactly opposite that expected of an autonomously expressed ascospore-color gene, where the wild type survives.

THE CYTOGENETICS OF

Neurospora

151

Moreover, all the ascospores from SK-1 x SK-I were viable and black. SK-I was discovered in crosses between N . sitophila strains from Nigeria and Virginia, U.S.A. (B. C. Turner, unpublished). Nigeria X Nigeria and Virginia X Virginia crosses resulted in a majority of black ascospores and 8B :OW asci, whereas asci from reciprocal crosses between strains from the two sources were all 4B:4W (with 95% first-division segregation). Progeny from the viable, black spores were almost all (>95%) of the Virginia type. Similar crosses with N . sitophila isolates from many other localities showed that each of them was either one type or the other-SK-1 (Virginia) or SK-In (Nigeria). A different Spore Killer, SK-2, was found in stocks that had been derived from a Borneo strain of N . intermedia by recurrent backcrosses to N . crassa (Blakely and Srb, 1962). In further crosses of these strains to N . crassa we noticed that the asci were exclusively 4B:4W, and almost all progeny from the viable, black spores repeated this behavior. SK-2 is located a t or near the centromere of linkage group 111, and it segregates independently of markers in the other six linkage groups. SK-2 makes many inviable spores when it is homozygous in the N . crassa background, but when SK-2 strains of N . intermedia are used, derived directly from the original Borneo strain, homozygous crosses produce mostly viable ascospores and 8B:OW asci. N. B. Raju (personal communication) has examined heterozygous crosses cytologically. Both SK-I and SK-R behave similarly. Meiosis and ascus development are normal, so far as can be seen with the light microscope. This is true of chromosome pairing, behavior of centrosomes, ascospore delimitation, and the fourth nuclear division in the spores. The nuclei in all eight ascospores look normal and identical for some time after the ascospore walls have formed. Then while four of the spores continue to enlarge and mature normally, the other four remain small and their nuclei shrink or slowly degenerate. A third Spore Killer has been found in N . intermedia strains collected in New Guinea. SK-3 from Rouna is similar to SK-I and SK-2 in its behavior, with the added feature th at some naturally occurring strains that do not themselves show the SK-3 killer effect are nevertheless resistant to killing by SK-3.

4. Bubble Asci I n many crosses with N . crassa laboratory stocks, numerous small sacs are found in the perithecia that contain chains of eight tiny round refractile bodies with a diameter about one-fourth the length of a normal mature ascospore. N. B. Raju (unpublished) has shown that these bubble asci originate by degeneration of asci that develop normally until the

152

DAVID D. PERKINS AND EDWARD G. BARRY

third division is completed and spores have been delimited. All the ascospores in an affected ascus begin to degenerate and shrink a t that time. The frequency of bubble asci varies from less than 10% to over 80% in different crosses, but is constant for the same combination of parents. A high frequency of bubble asci is partially correlated with inbreeding (D. Newmeyer and N. B. Raju, unpublished observations). Several widely used standard wild types show a high frequency. The abnormal asci reported by McNelly-Ingle and Frost (1965) probably include bubble asci as well as other abnormalities. The degenerated spores in bubble asci are apparently produced nonselectively and do not interfere with genetic analyses. They also do not interfere in the diagnosis of our typical identified rearrangements, because deficiency ascospores from heterozygous aberrations are &sually much larger than bubble ascospores. Bubble asci are probably never ejected from the perithecium, and the tiny bubble spores are readily overlooked when perithecia are opened. IV. The Wild-Type Genome

A. THECHROMOSOMES 1 . Chromosome Size

The meiotic chromosomes of Neurospora are small, but within the range of study with the light microscope. Lengths of the seven chromosomes a t pachynema range from 7 to 19 pm (Singleton, 1953). This is immense compared to somatic chromosomes, where the entire nucleus in the hyphae is only about 2 pm in diameter. However, it is small compared to maize, where the ten chromosomes range from 37 to 82 pm in pachytene length (Rhoades, 1950). At metaphase I, the Neurospora bivalents contract to lengths of 1.7 to less than 1 pm. The enormous differences in chromosome size between meiotic and somatic cells, and between stages of the cell cycle, are presumably not due to changes in DNA content [see Rossen and Westergaard (1966) for quantitative data from another ascomycete, Neottiella, where similar size differences are found]. 2. Chromosome Composition

a. D N A Content. Horowitz and Macleod (1960) showed by chemical analysis that haploid nuclei from microconidia of N . crassa contain 4.63 X gm of DNA (2.8 x 10'O daltons or 4.5 x lo7 nucleotide pairs): Other methods gave comparable amounts: 2.2 X 10'O daltons by DNA: DNA reassociation kinetics (Ray and Dutta, 1972), and 2.8 x 1O1O

THE CYTOGENETICS O F

Neurospora

153

daltons by sucrose density gradient centrifugation (Brooks and Huang, 1972). Other fungi have similar amounts of DNA. Neurospora is about midway between E. coli and Drosophila in DNA content, and much closer to coli (10 x ) than to higher plants or vertebrates. I n fact, the DNA content of a small chromosome in Neurospora is probably less than that of the E. coli chromosome. It is reduced even more in some rearrangement strains of Neurospora (for example, T (1V;VI)45502 and T [ I I + (1V;V)] AR179), where the smallest translocation chromosome is much smaller than any in the wild type. The DNA content per haploid genome for representative organisms can be seen in Table 1. b. Repetitive and Uniquesequence D N A . Brooks and Huang (1972) report that about 20% of Neurospora crassa DNA is repetitive or redundant. Their experiments indicate that little of the repetitive DNA comes from the mitochondria, which according to Luck and Reich (1964) contribute less than 1% of the total DNA of the cell. [The mitochondria1 DNA also contains repeated sequences (Wood and Luck, 1969).] However, Dutta (1973) reports that 10-12% of the DNA consists of repeated sequences, while about 90% behaves in reassociation kinetics as though it were composed of unique copies; and Dutta and Chaudhuri (1975) conclude that 3 4 % of the total comes from the mitochondria. i. D N A specifying t R N A and r R N A . The experiments of Brooks and Huang (1972) indicate that 2.3% of the repetitive DNA hybridizes with ribosomal RNA (rRNA) and 1.2% hybridizes with transfer RNA (tRNA). When their hybridization experiments were conducted with the total DNA of the cell, 1% hybridized with rRNA and 0.3% with tRNA. They calculate the complexity of the redundant DNA to be 2 x 10' daltons, and they suggest an average repeat frequency of about 60 copies per genome for all repeated genes. The 1% of the total DNA attributed to rRNA cistrons corresponds to 100-200 rRNA genes by their estimates, while the 0.3% tRNA cistrons corresponds to 3000 total copies. Chattopadhyah et al. (1972) calculate that about 100 copies of the rRNA genes exist in each nucleus, and that individual copies have essentially identical DNA sequences. When the tRNA genes were isolated by shearing DNA into fragments of 1.2 x 1 0 daltons and hybridizing with tRNA, Ray and Dutta (1972; Dutta and Ray, 1973) found 0.3% of the total Neurospora DNA to be composed of tRNA cistrons, in agreement with the report by Brooks and Huang (1972). Ray and Dutta (1972) calculate that if there are 60 different species of tRNA each may be represented an average of 44 times in the genome, giving a grand total of 2640 tRNA cistrons. ii. D N A specifying mRNA. Dutta (1973) reports that 35% of the

154

DAVID D. PERKINS AND EDWARD G. BARRY

TABLE 1 Comparative Genome Size of Representative Organisms

Organism

Haploid chromosome number

Total pachytene chromosome length (pm)* LM

EM

768

28b 46, 50-90#" 11-2op 3531

Escherichia coli Saccharomyces cerevisiae Neurospora crassa Drosophila melanogasler X-chromosome only Zea mays Lycopersicon esculenlum Mus musculus

1 17 7 4

-o

10 12 20

5521 387P 229'

Homo sapiens

23

-5

124 8" 234

Haploid DNA content (daltons X log) 2.8ta 9.2c 280 85,i l l O k 3300" 1200" 1500' 1800'

Total genetic map length* (centimorgans) 2600d > 1000$ 287." 70h 11190 969s 120018OOU 25307"

* The values in columns 3 to 6 are, in some cases, approximations, or they may be based on incomplete information. Other values may be found in the literature. These are presented only for the purpose of making a general comparison with Neurospora. LM: Measurement with the light microscope of conventionally fixed and stained material. EM: Measurement with the electron microscope of synaptonemal complexes. t Based on 1100 pm double-stranded DNA, measured with the electron microscope. $ Tentative because of high variability in crossing over. For calculations, see Section IV, B, 2, c. 0 Salivary gland polytene chromosome lengths are 2219 pm (entire complement) and 414 pm (X-chromosome) (Lindsley and Grell, 1968). # Euchromatic regions only, except for X-chromosome, which is to nucleolus organizer. The SC shortens as meiosis progresses. 7 The autosomes only. References: C a i r n s (1963). bByers and Goetsch (1978). CBicknell and Douglas (1970). dMortimer and Hawthorne (1975). +3ingleton (1953). 'Gillies (1972). UHorowitz and Macleod (1960). "indsley and Grell (1968). 'Carpenter (1975a). jLaird (1971). kRasch et al. (1971). 'Rhoades (1950). mGillies (1973). "Evans and Rees (1971). ONeuffer and Coe (1974). pBarton (19,50). CKhush and Rick (1968). 'Griffen (1955, 1960). .M. J. Moses (personal communication). 'McCarthy (1969). .Miller and Miller (1975). uHultBn and Lindsten (1973). wA. T. C. Carpenter (personal communication). unique sequence DNA hybridizes with RNA which has been transcribed during log phase mycelial growth. Bhagwat and Mahadevan (1973) have labeled RNA a t 8, 16 and 24 hours after the beginning of germination and growth of conidia. Identifying the mRNA (messenger RNA) component extracted from the hyphae, they find that the mRNA transcribed a t different times after the beginning of conidial germination and growth is different both by amount

THE CYTOGENETICS OF

Neurospora

155

of the total DNA to which it hybridizes and by the amount with which it overlaps the mRNA transcribed at the other times, thus indicating differential activity of genes in the maturing culture. c. Histones. Neurospora crassa appears to have very little histone protein associated with the chromosomes. Dwivedi et al. (1969) and Leighton et al. (1971) were unable to isolate and identify any histones, but Hsiang and Cole (1973) have isolated two “slightly lysine-rich” histones from Neurospora chromatin. Hsiang and Cole find that there is only 25% as much histone protein as in higher eukaryotes; and they believe that the earlier failures may have been due to the small quantity of histones, proteolytic degradation, and other isolation procedures that gave low yields of chromatin and histones. Other basic proteins may be present in Neurospora chromosomes, since there is a requirement for putrescine, which is a precursor of spermidine and other polyamines (Davis et al., 1970; Deters et al., 1974). 3. Techniques for Chromosome Cytology

Conventional techniques developed for eukaryotic plants and animals are not usually successful in fixing and staining Neurospora chromosomes. However, variations have been developed that stain chromosomes eff ectively during meiotic and mitotic divisions in the ascus. McClintock (1945), Singleton (1948, 1953), St. Lawrence (1950, 1953), Barry (19661, Phillips (1967), and Pincheira and Srb (1969) have obtained good results with orcein or carmine, and B. C. Lu (personal communication) and Raju (1976) have used hematoxylin effectively in Neurospora. Hematoxylin and carmine stain the nucleolus and centrosome intensely, while orcein stains the nucleolus only lightly and the centrosome not a t all. Good staining of chromosomes in the asci of numerous other ascomycete genera has also been obtained using Giemsa (Rogers, 1965, and later publications). The procedures we now follow and recommend for Neurospora are described by Barry (1966) for acetoorcein and by Raju (1976) for hematoxylin. Most of the observations reported in the following sections have employed ethanol-acetic acid-lactic acid fixation followed by staining with acetoorcein. A technique for producing giant conidia promises to make vegetative nuclei of Neurospora more accessible to observation. When conidia are suspended in medium containing 3.2 M ethylene glycol, they form spheres that grow without cell division (Bates and Wilson, 1974). N. B. Raju (unpublished observations) has examined these cytologically using hematoxylin. Nuclei divide, and chromosomes can be observed a t different mitotic stages. Some giant nuclei become polyploid.

156

DAVID D. PERKINS AND EDWARD G. BARRY

Particular genotypes also promise to be technically useful in cytology. For example, N. B. Raju (unpublished) has found that a dominant mutant discovered by Newmeyer has as one of its effects the induction of waves of synchronized mitoses in ascogenous hyphae. Thus, large numbers of nuclei in precisely the same stage of division may be observed together, and different stages are characteristic of different groups of hyphae. Attempts to stain Neurospora meiotic chromosomes with Feulgen have apparently not been successful. Weijer and McDonald (1965)have used a modified Feulgen reaction to obtain chromosome staining in the two mitotic divisions in the ascus. The Feulgen reaction has been used successfully for staining somatic nuclei (Horowitz and Macleod, 1960; Somers et al., 1960; Weijer, 1965), but other stains are probably preferred. I n Gillies’ (1972) studies of the synaptonemal complex, he reports difficulty in staining chromosomes and obtaining contrast between chromosomes and nucleoplasm for electron microscope observations of meiosis.

4. Chromosome Identification and Morphology a. Chromosome Number. McClintock (1945)showed the haploid number to be 7 in N . crassa, and all later observations of chromosomes in the ascus have been in agreement. [However, Somers et al. (1960)reported a minute eighth chromosome in somatic nuclei.] The same number has since been found in all the other Neurospora species: N . sitophila (Fincham, 1949; Dodge et al., 1950; Perkins et al., 1976),N . tetrasperma (McClintock and Weaver, reported by McClintock, 1945;Dodge et al., 1950),N . intermedia (Perkins et al., 1976), and the five homothallic species N . terricola (Nelson and Backus, 1968),N . africam, N . dodgei, N . lineolata, and N . toroi (Raju, 1976). [Early counts of n = 6 for N . tetrasperma by Colson (1934) and Cutter (1946) were probably incorrect.] b. Chromosome Morphology at Pachynema i. Idiograms. McClintock (1945) drew up karyotypes numbering the seven chromosomes from longest to shortest and showing relative lengths of the chromosomes measured at pachynema and a t mitotic metaphase stages in the ascus. She tentatively determined the probable centromere position of each chromosome by measurement a t mitotic metaphase in the third division when the chromosomes show a sharp bend a t the centromere. The centromere is not visible in the meiotic stages, although McClintock supposed it to be correlated with the position of the heaviest chromomere of each pachytene chromosome. Singleton (1948,1953) subsequently published more detailed idiograms showing the identifying morphological features of each chromosome. Each chromosome was

THE CYTOGENETICS OF

157

Neurospora

distinguished by its chromomere pattern and length a t pachynema, and by its centromere position and length a t mitotic metaphases in the ascus. Singleton's chromosome maps were assembled from observations of about 20 pachytene figures where most or all of the chromosomes could be identified. He regarded the maps as tentative. They are reproduced in the upper part of Fig. 3.

,satel Iite

a1

a

1

2

3

b"

b

a

I

4

a

a

5

b

6

7

I

FIG.3. Pachytene chromosome morphology of Neurosporu crassa. Above: The chromomere pattern as drawn by Singleton (1953). a and b identify the largest chromomeres of each chromosome. Bebw: The patterns that we now regard as most characteristic of each chromosome. Not all chromomeres are included in the diagram (compare with Fig. 4). In many nuclei the chromomeres identified here are not seen and chromosome identification depends on length, or will be in doubt. Even though the patterns of chromosomes 3, 4, and 5 appear to be very distinct, these chromosomes may be easily confused if the conspicuous chromomere does not stain or show clearly. The heteromorphic, double chromomere in 1 is visible in about 50% of nuclei. This may be due to stretching when the chromosome is spread out. The achromatic gap in 2 is not always observed. Singleton's chromosome 7 is our chromosome 6.

158

DAVID D. PERKINS AND EDWARD G. BARRY

The pachytene maps which we feel are more typical of the usual chromomere patterns and configurations are shown in the lower part of Fig. 3. These revised maps are also tentative working maps for our present studies. Probably one may expect to find other typical patterns, depending on the combinations of strains used. Most of our observations are now made on strains from the Oak Ridge wild-type (74-OR23-1A and 74-OR8-la) background. ii. Variability of chromomere patterns. It has been our experience that each chromosome is not always identifiable, and that the chromomere patterns are not distinctive for all the chromosomes, a t least throughout the pachytene stage when aberrations are analyzed and their breakpoints are located most effectively. There are two difficulties that contribute to the problem of correct chromosome identification. The first is that the chromosomes undergo change during pachynema, which is a stage of long duration, and the chromomere patterns shift as the stage progresses. Some chromosomes may also elongate faster than others. The second difficulty is that different strains probably differ slightly in their chromomere patterns. The strains now used as standards are apparently somewhat different in this respect from the combinations used by Singleton and McClintock. Probably for these reasons, Singleton may have omitted or confused some of the chromomere positions in his idiograms. For example, chromosome 1 has a conspicuous, central, heavy chromomere (the a-chromomere) surrounded by a heavily stained region in Singleton’s diagram. This largest chromomere is almost exactly in the middle of the chromosome, and there is a second, less-marked chromomere ( b ) about halfway between the a-chromomere and one end of the chromosome. I n most of our figures, and in some of Singleton’s diagrams reconstructing the figures he photographed (his Figs. 22-23A1 27A and 30A), the a and b chromomeres are about equally intense, and they divide the chromosome into approximate thirds (see Fig. 3). iii. Heterochromatin. Large and numerous chromomeres are a conspicuous feature of the pachytene chromosomes, with the largest and darkest blocks of chromomeres usually appearing in two sets in the midregion of chromosome 1. McClintock (1945) and Singleton (1953) recognized chromosome regions a t second telophase which they called heterochromatic. However, our convention has been simply to refer to the more darkly and intensely stained regions as “heavy” chromomeres. The chromomeres are observed during pachynema and other prophase stages, but variable chromatic regions are not observed a t metaphase of meiosis and mitosis in the ascus. Detecting heterochromatin by a possible differential time of onset of DNA synthesis by chromosome or chromosome region has not been feasible for technical reasons, mostly because of the inability to

THE CYTOGENETICS OF

Neurospora

159

obtain specific radioisotope labeling of DNA (Fink and Fink, 1962a,b; Baer and St. Lawrence, 1964), but also because the continuous formation of new waves of asci in the perithecium in asynchronous development prevents recognition of specific early or late synthesis by chromosome segments in any one ascus. None of the chromosome ends are marked by knobs of great contrasting size, except the satellite of the nucleolus chromosome. Variations in the size of the terminal chromomeres are seen among nonhomologous chromosomes, however. Nor have any striking heteromorphic chromosomes or chromomeres yet appeared among N . crassa stocks of different background, excepting the satellite and chromosome aberrations. Crosses between wild strains of N . intermedia, and interspecies crosses of N . crassa by N . intermedia have shown some differences in chromomere patterns (Perkins et al., 1976). N . intermedia chromosomes are much less well marked by heavy chromomeres than are N . crassa chromosomes, in the asci of both intraspecific and interspeoific crosses. iv. Spear ends. At pachynema, McClintock, Singleton, and St. Lawrence recognized “spear ends” of chromosomes 1 and &paired homologs appeared to be touching a t the tips while elsewhere the pairs were separated and lying parallel. I n our preparations, this seems not to be a consistent feature of any chromosome pair. Any apparent spear ends can equally well be attributed to a partial twist of the bivalent. I n studies of the synaptonemal complexes of Neurospora, Gillies (1972) found that all homologous ends of pachytene chromosomes are separated at their sites of attachment to the nuclear membrane. v. Appearance of univalent chromosomes. Sometimes unpaired chromosomes (univalents) or portions of unpaired chromosomes can be clearly recognized. At other times their identification is uncertain. The difficulty lies essentially with the small diameter of Neurospora chromosomes. If two paired homologs are closely associated or twisted about one another a t pachynema, i t may be impossible to resolve their double nature. Furthermore, unpaired and paired chromosomes differ in their morphology. The chromatids of an unpaired chromosome are unraveled, and appear to separate somewhat from one another, thus making a fuzzy doubleness and a width sufficient to suggest the diameter and other characteristics of an indistinct bivalent. For a synapsed pair, the chromatids of each chromosome are not visible with the light microscope, and each chromosome appears to be a single strand. Unpaired regions or chromosomes thus often mimic a normal paired region or chromosome. In paired regions, the chromomere patterns are usually distinct and sharp at midpachynema, but unpaired sections often show no such crisp pattern and may be darkly staining yet indistinct in fine detail. vi. Description of individual pachytene chromosomes. Chromosome 1

160

DAVID D. P E R S I N S AND EDWARD G. BARRY

can be identified with a high degree of certainty. It is the longest chromosome of the complement by a significant margin (one half again the length of chromosome 2). There are numerous distinctive chromomeres. Confusion regarding the actual position of the heavy a and b chromomeres, discussed above, does not interfere in practice with the identification. The pachytene morphology of an inverted chromosome 1 can be seen in Fig. 4. Chromosome 2 is usually easy to identify because it bears the nucleolus organizer region, and a t pachynema the nucleolus is a very conspicuous body in the nucleus. The region of chromosome 2 near the organizer in a typical nucleus is well marked by large chromomeres displaying a characteristic pattern. In some nuclei, chromosome 2 may be shorter than 3. Some strains of h'eurospora crassa (the St. Lawrence and Oak Ridge wild types and their derivatives) have a small satellite at the end of the nucleolus organizer region which may be identified in well stained, con-

FIQ.4. Pachynema in Neurospora crassa. The photograph is of one focal level of the nucleus, and the accompanying drawing shows the entire nuclear complement of seven chromosomes. (Chromosome 5 has been shifted to the right in the drawing to avoid a confusing overlap with parts of 2 and 4. The overlap in the actual nuclcus was not a problem because the arms were in different focal planes.) Chromomere detail is shown in the drawing, and each chromosome is identified; only 6 is not clear. Chromosome 1 is homosygous for inversion H4250. One parent contained the original inversion, but in the second parent the inversion sequence was derived by mei-3-induced somatic breakdown from an H4250 duplication (see Section V, E, 4, b). (Orcein staining, X4000. Stocks obtained from D. Newmeyer.)

THE CYTOGENETICS OF

Neurospora

161

trasting figures. At pachynema when the nucleolus is a t its greatest size, the satellites are seen on the surface of the nucleolus, usually in the hemisphere opposite the organizers. Photographs showing the organizers and satellites can be found in Barry and Perkins (1969); see also Fig. 4. Many stocks, both mutant and wild-type, lack the satellite. Chromosomes 3, 4,and 5 are all about the same size, and in the usual orcein-stained nucleus they do not develop constant and distinctive patterns. I n some figures, our comparisons by chromomere patterns match rather well with the McClintock-Singleton diagrams. In others they do not, and our identifications are then very tentative. Since the chromosomes are rarely flattened into two dimensions in our preparations, measurements of true length are not accurate enough to distinguish among the members of the 3-4-5group. Chromosomes 6 and 7 are about equal in size, and usually they are clearly smaller than the intermediate chromosomes 3,4, and 5. I n darkly stained nuclei they are generally not in doubt because the chromomere patterns are distinctive, but in lightly stained nuclei we have found these two chromosomes to be more similar than the McClintock-Singleton descriptions would suggest. Each has a large, shifting, median or submedian chromomere, the a chromomere. Singleton’s (1953) chromosome 7, which other workers (McClintock, St. Lawrence, Barry) call 6, has several additional large chromomeres arranged in a distinctive pattern. Singleton’s chromosome 6 (7 for McClintock, St. Lawrence, and Barry) is less well marked, by comparison with 7. However, in Singleton’s maps of chromosomes 6 and 7, the overall pattern is much the same, with the a and b chromomeres spaced along the chromosome a t about the same ratios from the ends, and with chromomeres a t the tips of each chromosome, although the tip chromomeres differ in size. c. Recognition of Chromosomes at Condensed Stages. Phillips (1967) was able to recognize interchanges involving chromosome 2 a t diakinesis and metaphase I by the rings and chains that indicate aberration pairing in translocation heterozygotes. Our experience has been that the small chromosome size at metaphase I, the possibjlity of overlapping bivalents, and the unsynchronixed anaphase-I disjunction make identification unreliable a t this stage, except for chromosome 2 if the nucleolus is still attached. d. Chromosome Morphology at Later Stages in the Ascus. The number, size, centromere position, and arm ratios of the mitotic chromosome complement have been diagrammed tentatively by McClintock (1945) and Singleton (1953).The mitotic karyotype is obtained a t metaphase of the third nuclear division in the ascus, when the chromosomes may be seen clearly in polar view on the metaphase plate. Each chromosome

162

DAVID D. PERKINS AND EDWARD G. BARRY

has a V or J shape, with the bend presumably located a t the centromere. The following fourth division, after the ascospores have been delimited, is also useful for observing chromosome morphology except that the chromosomes are smaller and more crowded inside the nucleus.

B. MARKERDISTRIBUTION AND GENETICMAPPING 1. The Markers

Over 400 gene loci have been mapped to the seven linkage groups of Neurospora crassa. The broad categories of phenotypes are listed in Table 2. TABLE 2 Numbers of Nonallelic Genes of Various Types That Have Been Mapped to Linkage Groups in Neurospora crassan Linkage group Type of mutant Nutritional Other biochemically defined Morphology Resistance to toxic agents Irreparable temperature-sensitive Suppressor Radiation-sensitive Recombination Meiotic Vegetative incompatibility Carotenoid Melanin Modifier Rhythm Lethal Sterility Growth rate Mating type Total genes

I

I1

I11

34 13

14 10

14 3

31 12

13

7

1

VI

27 5

21 12

10 3

9 11

129

10 4

24 2

18 5

5 5

17 2

118 30

3

3

3

2

1

20

2

2 1

4

17

1 2

1

1 -

-

-

1

-

38 (9%)

2 1

-

-

3 1 1

5 1 1 124 (29%)

Total mapped

V

IV

1 2 1 2

1 1

-

-

-

67

70

-

VII

-

-

2 1 2 -

-

33

2 1

1 1 1 1

-

51 (12%)

57

7

4 3 9 6 5 4 3 3 8 2 1 426

A mutant is listed under only one category, even though it might equally well fall under several categories. Uptake and permeability mutants are not listed separately, but are included under another heading, such as resistance, other biochemically dejined, or irreparable temperature-sensitive.

THE CYTOGENETICS OF

Neurospora

163

Several types of mutants have not been found, or are rare. There are few autonomous ascospore color mutants compared to Sordaria, Ascobolus or Podospora. This may be because white spores are usually inviable, or because they fail to survive the high temperature routinely used to break dormancy. White ascospores of one mutant type have been shown to germinate spontaneously (Johnson, 1975a). Several other categories of mutants are as yet underrepresented in Neurospora compared to other fungi-for example, radiation-sensitive, super-suppressor, meiotic, and inositol-requiring genes. 2. Genetic Maps and Marker Distribution

All the genes in Table 2 have been assigned to linkage groups, but by no means all are mapped accurately within groups. Useful maps have been published by Fincham and Day (1971), Davis and de Serres (1970), and Radford (1972). Uncertainties of map location are shown by Radford (1972), who has also listed known loci with brief descriptions of phenotypes, including known enzyme phenotypes (Radford, 1976). The maps presented in Fig. 5 have been prepared specifically to show the genes that are referred to in this review and its Appendix. These maps are not complete. Numerous useful, well mapped markers are not included because they do not happen to have been employed in the studies reported here. For special reasons, the positions of a few rearrangement breakpoints are shown, mainly those th at mark linkage-group ends. Gene symbols are identified in the legend to Fig. 5. Neurospora genetic nomenclature follows Drosophila usage more closely than that of maize, yeast, Aspergillus, or bacteria (Barratt and Perkins, 1965; Barratt, 1969). Different genes having the same basic symbol (“mimic genes”) are distinguished by numbers (eg., arg-I, arg-2) as in Drosophila, rather than by capital letters as in bacteria. This usage predates the introduction of the bacterial system and is more suited to an organism having mating types designated A and a. (The only exception in Neurospora is a d 3 A adSB, retained because o f long-standing use.) Neurospora suppressors are also symbolized as in Drosophila, with su for the mutant suppressor, su+ for its wild-type allele. The three-letter gene symbols of bacterial genetics are used for some classes of genes such as amino-acid auxotrophs (Perkins and Barratt, 1973). No scale is shown in Fig. 5, because uncontrolled variability of recombination is so great in Neurospora. Linkage group I is estimated to be about 200 map units (centimorgans) in length. Interval lengths in published maps may be based on recombination values from crosses with either high or low crossing over, or they may represent pooled values, or values felt to be most representative. Whatever their basis, their predictive value for distances is limited to within perhaps one order of magnitude for any strains that are not highly homogeneous. The gene orders, of course, should be invariant.

a. Distribution of Genes and Rearrangements. The distribution of mapped loci and rearrangements among the seven linkage groups is shown in Table 3. Both the number of genes in each group and the involvements

DAVID D. PERKINS AND EDWARD G . BARRY

FIG.5. Genetic map of Neurospora crmsa showing only loci named in this review. Parentheses indicate that order is uncertain

relative to outside markers; where vertical lines do not intersect the map, order within parentheses is uncertain. Distances are only roughly to scale; interval lengths can vary as much as 10-fold in crosses of different parentage. Because of the large number of loci, linkage group IV and the left half of I are both expanded to about 1.5x the scale of the other groups. The second line is a continuation of linkage group I. The following genes are not shown, though they are referred to: Linkage group I : ser-3 is near cys-5 and cys-11; cyt-1 is between ser-3 and leu-3; sn is near centromere, probably left; rg is near his-2; het-5 is right of thi-I; tre and mig are between ud-9 and al-2; uvs-6 is near and left of al-2. Linkage group 11: pi is probably an allele of col-10; pcon is an allele of nuc-2; cpt is between a r g d and pe. Linkage group 111: uvs-4 is near ad-4; het-7 is right of trp-1. Linkage group IV: r i b 4 is right of T(S4342); to1 is near trp-4. Linkage group V: a1-3, trp-5, and Me& are near inl; erg-1 and erg-2 are in VR. Linkage group VI: het-9 is in VIR; lys-5 is allelic with asco. Linkage group VII: thr-1 is near met-7; het-10 is right of for; the centromere position is uncertain relative to qa and met-7. Symbols: A, a-mating-type, ace-acetate, acr-acriflavine resistant, ad-adenine, abalbino, am-amination deficient, urgarginine, aro-aromatic biosynthesis, asco-ascospore color, an-asparagine, at-attenuated, aur-aurescent, bal-balloon, bis-biscuit, chol-choline, col-colonial, cot-colonial-temperature-sensitive, cpt-carpet, cr-crisp, c u t - c u t , cyh-cyloheximide (actidione) resistant, cys-cysteine, cyt-cytochrome, deddelicate, dow-downy, erg-ergosterol, eth-ethionine resistant, $-fissure, fl-fluffy, for-formate, fr-frost, het-heterokaryon (vegetative) incompatibility, his-histidine, hom-homoserine, ilv-isoleucine valine, In-inversion breakpoint, inl-inositol, leu-leucine, ly 3-lysine, m a t m a t , mei-meiotic, met-methionine, mig-migration of trehalase, mo-morphological, mt-mating type, &-niacin, nit-nitrate nonutilizer, nt-niacin or tryptophan, nuc-nuclease, 0s-osmotic, pub-p-aminobenzoate, pan-pantothenate, pcon-phosphatase control, pdz-pyridoxine, pe-peach, per-perithecial color, phe-phenylalanine, pi-pile, pk-peak, preg-phosphatase regulation, ptphenylalanine or tyrosine, pyr-pyrimidine, qaquinate catabolism, R-Round spore, rg-ragged, rib-riboflavin, ro-ropy, satsatelliteless, sc-scumbo, ser-rine, sk-skin, sn-snowflake, so-soft, sor-sorbose resistance, sp-spray, spco-spreading colonial, suc-succinate, T-translocation breakpoint, tatufted aerial, thi-thiamine, thr-threonine, todtolerant, tre-trehalase, trp-tryptophan, tyr-tyrosine, un-unknown heat-sensitive, uvs-ultraviolet-sensitive, vel-velvet, wc-white collar, ws-white spore, ylo-yellow. For full symbols of the translocations and inversions, see the Appendix.

e m M

c)

2

o

0

m

3 c)

rn

$ 2: 0

52

+

c-L Q,

01

166

DAVID D. PERKINS AND EDWARD G. BARRY

TABLE 3 Comparison of Cytological Lengths of the Seven Neurosporu Chromosomes with the Numbers of Genes and Rearrangements Mapped in Each Linkage group Corresponding chromosome

N0.a

Mapped genes ( N = 426)* Number of rearrangements in which involved ( N = 3 3 6 ) ~ Genes plus rearrangements ( N = 762) Measured length of synaptonemal complex (total = 45.5 pm)d

I

v

111

IV

VI

I1

VII

1

2

(3)

(4)

(5)

6

7

29% 27%

16% 12%

9% 11%

16% 17%

8% 12%

10% 12%

12% 9%

28%

15%

10%

16%

10%

11%

11 %

22%

16%

15%

13%

12%

10%

10%

a See Section IV, C and Table 4 for basis of linkage group-chromosome assignments. Chromosomes were numbered in the order of cytological length (McClintock, 1945; Singleton, 1953). b Includes all genes that have been mapped to linkage groups and that are thought t,o be nonallelic. See Table 2 for the distribution of genes by phenotype category and linkage group. c Based on the 167 rearrangements summarized in Table 5 and described in the Appendix. Values are not based on number of breakpoints; e.g., inversions are counted only once, and an insertional translocation is counted once for donor chromosome and once for recipient. d Gillies (1972). Average for three nuclei reconstructed from thin sections.

in gross rearrangements are approximately proportional to the cytological length of the corresponding chromosome a t pachynema. (The slight excess in I might be expected because this linkage group contains the mating-type alleles and other markers favorable for detecting linkage.) There is no obvious concentration of any phenotypic category of mutant in a particular linkage group. Rearrangement breakpoints do not appear to be distributed randomly within individual linkage groups. Centromere regions and chromosome tips seem more likely to be involved, and there is a suggestion of excess breaks in IVR. The distribution of genes within linkage groups can be seen from the genetic maps. Identified loci are the most crowded in a portion of I that includes mating type and the centromere, and in the proximal half of IVR. There is a n unexplained lack of markers in the left arms of I11 and V. Cytological observations indicate that the centromere is not near the end of 111,and the genetic map would thus be expected to have two arms. Nothing is known about recombination in IIIL, but crossing over is known not to be suppressed in VL, where 35% recombination occurs between a centromere marker, Zys-1, and the terminal cytological marker

THE CYTOGENETICS OF

Neurospora

167

sat, satelliteless (Barry and Perkins, 1969). There is thus no reason to suspect that the arm is genetically inert. VL bears the nucleolus organizer near its tip. There is no evidence of chromosome breakage from entanglement of a terminalized chiasma in the nucleolus. (In Neurospora, the nucleolus is usually detached from the chromosomes before anaphase I.) Rick (1971) summarized information on silent regions in the chromosomes of several organisms and noted that when the two arms of the nucleolus organizer chromosome are compared, mapped genes are preferentially more abundant in the arm opposite the nucleolus, in both tomato and maize. Neurospora apparently conforms to the same pattern. b. Clustering of Functionally Related Mutations. When nutritional markers originally became available in Neurospora, one of the first questions to be asked was whether functionally related genes would be linked or clustered. The answer was clearly no clustering, for one biosynthetic pathway after another (see Horowitz, 1965). However, there are several exceptions. Some of these appear to be single genes with multiple functions, others clearly involve separate genes. Where the products have been studied, enzyme aggregates or multifunctional enzymes are usually found. Histidine biosynthesis is specified by nine genes in the histidine operon of Salmonella. In Neurospora, the corresponding genes are scattered through four linkage groups. However, three of the enzyme activities are specified by a single region ( h i s s ) in Neurospora, and an enzyme or aggregate has been purified that possesses all three activities (see Ahmed et al., 1964; Ahmed, 1968; Catcheside, 1965; Catcheside and Angel, 1974; Webber, 1965; Minson and Creaser, 1969). Two distinct activities are both specified by the p y r - 3 locus (Davis and Woodward, 1962; Williams and Davis, 1968). Multiple enzymic functions are also specified by trp-3 (see Crawford, 1975) and trp-1 (DeMoss et al., 1967). The mutants mac and met-6 behave recombinationally as though they were a t different sites within a single gene locus (Murray, 1969). However, they differ in response when adenine as well as methionine is added, they complement one another (Murray, 1969), and they differ in the activity of two different enzymes related to folyl polyglutamate biosynthesis (Ritari et al., 1973). Other examples in Neurospora involve clustered genes that govern related functions. The aro cluster consists of five genes governing steps in the aromatic synthetic pathway (Gross and Fein, 1960; Giles et al., 1967; Rines et al., 1969). The aro genes are transcribed together and their products form an aggregate. The qa (quinate) cluster consists of one regulatory and three structural genes governing the aromatic catabolic pathway (Chaleff, 1974a,b; Case and Giles, 1975). The products are not aggregated.

168

DAVID D. PERKINS AND EDWARD G. BARRY

met-7 and met-9 appear to be adjacent genes (Murray, 1970). They specify different enzymes (Kerr and Flavin, 1970), yet mutant sites in the two loci are so close that they undergo coconversion. The Neurospora maps also reveal several other apparently adjacent pairs of genes with functionally related phenotypes-cys-1, cys-2 (Murray, 1965) ; thr-2, -3 (Perkins et al., 1962; Emerson, 1950) ; ilv-1, -2 (Kiritani, 1962) ; arg-10, -11 (Newmeyer, 1957) ; cys-5, -11 (Murray, 1965, 1968b) ; erg-1, -2 (Grindle, 1974) ; tre, mig (Sussman et al., 1971) ; pcon, p e g (Littlewood et al., 1975) ; and pyr-3, arg-2 (Reissig, 1960, 1963). Other related gene pairs are close but not contiguous-ad-3A, ad-3B (de Serres, 1969) and d-1, al-2 (Hungate, 1945; Subden and Threlkeld, 1970; Perkins, 1971b). It is not known whether there is any functional relation between the two distinct roles of the mating-type alleles-in sexual crossing and in vegetative incompatibility (Section 11). A functional relation has been suggested between mating type and the closely linked locus un-3, which is known to affect membranes (Kappy and Meteenberg, 1967). For a general review of genetic clustering of biochemically related functions in fungi, see Fink (1971). c. Estimates of Total Map Length. Because crossover frequencies are extremely variable in Neurospora (Section IV, B, 3), any estimate of overall genetic map length will be unreliable within wide limits. Nevertheless, rough estimates can be based either on cytological chiasma counts or on genetic recombination frequencies. An average of a t least two chiasmata are observed per bivalent (Singleton, 1953; E. G. Barry, unpublished observations), indicating that the total map length is a t least 700 units. Maps compiled from genetic data by Fincham and Day (1971) total about 750 units. Several linkage groups have since been extended by means of newly discovered genes or rearrangement breakpoints, bringing the total length to perhaps 900 units. This is in reasonable agreement with an independent estimate based on exchange frequencies in a long segment of I R which was multiply marked so that all exchanges could be detected (Perkins, 1962a). On this basis, the longest linkage group is estimated to be 200 units, and it (chromosome 1) represents 22-25% of the total genome, based on cytological measurement of synaptonemal complexes and pachytene chromosomes (see Gillies, 1972). Thus the total length in Neurospora is probably a t least 1000 genetic map units. How this value compares with genetic maps in other organisms can be seen in Table 1. 3. Crossing-over Variability and Its Basis Recombination between the same two markers may vary 2-fold, 4-fold, or even 10-fold in crosses of different parentage (see, for example, Stadler,

THE CYTOGENETICS OF

Neurospora

169

1956; Nakamura, 1966; Landner, 1971 ; Catcheside and Corcoran, 1973). Yet recombination values are reproducible within close limits in repeated crosses between the same two strains. It is not known whether an increase in crossing over in one region is accompanied by a compensating decrease elsewhere in the genome, except that chiasma interference is positive (see, for example, Perkins, 1962a). Structural heterozygosity and genic regulation have both been considered as possible causes of the variability of recombination. Abnormal crossing over and changed linkage relations have several times led to the recognition that chromosome rearrangements were present in heterozygous condition in particular crosses (for example, by Houlahan et al., 1949; de Serres, 1971). It may also be that paracentric inversions are present in some stocks and have gone undetected. (The problem of “silent” paracentrics will be discussed in Section V, C, 3.) However, most cases of crossing-over variability appear to be due to genic control. Recombination (rec) genes have been shown to regulate both intragenic and intergenic recombination in specific regions located a t a distance from the controlling gene (Jessup and Catcheside, 1965). Three rec systems have been described in studies by D. G. Catcheside and his associates B. R. Smith, K. K. Jha, D. E. A. Catcheside, D. R. Smyth, P. L. Thomas, T. Angel, B. Austin, and D. Corcoran. The experimental results and interpretations have been reviewed by D. G. Catcheside (1974) ; see also Catcheside and Corcoran (1973) and D. E. A. Catcheside (1974). Differences a t all three rec loci are present among commonly used laboratory wild-type stocks (Catcheside, 1975). More than one region may be under control by the same rec gene. High recombination is recessive to low for all the rec genes studied. One example is known of a different class of element, cog (recognition), which is closely linked to his-3. The dominant cog+ allele must be present in order for high recombination to occur in or near his-3 when the appropriate rec gene is homozygous recessive. The existence is inferred of a third class of genes with local effects on recombination. Other approaches have also been used in attempts t o clarify the genetic basis of crossing-over variability (Frost, 1961 ; Lavigne and Frost, 1964; Towe, 1958; Stadler and Towe, 1962; Nakamura, 1966; Landner, 1971, 1974; de Serres, 1971). These include selection and inbreeding. It was shown by Towe (1958), Stadler and Towe (1962), and Cameron et al. (1966) that inbreeding tends to increase crossing over. Other experiments showed that reciprocal recombination between the closely linked genes ad-3A and ad-3B was greater when markers originated in different genetic backgrounds than when they arose in the same background (de Serres, 1971). These results must now be evaluated in light of the findings on

170

DAVID D. PERKINS AND EDWARD G . BARRY

regulation of recombination. If high recombination requires that recessive rec genes be homozygous, inbreeding may be expected sometimes but not always to increase recombination, depending on what regions are being monitored, what rec alleles are initially present, and which of them are retained during inbreeding.

4. Mapping Methodology Genetic mapping consists of three phases-detection of linkage, determination of gene order, and estimation of interval length. In choosing methods to solve a practical mapping problem a t any of these stages, it is important not to confuse the objectives of mapping with investigations into the mechanism of recombination. a. Linkage Detection. I n terms of labor, random isolates are more efficient than tetrads for detecting linkage (Perkins, 1953), and unordered tetrads are more efficient than ordered tetrads. Effort can be minimized by using multiply marked tester strains such as alcoy (Perkins et al., 1969), mu2ticent (Perkins, 1972c) and related follow-up testers ( Perkins, 197213, 1973). Markers in these strains have been selected for ease and reliability of scoring. The multicent tester contains proximal markers for all seven linkage groups, in normal sequence: mt, bal, acr-2, pdx, at, ylo-1, wc. alcoy contains three independent reciprocal translocations, each associated with a marker: aZ-1 ( I ; I I ) , cot-1 (IV;V), ylo-1 (1II;VI). Other less complete multiple-group testers are also available from FGSC. If tetrad data are available, gene-to-gene linkage is indicated by an excess of Parental Ditype over Nonparental Ditype tetrads (Perkins, 1953). b. Determining Gene Order. The method of preference is to use random isolates from 3-point crosses, where gene order is established not from absolute recombination frequencies, but from the relative frequencies of single- and double-crossover classes. Chiasma interference is positive in Neurosporu (see, e.g., Perkins, 1962a), and this increases the efficiency of determining gene order by reducing double crossovers relative to singles. Because crossing over is so highly variable, map sequences are likely to be incorrect if gene order is determined solely by combining the recombination values of single intervals from different crosses. Many errors have resulted from using 2-point data in this way. Maps with completely reliable sequences can be built up by basing gene order on a series of overlapping 3-point crosses that have intervals in common. In special situations, gene order can be resolved most easily by testing for duplication-coverage (Perkins et ul., 1969). That is, a right-left test is made, using the breakpoint of a chromosome rearrangement as a reference point. Duplication-generating rearrangements such as insertional

THE CYTOGENETICS OF

Neurospora

171

translocations are employed. If rearrangements with appropriate breakpoints are available, determining the order of closely linked genes by duplication coverage can be far quicker and easier than by conventional 3-point tests. Centromere mapping is a special case. Location of the centromere is already known with varying degrees of certainty for each of the seven linkage groups. Historically, the centromere positions were based on critical crossovers in ordered tetrads. Only rarely now does the need arise to determine a gene-centromere sequence, and that occurs when a gene is found to map between the most proximal loci that are reliably positioned in opposite arms. When this occurs, the gene-centromere order can be established in any of three ways-by ordered tetrads, by unordered tetrads, or by random ascospores where an appropriate chromosome rearrangement makes mapping possible by duplication coverage. The use of ordered tetrads has been familiar since Lindegren’s work in the 1930s. Unordered tetrads are the usual method of centromere mapping in organisms such as yeast and Chlamydomonas, and they might well be used to advantage for this purpose in Neurospora, where the presence in a cross of gene markers at two or more known centromeres would enable ejected groups of eight ascospores to be treated as though they had been isolated in order. See Mortimer and Hawthorne (1975) for this and other aspects of mapping with unordered tetrads. The ability to determine centromere location by duplication-coverage of nearby genes depends upon availability of a duplication-generating rearrangement with one breakpoint near the centromere in question. Since centromere regions seem prone to be involved in rearrangements, a number of such strains are available (Fig. 19). (Combinations of overlapping reciprocal translocations can also be used, where one breakpoint adjoins the centromere.) The method is best illustrated in linkage group I, where the centromere has been shown to lie between the left-arm duplication 39311 and the right-arm duplication ARl73, hence between mei-3 (covered by 39311) which must be in the left arm, and wn-2 (covered by ARl7S) which must be in the right arm. This method is far less laborious than isolating and scoring the large number of ordered asci that would be required for conventional mapping. i. Appropriate uses of tetrad analysis. Tetrads have unquestionably played an important role in research on the mechanism of recombination, providing basic information on gene conversion, crossing over, and interference. Fungal tetrads have contributed in an essential way to molecular models of eukaryote recombination. Ascus analysis has also been very useful for distinguishing whether a variant was cytoplasmic or chromosomal, for constructing double-mutant stocks involving mimic genes or suppressors, and for identifying and characterizing chromosome rearrangements.

172

DAVID D. PERKINS AND EDWARD G. BARRY

The most familiar use of ordered tetrads has been to determine gene-centromere distances from second-division segregation frequencies. This was a novel and fascinating feature in the early years of Neurospora genetics, enabling a direct determination of what could be arrived a t only indirectly in higher organisms. Consequently, ordered-ascus analysis seems to have become associated with Neurospora as though it were a fundamental cornerstone of all genetic analysis with the organism. (This is the impression given by many textbooks.) Ordered asci have been a mixed blessing, however, because they have often been used unnecessarily where the needed information could have been obtained by a less laborious method, as has been shown for routine mapping. Tetrad analysis is certainly not the best or most economical method for gene-gene mapping, and gene-centromere mapping that would require tetrads is rarely necessary any longer.

ii. Estimation of interval length. Recombination frequencies are highly variable in different genetic backgrounds in Neurospora (Section IV, B, 3), and the marker genes used for mapping are of mixed ancestry. Thus there is usually little point to refining the statistical analyses of mapping data, or to applying corrections for undetected multiples, or to enlarging the data beyond a modest level, because the uncontrolled background variability of recombination is so large. The idea of a genetic map of Neurospora with standard interval lengths is illusory. For organisms or strains in which markers are all in the same background, so that recombination frequencies are more nearly constant, cumulative map lengths would ideally be built up using recombination frequencies in marked intervals short enough to preclude the occurrence of double crossovers (probably 10 or 15 units). I n an organism like Neurospora, this would be most economically and reliably accomplished using random isolates in sufficient numbers to minimize statistical error (Fig. 8 of Barratt et al., 1954, can be adapted for this purpose). The eight spores of an ascus yield far less information than eight ascospores isolated a t random (Mather and Beale, 1942). If tetrad data are already available for use, the best estimate of the map length of an interval between two genes is given by the formula map distance 50 (T 6 N P D ) / ( P D N P D T),where PD, N P D , and T are frequencies of Parental Ditype, Nonparental Ditype, and Tetratype asci (Perkins, 1949). This corrects for undetected 2-strand and 3-strand double exchanges on the basis of 4-strand doubles, which are manifested as Nonparental Ditypes. The validity of this formula across long distances has been shown in experiments where closely spaced intermediate markers enabled all multiple exchanges to be detected between the distant markers to which it was applied (Perkins, 1962a).

+

+

-

+

C. LINKAGE GROUP-CHROMOSOME CORREL,ATIONS Chromosomal assignment of particular linkage groups was first attempted by Singleton (1948) and by St. Lawrence (1953), a t a time when the genetic maps were still rudimentary and the number of chromosome structural variants was small. Now, 28 years later, assignment of the seven linkage groups to specific chromosomes is for all practical pur-

THE CYTOGENETICS OF

173

Neurospora

poses complete. Only a beginning has been made, however, toward locating genes within individual chromosomes. The present status of linkage group-chromosome correlations is shown in Table 4. Linkage groups are designated with Roman numerals, and chromosomes with Arabic numbers. General Methodology. The assignment of linkage groups to chromosomes has depended on chromosome aberrations, which can be recognized and localized both genetically and cytologically. An aberration is mapped genetically t o a linkage group (or groups) and the aberrant chromosome TABLE 4 Relation of Genetically Defined Linkage Groups to Cytologically Defined Chromosomes in Neurospora crassa ChromoLinkage some assogroupo ciationb

Aberrationc

I

1

R.55, 4637 4637 4637 4637, S1007, 17084, H42.50 AR190, S1325, 36703 39311

I1

6

R55, 4637 4637 4637 4637 NM149, 39311

I11 IV V

(3) (4) 2

VI

(5) 7

VI I

45502, AR211, S4342 C-1670, R2355, 36703, 46802 NM149, AR190, S1325, 36703, sat 46802 45502, 46802 S1229 S1007, 17084 5936

Referenced St. Lawrence (1953) McClintock (1955) Singleton (1948) Barry (1967) Barry and Perkins (1969) Barry (1 972) ; Perkins (1972a) St. Lawrence (1953) McClintock (1955) Singleton (1948) Barry (1967) E. G. Barry (unpublished; 1972)

E. G. Barry (unpublished) Phillips (1967) Barry and Perkins (1969) E. G. Barry (unpublished) E. G. Barry (unpublished) Barry (1960a) Barry (1967) E. G. Barry (unpublished)

Linkage group numbers (Roman) were assigned in chronological order of discovery (see Barratt et al., 1954). Chromosome numbers (Arabic) were assigned in descending order of length (McClintock, 1945). Parentheses indicate that the association is probably correct but is subject to some uncertainty. Or heteromorphic satellite (sat). d References are omitted to studies that do not support the assignments made in this table; all contrary findings are discussed in the text, however.

174

DAVID D. PERKINS AND EDWARD G . BARRY

(or chromosomes) is identified cytologically. Two rearrangements are examined cytologically which have one genetic linkage group in common and a second linkage group that is not in common. The chromosome altered in both strains must be the counterpart of the shared linkage group. Examples of this type of analysis will be given below. Aberrations are also the best means of determining the location of specific genes within a chromosome. This is because rearrangement break points can be placed precisely on linkage maps by genetic analysis (see Section V, A, 3 ) . However, attempts to use rearrangements for intrachromosomal correlations have not been very productive so far. There is often uncertainty in recognizing chromomere patterns of the aberrant chromosomes. Sometimes there may be mispairing in the region of the breakpoints or even “slippage” where the paired regions are nonhomologous. Consequently, only tentative intrachromosomal correlations have been possible using the usual structural rearrangements. One unambiguous intrachromosomal localization has been made, however, and this employed a heteromorphic chromosome deficient in the small satellite that lies distal to the nucleolus organizer in chromosome 2. The satellite is clearly located a t the left end of linkage group V (Barry and Perkins, 1969). Steps in Establishing the Linkage Group-Chromosome Correlations. Singleton

(1948) made a brief attempt to associate linkage groups and chromosomes using

four chromosome aberrations that had been only partially mapped genetically. He very tentatively suggested that chromosome 2, the nucleolus-organizer chromosome, is linkage group I. I n view of his limited observations, it is not surprising that his suggested correlation proved to be wrong. The first detailed effort to correlate linkage groups with chromosomes was made by St. Lawrence (1953). She concluded from cytogenetic studies that chromosome 1 is linkage group 11, 2 is IV, and 6 is I. (These are not quite right, as will be seen.) St. Lawrence’s correlations were based on examination of a complex aberration, R66, which had major rearrangements in three chromosomes (1, 2, 6) for which she located breakpoints in three linkage groups (I, 11, IV). Her determination that I was 6 was based on the argument that the R66 breakpoints found in chromosome 6 and linkage group I were both far out in their respective arms, while the breakpoints of chromosomes 1 and 2 and linkage groups I1 and IV were all near their respective centromeres. Observations on two other translocations supported and extended this conclusion. Her correlation of linkage group IV with chromosome 2 was based on an analysis of T(ZV;VZ)46602. This translocation is linked in groups IV and VI (Houlahan et al., 1949). B. McClintock (personal communication to St. Lawrence) identified an exchange between chromosome 2 and another, smaller chromosome, possibly 4. Since 46602 and R66 both involved only chromosome 2 and linkage group IV in common (R66 was shown not to be linked to ad-f in VI), St. Lawrence concluded that linkage group IV equals chromosome 2. Study of T(Z;IZ)4637 completed St. Lawrence’s analysis. This translocation has breakpoints in IR (Houlahan et a!., 1949) and IIR (St. Lawrence, 1953; Hagerty, 1952). B. McClintock (personal communication to St. Lawrence) determined that

THE CYTOGENETICS OF

Neurospora

175

the translocation was between chromosomes 1 and 6. Since translocations R66 and 4637 both showed involvement of chromosomes 1 and 6 and linkage groups I and 11, and since chromosome 6 by other evidence seemed to be linkage group I, St. Lawrence concluded that chromosome 1 should be linkage group 11. Barry (1962, 19671, however, proposed that the St. Lawrence correlations of I = 6 and I1 = 1 should be reversed with I = 1 and I1 = 6. This amendment was based on studies of translocations T(I;VII)17084 t h i l and T(I;VII)S1007 which

both involve linkage groups I and VII. Neither translocation has an abnormality of chromosome 6, whereas chromosome 1 is involved in both. Also, inversion strain In(IL+IR)H4260 involves both group I (Newmeyer and Taylor, 1967) and chromosome 1. Further confirmation that chromosome 1 is linkage group I was found in other studies (Barry and Perkins, 1969; Barry, 1973). Barry (1960a) proposed that VII = 7. A complex chromosome rearrangement in 51229 is linked in I, 11, IV, and VII, and it appeared to involve chromosomes 1, 2, 6, and 7. The involvement of a chromosome (7) and a linkage group (VII), in addition to those detected by St. Lawrence in R66, indicated that VII = 7. Supporting observations were reported by Barry (1967) on T(I;VII)S1007 and T(I;VII)17084 thi-1. Phillips (1967) examined four translocations that showed interchanges involving chromosome 2. The translocations (36‘703, R2366, 46802, and C-16‘70) all involved linkage group V, but only one of them involved IV. He therefore proposed that chromosome 2 corresponds to V rather than to IV. This was confirmed (Barry and Perkins, 1969) by using other translocations and mapping the satellite of chromosome 2 to linkage group V. The earlier errors can now be explained as follows. St. Lawrence did not test R66 for V linkage, presumably because she had already located a linkage group involvement for each of the chromosomes known to be aberrant. McClintock’s determination that translocation 46602 involved chromosome 2 was probably in error because of stock problems. The aberration is separable from a closely linked pvr-3 mutant which was present in the stock with the original aberration, but this may not have been realized a t first, and derived stocks with the mutant may have been thought to contain the aberration also. McClintock made several observations of supposed 46602 stocks. In 1945 she reported that the translocation “involved a very unequal exchange of segments of two nonhomologous chromosomes. The breaks appear to have occurred close to the end of the long arm of chromosome 1 and close to the centromere in the long arm of one of the chromosomes with a subterminal centromere, possibly 4.” In 1952, McClintock’s observations were as described by St. Lawrence (1953). In 1955 she again examined stocks derived from the original 46602, but found no gross structural rearrangements. It now seems probable that different aberration stocks, or suspected aberrations, were examined in the three studies. (It is not unusual for more than one rearrangement to be found in the same strain following mutagenesis, or for a second aberration to occur spontaneously. Examples of both are documented in the Appendix.) Singleton (1948) also examined a 46602 strain and interpreted i t as a reciprocal translocation between chromosomes 1 and 6. E. G. Barry (unpublished) has examined available strains of 46602 known genetically to involve IV and VI, and believes them to contain a translocation between chromosomes 4 and 5. Barry’s (1960a) initial analysis of 51229, which appeared to support the St. Lawrence correlation of 2 with IV, was rechecked, and the conclusion that 2 = IV was withdrawn (Barry and Perkins, 1969).

176

DAVID D. PERKINS AND EDWARD G . BARRY

This completes the chromosome assignments for four linkage groupsI, 11,V and VII. These are considered to be firmly established. The remaining three linkage groups, 111, IV and VI, are clearly associated with the three middle-sized chromosomes, 3,4, and 5. The tentative assignments shown in Table 4 are probably correct, although an error may possibly have occurred because the three chromosomes are similar in size and appearance, and their chromomere patterns tend to be inconsistent. It is doubtful if such an error would ever be of any practical consequence, because the experimental usefulness of the three is severely limited because of their similarity. It is proposed to designate the chromosome numbers permanently as shown in Table 4. Number 3 is the chromosome bearing linkage group 111, 4 carries IV, and 5 carries VI. If further refinement of techniques makes morphological distinctions practical, it is proposed that these numbers be retained, even if the chromosome lengths prove to be out of order. In order for all linkage groups to correspond exactly with chromosome numbers, three changes would now be required (11, V, and VII). However, the present linkage group numbers are so firmly established in general use that we do not propose to make any changes. To do so would only introduce confusion. Bask of the Tentative IIZ, IV, V I Assignments. From our cytological analysis of T(ZV;VI)46602, one breakpoint is probably near centromere in the long arm of chromosome 4, and the other is probably near the tip of 5. The resulting exchange has produced one very long chromosome (most of 5 plus three-fourths of 41, which is about the length of chromosome 1, and one very short chromosome (the centromere and short arm of 4, and the tip of 5 ) . In the genetic map, the positions of &602 breakpoints are close to centromere in IVR and far distal in VIR. I t can be concluded that IV = 4 and VI = 5 from the position of genetic and cytological breakpoints. This conclusion is based on the assumption that cytologically and genetically determined distances correspond. If IV = 4 and VI = 5, then chromosome 3 and linkage group I11 are associated by default, being the last chromosome and linkage group not otherwise designated. However, Griffiths, et al. (1974) tentatively proposed a correlation of chromosome 3 with linkage group IV. Their evidence is based on a cytogenetic study of T ( I ~ Z V ) Y l l B M l a6d d , which involves I and IV. They state that in one nucleus one of the chromosomes involved corresponds cytologically to McClintock’s description of 3.

I n no other fungus or eukaryotic microorganism has there been a well established correlation between a linkage group and a specific cytologically recognizable chromosome. However, Mu’Azu (1973) has tentatively suggested four correlations of chromosomes and linkage groups of Sordaria brevicollis, based on cytogenetic analyses of six translocations.

THE CYTOGENETICS OF

Neurospora

177

D. CYTOPLASMIC GENES Mutant strains of Neurospora are known whose differences from wild type are transmitted independently of the nuclear chromosomes, as first shown by Mitchell and Mitchell (1952) and Mitchell et al. (1953). The mutants show slow or abnormal growth, abnormal cytochromes, and respiratory defects. No drug-resistant cytoplasmic mutants have been reported. With one exception (Srb, 1963) transmission of the cytoplasmic determinant is strictly maternal, i.e., through the protoperithecial parent. The cytoplasmic factor carried by the paternal (fertilizing) parent is excluded or eliminated. Abnormal cytoplasm has been transmitted by microinjection, using techniques developed by Wilson (1961). The cellular locus of the cytoplasmic genes is probably, but not certainly, the mitochondria (Diakumakos et al., 1965). Measurements of individual molecules by electron microscopy (Agsteribbe et al., 1972) or by reassociation kinetics (Wood and Luck, 1969) show that the mitochondria1 DNA of Neurospora is long enough to contain a t least 50 genes. No evidence has been obtained of recombination between different cytoplasmic determinants. For a review and critique of cytoplasmic inheritance in Neurospora and other fungi, see Sager (1972, Chapter 5). V. Chromosome Rearrangements

Structural rearrangements occur frequently in Neurospora, are readily recognized, and can be used quite effectively. This is fortunate because the possibility of cytogenetic manipulation by means of numerical variants is extremely limited in Neurospora, as disomics and diploids are highly unstable and only one useful heteromorphic chromosome variant is known. Most cytogenetic experiments must depend therefore on chromosome aberrations. Each of the 167 rearrangements that have been characterized in Neurospora crassa is described in the Appendix. A sizable number of these rearrangements can be used to produce nontandem duplications-aneuploids of recombinational origin equivalent to partial diploids. These are valuable research tools, resembling the tertiary or telo-trisomics of plant cytogenetics in some of their applications. Appendix Table 1 gives a complete list of the reciprocal translocations, arranged according to linkage groups. All possible combinations of the linkage groups are represented among the 123 interchanges. Two mutual

178

DAVID D. PERKINS AND EDWARD

G.

BARRY

insertions are also listed. Appendix Table 2 lists the identified duplicationproducing rearrangements, 42 in all. These fall into three categoriesinsertions, quasiterminal rearrangements, and those still uncertain whether interstitial or terminal. The two Appendix tables are intended to serve as indexes, in which a rearrangement having desired specifications can be identified by isolation number. Information on it can then be found in the main part of the Appendix, where the aberrations are all arranged in a single sequence according to isolation numbers, without regard to aberration type. The main Appendix entry summarizes information on each rearrangement, describing its type, map relations, phenotype, fertility, genetic behavior, cytology, and origin. Rearrangements discovered and analyzed by other workers are included. Secondarily derived rearrangements (such as the duplications generated by meiotic recombination from each of the known duplication-generating aberrations) are not listed as separate entries in the Appendix or the Appendix tables. A. METHODSOF IDENTIFYING AND MAPPINGREARRANGEMENTS 1 . Detection

Almost all the Neurospora rearrangements were detected initially by noting that defective, white ascospores were produced in crosses heterozygous for the rearrangement, a situation exactly comparable to pollen abortion in flowering plants (Belling, 1914). Pollen abortion that originates from heterozygous rearrangements is termed semi-sterility or partialsterility in plant cytogenetics. These terms are best avoided in Neurospora because they would be confused with sterility of other types, such as barrenness of perithecia. Only a handful of the known Neurospora rearrangements were discovered because of altered linkage (e.g., T (I;VZI) 17084 thi-1 ) , and only one translocation was first recognized cytologically, by abnormal meiotic pairing ( T (V + V I I ) E B 4 ) . Standard methods for identifying and analyzing rearrangements have been described by Perkins (1974). The procedure will be summarized here. About 90 or 95% of ascospores from structurally homozygous crosses are viable and develop normal black pigment. I n contrast, heterozygous reciprocal translocations usually produce 50% defective, inviable spores, due to deficiencies, while insertional translocations and other aberration types that generate viable duplications typically produce 25% defective ascospores. The deficiency ascospores remain unpigmented or pale ; for simplicity they will be called white. (Point mutants that result in white ascospores are rare in Neurospora, compared to rearrangements.) I n

THE CYTOGENETICS OF

Neurospora

179

searching for new rearrangements, each strain to be tested is crossed to a normal-sequence tester strain on agar medium in a small (12 x 75 mm) tube. Ten days later, when ascospores have matured and been shot from the perithecia, the glass wall of each tube is examined, and the proportion of black and white spores is estimated. Strains producing less than 90% black spores are saved for further testing. Putative rearrangement strains are then subjected to a second step of analysis that employs unordered asci, which can be obtained in large numbers by the method of Strickland (1960). Each strain t o be tested is crossed on a petri dish to the same normal-sequence tester as before. After 10 days, unordered asci are collected as spontaneously shot groups of eight ascospores on an agar slab under the inverted cross plate. The collecting slab is exposed for a period ranging from a few seconds t o several minutes, depending on the rate of shooting, and is then scanned, classifying each well separated group according to number of black and white spores. The major classes are 8:0, 6:2, 4:4, 2:6, 0:8 (Black: White). 2. Diagnosis

Frequencies of the unordered ascus classes in crosses of Normal X Rearrangement immediately enable us to classify most rearrangements into two main categories, according to whether they do or do not generate viable duplication progeny. The frequencies also provide information on the positions of break points relative to centromeres. Frequencies of the unordered ascus types from structurally homozygous crosses are shown in Fig. 6. I n contrast, Fig. 7 shows the frequencies for heterozygous translocations. The top row is typical of translocations that do not produce viable duplications. Two examples are given, with breakpoints far from centromere (A), and close to centromere ( B ) . Examples in the bottom row are typical of duplication-producing translocations whose breakpoints are far from centromere (C) and close to centromere (D). The ascus frequencies from translocations that do not produce viable duplications are typically symmetrical around 4B:4W, with 8:O = 0:8. The ascus frequencies from duplication-producing translocations are usually symmetrical around 6B:2W, with 8:O = 4:4. The occurrence of 6B :2W asci in significant numbers usually indicates that viable duplications are being formed. The meiotic basis for these distributions will be described later for each specific type of rearrangement. Frequencies of unordered tetrad types which correspond to the histograms in Fig. 7 are given as part of the description of each aberration in the main part of the Appendix.

180

DAVID D. P E R K I N S AND EDWARD G . BARRY

l

W

I Tll;Vl36703 X Tll;V136703 IN = 1351

Normal lfIPl Normtl (fI’I

IN = 1391

75 -

75 I

_

5

K :

Di

8 -

8

5 50-

50-

E -

c

%4! 8

8 :

-

25 -

O

1

f 25 -

L

~

8:O

6:2

4:4

2:6

A5mr class (8lack:Whitel

0:8

0-

II 62

4:4

2:6

0:8

A r u r class 18lack:Whitel

Fro. 6. Results of structurally homozygous crosses, when the parents are both wild types (left), and when they both contain the same reciprocal translocation (right). In this and succeeding figures, histograms show the frequencies of unordered asci having various members of black (viable) and white (inviable) ascospores. There are five major classes, ranging from all black (leftmost histogram) through 6B:2W, 4B:4W, 2B:6W, to all white (rightmost). N is the observed number of asci. Rare asci with odd numbers of defective spores (5:3, 7:1, etc.) are not shown. Reproduced from Perkins (1974).

The rearrangements that do not produce viable duplications are predominantly reciprocal translocations. The rearrangements that are capable of making viable duplications are predominantly insertional and quasiterminal translocations. Other tests in addition to the ascus patterns are required in order to distinguish one type of duplication-producing rearrangement from another. Advantages and Limitations oj Unordered Tetrads jar Diagnosing Rearrangements. The examples given in Fig. 7 conform well to theoretical expectations. The arrays of ascus types are distributed symmetrically around the 4B:4W or the 6B:2W class. From such a result it can be confidently inferred that a strain is a rearrangement, and the type of rearrangement can be predicted with reasonable confidence. Because the contents of unripe asci are usually not ejected, the problem of distinguishing true 0:s asci from unripe asci of other types is avoided when the analysis is based on unordered asci that have been shot out of the perithecium. If a similar analysis is made by opcning perithecia, unripe as well as ripe asci are seen and may not readily be distinguished from one another, leading to an excess of asci in the OB :8W class.

THE CYTOGENETICS OF

Neurospora 70

67 %

IBI L

Tlf: Vl36703 X Normal IN=2451

a

181

a >

.

50-

TII: IVlNM137

X

Normal IN = 116)

E

D

i

I

r ’ 25.

7

-I 8

6

2:6

-

-

9%

08

A r u r class 1Biack:Whitel

,-

70 1

64% ICI

-c

-

a >

-

{ 50-

TI1 -I I 114540 X Normal IN = 941

1 T(l-11)39311

-

g

50

Normal IN = 3921

D

7 c

25-

m 8 0

8:0

6:2 4:4 26 Axus class l8lack:Whitel

I

0:8

Arcus claw (8lack:Whitel

FIG.7. Frequencies of unordered ascus types in crosses of Reciprocnl Translocation x Normal (A and B), and Insertioncil Translocation x Normal (C and D).

The rearrangements on the left have breakpoints far from centromere, while those on the right, have both breakpoints close to centromerc. Note that with reciprocal translocations, which produce no viable duplications, the frequencies are symmetrical around the 4B:4W class and effectively no 6B:2W asci are seen. With insertional translocations, which do produce viable duplications, symmetry is around the 6B:2W class, and effectively no OB:8W asci are seen. Reproduced from Perkins (1974). I n some situations, errors of diagnosis may occur when unordered tetrads are used. Nonblack ascospores may result from mutant ascospore-color genes rather than deficiencies. Conversely, lethal deficiencies from some rearrangements do not prevent the ascospores that contain them from becoming black. Asci of the 0:8 type mag be underrepresented because they disintegrate. Most of these exceptions are rarely encountered. The most common departure from ideal expectations occurs when one of the inviable duplication-deficiency classes from a nonterminal reciprocal translocation becomes pigmented. The resulting ascus array, and the patterns of black and white ascospores in individual asci, misleadingly resemble those of a n insertional or quasiterminal translocation. Fifteen of the 123 identified nonterminal reciprocal translocations are clearly of this typc (see T ( I I I ; V ) A R l 7 7 as an example). Unordered tetrads are so informative in analyzing aberrations that ordered tetrads are not required even to determine centromere positions. A few special applications

182

DAVID D. PERKINS AND EDWARD G. BARRY

where ordered asci are necessary or useful have been listed by Perkins (1974, p. 485). Anomalies that can lead to misdiagnosis are discussed and examples given by Perkins (1974, pp. 474-477). It is clear that the preliminary diagnosis based on ascus t.ypes should be confirmed by other methods.

3. Genetic Verification and Mapping

Following such a preliminary diagnosis based on abortion patterns in the asci, each rearrangement is then verified and analyzed genetically, using appropriately marked tester strains. First the aberration breakpoints are mapped to linkage groups. For this purpose a normal-sequence tester such as multicent is preferable to alcoy, which itself contains translocations (Section IV, B, 4, a ) . In mapping a rearrangement, segregants are scored for Rearrangement us. Normal by the incidence of white spores in test crosses. The breakpoint of a translocation is treated as though it were a mutant allele with the phenotype “50% black spores in test cross.” The “allelic” normal sequence has the contrasting phenotype “90% black spores in test cross.” If few or no ascospores are produced, the test cross is classed as barren. Usually the necks of barren perithecia are rudimentary or absent. It is characteristic of most or all duplications in Neurospora that crosses involving them are barren. In this way, the rearrangement is confirmed, linkage groups are identified, breakpoints are located, and if duplications are produced they are verified. For details of the identification and verification procedures, see Perkins (1974).

4. Cytological Verification Genetic analysis has usually been complemented by cytological examination only when a rearrangement is of special interest. Each type of chromosome rearrangement is recognized cytologically by its heterozygous meiotic pairing with standard-sequence chromosomes, or by the production of bridges and acentric fragments. Identification of Neurospora aberrations is very similar to the meiotic analyses developed in maize, with allowances for the smaller chromosome size. Pachynema is the most favorable stage for diagnosing Neurospora aberrations. 6. Simulation of Synthetic LethaE Genes b y Rearrangement Breakpoints The breakpoints in simple (nonterminal) reciprocal translocations and inversions are in fact formally equivalent to synthetic lethal genes (Dobzhansky, 1946) in normal sequence. Most Neurospora rearrangements clearly cannot be attributed to synthetic lethals. Either they are known to generate viable duplications, or they have been confirmed cytologically, or markers show new linkage relations in homoaygous aberration sequence. Where none of these tests has been made, there remains a remote possibility that a pair of synthetic lethal genes could be responsible for the properties attributed to a rearrangement. It might be especially difficult to dia-

THE CYTOGENETICS OF

Neurospora

183

tinguish short mutual insertions from synthetic-lethal point-mutants. However, this is no problem for the two Neurospora aberrations believed to be mutual insertional translocations-T(I V)S1326 and T ( I ZV)Y112M16 ad-3A. Genetic and cytological evidence shows that they are both chromosome rearrangements.

+

+

6. The Need for Quantitative Data on Induced Rearrangements

A committee of the Environmental Mutagenesis Society has listed quantitative studies of chromosome aberrations in microbial eukaryotes among major recommendations for future research : “Cytogenetic tests are now feasible only with the higher eukaryotes. It would be of great significance for determining comparative rates for point versus chromosomal mutation to possess a system capable of detecting chromosomal aberrations in a microbial eukaryote” (Drake, 1975). Among eukaryotes with small genomes, Neurospora seems the most likely practical source for both types of information. Point mutations can already be measured and characterized very precisely (see, for example, de Serres and Malling, 1971; Malling and de Serres, 1973). If Neurospora is to be considered as a test organism for chromosome aberrations, methods must now be developed for obtaining reliable quantitative measures of new rearrangements. Availability of such methods would also enable rearrangement frequencies to be compared in genotypes suspected of affecting chromosome stability and DNA repair. The rationale behind the quoted recommendation is as follows: When organisms in a phylogenetic series are examined, their sensitivity to radiation-induced forward mutation per rad per locus is directly proportional to DNA content per haploid genome (Abrahamson et al., 1973). Likewise cellular radiosensitivity is proportional to chromosome volume and nucleotide content (see Sparrow et al., 1967; Underbrink et al., 1968). This correspondence between sensitivity and DNA content allows extrapolations to be made with increased confidence from experimental organisms to man. I t is still not known whether a similar relation holds for induced chromosome rearrangements. Such information is needed in order to evaluate whether point mutations or chromosome rearrangements are the more hazardous in relation to environmental mutagens.

B. THEIDENTIFIED REARRANGEMENTS : TYPES, FREQUENCIES, ORIGINS,AND PHENOTYPES

I. T y p e s The numbers of rearrangements of various types that have been identified are summarized in Table 5 . Reciprocal translocations are the most frequent, followed by insertional translocations and quasiterminal (tip) translocations. There are no paracentric inversions and only three pericentric inversions, all of which involve a chromosome tip. The 167 rearrangements are probably a representative sample of those newly arisen aberrations that result in ascospore abortion. Some bias doubtless exists against aberrations that have very drastic effects on ascospore abortion; these may have been rejected as too complex for analysis. At the other extreme, a bias must exist against aberrations with slight or borderline effects. These may not have been recognized in the

184

DAVID D. PERKINS AND EDWARD G. BARRY

TABLE 5 Identified Rearrangements in Neurospora crassa, Summarized According to Type of Aberration Type

Number.

I. Rearrangements that do not generate viable duplications b y meiotic recombination i n crosses by Normal Reciprocal translocations 123 Mutual insertions 2 Inversions 0 125 (75%) 11. Rearrangements that regularly generate viable duplications b y meiotic recombination in crosses b y Normal Insertional translocations 19b Intrachromosomal transpositions 1 Quasiterminal translocations 12 Quasiterminal pericentric inversions 3 Type uncertain 7 42 (25%) Total rearrangements 167 Similar rearrangements from the same experiment, that could have had a common origin, are counted only once. Duplications or other secondarily derived aberrations are not included. * Including some that are complex.

first place, or they may have been abandoned because of scoring difficulties. Short intrachromosomal transpositions with small displacements would not be detected by our methods, nor would short inversions. Tandem or nontandom duplications that arose directly as deviants from wild type would escape detection. Deficiencies would be lethal in a haploid organism unless sheltered, and would not be transmitted. Deficiencies of considerable length have, in fact, been obtained somatically by de Serres (1969) using techniques that allowed them to be rescued in heterokaryotic condition. The properties of each of the rearrangement types will be described and discussed in separate sections to follow. The basis for recognition and diagnosis of each type will be shown, and a possible explanation will be suggested for the absence of paracentric inversions. 2. Origin and Frequency

The aberrations have come from a variety of sources. Some were encountered as newly arisen variants in routine crosses. Others were recognized as present in various laboratory stocks. A majority were obtained by screening the survivors of experiments primarily designed to recover

THE CYTOGENETICS OF

Neurospora

185

point mutants. Most of these experiments involved mild UV-irradiation followed by a procedure to enrich for particular mutant types. Less commonly, X-rays or chemical mutagens were used. The frequency of recovered rearrangements has not been determined as a function of treatment. It is our impression, based on several UV experiments where both point mutants and rearrangements have been sought, that the frequency of recovery is in the same order of magnitude for new gross rearrangements as for gene mutations. With UV doses giving 10 to 50% survival of treated conidia, from 5 to 20% of survivors have been found to contain new gross rearrangements. For representative recovery frequencies see Table 4 in Perkins (1974). Tector and Kafer (1962) have found new rearrangements in a high proportion of Aspergillus nuclei following commonly used radiation dosages. Neurospora strains have been collected from nature in such a way as to sample local populations from many geographical regions (Perkins et al., 1976). These were examined for structural differences within populations, and only two rearrangements have been found and confirmed. Both of them were reciprocal translocations, recovered as single isolates.

3. Fertility and Vegetative Phenotype

A vast majority of the 167 rearrangements are homozygous fertile and have no obvious mutant phenotype. I n Aspergillus also, most balanced rearrangements are morphologically wild type (Kafer, 1965 ; Nga, 1968). Eight of the rearrangements are barren in crosses where the rearrangement is homozygous-i.e., perithecia are formed that produce no ascospores, or few. Ascospore production is somewhat reduced in an additional five cases when rearrangement sequence is homozygous. Fourteen rearrangements have not been tested as homozygotes because of femalesterility (nine) or for lack of a recombinant with mating type (five). Twelve rearrangements acquired a t their origin a mutant phenotype that is allelic with a known gene locus and inseparable by recombination from the arrangement: HK5S cut, K79 me-7, Y l l 2 M 4 i ad-SB, Y l l b M 1 5 ad-SA, C161 aro, TM429 his-3, S1325 nic-2, C-1670 pk-1 (bis),4540 nic-2, 46S7 al-1, 17084 thi-I, and 46702 inl. Several other rearrangements are inseparable from a mutant phenotype that is not known to be allelic with a previously recognized gene locus-NM139 bs, 81229 arg, and T51M156 un are examples. To this list should be added 17 other rearrangements classified as morphologically mutant and nine as near-wild. These have not been tested for allelism with known morphological mutants. They include eight of the nine female-sterile rearrangements. At least 18 rearrangements arose simultaneously with a linked but separable point mutation: T54M94 (zcn-18), NM136 (arg-S), NM160 ( p h e - I ) ,A R l 7 4 ( p e r ) ,NM177 ( m o ) , AR216 (al-1 or - 2 ) , AR2dl (ilv),

186

DAVID D. PERKINS AND EDWARD G . BARRY

Y234M419 ( a d - 1 ) , 0 3 0 4 (morph), A420 ( t r p - 5 ) , H4250 (arg-1), S4342 ( p t ) , 5936 (leu-3), Y16329 (phe-2), 36703 ( a r g - l ) , 39311 ( S U C ) , 45502 ( p y r - S ) , 47711 (ilw). Also, five examples are known where two linked but separable rearrangements appear to have arisen simultaneously : T54M140b originated with another still unanalyzed rearrangement, Y l l 2 M 4 i originated with Y l l g M d r , AR180b with ARMOr, B362i with B362r, and STL384b with STL384r.

4. Position Efject No observations in Neurospora suggest a variegated-type position effect, but the growth habit might make a variegated phenotype difficult to detect. Rearrangements that originated simultaneously with mutant phenotypes have either been separable by recombination, or can be explained in terms of a breakpoint within a gene or within a single transcriptional unit. Several closely linked genes are simultaneously inactivated in T(ZZ;ZZZ)C161 aro (Gross and Fein, 1960). However, this is the result expected of a breakpoint within a cluster of genes that are coordinately transcribed (see Section IV, B, 2, b). All three functions specified by his-3 are missing in T (Z;VZZ)TM429 his-3, which has one break in the locus and no detectable deletion (Catcheside and Angel, 1974). Position effects might be anticipated when closely linked genes with coordinate functions are separated from each other by rearrangement. al-1 is separated from al-2 by T p ( I R + ZR)T54M94 and arg-2 is separated from pyr-3 in two translocations, Sl229 and S4342. The relevant phenotypes are not noticeably altered in any of the three rearrangements, however. A translocation in Aspergillus (VIII +=111) is characterized by a variegated morphological phenotype which is allelic with a known gene located a t or near one of the breakpoints (Clutterbuck, 1970). Evidence suggests but does not prove that the wild-type allele can be recovered from the rearrangement by crossing over; if so, i t would be the first example of a variegated-type position effect in fungi. C. REARRANGEMENTS THATDo NOTMAKEVIABLEDUPLICATIONS Within this category, no duplication progeny survive from crosses of Rearrangement X Normal, because whenever aneuploid segregants are duplicated for one chromosome segment they are deficient for another. Most of the Neurospora rearrangements that behave in this way are reciprocal translocations. Two are probably mutual insertions. Inversions behaving in this way would also be expected, but none has been found.

THE CYTOGENETICS OF

Neurospora

187

1 . Reciprocal Translocations

Simple reciprocal translocations are the most readily identified and the most commonly occurring rearrangements in Neurospora (Table 5 ) . Appendix Table 1 lists the 123 mapped reciprocals according to linkage groups, and they are described individually in the Appendix. a. Ascus Effects and Their Meiotic Basis. Unordered asci from crosses heterozygous for typical reciprocal translocations show a distinctive distribution of the frequencies of ascus types (Fig. 7A, B ) . The meiotic origin of these ascus types is shown in Fig. 8. Normal disjunction of the centromeres of the two chromosomes involved in an interchange should produce 8 : 0 and 0:8 asci with equal frequencies when there is no crossing over in the interstitial centromere-breakpoint intervals (top half of Fig. 8 ) . Occurrence of interstitial crossing over is expected to result in 4:4 asci (bottom half). 4:4 asci will thus be rare if interchange points are both close to their respective centromeres, but frequent if one or both interchange points are far out in a chromosome arm. The breakpoints of many translocations have been mapped genetically, and they conform generally to these expectations. The examples in Fig. 7 were selected to show the two extremes-T (I;V)36703, with breakpoints far out from centromere, and T ( I ; I V ) N M l S 7 ,with breakpoints close in to centromere. For additional examples showing intermediate amounts of interstitial crossing over, see Fig. 3 in Perkins (1974). b. Cytology of Reciprocal Translocations. Pachynema is by far the most reliable and informative stage for verifying and diagnosing interchanges. Completely paired heterozygotes show a typical cross-shaped arrangement of the four bivalent arms (for photographs, see Barry, 1967, Figs. 2, 5, 7 ; Barry and Perkins, 1969, Fig. 1 ; Fincham and Day, 1971, Fig. 26). Pachytene associations have been used to confirm the genetic analysis of numerous translocations and to relate linkage groups to specific chromosomes (Section IV, C ) . I n theory, the contracted stages following pachynema should be useful for verifying and diagnosing interchanges, which should appear as rings or chains of four chromosomes. I n practice in Neurospora, however, diakinesis and metaphase I are very limited in their usefulness. The chromosomes are so small that recognition of a quadrivalent may be uncertain, and overlying bivalents can easily be mistaken for an interchange complex (see, for example, Fig. 4 in Barry, 1967). I n spite of these difficulties, contracted stages have been used successfully t o study interchanges that involve the easily recognized nucleolus organizer chromosome (Section IV, A, 4, c). McClintock and Singleton (Singleton, 1948) attempted to analyze

188

DAVID D. PERKINS AND EDWARD G . BARRY

Ascus

PROPHASE I ORIENTATION AND CROSSING OVER

ADJACENT CENTROMERES TO SAMEPOLE

-

CONSTITUTION

...:::::r::: I

Q”’ 4 . I .;:.”””. ;””’+”’ .

f

0:8

.. .. .. .. ....

f >

l

Dup, Def N T

- 4 >

x

Def, Dup

H

CEN TROMERES TO SAME POLE

VIABLE: DEFICIENT ASCOSPORES

‘ 4 ‘ 4

.. .,. .....,... .. .. : :

H

f

4:4

FIG.8. The origin and constitution of asci containing various numbers of deficient spores, from crosses of Reciprocnl Trnnslocntion (black centromeres) x Normal (white centromeres). Segments originally in one of the Normal chromosomes are shown as solid lines, those in the other Normal chromosome as dotted lines. The consequences of segregation without crossing over are shown in the two top diagrams. Crossing over between either breakpoint and centromere is expected to produce 4:4 asci, as shown in the bottom two diagrams. Other linear orders of black and white spore-pairs are possible, depending on which chromatids were involved in crossing over; the resulting unordered tetrad is classed as 4:4 regardless of its original order. The defective spores are of two types, representing complementary duplicationdeficiency classes. These may or may not he recognizably different, depending on the particular translocation. If adjacent-2 segrcgations occurred (where homologous centromeres failed to disjoin), 0:s asci would result, with all spores deficient (not shown in the figure). Reproduced from Perkins (1974).

trans’locations by determining changes in chromosome lengths and arm ratios a t metaphase of the third division in the ascus. In our experience this stage is of doubtful practical value because of small chromosome

THE CYTOGENETICS OF

Neurospora

189

size and possible inaccuracies of measurement if a chromosome is tilted in the polar plane.

c. Some Research Applications of Reciprocal Translocations i. Mapping genes lo linkage group. The efficiency of assigning unmapped point mutants has been greatly increased by a tester strain (acronym alcoy) containing three independent translocations tagged by markers which can be scored visually (Section IV, B, 4, a ) . A majority of new point mutants readily show linkage to one of the markers, and linkage group assignment is then completed with a single follow-up cross (Perkins et a/., 1969; Perkins, 197213). ii. Production of duplications.
v. Centromere behavior and disjunction (a) Allernole and ndjljncent segregation. Measures of alternate versus adjacent segregation of centromrrcs in translocation hetcrozygotes are of interest because of the possibility of genetic control of crntromere disjunction, as in Oenothern. Information can he obtained from unordered asci in two ways, comparing 8B:OW with 0 : s asci, and comparing crossovers with noncrossovers among 4B:4W asci. 8:0 nntl 0 : s asci. With no interstitial crossing over, 8B:OW asci result when alternatc centromeres go to the same pole a t anaphase I. 0B:SW’s result when adjacent centromeres go together. This could happen in either of two ways, with homologous centromeres disjoining (called adjacent-1) or failing to disjoin (called adjacent-2) (Fig. 8 shows the first two types, but does not show adjacent-2). Adjacent-2 segregation is found in maize when interchange points are close t o the centromeres, hut not when they are far enough out for interstitial crossing over t o occur regularly (Burnham, 1949, 1950). Apparently adjacent-2 occurs in cither situation in the mouse (Searle et nl., 1971).

190

DAVID D. PERKINS AND EDWARD G. BARRY

McClintock (1945) opened perithecia and observed four ascus classes from Neurospora translocation T ( 1 V ; V I ) 45502, including one class with eight tiny, degenerate spores. These she attributed to adjacent-2 segregation. They may well have been bubble asci instead, as described in Section 111, C, 4. With most Neurospora translocations, the frequencies of 8:O and 0:8 asci are about the same. This 1:l ratio is consistent with either of two hypotheses: (1) alternate equals adjacent-1, with no adjacent-:! segregation occurring ; or (2) alternate segregations equal the sum of adjacent-1 plus adjacent-2. Endrizzi (1974) has shown cytologically that the second is true for two translocations in cotton, and that there are actually two subtypes, alternate-1 and alternate-2, corresponding to the two adjacent types. 4 : 4 asci. A different population of meioses is represented by the 4B:4W’s. These result from interstitial crossing over (Fig. 8, bottom half). Among the 4:4 asci, only parental combinations of markers survive following alternate segregation, and only crossover combinations survive following adjacent-1 segregation. Adjacent-2 segregation results in 0:8’s, and rannot produce 4:4 asci, even if crossing over has occurred interstitially. If Neurospora resembles cotton in having two equally frequent types of alternate segregation, and if all-1 aIt-2 = adj-1 adj-2, then an excess of the 4:4 class having parental marker combinations might be observed. The magnitude of the excess would depend on the frequencies of type-1 and type-2. If alt-1 = alt-2 = adj-1 = adj-2, the expected ratio would be 2:l for parenta1:recombinant 4:4 asci. Such an observation would be inconsistent with the traditional view that alt = adj-1, for which the predicted ratio of parenta1:recombinant 4:4 asci is 1:l. (b) 3 :1 segregation. Sometimes three chromosomes of an interchange complex are included in one daughter nucleus, and only one is included in the other nucleus. The disomics arising from translocations in this way, by 3: 1 segregation, are called tertiary disomics. They resemble normal-sequence (primary) disomics in their instability (Section 11). A few aneuploid progeny are commonly found in crosses of Translocation x Normal in Neurospora. These have been attributed to occasional 3: 1 segregations. With some translocations, 3: 1 segregations are apparently frequent. Actual frequencies are difficult to determine accurately because the extra chromosome is so rapidly lost, producing balanced fertile haploid nuclei that cannot be distinguished from one or the other parental type when they are test crossed. Disomics that originate from 3:1 segregation can be recognized, however, by the presence of translocation-linked markers in heterozygous or heterokaryotic condition. I n this respect they resemble the segmental duplications from duplication-generating rearrangements such as insertional translocations, to be discussed in sections that follow. Indeed, conventional reciprocal translocations can a t first be mistaken for insertional or quasiterminal translocations if 3: 1 segregations occur in significant numbers. Examples where some confusion has existed can be found in the Appendix (see translocations T61M168, T64Ml4Ob, NM12.9, ALS176, D306). The following criteria may be useful in distinguishing tertiary disomics from segmental duplications: (a) Heterorygosity is not limited to a single region in the products of 3:l segregation, as it is in the segmental duplications from insertional or quasiterminal translocations. Markers anywhere in the two interchanged chromosomes may be heterozygous in one or another disomic progeny. (b) There is no a priori expectation for a fixed frequency of aneuploids from 3:l’s, as there is from insertional or quasiterminal translocations, where one-third of viable progeny are segmental duplications. With 3:l’s, the frequency of disomy for markers in a given

+

+

THE CYTOGENETICS OF

Neurospora

191

segment is usually well below one-third. (c) Disomics are somatically less stable than all or most segmental duplications. (d) Most aneuploids from 3 : l segregation are not barren in backcrosses. (Occasionally an exceptional aneuploid is barren and highly stable in a cross known to undergo 3 : l segregation. This might result if different chromosomes were lost in separate nuclei of the disomic, and the two nuclei, each with a deficiency but complemented by the other, then displaced the original disomic type to form a stable balanced heterokaryon.) 3 :1 segregation in other organisms. Disomics in Aspergillus nidulans are relatively stable and can be recognized by characteristic morphologies (Kafer and Upshall, 1973). Single heterozygous translocations increase the frequency of disomics 50- t o 100-fold for the two chromosomes involved (Upshall and Kafer, 1974). A comparable increase may well occur in Neurospora. Trisomics from interchange heterozygotes are well known in plants (see Burnham, 1962). They have been put t o practical use in agriculture (Wiebe and Ramage, 1971). Over 50 cases of 47- or 45-chromosome offspring have been reported in man, that apparently originated by 3:l segregation from translocation heterozygotes (Lindenbaum and Bobrow, 1975).

2. Mutual Insertions

Two hTeurospora rearrangements appear to be of this type--T(IFz:

V)S1325 nic-2 and T(Z p Z V ) Y l l d M 1 5 ad-3A. Both are inverted inser-

tional translocations that differ from typical insertionals in producing no viable duplication progeny. With both rearrangements, the long inserted IR segments should be viable as duplications, because even longer IR duplications are known to be viable, that include the same intervals. Absence of duplications could be explained in both rearrangements if an unmarked, short interstitial segment of V (or IV, respectively) had been inserted into I, simultaneously with excision of the long I segment and its insertion into V (or IV). This explanation is inferential and is unproved for both strains, although observations on chromosome pairing a t pachynema suggest the presence in S1325 of a short V + I insertion (see Appendix for details and references). It should be noted that these strains resemble paracentric inversions in many respects, and both were diagnosed as inversions before a second linkage group was known t o be involved. 3. Inversions

a. Paracentric Inversions. There are no paracentric inversions among the 167 rearrangements that have been identified in Neurospora, nor are there any reliably established cases in other fungi. I n a critique of all published reports of supposed inversions in Neurospora and other fungi, it was concluded that the evidence either favors another type of rearrangement, or is inadequate to prove unequivocally that a paracentric is actually involved ( Perkins, 1974). A possible rationale has been offered for the failure to find paracentric

192

DAVID D. PERKINS AND EDWARD G. BARRY

inversions (Perkins, 1974). It is suggested that dicentric bridges abort the entire ascus in which they occur. This would prevent the detection of paracentrics by the spore-pattern method, because they would have no or little visible effect on the appearance of surviving asci. Evidence supporting this explanation comes from the study of inverted insertional translocations. These are detected by the spore-pattern method, despite the loss of asci with dicentric bridges, because they also produce white spores by centromere segregation without crossing over. There is both genetic and cytological evidence from inverted insertionals suggesting that bridge formation usually results in loss of the entire ascus [Section V, D, 1, a, (iii)]. If so, cryptic paracentric inversions may in fact be present in apparently normal Neurospora strains where their presence is unsuspected. Some parallels are suggested by the behavior of paracentric inversions in the mouse. Although numerous translocations had been identified, no inversions were known in mice until they were deliberately sought by looking for bridges a t anaphase I of meiosis (Roderick, 1971; Roderick and Hawes, 1974). Fertility is not reduced in either male or female mice by heteroeygous paracentric inversions, suggesting, as one hypothesis, that genetically unbalanced gametes are eliminated because of bridge formation.

A cytological approach may now be the most feasible way to identify a paracentric inversion in Neurospora, since other methods have failed. A paracentric inversion should be distinguishable from an inverted insertional translocation because dicentric bridges that resulted from crossing over in the paracentric inversion would be mostly limited to the first division, whereas bridges from inverted insertional translocations occur regularly also a t the 2nd, 3rd, and 4th divisions [described in Section V, D, 1, a, (iii)]. A possible complication would result if a breakagefusion-bridge cycle was initiated in the paracentric inversion by breakage of the anaphase-I bridge. This could result in bridges a t the 3rd and 4th divisions, but not a t the 2nd division. A second possible complication is an unexplained background of “spontaneous” anaphase-I1 bridges in some Neurospora crosses thought not to contain an aberration of any type, as well as in crosses with aberrations which should not make bridges. I n standard wild-type crosses, telophase11 bridges may be found in as many as 10% of the divisions. No acentric fragments accompany this bridge formation. If the asci containing them survive, such bridges might account for some part of the 5-1070 of white spores found in wild-type crosses. b. Pericentric Inversions. We have not succeeded in identifying a single nonterminal pericentric inversion. There is no obvious reason for

THE CYTOGENETICS OF

Neurospora

193

this failure. The rationale for failing to find paracentric inversions does not apply to pericentrics, because no dicentric bridges are produced when the centromere is located inside the inversion. Heterozygous pericentric inversions would be expected to produce complementary duplicationdeficiency products by crossing over, and should thus be recognizable visually by inspection of ascospores or asci. Single-crossover asci should be 4B:4W. The frequency of aborted spores would depend on length of the inversion, effectiveness of pairing, and frequency of crossing over. It is known that inversions can occur and survive in Neurospora. Three pericentric inversions have in fact been found and mapped, all of them quasiterminal-Zn(ZL + ZR) H4.250, In(ZL + ZR)NM176, and In(ZL + ZR)ARl6. (Because one breakpoint is a t a chromosome tip, each of these produces viable duplications by meiotic crossing over. They will therefore be described in Section V, D, 2, b.) It can be calculated from the behavior of these long-terminal pericentrics that a nonterminal pericentric inversion only half their length should still produce enough white ascospores to be readily detected as an aberration.

D. REARRANGEMENTS THATGENERATE VIABLENONTANDEM DUPLICATIONS VIA MEIOTICRECOMBINATION About one-fourth of the rearrangements known in Neurospora fall under this heading (Table 5 ) . The 42 that have been mapped are listed in Appendix Table 2 and are described individually in the Appendix. Nontandem duplications are regularly produced by meiotic recombination in three types of crosses, each with two subtypes according to whether two chromosomes are involved, or a single chromosome: Type 1: Normal Znsertional rearrangement (Fig. 9 ) , with subtype ( a ) , insertional translocations, and subtype ( b ) , transpositions within one chromosome. Type 2: Normal Quasiterminal rearrangement (Fig. lo), with subtype ( a ) , quasiterminal reciprocal translocations, and subtype (b), quasiterminal pericentric inversions. Type 3 : Rearrangement Partially overlapping rearrangement (Fig. 11), with subtype ( a ) , partially overlapping reciprocal translocations, and subtype ( b ), partially overlapping inversions. Duplication progeny from crosses of the first and third type are pure, in the sense that nothing is missing. Duplication progeny from crosses of the second type are presumably deficient for one chromosome tip; in order for them to survive, that tip must contain no essential gene. With translocations (subtypes a ) , duplication progeny result from independent segregation and ideally constitute one-third of the surviving progeny.

x

x

x

a lnsertiona I translocations betwoen chromosomes Origin: (normal sequence) 1 2 3 _u

(insertional)

1 2 3 ..... (or ................. 3 2 1)

.....0......................

-

I

0

Meiotic pairing, normal x translocation:

-

segregation cpn

give

P

9

3

h Transpositions within one chromosome Origin :

-

(transposed)

(normal sequence) 1 2 3

Meiotic pairing, normal

x

.................. 1

-0

4 2 3 ....... ........... (or 3 2 1)

transposad:

crossinq over can give

+

FIG.9. The origin of duplication progeny from crosses involving insertional rearrangements. a. Above: Genesis of an insertional translocation by rearrangement of the normal sequence. Below: Meiotic production of a duplication by independent segregation in Insertional Translocation X Normal. b. Above: Genesis of an intrachromosomal transposition by rearrangement of the normal sequence. Below: Meiotic production of a duplication by crossing over in Transposition X Normal.

0

a.Tip- nontip reciprocal translocations Origin: (translocated)

(normal) I:

4

3*'

..............0.............

t

4

-2ji...o

3

-c

i!

1 2 3 ............... ............. 0

Moiotic pairing, normatx translocation:

.......0......... .........

-=.-

b. Tip-nontip poricsntric inversions (normal)

Origin: 1

34

t

,.: ....b....L....?

t

......*.........

segregation

7 F

con give-

-

'

(invert-&) 2

3

..r. ....P......5 Q L . g

0

'21

Meiotic pairing, inversion x normal:

\..z

or

.....6.... 6 .........6.. 25 . a . .

-

crossing. ovar can grva

:*T

FIG.10. The origin of duplication progeny from crosses involving quasiterminal rearrangements. a. Above: Genesis of a quasiterminal reciprocal translocation by rearrangement of the normal sequence. Below: Meotic production of a duplication by independent segregation in Translocation X Normal. b. Above: Genesis of a quasiterminal pericentric inversion by rearrangement of the normal sequence. Below: Meiotic production of a duplication by crossing over in Inversion X Normal.

+,

a. Reciprocal translocation X Overlapping reciprocal (same arms) Omgins:

[normal)

.=-

1 2 3

0.

and 4

..... .................. 4 5 6 c

-

4

..........6 3 =-

.....0.. 4 5 2 1 .........

C'L (D 0,

(second tmnslocation)

.....0....A..S..e. .... 4

23

(Orst tronslocotjon)

............6 5 2 3 =-

.....o . . . 4 . L

Meiotic pairing, First x second trunslocation:

{'?'=

5..."\2

P

...... 4 i.. jl ......8:::::.-

........ ....... 6 6

~4

3m

4

w

5--..

b Inversion x overlapping inversion Origins:

(normal) -0

4

2 3

(first inversion)

..........$

...........s _ 4 . . 5 . a

4 5 6

and 4 2 3

4

&xond inversion)

.....4...5...6

0

.............

6 5 2 3

4

......-

4 4

Meiotic wiring, Cirst with second:

ye.*.*..($2

.......... 6 A?..::. ........ 6 5'..

crossin over con +

6 ........

4

5.-.. FIG.11. The origin of duplication progeny from intercrosses between overlapping rearrangements having breakpoints suitably placed in the same two chromosome arms. a. Aboue: Genesis of two partially overlapping reciprocal translocations, by two independently occurring rearrangements of the normal sequence. Below: Meiotic production of a compound duplication by independent segregation in the intercross. b. Aboue: Genesis of partially overlapping inversions. by two independently occnring rearrangements of the wild type. (The inversions could both be paracentric a.nd in the same arm.) Below: Meiotic production of a wrnpound duplication by crossing over in the intercross.

?

THE CYTOGENETICS OF

Neurospora

197

With intrachromosomal transpositions or inversions (subtypes b ) , duplication progeny result from crossing over, and their frequency depends upon the distance between breakpoints. All these types and subtypes have been observed in Neurospora, and they will be considered in sequence below. It is characteristic of them all to produce unordered asci of the 6B:2W type, which is diagnostic of duplication-producing rearrangements. I n symbols for rearrangements that produce viable duplication progeny, an arrow is placed between the two linkage groups (or arms). The linkage group (or arm) preceding the arrow is the source (donor) of the duplicated segment. For example, in the insertional translocation T ( I + II)39311, a segment of I has been inserted into 11, and duplication progeny will possess the group-I segment in two doses. Duplications from a particular rearrangement always contain the same loci. 1. Insertions

a. Insertional Translocations. At least 20 insertional translocations have been identified. The properties of one insertional, T ( I + II)39311, have been studied in especial detail, both genetically and cytologically (Perkins, 1972a; Barry, 1972). i. Ascus effects and their meiotic basis. Frequencies of the ascus types characteristic of insertionals are shown in Fig. 7C and D. Additional examples can be found in Fig. 5 of Perkins (1974). The meiotic basis for these proportions is shown in Fig. 12. When there is no crossing over, normal centromere disjunction results in 8:O and 4 : 4 asci with equal probability (top half). Asci of the 6:2 type are produced by interstitial crossing over. They will be rare if the proximal breakpoints are both close to their respective centromeres, and frequent if one or both of the interstitial regions are long. The breakpoints of the examples in Fig. 7C,D have been mapped genetically and their locations are consistent with the frequencies of 6 : 2 asci. The translocated inserted segment and its homolog are shown as unpaired loops in Figs. 9, 12, and 13. Alternatively, they might pair with each other, with consequences described below. ii. Cytology at prophase I . (a) Insertion not paired. The situation is simplest a t pachynema when pairing does not occur between the translocated segment and its normal homologous segment. The deleted or the inserted segment bulges or loops out as a univalent strand from a bivalent with paired ends (just as diagrammed in Figs. 9 or 12). Figure 13 shows the appearance of one such loop (see also photographs in Barry, 1972). This loop-pairing pattern is frequently seen when short insertional translocations are examined, and i t can be used to identify the specific chromo-

198

DAVID D. PERRINS AND EDWARD G . BARRY

PROPHASE I ORIENTATION AND CROSSING OVER

. -~

'

ASCUS VIABLE: DEFICIENT CONSTITUTION ASCOSPORES

m::::< 2

.....+ ..................

M

:::::I::..

........................ .....w: ..................

'-r

A

-

4

X -

-

L

-

M

>

I

T

T N

8:0

N

6:2

> -L

FIQ.12. The origin and constitution of asci containing various numbers of deficient spores from crosses of an Insertional Translocation (black centromeres) x Normal (white centromeres.) The consequences of segregation without crossing over are shown in the two top diagrams. Crossing over between either breakpoint and centromere is expected to produce 6:2 asci, as shown in the bottom two diagrams. The order of black and white spore-pairs is not necessarily as shown. The defective spores are all identical, containing the same deficiency and no duplication. Pairing and crossing over between the translocated segment and its normal homolog are not shown in the diagram. Reproduced from Perkins (1974).

somes involved in the exchange when the chromosome lengths are measurable and the chromomere patterns are clear. (b) Insertion paired. The situation a t pachynema is more complex when the interstitial translocated segment pairs with its normal homolog.

THE CYTOGENETICS OF

Neurospora

199

FIG.13. Insertional translocation pairing at pachynema in a cross of T(ZV + ZZZ) S44Z by wild type. The extent of the large translocation in S@@ is shown by the size of the loop. (Orcein staining, ~ 3 0 0 0 . ) Complexes of four chromosomes are seen sometimes during prophase and at metaphase I. The looped part of the complex a t pachynema should distinguish an insertional translocation from a reciprocal translocation and indicate the position and size of the translocated segment. Long insertions are known from both genetic and cytological evidence usually to be paired. Yet such paired chromosome complexes are rarely clear and interpretable cytologically. The paired loops are difficult to trace through three-dimensional angles with their abrupt changes in pairing partner a t the break points, just as is true for inversions (Section V, D, 3, b). Insertional translocations have the further complication that a second chromosome pair is involved. iii. Effects of crossing over in the inserted segment. When the translocated insertion pairs with its normal homolog, the genetic and cytological consequences of crossing over are critically different, depending on whether the translocated segment is inverted or noninverted with respect to its new centromere. (These orientations have been called dyscentric and eucentric. The simple terms inverted and noninverted will be used in this sense.) When crossing over occurs between the inserted segment and its normal homolog, dicentric bridges and fragments are produced if the insertion is inverted, but not if it is in its original orientation. Diagnosis of an insertion as inverted can thus be accomplished cytologically, by noting a heightened frequency of bridges and fragments. This is straightforward

200

DAVID D. PERKINS AND EDWARD G. BARRY

with long insertionals, but a reliable cytological determination is difficult when the insertion is short, because the background frequency of bridges and fragments is high, even in control crosses that are supposed to be structurally homozygous, and this makes any small increase difficult t o recognize. It is more difficult to be certain that an insertion is not inverted, because failure to detect increased bridge formation could be due to short genetic length or to absence of pairing rather than to a noninverted orientation. Orientation can be determined genetically in T X T crosses if two gene markers are present within the insertion (see Perkins, 1972a, for an example). Introducing markers by crossing over is practical only with long insertions, however. Despite these difficulties, a t least five of the 20 insertionals listed in Appendix Table 2 have been diagnosed as inverted. Not all the rest have been examined cytologically. Among those that have, a l o i frequency of bridges and fragments suggests that some are noninverted, but there is no positive evidence. Crossing over in noninverted insertions. With noninverted insertions, crossing over could result in viable duplications having chromosome segments interchanged that were distal to the insertion-a new rearrangement would result having the properties of both a duplication and a reciprocal translocation. Patau (1963) and Lejeune and Berger (1965) have pointed out how crossing over of this type could account for otherwise unexplained cases of human anomalies attributed to aneuploidy. Study of such crossover-duplications in Neurospora must await positive identification of a suitable long uninverted insertional. D. Newmeyer (unpublished) has calculated that single exchanges within a paired noninverted insertion should produce only 4B:4W and 0:8 asci in a ratio of 3: 1. Thus, ascus frequencies for a long insertional of this type might be quite different from those shown in Fig. 7C. Only one-third of the 4:4 asci from this source would contain viable Duplications, in contrast to 4 :4’s from unpaired insertionals, which theoretically consist entirely of Duplications. Crossing over in inverted insertions: Dicentric bridges. With inverted insertional translocations, crossing over between the inverted inserted segment and its normally placed homolog produces dicentric anaphase bridges and acentric fragments (81325 and 39311 are examples). This resembles the behavior of paracentric inversions, but with one important difference. Single crossovers in a heterozygous paracentric inversion should result in bridges at anaphase I, except that where a second exchange of 3-strand type occurred proximal to the inversion bridge formation would be deferred to anaphase 11. I n contrast, with an inverted insertional translocation as many as 50% of the bridges resulting from

THE CYTOGENETICS OF

Neurospora

201

single crossovers may occur a t anaphase I1 or later divisions in the ascus. This is a result of perfectly normal chromosome segregation and is ex7 pected if there is an equal probability that nonhomologous centromeres joined by a chromatid tie will go either to opposite poles (bridge) or to the same pole (no bridge) a t each division (Fig. 14). (After the second division, twisting of homologs around one another would nullify this expectation, but there is little relational coiling of chromosomes in Neurospora.) Bridges are in fact observed frequently in the second and later ascus divisions with T ( I +II)39311 (Barry, 1972) and with other inverted insertional translocations. It is unnecessary t o invoke breakage-fusion-bridge cycles to explain this behavior, which is expected as a simple consequence if centromeres

YST' ........... .........

.*:::.-J .....

...................... a::::: ...................... 4

e

1

f

3

1

d

OR

I 2

::e

3

FIG.14. Pairing of an inverted inserted scgment with its normal homolog, followed by crossing over and segregation surh as to result in a dicentric chromatid and an acentric fragment. Because centromeres shown solid are not homologous with centromeres shown open, the joined centrorneres may segregate to the same (lower left) or to opposite (lower right) poles with equal probability a t anaphase I. I n the former case the dicentric does not form a bridge, but may do so a t a later division.

202

DAVID D. PERKINS AND EDWARD

Q.

BARRY

joined by the chromatid tie fail to disjoin at anaphase I but do so later. However, if a breakage-fusion-bridge cycle does occur in Neurospora, then the bridges observed a t mitotic divisions in the ascus could be a consequence either of the cycle, or of the earlier formed but previously unbridged dicentric. The fate of asci with dicentric bridges. There is evidence suggesting that bridge formation usually results in loss of the entire ascus in which it occurs. I n crosses with the long inverted insertional T ( I + IZ)39311, many asci contain anaphase-I bridges, and these asci should contain four black and four white spores if they survived. But no excess asci of the 4:4 type were found above those expected from other causes (Perkins, 1972a). Consistent with this, the number of asci observed to contain bridges and fragments in the later divisions is less than expected from the frequency of anaphase-I bridges, suggesting differential ascus mortality (E. G. Barry, unpublished). Acentric fragments. Inverted insertional translocations produce a fragment only a t the first division, though it may not appear in the cytoplasm until the second division. The locus content of the fragment is constant and unique for each translocation. It comprises the inserted segment plus both distal segments, including the chromosome tips. The fragment from a specific rearrangement may possess a strikingly distinctive morphology, which is cytologically recognizable (J. R. Singleton, unpublished observations of translocation S1325). With T ( I -+ V)S1525, the acentric fragments were seen not to be degraded, but to persist in the cytoplasm in a micronucleus (J. R. Singleton, unpublished). Fragments from T ( I + V)S1325,T ( I + II)39311, and some other insertionals replicate and divide synchronously with the centric chromosomes of the regular nuclei of the ascus (Barry, 1972, 1973). Four micronuclei containing identical acentric fragments may thus be present in a single ascus following the fourth division, when two mitotic cycles have been completed. This is best documented for T ( I + II)39311. Fragments from some other insertionals do not persist and replicate, but become pycnotic. b. Intrachromosomal Transpositions. One transposition has been identified in Neurospora, T p ( I R + I R )T54M94. Viable duplications from Transposition x Normal have flanking markers always recombined in the direction that would be expected of an inverted insertion (B. C. Turner, personal communication). (A noninverted transposition could pair so as to form a double loop and give rise to duplications of two types, in the second of which the interstitial segment is duplicated and the flanking markers are in the complementary coupling phase.) See the Appendix for further details on T ( Z R + I R ) T64M94.

THE CYTOGENETICS OF

Neurospora

203

c. Research Applications of Insertional Rearrangements i. Production of duplications. Insertional translocations have been an important source of nontandem duplications for experimental use (Section V, E). ii. Simulation of paracentric inversions. In the absence of any identified paracentric in Neurospora, inverted insertions provide an alternative opportunity for obtaining acentric fragments and dicentric bridges, and for reversing polarity. We are attempting to find out what portion of the genome is responsible for the difference between acentric fragments that survive and replicate, and those that cannot, and to determine whether viable acentric fragments can be transmitted through the cytoplasm. Inverted insertionals might be used to see whether a breakage-fusionbridge cycle (McClintock, 1938, 1939) can be initiated in Neurospora following anaphase rupture of a dicentric chromosome, and if so, whether broken ends are capable of healing. Inverted insertionals might also serve as a source of tandem reverse-repeats (Wallace and Kass, 1974) and possibly of controlling elements (McClintock, 1950, 1951). iii. Polarity in recombination. An inverted insertion, 81326,was used by Murray (1968a) to demonstrate that polarity in intragenic recombination is an intrinsic property of the marked chromosome segment and does not depend on orientation of the segment with respect to the centromere. iv. Chromosome twisting and deferred bridging. The relative frequencies with which dicentric bridges occur in successive divisions in the ascus depend on the amount of chromosome twisting in the segment between the two nonhomologous centromeres. Differences in observed bridge frequencies might provide a sensitive measure of rigidity, twisting or coiling-properties of the chromosomes likely to be under genetic control and to be significant for such phenomena as chiasma interference. v. Uses of insertional rearrangements in other organisms and reasons f o r their neglect. Few or no insertionals have been identified in other organisms, with the striking exception of Drosophila, where identification of insertions has been facilitated by the salivary chromosomes. The first translocation ever found was in fact an insertional-Pale in Drosophila (discovered by C. B. Bridges in 1923, but shown to be an insertion only 12 years later). Over 70 Drosophila insertionals are now listed by Lindsley and Grell (19681, and they have been put to many uses, such as to study position effect, dosage compensation, synapsis, recombination, chromosome replication, genetic fine structure, effects of hyper- and hypoploidy, and the functioning of sperm. I n contrast to DrosophiEa, only one insertional translocation has been reported in maize (used to show the relation of crossing over to homologous pairing-Rhoades, 1968), and one in the mouse (used to study controlling elements and chromosome inactivation-review by Cattanach, 1975). For references on h e r tionals and their applications, see Muller (1967) and Perkins (1972a). In medical genetics, balanced insertional translocations have been implicated several times in the origin of birth defects associated with partial trisomy (see, for example, Rethore et al., 1972; Therkelsen et al., 1973; Grace et al., 1973). It now appears from Neurospora that insertional translocations are common in their occurrence, though somewhat less frequent than reciprocals. Why then have these important and useful rearrangements not been recognized more readily in other organisms, and why are they not even mentioned in most textbooks? The neglect is probably because both genetic and cytological detection are more difficult than for reciprocal interchanges, the consequences of segregation and recombination are more complex, and stocks may fail to be established or may be lost because aneu-

204

DAVID D. PERKINS AND EDWARD G. BARRY

ploid rather than euploid progeny are inadvertently retained. Further studies of insertionals should be rewarding, especially in haploid organisms, where they are relatively easy to recognize, manipulate, and preserve.

2. Quasiterminal Rearrangements

a. Translocations. At least 12 rearrangements have been identified where the breakpoints are a t the tip of one chromosome and in a nonterminal region of a second chromosome (Appendix Table 2 ) . Frequencies of the ascus types characteristic of a quasiterminal translocation are shown in Fig. 15A. (Additional examples can be found in Fig. 7 of Perkins, 1974.) The ascus frequencies for quasiterminal translocations resemble those for insertionals, and they result from similar events in meiosis, as diagrammed in Fig. 16. When there is no crossing over, normal centromere disjunction results in 8:O and 4 : 4 asci with equal probability (two top diagrams). Asci of the 6:2 type are produced by interstitial crossing over, and their frequency reflects the combined lengths of the two interstitial intervals. Since one of the breaks is necessarily a t a chromosome tip, and since even the shortest chromosome arms are known to be of substantial length, 6:2 frequencies should never be very low with this type of rearrangement.

7r

It is assumed without direct evidence that the tip breakpoint is subterminal rather than strictly terminal, and that the tip is translocated. Our diagrams and terminology TIIR-VL)ARlSO X Normal (N-2111

zil

50

-

>

@

6:O

AEU. clam (6bck:Whitel

8:2

AEus clsn I6lack:White)

FIQ.15. Frequencies of unordered ascus types in crosses of (A) Quasiterminal Translocation x Normal (left), and (B) Quasiterminal Pericentric Inversion x Normal (right). The translocation has one breakpoint at centromere, the other at the tip of VL, which is a moderately short arm; this is reflected in an intermediate frequency of 6B:2W asci. The inversion produces 6B:2W's from single exchanges and 4B:4W's from 4-strand doubles; symmetry is not expected. The inversion data are from Turner et al. (1969). Reproduced from Perkins (1974).

THE CYTOGENETICS OF PROPHASE I ORIENTATION AND CROSSING OVER

ALTERNATE CENTROMERES TOSAMEPOLE

ADJACENT CENTROMERES TOSAMEPOLE

t ((g .““4 I ,

ii .. .. ... ....

+ ~:

1

......... ‘-...w:...... :::

\. ‘””’#:.. 1

‘4

c

AL TERNA TE TO SAME POLE

Ascus

CONSTITUTION

VIABLE: DEFICIENT ASCOSPORES

8:0

f

....... ~.::::::a::: I

;. .; .; .i .. .. .. .. .. .. .. ..

205

Neurospora

4:4

6:2

.... .... ....: .:...

..

ADJACENT CENTROMERES TO SAME POLE

t ‘..‘:::::a::: t fl d ... ... ... ...

FIG.16. The origin and constitution of asci containing various numbers of inviable spores, from crosses between Normal ,and a Reciprocal Translocation in which one breakpoint is effectively terminal. Because one of the duplication-deficiency classes is viable, the asci resemble those from an insertional translocation (Fig. 7C,D; Fig. 12) rather than from an ordinary reciprocal translocation (Fig. 7A,B; Fig. 8). The order of black and white spore-pairs is not necessarily as shown in the diagram. Reproduced from Perkins (1974). conform with the impressive evidence in Drosophila, summarized by Muller and Herskowitz (1954), that breaks do not heal to form new stable ends, but must be capped by a preexisting telomere. LSee also Cavalier-Smith (1974) on the origin and molecular structure of telomeres, and Roberts (1974, 1975) for recent observations in DrosophiZa.1 Other models are not excluded, however. Healing to form new, stable chromosome ends has been reported by McClintock (1939, 1941) following a breakagefusion-bridge cycle in maize, and by T. C. Hsu (1963) following breakage by radiation or chemicals in the hamster.

206

DAVID D. PERKINS AND EDWARD G. BARRY

Some apparently terminal rearrangements could instead be noninverted insertions beyond the farthest known gene marker but short of the tip, so that the distal segment included essential loci. When there are no distal markers in either the donor or the recipient chromosome arm, the distinction between interstitial and terminal becomes difficult. Even then the two alternatives might be distinguished if there were appropriate markers within the transposed segment, because single crossovers there should’ be fully viable for a quasiterminal interchange, but one or both single crossover products might be inviable for an interstitial insertion. Where the two alternatives cannot be distinguished, it is simplest to assume that duplication-generating rearrangements are quasiterminal (two breaks required for origin) rather than insertional (three breaks). Especially clear cytological evidence indicates that T(ZR + VL)ARI9O is effectively terminal. The longest arm in the complement, IR, is translocated in its entirety (except for one known proximal gene) to the left tip of chromosome 2, distal to the nucleolus organizer (Barry and Perkins, 1969). Only a minute terminal satellite occupies this position in the normal sequence, and some fully viable strains even lack the satellite. No cytologically detectable nucleolus organizer activity has been removed to the IR break point in T(ZR + VL)ARIgO.

b. Quasiterminal Pericentric Inversions. The three known inversions are of this type, and they all involve linkage group I, where a substantial segment of the left arm has been transferred to the right tip. Frequencies of ascus types are shown in Fig. 15B. The consequences of meiotic crossing over are diagrammed in Fig. 17. All ascus types other than 8:O’s originate from crossing over within the long inverted segment. 6:2 asci come mainly from single crossovers and 3-strand doubles, 4: 4’s from 4-strand doubles, and 8 :0’s from noncrossovers and 2-strand doubles. Thus the frequencies of 8:0, 6:2 and 4:4 asci are not expected to be symmetrical around the 6 : 2 class. For data and a fuller discussion of inversions of this type, see Newmeyer and Taylor (1967) and Turner et al. (1969). Cytology of pericentrics. Only by making a loop can an inverted chromosome pair in complete register with its Normal homolog. We have observed such loops a t pachynema with two Neurospora inversions, In(1L + I R )H4260 and I n ( I L + I R ) NM176. Workable figures of this type are infrequent, however, because the pachytene nuclei are not flattened. The chromosomes tend to be oriented a t approximate right angles in three dimensions a t the breakpoint, making the inversion switches very difficult to trace, just as is true for paired insertions [Section V, D, 1, a, ii (b)]. The most convincing observations have been of nuclei where pairing was incomplete, with one or the other uninverted segment unpaired, as in Fig. 12 of Barry (1967) and the bottom left diagram in Fig. 10. These inversions do not form dicentric bridges or fragments. It should be possible to see altered arm ratios in chromosome 1 a t metaphase of the third and fourth divisions in the ascus, but this has not been examined.

THE CYTOGENETICS OF PROPHASE I ORIENTATION AND CROSSING OVER

. - ,

........ . 5 .. 6 *

f

I

ASCUS CONSTITUTION

‘ 4 ‘ 4

M 3

N N

H I I

‘43

In In

‘4

%.

............ ..5 . . .3

..:--1:::fi::::: *6 3 4

5

207

Neurospora VIABLE: DEFICIENT ASCOSPORES

8:0

f

*$ I

f ‘6

\

FIG.17. The origin and constitution of asci from crosses of Normal (white centromeres) x Pem’centric rnversion (black centromeres) in which one break point is effectively terminal. For clarity, terminal segments are shown unpaired rather than synapsed to form a typical inversion loop. Segments originally in the left arm are shown as solid lines, those in the right arm as dotted lines. Duplications and deficiencies occur only when crossing over occurs within the paired, inverted segment. Single exchanges and %strand doubles result in 6:2 asci, while 4-strand doubles result in 4:4 asci. The order of black and white spore-pairs is not necessarily as shown. Reproduced from Perkins (1974). c. Research Applications of Quasiterminnl Rearrangements i. Production of duplications. (Section V, E describes the characteristics and uses of duplications.) ii. Duplication instability. Many duplications in Neurospora are unstable to varying degrees. The mechanism of breakdown and factors affecting i t are most easily studied by means of the terminal duplications obtained as segregants from crosses of Normal with various quasiterminal rearrangements. Studies of this kind are described in Section V, E, 4. iii. Mapping chromosome ends. Appropriate quasiterminal translocations can be used as testers in a search for genes near unmarked chromosome tips. Distal interchange points of the known quasiterminal rearrangements are shown in Fig. 5. They serve to mark five of the 14 chromosome ends. The most distal known gene markers are not necessarily located terminally in the physical chromosomes. Recombination between such a marker and the interchange

208

DAVID D. PERKINS AND EDWARD G. BARRY

point of a quasiterminal rearrangement may indicate that a long distal region remains unmarked. For example, uvsd shows 10% recombination with interchanges involving the IVR tip. It can thus be anticipated that gene loci remain to be discovered in the IVR segment distal to uvs-2. T ( I R + VL)ARfSO carrying nic-8 has been used (so far unsuccessfully) in searching for genes near the nucleolus organizer a t the left end of V. iv. Chromosomes with altered morphology. Some rearrangement strains contain altered chromosomes that are very short or very long, chromosomes that are acrocentrics, and a t least one where the nucleolus organizer is no longer terminal. Base-tip translocations such as ALSi59, ARfSO, AR.809 alter chromosome size drastically. Further alterations can be produced by combining rearrangements. Altered chromosomes can be used to study pairing, recombination, or disjunction. d . Quasiterminal Rearrangements in Other Organisms. Translocations of this type were among the first to be described in maize (Brink and Cooper, 1932). Rearrangement breakpoints are commonly located near chromosome tips in Drosophila (Muller and Herskowitz, 1954), and this may also be true in man (Francke, 1972; Jacobs et al., 1974). Translocations that involve the terminal satellite are known in maize (Burnham, 1950) and barley (Hagberg and Hagberg, 1971) ; these resemble T(Z + V)ARISO in Neurospora. Application of quasiterminal translocations to linkage detection has been illustrated in maize (Phillips et al., 1971). For further examples of quasiterminal rearrangements in various organisms, see Muller and Herskowitz (19541, White (l973), Burnham (1902), and Clutterbuck (1970). For representative recent reports of quasiterminal translocations and inversions in man, and their role in the origin of partial trisomy, see Francke (1972), Dutrillaux et al. (1973), de la Chapelle and Schroeder (19731, Dallapiccola et al. (19741,and Rethore et al. (1974).

3. Partially Overlapping Rearrangements

a. Reciprocal Translocations. Viable nontandem duplications can also be obtained from crosses between two reciprocal translocations whose breakpoints involve the same two chromosome arms, as first proposed by Muller (1930). A condition for the production of viable duplications is that the translocations must overlap so that each has one breakpoint distal and one proximal, relative to the other, or they may have one breakpoint in common and their other breakpoints in a shared chromosome arm. If either of these conditions obtains, synapsis in the intercross between the two translocations resembles that of Insertional Translocation X Normal, as was seen in Fig. 11. One-third of the viable progeny from such a cross are duplicated for the segments between breakpoints. As with insertional translocations, the duplication progeny from partially overlapping reciprocals contain no deficiencies, and ascospores containing such duplications are viable and black (Perkins, 1971a, 1974). Frequencies of ascus types are shown in Fig. 18C for a cross between two partially overlapping reciprocal translocations. (Two other examples are given in Fig. 11 of Perkins, 1974.) The distribution resembles that of an insertional translocation x Normal.

THE CYTOGENETICS OF

Neurospora -

70-

IAl

T(IR; llRl4537 X

-

Normal IN 2371

I50I

-

I

i -

t

2

25-

-

209

TIIR; IIRISTL 76 X Normal (N = 1391

ICI

32%

I

; 0-

8:0

6:2

AEUS class

2:6

: Whitel

08

6:2 4:4 2:6 08 Ascur class (Black: Whitel

Fro. 18. Frequencies of unordered ascus types in a cros9 between reciprocal translocations that have overlapping breakpoints in the same two chromosome arms, as in Fig. 11. When each parent translocation is crossed to the normal-sequence tester, it produces unordered asci that are typical of a simple reciprocal translocation (diagrams A and B). Intercrosses between the two translocations result in the frequency distributions shown in C , which are typical of duplication-producing combinations.

Many reciprocal translocations in Neurospora have been intercrossed with other reciprocals whose breakpoints are known to fall in the same two chromosome arms or might do so, and over 30 different pairs of reciprocals have been shown to produce viable duplication progeny, indicating that they are partially overlapping. Evidence for this conclusion is of two types, consisting for each intercross of (1) frequencies of ascus types similar to Fig. 18C, and (2) identification of f, progeny as duplications (see Section, V, El 3 and 4 for characteristics of duplications). Pairs of translocations whose breakpoints do not overlap properly give negative tests (examples in Table 1 of Perkins, 1974). Intercrosses can be used in this way to determine the relative positions of breakpoints of pairs of reciprocal translocations, and if a particular pair overlaps so as to produce duplications, the breakpoints can be mapped precisely provided that suitable markers can be introduced so as to test for coverage. b. Partially Overlapping Inversions. Similar considerations apply to intercrosses between overlapping inversions. Crossing over produces viable progeny that are duplicated for the two segments between the left breakpoints and between the right breakpoints. This has been tested in h'eurospora by intercrossing the IL + IR inversions H4250 and NM176, which share a common breakpoint a t the IR tip. Ascus frequencies were 18% 8:0,64% 6:2, 16% 4:4, 1% 2:6, 0% 0:8 ( N = 2073, and viable duplications were recovered, as expected. c. Successive Rearrangements. If two successive rearrangements

210

DAVID D. PERKINS AND EDWARD G. BARRY

occurred a t different times in the same cell lineage, resulting in an overlapping double rearrangement, and this was crossed to the normal sequence, pairing and segregation would resemble that of an intercross between the two corresponding single rearrangements. d. Applications in Other Organisms. Overlapping rearrangements have been widely used, and for many purposes. They were employed in Drosophila by Muller and Prokofyeva (1935), Sturtevant and Beadle (1936), Kelstein (1938), Patterson et al. (19401, Lindsley et al. (19721, Rawls and Lucchesi (1974), and Steward and Merriam (1974); in Datum by Blakeslee et at. (1936); in maize by Gopinath and Burnham (1956) and Phillips and Springer (1972); and in barley by Hagberg (1962). For further examples, see Burnham (1962). Translocations that involve the Drosophila Y chromosome offer special advantages for the production of duplication gametes (Lindsley et al., 1972). Because the Y is largely inert, most breakpoints in it behave as though they were identical, and essentially all translocations between the Y and a given autosome arm produce viable duplications when they are intercrossed. A series of such intercrosses can produce a precisely contiguous set of nonoverlapping duplications. Translocations between an inert B chromosome and a chromosome of the normal complement might be used similarly in other organisms. [For B-chromosome translocations in maize, see Roman (1947) and Beckett (1972).1 Kafer (197513, 1977) has obtained interstitial duplications in Aspergillus by a more complicated method which involves intercrossing translocations that have only one chromosome arm in common.

E. NONTANDEM DUPLICATIONS AND THEIRCHARACTERISTICS Neurospora has proved to be nearly ideal material for identifying and investigating the rearrangements that recurrently generate nontandem duplications. Such aberrations are recognized visually by the production of asci with unique patterns of black and white spores, as described in Section V, A, 2. The methods of detecting duplication-generating rearrangements are just as effective for short duplications as for long. Theoretically, i t should be possible to recognize a rearrangement where only a single essential locus was translocated or inserted. At the other extreme, long duplications are quite viable in Neurospora. 1 . Mapped Duplications and Their Extent

Duplications are produced by each of the 41 extant rearrangements listed in Appendix Table 2. Information on each duplication will be found in the Appendix following the description of the rearrangement that produces it. The segments covered by these duplications collectively cover a t least half the genetic map (Fig. 19). Duplication strains that cover any of the desired intervals shown in Fig. 19 can be obtained from a cross with the appropriate rearrangement, usually as one-third of the progeny. Since markers can be introduced from the normal-sequence parent, it is possible to obtain duplications that are

Ill 1

/

-

&'

1

, d "

/

a,

8

' ' 4

/

. . . . . . . . . . . . . . . . . .D306 (. ..). I

,

------ AR17

$3 /

VI

/

............. /

1

--------------------V 1 6 m T39Mn7 .#*

VII I

d

&? I

1

/

1

a" i: ............................... ARZOB

- / (

@3

-

-_____________________T W M W --ALS179

'0

d

y

........ I

8

&-

¶$*

I

I

I

(

Isg=

FIG.19. Intervals covered by duplications from various duplication-generating aberrations. A broken line under the donor linkage group indicates the segment that is involved in the duplications produced by crossing each rearrangement by normal sequence. For clarity, short intervals have been greatly expanded. Interval lengths are thus grossly distorted. Dashed lines, - - - - _, indicate the extent of duplications as determined directly by marker coverage. Dotted lines, . . . . . , indicate segments not tested for coverage, but where duplication is inferred from other evidence. Dotted lines within parentheses, (. . . . .), indicate segments not tested for coverage. Rearrangements are designated by isolation numbers a t the right end of each duplicated segment. For the new location of each segment in rearranged sequence, and for other details, see Appendix. Duplications from overlapping rearrangements are not included in the figure. Since this paper was completed, new data show that ALS182 duplications include m e t d , ALSl76 duplications include un-16, Y16329 duplications include un-4, and AR33 duplications involve a terminal segment of VL that includes the nucleolus organizer. 0306 duplications do not include ro-2, and NM16d duplications do not include arg-2.

212

DAVID D. PERKINS AND EDWARD G . BARRY

heteroeygous for known genes, to test for coverage of markers located in the donor chromosome, or to vary the genetic background as desired, all while working with duplications of exactly the same segment.

6.Source and Mode of Origin All the identified Neurospora duplications are nontandem, and they were obtained exclusively as segregant progeny from crosses heteroeygous for duplication-producing rearrangements. Probably new duplications are also being produced directly as variants of the normal chromosome sequence, and are present among the survivors of mutant hunts when we examine them for new rearrangements. A stable new duplication would not be expected to produce the white, deficiency ascospores on which our recognition method depends for other rearrangement types. However, it would usually be expected to produce barren perithecia in a test cross. It is not clear how such an isolate could be tested routinely to prove that barrenness was due to a duplication rather than to a dominant gene mutation that affected fertility. New variant strains that make barren perithecia have indeed been found with a low frequency in some of our mutant hunts. Mutants of this type were obtained by Schroeder (1970) as spontaneous somatic variants in the ultraviolet-sensitive strains UVS-3.Because their barrenness resembled that of known duplications under similar conditions, she suggested that they might be new duplications. Similar strains, barren both as male and female, were found among sterility mutants obtained by Weijer and Vigfusson (1972). These were attributed to gene mutation.

With duplications of recombinational origin there is usually no problem of recognition, because an entire new class of Duplication progeny is present rather than a single unique isolate. I n the case of duplicationgenerating translocations, the new class makes up one third of the viable progeny (Section V, D ) . The Duplication class is typically barren in test crosses. Appropriate genes can be used to confer a distinctive marker genotype on the Duplications. Morphology of the Duplications may be grossly abnormal if vegetative incompatibility genes are heterozygous. Occasionally the Duplication class is intrinsically distinct in its vegetative morphology. Do nontandem duplications generally originate de novo, or b y recombination? Most textbooks assume that duplications usually arise directly from the normal chromosome complement, much as do other aberrations such as translocations. The probability of their arising indirectly by recombination is usually neglected. Yet simple considerations show that a recombinational origin is probably the more likely, because a new balanced duplication-generating rearrangement is at least as likely to occur originally as is the corresponding duplication, and once such a rearrangement is present, it is capable of producing a large number of identical duplications by meiotic recombination.

THE CYTOGENETICS OF

Neurospora

213

This is best seen when an aberration occurs in the somatic nucleus of a haploid organism. Figure 20 shows the origin of an insertional duplication and of the corresponding balanced insertional translocation. Similar considerations would apply to terminal duplications and translocations. The rearrangement is shown to occur between chromatids in the Gr period, following chromosome replication. The haploid nucleus is temporarily heterozygous for an insertional translocation, and this is resolved a t the following mitosis in either of two equally likely ways, depending on centromere orientation. One outcome yields a duplication, the other a balanced insertional translocation. If a similar aberration occurs in the GI period, before replication, only one outcome is possible-an insertional translocation. No duplication can arise at that stage in a haploid nucleus. Thus the balanced translocation is a t least as likely to occur as the duplication, and probably more likely, depending on what proportion of aberrations originate in GI. We suggest that this result is not trivial. A single newly arisen duplication probably has little expectation of survival in nature, whereas the chances are considerable that a balanced translocation will survive and be transmitted for a t least a few generations, because fitness of the

ORIENTATION I:

-e Xz---ORIENTATION II:

-b XI:----

=4

+

-&-----

DUPLICATION t

DEFICIENCY

-

--* ----* INSERTIONAL TRANSLOCATION

c

t

-*-----

NORMAL SEQUENCE

FIG.20. Suggested origin of ( I ) a nontandem interstitial duplication and (2) the corresponding insertional translocation, in Neurospora. It is seen that the outcome depends solely on orientation of centromeres at the division following occurrence of the rearrangcment, and lhat cach outcomc is equally probable when the rearrangement occurs between chromatids at a stage following chromosome replication. Before replication in a haploid nucleus, an insertional translocation could be produced, but not a duplication.

214

DAVID D. PERKINS AND EDWARD

G. BARRY

balanced individuals is independent of the fitness of the Duplications that will constitute one-third of their viable progeny. For these reasons, balanced duplication-producing rearrangements appear to deserve fully as much attention as do the duplications themselves whenever the origin of nontandem duplications is considered, either in experimental situations or in nature. 3. Vegetative Phenotype of Duplications; Fertility In general, Neurospora duplications deviate remarkably little from wild-type morphology and growth rate. However, the duplications from a few rearrangements are recognizably abnormal, and that abnormality cannot be attributed to heterozygosity for genes in the duplicated segment. (This is true, for example, of D p ( I I + I I I ) A R 1 8 . ) No general trend is evident relating normal or abnormal phenotypes to length of the duplication or to specific regions of the complement. The phenotype of any duplication culture can, however, be grossly abnormal if it is heterozygous for alleles of a vegetative (heterokaryon) incompatibility gene (Newmeyer and Taylor, 1967; Perkins, 1975; Mylyk, 1975). This is a result of the genic content rather than an expression of the duplicated condition; when het genes are homozygous in the same duplication, the phenotype is typically normal or nearly so. Fertility. When a duplication strain is crossed with a nonduplication strain of opposite mating type, perithecia are formed that are barren and produce few or no asci or ascospores [first observed by St. Lawrence (1953, and unpublished) with R55 and 45401. Typically the perithecial necks are rudimentary or short. This is true of nearly all the duplications known in Neurospora and does not depend upon whether the duplication strain is used as protoperithecial or as fertilizing parent. There is no obvious relationship between the degree of infertility and the length of the duplication or the specific segment involved. Crosses of Duplication X Duplication are also barren. We do not understand why Neurospora duplications result in barrenness. Duplications in Aspergillus are apparently fully fertile. Duplications in many plants are transmitted through the female but not the male because of pollen-tube competition. But there is no evidence of competition in laboratory fertilizations of Neurospora. Initiation of perithecial development is not delayed after fertilization with a Duplication, in crosses destined to be barren, and both reciprocal crosses are equally barren when one of the parents is a Duplication. The critical stage appears to be just when the conjugate nuclei fuse in the crozier, or are about to do so. In crosses with a series of duplications, N. B. Raju (unpublished) finds that karyogamy may or may not occur,

THE CYTOGENETICS OF

Neurospora

215

depending on the duplication. With some duplications, meiosis may be initiated but not completed. Rarely an ascus may be produced, with ripe ascospores. A few duplications are sufficiently stable and fertile so that both segments of the duplication are retained through meiosis and delivered together intact in the f, ascospores. This occurs regularly with interstitial duplication D p (IV -+ VII)S1229 (Barry, 1960a) and occasionally with terminal duplication D p ( I L + I R ) N M I 7 6 (Turner et al., 1969; D. D. Perkins, unpublished). The crosses in which this occurs are nevertheless highly unproductive, and are clearly classified as barren. In crosses with some duplications, no ascospores whatever are produced. With other duplications a small number of spores are regularly produced-perhaps ten to a few hundred in each small cross tube, where many thousands would be present in fertile euploid crosses. These crosses can readily be distinguished as nonfertile if scoring is properly timed. They are classified as barren. Usually when ascospores appear in substdntial numbers in the test cross of a duplication, this is because the duplication is unstable and has broken down, and it does not mean that the unchanged duplication is fertile.

4. Instability of Duplications The previous section concerns strains that are still intact duplications and have not undergone changes in constitution. Most duplications are not completely stable and may change spontaneously either somatically or in the perithecium. The degree and kind of instability depend both on the specific duplication and on the genetic background (Section d, below). Instability has been examined most carefully with terminal duplications, where a single event could result in euploidy or homozygosis. These seem to be less stable than insertional duplications, where interstitial deletion or double mitotic crossing over would be required. a. Somatic Instability. A few duplications are highly unstable even under nonselective conditions. Duplications from T ( I R + V I R )NMlOS (Turner, 1975, 1976) and T ( I R + VL)AR19O (D. D. Perkins, unpublished) are the best-studied examples. Vegetative cultures accumulate nuclei that have completely lost one of the duplicate segments, becoming euploid by all available criteria. When appropriate markers are present, instability may result in visible recessive patches in vegetative cultures, but heterokaryosis may prevent the results of instability from becoming visible. The euploid nuclei can best be detected by the appearance of recessive phenotypes among the isolates from conidial plating. Crosses of such conidial isolates are fully fertile, and segregation is as expected of a euploid cross.

216

DAVID D. PERKINS AND EDWARD G . BARRY

Most duplications are sufficiently stable that little variation is observed vegetatively under nonselective conditions. Somatic variants can be obtained, however, if the original duplication is placed a t a selective disadvantage. This happens automatically whenever a duplication is heterozygous for alleles at a heterokaryon (vegetative) incompatibility locus (het locus). Such heterozygotes have very abnormal “inhibited” growth. Under these conditions fast-growing somatic variants are found that have lost one of the het alleles, either by deletion or by recombination resulting in homozygosis. Most studies of somatic instability and factors affecting it have employed het genes (Newmeyer, 1965; Newmeyer and Taylor, 1967; Schroeder, 1970, 1975; Mylyk, 1972 ; Newmeyer and Galeazzi, 1974, 1976a,b). A similar selection can be achieved by using cycloheximide when duplications are heterozygous for a recessive gene that confers resistance (Turner, 1975, 1976). Some of the fast-growing somatic variants obtained by these selective systems are fertile in test crosses and therefore are apparently euploid, but most of them remain barren. The latter could be due to incomplete deletion of a duplicate segment or to a mitotic crossover that led to homozygosis for the selective marker. These alternatives are difficult to distinguish because the infertility interferes with progeny-testing. I n one system it has been shown that most of the barren variants express recessive markers throughout the duplicated region, indicating either mitotic crossing over or extremely long but incomplete deletion (Newmeyer and Galeazzi, 1976a). b. Instability after Fertilization. Perithecia from a Duplication parent are typically barren as a result of being aneuploid. When fertile perithecia appear in such crosses, they are found to contain nuclear types that have lost the duplicated segment. Segmental loss can occur either before or after fertilization. Distinction between the two is usually clear, and i t depends on the time and pattern of appearance of the fertile perithecia. Perithecia whose fertility is restored by postfertilization loss are recognized because they are initially barren and become fertile only after a significant lag. In contrast, perithecia from prefertilization loss are immediately fertile with no lag in the appearance of necks or ascospores. However, the distinction is difficult with very unstable duplications. The sexual phase automatically acts as a selective system enabling the results of postfertilization instability to be recognized by changes of individual perithecia from barren to fertile. Barrenness apparently results from the duplicated condition itself. Therefore, selective markers are irrelevant, and fertility cannot be restored by homozygosis, but only by loss of duplicated material. Chromosome losses that change the perithecium from barren to fertile

THE CYTOGENETICS OF

Neurospora

217

are usually such as to restore precisely the euploid condition, as though the parental chromosome tip has either been restored or is unnecessary. This is true within the limits of both genetic and cytological resolution for Dp ( I L + IR)H&60, where fertile derivatives have been examined genetically (Newmeyer and Galeazzi, 1976a,b), and in one instance cytologically. Pachytene pairing of a euploid derivative of D p ( I L + I R ) H4.250 that arose by mei-$-mediated breakdown so as to restore the inversion sequence is shown in Fig. 4. c. Nonrandomness of Spontaneous Breakdown. With Dp ( I +VI)NMIOS, the loss occurs with about equal probability either a t the interchange point in translocation sequence so that normal sequence remains, or a t the interchange point in normal sequence so that translocation sequence remains (Turner, 1975, 1976). Such an equally likely loss of either duplicated segment is not generally found, however, among the seven other duplications that have been tested adequately. For example, D p ( I + V)ARI90 reverts to euploid only by losing the duplicated segment from the translocated position, and it has never been observed to lose the segment from the normal position (Fig. 21). Several other duplications are known to resemble AR190 in this respect (for example, AR.209, ALSI76). Other duplications usually resemble AR190, but not always; these occasionally break down by losing the other segment from the normal position (H&60 and NMI76 are examples). No duplication is known where all or most of the losses are such as to restore the rearrangement sequence. IL

......................... nic-2

lys-1

.

+ al

+

I)

IR

a

TRANSLOCATION (NOT FOUND)

NORMAL (RECOVERED)

........o... lys-1

-.:

a

ar03

................ lyr-1 0

FIG.21. Alternate modes by which Duplication D p ( I R + VL)ARISO could lose a duplicated segment so as to restore the euploid condition.

218

DAVID D. PERKINS AND EDWARD G . BARRY

The foregoing examples are all quasiterminal duplications. Breakdown products from insertional duplication D p ( I I R + I L )NM177 have been examined by Metzenberg et al. (1974). When somatic variants occur, material is apparently lost from the inserted location. The loss is not complete, because the somatic variants are still barren, resembling in this respect many of the variants that originate vegetatively by spontaneous escape from het-incompatibility with the terminal duplications H4250 (Newmeyer and Galeazzi, 1976a) and NM149, and the interstitial duplication 39311 (D. D. Perkins, unpublished). Duplications from overlapping reciprocal translocations present a special problem which is still unsolved. When the perithecia change from barren to fertile, a simple deletion would be expected to restore the sequence of one or the other parental translocation. Unexpectedly, fertile perithecia from this source often produce 95% black ascospores, as though the normal sequence had been restored (D. D. Perkins, unpublished). d. Genes Affecting the Stability of Duplications. Five genes affecting radiation sensitivity have been tested with D p ( I L + I R )H.4250 or Dp ( I L + I I R )3931 1 . Escape from inhibition due to A / a heterozygosity was timed. uvs-2, -4 and -5 were without effect, but escape was speeded by uvs-3 and uvs-6 (Schroeder, 1970, 1974, 1975), indicating that they increase either mitotic crossing over or deletion. For a summary of the properties of these genes, see Schroeder (1975). A gene linked to Dp(II + V ) N M l @ affects the speed of escape from inhibition due to het-C/het-c heterozygosity (Mylyk, 1972). mei-3, a gene in IL which somewhat resembles uvs-3 and -6 (Section 111, C, l ) , markedly speeded the restoration of fertility in barren duplications from a t least four different rearrangements, and it also speeded vegetative escape from inhibition due t o het incompatibility (Newmeyer and Galeazzi, 1974, 1976b). Other factors, not mapped, each affect vegetative or sexual-phase instability in characteristic ways (Newmeyer and Galeazzi, 1976a). 6. Nontandem Duplications in Aspergillus and Higher Organisms

J. A. Roper and his colleagues (B. W. Bainbridge, B. H. Nga, and J. L. Azevedo) have studied the vegetative instability of duplications in A . nidulans, using one duplication that originated as a unique spontaneous variant, and others obtained as progeny of duplication-producing rearrangements. The results have been reviewed by Roper (1973a,b). Several resemblances to Neurospora duplications are apparent : All duplications are essentially unstable. Duplicated material is lost by one or more steps to give effectively haploid derivatives. Loss can be from either position, but with at least one duplication it is predominantly from the translocated position. Instability is increased by caffeine, trypan blue, and by a uvs mutant. A recessive factor reduces instability (Azevedo, 1975).

THE CYTOGENETICS OF

Neurospora

219

Some differences exist between Aspergillus and Neurospora. The Aspergillus duplications apparently do not affect fertility, and are readily transmitted through the sexual cycle. They are distinct from the wild type in morphology and growth rate. Because of its growth habit, uninucleate conidia, autonomously expressed color markers, and stability of vegetative diploids, Aspergillus is very favorable material for studying somatic instability, and the observations of Roper and his‘ associates on vegetative changes go beyond those in Neurospora in several respects. It has been shown, for instance, that the duplication itself provokes instability, limited largely to the duplicated segment. Not only losses but also gains of material are found, in “deteriorated” sectors. On the other hand, meiotic mapping and analysis are accomplished more readily with Neurospora, and a larger number of genetically characterized duplication-producing rearrangements are available for comparison. Also, studies of duplication instability are not limited to somatic stages, but can readily be extended to the sexual phase. In many higher plants, duplications are readily transmitted through the female but not the male, because pollen tubes bearing a duplication are unable to compete in growth down the style (see Burnham, 1962). In Drosophila, extensive hyperploidy usually results in decreased survival and abnormal morphology (Lindsley et al., 1972). Duplication in man (partial trisomy-aneusomie de recombinaison) characteristically results in developmental and neurological defects (see references cited following the sections on insertional and quasiterminal rearrangements). Segmental loss of duplicated chromosome material has been reported in barley, where deletion from a balanced tertiary trisomic reduces the extra chromosome to about one-third size. This has been called the “chopper” effect (Wiebe et al., 1974). The reduction increases plant vigor and increases transmittal of the chromosome through the pollen, compared tb the original trisomic. Segmental loss is one manifestation of the Ds-Ac controllingelement system in maize (McClintock, 1950). Conceivably a similar mechanism might be responsible for deleting duplicated segments in Neurospora. 6. Other Research Applications of Duplications

a. Mapping b y Duplication Coverage. Nontandem duplications produced by any of the methods described above provide a quick and easy way of mapping closely linked genes by a right-left test (comparable to deletion mapping in bacteriophages) (Section IV, B, 4, b ; Perkins et al., 1969). The same technique can be used for centromere mapping. b . Vegetative Incompatibility. Genes affecting the ability of somatic hyphae to form stable heterokaryons (het-genes) are difficult to study genetically if scoring depends on heterokaryon tests, because a difference a t any one of many het loci results in a similar incompatibility phenotype, and commonly used marker stocks differ a t several het loci. This difficulty is avoided by using for the analysis duplications that are short enough to include only one het locus. Duplications heterozygous for het alleles have characteristic abnormal phenotypes (Newmeyer and Taylor, 1967; Perkins, 1975; Mylyk, 1975). A series of duplications have been used i n this way to identify previously undetected het loci (Mylyk, 1975), and to show that natural populations of N. crassa are highly polymorphic for het genes (Mylyk, 1976). c. Regulation. The ability to produce duplications of desired allele content allows the same gene combinations to be compared within the same nucleus in a heterozygous duplication, and in separate nuclei in a heterokaryon. This type of comparison has been made by Metzenberg et al. (1974) in studies of the regulation of alkaline phosphatase synthesis, using duplications from T ( I I + I ) NMlYY.

220

DAVID D. PERKINS AND EDWARD G . BARRY

d . Mitotic Recombination. The nontandem duplications now available are far more stable than the diploids or disomics described in Section 11, and it should be possible to use duplications to study intra- and intergenic mitotic recombination in Neurospora, much as diploids are used in Aspergillus, yeast, or Ustilago. Such a system is being developed. A test system where only a small part of the genome is diploid should be of special value in searching for and scoring recessive genes that affect recombination. Heterorygous duplications have been used for this purpose in Aspergillus (Parag and Parag, 1975). Disomics provide similar test systems in yeast (Roth and Fogel, 1971; Rodarte-Ram6n and Mortimer, 1972). e. Tests of Dominance. Duplications provide a more reliable test of dominance than do heterokaryons, because a 1:1 allele ratio is assured. (Very often duplications are also more convenient because heterokaryon incompatibility genes can be ignored in most of the genome.) An example is provided by cyh-I, where resistance is clearly recessive in duplications from T ( I + VI)NMlOS (Turner, 1976). The resistant cyh-I allele had originally been found dominant in heterokaryons, in carefully conducted tests with forcing markers (K. s. Hsu, 1963). Skewed nuclear ratios in the heterokaryons probably affected the tests, however. A similar error could result with a duplication if it were so unstable that the dominant sensitive allele was usually lost during testing, and the resistance then expressed was mistakenly attributed to dominance rather than loss. In practice, instability has not interfered with such tests, using either D p ( I + VI)NMlOS or other, moderately stable, duplications. However, suitable controls should be done to guard against this error before concluding that a resistance allele is dominant.

F. REARRANGEMENTS I N OTHEREUKARYOTIC MICROORGANISMS The pigment patterns in Neurospora asci provide a model of the inviability patterns to be expected in other eukaryotic microorganisms where the meiotic products are unpigmented and where chromosome aberrations may be revealed only by the inviability of deficiency spores. 1 . Recognized Rearrangements

In spite of detection difficulties, evidence for rearrangements has been obtained in nine or ten cryptogamic organisms other than Neurospora. Information on these is given in Table 6. The nature and conclusiveness of the evidence varies, and is often tenuous. I n four organisms it depends solely on the numbers of defective spores. Only in Aspergillus is the genetic evidence extensive. Only in Coprinus and Sorduria has there been cytological confirmation. Cochliobolus appears in Table 6 because of observations made during a study of mating behavior and fertility. Some progeny were initially dual mating type, inhibited, and barren, and this condition was unstable, suggesting in retrospect that they may have been heterozygous Duplications and that one of the parental strains may possibly have been a rearrangement resembling T ( I L + 11)39311 or In(IL + I R ) H.4250 in Neurospora. Sphaerocarpos appears in the table

THE CYTOGENETICS OF

Newospora

221

TABLE 6 Chromosome Rearrangements in Eukaryotic Lower Organisms Other Than Neurospora Organism

Type of rearrangement”

Evidence only from spore abortion: Chlamydomonas (Reciprocal transeugametos locations) Saccharpmyces (Reciprocal transcerevursaae location) (Interstitial deletion) Sordaria macro- (Reciprocal transspora locations) Sphaerocarpos (Unknown) donnellii Evidence other than spore abortion: Ascobolus Translocation ammersus Aspergillus Reciprocal transnidulans locations

Coprinus radiatus Sordaria brcvicollis

Sordaria Jimicola Cochliobolus heterostrophus

No. of rearrangements 4 1 1

2 Several

1

Type of evidence* Tetrad patterns (viability) Tetrad patterns (viability) Marker loss Tetrad patterns (viability) Spore lethality in heteroa ygous crosses

Reference

I 0

*

Meiotic linkage

Numerous ( > 12 Mitotic linkage; mapped) meiotic linkage; dispmjcs from meiosis Nonreciprocal Numerous ( > 6 Mitotic linkage; (unilateral) mapped) duplications traneloca tions from meiosis Reciprocal trans1 Tetrad patterns location (viability); pachytene pairing (1nversionl)e 1 Tetrad patterns (pigment): meiotic bridges Translocat ions 6 Tetrad patterns (pericentric (pigment); inversion?) 1 meiotic linkage; CYtOlogY Redpro cal trans1 Tetrad patterns location (pigment) ; cytology (DuplicationI Unstable barren generating rearbisexual rangement? See progeny text)

i

p

Parentheses indicate that evidence is only presumptive. See Table 6 in Perkins (1974) for compilation of tetrad-pattern data. c See Perkins (1974) for a critique of the evidence for inversion. A derivative of the same aberration strain was later diagnosed as a reciprocal translocation (Mu’Aau, 1973). McBride and Gowans (1969). c McKey (1967). f Hawthorne (1963). 0 Heslot (1958). * W. 0. Abel (personal communication). F.-X. Francou (personal communication). iSee Kiifer (1975a, 1976). Ma and Kgfer (1974). and Kiifer (19741, and . .. UDshall references therein. . See Roper (1973a,b), Clutterbuck (1970), Bainbridge (1970), Upshall and K lfe r (1974), Lee and Nga (1973). Brygoo (1972). Ahmad (1970), Admad et al. (1972), Mu’Aau (1973). Mu’Aau (1973). 0 Cox and Gill (1967). p Nelson (1959). f

222

DAVID D. PERKINS AND EDWARD G. BARRY

even though it is a bryophyte, because methods of culture and genetic tetrad analysis resemble those used with fungi. A report of cytologically observed translocation heterozygosity in Phytopthora was inadvertently omitted from Table 6 (Sansome and Brasier, 1973).

2. Errors from Failure to Detect Rearrangements

There have been no reports of chromosome rearrangements in the other eukaryotic microorganisms and cryptogamic plants most used by geneticists-Coprinus lagopus, Schizophyllum commune, Ustilago maydis, U . violacea, U . hordei, Venturia inaequalis, Glomerella cingulata, Ophiostoma multiannulatum, Podospora anserina, Schizosaccharomyces pornbe, or Chlamydomonas reinhardi. It is difficult to imagine that rearrangements do not occur frequently in these organisms, in view of the fact that detectable rearrangements in Neurospora and Aspergillus are extremely frequent (Section V, B, 2). Even in Neurospora, the presence of deficiencies may not always be detected if defective spores or asci disintegrate, or if deficiency spores become pigmented (as can happen with some rearrangements), or if microscope illumination is such that white ascospores cannot be seen. We know from experience in Neurospora that failure to recognize an aberration can lead to serious errors or misinterpretations. Some examples from Neurospora are given below. a. Errors with Random-Isolated Ascospores. Markers in different chromosomes linked to a translocation will, of course, show pseudolinkage. A mutant trait that is linked to an unsuspected aberration may thus be assigned to an incorrect linkage group. (This happened with T(IL;IVR) HK63 cut. See Appendix.) Variability in crossing over from cross to cross could result from unrecognized rearrangements, and this would depend on whether the strains used as parents were homozygous or heterozygous for the rearrangement. Undetected aberrations such as small inversions or transpositions might well account for some of the crossing-over variability in Neurospora; if so, their identification will be complicated by the presence of different rec genes. Progeny from a rearrangement that generates viable duplications may show distorted, apparently non-Mendelian, allele ratios for linked genes, depending upon the coupling phase and map location. Among viable progeny, allele ratios from M x m can range from 2M:Im to 1M:2m. If two closely linked recessive genes are present when a duplication-generating rearrangement is crossed by normal sequence, and only one of them is covered by the duplication. one-third of the surviving progeny (the duplications) will appear to have undergone recombination, while the complementary recombination class is absent. (For data examples, see Table 3 of Perkins, 1972a.) Unorthodox results of this type might be interpreted invalidly in terms of selection, synthetic lethal genes, preferential segregation, or directed recombination. b. Errors Due to Selective Tetrad Analysis. If tetrads containing fewer than four viable products fail to survive, or if they are excluded from analysis by the investi-

THE CYTOGENETICS OF

Neurospora

223

gator, the results for the remaining, complete tetrads may simulate chromatid interference for marked intervals linked to an undetected rearrangement. Specifically, an excess of 2-strand double exchanges relative to 3- or 4-strand might be expected. This is true of interstitial regions between centromeres and breakpoints when reciprocal translocations arc heterozygous, and of regions included in heterozygous inversions. Emerson (1963, Addendum 11) has considered the consequences in detail for translocations, with a probable example from yeast. Undetected rearrangements are undoubtedly responsible for some of the deviations attributed to chromatid interference in the literature. c. Errors jrom Unstable Duplicnlions. Because the Duplication progeny derived from duplication-generating aberrations by meiotic recombination are often unstable, heterozygous, cryptic markers from a recessive parent may appear as somatic sectors, or may reappear among the sexual progeny of an unrecognized duplication. Such behavior could be spuriously attributed to mutable genes or mutators, or interpreted as directed mutation. d. Errors Due to Compound Rearrangements. The risk of failing to recognize a new rearrangement is not limited to the naive beginner, but is a problem even for the investigator who is experienced with rearrangements, because new chromosome breaks and rearrangements seem to occur with an appreciably greater frequency in strains and crosses where a simple rearrangement is already present. Progeny containing a new and unsuspected rearrangement may be encountered, or a stock known originally to be simple may spontaneously produce a derivative that is compound or complex. In our experience a sizable portion of newly detected rearrangements are complex or compound, even with very mild mutagen treatment. Failure to recognize extra complexities could result in false diagnoses or spurious conclusions regarding the correspondence between cytological chromosomes and genetic linkage groups.

VI. Summary

Progress in Neurospora cytogenetics has been speeded by advances in the cytology and genetics of chromosome rearrangements. Although DNA content is only ten times as great as in E. coli, individual chromosomes can be identified and their behavior readily observed by light microscopy during meiosis and related divisions in the ascus. Synapsis follows karyogamy. The largest bivalent elongates to 19 pm by pachynema. Synaptonemal complexes are present. Postmeiotic mitoses in the ascus are conventional. The haploid number is 7 and meiosis is similar throughout thc genus, which includes hetero-, homo- and pseudohomothallic species. Only two mating-type alleles are known. Several known genes specifically affect meiosis. Three Spore-Killer genes are known which kill the meiotic products that do not receive them. I n N . crassa, over 400 genes and 167 rearrangements have been mapped to the seven linkage groups, which have been assigned cytologically to specific chromosomes. Functionally related genes are usually not closely linked, but several pairs or clusters are knovn, and several bifunctional

224

DAVID D. PERKINS AND EDWARD G. BARRY

genes. Crossing over is highly variable in different genetic backgrounds. rec genes regulate meiotic recombination in one or more specific unlinked regions. Ascospores containing deficiencies are inviable and white, while euploid ascospores and those containing duplications are viable and black. This greatly facilitates the detection and analysis of rearrangements. New aberrations can be diagnosed by determining the frequencies of asci that contain different numbers of white, deficiency spores. Typical reciprocal translocations are readily distinguished from insertional and quasiterminal translocations, because only the latter two produce viable duplications, and these result in distinctive frequencies of ascus types. Each known rearrangement is described. Nearly all are phenotypically normal and homozygous fertile. Reciprocal translocations are most common. There are two mutual insertions. The remainder produce viable nontandem duplications by meiotic recombination. These duplication generators are mostly insertional and quasiterminal translocations, plus three quasiterminal pericentric inversions and one transposition. Crosses between partially overlapping rearrangements also produce nontandem duplications. Some of the insertional translocations are inverted ; these produce bridges and fragments. No paracentric inversion has been found. Duplications obtainable from the duplication-generating rearrangements collectively cover more than half the genetic map. Most duplications are phenotypically wild but barren in crosses, and are unstable, reverting to euploidy by segmental loss. With most but not all of eight duplications tested, the segment in rearranged position is lost preferentially so as to precisely restore the normal sequence. Genetic variants affect the instability. When new aberrations arise directly from wild type, the probability for a new nontandem duplication may be less than for a new balanced rearrangement which can later produce many Duplication progeny by recombination. Except for Aspergillus, only a few rearrangements have been recognized in other eukaryotic microorganisms, where usually they are more difficult to detect than in Neurospora, with its pigmented spores. Failure to recognize an existing rearrangement can lead to spurious conclusions regarding linkage, recombination, interference, preferential segregation, or the presence of synthetic lethal genes. ACKNOWLEDGMENTS The importance of Barbara McClintock's pioneering contributions to Neurospora cytogenetics cannot be overstated. The direction of our work has been influenced especially by her ideas and research, and by those of J. R. Singleton, Patricia St. Lawrence, and Dorothy Newmeyer. Dorothy Newmeyer has critically reviewed the

THE CYTOGENETICS OF

Neurospora

225

entire text. Her suggestions have contributed in a major way to its content, accuracy, and clarity. Unpublished information has been generously provided by all these individuals and by Barbara C. Turner, Anna Kruszewska, N. B. Raju, B. C . Lu, 0. M. Mylyk, D. A. Smith, F. J. de Serres, A. Radford, R. L. Metzenberg, S. Brody, Carol J. Smarr, M. J. Moses, A. T. C. Carpenter, W. 0. Abel, and F.-X. Francou. In addition to the Fungal Genetics Stock Center, we are indebted to many individuals for providing strains: Patricia St. Lawrence, E. L. Tatum, Laura Garnjobst, Mary B. Mitchell, V. W. Woodward, S. R. Gross, Noreen E. Murray, Alice L. Schroeder, A. Radford, T. Ishikawa, R. W. Barratt, A. M. Srb, B.arbara Maling, D. G. Catcheside, Teresa M. Angel, F. J. de Serres, J. C. Murray, D. P. Mahoney, J. F. Wilson, and 0. M. Mylyk. This review was completed while the first author was a guest in the Department of Genetics, University of Washington, Seattle. The hospitality of David R. Stadler and Herschel L. Roman is greatly appreciated. Research of the authors has been supported primarily by Public Health Service Research Grant A141462 from the National Institutes of Health. It has also been aided by Research Career Award K6-GM-4899 (DDP), Research Grant GM-14263, and Genetics Training Grant 2G158, all from the Public Health Service.

226

DAVID D. PERKINS AND EDWARD G . BARRY

Appendix: Chromosome Aberrations of Neurospora crassa

EXPLANATORY FOREWORD

Organization. The Appendix consists chiefly of summary descriptions of all analyzed rearrangements arranged in a single numerical sequence according to isolation number. The descriptions are preceded by two index tables (Appendix Tables 1 and 2) where the rearrangements are grouped in categories, according to type and linkage group. Appendix Table 1 lists rearrangements that do not produce viable duplications. These are mainly reciprocal translocations. Appendix Table 2 lists duplication-generating rearrangements under three subheadings : (1) Insertions, (2) Quasiterminal rearrangements, and (3) Less completely analyzed duplication-generators: aberrations about which knowledge is still inadequate for placing in (1) or (2). Duplications are not listed separately, but each is described under the rearrangement that produces it. Isolation Numbers. Each rearrangement is permanently identified by a unique isolation number, which is usually the number of the strain in which the rearrangement originated or was first recognized. This may or may not include letters, depending on the usage of the discoverer. I n all symbols for chromosome rearrangements the isolation number appears as the terminal item of the symbol, immediately following the parentheses. For example, in the symbols T(ZZ + Z ) N M l 7 7 and T(11Z;VZI)Y112M4r, the isolation numbers are NM177 and Y112M4r. I n arranging the descriptions according to isolation number, letters preceding the numerals are ignored unless the numerals are identical. If digits of the isolation number are interrupted by a letter, the order of listing is based on the digits preceding the letter. The initial letters preceding the numerals in an isolation number usually indicate the worker or institution of origin of the isolate. I n the rearrangements listed here, these are : ALS-Alice L. Schroeder ; A R A . Radford ; B-Brookhaven National Laboratory (V. W. Woodward) ; C-California Institute of Technology; C- (with hyphen)-Cornell University (J. C. Murray, A. M. Srb); CJS-Carol J. Smarr; D-Duke University (S. R. Gross); EB-E. G. Barry; H-F. P. Hungate; HKH. Kuwana; J H - J o h n s Hopkins University (Silver and McElroy, 1954) ; K-D. G. Catcheside ; NM-Noreen E. Murray; P-D. D. Perkins ; R-Rockefeller University (E. L. Tatum laboratory) ; S-Stanford University (Tatum laboratory after 1947) ; STL-Patricia St. Lawrence; T-Tokyo (Inoue and Ishikawa, 1970) ; Y-Yale University (Tatum or N. H. Giles laboratories) ; TM-Teresa M. Angel. Isolation numbers without letter prefixes refer to mutants in the original Beadle and Tatum (1945) numbering system, obtained chiefly at Stanford University before 1945. Strains prefixed ALS, NM, and AR were mostly discards from mutant hunts that were given to the first author to check for rearrangements.

THE CYTOGENETICS OF

Neurospora

227

Symbols and Conventions. The identifying symbol for each rearrangement consists of three elements, (1) an initial letter or combination of letters specifying the type of aberration (T-translocation, In-inversion, Dp-duplication, Tp-intrachromosomal transposition) ; (2) Roman numerals in parentheses indentifying the linkage groups involved ; and (3) the isolation number-a unique number (or combination) identifying the specific rearrangement. The entire 3-element symbol is written in italics without spacing between elements. Linkage groups are separated by a semicolon for ordinary reciprocal translocations, but by an arrow for insertional and quasiterminal rearrangements. A single arrow indicates that viable duplications are produced, and its direction indicates which component is the donor and which the recipient of the segment subject to duplication. The arrow is used in similar fashion in the genotype symbols for duplications, each of which bears the isolation number of the rearrangement from which it was obtained. Symbols for insertions do not specify whether the inserted segment is inverted with respect to the centromere. Double arrows are employed in symbols for mutual insertions. Linkage group arms (R and L) may or may not be shown in symbols, depending on the type of rearrangement and the context. Where a mutant trait is inseparable from the rearrangement, an appropriate symbol for the trait follows the aberration symbol, separated by a space but not a comma, as for example T(I;II)4637 a2-1. The trait may or may not be allelic with a known gene. Standard chromosome sequence is designated Normal and symbolized N . Genetic linkage groups are designated by Roman numerals I-VII. Chromosomes are designated by Arabic numerals 1-7. For specific gene symbols, see the legend to Fig. 2. Abbreviations: A , a : alleles a t the mating-type locus m t ; Ab: aberration of undefined or unspecified nature, or as a general term for rearrangements where the specific type is clear in context; B: black ascospore (usually viable) ; D p : duplication (terminal or interstitial, inverted or noninverted) ; EMS : ethyl methanesulfonate ; FGSC : Fungal Genetics Stock Center; In: inversion; L: left arm of linkage group (follows Roman numeral) ; m t : mating-type locus; N : number of asci scored for patterns of aborted ascospores ; N : normal (standard) chromosome sequence; R : right arm of linkage group (follows Roman numeral); T: translocation ; Tp : transposition (intrachromosomal) ; UV: ultraviolet light; W: white ascospore (usually inviable).

228

DAVID D, PERKINS AND EDWARD G. BARRY

APPENDIX TABLE 1 Rearrangements That Do Not Produce Viable Duplications, Listed by Linkage Groups"

1;II T(Z,ZZ)B66 T ( Z R;ZZ R )STL76 *T(Z;ZZ)NM 129 T (Z;II)N M 135 T (Z;ZZR)NM168 T ( Z R;ZZL)A RZl6 T(ZR;ZZR)4637 al-1 T (Z;ZZR)P4704 T (1L;I ZL)P5390 I $11 T (ZR;ZIZR)P7SBlOl T(Z;ZZZ)NMlO7 T(1L;ZZZR)NM109 T (1;I1 I ) N M127 T(Z;ZZI)NMlS6 T(Z;ZZI)NM146 T (ZR;IZZR)A R180r T(1R;ZZZ)A R208 T ( I R;III)P2648 T(Z;ZZZR)S717vis

1;IV

T(Z;ZVL)HK53cut T(ZR;ZVR)T54M19 T(Z;IV R)N M128 T(ZR;IV )NMlS2 T (Z;ZV )N M137 T (IR;ZVR)NMl39 bs ( = NM147 bs, NM187 bs) T ( IR;I V R)N M140 T (ZR;ZVR)N M144 T ( Z R;Z V R )NM160 ( = NM162, NM167) T ( IR;I V R )N MI 64 T (1;IV )N M170 T(ZR;IV R )NM1 72 (Probably = NM119) T (Z;IV R )ARl80b T(Z;ZV R )NM181 T(I;I V )ARl93 T(ZR;ZVR)ARSl.2 T(Z;IV)D304 1;v T (ZL;VR)A R12 T(ZL;V)T97M9

T(ZR;VR)ALSl11 T (Z;VL)NM130 T (ZR;V R )N M14S T ( I ;V )ARl75 T (ZR;VR )C-1670 Qk-1 T (ZR;V R )P5166 T(ZL; VR)P5401 T(ZR;VR)S6703 T(ZL;VR)47711

1;VI

T (ZR;VZ)A R13

* T(Z;VZ)T61M158

T(Z;VZ)T51M166 T ( Z R; VZ)P54 T (Z;VZL)N M 163 T ( I ;V I )A R182 T(Z;VZ)Y934M410 T(ZR;VI)P649

1;VII

T(Z;VZZ)K79met-7 T (ZR;VZZR)N M165 T ( I ;VZZ)ALSl67 T ( IR; VZZ)TM429 h15-3 T ( I ;VZZ)S1007 T(Z;VZZ)P1676 T(ZR;VZZ)17084thi-1

1I;III

*T(ZZ;IZZ)T54M140b T (ZZ;ZZZR)A R62 T(ZZ;ZZZR)NM150 T ( Z Z R;ZZZ)C161 aro T(ZZ;ZZZR)NM161 T(ZZ;IZZ)36703b

1I;IV

T (ZZ;ZV )0 5 T(IZR;ZVR)NM126 T(ZZ;IV)Y256MBSO

1I;V

T(Z1;VL)AR3O T ( II R;V R)ALS154 T (ZZR;VR)NM180

I1 ;VI

T (IZR;VZL)A R9r T(ZZR;VZ)AR181 T ( I1;VZ)B362r

THE CYTOGENETICS OF

Neurospora

229

APPENDIX TABLE 1 (continued)

1I;VII

T(ZlR;VZZR)T61M14S T(Z1R;VZIR)P7SB168 T (1I;VZI)NM134

1II;IV

T(Z1Z;IV)T42M36 T (ZZZ;ZV )NM118 T ( I I I ; I V ) N M f31 T ( l l l ; l V ) AR211

1II;V

T(III;VR)NMlOl ( = NM102, NM104, NMl11, NM112, NM114, NMll.5) T(ZI1;V)AR177 T(ZZ1;V)N M 183

1II;VI

T (ZZZR;V I )1 T ( I l l ;VZ)A R f 86 T (ZZIR;VZ)P6070

I11jVI1

T (IZI;VII)A Rl9 T(Z1Z;VZI)YlldM4r T (ZZZR;VZIR )N M I 69r

IV ;v

T ( Z VR;VR)A R1 1r T ( IV ;V )TSSM8 T ( Z V R ;V R )N M126 T(ZVR;VR)NM14l T ( Z VR;V R )NM146 T ( I VR;VR)AR221 T(ZVR;VR)R2366

1v;VI

T ( I V;VZ)B8 T ( Z V;VZL)P7SB12 T ( I VR;VlR )N M176 T ( Z V R ;VZ)A m 0 7 T (ZVR;VZ)STLS84r T ( Z VR;VZR)46602

IV ;VII

T (ZVR;VZIR)A R10 T ( IV;VZI)N M 1 f 3 T (ZV;VZZ)NIkf166 T(ZVR;VIIR)NMIBB T ( Z VR;VZZR)STLS84b

V;VI

T ( V R ;VZR )NMI 67 T (V R;VI)N M16db T (V ;VZ)NM171 T(VR;VZ)A R174 T ( V;VZ)A R f 84 T ( V;VI)A480 T (V;VZ)JH2003 T(VR;VZL)46802in1

V;VII

T(VL;VZI)AR46 T ( V ;VZI)N M169

VI ;VII

T (VZR;VZZR)ALS7 T (VZ;Vll)NM124 Mutual insertions T ( I R e ZV)Y112MfS ad-SA T ( l R e VR)SlS36 nic-2

Right and left arms are indicated only where they are known with certainty. Strains in parentheses appear to be identical with the translocation after which they are listed, and were probably derived from the same original mutational event. They are not included in the total count of 123 interchanges. * Aneuploids are found among the progeny of T x N , probably as a result of 3: 1 segregation. Except for these three exceptions, the translocations do not produce viable aneuploid progeny in substantial numbers.

230

DAVID D. PERKINS AND EDWARD G . BARRY

APPENDIX TABLE 2 Duplication-Generating Rearrangements, Listed by Type and Linkage Groupo

1. Insertions 2. Quasiterminal rearrangements *T(ZL ZZR)393ll Zn(ZL -+ ZR)ARl6 ( = ARSi, A R l l , AR15) T ( Z L - +VZL)T61M166 un Zn(ZL-+ ZR)NMl76 *Tp(ZR-+ ZR)T64M94 Zn(ZL -+ ZR)H4.260 T(ZR-+ ZZZR) YllZ?M4iad-SB T(ZR-+ ZZZR)4640 nic-2 T(ZR VL)ARlSO T(ZR + VZR)NMlOS T(ZR + VZZ;Z;V;VZZ)ARl73 T(ZR + ;ZZ;VZZ)AR.217 T(ZR+ )NM169d *T(ZR-+ )ALSl82 I T(ZZL -+ VR)NM149 T(ZZL --* ZZZR)AR18 T(ZZR + V)ALS176 T(ZZL + [ZV;V])ARl79 T(ZZZR -+ ;ZZZR;VZL)DS05 *T(ZZL-+ VZ)PS869 T(ZVR + VZR)ALS169 T(ZZR-+ ZL)NMl77 T(VZL-+ ZR)TSSM777 T(VZR -+ IVR)ARBOS T ( Z Z + ZV )R.2394 T(ZZZR [ZR;ZZR])ARl7 T ( V Z Z L - +ZVR)T64M60 T(ZVR + Z)NM16.2 T(VZZL+ ZVR)ALS179 *T(ZVR-+ ZZZR)S4346 3. Less completely analyzed *T(ZVR-+ VZZ;ZL;ZZR;ZVR)Sl.2~9 duplication-generators T ( V + ZVL)ARSS arg T(VR-+ VZZ)EB4 T ( Z R;ZZ R;Z V R )R65 T(VZL-+ [Z;ZZZR]) Y163.20 T(VZZ-ZV)ALSl76 T(VZR -+ ZVR)CJSl T (Z;ZZZ;VZ;VZZ)ARl76 T(VZZR + ZL)6936 T(ZV Z)BSSXi T (ZZZR;VR;VZZL)Pll56 T( Z)R.247.2arg -+

-+

-+

-+

-+

When these rearrangements are crossed by normal sequence, a class of viable Duplication progeny is produced regularly by meiotic recombination. Rearrangements are listed here in order of the linkage group from which the duplicated segment originated (the donor). Information on each aberration and duplication is summarized in the Appendix, where rearrangements of all types are given in a single numerical order. Figure 19 shows what is known of the segments included in the duplications. Some of the strains under 2 could be subterminal insertional translocations, but interchange of a terminal segment with a tip is the simplest hypothesis on present data. * Production of bridges and fragments in meiosis indicates that the insertion is inverted with respect to centromere. Not all insertions have been examined for meiotic bridges and fragments.

DESCRIPTIONS OF INDIVIDUAL IDENTIFIDD REARRANGEMENTS Rearrangements are listed in a single numerical sequence according to isolation number (the number that follows the parentheses in the symbol). Information is given in the following order. Identifying symbol Genetic basis (terse description ; breakpoints) Vegetative phenotype

THE CYTOGENETICS OF

Neurospora

231

Fertility of A b x A b crosses A b x N crosses: Percent, black ascospores Unordered asci : Percent asci of the types: 8:0,6:2, 4:4,2:6,0:8 (B1ack:White) Total number of asci (in parentheses) Cytology Origin: strain, mutagen, discoverer Other information FCSC Numbers of A and a stocks References

Additional information is given on the viable duplications from duplication-producing rearrangements : Duplication symbol Origin Vegetative phenotype and stability Fertility in crosses x non-Duplication Derived products if unstable Markers shown covered Markers shown not covered

T( 1II;VI)l Reciprocal translocation. I I I R (near vel and tyr-1) interchanged with VI (near

ylo-1). Wild phenotype. Homozygous-barren. T x N ascospores 50% black; unordered asci 15:4:55:7:19 (N = 223). Cytology: Observed as a component of ulcoy linkage-tester stock. Reciprocal translocation confirmed (Barry). Origin; Spontaneous, from 74A x rg CT a. Detrcted by P. St. Lawrence. Analyzed genetically by Perkins. FCSC 976,975. Reference : Perkins et al., 1969.

T(VI+ 1V)CJSl Insertional translocation. VIR segment distal to trp-2 is inserted in IVR (near pun-1). Wild phenotype. Homozygous-fertile. T x N ascospores 25% black; unordered asci 1:1:34:27:37 ( N = 164). Basis of the excessive defective ascospores is not understood. Cytology: Chromosome 2 is not abnormal. No bridges or fragments observed. Origin: Spontaneous as sector in a cross of p’ x Normalsequence fr from OR wild-type x T(ZV + IIZ)S4342. Discovery and preliminary analysis by Carol J . Smarr. Further genetic analysis by Perkins. FGSC 2676, 2677. Duplications: Dp(V I R + ZVR)CJSl. Less than one-third of surviving progeny from T x N . Vegetatively scant or feeble. Barren in cross by nonduplication. Markers shown covered: None. Markers shown not covered: ylo-f, rib-1, trp-2, trp-4, pan-1.

T(V + VII)EB4

Insertional translocation. A short VR segment near al-3 and his-l is inserted into VII, probably left of wc. Wild phcnotype. Homozygous-fertile. T x N ascospores 90% black; a few percent arc round. Unordered asci 53:20:25:2:0 ( N = 93). Cytology: Asynnpsis of 2R is common a t pachynema in T x N . Breakpoint is

232

DAVID D. PERRINS AND EDWARD G. BARRY

far out in long arm of 2. Second chromosome unidentified cytologically. Origin: Detected cytologically in 1971 by Barry as a minor component of a stock of 74-0R8-1 a, which had been preserved on anhydrous silica gel s h e received from F. J. de Serres in 1961. Genetic analysis by Perkins. FGSC 2179, 2180. Reference: Barry et al., 1972. Duplications: D p ( V R +- VII)EB4. One third of surviving progeny from T x N. Vegetatively normal and stable. Crosses of duplication by nonduplication are ecorable as barren ; however, all produce ascospores, although in much smaller numbers than euploid crosses. Of the spores produced, some are round. Markers shown covered: None. Markers shown not covered: at, his-1, inl, a l 9 , bk, pub-i, m e t d , his-6.

T(II;IV)D5

Reciprocal translocation. I1;IV interchange with 17% recombination between bal and pdx in T x N. Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 25:3:24:9:39 ( N = 103). Origin: inl (89601), W. Detected and analyzed by Perkins. FGSC 2393, 1554.

T ( V1;VII ) ALS7

Reciprocal translocation. VIR (right of trp-2) interchanged with VIIR (right of arg-10). Wild phentoype. Homozygous-fertile. T x N ascospores 5045% black; unordered asci 31:21:37:5:7 (N = 272). Many 5B:3W asci; evidently one duplication-deficiency class makes inviable black ascospores. Cytology : Produces acentric chromosome fragments (Barry). Origin: rg cr a, W. Detected and analyzed genetically by Perkins. No viable duplications produced. FGSC 1993, 2016.

T(N;VI)B8

Reciprocal translocation. 1V;VI interchange with 9% recombination between pdx and ylo-I. Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 23:19:19:14:24 (N = 78). Origin: STA, UV. Detected and analyzed by Perkins. Originated with unlinked morphological mutant. FGSC 2394, 2395.

In( IL + IR)AR9i See In(IL + IR)ARlG.

T(1I;VI)ARgr

Reciprocal translocation. I I R (between argd and fl) interchanged with VIL (near choL.9). Growth slower than wild type. Homozygous-fertile, but ascospores ooze rather than shoot from perithecia. T x N ascospores 50% black; unordered asci 18:0:68:3:11 (N = 238). Origin: pyr-1 met-1 a (H263, 38706), W. Detected and analyzed by Perkins. Strain of origin also contained unlinked In(IL+ IR)ARQi.FGSC 2131, 2132.

T(IV;VII)ARlO

Reciprocal translocation. IVR (near cot-1) interchanged with VIIR (linked arg-10). Wild phenotype. Homozygous-fertile. T x N ascospores 75% black; unordered asci 15:52:22:6:4 ( N = 208). Origin: pyr-1 met-i a, W. Detected and malyzed by Perkins. Apparently one duplication-deficiency class is represented

THE CYTOGENETICS OF

Neurospora

233

by inviable black ascospores; a t least one-third of black spores from T x N are inviable, and no viable duplications are recovered. Generates viable duplications from intercross with T(IVR;VIlR)NM168.FGSC 2007, 2008. In( ZL + 1R)ARlli See I n ( l L + IR)ARlG. T(1V;V)ARllr Reciprocal translocation. IVR (proximal to p&) interchanged with VR (distal inl). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 14:2:42:16:26 (N = 520). Origin: pyr-1 met-1 a, W. Detected and analyzed by Perkins. Generates viable duplications from intercrosses with T ( I V R ; V R )NM126, N M l d l , NM146, and R2366. Duplications are pdx+ from A R l l r x N M l 4 l ; therefore A R i l r break is proximal to pdx. Strain of origin also contained In(IL + I R ) A R l l i . FGSC 2093, 2094. T ( 1;V)ARIP Reciprocal translocation. IL (proximal to m t ) interchanged with VR (near id). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 11:13:53:13:11 (N = 191). Origin: pyr-i met-1 a, UV. Detected and analyzed by Perkins. Beske and Phillips (1968) first showed linkage in I or I1 and IV or V. Generates viable duplications from intercrosses with T(IL;VR)P6401 and 47Yll. These have an inhibited Dark-Agar phentoype typical of A l a ; therefore ARla breakpoint is proximal to m t . FGSC 2006, 1462. Reference: Perkins, 1975. T ( I;VI)AR13 Reciprocal translocation, I R (distal to cr) interchanged with VI (near ylo-1, to the right). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 38:1:39:1:22 ( N = 150). Origin: pyr-1 met-1 a, W. Detected and analyzed by Perkins. Generates viable duplications from intercross with T(IR;VI)'I)[email protected] 1913, 1914.

In( IL + 1R)ARlS See I n ( l L + I R ) A R l 6 . In( IL + IR)AR16 Pericentric inversion. A distal segment of IL (including ser-3 but not un-3 and m t ) is interchanged with the I R tip. No recombinant has been obtained with mating type. Wild phenotype. In x N ascospores 80% black; unordered asci 25:54:19:1:2 (N = 177). Origin: pyr-1 met-1 a, TJV. Discovered by Perkins, analyzed by Turner. In(IL + IR)ARSi, A R l l i , AR16, AR16, all from the same experiment, apparently have a common origin. Breakpoints and genetic behavior resemble In(IL + IR)NM17e. The structure is formally similar to In(1LR)sc" in Drosophila. FGSC 1614. Reference: Turner et al., 1969.

Duplications: D p ( I L + IR)ARlG. About one-fourth of surviving progeny from In x N . Slightly abnormal morphology of aerial mycelia, resembling D p ( I L 3 I R )N M m . Barren in crosses with nonduplication ; fertile normal-sequence derivatives are produced by loss of the duplicated IL segment from the translocated

234

DAVID D. PERKINS AND EDWARD G . BARRY

position. Markers shown covered: ser-3, leuJ, fr. Markers shown not covered: un-3, mt, arg-1.

T(I11 -j [I;II] )AR17 Complex insertional translocation involving IR near aZ-1, I I R between a r g d and pe and IIIR between tyr-1 and dow. Duplications are generated of a distal IIIR segment. Wild phenotype. Homozygous-fertile. T x N ascospores < 50% black; unordered asci 16:11:41:17:15 ( N = 181). Cytology: Pairing a t pachynema resembles a reciprocal translocation between 1 and 6. The I11 abnormality is not obvious, thus is probably small. Spore development is abnormal in some asci. Origin: pyr-1 met-1 a, UV. Recognized aberrant by Perkins. Analysis and I11 linkage by B. C. Turner. FGSC 2442, 1463. Duplications: Dp(l1lR + [ I ; I I l )AR17. One-third of surviving progeny from T x N . Wild phenotype, stably barren. Many perithecia produce a few spores each. A high proportion of the spores are peculiar in shape, including some oversized. Markers shown covered: dow. Markers shown not covered: tyr-1, uel, trp-1, acr-2.

T ( I 1 - j 1II)ARlS Insertional translocation. A IIL segment between pi (col-10) and pyr-4 is inserted distal to dow in IIIR. Wild phenotype. Homozygous-fertile. T x N ascospores 70% black; unordered asci 11:60:25:3:2 (N = 500). Origin: pyr-1 met4 a, UV. Detected by Perkins. Genetic analysis by A. Kruszewska. Used as het-6 tester to study vegetative incompatibility. FGSC 2643, 2644 (het-GOR).Reference : Mylyk, 1975.

Duplications: Dp(IIL += 1IIR)ARlB. One-third of viable progeny from T x N. Very slow, feeble growth initially after ascospore germination, which cannot be attributed to heterozygous vegetative incompatibility alleles, but appears to be an intrinsic property of this duplication. Barrenness is exceptionally stable in crosses to nonduplication. The barren perithecia, with no beaks, progress only occasionally to form a few croziers (N. B. Raju). Marken shown covered: het-6. Markers shown not covered: p i , cys-3, pyr-4, het-c, ro-3, thr-2, arg-6.

T ( III;VII)AR19 Reciprocal translocation. I11 (linked acr-2) interchanged with VII (near W C ) . Late conidiating. Homozygous-fertile. T x N ascospores 50% black. Unordered asci 49:4:19:1:26 ( N = 146). Origin: pyr-1 met-1 a, UV. Detected and analyzed by Perkins. FGSC 1915, 1916. T ( I;V)T27M9 Reciprocal translocation. IL (near m t ) interchanged with V (near lys-1). Wild phenotype. T x T test not made-not recombined with mt. T x N ascospores < 50% black; unordered asci 10:4:20:15:51 (N = 106). Origin: 74A, UV. Detected and analyzed by Perkins. FGSC 2095. T ( 1I;V ) AR30 Reciprocal translocation. I1 (probably L, near pi) interchanged with VL (unmarked segment far left of a t ) . Wild phenotype. Homozygous-fertile. 2’ x N

THE CYTOGENETICS OF

Neurospora

235

ascospores 85-90% black ; blackening increases with age ; unordered asci 49:30:19:2:1 ( N = 177). Origin: rg cr; pyr-1 met-1 A, W. Detected and analyzed by Perkins. ‘Because duplication-deficiency ascospores become black, scoring of translocation by presence of nonblack ascospores is difficult. Less than half of black ascospores are viable. No viable duplications are recovered. FGSC 2004, 2005. T ( V + IV)AR33 Translocation, either quasiterminal or insertional. An unmarked segment of V is translocated to IVL (near cys-10). (If translocation is quasiterminal, the segment is VL.) Wild phenotype. Homozygous-fertile. T x N ascospores 70% black or less; unordered asci 36:32:28:2:2 (N = 117). Origin: rg cr; pyr-1 met-1 A, W. Detected and analyzed genetically by Perkins. FGSC 2021, 2396. Note added in proof: A distal segment of the short arm of chromosome 2, which carries the nucleolus organizer, appears cytologically to be translocated to the tip of another chromosome.

Duplications: D p ( V + IVL)ARSS. One-third of viable progeny from T x N . Wild morphology. Barren in crosses by nonduplication, where perithecia contain all stages of meiosis, but few or no spores (N. B. Raju). Duplications are &able and may be transmitted through crosses. Markers shown covered: None. Markers shown not covered: lys-1, at, leu-6, a@, his-6.

T( IV;V)T33M8 Reciprocal translocation. IV (loosely linked p d z ) interchanged with V (loosely linked at). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 14:3:48:10:24 ( N = 147). Origin 74A, UV. Detected and analyzed by Perkins. FGSC 2397, 2398. T ( VI + I ) T39M777 Quasiterminal translocation. A VIL segment including un-4 and distal markers is translocated to the right end of I near R. Wild phenotype. Homozygous-fertile. T x N ascospores 75% black; unordered asci 18:46:29:5:3 ( N = 114). Origin: 74A, W. Detected and analyzed by Perkins. Used as het-8 tester to study vegetative incompatibility. FGSC 2133, 2134 (het-SoR).References : Mylyk, 1975, 1976. Duplications: D p ( V I L + I R ) T39M777. One-third of surviving progeny from T x N . Wild phenotype, but with slowly developing diffuse patchy dark pigment on surface of lower part of minimal slant. Crosses by nonduplication are scorable as stably barren; however, all produce some ascospores, but far fewer than euploid crosses. Made unstable by mei-3 (Newmeyer and Galeazzi, 1976b). Vegetative escapes from het-S inhibition remain barren (Mylyk). Markers shown covered: chol-2, lys-6, un-4, het-S. Markers shown not covered : cys-1, yb-1.

T(III;IV)T4fZM36

Reciprocal translocation. I11 (probably left of acr-2) interchanged with IV (linked

p d z ) . Wild phenotype. Homozygous-fertile. T x N ascospores 75% black; unordered asci 16:53:23:7:1 ( N = 100). Origin: 74A, UV. Detected and ana-

lyzed by Perkins. Evidently one duplication-deficiency class becomes black. Ger-

236

DAVID D. PERKINS AND EDWARD G. BARRY

mination of black ascospores is low. No viable duplications are recovered. FGSC 2443, 2444.

T ( V;VII ) AR45 Reciprocal translocation. VL (near a t ) interchanged with VII (probably L, between thi-3 and met-7). Wild phenotype. Homozygous-fertile. T x N ascospores 75% black; unordered asci 37:25:34:2:2 ( N = 92). Origin: rg cr; pyr-1 met-1 A, W. Detected and analyzed by Perkins. Evidently one duplication-deficiency class makes inviable black ascopores. Germination of black ascospores is low. No viable duplications are recovered. FGSC 1760, 1761. T ( II;VII)T51M143 Reciprocal translocation. I I R (between arg-6 and p e ) interchanged with VIIR (linked arg-10). Wild phenotype. Homozygous-fertile. T x N ascospores 6040% black; unordered asci 26:39:31:2:3 ( N = 376). Origin: 74A, X-rays. Detected and analyzed by Perkins. Evidently one duplication-deficiency class makes inviable black ascospores. Germination of black spores is low (about 2/3). No viable duplications are recovered. FGSC 2399. 2400. T ( I + VI)T51M156 un Insertional translocation. An unmarked segment of I L near mt is inserted in VIL between chol-2 and ad-8. Both the translocation and its duplication progeny are un phenotype, unable to grow a t 34°C regardless of supplement, but capable of growth with wild-type morphology on minimal a t 25". Fertility is limited in T x T crosses, but viable progeny are produced. T x N ascospores > 75% black; unordered asci 22:33:30:12:3 ( N = 366). Origin: 74A, X-rays. Detected and analyzed by Perkins. S. Brody (personal communication) has shown that the closely linked chol-2+ and a d a t functions are not impaired in the rearrangement sequence. FGSC 2270, 2271. Duplications: Dp(ZL + VZL)T61M166 un. One-third of viable progeny from T x N . Phenotypically un. Barren in crosses. Markers shown covered: None. Markers shown not covered: f r , leu-3, ser-3, m t , arg-3. Probably not covered: ta, suc, arg-1.

T(I;VI)T51M158 Probably a reciprocal translocation that undergoes frequent 3: 1 segregation at anaphase I. I (near centromere, linked arg-1 and cr) interchanged with VI (near rib-1 and ylo-1). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black, or less; unordered asci 20:7:40:9:25 (N = 258). Origin 74A, X-rays. Detected and analyzed by Perkins. FGSC 2759,2760. Duplications: 5%-10% or more progeny from T x N are duplications that can carry I and VI markers in heterozygous condition. Markers shown covered: rib-1, pan-2, del, trp-2 (when m t not heterozygous). $0-1 (when m t heterozygous). Markers shown not covered: ad-8, cys-1, ylo-1, ad-1 (when m t not heterozygous), del, t r y 4 (when m t heterozygous). Some but not all duplications are detected as barren in crosses x Normalsequence testers. The hypothesis of origin by 3 : l segregation is consistent with (a) a higher frequency of 4B:4W asci than ex-

THE CYTOGENETICS OF

Neurospora

237

pected when both break points are near centromeres, and (b) shifting patterns of marker coverage, depending on which member of the translocation complex is lost. T( I;VI)T51M166 Reciprocal translocation. I (linked mt ) interchanged with VI (near ylo-1). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black, or more; unordered asci 44:8:21:9:18 ( N = 101). Origin: 74A, X-rays. Detected and analyzed by Perkins. FGSC 2401, 2402. T(I;IV)HK53 cut Reciprocal translocation. I (near arg-1) interchanged with IVL (distal to fi). Phenotype cut (osmotic-sensitive), scorable by morphology on minimal at 34", or by no growth on minimal 4% sodium chloride. Homozygous-fertile. T x N ascospores 50% black; unordered asci 55:1:6:0:37 (N = 231). D. A. Smith (unpublished) has confirmed cytologically an exchange involving chromosome 1. Origin: Strain 4A (probably Abbott 4A), UV. Described as morphological mutant by Kuwana. Translocation recognized by Perkins. Original mutant strain also contained linked gene mei-1 as well as a tol-like suppressor of the heterokaryon incompatibility associated with mating type, both separable from the translocation. An allelic point mutant, cut (LLMl), maps in IVL. Kuwana's cut has not previously been numbered. It is proposed from now on to use HK53 as isolation number, for convenience and to avoid confusion with allelic cut mutations. FGSC 2272, 2068. References: Kuwana, 1953, 1960; Smith and Perkins, 1972; Smith, 1975.

+

T( I;VI)P54 Reciprocal translocation. IR (near al-2) interchanged with VI (near ylo-1). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 25:2:62:2:9 ( N = 125). Origin: Recognized by Perkins in fl from cross aur cr A (Fincham stock F945-78) x thi-1 a. FGSC 2445,2446. T( I;IV)T54M19 Reciprocal translocation. IR (linked m t ) interchanged with IVR (linked p d z ) Flat pe-like morphology. Female-sterile, with no perithecia. T x N ascospores 50% black; unordered asci 26:6:39:5:24 ( N = 217). Origin: 74A, W. Detected and analyzed by Perkins. Generates viable duplications from intercross with T ( Z R ; I V R ) N M 1 6 4 .Therefore breaks must be in right arms. FGSC 2135, 2136.

.

T ( VII + IV ) T54M50 Quasiterminal translocation. A VIIL segment including thi-3 and markers distal to it is translocated to the right end of IV. Wild phenotype. Homozygous-fertile. T x N ascospores 75% black, or more; unordered asci 17:32:32:8:10 (N = 220). (Interpreted to be 21:58:21:0:0.) Origin: 74A, UV. Detected and analyzed by Perkins. Used for studying vegetative incompatibility. Translocation stocks containing het-e and het-E are used as testers. FGSC 2466, 2467 (het-el; 2603, 2604 ( h e t - E ) . References: Perkins, 1975; Mylyk, 1975. Duplications: D p ( VZZL + ZVR) T54M50. One-third of viable progeny from T X N . Wild phenotype unless heterozygous for het-e alleles. Duplications are

238

DAVID D. PERKINS AND EDWARD G . BARRY

stable. Barren in crosses. Few ascospores produced, even after long incubation. Markers shown covered : het-e, spco-4, nic-S, thi-3. Markers shown not covered: met-7, wc. Tp(1R + IR)T54M94 Transposition. A segment of I R including nit-1 through aL2 is transposed proximally and inserted in inverted order near nic-2. Wild phenotype. Homozygousfertile. T X N ascospores 80% black, or more; darken with age. Unordered asci 47:35:14:3:2 (N = 229). Origin: 74A, UV. Aberration detected and analyzed by Perkin8 and B. C. Turner. Strain of origin also contained a linked but separable mutant gene, un-18 (T54M94), located near IR tip. FGSC 2943,2928.

Duplications: D p U R + ZRIT54M94. Usually one-fifth or less of surviving progeny from T X N. Wild phenotype. Highly barren in crosses by nonduplication. Barren condition is exceptionally stable. Markers shown covered : nit-1, cyh-1, 05-6, al-2. Markers shown not covered: cr, thi-1, met-6, arg-6, al-1, hom. T ( II;III)T54M140b Reciprocal translocation (probably). I1 (near arg-5) interchanged with I11 (between acr-2 and thi-4). Wild phenotype. Homozygous-fertile. T x N ascospores > 50% black; unordered asci 49:5:35:2:9 (N = 162). Origin: 74A, W . Detected and analyzed by Perkins. No barren duplications are produced. Cryptic aneuploids have been obtained from several crosses. These segregated acr-2, thi-4, or bal and are thought to originate as disomics, from 3 : l segregations. Black ascospores show good viability. Stock of origin also contained another probable rearrangement, tentatively T ( I + 111)T54M150 un ; this has probably been separated, but is inadequately analyzed. FGSC 2941, 2942. T ( I;II;IV)R55 A complex translocation showing linkage in I R near Zys-3, in I1 near pe, and in IVR. (Not tested for linkage in group V.) Wild morphology, but slower growing. Homozygous-fertile. Cytology: Chromosomes 1, 2 and 6 are involved in the translocation. Breaks in 1 and 2 near centromere with arms exchanged, and in 2 through the nucleolus organizer with the tip of the organizer translocated to the long arm of 6. Origin: pe fl, X-rays. Detected and analyzed cytologically and genetically by P. St. Lawrence. Stock now lost. Reference: St. Lawrence (1953).

Duplications: Two types of probable duplications observed, called abnormal ( a b n ) and abnormalsterile (abn-s). The abn type crossed by standard chromosome sequence, R55 sequence, or other abn’s produced asci developing to late meiotic prophase stages, but chromosome pairing appeared to be abnormal. Few asci completed the third division, and spore formation was rare and irregular. ablzrs crossed to standard was barren, with perithecial development arrested prior to ascus formation.

T ( 1I;III ) AR62 Reciprocal translocation. I1 (linked a r g d ) interchanged with IIIR (linked trp-1) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 36:1:40:1:22 (N = 306). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. Generates viable duplications from intercross with

THE CYTOGENETICS OF

Neurospora

239

T(II;IIIR) NM161. Strain of origin also contained an unlinked ascospore-color gene bs (AR62, in IR). FGSC 1545, 1546. T( I;II)B66 Reciprocal translocation. I (linked mt) interchanged with I1 (linked p e ) . Wild phenotype. Homozygous-fertile. T x N ascospores 5070 black ; unordered asci 14:9:52:7:18 ( N = 88). Origin: 74A, UV. Detected and analyzed by Perkins. FGSC 1464, 1465. T( IV;VI)P73B12 Probably reciprocal translocation. IV (near p d z ) interchanged with VIL (near choZ-2). Wild phenotype. Homozygous-fertile. .Tx N ascospores > 50% black; unordered asci 16:13:56:6:9 (N = 111). (Interpreted 16:0:69:0:15.) Origin: sn cr; aG3 inl, EMS. Detected and analyzed by Perkins. Evidently some inviable duplication-deficiency ascospores become black. FGSC 2623, 2624. T( I;III)P73BlOl Reciprocal translocation. I R (linked aur) interchanged with IIIR (linked trp-1). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 18:25:41:11:6 ( N = 175). Origin: sn cr; a M in!, EMS. Detected and analyzed by Perkins. FGSC 2645, 2646. T( II;VII)P73B169 Reciprocal translocation. I I R (right of p e ) interchanged with VIIR (right of nt). Wild phenotype. Homozygous-fertile. T x N ascospores 75% black; unordered asci 21 :49:27:2:1 (N = 182). No viable duplications recovered. Origin: sn c r ; a14 inl, EMS. Detected and analyzed by Perkins. Evidently some inviable duplicatios-deficiency ascospores become black. Less than two-thirds of black ascospores are viable. FGSC 2625, 2626. T ( 1;II ) STC76 Reciprocal translocation. IR (between cyh-1 and p e l ) interchanged with IIR (between p e and ace-1). Wild phenotype. Homozygous-fertile. T x N ascospores > 50% black, unordered asci 29:8:32:6:24 ( N 139). Origin: 76A, spontaneous. Aberration detected by St. Lawrence, analyzed by Perkins. Generates viable duplications from intercross with T ( I R ;ZIR)4837 d-1. Markers shown covered by progeny test of duplications: os-6, un-7, al-2, arg-6, p e . Loci shown not covered because duplications from intercross are of recessive marker phenotype : ace-1, trp-3. FGSC 2096, 2097.

-

T( I;VII)K79 met-7 Reciprocal translocation. I (linked mt) interchanged with VII (at met-7). Phenotype met-7, wild morphology. Homozyqous-fertile. T x N ascospores 50% black; unordered asci 47:4:7:2:41 (N = 189). Origin: E m a. Translocation detected and linkage groups identified by N. E. Murray. Generates viable duplications from intercross with T(I;VIZ)S1007.FGSC 2297,2298. T( 1II;V)NMIOL Reciprocal translocation. I11 (linked acr-2, trp-1) interchanged with VR (linked id). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; un-

240

DAVID D. PERKINS AND EDWARD G. BARRY

ordered asci 31:9:44:4:13 (N = 231). Origin: E m a, W. Detected and analyzed by Perkins. Intercrosses indicate probably identical to I11;V translocations NMlO.3, 10.4, 111, 112, 114, 116 isolated from the same experiment. FGSC 1879, 1880.

T(III;V)NM102

See T (111; V )NMlOl .

T(I + V1)NMlOJ

Quasiterminal translocation. A segment of IR including met-6 and distal markers is translocated to the right end of VI beyond trp-g. Wild phenotype. Homozygousfertile. T x N ascospores 75% black, or more; unordered asci 26:42:27:4:1 (N = 219). Origin: Em a. W. Detected by Perkins. Analyzed by B. C. Turner. Used as het-6 tester to study vegetative incompatibility. FGSC 2137, 2138 ( h e t b o R ) . References: Turner, 1975, 1976; Mylyk, 1975, 1976. Duplications: D p ( 1 R + V1R)NMlOS. In one-third of surviving progeny from T x N . Duplications make relatively large, diffuse colonies on sorbose medium. In tubes, duplication resembles a slow wild type a t 25°C but resembles fZ: fluffy at 34". Sectoring (uncovering of recessive heterozygous markers) is apparent in some backgrounds. Initially highly barren. Marked crosses give no recovery of intact duplications. However, in many crosses a few perithecia are fully fertile through loss of one duplicated segment. Either segment-that in N or in T sequencemay be lost with equal probability. Euploid derivatives are always found in platings of such partially fertile duplications. There is no evidence of delayed fertility, which might be expected if loss were stepwise. Markers shown covered: met-6, ad-9, nit-1, cyh-1, al-2, arg-13, R , nn-18, het-6. Markers shown not covered: thi-1, un-1, cr, his-3, mt, fr.

T(III;V)NM104

See T ( I I I ; V ) N M l O l ,

T(I;III)NM107

Reciprocal translocation. I (linked m t ) interchanged with I11 (linked acr-2). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 20:1:55:1:23 (N = 82). Origin: Em a, UV. Detected and analyzed by Perkins. Intercrosses show not identical with T(I;III)NMlOS, NMlgY, NM136, or N M l 4 6 . FGSC 2058, 2059.

T(I;III)NM109

Reciprocal translocation. I L (between un-6 and m t ) interchanged with IIIR (between acr-2 and dow). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 20:0:63:2:15 (N = 54). Origin: Em a, W. Detected and analyzed by Perkins. Intercrosses show not identical with T ( 1 ; I I I )NMlO7, NM127, NM136, NM146. FGSC 2627, 2628.

T(1;V)ALSlll

Reciprocal translocation. I R (near cr) interchanged with VR (between al-3 and his-6). Wild phenotype. Homozygous-fertile. T x N ascospores 70% black; unordered asci 39:16:35:3:6 ( N = 148). Origin: rg cr a, UV. Detected and analyzed

THE CYTOGENETICS OF

Neurospora

241

by Perkins. No viable duplications are produced in crosses by Normal. Frequencies of ascus types suggest the one inviable duplication-deficiency class darkens. However, germination is good among black ascospores. Possibly white spores degenerate. Produces viable duplications from intercrosses with T(1;V)SSrOS and C-1670 pk-1. FGSC 2629, 2630. T ( III;V)NMlll, T ( III;V)NM112 See T ( l 1 1;V)N M l O l . T ( I + 111)Y112M4i adSB Insertional translocation. I R segment including nic-2 is inserted in IIIR near vel. a d S B phenotype: requires adenine, accumulates purple pigment. T x T crosses infertile, perhaps because of adenine requirement. T x N ascospores 7570 black; unordered asci 15:50:26:6:3 ( N = 185). Origin: 74A, X-rays. Discovered and first analysis by de Serres. I11 linkage identified by P. St. Lawrence. Separated from T ( Z l l ; V l l )Y112M4r which was also present in original strain. FGSC 2637, 2638. Reference: de Serres, 1957. (This is the first published account of an insertional translocation in Neurospora.)

Duplications: D p ( l R + I I I R ) Y112M4i. One-third of surviving progeny from T x N . ad+ phenotype. Highly barren in crosses by nonduplication. Markers shown covered: nic-2. Markers shown not covercd: his-2, ad-3A, cr, thi-1, at-2. T ( 1II;VII) Y 112M4.r Reciprocal translocation. I11 (near acr-2) interchanged with VII (linked W C ) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 25:3:40:3:29 ( N = 177). Origin: 74A, X-rays (de Serres, 1957). Strain of origin also contained T ( I + I l l ) Y l l d M 4 i ad-3B. Extracted, mapped and analyzed by Perkins. FGSC 2631, 2632. T(I IV)Y112M15 ad-3A Translocation with breakpoints a t or near ad-SA in IR and near pdx in IV. Probably an inverted insertional I + IV, with mutual IV + I insertion postulated to explain absence of viable duplications among progeny of T x N . Phenotype n&SA : requires adenine and makes purple pigment. Cytology: Chromosomes 1 and 3 reported aberrant by Griffiths et nl. (but see Section IV, C). Acentric chromosome fragments are formed, which persist in micronuclei and replicate (Barry). Origin: 74A, X-rays. Aberrant recombination noted by de Serres. Aberration analyzed by Griffiths. References : de Serres, 1971 ; Griffiths, 1970, 1972; Griffiths, et nl., 1974. T ( IV;VII)NM113 Reciprocal translocation. IV (linked pdx) interchanged with VII (linked met-7). Sub-wild phenotype, pale pigment. Lysis and exudate at top of agar slant. Homozygous-fertile. T x N ascospores 50% black; unordered asci 31:13:20:1:34 ( N = 70). Origin: Em a, UV. Detected and analyzed by Perkins. Intercrosses show not identical with T(IV;VII)NM16G or NM158, which originated from a different experiment. FGSC 1917, 1918. T ( III;V)NMI14, T(III;V)NM115 See T ( I I I ; V ) N M l O l .

242

DAVID D. PERKINS AND EDWARD G . BARRY

T ( III;IV)NM118 Reciprocal translocation. 111 (near ncr-2) interchanged with IV (near p d z ) . Slow to conidiate. Homozygous-fertile. T x N ascospores 50% black; unordered asci 31:5:35:2:27 ( N = 213). Origin: Em a, W. Detected and analyzed by Perkins. Intercross shows not identical with T(I1l;ZV)N M i 3 1 . FGSC 2403, 2404. T ( 1;IV) NMl19 Reciprocal translocation. Probably identical structure with T(I;ZV)NMI72, q.v. Morphology is ropylike, variable. Homozygous fertility reduced. Cytology: Chromosome 2 is not part of the rearrangement. Generates viable duplications in crosses with T(IR;ZVR)NM140. FGSC 1447. 1334. References: Barry and Perkins, 1969; Kowles, 1972, 1973. T ( VI;VII)NM124 Reciprocal translocation. VI (linked ylo-1) interchanged with VII (near met-7). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 46:3:5:1:45 ( N = 361). Origin: Em a, UV. Detected by Perkins. Analyzed and linkages established by Anna Kruszewska. Shot asci shift with time from excess OB:8W to excess 8B:OW. FGSC 2214, 1472. T(IV;V)NM125 Reciprocal translocation. IVR (linked pdx) interchanged with VR (linked a t ) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 13:6:41:3:36 (N = 63). Origin: Em a, UV. Detected and analyzed by Perkins. Generates viable duplications from intercross by T ( I V R ; V R )A R l l r . Arm assignments were made on this basis. FGSC 2447,2448. T(II;IV)NM126 Reciprocal translocation. IIR (near tip-3, probably distal) interchanged with IVR (near col-4). Wild phenotype. Homozygous-fertile. T x N ascospores 50-75% black; unordered asci 21:22:49:7:1 (N = 295). Origin: Em a, UV. Detected and analyzed by Perkins. Evidently some inviable duplication-deficiency ascospores become black. Germination of black ascospores is low. No viable duplications are recovered. cot-1 and 11 are linked in T x T cross. FGSC 1611, 1612. T ( 1;IJ.I )NM127 Reciprocal translocation. I (near m t ) interchanged with 111 (probably R ; loosely linked ncr-2). Wild phenotype. Homozygous-fertile. T X N ascospores 50% black; unordered asci 17:1:53:3:25 ( N = 217). Origin: E m a, UV. Detected and analyzed by Perkins. Generates viable duplications from intercross with T(I;IIZ)NMlS(S. Arm assignments are based on this fact. Intercrosses show not identical with T(I;ZII)NMlO7,NM109 or NM146. FGSC 2405,2406. T ( I;IV)NM128 Reciprocal translocation. I (linked ml) interchanged with IVR (near p t ) . pe-like morphology. T x T crosses nearly infertile. T x N ascospores 50% black; unordered asci 50:8:25:3:15 ( N = 93). Origin: Em a, UV. Detected and analyzed by Perkins. Not tested for identity by crosses with NMl4O or other pe-like I ;IV translocations from the same source.

THE CYTOGENETICS OF

Neurospora

243

T(I;II)NM129

Reciprocal translocation. I (near his-2, probably right) interchanged with I1 (near

arg-5). Morphology not wild: flat growth habit. Homozygous-barren (empty perithecia). T X N ascospores 50% black; unordered asci 30:5:9:6:51 ( N = 661). Origin: Em a, UV. Detected and analyzed by Perkins. Progeny from

T x N include a few percent of barrens, “Dark Agar” phenotypes (attributed to A / a heterozygosity), and in some crosses “Brown-flats” (attributed to het-Clhet-c) . These are thought to arise from 3 : l segregations. FGSC 2330, 2331. T(1;V)NMlSO

Reciprocal translocation. I (linked arg-1) interchanged with VL (26 units left of a t ) . Wild phenotype. Homozygous-fertile. T x N ascospores > 50% black; unordered asci 36:16:30:4:15 ( N = 230). Origin: Em a, UV. Detected and analyzed by Perkins. Not overlapping with T ( I R += VL)ARISO, because no viable duplications are recovered from intercrosses. This favors IL location of NMiSO break point. Evidently some duplication-deficiency ascospores darken. FGSC 2407, 2408.

T(III;IV)NM131

.

Reciprocal translocation. I11 (linked acr-2) interchanged with IV (linked pdz) Wild phenotype. Homozygous-fertile. T X N ascospores 50% black; unordered asci 17:3:69:0:11 (N = 36). Origin: Em a, UV. Detected and analyzed by Perkins. Intercross shows not identical with T(IZI;IV)N M i 1 8 . FGSC 2409, 2410.

T(I;IV)NM132

Reciprocal translocation. I R (right of aG2) interchanged with IV (near p d z ) . “Creamy” morphology. Female-sterile. T x N ascospores 50% black; unordered asci 17:10:30:10:34 (N = 95). Origin: Em a, UV. Detected and analyzed by Perkins. Not tested for identity by intercrossing with similar 1;IV translocations from. the same source.

T(II;VII)NM134

Reciprocal translocation. I1 (near arg-6) interchanged with VII (linked met-7). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 46:2:11:2:39 (N = 54). Origin: E m a, UV. Detected and analyzed by Perkins. FGSC 1919, 1920.

T ( 1;II) NM135 Reciprocal translocation. I (linked m t ) interchanged with I1 (linked arg-5). Slightly flat, pe-like morphology. Homozygous-fertile. T x N ascospores 50% black; unordered asci 43:6:30:3:17 ( N = 178). Origin: Em a, UV. Detected and analyzed by Perkins. FGSC 2023, 2024.

T(I;III)NM136

Reciprocal translocation. I (near m t ) interchanged with I11 (probably R ; linked t r p - f ) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 29:4:46:2:19 ( N = 94). Origin: Em a, UV. Detected and analyzed by Perkins. Original isolate contained a linked but separable arg-3 mutation. Generates viable but morphologically distinct duplications from intercross with

244

DAVID D. PERKINS AND EDWARD G. BARRY

T ( I ; I I I ) N M l 2 7 . Intercrosses show not identical with T(I;III)NMlOY, NM109, or NM146. FGSC 2639,2588.

T ( I;IV)NM137 Reciprocal translocation. I (linked m t ) interchanged with IV (linked col-4). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 41:5:9:3:42 ( N = 116). Origin: Em a, W. Detected and analyzed by Perkins. FGSC 1874. 1875.

T( I;IV)NM139 bs

Reciprocal translocation. I R (near nl-2 and proximal) interchanged with IVR (near p d z and distal). Wild vegetative morphology. T ascospores are brown, yet viable. Homozygous-fertile. T x N ascospores 2570 black; 25% viable brown, 50% white. Unordefed asci 22:0:53:0:25 ( N = 271), when brown ascospores are counted with black. Origin: Em a, UV. Detected and analyzed by Perkins. Allelic and identical with T(I;IV)NM147 and NMlBY, both of which show the same brown-ascospore phenotype. Not allelic with I R point mutant bs-1 : brown ascospore (AR62). Generates viable duplications from intercrosses with T ( I R ; IVR)NMl4O and NMlY2. Intercross shows not structurally identical with T ( I R ; I V R ) N M 1 6 0 . FGSC 1565, 1566.

T(I;IV)NM140

Reciprocal translocation. I R (near 08-1, distal) interchanged with IVR (proximal to t i p - 4 ) . Smooth pe-like morphology. Female-sterile. T X N ascospores 50% black; unordered asci 20:3:55:4:18 ( N 383). (Values in Fig. 11 of Perkins, 1974, are incorrect.) Also 29:2:45:2:22 ( N = 121-Kowles). Cytology: Chromosome 2 is not part of the rearrangement (Barry). Origin: E m a, UV. Detected and linkage groups identified by Perkins; further data by Kowles. Generates viable duplications from intercrosses with T ( I R ; I V R ) N M l l 9 , NM139, NM160, and NM172. FGSC 1759, 1548. References: Barry and Perkins, 1969; Perkins, 1971a; Kowles, 1972, 1973.

-

T(IV;V)NM141 Reciprocal translocation. IVR (linked p d z ) interchanged VR (near al-3). “Creamy” morphology. Homozygous-fertile, T x N ascospores 50% black ; unordered asci 16:1:64:5:14 ( N = 163). Origin: Em a, UV. Detected and analyzed by Perkins. Generates viable duplications from intercrosses with T ( I V ; V R ) A R l l r , NM146 and R2366. N M l 4 l break point assigned to IVR because R2366 maps in IVR. FGSC 2025, 1479.

T( I;V)NM143 Reciprocal translocation. IR (near nur) interchanged with VR (near i d ) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 22:2:63:3:11 (N = 139). Origin: Em a, UV. Detected and analyzed by Perkins. Generates viable duplications from intercroes with T ( I R ;VR)P6166. FGSC 1549, 1550.

T ( I;IV)NM144 Reciprocal translocation. I R (linked 0s-1, distal) interchanged with IVR proximal to trp-4. “Creamy” morphology. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 57:4:31:4:4 ( N = 721, and 22:4:63:0:12 ( N = 8LKowles). Origin:

THE CYTOGENETICS OF

Neurospora

245

Em a, UV. Detected by Perkins, analyzed by Perkins, Kowles. Generates viable duplications from crosses with T ( I R ; I V R )NMlGO, NM164, and NM172. Assignment of NM144 to riglit arm of IV is thus confirmed. Intercrosses infertile with T ( I ; I V ) N M 1 4 0 , NM167, T64M19. FGSC 1336, 1335. References: Kowles, 1972, 1973.

T(IV;V)NM145

Reciprocal translocation. IVR (linked p d z ) interchanged with VR (between at and id).Wild phenotype. Homozygous-fertile. T x N ascospores 60-75% black; unordered asci 18:50:27:3:2 ( N = 277). Origin: Em a, UV. Detected and analyzed by Perkins. Evidently some inviable duplication-deficiency ascospores from T x N become black. Germination of black ascospores is 6O-75p/o0.No viable duplications are recovered from T x N . Generates viable duplications from intercrosses with T ( I V R ; V R ) A R l l r , N M l 4 1 , and RI355. Breakpoint assigned to IVR on this basis. F W C 2098, 2099.

T(I;III)NM146

Reciprocal translocation. I (loosely linked m t ) interchanged with I11 (loosely linked acr-2). Wild phenotype. Homozygous-fertile. 2' X N ascospores 50% black; unordered asci 15:5:62:5:13 ( N = 61). Origin: Em a, UV. Detected and analyzed by Perkins. Intercrosses show not identical with T ( I ; I I I ) N M l O 7 , NM109, N M l U , or NM13G. FGSC 2449, 2450.

T(I;IV)NM147 bs

See T ( I ; I V ) N M l 3 9 bs

T(I1 --j V)NM149

Translocation, probably quasiterminal. A IIL segment including ro-3 and distal markers is translocated to the tip of VR near his-G. Wild phenotype. Homozygousfertile. T x N ascospores 757'0 black or less; unordered asci 13:46:3?3:3:0 ( N = 206). Cytology: Chromosome break points identified near the end of the long arm of 2, and in chromosome 6 (Barry). Origin: Em a, W. Detected and analyzed genetically by Perkins. Used as het-c tester for studying vegetative incompatibility. FGSC 1483, 1482 (het-c) ; 2011, 2012 ( h e t - C ) . References: Perkins, 1969a,b, 1975; Barry and Perkins, 1969; Mylyk, 1972, 1975. Duplications: D p ( I I L + VRINM149. One-third of surviving progeny from T x N . Wild phenotype unless heteroaygous for vegetative incompatibility genes. The speed of somatic escape from inhibition in het-clhet-C duplications is increased by mei-3 (Newmeyer and Galeaazi, 1976b). After escape, individual duplications vary from highly barren to relatively fertile, when crossed by nonduplication. Markers shown covered: ro-3, het-c, pyr-4, het-6, cys-5, pi, col-10. Markers shown not covered : thr-2, thr-3, arg-5. There is evidence also for a second class of duplications that are not heterozygous for I I L markers. Possibly these arise by 3 : l segregation, and they may involve VR.

T(II;III)NM150

Reciprocal translocation. I1 (linked arg-6) interchanged with IIIR (linked un.6). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 11:0:63:6:20 ( N = 54). Origin: Em a, UV. Detected and analyzed by Perkins. Intercross shows not identical with T ( I I ; I I I R ) N M 1 6 1 . FGSC 2060, 2061.

246

DAVID D. PERKINS AND EDWARD G. BARRY

T ( I V + I)NM152 Insertional translocation. An extensive segment of IVR, including loci from pyr-3 through pyr-8, is inserted in I, ten units from arg-3 (probably IR). Wild phenotype. Homozygous-fertile. T x N ascospores, 75% black, or less; unordered asci 18: 16:44:9: 13 ( N = 324). Cytology: Preliminary cytological examination. Acentric fragments are produced which become pycnotic (Barry, 1973). However, their frequency is lower than would be expected if such a long insertion were inverted. Origin: Em a, UV. Detected and analyzed by Perkins. Data on IV breakpoint obtained by A. Radford. FGSC 1752, 1753. Duplications: Dp(IVR + I)NM162. One-third of surviving progeny from T x N. Wild phenotype. Duplications scorable as barren with 90% confidence. Markers shown covered: pyr-3, trp-4, met-2, cot-1, pyr-2. Markers shown not covered: pdx-1, COG,$, cys-4.

T ( 1I;V ) ALS154 Reciprocal translocation. IIR (near fl) interchanged with VR (near id).Wild phenotype. Homozygous-fertile. T x N ascospores > 50% black; unordered asci 11:40:32:6:11 ( N = 88). Origin: rg cr a, W. Detected and analyzed by Perkins. Poor germination among black ascospores and asymmetrical unordered ascus distribution suggest that some duplication-deficiency ascospores become black. FGSC 2062, 2063.

T(I;VII)NM155 Reciprocal translocation. I R (near our, probably proximal) interchanged with VIIR (linked mo(P11G3) and met-7). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 36:3:22:8:31 ( N = 74). Origin: Em a, UV. Detected and analyzed by Perkins. FGSC 1877, 1878. T ( IV;VII)NM156 Reciprocal translocation. IV (linked pdz) interchanged with VII (probably R ; linked wc and arg-10). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 42:7:33:7:11 ( N = 272). Origin Em a, UV. Detected and analyzed by Perkins. Intercrosses show not identical with T(IV;VII) NMllS or NM168. FGSC 1921, 1922. T ( V;VI)NM157 Reciprocal translocation. VR (between at and al-3) interchanged with VIR (near trp-8, probably distal). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 19:3:46:6:26 ( N = 72). Origin: Em a, UV. Detected and analyzed by Perkins. Intercross shows not identical with T(VR ;VI)NM162b. FGSC 2648, 2649. T ( IV;VII)NM158 Reciprocal translocation. IVR (linked cot-1) interchanged with VIIR (near arg-10, probably distal). Wild phenotype. Homozygous-fertile. T X N ascopores > 50% black; unordered asci 37:11:35:9:9 ( N = 151). Origin: Em a, W. Detected and analyzed by Perkins. Generates viable duplications from intercross with T(IVR ;VII)ARlO. Assignment to IVR is based on this observation. Intercross shows not identical with T(IV;VII)NMlBG. FGSC 2026, 2027.

THE CYTOGENETICS OF

Neurospora

247

T(IV + VI)ALS159 Quasiterminal translocation. All IVR markers are translocated to the right end of VI beyond trp-2. Wild phenotype. Homozygous-fertile. T x N ascospores 75% black; unordered asci 33:22:32:5:8 ( N = 259). Origin: rg cr a, UV. Detected and analyzed by Perkins. FGSC 2100,2101. Duplications: D p ( I V R + VIR)ALS169. One-third of viable progeny from T x N . Usually recognizable by reduced conidiation on minimal slants at 34°C or patches of hyphae without conidia. Barrenness of duplications is exceptionally stable in crosses to nonduplications. A few individual perithecia become fertile. Markers shown covered: pyr-1, un-8, pdx, pur-3, cot-1, cys-4, uvs-2. T ( V;VII ) NM159 Reciprocal translocation. V (near a l ) interchanged with VII (near wc). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black (defective spores become brown) ; unordered asci 25:2:44:3:25 ( N = 88). Origin: Em a, W. Detected and analyzed by Perkins. FGSC 2411, 2412.

T ( 1;IV ) NM160 Reciprocal translocation. I R (right of nic-2) interchanged with IVR (near COG,$). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 31:6:15:6:42 (N = 209). Origin: Em a, UV. Detected and analyzed by Perkins. Intercrosses indicate identical sequence with T ( I ; I V ) N M l 6 2 and with N M i 6 7 , which is female-sterile. Generates viable duplications from crosses with T ( I R ; IVR)NM140, NMIQQ, and NM172. Strain of origin also contained linked but separable point mutant phe-1 (NM160). FGSC 1338, 1337. T( II;III)C161 aro Reciprocal translocation. IIR (in nro cluster) interchanged with I11 (linked acr-2). Multiple deficiencies in aromatic synthetic enzymes. Homozygous fertility not tested. T x N ascospores 50% black; unordered asci 20:2:48:4:27 (N = 157). Origin: Recognized aberrant by Gross and Fein (1960). I11 linkage found by Perkins. Called arom-2 by Gross and Fein. This differs from the point mutant Y306M81 called arom-2 by Giles et al. (1967). FGSC 2106, 2107. T ( II;III)NMl61 Reciprocal translocation. I1 (linked arg-6) interchanged with IIIR (linked t i p - I ) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 18:4:46:7:26 ( N = 179). Origin: Em a, UV. Detected by Perkins. Beske and Phillips (1968) showed I or I1 linked with I11 or VI. Further analysis by Perkins. Generates viable duplications from cross with T ( I I ; I I I R ) A R 6 2 . Intercross shows not identical with T ( I I ; I I I R ) N M 1 6 0 .FGSC 2028, 2029. T ( I;IV)NM162 Structurally identical with T ( 1 ; I V )NM160, q.v. Found in same isolate with T ( V ; V I ) N M 1 6 2 b .FGSC 2589, 2590. Reference: Perkins, 1974. T ( V;VI)NM162b Reciprocal translocation. VR (linked id) interchanged with VI (linked ylo-1). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered

248

DAVID D. PERKINS AND EDWARD G. BARRY

asci 15:2:52:6:25 (N = 182). Origin: Em a, UV. Detected and analyzed by Perkins. Found in same isolate with T ( 1 ; I V )NM162. Intercrosses show not identical with T ( V R ; V I R ) N M 1 6 7 or N M 1 7 l . FGSC 2.591, 2592. Reference: Perkins, 1974.

T(I;VI)NM163 Reciprocal translocation. I (linked mt) interchanged with VIL (near chol-2). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 13:0:67:2:18 ( N = 45). Origin: Em a, W. Detected by Perkins. Beske and Phillips (1968) showed I or I1 linked with I11 or VI. Further analysis b y Perkins. FGSC 2030, 2756.

T(1;IV)NMlB.I

Reciprocal translocation. I R (linked al-2, proximal) interchanged with IVR (proximal to trp-4). Wild phenotype. Homozygous-fertile. T X N ascospores 50% black; unordered asci 29:4:27:3:36 ( N 294). Also 32:12:45:1:9 (N = 106Kowles). Cytology: Chromosome 2 is not part of the rearrangement (Barry). Origin: Em a, UV. Detected by Perkins. Genetic analysis by Perkins, Kowles. Generates viable duplications from intercrosses with T ( 1 ; I V )T64M19, NM144, and NM172. FGSC 1341, 1340. References: B'arry and Perkins, 1969; Kowles, 1972,

-

1973.

T(I;VII)ALS167

Reciprocal translocation. I (not separated from rg or cr) interchanged with VII (near w c ) . T x T cross infertile because of markers. T x N ascospores 50% black or more; unordered asci 58:9:6:1:27 ( N = 144). Origin: rg cr a, UV. Detected and analyzed by Perkins. FGSC 2413,2529.

T(1;IV) NM167

Structurally identical with T ( 1 ; l V )NMi60, q.v. Female-sterile. FGSC 1343, 1342.

T ( I;II)NMlB8 Reciprocal translocation. I (probably L-near m t ) interchanged with I I R (near $). Wild phenotype. Homozygous-fertile, T x N ascospores > 50% black; unordered asci 25:24:40:6:5 (N = 185). Origin: Em a, UV. Recognized aberrant by Perkins. Analyzed by Anna Kruszewska. Evidently some inviable duplicationdeficiency ascospores become black. Germination of black ascospores is low. No viable duplications are recovered. FGSC 1923, 1924. T ( I + )NM16Qd Duplication-generating rearrangement in which a distal I R segment containing un-18 is covered in the duplications. Location of the segment is not known in rearranged sequence. Wild phenotype. Homozygous-fertile. T x N ascospores > 75% black; unordered asci 29:54:10:6:1 ( N = 124). Origin: Em a, UV. Detected by Perkins. Analyzed by Perkins and B. C. Turner. Strain of origin also contained T ( I I I R ; V I I ) N M 1 6 9 r .FGSC 2279, 22fUl. Duplications: D p ( I R + )NM169d. Stably barren in crosses by nonduplication. Markers shown covered: un-18. Markers shown not covered: arg-13, so, aro-8, R . Duplications possess the vegetative morphology characteristic of R , and this is known from other rearrangements to be recessive when a duplication is heterozygous R / R + .

THE CYTOGENETICS OF

Neurospora

249

T(II1;VII)NMISSr

Reciprocal translocation. IIIR (linked leu-1 trp-1) interchanged with VIIR (near wc to right). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 34:4:30:1:31 (N = 143). Origin: Em a, W. Detected and analyzed by Perkins. Original isolate also contained T ( I R + )NM169d. FGSC 1816, 1817. Reference : Perkins, 1974.

T(I;IV)NMl7O

Reciprocal translocation. I (linked m l ) interchanged with IV (linked cot-1). “Creamy” morphology. Female-sterile. T x N ascospores 50% black ; unordered asci 11:4:54:9:22 ( N = 46). Origin: Em a, UV. Detected by Perkins. Beske and Phillips (1968) showed I or I1 linked with I V or V. Further analysis by Perkins. FGSC 1489.

T ( V;VI ) NM171

Reciprocal translocation. V (near a t ) interchanged with VI (near ylo-1). Wild phenotype. Homozygous-fertile, T x N ascospores 50% black ; unordered asci 24:3:17:0:55 ( N = 29). Origin: Em a, W. Detected and analyzed by Perkins. Intercross indicates not identical with T ( V R ; V I )NM162b. FGSC 2451, 2452.

T(I;IV)NM172 Reciprocal translocation. I R (linked 0s-1, distal) interchanged with IVR (proximal to t7p-4). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black or more; unordered asci 10:17:62:5:6 ( N = 314). Also 30:6:46:4:15 (N = 139Kowles). Cytology: Chromosome 2 is not part of the rearrangement (Barry). Origin: Em a, UV. Detected by Perkins. Genetic analysis by Perkins, Kowles. Apparently one class of duplication-deficiency ascospores may become pigmented. Identical structure with T ( I R ;ZVR) NM119 by intercross. Generates viable duplications from intercrosses with T ( l R ; l V R ) N M 1 3 9 , NMl40, NM144, NM160, and NM164. FGSC 1345, 1518. References: Barry and Perkins, 1969; Perkins, 1971a; Kowles, 1972, 1973.

T(I + VII;I;V;VII)AR173

Complex insertional translocation involving I R (near centromere), V (near a t ) and VII (near w c ) . A short proximal I R segment including un-2 and h i s 4 is inserted in VII. Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 41:8:14:7:31 ( N = 212). Origin: 74-OR23-1A,UV. Detected and analyzed by Perkins. FGSC 2468,2469.References: Perkins, 1974. Duplications: D p ( l R + V I I ; I ; V ; V I I ) A R l 7 3 . In one-third of surviving progeny from T x N . Stably barren in crosses. Markers shown covered: un-2 his-2. Markers shown not covered: j r , nit-2, leu-3, m t , arg-1, arg-3, sn, rg, nuc-1, lys-4, met-10, his-3; lys-1, at, al-3, m o ( M 1 8 4 ) , mo(M193-1). (Data on nuc-1-his-3 from R. L. Metzenberg.)

T ( V;VI ) A m 7 4

Reciprocal translocation. VR (linked i d ) interchanged with VI (linked ~ Z O - 1 ) . Wild phenotype. Homozygous-fertile. T x N ascospores > 50% black; unordered asci 9:12:53:12:14 ( N = 58). Origin: 74-OR23-1A,W.Detected and analyzed by

250

DAVID D. PERKINS AND EDWARD G. BARRY

Perkins. Original isolate also contained linked mutant gene resulting in nonblack perithecia and ascospores. FGSC 2678, 2679.

T(VII-IV)ALS175

A duplication-generating translocation involving VII (linked wc) and IV (linked pdz). Either IV is the recipient or pdz is covered by the duplication. Wild phenotype. Homosygous-fertile. T x N ascospores < 50% black; unordered asci 0:3:28:19:51 ( N = 75). Origin: rg cr, W. Detected by Perkins and early analysis by D. A. Smith. FGSC 2931, 2932. Duplications: Dp(Vll-ZV)ALSlT6. Sub-wild vegetative phenotype. Barren in crosses with nonduplication.

T(I;V)AR175

Reciprocal translocation. I (linked mt) interchanged with V (near a t ) . Near-wild phenotype (bleeds high in slant), Homozygous-fertile. T X N ascospores 50% black; unordered asci 39:8:13:5:34 ( N = 165). Origin 74-OR23-1A, W. Detected and analyzed by Perkins. FGSC 2593, 2594.

T(IV;VI)NM175

Reciprocal translocation. IVR (linked pdz) interchanged with VIR (linked ylo-I). Wild phenotype. Homozygous-fertile. T x N ascospores 5oq0 black; unordered asci 49:0:5:5:42 ( N = 41). Origin: Em a, UV. Detected and analyzed by Perkins. Generates viable duplications from intercross with T(IVR;VIR)46602. Arm assignments are on this basis. FGSC 2295, 2293.

T ( I I + V)ALS176 Translocation involving I I R (between bal and arg-6) and V (linked at; probably L) . Probably quasiterminal. Wild phenotype. Homozygous-fertile. T x N ascospores 75% black, or less; unordered asci 24:43:28:3:2 ( N = 126). Origin: rg cr a, W. Detected and analyzed by Perkins. FGSC 2414, 2415. Duplications: Dp(ZIR + VL)ALSlTG. In one-third of viable progeny from T x N . Initially thin, transparent, slow growth. Not stably barren in crosses by nonduplication, mostly behaving like normal sequence. Markers shown covered : arg-6, m o d , cpt, arg-12, fl. (Other markers, in IIL, may be heterozygous in disomics from 3: 1 segregations, which occur with frequencies of several percent.) Markers shown not covered in simple duplications : bnl, pyr-4.

T(1;III;VI;VII)AR176

Complex rearrangement. Linked closely to acr-2 (III), ylo-1 (VI), and wc (VII) and less closely to mt (I). The structural basis is not understood. Wild phenotype. Homozygous-fertile. Ascospores oozed, few shot. T x N ascospores < 25% black; unordered asci 8:6:30:20:36 ( N = 154). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. FGSC 2708, 2709. Duplications: Dp(V1II + VZt)ARlTG. In less than one-third of surviving progeny from T x N . Duplications are recognizable by flat morphology. Very stably barren. Markers shown covered: None. Markers shown not covered: mt, acr-2, rib-1, met-7, nt.

THE CYTOGENETICS OF

Neurospora

251

In( IL + IR)NM176 Pericentric inversion. A distal segment of IL (including ser-3 but not un-3 or m t ) is interchanged with the I R tip. No bona fide euploid recombinant with mating type has been obtained. Wild phenotype. I n x N ascospores 75% black, or more ; unordered asci 43:43: 11: 1 :1 ( N = 1362). Cytology : Inversion id chromosome 1 confirmed cytologically (Barry). Origin: Em a, W. Recognized aberrant by Perkins ; genetic analysis by Turner and Taylor. Generates viable duplications from intercross with In(IL + IRIH4260, and these are A / a heterozygotes which are phenotypically inhibited Dark-Agar types. Break points and genetic behavior resemble In(IL + IR)ARlG. The structure is formally similar to In(lLR)scv’ in Drosophila. FGSC 1613. Reference: Turner et al., 1969. Duplications: Dp(IL + IR)NMlYG. About one-fourth of viable progeny from I n x N . Phenotype nearly wild, but with characteristic growth habit of aerial hyphae. Barren in crosses with nonduplications, eventually becoming fertile by loss of one duplicated I L segment, usually but not always that in the translocated position. Occasionally, duplication may be transmitted to progeny without breakage. Instability increased by mei-3 (Newmeyer and Galeazzi, 1976b). Markers shown covered: fr, un-5, leu-3, cyt-1, cys-6, ser-3. Markers shown not covered: un-3, mt, sue.

T(III;V)AR177

Reciprocal translocation. I11 (linked acr-2) interchanged with V (near at, probably VR). Wild phenotype. Homozygous crosses produce only a few spores. T x N ascospores > 50% black; unordered asci 37:16:34:4:9 ( N = 153). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. Apparently one duplicationdeficiency class consists of inviable black ascospores. Germination of black ascospores is 66%. No viable duplications recovered. FGSC 2680, 2681.

T ( I I + I)NM177 Insertional translocation. A segment of I I R between aro-3 and aro-1 is inserted in IL between leu4 and mt. Subtle morphological phenotype. Homozygous-fertile. T x N ascospores 75% black; unordered asci 28:41:29:1:1 ( N = 179). Origin: E m a, UV. Discovered and linkage groups identified by Perkins. Mapping and genetic analysis by A. Kruszewska. Separated with difficulty from closely linked morphological factor originally present. FGSC 1610, 2003. References : Perkins, 1974; Metzenberg et al., 1974; Littlewood et al., 1975. Duplications: Dp(IIR + IL)NM177. I n one-third of surviving progeny from T x N . Duplications are highly stable but sectoring may occur, with loss of material usually from the inserted location (Metzenberg et al., 1974). Vegetative “escapes” are still barren. Markers shown covered: peon (nuc-Z), pe, arg-12. Markers shown not covered: aro-3, aro-I. T(VI1 + IV)ALS179 Translocation, probably quasiterminal. An unmarked distal segment of VIIL is translocated to IVR near uvs-2. Wild phenotype. Homozygous-fertile. 2’ X N ascospores 75% black; unordered asci 18:66:13:2:1 (N = 141). Origin: c r r g a, UV. Detected and analyzed by Perkins. FGSC 2264,2265.

253

DAVID D. PERKINS AND EDWARD G . BARRY

Duplications: Dp(VIZL + IVR)ALSl79. In one-third of surviving progeny from T x N . Wild phenotype; ecorable as barren. Markers shown covered: None. Markers shown not covered: het-e, nic-3, thid.

T(11+ [IV;V] )AR179

Complex insertional translocation. A IIL segment is inserted, with break points closely linked to pdx (IV) and at (V) in T x N crosses. Near-wild phenotype, slightly less vigorous. Homosygous-barren. Forms perithecia but no ascospores. T x N ascospores < 50% black; unordered asci 0:5:9:34:52 ( N = 65). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. FGSC 2595,2596.

Note added in proof: The short arm of chromosome 2, which carries the nucleolus organizer, is clearly mvolved cytologically. Duplications: Dp(ZIL + [ZV;Vl)AR17Q.In one-third of surviving progeny from T x N . Markers may come uncovered somatically. Barren in crosses. Some are fully stable. Markers shown covered: pyr-4, het-c, rod, thr-2. Markers shown not covered : arg-6.

T(1;IV)ARlSOb

Reciprocal translocation. I (linked m t ) interchanged with IVR (near col-4). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 42:6:29:4:19 ( N = 195). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. Separated from T(ZR;IZIR)AR180r, which was present in the same isolate. FGSC 2754, 2755.

T(I;III)AR180r

Reciprocal translocation. I R (linked aur) interchanged with IIIR (linked trp-I). Wild phenotype. T x T crosses show reduced fertility. T x N ascospores 50% black; unordered asci 13:0:55:4:28 ( N = 127). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. Separated from T(I;IVR)ARlBOb, which waa present in strain of origin. FGSC 2939, 2940.

T(1I;V)NMlSO

Reciprocal translocation. I I R (near arg-12, to right) interchanged with VR (between lys-1 and inl). Flat vegetative morphology. T x T crosses sterile with no perithecia. T x N ascospores 50% black; unordered asci 25:9:41:4:22 ( N = 69). Origin: Em a, UV. Detected and analyzed by Perkins. FGSC 2031, 1491.

T(1I;VI)ARlSl

Reciprocal translocation. IIR (linked bal and fl) interchanged with VI (linked ylo-1). Wild phenotype. Homozygous-fertile. T x N ascospores CO% black; unordered asci 21:7:42:11:20 ( N = 189). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. FGSC 2453, 2454.

T(1;IV)NMlSl

Reciprocal translocation. I (linked m t ) interchanged with IVR (near cot-1 1. Wild phenotype. Homosygous-fertile. T x N ascospores 50% black ; unordered asci 37:2:35:1:24 (N = 241). Origin: Em a, W. Detected and analyzed by Perkins. FGSC 2933, 2934.

THE CYTOGENETICS OF

Neurospora

253

T ( I + )ALS182 Duplication-generating rearrangement, incompletely analyzed but probably with inverted insertion. A breakpoint in I R is between thi-1 and cyh-1. Wild phenotype. Homozygous-fertile. T x N ascospores 75% black; unordered asci 29:37:26: 4:5 ( N = 164). Cytology: Bridges and fragments observed a t meiotic divisions, but abnormal chromosomes not identified. Origin: r g cr a, UV. Detected and analyzed genetically by Perkins. Note added in proof: The IR segment of ALS182, which includes meld, is translocated to the tip of VL, distal to the nucleolus organizer. Viable duplications covering nic-2, cr, and thi-l are produced in crosses of ALSl82 x AR190. Duplications: D p ( I R += )ALS182. In one-third of viable progeny from T x N . Vegetative morphology of duplications is perhaps flat initially. If barren, only fleetingly; quickly become fertile and behave as normal sequence. When gene 1c is heterozygous, both R and R' ascospores result. When a1 is heterozygous, i t sectors vegetatively. Markers shown covered : cyh-1, 01-2, al-1, R, un-18. Markers shown not covered: thi-2, fr, un-6.

T( I;VI)AR182

Reciprocal translocation. I (linked cr) interchanged with VI (linked ylo-I). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 37:8:29:9:17 ( N = 194). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. FGSC 2597. 2598.

T( III;V)NM183 Reciprocal translocation. I11 (linked acr-2) interchanged with V (linked at). Wild phenotype. Homozygous barren. T x N ascospores > 50% black; unordered asci 45:11:29:4:11 ( N = 160). Origin: Em a, UV. Detected and analyzed by Perkins. Intercross shows not identical with T(1ZZ;V)N M l O l . FGSC 2633,2634. T( V;VI)AR184 Reciprocal translocation. V (near a t ) interchanged with VI (near y20-1). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 35:13:20:8:24 (N = 143). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. FGSC 2416, 2417. T( III;VI)AR186 Reciprocal translocation. I11 (linked acr-2) interchanged with VI (linked Y~O-1). Wild phenotype. (Slightly flat on synthetic cross medium.) Homozygous-fertile. T x N ascospores < 75% black; unordered asci 44:12:26:6:12 ( N = 173). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. Evidently some inviable duplication-deficiency ascospores darken. FGSC 1925, 1926. T( I;IV)NM187 bs See T ( 1 ; I V ) N M I S Qbs. T ( I + V)ARlSO Quasiterminal translocation. Nearly the entire long arm of I, with all known I R markers except un-2, is attached 35 units left of centromere in V. Wild phenotype. Homozygous-fertile. T x N ascospores 75% black or more; unordered asci 32:29:

254

DAVID D. PERKINS AND EDWARD G . BARRY

31:5:3 (N = 211). Cytology: Break point in chromosome 1 at a or b chromomere, and in 2 in satellite distal to the nucleolus organizer. For pachytene cytology see Fig. 3 in Barry and Perkins (1969). Origin : 74-OR23-1A, W. Detected and analyzed genetically by Perkins. FGSC 1951, 1952. References: Barry and Perkins, 1969; D. E. A. Catcheside, 1969. Duplications: D p ( I R + VL)ARIQO. One-third of viable progeny from T x N . Duplications grow slowly after germination from ascospores. Somatic sectoring is often seen when albino is heterozygous. Duplications break down somatically and premeiotically to give normal sequence by complete loss of the I R segment from the translocation sequence. Barrenness is transient, making duplications difficult to score. The duplication is not transmitted to progeny. D. E. A. Catcheside has used ARl90 to test complementation among albino mutants. Markers shown covered: his-2, nuc-1, me-10, ad-3A, nic-2, cr, cyh-1, aG2, al-1, R . Markers shown not covered: un-2, sn, urg-1, m t . (Information on nuc-1 from R. L. Metzenberg.)

T(I;IV)AR193

Reciprocal translocation. I (linked m t ) interchanged with IV (near p d z ) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 41:9:26:5:20 (N = 189). Origin: 74-OR23-1A, W. Detected and analyzed by Perkins. FGSC 2470, 2471.

T(IV;VI)AR207

Reciprocal translocation. IVR (linked pan-1, cot-1) interchanged with VI (near ylo-1). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black (variable); unordered asci 40:9:18:8:24 ( N = 312). Origin: 74-OR23-1A, W. Detected and analyzed by Perkins. FGSC 1927, 1928.

T ( 1;III ) AR208 Reciprocal translocation. I R (right of cr) interchanged with I11 (near acr-2). Wild phenotype. Homozygous-fertile. T x N ascospores 75% black or more; unordered asci 48:21:25:2:4 ( N = 468). Origin: 74-OR23-1A, W. Detected and analyzed by Perkins. Evidently one duplication-deficiency class makes inviable black ascospores. Germination is low among black ascospores. No viable duplications. FGSC 1929. 1930.

T(VI + IV)AR209

Quasiterminal translocation. Right arm of V I with p u n 4 and distal markers translocated tc iight end of IV. Wild phenotype. Homozygous-fertile. T x N ascospores < 75% black; unordered asci 16:33:28:10:13 ( N = 162). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. Used as het.9 tester to study vegetative incompatibility. FGSC 1931, 1932 (het-goR).Reference : Mylyk, 1975. Duplications: D p ( V I R + IVR)AR209. In one-third of surviving progeny from T x N . Wild phenotype. Not detectably barren in crosses to non-duplication; duplications behave like fertile normal sequence, apparently by rapid loss of segment in T sequence. Markers shown covered: pun-2, trp-2, het-9, probably rib-1. Markers shown not covered: chol-2, lys-6, ylo-1, ad-1.

THE CYTOGENETICS

OF

Neurospora

255

T ( III;IV)AR211 Reciprocal translocation. I11 (near acr-2) interchanged with IV (linked col-4, probably left). Wild phenotype. Homozygous-fertile. T x N ascospores > 50% black; unordered asci 55:5:33:2:5 ( N = 677). Cytology: Preliminary observations of pachytene chromosomes have indicated abnormalities of 3 and 4. Origin: 74OR23-1A, UV. Detected and analyzed by Perkins. Apparently many duplicationdeficiency ascospores darken. No barren progeny are found, and germination is usually low among black ascospores. FGSC 1933, 1934. T(I;IV)AR212 Reciprocal translocation. IR (near al-1) interchanged with IVR (between pdz, m a t ) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 26:2:50:3:19 ( N = 124). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. FGSC 1521, 1522. T ( I;II)AR216 Reciprocal translocation. I R (proximal to a,!+?) interchanged with IIL (between pyr-4 and bal). Wild phenotype. Homozygous-fertile. T x N ascospores > 50% black; unordered asci 44:8:16:0:32 (N = 262). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. Original isolate also contained a linked but separable albino mutant. FGSC 1950, 1607.

T(I + ;II;VII)AR217

Complex insertional translocation. A central segment of IR, including ad-9 and nic-1, is inserted elsewhere. Other breakpoints are in I1 (near arg-6) and VII (near w c ) . Wild phenotype. Homozygous-barren. T x N ascospores 5070black or more; unordered asci 46:10:13:5:27 ( N = 166). Origin: 74-OR23-1A, W. Detected and analyzed genetically by Perkins. FGSC 2418.

Duplications: D p ( I R + ; I I ; V I I ) A R 2 1 Y . In one-third of surviving progeny from T x N . Slow t o conidiate, pale pigment. Partially barren, with few ascospores shot from perithecia. Markers shown covered. ad-9, cyh-1, al-1, nic-I. Markers shown not covered: his-2, cr, 0s-1.

T ( IV;V)AR221 Reciprocal translocation. IVR (near COZ-4) interchanged with VR (between a t and ilv). Wild phenotype. Homozygous-fertile. T x N ascopores 50% black; unordered asci 37:6:8:7:43 ( N = 183). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. Strain of origin also contained linked but separable mutation, ilv (ARBl). FGSC 2034, 2035.

T(I;VI)Y234M419

Reciprocal translocation. I (near m t ) interchanged with VI (between Yl0-1 and trp-2). Wild phenotype. Homozygous-fertile. Translocation ascospores are slow t o blacken. T N ascospores < 50% black because of slow ripening. Unordered asci 19:4:25:8:44 (N = 167). Origin: 74A, UV. Detected and analyzed by Perkins. Strain of origin also contained closely linked point mutant ad-1 (74A-Y234-M419). FGSC 2635, 2636.

x

T ( II;IV)Y256M230 Reciprocal translocation. I1 (near arg-5) interchanged with IV (near Col-4). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci

256

DAVID D. PERKINS AND EDWARD G. BARRY

45:5:11:5:33 ( N = 150). Origin: 74A,W. Detected and analyzed by Perkins. Strain of origin also contained unlinked point mutant ylo-2. FGSC 917,1556.

T( I;IV)D304 Reciprocal translocation. I (linked mt, probably right) interchanged with IV (between cys-10 and trp-4). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 46:3:11:3:36 (N = 351). Also 50:4:19:1:26 (N =102Kowles). Intercross with T(ZR;IVR)NMl?Z gives ascus patterns expected if viable duplications are generated, but no such duplications were recovered. Origin : inl (89601), W.Detected by Perkins. Mapped and analyzed by Anna Kruszewska, further data by Kowles. Strain of origin also contained closely linked but separable morphological mutant. FGSC 1443,1444.Reference : Kowles, 1972, 1973. T( III + jIII;VI)D305 A complex translocation involving IIIR distal to trp-1 and VIL near chol-2. Generates viable duplications of a I11 segment. Wild phenotype. Homozygousbarren. T X N ascospores < 50% black; unordered asci 1:8:49:26:17 ( N = 211); T progeny are recovered less frequently than N . Origin: inl (89601), W. Detected and analyzed by Perkins. Used as het-7 tester to study vegetative incompatibility. FGSC 2139,2140. Reference : Mylyk, 1975. Duplications: Dp(ZZZR + )0306. From T x N. Slow or fluffy-like morphology. Variable fertility. Semibarren. Identification difficult, frequency uncertain. 3: 1 segregations are not excluded as the cause of some duplications. Markers shown covered: phe-2, tyr-1, dow, het-7; vet ( 1 ) . Markers shown not covered: acr-2, thi-2, trp-1.

T( IV + I)B362i Duplication-generating translocation. A segment of IV (near p d z ) is translocated to I (linked mt). Wild phenotype. Homozygous fertility not tested. T x N ascospores 80% black; unordered asci 61:20:16:3:0 ( N = 148). Origin: STA, gamma rays. Aneupoidy noted by D. Newmeyer. Extracted, diagnosed, and I linkage shown by D. A. Smith. Originated in same strain with unlinked T(ZZ;VZ)B362r and arg-10 (B362). FGSC 2935. Duplications: Dp(ZV+ Z)B362i. I n one third of viable progeny from I' x N. Stably barren. Markers shown covered: None (only p d z tested). Markers shown not covered: p d z .

T( IIjVI)B362r Reciprocal translocation. I1 (linked bal) interchanged with VI (linked ylo-1). Wild phenotype. Homozygous-fertile. T X N ascospores 50% black ; unordered asci 23:10:31:16:20 (N = 260). Origin: STA, gamma rays. Extracted and analyzed by D. A. Smith. Strain of origin also contained unlinked T(ZV + Z)B362i and arg-10 (B362). T( IVjVII)STL384b Reciprocal translocation. IVR (near col-4 and to right) interchanged with VIIR (near sk). Wild phenotype. T x T sterile with no perithecia. T x N ascospores 75% black; unordered asci 23:42:28:4:3 ( N = 803). Evidently one duplication-

THE CYTOGENETICS OF

Neurospora

257

deficiency class makes inviable black ascospores. Germination among black ascospores is low. No viable duplications. Origin: Detected by P. St. Lawrence in a single aberrant perithecium from a cross mi-1 384a 9 (maternal inheritance) x 74-ORB-1A 8 . Ascus of origin also contained linked but separable T(ZV;VZ) STL384r. Rearrangements resolved and mapped by Perkins. FGSC 2421, 2422.

T( IV;VI)STL384r Reciprocal translocation. IVR (near cot-1) interchanged with VI (linked y b - 1 ) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 25:6:53:4:12 (N = 362). Origin: Detected by P. St. Lawrence in a single aberrant perithecium from a cross mi-1 384a 0 (maternal inheritance) x 74-0R231A 8 . Ascus of origin also contained T(lV;VlZ)STLS84b.Rearrangements resolved and mapped by Perkins. FGSC 2419,2420. T ( V;VI ) A420 Reciprocal translocation. V (near a t ) interchanged with VI (near yb-1). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 55:2:9:0:34 ( N = 170). Origin: Em a, UV. Detected and analyzed by Perkins. Original isolate contained linked but separable mutant trp-6 (Ahmad et al., 1968). FGSC 2334, 2335. T(I;VII)TM429 his3 Reciprocal translocation. I R (at his-!?) interchanged with VII (near met-7). Phenotype his-!?; all three activities are missing. Wild morphology. Homozygous-fertile. T x N ascospores 50% black, or more; unordered asci 30:9:23:7:31 (N = 627). Origin: 429 cot-1 a, W. Translocation identified and his-3 association shown by Angel. VII breakpoint mapped by Perkins. Inviable duplication-deficiency spores darken with aging. FGSC 2530, 2531. References: Angel et al., 1970; Angel, 1971; Catcheside and Angel, 1974. T( I;VI)P649 Reciprocal translocation. I R (near aur) interchanged with VI (near ylo-1). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 33:3:50:5:10 (N = 224). Origin: Spontaneous in backcross of cr thi-1 nit-1 aur nic-1 as-1 a x STA4. Detected and analyzed by Perkins. Generates viable duplications from intercross with T(ZR;VI)ARlS.FGSC 1608, 1609. T( I;VII)S1007 Reciprocal translocation. I (near his-$) interchanged with VII (near met-7). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 58:5:2:2:33 ( N = 205). Cytology: Reciprocal translocation confirmed. Break points are in the long arm of chromosome 1 and near the middle of chromosome 7 (Barry). Origin: Detected by Perkins in as71 strain S1007-2(1-4)a (unlinked asn mutant was X-ray induced). Generates viable duplications from intercross with T(I;VII)K79 met-7. FGSC 227, 224. References: Barry, 1967; Perkins el at., 1962.

T(III;V;VII)P1156

Complex interchange, Break points near leu4 (IIIR), al-3 (VR), thi-3 (VIIL). Wild phenotype. Homozygous-fertile. T x N ascospores < 50% black; unordered

258

DAVID D. PERKINS AND EDWARD G. BARRY

asci 14:1:49:19:18 ( N = 125). Origin: Detected among progeny of a cross n i c J roS, and analyzed by Perkins. FGSC 2599,2800.

x

Duplications: About one-fifth of progeny from T x N are stably barren. Their constitution is not understood. Failure to obtain consistent marker ratios among barrens suggests origin by nondisjunction (3: 1 segregations).

T(IV + VII;I;II;IV)S1229 arg pe

Complex translocation. Interchange points in IL (near arg-3 and m t ) ; in I I R at p e ; in IVR, with markers between pdx and p y r S inserted into VII near thi-3. Arms of groups I, 11, and IV translocated in progressive interchange. Inseparable from pe, which was present in the stock of origin, and from requirement for arginine, citrulline, or ornithine, which arose simultaneously with the aberration. Homozygous-barren. A few ascospores are produced, however. T x N ascospores 40% black; unordered asci 24:2:27:10:36 ( N = 704). Cytology: Initial cytological examination (1960) found chromosomes 1, 2, 6, and 7 aberrant. However, chromosome 2 was later shown not to be involved (1969). Origin: pe fl Y8743-21-(13-7)a, X-rays. Identified as aberrant by Barratt and Garnjobst. Genetic and cytological analysis by Barry. FGSC 2946, 268. References: Barratt et a l , 1954; Barry, 1960a,b; Barry and Perkins, 1969. Duplications: D p ( I V R + VII)S1229. One third of viable progeny from T x N . Wild, but grows at slower rate. Crosses with either duplication or non-duplication strains are almost barren, producing very few spores. Duplications are stable through crosses, and segregate 1:1 in progeny from D p x N . Markers shown covered: p t , met-1, cys(oxD'), col-4, arg-2. Markers shown not covered: pyr-1, pdx, pyr-3, h i s d , trp-4, pan-i, cot-1, m e t d , his-4, pyr-2. (FGSC 264, 265 are Si229 Duplications.)

T(I S V)S1325 nic-2

Insertional translocation. A long central segment of I R is inserted in inverted order into VR between his-1 and inl. No viable duplications are produced; thus a short mutual VR + IR insertion is postulated. The rearrangement is inseparable from the nic-2 phenotype with which it arose. Wild morphology. Homozygousfertile. T x N ascospores 50% black or less; unordered asci 5:0:71:3:21 ( N = 223). Cytology : Singleton observed acentric chromosome fragments and dicentric bridges in the meiotic divisions in the ascus and concluded a paracentric inversion was present, thus confirming St. Lawrence's genetic interpretation of an inversion in linkage group IR. Further genetic and cytological investigation by N. E. Murray, Perkins, and Barry showed the aberration to be actually an inverted insertion of a long segment from I R (chromosome 1) into VR (chromosome 2). The acentric fragments persist in micronuclei and replicate. Origin: pe fl Y874321(13-7)a, X-rays. Detected and I involvement analyzed by St. Lawrence. V involvement shown by Perkins and Murray following evidence of Newmeyer that another chromosome was involved. The inserted segment includes thi-1, met-6, ad-9, a M , and al-1. FGSC 1558, 1557. References: St. Lawrence and Singleton, 1963; Murray, 1968a; Barry and Perkins, 1969; Barry, 1973.

T(1;V)C-1670 pk-1

Reciprocal translocation. IR (near centromere) interchanged with VR (at bis) . Phenotype pk-1 ( p e a k 4 morphology (allele of bis) . Homozygous-fertile. T x

THE CYTOGENETICS OF

Neurospora

259

N ascospores 50% black; unordered asci 33:3:46:3:15 ( N = 270). Cytology: Reciprocal translocation involving chromosome 2 shown by Phillips. Origin : 74A, p propiolactone. Detected and linkage groups identified by J. C. Murray. Generates viable duplications from intercrosses with T ( I R;VR )36703 and ALSIII. FGSC 483, 2761. References: J. C. Murray, 1959; Phillips, 1967; Perkins, 1971a.

T(I;VII)P1676

Reciprocal translocation. I (linked m t ) interchanged with VII (near W C ) . Wild phenotype. Homozygous fertile. T x N ascospores 50% black; unordered asci 61 :8 :10: 1 :20 ( N = 277). Origin : Spontaneous in cross 74-OR8-la x trp-I. Detected and analyzed by Perkins. FGSC 1935, 1936.

T ( V;VI) JH2003

Reciprocal translocation. V (linked at) interchanged with VI (near ylo-I). Wild phenotype. Homozygous-fertile. T x N ascospores > 50% black (probably because some defective white spores disintegrate) ; unordered asci 53: ll :16:3:16 (N = 220). Origin: Detected by Perkins in nit(JH.2003) (Silver and McElroy, 19541, which was obtained by FGSC from J. R. S. Fincham. Freed of nit mutation. FGSC 2423,2424.

T ( 1V;V) R2355

Reciprocal translocation. IVR (near cot-I) interchanged with VR (proximal to his-I). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 17:0:65: 1:17 ( N = 155). Cytology: Cytological examinations by R. L. Phillips and by J. R. Singleton (unpublished) confirmed the reciprocal translocation, and found chromosome 2 involvement. Origin: inl (896011, UV. Detected and analyzed genetically by Perkins et al. A component of alcoy linkage tester. Generates viable duplications from intercrosses with T(IV;V)ARIIT,NM141, and NM146. FGSC %I, 222. References: Phillips, 1967; Perkins et al., 1962, 1969; Perkins, 1971a.

T(I1 + IV)R2394

Insertional translocation. A I1 segment near bal (probably IIL) is translocated to IV (linked pdx). Wild phenotype. Homozygous-fertile. T x N ascospores > 7570 black; unordered asci 31:49:17:3:0 ( N = 121). Origin: inE (89601), UV. Recognized aberrant by Garnjobst and Tatum (1967). Analyzed by Perkins. FGSC 2757, 2758. Duplications: D p ( I I + I V ) R 2 9 4 , present in one third of viable progeny from T x N . Barren. Markers shown covered: None. Markers shown not covered: bal,

Pyr-4,

11.

T( + I)R2472 arg

Duplication-generating translocation. A segment of unknown constitution is translocated to I (linked m t ) . Flat vegetative growth; not separated from a requirement for arginine or aspartate or asparagine. Not homozygous-fertile. T x N ascospores 50% black; unordered asci 4:30:23:26:17 ( N = 100). Origin: inl (89601), W. Recognized aberrant by Garnjobst and Tatum (1967). Preliminary analysis by Perkins.

<

Duplications: Dp( + ILIR2479, present in one-third of viable progeny from T x N . Barren in crosses with nonduplication.

260

DAVID D. PERKINS AND EDWARD G. BARRY

T(I;III)P2848

Reciprocal translocation. I R (proximal to aur) interchanged with I11 (linked trp-I). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 18:3:59:4:16 ( N = 192). Origin: Found in a single f, from 74-ORB-1 A x arg-I2 a. Beske and Phillips (1968) showed I or I1 and TI1 or VI. Further analysis by Perkins. FGSC 1492, 2032.

T(11 + VI)P2889

Insertional translocation. A IIL segment including cys-3 and pi is inserted in inverted order into VI near ylo-1. Wild phenotype. Homozygous-fertile. T x N ascospores 75% black; unordered asci 29:35:34:1:1 ( N = 413). Cytology: Aberrant chromosomes not identified. Acentric chromosome fragments persist in micronuclei but do not replicate. Origin: Apparently originated spontaneously in a cross by Perkins of T(VZ + [Z;ZZZl) YI6329 x T(Z;ZZ)P6390. Called T ( I 1 + VI) Y1632Qiuntil 1974. Used as het-6 tester to study vegetative incompatibility. FGSC 1828, 1829 (het-GOB).References: Barry, 1973; Mylyk, 1975, 1976. Duplications: Dp(ZZL + VI)P2869. In one-third of viable progeny from T x N. Wild phenotype, barren in crosses. Markers shown covered : pi, cys-9, het-6. Markers shown not covered : pyr-4, het-c, ro-3, arg-6.

T(I;II1)3717 vis

Reciprocal translocation. I (probably R) interchanged with IIIR (near trp-I). Aconidial flat morphology, called vis (visible) and mapped in I by Houlahan et al. (1949). Homozygous-sterile with no perithecia. T x N ascospores 50% black; unordered asci 24:0:54:3:20 ( N = 119). Origin: LA x La, X-rays. Aberration recognized and analyzed by Perkins. FGSC 2882,2683.

In(1L + IR)H4250 Pericentric inversion, quasiterminal. A segment of I L including suc and mt is translocated to the right tip. Wild phenotype. Homozygous-fertile. T x N ascospores 80% black or more, darkening with age. Unordered asci 17:64:13:4:0 (N = 220). Cytology: Inversion of chromosome 1 confirmed cytologically (Barry). Origin: pe fl Y8743-21(13-7)a, S” (Hungate and Mannell, 1952). Detected and diagnosed by Newmeyer. Generates viable duplications from intercross with Zn(ZL + ZR)NMlTB. H4260 provided the first example of duplications heterozygous for mating type or for vegetative incompatibility genes. Strain of origin contained linked point mutant arg-1 (H4250). The structure is formally similar to Zn(lLR)scY’ in Drosophila. FGSC 1563, 1564. References: Newmeyer, 1965, 1970; Newmeyer and Taylor, 1967; Barry, 1967; Newmeyer and Galeazzi, 1974, 1976a,b; Schroeder, 1970, 1974.

Duplications: Dp(ZL + ZR)H4260. About one-fourth of viable progeny from

Zn x N (range 1535%). Usually growth is drastically inhibited because of Ala heterozygosity, with spidery morphology and brown pigment ; this “Dark

Agar” phenotype is suppressed by the gene to2 (Newmeyer, 1970). Duplications with tol are nearly wild, but with subtle (‘square’’ morphology. The speed of somatic escape from inhibition is increased by uvs-3 and meid. Duplications homozygous for mt alleles result occasionally from meiotic crossing over; these are not inhibited, but are subtly different from wild type in morphology, and

THE CYTOGENETICS OF

Neurospora

261

usually recognizable on this basis. Barren in crosses with non-duplications, eventually becoming fertile through loss of one duplicated IL segment, usually that in the translocated position. Barren instability increas2d by mei-3 and by factorb) from wild strain Adiopodoume A (Newmeyer and Galeazzi). Markers shown covered: fr, u n d , nit-2, leu-3, cut-1, ser-3, Zln-3, m t , ta, acr-3, S U C . Markers shown not covered: phe-1, sor, a d d , eth-1, arg-3, Tin-2, mei-3, sn, rg, his-2, lys-4, R .

T(IV + III)S4342

Insertional translocation. A long IVR segment distal to arg-2 and including markers pyr-3 through uvs-2 is inserted into IIIR proximal to ro-2, in inverted order. Wild phenotype. Homozygous-fertile. T x N ascospores 75% black, or leas; unordered asci 15:47:28:6:3 (N = 467). Cytology: Chromosome 4 probably aberrant. About 50% of asci have bridges. Acentric fragments are frequent and persist in rpicronuclei but do not replicate as do fragments from some other insertional translocations. Origin : pe fl Y8743-21(13-7)a,repeated X-rays (Colburn and Tatum, 1965). Translocation detected and analyzed by Perkins. Attempts to insert markers into the translocated segment have been unsuccessful. Strain of origin contained linked but separable mutation p t (S4342). FGSC 2061, 2065. Reference: Barry, 1973. Duplications: Dp(IVR + IIIR)S4342. I n one-thrid of surviving progeny from T x N . Lighter growth than wild type at 2 days, and tend to have yellowish aerial growth. Barrenness is exceptionally stable in crossos to non-duplications; perithecia have no beaks or ascospores. Markers shown covered: pyr-3, rib-2, trp-4, leu-2, pan-1, cot-1, his-4, cys-4, uvs-8. Markers shown not covered: pyr-1, p t , c y s ( o z D 1 ) ,coG4, arg-5'.

T(I + 111)4540 nic-2

Insertional translocation. A short segment of I R extending from nic-2 through cr is inserted between vel and tyr-1 in IIIR. Rearrangement inseparable from the n i c d phenotype with which it arose. Wild morphology. Homozygous-fertile. T x N ascospores 75% black; unordered asci 16:64:18:1:1 ( N 275). Cytology: Preliminary cytological examination by St. Lawrence and by Barry, but chromosomes involved were not identified. Origin: LA x La, X-rays. Detected and analyzed by St. Lawrence. FGSC 766,767.References: St. Lawrence, 1953,1959.

-

Duplications: Dp(IR + IIIR).46400. One-third of viable progeny from T x N . Duplications are nic'. Barren in crosses of duplication by non-duplication, where perithecial development is arrested prior to ascus development (P. St. Lawrence, unpublished observations, 1957). Markers shown covered : cr, cys-9, un-1. Markers shown not covered: thi-1, al-2.

T(I;II)4637 al-1

Reciprocal translocation. I R (at al-1) interchanged with I I R (near p e l . Phenotype al-1 (carotenoid deficient). Wild morphology. T x T perithecia are barren, but produce some ascospores which are viable but commonly malformed. T X N ascospores 50% black; unordered asci 20:1:63:2:14 ( N = 237). Cytology: Reciprocal translocation confirmed. Break points, identified by McClintock, are far out in the long arm of chromosome 1, and near centromere in 6. Origin: LA X La, X-rays. Linkages determined by Houlahan el al. (1949), Hagerty (1952), and

262

DAVID D. PERKINS AND EDWARD G. BARRY

St. Lawrence (1953). Generates viable duplications from intercross with T ( I R ; IIRISTL76, q.v. A component of alcoy linkage tester. FGSC 253, 252. References: McClintock, 1945, 1955; Singleton, 1948; St. Lawrence, 1953; Barry, 1967; Perkins et al., 1969.

T(I;II)P4704

Reciprocal translocation. I (linked m t ) interchanged with IIR (linked fl). Darkens synthetic cross medium. Wild morphology. Homozygous-fertile. T x N ascospores 50% black; unordered asci 32:9:25:4:30 (N = 142). Origin: From a cross of T(III + [I;IIl)ARl7 A x aur nic-1 0s R a. Detected and mapped by B. C. Turner. FGSC 2425, 24%.

T(I;V)P5166

Reciprocal translocation. I R (linked mt) interchanged with VR (distal to al-3). Wild phenotype. Homozygous-fertile. T x N ascospores 70% black ; unordered asci 23:2:70:2:3 ( N = 230). Origin: Detected by Perkins in cross of T ( I + IV)T61M166 un A x id; ylo-1; nt a. Generates viable duplications from intercross with T(IR;VR)NM143.IR arm assignment made on this basis. FGSC 2185, 2186.

T ( 1;II ) P5390 Reciprocal translocation. I L (near m t ) interchanged with I I L (distal to ro-3). Wild phenotype. Homozygous-fertile. T x N ascospores < 50% black; unordered asci 18:13:30:8:32 (N = 368). Origin: Found in a leu-1 trp-1 a stock, and analyzed by Perkins. Barren progeny were produced in cross of original '2 x N , but not in subsequent crosses using extracted translocation. FGSC 2455, 2456.

T(I;V)P5401

Reciprocal translocation. I L (near un-6, proximal) interchanged with VR (near aG3). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black with defective spores darkening with age; unordered asci 20:2:60:5:12 ( N = 129). Origin: Found in a stock of his-4 (C141, FGSC No. 78), and analyzed by Perkins. Generates viable duplications from intercrosses with T ( I L ; V R )ARl8 and $7711. These have an inhibited Dark Agar phenotype typical of A / a heterozygotes. FGSC 2427, 2428. Reference: Perkins, 1975.

T ( VII + I )5936 Insertional or quasiterminal translocation. A VIIR segment including arg-10 and distal markers is translocated in eucentric order to the left end of I, near fr and probably distal. Wild phenotype. Homozygous-fertile. T x N ascospores 7570 black; unordered asci 20:61:18:1:0 (N = 540). Translocation confirmed by Singleton. Translocation involves short arm of chromosome 1, and chromosome 7, which has a segment missing (Barry). Origin: LA x La, X-rays. Aberrant behavior first noted by D. R. Regnery. Subsequent genetic analysis by Perkins. Deficiency ascospores tend to become brown. Used as het-10 tester to study veget,ative incompatibility. Strain of origin contained linked point mutation Zeu-3 (5936). FGSC 2104, 2105 ( het-1OoR).References : Regnery, 1947 ; Singleton, 1948 ; Mylyk, 1975, 1976.

Duplications: Dp(VIIR + IL)6936. One third of viable progeny from T x N . Normal vegetative morphology. Duplications are barren and highly stable in

THE CYTOGENETICS OF

Neurospora

263

crosses. Markers shown covered: org-10, nl, sk, het-10. Markers shown not covered: wc, for.

T(III;VI)P6070

Reciprocal translocation. IIIR (near tyr-1) interchanged with VI (near ylo-1) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 12:3:60:5:20 (N = 192). Origin: Spontaneous, found in one segregant from his-2 un-2 A x met-7 a. Detected and analyzed by Perkins. FGSC 2601, 2602.

T(VI+ [I;III])Y16329

Complex translocation, probably insertional. Breakpoints in I (near mt), IIIR (near trp-11, and VIL (between lys-5 and cys-1). A segment of VIL including chol-2 and lysd is translocated to another interstitial or quasiterminal position. Vegetative growth sub-wild, with pale pigmentation and conidia in flecks. Femalesterile. No perithecia from T x T crosses. T x N ascospores < 50% black; unordered asci 6:4:35:25:29 ( N = 996). Origin: coC1; p e ; nL2 Y8743-6A, X-rays (Tatum et nl., 1950). Rearrangement detected and analyzed by Perkins. Strain of origin contained linked but separable mutation phe-2 (Y16329). FGSC 2710, 2711. Duplications: D p ( V I L + [ I ; I I I R I )YliZs29. In one-third of surviving progeny from T x N . Wild phenotype, vigorous. Grow better than the parental translocation. Stably barren. Markers shown covered : chol-2, Zys-6. Markers shown not covered : cys-1, ylo-1, trp-2.

T(1;VII)17084 thi-1

Reciprocal translocation. IR (at t h i - I ) interchanged with VII (left of met-7). Phenotype thi-1 (thiamine deficient). Wild morphology. Homozygous-fertile. T x N ascospores 50% black; unordered asci 30:1:42:2:25 ( N = 104). Cytology: Reciprocal translocation confirmed. Break points are in the long arm of 1, and near the middle of 7 (Barry). Origin: La x LA, X-rays. Detected genetically by pseudolinkage (Houlahan el al., 1949). FGSC 216, 215. References: Barry, 1967; Perkins et al., 1962.

T ( I;V ) 36703

Reciprocal translocation. I R (proximal to nnr) interchanged with VR (proximal to bis). Wild phcnotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 16: 1:67:2: 14 ( N = 245). Cytology: Chromosome 2 involvement (Phillips). Reciprocal translocation hetwcen chromosomes 1 and 2 determincd by examination of pachytene chromosomes (Barry). Origin: 1A x 25a, UV. 1;V linkage detected by A. M. Srb. Generates viable duplications when intercrosscd with T ( I R ; V R ) A L S l l l , and C-lG"?O pk-1. Strain of origin contained linked mutant nrg-I (36703), and possibly also unlinked T(II;III)36703b, q.v. FGSC 1445, 1446. References : Singleton, 1948; Phillips, 1967 ; Barry and Perkins, 1969 ; Perkins, 1971a.

T(II;III)36703b

Reciprocal translocation. I1 (near nrg-5) interchanged with I11 (nenr acr-2). Arms not determined. Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 32:2:28:9:30 (N = 222). Cytology: This may be tlie 36703 translocation examined by Singleton ; see Barry and Perkins (1969) for discrission. Origin: Uncertain. May have been prescnt with Z'(I;V)3G?O3 in strain 36703

264

DAVID D. PERKINS AND EDWARD G. BARRY

lineage (W),or may have arisen anew in a cross of 36703-5-9a x aur; bis A. Genetic analysis by Perkins. FGSC 1552, 1553. References: Singleton, 1948; Barry and Perkins, 1969.

T(I + II)39311 Insertional translocation. A long segment of IL, including nit-,?, mt and arg-9, is inserted in inverted order in I I R between aro-3 and pe. Wild phenotype. Homozygous-fertile. T x N ascospores 75% black; unordered asci 44:10:41:3:2 (N = 392). Cytology : Confirmed cytologically as inverted insertion from chromosome 1 into chromosome 6. Acentric fragments and dicentric bridges observed in divisions in the ascus (Barry). Acentric fragments persist in micronuclei and replicate. Origin: 1A x 25a, UV. Detected and analyzed genetically by Perkins. Strain of origin contained linked but separable point mutant suc (39311). FGSC 1245, 1246. References: Perkins, 1972a; Barry, 1972; Newmeyer, 1970 ; Schroeder, 1970; Metzenberg and Ahlgren, 1973. Duplications. DpUL + ZZR)99311. One-third of viable progeny from T x N. Wild morphology. Barren in crows. Duplications are usually drastically inhibited because of A / a heterozygosity, with restricted spidery morphology and dark brown pigment on complete medium; this “Dark Agar” phenotype is suppressed by the gene to1 (Newmeyer, 1970). Duplications with tot are nearly wild, but slow-growing with subtle “square” morphology. The speed of somatic escape from inhibition is increased by uvsd. Markers shown covered: nit-2, leud, cyt-1, ser-9, und, mt, suc, phe-1, arg-1, eth-1, arg-3, mei-3. Markers shown not covered: fr, un-6, sn, rg, un-2, his-,?.

44105 thr-1 Probably not aberrant. McClintock (1945) examined this strain, regarded as showing genetic evidence of an aberration. Her cytological observations were inconclusive. Subsequent attempts have failed to show abnormal recombination of markers in VII, pseudolinkage of thr-1 with markers in other linkage groups, or other evidence of a rearrangement. References : McClintock, 1945; Singleton, 1948; Perkins et al., 1962. T ( 1V;VI ) 45502 Reciprocal translocation. IVR (near pyr-3) interchanged with VIR (distal to trp-2). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 21:2:56:0:20 ( N 225). Cytology: Cytological information conflicting. Examined by McClintock, Singleton, and Barry. For discussion see Section IV, C. Origin: Abb 4A x 25a, W. Strain of origin also contained closely linked point mutant p y r d (45502); 1V;VI linkage of pyrd was reported by Houlahan et al. (1949). Generates viable duplications from intercross with T(ZVR ;VZR) NM176. FGSC 1067, 1876. References: McClintock, 1945, 1955; Singleton, 1948; Mitchell et a l , 1952; St. Lawrence, 1953; Perkins et al., 1962; Murray, 1968a; Barry and Perkins, 1969; Perkins, 1974.

-

T ( V;VI ) 46802 in1 Reciprocal translocation. VR (at inl) interchanged with VIL (between chol-2 and ad-8). Phenotype inl (inositol requiring). Nonrevertable, even with mutagens (Giles, 1951). Wild morphology. Homozygous-fertile. T x N ascospores 50% black;

THE CYTOGENETICS OF

Neurospora

265

unordered asci 12:2:64:4:18 (N = 107). Cytology: Demonstration of reciprocal translocation involving chromosome 2 by Phillips. Origin: Abb 4A x 25a, W. Recognized as aberrant by Giles on the basis of ascospore patterns and cytology. VI linkage detected by Perkins. FGSC 670, 1199. References: Giles, 1951; Perkins and Murray, 1963; Phillips, 1967.

T(I;V)47711

Reciprocal translocation. IL (near arg-I) interchanged with VR (near id).Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 30:0:44:2:24 (N = 235). Origin: Abb 4A x 25a. Detected by spore-abortion patterns, and linkage groups identified, by Pittenger. Generates viable duplications from intercrosses with T(IL;VR)P6401 (but not with T(ZL;VR)ARI??); these are all inhibited Dark-Agar phenotype, because of A / a heterozygosity. Strain of origin contained linked point mutant ilv (47711). FGSC 226, 223. References: Pittenger, 1954; Perkins et al., 1962; Perkins, 1975.

266

DAVID D. PERKINS AND EDWARD G. BARRY

REFERENCES Abrahamson, S., Bender, M. A., Conger, A. D., and Wolff, S. 1973. Uniformity of radiation-induced mutation rates among different species. Nature (London) 245, 460462. Agsteribbe, E., Kroon, A. M., and Van Bruggen, E. F. J. 1972. Circular DNA from mitochondria of Neurospora crassa. Biochim. Biophys. Acta 269, 299-303. Ahmad, A. F. 1970. Cytogenetic studies in Sordaria brevicollis. Ph.D. Dissertation, University of Cambridge. Ahmad, A. F., Bond, D. J., and Whitehouse, H. L. K. 1972. The effect of an inverted chromosome segment on intragenic recombination in another chromosome of Sordaria brevicollis. Genet. Res. 19, 121-127. Ahmad, M., Mozmadar, A., and Hendler, S. 1968. A new locus in the tryptophan pathway of Neurospora crassa. Genet. Res. 12, 103-107. Ahmed, A. 1968. Organization of the histidine-3 region of Neurospora. Mol. Gen. Genet. 103, 185-193. Ahmed, A., Case, M. E., and Giles, N. H. 1964. The nature of complementation among mutants in the histidine-3 region of Neurospora crassa. Brookhaven S y m p . Biol. 17, 53-65. Angel, T. 1971. Genetic control of recombination in the histidine-3 region of Neurospora crassa. Ph.D. Thesis, Australian National University, Canberra. Angel, T., Austin, B., and Catcheside, D. G. 1970. Regulation of recombination a t the his-3 locus in Neurospora crassa. Aust. J. Biol. Sci. 23, 1229-1240. Azevedo, J. L. 1975. Altered instability due to genetic changes in a duplication strain of Aspergillus nidulans. Genet. Res. 26, 55-61. Bachmann, B. J. 1970. Neurospora bibliography and index: Number two 1964-1969. Neurospora Newsl. 17, 3-80. Bachmann, B. J., ed. 1971-1976. Neurospora bibliography. (Published annually in Neurospora Newsl.) Bachmann, B. J., and Strickland, W. N. 1965. “Neurospora Bibliography and Index.” Yale Univ. Press, New Haven, Connecticut. Baer, D., and St. Lawrence, P. 1964. Autoradiographic determination of the location of radioactivity in asci grown on some tritiated pyrimidines. Neurospora Newsl. 6, 5-6. Bainbridge, B. W. 1970. Genetic analysis of an unequal chromosomal translocation in Aspergillus nidulans. Genet. Res. 15, 317-326. Barratt, R. W. 1962. Origin of important wild type stocks of N . crassa. Neurospora Newsl. 2, 24-25. Barratt, R. W. 1969. Further notes on nomenclature : Suppressors. Neurospora Newsl. 14, 13. Barratt, R. W., and Garnjobst, L. 1949. Genetics of a colonial microconidiating mutant strain of Neurospora crassa. Genetics 34, 351-369. Barratt, R. W., and Perkins, D. D. 1965. Neurospora genetic nomenclature. Neurospora Newsl. 8, 23-24. Barratt, R. W., Newmeyer, D., Perkins, D. D., and Garnjobst, L. 1954. Map construction in Neurospora crassa. A d v . Genet. 6, 1-93. Barry, E. G. 1960a. A complex chromosome rearrangement in Neurospora crassa. Ph.D. Thesis, Stanford University, Stanford, California ; Abstracted in Diss. Abstr. 21, 3233-3234 (1961).

THE CYTOGENETICS OF

Neurospora

267

Barry, E. G. 1960b. Genetic analysis of an insertional translocation in Neurospora crassa. Genetics 45, 974 (abstr.). Barry, E. G. 1962. Genetic and cytologic correlation of some Neurospora c r a m chromosome rearrangements. Genetics 47,942 (abstr.). Barry, E. G. 1966. Cytological techniques for meiotic chromosomes in Neurospora. Neurospora Newsl. 10, 12-13. Barry, E. G. 1967. Chromosome aberrations in Neurospora, and the correlation of chromosomes and linkage groups. Genetics 55, 21-32. Barry, E. G. 1969. The diffuse diplotene stage of meiotic prophase in Neurospora. Chromosoma 26, 119-129. Barry, E. G. 1972. Meiotic chromosome behavior of an inverted insertional translocation in Neurospora. Genetics 71, 53-62. Barry, E.G. 1973. Introducing chromosomal genes into the cytoplasm of Neurospora. Genetics 74, s16-sl7 (abstr.). Barry, E. G., and Perkins, D. D. 1969. Position of linkage group V markers in chromosome 2 of Neurospora crassa. J . Hered. 60, 120-125. Barry, E. G.,Newmeyer, D., Perkins, D. D., and Turner, B. C. 1972. Genetically determined round ascospores in N . crassa. Neurospora Newsl. 19, 17. Barton, D. W. 1950. Pachytene morphology of the tomato chromosome complement. A m . J . Bot. 37, 639-643. Bates, W. K.,and Wilson, J. F. 1974. Ethylene glycol-induced alteration of conidial germination in Neurospora crassa. J . Bacteriol. 117, 560-567. Beadle, G. W.1945. Genetics and metabolism in Neurospora. Physiol. R e v . 25, 643663. Beadle, G. W., and Coonradt, V. L. 1944. Heterocaryosis in Neurospora crassa. Genetics 29, 291-308. Beadle, G. W.,and Tatum, E. L. 1941. Genetic control of biochemical reactions in Neurospora. Proc. Natl. Acad. Sci. U.S.A. 27, 499-506. Beadle, G. W., and Tatum, E. L. 1945. Neurospora. 11. Methods of producing and detecting mutations concerned with nutritional requirements. A m . J . Bot. 32, 678-686. Beckett, J. B. 1972. An expanded set of B-type translocations in maize. Genetics 71, s3-4. Belling, J. 1914. The mode of inheritance of semi-sterility in the offspring of certain hybrid plants. Z . Indukt. Abstamm.-Vererbungsl. 12, 303-342. Beske, J. L.,and Phillips, R. L. 1968. Preliminary mapping of nineteen new translocations with the alcoy multiple translocation tester strain. Neurospora Newsl. 13, 19-21. Bhagwat, A. S., and Mahadevan, P. R. 1973. Differential gene action in Neurospora crassa. J . Bacteriol. 113, 572-575. Bicknell, J. N., and Douglas, H. C. 1970. Nucleic acid homologies among species of Saccharomyces. J . Bactem'ol. 101, 505-512. Blakely, R. M.,and Srb, A. M. 1962. Studies on the genetics and physiology of a nitrate non-utilizing strain of Neurospora. Neurospora Newsl. 2, 5-6. Blakeslee, A. F., Bergner, A. D., and Avery, A. J. 1936.A new method of synthesizing pure breeding types with extra chromosomal material in Datura. A m . N u t . 70, 255-257. Bole-Gowda, B. N., Perkins, D. D., and Strickland, W.N. 1962. Crossing-over and interference in the centromere region of linkage group I of Neurospora. Genetics 47, 1243-1252.

268

DAVID D. PERKINS AND EDWARD G. BARRY

Bresch, C., Muller, G., and Egel, R. 1968. Genes involved in meiosis and sporulation of a yeast. Mol. Gen. Genet. 102,301-306. Brink, R. A., and Cooper, D. C. 1932. A structural change in the chromosomes of maize leading to chain formation. A m . Nut. 66,310-322. Brooks, R. R., and Huang, P. C. 1972. Redundant DNA of Neurospora crassa. Biochem. Genet. 6, 41-49. Brygoo, Y. 1972. Mise en kvidence gknktique et cytologique d’une translocation dciproque chez Coprinus radialus. C . R . Hebd. Seances Acad. Sci., Ser. D 275, 2115-21 17.

Burnham, C. R. 1946. An “Oenothera” or multiple translocation method of establishing homozygous lines. J. A m . Soc. Agron. 38,702-707. Burnham, C. R. 1949. Chromosome segregation in maize translocations in relation to crossing over in interstitial segments. Proc. Natl. Acad. Sci. USA. 35, 34% 356.

Burnham, C. R. 1950. Chromosome segregation in translocations involving chromosome 6 in maize. Genetics 35, 446481. Burnham, C. R. 1962. “Discussions in Cytogenetics.” Burgess, Minneapolis, Minnesota. (Third printing, 1972, by the author, 1539 Branston St., St. Paul, Minn. 55108.)

Burnham, C. R. 1968. A new method of using interchanges as chromosome markers. Crop Sci. 8, 357-360. Burnham, C. R. 1973. Progress report on establishing a line with all chromosomes interchanged. Maize Genet. Coop. News Lett. 47, 132-133. Byers, B., and Goetsch, L. 1975. Electron microscopic observations of the meiotic karyotype of diploid and tetraploid Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA. 72, 5056-5060. Cairns, J. 1963. The chromosome of Escherichk coli. Cold Spring Harbor Symp. Quant. Biol. 28, 43-46. Cameron, H. R., Hsu, K. S., and Perkins, D. D. 1966. Crossing over frequency following inbreeding in Neurospora. Genetica 37, 1-6. Carpenter, A. T. C. 1975a. Electron microscopy of meiosis in Drosophila melanogaster females. I. Structure, arrangement, and temporal change of the synaptonemal complex in wild-type. Chromosoma 51, 157-182. Carpenter, A. T. C. 1975b. Electron microscopy of meiosis in Drosophila melanogaster females. 11. The recombination nodule-a recombination-associated structure a t pachytene? Proc. Natl. Acad. Sci. U S A . 72, 3186-3189. Case, M. E., and Giles, N. H. 1962. The problem of mitotic recombination in Neurospora. Neurospora Newsl. 2, 6-7. Case, M. E., and Giles, N. H. 1975. Genetic evidence on the organization and action of the qa-1 gene product: A protein regulating the induction of three enzymes in quinate catabolism in Neurospora crassa. Proc. Natl. Acad. Sci. U S A . 72, 553-557.

Catcheside, D. E. A. 1969. A new carotenoid mutant of Neurospora. Neurospora Newsl. 15, 3-4. Catcheside, D. E. A. 1974. A second locus subject to recombination control by the r e d + gene in Neurospora crassa. Aust. J . Biol. Sci. 27, 561-573. Catcheside, D. G. 1965. Multiple enzymic functions of a gene in Neurospora crassa. Biochem. Biophys. Res. Commun. 18, 648-651. Catcheside, D. G. 1966. Behaviour of flanking markers in allelic crosses. Aust. J . Biol. SCi. 19, 1047-1059.

THE CYTOGENETICS OF

Neurospora

269

Catcheside, D. G. 1973. Neurospora crassa and genetics. Neurospora Newsl. 20, 6-8. Catcheside, D. G. 1974. Fungal genetics. Annu. Rev. Genet. 8, 279-300. Catcheside, D. G. 1975. Occurrence in wild strains of Neurospora crassa of genes controlling genetic recombination Aust. J . Biol. Sci. 28, 213-225. Catcheside, D. G., and Angel, T. 1974. A histidine-3 mutant, in Neurospora crassa, due to an interchange. Aust. J . Biol. Sci. 27, 219-229. Catcheside, D. G., and Corcoran, D. 1973. Control of non-allelic recombination in Neurospora crassa. Aust. J . Biol. Sci. 26, 1337-1353. Cattanach, B. M. 1975. Control of chromosome inactivation. Annu. Rev. Genet. 9, 1-18. Cavalier-Smith, T. 1974. Palindromic base sequences and replication of eukaryote chromosome ends. Nature (London) 250, 467-470. Chaleff, R. S. 1974a. The inducible quinate-shikimate catabolic pathway in Neurospora crassa: Genetic organization. J . Gen. Microbwl. 81, 337-355. Chaleff, R. S. 197413. The inducible quinate-shikimate catabolic pathway in Neurospora crassa: Induction and regulation of enzyme synthesis. J . Gen. Microbiol. 81, 357472. Chattopadhyah, S. K., Kohne, D. E., and Dutta, S. K. 1972. Ribosomal RNA genes of Neurospora: Isolation and characterization. Proc. Natl. Acad Sci. U S A . 69, 3256-3259. Clutterbuck, A. J. 1970. A variegated position effect in Aspergillus nidulans. Genet. Res. 16, 303-316. Colburn, R. W., and Tatum, E. L. 1965. Studies of a phenylalanine-tyrosine requiring mutant of Neurospora crassa (strain 54342). Biochim. Biophys. Acla 97, 442448. Colson, B. 1934. The cytology and morphology of Neurospora tetrasperma Dodge. Ann. Bot. (London) 48, 211-224. Coyle, M. B., and Pittenger, T. H. 1965. Mitotic recombination in pseudo-wild types of Neurospora. Genetics 52, 609-625. Cox, B. S., and Gill, J. J. B. 1967. A chromosomal translocation in Sordaria fimicola and irregular segregation of chromosomes. New Phytol. 66, 653-664. Crawford, I. P. 1975. Gene rearrangements in the evolution of the tryptophan synthetic pathway. Bacteriol. Rev. 39, 87-120. Cutter, V. M. 1946. The chromosomes of Neurospora tetrasperma. Mycologia 38, 693-698. Dallapiccola, B., Capra, L., Preto, G., Covic, M., and Dutrillaux, B. 1974. Inversion pkricentrique du 4 : inv(4) (p13q35) et trisomie du bras court du 4 par aneusomie de recombinaison. Ann. Genet. 17, 115-118. Davis, R. H. 1966. Mechanisms of inheritance. 2. Heterokaryosis. In “The Fungi : An Advanced Treatise” (G. C. Ainsworth and A. S. Sussman, eds.), Vol. 2, pp. 567-688. Academic Press, New York. Davis, R. H., and de Serres, F. J. 1970. Genetic and microbiological research techniques for Neurospora crassa. In “Methods in Enzymology” (H. Tabor and C. W. Tabor, eds.), Vol. 17A, pp. 79-143. Academic Press, New York. Davis, R. H., and Woodward, V. W. 1962. The relationship between gene suppression and aspartate transcarbamylase activity in pyr-3 mutants of Neurospora. Genetics 47, 1075-1083. Davis, R. H., Lawless, M. B., and Port, L. A. 1970. Arginaseless Neurospora: Genetics, physiology and polyamine synthesis. J . Bacteriol. 102, 299-305. de la Chapelle, A., and Schroder, J. 1973. Apparently non-reciprocal balanced human

270

DAVID D. PERKINS AND EDWARD G . BARRY

(3q- ;Xq+) translocation : Late replication of structurally normal X. Chromosomes Today 4, 281-265. DeMoss, J. A., Jackson, R. W., and Chalmers, J. H., Jr. 1967. Genetic control of the structure and activity of an enzyme aggregate in the tryptophan pathway of Neurospora crassa. Genetics 56, 413-424. de Serres, F. J. 1957. A genetic analysis of an insertional translocation involving the ad-3 region in Neurospora crassa. Genetics 42, 366367 (abstr.). de Serres, F. J. 1969. Comparison of the complementation and genetic maps of closely linked nonallelic markers on linkage group I of Neurospora crassa. Mutat. Reu. 8, 43-50. de Serres, F. J. 1971. Studies with purple adenine mutants in Neurospora crassa. VI. The effects of differences in genetic background on a d J A x a d 3 B crosses. Genetics 88, 383400. de Serres, F. J., and Malling, H. V. 1971. Measurement of recessive lethal damage over the entire genome and a t two specific loci in the a & ¶ region of a twocomponent heterokaryon in Neurospora crassa. In “Chemical Mutagens: Principles and Methods for Their Detection” (A. Hollaender, ed.), Vol. 2, pp. 311342. Plenum, New York. Deters, J., Miskimen, J., and McDougall, K. J. 1974. A putrescine-requiring mutant of Neurospora crassa. Genetics 77, s l 6 s l 7 (abstr.). Diakumakos, E. G., Garnjobst, L., and Tatum, E. L. 1965. A cytoplasmic character in Neurospora crassa. The role of nuclei and mitochondria. J . Cell Biol. 28, 427-443. Dobzhansky, T. 1946. Genetics of natural populations. XII. Recombination and variability in populations of Drosophila pseudoobscura. Genetics 31, 269-290. Dodge, B. 0. 1927. Nuolear phenomena associated with heterothallism in the ascomycete Neurospora. J . Agric. Res. 35, 289-305. Dodge, B. 0. 1929. Nature of giant spores and segregation of sex factors in Neurospora. Mycologia 21, 222-231. Dodge, B. 0. 1931. Inheritance of the albinistic non-conidial characters in interspecific hybrids in Neurospora. Mycologia 23, 1-50. Dodge, B. 0. 1937. Spindle orientation and spore delimitation in Gelasinospora tetrasperma. Cytologia, Fujii Jubilaei Vol. pp. 877-887. Dodge, B. O., Singleton, J. R., and Rolnick, A. 1950. Studies on lethal E gene in Neurospora tetrasperma, including chromosome counts also in races of N . sitophila. Proc. A m . Philos. SOC.94, 38-52. Drake, J. W., chairman. 1975. Environmental mutagenic hazards. (Report of a Committee of the Environmental Mutagen Society.) Science 187, 503-514. Dutrillaux, B., Laurent, C., Robert, J. M., and Lejeune, J. 1973. Inversion p6ricentrique, inv(l01, chez la mere et aneusomie de recombinaison, inv(l01, rec(lO), chez son fils. Cytogenet. Cell Genet. 12, 245-253. Dutta, S. K. 1973. Transcription of non-repeated DNA in Neurospora crassa. Biochim. Biophys. Acta 324, 482-487. Dutta, S. K., and Chaudhuri, R. K. 1975. Transcription of repetitive DNA in Neurospora crassa. Mol. Gen. Genet. 136,227-232. Dutta, S. K., and Ray, R. 1973. Partial characterization of transfer RNA genes isolated from Neurospora crassq. Mol. Gen. Genet. 125, 295300. Dwivedi, R. S., Dutta, S. K., and Bloch, D. P. 1969. Isolation and characterization of chromatin from Neurospora crassa. J . Cell Biol. 43, 51-58.

THE CYTOGENETICS OF

Neurospora

271

Egashira, T., and Nakamura, K. 1972. Genes influencing selective fertilization in Neurospora crassa. Genetics 70,511-519. El Ani, A. S., and Olive, L. S. 1962. The induction of balanced heterothallism in Sordaria fimicola. Proc. Natl. Acnd. Sci. U S A . 48, 17-19. Emerson, S. 1950. Competitive reactions and antagonisms in the biosynthesis of amino acids by Neurospora. Cold Spring Harbor Symp. Quant. Biol. 14, 40-48. Emerson, S. 1955. Biochemical genetics. I n “Handbuch der physiologisch- und pathologisch-chemischen Analyse” (F. Hoppe-Seyler and H. Thierfelder, eds.) , 10th ed., Vol. 2, Part 2, pp. 443-537. Springer-Verlag, Berlin and New York. Emerson, S. 1963. Meiotic recombination in fungi with special reference to tetrad analysis. In “Methodology in Basic Genetics” (W. J. Burdette, ed.), pp. 167208. Holden-Day, San Francisco, California. Emerson, S. 1966. Mechanisms of inheritance. 1. Mendelian. I n “The Fungi: An Advanced Treatise” (G.C. Ainsworth and A. S. Sussman, eds.), Vol. 2, pp. 513-566. Academic Press, New York. Emerson, S. 1969. Linkage and recombination at the chromosomal level. In “Genetic Organization: A Comprehensive Treatise” (E. W. Caspari and A. W. Ravin, eds.), Vol. 1, pp. 267-360. Academic Press, New York. Endrizzi, J. E. 1974. Alternate-1 and alternate-2 disjunctions in heterozygous reciprocal translocations. Genetics 77, 55-60. Esposito, M. S., Esposito, R. E., Arnaud, M., and Halverson, H. 0. 1970. Conditional mutants of meiosis in yeast. J. Bacteriol. 104, 202-210. Esser, K., and Straub, J. 1958. Genetische Untersuchungen an Sordaria mcrospora Auersw., Kompensation und Induktion bei genbedingten Entwicklungsdefekten. 2. Vererbungsl. 89, 729-746. Evans, G. M., and Rees, H. 1971. Mitotic cycles in dicotyledons and monocotyledons. Nature (London) 233,350-351. Fincham, J. R. S. 1949. Chromosome numbers in species of Neurospora. Ann. Bot. (London) 13, 23-28. Fincham, J. R. S. 1951. A comparative genetic study of the mating type chromosomes of two species of Neurospora. J. Genet. 50,221-229. Fincham, J. R . S. 1974. Negative interference and the use of flanking markers in fine-structure mapping in fungi. Heredity 33, 116-121. Fincham, J. R. S., and Day, P. R. 1971. “Fungal Genetics,” 3rd ed. Blackwell, Oxford. Fincham, J. R. S., and Pateman, J. A. 1957. Formation of an enzyme through complementary action of mutant “alleles” in separate nuclei in a heterocaryon. Nature (London) 179, 741-742. Fink, J . R. 1971. Gene clusters and the regulation of biosynthetic pathways in fungi. Metab. Pathways, 3rd Ed. 5, 200-223. Fink, R. M., and Fink, K. 1962a. Utilization of radiocarbon from thymidine and other precursors of ribonucleic acid in Neurospora crassa. J. Biol. Chem. 237, 2289-2290. Fink, R. M., and Fink, K. 196213. Relative retention of Ha and C“ labels of nucleosides incorporated into nucleic acids of Neurospora. J. Biol. Chem. 237, 2889-2891. Francke, U. 1972. Quinacrine mustard fluorescence of human chromosomes : Characteristics of unusual translocations. A m . J. Hum. Genet. 24, 189-213. Frederick, L., Uecker, F. A., and Benjamin, C. R. 1969. A new species of Neurospora from the soil of West Pakistan. Mycologiu 61,1077-1084.

272

DAVID D. PERKINS AND EDWARD G. BARRY

Frost, L. C. 1961. Heterogeneity in recombination frequencies in Neurospora crassa. Genet. Res. 2,43-62. Garnjobst, L. 1953. Genetic control of heterocaryosis in Neurospora crassa. Am. J. Bot. 40, 607-614. Garnjobst, L. 1955. Further analysis of genetic control of heterokaryosis in Neurospora crassa. Am. J. Bot. 42, 444-448. Garnjobst, L., and Tatum, E. L. 1967. A survey of new morphological mutants in Neurospora crassa. Genetics 57, 5 7 9 4 4 . Garnjobst, L., and Wilson, J. F. 1956. Heterocaryosis and protoplasmic incompatibility in Neurospora crassa. Proc. Natl. Acad. Sci. USA. 42, 613-618. Giles, N. H. 1951. Studies on the mechanism of reversion in biochemical mutants of Neurospora crassa. Cold Spring Harbor Symp. Quant. Biol. 16,283-313. Giles, N. H., Partridge, C. W. H., and Nelson, N. J. 1957. The genetic control of adenylosuccinase in Neurospora crassa. Proc. Natl. Acad. Sci. USA. 43, 305-317. Giles, N. H., Case, M. E., Partridge, C. W. H., and Ahmed, S. I. 1967. A gene cluster in Neurospora crassa coding for an aggregate of five aromatic synthetic enzymes. Proc. Natl. Acad. Sci. USA. 58, 1453-1460. Gillies, C. B. 1972. Reconstruction of the Neurospora crassa pachytene karyotype from serial sections of synaptonemal complexes. Chromosoma 36, 119-130. Gillies, C. B. 1973. Ultrastructure analysis of maize pachytene karyotypes by three dimensional reconstruction of the synaptonemal complexes. Chromosoma 43, 145-176.

Gopinath, D. M., and Rurnham, C. R. 1956. A cytogenetic study in maize of deficiency-duplication produced by crossing interchanges involving the same chromosomes. Genetics 41, 382-395. Gottschalk, W. 1973. The genetic control of meiosis. Genetics 74, s99 (abstr.). Grace, E., Sutherland, G. R., Stark, G. D., and Bain, A. D. 1973. Partial trisomy of 7q resulting from a familial translocation. Ann. Genet. 16,51-54. Griffen, A. B. 1955. A late pachytene chromosome map of the male mouse. J. Morphol. 96, 123-141. Griffen, A. B. 1960. Mammalian pachytene chromosome mapping and somatic chromosome identification. J. Cell. Comp. Physiol. 56, Suppl. 1, 113-121. Griffiths, A. J. F. 1970. Genetic effects attributable to a paracentric inversion in Neurospora crassa. Genetics 64, s26s27 (abstr.). Griffiths, A. J. F. 1972, Ascus analysis of a chromosomal rearrangement in Neurospora crassa. Can. J. Genet. Cytol. 14, 727-728 (abstr.). Griffiths, A. J. F., Delange, A. M., and Jung, J. H. 1974. Identification of a complex chromosome rearrangement in Neurospora crassa. Can. J . Genet. Cytol. 16, 805-822.

Grindle, M. 1974. The efficacy of various mutagens and polyene antibiotics for the induction and isolation of sterol mutants of Neurosposa crassa. Mol. Gen. Genet. 130, 81-90.

Gross, S. R. 1952. Heterokaryosis between opposite mating types in Neurosposa crassa. Am. J. Bot. 39, 574-577. Gross, S. R., and Fein, A. 1960. Linkage and function in Neurospora. Genetics 45, 885-904.

Hagberg, A. 1962. Production of duplication in barley breeding. Hereditas 48, 243-246. Hagberg, A., and Hagberg, G. 1971. Chromosome aberrations and their utilization. Barby Genet. 2, Proc. Int. Symp., %nd,pp. 65-71. Hagerty, J. E. 1952. A correlation of genetic and ‘cytological observations of the

THE CYTDGENETICS OF

Neurospora

273

albino-1 mutant of Neurospora crassa. M.A. Thesis, Stanford University, Stanford, California. Hartl, D. L., and Hiraizumi, Y. 1976. Segregation distortion. In “The Genetics and Biology of Drosophila” (M. Ashburner and E. Novitski, eds.), Vol. lb, pp. 616-666. Academic Press, New York. Hawthorne, D. C. 1963. A deletion in yeast and its bearing on the structure of the mating type locus. Genetics 48, 1727-1729. Heslot, H. 1958. Contribution B 1’Btude cytogCnBtique et gknCtique des SordariacBes. R e v . Cytol. Biol. Veg. 19, Suppl. 2, 1-209. Holliday, R. 1974. Molecular aspects of genetic exchange and gene conversion. Genetics 78, 273-287. Holloway, B. W. 1953. Heterocaryosis in Neurospora crassa. Ph.D. Thesis, California Institute of Technology, Pasadena. Holloway, B. W. 1955. Genetic control of heterocaryosis in Neurospora crassa. Genetics 40, 117-129. Horowitz, N. H. 1965. The evolution of biochemical syntheses-retrospect and prospect. I n “Evolving Genes and Proteins” (V. Bryson and H. J. Vogel, eds.), pp. 15-23. Academic Press, New York. Horowitz, N. H. 1973. Neurospora and the beginnings of molecular genetics. Neurospora Newsl. 20, 4-6. Horowitz, N. H., and Macleod, H. 1960. The DNA content of Neurospora nuclei. Microb. Genet. Bull. 17, 6-7. (Cited with permission.) Hotchkiss, R. D. 1974. The evolution of recombination models. I n “Mechanisms in Recombination” (R. F. Grell, ed.), pp. 439-443. Plenum, New York. Houlahan, M. B., Beadle, G. W., and Calhoun, H. G. 1949. Linkage studies with biochemical mutants of Neurospora crassa. Genetics 34, 493-507. Howe, H. B., Jr. 1963. Markers and centromere distances in Neurospora tetrasperma. Genetics 48, 121-131. Howe, H. B., Jr. 1964. Sources of error in genetic analysis in Neurospora tetrasperma. Genetics 50, 181-189. Howe, H. B., Jr., and Haysman, P. 1966. Linkage group establishment in Neurospora tetrasperma by interspecific hybridization with N . crassa. Genetics 54,293-302. Hsiang, M. W., and Cole, R. D. 1973. The isolation of histone from Neurospora crassa. J . B w l . Chem. 248, 2007-2013. Hsu, K. S. 1963. The genetic basis of actidione resistance in Neurospora. J . Cen. Microbiol. 32, 341-347. Hsu, K. S. 1965. Acriflavin resistance controlled by chromosomal genes in Neurospora. Neurospora Newsl. 8, 4-6. Hsu, T. C. 1963. Longitudinal differentiation of chromosomes and the possibility of interstitial telomeres. Exp. Cell Res., Suppl. 9, 73-85. Hulten, M., and Lindsten, J. 1973. Cytogenetic aspects of human male meiosis. Adv. Hum. Genet. 4, 327-387. Hung, C.-Y. 1972. Notes on meiosis in Neurospora tetrasperma. Mycologia 64, 631-634. Hungate, F. P., and Mannell, T. 1952. Sulfur-35 as a mutagenic agent in Neurospora. Genetics 37, 709-719. Hungate, M. V. G. 1945. A genetic study of albino mutants of Neurospora crassa. M.A. Thesis, Stanford University, Stanford, California. Inoue, H., and Ishikawa, T. 1970. Macromolecule synthesis and germination of

274

DAVID D. PERKINS AND EDWARD G . BARRY

conidia in temperature sensitive mutants of Neurospora crassa. Jpn. J . Genet. 45, 357-369.

Jacobs, P. A., Buckton, K. E., Cunningham, C., and Newton, M. 1974. An analysis of the break points of structural rearrangements in man. J . Med. Genet. 11,5044. Jessup, A. P., and Catcheside, D. G. 1965. Interallelic recombination a t the his-l locus in Neurospora crassa and its genetic control. Heredity 20,237-256. Johnson, T. E. 1975a. Perithecial formation and pattern formation in Neurospora crassa. Ph.D. Thesis, University of Washington, Seattle. Johnson, T. E. 1975b. Pattern formation in Neurospora crassa perithecial development : A mosaic analysis. Genetics 80, s45-s46 (abstr.) . Kafer, E. 1965. Origins of translocations in Aspergillus nidulans. Genetics 52,217-232. Kafer, E. 1975a. Reciprocal translocations and translocation disomics of Aspergillus and their use for genetic mapping. Genetics 79, 7-30. Kiifer, E. 1975b. Reciprocal mitotic crossing over in stable disomics from crosses between reciprocal translocations of Aspergillus. Genetics 80,s46-s47 (abstr.) . Kafer, E. 1977. Meiotic and mitotic rerombination in Aspergillus and its chromosomal aberrations. A d v . Genet. 19, 33-131. Kafer, E., and Upshall, A. 1973. The phenotypes of the eight disomics and trisomics of Aspergillus nidulans. J . Hered. 64,35-38. Kappy, M. S., and Metzenberg, R. L. 1967. Multiple alterations in metabolite uptake in a mutant of Neurospora crassa. J . Bactenol. 94, 1629-1637. Kelstein, L. V. 1938. The influence of minute deficiencies and duplications on the individual development of Drosophila melanogaster. B w l . Zh. 7 , 1145-1166 (cited by Lindsley et al., 1972). Kerr, D. S., and Flavin, M. 1970. The regulation of methionine synthesis and the nature of cystathionine y-synthase in Neurospora. J . Biol. Chem. 245, 1842-1855. Khush, G. S., and Rick, C. M. 1968. Cytogenetic analysis of the tomato genome by means of induced deficiencies. Chromosoma 23,452-484. Kiritani, K. 1962. Linkage relationships among a group of isoleucine and valine requiring mutants of Neurospora crassa. Jpn. J . Genet. 37, 42-56. Kowles, R. V. 1972. The synthesis and behavior of two-chromosome double interchanges in maize and Neurospora crassa. Ph.D. Thesis, Universiky of Minnesota, Minneapolis; abstracted in Diss. Abstr. Znt. B 33, 60-61 (1972). Kowles, R. 1973. Synthesis of two-chromosome double interchanges in N . crassa. Neurospora Newsl. 20, 39-40. Kuwana, H. 1953. Studies on a morphological mutant “cut” in Neurospora crassa. Cytologia 18, 235-239. Kuwana, H. 1960. Studies on a temperature-sensitive irreparable mutant of Neurospora crassa. Jpn. J . Genet. 35, 49-57. Laird, C. D. 1971. Chromatid structure: Relationship between DNA content and nucleotide sequence diversity. Chromosoma 32, 378406. Landner, L. 1971. Genetic control of recombination in Neurospora crassa: Correlated regulation in unlinked chromosome intervals. Heredity 27, 385-392. Landner, L. 1974. Genetic control of recombination in Neurospora crassa. 111.Selection for increased and decreased recombination frequency. Hereditas 78, 185-200. Lavigne, S., and Frost, L. C. 1964. Recombination frequency and wild-type ancestry in linkage group I of Neurospora crassa. Genet. Res. 5 , 3 6 3 7 8 . Lee, Y . T., and Nga, B. H. 1973. Meiotic segregation behavior of translocation haploids in Aspergillus nidulans. Genetics 74, s153-s.154 (abstr.).

THE CYTOGENETICS OF

Neurospora

275

Leighton, T. J., Dill, B. C., Stock, J. J., and Phillips, C. 1971. Absence of histones from the chromosomal proteins of fungi. Proc. Natl. Acad. Sci. U 5 . A . 68, 677-680.

Lejeune, J., and Berger, R. 1965. Sur deux observations familiales de translocations complexes. Ann. Genet. 8, 21-30. Lewis, L. A. 1969. Genetics of an interspecific cross in the genus Sordaria. I. Analysis of 8-spored asci homozygous for the mating type factor. Genetics 62,79-101. Lindegren, C. C. 1933. The genetics of Neurospora. 111. Pure bred stocks and crossing over in N . crassa. Bull. Torrey Bot. Club 60, 133-154. Lindegren, C. C. 1936a. A six point map of the sex chromosome of Neurospora crassa. J . Genet. 32,243-256. Lindegren, C. C. 193613. The structure of the sex chromosomes of Neurospora crassa suggested by genetical analysis. J . Hered. 27, 251-259. Lindegren, C. C. 1973. Reminiscences of B. 0. Dodge and the beginnings of Neurospora genetics. Neurospora Nezusl. 20, 13-14. Lindegren, C. C., and Lindegren, G. 1937. Non-random crossing over in Neurospora. J . Hered. 28, 105-113. Lindegren, C. C., and Lindegren, G. 1939. Non-random crossing over in the second chromosome of Neurospora crassa. Genetics 24, 1-7. Lindegren, C. C., and Lindegren, G. 1941. X-ray and ultra-violet induced mutations in Neurospora. I. X-ray mutations. J . Hered. 32,404-412. Lindegren, C. C., and Lindegren, G. 1942. Locally-specific patterns of chromatid and chromosome interference in Neurospora. Genetics 27, 1-24. Lindenbaum, R. H., and Bobrow, M. 1975. Reciprocal translocations in man. 3:1 meiotic disjunction resulting in 47- or 45-chromosome offspring. J . Med. Genet. 12, 29-43.

Lindsley, D. L., and Grell, E. H. 1968. Genetic variations of Drosophila melanogaster. Carnegie Inst. Washington Publ. 627. Lindsley, D. L., Sandler, L., 'Baker, B. S., Carpenter, A. T. C., Denell, R. E., Hall, J. C., Jacobs, P. A., Miklos, G . L. G., Davis, B. K., Gethmann, R. C., Hardy, R. W., Healer, A., Miller, S. M., Nozawa, H., Parry, D. M., and Gould-Somero, M. 1972. Segmental aneuploidy and the genetic gross structure of the Drosophila genome. Genetics 71, 157-184. Littlewood, B. S., Chia, W., and Metzenberg, R. L. 1975. Genetic control of phosphatemetabolizing enzymes in Neurospora crassa: Relationships among regulatory mutants. Genetics 79, 419-434. Lu, B. C. 1967a. Meiosis in Coprinus Zagopus: A comparative study with light and electron microscopy. J. Cell Sci. 2,529-536. Lu, B. C. 1967b. The course of meiosis and centriole behaviour during the ascus development of the ascomycete Gelasinospora calospora. Chromosoma 22, 210226.

Lu, B. C., and Raju, N. B. 1970. Meiosis in Coprinus. 11. Chromosome pairing and the lampbrush diplotene stage of meiotic prophase. Chromosoma 29, 305-316. Luck, D. J. L., and Reich, E. 1964. DNA in mitochondria of Neurospora crassa. Proc. Natl. Acad. Sci. U S A . 52,931-938. Ma, G . C. L., and Kafer, E. 1974. Genetic analysis of the reciprocal translocation TZ(I;VIII) of Aspergillus using the technique of mitotic mapping in homozygous translocation diploids. Genefics 77, 11-23. McBride, A. C., and Gowans, C. S. 1969. The induction of gene mutation and chro-

276

DAVID D. PERKINS AND EDWARD G . BARRY

mosome aberration in Chlamydomonas eugametos by a phenylalanine analog. Genet. Res. 14, 121-126. McCarthy, B. J. 1969. The evolution of base sequences in nucleic acids. In “Handbook of Molecular Cytology” (A. Lima-de-Faria, ed.), pp. 3-20. North-Holland Publ., Amsterdam. McClintock, B. 1938. The fusion of broken ends of sister half-chromatids following chromatid breakage at meiotic anaphases. Mo., Agric. Exp. Stn., Res. Bull. 290, 1-48.

McClintock, B. 1939. The behavior in successive nuclear divisions of a chromosome broken at meiosis. Proc. Natl. Acad. Sci. U.S.A. 25, 405-416. McClintock, B. 1941. The stability of broken ends of chromosomes in Zea mays. Genetics 26, 234-282. McClintock, B. 1945. Neurospora. I. Preliminary observations of the chromosomes of Neurospora crassa. A m . J. Bot. 32, 671-878. McClintock, B. 1947. Cytogenetic studies of maize and Neurospora. Carnegie Inst. Washington, Yearb. 46, 146-152. McClintock, B. 1950. The origin and behavior of mutable loci in maize. Proc. Natl. Acad. Sci. U S . A . 36, 344-355. McClintock, B. 1951. Chromosome organization and gene expression. Cold Spring Harbor Symp. Quant. Biol. 16, 13-47. McClintock, B. 1955. Mutations in maize and chromosomal aberrations in Neurospora. Carnegie Inst. Washington, Yearb. 53, 254-261. McKey, T. J. 1967. A reciprocal translocation in Saccharomyces cerevisiae. Lawrence Radiat. Lab. UCRL-18066,38-42. McNelly-Ingle, C. A., and Frost, L. C. 1965. The effect of temperature on the production of perithecia by Neurospora crassa. J. Gen. Microbiol. 39, 33-42. Malling, H. V., and de Serres, F. J. 1973. Genetic analysis of purple adenine ( a d 4 mutants induced by methyl methanesulfonate in Neurospora crassa. Mutat. Res. 18, 1-14. Mather, K., and Beale, G. H. 1942. The calculation and precision of linkage values from tetrad analysis. J. Genet. 43, 1-30. Meselson, M. S., and Radding, C. M. 1975. A general model for genetic recombination. Proc. Natl. Acad. Sci. U S A . 72,358-361. Metzenberg, R. L., and Ahlgren, S. K. 1973. Behavior of Neurospora tetraspema mating-type genes introgressed into N . crassa. Can. J. Genet. Cytol. 15,571-576. Metzenberg, R. L., Gleason, M. K., and Littlewood, B. S. 1974. Genetic control of alkaline phosphatase in Neurospora : The use of partial diploids in dominance studies. Genetics 77, 25-43. Miller, 0. J., and Miller, D. A. 1975. Cytogenetics of the mouse. Annu. R e v . Genet. 9, 285-303.

Minson, A. C., and Creaser, E. H. 1969. Purification of a trifunctional enzyme, catalysing three steps of the histidine pathway, from Neurospora crassa. Biochem. J . 114, 49-56. Mishra, N. C. 1971. Heterokaryosis in Neurospora sitophila. Genetics 67, 55-59. Mitchell, M. B. 1955. Aberrant recombination of pyridoxine mutants of Neurospora. Proc. Natl. Acad. Sci. U S A . 41,215-2X1. Mitchell, M. B. 1966. A round-spore character in N . crassa. Neurospora Newsl. 10, 6. Mitchell, M. B., and Mitchell, H. K. 1952. A case of “maternal” inheritance in Neurospora crassa. Proc. Natl. Acad. Sci. U.S.A. 38, 442-449.

THE CYTOGENETICS OF

Neurospora

277

Mitchell, M. B., Pittenger, T. H., and Mitchell, H. K. 1952. Pseudo-wild types in Neurospora crassa. Proc. Null. Acad. Sci. U S A . 38,569-580. Mitchell, M. B., Mitchell, H. K., and TissiBres, A. 1953. Mendelian and non-Mendelian factors affecting the cytochrome system in Neurospora crassa. Proc. Natl. Acad. Sci. USA. 39, 606-613. Mortimer, R. K., and Hawthorne, D. C. 1975. Genetic mapping in yeast. Methods Cell Biol. 11, 221-233. Mu’Azu, S. 1973. Cytogenetic studies on Sordaria brevicollis. Ph.D. Dissertation, University of Cambridge. Muller, H. J. 1930. Types of visible variations induced by X-rays in Drosophila. J . Genet. 22, 299-334. Muller, H. J. 1967. Translocational Pale Drosophila and Snaker mice, a semicentennial parallel. Mutat. Res. 4, 201-205. Muller, H. J., and Herskowitz, I. H. 1954. Concerning the healing of chromosome ends produced by breakage in Drosophila melanogaster. Am. Nut. 88,177-208. Muller, H. J., and Prokofyeva, A. A. 1935. The individual gene in relation to the chromomere and the chromosome. Proc. Natl. Acad. Sci. U S A . 21,16-26. Murray, J. C. 1959. Studies of morphology, genetics, and culture of wild-type and morphological mutant strains of Neurospora crassa. Ph.D. Thesis, Cornell University, Ithaca, New York; abstracted in Diss. Abstr. 20,3480 (1960). Murray, N. E. 1965. Cysteine mutant strains of Neurospora. Genetics 52, 801-808. Murray, N. E. 1965a. Polarized intragenic recombination in chromosome rearrangements of Neurospora. Genetics 58, 181-191. Murray, N. E. 1968b. Linkage information for cysteine and methionine mutants. Neurospora Newsl. 13, 19. Murray, N. E. 1969. Reversal of polarized recombination of alleles in Neurospora as a function of their position. Genetics 61, 67-77. Murray, N. E. 1970. Recombination events that span sites within neighbouring gene loci of Neurospora. Genet. Res. 15, 109-121. Mylyk, 0. M. 1972. The breakdown of duplications heterozygous for the het-c locus in Neurospora crassa. Genetics 71, s43 (abstr.). Mylyk, 0. M. 1975. Heterokaryon incompatability genes in Neurospora crassa detected using duplication-producing chromosome rearrangements. Genetics 80, 107-124.

Mylyk, 0. M. 1976. Heteromorphism for heterokaryon incompatibility genes in natural populations of Neurospora crassa. Genetics 83, 275-284. Mylyk, 0. M., and Threlkeld, S. F. H. 1974. A genetic study of female sterility in Neurospora crassa. Genet. Res. 24, 91-102. Nakamura, K. 1966. Heterogeneity in crossing-over frequency in Neurospora. Genetica 37, 235-246.

Nakamura, K., and Egashira, T. 1961. Genetically mixed perithecia in Neurospora. Nature (London) 190, 1129-1130. Nelson, A. C., and Backus, M. P. 1968. Ascocarp development in two homothallic Neurosporas. Mycologia 60, 1628. Nelson, R. R. 1959. Genetics of Cochliobolus heterostrophus. 11. Genetic factors inhibiting ascospore formation. Mycologia 51, 24-30. Neuffer, M. G. and Coe, E. H., Jr. 1974. Corn (maize). I n “Handbook of Genetics” (R. C. King, ed.), Vol. 2, pp. 3-30. Plenum, New York. Newmeyer, D. 1957. Arginine synthesis in Neurospora: Genetic studies. J . Gen. Microbiol. 16, 449-462.

278

DAVID D. PERKINS AND EDWARD G. BARRY

Newmeyer, D. 1965. Somatic instability in Neurospora resulting from a pericentric inversion. Genetics 52,462-463 (abstr.) , Newmeyer, D. 1970. A suppressor of the heterokaryon-incompatibility associated with mating type in Neurospora crassa. Can. J . Genet. Cytol. 12, 914-926. Newmeyer, D., and Galeazzi, D. 1974. A genetic factor which causes deletion of duplications in Neurospora. Genetics 77, s48 (abstr.). Newmeyer, D., and Galeazzi, D. R. 1976a. The instability of Neurospora duplication D p U L + ZR)H4260, and its genetic control. (In preparation.) Newmeyer, D., and Galeazzi, D. R. 1976b. A meiotic mutant which causes deletion of duplications in Neurospora. (In preparation.) Newmeyer, D., and Taylor, C. W. 1967. A pericentric inversion in Neurospora, with unstable duplication progeny. Genetics 56, 771-791. Newmeyer, D., Howe, H. B., Jr., and Galeazzi, D. R. 1973. A search for complexity at the mating-type locus of Neurospora crassa. Can. J . Genet. Cytol. 15,577-585. Nga, B. H. 1968. Production and analysis of new translocations in A . nidulans. Aspergillus Newsl. 9, 16-18 (cited by Clutterbuck, 1970). Nilan, R. A. 1974. Barley (Hordeum vulgare). In “Handbook of Genetics” (R. C. King, ed.), Vol. 2, pp. 93-110. Plenum, New York. Novak, D. R., and Srb, A. M. 1973. A dominant round spore mutant in Neurospora tetrasperma and its implications for ascospore development. Can. J . Genet. Cytol. 15, 685-693. Olive, L. S. 1965. Nuclear behavior during meiosis. In “The Fungi: An Advanced Treatise” (G. C. Ainsworth and A. S. Sussman, eds.), Vol. 1, pp. 143-161. Academic Press, New York. Parag, Y., and Parag, G. 1975. Mutations affecting mitotic recombination frequency in haploids and diploids of the filamentous fungus Aspergillus nidulans. M o l . Gen. Genet. 137,109-123. Patau, K. 1963. The origin of chromosomal abnormalities. Pathol. Biol. 11, 1163-1170. Pateman, J. A. 1959. The effect of selection on ascospore size in Neurospora crassa. Heredity 13, 1-21. Patterson, J. T., Brown, M. S., and Stone, W. S. 1940. Experimentally produced aneuploidy involving the autosomes of Drosophila melanogaster. Univ. Tex. Publ. 4032, 167-189. Peacock, W. J., and Miklos, G. L. G. 1973. Meiotic drive in Drosophila: New interpretations of the Segregation Distorter and sex chromosome systems. Adv. Genet. 17, 361-409. Perkins, D. D. 1949. Biochemical mutants in the smut fungus Ustilago maydis. Genetics 34,607-626. Perkins, D. D. 1953. The detection of linkage in tetrad analysis. Genetics 38, 187-197. Perkins, D. D. 1962a. Crossing over and interference in a multiply marked chromosome arm of Neurospora. Genetics 47, 1253-1274. Perkins, D. D. 196213. The frequency in Neurospora tetrads of multiple exchanges within short intervals. Genet. Res. 3, 315-327. Perkins, D. D. 1966a. Preliminary characterization of chromosome rearrangements using shot asci. Neurospora Newsl. 9, 10-11. Perkins, D. D. 1966b. Details for collection of asci as unordered groups of eight projected ascospores. Neurospora Newsl. 9, 11. Perkins, D. D. 1967. Manifestation of reciprocal and insertional translocations in unordered tetrads, and their frequency in Neurospora. Genetics 56, 580-581 (abstr.) ,

THE CYTOGENETICS OF

Neurospora

279

Perkins, D. D. 1969a. Heterozygosity for the C/c heterokaryon compatibility alleles in duplications generated by a translocation in Neurospora. Proc. Znt. Congr. Genet., 12th, 1968 Vol. 1, p. 67 (abstr.). Perkins, D. D. 1969b. Multiple alleles a t a heterokaryon-compatibility locus in Neurospora. Genetics 61, s47 (abstr.). Perkins, D. D. 1971a. Viable duplications from intercrossing partially overlapping reciprocal translocations in Neurospora. Genetics 68, s50 (abstr.) . Perkins, D. D. 1971b. Gene order in the albino region of linkage group I. Neurospora Newsl. 18, 14-15. Perkins, D. D. 1972a. An insertional translocation in Neurospora that generates duplications heterozygous for mating type. Genetics 71,25-51. Perkins, D. D. 1972b. Special-purpose Neurospora stocks. Neurospora Newsl. 19, 30-32. Perkins, D. D. 1972c. Linkage testers having markers near the centromere. Neurospora Newsl. 19, 33. Perkins, D. D. 1973. Additional special-purpose Neurospora stocks. Neurospora Newsl. 20, 40. Perkins, D. D. 1974. The manifestation of chromosome rearrangements in unordered asci of Neurospora. Genetics 77, 459489. Perkins, D. D. 1975. The use of duplication-generating rearrangements for studying heterokaryon incompatibility genes in Neurospora. Genelics 80,87-105. Perkins, D. D. 1976. Behavior of Neurospora silophila mating-type alleles in heterozygous duplications after introgression into N . crassa. (In preparation.) Perkins, D. D., and Barratt, R. W. 1973. A modest proposal regarding gene symbols. Neurospora Newsl. 20, 38. Perkins, D. D., and Murray, N. E. 1963. New markers and linkage data. Neurospora Newsl. 4, 2627; see also Errata in Neurospora Newsl. 6, 27 (1964). Perkins, D. D., Glassey, M., and Bloom, B. A. 1962. New data on markers and rearrangements in Neurospora. Can. J. Genet. C y t o l . 4, 187-205. Perkins, D. D., Newmeyer, D., Taylor, C. W., and Bennett, D. C . 1969. New markers and map sequences in Neurospora crassa, with a description of mapping by duplication coverage, and of multiple translocation stocks for testing linkage. Genetica 40, 247-278. Perkins, D. D., Newmeyer, D., and Turner, B. C. 1972. Nontandem duplications in Neurospora, and restoration of the euploid condition by chromosome breakage. Genetics 71, s46-s47 (abstr.). Perkins, D. D., Turner, B. C., and Barry, E. G. 1976. Strains of Neurospora collected from nature. Evolution 30, 281-313. Petes, T. D., Byers, B., and Fangman, W. 1,. 1973. Size and structure of yeast chromosomal DNA. Proc. Natl. Acnd. Sci. U 2 . A . 70, 3072-3076. Phillips, R . 1,. 1967. The association of linkage group V with chromosome 2 in N e w o spora crassa. J . Hered. 58,263-265. Phillips, R. L. and Magill, J. 1969. Synthesis of a 7-chromosome (complete) interchange in Neurospora crassa: A comparative study with maize. Maize Genet. Coop. News L e t t . 43, 115-116. Phillips, R. L., and Springer, W. D. 1972. A cytogenetic method for utilizing nuclear male-sterility in hybrid corn production. Maize Genet. Coop. News L e t t . 46, 124-125. Phillips, R. L., Burnham, C. R., and Patterson, E. B. 1971. Advantages of chromo-

280

DAVID D. PERKINS AND EDWARD G. BARRY

soma1 interchanges that generate haplo-viable deficiency-duplications. Crop Sci. 11, 525-528. Pincheira, G., and Srb, A. M. 1969. Genetic variation in the orientation of nuclear spindles during the development of asci in Neurospora. Amer. J. Bot. 56,846-852. Pittenger, T. H. 1954. The general incidence of pseudo-wild types in Neurospora crassa. Genetics 39,326-342. Pittenger, T. H. 1958. Mitotic instability of pseudo-wild types in Neurospora. A o c . Int. Congr. Genet., loth, 1965 Vol. 2, pp. 218-219 (abstr.). Pittenger, T. H., and Brawner, T. G. 1961. Genetic control of nuclear selection in Neurospora heterokaryons. Genetics 46, 1645-1663. Radford, A. 1972. Revised linkage maps of Neurospora crassa. Neurospora Newsl. 19, 25-26. Radford, A. 1976. Linkage maps of Neurospora crassa. In “Handbook of Biochemistry and Molecular Biology,’’3rd ed. (G. D. Fasman, ed.), Nucleic Acids, Vol. 2, pp. 739-761. Chem. Rubber Publ. Co., Cleveland, Ohio. Radu, M., Steinlauf, R., and Koltin, Y. 1974. Meiosis in Schizophyllum commune. Chromosomal behavior and the synaptinemal complex. Arch. Microbiol. 98, 301310. Raju, N. B. 1976. Ascus development and nuclear behavior in five homothallic species of Neurospora. (In preparation.) Rasch, E. M., Barr, H. J., and Rasch, R. W. 1971. The DNA content of sperm of Drosophila melanogaster. Chromosoma 33, 1-18. Rawls, J. M., Jr., and Lucchesi, J. C. 1974. Regulation of enzyme activities in Drosophila. I. The detection of regulatory loci by gene dosage responses. Genet. Res. 24, 59-72. Ray, R., and Dutta, S. K. 1972. Isolation of transfer RNA genes from Neurospora crassa. Biochem. Biophys. Res. Commun. 47, 1458-1462. Regnery, D. C. 1947. A study of the leucineless mutants of Neurospora crassa. Ph.D. Thesis, California Institute of Technology, Pasadena. Reissig, J. L. 1963. Spectrum of forward mutants in the p y r J region of Neurospora. I. Mutations from arginine dependence to prototrophy. Genet. Res. 1, 356-374. Reissin, J. L. 1963. Spectrum of forward mutants in the pyr-3 region of Neurospora. J . Gen. Microbiol. 30, 327-337. RethorB, M A . , Lejeune, J., Carpentier, S., Prieur, M., Dutrillaux, B., Seringe, P., Romier, A., and Job, J.-C. 1972. Trisomie pour la partie distale du bras court du chromosome 3 chez trois germains. Premier exemple d’insertion chromosomique: ins(7;3) (q31;p21p26). Ann. Genet. 15, 159-165. RethorB, M.-O., Dutrillaux, B., Job, J.-C., and Lejeune, J. 1974. Trisomie 4p par aneusomie de recombinaison d’une inv(4) (p14q35). Ann. Genet. 17, 109-114. Rhoades, M. M. 1950. Meiosis in maize. J . Hered. 41, 58-67. Rhoades, M. M. 1955. The cytogenetics of maize. In “Corn and Corn Improvement” (G. F. Sprague, ed.), pp. 123-219. Academic Press, New York. Rhoades, M. M. 1968. Studies on the cytological basis of crossing over. In “Replication and Recombination in Genetic Material’’ (W. J. Peacock and R. D. Brock, eds.), pp. 229-241. Aust. Acad. Sci., Canberra. Rhoades, M. M., and Dempsey, E. 1966. Induction of chromosome doubling at meiosis by the elongate gene of maize. Genetics 54,505-522. Rick, C. M. 1971. Some cytogenetic features of the genome in diploid plants species. Stadler Genet. Symp. 1/2, 153-174.

THE CYTOGENETICS OF

Neurospora

281

Rick, C. M. 1974. The tomato. In “Handbook of Genetics” (R. C. King, ed.), Vol. 2, pp. 247-280. Plenum, New York. Rick, C. M., and Butler, L. 1956. Cytogenetics of the tomato. Adv. Genet. 8, 267382.

Rines, H. W., Case, M. E., and Giles, N. H. 1969. Mutants in the arom gene cluster of Neurospora crassa specific. for biosynthetic dehydroquinase. Genetics 61, 789-800.

Ritari, S. J., Sakami, W., and Black, C. W. 1973. Identification of two genes specifying folylpolyglutamic synthase. Neurospora Newsl. 20, 27. Rizet, G., and Engelmann, C. 1949. Contribution It 1’Btude genhtique d’un Ascomyc6te tBtrasporB: Podospora anserina (Ces.) Rehm. Rev. Cytol. Biol. Veg. 11, 201-304.

Roberts, P. A. 1974. Rapid evolutionary changes in chromomeric patterns of chromosome tips in wild-type laboratory strains of Drosophila melanogaster. Genetics 77, 64-955 (abstr.). Roberts, P. A. 1975. In support of the telomere concept. Genetics 80, 135-142. Robinow, C. F., and Bakerspigel, A. 1965. Somatic nuclei and forms of mitosis in fungi. In “The Fungi: An Advanced Treatise” (G. C. Ainsworth and A. S. Sussman. eds.), Vol. 1, pp. 119-142. Academic Press, New York. Rodarte-Rambn, U. S., and Mortimer, R. K. 1972. Radiation-induced recombination in Saccharomyces: Isolation and genetic study of recombination-deficient mutants. Radiat. Res. 49, 133-147. Roderick, T. H. 1971. Producing and detecting paracentric chromosomal inversions in mice. Mutat. Res. 11, 59-69. Roderick, T. H., and Hawes, N. L. 1974. Nineteen paracentric chromosomal inversions in mice. Genetics 76, 109-117. Rogers, J. D. 1965. Hypoxylon fuscum. I. Cytology of the ascus. Mycologia 57, 789-803.

Roman, H. 1947. Mitotic nondisjunction in the case of interchanges involving the B-type chromosome in maize. Genetics 32, 391-409. Roper, J. A. 1952. Production of heterozygous diploids in filamentous fungi. Experientk 8, 14-15. Roper, J. A. 1973a. Vegetative instability in fungi. The role of chromosome aberrations. Zn “Symposium on the Use of Radiation and Radioisotopes for Genetic Improvement of Industrial Microorganisms,” pp. 113-121. IAEA, Vienna. Roper, J. A. 1973b. Mitotic non-conformity in the fungus Aspergillus nidulans. Chromosomes Today 4 , 297-305. Rossen, J. M., and Westergaard, M. 1966. Studies on the mechanism of crossing over. 11. Meiosis and the time of meiotic chromosome replication in the ascomycete Neottiella rutilans (Fr.) Dennis. C . R . Trav. Lab. Carlsberg 35, 233-260.

Roth, R., and Fogel, S. 1971. A system selective for yeast mutants deficient in meiotic recombination. Mol. Gen. Genet. 112, 295-305. Ryan, F. J. 1950. Selected methods of Neurospora genetics. Methods Med. Res. 3, 51-75.

Sager, R. 1972. “Cytoplasmic Genes and Organelles.” Academic Press, New York. St. Lawrence, P. 1950. Cytologic preparations (in Ryan, 1950, p. 57). St. Lawrence, P. 1953. The association of particular linkage groups with their respective chromosomes in Neurospora crassa. Ph.D. Thesis, Columbia University, New York; abstracted in DGs. Abstr. 14, 7-8 (1954).

282

DAVID D. PERKINS AND EDWARD G . BARRY

St. Lawrence, P. 1959. Gene conversion at the nic-2 locus of Neurospora crassa in crosses between strains with normal chromosomes and a strain carrying a translocation a t the locus. Genetics 44,532 (abstr.). St. Lawrence, P., and Singleton, J. R. 1963. The cytogenetic effects of a paracentric inversion in Neurospora crassa. Genet. Today, Proc. I n t . Congr., I l t h , 1963 Vol. 1, p. 119 (abstr.). Sandler, L., Lindsley, D. L., Nicoletti, B., and Trippa, G. 1968. Mutants affecting meiosis in natural populations of Drosophila melanogaster. Genetics 80, 525558. Sansome, E. R. 1946. Heterokaryosis, mating-type factors, and sexual reproduction in Neurospora. Bull. Torrey Bot. Club 73, 397-409. Sansome, E. R. 1956. Camphor-induced gigas forms in Neurospora. Trans. Br. Mycol. SOC.39, 67-78. Sansome, E., and Brasier, C. M. 1973.Diploidy and chromosomal structural hybridity in Phytopthora infestans. Nature (London) 241,344-345. Sansome, E. R., Demerec, M., and Hollaender, A. 1945. Quantitative irradiation experiments with Neurospora crassa. I. Experiments with X-rays. A m . J. B o t . 32, 218-226. Schrantz, J.-P. 1970. Etude cytologique, en microscopie optique et Clectronique, de quelques Ascomychtes. I. Le noyau. R e v . Cytol. Biol. Veg. 33,l-100. Schroeder, A. L. 1970.Ultraviolet-sensitive mutants of Neurospora. I. Genetic basis and effect on recombination. M o l . Gen. Genet. 107,291-304. Schroeder, A. L. 1974. Properties of a W-sensitive Neurospora strain defective in pyrimidine dimer excision. Mutat. Res. 34, 9-16. Schroeder, A. L. 1975. Genetic control of radiation sensitivity and DNA repair in Neurospora. I n “Molecular Mechanisms for Repair of DNA” (P. C. Hanawalt and R. B. Setlow, eds.), Part B, pp. 567-576. Plenum, New York. Searle, A. G., Ford, C. E., and Beechey, C.V. 1971. Meiotic disjunction in mouse translocations and the determination of centromere position. Genet. Res. 18, 215-253. Shaffer, B., Brearly, I., Littlewood, R., and Fink, G. R. 1971. A stable aneuploid of Saccharomyces cerevisiae. Genetics 87, 483-495. Shear, C. L., and Dodge, B. 0. 1927. Life histories and heterothallism of the red bread-mold fungi of the Monilia sitophila group. J . Agric. Res. 34, 1019-1042. Silver, W.S., and McElroy, W. D. 1954. Enzyme studies on nitrate and nitrite mutants of Neurospora. Arch. Biochem. Biophys. 51, 379-394. Simonet, J. M., and Zickler, D. 1972. Mutations affecting meiosis in Podospora anserina. I. Cytological studies. Chromosoma 37, 327-351. Singleton, J. R. 1948. Cytogenetic studies of Neurospora crassa. Ph.D. Thesis, California Institute of Technology, Pasadena. Singleton, J. R. 1953. Chromosome morphology and the chromosome cycle in the ascus of Neurospora crassa. A m . J. Bot. 40, 124-144. Smith, D. A. 1974. Unstable diploids of Neurospora and a model for their somatic behavior. Genelics 78, 1-17. Smith, D. A. 1975. A mutant affecting meiosis in Neurospora. Genetics 80, 125-133. Smith, D. A., and Perkins, D. D. 1972. Aneuploidy associated with the osmoticsensitive cut strain in Neurospora. Genetics 71,s60 (abstr.). Sobell, H. M. 1974. Concerning the sterochemistry of strand equivalence in genetic recombination. In “Mechanisms in Recombination” (R. W. Grell, ed.), pp. 433438. Plenum, New York.

THE CYTOGENETICS OF

Neurospora

283

Somen, C. E., Wagner, R. P., and Hsu, T. C. 1960. Mitosis in vegetative nuclei of Neurospora crassa. Genetics 45, 801-810. Sparrow, A. H., Underbrink, A. G., and Sparrow, R. C. 1967. Chromosomes and cellular radiosensitivity. I. The relationship of Do to chromosome volume and complexity in seventy-nine different organisms. Radiat. Res. 32, 915-945. Srb, A. M. 1963. Extrachromosomal factors in the genetic differentiation of Neurospora. S y m p . SOC.Exp. Biol. 17, 175-187. Srb, A. M., Bad, M., Bobst, M., and Leary, J. V. 1973. Mutations in Neurospora crmsa affecting ascus and ascospore development. J. Hered. 64, 242-246. Srb, A. M., Nasrallah, J. B., and Basl, M. 1974. Genetic control of the development of the sexual reproductive apparatus of Neurospora. Brookhaven S y m p . Biol. 25, 40-50. Stadler, D. R. 1956. A map of linkage group VI of Neurosporu crassa. Genetics 41, 528-543. Stadler, D. R. 1973. The mechanism of intragenic recombination. Annu. R e v . Genet. 7, 113-127. Stadler, D. R., and Towe, A. M. 1962. Genetic factors influencing crossing-over in Neurospora. Genetics 47, 839-846. Stanford Neurospora Methods. 1963. Neurospora Newsl. 4, 21-25. Stewart, B. R., and Merriam, J. R. 1974. Segmental aneuploidy and enzyme activity as a method for cytogenetic localization in Drosophila melanogaster. Genetics 76, 301-309. Strickland, W. N. 1960. A rapid method of obtaining unordered Neurospora tetrads. J. Gen. Microbiol. 22, 583-588 (see also Perkins, 1966b). Sturtevant, A. H., and Beadle, G. W. 1936. The relations of inversions in the X chromosome of Drosophila melanogaster to crossing over and disjunction. Genetics 21, 554-604. Subden, R. E., and Threlkeld, S. F. H. 1970. Genetic fine structure of the albino (all region of Neurospora erassu. Genet. Res. 15, 139-146. Sussman, A. S., Garrett, M. K., Sargent, M., and Yu, S.-A. 1971. Isolation, mapping and characterization of trehalaseless mutants of Neurospora crassa. J . Bacteriol. 108, 59-68. Tatum, E. L., Barratt, R. W., Fries, N., and Bonner, D. 1950. Biochemical mutant strains of Neurospora produced by physical and chemical treatment. A m . J. Bot. 37, 38-46. Tector, M. A,, and Kafer, E. 1962. Radiation-induced chromosomal aberrations and lethals in Aspergillus nidulans. Science 136, 1056-1057. Therkelsen, A. J., HultBn, M., Jonasson, J., Lindsten, J., Christensen, N. C., and Iversen, T. 1973. Presumptive direct insertion within chromosome 2 in man. Ann. Hum. Genet. 36, 367-373. Threlkeld, S. F. H., and Stoltz, J. M. 1970. A genetic analysis of non-disjunction and mitotic recombination in Neurospora crassa. Genet. Res. 16, 29-35. Towe, A. M. 1958. Factors influencing crossing over in Neurospora. Microb. Genet. Bull. 16, 31-32. Turner, B. C. 1975. Duplications in Neurospora produced meiotically by a translocation T ( I + VI)NM103, and their breakdown to produce euploid derivatives. Genetics 80, s81 (Abstr.). Turner, B. C. 1976. Euploid derivatives of duplications from a translocation in Neurospora. (In preparation.)

284

DAVID D. PERKINS AND EDWARD G. BARRY

Turner, B. C., Taylor, C. W., Perkins, D. D., and Newmeyer, D. 1969. New duplication-generating inversions in Neurospora. Can. J . Genet. Cytol. 11, 622-638. Underbrink, A. G., Sparrow, A. H., and Pond, V. 1968. Chromosomes and cellular radiosensitivity. 11. Use of interrelationships among chromosome volume, nucleotide content and Do of 120 diverse organisms in predicting radiosensitivity. Radiat. Bot. 8, 205-238. Upshall, A., and Kafer, E. 1974. Detection and identification of translocations by increased specific nondisjunction in Aspergillus nidulans. Genetics 7 6 , 19-31. Vigfusson, N. V., and Cano, R. J. 1974. Artificial induction of the sexual cycle of Neurospora crassa. Nature (London) 248, 383-385. Vigfusson, N. V., Walker, D. G., Islam, M. S., and Weijer, J. 1971. The genetics and biochemical characterization of sterility mutants in Neurospora crassa. F o l k Microbial. (Prague) 16, 166-196. Wagner, R. E., Jr., and Radman, M. 1975. A mechanism for initiation of genetic recombination. Proc. Natl. Acad. Sci. USA. 7 2 , 3619-3622. Wallace, B.,and Kass, T. L. 1974. On the structure of gene control regions. Genetics 77, 541-558. Webber, B. B. 1965. Genetical and biochemical studies of histidine-requiring mutants of Neurospora crassa. 4. Linkage relationships of his-3 mutants. Genetics 51, 275-283.

Weijer, J. 1965. Feulgen staining of somatic nuclei of N . crassa. Neurospora Newsl. 7 , 25-26. Weijer, J., and McDonald, B. R. 1965. Karyokinesis of somatic nuclei of Neurospora crassa. IV. The occurrence of the juvenile ring phase during division IV of ascosporogenesis. Can. J. Genet. Cytol. 7 , 519-522. Weijer, J., and Vigfusson, N. V. 1972. Sexuality in Neurospora crassa. I. Mutations to male sterility. Genet. Res. 19, 191-204. Wells, K. 1970. Light and electron microscope studies of Ascobolus stercorarius. I. Nuclear divisions in the ascus. Mycologia 62, 761-790. Westergaard, M. 1964. Studies on the mechanism of crossing over. I. Theoretical considerations. C. R . Trav. Lab. Carlsberg 34, 359-405. Westergaard, M., and von Wettstein, D. 1966. Studies on the mechanism of crossing over. 111. On the ultrastructure of the chromosomes in Neottiella rutilans (Fr.) Dennis. C . R . Trav. Lab. Carlsberg 35, 261-286. Westergaard, M., and von Wettstein, D. 1970. The nucleolar cycle in an ascomycete. C. R . Trav. Lab. Carlsberg 37, 195-237. Westergaard, M., and von Wettstein, D. 1972. The synaptinemal complex. Annu. R e v . Genet. 6, 71-110. Wheeler, H. E., and Driver, C. H. 1953. Genetics and cytology of a mutant, dwarfspored Glomerella. A m . J. Bot. 40, 694-702. White, M. J. D. 1973. “Animal Cytology and Evolution,” 3rd ed. Cambridge Univ. Press, London and New York. Wiebe, G. A., and Ramage, R. T. 1971. Hybrid barley. Barley Genet. 2, Proc. Znt. Sump., h d , 1969 pp. 287-291. Wiebe, G. A., Ramage, R. T., and Eslick, R. E. 1974. Eight paired barley lines. Barley Genet. Newsl. 4, 93-95. (Cited by permission.) Wilcox, M. S. 1928. The sexuality and arrangement of the spores in the ascus of Neurospora sitophila. Mycologia 20, 3-17. Wildenberg, J., and Meselson, M. 1975. Mismatch repair in heteroduplex DNA. Proc. Natl. Acod. Sci. U.S.A. 72, 2202-2206.

THE CYTOGENETICS OF

Neurospora

285

Williams, L. G., and Davis, R. H. 1968. Genetic and physical relationships between two early steps of pyrimidine synthesis. Genetics 60, 238 (abstr.). Wilson, J. F. 1961. Micrurgical techniques for Neurospora. A m . J. Bot. 48, 46-51. Wood, D. D., and Luck, D. J. L. 1969. Hybridization of mitochondria1 ribosomal RNA. J. M o l . Biol. 41, 211-224. Worthy, T. E., and Epler, J. L. 1972. Repair of ultraviolet light-induced damage to the deoxyribonucleic acid of Neurospora crnssn. J. Bacterial. 110, 1010-1016. Worthy, T. E., and Epler, J. L. 1973. Biochemical basis of radiation-sensitivity in mutants of Neurospora crassa. Mutat. Res. 19, 167-173. Zickler, D. 1967. Analyse de la m6iose du champignon discomychte Ascobolus immerzis (Pers.). C . R . Hebd. Seances Acad. Sci., Ser. D 265, 198-201. Zickler, D. 1970. Division spindle and centrosomal plaques during mitosis and meiosis in some ascomycetes. Chromosoma 30, 287-304. Zickler, D. 1971. DBroulement des mitoses dans les filaments en croissance de quelques ascomychtes. C. R . Hebd. Seances Acatl. Sci., Ser. D 273,1687-1689. Zickler, D. 1973. Fine structure of chromosome pairing in ten ascomycetes: Meiotic and premeiotic (mitotic) synaptonemal complexes. Chromosoma 40, 401416.

GENETIC ANALYSIS OF CHLOROPLAST DNA IN Ch/amydomonas Ruth Sager Department of Microbiology and Molecular Genetics. Harvard Medical School. and Sidney Farber Cancer Institute. Boston. Massachusetts

I . Introduction . . . . . . . . . . . . I1. Identification of Cytoplasmic Genes in Chlamydomonas

. . . . . . . . . . . . A. Terminology . . . . . . . . . . . . . . . . . B . Criteria for Identification of Cytogenes . . . . . . . . . C . Phenotypic Classes of Cytoplasmic Mutants in Chlamydomonas . . I11. Mechanism of Maternal (4:O) Inheritance in Chlamydomonas . . . . A . Effects of UV Irradiation and Drug Pretreatments of Gametes . . . B . Differential Fates of Chloroplast DNAs in Zygotes . . . . . . C . The mat Mutations . . . . . . . . . . . . . . . IV . Methods of Genetic Analysis . . . . . . . . . . . . . A . Pedigree Analysis . . . . . . . . . . . . . . . . B . Liquid-Culture Kinetic Analysis . . . . . . . . . . . . C . Statistical Analysis of Large Undissected Zygote Colonies . . . . V . Results of Genetic Analysis of the Chloroplast Genome in Chlamydomonas . A . Introduction . . . . . . . . . . . . . . . . . B. The Segregation Process in Zoospore Clones . . . . . . . . C . Mapping by Means of Cosegregation Frequencies . . . . . . D . Closing the Circle . . . . . . . . . . . . . . . . E . Persistent Cytoplasmic Heterozygotes . . . . . . . . . . F. Genetic Diploidy of the Chloroplast Genome in Zoospores . . . . VI . Association of Cytoplasmic Genes with Organelle DNAs and Structures . A . Is the Cytoplasmic Linkage Group Located in Chloroplast DNA? . . B . Is sd a Mitochondria1 Gene? . . . . . . . . . . . . C. Is spc 1-27-3 a Chloroplast Gene? . . . . . . . . . . .

. .

D Cell-Wall Mutants E Concluding Remarks References . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . .

287 289 289 289 293 29.5 29.5 296 300 304 305 309 310 311 311 314 317 317 321 323 329 330 331 332 333 335 336

.

1 Introduction

The first genetic evidence for the existence of cytoplasmic genes regulating chloroplast development was published in 1909 (Baur. 1909; Correns. 1909a.b) ; the identification of high-molecular-weight DNA in chlo287

288

RUTH SAGEB

roplasts was first reported in 1963 (Sager and Ishida, 1963; Chun et al., 1963). During the intervening 54 years, evidence slowly accumulated showing that eukaryotic cells contain cytoplasmic genes (Correns, 1937; Caspari, 1948; Rhoades, 1955), but interest was scant until presence of DNA in chloroplasts (Sager and Ishida, 1963; Chun et al., 1963) and in mitochondria (Luck and Reich, 1964; M. M. K. Nass and Nass, 1963; S. Nass and Nass, 1963) was well established. I n the past 10 years, research into the genes, DNAs, and functions of chloroplast and mitochondria1 genomes has burgeoned. We now know that chloroplasts and mitochondria contain unique DNA genomes that are transcribed within the organelle, and mappable by recombination analysis; and that these genomes function in the biogenesis of the corresponding organelles. However, present-day knowledge provides only a sketchy framework for understanding the tightly coordinated interactions between organelle and nuclear genomes that are essential for regulation of cellular metabolism and growth (reviewed in Sager, 1972). The study of cytoplasmic genes, as of nuclear genes, can be roughly divided into two sectors: experiments focusing on the genetic material itself; and experiments focusing on the functions of the cytoplasmic genomes. There is also a dichotomy between experiments designed primarily to examine the DNA, and those designed to dissect out the genes. The situation is reminiscent of the early development of classical cytogenetics, in which a similar dichotomy existed between formal genetic and chromosome cytology. Many of the classical experiments combined genetic and cytological approaches, and similarly today, some of the best insights into the behavior of cytoplasmic genes are coming from an integrated approach combining genetic and molecular studies of organelle DNAs. Also, following the precedents of microbial genetics, understanding of genomic function is heavily dependent upon the identification of individual cytoplasmic genes. Thus despite the formidable advances in molecular biology and its technology, genetically defined mutations and recombination analysis still play a central role in the investigation of cytoplasmic genetics. The book “Cytoplasmic Genes and Organelles” (Sager, 1972) attempted to summarize the subject in 400 pages: cytoplasmic DNAs, formal genetic analysis, and functions of cytoplasmic genes, especially in organelle biogenesis. Since then, the subject has continued to grow, and it cannot effectively be discussed in a single review chapter. Aspects of the subject have been presented in a number of recent reviews, of which the most specifically genetic are those of Preer (1971) and Gillham (1974). Here I have chosen to focus on genetic analysis and correlated physical studies of the chloroplast DNA of Chlamydomnas, in particular

GENETIC ANALYSIS OF CHLOROPLAST DNA

289

those aspects of the subject that have progressed in the past few years. This review was completed in December, 1975.

II. Identification of Cytoplasmic Genes in Chlamydomonas

A. TERMINOLOGY Cytoplasmic genes were first identified by the failure to follow Mendel’s laws, and were therefore called non-Mendelian genes. This negative terminology was appropriate to a time when neither the molecular identity nor cellular location of these “factors” were known. With the identification of chloroplast and mitochondrial DNAs, it became a strong inference (Platt, 1964) that some, if not all, non-Mendelian genes were located on these DNAs and could therefore reasonably be called organelle genes or cytoplasmic genes-more concisely cytogems. I n yeast, some cytoplasmic genes have been identified with mitochondrial DNA by a number of criteria and are generally called mitochondrial genes (Mounolou e t al., 1966). In Chlamydomonas, the presence of both chloroplast and mitochondrial DNAs presents a special problem in the attribution of cytoplasmic genes to one or the other organelle. As will be discussed below, the cytoplasmic genes that fall into a single linkage group have been shown by a number of criteria to be located in chloroplast DNA, and they are therefore referred to as chloroplast genes. Use of the term UP (uniparental) is to be discouraged, since it represents no conceptual advance over non-Mendelian ; furthermore it is inappropriate, since cytoplasmic genes are inherited biparentally in zygotes used for genetic analysis.

B. CRITERIAFOR IDENTIFICATION OF CYTOGENES 1.

4

0 Segregation

The first cytoplasmic mutation discovered in Chlamydomonas was detected by its special pattern of inheritance (Sager, 1954). A streptomycinresistant mutant strain, selected on streptomycin-agar and resistant to 500 pg of the drug per milliliter, was crossed with a wild-type str.ain, and the four meiotic products recovered from each zygote were found to be streptomycin-resistant (sr). Test crosses of F, and F, progeny showed that transmission of the sr mutation was regulated by the nuclear matingtype locus; that the mutant sr and the wild-type ss behaved as alleles; and that whichever allele was present in the mt+ parent was transmitted

290

RUTH SAGER

to all four progeny, while the allele in the mt- parent disappeared in the zygote, and did not reappear among any of the progeny in further testcrosses. The mt+ has been designated the female parent because of the similarity of this pattern of inheritance with maternal transmission of cytogenes in higher plants (Sager, 1972). The cytogene mutations so far reported in Chlamydomonus, numbering close to 100 (Table 1) have all shown this same pattern of 4:O segregation regulated by the mt locus. Thus, in Chlamydomonas, the pattern of 4 :0 segregation is the first criterion for identifying cytoplasmic mutant genes. Exceptions to the 4:O rule have been reported; they will be mentioned here, and discussed in later sections as noted: (1) partial preservation of the male cytoplasmic genome under the influence of the mat-1 mutation (Section 111, B) ; (2) persistent cytoplasmic heterozygotes, an apparent exception to the rule (Section V, E) . In addition, Davies (1972) has described cell-wall mutants that may be nuclear but exhibit aberrant segregation ratios in backcrosses. They do not fit clearly either nuclear or cytoplasmic criteria and will be discussed below. 2. Mitotic Segregation and Recombination

A second means of distinguishing nuclear from cytoplasmic genes is based on the occurrence of segregation and recombination in mitotic cells that are cytoplasmic heterozygotes (cytohets) . In maternal inheritance, of course, the mitotic cells issuing from a cross are homozygous for all cytoplasmic genes. However, spontaneous exceptions to the rule of maternal inheritance occur with a low frequency (about 0.1% in our stocks; higher in some strains). When these exceptions were examined, they were found to contain zoospores that were heterozygous for all the cytoplasmic markers in the cross (Sager, 1954, 1972; Sager and Ramanis, 1970). These cytohets were not persistent ; the homozygous pure types segregated out during the first several mitotic doublings of each zoospore clone. This process of segregation and recombination, seen with either spontaneous (low frequency) (Sager and Ramanis, 1965) or UV-induced (high frequency) (Sager and Ramanis, 1967) biparental zygotes, has provided the basic data for genetic analysis. Thus, a second key distinction between nuclear and cytoplasmic genes of Chlamydomonas lies in the mitotic recombination and allelic segregation of cytoplasmic, but not of nuclear, alleles. The criteria of 4:O segregation in crosses and allelic segregation in mitosis have been found to be applicable to identification of cytoplasmic genes in many organisms (Sager, 1972). In addition, with each organism

GENETIC ANALYSIS OF CHLOROPLAST DNA

291

TABLE 1 Cytoplasmic Gene Mutation in Chlamydomonas Gene acl ad acS ac4 tml tmd

Seven tm mutants ti1 thru t i 6 eryl kanl spcl spil thru 6 ole1 t h r u 3 curl clel eryS

eryll sm2 smS sm4 SmS

D-371 and D-310

Four D mutants Eleven D mutants

D-769 Three D mutants UV-16 UV-17

Origina

SM induced S M induced SM induced S M induced S M induced SM induced S M induced NG SM induced S M induced SM induced SM induced SM induced S M induced S M induced S M induced

S M induced

S M induced SM induced S M induced S M induced Induced by growth of strain sm4 without SM Induced by growth of strain sm4 without S M Induced by growth of strain sm4 without SM Induced by growth of strain sm4 without SM Induced by growth of strain sm4 without SM UV induced in strain sm4

Phenotype

Mappedb

Requires acetate (leaky) Requires acetate (stringent) Requires acetate (stringent) Requires acetate (leaky) Cannot grow a t 35°C Conditional: grows at 35°C only in the presence of streptomycin Cannot grow at 35°C

Yes Yes Yes Yes Yes No

Tiny colonies on all media Resistant to 50 pg/ml erythromycin Resistant to 100 pg/ml kanamycin Resistant to 50 pg/ml spectinomycin Resistant to 100 pg/ml spiramycin Resistant to 50 pg/ml oleandomycin Resistant to 50 pg/ml carbamycin Resistant to 50 pg/ml cleosine Resistant to erythromycin, carbamycin, oleandomycin, spiramycin (same concentrations as single mutations above) Same as for eryS Resistant to 500 pg/ml SM Resistant to 50 pg/ml S M SM dependent Resistant to 500 pg/ml SM; recombines with sm2 Resistant to 500 pg/ml S M

No Yes No Yes Yes Yes Yes Yes No

Resistant to 500 pg/ml SM; segregate like persistent hets (sd/sr) Resistant to various low levels of SM: 20 pg/ml; 50 pg/ml; 100 pg/ml. Segregate like persistent hets (sdllow sr) Conditional sd

No

Conditional sd; segregate like persistent HETS (sdlcond. sd)

No

Resistant to 500 pg/ml SM

No

No

No Yes Yes Yes Yes No

No

Yes

(continued)

292

RUTH SAGER

TABLE 1 (continued) Phenotype

Gene

Origino

Four U V mutants Three UV mutants

UV induced in strain sm4 UV induced in strain sm4 Spontaneous mutations selected on SM

Resistant to 20 pg/ml SM

No

Resistant to 20 pg/ml SM; segregate like persistent HETS (sdllow sr) Resistant to SM 500 pg/ml

No

Spontaneous; selected on kanamycin NG NG NG NG NG.

Resistant to kanamycin 50 pg/ml

No

Resistant to erythromycin 100 pg/ml Resistant to erythromycin 100 pg/ml Resistant to spectinomycin 100 pg/ml Dependent on at least 20 pg/ml SM Resistant to 1 mg/ml neamine

No No No No Yes

sr-8-1 ST-2-60 ST-2-280 ST-8-21 8 kan-1

ery-2-y ery-3-6 spr-1-27 sd-3-18 nea-2-1 a

Mappedb

No

SM = streptomycin; NG = nitrosoguanidine. Mapping references (see Sager, 1972).

studied, special criteria have been developed, derived either from special properties of the organism or special techniques of investigation. In Chlamydomonas, three additional criteria have been utilized : mutagen specificity, properties of persistent cytohets, and UV effect on maternal inheritance.

3. Streptomycin as a Mutagen Streptomycin (SM) was shown to act in Chlamydomonas as .a specific mutagen, mutageniaing cytoplasmic genes with a high efficiency, while having little or no mutagenic effect on nuclear genes (Sager, 1962). Cytoplasmic mutations to streptomycin resistance were shown by fluctuation analysis to be induced by growth in the presence of streptomycin (Sager, 1960, 1962; Sager and Tsubo, 1962; Gillham and Levine, 1962). Most of the markers used in genetic analysis were selected after SM mutagenesis (Table 1 ) . The mechanism of its mutagenic action and the basis of its specificity for cytoplasmic, but not for nuclear, DNAs are unknown. Since the drug binds to DNA, its mutagenic action may result from a direct interaction a t the DNA level, with chloroplast DNA more available to the drug than nuclear DNA. Another possibility is that the mutagenic action is a second.ary effect resulting from streptomycin-in-

GENETIC ANALYSIS OF CHLOROPLAST DNA

293

duced miscoding, leading to phenotypically altered chloroplast DNA polymerase or repair enzyme. Cytoplasmic but not nuclear mutations have also been induced in a streptomycin-dependent ( s d ) strain by streptomycin withdrawal (Sager and Ramanis, 1 9 7 6 ~ )When . growth of an sd strain in streptomycin-free medium ceases, a burst of mutants appear, including apparent revertants to sensitivity, as well as other types of mutations: various levels of streptomycin resistance, temperature sensitivity, and acetate requirement. I n contrast to streptomycin, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) has been found to induce mutations in every system to which it has been applied. In Chlamydomonas, both nuclear and cytoplasmic gene mutations are induced by this drug (Gillham, 1965). Its drawback as a mutagen of choice for cytoplasmic genes is the preponderance of nuclear mutations that are simultaneously induced. Lee and Jones (1973) showed that treating synchronous cultures during the period of nuclear DNA replication with MNNG yielded a 15-30-fold increase in nuclear gene mutations from streptomycin sensitivity to resistance but no increase in chloroplast mutations to streptomycin resistance. Treating cultures with this mutagen during replication of chloroplast DNA yielded an increase of 1.5-1.6-fold in both chloroplast and nuclear mutations, an increase that Lee and Jones consider statistically significant.

4. UV Effect on Maternal Znheritance The dramatic effect of UV irradiation upon maternal inheritance (Sager and Ramanis, 1967) provides another special criterion for identifying cytoplasmic genes in Chlamydomonas. As discussed below (Section 1111, UV irradiation of female gametes just before mating leads to an inhibition of maternal inheritance, i.e., transmission to progeny of male as well as female cytogenes. This response clearly distinguishes nuclear from cytoplasmic genes, since nuclear gene transmission is unaffected by the low dose of UV that is effective in inhibiting maternal inheritance. Pretreatment of gametes before mating with various inhibitors of protein synthesis (Sager and Ramanis, 1973) or the presence of a single nuclear mutant gene mat-1 (Sager and Ramanis, 1974) also inhibit maternal inheritance in a manner similar to the UV effect. The mechanism of these effects is discussed in Section 111,C. C. PHENOTYPIC CLASSES OF CYTOPLASMIC MUTANTSIN Chlamydomonas Four general classes of mutants that show 4:O segregation in crosses have been reported and are listed in Table 1. Close to 100 mutations have

294

RUTH SAGER

been crossed to distinguish them from nuclear gene mutations. Of these, the 20 that have been mapped (Sager, 1972; R. Sager, unpublished; Sager and Ramanis, 1976b) fall into a single linkage group (Section V, C) * The acetate-requiring mutants (Sager, 1972) are green when grown in the light with acetate as carbon source; they grow for a few doublings (stringent) or slowly for many doublings (leaky) on minimal agar without acetate. Temperature-sensitive mutants grow a t 25W, but not a t 35O-37OC. (R. Sager, unpubl.) . Conditional temperature-sensitive mutants have been isolated that can grow a t the nonpermissive temperature if the medium is supplemented (Sager and Ramanis, 1970, and unpublished). A third class of mutants, giving tiny colonies on agar, have been crossed (R. Sager, unpublished), but they are very difficult to handle and therefore have not yet been mapped. Mutants of the most popular class, the only class that has been examined genetically by other investigators (reviewed in Sager, 1972; Gillham, 1974) as well as ourselves, are drug resistant. The drugs commonly employed are those that inhibit protein synthesis on 70 S but not on 80 S ribosomes: streptomycin, neamine, spectinomycin, kanamycin, erythromycin, spiromycin, cleocin, oleandomycin, and carbomycin. Chloroplast ribosomes, which are 70 S (Sager and Hamilton, 1967; Hoober and Blobel, 1969), show many similarities to bacterial ribosomes (Nomura, 1970). Biochemical studies of some of these mutants have shown that several mutational alterations are a t the chloroplast ribosome level (reviewed in Sager and Schlanger, 1976). Three spectinomycin-resistant strains have been described, the SPC mutant from our laboratory (Sager, 1972; Sager and Ramanis, 1976c), spr 1-27-3 (Boynton et al., 1973), and spr 1-6-2 (Gillham, 1969). The spc mutant studied by Burton (1972) is now reported to be identical to spr 1-27-3 (Gillham, 1974). The physiological and biochemical studies described in Boynton et al. (1973) were carried out with spr 1-27-3, whereas the genetic analyses (Gillham et al., 1974) were done with spr 1-6-2. The mutant spr 1-6-2 appears to be identical with our spc mutant, giving no spc-sensitive recombinants in crosses (R. Sager and 2. Ramanis, unpublished) and showing a similar phenotype: high-level resistance in liquid culture and on agar, resistance a t the 70 S ribosome level in poly(U) -directed in vitro polypeptide synthesis. The spr 1-27-3 mutant, on the contrary, is phenotypically distinguishable on agar, by its low level resistance to the drug, from our spc mutant and genetically different as shown by recovery of spc-sensitive recombinants (R. Sager and Z. Ramanis, unpublished). The cytoplasmic mutations to drug resistance so far investigated have

GENETIC ANALYSIS OF CHLOROPLAST DNA

295

shown little or no cross-resistance to other antibiotics a t the ribosomal level (Schlanger and Sager, 1974b). However, some chloroplast mutants have also been isolated (R. Sager, unpublished) that show cross-resistance to two or more of the macrolide antibiotics erythromycin, carbomycin, oleandomycin, and spiromycin. Some of them also cross-react with cleocin, a lincomycin derivative, which is not a macrolide. Similar patterns of cross-resistance have been reported in bacteria (Pestka, 1971). No cross-reaction has been found between any of these mutants and either streptomycin or spectinomycin. The neamine-resistant mutant is cross-resistant to kanamycin, but the kanamycin-resistant mutants are not cross-resistant to neamine or any other tested drugs. One of the lcanamycin-resistant mutants has been shown to have drug-sensitive chloroplast ribosomes (N. Ohta and R. Sager, unpublished).

Ill. Mechanism of Maternal (4:O)Inheritance in Chlamydomonas

The events responsible for loss of the paternal chloroplast genome leading to maternal inheritance occur after zygote formation, since the pairs of isogamous (equal-size) gametes contribute their entire cellular contents whcn they fuse to form zygotes. The process differs from fertilization in higher plants and animals, in which the maternal cytoplasmic contribution is very much larger than the paternal one. Yet the genetic outcome is the same. What is the mechanism of maternal inheritance in Chlarnydonionas? And is it fundamcntally different a t the molecular level from that occurring in higher organisms? Three lines of experimentation have been reported that bear on the mechanism of maternal inheritance in Chlamydomonas: (1) effects of UV irradiation and of various inhibitors of protein synthesis upon the pattern of cytogene transmission (Sager and Ramanis, 1967, 1973) ; (2) behavior in zygotes of chloroplast DNAs, differentially labeled, from the two parental strains (Chiang, 1968, 1971; Sager and Lane, 1969, 1972; Sager, 1972; Schlanger and Sager, 1974a); (3) effects of two mutant genes, mat-1 and mat-2, altering the transmission pattern (Sager and Ramanis, 1974). A. EFFECTS OF UV IRRADIATION AND DRUG PRETREATMENTS OF GAMETES

UV irradiation of the mi+ (female) parent just before mating increases the frequency of exceptional zygotes from the spontaneous frequency of about 0.1% up to 40-100%, depending upon the UV dose

296

RUTH SAGER

(Sager and Ramanis, 1967). At higher doses, the exceptional zygotes include an increasing number of paternal zygotes, in which the maternal cytogene complement is absent. Thus the ratio of maternal: biparental: paternal zygotes is UV-dose dependent. Furthermore, the UV effect is photoreactivable (see Figs. 4 and 5a). UV irradiation of the mt- (male) gametes has no effect on the pattern of cytogene inheritance, and UV irradiation of zygotes is highly lethal. The UV doses that give a maximum yield of biparental zygotes show 30-60% survival of photoreactivated gametes, and little or no killing of photoreactivated zygotes, presumably in part because of rescue by enzymes of the unirradiated parent after mating, as well as by photoreactivation. The UV experiments demonstrate that some factor produced by the female parent is required for effective loss of the male cytoplasmic genome, i.e., for maternal inheritance. A number of drugs known to block chloroplast DNA replication, transcription, and protein synthesis were examined for their effects on cytogene inheritance (Sager and Ramanis, 1973). Ethidium bromide (10 pg/ml) was found t o produce an effect similar to that of UV: when female gametes were treated for 6 hours just prior to zygote formation, a great increase (about 70-fold) in exceptional zygotes resulted, whereas a similar treatment of male gametes had little effect. Rifamycin SV, erythromycin, and spiramycin were each more effective (about 30-fold increase in exceptional zygotes over controls) when the male gametes were treated than when the female gametes were treated (approximately 5-fold increase over controls). Effects of cycloheximide were inconclusive, since extensive lethality occurred a t all effective concentrations. These studies provide evidence that both parental strains contribute different but essential components to the process of maternal inheritance. The UV and ethidium bromide effects suggest that, for maternal inheritance to result, transcription of DNA must occur in the female gametes; and the inhibitor studies indicate that protein synthesis must occur in the male gametes.

B. DIFFERENTIAL FATESOF CHLOROPLAST DNAs

IN

ZYGOTES

When total DNA from vegetative cells of Chlamydomonus is centrifuged to equilibrium in CsCl, two major bands are seen: nuclear DNA a t 1.723-5 gm/cm3 and chloroplast DNA a t 1.695 gm/cm3, the latter about 15% of the total DNA extracted from cells in log-phase growth. When total DNA is extracted from young zygotes, 6-24 hours after mating, the nuclear DNA appears unchanged, but the chloroplast is now 0.005 gm/cm3 lower in buoyant density, banding a t 1.690 gm/cm3 (Sager

GENETIC ANALYSIS OF CHLOROPLAST DNA

297

and Lane, 1972) as shown in Fig. 1. After replication this DNA returns to its former density. We have shown that chloroplast DNA in zygotes banding a t 1.690 gm/cm3 originates in the female (mt+) parent and that the homologous chlDNA from the male (mt-) parent is degraded soon after mating. The different fates of chlDNAs from the two parents were followed by prelabeling the parental DNAs differentially with I5N/l4N in some experiments (Sager and Lane, 1969, 1972) and with 3H/14Cadenine in others (Schlanger and Sager, 1974a). A typical preparative gradient is shown in Fig. 2, in which the chlDNA of male origin is no longer present. What causes the density shift? We infer that it results from covalent addition of some component with a lighter buoyant density in CsCl than chloroplast DNA, covalent because the density shift withstands detergent extraction with sodium lauryl sulfate, sarkosyl, Triton X-100, and deoxycholate, as we11 as enzymic digestion with Pronase, ribonucleases, and amylase. If the density shift results from methylation, as in bacterial 1.724

FIG.1. Density shift of chloroplast DNA in zygotes of Chlamydomotaas. Microdensitometer tracings of DNAs extracted from (a) mt' gametes and (b) zygotes kept in nitrogen-free medium for 24 hours after mating. Nuclear DNA bands a t 1.724 gm/cm3. I n (a) chloroplast DNA bands a t 1.694 gm/cm3, but in (b) it has shifted to a new density of 1.689 gm/cm3. Density markers are crab poly(dAT1 (from N. Sueoka) a t 1.680 gm/cm' and Bacillus aubtilh phage-15 DNA (from J. Marmur) at 1.761 gm/cm3. (From Sager and Lane, 1972.)

RUTH SAGER

FIG.2. Preparative CsCl gradient of DNA extracted from zygotes 6 hours after mating. mt+ cells were pregrown with “C-adenine and mt- cells with 3H-adenine to prelabel DNA. (From Schlanger et at., manuscript in preparation.)

modification, about 5% methylation would be required (Szybalski and Szybalska, 1971; Kirk, 1967) and should be detectable; experiments are in progress to look for it. I n the reciprocal crosses between parents grown in 15N- and in 14Nlabeled medium, a single chloroplast peak (Fig. 3) was found in DNA from zygotes sampled a t 6 hours and at 24 hours after mating: a t 1.692 gm/cm3 in the 14N(mt+)X 16N(mt-) cross, and 1.698 gm/cm3 in the reciprocal cross. As listed in Table 2, the densities were similar to but

GENETIC ANALYSIS O F CHLOROPLAST DNA

299

not identical with those from 15Nx 15N and from 14NX 14N crosses. The 14NX 16Naverage value was about 0.002 gm/cm3 heavier than the I4N X 14N average, and similarly, the 15N X 14N value was about 0.002 gm/cm3 lighter than the 15NX 16Nvalue. These small but significant differences could be the result of limited recombination, except that we have genetic evidence indicating that recombination of our markers, situated all around the map (Section V, D) , does not occur in unirradiated crosses a t a frequency that would be detectable in the DNA. Another possibility, which we favor, is that the 0.002 gm/cm3 density difference reflects either repair or partial replication in which the nucleotides come from a common 15N-14N pool (Sager, 1'975). Another point to be noted from Table 2, is that the density shift in the 15NX 15N chloroplast DNA is greater than in the 14NX 14N DNA, and similarly in the pairs of reciprocal crosses. This difference could be the result of a heavy-isotope effect, in which the presence of 15N influences the extent of covalent addition of whatever component is responsible for the density shift of maternal chloroplast DNA in zygotes (Sager, 1975).

1.761

b

FIG.3. CsCl density equilibrium centrifugation. (a) DNA extracted from "N x "N zygotes, 6 hours after mating. (b) DNA from "N x I4N zygotes, extracted a t 6 hours, as in (a). Nuclear peaks are overloaded; positions of single chloroplast peaks a t 1.692 gm/cms in (a) and a t 1.699 gm/cm' in (b) are estimated from poly(dAT) a t 1.680 gm/cm3. DNAs from extraction fraction (a) (44,000rpm, 20 hours, 25°C). (From Sager and Lane, 1972.)

300

RUTH SAGER

TABLE 2 Buoyant Densities of Chloroplast DNAs from Zygotes" Crossb

14N

x

1"

X 16N 1sN X 14N 16N X 16N 14N

vegetative cells gametes 16N vegetative cells 16N gametes 1"

1"

Observed density"

Computedd

Discrepancy

1.690 f 0,0009 1.692 f 0.001 1.698 f 0.0009 1.7005 1.695 f 0.0005 1.695 f 0.0005 1.709 f 0.0005 1.709 f 0.0005

1.690 1.6935 1.7005 1.704

0 0.0015 0.0025 0.0035

-

From Sager a.nd Lane (1972); R. Sager, unpublished. Female (mt+) parent given first. Data from 5 or more preparations except '6N X 16N from two preparations. d Computation assumes one round of semiconservative replication: one strand conserved and one strand replicated from equal pools of 14N and 16N precursors in reciprocal crosses; both strands modified (0.005gm/cma lighter). a

Chiang (1968) has reported the results of experiments using parents pregrown with 3H- and 14C-labeled adenine to distinguish the parental DNAs in zygotes, and later (1971) using both density and radioisotope labeling simultaneously. He reported equal contributions of label from both parents in DNAs extracted several days after mating, and from zoospores after meiosis and zygote germination, and concluded that chloroplast DNAs from both parents were conserved in the zygote. I n the double-isotope experiments, Chiang extracted DNA as early as 30 hours after mating, but still was unable to recover discretely banding DNA in the chloroplast DNA region. Nonetheless, he again concluded that the chloroplast DNAs from the two parents were both conserved. Chiang's erroneous conclusion is primarily the consequence of two experimental difficulties: (1) his inability to recover chloroplast DNA from zygotes in a sufficiently undegraded condition to identify peak positions in preparative gradients ; and ( 2 ) his sampling times, which were too late in zygote development to see the significant events that determine maternal inheritance of chloroplast DNA, and occur within a few hours after zygote formation.

c. THEmat

MUTATIONS

An independent line of evidence on the mechanism of maternal inheritance comes from our discovery of two nuclear mutations, mat-1 and mat-2, that alter the pattern of cytogene transmission. The mat-1 muta-

301

GENETIC ANALYSIS O F CHLOROPLAST DNA

tion was identified by the very high frequency of exceptional zygotes, both biparental and paternal, occurring in crosses not UV-irradiated. The mat-2 mutation was identified by the very low frequency of exceptional zygotes occurring in crosses that were UV-irradiated. The principal properties conferred by these mutations are listed in Table 3 (Sager and Ramanis, 1974). The responses of mat-1 and mat-2 mutants to UV irradiation are shown in Figs. 4 and 5. The mat genes are closely linked to mating type: mat-1 to mt- and mat-2 to mt+. The linkage is not suprising, since the maternal transmission of cytogenes is regulated by the mating type locus or factors tightly linked to it (Sager, 1954). As pointed out by Gillham (1969), it seems likely that several genes are localized in the mating-type region; the mating-type difference appears to govern a number of coupled molecular events. We have interpreted the functions of the mat genes in terms of a model TABLE 3 Properties of mat-1 and mat-,??Mutant Genes. Property 1. Phenotype a

wt

mat-1

Frequency of spontaneous exceptional zygotes 0.1 %-LO%

High frequency of spontaneous exceptional zygotes (20-100 %)

b

UV increases frequency of exceptional zygotes, dose dependent

C

Both biparental and paternal among exceptional zygotes (paternal rare)

UV low dose further increases frequency of exceptional zygotes to 100 % Both biparental and paternal among exceptional zygotes (paternal frequent) Expressed only in zygotes with mat-1- mtparents Linked to mtVariable

d

2. Inheritance 3. Zygote viability

Close t o 100% ~

From Sager and Ramanis (1974).

mat-,??

Lower than wildtype frequency of spontaneous exceptional zygotes (0.02-0.09 %) UV high dose slightly increases frequency exceptional zygotes to ca. 10% Only biparental; no paternal zygotes

Expressed only in zygotes with mat-,??-mt+ parents Linked to mtf Close to 100%

RUTH SAGER

T

e

a B P

FIG.4. Comparison of percent maternal, biparental, and paternal zygotes recovered in crosses with and without mat-1-. Frequency of maternal (M), biparental (B), and paternal (P) zygotes in two crosses: open bars: mat-I+ mt+ X m5t-l' m t - ; filled bars: mat-l+mt+x mat-l- m t - ; NI, not irradiated; W - D k , 50 seconds UV; UV-Lt, to seconds UV plus photoreactivation. (a) N I 0 x N I 8 ; (b) UV-Dk 0 x N I 8 ; (c) UV-Lt 0 x N I 8 ; (d) N I 0 x UV-Dk 8 ; (el NI 0 x W - L t 8 . (From Sager and Ramanis, 1974.) (Fig. 6) previously proposed (Sager and Ramanis, 1973) to explain the mechanism of maternal inheritance in Chlamydomonas. The model is based on our hypothesis that maternal inheritance of cytogenes is regulated by modification and restriction of chloroplast DNA occurring in zygotes. On this model, an inactive modification enzyme [MI is postulated to be present in the chloroplast of mt+ gametes, and an inactive restriction enzyme [R] in the chloroplast of mt-gametes. Both enzymes are postulated to be activated a t the time of mating; their effects are seen only in zygotes. The activation of these two enzymes is regulated by a pair of postulated compounds G, and G,, both synthesized in the mt+ cells, but acting in zygotes. The fact, noted in light microscope (R. Sager, unpublished) and electron microscope (Friedmann et al., 1968; CavalierSmith, 1970) studies that chloroplast fusion occurs some 6 hours after zygote formation, provides time for modification to occur within the chloroplast from the female parent, and degradation of chloroplast DNA to occur within the plastid from the male parent, while both coexist within the zygote. In summary, the mechanism of maternal inheritance of chloroplast

GENETIC ANALYSIS OF CHLOROPLAST DNA

,

1

Exposure to UV(sec)

.

0

0

303

30 60 90 Duration of UV irradiation (sac)

FIG.5A. Effect of UV irradiation on maternal inheritance. Percent of exceptional and biparental zygotes recovered in crosses after irradiating mt+ cells. Curves: (1) mat-2' mt+ x mat-2' mt-, exceptional zygotes ( 2 ) mat-2' mtc x mat-2' mt-, biparental zygotes (3) mat-2- mt+ x mat-2' mt; exceptional zygotes, all biparental. FIG.5B. UV survival curves for exponentially growing cultures. 0-0 mat-2; 0-0 21 gr (wild-type). (From Sager and Ramanis, 1974.)

genes in Chlamydomonas is postulated to be the modification and restriction of chloroplast DNA. Direct evidence in support of this hypothesis comes from the disappearance of paternal chloroplast genes. Indirect supporting evidence comes from the effects of UV irradiation and drug pretreatments of gametes, and from the phenotypes conferred by the mat-1 and mat-2 mutations. All these lines of evidence support the thesis that the female (mt+)parent regulates maternal inheritance by contributing a component (G2) that activates the restriction enzyme R, present in the male chloroplast after mating. This activation leads to the degradation of male chloroplast DNA, and a t the same time the female chloroplast DNA is modified so that it becomes resistant to the restriction enzyme. Of the four postulated components, two (i.e., the restriction enzyme R and its activator, G,) have been tentatively identified as products of the mat-1 and mat-2 genes respectively, which are closely linked to mating type. It seems likely that the other two, the modification enzyme M and its activator GI, are also coded by genes in this mating type complex. However, mutations leading to loss of modification activity

304

RUTH SAGER

-

newly formed zygote

maturing zygote

FIG. 6. Postulated control of maternal inheritance of chloroplast DNA in Chlamydomonas by a modification-restriction mechanism. Female (mt') gamete contains inactive modification enzyme M in chloroplast, and two regulatory substances, GI and G,, in the cell sap. The male ( m t - ) gamete contains inactive restriction enzyme R in its chloroplast. After zygote formation and before fusion of chloroplasts, the modification enzyme is activated by G, to modify chloroplast DNA in the female chloroplast, and the restriction enzyme is activated by G, to degrade chloroplast DNA in the male chloroplast. The two chloroplasts then fuse, and only the chloroplast DNA from the female parent is available for replication. (Nuclei are not shown for sake of clarity.) Mat-I gene is postulated to code for R. M a t 4 gene is postulated to code for G, and G?. (From Sager and Ramanis, 1973.)

would presumably be lethal and could only be studied in cells with no restriction activity. IV. Methods of Genetic Analysis

Methods have been developed over the past 20 years in our laboratory for genetic analysis of cytoplasmic genes. Genetic analysis based on segregation and recombination has been carried out with biparental zygotes, either of spontaneous origin or UV-induced. A comparison of results from zygotes of bath types will be discussed below (p. 311). The analytical methods were developed in response to the problems encountered as we went along. Initially, we tried to devise methods that were as unbiased as possible, taking nothing for granted beyond our (tested)

GENETIC ANALYSIS OF CHLOROPLAST DNA

305

ability to score accurately the progeny recovered from crosses. We investigated (1) the events affecting cytoplasmic gene markers occurring in the zygote, and the distribution of cytogenes t o the four meiotic products; (2) the events occurring in the first few mitotic doublings, examining each doubling independently by means of pedigree analysis; and (3) the events occurring over the range of doublings (about 10) required before most of the heterozygous genes had segregated, examined in liquid cultures of zoospore clones (Singer et 1976).

A. PEDIGREE ANALYSIS 1. Preparation of Zygotes

Parental strains, started from single colonies, are grown on a nitrogendeficient minimal agar medium in continuous light until they approach stationary growth. Cells are washed off into a 5-fold diluted nitrogenfree minimal medium (Sager and Granick, 1954) a t p H 8 and left overnight on a table top a t room temperature. When the parental strains are mixed the next day, zygote formation is fast (about 1 hour to completion) and the yield is usually over 90%. Zygotes are plated a t dilution onto minimal medium plus 0.1% acetate-containing agar, incubated overnight in the light, and then kept in dark 5-8 days. When 7nt+ cells are UV irradiated before mating, the mating mixture is kept in the dark for 2 hours, then plated and incubated overnight in the light, and stored in the dark as above. Zygotes germinate synchronously following incubation for about 18 hours in continuous light. Vegetative cells contaminating zygote plates are killed by 30 seconds of exposure to chloroform vapor, usually the day before zygote germination. 2. Preparation of Pedigrees

Germinating zygotes are transferred individually to fresh minimal agar a t the four-cell (tetrad) stage, and a t the 8-cell stage, cells are spread over the petri dish. After one further doubling, each pair is locally respread, so that, after growth, 16 colonies are expected from each zygote. Occasional losses are more than offset by the ease of the mcthod, which permits routine preparation of about 40-50 zygotes per hour. The four products of meiosis, the zoospores, are distinguished from each other by the segregation of three pairs of unlinked nuclear genes act (actidione resistance), 7ns (methionine sulfoximine resistance), and vat (mating type) which also identify the zoospore sisters or octospores. The pairs of octospore daughters are recognized by their position on the plates, following local respreading. Thus, as shown in Fig. 7, the pedigree

306

RUTH SAGER

0 Zygote

I I

0

0

2

1

0

0 3

4

Zoospores

la

la1 l a 2

Ib

20

l b l l b 2 201 2 0 2

2b 3a Oc tospores

3b

4a

4b

2bl 2b2 3al 3 0 2 3bl 3b2 401 4 a 2 4bl 4 b 2 Octospore daughters

FIG.7. Procedure for pedigree analysis. After germination, zoospores are allowed to undergo one mitotic doubling and then the eight cells (octospores) are transferred to a fresh petri plate and respread. After one further doubling, each pair of octospore daughters is separated and allowed to form colonies. The sixteen colonies, derived from the first two doublings of each zoospore are then classified for all segregating markers. (From Sager and Ramanis, 1970.) is fully established for the first two postmeiotic divisions (Sager and Ramanis, 1963, 1965,1967, 1968, 1970, 1976a). 3. Importance of Pedigree Analysis

The value of pedigree analysis in delineating the segregation and recombination behavior of cytoplasmic genes can hardly be overstated. We began cytoplasmic genetic analysis in total ignorance of the number of genetic copies involved, of whether the contributions of the two parents were equal, and above all ignorance of the mechanism of distribution of cytogenes to daughter cells. I n this circumstance, every bit of information that could possibly be gleaned from the classification of progeny needed to be considered. Fortunately, the Chlamydomonas life cycle lends itself readily to tetrad and postmeiotic pedigree analysis, as outlined above. The use of pedigree analysis permits one to examine events occurring in the zygote

307

GENETIC ANALYSIS OF CHLOROPLAST DNA

as expressed in the phenotypes of the four zoospores, and to distinguish them from events occurring a t each of the postmeiotic divisions. By events we mean exchanges or conversions a t the DNA level, which are scored phenotypically as allelic segregation or recombination. With this method we have been able to determine the extent of segregation and recombination occurring in zygotes and to compare these results with the data from postmeiotic divisions. This distinction has turned out to be critically important in evaluating the number of genetic copies present, as will be discussed below. I n our studies (Sager and Ramanis, 1963, 1965, 1967, 1968, 1970, 1976a,b; Singer et al., 1976), we used the frequency and pattern of distribution of cytogenes in postmeiotic divisions as the basis for mapping. In our initial examination of pedigrees, we found that a t each division of cells containing heterozygous cytogene markers, one of three patterns of distribution occurred: type I, type 11, or type 111 (Fig. 8). In type I events, both daughter cells are heterozygous like the parent cell, showing that no exchange event has affected the gene being observed. I n a type 111 event, one daughter becomes homozygous for one of the parental alleles, and the other daughter for the other one, suggesting the occurrence of a reciprocal exchange event in the parent cell before cell division. I n a type I1 event, one daughter cell remains heterozygous and the other HET -1:

t

m + m

TypeII:

r n + P 0rHET+P2 1

Type 111:

PI

+

P2

sr + sr

sr sr

sr

Qf.

SS

+

ss

FIG.8. Alternative segregation patterns of chloroplast allelic pairs a t mitosis. H E T refcrs to any vegetative cell containing a pair of chloroplast alleles, e.g., sr (streptomycin resistant) and ss (streptomycin sensitive). Type I segregation preserves the heterozygous condition; type I1 gives rise to one daughter cell that is HET and one that is pure for either of the two parental alleles; Type I11 gives rise to two daughter cells, each pure type for one of the parental alleles. See Fig. 11 for proposed mechanisms of segregation.

308

RUTH SAGER

becomes homozygous for one or the other of the parental alleles, suggesting the occurrence of a nonreciprocal event. Pedigree analysis provides the only means for distinguishing among these types of events, determining their relative frequencies, and tracing the individual DNA strands for one, two, or three rounds of replication, back to the zygote. With multiply marked stocks, it has been possible to obtain a fairly detailed picture of the type I1 and type 111 events occurring a t the DNA level, as they are reflected in allelic segregation and recombination. A further advantage of pedigree analysis is that no selection is involved: the entire “family” is recovered on nonselective media, and aliquots of each clone are subsequently scored on selective media. A typical pedigree is shown in Fig. 9. In addition to providing for an unequivocal distinction between zygotic and postzygotic events, pedigree analysis makes possible the distinction between reciprocal and nonreciprocal segregational events. For example, in clone 1, a t the first mitotic doubling, the genes sm2, ery, and car showed a type 11 segregation pattern, and in clone 2, the gene smd showed type I1 segregations in each pair of progeny a t the second doubling, and so did spc. In clone 3, ery and car showed type I11 segregations a t the second doubling of one pair of daughter cells, and type I1

Genes

acl ac2 sm2 9

car tml

ZygOtlC

-

-

Clone 2

Zoos~ores’

1

2

3

4

1

2

3

4

1

2

3

4

1

2

3

4

A

B

C

D

0

3

0

3

0

0

1

2

0

0

0

0

0

0

1

1

0

0

0

0

0

3

0

3

0

0

1

2

0

0

0

0

0

0

1

1

0

0

0

0

0

3

2

3

0

1

1

0

0

2

0

0

2

0

0

0

0

0

0

0

0

3

2

3

1

1

1

1

1

2

2

0

2

1

0

0

0

1

0

0

0

3

2

3

1

1

1

1

1

2

0

1

0

0

0

0

0

1

0

0

2

3

2

3

2

0

o

i

2

z

2

1

o

o

2

o

2

0

0

0

2

3

2

3

0

0

0

0

0

0

0

1

0

0

2

0

2

0

0

0

s,

=,

I

I

GENETIC ANALYSIS OF CHLOROPLAST DNA

309

events a t the second doubling in the other pair. However, the type I1 events occurred on different strands and involved different parental alleles (Sager and Ramanis, 1976a).

B.

LIQUID-CULTURE

KINETICANALYSIS

For some purpose it is useful to examine segregation and recombination events in large populations. We developed a simple method to follow the segregation of individual markers in multigene crosses over many cell generations (Sager and Ramanis, 1968). The method was originally developed to follow segregational events beyond the first few doublings accessible in pedigrees. We have subsequently adapted the method to assess the map position of new markers relative to those already established (Sager, 1972; Singer et at., 1976). The method can also be used to sample a population of exponential or synchronously growing zoospore clones at any desired time after zygote germination. Zoospores a t the 4-cell stage from synchronously germinating zygotes are washed off of agar and grown in liquid culture. Cells are sampled a t hourly intervals, plated on nonselective media, and subsequently replicaplated to selective media to determine the frequency of each phenotypic class. The data are presented as a set of survival curves, plotting the log percent of each gene surviving in heterozygous condition against time (Fig. 10). The results, discussed in Section V, C, have shown that each gene segregates with a constant frequency per unit time and has a different and characteristic survival rate, expressed as the slope of the survival curve. The ratios of slopes determined for each of a set of genes segregating in a single cross, bear a constant relationship, which provides a basis for mapping. The differential slopes reflect the differential frequencies of type I11 events for each gene. I n this method, type I1 and type I11 segregation events cannot be distinguished, but type I1 events, which occur with equal frequency for all genes (Table 5 ) do not contribute to the differcntial segregation rates. The success of this method is demonstrated by the excellent agreement with the data obtained by pedigree analysis (Singer et al., 1976). The reliability and usefulness of slope data for mapping has been greatly strengthened by establishing best slope values for five key genes that are well spaced around the map. Data were pooled from 16 experiments involving 11 different multifactor crosses and evaluated by a powerful statistical method (an adaptation of jackknife methodology to a regression problem) especially developed for the purpose (Singer et al., 1976). The method provides a way to assess the relative slopes of

310

RUTH SAGER

1

I

1

I

I

I

3

6

0

I2

IS

IS

I

21

1

I

I

24

27

30

TIME (HOURS)

FIG.10. Segregation ratios for five key genes. The slopes were computed from pooled data of 15 experiments. Slope values and variability are given in Tablc 6. For method of computation see text and Singer et al. (1976).

new markers tested in single crosses in relation to the pooled data for the five key markers.

C. STATISTICAL ANALYSISOF LARGEUNDISSECTED ZYGOTECOLONIES Recently, Gillham et al. (1974) have used a third method of analysis. They start with large zygote colonies containing about lo6 cells, growing on nonselective media, replicate them to determine which are maternal and which exceptional, and then sample the original colonies from exceptional zygotes to determine the ratios of maternal and paternal alleles of each of the three drug-resistance markers segregating in the cross. This method is good for distinguishing exceptional from maternal zygotes, with respect to whichever gene is used for identification purposes. The method does not allow distinction between segregation events oc-

GENETIC ANALYSIS OF CHLOROPLAST DNA

31 1

curring in zygotes and in postmeiotic divisions, or between reciprocal and nonreciprocal exchanges, nor does it protect the investigator from differential growth rates of progeny clones. Furthermore, the method provides no check on aberrant nuclear events, since the four products of meiosis are not identified. V. Results of Genetic Analysis of the Chloroplast Genome in Chlamydomonas

A. INTRODUCTION The principal conclusions derived from a large number of crosses, some published and some unpublished, were summarized in the book “Cytoplasmic Genes and Organelles” (Sager, 1972). The purpose of the summary was to provide students and colleagues with an overall picture of the segrcgation and recombinational behavior of the linked set of chloroplast genes we had been investigating, without waiting for the full analysis, which was still in process of preparation. Since that time, with the analysis of more crosses involving new markers, the conclusions presented there have been confirmed and strengthened. I n the present review, the overall picture will again be summarized, and supporting data, including the results of a set of 25 multigene crosses (Table 4) will be included. It should be pointed out that a computer program for fuller data analysis is still being developed; and many new insights may be anticipated to result from that analysis. The principal conclusions so far reached from our analysis of the 25 crosses listed in Table 4 are the following. 1. Chloroplast genes of Chlamydomonas show maternal inheritance and can be identified by this transmission pattern (Sager, 1954). 2. Spontaneous exceptions to the rule of maternal inheritance, in which chloroplast genes from the male parent are transmitted to progeny, occur a t frequencies that are genetically determined, e.g., about 0.1% in our wild-type stocks, up to 20% in some of Gillhams’ stocks (Gillham, 19691, and up to 100% in crosses involving the gene mat-1 (Sager and Ramanis, 1967, 1974). 3. The frequency of exceptional zygotes can be increased to 50% or more by UV-irradiation of the female gametes just before mating. The yield of exceptional zygotes can also be increased by pretreatment of gametes with drugs that inhibit transcription or protein synthesis in the chloroplast (Sager and Ramanis, 1967, 1973). 4. Exccptional zygotes are of two types: biparental zygotes that transmit the chloroplast genome from both parents ; and$paternal zygotes, in which the maternal complement is lost.

TABLE 4 Crosses Used in These Studiesa Cross 1 2 3 4b 5b 6 7 8 9 10 11

12

PI (mi+) acl ac2+ sm2-s sm3-s csd-s 6978a ac2+ sm2-s ery-r 11,108-5 acl ac2+ sm4d sm2-s ery-s 229-3-4 acl s m 4 d nea-s sm2-s 229-3-4 ac2+ sm2-s ery-s nea-r 12-4 [from S X (G X S)ld acl+ ac2 sm2-s ery-s spc-r tm-r 11,554-4 acl ac2+ sm2-s ery-s spc-r car-s tm-r 11,932-4 acl ac2+ sm2-s ery-s cle-s spc-r tm-r 11,932-4 acl+ ac2 sm2-r ery-r ole-r spc-s tm-s 11,968-6 acl+ ac2 sm2-r ery-r spi-r spc-s tm-s 11,978-3 acl ac2+ sm2-r ery-r car-r spc-s tm-s 11,925-5 acl+ ac2 sm2-r ery-s spc-s tm-s 13,363-4

Pdm-) acl+ ac2 sm2-r sm3-r csd-r 7018g ac2 sm2-r ery-s 7018g acl+ ac2 s m 4 s sm2-r ery-r 11,209-5 acl+ sm4-s nea-r sm2-r 12-62 [from s x (G x S)I ac2 sm2-r ery-r nea-s 11,154-4 acl ac2+ sm2-r ery-r spc-s tm-s 11,358-3 acl+ ac2 sm2-r ery-r spc-s c a w tm-s 11,927-1 acl+ ac2 sm2-r ery-r cle-r spc-s tm-s 11,907-3 acl ac2+ sm2-s ery-s ole-s spc-r tm-r 11,909-3 acl ac2+ sm2-s ery-s spi-s spc-r tm-r 11,909-3 acl+ ac2 sm2-s ery-s car-s spc-r tm-r 11,927-2 acl ac2+ sm2-s ery-r spc-r tm-r 13,363-7

Number of zygotes

Number of zoospores

159

571

62

219

42

140

42

145

53

186

47

170

7

35

16

63

12

43

15

57

41

163

63

242

L-d

9 9

cl M L-d

13 14 15" 16c 17c

18* 20

21

22b 23b 24 25

ac2+ sm2-s spc-s mrl-r tm-r 12,632-6 ac2+ sm2-s spc-s mr2-r tm-r mt+ 12,674-1 ac2+ ac3 sm2-s ery-s spc-s tm-r 7788-12 ac4 sm2-r ery-s spc-s tm-s 7860-19 a c l sm2-r ery-r kan-s tm-s 11,925-5 ac2+ sm2-s spcl-s spc2-r tm-r spc-r 1-27-3 (G) a c l ac2+ sm2-s ery-r spc-r mrl-s tm-r 14,678- 1

ac2+ spc2-r ery-r sm2-s mrl-s tm-r 14,704-1 (FI from cross 18) acl smZ-r spc-r ery-r nea-s tm-s 11,925-1 ac2 sm2-r spc-r ery-r mrl-r nea-s tm-s 14,761-2 ac2+ sm5-r ery-s spc-s tm-s sr 643 ac2 sm3-r ery-s spc-s tm-s 11,054-1 (F1from G X S)

ac2 sm2-r spc-r mrl-s tm-s 13,181-6 ac2 sm2-r spc-r mr2-s tm-s mt13,181-6 ac2 ac3+ sm2-r ery-r spc-r tm-s 13,181-6 ac4+ sm2-s ery-r spc-r tm-r 11,932-4 a c l + sm2-s ery-s kan-r tm-r 11,482-4 ac2 sm2-r spcl-r spc2-s tm-s 13,181-6 a c l + ac2 sm2-r ery-s spc-s mrl-r tm-s 14,689-1 15 sec UV 30 sec UV 50 sec UV ac2 spc2-s ery-s sm2-r mrl-r tm-s 14,689-1 acl+ sm2-s spc-s ery-s nea-r tm-r 13,971-4 (F, from G X S) ac2+ sm2-s spc-s ery-s mrl-s nea-r tm-r 13,971-4 (F1from G X S) ac2 sm5-s ery-r spc-r tm-r 14,681-1 ac2+ sm3-s ery-r spc-r tm-r 14,679-1

From Sager and Ramanis (1976a). Crosses 1-5 were previously described (Sager and Ramanis, 1970). (R. Sager and Z. Ramanis, unpublished). c Liquid-culture sampling only. See Singer et al. (1976). d G: stock from Gillham; S: stock from Sager.

32

125

55

211

-

-

-

-

-

-

20

71

n

2

2

0

&*

F

22 35 31 24

88 139 123 87

54

214

27

101

19

72

17

65

$

0

r 0

E

B

P

5

U

2

a

* Crosses include spe2-r and nea-r markers from Gillham

w c. w

314

RUTH SAGER

5. Zoospores from biparental zygotes are usually, heterozygous for the entire genome, and are referred to as cytohets. (See Table 8 for the frequency. of zoospores in which the individual markers have segregated.) (Sager and Ramanis, 1976a). 6. I n cytohet progeny of biparental zygotes, whether spontaneous or UV induced, two types of exchanges occur: reciprocal (type 111) and nonreciprocal (type 11). The reciprocal event produces two daughter cells, each carrying one (of the parental alleles. The nonreciprocal event, resembling gene conversion, produces one daughter cell which is a pure parental type, and another which is still a cytohet (Figs. 8 and 11).Different genes in the same strand (i.e., double-stranded DNA molecule) undergo type I1 and type I11 segregations in the same round of cell division (Sager and Ramanis, 1976b). 7. Alleles segregate approximately 1:l in zoospore clones that are initially heterozygous. Deviations from 1:1 are allele-specific ; some mutant alleles show nonreciprocal segregation more frequently than do their wild-type counterparts (Sager and Ramanis, 1976a). 8. The gene-specific frequencies of reciprocal exchanges (measured as type I11 events) provide evidence for the existence of a centromere-like attachment point ( u p ) which governs the distribution,of the genomes a t cell division (see Tables 5 and 6 ; Fig. 11) (Sager and Ramanis, 1970, 1976b). 9. Exchanges occur a t each cell division beginning with the first postmeiotic doubling, and continue indefinitely. We have followed the process as long as any heterozygous markers remain to detect it (Sager and Ramanis, 1968; Singer et ul., 1976). 10. Genes can be mapped by several parameters: (a) the frequency of type I11 events affectingleach gene in progeny of multigene crosses; (b) the relative slopes of cytohet survival curves for each of the genes in progeny of multigene crosses; (c) the frequencies of simultaneous type I1 segregation and type I11 segregation of two or more markers; and (d) recombination frequencies. 11. A special condition exists in which cytohets, so-called persistent cytohets, rarely segregate in vegetative growth, but do segregate in meiosis (Sager and Ramanis, 1976d).

B. THESEGREGATION PROCESS IN ZOOSPORE CLONES The process of segregation of cytogene pairs has been studied both in pedigrees and in liquid-culture populations of zoospore clones. Pedigrees are essential for examining the pattern of segregation: to distinguish events occurring in zygotes from those in zoospore clones, and to dis-

315

GENETIC ANALYSIS OF CHLOROPLAST DNA

tinguish between reciprocal and nonreciprocal exchanges. The liquid culture method is preferable for measuring the rate of segregation and for examining the kinetics over several cell generations. The mean frequencies of reciprocal (type 111) and nonreciprocal (type 11) exchanges for each gene pair that occurred in the first two doubIings in the 25 crosses of Table 4 are given in Table 5. The frequencies of type I1 events were relatively constant from cross to cross and from marker to marker. Thus, type I1 events do not seem to be gene specific, and consequently do not, as such, contribute information for mapping purposes. However, the frequencies of simultaneous coconversions of two or more genes do provide valuable mapping data, as will be discussed below. Type 111 segregations have been interpreted, as shown in Fig. 11, to represent reciprocal exchange events, occurring between the gene and the postulated attachment point, ap. The frequencies of type I11 events can be used as measures of gene-ap distance, on the assumption of spatial randomness of occurrence of exchanges along the DNA. The liquid-culture sampling method was developed to measure gene-up distances with greater precision, using much larger numbers of cell and more cell generations than is possible with pedigrees. The slopes shown in Fig. 10 have been computed as for exponential decay curves of the form (-dN/dt) = k N , where Ic is the first-order rate constant, and N is the log of the number of surviving hets a t time t. The slopes have been computed using a statistical method designed to provide the best summary slope from data pooled from many experiments (Singer et al., 1976). These summary slopes and the variability assessment show in Table 6 have been used to estimate the relative distances from ap of a set of five genes, each present as heterozygous markers in many crosses. These highly reliable values have provided a global framework for mapping additional markers, each present in one or two crosses. Comparison with Table 5 shows that the two methods agree quite well in TABLE 5

Type I1 (non- 39.11 reciprocal) Type I11 5.89 (reciprocal)

* 1.18 + 0.56

Gene Segregation Frequenciesaa 39.38

* 1.34

39.58 f 1.40 38.45

+_

1.57 43.86 f 1.18

9 . 7 4 f 1 . 2 3 12.27 f 1.25 11.75 f 1 . 2 0

6.70 f 0.58

From Sager and Itamanis (197613). Number of segregation events/total segregation events possible at first and second zoospore doublings. Data: 2857 zoospore clones from 16 multifactor crosses. a

b

316

RUTH SAGEB

TYPE

I

-Q-+Qs

ery-s

S

NO SEOREQATION

TYPE

It

-Q+Qr s

ery-s

S

s

NONRECIPROCAL

RECIPROCAL

FIO.11. Segregation patterns in circular molecules. Each line represents a double-

stranded DNA. The homologs from the two parents are distinguished by thick and thin lines, with their respective black and white attachment points. Exchanges occur after replication but before cell division at the 4-strand stage. Attachment points regulate distribution of sister strands to different daughter cells as in mitosis. If no exchanges occur, both daughters are heterozygous (type I). Nonreciprocal (type 11) exchange is the result of a miscopying (conversion) event. Reciprocal (type 111) exchange requires two events, one on either side of the recombined region. (From Singer et al., 1976.)

establishing a relative gene order for the five genes ac, sm2, spc, tm, and ery. (In this summary, data have not been presented on other markers present in some of the crosses, and the closely linked markers acl and acd have been treated as a single locus ac.) The data obtained from these two mapping procedures do not dis-

GENETIC ANALYSIS OF CHLOROPLAST DNA

317

TABLE 6 Summary Slopes and Variability Assessment for Principal Markersa

Gene

Slope rf: variabilityb

Number of crossesc

tml

-0.01204 rf: 0.0021 -0.01709 0.0084 -0,02459 rf: 0.0027 -0.02513 0.0138 -0,04512 k 0.0060

15 13 13 14 10

ac sm2 SPC

ery

+

From Singer d al. (1976). See original paper for methods of computation. c Crosses 7 and 8 were done twice; cross 12 three times; all others listed in Table 1 once each. Ac segregation was not scored in cross 8A or cross 16. Sm2 was not scored in cross 7A and 7B; spc was not scored in cross 17; ery was not scored in crosses 7A, 8A, 13, 14, and 15.

tinguish one-armed from two-armed or circular arrangements. For this purpose, it is necessary to apply other methods. We have used cosegregation frequencies as an independent means of evaluating gene order and linkage (Sager and Ramanis, 197613). C.

MAPPING BY MEANSOF COSEGR~ATION FREQUENCIES

The frequencies of segregation events involving two or more genes simultaneously were computed from data of zoospores in crosses scored as shown in Fig. 9. Only events involving alleles from the same parent (i.e., nonrecombinant) and occurring a t the same doubling were included. The results for five key genes considered in all pairwise combinations are shown in Table 7. The values in Column A are based upon type I1 cosegregation events alone, whereas those in column B include types 11 and I11 and zygotic frequencies. As shown in Fig. 12A and B, the maps generated by these two sets of data do not differ in gene order or relative gene distances. Figure 12C shows the map including other genes that were present in only one or two of the crosses examined in this study. Cosegregation runs of three or more genes also examined in this study provided additional support for the maps shown in Fig. 12 (Sager and Ramanis, 1976b).

D. CLOSING THE CIRCLE The maps generated both by type I1 segregation frequencies of individual genes (gene-up distances) and by cosegregation frequencies (gene-

318

RUTH SAGER

TABLE 7 Cosegregation Frequencies0 for Pairw ise Gene Combinationsb Gene pair

No. of crossese

Ad

I. Genes present in several crosses ac-sm2 ac-ery ac-spc ac-tml sm%-ery sn&spc sm%-tml ery-spc ery-tml spc-tml

8 10 12 12 6 8 8 10 10 12

7.36 f 0.5.58 4.64 k 0.510 3.55 f 0.337 Fj.98 f 0.678 16.14 f 1.648 9.69 f 2.058 6.42 f 1.082 15.11 5 2.644 7.06 k 1.258 12.24 f 1.179

9.445 6.62 5.09 7.27 26.44 18.36 8.79 28.68 10.91 18.18

f 0.718 f0.576 f 0.401 f0.721 f 3.564 f 3.516 f 0.921 f 4.183 f 1.472 f 1.870

11. Genes present in single crosses sm3-ac smS-ery sm3-spc sm3-tml sm6-ac sm6-ery sm6-apc smb-tml sm6-ac sm6-ery sm6-spc sm6-tml car-ac

car-sm%

car-ery car-spc car-tml cle-ac cle-sm% cle-ery cle-spc cle-tml ole-ac ole-sm2 ole-erg ole-spc ole-tml spi-ac spi-sm8 spi-ery

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1

1 1 1 1 1 1 1 1

3.3 8.9 5.6 3.3 14.9 4.0 4.0 1.0 18.8 9.4 6.9 5.5 3.88 12.74 21.88 6.09 4.43 2.5 15.1 21 .o 11.a 4.2 2.8 14.1 25.4 11.1 1.4 3.2 19.4 23.4

4.4 16.7 13.3 7.7 17.8 13.9 10.9 1.o 24.3 14.9 8.9 8.4 4.11 15.07 28.77 9.86 5.48 5.0 36.1 51.3 31.9 8.4 4.2 25.4 40.9 18.3 8.5 4.8 25.8 35.5

319

GENETIC ANALYSIS OF CHLOROPLAST DNA

TABLE 7 (continued) Gene pair spi-spc spi-tmi mrl-ac mrl-sml mrl-sm6 mrl-ery mrl-spc mrl-tml mr2-ac mr2-sml mr2-spc mr2-tml

No. of crossesc 1 1 2 1 1 1 2 2 1 1 1 1

Ad

B'

15.3 7.3 6.87 10.7 8.9 7.9 14.2 20.3 7.8 8.6 8.2 9.4

26.6 9.7 8.33 15.1 11.4 9.9 17.1 26.9 8.6 12.7 14.8 12.3

Computed as number of cosegregation events per total first and second doublings. From Sager and Ramanis (197613). Data of crosses 6-25 listed in Table 1. Data from type I1 segregations only. c Data from type 11, type 111, and zygote segregations. a

J

gene distances) are linear. (However, the gene-ap distances provide no information per se for distinguishing between a linear arrangement, onearmed or two-armed, and a circular arrangement. Apparent linearity of a circular map based on cosegregation frequencies would result if cosegregation was rare across the attachment point.) When the maps generated by these two methods are compared, circularity of the genome becomes evident. The genes ac and tmd are closest to ap but farthest apart on the cosegregation map, so they must be located on opposite sides of ap. Since ac is linked to smd more closely than is spc and tm is closer to spc than to smd by cosegregation, their positions are established vis-8-vis uc and tm. By cosegregation, ery lies between smd and spc, but it is farthest from up. Only the closed circular arrangement shown in Fig. 13 will satisfy the instructions implicit in the data derived by the complementary mapping methods (Singer et al., 1976). Thus, the three methods of mapping by type 111 frequencies in pedigrees, by differential slopes, and by type I1 cosegregation frequencies, taken together, establish circularity and provide a unique gene order and set of relative distances. It is anticipated that the appropriate use of recombination data (not to be discussed in this review) will provide further quantitation. The principal difficulty with recombination data is that the type I1

320

RUTH SAGER

A 1

I

I

,

sm2 ery spc

oc

e l

I

I

4.6

6.4

i

3.6

9.5

-I

w

6.0

oc

tml

116.11 15.11 1221 9.7 j

7.4

sm2 ery spc

9

(26.4 29

I

18

tml

10.9

I

w

I

I

i

8.8

5.I

i

7.3

c 1

oc2 acl

,

sm2 ery lii, I sm5 sm3 car sm6 cle ole spi

' I 11

spc

mL

tml

mh

i

FIG.12. Maps of chloroplast genome based upon relative cosegregation frequencies. (From Sager and Ramanis, 1976b.) (A) Linear arrangement of five key markers based upon the data of Table 7, column A, from frequencies of type I1 cosegregation. (B) Linear arrangement of five key markers based upon the data of Table 7, column B from frequencies of type 11, type 111, and zygote cosegregation. ( C ) Relative positions of 15 genes, based upon the data of Sager and Ramanis (1970, 197613).

segregation frequencies are much higher than the type 111 frequencies (Table 5 ) . Thus, if one scores both classes of events as exchanges, and counts up exchange frequencies in each interval, very little discrimination between intervals is obtained except for closely linked markers. (No difficulty is encountered in scoring recombination between acl and ac2, for example, which show about 1% recombination.) One solution is to obtain more closely linked markers for detailed mapping purposes; another is to look for mutations that decrease the frequency of type I1 events relative to type I11 events. A third approach is the development of a computer program to test many alternative scoring procedures. We are currently engaged in all these lines of approach.

GENETIC ANALYSIS O F CHLOROPLAST DNA

321

car

FIG.13. Relative map positions of 15 chloroplast genes. (From Singer et al., 1976.)

E. PERSISTENT CYTOPLASMIC HETEROZYGOTES The discovery of persistent cytohets resulted from our studies of mutant strains recovered after growth of a streptomycin-dependent mutant in the absence of the drug (Sager and Ramanis, 1 9 7 6 ~ )Rever. tants were recovered that displayed a wide range of mutant phenotypes in addition to the streptomycin-resistance or -sensitivity that permitted their survival in the absence of streptomycin. A set of 25 individually isolated mutants of this origin, so-called D mutants, were crossed to tester mt- stocks, and were found to be of two classes. Class A mutants were genetically indistinguishable from other cytogene mutations, showing maternal inheritance with a low frequency (about 0.1%) of biparental exceptions. Class B mutants when crossed to tester mt- stocks, segregated 2sd: 2 mutant phenotype in tetrads from unirradiated crosses, with the usual low frequency of biparental zygotes in which markers from the mt- parent were transmitted. Backcrosses of F, mutant mt+to the same tester mt- gave a similar result, showing that the mutant phenotype masked the presence of a cryptic copy of the sd gene. We postulated that the D mutants of class B were persistent cytohets, containing one copy of the mutant gene and one copy of the original sd gene. However, we could not distinguish whether the entire genome was present in the cytohet form, or only some fragment of it carrying the extra gene. If the entire genome were present in cytohet form in the class B mutants, then persistent cytohets might be normally present a t a low frequency in all stocks, and could be selected for among the progeny of any suitably constructed cross. To test this possibility, we crossed a pair

322

RUTH SAGER

of mating strains differing in six linked chloroplast markers (Cross 11 of Table 4) and screened for potential heterozygotes among the progeny by plating cells after about 20 doublings (when postmeiotic segregation was no longer detectable) on multiple-drug-containing agar (Sager and Ramanis, 1976d). Only cells containing genes for resistance to all four antibiotics would form colonies on such plates. (The success of this experiment depends on the fact that each of these drug resistances is dominant to sensitivity in Chlamydomonas.) Both homozygous recombinants and persistent cytohets were recovered, distinguished first by the fact that persistent cytohets are not 100% persistent, but do throw off segregants during clonal growth, and then distinguished critically by test crosses: het mt+ x tester mt-. Eleven of nineteen tested clones proved, on crossing to a tester mtstrain, to be persistent cytohets, as shown by the phenotypes of the progeny. The recovery of both parental cytogenomes (from cross 11) from single zygotes, as shown in Fig. 14, provides the unambiguous evidence sought in this experiment: i.e., the existence of persistent cytohets that transmit two complete cytogenomes to their progeny in sexual crosses of the type het mt+ x tester mt-.

g Parental

+

mtt x

0 mt-

recombinant classes of progeny, including Persistent HET

0 Cross

1 ""

growth

No

Subclones 1.

2. 3.

0

g

+

Major class

Zoospore progeny clones

1.

Few %

2.

Few %

3.

0 0

+ 0

Few% Major class Major class

FIO.14. Origin and transmission of a persistent cytohet. Key, 0, sm2-s ery-s apc-r sm2-r ery-r car-r spc-s t s ; A , sm2-s erg-s car-s apc-a ts. Note that (1) the t r ; 0, persistent HET rarely segregates during vegetative growth, but usually segregates in meiosis; (2) persistent HET shows maternal inheritance; and (3) recombination is very rare. (From Sager, 1972.)

GENETIC ANALYSIS OF CHLOROPLAST DNA

323

I n addition to the homozygotes of the two parental classes (from cross l l ) , recombinants, and partial hets, as well as progeny that were still

heterozygous for all the markers, were recovered from some zygotes. The reason for the heterogeneity is that “clones” of persistent hets continue to segregate and recombine occasionally during vegetative growth. Thus, on crossing such a “clone,” different zygotes are in fact of different parentage. We have proposed (Sager and Ramanis, 1976d) that this identification of persistent cytohets from cross 11, provides an explanation for the 2:2 segregations of the D mutants of class B. On this basis, the D mutants contain two copies of the chloroplast genome, one containing the sd gene, and the other containing a mutation to streptomycin-resistance or sensitivity, that phenotypically masks the presence of sd. The physical basis for persistent heterozygosis during vegetative growth is postulated to be physical separation of the two chloroplast genomes in mature vegetative clones. As a result, the pairing that leads to segregation and recombination is rare, compared to the extensive recombination normally seen in the first few doublings of zoospore clones. On the other hand, in zygotes, the two chloroplast genomes from the mt+ parent do segregate, giving rise to the 2:2 segregation ratios seen in the progeny. The 2 :2 segregation of the genetically different chloroplast genomes present in the het mt+parents provides further evidence for the genetic diploidy of the chloroplast genome. A similar conclusion was reached by Gillham (1969) on the basis of mixed colonies of streptomycin-resistant and wild-type cells found after mutagenesis.

F. GENETICDIPLOIDY OF THE CHLOROPLAST GENOMEIN ZOOSPORES 1. Evidence Favoring Genetic Diploidy The methods of mapping discussed above are empirical and do not depend upon any assumptions concerning the numbers of copies of each chloroplast genome present in the zoospore pedigrees. Nonetheless, strong evidence of genetic diploidy has emerged from our studies, in particular from the zoospore pedigree analysis. By means of pedigree analysis, segregations occurring in the zygote have been distinguished from those occurring in zoospore clones (Sager and Ramanis, 1967s). The genes acl, ac2, and t m l showed little if any deviation from 1 : 1 either in zygotic segregation or in zoospore clones. The genes sm2, ery, and spc segregating in the same crosses as acl, ac2, and tml did show an excess of the allele from the mt’ parent in zygotes, but in zoospore pedigrees, mt+excess was

324

RUTH SAGER

seen only when the allele coming from the mt+ parent was the mutant (resistant) form but not when it was the wild type (sensitive). Thus, deviations from 1 :1 are either allele-specific (in zoospore clones} or location-specific (in zygotes), and in both stages provide no evidence of multigenome copies since the linked markers acl, ac2, and tml always show 1: 1 segregation. Genetic diploidy of the chloroplast genome is further supported by the pattern of exchange events occurring a t a “four-strand stage,’’ during association of four double-stranded DNA molecules. This four strand stage is comparable to the association of chromosomes inferred to occur in nuclear mitoses leading to mitotic recombination, e.g., in Aspergillus (Pontecorvo, 1958; Kiifer, this volume), Abundant evidence from pedigree analyses supports this model, in particular the frequent involvement of different linked markers in types I, 11, and 111 segregation events a t the same doubling. These patterns of segregation exclude the presence of more than two copies (after replication) of the chloroplast genome, one coming from each of the parental cells. The kinetics of gene segregation in zoospore clones (Fig. 10) provides additional support for the diploid model by showing that segregation rates are constant per doubling and much too high to be accounted for by any multicopy model. A different line of evidence in support of the diploidy model comes from the study of persistent cytohets discussed above. The results of crosses with multiply labeled cytohet progeny from cross 11 used as the mt+parent are particularly instructive. As shown in Fig. 14, in crosses of the type persistent het mt’ X tester stock rnt-, the progeny of some zygotes corresponded to the two differentially labeled genomes from the mt+ parent (the progeny of other zygotes included some recombinant types), while the tester genome from the mt- parent was not transmitted (except in rare exceptional zygotes). Thus, the mt+ persistent cytohets carry two distinguishable chloroplast genomes and show maternal inheritance by eliminating the genome from the mt- parent while transmitting both of their own two genomes. 2. Presence of Reiterated Sequences in Chloroplast D N A

Measurements of the kinetics of reannealing of denatured chloroplast DNA were undertaken to look for evidence of reiterated sequences and to estimate the genomic size of this DNA fraction (Wells and Sager, 1971; Bastia et al., 1971). The chloroplast DNA was identified by its bouyant density in CsCl of 1.695 gm/cm3 and purified by two successive rounds of centrifugation to equilibrium in CsC1 (Wells and Sager, 1971) or by a single centrifugation (Bastia et al., 1971).

GENETIC ANALYSIS O F CHLOROPLAST DNA

325

The major component of chloroplast DNA reannealed a t a rate close to that of phage T4 DNA, indicating a genomic complexity of about 2 X lo8 daltons. A fast-renaturing fraction was also detected (Wells and Sager, 1971) with an estimated size of 1 to 10 x lo6 daltons, comprising about 10% of the total DNA. If the 2 x 10’ dalton component comprises 90% of the total DNA, then it would be present in about 24 copies, assuming the analytical size of chloroplast DNA as 4.3 x lo9 daltons (Wells and Sager, 1971). However, reannealing was not observed for technical reasons, beyond about 50% of the total hyperchromicity, leaving open the possibility that a slow-annealing fraction may also be present, unobserved in these experiments. If only 20% of the total DNA represented genes present in one copy, they would constitute an additional genomic content of 8 X loS daltons but would be invisible by standard methods of reannealing kinetics. The results to date are therefore compatible with a model in which single-copy DNA is present and contains the cytogenes identified by mutation and studied by segregation and recombination analysis. The reiterated DNAs might then be interspersed between the single-copy material, the entire assemblage being a single molecule of about 2 X lo9 daltons. This interpretation is not only compatible with the reannealing data and best fits the genetic data, but also is supported by the cytochemical evidence of Ris and Plaut (1962), who reported the presence of two DNA bodies in the chloroplast. Their methods could have detected molecules in the size range of 2 to 5 X loo daltons but could not have seen molecules of 2 x lo8 daltons. Two further lines of evidence bear on this question. Flechtner and Sager (1973) found that the replication of chloroplast DNA is preferentially inhibited in cells grown in the presence of 10 pg/ml of ethidium bromide for 8-10 hours. At the end of this time, virtually no new synthesis of chloroplast DNA has occurred, and that initially present has been degraded to less than 15% of the original content. Nonetheless, virtually all the cells are viable a t this time as shown by removing the dye and replating the cells a t suitable dilution on agar-containing medium. We interpret this result as evidence of reiteration in chloroplast DNA, but not necessarily reiteration of the entire chloroplast genome. Rochaix (1972) used the method of Thomas et al. (1970) to look for evidence of reiterated sequences in chloroplast DNA of Chlamydomonas. In this method, the DNA is sheared, the exposed ends are nibbled back by a 3’ exonuclease, and the mixture of DNA molecules with singlestranded ends are then reannealed and examined by electron microscopy to determine the extent of circularization. Rochaix found up to 20% of the DNA fragments to be rings or lariats, the majority of which were

326

RUTH SAGER

0.75-1.0 pm in length. On the basis of these data plus calculations of the length of the reiterated sequences from melting curves, Rochaix concluded that chloroplast DNA has interspersed reiterated sequences arranged similarly to those described in nuclear DNAs from Xenopus and sea urchins (Davidson et al., 1974). He raised the question of how recombination is regulated to ensure the absence of pairing between these reiterated sequences, to avoid scrambling the genome. Since our genetic data indicate that scrambling is rare if it occurs a t all, we suggest that pairing may be sequential, starting from a unique place in the molecule, perhaps the attachment point, and moving either unidirectionally or bidirectionally around the circle. In summary of this section, we conclude that the evidence of reiteration in chloroplast DNA is not inconsistent with genetic diploidy. Further experiments are needed to establish the existence of single-copy regions and to locate them vis-it-vis the reiterated sequences. 3. Gillham’s Multicopy Model

Our model of genetic diploidy has recently been challenged by Gillham et al. (1974), on the basis of their data from crosses with the markers spc, sr, and ery, independently isolated by them in strains that are cross fertile with ours but have been kept separate for many years. They find an excess of progeny carrying markers from the maternal parent in biparental zygotes recovered from unirradiated crosses, and further, as they increase the UV dose to the maternal parent before mating, the aberrant ratios gradually approach 1 :1. They have proposed a multicopy model to a.ccount for the excess of maternal genes in progeny of unirradiated crosses, and have further proposed that the diploidy we find is the result of UV irradiation, and that our data too can be made to fit their multicopy model. Furthermore on the basis of their results, Gillham et al. (1974) propose that the Visconti-Delbriick method developed for analysis of phage recombination is a useful methodology of analysis of genetic recombination of the chloroplast genome. Briefly stated, we consider that Gillham et al. have been misled by their method of genetic analysis, in which they score samples taken from whole zygote colonies instead of zoospore clone pedigrees, and by their use of markers located in a very small and restricted sector of the genome. We consider their analysis of our data to be invalid, and the use of the Visconti-Delbruck method or some variant of it not only unnecessary but misleading for reasons to be discussed now. 1. Gillham et al. (1974) have not distinguished between events occurring in the zygote and those in zoospore clones, either conceptually or experimentally. Conceptually, the distinction is very important, because

GENETIC ANALYSIS O F CHLOROPLAST DNA

327

the mechanisms regulating pairing and exchange events a t these two stages of the life cycle may be different: the zygote contains a t least twice as many double-stranded chloroplast DNA molecules as do zoospores and vegetative cells after replication. Special mechanisms may be present to distribute the chloroplast DNA copies to the four zoospores. It is essential therefore to distinguish between events occurring a t these different stages rather than lumping them together in a single observation. Experimentally, the demonstration that all four meiotic products have been recovered is an essential control in crosses with cytogenes, since meiotic aberrations may affect cytogene transmission. No evidence is presented by Gillham et nl. (1974) that the zygotes they sampled for cytogene allelic ratios contained the four meiotic products. 2. The data of Gillham et al. (1974) are limited because they used only three drug-resistance markers, which, if similar to ours, are rather closely linked. The lack of sufficient markers located around the genome severely limits the conclusions to be drawn from the data, since the behavior of the entire genome cannot be inferred from data restricted to one small region. The Gillham et al. proposal that multiple copies of the chloroplast genome are present in zoospores could only be validated by evidence that sets of markers all around the genome regularly segregate together, with all the maternal markers simultaneously in excess. 3.The crosses reported by Gillham et al. (1974) were done with strains giving a high frequency of spontaneous exceptional zygotes, about 20% in contrast to frequencies of about 0.1% in our wild-type stocks. This high frequency of exceptional zygotes has permitted them to examine the progeny of unselected biparental zygotes from unirradiated crosses, which we cannot do, but it raises the question of the mechanism responsible. The frequencies of exceptional zygotes in the Gillham et al. (1974) paper approach those given by stocks carrying mat-1 (Section 111, C) in which an altered restriction enzyme has been postulated as the effector. In Gillham’s stocks, the high frequency of exceptional zygotes may also involve some special nuclease activity, leading to partial degradation in zygotes of the chloroplast DNA from the male parent followed by “marker rescue” or copying of pieces of the genome. Thus the so-called “biparental zygotes” described by Gillham et al. (1974) may not contain the two full sets of the chloroplast genome that we recover, but rather some fraction of the male genome rescued after initial degradation by the postulated nuclease. Pedigree analysis and additional markers are necessary to test this postulated explanation of the Gillham et al. results. If our explanation is correct, it would fully account for the excess of alleles of maternal origin seen in spontaneous biparental zygotes in

328

RUTH SAGER

Gillham’s strains, but would be irrelevant to the explanation of our results, in which complete sets of cytogene alleles from both parents are recovered in most zoospore clones. The key observations that distinguish our data from those of Gillham et al. are (1) that we recover all four products of meiosis, and (2) that most zoospores we recover from biparental zygotes of UV-irradiated crosses are heteroaygous for all the markers present in the cross. Some marker segregation does occur in the zygote, as shown by the average frequencies of zygotic segregation of individual genes listed in Table 8. In these instances, the frequencies of cosegregation for two or more genes is very low; most segregations involve a single allele in a cell that is predominantly heterozygous. The effect of UV irradiation upon allelic segregation ratios and upon cosegregation frequencies was examined with progeny of cross 20 from

TABLE 8 Extent of Gene Segregation in Zygotes0 Frequency of homozygous genes per zoosporeb

Cross 2 5 6 7 8 9 10 11 12 13

14 20-15 sec 20-30 sec 20-50 sec 24

25

a

None

One

58.1 51.6 63.5 54.3 55.6 34.9 66.7 50.9 61.6 52.8 34.6 74.7 51.8 60.2 39.7 48.5 av. 53.7

23.7 27.7 13.5 18.9 6.4 18.6 10.5 21.5 14.9 16.0 19.0 14.9 24.5 17.9 16.4 21.2 av. 17.9

From Sager and Ramanis (1976a).

Total (one or none) 81.8 79.3 77.0 73.2 62.0 53.5 77.2 72.4 76.5 68.8 53.6 89.6 76.3 78.1 56.1 69.7 av. 71.6

* Computed as the number of zoospores with zero or one segregated genes per total

zoospores analyzed.

GENETIC ANALYSIS OF CHLOROPLAST DNA

329

biparental zygotes formed after zero, 15, 30, and 50 seconds of UV irradiation of the mt+parent before mating. No significant differences in allelic ratios were seen for any of the six markers present in the cross under any of the four irradiation conditions of the experiment (Sager and Ramanis, 1976a). Similarly, cosegregation frequencies were not influenced by UV irradiation (Sager and Ramanis, 1967b). The attempt by Gillham et al. (1974) to fit their multicopy model to our data is invalid because no account was taken of the fact that types I, 11, and I11 segregational events frequently occur simultaneously. No multicopy model we have seen can account for segregations of the sort shown in Fig. 9. Any proposed model must account for the simultaneous behavior of the entire set of available markers in the chloroplast genome. Use of the Visconti-Delbruck methodology is unnecessary because we are not dealing with an unknown number of rounds of mating. On the contrary, pedigree analysis with well marked genome provides evidence of exchange events occurring a t individual doublings. All statistical methods that do not distinguish reciprocal from nonreciprocal events are misleading in this system because the nonreciprocal events occur with equal frequency all around the map and are much more frequent than the reciprocal events that occur with frequencies characteristic of each gene. VI. Association of Cytoplasmic Genes with Organelle DNAs and Structures

Chlamydornonas contains one large cup-shaped chloroplast and a number of mitochondria as well as Golgi apparatus, lysosomes, flagellar basal bodies, and other differentiated structures of a typical eukaryotic cell (Sager and Palade, 1957; Johnson and Porter, 1968). I n addition to nuclear DNA banding a t 1.725 gm/cm3 in CsCI, chloroplast DNA banding a t 1.695 gm/cm3 has been identified (Sager and Ishida, 1963; Sueoka et aZ., 1967) and extensively examined (Wells and Sager, 1971; Bastia et al., 1971). Mitochondria1 DNA has recently been reported as banding a t 1.706 gm/cm3 in CsCl (Ryan e t al., 1973). No other cytoplasmic DNAs have been identified in Chlamydomonas, but the reports of small DNA circles (Smith and Vinograd, 1972) and membrane-associated DNA (Meinke et al., 1973) in other organisms, as well as the reports of cytoplasmic genes not associated with mitochondria in yeast (Griffiths et aE., 1975), raise the possibility of additional cytoplasmic DNAs and genetic systems yet to be identified. I n view of these uncertainties, can the cytogenes of Chlamydomonas be rigorously identified with a particular cytoplasmic DNA? (Also see Guerineau et aZ., 1974.)

330

RUTH SAGER

A. Is THE CYTOPLASMIC LINKAGE GROUPLOCATED IN CHLOROPLAST DNA? The attempt to answer this question definitively has been an aim of several research efforts including our own. As yet no single definitive experiment has been reported, but one may appear before this review is published. In lieu of one definitive experiment, however, a number of lines of evidence have been developed that associate the cytoplasmic linkage group of Chlamydomonas with chloroplast DNA, and these will now be summarized. 1. The most direct evidence associating chloroplast genes with chloroplast DN-4 is based upon the parallel behavior of genes and DNA in zygotes, i.e., maternal inheritance as discussed above (Sections II1,A and B) and in recent publications (Sager and Lane, 1972; Sager and Ramanis, 1973, 1974; Schlanger and Sager, 1974a). We have concluded that chloroplast DNA itself exhibits maternal inheritance, in parallel with the chloroplast genes it contains. I n a recent review, Giliham (1974) has reached a similar conclusion, listing the maternal inheritance of chloroplast DNA as one of four lines of evidence for locating chloroplast genes in chloroplast DNA. 2. A second strong line of evidence is based upon the phenotypes conferred by several mutant genes located in the cytoplasmic linkage group. The mutations, each conferring resistance to a different antibiotic, lead to mutationally induced alterations in chloroplast ribosome function (Schlanger and Sager, 1974b) and mutationally induced changes in a t least two chloroplast ribosomal proteins (Mets and Bogorad, 1972; Ohta et al., 1974). Clearly, the correlation of genotype and phenotype in the same organelle is not a formally critical demonstration that the genes of the organelle determine the phenotype (Rhoades, 1955). Nonetheless, the only postulated alternative in Chlamydomonas is that these chloroplast phenotypes are coded by mitochondrial DNA. Nuclear DNA has been eliminated by formal genetic analysis, and no other DNAs have been identified. 3. I n terms of genomic size, the chloroplast genome is minimally 1 to 2 X lo8 daltons, and possibly considerably larger (see Section V, F, 2), while the mitochondrial genome is reported to be about 1 X lo7 daltons (Borst, 1972). Only Maxwell’s demon could have ensured that all the cytogene mutations so far identified would be part of the small mitochondrial genome, hardly large enough to contain them, while not a single mutation appeared that was part of the much larger chloroplast genome. 4. The genetic evidence has demonstrated that the cytoplasmic linkage group is diploid, or behaves genetically as if it were diploid. Diploidy

GENETIC ANALYSIS O F CHLOROPLAST DNA

331

is consistent with the report by Ris and Plaut (1962) of two Feulgenpositive bodies in the chloroplast. Diploidy is also much easier to reconcile with the presence of one chloroplast per cell than with the numerous mitochondria. I n my view, these four lines of evidence are sufficient to assign the cytoplasmic linkage group to chloroplast DNA. B. Is sd

A

MITOCHONDRIAL GENE?

Arnold, Schimmer, and Behn (Schimmer and Arnold, 1970a,b,c; Behn and Arnold, 1972, 1973) in a series of papers have described studies with a streptomycin-dependent strain, a neamine-dependent strain, and their drug-resistant and sensitive revertants. On the basis of their observations, they have suggested that the sd mutation obtained from Gillham is mitochondrial, while the nd mutation, also from Gillham, is located in the chloroplast. Their line of reasoning is the following. The sd strain was grown in the absence of streptomycin, and a number of ss-revertants were obtained. On subculturing, these revertants were found to segregate out some sd clones, indistinguishable phenotypically from the original sd. When some of these newly obtained sd strains were grown without the drug, a new series of ss-revertants were again obtained. In crosses of wild-type mt+ with mt-revertants, only wild-type progeny were recovered (Schimmer and Arnold, 1970a,b,c). Schimmer and Arnold interpreted these findings as evidence that the sd gene was present in multiple copies, and that the ss-revertants still contained some copies of sd, which segregated out during vegetative growth. Subsequently, Behn and Arnold did analogous experiments with the nd strain (Behn and Arnold, 1972). When grown in the absence of the drug, nd cells threw off both nr and ns revertants, a t a mutant frequency of about However, these revertants did not subsequently give rise to any nd cells, but remained stably nr and w. I n backcrosses of wildtype mt+ with mt-nr-revertants, all progeny were wild-type, indicating that the nr revertant genes were non-Mendelian. Behn and Arnold (1973) then examined some of these strains in the electron microscope comparing cells grown with and without either streptomycin or neamine. They found that streptomycin-dependent cells grown without the drug show mitochondrial anomalies before any effects upon the chloroplast can be seen whereas neamine-dependent cells, grown without neamine, show chloroplast aberrations within 2 days and mitochondrial aberrations only after 5-7 days. They propose th a t the cytological evidence supports their hypothesis locating the sd gene in the mitochondrial genome and the nd gene in the chloroplast genome. In our view, the cytologica1 evidence is interesting but difficult to in-

332

RUTH SAGER

terpret. When more becomes known about interactions between mitochondrial and chloroplast protein-synthesizing systems, it may become possible to use these observations in the analysis of organelle interactions. The central issue here concerns the sd mutation. In experiments similar to those of Arnold and Schimmer, we recovered “revertants” following growth of our sd strain (which may or may not be identical with Gillham’s strain) including not only sensitive strains but also many levels of streptomycin resistance (Sager, 1972; Sager and Ramanis, 1 9 7 6 ~ )When . crossed with wild-type mt-, some of our mutant strains segregated 4:O as expected for cytoplasmic mutations, and some of them segregated 2 sd : 2 sr (or ss) revertant. The strains giving 2:2 segregation still showed non-Mendelian inheritance, since both the sd and sr (or ss) genes were of maternal origin. The cytoplasmic markers from the mt- parent were not transmitted. On further study, these strains were shown to be persistant cytoplasmic heterozygotes (cf. Section V, E) and the occasional segregation of sd clones during vegetative growth was precisely as described by Schimmer and Arnold (1970a). It should be stressed that (1) the sd gene involved in these studies has been mapped in the linkage group located in chloroplast DNA, and (2) any genes of that linkage group may be involved in persistent cytohet strains. Thus, in our view the behavior described in such detail by Schimmer and Arnold (1970a,b,c) results not from the presence of multiple copies of the gene, but rather from the special behavior of persistent cytohets.

C. Is spc 1-27-3

A

CHLOROPLAST GENE?

The fact that no mitochondrial genes have yet been identified complicates the problem of examining the interaction between chloroplast and mitochondrial genomes and their protein-synthesizing systems. One problem is posed by the observation that many antibiotic-resistant mutants have been isolated in which the mutational lesion, located in chloroplast DNA, confers cellular-level drug resistance. The antibiotics are of the class that kill bacteria by blocking prot,ein synthesis a t the ribosome level; in various eukaryotes both chloroplast and mitochondrial ribosomes have been found to be sensitive to these drugs. How then do chloroplast mutations conferring drug-resistance a t the chloroplast ribosome level confer cellular resistance? Why are the cells not killed because of mitochondrial drug sensitivity? Four explanations have been proposed: (1) mitochondria are impermeable to these antibiotics, and therefore are resistant a t the organelle level; (2) mitochondrial ribosomes of Chlamydomoms are resistant to these drugs; (3) chloroplasts and mitochondria share the same ribosomes

GENETIC ANALYSIS OF CHLOROPLAST DNA

338

for protein synthesis ; and (4) chloroplast and mitochondrial ribosomes are different, but some ribosomal proteins coded by chloroplast DNA are present in both. No direct evidence concerning the drug-resistance of isolated mitochondria or mitochondrial ribosomes has yet been reported, so there is no experimental basis for choosing among these (or other) alternatives. Surzycki and Gillham (1971) attempted to distinguish between chloroplast and mitochondrial mutations to drug resistance by comparing the resistance level of wild-type cells grown phototrophically and heterotrophically. Of all the drugs tested (streptomycin, paromomycin, kanamycin, lincomycin, pactamycin, chloramphenicol, spectinomycin, and erythromycin), only spectinomycin evinced a different resistance level under different growing conditions. From the greater sensitivity of phototrophically grown cells to spectinomycin than heterotrophically grown cells, they proposed that spectinomycin has a preferential effect on the chloroplast system, and that chloroplast and mitochondrial proteinsynthesizing systems are equally sensitive to all the other antibiotics tested. I n a further study, Boynton et al. (1973) described a non-Mendelian mutation, spc 1-27-3. A single protein of the small chloroplast ribosomal subunit was reported missing in a mutant strain but present in the wild type. They suggested that it might be the spectinomycin-binding protein and further proposed that cellular resistance to spectinomycin in this mutant is conferred by mutation of the spectinomycin-binding protein of the chloroplast ribosome. However, cells carrying the spc 1-27-3 mutation are more resistant to spectinomycin when grown heterotrophically than phototrophically. Therefore, the authors proposed that this postulated spectinomycin-binding ribosomal protein is coded by chloroplast DNA and shared by chloroplast and mitochondrial ribosomes. The spc 1-27-3 mutation is of interest because it confers differential sensitivity to the drug in light- and dark-grown cells, just as in the wildtype cell response to spectinomycin. This mutation merits careful genetic analysis, but none has yet been reported. It should be noted that this mutation spc 1-27-3 is different from the one studied genetically by Gillham et al. (1974), spc 1-6-2, which does not show this differential sensitivity and is probably genetically allelic with our spc mutation, with which it shows no recombination (R. Sager, unpubl.).

D. CELL-WALL MUTANTS In a series of papers (Davies and Plaskitt, 1971; Davies, 1972; Hills

et al., 1973; Roberts et al., 1972; Hyams and Davies, 1972; Davies and

334

RUTH SAGER

Lyall, 1973) Davies and his students and colleagues have described cellwall (CW) mutants of Chlamydomonas, recovered after NG mutagenesis, most of which showed Mendelian 2 : 2 segregation in tetrads from crosses of mutant and wild type. The CW mutations involve several gene loci as judged by the recovery of wild type (WT) recombinants in pairwise crosses of mutant X mutant. Two mutants, CW 17 and CW 18, showed anomalous patterns of segregation in crosses with wild type and were studied in further generations of backcrosses to WT and intercrosses to CW relatives. The phenotypes of CW 17 and CW 18 are somewhat different, in extent of attachment of wall to plasma membrane, but the genetic behavior of the two strains is similar. I n the initial crosses of CW 18 (mt+)with wild type (mt-),all tetrads segregated 2: 2. Both mutants showed irregular segregation patterns in backcross generations with 2:2, 3:1, and 4:O ratios seen in crosses between CW and WT, unrelated to mating type, and not affected by prior UV irradiation of either parent before mating. The progeny did not segregate out CW and WT types during growth of zoospore clones although rare reversions to a “pseudo wild type” were noted. In crosses of CW X WT, with either mating type CW, the majority of zygotes segregated 2:2, and in crosses of CW x CW the majority segregated 4:O. The frequency of exceptions in CW X WT was of the order of 12% for 3 : l and 3% for 4:O; and most remarkable, in crosses of CW X CW 44% of the zygotes produced one or more W T progeny. It should be emphasized that in all crosses giving aberrant ratios, one or both of the CW parents were descendents of a previous cross of CW X WT. Davies suggested three alternative interpretations. (1) CW 17 and CW 18 are nuclear genes, but their expression may be impaired by other genes, which regulate wall formation specifically in the diploid zygote and contribute W T information, during zygote maturation and zoospore formation leading to a WT phenotype that persists in the CW progeny until the next sexual generation. (This interpretation accounts for excess WT progeny, but not for excess CW progeny, e.g., in crosses of CW X WT giving 4 CW:O WT.) (2) CW 17 and CW 18 are autonomous extranuclear genes. (3) A dual information system is operating. Subsequent studies of environmental conditions promoting wall formation have provided some insight into the system. The wall is a 7-layered structure, in which layers 2-6 comprise a lattice structure, consisting of 6-8 glycoproteins. The original CW 18 mutant does not produce lattice under any growing conditions, whereas a CW 18 mt- (F, from the cross of CW 18 X WT) can produce the lattice when grown on agar but not in liquid. This observation shows that the CW 18 F, clone (coming from a cross

GENETIC ANALYSIS O F CHLOROPLAST DNA

335

of CW X WT) is not phenotypically the same as the original mutant strain. The possibility is raised, thus, that the other genes or gene products coming from the WT parent are contributing to the phenotype of the F, progeny, and perhaps to the genotype as well. This possibility is consistent with the fact that aberrant ratios were seen only in crosses in which the CW parent had a CW X W T cross in its pedigree. The evidence is strong favoring aberrant segregation of some factor influencing phenotype and genotype. For example, a tetrad was analyzed from a cross of the original CW 18 mt+X CW 18 mt- (F1). One product was pseudo-wild in phenotype and could produce lattice in liquid; the other three were CW in phenotype and could not produce lattice. However, two of the three produced glycoprotein banding patterns indicating the presence of lattice components, but the third had no detectable proteins, like the CW 18 mt+parent. I n summary, the results to date have led Davies to favor the hypothesis of an extranuclear system responsible for the genetic and phenotypic properties of CW 18. He writes: “It remains to be seen whether the extra-nuclear system and the lattice template or primer could be synonomous” (Davies and Lyall, 1973). The genetic behavior of the CW mutants differs greatly from the usual pattern of cytogene transmission in Chlarnydomonas: in particular, the 2 : 2 segregation in the initial crosses, the lack of mating-type effect, the lack of a UV effect on transmission ratios, and the absence of postmeiotic segregation. Consequently I favor the interpretation of these aberrant segregations as evidence of a non-DNA-based, long-lived, and erratically distributed cell-wall template laid down in the zygote, that superimposes a phenotype not always in accord with the gene products supplied by the CW 17 and CW 18 mutant genes, which I suggest as nuclear. The system is of great continuing interest, both in the working out of its formal genetics and in the genetic control of cell wall biogenesis.

E. CONCLUDING REMARKS This section has considered the evidence so far available on the association of cytogenes of Chlamydoinonas with specific DNAs. All the mutations so f a r described and examined genetically appear to fall within a single linkage group located in chloroplast DNA, with two notable exceptions. The genetic analysis of spc 1-27-3 has not been reported. The cell-wall mutants CW 17 and 18 show anomalies of transmission not yet resolved, but apparently dissimilar from the chloroplast genome. It is anticipated that further studies will uncover new classes of cytogenes, some mitochondria1 and some perhaps associated with other as yet un-

336

RUTH SAGER

known cytoplasmic genetic systems. Alexander et al. (1974) have proposed that a class of acriflavin-induced minute colonies are mitochondrial.

REFERENCES Alexander, N. J., Gillham, N. W., and Boynton, J. E. 1974. The mitochondrial genome of Chlamydomonas: Induction of minute colony mutations by acriflavin and their inheritance. Mol. Gen. Genet. 130, 275-290. Bastia, D., Chiang, K. S., Swift, H., and Siersma, P. 1971. Heterogeneity, complexity and repetition of chloroplast DNA of Chlamydomonas reinhardii. Proc. Natl. Acad. Sci. U 9 . A . 68, 1157-1161. Baur, E. 1909. Das Wesen und die Erblichkeitsverhaltnisse der “varietates albomarginatae hort” von Pelargonium conale. Z . Vererbungst. 1, 330-351. Behn, W., and Arnold, C. G. 1972. Zur Lokaliaation eines nichtmendelnden Gen von Chlamydomonas reinhardii. Mol. Gen. Genet. 114, 266-272. Behn, W., and Arnold, C. G. 1973. Localization of extranuclear genes by investigations of the ultrastructure in Chlamydomonas reinhardii. Arch. Mikrobiol. 92, 85-90. Blamire, J., Flechtner, V. R., and Sager, R. 1974. Regulation of nuclear DNA replication by the chloroplast in Chlamydomonas. Proc. Natl. Acad. Sci. USA. 71, 2867-2871. Borst, P.1972.Mitochondria1 nucleic acids. Annu. R e v . Biochem. 41, 333-376. Boynton, J. E., Burton, W. G., Gillham, N. W., and Harris, E. H. 1973. Can a non-Mendelian mutation affect both chloroplast and mitochondrial ribosomes? Proc. Natl. Acad. Sci. U 8 . A . 70, 3463-3467. Burton, W. G. 1972. Dehydrospectinomycin binding to chloroplast ribosomes from antibiotic-sensitive and -resistant strains of Chlamydomonas reinhardii. Biochim. Biophys. Acta 272, 305-311. Caspari, E. 1948.Cytoplasmic inheritance. A d v . Genet. 2, 1-66. Cavalier-Smith, T. 1970. Electron microscopic evidence for chloroplast fusion in zygotes of Chlamydomonas reinhardii. Nature (London) 228, 333. Chiang, K. S. 1968. Physical conservation of parental cytoplasmic DNA through meiosis in Chlamydomonas reinhardii. Proc. Natl. Acad. Sci. U.S.A. 60, 194-200. Chiang, K. S. 1971. Replication, transmission, and recombination of cytoplasmic DNAs in Chlamydomonas reinhardii. I n “Autonomy and Biogenesis of Mitochondria and Chloroplasts” (N. K. Boardman, A. W. Linnane, and R. M. Smillie, eds.), pp. 235-249. North-Holland Publ., Amsterdam. Chun, E. H. L., Vaughan, M. H., and Rich A. 1963. The isolation and characterieation of DNA associated with chloroplast preparations. J. MoZ. Biol. 7, 130-141. Correns, C. 1909a. Vererbungsversuche mit blass(ge1b)-grunen und buntblattrigen Sippen bei Mirabilis galapa, Urtica pilulifera und Lunaria annua. Z . Vererbungsl. 1, 291-329. Correns, C. 1909b. Zur Kenntnis der Rolle von Kern und Plasma bei der Vererbung. Z . Vererbungsl. 2, 331-340. Correns, C. 1937. I n “Nicht mendelnde Vererbung” (F. von Wettstein, ed.). Borntraeger, Berlin. Davidson, J . N., Hanson, M. R., and Bogorad, L. 1974. An altered chloroplast ribosomal-protein in ery-M1 mutants of Chlamydomonas reinhardii. Mol. Gen. Genet. 132, 119-129.

GENETIC ANALYSIS OF CHLOROPLAST DNA

337

Davies, D. R. 1972. Cell wall organization in Chlamydomonas reinhardii. The role of extra-nuclear systems. Mol. Gen. Genet. 115, 334-348. Davies, D. R., and Lyall, V. 1973. The assembly of a highly ordered component of the cell wall: The role of heritable factors and of physical structure. Molec. Gen. Genet. 124, 21-34. Davies, D. R., and Plaskitt, A. 1971. Genetical and structural analyses of cell-wall formation in Chlamydomonas reinhardii. Genet. Res. 17, 33-13. Dujon, B., Slonimski, P. P., and Weill, L. 1974. Mitochondria1 genetics. IX. A model for recombination and segregation of mitochondrial genomes in Saccharomyces cerevisiae. Genetics 78, 415-437. Flechtner, V. R., and Sager, R. 1973. Ethidium bromide induced selective and reversible loss of chloroplast DNA. Nature (London), New Biol. 241, 277-279. Friedmann, I., Colwin, A. L., and Colwin, L. H. 1968. Fine-structural aspects of fertilization in Chlamydomonas reinhardii. J . Cell Sci. 3, 115-128. Gillham, N. W. 1965. Induction of chromosomal and nonchromosomal mutations in Chhmydomonas reinhardii with N-methyl-N'-nitro-N-nitrosoguanidine. Genetics 52, 529-537. Gillham, N. W. 1969. Uniparental inheritance in Chlamydomonas reinhardii. Am. Nut. 103, 355-388. Gillham, N. W. 1974. Genetic analysis of the chloroplast and mitochondrial genomes. Annu. Rev. Genet. 8, 347-391. Gillham, N. W., and Levine, R. P. 1962. Studies on the origin of streptomycin resistant mutants in Chlamydomonas reinhardii. Genetics 47, 1463-1474. Gillham, N. W., Boynton, J. E., and Lee, R. W. 1974. Segregation and recombination of non-Mendelian genes in Chlamydomonas. XIII. International Congress of Genetics Symposia. Genetics 78, 439457. Griffiths, D. E., Lancashire, W. E., and Zanders, E. D. 1975. Evidence for an extra-chromosomal element involved in mitochondrial function : A mitochondria1 episome? FEBS Lett. 53, 126-130. Cuerineau, M., Slonimski, P. P., and Avner, P. R. 1974. Yeast episome: Oligomycin resistance associated with a small covalently closed non-mitochondria1 circular DNA. Biochem. Biophys. Res. Commun. 61, 462469. Hills, G. J., Gurney-Smith, M., and Roberts, K. 1973. Structure, composition and morphogenesis of the cell wall of Chlamydomonas reinhardii. 11. Electron microscope and optical diffraction analysis. J . Ultmstruc. Res. 43, 179-192. Hoober, J. K., and Blobel, G. 1969. Characterization of the chloroplastic and cytoplasmic ribosomes of Chlamydomonas reinhardii. J . Mol. B i d . 41, 121-138. Hyams, J., and Davies, D. R. 1972. The induction and characterization of cell wall mutants of Chlamydomonas reinhardii. Mutat. Res. 14, 381-389. Johnson, U. G., and Porter, K. R. 1968. Fine structure of cell divisions in Chlamydomonas reinhardii. Basal bodies and microtubules. J . Cell Biol. 38, 403-425. Kirk, J. T. 0. 1967. Effect of methylation of cytosine residues on the buoyant density of DNA in caesium chloride solution. 1. Mol. Biol. 28, 171-172. Lee, R. W., and Jones, R. P. 1973. Induction of Mendelian and non-Mendelian streptomycin resistant mutants during the synchronous cell cycle of Chlamydomonas reinhnrdii. Mol. Gen. Genet. 121, 99-108. Lee, R. W., Gillham, N. W., Vanwinkle, K. P., and Boynton, J. E. 1973. Preferential recovery of uniparental streptomycin resistant mutants from diploid Chlamydomonas reinhardii. Mol. Gen. Genet. 121, 109-116.

338

RUTH SAGER

Luck, D. J. L., and Reich, E. 1964. DNA in mitochondria of Neurospora crassa. Proc. Natl. Acad. Sci. US.A. 52, 931-938. Meinke, W.,Hall, M. R., Goldstein, D. A., Kohne, D. E., and Lerner, R. A. 1973. Physical properties of cytoplasmic membrane-associated DNA. J. Mol. Biol. 78, 43-56. Mets, L. J., and Bogorad, L. 1971. Mendelian and uniparental alteration in erythromycin binding by plastid ribosomes. Science 174, 707-709. Mets, L.J., and Bogorad, L. 1972. Altered chloroplast ribosomal proteins associated with erythromycin-resistant mutants in two genetic systems of Chlamydomonas reinhardii. Proc. Natl. Acad. Sci. U S A . 69,3779-3783. Mets, L. J., and Bogorad, L. 1974. Two-dimensional polyacrylamide gel electrophoresis: An improved method for ribosomal proteins. Anal. Biochem. 57, 200-210. Mounolou, J. C., Jakob, H., and Slonimski, P. P. 1966. Mitochondria1 DNA from yeast “petite” mutants: Specific changes of buoyant density corresponding to different cytoplasmic mutations. Biochem. Biophys. Res. Commun. 24, 218-224. Nass, M. M. K., and Nass, S. 1963. Intramitochondrial fibers with DNA characteristics. I. Fixation and electron staining reactions. J . Cell B i d . 19, 593-611. Nass, S., and Nass, M. M. K. 1963. Intramitochondrial fibers with DNA characteristics. 11. Enzymatic and other hydrolytic treatments. J. Cell Biol. 19, 613-629. Nomura, M. 1970.Bacterial ribosome. Bacteriol. Rev. 34, BS. Ohta, N., Sager, R., and Inouye, M. 1974. Identification of a chloroplast ribosomal protein altered by a chloroplast mutation in Chlamydomonas. J. Biol. Chem. 250, 3655-3659. Pestka, S. 1971.Inhibitors of ribosome functions. Annu. Rev. Microbiol. 25, 488-562. Platt, J. R. 1964.Strong inference. Science 146,206-207. Pontecorvo, G. 1958. “Trends in Genetic Analysis.” Columbia Univ. Press, New York. Preer, J. R., Jr. 1971. Extrachromosomal inheritance : Hereditary symbionts, mitochondria, chloroplasts. Annu. R e v . Genet. 5, 361-406. Rhoades, M. M. 1955. Interaction of genic and non-genic hereditary units and the physiology of non-genic inheritance. Encycl. Plant Physiol. 1, 19-57. Ris, H., and Plaut, W.1962. Ultrastructure of DNA-containing areas in the chloroplast of Chlamydomonas. J . Cell Biol. 13,383-391. Roberts, K., Gurney-Smith, M., and Hills, G. J. 1972. Structure, composition and morphogenesis of the cell wall of Chlamydomonas reinhardii. I. Ultrastructure and preliminary chemical analysis. J. Ultrastruc. Res. 40, 599-613. Rochaix, J. D. 1972. Cyclization of DNA fragments of Chlamydomonas reinhardii. Nature (London),New Biol. 238, 76-78. Ryan, R. S., Grant, D., Chiang, K. S., and Swift, H. 1973. Isolation of mitochondria and characterization of the mitochondria1 DNA of Chlamydomonas reinhardii. J. Cell Biol. 59,297a. Sager, R. 1954. Mendelian and non-Mendelian inheritance of streptomycin resistance in Chlamydomonas reinhardii. Proc. Natl. Acad. Sci. U S A . 40, 356363. Sager, R. 1960. Genetic systems in Chlamydomonas. Science 132, 1459-1465. Sager, R. 1962. Streptomycin as a mutagen for non-chromosomal genes. Proc. Natl. Acad. Sci. U S A . 48,2018.2026. Sager, R. 1972. “Cytoplasmic Genes and Organelles.” Academic Press, New York. Sager, R. 1975. Patterns of inheritance of organelle genomes: Molecular basis and evolutionary significance. In “Genetics and Biogenesis of Mitochondria and

GENETIC ANALYSIS OF CHLOROPLAST DNA

339

Chloroplasts” (C. W. Birky, Jr., P. S. Perlman, and T. J. Byers, eds.), p. 252. Ohio State Univ. Press, Akron. Sager, R., and Granick, S. 1954. Nutritional control of sexuality in Chlamydomonas reinhardii. J. Gen. Physiol. 37, 729-742. Sager, R., and Hamilton, M. G. 1967. Cytoplasmic and chloroplast ribosomes of Chlamydomonas: Ultracentrifugal characterization. Science 157, 709-711. Sager, R., and Ishida, M. R. 1963. Chloroplast DNA in Chlamydomonas. Proc. Natl. Acad. Sci. US.A. 50, 725-730. Sager, R., and Lane, D. 1969. Replication of chloroplast DNA in zygotes of Chlamydomonas. Fed. Proc., Fed. Am. SOC.Exp. Biol. 28, 347 (abstr.). Sager, R., and Lane, D. 1972. Molecular basis of maternal inheritance. Proc. Natl. Acad. Sci. U S A . 69, 2410-2414. Sager, R., and Palade, G . E. 1957. Structure and development of the chloroplast in Chlarnydomonas. I. The normal green cell. J. Biophys. Biochem. Cytol. 3, 463488. Sager, R., and Ramanis, Z. 1963. The particulate nature of non-chromosomal genes in Chlamydomonas. Proc. Natl. Acad. Sci. U S A . 50, 260-268. Sager, R., and Ramanis, Z. 1965. Recombination of non-chromosomal genes in Chlamydomonas. Proc. Natl. Acad. Sci. USA. 53, 1053-1061. Sager, R., and Ramanis, Z. 1967. Biparental inheritance of nonchromosomal genes induced by ultraviolet irradiation. Proc. Natl. Acad. Sci. US.A. 58, 931-937. Sager, R.,and Ramanis, Z. 1968. The pattern of segregation of cytoplasmic genes in Chlarnydornonas. Proc. Natl. Acad. Sci. U S A . 61, 324-331. Sager, R., and Ramanis, Z. 1970. A genetic map of non-Mendelian genes in Chlamydomonas. Proc. Natl. Acad. Sci. U S A . 65, 593-600. Sager, R., and Ramanis, Z. 1973. The mechanism of maternal inheritance in Chlamydomonas: Biochemical and Genetic Studies. Theor. Appl. Genet. 43, 101-108 Sager, R., and Ramanis, Z. 1974. Mutations that alter the transmission of chloroplast genes in Chlamydomonas. Proc. Natl. Acad. Sci. USA. 71, 46984702. Sager, R., and Romanis, Z. 1976a. Chloroplast Genetics of Chlamydomonas. I. Allelic segregation ratios. Genetics 83, 303-321. Sager, R., and Ramanis, Z. 1976b. Chloroplast Genetics of Chlamydomonas. 11. Mapping by Cosegregation Frequency Analysis. Genetics 83, 323-340. Sager, R., and Ramanis, Z. 1976c. Mutations induced by streptomycin starvation of a streptomycin-dependent strain of Chlamydomonas. (In preparation.) Sager, R., and Ramanis, Z. 1976d. Persistant cytoplasmic heterozygotes in Chlamydomonas. (In preparation.) Sager, R., and Schlanger, G. 1976. Chloroplast DNA: Physical and genetic studies. In “Handbook of Genetics” (R. C. King, ed.), Vol. 5, pp. 371424. Plenum, New York. Sager, R., and Tsubo, Y. 1962. Mutagenic effects of streptomycin in Chlamydomonas. A x h . Mikrobiol. 42, 159-175. Schimmer, O., and Arnold, C. G. 1970a. Untersuchungen uber Reversions- und Segregationsverhalten eines ausserkaryotischen Gens von Chlamydomonas reinhardii zur Bestimmung des Erbtragers. Mol. Gen. Genet. 107, 281-290. Schimmer, O., and Arnold, C. G. 1970b. Uber die Zahl der Kopien eines ausserkaryotischen Gens bei Chlamydomonas reinhardii. Mol. Gen. Genet. 107, 366-371. Schimmer, O., and Arnold, C. G. 1970c. Hin- und Rucksegregation eines ausserkaryotischen Gens bei Chlamydomonas reinhardii. Mol. Gen. Genet. 108, 33-40.

340

RUTH SAGER

Schlanger, G., and Sager, 1974a. Correlation of chloroplast DNA and cytoplasmic inheritance in Chlamydomonas zygotes. J . Cell Biol. 63,301a. Schlanger, G., and Sager, R. 1974b. Localization of five antibiotic resistance at the subunit level in chloroplast ribosomes of Chlamydomonas. Proc. Natl. Acad. S C ~ . USA. 71, 1715-1719. Singer, B., Sager, R., and Ramanis, Z. 1976. Chloroplast genetics of Chlamydom6nas. 111. Closing the circle. Genetics 83, 341-354. Smith, C. A., and Vinograd, J. 1972. Little DNA circles in animal cell cytoplasm. J. Mol. Biol. 69, 163. Sueoka, N., Chiang, K. S., and Kates, J. R. 1967. Deoxyribonucleic acid replication in meiosis of Chlamydomonas reinhardii. I. Isotopic transfer experiments with a strain producing eight zoospores. J. Mol. Biol. 25, 47-66. Surzycki, S. J., and Gillham, N. W. 1971. Organelle mutations and their expression in Chlamydomonas reinhardii. Proc. Natl. Acad. Sci. U8.A. 68, 1301-1306. Szybalski, W., and Szybalski, E. H. 1971. Equilibrium density gradient centrifugation. Proced. Nucleic Acid Res. 2, 311-354. Thomas, C. A., Jr., Hamkalo, B. A., Misra, D. N., and Lee, C. S. 1970. Cyclization of eucaryotic deoxyribonucleic acid fragments. .I. Mol. Biol. 51, 621-632. Wells, R., and Sager, R. 1971. Denaturation and renaturation kinetics of chloroplast DNA from Chlamydomonas reinhardii. J. Mol. Biol.58,611-622.

GENETICS AND BIOCHEMISTRY OF MORPHOGENESIS IN Neurosporu N.

C. Mishra

Department of Biology, University of South Carolina, Columbia, South Carolina

. . . . . . . . . . . . . . . . . . 34 1 . . . . . . . . 343 A. Role of the Cell Wall in Morphogenesis . . . . . . . . . 343

I. Introduction

11. Biochemical Genetics of Hyphal Development

B. Role of the Cell Membrane in Determining the Morphology of

Neurospora . . . . . . . . . . . . . . . . . . C. Gene, Enzyme, and Morphology . . . . . . . . . . . 111. Conidiogenesis . . . . . . . . . . . . . . . . . A. Ultrastructure and Surface Architecture of Conidia . . . . . . B. Biochemistry and Genetics of Conidiogenesis . . . . . . . . C. Circadian Rhythm . . . . . . . . . . . . . . . IV. Morphogenesis during Sexual Development . . . . . . . . . A. Sexual Development and the Mating-Type Locus . . . . . . B. Genetics and Biochemistry of Perithecial Development . . . . . C. Genetics of Ascus and Ascospore Development . . . . . . . V. Conclusions . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

356 357 380 380 38 1 385 386 387 388 390 394 396

I . Introduction

The development of an organism involves a series of genetically controlled biochemical reactions. Elucidation of these biochemical reactions has been the main theme of past and current research in genetics, biochemistry, and developmental biology. Neurosporu, a filamentous haploid fungus, offers several advantages in biochemical genetic studies of development; some of the attributes of Neurosporu, as an organism of choice, arise from the facts that (1) its biochemistry, cytology, and genetics are well investigated, more so than those of any other eukaryotic microorganisms (Fincham and Day, 1971) ; (2) tetrad and heterokaryon analyses are possible; (3) selective and other techniques of microbial genetics are applicable; (4)it also undergoes a certain degree of differentiation, producing during its life cycle an array of developmentally linked fungal structures that can be biochemically analyzed; and ( 5 ) 34 1

342

N. C. MISHRA

above all, a large number of mutants defective in a particular stage of morphological development have been available for biochemical genetic studies (Garnjobst and Tatum, 1967; Srb et al., 1974; Siegel et al., 1974). Therefore, the morphogenesis of Neurospora has been extensively studied as a model example to understand the biochemical basis of differentiation among eukaryotic microorganisms. The study of morphogenesis in Neurospora has been carried out by two distinct approaches: (1) The biochemical genetic approach: In this approach, the isolation of a single-gene mutant and its biochemical analysis have been utilized to understand the biochemical basis of the processes involved therein. This approach was first initiated by Beadle and Tatum (1941) to elucidate the mechanism of gene action and has later been successfully applied in the understanding of metabolic pathways (Srb and Horowitz, 1944), genetic regulatory mechanisms (Jacob and Monod, 1961), bacteriophage assembly (Wood et al., 1968), behavioral phenomena in Drosophila (Benzer, 1967) , and morphogenesis in Neurospora (Tatum, 1973). Neurospora, a filamentous fungus, can undergo mutations to bizarre morphological forms (see Fig. 1) (Garnjobst and Tatum, 1967) or mutations that can block a particular developmental stage (Srb et al., 1974; Siegel et al., 1974) ; these can be analyzed to reveal the biochemical (or molecular) basis of a particular morphological or developmental change. The production and study of a phenocopy (of a genetic mutant) by chemical manipulation has been an essential supplement to the biochemical genetic approach mentioned above. The production of a phenocopy first described by Tatum et al. (1949) was later extended by different authors (Reissig and Glasgow, 1971; N. C. Mishra and E. L. Tatum, unpublished ; Scott and Soloman, 1975 ; Rand, 1975). (2) The comparative biochemical approach: I n this approach distinct fungal structures were analyzed to understand the biochemical basis of the differences in their structure and function. As mentioned earlier, Neurospora undergoes a certain degree of differentiation in its life cycle and produces distinct structures, such as vegetative hyphae, aerial hyphae, macroconidia, microconidia, ascogonia, perithecia, asci, and ascospores ; each of these fungal structures can now be obtained as homogeneous preparation and analyzed biochemically and by electron microscopy. Both these approaches have been adopted in the study of morphogenesis in Neurospora, and a vast amount of literature has recently appeared on this subject; the purpose of this review is to present a comprehensive account of these works and to assess the current status of our knowledge on this subject, which should form a basis for future work. There have appeared three reviews on different aspects of this subject (Brody, 1973; Scott et al., 1974; Srb et al., 1974). The first review, by

BIOCHEMICAL GENETICS OF

Neurospora

MORPI-IOGENESIS

343

Brody, was written a t a time when the biochemical genetics of Neurospora morphogenesis was in its infancy, as described by Metzenberg (1972) ; the other two reviews were limited by the scope of the symposium where these were presented, and therefore are very specialized in their content. Here I have attempted to present an account of the different aspects of Neurospora morphogenesis as elucidated by the different laboratories all over the world and to present an overall picture that emerges from the discussion of these works. This review should be helpful in providing a unified picture of the current status of our knowledge on this subject, particularly to beginners in this area of meaningful research. It. Biochemical Genetics of Hyphal Development

Neurospora is a filamentous fungus, and the wild-type strain grows as a spreading mycelium on agar medium. Lindegren first described a morphological mutant called button (Lindegren and Lindegren, 1941) ; this mutant has very slow and restricted growth. Since then a large number of mutants have become known (Garnjobst and Tatum, 1967) which have altered morphology and mycelial growth pattern. Mutation in any of approximately 120 of the known 500 loci of Neurospora can cause a morphological change. The different morphological mutants have been classified into six groups by Garnjobst and Tatum (1967) depending on their growth pattern as discussed later in this paper. A large number of these morphological mutants have been biochemically analyzed ; these were found to be altered in their cell wall composition, and in some cases the defective enzymes responsible for changes in cell wall composition have been identified and characterized as discussed below. Besides genetic mutation, changes in colonial morphology can be chemically induced in Neurospora (Tatum et al., 1949) ; phenocopy of genetic mutation has been utilized in understanding the biochemical changes underlying the colonial morphology in Neurospora. These studies also lead to the conclusion that Neurospora morphology is mainly determined by the chemical composition of its cell wall. The works discussed below provide evidence on the role of the cell wall in the determination of Neurospora morphology.

A. ROLEOF

THE

CELLWALLIN MORPHOGENESIS

It has been suggested that the rigid cell wall of Neurospora must obviously play an important part in determining the morphology of this organism (deTerra and Tatum, 1961, 1963). This idea is based in part on

344

N. C. MISHRA

FIG. 1. External appearance of different morphological mutants of Neurospora (Garnjobst and Tatum, 1967) after 48 hours of growth on minimal medium at 30°C. ( A X ) colonial (cot) mutant; (D-F) spreading colonial (spco) mutants; (G-I)

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

345

semicolonials (smeo) mutants; (J-L) spreading morphologicals (mo) ; (M, N, and P) ropy mutants; (Q) ropylike; (0,R) medusa; (S-N) environment-sensitive morphological (moe) mutants. (After Garnjobst and Tatum, 1967.)

346

N. C. MISHRA

the findings that Neurospora forms osmotically sensitive spherical protoplasts on treatment with carbohydratases (Bachman and Bonner, 1959) and assumes the colonial form of growth if treated with lower concentrations of these enzymes (Tatum, quoted in Scott et al., 1974); it is based also on the finding that morphological changes in Neurospora are always accompanied by changes in chemical composition of the cell wall (deTerra and Tatum, 1961; Mahadevan and Tatum, 1965). 1. Biochemistry, Ultrastructure, and Surface Architecture of the Cell Wall

The chemical composition of the wall of many fungi has been studied in an attempt to elucidate the relationship between the hyphal wall and fungal morphology (Bartnicki-Garcia, 1968). The biochemistry and ultrastructure of Neurospora, has been, therefore, investigated in detail (Shatkin and Tatum, 1961; Mahadevan and Tatum, 1965, 1967; Hunsley and Burnett, 1970; Manocha and Calvin, 1967; Wrathal and Tatum, 1973) in order to understand its role in the morphogenesis of this organism (see Fig. 2). The main components of the Neurospora wild-type cell wall are (1) glucan, (2) chitin, (3) peptides. The glucan layer surrounds the internal layer of chitin; the peptides are interspersed in between these two layers. Composition of the Neurospora cell wall has been determined by selective removal of the different components either by chemical or enzymic degradation (Mahadevan and Tatum, 1965; Hunsley and Burnett, 1970). Mahadevan and Tatum (1965) have shown that the Neurospora cell wall contains four components called fractions I, 11, 111, and IV (see Fig. 3 for the fractionation procedure used by these authors). Of these different components, fraction I is alkali soluble and is essentially a peptide-polysaccharide (glucan) complex; fraction I1 is an alkali-insoluble but acid-soluble fraction of the cell wall. Both fractions I and I1 are glucans, but the precise linkage of the hexose molecules has not yet been determined. Fraction I11 is definitely p-1,3-glucan, and it can easily be digested by laminarinase. Fraction IV is chitin. I n their chemical analysis Mahadevan and Tatum (1965) have shown in fraction I the presence of several amino acids that might have been released by the hydrolysis of peptides present there. Fraction I also contained a galactosamine polymer that was earlier identified by Harold (1962). The role of this component in morphogenesis has been discussed by Reissig and Glasgow (1971) as described in Section 11, C, 1, b. Hunsley and Burnett have found the chemical composition of the Neurosporu cell wall similar to that described by Mahadevan and Tatum (1965). The localization and orientation of the different glucan and chitin polymers in the Neurospora cell wall have been studied by electron microscopy. For this purpose, the

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

347

cell wall was digested either chemically or enzymically or by both methods to remove a particular component. Such studies by Mahadevan and Tatum (1967) and later by Hunsley and Burnett (1970) show the Neurospora cell wall to be composed of two distinct layers: (a) The outer layer: This is a wide region made up of fibrillar structures and divisible into outer and inner subregions. The outer fibrils are made up of glucan peptide complexes which correspond to fractions I and I1 (Mahadevan and Tatum, 1965) whereas the inner fibrils are made up of P-ll3-glucan (fraction 111). The outermost fibrils can be effectively digested by treatment with a combination of Pronase and laminarinase suggesting its glycoprotein complex nature. (b) The inner layer: This is made up of chitin (fraction I V of Mahadevan and Tatum). This is thicker near the inner wall (septum) (Mahadevan and Tatum, 1965; Hunsley and Goodday, 1974). This layer, however, does not seem to have much effect in determining the morphology of Neurospora, as discussed in Section 11, A, 2. The extent to which protein permeates the different cell wall regions and the manner in which i t is chemically attached to them is not wholly resolved. Hunsley and Burnett have suggested the occurrence of the proteins in the interstices of the reticulum and that the wall becomes progressively rich in protein from the outside of this region inward until the latter (protein) forms a discrete layer on the surface of the chitinous region. It is further suggested that protein may well permeate this innermost coaxial region since the microfibrils, once revealed, become more distinct with Pronase treatment (Hunsley and Burnett, 1970). This widespread distribution of protein is compatible with claims that protein accounts for 14% of the wall material as estimated from total nitrogen values (deTerra and Tatum, 1961). The presence of peptide protein in the Neurospora cell wall was first evidenced by the work of deTerra and Tatum (1963) and by Mahadevan and Tatum (1965) and has been established and elucidated by Wrathal and Tatum (1973) as discussed below. Of the three polymeric components of the Neurospora cell wall, glucan and chitin have been studied in more detail, whereas the third component-peptide-received little attention in earlier work. This was mainly because carbohydrate comprises a major component of the cell wall. Earlier, proteins were thought to be only those that were trapped within the growing cell wall structure (Chang and Trevithick, 1972) or were in transit (Trevithick and Metzenberg, 1966). However, recently Wrathal and Tatum (1973) have reported on the quantitation and partial characterization of the peptides that occur in the cell wall as its structural component and that the peptides have a significant role in morphogenesis. The peptides of this hyphal wall of wild-type Neurospora crassa

348 N. C. MISHRA

FIG.2. Electron micrograph of the different strains of Neurosporu erassa. (A) Wild-type strain (74A) (x38,OOO). (B) Mutant strain (snow flake, 507) (x24,OOO). CW, cell wall, ER, endoplasmic reticulum, M, mitochondria, N, nucleus, and MF, microfilaments (courtesy of Dr. Edward D. Allen).

Cel'r''

350

N. C. MISHRA 2 N sodium hydroxide 16 hr a t room temperature

Centrifuged I

Resihue 1 N sulfuric acid 90°C. 16 hr

SupeAatant

I

Centrifuged

I

m

Suoernatant

Barium hydroxide to pH 7.0 Precipitate

I

Cenirifuged

I

I

Precioitate discarded

I

60%saturation ammonium sulfate or 2 volumes of ethyl alcohol

-

Residue

After washing several times, 2 N sodium hydroxide 30 min, room temperature

Precipitate

Resuspended in water, dialyzed against water for 24 hr, and lyophilized

Cenlrifuged

FRACTION I

Residue

Suoernatant

2 volumes of

ethyl alcohol

Supernatant

FRACTION II

Resuspended i n water, dialyzed, and lyophilized

FRACTION I V

Preciiitate Centrifuged, resuspended, dialyzed 24 hr against water, and lyophilized FRACTION III

FIQ.3. Procedure for the fractionation of major Neurosporu cell-wall components. (After Mahadevan and Tatum, 1965.)

were shown to comprise approximately 10% of the hyphal wall by weight and to consist of a t least five major fractions, which were separated by ion-exchange chromatography. These peptides were found to be covalently linked to the carbohydrate portion of the wall by o-glycosylserine linkages that are labile to strong alkali; the peptides were thus separated from the remainder of the wall by treatment with NaOH and then fractionated by DEAE-cellulose chromatography. The molecular weight of the cell wall peptide was approximately 3800 daltons as estimated by gel filtration. These authors (Wrathal and Tatum, 1974) have also compared the cell wall peptide content of 23 colonial mutants strains with the wildtype strain of N . crassa. The hyphal wall peptides prepared by 0.5 M NaOH treatment of the wall were further fractionated by DEAE chromatography, and five peaks of peptides were obtained. The amino acid composition of these hyphal wall peptides shows the presence of all 20 amino acids besides two unidentified compounds that were acidic and basic in character and have not yet been characterized. Peptides were found i , ~ be linked to sugar through either a serine or a threonine residue

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

35 1

(o-glycosylserine or glycosylthreonine) . Both chitinase and glusulase were used to digest the other major components of the cell wall and on such digestion the peptide was found to be located with the cell wall. All the morphological mutants analyzed showed quantitatively a lesser amount of cell wall peptides. Of the different mutants, only doily showed a qualitative difference in the profile of cell wall peptides on DEAE cellulose chromatography; the bulk of the mutant peptide was found to elute in the void volume of the column, suggesting that the mutant peptides did not bind to the coIumn. Amino acid composition of the wildtype peptide has been shown to include aspartic and glutamic acids; a significant reduction in the amount of these amino acids would result in loss of the ability of the mutant peptide to bind with DEAE. Preliminary studies by the authors (Wrathal and Tatum, 1974) do show a dramatic reduction in the amount of these amino acid residues in the mutant peptides. The chemical composition of the Neurospora cell wall and the organization of the different components as described by Mahadevan and Tatum (1965) was soon confirmed by Hunsley and Burnett (1970). These workers have subjected Neurospora cultures to single or sequential treatment with enzymes including laminarinase, Pronase, cellulase, and chitinase and various other chemical treatments and then compared the normal (untreated) and treated cultures by electron microscopic examination. From such a study, they have inferred the coaxial distribution of different polymers, such as glucose, glycoprotein, and chitin. A mutant (sl) that lacks the rigid cell wall has been described (Schulte and Scarborough, 1975); Scarborough has found that the sl mutant or the spheroplast of the wild-type strain contains only chitin (6%) as compared t o different components of the normal hyphal cultures (Scarborough and Schulte, 1974). a. The Mechanical Integrity of Mature Walls. According to Hunsley and Burnett the mechanical integrity of the mature hyphal wall does not seem to reside in any one major wall component. Their evidence for this is based on the fact that hyphal disintegration occurred only after enzymic sequences attacked all the main components in Neurospora. However, according to Mahadevan and Tatum (1965) the glucan and peptide complex is critical in maintaining the normal morphology of Neurospora; their conclusion is based on the fact that in morphological mutants the glucan-peptide complex is the principal cell wall component that suffers any change. Nevertheless, the apical region of Neurospora hyphae seems to be more susceptible to rupture than the mature walls (Robertson and Rizvi, 1965 ; Robertson, 1968). Thus the mechanical integrity of the apical and subapical regions could be related to differ-

352

N. C. MISHRA

ences in cell wall composition as evidenced by work of Hunsley and Burnett (1970) and Mishra (1975). The electron microscopy studies of Hunsley and Burnett (1970) clearly show lack of development of the reticulum and interstitial protein in the apical region. That is, the apical region of Neurospora consists of chitin and protein and little glucan and is mechanically nonrigid. Thus, the apical region has great plasticity, as suggested by Robertson (1959). It is suggested that a greater degree of rigidification is provided progressively farther back from the subapical region by the development of reticulum and the outer glucan (Hunsley and Burnett, 1970). A similar picture regarding the differences in the structure of apical and subapical region of Neurospora hyphae also emerges from the recent scanning electron microscopic studies (Mishra, 1975) as described below (see Fig. 4). b. Surface Architecture of Neurospora. Mishra has recently studied different strains of Neurospora in an attempt to reveal the differences in their three-dimensional structure. The wild-type hypha was found to possess several parallel foldings on its surface. The apical region, however, was found to lack the characteristics parallel folding and instead to have a very smooth surface. These scanning electron microscopic pictures reflect the differences in the chemical composition of these fungal structures. The lack of parallel folding in the apical region marks the absence of the outermost glucan region, which is responsible for the plasticity of the apical region. The difference in the chemical composition and, in the ultrastructural and surface architecture of the apical and subapical regions of Neurospora hyphae provides a structural basis for a balance between the hypothetical processes of extension growth and rigidification postulated by Robertson (1959). This balance is internally controlled, and forces that can cause any imbalance also lead to abnormal morphological development (deTerra and Tatum, 1961). 2. Changes in the Cell Wall’Composition of

the Morphological Mutants The idea that the cell wall is the principal determinant of Neurospora morphology was first tested by deTerra and Tatum (1961). These authors found that the ratio of glucose: glucosamine in a number of morphological mutants was changed as compared to the wild type. Later, Mahadevan and Tatum (1965) defined the different components of the wild-type cell wall and compared the levels of the different cell-wall components (fractions I-IV) of the wild type with those of the mutants. In each instance of morphological change caused either by a gene mutation or by chemical induction, the altered morphology was found to be related to either a n

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

353

increase in the level of fraction I (glucan) or a decrease in fraction I11 (P-1,3-glucan) or both. These authors also examined the cell wall of certain conditional mutants found to have normal morphology at 25OC but a colonial morphology a t 35OC. On chemical analysis of these temperature-sensitive mutants grown a t permissive (wild-type morphology) and nonpermissive (colonial morphology) temperature, in each case altered morphology was found t o be related with an increase in fraction I or a decrease in fraction 111. Mahadevan and Tatum (1965) also showed similar changes in the cell wall composition of the sorbose-grown wild-type strain having colonial morphology. Despite the dramatic changes in the cell-wall composition of the sorbose-grown wild-type strain, these authors found no difference in a sorbose-grown mutant strain called patch. This mutant strain (patch) was earlier shown by Stadler (1959) to resist the paramorphogenic effects (Tatum et al., 1949) of sorbosc (i.e., patch has a normal morphology on a growth medium containing sorbose). Later, Wrathal and Tatum (1974) found that in some other morphological mutants, the nature of the cellwall peptide was changed. Thus these studies clearly showed the role of glucan, P-1,3-glucan, and peptide to be critical in determining the morphology of Neurospora. I n no instance was there any change in the level of chitin, which is a major portion of the cell wall of Neurospora. Therefore, it was concluded by Mahadevan and Tatum (1965) that chitin perhaps has no role in determining the morphology of this organism. The possible role of chitin was examined by Mahadevan and Tatum (1965) using a slightly diflerent approach. Since glutamine is the specific amino group donor in the biosynthesis of glucosamine (LeLoir and Cardini, 1953) , Mahadevan and Tatum examined the effect of glutamine on the morphology of the glutamine-requiring mutants of Neurospora. It was thought that if chitin has any role in determining the morphology of Neurospora, the limiting amount of glutamine in the growth medium would lead t o changes in morphology. Such an expectation was analogous to the effect of limiting the amount of inositol in producing colonial morphology in inositolrequiring mutants of Neurospora, to be discussed later on in this paper. However, no morphological changes were seen to result in the glutamine mutants from the limiting amount of glutamine in the growth media. Mahadevan and Tatum also found that there was no significant difference in the level of chitin (or in other cell wall fractions) when the glutaminerequiring mutant was grown on medium containing limiting amounts of glutamine. A similar conclusion regarding the nonessential role of chitin in determining morphology of Neurospora was arrived at by other workers (Endo and Misato, 1969). These authors found that polyoxin D,

354

N. C. MISHRA

FIQ.4. Scanning electron micrographs of the different Neurospora crassa strains. (A-G) Different stages in the life cycle of the wild-type strain (RL3-8A). (A) Vegetative hyphae, (B) apical region of the hyphae, (C) macroconidia, (D,E) microconidia, (F) ascogonium, (G) ascospores, (HJ) surface architecture of the wild-type strain (RL3-8A) grown on sucrose medium (H) and on medium with L-sorbose (I), (J,K,L) different stages in the life cycle of the mutant strain (ragged), (J) vegetative hyphae, (K) macroconidia, (L) ascospore, (M-P) surface architecture of an osmotic mutant under different growth conditions, (M) vegetative hyphae grown on minimal medium, ( N ) vegetative hyphae grown on minimal medium containing 1N NaCI, (0)macroconidia of the mutant culture grown on minimal medium, (P) a magnified view of the macroconidium (as in 0) (After Mishra, 1975).

356

N . C. MISHRA

an inhibitor of chitin synthetase, was ineffective in producing any morphological changes in Neurospora when the latter was grown on media cont,aining various concentrations of the inhibitor (polyoxin D) . These findings regarding the essential and nonessential roles of different cell wall components in determining the morphology of Neurospora affirm the validity of the concept of internal imbalance leading to morphological changes. It also fits with the ideas that glucan and peptides are laid down subsequent to the growth by elongation a t the apical region (Robertson, 1959). It can also be argued that since chitin is the innermost layer of the Neurospora cell wall, any significant change would be deleterious to its survival. This conjecture is supported by studies in which the inhibitor acts as a fungicide a t particular concentrations. HOWever, recent work of Russell and Srb (1974) casts doubt on the assumption regarding the nonessential role of chitin in determining the morphology of Neurospora. These authors have found elevated levels of an enzyme concerned with chitin biosynthesis in peak-2 and other morphological mutants as discussed later in this review article. B. ROLEOFTHE CELL MEMBRANE IN DETERMINING THE MORPHOLOGY OF Neurospora Both inositol and choline are major components of the cell membrane of living systems. Earlier, Fuller and Tatum (1956) had reported the role of inositol in determining the morphology of Neurospora. These authors found that inositol-requiring mutants had colonial morphology when grown on a limiting amount of inositol in the growth medium. Later Shatkin and Tatum (1959) showed differences in the structure of the membrane of the mutant strains. The membrane structure was impaired in the mutant hyphae grown on media with a limiting amount of inositol as compared to a culture grown on an optimal amount of inositol. Such impairment in the membrane structure of the mutants was suggested to cause the morphological changes. Since the cell membrane and cell wall are very close to each other in Neurospora, its role in morphology can be easily visualized. However, later Matile (1966) provided another explanation for the effect of inositol in causing morphological changes in the mutants. According to Matile, the lysosomes are impaired in a mutant grown on limiting amounts of inositol, several hydrolyzing enzymes escape from the lysosomes, and the hydrolysis of the cell wall leads t o morphological changes. On the basis of aforementioned studies mainly by Tatum’s group, it can be concluded that a complex of cell wall and membrane is instrumental in causing morphological changes in Neurospora.

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

357

C. GENE,ENZYME, AND MORPHOLOGY An important approach to understanding the morphogenesis of iVeurospora has been to elucidate the biochemical lesions underlying the morphological changes in the mutants of Neurospora or their suppressors and phenocopies. The rationale behind this approach has been similar to that applied to biochemical mutants in the 1940s as elucidated by Beadle and Tatum (1941). Following this approach, Tatum's group a t the Rockefeller University has been ablc to elucidate the biochemical lesions underlying the niorphological changes in several genetic mutants, their phenocopies and suppressors. I n Neurospora, over a hundred genes are now known (Garnjobst and Tatum, 1967) ; a mutation in any of them can cause a specific morphological change. These mutations are mainly point mutations from several criteria (such as recombination, reversion, and suppression) ; most of the mutations have been mapped with various degrees of refinement (Garnjobst and Tatum, 1967; Morgan et al., 1967). These mutations are distributed among all 7 chromosomes ; however, interestingly enough, the genes controlling morphological development were found to be localized to the centromeric and to the telomeric regions (Garnjobst and Tatum, 1967). These Neurospora mutants, on the basis of their growth pattern, have been divided into several groups (Garnjobst and Tatum, 1967). The main criterion taken into consideration during the classification of the morphological mutants was the type of growth (a) during development of mycelium from the ascospore, (b) in a test tube on transfer, and (c) a t different temperatures, such as 25OC and 35°C.On this basis, the morphologicals have been grouped into six classes; these are: (1) true colonials-these retain their restricted colonial-type growth a t optimum temperature and have been designated col-1, col-2, etc. ; (2) spreading colonials (spco)-these begin as colonials upon germination of the ascospore, but growth does not remain restricted and soon spreads over the agar surface; (3) semicolonial (smco)-these begin as a restricted colony but soon produce flares of wild-type-like hyphae (with or without conidia) . The wild-type-like growth of smco is influenced by the moisture and CO, content of the environment; (4) this class includes a group of spreading morphological (mo) strains, which are readily distinguishable from the wild type (see Fig. 1 ) by virtue of scanty growth or fine hyphae and reduced conidiation; ( 5 ) this class also includes a number of miscellaneous types of strains each having a distinct type of mycelial growth ; (6) this last group includes colonial temperature-sensitive strains (cot) and other morphologicals environmentally influenced in their growth pattern-therefore, this group has been designated as ( m o e ) .

358

N. C. MISHRA

Most of these morphological mutants have no specific biochemical requirement, and their pattern is not influenced by supplementation to their growth media ; however, there are certain exceptions (Garnjobst and Tatum, 1967). The fact that these mutants show similar growth patterns on minimal or supplemented media in general, and that the morphological changes were always accompanied by changes in cell-wall composition, suggested that a t least some of these morphological mutants were defective for enzymes of Carbohydrate metabolism. On biochemical screening, over a dozen of these mutants have now been found to be defective for enzymes of carbohydrate metabolism (see Tables 1-3). The validity of this approach was further shown by Murayania and Ishikawa (1975), who isolated several morphological mutants of Neurospora that could grow very poorly on glucose but could not grow on fructose and were found to lack hexoseisomerase. The rationale behind this approach was that a lesion in carbohydrate metabolism would lead to morphological changes. However, such an approach to isolate phosphoglucomutase mutants that were able to grow on glucose but not on galactose has not been successful in Neurospora; although such mutants are known in yeast (Tsoi and Douglas, 1964). 1. Genetics and Biochemistry of Morphological Mutants and

Their Phenocopies and Suppressors The gene-enzyme relations in a number of morphological mutants have been elucidated. All these mutants are defective for enzymes of cell wall or membrane biosynthesis ; their detection and characterization are discussed below. Detection of the biochemical lesion. Different screening procedures were employed to detect the biochemical lesions in the carbohydrate metabolism in the morphological mutants. The following procedures were included: (1) Determination of the steady state level of the different phosphorylated sugars. For example, mutants defective for glucose-6phosphate dehydrogenase would accumulate glucose 6-phosphate (Brody and Tatum, 1966). (2) Determination of the specific activity of the enzymes and their heat stability in crude extracts (Brody and Tatum, 1966; Mishra and Tatum, 1970a; Scott and Tatum, 1970; Abramsky et al., 1971). Heat inactivation has proved to be the most useful method for detecting enzymic lesions in several mutant strains. (3) Growth pattern of a particular mutant strain on different sugars (Murayama and Ishikawa, 1975; Rand, 1975; Abramsky and Tatum, 1976). For example, a strain that did not grow on fructose or on galactose but showed growth on glucose would be suspected to be defective in a metabolic pathway that leads to utilization of these sugars; similarly, a mutant that grows

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

359

TABLE 1 Gene-Enzyme Relationships in Different Morphological Mutants of Neurospora Mutation (linkage group) 1. col-2 (1711) 2 . balloon (11) 3. frost (I) 4. ragged-1 (I)* 6. ragged4 (I) 6. COl-7 (I)* 7. COZ-3 (VII) 8 . col-10 (11) 9. choi-1 (IY)

10. Chol-2 (VI) 11. inos (V)c 12. gpi-f (IV) 13. gpi-2 (IV) 14. T9 (I) 15. cr-1 (I) 16. C8p-f (I) 17. csp-2 (VII) 18. spco-1 19. peak-2 (V) 20. gal-f (I\.) 21. g a l 4 (IV)

Defective enzyme"

References

G6PD G6PD G6PD

Brody and Tatum (1966) Scott and Tatum (1970) Scott and Tatum (1970) PGMI Brody and Tatum (1967a) PGMII Mishra and Tatum (1970a) PGMI N. C. Mishra (unpublished) 6PGD Abramsky et al. (1971); Scott and 6PGD Abramsky (1973b) Methylation of phosphatidyl- Crocken and Nyc (1964) ethanolamine Methylation of phosphatidyl- Crocken and Nyc (1964) mono(di-)methyl ethanolamine Pina and Tntum (1967) Glucocy cloaldolase Glucose isomerase Murayama and Ishikawa (1975) Glucose isomerase Glucoamylase Murayama and Ishikawa (1973) Adenyl cyclase Terenzi et al. (1974) Selitrennikoff et al. (1974) Cell-wall autolyzing enzyme Selitrennikoff et al. (1974) Cell-wall autolyzing enzyme Ccll-wall autolyzing enzyme Mahadevan and Mahadkar ( 1970a) L-Glutamine ~-Fructose-6Russell and Srb (1974) phosphate amidotransferase Galactokinase Rand (1975) UDPG pyrophosphorylasc

a Defective enzyme or biochemical reactions. Nomenclature for enzymes; GGPD, glucose-&phosphate dehydrogenase; PGM, phosphoglucomutase ; GPGD, 6 phosphogluconate dehydrogenasc. * cof-7 (54357) is allelic to rugged-1 (B-53) (N. C. Mishra, unpublished). Enzymes responsible for the defective biochemical reactions are not yet characterized.

as a colonial on glucose, sucrose, and maltose but as a wild type on isomaltose should be defective for the enzyme that catalyzes the synthesis of w1,6 linkages (present in isomaltose) as elucidated by Abramsky and Tatum (1976) ; Rand (1975) has examined a large number of the morphological mutants for their ability to grow on different sugars as discussed later in this paper. (4) Last, the determination of the enzyme activity in situ. This was carried out by coupling NADPH generated by the dehydrogena,se activity to phenazine methosulfate-nitroblue tetrazolium and

360

N. C. MISHRA

then scoring for formazan deposits in cells dried on microscope slides (Mishra and Taturq, see Scott and Abramsky, 197313). By this approach both col-3 and col-10 were found to be defective for 6-phosphogluconate dehydrogenase (Scott and Abramsky, 1973b). Another mutant C O ~ -was 7 found to be defective for in situ phosphoglucomutase activity by the formazan technique (N. C. Mishra, unpublished). On subsequent genetic analysis col-7 (S4357) was found to be allelic to rg-I (B53) ; also col-'7 was found to have a reduced level of phosphoglucomutase and a heatlabile form of this enzyme (N. C. Mishra, unpublished). I n most cases a combined approach was applied in the screening of a particular morphological mutant for possible biochemical lesion, and the primary biochemical lesions in a number of morphological mutants have been described and elucidated; these are summarized in Table 2. a. Genetics and Biochemistry of the Morphological Mutants. The primary enzyme defect in a number of morphological mutants is now known; defects are listed in Table 1, and their characteristics are presented in Tables 2 and 3. The particular enzyme defect of the different mutants listed in the Tables 1-3 has been described in terms of a number of parameters ; these included specific activity, thermal stability, electrophoretic and electrofocusing behaviors, and the substrate affinity of the enzyme in question. Each mutant enzyme was found to differ from that of the wild type by a t least three of these criteria (Brody and Tatum, 1966; Scott and Tatum, 1970; Mishra and Tatum, 1970a; Abramsky et al., 1971; Lechner et al., 1971). Similar data have not yet been available for the mutant enzymes listed in Table 3. The altered characteristics of these enzymes are listed in Table 3. The conclusion that the altered morphology is the phenotypic consequence of a single enzyme defect has been established on the basis of the following facts. (1) Both the enzyme defect and altered morphology were found to be transmitted together to the progeny of the cross involving the morphological mutant and a wild-type strain. (2) A single reversion in the mutant leads to complete and simultaneous restoration to the wild-type forms of both the enzyme and the morphology. Revertants of most of the mutants listed in Table 1 are known, and each has been found to have the wild-type form of the enzyme and the filamentous form of growth (Brody and Tatum, 1966; Murayama and Ishikawa, 1975). Additional evidence is provided by the study of the temperature-sensitive revertants of col-2, which has colonial morphology and altered glucose-6-phosphate dehydrogenase when grown a t high temperatures (Brody and Tatum, 1966). (3) A suppressor of any of these mutations again leads to restoration of both the wild-type form of the enzyme and of morphology; suppressors of balloon (Scott and Brody,

TABLE 2 Characteristics of Some of the Enzvmes Involved in Moruholoeical Chanees in Nrurosvora (see Table 1)

Enzyme 1. G6PD (EC 1.1.1.49)

Electrophoresis

Electrofocusing

No. of enzyme activities

3 Bands

6.32, E.44,6.54

3

3.1

3 Bands

6.30,6.33,6.41

3

2.1

2.9

3Bands 6.55,6.€2,6.72

3

1.3 0.66 1.o 2.2 1.1

2.9

3 Bands

6.35,6.46,6.57 6.33,6.42

3 2

6.33,6.47,6.57 6.22

3

6.23,6.33,6.48 6.54,6.70 4.23,4.43

3

Substratem

Cosubetrateb

Wild type

0.24

2.9

1.3

balloon

0.24

10.0

1.3

col-2

0.24

10.0

frost

0.24

10.0 3.1 3.1 10.0 5.0

SU-B su-c-col-2 Su-B-bal rol-2, bal Het, col-2 balloon Wild type

0.35

ragged-1

0.035

ragged-2 rg-2 su-2

+

3. 6PGD Wild type (EC 1.1 1.1.1.47) col-3 col-lo H d , COI-3 COG1 0 "-I

+

a

x

Strain

su-c

2. PGM (EC 2.7.5.1)

b( M

spe cific activity

~

,

105)

Heat stability (min) 10

Enzyme pattern on

2.0 6.0

0.18

150 (PGMI)

35

1.1

210 (PGMII) 10 (PGMI) 2 Bands

0.035

35

1.3

0.25 0.66

16 3

0.37 1

210 (PGMII) 150 (PGMI) 2 Bands 10 (PGMII)

0.66 ..._ 0.66

9 9

7.8

6.0

3

1

L.8

2.5

2 Bands

2

Brody and Tatum (1966) Scott and Tatum (1970) Scott and Brodv (1973)

Brody and Tatum (1967a) Mishra and Tatum (1970a)

2 Bands 4.93,5.49

2

4.84,5.42 4.93,5.40 4.93, 5.40

2

5 54 10.8

References

Scott and Abramsky (1973a,b)

2 2

GGPD, glucose-6-phosphate dehydrogenase; PGM, phosphoglucomutase; 6PGD, gphosphogluconate dehydrogenase.

* Cosubstrates used were NADP for 6GPD and glucose 1,6-diphosphate for PGM.

362

N. C. MISHRA

TABLE 3 Partial Characterization of Enzyme Defects in Some Morphological Mutants of Neurospora ~

Enzyme

Strain

Specific activity

References

Pina and Tatum (1967) 3.3 0.0 Murayama and Ishikawa Wild type 3.8 gpi-1 (197.3 0.0 gpi-8 0.3 Terenzi et al. (1974) 3. Adenyl cyclase (EC 4.6.1.1) Wild type 40.0 1.3 cr-1 Selitrennikoff et al. (1974) 4. Cell wall autolyzing cnzymes Wild type 27.9 5.5 csp-1 10.8 CSp-8 Mahadevan and Cell wall autolyzing enzymes Wild type 100.00 Mahadkar (197Ca) 600.00 speo-1 0.00168 Rand (1975) Wild type Galactokinase (EC 2.7.1.6) 0.00069 gal-1 Itand (1975) 1.2 UDP or pyrophosphorylase Wild type (EC 2.7.7.9) 0.54 gal4 Russell and Srb (1974) LGlutamine D-Fructose-6Wild type 0.58 phosphate amidotransferase peak4 1.01 clock (EC 2.6.1.16) 0.93 1. Glucocycloaldolase (NAD+dependent) (EC 5.5.1.4) 2. Glucose isomerasc (EC 5.3.1.9)

Wild type inos

1973) and of ragged4 (Mishra and Threlkeld, 1967; Mishra and Tatum, 1970a; Mishra, 1975) have been described to have the wild-type morphology and also wild-type characteristics of the respective enzymes, i.e., glucose 6-phosphate dehydrogenase (balloon) and phosphoglucomutase (ragged-2). (4) There is a distinct correlation between the severity of the enzyme defect and the degree of morphological abnormality in heterokaryons and double mutants. The data presented in Tables 2 and 3 clearly suggest that proper functioning of the enzymes listed therein is essential for the maintenance of the wild-type morphology. The multigenic control of all these enzymes further suggests that each of these enzymes should consist of more than one identical subunit. Thus, based on the number of unlinked genes that affect these enzymes, G6PD" should contain 4 nonidentical polypeptide chains, whereas both the PGM and 6PGD should have a t least two nonidentical polypeptide chains. The sodium dodecyl sulfate (SDS) poly-

* Abhreviations : G-1-P, glucose 1-phosphate ; G-6-P, glucose 6-phosphate ; G6PD, glucose-bphosphate dehydrogenase ; GPCD, 6-phosphogluconate dehydrogenase ; PGM, phosphoglucomutase ; UDPG, uridine diphosphate glucose ; UDPGNAC, uridine diphosphate-N-acetylglucosamine; SEM, scanning electron microscopy.

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

363

acrylamide gel electrophoresis of purified preparations of the enzymes G6PD and 6PGD (Scott and Tatum, 1971; Scott, 1971; Scott and Abramsky, 1973a,b) provides evidence regarding the multimeric nature of these proteins having subunits of 57,000 daltons. Neurospora phosphoglucomutase, glucose-6-posphate dehydrogenase, and 6 phosphogluconate dehydrogenase are of interest because of their influence on morphology. The characteristics of the mutant and wild-type enzymes have been described (Brody and Tatum, 1966, 1967a; Mishra and Tatum, 1970a; Scott and Tatum, 1970, 1971; Scott, 1971; Scott and Abramsky, 1973a,b) ; these have been summarized in Table 2 and are discussed below. i. Phosphoglucornutase ( P G M ) mutants of Neurospora. The wild-type strain of Neurospora seems to have two isoeymic forms of this enzyme (PGMI and PGMII) , which are detectable by electrophoresis, electrofocusing, and DEAE-cellulose chromatography. The two isozymes detected on electrophoresis are designated PGMI and P G M I I ; PGMI is the anodically migrating fast band whereas PGMII is the slow band of activity; on gel filtration each has a molecular weight of 64,000. In the mutant, although the specific activity of the enzyme is very much reduced, physically only one form of the PGM isozymes is altered in a particular ragged mutant; in ragged-1 PGMI is altered, and in ragged-2 PGMII is altered (see Table 2 ) . It seems that PGMI and P G M I I may occur as a complex under certain physiological conditions; in vitro such a complex may be formed by increasing the Mg2+ ion concentration (Mishra and Tatum, 1970a). These authors have also reconstituted such n complex betwccn mutant PGhlI (obtained from ragged-1 strain) and wild-type PGMII (obtained from the wild-type strain). Such a complex between mutant PGMI wild-type PGMII has the properties of mutant enzyme (Mishra and Tatum, 1970a), and this supports the idea of negative complementation. The data presented in Table 2 clearly show that the genes rg-1 and rg-2 control the structure of the two isoeymes P G M I and PGMII in Neurospora. Phosphoglucomutase is an enzyme at the metabolic branch point, and its role in causing altered morphology in the mutant strains seems straightforward as discussed later in this paper. ii. Glucose-6-phosphate dehydrogenase mutants of Neurospora. The wild-type glucose-6-phosphate dehydrogenase has been purified to homogeneity and was found to contain three bands of enzyme activity on polyacrylamide gel electrophoresis and also on electrofocusing. The Neurospora enzyme has a molecular weight of 206,000 with 4 subunits having a molecular weight of 57,000 as determined by SDS-gel electrophoresis. Both dimeric and tetrameric forms of the enzyme have catalytic activity (Scott, 1971). The existence of the molecular forms (i.e., dimeric

+

364

N. C. MISHRA

and tetrameric) is supported by electron microscopic studies by Scott (1971). The proportion of the two molecular forms can be varied by NADP concentration. Increasing concentration of this cofactor was found to convert a mixture of dimers and tetramers to tetramers with corresponding increase in specific activity. This effect of NADP on the nature of the native enzyme has been implicated in the in vivo regulation of its activity and the pleiotropic effects of mutant enzymes (Scott and Tatum, 1970, 1971). The mutant G6PD was found to have increased thermolability, altered kinetic properties, and different electrofocusing patterns than the wild-type enzyme (Scott and Tatum, 1970). Although the wild-type enzyme showed three electrophoretic bands of activity on polyacrylamide gel, there is no evidence as to whether or not one specified band is qualitatively or quantitatively altered in a particular morphological mutant (col-2, or balloon, or frost). The fact that in the mutants all three peaks of enzyme activity obtained on isoelectric focusing are changed in either of these mutants suggests a multigenic control of this enzyme (Scott and Tatum, 1970). Electron-microscopic studies of the wild-type G6PD do show that the enzyme is composed of four subunits; it is suggested that these subunits are nonidentical and a t least three of them are specified by the genes col-2, balloon, and frost (Scott and Tatum, 1971). Scott and Brody have also implied that a fourth nonlinked gene, su-C, is the structural gene for the fourth subunit of the enzyme. The nature of the su-C mutation has been further discussed in this paper. iii. 6-Phosphogluconate dehydrogenase mutants of Neurospora. The wild-type Neurospora seems to have two 6PGD enzymes, as shown by Scott and Abramsky (1973a). These authors found two coincident bands of protein and enzymic activity on electrofocusing carried on polyacrylamide gels. However on polyacrylamide gel electrophoresis only one band of activity was seen. The elution profiles on gel filtration reveded a single coincident peak of activity and protein suggesting a molecular weight of 110,000-120,000 daltons. SDS-polyacrylamide gel electrophoresis revealed the enzyme to be made up of two subunits each having a molecular weight of 57,000. The purified col-3 and col-I0 enzymes were found to differ from the wild type in terms of their kinetic parameters, heat stability, and isoelectric points ; however, there was no difference in the molecular weights of the mutant and wild-type enzymes or in their subunits (Scott and Abramsky, 1973b). It further seems that the two enzyme activities obtained on electrofocusing of the mutant strains are heteropolymers specified by the col-3 and col-10 enzymes (Scott and Abramsky, 197313). The second peak of enzyme activity may be an artifact, since it does not show any complementarity in the heterokaryon and its proportion to total enzyme varies widely on repetitive separation

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

365

of the different samples of the same preparations. Thus, according to these authors the Neurospora 6-phosphogluconate dehydrogenase consists of a single peak of activity as suggested by gel filtration and electrophoresis and is a dimer of two nonidentical subunits. The nature of the second peak of enzyme activity remains to be elucidated. iv. Other mutants. Other morphological mutants in which the geneenzyme relationship has been elucidated are presented in Tables 1 and 3. However, in these cases it remains to be established whether or not the gene affecting the enzyme is a structural gene for thc defective enzyme. Studies by Pina and Tatum (1967) show lack of glucosephosphate aldocyclase in inos- strains of Neurospora. These authors have also shown that alternative pathways for inositol formation from epi-inosose are not usually utilized in Neurospora (Pina and Tatum, 1967). Crocken and Nyc (1964) have shown that chol-1 and chol-2 mutants of Neurospora are blocked for the first and second (or third) methylation of phosphatidyl ethanolamine leading to formation of lecithin (see Fig. 5 ) . I n wild-type hreurospora, the stepwise methylation of phosphatidyl ethanolamine is the major pathway for the de novo synthesis of lecithin. However, the mold uses an alternative route (via the cytidine nucleotide system see Fig. 5B) for the reutilization of phospholipid bases released by the tissue or when a genetic block prevents the methylation of phosphatidyl ethanolamine. Thus in the choline-requiring mutants (which accumulate ethanolamines or can utilize choline or other methylated ethanolamines) growth is dependent on the utilization of the pathway via cytidine nucleotide (see Fig. 5B).The defective enzyme in chol-I and chot-2 mutants has not yet been characterized. In crisp mutants ( c r - I ) it is not obvious from the work of Terenzi et al. (1974) that the mutation causes a change in the structure of adenyl cyclase. The defect in adenyl cyclase could be explained via a defective membrane produced by low NADPH level [as discussed in the case of frost adenyl cyclase by Scott and Soloman (1975) 1. There is some indication that crisp mutants are defective for isocitrate dehydrogenase (N. C. Mishra, unpublished). If this is true, it can explain the low specific activity of adenyl cyclase in the cr-1 mutant reported by Terenzi et al. (1974) via an impaired membrane and also the change in the amino acid composition of cell wall peptide seen in the crisp mutants (R. Wrathal, unpublished) of Nezwospora since the TCA cycle is the main route for the production of amino acids by transamination. However, in the cr-1 mutant the effect of low adenyl cyclase seems to be direct, since the addition of CAMP to the growth medium seems to reverse the morphological aberrations of the genetic mutation (Terenzi et al., 1974). The colonial morphology of Neurospora is associated with a higher

366

N. C. MISHRA

frequency of branches per unit length of mycelium (deTerra and Tatum, 1963). An increase in the frequency of branching can be induced by treatment of Neurospora cultures with snail digestive enzymes (deTerra and Tatum, 1961) or with amylase (E. L. Tatum, unpublished results, see Scott et al., 1974). Similar effects could be produced by a gene mutation that can increase the production of enzymes involved in the composition CHIOR

I

ROCH

I

0

1' I

CHIOP-OOCHICH~NH~

OH Phorphatidyl ethanolamine

ic

CHiOR

S-Adenoryl methionine S-Adenoryl homocyrteine

I I

nocn 0 CH~OPOCH~CH~YHCH~

I

OH

L

Phosphatidyl monomethylethanolamine S-Adenosylmethioriine

CHiOR

I

R'OCH

I

S-Adenoryl hornocyrteine

0 I!

CHIOPOCH~CHIN(CHI)~

I

OH Phorphatidyl dimethylethanolamine

L

S-Adenoryl methionine

S-Adenoryl homocyrteine

CHlOR

I I

R'OCH

0

II

LHi0;

CHIOPOCH~CH$J(CHI)I

I

OH Phorphrridyl choline (Leclthin) Phorphiiidyl choline phosphscidohydrolare Phorpholipase

3.1.4.4

CH~OR

R'OCH

Phorphatidate

CHiO P

(A)

HOCH~CH~N(CH~)~ Choline

FIG.5. Metabolic pathways involved in biosynthesis (A) and utilization (B) of choline. (After Dagley and Nicholson, 1970.)

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

367

HOCH~CH&(CH~), Chollne ATP: cholina phoaphocnnakrua Cholina kin-

1.7.1.32

1

ADP

@OCHICH~&(CHI)I Phorphocholine CTP: cholinephosphatacytidylylrranafanu choline phoaph8co cyeidylylcnn~~arua

1.7.7. I 5

1

np ppi t

CPP-OCH~CH~N(CH~)I Cytidine diphorphochollne (CDP-chollne)

I

CDP cholina: I.>di#lycarida choline phonphotnnrferasa

CHiOR

R’O$H

1.2-Diglyceridc

CHiOH

1.7.8.1

CMP

FHiOR R’OfH

7

+

CH@--P--OCH~CH~N(CH,)3

I

OH Phoophrrldylcholine (Lecithin)

FIG.5 (continued)

of the cell wall. Mahadevan and Mahadkar (1970a) have described that, in a particular morphological mutant called spco-I, the earlier and higher frequency of branching was correlated with a greater amount of these autolyzing enzymes as compared to wild-type strains (see Table 3). These authors have described both glucanase and protease activity associated with the cell wall of hleurospora, which can be extracted with p-mercaptoethanol. The activity of these enzymes in the mutant was almost 6 times higher than that found in the wild-type strain (Mahadevan and Mahadkar, 1970a). Of these two enzymes the activity of proteases was found to be much higher than that of the glucanase. The fact that the increased activity of proteases in spco-I lead to morphological changes suggests the role of cell wall peptide in morphogenesis (Wrathal and Tatum, 1974). Abramsky and Tatum have recently described changes in the morphology of a particular mutant strain (smco-9) due t o the inactivation of an enzyme commonly called “branching enzyme” (a-lj4-glucan: a-1,4glucan-6-glycosyltransferase, EC 2.4.1.18), which catalyzes the synthesis of a-1,4 linkages during the biosynthesis of polysaccharides. The authors have characterized a thermolabile inhibitor (MW = 100,000) from the

368

N.

C. MISHRA

mutant strain which caused a drastic reduction in the activity of the branching enzyme. In the mutant cell extract the activity of the branching enzyme could not be detected unless the inhibitor was removed by DEAE chromatography. The inhibitor, which was present in the mutant strain only, was found to affect the activity of other enzymes of polysaccharide biosynthesis. The branching enzyme characterized from the mutant and wild-type strains had a molecular weight of 140,000 and was similar in other physicochemical properties. These authors have further found that the inability of the mutant to synthesize a-1,6 linkages due to reduced activity as branching enzyme could be overcome, however, by adding sugars (having such ~ 1 , link6 ages) to the growth medium as shown by the wild-type-like growth of the mutant strain on media containing isomaltose or potato starch as the sole carbon source (see Table 4) (Abramsky and Tatum, 1976). I n the morphological mutant called snowflake (sn),the biochemical defect seems to involve cellular accumulation of microfilaments 78 A in diameter and up to several micrometers in length (Allen et al., 1974). The microfilament in this mutant strain (sn) seems to run in all directions TABLE 4 Differences in the Growth Characteristics of Different Neurospora Strains on Different Sugars as Sole Carbon Source Morphology and growth pattern on media with ~~

Strains

Glucose

1. Wild type 2. gal-1

Wild typeWild type

3. gab8

Wild type

4. smco-8

Wild type

5. smco-9

Colonial

6. Osmotic mutants (R2361, R2473)

Wild type

Galactose

~~

Iomaltose

References

Colonial __ Rand (1975) Colonial or no Rand (1975) growth Rand (1975) Colonial or no growth Rand (1975) Colonial or no growth Wild type Abramsky and Tatum (1976) Wild type or Rand (1975) even better growth

The spreading mycelial growth characteristic of the wild-type strain (RL3-8A) ati seen on glucose medium. smco-9 was found to have colonial morphology on glucose, sucrose, and maltose but wild-type-like morphology on potato starch and isomaltose (Abramsky and Tatum, 1976).

BIOCHEMICAL

GENETICS OF

Neurospora

MORPHOGENESIS

369

(see Fig. 2B) and sometimes up to the plasma membrane and even inside the nuclei. According to Sussman and his collaborators, the altered morphology of snowflake is related to accumulation of these microfilaments which may affect several physiological processes. Microfilaments are known to control cytoplasmic streaming (O’Brien and Thimann, 1966), and also morphogenesis of animal cells (Wessells et al., 1971). Wood and Luck ( 1971) have reported the occurrence of “paracrystalline inclusions’’ in the mitochondrial mutant (mi-1) of Neurospora or induction of similar microfilaments in the wild-type strain when ethidium bromide or euflavin was added to the growth medium. According to Wood and Luck (1971), these “paracrystalline inclusions” are not products of a mitochondrial gene, but their cellular crystallization is due to a mitochondrial defect. Another interesting mutant is strain 4520% isolated by Houlahan et al. (1949) and described by Dubes (1953) as a temperature-sensitive mutant that required leucine for growth a t low and intermediate temperatures; however, a t high temperature (37°C) its growth was severely restricted regardless of leucine supplementation ; it was designated leu-5. The biochemical lesion of this mutant has been elucidated by Gross and his collaborators a t Duke University (Printz and Gross, 1967; Weeks and Gross, 1971). According to these authors, a number of proteins synthesized during the growth of this mutant (4520%) were structurally altered and enzymically defective (some of the enzymes examined were trytophan synthetase, alcohol dehydrogenase, NADase, and 5-dihydroshikimic reductase, all of which were rendered more thermolabile in the mutant). On the basis of these results authors concluded that the phenotype of strain 4520% resulted from a decrease in the fidelity with which leucine is incorporated into protein due to a defective leucyl-tRNA synthetase with altered binding properties. This conclusion was supported by their finding that the mutant strain 4520% lacked the mitochondrial species of this enzyme whereas the cytoplasmic enzyme was found to have reduced affinity for leucyl tRNA (Weeks and Gross, 1971). Rand (1975) has attempted to identify the primary biochemical lesions in a number of mutants based on their growth characteristics on galactose or glucose as a carbon source. As discussed earlier he has shown that the wild-type strain of Neurospora grew as tightly restricted pellets in galactose medium as opposed to its filamentous growth in glucose medium. He has tested a large number of morphological mutants described earlier by Garnjobst and Tatum for their ability to grow on galactose. Among the mutants tested only semicolonial-8 (snico-8) was found to be unable to grow on galactose (or grow as restricted colonies) whereas all the osmotic mutant strains tested were found to grow better on galactose than on glucose. Rand has also described two new mutations, gal-1 and gal-2, that were

3 70

N. C. MISHRA

unable to grow on galactose (or grew as restricted pellets). Although he could not identify the primary biochemical defect of the semicolonial-8 or the osmotic mutations, he found that gal-1 and gal-2 were defective for the enzymes galactokinase and UDPG pyrophosphorylase, the enzymes of the Leloir pathways (Leloir, 1951). I n the gal-1 mutant galactokinase specific activity and its heat stability were much reduced although there was no change in K , values of the mutant enzyme as compared to the wild type (Rand, 1975). The gab2 enzyme (UDPG pyrophosphorylase) has reduced specific activity, altered Iilr,sand lowered heat stability (see Table 3 ) . On the basis of these data, the author has suggested that gal-1 and gal-2 may represent mutation in the structural genes for the enzymes galactokinase and UDPG pyrophosphorylase, respectively. Furthermore, the g a l 4 mutant seems to respond to arginine supplementation, Thus the behavior of gal-2 seems to be similar to that of the mutant described by Houlahan et al. (1949), which has been elucidated by Gross and his collaborators (Printz and Gross, 1967) ; Weeks and Gross, 1971). Therefore, the arginine response of gal-2 can be explained on a similar basis: i.e., gal-2 mutation may cause a change in the fidelity of the translation mechanism. In case of the mutants listed in Table 4, although the specific activity of the enzymes involved is lowered owing to genetic mutations, it is not yet known whether or not this is due to changes in the structure of the enzymes involved. I n all these cases of enzyme defects the unequivocal proof (see Tables 1-3) for a change in the structure of an enzyme could be established only through amino acid sequence analysis of the polypeptide in question. However, the changes in the physical parameters (heat stability, electrophoretic, and K,,, studies) and the effect of reversion and suppressor mutation do indicate that the enzyme defect a t least in PGM, GGPD, and 6PGD mutant is due to a structural change. However, no such data are available for a large number of morphological mutants (see Table 4). b. Genetics and Biochemistry of the Phenocopy of Genetic Mutants. Chemical induction of morphological changes provides another interesting and useful approach to the understanding of colonial morphology in Neurospora. Tatum, Barratt, and Cutter were the first to induce colonial growth in Neurospora by the addition of L-sorbose to the medium (Tatum et al., 1949). Their discovery was very useful, as it led to the development of a methodology for high-resolution genetics of Neurospora besides its role in the understanding of morphogenesis in Neurospora. Soon it was demonstrated by Stadler (1959) that this sorbose effect was genetically controlled ; Stadler described a particular mutation called patch, which can grow as spreading filaments even in the presence of sorbose. The sorbose-grown culture of the wild-type strain (with colonial morphology)

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

371

was found to have altered cell-wall composition (deTerra and Tatum, 1963; Mahadevan and Tatum, 1965). Mahadevan and Tatum showed that in the sorbose-grown culture, there was a decrease in the level of fraction I11 with a concomitant increase in the level of fraction I. Such an increase in the amount of fraction I in a sorbose-grown culture of the wild-type Neurospora has been visualized by scanning electron microscopy (Mishra, 1975). SEM studies by Mishra have shown the presence of several facetlike structures on the surface of the sorbose-grown culture whereas the normal culture showed a smooth surface. The chemical analysis of the cell wall of the patch mutant by Mahadevan and Tatum revealed no significant changes in its cell wall composition when this mutant (patch) was grown in the presence or in the absence of L-sorbose. Likewise, the surface architecture of the mutant strain (patch), when examined by SEM, was found to remain unchanged in the presence of sorbose (Mishra, 1975). Thus, the fact that sorbose cac cause a dramatic effect in the morphology, cell-wall con~position,and the surface architecture of the wild type but not of the mutant (patch) strain strengthens, the conclusion that the cell-wall composition is the main determinant of the morphology of Neurospora. Mechanism of sorbose effects. Crocken and Tatum (1968) have described that sorbose is taken up by the same transport system as that utilized by glucose. These authors have further provided evidence for the counterflow of sorbose in the presence of high concentrations of glucose in the growth medium. The fact that sorbose and glucose can be transported by the same transport system might explain the role of increasing concentration of glucose on the reversal of the sorbose effect. Scarborough has confirmed the presence of a common transport system for both glucose and sorbose in Neurospora, using spheroplasts or mutant ( s l ) in Neurospora (Scarborough and Schultz, 1974) lacking a cell wall. Later Mishra and Tatum (1972) showed that sorbose causes morphological changes in the wild-type strain by inhibiting the activity of polysaccharide synthetase in Neurospora (1972). Both in vivo and in vitro activities of the wildtype p-1,3-glucan and glycogen synthetases were found to be inhibited by sorbose whereas the activity of these enzymes from the mutant strains (patch) remains unaffected. The idea that a chemical which can interfere with the activity of the enzymes that synthesize the cell wall polymers was further investigated by examining the role of CAMP on morphogenesis. The rationale behind this approach was that excess of CAMP, which is known to regulate the level of carbohydrate synthetase, would lead to morphological changes. Mishra (1976) has observed such morphological changes in Neurosporu when CAMP was added to the growth medium (see Fig. 6). Such a para-

372

N. C. MISHRA

FIQ.6. The effect of cAMP on the morphology of the wild-type strain (RL 3-8A) of Neurospora crassa. The wild-type strain was grown on solid medium containing glucose (0.1%) without or with cAMP a t 25°C in 5-cm petri plates. (A) The wildtype strain showing normal morphology on medium without CAMP. (B) The wildtype strain showing a colonial morphology (with restricted growth) on a medium with cAMP (6 mg/ml).

morphogenetic effect of cAMP can also be elicited by its dibutyryl derivative, but not by 3’-AMP, B’-AMP, or 2’-3’-cyclic AMP (Mishra, 1976). Similarly, theophylline (an inhibitor of phosphodiesterases) should cause morphological changes by increasing the in vivo level of CAMP; such an effect of theophylline has also been observed (N. C. Mishra, unpublished; Scott and Soloman, 1975). The insensitiveness of patch to sorbose can be explained by either of the assumptions that ( 1 ) the uptake of sorbose in patch is decreased as a result of the mutation or (2) the enzymes of cell wall biosynthesis have been so changed as to become insensitive to sorbose. I n sorbose uptake using l*C-labeled sorbose, no significant difference was seen between the wild-type and the mutant strains (Mishra and Tatum, 1972). I n view of this fact, the latter hypothesis seems more plausible (i.e., the nature of the cell wall synthesizing enzymes has changed). Although, the sorbose uptake remains unimpaired in the patch mutant. Klingmuller has described a number of mutations that cause sorbose-resistance in mutant strains of Neurospora which appear to be impaired in the sorbose transport system and have decreased uptake of sorbose (Klingmuller, 1971). In these mutants a lipid component of the cell wall-membrane complex seems to be impaired. Recent work by Scarborough (1971) has led to a similar conclusion

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

373

regarding the involvement of a membrane component in the uptake of sorbose in Neurospora. Scarborough found that, in the inositol-requiring mutants of Neurospora, sorbose uptake was inhibited under limiting concentration of inositol in the growth medium (Scarborough, 1971). Recently, Murayama and Ishikawa (1973) have described a sorboseresistant mutation (T9) in Neurospora crassa with pleiotropic effects ; the pleiotropy of this mutation includes sensitivity to high osmolarity of the growth medium, altered elution profile of the enzyme glucoamylase on gel filtration, and a high activity of extracellular acid phosphatase. These authors have explained the pleiotropy of this sorbose-resistant mutation on the assumption that the mutation (T9)causes a primary alteration of the cell wall or membrane and that secondarily this leads to other effects. The fact that these authors found no difference in the cell wall composition of the wild type and the mutant (T9) excludes the idea of a change in the cell wall (Murayama and Ishikawa, 1973). Recently, Rand (1975) has initiated an exciting new aspect of Neurospora morphology by describing the induction of morphological changes in the wild-type strain by galactose. The wild-type strain was found to grow as tightly restricted colonies (Rand, 1975) when grown in a medium containing galactose as the sole carbon source. It has also been shown that the galactose effect was maximal in the presence of xylose, but glucose reversed the effect of galactose. The biochemistry of the galactose effect can be understood in terms of the inhibitory effect on certain enzymes of cell wall biosynthesis ; however, this remains to be elucidated. Mahadevan and Tatum (1965) have indicated the presence of galactosamine in fraction I of the Neurospora cell wall and a galactosamine polymer has been isolated from Neurospora cell wall (Harold, 1962). Recently, Mishra and Forsthoefel (1976) have described an enzyme in Neurospora that can synthesize a polymer of galactose. Reissig and his collaborators (Reissig and Glasgow, 1971 ; Glasgow and Reissig, 1974) have approached the problem of Neurospora morphogenesis in a novel way by examining the effect of a purified cell wall component on the growth of this mold. These workers have isolated a phasespecific mucopolysaccharide called MP from the wild-type culture of Neurospora. The M P is characteristically present in the growth medium of the wild-type culture at stationary phase. This mucopolysaccharide was found to inhibit growth and agglutination of Neurospora cells and was also to precipitate purified membrane protein. I n certain morphological mutants a phase of slow growth was markcd by the production of this mucopolysaccharide (Reissig and Glasgow, 1971). The M P is a polymer of galactosamine (galactosaminoglycan) and has a molecular wcight of approximately 1 x lo6; its biological activity can be abolished

374

N. C. MISHRA

by its complete acetylation. Polyphosphate was also found to bind MP and found to cause its inactivation in vitro. Harold has earlier described a polymer of galactosamine from Neurospora which binds with phosphates (Harold, 1962). On the basis of these characteristics of MP, Reissig and Glasgow have suggested its role in the regulation of growth and morphogenesis in Neurospora. Additional evidence is provided by the results of an experiment in which these workers have utilized the antagonistic effect of polyphosphate on M P produced by mutant cultures. At lower concentrations (50-400 pg/ml) polyphosphate was found to stimulate the growth of the mutant culture ( c o t ) . After 8 hours of growth the mutant (cot) which produced M P showed signs of agglutination. However, when the growth medium was supplemented with polyphosphate (100 pg/ml) the agglutination was prevented. These authors have also shown that M P can cause inactivation of conidia as evidenced by the inability of the treated conidia to germinate and to take up radioactive metabolites from the growth medium. Studies with radioactive M P showed that a t least lo” molecules of M P bind per cell to cause a lethal effect (Glasgow and Reissig, 1974). Thus the results of their studies clearly suggest a physiological role for M P and polyphosphate in the growth of Neurospora. The role of galactosamine polymer in determining the morphology of Neurospora is further revealed by the fact that certain morphological mutants have deficient levels of UDP-N-acetyl glucosamine-4-epimerase (Edson, 1972) ; this enzyme is necessary for the synthesis of galactosamine, a component of the hyphal wall (Harold, 1962; Mahadevan and Tatum, 1965). However, Schmidt and Brody (1975) have suggested that the galactosamine polymer cannot be a major shape determinant of Neurospora hyphae. This conclusion is based on their finding that the level of galactosamine was always much lower in the growing front than in the older region of the band ( b d ) mutant of Neurospora. This mutant strain ( b d ) possesses a circadian conidiation and shows zonation in its growth as band (conidiating) and interband (nonconidiating) regions (Sargent and Woodward, 1969), which has been found suitable for elucidation of morphogenesis and circadian rhythm as discussed later in this paper. c. Genetics and Biochemistry of the Suppressor of the Morphological Mutants. The study of suppressor mutations has been extremely useful in understanding the genetics and biochemistry of gene structure and function (Yanofsky, 1966; Gorini, 1970). Therefore, the study of such mutations has received much attention in the elucidation of the biochemistry of morphogenesis in Neurospora. The suppressor of morphological mutants was first described by Srb (1957). Srb found that a particular morphological mutant mel-3 usually reverts to the wild-type growth form,

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

375

and this change was found to result from a mutation in a gene unlinked to mel-3. Later Giles (1951) described several suppressors of the inositolrequiring (inos) mutants of Neurospora; inos strains were shown to have colonial morphology when grown on medium containing limiting amounts of inositol. Mishra and Threlkeld (1967) have described an allele specific suppressor of the ragged mutation in Yeurospora. The suppressor mutation su-2 could act only on rg-2, not on the rg-1 mutation. The double mutant rg-2-su-2 has a wild-type-like phosphoglucomutase in Neurospora (Mishra and Tatum, 1970a). Brody has isolated two suppressor mutations, su-B and su-C, which are allele-specific to the morphological mutants balloon and col-2, respectively (Scott and Brody, 1973). Both suppressors are unlinked to the mutations that they suppress. Since the primary defect in the morphological mutants balloon and col-2 was elucidated by Tatum's group a t Rockefeller University, it provided a unique opportunity to investigate the biochemistry of these suppressor mutations. Such biochemical studies were carried out by Scott and Brody. The double mutants su-B, bal and su-C, col-2 have a more spreading morphology and were found to have altered kinetic parameters of the enzyme involved (i.e., G6PD) (see Table 2.). The su-C mutation was found, however, to alter the nature of the enzyme G6PD even when present alone in the wild-type background (see Table 2 ) , although the su-B mutation by itself did not show any effect on the morphology. Both su-C and su-B were found to be dominant over their respective alleles su-C' and su-B' in the balanced heterokaryon. These authors have invoked several possible assumptions in order to explain the unusual behavior of the su-C gene in altering the properties of G6PD in the wildtype background. Some of these explanations are: (1) su-C is a structural gene for one of the subunits to GGPD enzyme. The mutation by itself does not impair the function of the enzyme to an extent that can lead to morphological change; however, this mutation su-C, when in combination with col-2 perhaps can overcome the impairment caused by the col-$2 mutation and thus cause the suppressor effect; (2) the su-C mutation may cause mistranslation of a codon other than the one altered by col-2; (3) su-C may code for an enzyme that can cause some posttranslational modification (DeLange et al., 1968; Kingdon et al., 1967) of G6PD; (4) su-C may be a regulatory gene that can control the appearance of a GGPD subunit. Future work is required to support one or another of these possibilities regarding the su-C function. Modifiers of crisp. Lindegren (1936) first described the crisp (cr)mutation in Neurospora; since then Garnjobst and Tatum (1967) have obtained 14 c ~ i s pstrains of independent origin. On the basis of linkage and complementation analyses, these authors found that crisp mutations were

376

N. C. MISHRA

in either of three closely clustered loci designated cr-1, cr-2, and 0-3 (their gene order being centromere-cr-1-cr-3-cr-2) (Garnjobst and Tatum, 1967). Garnjobst and Tatum have suggested that these three loci may be involved in a sequence of reactions biochemically related to the crisp phenotype; there is some indication that crisp morphology may be associated with defective functioning of the enzymes of the Krebs cycle. Garnjobst and Tatum have described genetic modification of the crisp phenotype controlled by five unlinked genes ; these mutant genes either merely modify the crisp phenotype (m-1, m-2, and m-3 genes) or completely suppress the crisp phenotype (m-4 and m-5 genes). The m-1 mutation shows no locus specificity and acts on all CT-1,cr-2, and m-3 alleles. It is not known whether other modifiers, especially m-4 and m-5, show some nonspecificity. Further work is required to elucidate the biochemical basis of the crisp mutants and their modifiers; it would be of interest to see the effect of these modifiers on the enzyme adenyl cyclase, since its level is reported to be drastically reduced in the cl-isp mutant (Terenzi et al., 1974). 2. Altered Enzyme to Altered Morphology and Pleiotropy

Data presented in the Tables 1-3 clearly establish the enzymic defect of a particular morphological mutant whose normal functioning is required for the maintenance of the wild-type morphology. The pleiotropic effects of altered morphology in the morphological mutants seem to arise from the primary enzyme defect due to a gene mutation. At first glance it does not seem obvious how an altered enzyme leads to an altered morphology. However, after a closer look, a meaningful relation between changes in enzyme and in morphology can be established. It seems that in all these cases the morphology is altered via a defect in cell wall or cell-membrane biosynthesis. a. MorphoEogical Changes via Direct Effects on Cell-Wall and CellMembrane Biosynthesis. This is obvious in the case of the PGM mutants, since PGM catalyzes an enzymatic reaction a t the branch point in carbohydrate biosynthesis as shown below: C-1-P -+ UDPG -+ glucan I

4

C-6-P

1

Glycolysis

Thus it can be conceived that a defective PGM would lead to a reduction in the level of cell-wall polymers. The ragged mutants have indeed been shown to have a reduced level of fraction I11 (p-1,3-glucan) (Mahadevan and Tatum, 1965; N. C. Mishra, unpublished)-a major component of

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

377

the Neurospora cell wall. Much insight has been obtained about the regulation of PGM in Neurospora (Mishra and Tatum, 1970a) by the studies of the ragged mutants. The study of the behavior of PGMI and PGMII a t different ionic concentrations of Mg2+showthat they exist as two distinct molecular species a t low Mg2+concentration whereas a t high Mg2+ concentration they interact to form a single complex molecular species with 2-fold increase in molecular weight and a change in the structural conformation of the enzyme. The result of gel filtration does provide evidence for such interaction in PGMI and PGMII (Mishra and Tatum, 1970a). Mishra and Tatum have also shown that in vitro mutant PGMI interacts with wild-type PGMII a t high Mg2+concentration, and the resultant enzyme complex has the properties of the mutant enzymes as determined by their increased thermolability and K m values for substrate (Mishra and Tatum, 1970a). No interaction between two isozymes as seen in Neurospora PGMs had been reported previously. Such an interaction between PGMI and P G M I I under physiological conditions would provide a regulatory mechanism for control of the production of the two isozymes in vivo. A common regulation of the two isozymes is evidenced by the fact that in the ragged mutants (although each is defective for only one form of P G M ) , the level of the wild-type-like isozyme is also significantly reduced. The fact that both PGMs are required for the expression of the wild-type morphology supports the idea that a complex PGM molecule is the physiologically active form of the enzyme. The nature of the agent promoting such a complex formation in vivo remains to be determined. The role of Mg2+ in vivo is uncertain since a high concentration is required for the in vitro interaction (Mishra and Tatum, 1970a). However, cations like MgZ+, spermine, spermidine, and Tris-HC1 are known to cause aggregation of yeast UDP-galactose-4-epimerase (Darrow and Rodstrom, 1966). The polyamine cations may be involved in vivo in such interactions of PGM (s) ; spermidine has been found to occur in Neurospora in abundance (Bachrach and Cohen, 1961) and has been found to stimulate the in vitro activity of PGM (N. C . Mishra, unpublished). The decrease in the cell wall polymer (p-1,3-glucan) in rugged mutants seems to be a direct consequence of the defective PGM. However, modification of glucan biosynthesis may also arise in part from the allosteric effects of the different phosphorylated sugars (such as G-6-P) which accumulate in significant excess in the mutants owing to altered P G M (Brody and Tatum, 1967b; N. C. Mishra, unpublished) as reported by Fiengold et al. (1958). Interaction of metabolic pathways has long been known to produce multiple effects (Chaikoff, 1953). Such morphological changes via a direct effect on biosynthesis of cell wall polymers is further

378

N. C. MISHRA

evidenced by the study of sorbose; sorbose, which leads to the production of a phenocopy of the morphological mutation in the wild-type strain of Neurospora, also causes changes in the levels of cell wall polymers by inhibition of polysaccharide synthetases. The pleiotropy of the sorbose effect is not seen, however, in the mutant strain patch; none of the enzymes of cell wall biosynthesis in this mutant (patch) are affected by sorbose (Mishra and Tatum, 1972). Changes in cell membrane. Work with the choline- and inositol-requiring mutants of Neurospora has indicated the role of lipid metabolism and membranes in the morphogenesis of Neurospora (Crocken and Nyc, 1964; Pina and Tatum, 1967). The choline- and inositol-requiring mutants behave as conditional morphologicals, and the morphological alterations are caused only under suboptimal conditions of choline and inositol in the growth medium. Both choline and inositol are integral parts of the cell membrane, and a defective membrane formed under suboptimal conditions of these chemicals may disrupt the function of the membranebound enzymes in cell-wall biosynthesis. In both cases the inability of these mutant strains to synthesize a particular lipid has been related to particular enzyme deficiencies (see Table 1) (Crocken and Nyc, 1964; Pina and Tatum, 1967). The nystatin-resistant mutants of Neurospora with altered membrane composition (Grindle, 1974) may cause morphological changes in a similar manner by the disruption of the function of membrane-bound enzymes in cell-wall biosynthesis. However, the Neurospora mutants that require fatty acids for growth were found to have normal morphology although growth was much slower (Lein and Lein, 1949; Henny and Keith, 1971). b. Morphological Changes via Indirect Effect on Cell-Wall and CellMembrane Biosynthesis. The effect of a defective G6PD on the changes in the morphology of the col-2, balloon, and frost mutants is not as direct as that of PGM in the ragged mutants. It has been suggested that a defective G6PD would cause a reduction in the amounts of NADPH that are essential for the synthesis of the components of cell membrane. Therefore, it can be visualized that a defect in cell membrane due to reduced NADPH level in these mutants (col-2, balloon, and frost) might lead to improper functioning of the enzymes of cell wall biosynthesis since some of these enzymes are membrane bound. Much evidence for the above conceptual scheme of the pleiotropic effects of mutations in the pentose pathways has been provided by the work of Brody (1970, 1972). Brody has shown that in the G6PD and 6PGD mutants of Neurospora the levels of NADP and NADPH are reduced. Since NADPH is used primarily for synthesis of fatty acids and their desaturation, a deficiency in the cellular amount of NADPH would cause considerable reduction in the fatty acid biosyn-

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

379

thesis; Brody and Nyc (1970) have found changes in the composition of unsaturated fatty acids in balloon, col-2, and col-3 mutants of Neurospora. However, supplementation with precursors of NADP (such as tryptophan and nicotinic acid) has failed to relieve the morphological abnormalities of col-2 mutants or to increase the level of pyrimidine nucleotide in either wild-type or the col-2 mutant (Brody, 1972). However, this may be explained on the basis of different nucleotide pools and their compartmentalization. The other possible manner in which the cell wall biosynthesis may be impaired in these mutants is by modification of the enzymes by phosphorylated sugars that accumulate in large excess in the mutants (Brody and Tatum, 1966; Scott and Tatum, 1970). Recently, Scott has found that frost, a G6PD mutant has also altered adenyl cyclase; this has created an apparent paradox since i t implies that the single mutation (frost) affects both unrelated enzymes, such as G6PD and adenyl cyclase (Scott and Soloman, 1974). This difficulty has been overcome by the assumption that the altered adenyl cyclase of the frost mutant is a secondary consequence of the G6PD defect mediated by a defective membrane. As discussed earlier in this section, these G6PD mutants have impaired membrane due to a reduced level of available NADPH. The fact that addition of linolenic acid to the growth medium can alleviate the severity of the morphological changes in the frost mutant and leads to reversal toward wild-type-like growth supports this assumption. It can further be argued that the reduced level of adenyl cyclase reported by Terenzi et al. (1974) in the crisp-1 mutant may also have arisen as a secondary effect of membrane impairment. c . Cyclic-AMP, the Ubiquitous Regulatory Molecule. The role of this ubiquitous molecule was conceptualized in view of the following facts: (1) The morphology of Neurospora is determined by the chemical composition of its cell wall, and the latter can be influenced by the regulation of the enzymes of cell-wall biosynthesis, which are controlled by cAMP (Traut and Lipmann, 1963). (2) The addition of cAMP to the growth medium can influence the growth pattern of the wild-type strain of Neurospora (Mishra, 1976) as seen in Fig. 6. Further evidence regarding the role of cAMP in morphogenesis has been reported. (1) Addition of theophylline to the growth medium can cause morphological changes in Neurospora. Theophylline is an inhibitor of phosphodiesterase and thus can lead to excessive accumulation of cAMP in vivo. (2) Various effectors of CAMP (such as histamine, caffeine, quinidine, and serotonin) can cause semicolonial to colonial growth in Neurospora (Scott and Soloman, 1975), although, in general higher concentrations of these chemicals are required to cause morphological changes in Neurospora than in a mammalian system (Robinson et al.,

380

N. C. MISHRA

1971). (3) In a particular morphological mutant frost, a low level of CAMP and a defective adenyl cyclase have been reported (Scott, in Scott et al., 1974). (4) In the mutant cr-1 with reduced specific activity of adenyl cyclase, addition of CAMPto the growth medium was found to overcome the pleiotropic effect of this mutation (Terenzi et al., 1974). ( 5 ) The presence of a membrane-bound adenyl cyclase and of a particulate phosphodiesterase has been reported in Neurospora (Flawia and Torres 1972a,b; Scott and Soloman, 1974). These studies of the gene-enzyme relationships among morphological mutants, their phenocopies and suppressors clearly suggest that the morphology of Neurospora is determined by cell-wall-membrane complex in this organism. Any direct or indirect disruption in the structure and function of this complex may cause changes in morphology of Neurospora. Recent studies have shown that some hydrolysis of cell-wall structure is an essential part of the normal development of the wild-type strain ; an increased activity of certain cell-wall hydrolyzing enzymes in Neurospora during hyphal development has been demonstrated (Mahadevan and Mahadkar, 1970a; Mahadevan and Rao, 1970; Gratzner and Sheehan, 1969; Gratzner, 1972; Sternberg and Sussman, 1974). Ill. Conidiogenesis

Formation of conidia and their development marks a distinct stage in the differentiation of Neurospora. The conidial development has been extensively studied in order to understand the biochemical basis of differentiation in Neurospora. Some of the earlier studies were directed toward structure and function analysis of conidia (Lowry et al., 1967) or describing the physiological conditions that stimulate conidial development (Weiss and Turian, 1968; Turian and Bianchi, 1971) ; soon genetic mutants were described that can interrupt or disturb conidiation ; peach ( p e ) , fluffy (flu), and aconidial were such mutations (Barrat and Garnjobst, 1949). Recently, some other interesting phase-specific mutants have been described (Selitrennikoff et al., 1974; Matsuyama et al., 1974), and it is believed that a biochemical genetic approach will soon be adopted in the study of Neurospora conidiogenesis. AND SURFACE ARCHITECTURE OF CONIDIA A. ULTRASTRUCTURE

Electron microscopic studies carried out by Sussman’s group at the University of Michigan described the complex nature of the conidial

BIOCHEMICAL

GENETICS OF

Neurospora

MORPHOGENESIS

381

structure (Lowry et al., 1967). Their studies also revealed the differences in the origin of the micro- and macroconidia. The outermost layer of the hyphal wall was found to be dissolved during the formation of the microconidia. Thus the microconidial wall was found to be different in constitution from that of the macroconidial wall. Such differences in the surface of the micro- and macroconidia (due to their different origin) has been visualized by scanning electron microscopy (see Fig. 4) (Mishra, 1975). The macroconidial surface, although more sculptured, was found to be very similar in its surface architecture to the vegetative hyphae, whereas the surface architecture of microconidia lacked the sculptured structure and instead showed several fibrillar structures (Mishra, 1975). The macroconidia also showed the presence of a collarlike structure that delimited the chain of macroconidia from the conidiophore (Mishra, 1975). The ragged mutant and some of the osmotic mutants were found to show significant differences in their surface architecture as compared to the wild-type macroconidia (Mishra, 1975). B. BIOCHEMISTRY AND GENETICSOF CONIDIOGENEYIS The study of the conidial formation and its eventual germination into a mycelial structure provides a good model system to probe into molecular processes underlying differentiation in Neurospora. The biochemical processes underlying these morphogenetic developments are distinct and just being elucidated. I n general, the biochemistry of the cellular content of conidia seems to be different from that of the vegetative hyphae, as one would expect. Therefore, one of the approaches to understand conidial differentiation has been to compare the mycelia and conidia in order t o identify the possible biochemical differences. A number of enzymatic activities have been found to be greatly increased in conidia as compared with mycelia. The enzymes whose elevated levels have been established in conidia are: NAD nucleotidase (Zalokar and Cochrane, 1956), p-galactosidase (Zalokar, 1954), p-glucosidase (Eberhardt, 1961), isocitrate lyase (Turian, 1961), trehalase (Hill and Sussman, 1963). Also the level of certain low-molecular-weight substances has been found to increase during conidiation (Fahey et al., 1975). Tuveson et al. (1967) have shown that NADP-dependent glutamic dehydrogenase cannot be detected in Neurospora conidia before germination starts. Zalokar (1954) found that conidial formation can be prevented by addition of Tween-80, a surface-active agent, which inhibits the formation of aerial hyphae. In a related study, Turian (1957) found marked reduction in conidial formation when diphenylamine was added to the growth medium to

382

N . C. MISHRA

inhibit carotenoid production. Later, Weiss and Turian (1966) found that the relative activities of glycolytic and oxidative pathways in the cell regulate both conidiation and pigment synthesis. When glycolysis was inhibited, conidiation occurred , whereas any disruption of oxidative metabolism in normally conidiating cultures by iodoacetate, sodium fluoride, and PCMB resulted in suppressed conidiation. These authors also suggested that the control is exerted a t the stage of pyruvate, a common branch point of these two pathways. They also found a lower concentration of pyruvate carboxylase, but a higher concentration of NADase activities, in conidia (Weiss and Turian, 1966). The role of NADase in conidiation was suggested earlier by Zalokar and Cochrane (1956). I n a particular mutant called fluffy, which lacks conidiation, the NADase activity was found to be much reduced (Urey, 1971) , indicating the role of NADase in conidiation. However, its role in conidiation has been disputed by recent biochemical genetic studies of Siegel’s group a t UCLA. These workers have isolated some NADase-less mutants of Neurospora that have normal conidiation (Selitrennikoff et al., 1974). Siegel has recently described the series of events that leads to the formation of conidia in Neurospora (Siegel et al., 1974). Siegel has made a distinction among three cell types: vegetative hyphae, aerial hyphae, and conidia. According to Siegel’s group, the vegetative hyphae can be called upon to form the intermediary cell type, the aerial hyphae which then will produce conidiophore and asexual spores known as macroconidia. Matsuyama et al. (1974) have described the stages in the formation of conidiophores and emphasized the differences between aerial hyphae and conidiophores. The role of aerial hyphae in conidiation was also evidenced by Zalokar (1954). He showed that conidiation was inhibited by preventing the formation of aerial hyphae after treatment with Tween-80, a surface-active agent. Siegel and his collaborators (1974) have also developed the methodology for obtaining these three cell types, and thus their biochemistry can be compared. The three cell types differ in their cell-wall composition; Mahadevan and Mahadkar found that the level of fraction I in conidial wall was almost 40% higher than that in the hyphal wall. Also, the amino sugar content of the conidia was much less as compared to the mycelial mat which produced these conidia in the bd mutant of Neurospora; on the basis of this finding, Schmidt and Brody (1975) have suggested the presence of a mechanism for spatial distribution of galactosamine in Neurospora. Siegel et al. (1974) found a difference in the chemical composition of the cell wall of vegetative and aerial hyphae. The development of micro- and macroconidia are controlled by different genes (Barratt and Garnjobst, 1949). Several mutations are known to affect conidiation in Neurospora. Most

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

383

of the conidiation mutants have effects on other phases of the cell cycle. However, recently Siege1 and his collaborators described mutations that are phase specific and affect conidiation only. Matsuyama et al. (1974) have detailed the developmentally linked events during conidiogenesis. According to these authors, formation of conidia includes the following steps: (1) formation of conidiophores from aerial hyphae, (2) migration of nuclei to the top of conidiophore, (3) swelling and budding of the conidiophore tip, and (4) septation. These authors (Matsuyama et al., 1974; Selitrennikoff e t al., 1974) have isolated and characterized several phase-specific mutants that are defective in one or other of the developmental steps. The authors have mainly studied fl ( f l u f f y ) ,acon-2, aeon-3, csp-1, csp-2 mutations, which are located in separate chromosomes of Neurospora. They have tried to map the sequence of developmentally linked events by the phenotypic characteristics of the double mutants. Besides these phase-specific mutants, these authors have studied the effect of other mutations, such as flufly (fl) on conidiation. According to these authors, the fluffy strain is blocked a t step 3 and is also less efficient in earlier steps as compared to the wild type; the mutant acon-3 can successfully complete steps 1 and 2, although i t rarely produces a swollen tip ; acon-2 is expressed earlier and nuclei are seldom seen in the conidiophore tip; and csp-1 and csp-2 are blocked in the last step, i.e., separation of conidia. Thus the sequence of developmental steps for which these mutants are defective can be mapped as acon-2, acon-3, f l u f f y ,and csp-1 or csp-2. Among all these mutants, csp-1 and csp-2 have been studied in greater detail (Selitrennikoff e t al., 1974). These conidial separation mutants (csp-1 and csp-2) complement in heterokaryon to produce conidia like the wild-type strain, and therefore these mutations are recessive. These authors have also found that the cell wall-associated autolytic activity of the aerial hyphae was directly related with the conversion of the preconidial chains into free conidia, and they have measured the autolytic activity of wild-type aerial hyphae and compared it with that of the csp-1 and csp-2 mutants. The csp-1 and csp-2 were found to have only about 18% and 36% of the wild-type autolytic activity (Selitrennikoff et al., 1974). The nature of the autolytic enzymes involved in conidial separation, which are presumably controlled by csp-1 and csp-2 genes, has not yet been characterized. Conidial germination is very poorly understood. Recently Horowitz’s group a t California Institute of Technology has described and identified a substance essential for the germination of conidia (Charlang and Horowitz, 1971). The germination factor (GF) is heat stable and dialyzable; it is present in the growth medium and can be extracted from the mycelium (Charlang and Horowitz, 1971), and has now been identi-

384

N. C. MISHRA

fied as a polyalcohol (Horowitz, 1974). I n the medium of low water content, conidial germination was inhibited by loss of the germination factor from the conidia. The germination factor is very active, and a very low concentration 1-0.1 pg/ml can stimulate conidial germination (Charlang and Horowitz, 1971). Surface-active agents like tyrocidin and Tween-80 were also found to induce loss of the germination factor from conidia. When tyrocidin (20 pg/ml) was added to a dense conidial suspension ( los conidia/ml) , there was immediate and rapid loss of the germination factor to the medium. However, after 1 hour of incubation this was reversed; the germination factor began to disappear from the medium, and the germination of conidia could occur after 3 hours of incubation. When 20 pg/ml of tyrocidin was added to a conidial suspension containing only lo7 conidia/ml, there was no recovery of the loss of GF, and these conidia failed to germinate ; however, 2 pg/ml of tyrocidin was ineffective when added to a similar conidial suspension (i.e., lo7 conidia/ml). Mack and Slayman (1966) earlier reported that a concentration of tyrocidin of 0.5 pg/ml could inhibit the log-phase culture; therefore, the conidia seem to be less sensitive to tyrocidin than are hyphal cells. The difference in sensitivity toward tyrocidin of these two cell types (hypae and conidia) again points to the differences in the biochemical makeup discussed earlier. This discovery of germination factor by Charlang and Horowitz (1971) seems to lead to a better understanding of the molecular processes involved in conidial differentiation. Bhagwat and Mahadevan (1970) have described the presence of long-term mRNA in the conidia of Neurospora; these authors have also reported differences in the profile of RNA during different stages of germination. These findings of Bhagwat and Mahadevan have been recently confirmed (Mirkes, 1974). It is believed that soon the molecular aspects of conidial differentiation will begin to be revealed. A start in this direction has been made by the work of Loo (1975a,b). She has recently isolated a number of temperature-sensitive mutants ; these have normal germination and hyphal growth a t 25OC, but both are greatly inhibited a t 35OC. The temperature-sensitive phenotype was found to be due to a single gene mutation which behaved as a recessive in the heterokaryon. Of the several mutants studied, two have been biochemically analyzed ; these were found to have altered polysome profiles as compared to the wild-type strain. These mutations seem to affect either the initiation of protein synthesis (as in mutant 34Cts, now designated as p s i - I ) or the ribosomal processing [as in another mutant designated rip-1 (Loo, 1975a,b) 1. Earlier, Inoue and Ishikawa (1970) also described several temperature-sensitive mutants that were unable to germinate a t high temperature, presumably because of disruption in macromolecular synthesis.

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

385

C. CIRCADIAN RHYTHM Conidiation in Neurospora is a manifestation of circadian rhythm. Pittendrigh et al. (1959) and Sussman et al. (1964) independently recognized the significance of the circadian rhythm and the utilization of Neurospora mutants in the elucidation of the biochemical mechanism underlying this biological phenomenon. Circadian rhythm can be expressed as periodic formation of conidia as in certain mutant strains, patch (Stadler, 1959), timex (Sargent et al., 1966; Sargent and Woodward, 1969), or band (Sargent and Woodward, 1969), whereas in other mutant strains e.g., ( c l o c k ) , perodicity can be seen as changes in the hyphal branching frequency (Sussman et al., 1964). It was soon discovered that certain wild-type strains of Neurospora could phenocopy the periodicity in conidiation or branching of these mutants under certain growth conditions, such as aeration of the culture or proper concentration of sorbose in the growth medium (Sargent and Woodward, 1969; Sussman et al., 1964; Feldman and Hoyle, 1974). The conidiation of Neurospora is truly a circadian rhythm because it is not affected by temperature, it can be maintained in the dark and entrained by light and has a periodicity of approximately a day. The fact that a particular strain showing periodicity of conidiation also shows a single gene difference suggests that circadian rhythm is genetically controlled in these strains of Neurospora. Although the periodicity of conidiation is approximately 24 hours, Feldman and Hoyle (1974) have recently reported mutations (frq-I, frq-2, frq-3, and frq-4), which can decrease the length of periodicity in the band mutants of Neurospora. The mutations of chromosome VII (frq-I, frq-2, and frq-3) are closely linked to each other, whereas frq-4 is unlinked to either of them (Feldman and Hoyle, 1974). Since the length of periodicity is intrinsic t o the timing mechanism, these authors (Feldman and Hoyle, 1974) have concluded that these mutations affect the clock mechanism itself. Following a biochemical genetic approach, these mutants have been biochemically analyzed to elucidate the molecular mechanism behind the biological clocks. Among the different mutants showing circadian rhythm, band ( b d ) has been extensively studied because i t shows clear zonation during its growth and the conidiating (band) and the nonconidiating (interband) regions can easily be separated for chemical analyses. Brody, in an attempt to elucidate the biochemical basis of the biological rhythm, has shown that in the bd mutant the level of NADH, and NADPH, and NADP was lower in the conidiating region while the NAD level was higher as compared to the nonconidiating region (Brody and Harris, 1973). The exact nature of the spatial difference in the concentration of different

386

N. C. MISHRA

pyrimidine nucleotides in the band ( b d ) mutant is not yet known. Hochberg and Sargent (1974) have reported the rhythmic activity of several enzymes during circadian conidiation in the band ( b d ) mutant of Neurospora. I n the band ( b d ) mutant a number of enzymes (such as NADase, isocitrate lyase, glyceraldehydephospate dehydrogenase, 6PGD and G6PD) were found to oscillate with the visible morphological changes. Some wild-type strains and the flufll~mutant, which showed a circadian osciliation of nucleic acid metabolism (Martens and Sargent, 1974), was, however, found to lack the oscillation of these enzymes. The role of NADase activity in circadian conidiation is aiso refuted by the fact that NADaseless mutant of Neurospora are not impaired in conidiation and also that the fluffy strain which shows circadian morphological alterations lacks any detectable NADase activity. These data regarding the fluctuation in enzyme activity during conidiation could therefore be interpreted to suggest that they are not part of the time keeping mechanism of the clock per se. However, Brody has recently observed circadian oscillations in the adenylate energy charge and in NAD:NADH redox ratio. The adenylate energy charge was found to decrease from 0.93 to 0.65 whereas the NAD:NADH redox ratio increased from 1.6 to 3.6 in the growing front of the band mutant strain (Brody, 1974). Since these parameters (i.e., high redox ratio and low energy charge) are associated with partial uncoupling of mitochondrial oxidative phosphorylation, Brody has proposed the latter (uncoupling of mitochondria1 oxidative phosphorylation) to be a critical process for the cellular biological clock. Support for the above scheme (Brody, 1974; Delmar and Brody, 1975) is provided by experiments with dinitrophenol ( D N P ), an uncoupler of oxidative phosphorylation ; in such experiments D N P was found to raise the redox ratio (from 1.8 to 3.1) and to lower the energy charge (0.93 to 0.80) of the growing front (nonconidiating region) only. No such effect of D N P was seen, however, on the conidiating region of the band ( b d ) strain (Delmar and Brody, 1975). The involvement of oxidative phosphorylation in the biological clock implies the role of the cell membrane in this process. Presumably, the cell membrane can act as a receptor or transducer of biological signals or both, as suggested earlier (Sweeney, 1969) ; however, its precise role remains to be elucidated. IV. Morphogenesis during Sexual Development

Neurospora undergoes elaborate differentiation during development of its sexual reproductive apparatus. The studies carried out by Srb’s group

BIOCHEMICAL

GENETICS OF

Neurospora

MORPHOGENESIS

387

a t Cornell University have been mainly from the genetic point of view and only recently some biochemistry of this significant aspect of development in Neurospora is being elucidated by their group. Another important biochemical study has been carried out by Horowitz and his collaborators a t Cal Tech. Some of these aspects of morphogenetic development in Neurospora have been recently reviewed by Srb et al. (1974). The suitability of Neurospora for developmental genetic studies of the sexual reproductive apparatus has been discussed by Srb et al. (1974). Some of the attributes of Neurospora in such studies are as follows: (1) the cytology of ascus development is well established (McClintock, 1945; Singleton, 1953) ; (2) the different Neurospora species, although similar in physiology and morphology have significant differences in the genetic implications of their sexual reproductive processes in view of the fact that N . crassa and N . sitophila are heterothallic with 8-spored asci ; N . tetrasperma is pseudohomothallic with 4-spored asci whereas N . terricola is a true homothallic species with 8-spored asci; (3) the cytological differences between different species leading to different ascus patterns have been established by Dodge (1942) ; (4) genetic material can be routinely transferred among these Neurospora species.

A. SEXUAL DEVELOPMENT AND THE MATING-TYPE LOCUS The nature of the mating-type locus in Neurospora has remained elusive. N . crassa is heterothallic and occurs as two distinct mating types called A and a. Ordinarily these mating types have two functions, which control ( 1 ) crossing compatibility during sexual reproduction that leads to the formation of perithecia, asci and ascospores and (2) heterokaryon incompatibility; i.e., strains of the opposite mating types cannot form heterokaryons during vegetative growth. Besides the mating-type locus there are several other genes that control heterokaryon incompatibility in Neurospora crassa (Garnjobst, 1955; Garnjobst and Wilson, 1956). This heterokaryon incompatibility of the mating-type locus is never complete, and in N . crassa, heterokaryons betwecn opposite mating types grow poorly and usually segregate out as homokaryon sectors (Gross, 1952). I n N . sitophila the mating-type locus does not inhibit heterokaryon formation between the opposite mating types (Mishra, 1971) whereas in N . tetrasperma the nuclei of opposite mating types can exist together throughout its life cycle; and this N . tetrasperma is a pseudohomothallic species (Dodge, 1941) as opposed to N . dodgei a truly homothallic species (Nelson et al., 1964). I n an extensive study by Newmeyer et al. (1973) the two above-mentioned functions of the N . crassa mating-type locus (ie., crossing compatibility and heterokaryon incompatibility) could

388

N. C. MISHRA

never be separated. Recently, Newmeyer (1970) has described a mutation called tol for “tolerance”), which allows the two mating-type loci ( A and a ) to coexist in heterokaryon in N . crassa. This locus (tol) is not linked to the mating-type locus. Similar conclusions have been arrived a t by Metzenberg and Ahlgren (1973) regarding the coexistence of the nuclei of opposite mating locus in N . tetrasperma. When the N . tetraspemna mating type loci were separately introduced into a largely N . crassa background by introgression, the organisms, were unable to form heterokaryons between strains of opposite mating types (Metzenberg and Ahlgren, 1973). Other preliminary studies show the involvement of a single mycelium in ascogonal development (Bhattacharya and Feldman, 1974) and the possible involvement of certain hormones in sexual differentiation (Islam, 1973). B. GENETICSAND BIOCHEMISTRY OF PERITHECIAL DEVELOPMENT The sexual differentiation in Neurospora is always accompanied by the characteristic appearance of certain kinds of proteins ; these are tyrosinase, L-amino acid oxidase, and a phase-specific perithecial protein. Much of the work in this area has been directed toward the understanding of the specific role of these proteins and their characterization. These works, mostly by Horowitz’s group a t Cal Tech and by Srb’s group a t Cornell University have been discussed below.

i. Induction of Tyrosinase Activity Production of melanin is an essential part of sexual differentiation in Neurospora. The enzyme tyrosinase, which converts L-tyrosine into melanin, the main pigment of perithecia and ascospores, has been found to be absent from the vegetative cell cycle (Hirsch, 1954). Earlier, Horowitz and his collaborators showed that sexual differentiation as well as the derepression of tyrosinase and L-amino acid oxidase could be induced by starvation conditions (Horowitz et al., 1960; Horowitz, 1965). These workers have described three genes, T, ty-1, and tu-2, that are responsible for tyrosinase activity in Neurospora. Recently, another strain, T-22 has been described, which cannot be induced for tyrosinase production although the gene is not a structural gene for tyrosinase (Horowitz and Macleod, in Feldman and Thayer, 1974) whereas the wild-type strain, 69-113a has a high level of tyrosinase production. All the tyrosine mutants ty-1, ty-2, and T-22 are female sterile. According to Horowitz et al. (1960) , both ty-1 and ty-2 are regulatory genes whereas the structure of tyrosinase is controlled by the alleles of T gene. Horowitz and his collaborators have also shown that inducibility of sexual differ-

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

389

entiation as well as the derepression of tyrosinase and several L-amino acid oxidase depends on starvation condition. They have also found that the de novo synthesis of tyrosinase or L-amino acid oxidase can be stimulated by inhibition of protein biosynthesis or by D-amino acids {Horowitz, 1966; Horowitz et al., 1970). The apparent paradox that an inhibitor of protein biosynthesis stimulates tyrosinase activity in Neurospora was resolved by these workers on the assumption that the partial inhibition of protein synthesis causes a decrease in the level of an unstable protein which acts as a repressor of the tyrosinase genes (Horowitz et al., 1970). However, soon it was realized that the inducer of tyrosinase activity can act only during the stationary phase, never when added to an actively growing culture. These effects are reminiscent of the catabolite repression in bacteria (Magasanik, 1961) and thus suggest an involvement of CAMPin this process. Feldman and Thayer (1974) have reported the effect of cAMP on derepression of tyrosinase activity in different strains of Neurospora. These authors found that cAMP can increase tyrosinase production by 10 to 12-fold in the wild-type strain. They also found that caffeine or theophylline (at concentrations able to inhibit in vitro phosphodiesterase activity) can also increase tyrosinase production by increasing the in vivo level of CAMP. Only 3‘,5‘-cAMP or its dibutyryl or 8-bromo derivatives were active in inducing tyrosinase production ; 2’,3’-cAMP, 3’-AMP, or 5’-AMP were ineffective in this manner. Also, 3’5’-cAMP was found to increase the production of tyrosinase in the ty-1 mutant but not in ty-2 or T-22 strains. These data suggest functional differences among these three mutations (ty-I, ty-2, and T-22) controlling tyrosinase production. I n this relation, an interesting finding is that the inducer of tyrosinase activity cannot overcome the female sterility of the mutant strains ty-1 and ty-2; this suggests that these mutant loci regulate more than one function during sexual development. This conclusion is further supported by the fact that heterokaryons between ty-I and ty-2 are still female sterile although capable of tyrosinase production (Horowitz et al., 1965). 2. Phase-Specific Perithecial Protein

Srb and his collaborators have discovered the presence of a phasespecific protein in different species of Neurospora, which is characteristically present only in perithecia and is absent from vegetative cells and also from ascospores. The concentration of the perithecial proteins was found to increase to maximal level during 5-6 days after fertilization. These authors have found two electrophoretic variants (slow and fast) of the perithecial protein; in both cases there was a parallel increase in

390

N. C. MISHRA

their amount after fertilization. The two variants of the perithecial protein found in ATeurospora strains of different origin were shown by tetrad analysis to be controlled by members of an allelic pair (Nasarallah and Srb, 1972). The role of this newly discovered phase-specific protein found only in perithecial extract has yet to be understood. Srb and his collaborators are trying to understand whether a t fertilization the synthesis of this protein is induced or whether it is released from some other form of protein that remains undetected by their present technique. The fact that synchronous production of ascogonia can be induced (Vishwanatha-Reddy and Turian, 1972) could be utilized in the study of different proteins or enzymes associated with sexual differentiation in Nezsrospora. These studies could also be aided by the utilization of different female-sterile mutants or a particular perithecial mutant that lacks melanin and instead has orange pigments (Howe and Benson, 1974). It is known that the ascogonia are produced only at 25OC, not at 37OC (Hirsch, 1954 ; Westergaard and Hirsch, 1954; Barbesgaard and Wagner, 1959). However, Vishwanatha-Reddy and Turian have found that the effect of temperature is reversible; i.e., if a 37OC culture is transferred back to 25OC, ascogonia are produced. Therefore, it appears that the increased temperature only prevents the production of a component necessary for ascogonial development rather than destroying this component. These authors have also found that this component is synthesized between 36 and 48 hours of incubation a t 25OC, when i t is no longer affected by transfer to 37OC (i.e., it suggests that the biosynthesis of this component, not the component itself, is temperature sensitive).

C. GENETICSOF Ascus

AND

ASCOSPORE DEVELOPMENT

The different aspects of ascus development (such as its morphology, cytology, and genetic control) have been described by Dodge (1927, 1939, 1942) and others (McClintock, 1945; Dodge et al., 1950; Singleton, 1953). These authors have also discussed the role of the specific spindle behavior responsible for the heterothallic 8-spored asci of N . crassa and the homothallic 4-spored asci of N . tetrasperma. Dodge and later Srb have shown that the behavior of the spindle, which is of such profound genetic consequence, is itself genetically controlled (Dodge, 1939 ; Pincherra and Srb, 1969). The significance of ascus development in biochemical genetic studies have been emphasized by Srb and his collaborators a t Cornell University, and these authors have carried out a systematic study of the genetic control of development in Neurospora. Srb and his collaborators have described a large number of mutants that are defective for shape and size of ascus or ascospores or for delimitation of ascospores. Srb and

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

391

Base (1969) have also developed a unique technique of zygotic complementation to analyze the number and kind of genetic functions controlled by a particular mutation. I n such a test, a cross between two mutants defective for distinct functions yielded wild-type asci or spores, whereas asci or spores with mutant phenotypes were seen when the mutants involved in the cross were defective for the same function (Srb and Base, 1969). Further, the recessiveness or dominance of a mutation was decided by whether or not a cross between the mutant and the wild type led to wild-type or mutant phenotype. These authors have also determined the sequence of developmentally linked mutations by determining the effect of double mutations. I n this elegant manner Srb and his collaborators have initiated what may be called a formal genetics of development in Neurospora. The biochemical changes underlying these developmental mutants of Neurospora, however, remain to be elucidated. A number of mutants defective for a specific ascus or ascospore feature have been listed in Table 5. 1. Ascus Development and Genetic Control of Spindle

The different wild-type strains of hreurospora produce asci with 8 or 4 ascospores in a linear arrangement, these are either heterothallic or homothallic. The specific orientation of the spindle (longitudinal or transverse) during nuclear division in an ascus is known to determine the different kinds of asci present in hr.crassa and N . tetrasperma (Dodge, 1927, 1939, 1942; Colson, 1934; McClintock 1945; Singleton, 1953). According to these authors, the orientation of the spindle parallel to the transverse axis of the ascus during the second and third nuclear divisions in its development leads to the formation of 4 heterokaryotic but homothallic spores in N . tetrasperma; whereas, in hi. crassa (or N . sitophila) the spindle is parallel to the longitudinal axis of the ascus and thus leads to the formation of an ascus with 8 homokaryotic spores, each of which is capable of producing mycelia of only onc mating type. However, the parallelism in spindle orientation in ill. tetraspernza is never complete, and an occasional disruption (in the spindle orientation mechanism) leads to the formation of small homokaryotic ascospores, which belong to either A or a mating types. The orientation of the spindle is genetically controlled; Dodge (1939) described a dominant lethal mutation ( E ) in N . tetrasperma that causes the formation of %spored asci in N . tetraspernaa. Pincherra and Srb (1969) have also shown that a particular N . crassa mutation called peak ( p k ) when introduced into N . tetrasperma also causes formation of asci with 8 spores. These studies are of profound genetic consequence in the sense that a single gene mutation is shown to wipe out the very primary morphological criterion that makes N . tetra-

392

N. C. MISHRA

TABLE 5 Characteristics of Some of the Neurospora Mutants That Cause Morphological Changes in Ascus o r Ascospore Development

Mutation 1. peak (pk)"

Origin or genetic background

N . crassa N . crassa in N . tetrasperma N . tetrasperma

2. E

N . tetrasperma

3. scumbo (sc)

N . crassa

4 . semicolonial-9 (smco-9) 5 . Round spores (R or r) 6. triangular ( p k t ) 7 . giant (G) 8. indurated (I) 9. lethal ascospore (le-1) 10. lethal ascospore (le-8)

N . crassa

N . crassa N . crassa N . t etrasp erma

N . tetrasperma N . crassa

N . crassa

Characteristics

References

Balloon-shaped ascus with 8 spores in nonlinear arrangement

Murray and Srb (1962)

Balloon-shaped ascus with only 4 spores in nonlinear arrangement 8 Linear spores in each ascus 8 Linear spores in each ascus 8 Linear spores in each ascus Round spores Triangular spores Giant spores Indurated ascus Ascospore fails to germinate Ascospore fails to germinate

Pincherra and Srb (1969) Novak and Srb (1973) Dodge (1939) Srb et al. (1974) Srb et al. (1974) Mitchell (1966) Srb et al. (1973) Dodge (1927) Dodge (1927) Garnjobst and Tatum (1967) Garnjobst and Tatum (1967)

apeak-8, biscuit, and clock are allelic and zygote recessive and peak-1 through peak-6 are zygote dominant.

sperma a distinct species. Studies by Topper (1972) suggest that the longitudinal orientation of the spindle is dominant over the transverse orientation; also the ascus pattern depends not entirely on the spindle orientation, but also on the movement of the centrosome and the attached nuclei. 2, Developmental Mutants, Phenotypic Interaction, and

Biochemical Lesions

Murray and Srb (1962) have described a number of mutations called peak ( p k ) in N . crassa, which leads to a colonial hyphal morphology and ballon-shaped asci with nonlinear arrangement of the ascospores. Srb and Base (1969) have described a large number of other recessive muta-

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

393

tions that affect the ascus shape and spore pattern; these mutations were found to belong to several groups by zygotic complementation tests and were distributed well over the seven chromosomes of N . crassa. Some of these mutants were found to be allelic to previously known mutations, such as peak; however, a great majority were non-allelic to each other and to peak. Other mutations like scumbo (sc) and semicolonial-9 (srnco-9) also give rise to asci of different shapes with nonlinear spores. These mutations (i.e., sc and smco-9) are complementing, and a cross between them produces a linear ascus. Also the peak mutations of independent origin from N . crassa and N . tetrasperma, were found t o be allelic although these have distinct phenotypes in different genetic backgrounds (Novak and Srb, 1973). The peak mutation of N . tetrasperma origin causes the formation of the balloon-shaped asci with 4 nonlinear spores whereas the N . crassa peak mutation causes production of asci with 8 nonlinear spores in both genetic backgrounds (i.e. N . crassa and N . tetraspemna) . Russell and Srb (1972) have described several mutations that modify the phenotypic expression of the peak mutation. Preliminary studies by Srb and his collaborators also suggest a distinct correlation between the abnormal morphology and the behavior of certain orangelles (like centrosome and spindle). Recently, the biochemical defect of peak-2 mutation has been elucidated by Russell and Srb (1974). These authors found the activity of an enzyme involved in chitin biosynthesis [L-glutamine: ~-fructose-6-phosphateamidotransferase (GFAT)] was increased in the peak-2 mutant strain as compared to the wild type. The pealc-2 enzyme (GFAT) activity was found to be more thermolabile a t 35OC. Recently it has been shown that GFAT is subject to feedback inhibition by UDP-GlUNAC (Endo et al., 1970) ; however, the increased enzyme activity seen in peak-2 mutants was not due to differential feedback inhibition (Russel and Srb, 1974). Other Mutations. Heslot (1958) has described a large number of mutations that can alter the shape of the ascus, its size, and the number of spores in Sordariaceae. Mitchell (1966) first described the dominant mutation ( R ), which produces round spores in N . crassa. Srb et al. (1974) have described additional such mutations in A’. crassa. Mutations that cause production of giant spores or indurated asci in N . tetrasperma have been described by Dodge (1927). I n the case of giant spore mutations, the delimination of spores is defective and in eech ascus a single oversized spore is produced which germinates into a heterokaryotic mycelium. Leary and Srb (1969) have described several such recessive mutations in N . crassa. In the case of indurated asci, mutation causes fusion of the ascus wall with the spores, which fail to be delimited. Garnjobst and Tatum (1967) have described ascospore lethal mutations (le-1 and Ze-2).

394

N. C. MISHRA

These ascospores fail to germinate; these authors also found that some mutant spores could be germinated after treatment with Clorox. Some of these mutants are listed in Table 5. 3. Phenotypic Interaction

Srb and his collaborators (1973, 1974) determined the sequence of developmentally linked events in ascus morphogenesis by examining the effect of a particular mutation on the expression of another mutation controlling a distinct function. I n such studies these authors found that, in the double mutant R, p k (i.e., round spore and balloon-shaped ascus), the spores were round in a balloon-shaped ascus, suggesting that the delimitation of the ascospore was not affected by the geometry of the ascus. However, the double mutant (giant and peak) produced a balloonshaped giant spore, suggesting that spore shape was determined by the ascus shape when the delimitation of spore was defective. At present, the biochemical basis of these mutants that affect the different aspects of ascus and ascospore development is not known ; however, it is imperative that a t least some of these mutants are defective for cell wall membrane metabolism since several colonial mutants have aberrant ascus formation, while others may be defective for certain organelle structures and functions (Srb et al., 1974). However, a start has been made in this direction by Russell and Srb (1974). These authors have compared the wild type and several mutant strains with altered mycelial and ascus morphology for the specific activity of the enzyme L-glutamine: D-fructose 6-phosphate amidotransferase (GFAT) , which is involved in the synthesis of chitin (Leloir and Cardini, 1953). GFAT activity was found to be significantly increased in the crude extracts of six mutants; of these six mutants, two were allelic to pealc-2 and clock while others mapped a t different loci (Russell and Srb, 1974). According to these authors, the peak locus is not a structural gene for the enzyme GFAT, since only two out of the eleven mutants allelic to the peak locus were found to show elevated GFAT activities as compared to that of the wild type. Further work is required to determine whether or not the other four nonallelic mutants showing higher GFAT activities control the structure of this enzyme (Russell and Srb, 1972).

V. Conclusions The cell wall-membrane complex of Neurospora seems to be the main determinant of its morphology. A genetic mutation or a chemical manipulation that directly or indirectly causes changes in this complex (cell

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

395

wall-membrane) leads to an altered morphology. Such changes can be affected in any one of the several ways as summarized: 1. A defect in the enzyme which is directly or indirectly involved in the biosynthesis of the components of the cell wall-membrane complex (Tatum, 1973; Crochen and Nyc, 1964; Pina and Tatum, 1967). 2. An inhibition in the activity of the enzymes of cell-well biosynthesis (Mishra and Tatum, 1972). 3. An overproduction of certain cell wall autolyzing or other enzymes (Mahadevan and Mahadkar, 1970; Russell and Srb, 1974). 4. A disruption in the membrane function either by a mucopolysaccharide (Reissig and Plasgow, 1971) or by an excessive accumulation of microfilament (Allen et al., 1974). 5. Changes in the in v i m level of cAMP (Mishra, 1976; Scott and Soloman, 1975; Terenzi et al., 1974). It is also implied that the development of the conidia and the asci should involve changes in this complex (i.e., cell wall-membrane) of Neurospora. This idea is supported by the observed changes in the cell-wall composition (Mahadevan and Mahadkar, 1970b) and in the surface architecture of some of these fungal structures (Mishra, 1975). Although the primary enzyme defects in a number of the morphological mutants or their phenocopies and suppressors are now established (see Tables 1-3), this still remains to be elucidated for a large number of mutants. Such elucidation of gene-enzyme defects would not only increase our knowledge of the enzymes involved therein, but would also reveal their role in the morphogenesis of Neurospora. Such studies may lead to the discovery of the kinds of defects already described or to some novel kinds. Some of these may involve defects in protein biosynthesis or ribosome assembly or general changes in the fidelity of the translation which are now being described (Loo, 1975a,b; Printz and Gross, 1967). This information not only would provide a better understanding of the morphogenesis and development of Neurospora, but would add to our much needed knowledge of the molecular biology of differentiation in this organism. The recent studies have been successful in determining the sequence of morphogenetic events during conidiogenesis and ascus development (Siege1 et al., 1974; Srb et al., 1974). The characterization of the germination factor described by Charlang and Horowitz (1971) would be of great aid in understanding the conidial germination processes. Further study of the genetic control and regulation of different inducible or repressible proteins during sexual differentiation would lead to a better understanding of the complex processes involved (Horowitz et aE., 1965) and the role of cAMP therein. Also, further characterization of the perithecial pro-

396

N. C. MISHRA

tein (Srb et aZ., 1973) from wild-type and mutant strains (female sterile and/or other phase-specific mutants) would reveal its role in the sexual development of Neurospora. A biochemical genetic approach would soon be available to reveal the different aspects of conidiation and sexual differentiation (including mating-type reaction and development of ascus and ascospores) . The study of circadian mutants in Neurospora has provided a better understanding of the biochemical basis of the biological clock (Brody, 1974). The role of cell membrane either as acceptor or transducer of signals for biological rhythms would soon be understood by employing appropriate mutant strains in future studies. A number of mutants that are blocked in different developmental pathways during conidiation (Siege1 et al., 1974), sexual differentiation (Horowita et aZ., 1960), and ascus development (Srb et al., 1974) are already known, and their use in future biochemical genetic study would prove fruitful in providing a better insight into the complex processes involved in the development of Neurospora. ACKNOWLEDGMENTS I wish to express my sincere appreciation to Dr. E. L. Tatum for his interest and also to thank my colleagues for helpful discussions and Barbara Kay O'Dowd for her patient typing of the manuscript.

REFERENCES Abramsky, T., and Tatum, E. L. 1976. Differential inhibition of branching enzyme in a morphological mutant and in wild type Neurospora. Influence of carbon source in the growth medium. Biochim Biophys. Acta 421, 106-114. Abramsky, T., Scott, W. A., and Tatum, E. L. 1971. Neurospora morphology and 6phosphogluconic acid dehydrogenase. Fed. Proc., Fed. A m . SOC.Exp. Biol. 30, 1069.

Allen, E. D., Lowry, R. J., and Sussman, A. S. 1974. Accumulation of microtilaments in a colonial mutant of N . crassa. J. Ultrastruct. Res. 48, 455-464. Bachman, B. J., and Bonner, D. M. 1959. Protoplasts from Neurospora crassa. J. Bacteriol. 78, 550-556. Bachrach, V., and Cohen, I. 1961. Spermidine in the bacterial cell. J. Gen. Microbiol. 26, 1-9. Barbesgaard, P. Q., and Wagner, S. 1959. Further studies on the biochemical basis of protoperithecia formation in Neurospora crassa. Hereditas 45, 564-572. Barratt, R. W., and Garnjobst, L. 1919. Genetics of a colonial microconidiating mutant strain of Neurospora crassa. Genetics 34, 351-369. ' Bartnick-Garcia, S. 1968. Cell wall chemistry, morphogenesis and taxonomy of fungi. Annu. R e v . Microbiol. 22, 87-108. Beadle, G. W., and Tatum, E. L. 1941. Genetic control of biochemical reactions in Neurospora. Proc. Natl. Acad. Sci. U.S.A. 27, 499-506.

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

397

Benzer, S. 1967. Behavior mutants of Drosophila isolated by countercurrent distribution. Proc. Natl. Acad. Sci. U.S.A. 27, 499-506. Bhagwat, A. S., and Mahadevan, P. R. 1970. Conserved mRNA from the conidia of Neurospora crassa. M o t . Gen. Genet. 109, 142-151. Bhattacharya, L., and Feldman, J. 1974. Sexual differentiation 11. Neurospora Newsl. 21, 6. Brody, S. 1970. Correlation between reduced nicotinamide adenine dinucleotide phosphate levels and morphological changes in Neurospora crassa. J . Bacterial. 101, 802. Brody, S. 1972. Regulation of pyrimidine nucleotide levels and ratios in Neurospora crassa. J . Biol. Chem. 247, 6013-6017. Brody, S. 1973. Metabolism, cell walls, and morphogenesis. I n “Developmental ReguIation: Aspects of Cell Differentiation” ( S . J. Coward, ed.), pp. 107-154. Academic Press, New York. Brody, S. 1974. Mitochondria] uncoupling and circadian biochemical oscillations in Neurospora. Fed. Proc., Fed. A m . Sac. Exp. Biol. 33, 1271 (abstr.). Brody, S., and Harris, S. 1973. Circadian rythms in Neurospora: Spatial differences in pyridine nucleotide levels. Science 180, 498-500. Brody, S., and Nyc, J. F. 1970. Altered fatty distribution in mutants of Neurospora crassa. J . Bacterial. 104, 780-786. Brody, S., and Tatum, E. L. 1966. The primary effect of a morphological mutation in Neurospora crassa. Proc. Natl. Acad. Sci. U.S.A. 56, 1290-1297. Brody, S.,and Tatum, E. L. 1967a. Phosphoglucomutase mutants and morphological changes in Neurospora crassa. Proc. Natl. Acad. Sci. USA. 58, 923-930. Brody, S., and Tatum, E. L. 1967b. On the role of glucose-6-phosphate dehydrogenase in the morphology of Neurospora In “Organizational Biosynthesis” (H. J. Vogel, J. 0. Lampen, and V. Bryson, eds.), pp. 295-301. Academic Press, New York. Chaikoff, I. L. 1953. Metabolic blocks in carbohydrate metabolism in diabetes. Harvey Lect. 47, 99-125. Chang, P. L. V., and Trevithick, J. R. 1972. Release of wall-bound invertase and trehalase in Neurospora crassa by hydrolytic enzymes. J . Gen. Microbial. 70, 13-22. Charlang, G. W., and Horowitz, N. H. 1971. Germination and growth of Neurospora a t low water activities. Proc. Natl. Acad. Sci. U.S.A. 68, 260-262. Colson, B. 1934. The cytology and morphology of Neurospora tetrasperma Dodge. Ann. Bat. (London) 48, 211-224. Crocken, B., and Nyc, J. F. 1964. Phospholipid variations in mutant strains of Neurospora crassa. J . Biol. Chem. 239, 1727-1730. Crocken, B., and Tatum, E. L. 1968. The effect of sorbose on metabolism and morphology of Neurospora. Biochim. Biophys. Acta 156, 1-8. Dagley, S., and Nicholson, D. E. 1970. “An Introduction to Metabolic Pathways,” pp. 178-179. Wiley, New York. Darrow, R. A., and Rodstrom, R . 1966. Subunit association and catalytic activity of uridine diphosphate galactose-4-epimerase from yeast. Proc. Natl. Acad. Sci. U S A . 55, 205-211. DeLange, R. J., Kemp, R. G., Riley, W. D., Cooper, R. A., and Krebs, E. G. 1968. Activation of skeletal muscle phosphorylase kinase by adenosine triphosphate and adenosine 3’,5’ monophosphate. J . Biol. Chem. 243,2200-2205.

398

N. C. MISHRA

Delmar, D. P., and Brody, S. 1975. Circadian rhythms in Neurospora crassa: Oscillation in the level of adenine nucleotide. J. Bacteriol. 121, 548-553. deTerra, N., and Tatum, E. L. 1961. Colonial growth of Neurospora. Science 134, 1066-1068. deTerra, N., and Tatum, E. L. 1963. A relationship between cell wall structure and colonial growth in Neurospora. Am. J. Bot. 50, 669-677. Dodge, B. 0. 1927. Nuclear phenomena associated with heterothalliam in the ascomycete Neurospora, J. Agric Res. 35, 289-305. Dodge, B. 0. 1939. A new dominant lethal in Neurospora. The E locus in N. tetrasperma. J. Hered. 30, 467-474. Dodge, B. 0. 1941. A heritable factor complex for heterokaryotic vigour in Neurospora Genetics 27, 140-141. Dodge, B. 0. 1942. Conjugate nuclear division in the fungi. Mycologia 34, 302307. Dodge, B. O., Singleton, J. R., and Rolnick, A. 1950. Studies on lethal E gene in Neurospora tetrasperma including chromosome counts also in razes of N . sitophila. Proc. Am. Philos. Soc. 94, 38-52, Dubes, G. R. 1953. Investigations of some “unknown” mutants of Nezirospora crassa. Ph.D. Thesis, California Institute of Technology, Pasedena. Eberhardt, B. M. 1961. Exogenous enzymes of Neurospora conidia and mycelia J. Celt. Comp. Physiol. 58, 11-16. Edson, C. M. 1972. The biosynthesis of cell wall structural components in Neurospora. Ph.D. Thesis, University of California, San Diego. Endo, A., and Misato, T. 1969. Polyoxin D: A competitive inhibitory of UDPN-acetylglucosamine chitin N-acetyglucosaminyl-transferase in Neurospora crassa. Biochem. Biophys. Res. Commun. 37, 718-722. Endo, A., Kekiki, K., and Misato, T. 1970. Feedback inhibition of L-glutaminen-fructose-6-phosphate o-amido transferase by uridine diphosphate n-acetylglucosamine in Neurospora crassa. J. Bacteriol. 103, 588-594. Fahey, R. C., Brody, S., and Mikolajcyk, S. D. 1975. Changes in the glutothione thiol-disulfide status of Neurospora crassa conidia during germination and aging. J. Bacteriol. 121, 144-151. Feldman, J. F., and Hoyle, M. N. 1974. A direct comparison between circadian and noncircadian rhythms in Neurospora crassa. Plant Physiol. 53, 928-930. Feldman, J. F., and Thayer, J. P. 1974. Cyclic AMP-induced tyrosinase synthesis in Neurospora crassn. Biochem. Biophys. Res. Commun. 61, No. 3, 977-982. Fiengold, D. S., Neufeld, E. F., and Hassid, W. Z. 1958. Synthesis of a p-1,3linked glucan by extracts of PhaseoEus avieus seedlings. J . Bid. Chem. 233, 783-788. Fincham, J. R. S., an’d Day, P. 1971. “Fungal Genetics,” 3rd ed. Blackwell, Oxford. Flawia, M. M., and Torres, H. N. 1972a. Adenyl cyclase activity in Neurospora crassa. J. Biol. Chem. 247,6873-6879. Flawia, M. M., and Torres, H. N. 1972b. Adenyl cyclase activity in Neurospora crassa 11. J . Biol. Chem. 247, 6880-6883. Fuller, R. C., and Tatum, E. L. 1956. Inositol phospho lipid in Neurospora and its relationship to morphology. Am. J . Bot. 43, 361-365. Garnjobst, L. 1955. Further analysis of genetic control of heterokaryosis in Neurospora crassa. A m . J. Bot. 42, 444-448. Garnjobst, L., and Tatum, E. L. 1967. A survey of new morphological mutants in Neurospora crassa. Genetics 57, 579-604.

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

399

Garnjobst, L., and Tatum, E. L. 1970. New crisp genes and crisp-modifiers in NeuTospora crassa. Genetics 66, 281-290. Garnjobst, L., and Wilson, J. F. 1956. Heterokaryosis and protoplasmic incompatability in Neurospora crassa. Proc. Natl. Acad. Sci. U S A . 42, 613-618. Giles, N. H. 1951. Studies on the mechanism of reversion in biochemical mutants of Neurospora crassa. Cold Spring Harbor Symp. Quant. Biol. 16, 283-313. Glnsgow, J. E., and Reissig, J. L. 1974. Interaction of galactosaminoglycan with Newospora conidk. J . Bncteriol. 120, 759-766. Gorini, L. 1970. Informational suppressor. Annu. Reu. Genet. 4, 107-134. Gratzner, H. G. 1972. Cell wall alterations associated with hyper production of extracellular enzymes in Neurospora crassa. J . Bacteriol. 111, 443-446. Gratzner, H., and Sheehan, D. N. 1969. Neurospora mutant exhibition hyper production of amylase and invertas.. J . Bacteriol. 97, 544-549. Grindle, M. 1974. The efficacy of various mutagens and polyene antibodies for the induction and isolation of sterol mutants of Neurospora crassa. M o l . Gen. Genet. 130, 81-90. Gross, S . R. 1952. Heterokaryosis between opposite mating types of Neurospora crassa. A m . J . Bot. 39, 574-577. Harold, F. M. 1962. Binding of inorganic polyphosphate to the cell wall of Neurospora crassa. Biochim. Biophgs. Acta 57,59-66. Henry, S. A., and Keith, A. D. 1971. Saturated fatty acid requirer of Neurospora crassa. J . Bacteriol. 106, 174-182. Heslot, H. 1958. Contribution B l’ktude cytogknktique et gknCtique des Sordariacees. Dev. Cytol. Biol. Veg. Italog Snppl. 2, 1-234. Hill, E. P., and Sussman, A. S. 1963. Purification and properties of trehalase(s) from Neurospora. Arch. Biochem. Biophys. 102, 389-396. Hirsch, H. M. 1954. Melanogenesis and the differentiation of protoperithecia in Neurospora crassa. Bncteriol. Proc. 1054, 88 (abstr.). Hochberg, M. L., and Sargent, M. L. 1974. Rhythms of enzyme activity associated with circadian conidiation in Neurospora crassa. J . Bacteriol. 120, 1164-1175. Horowitz, N. H. 1965. Evidence for common control of tyrosinase and L-amino acid oxidase in Neurospora. Biochem. Biophys. Res. Commun. 18, 68fj-692. Horowitz, N. H. 1966. Evidence for common control of tyrosinase and L-amino acid in Neurospora. Biochem. Biophys. Res. Commun. 18, 686-692. Horowitz, N. H. 1974. Membrane and transport: A conidial factor necessary for germination. Neurospora Newl. 21, 5-6. Horowitz, N., Fling, M., McLeod, H. L., and Sueoka, N. 1960. Genetic determination and enzymatic induction of tyrosinase in Neurospora. J. Mol. Biol. 2 , 9 6 1 0 4 . Horowitz, N. H., Fling, M., McLeod, H., and Watanabe, Y. (1965). Structural and regulatory genes controlling tyrosinase systhesis in Neurospora. Cold Spring Harbor Symp. Quant. Biol. 26, 233-238. Horowitz, N. H., Feldman, H. M., and Pall M. L. 1970. Derepression of tyrosinase synthesis in Neurospora by cycloheximide, actinomycin D, and puromycin. J. Biol. Chem. 245, 27842788. Houlahan M. B., Beadle, G. W., and Calhoun, H. G. 1949. Linkage studies with biochemical mutants of Neurospora. Genetics 34, 493-507. Howe, H. B., and Benson, E. W. 1974. A perithecial color mutant of Neurospora crassa. Mol. Gen. Genet. 131, 79-83. Hunsley, D., and Burnett, J. H. 1970. The ultrastructural architecture of the walls of some hyphal fungi. J. Gen. Microbial. 62, 203-218.

400

N. C. MISHRA

Hunsley, D., and Goodday, G. W. 1974. The structure and development of Septa in Neurospora crassa. Protoplasma 82, 125-146. Inoue, H., and Ishikawa, T. 1970. Macromolecule synthesis and germination of conidia in temperature sensitive mutants of Neurospora crassa. Jpn. J . Genet. 45, 357-369. Islam, M. S. 1973. Sex hormones in Neurospora crassa further studies on its biological properties. Mycopathol. Mycol. Appl. 51, 87-97. Jacob, F., and Monod, J. 1961. Genetic regulatory mechanisms in the synthesis of proteins. J . M o l . Biol. 3, 318-356. Kingdon, H. S., Shapiro, B. M., and Stadtman, E. R. 1967. Regulation of glutamine synthetase VIII. Proc. N a t . Acad. Sci. 58, 1703-1710. Klingmuller, W. 1971. Cellular components specifically labeled during sorbose stimulation of sugar transport in Neurospora. 2. Naturforsch. Teil B 26, 409413. Leary, J. V., and Srb, A. M. 1969. Giant spores, a new developmental mutant of N . crassa. Neurospora Newsl. 15, 22-23. Lechner, J. F., Fuscaldo, K. E., and Bazinet, G. 1971. Genetic and biochemical studies of the hexose monophosphate shunt in Neurosporu crassa. 11. Characterization of biochemical defects of the morphological mutants colonial-2 and colonial-3. Can. J. Microbiol. 17, 784-794. Lein, J., and Lein, P. S. 1949. Studies on a Neurospora mutant requiring unsaturated fatty acid for growth. J. Bacterial. 58, 595-599. Leloir, L. F. 1951. The enzymatic transformation of uridine diphosphate glucose into galactose derivative. Arch. Biochem. 33, 186-190. Leloir, L. F., and Cardini, C. E. 1953. The biosynthesis of glucosamine. Biochim. Biophys. Acta 12, 15-22. Lindegren, C. C. 1936. A six point map of the sex chromosomes of N . crassa. J . Hered. 27, 251-259. Lindegren, C. C., and Lindegren, G. 1941. X-ray and ultraviolet induced mutations in Neurospora I. J . Hered. 32, 404-412. LOO, M. 1975a. Neurospora crassa temperature-sensitive mutant apparently defective in protein synthesis. J. Bacteriol. 121, 286-295. LOO, M. 1975b. A temperature-sensitive mutant of Neurospora defective in ribosome processing (rip-1). Neurospora Newsl. 22, 10-11. Lowry, R. P., Durkee, T. L., and Sussman, A. S. 1967. Ultrastructural studies of microconidium formation in Neurospora crassa. Am. J . Bot. 45, 397403. McClintock, B. 1945. Neurospora. I. Preliminary observations of the chromosomes of Neurospora crassa. Am. J. Bat. 32, 671-678. Mack, B., and Slayman, C. W. 1966. Mode of action tyrocidine on Neurospora. Biochim. Biophys. Acta 124, 351-361. Magasanik, B. 1961. Catabolite repression. Cold Spring Harbor S y m p . Quant. Biol. 26, 249-256. Mahadevan, P. R., and Mahadkar, V. R. 1970a. Role of enzymes in growth and morphology of Neurospora crass%. Cell-wall-bound enzymes and their possible role in branching. J . Bacteriol. 101, 941-947. Mahadevan, P. R., and Mahadkar, V. R. 1970b. Major constituents of the conidial wall of Neurospora crassa. Indian J. E x p . Biol. 8, 207-210. Mahadevan, P. R., and Rao, S. R. 1970. Enzyme degradation of conidial wall during germination of Neurospora crassa. Indian J . E x p . Biol. 8, 293-297. Mahadevan, P. R., and Tatum, E. L. 1965. Relationship of the major constituents

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

401

of the Neurospora crassa cell wall to wild-type and colonial morphology. J. Bacteriol. 90, 1073-1081. Mahadevan, P. R., and Tatum, E. L. 1967. Localization of structural polymers in the cell wall of Neurospora crassa. J. Cell Biol. 35, 295-302. Manocha, M. S., and Colvin, J. R. 1967. Structure and composition of the cell wall of Neurospora crassa. J. Bacteriol. 94, 202-212. Martens, C. L., and Sargent, M. L. 1974. Circadian rhythms of nucleic acid metabolism in Neurospora crassa. J. Bacteriol. 117, 1210-1215. Matile, P. 1966. Inositol deficiency resulting in death: An explanation of its occurrences in Neurospora crassa. Science 66, 86-88. Matsuyama, S. S., Nelson, R. E., and Siegel, R. W. 1974. Mutations specifically blocking differentiation of microbonidia in Neurospora crassa. J. Dev. Biol. 41, 27S287.

Metzenberg, R. L. 1972. Genetic regulatory systems in Neurospora. Annu. Rev. Genet. 6, 111-132. Metzenberg, R. L., and Ahlgren, S. K. 1973. Behavior of Neurospora tetrasperma mating-type genes introgressed into N . crassa. Can. J. Genet. Cytol. 15, 571576.

Mirkes, P. 1974. Polysomes, ribonucleic acid and protein synthesis during germination of Neurospora crassa conidia. J. Bacteriol. 117, 196-202. Mishra, N. C. 1971. Heterokaryosis in Neurospora. Genetics 67, 55-59. Mishra, N. C. 1975. The surface architecture of different Neurospora strains as revealed by scanning electron microscopy and their relation to morphology. J. Microsc. Biol. Cell. 26, 151-159. Mishra, N. C. 1976. The effect of cyclic adenosine monophosphate on the growth of Neurospora crassa. Naturwissenschaften 63,485-486. Mishra, N. C., and Forsthofel, A. 1976. Galactosyl transferases of Neurospora. In preparation. Mishra, N. C., and Tatum, E. L. 1970a. Phosphoglucomutase mutants of Neurospora sitophila and their relation to morphology. Proc. Natl. Acad. Sci. U.S.A. 60, 638-645.

Mishra, N. C., and Tatum, E. L. 1970b. Pleiotrophy of gene mutations in Neurospora. J. Cell Biol. 470, 141a. Mishra, N. C., and Tatum, E. L. 1972. Effects of L-sorbose in polysaccharide synthetase of Neurospora. Proc. Natl. Acad. Sd.U.S.A. 69, 313-317. Mishra, N. C., and Threlkeld, S. F. H. 1967. Variation in the expression of rg mutants in Neurospora. Genetics 55, 113-121. Mitchell, M. B. 1966. Round spore character in N. crassa. Neurospora Newsl. 10, 6. Morgan, M. F., Garnjobst, L., and Tatum, E. L. 1967. Linkage relations of new morphological mutants in linkage group V of Neurospora crassa. Genetics 57, 605-612.

Murayama, T., and Ishikawa, T. 1973. Mutation in Neurospora crassa affecting some of the extracellular enzymes and several growth characteristics. J. Bacteriol. 115, 796-804. Murayama, T., and Ishikawa, T. 1975. Characterization of Neurospora crassa mutants deficient in glucosephosphate isomerase. J. Bacteriol. 122, 54-58. Murray, J. C., and Srb, A. 1962. The morphology and genetics of wildtype and seven morphological mutant strains of Neurosporn crassa. Can. J. Bot. 40, 337-349.

402

N . C. MISHRA

Nasarallah, J., and Srb, A. 1972. Genetically related protein variants specifically associated with fruitlng body maturation in Neurospora. Proc. Natl. Acad. Sci. U 8 . A . 70, 1891-1893. Nelson, A. C., Novak, R. O., and Backus, M. P. 1964. A new species of Neurospora from soil. Mycologia 56, 384-392. Newmeyer, D. 1970. A suppressor of the heterokaryon-incornpatability associated with mating type in Neurospora crassa. Can. J . Genet. Cytol. 12, 914-926. Newmeyer, D., Howe, H. B., and Galeazzi, D. R. 1973. A search for complexity at the mating type locus of Neurosporn crassa. Can. J . Genet. Cytol. 15, 577-585. Novak, D. R., and Srb, A. 1973. A dominant round spore mutant in Neurospora tetrasperma and its implications for ascospore development. Can. J . Genet. Cytol. 15, 685-693. O’Brien, T. P., and Thimann, K. U. 1966. Intracellular fibers in oat coleoptile cells and their possible significance in cytoplasmic streaming. Proc. Natl. Acad. Sci. U.S.A. 56, 888-894. Pina, E., and Tatum, E. L. 1967. Inositol biosynthesis in Neurospora crassa. Biochim. Biophys. Acta 136, 265-271. Pincherra, G., and Srb, A. 1969. Genetic variation in the orientation of nuclear spindles during the development of asci in Neurospora. A m . J. Bot. 56, 846-852. Pittendrigh. C. S., Bruce, V. G., Rosenweig, N. S., and Rubin, M. L. 1959. A biological clock in Neurosporn. Nature (London) 184, 169-170. Printz, D. B., and Gross, S. 1967. An apparent relationship between mistranlation and an altered leucyl-tRNA synthetase in a conditional lethal mutant of Neurospora crassa. Genetics 55, 451-467. Rand, J. B. 1975. The regulation of sugar transport and galactose metabolism in Neurospora crassa. Ph.D. Thesis, Rockefeller University, New York. Reissig, J. L., and Glasgow, J. E. 1971. Mucopolysaccharide which regulates growth in Neurosporn. J . Bactejiol. 106, 882-889. Robertson, N. F. 1959. Experimental control of hyphal branching and branch form in hypomycetous fungi. J. Linn. Soc. London 56,207-211. Robertson, N. F. 1968. The growth process in fungi. Annu. R e v . Microbiol. 6, 113136. Robertson, N. F., and Rizvi, 5. R. H. 1965. Apical disintegration of hyphae of N . crassa as a response to L-sorbose. Trans. Br. Mycol. Soc. 48, 467-477. Robinson, G. A., Butcher, R. W., and Sutherland, E. W. 1971. “Cyclic AMP.” Academic Press, New York. Russell, P. J., and Srb, A. M. 1972. Dominance modifiers in Neurospora crassa: Phenocopy selection and influence on certain ascus mutants. Genetics 71, 233245. Russell, P. J., and Srb, A. M. 1974. A study of L-glutamine: D-fructose-6-phosphate amidotransferase in certain developmental mutants of Neurospora crassa. Mol. Gen. G e n e f . 129, 77-86. Sargent, M. L.,and Woodward, D. 0. 1969. Genetic determinants of circadian rhythmicity in Neurospora. J. Bncteriol. 97, 861-866. Sargent, M. L., Briggs, W. R., and Woodward, D. 0. 1966. The circadian nature of a rhythm expressed by an invertaseless strain of Neurosporn crnssa. Plant Physiol. 41, 1343-1349. Scarborough, G. A. 1971. Sugar transport in Neurospora crassn. 111. An inositol requirement for the function of the glucose active transport system. Biochem. Biophys. Res. Commun. 43, 96tL974.

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

403

Scarborough, G. A,, and Schulte, T. H. 1974. An improved method for the production of Neurospora crassa spheroplasts and a new method for measuring transport in fragile cells. Anal. Biochem. 61, 441447. Schmidt, J. C., and Brody, S. 1975. Developmental control of glucosamine and galactosamine levels during conidiation in Neurospora crassa. J . Bacteiiol. 122, 1071-1075. Schulte, T. H., and Scarborough, G. A. 1975. Characterization of the glucose transport systems in Neurospora crassa sl. J . Bacteriol. 122, 10761080. Scott, W. A. 1971. Physical properties of glucose 6-phosphate hydrogenase from Neurospora crassa. J . Biol. Chem. 246, 6353. Scott, W. A., and Abramsky, T. 1973a. Neurospora 6-phosphogluconate dehydrogenase. 11. Properties of two purified mutant enzymes. J . Biol. Chem. 248, 3542-3545. Scott, W. A., and Abramsky, T. 197313. Neurospora 6-phosphogluconate dehydrogenase. I. Purification and characterization of the wild-type enzyme. J . BioE. Chem. 248, 3535-3542. Scott, W. A., and Brody, S. 1973. Effects of suppressor mutations on nonallelic glucose 6-phosphate dehydrogenase mutants of Neurospora crassa. Biochem. Genet. 10, 285-295. Scott, W. A., and Soloman, B. 1974. Cyclic 3’,5’-AMP phosphodiesterase of Neurospora crassa. Biochem. Biophys. Res. Commun. 53, 1024-1030. Scott, W. A., and Soloman, B. 1975. Adenosine 3’,5‘-cyclic monophosphate and morphology in Neurospora crassa: Drug indured alterations. J . Bacteriol. 122, 454-463. Scott, W. A., and Tatum, E. L. 1970. Glucose-6-phosphate dehydrogenase and Neurospora morphology. Proc. Ngtl. Acad. Sci. U S A . 66,515-522. Scott, W. A., and Tatum, E . L. 1971. Purification and partial characterization of glucose 6-phosphate dehydrogenase from Neurospora crassa. J . Biol. Chem. 246, 6347-6352. Scott, W. A., Mishra, N. C., and Tatum, E . L. 1974. Biochemical genetics of morphogenesis in Neurospora. Brookhaven Syrnp. Biol. 25, 1-18. Selitrennikoff, C. P., Nelson, R. E., and Siegel, R. W. 1974. Phase-specific genes for macroconidiation in Neurospora crassa. Genetics 78, 679490. Shatkin, A. J., and Tatum, E . L. 1959. Electron microscopy of Neurospora crassa mycelia. J . Biophys. Biochem. Cytol. 6, 423-426. Shatkin, A., and Tatum, E. L. 1961. The relationship of m-inositol to morphology in Neurospora crassn. Am. J . Bat. 48, 760, 771. Siegel, R. W., Selitrennikoff, C. P., and Nelson, R. 1974. Cell wall changes in Neurospora. I n “Results and Problems of Cell Differentiation.” Spring-Verlag, Berlin and New York (in press). Singleton, J. R. 1953. Chromosome morphology and the chromosome cycle in the ascus of Neurospora crassn. Am. J . Bot. 40, 124-143. Srb, A . M. 1957. Exotic growth forms in Neurospora. J . Heredity 48, 146153. Srh, A. M., and Basl, M. 1969. The isolation of mutants affecting ascus development in Neurospora crassa and their analysis by a zygote complementation test. Genet. Res. 13, 303-311. Srb, A. M., and Horowitz, N. H. 1944. The ornithine cycle in Neurospora and its genetic control. J . Biol. Chem. 154, 129-139. Srh, A. M., Bad, M., Bolist, M., and Leary, J. V. 1973. Mutations in Neurospora crassa affecting ascus and ascospore development. J . Hered. 64, 242-246.

404

N. C. MISHRA

Srb, A. M., Nasarallah, J., and Basl, M. 1974. Genetic control of the development of the sexual reproductive apparatus of Neurospora. Brookhaven Symp. Biol. 25, 40-50.

Stadler, D. R. 1959. Genetic control of a cyclic growth pattern in Neurospora. Nature (London) 184, 170-171. Sternberg, D., and Sussman, A. S. 1974. Hyperproduction of some glycosidases in Neurospora crassa. Arch. Microbial. 101, 303-320. Sussman, A. S., Lowry, R. J., and Durkee, T. 1964. Morphology and genetics of a periodic colonial mutant of Neurospora crassa. Am. J . Bat. 51,243-253. Sweeney, B. M. 1969. “Rhythmic Phenomena in Plants.” Academic Press, New York. Tatum, E. L. 1973. Biochemische Genetik der Morphogenese bei Neurospora. Naturwiss. Rundsch. 86, 140-142. Tatum, E. L., Barratt, R. W., and Cutter, U. M. 1949. Chemical induction of colonial garamorphs in Neurospora and syncephalastrum. Science 109, 509-511. Terenzi, H. F., Flawia, M. M., and Torres, H. N. 1974. A Neurospora crassa morphological mutant showing reduced adenylate cyclase activity. Biochem. Biophys. Res. Commun. 58, 990-996. Topper, V. M. 1972. A cytological study of the sexual cycle of Neurospora tetraspermu. Cornell University. M.S. Thesis. Traut, R. R., and Lipmann, F. 1963. Activation of glycogen synthetase by glucose 6-phosphate. J. Biol. Chem. 238, 1213-1221. Trevithick, J. R., and Metzenberg, R. L. 1966. Molecular sieving by Neurospora cell walls during secretion of invertase isozymes. J. Bucteriol. 92, 1010-1015. Tsoi, A., and Douglas, H. C. 1964. Phosphoglucomutase mutants of Saccharomyces cerevisiae. Biochim. Biophys. Acta 92, 513-917. Turian, G. 1957. Recherches sur l’action anticarotknogene de la diphknylamine et ses conskquences sur la morphogknese productive chez Allomyces et Neurospora. Physiol. Plant. 10, 667-680. Turian, 0. 1961. L’acBtate et son double effet d’induction isocitratasique et de diff6renciation conidienne chez les Neurospora. C. R. Hebd. Seances Acad. S C ~ . 252, 1374-1376.

Turian, G., and Bianchi, D. E. 1971. Conidiation in Neurospora crassa. Arch. Microbial. 77, 262474. Tuveson, R. W., West, D. J., and Barrat, R. W. 1967. Glutamic acid dehydrogenase in quiescent and germinating conidia of Neurospora crassa. J . Gen. Microbial. 48, 235-248. Urey, J. C. 1971. Enzyme patterns and protein synthesis during synchronous conidiation in Neurospora crassa. Dev. B i d . 26, 17-27. Vishwanatha-Reddy, M., and Turian, G. 1972. Temperature-induced synchronous differentiation of ascogonia in Neurospora. Experientia 28, 99-100. Weeks, O., and Gross, S. 1971. Mutation and “reversion” a t the l e u 4 locus of Neurospom and its effect on the cytoplasmic and mitochondria1 leucyl-tRNA synthetases. Biochem. Genet. 5, 505-516. Weiss, B., and Turian, G. 1966. A study of conidiation in Neurospora crassa. J. Gen. Microbial. 44, 407-418. Wessells, N. K., Spooner, B. S., Ash, J. F., Bradley, M. O., Luduena, M. A., Taylor, E. L., Wrenn, J. T., and Yamada, K. M. 1971. Microfilaments in cellular and developmental processes. Science 171, 136143.

BIOCHEMICAL GENETICS OF NeUTOSpOTU MORPHOGENESIS

405

Westergaard, M., and Hirsch, H. 1954. Environmental and genetic control of differentiation in Neurospora. Proc. S y m p . Colston Res. Sac. 7, 171-183. Wood, D. D., and Luck, D. J. L. 1971. A paracystalline inclusion in Neurospora crassa. Induction by ethedium and acridine, isolation and characterization. J. Cell Biol. 51, 249-264. Wood, W. B., Edgar, R. S., King, J., Lielausis, I., and Henninger, M. 1968. Bacteriophage assembly. Fed. Proc., Fed. Am. SOC.E z p . Biol. 27, 1160-1166. Wrathal, R., and Tatum, E. L. 1913. The peptides of the hyphal wall of Neurospora crassa. J . Gen. Microbial. 78, 139. Wrathal, R., and Tatum, E. L. 1974. Hyphal wall peptides and colonial morphology in Neurospora crassa. Biochem. Genet. 12, 59-68. Yanofsky, C. 1966. Amino acid replacements associated with mutations and relationship to in vitro coding data I n “Papers on Bacterial Genetics” (E. A. Adelberg, ed.), 2nd ed., pp. 163-176. Little, Brown, Boston, Massachusetts. Zalokar, M. 1954. Studies on the biosynthesis of carotenoids in Neurospora crassa during development. J. Gen. Microbial. 39, 85. Zalokar, M., and Cochrane, V. W. 1956. Diphosphopyridine nucleotidase in the life cycle of Neurospora crassa. Am. J . Bat. 43, 107.

GENETIC CONTROL OF THE CONTENT, AMINO ACID COMPOSITION, AND PROCESSING PROPERTIES OF PROTEINS IN WHEAT* Calvin F. Konzak Deportment of Agronomy and Soils, and Program in Genetics, Washington Stote University, Pullmon, Washington

I. Introduction , . , . . . . . . . . . . . . . . . 11. Structure of the Wheat Kernel; Genetic Origin of the Tissue Components A. Structure, . . . . . . . . . . . . . . . . . B. Origin, Development, and Ontogenetic Relationships among Tissues. C. Intracellular Sites of Protein Synthesis and Storage . . . . . D. Genetics of Endosperm Proteins . . . . . . . . . . . 111. Origin and Genetic Relationships among Triticum and Closely Related Species . . . . . . . . . . . . . . . . . . IV. Genetic Relationships and Homoeologies among Chromosomes of Cultivated Wheats . . . . . . . . . . . . . . . . V. Role of Environmental Factors in Protein Synthesis and Accumulation in Wheat Endosperm . . . . . . . . . . . . . . . A. Influence on Amino Acid Composition . . . . . . . . . B. Influence on Protein Solubility Fractions . . . . . . . . C. Genetic Regulation and Control of Protein Content, Composition, and Processing Properties . . . . . . . . , . . . . . VI. Role of the Cytoplasm in the Hereditary Control of Protein Content and Composition . . . . . . . . . . . . . . . . . . A. Cytoplasmic Organelle-Nuclear Gene Interactions . . . . . . B. Joint Chloroplast-Nuclear Gene Control of Ribulose-1,5-Diphosphate Carboxylase . . . . . . . . . . . , . . . . . C. Chloroplast-Nuclear Gene Interactions in Nitrogen Reduction . . D. Cytoplasm Source and Protein Composition in Wheat. . . . . VII. Nuclear Gene Control of Protein Content and Composition . . . . A. Genetic Control of 80 S Ribosomal Proteins. . . . . . . . B. Nitrate Reductase Synthesis and Genetic Control of Nitrogen Reduction . . . . . . . . . . . . . . . . . ~

~

408 411 411 411 415 417 417 426 427 428 430 430

432 432 434 435 435 436 436 437

* Information paper. College of Agriculture Research Center, Washington State University, Pullman, Project No. 1568. Research conducted in part while on Professional Leave September, 1973 to September, 1974 as Special Service Advisor to the Plant Breeding and Genetics Section of the Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture, Vienna, and completed at Washington State University. 407

408

CALVIN F. KONZAK

VIII. Genetic Variability in the Nature and Properties of Wheat Proteins. . A. Wheat Endosperm Protein Composition . . . . . . . . . B. Major Factors Affecting Processing Characteristics and Nutritional Value of Wheat Flour . . . . . . . . . . . . . . IX. Genetic Variation for Protein Content and Lysine Composition in Wheat A. Amino Acid Composition of High-Lysine and High-Protein Wheats . B. Site of the High-Protein and High-Lysine Composition in the Wheat Grain. . . . . . . . . . . . . . . . . . . X. Chromosomal Location of Genes Affecting Protein Content, Composition, and Use Properties . . . . . . . . . . . . . . . . XI. General Discussion . . . . . . . . . . . . . . . . XII. Role for Induced Mutations . . . . . . . . . . . . . A. Principles. . . . . . . . . . . . . . . . . . B. Mutations in Protein Structure and the Genetic Code. . . . . C. Efficient Induction of Improved Protein Mutants . . . . . . XIII. Directions for Future Research . . . . . . . . . . . . XIV. Summary and Conclusions. . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

440 440 463 476 481 481 487 528 535 535 539 540 54 1 544 546

I. Introduction

I n the years ahead, wheat, perhaps more than other cereals, can be expected to assume increasingly greater importance as a source of protein for much of the world’s increasing population. Even today, wheat provides more nourishment for more people than does any other food source (Inglett and Anderson, 1974). As the world’s population continues to increase, stresses on available food supplies will make it desirable, if not imperative, that the nutritional value of foods be improved. The nutritional quality of wheat protein, like that of other cereals (except possibly rice), is primarly limited by the amount and balance of four essential amino acids : lysine, threonine, isoleucine, and methionine (Kasarda et al., 1971; Kies and Fox, 1970a,b; Olson and Sander, 1975; ProteinCalorie Advisory Group (PAG) of the United Nations System, 1976). With a better balance of these essential amino acids, particularly of lysine, wheats would contribute appreciably more to the proper nutrition of those people for whom wheat is a main dietary source of protein. Since no wheat cultivar known today has a protein composition approaching the level desired (Schmidt et UI!.,1974; V. A. Johnson et al., 1975b), the development by breeding of high-yielding varieties with an increased quantity of protein with improved nutritional quality is an important scientific challenge. This challenge has stimulated much new research into the biophysical and chemical nature, functional properties, and inheritance of proteins in wheat, to build upon and expand an already solid foundation of information gained from years of intensive research in many countries by geneticists, biochemists, cereal tech-

GENETIC CONTROL O F PROTEINS IN WHEAT

409

nologists, plant physiologists, and agronomists. A number of recent findings point to further opportunities for gaining insight into the mechanisms and nature of the biochemical genetic control of protein synthesis in wheat, into the genetic basis for improving the nutritional value of wheat protein, and into the physical and chemical nature of those protein components responsible for the unique rheological (functional, dough-forming) properties of wheat flour. Much of the new genetic knowledge of wheat has been achieved via the application of recently developed research technology, e.g., electrophoresis, electrofocusing, to analyses of the several series of genetic stocks (termed aneuploids) generously made available by E. R. Sears and colleagues, and through the concerted efforts of the European Wheat Aneuploids Cooperation. Use of these special genetic stocks markedly reduces the genetic variables, adding greater precision to the necessary comparative experiments. Because these stocks also provide the foundation for classical genetic analyses (Sears, 1953, 1954, 1959a, 1966, 1975a; Unrau, 1958; Law, 1968; Law and Worland, 1972, 1973), the genetic engineering of the various aneuploid stocks in ‘Chinese Spring’ wheat is now widely recognized as one of the most creative achievements in modern crop science. An increasing array of chromosome and gene addition, deletion, and substitution derivatives is greatly extending the research potential of these special genetic stocks (Law, 1968; Sears, 1969, 1975a,b). This paper summarizes current information about the inheritance, properties, and amino acid composition of wheat proteins and provides a compilation of evidence for the control of specific proteins and protein subunits, regulatory mechanisms, and quality characteristics by genetic factors located on particular wheat chromosomes. Much of this genetic evidence has been established through the use of aneuploid and related techniques. The purpose of this review is t o examine the genetic basis and potential for increasing the protein content of wheat and improving the nutritional value of wheat protein, by breeding and genetic manipulations, while retaining those processing properties (product-use qualities) required for making such traditional foods as bread, cakes, pastry, noodles, chapattis, and pasta. Although the emphasis here is focused on evidence for genetic control of proteins in the wheat endosperm, consideration also is given to the genetic aspects of the composition and technological properties of the wheat grain and to the roles of environmental factors and cytoplasmic organelles in the synthesis and accumulation of wheat proteins. Knowledge about those areas is no less important t o an understanding of the problems involved in wheat protein improvement, or to the effective and efficient deployment of resources toward

GENETIC CONTROL OF PROTEINS IN WHEAT

411

that goal. More detailed information about the chemical and physical structure of wheat proteins and their function in breadmaking are found in the recent reviews by Hoseney and Finney (1971), Kasarda et al. (l971,1976a), Pomeranz (1971), Simmonds and Orth (1973), and Bushuk and Wrigley (1974). 11. Structure of the Wheat Kernel; Genetic Origin of the Tissue Components

Knowledge of the basic structure of the wheat kernel, its component parts and genetic origin and ontogenetic relationships provides a better foundation for understanding the nature and location of tissues in which proteins have been identified through the exploitation of genetic stocks and modern methods of analysis.

A. STRUCTURE I n botanical terms, the wheat kernel, like that of other cereal grains, is a caryopsis or single-seeded, indehiscent dry fruit. The true seed consists of the embryo, or germ, and endosperm, which are encased within the nucellar epidermis (hyaline layer), seed coat (testa), and fruit coat (pericarp), to form the kernel or caryopsis. The arrangement and composition of the component tissues in the wheat kernel (Fig. 1) are of importance to the nutritional and processing properties of the grain in commerce, while the genetic and ontogenetic origins of the tissue components are of importance in genetic and biochemical genetic analyses. Notably, the aleurone (a layer of single cells, which is removed with the bran upon milling of white flour) contains the highest proportion of the B vitamins and minerals, as well as a significant portion of the protein present in whole wheat grain (Fig. 2). Further details about the component tissue structure and nutrient composition of the wheat kernel can be found in the excellent review by MacMasters et al. (1971). A bibliography on the industrial utilization of cereals also has been published (Pomeranz, 1973a).

B. ORIGIN,DEVELOPMENT, AND ONTOGENETIC RELATIONSHIPS AMONG TISSUES The wheat kernel develops by a process called “double fertilization,” in which two identical nuclei from a single pollen grain enter the embryo sac within the ovary. One pollen nucleus fuses with the egg cell, the FIG. 1. Gross structure of the wheat kernel. (Courtesy of the Wheat Flour Institute, Chicago, Illinois.)

412

CALVIN F. KONZAK

VITAMINS THIAMINE

RIBOFLAVIN

ALEURONE LAYER

ALEURONE LAYER

E

PYRIDOXINE ALEURONE LAYER ALEURONE LAYER

-

EMBRYO

PANTOTHENIC A L D

PROTEI N

MINERALS

ALEURONE LAYER

FIQ.2. Vitamin, protein, and mineral distribution in parts of the wheat kernel. (Reproduced from MacMasters, et a l , 1971.)

second forms a triple-fusion nucleus with the two polar nuclei. The two polar nuclei are genetically identical to the egg nucleus as the result of cell divisions that occur during the development of the embryo sac and its contents within the ovary. The zygote formed from the fusion of the egg cell and one pollen nucleus becomes the embryo, or young plant, while the triple-fusion nucleus develops into the endosperm of the mature seed (Yampolsky, 1957; Mares et al., 1975). The fruit coat, seed coat, vascular bundle, and nucellus remnant tissues are derived entirely from ovary tissues formed by the “mother” plant and thus are genetically identical, but may be different in genetic composition from the embryo and endosperm contained within

GENETIC CONTROL OF PROTEINS IN WHEAT

413

(MacMasters et al., 1971 ; Simmonds, 1974). However, these tissues serve an important role in the nutrition of the seed during early grain development (Rijven and Banbury, 1960). I n a hosnozygous, or true-breeding line, the ovary and embryo tissues are genetically identical. I n a heterozygous plant the embryo and endosperm may be genetically different from the ovary tissues. For example, seed color in wheat shows maternal inheritance, since the red-brown phlobaphene color pigment of red wheats is derived from the integument cells of the ovary and is located in the testa portion of the seed-coat tissue. Consequently, the genetic constitution of the plant on which the seeds grow is reflected in the seed-coat color, and all seeds produced on any wheat plant normally have the same seed-coat color. However, the seed embryos (on the same heterozygous pIant) derived from self-pollination will be of three classes with regard to a single red seed coat color gene: 2:0, O:O, and 1 : l ; but the endosperms of the same seeds would carry, respectively, 3:0, 0:O; 1 :2, o r 2 :1 doses of this gene. Endosperm development following fertilization is extremely rapid. I n the first period of endosperm development the early mitoses are not accompanied by cytokinesis. Cell divisions begin with the single large uninucleate, membrane-limited primary endosperm cell as a syncytium. This central cell divides numerous times, producing a multinucleate cell consisting of a peripheral zone of cytoplasm with a large central vacuole. Cytokinesis then occurs, leading to the development of a cellular structure. Disintegration of the nucellus tissue occurs concurrently with cellularization of the multinucleate endosperm; both processes are completed within 1 or 2 days after fertilization. The cellularization first involves the subdivision of the large central vacuole into a number of small vacuoles surrounded by cytoplasmic network and associated nuclei. Cellularization of the endosperm by cytokinesis occurs a t about 4-5 days after anthesis, initiated a t both the dorsal and ventral surfaces of the dense cytoplasmic mass (Simmonds, 1974; Mares et al., 1975), or first near the proembryo (Shealy and Simmonds, 1973), or near the chalaza1 end of the endosperm (Bennett and Smith, 1973). The ventral segment of the central cell is transformed into a layer of thick-walled cells with dense cytoplasm, giving rise to the modified and differentiated aleurone cells of the crease region, which then are no longer meristematic but develop into the pigment strand (Mares et al., 1975). I n the dorsal segment of the endosperm, cytoplasmic lamellae arising a t the plasma membrane project out and grow toward the center of the endosperm. Cell wall material is deposited along the lines of the lamellar projections and within the cytoplasmic strands. The large central vacuole is thus replaced by a mass of thin-walled, highly vacuolated cells. The cells a t the dorsal surface of the endosperm form a meristematic zone identifiable by 7 to

414

CALVIN F. XONZAR

8 days after anthesis, the zones of meristematic cells functioning in the

further development of the endosperm. Division of these cells both radially and tangentially leads to the formation of both peripheral and prismatic cells, and eventually the outer cell layer becomes the thickwalled aleurone which is found all around the outer surface of the endosperm of the mature kernel, except in the region of the crease (Simmonds, 1974; Mares et al., 1975). Cell formation in the endosperm of the wheat kernel is completed at about 12-14 days after anthesis (Sandstedt, 1946; Evers, 1970 ; Simmonds, 1974). Subsequent development of the endosperm occurs by cell enlargement, each cell expanding in size and developing starch grains and protein bodies according to a sequence related to its time of formation; i.e., the inner endosperm cells begin forming starch granules before the final cell divisions in the outer regions are completed, while the aleurone cell layer differentiates further to become thick-walled (Sandstedt, 1946; Buttrose, 1963 ; Evers, 1970; Simmonds, 1974; Mares et al., 1975). The subaleurone cells (immediately inside the thick-walled aleurone layer) are smaller, have thinner cell walls, and contain smaller starch grains than aleurone cells or cells located in the more central portion of the endosperm. Differences in endosperm cell size and development time are considered to be the principal factors responsible for the often-observed high prote'in content of cells located near the aleurone layer as compared to that of cells of the inner endosperm (Evers, 1970; Kent, 1966). Wheats with high protein content normally have higher amounts of residual organelle materials in subaleurone cells than do wheats with low-protein content (Barlow et al., 1974; Simmonds, 1974). Major genes for high protein content appear to proportionately increase protein subaleurone and inner endosperm cells, but may differentially increase protein in the subaleurone region (C. F. Konzak and N. V. Mung, 1976, unpublished). The crease of the kernel forms as a result of the development and expansion of the endosperm within the ovary wall tissues, to surround the relatively rigid "pigment strand" segment of thick-walled cells located toward the inner side of the ovary wall and ventral side of the kernel (Evers, 1970). In the crease, the seed coat joins with the pigment strand to form a complete covering around the endosperm and embryo (MacMasters et al., 1971). The vascular tissues are also located in the crease area of the kernel, all along the length of the pigment strand (Zee and O'Brien, 1970a). There are four vascular tracheids in the ovary, branching from a basal vascular bundle. Three of the tracheids, two lateral, and one dorsal to the embryo sac, extend through the pericarp in the ovary, but disintegrate during early grain development, leaving only the fourth tracheid, which extends through the funiculus and supplies the

GENETIC CONTROL O F PROTEINS I N WHEAT

415

developing ovule (Simmonds, 1974). The thick-walled tissues of the pigment strand, which are pigmented only in “red” wheats, may serve as a supporting structure. However, the porous, thick-walled pigment strand is also thought to have a role in terminating starch accumulation and may aid in water loss during grain ripening (Zee and O’Brien, 1970a,b; Zee, 1975). The vascular tissues which serve as the supply system for the kernel in the wheat spikelet are described by Hanif and Langer (1972).

C. INTRACELLULAR SITES OF PROTEIN SYNTHESIS AND STORAGE The principal site of soluble protein synthesis appears to be the cells of the aleurone layer of the endosperm, which contain few insoluble protein storage bodies and only small starch grains (Sandstedt, 1946; Sandstedt and Beckord, 1946; Stevens, 1973). The aleurone cells are the last cells of the endosperm with a meristematic capability. They evidently terminate their development according to a predetermined ontogenetic sequence involving cell-wall thickening and accumulation of greater concentrations of soluble proteins (enzymes, inhibitors, etc.) than other cells of the endosperm. Protein synthesis appears to involve various classes of ribosomes, some of which are concerned with soluble protein synthesis while others are involved with the synthesis of storage proteins that are accumulated in protein bodies, clearly distinguishable by electron microscopy (EM), until near maturity. At maturity the dehydration processes compress the thin-walled inner endosperm cells and their inclusions, causing considerable distortion and rupturing of membranes that appear to surround the protein bodies (see Graham e t al., 1962a,b; Aranyi and Hawylewicz, 1969; Simmonds, 1972a; Simmonds e t al., 1973; Barlow et al., 1974; Adams e t al., 1976). The ribosomes associated with storage protein synthesis are attached to or enclosed within a rough endoplasmic reticulum (RER) distinguishable in EM photographs. At 14 days past flowering, well-developed protein bodies occurring singly or in groups are identifiable in vacuolar structures. The protein bodies (one or several) appear to be surrounded by a membrane (Graham et al., 1962b; Barlow e t al., 1974). Granular-appearing osmiophilic areas associated with the protein bodies are thought to be ribosomes. The RER shows little development, but there is evidence of free ribosomes. The R E R shows greater development by 21 days, and there is an increase in the number of mitochondria, which together with the R E R create a potential for rapid protein synthesis.

416

CALVIN F. XONZAK

The type of protein body synthesized a t 21 days and beyond differs in appearance from that in younger cells, although the gliadin proteins are identical throughout endosperm development (Barlow e t al., 1974). Bushuk and Wrigley (1971) observed changes in the proportion of glutenin proteins toward endosperm maturity. The synthesis of protein bodies after 21 days past flowering appears to involve secretion of the storage protein into swollen lumina of the RER, and as synthesis proceeds the two parts of a double membrane separate and the ends join to form a single membrane around the protein body, outside of which are ribosomes. The protein bodies remain within the lumina of the RER and are not transported elsewhere in the cell. These observations of Barlow et al. (1974) suggest that the origin of protein bodies in wheat is similar to that observed for Capsella and Arachis (Dieckert and Dieckert, 1972), while confirming the occurrence of a lipoprotein membrane around the protein bodies as observed by Buttrose (1963). On the other hand, scanning EM studies of storage proteins of several cereals isolated using a centrifuge technique showed wheat proteins to be unique in their lack of identity in the mature endosperm, suggesting that a t maturity the protein bodies have been replaced in the endosperm by matrix-type material with a more amorphous characteristic. In all cereals, barley, and sorghum, proteolytic activity appears to be associated with insoluble protein, and isolated protein bodies contain several other enzymes possibly associated with surrounding membrane material (Ory and Henningsen, 1969; Tronier and Ory, 1970; Adams and Novellie, 1975; Adams et al., 1976). The protein bodies of immature wheat endosperms are comprised of protein fibrils deposited in a layered or laminar structure (Bernardin and Kasarda, 1973a). I n the EM photographs of Seckinger and Wolf (1973) , the protein bodies of sorghum endosperm were shown to have a nonprolamine core. The sorghum protein bodies were embedded in a cytoplasmic protein matrix, but only a few seemed to be surrounded completely by matrix protein. The protein bodies of developing wheat endosperm vary in size from a fraction of 1 pm to 15 pm. Soluble proteins also formed in the cytoplasm were separable in a centrifuge supernatant fraction, indicating their association with smaller organelles, or free (unattached) ribosomes, but elements of the RER were also isolated (Graham et al., 1962a). Recent EM and solubility fractionation studies of Ingversen (1975) showed, however, that both prolamine (isopropanol soluble) and glutelin (alkali soluble) components are present in the protein bodies of Bomi barley, but the glutelin component occurs primarily in the high-lysine mutant Bomi 1508. Those observations indicate that glutelins may be integral components of protein bodies, not just RER or matrix material.

GENETIC CONTROL OF PROTEINS I N WHEAT

417

D. GENETICSOF ENDOSPERM PROTEINS Because of the “tri-ploid” nature of the endosperm tissues compared with the “di-ploid” embryo, differences in the expression of endosperm proteins with relation to the genetic constitution of the embryo can be observed for individual seeds formed on plants heterozygous for structural or regulatory genes controlling the content, composition, and/or physical properties of proteins. Those observed differences in expression are primarily due to effects of the different dosages of genes contributed by the polar nuclei ( = twice the same gene content as the egg nucleus) us a single dose of genes contributed by the pollen nucleus. Thus, endosperm cells of the F, generation (in seed formed on F, plants) may contain 0, 1, 2, or 3 doses of proteins unique to one of the parents, whereas the embryos will contain 0, 1, or 2 doses; those with one dose represent heterozygotes corresponding to endosperms with one or two doses of the unique proteins. The seeds produced on some of the F, plants from such a cross will contain either 0 or 3 doses of the proteins, expressing the homozygosity of the embryos and the F, plant, while seeds produced on other plants will be genetically similar to those from the F, plant. When the parents differ by more than one protein, various genetic phenomena including linkage and gene interactions will complicate genetic analyses. Solari and Favret (1968), Favret e t al. (1970), Manghers et al. (1973), and Wrigley (1976a) have illustrated and described the different modes of inheritance of prolamine proteins in wheat and the influence of regulatory mechanisms and gene interactions on the quantitative aspects of protein inheritance. With such analytical procedures as electrophoresis and electrofocusing (Fig. 3) of proteins (Wrigley and Shepherd, 1973; Bietz et al., 1975) as well as less specific single-kernel protein estimation techniques (Brunckhorst e t aE., 1974a,b; Mertz et al., 1974), it is even possible to identify protein phenotypes after growing only the F, plant. However, when protein differences are more complex and genetic linkages as well as gene interactions are important t o the identification of desired recombinants, it may be necessary to conduct analyses on seeds from F2or FSplants or their bulk progeny. 111. Origin and Genetic Relationships among Trificum and Closely Related Species

The modern commercially important wheats are allopolyploids with either two (A and B) or three (A, B, D) genomes or sets of seven chromosomes derived from their diploid (AA, BB, and DD genome) progenitor

418

CALVIN F. KONZAH

FIG.3. Combined gel isoelectric focusing and electrophoresis of proteins from wheat endosperm, by the method of Wrigley and Shepherd (1973). The pattern obtained by electrofocusing in the first dimension appears across the top. The strip a t left shows the pattern given by gel electrophoresis (pH 3) alone. Starch gel was used for the second stage (electrophoresis) in preference to polyacrylamide, since starch gel has proved to be better for resolving this particular mixture of proteins. In general, however, polyacrylamide is preferred. (Reproduced from Wrigley, 1976a.)

species (Table 1).Two main approaches to taxonomic classification of the Triticinae are in use today. The approach followed in this review recognizes Triticum as an evolved hybrid genus, distinguishing Aegilops as a separate genus-mainly because all Aegilops species are wild forms that have followed separate paths of evolution, even though possibly two Aegilops species have contributed whole or parts of whole genomes to progenitors of the cultivated polyploid wheats (MacKey, 1966, 1968b, personal communication, 1975). The second classification system combines all the Aegilops forms into the single genus Triticum on the basis of evidence that the diploid Aegilops forms have contributed equally or more than the diploid Triticum to the genome composition of the polyploid wheats (Bowden, 1959; Morris and Sears, 1967; Sears, 1975a). All the common bread wheats in cultivation are considered subspecies of a single major hexaploid (AABBDD) species Triticum aestivum (L.) Thell. Among the tetraploid (AABB) wheats, only the convariety durum (Desf.) M K of T. (urgidum (L.) Thell. is commercially important today,

GENETIC CONTROL OF PROTEINS IN WHEAT

419

TABLE 1 Subdivision of the Genus Triticum (L.) Sum. According to Genetic Conceptions (MacKey 1975) Monococca Flaksb. 2n = 14

T. monococcum (L.) MK einkorn wheat ssp. boeolicum (Boiss.) MK wild einkorn wheat var. aegilopoides (Bal. ex Korn.) MK var. thaoudar (Reut.) Perc. ssp. monococcum einkorn wheat T . urartu Tum. zweikorn wheat Dicoccoidea Flaksb. 2n = 28

.'2 timopheevi (Zhuk.) MK zanduri wheat ssp. araraticum (Jakubz.) MK wild zanduri wheat ssp. timopheevi zanduri wheat T. turgidum (L.) Thell. emmer wheat ssp. dicoccoides (Korn.) Thell. wild emmer wheat ssp. dicoccum (Schrank) Thell. (true) emmer wheat ssp. paleocolchicum (Men.) NK kolchis wheat ssp. turgidum conv. turgidum Rivet wheat conv. durum (Desf.) MK macaroni wheat conv. turanicum (Jakubs.) MK khorosan wheat conv. polonicum (L.) MK Polish wheat ssp. carthticum (Nevski) MK Persian wheat

Speltoidea Flaksb. 2n = 42

T . zhukovskyi Men. et Er. timon wheat T . aestivum (L.) Thell. dinkel wheat ssp. spelta (L.) Thell. spelt wheat ssp macha (Dek. et Men.) MK macha wheat ssp. vulgare (Vill.) MK bread wheat ssp. compactum (Host) MK club wheat ssp. sphaerococcum (Perc.) MK Indian dwarf wheat

mainly for it.s use in alimentary paste products-spaghetti, macaroni, etc., although in a few areas (southern Italy and North Africa, especially), the flour is used also for flat or leavened breads. The T.aestivum

420

CALVIN F. ICONZAK

T

E T R A

P L

0 I

D

Common Bread Wheals

GENETIC CONTROL OF PROTEINS I N WHEAT

421

wheats may have unique dough functionality properties for breadmaking. Certain of these unique properties are identified with proteins controlled by genes located in the D genome, which is absent from durum and other tetraploid wheats (La Clerc et al., 1918; Yamashita, 1953; Welsh and Hehn, 1964; Wrigley and Moss, 1968; Kaltsikes et al., 1968a; Kerber and Tipples, 1969; Simmonds and Orth, 1973; Schmidt et al., 1974; Bietz et al., 1975; Joppa et al., 1975; Kasarda et al., 1976a). Analyses of the protein composition and properties of wheats are becoming increasingly important not only in research (to identify new sources of genetic variability for the nutritional and agronomic improvement of wheats by breeding, and to better understand the biochemical genetic basis for the different processing quality properties of wheats), but also to trace the evolutionary history of the cultivated species. Certain proteins soluble in 70% ethanol (EtOH), for example, show little intraspecific variation but show distinctive interspecific variations. These EtOH-soluble proteins serve as markers useful for investigations into the phylogenetic and genetic relationships among wheat species and relatives (Hall et al., 1966; B. L. Johnson and Hall, 1966; B. L. Johnson, 1972a,b; Rodriguez-Loperena et al., 1975). A form of diploid einkorn wheat, T. monococcum (L.) MK, ssp. boeoticum (Boiss.) MK, or more likely T. urartu Tum., has contributed the A genome (Chapman et al., 1976), but not the cytoplasm (Suemoto, 1968, 1973; Kihara, 1974) or chloroplasts (Chen et al., 1975), to both tetraploid and hexaploid common wheats (Fig. 4). The “zanduri” group of wheats has evolved parallel to the common wheats. The hexaploid T. zhukovskyi Jakubz. (Genomes AAAAGG) is of most recent origin (B. L. Johnson, 1968), and is cultivated to only a small extent in Georgia, U.S.S.R. The donor of the D genome, Aegilops squarrosa (L.), which differentiates the common hexaploids from the tetraploid wheats, is the one other diploid progenitor of common wheats that is known with relative certainty (Kihara, 1944, 1968, 1974, 1975; Sears, 1975a). In evolutionary history, the D genome addition to the tetraploid genome (AABB) wheats is the most recent of the polyploidization changes that have occurred. The T . aestivum (L.) Thell., ssp. spelta (L.) Thell. (true spelt) progenitor

FIG.4. Probable origin and evolution of cultivated wheats and closely related wild relatives. Parallel evolution of common and zanduri wheat lines of descent. Taxonomy according to MacKey (1975). Schematic incorporates information from B. L. Johnson (1968, 1975, 1976), Maan and Lucken (1972), Shands and Kimber (1973), Xihara (1974), Chen et al. (1975), Chapman et al. (1976), and E. R. Sears (personal communication, 1976). 2’. = Triticum; Ae. = Aegilops; conv. = convariety ; RuDPCase = ribuIose-1,5-diphosphate carboxylase.

422

CALVIN F. KONZAK

of all common wheats arose cataclysmically through the union of unreduced gametes from a natural hybrid (McFadden and Sears, 1944, 1946; Kihara and Lilienfeld, 1949; Stebbins, 1951) between plants of cultivated tetraploid emmer species, T . turgidum (L.) Thell. ssp. dicoccum (Schrank) Thell. in the Caucasus-Eastern Turkey region, where it is believed that the natural hybridization and polyploidization took place. The putative primitive hexaploid T . spelta, which was created under natural conditions, has been artificially synthesized (McFadden and Sears, 1944,1946; Kihara and Lilienfeld, 1949). The main area of uncertainty today in wheat evolution studies concerns the identity of the B genome donor for the emmer progenitor of modern cultivated wheats. The B genome donor species is still unknown, although many of its characteristics have been determined through genetic and cytogenetic analyses. The most widely accepted hypothesis suggests that the diploid B genome donor is taxonomically identified as belonging to the Sitopsis section of Aegilops (Kihara, 1954, 1959, 1974; Sarkar and Stebbins, 1956). Prime candidates considered are Ae. speltoides Tausch. or subspecies Auscheri, Ae. longissima Schweinf. et Munsch, or Ae. bicornis (Forsk.) Jaub et Sp., or an as yet unidentified species or subspecies form. It is now well established that the cytoplasm of cultivated tetraploid and hexaploid wheats is similar to and derived from their B genome progenitor (Kihara, 1966; Maan and Lucken, 1972; Maan, 1973a,b,c). Certain experiments indicate that there is considerable similarity between the cytoplasms of the common wheats and that of Ae. speltoides (Suemoto, 1968, 1973) while other experiments indicate that their cytoplasms are distinctly different (Maan and Lucken, 1972; Maan, 1973a,b,c; Shands and Kimber, 1973). Recently, T. Mello-Sampayo (personal communication, 1976) has obtained evidence of a high degree of chromosome pairing between B genome chromosomes in tetraploid wheat and a variant of Ae. longissima which shows an intermediate amount of chromosome pairing with T . aestivum chromosomes (possibly due t o suppression of the 5B Ph locus (Larsen and Kimber, 1973 ; E. R. Sears, personal communication, 1976). Aegilops longissima appears to have cytoplasm similar to that of Ae. speltoides as well as a high degree of cytogenetic identity with individuals of that species (Maan and Lucken, 1972; Kimber, 1973) and is in fact considered a variety of Ae. speltoides by Morris and Sears (1967). However, in nature the two forms appear to have different mating systems and population structures (Hillel and Simchen, 1973). Aegilops speltoides is an outbreeder (cross-pollinating) while Ae. longissima is self-pollinated, like the wheats. Since the large subunit proteins of chloroplast fraction I protein ribulose-1,5-diphosphate carboxylase (RuDPCase) located in

GENETIC CONTROL O F PROTEINS I N WHEAT

423

the chloroplasts of a t least one Ae. speltoides collection (and presumably, Ae. longissima and Ae. bicornis) are similar to those of T . turgidum ssp. dicoccum and T . aestivum (L.) Thell. (Chen et al., 1975), it seems quite plausible that a form of Ae. longissima may be the B genome donor to the progenitors of cultivated wheats. The evidence that the Ae. speltoides cytoplasm is the same as that of T . timopheevi (Zhuk.) M K (zanduri wheat) and that both cytoplasms bring about cytoplasmic male sterility in common wheats (Maan and Lucken, 1972) is contradicted by the evidence of Suemoto (1968,1973) indicating that the cytoplasm of another strain of Ae. speltoides does not induce cytosterility t o a tetraploid wheat when the genome transfer is made via backcrossing. The basis for this contradiction merits investigation, since i t suggests that cytoplasm variation exists within Ae. speltoides. This is not unexpected, however, since considerable variation appears to occur also among wild tetraploid wheats (Tanaka and Ishii, 1973). Aegilops speltoides was ruled out as the B genome donor for common wheats by both cytogenetic and protein electrophoresis studies (Kimber and Athwal, 1972; Shands and Kimber, 1973). However, the cytogenetic and biochemical genetic studies and cytoplasm-genome substitution experiments indicate that Ae. speltoides is the source of the second or G genome, recently reclassified as S, and of the cytoplasm for the T . timopheevi (Zhuk.) M K group of zanduri wheats, including cultivated form ssp. timopheevi as well as the wild form, ssp. araraticum (Jakubz.) MK = T. dicoccoides nudiglumis Nebalek (Maan and Lucken, 1972; Shands and Kimber, 1973; Kimber, 1973 ; MacKey, 1966, personal communication, 1975). An alternative hypothesis put forth by B. L. Johnson (1975) is that T. urartu Turn. is the source of the B genome while a form of T. boeoticum is the source of the A genome of both the T. araraticum and T. dicoccoides-T. diwccum lines of descent in wheat. Cytogenetic and morphological evidence shows that even though bivalent chromosome pairing is complete, the F, hybrid of T. boeoticum x T . urartu is sterile (B. L. Johnson, 1975; B. L. Johnson and Dhaliwal, 1976). However, fertility in the F, hybrid is restored in the amphiploid, in accord with widely accepted genetic principles behind the creation of successful polyploid species (Stebbins, 1947, 1951). Except for the presence of two quadrivalents (considered by B. L. Johnson, 1975; B. L. Johnson and Dhaliwal, 1976 as two chromosomal interchanges), normal closed bivalent pairing occurs in the amphiploid. Morphological features of the amphiploid closely resemble those of T. dicoccoides types (B. L. Johnson, personal communication, 1976). The cross of T . boeoticum X T. urartu is only successful with T . boeoticum as the female parent, based on over 700 attempts including crosses with a derivative with a largely

424

CALVIN F. KONZAK

T. urartu genome in T . boeoticum cytoplasm. The failure of T . urartu x T . boeoticum crosses to yield viable F,, except in culture, suggests that T. urartu cytoplasm may carry a factor that serves as an evolved genetic barrier causing failure of endosperm development, hence preventing the reciprocal combination (B. L. Johnson and Dhaliwal, 1976; Dhaliwal and Johnson, 1976a,b,c ; Dhaliwal, 1976). Thus, T . boeoticum is considered to be the donor of both the A genome and the cytoplasm to the tetraploid wheats. Electrophoresis studies indicate the presence in T. urartu of proteins found in polyploid wheats but not in any of a large number of T . monococcunz (L.) MK. forms examined. The cultured T. urartu X T. boeoticum hybrid is viable and similar to the reciprocal cross. Crosses of the synthetic tetraploid with T. boeoticuwa or T. urartu generally show six or more bivalents, rarely trivalent or other multivalent chromosomal associations (Dhaliwal and Johnson, 1976a,b,c; B. L. Johnson and Dhaliwal, 1976; Dhaliwal, 1976). However, according to new results of Chen et al. (1975) the chloroplast fraction I (RuDPCase) proteins of T. monococcum, ssp. boeoticum and ssp. monococcum are different from those of polyploid wheats, but surprisingly they are similar to those of Ae. squarrosa. Also, when both tetraploid and hexaploid wheat genomes are transferred to T . monococcum ssp. cytoplasms, the plants produced are both male sterile and weak in vigor (Maan, 1973b). Vigor and male fertility are restorable by crossing with R (male fertility restorer) lines carrying genes for fertility restoration from T. monococcum ssp. boeoticum or from T . zhukovskyi (Maan and Lucken, 1970, 1971a). The results demonstrate that these strong cytoplasmically controlled genetic isolation mechanisms are in fact manifestations of nuclear gene-cytoplasm interaction systems. Separate (possibly linked) genes may be concerned with the restoration of vigor and with male fertility. On the basis of the evidence reviewed above and on the facts that (1) cytoplasm variation (Kihara and Tsunewaki, 1968; Maan and Lucken, 1972; Suemoto, 1973; Mukai and Tsunewaki, 1975; Tsunewaki and Endo, 1973) and (2) chloroplast RuDPCase protein variation (Chen et al., 1975) occurs among related Aegilops species, (3) T . boeoticum and T . uartu show the same RuDPCase large subunit proteins as Ae. squarrosa, and (4) T . boeoticum is an old and widely distributed species showing considerable genetic variability, as does T . urartu, it seems possible that variants of T . boeoticum with chloroplast RuDPCase proteins like the bread wheats yet may be found (B. L. Johnson, 1976, and personal communication). Still other evidence, however, tends to contradict the hypothesis that a form of T . urartu provided the B genome, since a high degree of pairing homology was obtained for T. urartu

GENETIC CONTROL OF PROTEINS IN WHEAT

425

chromosomes with the ditelocentric chromosomes of the A genome, but little association with the B genome ditelocentric chromosomes of hexaploid wheat (Chapman et d , 1976) . Electrophoretic analyses indicate that the esterase and acid phosphatase isozymes of T. urartu are different from those in any other diploid wheat species (Jaaska, 1974). The value of isozyme variations for distinguishing genome homoeologies is questionable (Nakai, 1973), although Tang and Hart (1975) and Hart et al. (1976) have demonstrated homoeology of genes on different genomes using isozymes. The reduction of SS bonds by mercaptoethanol prior to sodium dodecyl sulfate (SDS) electrophoresis has revealed the several expected albumin protein bands of T. boeoticum, which complement those of T . urartu to produce the typical isozyme pattern of the tetraploid wheats (B. L. Johnson and Dhaliwal, 1976, and personal communication). The certainty with which chromosome pairing is viewed as being an indication of homoeologies, is also questionable since chromosome pairing itself is controlled by specific genes (Kimber, 1966; Mello-Sampayo, 1971a,b ; B. L. Johnson, personal communication, 1976). A third concept for the origin of the B genome which is currently being investigated involves the possibility that the B genome may be a complex involving whole chromosomes or parts from one or more donors, brought about following the creation of a primary tetraploid form. The primary tetraploid form would produce triploid hybrids with one or several diploid species growing in close association. Fixation of similar tetraploid forms which also hybridize freely to exchange genes would eventually occur, giving rise t o one or a few related forms able to compete successfully with diploids. These new tetraploids could then carry similar A and B genomes and cytoplasm, but their B genome chromosomes might be a mixture or a complex unlike any one of the diploid parental forms contributing chromosomes or parts of chromosomes to that complex. The occurrence of sufficient geographic separation and/or evolution of genetic isolation mechanisms (via mutation or hybridization) would prevent the breakdown of new tetraploid forms by free hybridization with diploid species. The increased adaptability and larger size of the polyploid forms may have made them successful in a wider range of environments, and eventually they became attractive to man as food sources. This concept, essentially of a pivotal form, is embodied in works by Kimber (1973), Kimber and Athwal (1972), Harlan and Zohary (1966), Zohary (1966), Tanaka and Ishii (1973), Vardi (1973), and Harlan (1975). The various cultivated forms of the tetraploid and hexaploid wheats have evolved naturally along separate paths, in part through conscious (though unplanned) selection by man for adaptability to cultivation and

426

CALVIN F. KONZAK

for specific food and feed uses, as well as for their interesting and attractive differences in plant, spike, and grain morphology (Harlan, 1970, 1975; Harlan and Zohary, 1966; Harris, 1967; Zohary, 1973). With regard to protein content of the various species, Dunstone and Evans (1974) compared cell size, grain size, plant and leaf structure, and percentage nitrogen us grain weight of a number of lines of diploid, tetraploid, and hexaploid wheat species. They noted increases in cell size with polyploidy, but cell size did not increase proportionately with genome content. They suggested that protein content decreased with increasing ploidy. However, recalculation of their data shows that N content per seed is considerably greater for the polyploids and for the cultivated forms. All polyploid cultivated forms have a similar N (protein) content per seed. Recent analyses of a large number of varieties in the USDA World Wheat Collection showed that T. monococcum accessions did not possess a superior percentage of protein as compared with that available in tetraploid and hexaploid wheats (V. A. Johnson and Mattern, 1975; V. A. Johnson et al., 1975b). IV. Genetic Relationships and Homoeologies among Chromosomes of Cultivated Wheats

The cytogenetic, genetic, and biochemical genetic relationships established among and between the 21 pairs of chromosomes in hexaploid wheats show that sufficient homology and genetic compensating ability still exists between the chromosomes of the A, B, and D genomes to consider the three sets of seven chromosomes as nearly homologous or homoeologous series, making possible the construction of unique sets of aneuploid and related stocks useful for genetic analyses. Thus, chromosomes lA, lB, and 1D; 2A, 2B, and 2D, etc., form different homoeologous series, in which any one pair will nearly compensate for the loss or absence of any pair of the other two homoeologous chromosomes in the genome. Evolutionary changes occurring prior to and after and natural synthesis of the tetraploid or hexaploid wheats have differentiated the genetic content of the chromosomes in each homoeologous series such that the compensation is incomplete, and the degree of compensation differs for different homoeologous series (Sears, 1954, 1969). Possibly because of their longer evolutionary history, the chromosomes of tetraploid wheats have become so differentiated that the monosomic series (lacking one chromosome of a pair) were difficult to construct, and the fertility of such plants is comparatively poor (Longwell and Sears, 1963; NoronhaWagner and Mello-Sampayo, 1966; Sears, 1975a). All 14 monosomics of

GENETIC CONTROL OF PROTEINS IN WHEAT

427

durum wheat now have been produced; all are viable, grow well, and set some seeds (Mochizuki, 1968; Joppa, 1973; Sears, 1969, 1975a). Even in hexaploid wheat, many nullisomics and ditelosomics are subnormal, nearly or completely sterile, while nearly all the 42 nullitetrasomics (i.e., lacking a pair of homoeologous chromosomes from one genome but carrying two pairs of a homoeologous chromosome from one of the other two genomes) are reasonably fertile and have essentially normal growth. Likewise, several members of ditelocentric series in hexaploid wheats are viable, but are nearly or completely sterile. These are maintained as heterozygous individuals carrying one or two doses of the other telocentric chromosome arm (Sears, 1954, 1974, 1975a, and personal communication, 1976). Such results demonstrate that, although genetic compensation exists, evolution has produced differentiation in the form of male or female sterility controlled by genes on a particular arm of a chromosome of the series. An extensive array of special genetic stocks has been developed by E. R. Sears and colleagues, not only a t the University of Missouri, but also a t Cambridge, England, and more recently a t many other research laboratories over the world. Lists of the available stocks and progress reports on the construction of new genetic stocks can be found in the European Wheat Aneuploid Cooperation Newsletter published a t the Plant Breeding Institute, Cambridge, e.g., E. W. A. C. Newsletter 4: 1974. Included among the interesting recent developments are the addition and substitution of specific chromosomes from rye (Secale), Agropyron, and Aegilops umbellulata, into wheat, and the progress toward determining their homoeologies (Sears, 1975a). More recently, chromosomes from barley (Hordeum) have been introduced into wheat (Islam et d., 1976). The many diverse genetic stocks constructed already have made wheats unique for genetic, cytogenetic, biochemical genetic, and allied research. Ways in which these stocks may be used in genetics investigations have been described by Kuspira and Unrau (1957, 1958), Law (1968), Law and Worland (1972, 1973), Sears (1954, 1975a), Schmidt et al. (1966), Unrau (1958), Tang and Hart (1975), Hart et al. (1976), and H a r t (1976). V. Role of Environmental Factors in Protein Synthesis and Accumulation in Wheat Endosperm

Some of the nutritional, and most processing characteristics of wheat grain are determined by the quantity and composition of proteins. Although all wheat quality characteristics are genetically controlled, it has

428

CALVIN F. XONZAK

long been known that the cultivation environment is an important modifier. Numerous studies show N availability and water supply to be the most important environmental factors, but soil and atmospheric conditions, especially temperature, light intensity, and photoperiod, as well as prevailing diseases, may also exert strong effects (Finney and Fryer, 1958; Hehn and Barmore, 1965; Kolderup, 1975; Reitz, 1964). Nitrogen availability, temperature, and water supply influence mainly the quantity of proteins accumulated by a genotype, but those factors also may affect the relative proportions of the different amino acids and protein types (Bushuk and Wrigley, 1974; Jahn-Deesbach and Jurgens, 1973; J. A. Johnson et al., 1972; V. A. Johnson et al., 1973; Kasarda et al., 1971; Kolderup, 1975; Mitra and Bhatia, 1973; Orth and Bushuk, 1973b, Pomeranz et al., 1966b, 1974, 1975a; Rhodes and Jenkins, 1975). A. INFLUENCE ON AMINOACID COMPOSITION Lawrence et al. (1958) observed that the lysine composition of wheats varied inversely with protein content and noted differences in the protein composition among wheat varieties and species in response to the cultural environment. A negative curvilinear relation of lysine to protein content was observed in analyses of over 12,600 common wheats in the United States Department of Agriculture (USDA) World Wheat Collection by V. A. Johnson, Mattern, and colleagues of the University of Nebraska, USDA-ARS: United States Agency for International Development (AID) Program (V. A. Johnson et al., 1968a,b, 1975a,b; Vogel et al., 1973; V. A. Johnson and Mattern, 1972, 1975). The negative curvilinear relation for lysine was most pronounced in the range between 7 and 15% protein. I n the range above 16% protein there was little influence of protein and lysine content on lysine per unit protein. Juliano (1972) showed a similar relationship of lysine per unit protein for protein variability above 10% in rice. The variability in protein and lysine content among 6000 durum wheats in the USDA collection was much like that found for common wheats (V. A. Johnson et al., 1973, 1975b). In a later study, less than 1% of 3399 durums analyzed showed significantly higher lysine/protein than the average, while about 4% showed higher than average protein (V. A. Johnson and Mattern, 1975). The nonlinear relations of lysine to protein content complicate the identification of the genetic vs environmental components of lysine variation. Over 50% of the variation in lysine content per unit protein in wheats from the USDA World Wheat Collection is attributable to variation in protein content, largely due to environmental or

GENETIC CONTROL OF PROTEINS I N WHEAT

429

nongenetic causes (V. A. Johnson et al., 1973; Vogel et al., 1973). Since nitrate availability of the cultural environment (Chmeleva and Medredev, 1973; Gunthardt and McGinnis, 1957; Jahn-Deesbach and Jiirgens, 1973; Mitra and Bhatia, 1973) or chemical treatments using Terbacil and Simazine (Strbac et al., 1974) may disproportionately increase the amounts of the lower lysine gluten proteins, it is not surprising that a nonlinear relation of lysine to total protein was observed for a wide range of genotypes (V. A. Johnson and Mattern, 1975; V. A. Johnson et al., 1975b). Heritability estimates for dibasic amino acid (DBAA) content of wheat were only about 27% for F,/F, and F,/F, generation comparisons for 51 wheat lines grown in 6 environments, but they were 96% for thousand-kernel weight (TKW) and 77% for flour recovery (Sharma et al., 1973). A negative relation of lysine to protein (and differences in genotype-environment interactions), also was observed for a group of winter wheats grown in an international array of environments (Stroike and Johnson, 1972). Analyses of essentially single-background genotypes grown in a single environment (location/year) in some cases show a nearly linear relationship of Udy protein or DBC values (based on pH 2.0 buffered acilane orange dye-binding capacity, a measure of dibasic amino acids, including lysine; Udy, 1971) relative to total protein (Siddiqui and Doll, 1973; C. F. Konzak, unpublished data on induced mutations, 1974). Siddiqui (1972) also observed different regression values, but essentially linear relations of DBC value us protein for substitution lines of ‘Hope’ and ‘Timstein’ us S-615 in the same (Chinese Spring) background. Thus, it seems that the variability of genotypeenvironment interactions for protein and lysine may be appreciably less for materials of similar genetic background (as induced mutants) if the cultural environment is also relatively uniform. The increased content of gluten proteins induced by higher nitrate availability may improve the quality of the protein for breadmaking (Kasarda et al., 1971; P. J. Mattern, personal communication, 1974). However, it is not yet clear whether culture under high nitrate availability will increase or reduce the chances for identifying high lysine-producing genotypes. Presumably the total protein production and the amino acid composition of the proteins are controlled by different genetic systems. Results of a study with barley varieties ‘Hiproly’, ‘Risg mutant 1508’, and ‘Maris Mink’, suggest that the greatest differences in grain protein content will be found under conditions of relatively low N supply, while lysine content differences may be relatively stable and unaffected by N supply. However, such results should not be taken as representative of all potential genetic sources for protein and lysine improvement, as they concern only one high-protein and two high-lysine “drastic” mutant gene sources in one crop, barley.

430

CALVIN F. KONZAK

B. INFLUENCE ON PROTEIN SOLUBILITY FRACTIONS Solubility fractionation studies indicate that the protein components of wheat flours may be somewhat disproportionately affected by culture under different environmental regimes (see Kasarda et al., 1971; Mitra and Bhatia, 1973; Orth and Bushuk, 1972, 1973b). This may to a certain extent result also from a hastened or extended maturity, due to weather conditions in particular locations and years. However, Tanaka and Bushuk (1972) observed little influence of protein content on the proportion of proteins in five modified “Osborne” solubility groups (albumins = water soluble, globulins = salt water soluble, gliadins = alcohol soluble, glutenins = acetic acid soluble, insoluble residue). Endosperm solubility fractionation analyses of 70 wheat samples from the Northern Regional Performance Nursery showed that, as protein content increased, saltsoluble proteins decreased according to similar but opposite trends. However, the percentage of acid-soluble proteins decreased slightly and alkaline-soluble proteins decreased moderately with increasing protein content (Ulmer, 1973; V. A. Johnson and Mattern, 1975). These results suggest definite trends toward an increased proportion of prolamine proteins as protein content increases over different environments and are consistent with the observation of lower 1ysine:protein ratios a t higher protein contents. C. GENETICREGULATION AND CONTROL OF PROTEIN CONTENT, COMPOSITION, AND PROCESSING PROPERTIES The functional or processing properties of wheat and wheat flour also may show responses to environmental conditions, and these may in part be a reflection of the influence of environment on the quantity and composition of wheat proteins (Kasarda et al., 1971, 1976a; Pomeranz, 1971). Some of the environmental interactions involved are variety dependent, and thus genotype dependent (G. Rubenthaler, personal communication, 1974; J . A. Johnson et al., 1972; V. A. Johnson et al., 1972a,b; Orth and Bushuk, 197313; V. A. Johnson and Mattern, 1975). Sharma et al. (1973) compared thousand-kernel weight (TKW) , DBC value, sedimentation value (SV) and flour recovery (FR) for 16 varieties and 51 line selections grown under 6 environments over 2 crop seasons in India. They found that the varieties and selections differed significantly from one another with regard to the reEponse of each trait to environments favoring increased response. Of the four traits studied, TKW and FR were most stable and had high heritability as calculated from the regression of F, upon F, values. Least stable was DBC value, which also had the lowest

GENETIC CONTROL O F PROTEINS I N WHEAT

431

heritability. Biuret protein values for the 16 varieties indicated that protein content was consistently higher for some varieties than for others. Jain et al. (1975, 1976) determined that high TKW has great selective value for advancing protein yield in wheat. I n a similar, but more detailed study, McGuire and McNeal (1974) showed that bread wheat varieties diflered with respect to their loaf volume, mixing-time requirements, and flour yields in response to Montana State environments favoring increases in those quality parameters. The comparably high us low flour yields characteristic of ‘Fortuna’ and ‘Centana’, for example, were relatively stable to culture under environments in which other varieties showed either increased or decreased flour yields. Likewise, the variety ‘Manitou’ showed a low response to environments favoring increases in mixing time, while the variety ‘Sheridan’ showed a relatively high response to the same environments. Such similar results from studies conducted continents apart strongly indicate that the responsiveness us stability of kernel characteristics and protein composition and functional (breadmaking) properties to environmental influences is genetically controlled. Environmental factors may assert considerable influence on the lysine composition, but the genotype-environment interactions also may be subject to genetic regulation. The environmental responsiveness of protein and lysine/protein composition of several wheat accessions selected for lysine content from the USDA World Collection was found by V. A. Johnson et al. (1972) and V. A. Johnson and Mattern (1975) to vary rather widely over different years and environments, but certain varieties had high protein or high lysine and high protein more consistently than others. Genotype-related differences in grain yield, protein, and lysine also were observed for wheat and triticale varieties grown a t several test locations (Ruckman et al., 1973). Likewise, durum wheat selections from local Sicilian populations showed marked differences in protein content stability when grown a t 3 different locations in Italy (Porceddu et al., 1973). High molecular weight (MW) glutenin is already present in the endosperm at 14 days past flowering, and the accumulation rates of different solubility classes of proteins vary for different wheats grown under glasshouse conditions. Ratios between gliadin and glutenin proteins change during endosperm development, suggesting that genetic factors also control the rates a t which the various storage proteins are accumulated in the endosperm (Bushuk and Wrigley, 1971). High-protein wheats synthesize greater amounts of protein in their subaleurone tissues (Barlow et al., 1974). The results indicate that there is ample opportunity for environment to modify the final protein composition, depending on the degree of genetic control (regulation) asserted over individual proteins

432

CALVIN F. KONZAK

by varieties (see also Koenig et al., 1964; Mitra and Bhatia, 1973; Manghers et al., 1973). However, a large number of other studies, mainly involving electrophoresis techniques, show that the structure, production (presence or absence), and dosage of specific proteins are under very strong, if not absolute, control of genes (Barber et al., 1968a,b; Bietz and Wall, 1972, 1973a; Bietz et al., 1975; Doekes, 1968, 1973; Doekes and Hack, 1971; Elton and Ewart, 1962; Ewart, 1966a,b; Feillet and Bourdet, 1967; Kasarda et al., 1976a; B. L. Johnson et al., 1967; B. L. Johnson, 1972a,b, 1975; J. A. Johnson et al., 1972; V. A. Johnson et al., 1972; May et al., 1973; Orth and Bushuk, 1973b; Shepherd, 1968; Silano et al., 1969; Wrigley and Shepherd, 1973; Tang and Hart, 1975; Hart et al., 1976). Even in a synthetic polyploid like triticale, it has been shown that the genetic systems for protein synthesis derived from the two parents, durum and rye, both function throughout plant development, essentially affirming the independence and additivity of the genetic systems combined in one nucleus and working in one cytoplasm (Riley and Ewart, 1970; Dexter and Dronzek, 1975a,b; Tang and Hart, 1975). VI. Role of the Cytoplasm in the Hereditary Control of Protein Content and Composition

An understanding of the important cellular components and mechanisms involved in the synthesis of amino acids and proteins may prove to be useful in devising new approaches to the nutritional improvement of proteins (see also Woodbury, 1972). The cytoplasmic proteins are generally among the water- and salt-soluble classes of proteins, some of which contain a high proportion of lysine. Recognized control mechanisms and interrelationships between nuclear genes and cytoplasmic organelles suggest some interesting possibilities and novel approaches toward the goal of improved protein nutritional value. A. CYTOPLASMIC ORGANELLE-NUCLEAR GDNBINTERACTIONS Nuclear-cytoplasmic interactions involved in the synthesis of plant proteins are indicated in a wide variety of investigations, but only in a few cases is there strong documentary evidence. Beck et al. (1971) have reported that 63 nuclear gene loci control the biogenesis, structure, and function of yeast mitochondria, while von Wettstein et al. (1971) have described 198 chloroplast mutations controlled by 86 genetic loci in barley. Nuclear genes in barley maintain a very strong control over both

GENETIC CONTROL OF PROTEINS IN WHEAT

433

pigment synthesis and chloroplast biogenesis (Henningsen et al., 1973 ; von Wettstein et al., 1974). On the other hand, several maternally inherited chlorophyll mutants in barley are well known (Nilan, 1964; Robertson, 1971). In wheat, the genome of the cultivar ‘Salmon’ was found to perform better in Aegilops caudata cytoplasm than in its own (Kihara and Tsunewaki, 1964). Similarly, field trials of several alloplasmic lines derived by substituting the genomes of two hard red spring T . aestivum cultivars ‘Chris’ and ‘Selkirk’ into cytoplasms from T . macha, T . dicoccoides, or Ae. squarrosa, showed some alloplasmic lines t o be significantly earlier heading and higher yielding than the original cultivars (Busch and Maan, 1974). Defective chloroplasts in a variegated mutant of Nicotiana tabacum L. carry a mutation in chloroplast DNA (Wong-Stahl and Wildman, 1973). The DNA alteration was identified by a difference in DNA buoyant density in CsC1, and by EM analyses which showed a mismatched DNA region corresponding to about 500-1000 base pairs. At least 40% of the genetic information in chloroplast DNA is expressed in mature chloroplasts ; but this genetic information accounts for only four proteins-about 10% of the identifiable products ; i.e., the results suggest that chloroplast DNA codes for relatively few structural and enzymic proteins and that its major function is concerned with regulation. Close associations between the informational contents of chloroplast DNA and nuclear DNA have been demonstrated in several other systems (see Kawashima and Wildman, 1971; Wildman et al., 1973, 1975). I n Nicotiana, nuclear DNA is known to code for (1) two of the numerous ribosomal proteins composing the 50 S subunit of the chloroplast ribosome (Bourque and Wildman, 1972), (2) a thylakoid membrane protein to which chlorophyll pigments of photosystem I1 are attached for ferredoxin (about 12,000 MW) which is involved in electron transport and is required for the photosynthetic function of chloroplasts, and (3) the large subunit of fraction I (RuDPCase) protein (Wildman et al., 1975). It is known, however, that RuDPCase is rapidly converted in the dark to membrane proteins (Wildman et al., 1973; Kannangara, 1969; Huffaker and Peterson, 1974). RuDPCase catalyzes the critical step of CO, fixation and also serves as an important temporary protein storage system in the plant (Kannangara, 1969; Huffaker and Peterson, 1974). About 40% of the soluble protein in the green plant leaves appears to be in the form of RuDPCase (Chan et al., 1972; Wildman et al., 1973). The mechanism for breakdown of this protein, the nature of the proteases responsible, the size of the peptide units released and their method of transport to the seed have not been determined. The chloroplast DNA also has been shown to carry two genes that

434

CALVIN F. KONZAK

code for its ribosomal RNA as determined from analyses of the buoyant density of chloroplast DNA and analyses of 23 S and 16 S rRNAs (Thomas and Tewari, 1974; see, also, Whitfeld et al., 1973). There also may be a physical interaction between chloroplasts and the cytoplasm via the vehicle of mitochondria. The movement of mitochondria into and out of the chloroplasts and their apparent involvement in starch granule accumulation in living cells has been photographed (Wildman et al.. 1974). CHLOROPLAST-NUCLEAR GENECONTROL OF B. JOINT RIBULOSE-1,5-DIPHOSPHATE CARBOXYLASE In tobacco species hybrids, chloroplast and nuclear DNA each code for different subunits of the dimeric fraction I protein, RuDPCase (Singh and Wildman, 1973; McFadden, 1973; McFadden and Tabita, 1974). A similar system exists in peas (Boulter et al., 1972). The large subunit of RuDPCase is made on chloroplast ribosomes in peas and in wheat (Roy and Jagendorf, 1974). RuDPCase is the enzyme responsible for photorespiration, and the oxygenase function is located in the large subunit (Andrews et al., 1973; Bowes et al., 1971; Bowes and Ogren, 1972; Lorimer et al., 1973; Ogren and Bowes, 1970). The large subunit of RuDPCase of N . tabacum has a molecular weight (MW) of 56 kilodaltons (Kung et al., 1974a). The small subunit of this enzyme is comprised of two polypeptides of 125 kilodaltons and is made in the cytoplasm on 80 S ribosomes. The large subunit of the RuDPCase of different tobacco species hybrids is comprised of three polypeptides, and the small subunit is comprised of one to four polypetides (Kung et al., 1974b). Each of 15 species of Nicotiana has a different combination of polypeptides, as indicated by the isoelectric points of the proteins on electrophoresis. I n the hybrid N . glutinosa P x N . tabacum 8 , the three large subunit polypeptides were the same as in the female parent (two from each in the doubled hybrid). Changes in the “Osborne” solubility properties of the fraction I proteins are correlated with changes in the composition of the small subunit (Sakano et al., 1974a,b). Fraction I protein thus appears to be an unique genetic marker (Kung, 1976). The large RuDPCase subunit proteins coded for by chloroplast DNA are similar or identical for an Ae. speltoides accession, the tetraploid wheats, T . dicoccum, T . turgidum, T . timopheevi, and the hexaploid wheat T. aestivum, but are distinctly different in electrophoretic mobility from those of T . boeoticum, T. monococcum, T. urartu, and Ae. squarrosa (Chen et al., 1975). Their results (albeit based on single accessions of each species) suggest that the chloroplast genome is similar for Ae.

GENETIC CONTROL O F PROTEINS I N WHEAT

435

speltoides and cultivated polyploid wheats, supporting other evidence that a n Aegilops species similar to Ae. speltoides may be the B genome progenitor of our modern wheats. The results also demonstrate th a t the products of ribosomes under the genetic control of chloroplast DNA (several genes) may interact with the products of ribosomes under the control of nuclear genes to synthesize the important plant enzyme RuDPCase (Wildman et al., 1975). C. CHLOROPLAST-NUCLEAR GENEINTERACTIONS IN NITROGEN REDUCTION Activity of the enzyme nitrate reductase, which reduces nitrate (NO,) to nitrite (NO,), may be associated with grain protein content of wheat. Nitrate reductase is the first, and possibly the main, limiting enzyme involved in the synthesis of protein from nitrate, the primary form of N utilized by plants. Cereals can also utilize some ammonium N, but the ammonium form of N is rarely available in soils. The nitrite reductase function (NO,+NH,) seems to be located within the chloroplasts (Polya and Jagendorf, 1971a,b ; Jaworski and Key, 1972; Dalling et al., 1972; Boulter et al., 1972; Magalhaes et al., 1974; Neyra and Hageman, 1974). The control of nitrite reductase by chloroplasts may be one of regulation via chloroplast DNA, although the requirement for energy and great efficiency of nitrite reductase suggests that this enzyme could be localized in chloroplast ribosomes. Ferredoxin, also located in the chloroplasts, is required for nitrite reductase activity and for RuDPCase activity (Paneque et al., 1964; Miflin, 1974; Warner and Kleinhofs, 1974; Neyra and Hageman, 1974; Wildman et al., 1975; Kwanyuen and Wildman, 1975 ; Klepper, 1975).

D. CYTOPLASM SOURCE AND PROTEIN COMPOSITION IN WHEAT Relatively little specific information is available about the effects of nuclear-cytoplasmic interactions on wheat protein content and amino acid composition. Apparently the T. timopheevi cytoplasm, extensively used in hybrid wheat development, causes no appreciable modification in use quality properties of the grain from tetraploid or hexaploid wheats. Electrophoretic studies are needed to prove that protein differences do not exist. It is known that several restorer lines in T. timopheevi cytoplasm have high protein (G. Rubenthaler, personal communication, 1974; V. A. Johnson and Mattern, 1975). Differences between the electrophoretic patterns of acid phosphatase proteins produced by triticales with cytoplasm from wheat us cytoplasm from rye, have been observed

436

CALVIN F. KONZAK

recently, providing further evidence that the control of certain proteins in the Gramineae involves nuclear-cytoplasmic “gene” interactions (J. Rimpau and G. Robbelen, personal communication, 1974). Marked differences in the effects of different cytoplasm sources on gene expression in hexaploid and tetraploid wheats, including effects on spike fertility and characteristics as well as on plant development and vigor, have been reported (Maan and Lucken, 1971a,b, 1972; Maan, 1973a,b, 1975; Suemoto, 1973; Tsunewaki and Endo, 1973; Endo and Tsunewaki, 1975; Mukai and Tsunewaki, 1975). Two malate dehydrogenase (MDH) isozymes of rye were absent when single chromosomes of rye were added to wheat, suggesting that rye cytoplasm plus two rye chromosomes may be required for their expression, or that the rye M D H genes were epistatic to those of wheat (Bergman and Maan, 1973). VII. Nuclear Gene Control of Protein Content and Composition

Nuclear genes appear to carry the codons specific for nitrate reductase and for many other enzymes, as well as for enzyme inhibitors and storage proteins, although there are indications that some structural proteins are coded for by mitochondrial DNA. A. GENETICCONTROL OF 80 S RIBOSOMAL PROTEINS The number of 80 S ribosomal proteins identifiable by two-dimensional gel electrophoresis is correlated strongly with chromosome numbers when the protein extractions were prepared from hexaploid and tetraploid wheat, diploid barley, diploid rye, common bean, garden peas, or spinach (Nagabhushan et al., 1974). The correlations between chromosome number and 80 S ribosomal proteins were highest for the hexaploid and tetraploid wheats and diploid barley, as compared to the other crop plants, and strongest for the number of basic us acidic or total proteins. The lower correlation of acidic proteins with chromosome number suggests a stronger contribution of cytoplasmic organelles to their control. Evidence for nuclear gene control over mitochondria has been reported recently ; the mitochondrial mass and mitochondrial DNA in diploid yeast is nearly double that in haploids (Grimes et al., 1974), the nucleolus organizer appears to be an important site for RNA synthesis (Jain et al., 1968a,b). Group 1 chromosomes are involved in the control of RNA genes in wheat (Flavell and Smith, 1974a,b). Further details about the cytoplasmic systems and organelles involved in protein synthesis are described by Boulter e t al. (1972) and Hertel (1974).

GENETIC CONTROL O F PROTEINS I N WHEAT

437

Current information about ribosome structure has been reviewed recently (Maugh, 1975). A summary of the proteins now known to be controlled by genes in wheat is presented later.

B. NITRATEREDUCTASE SYNTHESIS AND GENETICCONTROL OF NITROGEN REDUCTION Nitrate reductase is synthesized on the 80 S ribosomes and appears to be localized in the cytoplasm of plant cells. Nitrite reductase which further reduces the nitrite to ammonia is localized in chloroplasts. Nitrate reductase activity (NRA) is NADH (reduced nicotinamide adenine dinucleotide) dependent with requirements both for photosynthesis and respiration ; whereas increased CO, concentration causes an increase in malate but a reduction both in nitrate accumulation and NRA (Wells and Hageman, 1974; Aslam et al., 1973; Purvis et al., 1974), illustrating the competition for photosynthetic energy by carbohydrate production and nitrogen metabolism. The pivotal compound involved in the energy (electron) supply is ferredoxin. Ferredoxin donates electrons directly to nitrate reductase, but to fix C02 in photosynthesis, ferredoxin first reduces NADP to NADPH. Nitrate reduction utilizes 3-phosphoglyceraldehyde, the first sugar formed by photosynthesis, which passes from the chloroplast to the cytoplasm, where it is oxidized by NAD-dependent triose phosphate dehydrogenase, resulting in the reduction of NAD to NADH, which also is used for nitrate reduction. Nitrite formed in the first reduction enters the chloroplast for further reduction by direct photosynthetic energy as indicated above. I n the reduction of a single nitrate molecule to NH,, 8 electrons may be used in reducing 4 molecules of CO, to sugar (Klepper, 1975). NRA in wheat increases with N fertilization (Hernandez et al., 1974), indicating that the synthesis of nitrate reductase is induced by nitrate. Both nitrate and nitrite reductase from wheat are quite unstable in vitro but have been electrophoresed (Upcroft and Done, 1974). 1. Genetic Regulators or Inhibitors of N R A

A low-MW protein isolated from several plant species apparently serves a regulatory function in stimulating in vivo NRA (Ku, 1973). NRA inhibitors occur in leaves of several plant species-corn, beans, and spinach (Ku et al., 1973). The low-MW type inhibitors are heat stable. The lowMW inhibitors appear to be organic acids. One high-MW inhibitor is heat labile, another is heat stable. The heat-labile, high-MW inhibitor appears to be a protein, the heat-stable high-MW inhibitor has not yet been characterized (Purvis et al., 1974; Ku et al., 1973). Malate con-

438

CALVIN F. KONZAK

centration in eight maize genotypes was negatively correlated with nitrate concentration. An enzyme isolated from maize roots causes the inactivation of NRA, and the in vitro inactivation of NRA by the enzyme was prevented by including phenylmethyl sulfonyl fluoride (but not by other trypsin inhibitors) in the extraction medium, suggesting the involvement of a serine residue a t the active site of the inhibitor enzyme. The enzyme involved may be a protease (estimated MW 44,000) which, like NRA, is located in the cell cytoplasm and shows optimum activity a t neutral pH. An extract containing the NRA-inactivating enzyme was found to degrade casein but had no peptidase activity (Wallace, 1974). The increased stability of NRA by the use of 3% (w/v) bovine serum albumin or casein in the NRA extraction medium (Schrader e t al., 1974) suggests involvement of the same enzyme studied by Wallace (1974). The biochemical evidence for regulators and inhibitors of NRA also supports wheat genetics data. Nitrate reduction in wheat is under the control of genes on several chromosomes from each of the three genomes; chromosomes 2A, 7A, and 7B of Chinese Spring were found to carry inhibitors or regulators of NRA (Edwards, 1973,1974).Some of the results reported by Edwards (1974) were not confirmed by E. L. Deckard (personal communication, 1975), although those for chromosome 2A have been confirmed by Warner and Konzak (1975). The substitution of Chinese Spring chromosomes 2A, 2B, and 2D by the homoeologous chromosome from rye significantly increased grain protein content (Jagannathan and Bhatia, 1972; N. Darvey, personal communication, 1974), suggesting that a homoeologous series of NRA inhibitors may be located in the group 2 chromosomes of common wheat. The evidence for inhibitors of NRA, and their identification with particular chromosomes, provides a basis for the genetic removal of these factors as a means to increase the genetic potential for protein production in wheat. Analyses of the group 2 and 7 nullitetrasomie and ditelosomic lines for the inhibitor enzyme may permit greater resolution of the cellular function of the enzyme and its role as an inhibitor of NRA. A number of mechanisms for enzyme regulation have been suggested (Hammes and Wu, 1971). Wheat varieties differ in their response to additional nitrate, in their ability to utilize available soil nitrate, and in their ability to translocate the N into grain protein (V. A. Johnson e t al., 1967, 1973, 197513; V. A. Johnson and Mattern, 1975; Edwards, 1973, 1974; Hernandez et al., 19741, as do oat varieties (Cataldo et al., 1975). Genetic variation for NRA apparently also occurs between individuals within a cultivar (Klepper, 1975), indicating that selection to improve protein productivity may be effective in cultivars derived from F2 to F, populations or from bulks of lines.

GENETIC CONTROL OF PROTEINS I N WHEAT

439

2. Genetic Variation in Response to N and in Protein Content

Seasonal levels of NRA among several wheat genotypes may vary 2-fold or more, and there may be marked differences in the amounts of N translocated to the grain (Edwards, 1973, 1974; Dalling et al., 1975). Nitrogen translocation efficiency also differed among wheat varieties and was correlated with grain protein yield, but not with percent grain protein. Genetic studies demonstrated that NRA was highly heritable but polygenically controlled. Predicted responses to selection for high NRA were 2- to 3-fold greater than the predicted response for yield on protein. Sampling for NRA a t the onset of tillering appeared to be highly effective for selecting high-yielding lines, but was less effective for protein content. Similarly, NRA was associated with vegetative N in wheat (Eilrich and Hageman, 1973; Hernandez et al., 1974; Dalling et al., 1975) and in oats (Cataldo et al., 1975), but not necessarily to grain N. Wheat varieties differ not only with relation to the level of nitrate necessary to induce NRA but also with relation to the rate of response of NRA to increasing nitrate and with relation to the maximum level of nitrate able to induce an increase of NRA (A. Brunetti, personal communication from A. Bozzini, 1974). Wheat varieties may show different seasonal patterns for total NRA per plant and total N content per plant. Dalling et al. (1975) found the cumulative seasonal NRA to be highest for the cultivar ‘Olympic,’ followed by ‘Petit Rojo’ and lowest for ‘Gatcher.’ The selection ‘Argentine IX’ showed the steepest regression slope for total N/per plant with total NRA, with Olympic and Petit Rojo next and about equal. However, the varieties also differed considerably in their ability to translocate N to the grain. Olympic had the lowest translocation efficiency, and Petit Rojo and Gatcher had about equal efficiency. Two groups of varieties showing parallel variation for total seasonal NRA, were distinguishable on the basis of above and below 30% N translocation efficiency (Dalling et al., 1975). The results suggest the independence of NRA and N translocation to the grain, with perhaps fewer genetic factors controlling N translocation. High grain protein thus requires high NRA, plus ability for high N translocation from vegetative to storage tissues (Klepper, 1975). Nittler and Jensen (1974) also observed differences in the ability of barley varieties to respond to available N after being deprived of nitrate in culture solutions for 2 weeks, suggesting that the varieties may differ in their ability to grow under or tolerate conditions of low nitrate availability. The high-protein, high-lysine barley variety ‘Hiproly,’ was found to transfer more N to the grain (per grain basis) and do so longer during grain development than ‘Maris Mink,’ a standard low-protein cultivar (Rhodcs and Mathers, 1974). Hiproly was

440

CALVIN F. KONZAK

found to take up more nitrate from sand culture media than ‘Ymer’ barley, and to translocate more nitrate to the seedling tops in pots over a range of N nutrition levels (Edgar and Draper, 1975). When grown to maturity under gravel culture conditions in a glasshouse, the grain of Hiproly was found to have double the protein content of Risg 1508 and Maris Mink a t the lowest N level, whereas all had similar protein (gm/100 gm dry matter) a t the high N level. The protein of RisZ 1508 contained significantly more lysine than either Hiproly or Maris Mink a t all N levels. Wheat varieties also may differ markedly in their relative yield response to low vs high inputs of N fertilizer (S. A. Qureshi and A. Hafiz, personal communication, 1974). The results suggest that varieties able to produce well a t low N levels may lack NRA inhibitors, hence the genetic mechanisms involved in the response of varieties to low nitrate levels may be general in nature, and can be exploited by breeding to develop cultivars with greater N use efficiency.

VIII. Genetic Variability in the Nature and Properties of Wheat Proteins

A. WHEATENDOSPERM PROTEIN COMPOSITION Among the cereal grains, polyploid wheats have unique properties for making leavened bakery products. These unique properties are largely due to their protein composition. Hexaploid wheats are usually superior for breadmaking (Kerber, 1964; Kaltsikes et al., 1968a,b; Dronzek et al., 1970; Kasarda et al., 1971, 1976a; Bietz et al., 1975; Irvine, 1971). An extracted tetraploid that retained part of the D genome ( l A / l D substitution) had greatly improved baking quality (Kaltsikes et al., 1968a ; Konarev et al., 1972). A chromosome 1D addition line of tetraploid wheat has higher protein content and similar characteristics (Joppa et al., 1975). The uniqueness of the wheat endosperm proteins is reflected in their functional properties and in compositional differences as determined by protein fractionation, amino acid analyses, electrophoresis, electrofocusing, and electron microscope studies. Such differences are also reflected in the range of genetic variability for the species (B. L. Johnson et al., 1967; Kasarda et al., 1971, 1976a,b; Huebner and Wall, 1975; Bietz et al., 1975; Simmonds and Orth, 1973; Rodriguez-Loperena et al., 1975).

1. Protein Composition a. Solubility Fractionation of Wheat Proteins. Various modifications have been made of the procedure of Osborne (1907, 1924) for fraction-

GENETIC CONTROL OF PROTEINS IN WHEAT

441

ating proteins by their extraction in specific solvents. The most widely used solvents are water for albumins, saline for globulins, alcohol for gliadins or prolamines, and acetic acid for glutelins (wheat = glutenins), or NaOH for residue. Numerous protein extraction and fractionation methods have been reported in the literature (see Finney, 1943; Lee and Wrigley, 1963; Jones et al., 1963; Maes, 1966; Mattern et al., 1968a; Huebner, 1970; Landry and Moureaux, 1970; Chen and Bushuk, 1970a,b; Orth and Bushuk, 1972; Gavrilyuk et al., 1973; Bietz and Wall, 1975; Bietz et al., 1975). Most extraction methods show differences between varieties and species of wheats and other crops, but still more standardization is required ; consequently, the results from many experiments are difficult to compare. The greatest difficulties and variations in results have been encountered in the extraction and characterization of the "insoluble" fraction of the glutelin (glutenin) protein as well as in separating from among the glutelins those protein components soluble in alcohol or salt solutions (Finney, 1943; Inamine et nl., 1967; Hoseney et al., 1969c, 1970a; Mecham et al., 1972; Bieta e t al., 1973, 1975; Bietz and Wall, 1975; Huebner and Wall, 1974, 1975). But it should be recognized a t the outset that the solubility group method of fractionation is a completely artificial, very crude and preliminary means for proteh classification. Many proteins have multiple solubilities. Some proteins are complexes associated with lipids or carbohydrates ; others are polypeptides with different amounts of polar us nonpolar components. Some protein subcomponents differ from others only in electrical charge and often may be temporarily complexed until separated by solvent sequences, or by chemical treatment, e.g. reduction, alkylation, or electrofocusing (Kanazawa and Yonezawa, 1973, 1974a,b; Bietz and Wall, 1975). Even the albumins do not all conform to the classical solubility concepts. Some albumin proteins remain in flour after repeated water extraction, but can be extracted by salt solutions, with 0.26 M (NH,) .SO, most suitable (Minetti et nl., 1975). Those results indicated presence of two closely related albumin pools, one bound by salt linkages to some water-insoluble components. Both alcohol and water extracts of flour include such carbohydrate-protein complexes as arabinogalactan-peptides which are present in amounts up to 0.1% (w/w) in wheat endosperm (Fincher et al., 1974; Kasarda et al., 1976a). a-Amylase enzyme appears concentrated in aleurone cells and apparently is contained in sedimentable (lysosomal) fractions, sometimes difficult to isolate (Gibson and Paleg, 1975). However, there is abundant a-amylase in the inner endosperm cells. Protein fractionation studies generally indicate that there are wide differences in the relative proportions of albumins, globulins, prolamines, and glutelins comprising the proteins of the different major cereals

442

CALVIN F. KONZAK

TABLE 2 Protein Content and Solubility Fractions of Cereal Grainsa Protein fractions ( % of total protein) Cereal grain

Protein ( % dwb)*

Wheat Rice Maize Sorghum Rye Barley Oats

10-15 8-10 7-13 9-13 9-14 10-16 8-14

a

Albumins, Globulins, water salt soluble soluble 3-5

Trace Trace Trace 5-10 3-4 1

610 2-(8)? 5-6

Trace 5-10 10-20 -80

Prolamines, alcohol soluble

Glutelins, alkali soluble

40-50 (gliadin) (1-5) (oryein) 50-55 (zein) >60 (kafirin) 30-50 (secalin) 35-45 (hordein) 10-1.5 (avenin)

30-40 85-90 30-45

Considerable 30-50 35-45 5

Source: Brohult and Sandgren (1954); ? and orysin added by author.

* Percent, dry weight basis.

(Table 2 ) . The higher proportions of globulin or glutelin us prolamine proteins in oats and rice are in marked contrast to those of wheat, rye, barley, sorghum, and maize. The amino acid composition of the different cereal proteins shows considerable variation (Table 3) , but analyses in greater detail are necessary to reveal the genetic basis for the differences. Ewart (1967a,b, 1968) compared electrophoretic patterns of several different cereal proteins and amino acid compositions of the various proTABLE 3 Protein Content and Amino Acid ComDosition of Selected Cerealsa

Food Oats Barley Wheat Rye Sorghum Maize Rice References Whole egg Beef (round) Pork (ham, fresh)

Amino acid composition ( % of total protein)

Protein (%)

Lys Met Thr Trp

14.2 12.8 12.3 12.1 11.0 10.0 7.5

3.7 1 . 5 3.4 1.4 2.8 1.5 4 . 1 1.6 2.7 1.7 2.9 1.9 4.0 1.8

12.8 19.5 15.2

6.4 3 . 1 5.0 8.7 2.5 4.4 8.2 2.5 4.6

3.3 3.4 2.9 3.7 3.6 4.0 3.9

1.3 1.3 1.2 1.1 1.1 0.6 1.1

Ile

Leu

Tyr Phe Val Arg

5.2 4.3 4.3 4.3 5.4 4.6 4.7

7.5 6.9 6.7 6.7 16.1 13.0 8.6

3.7 5 . 3 3.6 5.2 3.7 4 . 9 3.2 4.7 2.8 5.0 6 . 1 4.5 4.6 5 . 0

1.7 6 . 6 1 . 2 5.2 1.3 5 . 1

6.0 5.0 4.6 5.2 5.7

6.6 5.1 4.8 4.9 3.8 lj.1 3.5 7.0 5.8

8.8 4.3 5.8 7.4 6.6 8 . 2 3.4 4.1 5.6 5.4 7.4 3.6 3.9 5.2 6 . 1

,. Source: Orr and Watt (1957): ? and oryein added by author.

GENETIC CONTROL O F PROTEINS I N WHEAT

443

tein solubility fractions. His observations indicated that the proteins of oats and maize were markedly different from those of wheat, with only wheat proteins having the composition and properties necessary for bread dough. Some rye proteins were similar to those of wheat. The close relation of rye to wheat is indicated also by the ease with which rye chromosomes appear to compensate genetically for individual wheat chromosomes (Shepherd, 1968, 1973; Sears, 1969). The solubility properties of proteins from the various species appear to be related to a number of chemical, physical and physical-chemical factors. Differences in molecular weight, conformation, electrochemical charges, number and exposure of hydrophophilic vs hydrophobic groups, intra- and intermolecular disulfide bonding, and associations with carbohydrates and lipids, all affect the ease with which proteins can be dispersed in various solvents. However, solubility characteristics also reflect genetically controlled alterations in protein structure, as has been shown for changes in the gene-controlled small subunit of crystalline fraction I (RuDPCase) protein (Sakano et al., 1974a). Until recently, a large portion of the glutenin proteins from wheat has remained unextracted and reported as an insoluble residue, or the total glutenin fraction has been dissolved in urea, which disrupts hydrogen bonding (Simmonds, 1962; Ewart, 196713; Orth and Bushuk, 1972; Bietz and Wall, 1975; Mecham et al., 1972; Cole et al., 1973; Bietz et al., 1975; Huebner e t al., 1974). The conversion of disulfide bonds from intra- to interchain positions occurs by SH-SS exchange reactions (Kanazawa and Yonezawa, 1974a,b). The removal of lipids by petroleum ether improves the proportion of the protein components extractable from wheat flour (Simmonds and Wrigley, 1972). However, petroleum ether also removes certain lipoproteins, and decreases the solubility of the high-MW fraction extractable from flour by 55% ethanol (Garcia-Olmedo et al., 1968; Charbonnier, 1973). The use of a detergent in the extracting solution also helps to release molecules of lower MW, e.g., albumins, globulins, and gliadins, which may be bound among the non-water-soluble and insoluble material (Bietz and Wall, 1973a,b, 1975; Huebner and Wall, 1975; Cole et al., 1973). Wheat amylases are sometimes tightly bound to glutenin (possibly membrane) proteins, but the catalytic sites of these enzymes are evidently located some distance away from the binding site (Rothfus and Kennel, 1970). Reduction and alkylation of the disulfide groups and the use of HgC1, also has allowed separation and electrophoresis of many relatively insoluble high M W glutenin components (Rothfus and Crow, 1968; Mecham et al., 1972; Cole et al., 1973; Huebner et al., 1974; Bietz and Wall, 1975).

444

CALVIN F. KONZAK

It is now clear that the solubility characteristics of proteins also may be changed in some way by the solubilization process, since proteins insoluble in alcohol, for example, become soluble in alcohol after removal from their native tissues or after extraction in another solvent and drying (Bieta and Wall, 1975; Kasarda et al., 1976a). Minetti et al. (1975) likewise found that a large portion of albumins could not be extracted by water, but after extraction by salt solutions they proved to be soluble in distilled water. Bieta and Wall (1975) have defined as glutenins only those proteins that prove to be insoluble in alcohol even after reduction and alkylation, and are soluble in dilute acid. Alcoholsoluble proteins are considered to be gliadins, although some gliadin components prove to be water soluble (Preston and Woodbury, 1975, 1976; Kasarda et al., 1976a). b. Amino Acid Composition of Solubility Fractions. Amino acid analyses of several solubility group fractions from wheat flours have been made by Simmonds (1962) and by Ewart (1968). Simmonds (1962) analyzed the fractions of flours from only two varieties (one low and one higher in protein), but observed only minor variations in amino acid composition. In both varieties the soluble (albumin globulin) proteins were highest in lysine, while protein of the gluten group (gliadin, glutenin, and insoluble residue) was higher in glutamic acid and lower in lysine. Low-protein flour had a higher proportion of soluble proteins. More detailed fractionations of wheat flour by Bushuk and Wrigley (1974) showed that of the soluble proteins, the globulins were highest in lysine. Of the gluten proteins, the “insoluble” residue protein (which remained after extraction of flour by aIcohol and acetic acid) contained more lysine than the lower MW glutenin and gliadin fractions. More recent work of Mecham et al. (1972), Cole et al. (1973), Bietz and Wall (1975), and Bietz et al. (1975) suggest that the “insoluble” proteins are high-MW glutenins. Numerous results reviewed here exemplify the complexities of problems faced by cereal chemists even in the first phase of protein identification: extraction of the proteins from within the storage tissues of the endosperm. A more extensive analysis of the problems involved and progress made in the extraction and identification of individual wheat proteins can be found in a recent review on wheat proteins by Kasarda et al. (1976a). c. Molecular Weight of Protein Components. The water-soluble albumins are of MW under 20,000; salt-soluble globulins of MW 29,000 to 200,000; alcohol soluble gliadins of MW about 25,000-100,000 with most in the range of 36,000; and the relatively less-soluble glutenins of MW to lo6 (Meredith and Wren, 1966). Jones et al. (1961) showed the MW

+

GENETIC CONTROL OF PROTEINS I N WHEAT

445

of purified y - and p-gliadins to be 31,000-37,000 and glutenin M W to average 1.5 t o 2 X loG. The gliadin components of wheat have been generally classified into four main groups (a,alpha; p, beta; y , gamma; and O , omega) based on their characteristics on gel electrophoresis in aluminum lactate buffer. There appear to be several different protein components in each gliadin fraction and with more recent electrofocusing-electrophoresis methods their genetic relationships should soon be clarified (Wrigley, 1968a,b, 1969, 1970a,b, 1971a,b, 1972a,b, 1976a,b; Wrigley and Shepherd, 1973; Leaback and Wrigley, 1976). The size of gliadin molecules appears to be variable. After reduction of the disulfide bonds in gliadin, using sodium dodecyl sulfate (SDS) , and electrophoresis on SDS polyacrylamide gels, Bietz and Wall (1972) obtained components of MW 11,400, 44,200, 69,300, and 78,100 in moderate amounts, while components of M W 25,600, 48,800, and 57,300 were present in trace amounts. SDS-polyacrylamide electrophoresis is widely used for molecular weight determinations and appears to be reasonably reliable (Weber and Osborn, 1969). Ewart (1973) obtained M W values of 32,000-36,200 for a-gliadins, M W 37,800-38,000 for Pgliadins, and MW 38,20044,100 for 7-gliadins. Charbonnier (1974) reported the "-gliadins to have M W values of 64,500-73,000 by gel filtration methods, while Booth and Ewart (1969) found M W 76,000 and 79,000 for these proteins by sedimentation equilibrium measurements. Hamauzu et al. (1974) suggested that the high protein content of the "-gliadins interfered with the complexing by SDS, and the absence of disulfide might allow these proteins to assume conformations that are more expanded than other gliadins in the SDS solvent. Hence, the gel filtration method would show those proteins to have apparently high MW in comparison to other gliadins. Bietz and Wall (1972) determined M W of 36,500 for yl-gliadin and 44,000 for 7,-gliadin by SDS-gel electrophoresis whereas Sexson and Wu (1972) obtained values of 30,300 and 34,700 for these same components by the sedimentation equilibrium method. Platt et al. (19741, Kasarda (1976), and Kasarda et al. (1976b) found that the aggregatable fraction of a-gliadin, which included 4 or 5 subcomponents, appears as a single protein of MW 36,000 on SDS electrophoresis, whereas Platt and Kasarda (1971) obtained a value of 32,000 by amino acid analyses. Stevens (1973) obtained results suggesting that gliadins are comprised of single polypeptide chains containing a n average of four SS bonds each. Other gliadin components have not been described in detail, nor have the more recently developed methods of electrofocusing been widely applied to their study. It is likely that several homoeologous gliadins of the subcomponents observed by Wrigley and Shepherd (1973) will have different M W values and/or differences in molecular charge.

446

CALVIN F. KONZAK

Interestingly, a low-MW component (MW 18,000),considered to be gliadin on the basis of solubility and amino acid composition, also had unusually low amounts of glutamic acid and unusually high amounts of methionine and cystine residues. At acid pH the mobility was less than that of a-gliadin, and a t pH 8.9 the molecule was still positively charged (Ewart, 1975). Because of its small size, this gliadin may represent a protein form resulting from an intramolecular chain termination mutation, or may be the 18,000 MW component found by Bietz and Wall (1972) in reduced glutenin. More recently, a protein of 10,000 MW classified as gliadin because of EtOH solubility was shown to have an amino acid composition similar to albumins (Preston and Woodbury, 1976). Wheat varieties also may have different numbers and relative quantities of gliadin components, although no clear relation with processing properties has been observed (Doekes, 1973; Elton and Ewart 1962; Khrabrova et al., 1973; Tanaka and Bushuk, 1973a; Wrigley and Shepherd, 1974), and may have homoeologous or homologous proteins differing in structure and amino acid composition. The definition of glutenin proteins is an even greater problem, because preparations isolated by many different workers and by many different methods often show considerably less similarity than gliadin or albumin preparations. However, this research area is currently receiving considerable attention by cereal chemists. The most widely applicable definition of glutenin is that this fraction includes less-soluble components, primarily proteins of high MW. Bietz and Wall (1975) indicate that, by the use of various extractants, glutenins from flours sequentially extracted in 0.04 M NaCI, 70% ethanol, 0.1 N acetic acid (HOAC), 0.2 M HgC1, and 0.1% 2-mercaptoethanol ( ME ) differed significantly in their genotype-dependent subunit composition. Upon further fractionation of the HOAC and HgCl, extracts with 70% ethanol, 3&50% gliadinlike proteins were obtained from the different wheats. Huebner and Wall (1975) identified an unreduced glutenin with MW over 20,000,000. Gluten also contains a “high MW gliadin” or “low MW glutenin” fraction which has a MW of 104,000-125,000 (Nielsen et al., 1968; Meredith, 1965a,b; Beckwith et al., 1966; Kanazawa and Yonezawa, 1973). However, the degree of purification, reduction, and chemical modification, as well as the methods of analysis, plays a role in the MW estimates obtained. Bieta and Wall (1973b) found that, after reduction and alkylation, 62% of the glutenin that was first extracted by acetic acid was soluble in 70% ethanol. The ethanol fraction was composed of subunits of MW 44,000 and 36,000 estimated by SDS electrophoresis. The fraction insoluble in 70% ethanol had a higher MW. Bietz and Wall (1972) identified

GENETIC CONTROL OF PROTEINS IN WHEAT

447

15 subunit fractions in reduced glutenin with MW 133,000, 124,000, 102,000, 87,200, 79,100, 71,000, 64,300, 49,400, 44,600, 42,200, 36,000, 32,600, 27,500, 18,000, and 11,600. Other investigators have found the highest MW subunits of glutenin to be 152,000 (Orth and Bushuk, 197313) and 104,000 (Hamauzu et al., 1974), or 98,000 (Kasarda et al., 1976a). These variations in highest M W values for glutenin subunits obtained by SDS-gel electrophoresis apparently are dependent somewhat upon the experimental procedures used, i.e., run time, techniques of gel and buffer preparations, etc. (Cole et al., 1973, 1976). The major portion of glutenin is made up of subunits near M W 44,000 and 36,000. The smaller subunits appear to have MW near 12,000, similar to albumins, and may in fact be albumins that wcre bound in some way with the glutenin complex (see Rothfus and Kennel, 1970). Most important, the proportion of glutenin subcomponents in the MW range 35,000-50,000 is sufficient for them to have a considerable influence on the amino acid composition and properties of native gluten. Bietz and Wall (1973b, 1975) noted the gliadinlike character of the 44,000 MW subunits found in the ethanol-soluble proteins of reduced and alkylated glutenin, and in acetic acid or mercuric chloride extracts of flour. They considered these subunits to be distinct from the high M W “purified” glutenin. Wheat flours contain many proteins differing widely in MW and other properties. Results from studies on peptic enzyme hydrolyzates provide evidence that components of gliadin and glutenins of wheat also may differ in primary structure, i.e., amino acid sequence (Bietz and Rothfus, 1970, 1971; Bieta and Wall, 1975; Huebner et al., 1974). The amino acid composition and sequence in “purified” glutenin preparations is currently under investigation (J. A. Bietz, personal communication, 1975). Bietz and Wall (1975) consider that it is no longer adequate to define glutenin by solubility in the classical manner, since it is a complex of proteins with MW ranging into the millions, and with characteristic spectra of disulfide-bonded subunits, some similar t.0 gliadins and others over MW 100,000. They noted that after extraction with acetic acid much of the glutenin remains undissolved either as gel or residue protein, which they found could be solubilized by HgCl,, surfactants, hydrogen bond-breaking agents, or reducing agents. The classical extracts of glutenin were found to contain other proteins separable by reprecipitation upon adjustment of pH, gel filtration chromatography, or SE Sephadex C-50 procedures. Such impurities were considered responsible for inconsistencies in reported properties and composition of glutenin. The observations by Bietz and Wall (1975) appear to support those of Bernardin and Kasarda

448

CALVIN F. KONZAK

(1973a) and Bernardin (1975), which suggest that proteins of the gliadin proteins may exist in unusual conformations or aggregations, which affect their solubilities in water, ethanol, or acetic acid. Thus, Bieta and Wall (1975) found that gel proteins ( = glutenins) first extracted in acetic acid (HOAC) or HOAC-HgCl, could then be solubilized in 70% ethanol, proving to be gliadinlike. Bernardin (1975) considers that most of the gluten proteins may be gliadins, but artifact changes caused by the common protein fractionation procedures temporarily alter their water and ethanol solubilities, which may be reversed once they are separated out by other procedures. Isoelectric focusing also has been applied recently to the separation of component polypeptides of glutenin, and should be useful in biochemical genetics studies (Mita and Yonezawa, 1971). The functional (breadmaking) properties of wheat flour are due largely to the properties and interrelations among the gliadin and glutenin components of gluten (Hoseney and Finney, 1971; Pomeranz, 1971, 1973b,c; Kasarda et al., 1971, 1976a; Bushuk and Wrigley, 1974). Similarly, the glutenin: gliadin ratio is important to pasta-making quality (Wasik and Bushuk, 1975a,b; Matsuo and Irvine, 1975). Other wheat flour proteins and lipids, including glycolipids may exert important modifying effects (Pomeranz et al., 1966a, 1970; Pomeranz, 1971, 1973b,c; Pomeranz and Finney, 1973a,b). Much of the recent research on solubility fractionation indicates that characterization of protein components in terms of the Osborne method does not adequately describe the chemical and physical nature of proteins or their interrelationships (Silano et al., 1969; Silano and Pocchiari, 1971; Mecham et al. 1972; Mecham, 1973; Minetti et al., 1973; Wasik and Bushuk, 1974; Huebner et al., 1974; Huebner and Wall, 1975; Bietz et al., 1975; Bietz and Wall, 1975; Kasarda et al., 1976a). Rather, a number of criteria may be needed, and even then it may not be possible to classify all protein components in simple terms. Moreover, Bernardin and Kasarda (1973a,b), Mecham (1973), and Bernardin (1975) suggested that many features, especially the relative insolubility and molecular sizes of many gluten proteins, may be a consequence and, therefore, are artifacts of chemical and physical changes that occur upon rewetting and during the solubility fractionation processes. In this regard, Charbonnier (1974) found that several protein bands revealed a t p H 8.9 by electrophoresis of a fraction which gave only one band a t pH 3.2 correspond to different amidation levels of the same protein. Likewise, Mosse (1973) showed that partial hydrolysis of amide groups occurs during storage of gliadin in buffer. Torres and Hart (1976) recently found that several homoeologous acid phosphatase isozymes distinguishable by elec-

GENETIC CONTROL O F PROTEINS I N WHEAT

449

trophoresis had essentially the same MW of 58,000 but were distinguishable because of differences in molecular charge. Bernardin and Kasarda (1973b) and Bernardin (1975) observed that marked changes in the physical and chemical characteristics of gliadin take place when high concentrations of those proteins reabsorb water. Rehydration of gluten proteins in flour or in sections of wheat endosperm was observed to be extremely rapid, producing a two-phase system of protein fibrils in highly concentrated form. Also, Shogren et uZ. (1969) showed that pH of the solvent for separating proteins has a major influence on the proportion of gliadins us glutenins extracted from flours, and that increasing amounts of proteins could be extracted with additional water extractions of gluten. Recent work by Bietz and Wall (1973a,b) indicates that as much as 757'6 of wheat protein may be gliadin or gliadinlike components. Bernardin (1975) considers the solubility properties of the protein fibrils to be largely those of gliadin, as expressed at high protein concentrations. When wet with aqueous solvents, gliadin proteins form a hydrated mass, which dissolves to varying degrees depending on the ability of the solvent to dissociate the aggregated proteins in the form of microfibrils lying parallel to one another. The liquid mass is considered to be a liquid crystalline form of the protein where the aggregate exposes a minimal surface to the solvent, while the degree of dissolution reflects the equilibrium concentration of the two phases. Variations in solubility and other properties of proteins may be affected also by small differences in amino acid composition as well as by variations in component subunits comprising the molecules (Kasarda et al., 1971 ; Bernardin, 1975). Binding with lipids, carbohydrates, and other components can also modify solubility. Garcia-Olmedo et uZ. (1975 ; personal communication) have found that the lipoprotein purothionins are soluble either in petroleum ether or in saline, whereas dilute sulfuric acid preferentially extracts the protein moiety, and ether the lipid moiety. The components of glycoproteins, and other carbohydrate protein complexes found in flour, can likewise be expected to be affected by solubility fractionation and chemical modification procedures. p-Amylase has been found adsorbed on glutenin, possibly representing its natural association in a n organelle membrane, although i t is abundant in cell walls and in bran (Kruger, 1973). The complexing of protein subunits can occur in vitro as well as in vivo. Mitra and Bhatia (1971) and Irani and Bhatia (1972) have shown that mixtures of wheat and rye proteins form the same dimer isozymes of alcohol dehydrogenase as appear in protein extracts of triticale or of wheat with a disomic rye chromosome addition. Some of the isozymes formed are combinations of the distinct

450

CALVIN F. RONZAK

monomeric components from wheat with component from rye. H a rt (1971) has obtained similar results for homoeologous isosyme monomers of wheat. 2. Physical Structure of Wheat Gluten Proteins

A number of wheat proteins have been isolated and studied extensively (see Wu and Cluskey, 1965; Wu et al., 1967; Kasarda, 1970; Kasarda et al., 1967, 1971, 1974a,b, 1975, 1976a,b; Mecham, 1973; Bernardin and Kasarda, 1973a,b; Silano et at., 1973; Nimmo et al., 1974; Bedetti et al., 1974). Using transmission EM, Seckinger and Wolf (1970) observed that dispersions of gliadins produced filmlike structures, while glutenins formed strands. Likewise, Crozet et al. (1974) observed that gliadins and four protein fractions high.in gliadin had a smooth, compact, and nearly electron-translucent structure, whereas glutenin and fractions high in glutenin had a granular and fibrillar structure. The fibrils of glutenins had a diameter of 100-700 A and formed a dense network. The structures of albumins and globulins appeared to be much more like glutenins than gliadins. Kasarda et al. (1967) observed reversible aggregation of a-gliadin to fibrils, depending on p H and ionic strength. The fibrils are about 80 A thick and several thousand angstroms long; they dissociate to globular protein subunits at very low ionic strength and pH 3 (0.001 M HCl), but are re-formed by increasing the pH to 5 and the ionic strength to 0.005 M . Bernardin and Kasarda (1973a,b) showed that wheat endosperm protein is present in sheet form, and they suggested that the sheets result from a laminar deposition of the storage protein in the protein bodies of the developing wheat grain. The sheets of endosperm protein in the protein bodies rupture under stress and, when wetted, form webs of protein composed of different-sized fibrils. The larger fibrils proved to be aggregates of the smaller fibrils interacting laterally. The observations suggest that the fibrillar wheat gluten proteins are comprised of subunits bonded together and stabilized by intramolecular disulfide bonds, and that there are no intermolecular bonds or cross-links joining the subunits of the fibrils to one another (Mecham, 1973). Complexing and conformational changes affecting the solubility of proteins may be introduced by disulfide interchange reactions involving glutathione or similar SH-bearing amino acids or SH-peptides. The SH-molecules are brought in contact with the proteins of protein bodies upon rehydration of the flour during dough formation or protein solubility fractionation.

GENETIC CONTROL OF PROTEINS IN WHEAT

451

Rather extensive recent work by Orth e t al. (1973, 1974b), using scanning EM, clearly shows differences in the ultrastructure of glutenin proteins that may provide a basis for the different rheological properties of flours from rye, triticale, durum, and common wheats. I n their studies, the glutenin from a hard red spring wheat cultivar ‘Manitou’ had a characteristic fibrous structure, including many thick strands (10 pm in diameter) intertwined with thin strands (1 pm). I n addition there was a general alignment in which cross sections of the fibers appeared cylindrical, suggesting a major unidirectional organization of the fibers ; and there was filmlike material, especially at the point of contact between the strands. On reduction by p-mercaptoethanol and 6 M urea, all the fibrous structure was lost, leaving only an amorphous mass. Four fractions of unreduced glutenin separated by gel filtration had different heterogeneous structures (Orth et al., 1974b). The fraction with highest M W contained disk-like particles connected with fibrous strands. The next highest MW component had large, platelike protein particles of different sizes. The third of the four fractions contained many small particles associated with few fibrous protein strands, and the fourth fraction had small aggregates of rather amorphous particles. The highest MW fraction was much more heterogeneous than the unfractionated, purified glutenin preparation obtained by pH precipitation (Orth and Bushuk, 1973a). I n the same study, the purified glutenin from ‘Canthatch’ appeared similar to that from Manitou, but had more intertwined, stringy fibers containing also some filmy materials. The tetraploid wheats, including a derived tetraploid with Canthatch A and B genomes (‘Tetra-Canthatch’) , contained a large amount of filmy material and large, flat ribbonlike structures quite unlike those of the bread wheats. The structure of the durum and Tetra-Canthakh glutenins was consistent with their lower elasticity and poorer breadmaking properties. Studies also of purified glutenin from Ae. squarrosa, the D genome source for hexaploid wheat, showed this protein to have the fibrous structure typical of the bread wheats; whereas removal of the D genome from the hexaploid wheat, as in Tetra-Canthatch, caused the purified glutenin to lose the more fibrous structure and take on a more amorphous appearance. Purified glutenin from rye had characteristic rodlike structures, but there were very few of the thin fibers typical of bread wheats. Rye glutenin differed from that of durum wheats and Ae. squarrosa. Glutenin from triticale (eelection 6A-190) had structures expected from the combined contributions of its parents (Orth e t al., 1974a), consistent with the relatively lower elasticity and relatively poor rheological properties of its flour. These results are strikingly similar to those reported by Bernardin and Kasarda

452

CALVIN F. KONZAK

(1973a,b) and Bernardin (1975) in studies on seed and flour proteins and on purified fractions of a-gliadin. A current concept of glutenin molecules is that it is comprised of a mixture of subunit proteins linked together by intrasubunit SS bonds in the form of concatenations (Greenwood and Ewart, 1975). End-to-end linkage of different numbers and sizes of monomer subunits into rather high polymers with MW into the millions may occur, depending probably on the genetic characteristics of the variety. The variety ‘Red River 68’, for example, appeared to contain extremely high MW glutenins compared with other varieties (Huebner and Wall, 1975). Any disulfide cross-linking between subunits is restricted to that permissible in linear polypeptides, which probably are subject to bonding together by secondary forces, such as may involve hydrogen ionic and hydrophobic bonds. The shape of the molecules and their intermolecular associations of the concatenations also depend on p H and other factors, such as hydration and association with free thiols (Kasarda et al., 1976a). Sulfhydryl bonding between glutenin molecules was thought by many to be necessary t o account for the alterations achieved by various chemical treatments and the actions of oxidizers and thiols in dough. However, the appearance of purified glutenin as fibrillar components rather than a more complex or amorphous mass in scanning EMS is not consistent with the concept of intermolecular disulfide bonding, and it is now thought that oxidizers and dough improvers mainly inactivate the free thiols in flour and dough (Greenwood and Ewart, 1975; Kasarda et al., 1976a). 3. Relative Increase of Endosperm Proteins during

Grain Development and Maturation Proteins of the different Osborne solubility classes are laid down in the cereal endosperm a t different rates during the various stages of grain development and maturation (Fig. 5 ) . I n the early stages of endosperm development, the highest proportion of the N is in the form of albumins, globulins, and nonprotein nitrogenous substances (Mitra and Bhatia, 1973; Manghers et al., 1973). As development proceeds, prolamine synthesis increases a t a very rapid rate, while glutenin synthesis increases a t a somewhat slower rate. I n the later stages of development, the larger proportion of N is incorporated into prolamines, while N in the form of soluble and insoluble proteins increases a t slower rates. The relative proportions of different protein components in different wheats appears to be similar during all development stages, with the maximum amount of low-MW materials occurring a t 14 days past flowering, and the minimum a t maturity (Bushuk and Wrigley, 1971). They found the proportion of albumins to be somewhat higher a t 14 days past flowering in the hard red

GENETIC CONTROL O F PROTEINS I N WHEAT

0.9

K

A

453

L NITROGEN

RESIDUAL PROTEINS

LUBLE NITROGEN DAYS OF DEVELOPMENT

FIG.5. Accumulation of different solubility classes of wheat proteins with relation to endosperm development. (Redrawn from Mitra and Bhatia, 1973.)

spring (HRS) wheat cv. RIanitou than in the soft white winter (SWW) cv. ‘Talbot’ or in the durum cv. ‘Stewart 63’, but a t maturity the proportions were the same in all three wheats. Acetic acid-soluble glutenin was found to comprise 2070 of the total protein as early as 14 days past flowering. During the final stages of endosperm development, the glutenin fraction increased markedly in the HRS wheat, less in the SWW wheat, and little, if a t all, in the durum. In maize, mutants opaque-:! ( o p ) and brittle-2 (bt,) and in sorghum,. mutant hily ( h l ) greatly affect the proportions of proteins synthesized during endosperm development (Singh and Axtell, 1973; Misra and Mertz, 1975a,b; Jambunathan et al., 1975). Both single mutants (0202 and bt,bt,) and the double mutants ( orozbtrbtp)had higher levels of albumins and globulins throughout development. Synthesis of the low lysine zein proteins was evident a t 14 days in normal endosperm, but only a t 18 and 21 days for bt,bt, and 0 2 0 y .The net zein synthesis rate was also slower, resulting in 50% less zein a t maturity in each single mutant, while no zein a t all was synthesized by the double mutant o,o,btzbt, (Misra and Mertz, 1975a,b; Misra et al., 1975a,b,c). The rates of growth and dry matter accumulation in developing wheat grains have recently been found t o show periodic fluctuations, with

454

CALVIN F. KONZAK

frequency maxima of about 3 4 , 6 7 , and 10 days (Jenkins et al., 1974). Another concept beginning to emerge from several studies is that the different isozymes of a given enzyme change markedly in intensity and presence in various tissues of the developing plant and kernel during development. The extent of genetic control over formation us inactivation of the enzymes is still not clear (Glasziou, 1969). Up to 12 peroxidase isozymes occur in different anatomical parts (pericarp, green layer, ovary wall, aleurone, embryo, scutellum) of the kernel, changing in intensity during kernel development with different distributions of total enzyme activity over time for a durum as compared with a bread wheat variety. Similar peroxidase isozymes were present in both species of wheat, although one variant appeared to be absent in the hexaploid (Kruger and La Berge, 1974). Four types of amylase activity including that of P-amylase are distinguishable in developing wheat grain (Meredith and Jenkins, 1973). High levels of amylase activity are associated with outer tissues during early grain development. These appear to be P-amylases (Kato et al., 1974). Thermal stability also varied for two other activities associated with breakdown of soluble starch, with patterns associated with specific cultivars. Proteolytic enzyme levels also change during growth and maturation (Kruger, 1973). Likewise, mono- and o-diphenolase activities varied in hexaploid wheats during kernel development, although the relations of activity to different tissues were not determined. Monophenolase activity was low until about 33-36 days after anthesis after which it rose sharply to a peak a t 42 days to fall again thereafter. Monophenolase was considered to have a role in the formation of seed-coat pigment. Twelve o-diphenolase isozymes were identified in the developing kernel, with the relative intensities (strong to none) varying throughout the development period. No one isozyme was found at all sampling times, and nearly all showed increases and/or decreases in intensity (Taneja et al., 1974). Presumably, the same isozymes described as polyphenolases (PPO) also were observed in different parts of the developing grain, with a large part of the activity in the endosperm (Kruger, 1976). The activities of seven acid phosphatase isozymes also showed tissue and developmental variation in relative intensity as measured in 7-day shoots, leaves, leaf sheaths, stems, peduncles, and in the endosperm and scutellum of kernels. Five other enzymes (anthranilate synthetase, anthranilate-5 phosphorylalase-1 pyrophosphate phosphoribosyl transferase = PR transferase, N-5’-phosphoribosylanthranilate isomerase = PRA isomerase, indole-3-glycerol phosphate synthetase = InGP synthetase, and tryptophan synthetase) in wheat differ in relative intensity in different plant tissues and a t different plant developmental stages (Singh and Widholm, 1974). Isozymes homoeologous

GENETIC CONTROL O F PROTEINS IN WHEAT

455

to each of the major isozymes in hexaploid wheat which are present in one or another of the progenitor species also showed similar developmental and/or tissue specificity (Torres and Hart, 1976). The further identification of isozymes with regard to substrate specificities and responses to inhibitors, should eventually clarify the role of these enzymes in plant biochemistry. Some progress in this regard has been reported for maize esterases (MacDonald and Brewbakcr, 1975), for wheat &-amylases (Tkachuk and Kruger, 1974; Tkachuk, 1975), and for wheat esterases (Cubadda and Quattrucci, 1974). The various molecular forms of enzymes appear to have a role in differentiation, but no clear understanding of their role has yet emerged (Scandelios, 1969, 1974; Hart, 1976, personal communication).

4. Specific High-Lysine Proteins The lipoprotein purothionins from wheat and barley (so named because of their high sulfur content, i.e., cysteine 16%) also have an exceptionally high lysine content and contain about 18% N (Balls et al., 1942; Nimmo et al., 1968, 1974). a. Occurrence and Concentration. Purothionin from barley is similar to, but distinct in composition from that of wheat (Nimmo et al., 1974). Purothionins were not found in rye, maize, or oats (Redman and Fisher, 1968, 1969; Fisher et al., 1968). I n polyploid wheats these lipoproteins occur as two electrophoretically distinct, but analogous forms. Both forms (a,p ) are present in the hexaploid and tetraploid wheats, whereas only the /3 form occurs in the diploid T . monococcum, and the form occurs in several Aegilops species, including Ae. squarrosa and Ae. speltoides (Carbonero and Garcia-Olmedo, 1969; Mecham, 1971). The concentration of purothionins was reported to be appreciably higher in certain varieties of hexaploid wheat than in durum wheat. All of 26 varieties of durum had less than 20 mg of purothionin/100 gm of flour, while all but four out of 40 T. aestivum varieties had over 20 mg/100 gm of flour, and some had as high as 78 mg/100 gm of flour (Carbonero and Garcia-Olmedo, 1969). However, as will be noted later, these differences may be artifacts of the extraction technique, since only about one-tenth as much purothionin could be extracted by petroleum ether as by dilute sulfuric acid (F. GarciaOlmcdo, personal communication, 1975). However, the purothionins may account for more than 0.07 to 0.09 gin of lysine per 16 gm of N, or about 0.1 gm of lysine per 100 gm total protein, in 11.4% protein flour from hexaploid wheat (based on values from Garcia-Olmedo et al., 1968; F. Garcia-Olmedo, personal communication, 1975). This suggests that compared with other seed proteins, purothionins contribute disproportionately to the total lysine composition and, as individual proteins, are a major (Y

456

CALVIN F. KONZAK

source of the lysine present in wheat. New evidence, however, indicates that the purothionins themselves may possibly be extraction artifacts (Garcia-Olmedo, personal communication, 1976). b. Physical Properties. Recently, Nimmo et al. (1974) presented further data on the physical characterization of wheat purothionins. They found that the LY and p forms, which differ slightly in amino acid composition and have different mobilities in gel electrophoresis, do not differ in other physical properties. Molecular weights for the LY and p forms as determined by sedimentation equilibrium were 5100 and 5300, respectively, whereas MW values for both forms determined were 6100 (by osmotic pressure) and 7500 by gel permeation chromatography. Studies of the circular dichroism spectra of the purothionins suggested a 40% a-helical structure, while intrinsic viscosity data suggested a compactly folded spherical globular structure. The amino acid sequencing studies (60% completed) indicate that LY purothionin has a MW of 5500, with a high cysteine content (four SS bonds/mole) and lysine located in the N-terminal position (Mak and Jones, 1974). Recent evidence of Mak and Jones (cited by Kasarda et al., 1976a) suggests that the remaining residues of apurothionin may correspond to the components identified by HernandezLucas et al. (1974)-one related to the B genome and containing isoleucine, the other related to the D genome and containing no isoleucine. G. Differential Afjinity for Protein Dyes. Purothionins have a strong affinity for acidic protein dyes (Redman and Fisher, 1968; Nimmo et al., 1968; Silano et al., 1969; Lawrence et nl., 1970). Lysine-containing proteins stain green, albumins stain black, and gliadins stain red with aniline blue-black dye (Silano et nl., 1969; Silano and Pocchiari, 1971). This differential staining results from differences in coprecipitation tendencies of the proteins with impurities vs normal components in the dyes (Minetti et al., 1973). The differential staining of electrophoretically separated proteins may be related to their content of free basic amino groups and thus to the lysine and other dibasic amino acid (DBAA) composition (Lawrence et al., 1970). Varieties of durum and bread wheat differ in their content of water-soluble gliadin and albumin proteins detected using aniline blue-black stain on electrophoretic gels (Minetti et al., 1971). Nigrosine dye was found to be useful for identifying proteolipid CM (chloroform-methanol extract) proteins in wheat (Aragoncillo et al., 1975b). Studies using flourescamine (Fluram) and carbocyanine dyes for staining and identification of proteins also are pertinent here: (1) A fluorescent antibody technique using fluorescamine showed differences in the local concentrations of soluble proteins in sections of wheat endosperm (Barlow et al., 1973a). (2) Staining of proteins with fluorescamine prior to separation on polyacrylamide-gel electrophoresis avoided fixation and

GENETIC CONTROL O F PROTEINS I N WHEAT

457

destaining procedures (Zak and Keency, 1974). (3) Fluorescamine labeling did not affect the mobility of proteins on polyacrylamide gels (Pace et al., 1974). (4) Amino acid analyses could successfully be made of Fluram-stained protein bonds cut from polyacrylamide gels (Stein et al., 1974). (5) The cationic carbocyanine dye, “Stains All,” 1 ethyl-2-[3(ethyl -naphtho [ 1,2d]thiazolin - 2 - ylidene) - 2 - methylpropenyl] -naphtha[ 1,2d]thiazolium bromide, differentially stained phosphoproteins, carbohydrate and sulfate carbohydrate-containing proteins of histological sections (Green and Pastewka, 1974a,b). “Stains All” is a sensitive stain for nucleic acids separated electrophoretically on polyacrylamide-agarose composite gels. Ribonucleic acid (RNA) stains bluish-purple, deoxyribonucleic acid (DNA) stains blue, and protein stains red (Dahlberg et al., 1969). The blue-stained proteins of Dahlberg et al., (i.e.) proved to be phosphoproteins (Singer, 1952; Haag et al., 1971 ; Green et al., 1973). The development of differential staining techniques for laboratory identification of higher lysine-containing endosperm proteins may be feasible. Such techniques may prove useful in genetics and breeding experiments and for the identification of specific proteins upon electrophoresis. d . Role in Processing Quality and Biological Properties. Purothionins apparently have no function in breadmaking (Hoseney et al., 1970b), but their high cysteine (SS) composition, relatively small molecular size, and relative abundance suggest that they could play a role in the SS-SH exchange reactions during dough development. Stuart and Harris (1942) , Fernandez de Caleya et al. (1972), and Hernandez-Lucas et al. (1974) showed that the purothionins have in vitro antibiotic or antifungal activity, but their biological function in wheat remains a mystery (Nimmo et al., 1974). Evidently their influence on human and animal nutrition is also unknown, although Stuart and Harris (1942) found purothionin to be toxic when intraperitoneally injected into mice. e. Genetic Control of Synthesis. The synthesis of the (Y and p holoprotein components of the purothionins is controlled by chromosomes of group 1 in wheat (Garcia-Olmedo et al., 1975). The different electrophoretic mobilities characteristic of the and p analogs are evidently due to evolutionary changes that occurred in one of the diploid ancestors (probably the A genome donor) prior to the natural origin of the tetraploid wheats. Synthesis of the lipid moiety of these purothionins is controlled by group 5 chromosomes. Using dilute sulfuric acid instead of petroleum ether to extract purothionins of Chinese Spring wheat, Garcia-Olmedo et al. (1975) observed that the same relative proportion of the protein component was produced by the group 1 chromosomes in each of the three genomes, whereas the quantity of lipid moiety produced by chro(Y

458

CALVIN F. KONZAK

mosome 5A was less than for 5B, the most being produced by 5D. These results suggest that the differences in purothionin content of wheats reported earlier (Carbonero and Garcia-Olmedo, 1969) may have been due to differences in the degree of extraction of the lipid-protein complex. The lipid components appear to have been identified as phosphatidylcholine (Redman and Fisher, 1968), mono- and digalactosyl diglycerides, and phosphatidyl ethanolamine (Hoseney et al., 1970b). 5. Lysine Content Variation in Other Proteins

Different gliadin components in wheat also appear to vary in lysine content, with 2-fold or greater differences (Booth and Ewart, 1970). HOWever, the entire prolamine fraction (gliadin) contains only about 0.1 gm of lysine per 100 gm of protein (about equal to the contribution of the purothionins) . Because of its greater abundance (25-75%), the prolamine fraction has a marked influence on the average lysine/protein composition of the endosperm. I n several high-lysine maize mutants and in the barley mutant ‘Bomi 1508’ (Ingversen et al., 1973), the prolamine fraction is reduced in proportion to the total with an increase of the salt-soluble (globulin) fraction, which is higher in lysine (Misra et al., 1972). The high prolamine composition of barley, rye, sorghum, maize, and wheat indicates that these crops have a similar potential for genetic alterations to improve the nutritional balance of lysine (Tables 1 and 2). The possibilities of reducing the proportion of low-lysine proteins are greater for maize and barley than for oats or rice, which contain much smaller amounts of prolamines. I n oats, strains with up to 5% lysine in the protein already have been identified (Robbins et al., 1971). Data on the amino acid composition of proteins indicate that variations in content of the four important amino acids occur among specific wheat gluten proteins (Kasarda et al., 1976a). Gliadins (a, p, y, 0 ) generally have low-lysine, high-glutamic acid, and high-proline composition (Ewart, 1973). Tryptophan composition is similar t o that of lysine, but methionine content varies and threonine composition is about three times that of lysine. High MW gliadin (variety not specified) contained about twice as much lysine and methionine, slightly more threonine, and less prolamine than the lower MW types. Several glutenin fractions contained two to four times as much lysine, about 1.5 times as much threonine, about twice as much methionine, and half as much proline as low MW gliadins. However, some glutenin fractions contained only as much lysine as the low MW gliadin, but were generally similar in composition to other glutenins. Recent studies by Huebner et al. (1974) indicate that two higher-lysine components (A, and A,) are present in the glutenin fraction of one hexa-

GENETIC CONTROL O F PROTEINS I N WHEAT

459

ploid wheat. The fraction A, also had a relatively lower leucine content; while A? had a much higher leucine content than the whole glutenin or other glutenin fractions. However, the differences also may represent variations in the relative purity of the two fractions which should be resolved by the amino acid sequencing of purified glutenin protein subunits currently in progress (J. A. Bietz, personal communication, 1975). The prospects of finding or inducing higher lysine variants in wheat may be indicated from evidence about the composition of mutants already identified in maize and barley. Misra et al. (1972) compared the amino acid composition of protein solubility fractions from normal maize with those of several mutants isogenic for genes s u, sh2, shr, bt, and bt, alone or genetic combinations also carrying 0, in inbreds ‘OH 43’, and ‘W22’. The range in Iysine (gm/100 gm of protein) was 3.7 to 6.3 for albumins globulins, 0.1 to 0.5 for zein (prolamine), 0.4 to 0.8 for glutelin 1, 1.4 to 2.8 for glutelin 2, and 6.4 to 7.1 for glutelin 3. The higher lysine composition in f12, bt,, o,, 02bt2,and o1 was due to proportionate increases in the albumin-globulin and glutelin-3 fractions. The 0, gene invariably raised the free amino acid content in the endosperms above the levels achieved by other genes present (Misra et al., 1975). However, the combination of o2 and fi2 or o7 mutant genes did not produce additive effects on lysine (Misra and Mertz, 1975a). I n some maize mutants the lysine increases were associated with the soluble protein fractions, others with the glutelin fraction (Jimenez, 1968; M a and Nelson, 1975). Ingversen and Kgie (1973) showed that the high lysine composition of ‘Hiproly’ barley is due to disproportionate increases in the globulin and glutelin fractions, a t least one or more proteins in each having a higher than normal lysine composition. One of the high-lysine globulin protein subfractions makes up about 46-50% of the salt-soluble protein of Hiproly. Different proportions of four globulin proteins appeared to account for the different higher lysine compositions of Hiproly, CI7115, and ‘Carlsberg’ mutants 20 and 86, but in all mutants the same protein subfraction is proportionately increased. Analyses of Bomi Mutant 1508 indicate that the nutritional composition of proteins can be increased also via another genetic route, since Ingversen et al. (1973) found that there was 300% more lysine in the prolamine fraction of this mutant than in Carlsberg without any change in lysine composition of either the albumin-globulin or insoluble fractions. This seemingly large increase, nevertheless, only raised the lysine in the prolamine (hordein) fraction from 1.0 to 2.9 gin per 100 gm of protcin. Bomi Mutant 1508 also has a significantly increased amount of lysine in its glutelin fraction. The Bomi Mutant 1508 has a genetically stable 44% increased lysine composition controlled by a recessive gene, which is inherited independently from

+

460

CALVIN F. KONZAK

the hily gene in Hiproly and has an additive effect on lysine content (Muench et al., 1976). Recent studies of Ingversen (1976) indicate that the proportion of electron-dense protein bodies (as estimated by E M after sucrose density sedimentation) differs for Bomi and Bomi 1508. Bomi 1508 has far fewer electron-dense protein bodies, and on electrophoresis several protein bands not found in Bomi were identified in the high-lysine mutant Bomi 1508. Bomi 1508 has a substantially increased level of a-amylase (Munck, 1976). Oram et al. (1975) found that two co-dominant alleles at one locus on chromosome 5 controlling the electophoretic pattern of a group of hordeins in the Bomi and Sultan barleys were not linked with and hypostatic to the recessive locus of Bomi 1508, which suppresses these and certain other hordeins. E M studies of Ingversen (1975) showed further that the protein bodies of Bomi contained both prolamine and glutelin proteins while those of Bomi 1508 contained mostly glutelin. The prolamine component appeared homogeneous in E M while the glutelin component was granular. Similar compositional changes in wheat might improve the nutritional quality of gluten proteins. However, it must also be pointed out that the mutant gene caused the prolamine fraction to decrease from the 29% in Bomi barley to 9% in Mutant 1508, while the albumin-globulin fraction increased from 27% to 4670, respectively, of the total protein. Such changed proportions of prolamine and soluble proteins would probably destroy the breadmaking potential of wheat. Nevertheless, the inexactness of the solubility fractionation procedures leaves the lysine-content data open to question until the isolation and analysis of the specific genecontrolled protein subunits with high lysine can be demonstrated. Although the high-lysine sources investigated so far in barley, maize, and sorghum have shrunken endosperms, one of six recently induced high lysine barley mutants has a nonshrunken endosperm, indicating that the defective shrunken endosperm, which reduces grain yield potential, is not an essential feature of all high-lysine sources (Doll, 1976). The shrunken endosperm of a t least some of the high-lysine sources appears to be due to the effects of a gene closely associated with the high-lysine character (Eslick and Hockett, 1976). 6. Enzyme Inhibitors and Toxic Components of Wheat Endosperm Wheat, like other cereals, appears to contain a number of components shown to act as antimetabolites or toxins in various organisms tested (see also, Kakade, 1974). a. a-Amylase Inhibitors. Among the components in wheat endosperm are albumins shown to have specificities toward a-amylases of different animal species (Kneen and Sandstedt, 1943), but not of cereal a-amylases

GENETIC CONTROL OF PROTEINS IN WHEAT

46 1

(Sandstedt and Beckord, 1946; Silano et nl., 1975). One, AmI, is active against insect a-amylases (Shainkin and Birk, 1970). Similar a-amylase inhibitors were isolated and studied by Ewart (1969), Fish and Abbott (1969), Feillet and Nimmo (1970), Sodini e t al. (1970), Silano e t d. (1973, 1975) , Saunders and Lang (1973), Petrucci et al. (1974, 1975, cited by Kasarda et al., 1976a). The a-amylase inhibitors of wheats are classified in two families, designated 0.19 and 0.28 on the basis of the mobility of the major component relative to bromophenol blue dye upon gel electrophoresis a t pH 8.5 (Silano e t al., 1973). The 0.28 albumin was the same as an albumin described by Ewart (1969) as well as the 13B albumin of Feillet and Nimmo (1970) and probably the same as AmI, of Shainkin and Birk (1970). The 0.19 inhibitor was the same as the albumin of Fish and Abbott (1969), the 13A albumin of Feillet and Nimmo (1970) and the AmI, inhibitor of Shainkin and Birk (1970). Recent analyses by Redman and Ewart (1973) suggest a close identity between ALB13A and CM2 and ALB13B and CM3 of Garcia-Olmedo and Carbonero (1970). Proteins CMI and CM2 were found to be similar even to amino acid sequence. The 0.19 family of albumins was found to have an M W of approximately 24,000 whereas those of the 0.28 family had an M W of approximately 12,500 (Petrucci et al., 1974). A 60,000-MW component with a-amylase inhibitory activity was also identified. When placed in dissociating solvents, the proteins of 24,000 MW were reduced to about 10,000 MW, accompanied by loss in activity which was regained after removal of the dissociating solvent. The reassociation active a-amylase inhibitors also regained their original electrophoretic characteristics. Inhibitors of the 12,500-MW group were found to inhibit a-amylases from several other insects, particularly those that attack grain or grain products (Silano e t al., 1975). The 60,000-MW inhibitors were active also against mammalian and avian species and all three aamylase inhibitors were active against marine species. The inhibitors were completely inactive against a-amylases from immature or germinating wheat grains of all three ploidy levels, suggesting that these aamylase inhibitors either evolved in nature or were selected by man as protective mechanisms to restrict losses caused by grain insects as well as losses due to birds, etc., since both nature and man would likely retain for further propagation those genotypes not previously eaten by other species, and man would likely have available to reproduce only those grains that survived insect attacks during storage. Petrucci et al. (cited by Kasarda e t al., 1976a) have found that 0.19 a-amylase inhibitor was sensitive to pepsin degradation but was not inactivated by trypsin. Puls and Keup (1973) reported that &-amylase inhibitors from wheat retarded the digestion of raw starch in animals and man, but were relatively in-

462

CALVIN F. KONZAK

effective on cooked starch. No symptoms were produced by feeding concentrated a-amylase inhibitors to celiac paticnts (Auricchio et al., 1974). However, the influence of the a-amylase inhibitors on starch utilization in animals and man would depend upon their relative abundance in the feed or food. I n any case, it is unlikely that active amylase inhibitors would be beneficial in animal or human nutrition. b. Protease Inhibitors. Trypsin inhibitor albumin proteins are present in wheat endosperm and embryo (Hochstrasser et al., 1969; Mikola and Kirsi, 1972; Petrucci et al., 1974; Minetti et al., 1975). I n one study the trypsin inhibitors made up about 9% of the water-soluble proteins of the endosperm (Mikola and Kirsi, 1972), 0.3% of the seed weight or about 1% of the kernel weight, were about one-third of the total albumins (Petrucci e t al., 1974). The trypsin inhibitors of wheat endosperm are quantitatively important components with MWs similar to the 0.19 and 0.28 a-amylase inhibitors, and both separate out in the water-soluble protein fraction. The trypsin inhibitors are more basic proteins separable from the a-amylase inhibitors by gel electrophoresis a t pH 8.5 (Petrucci et al., 1974). Madl and Tsen (1974a,b) identified trypsin and chymotrypsin inhibitor activities in extracts of rye, wheat, and triticale flours. The trypsin inhibitor content that was high in rye appeared to be intermediate in triticale and comparatively low in wheat. The chymotrypsin inhibitor activity of the extracts was equally high in all three cereals. The trypsin inhibitors from triticale were highly heat stable, retaining considerable activity even after cooking for 1 hour in a boiling water bath. The chymotrypsin inhibitor activity of the triticale extract was destroyed by heating to 5OOC. Trypsin inhibitors may be important components affecting the feeding value of triticales, and selection for low inhibitor activity may prove effective for increasing feed or food nutritional value (Madl and Tsen, 1974b). It is also possible that a similar basis may be found for nutritional differences in wheats. Protease inhibitors may function as regulators of protease activity a t certain stages of plant development, but also appear to have evolved as protective mechanisms against insect damage, etc. (Ryan, 1973). c. Gluten. Certain components of wheat gluten, now tentatively identified as fractions of a-gliadin, appear to be the agents responsible for a human ailment called celiac disease, which affects about one in 2000 people in the United States and about one in 400 in Ireland (Kendall et al., 1972; Mylotte et al., 1973). Celiac disease is due to a genetically controlled specific immune response derived by digestion of a gliadin fraction to a peptide (or peptides) . The immune reaction is localized in the tissues of the small intestine and results in a reduction of nutrient absorbing

GENETIC CONTROL O F PROTEINS I N WHEAT

463

capacity (Kasarda, 1975; Kasarda et al., 1976a,b). The aggregatable 0 gliadins, or A-gliadins, which comprise about 4-5 polypeptides are among the gliadins active in celiac disease and may be the only fraction involved (Kendall et al., 1972), but this is not yet certain (D. D. Kasarda, personal communication, 1976). The A-gliadins are absent in the wheat cultivars ‘Justin’, ‘Anza’, ‘Ramona 50’, ‘Calidad’, ‘Atlas 66’, ‘Siete Cerros 66’’ ‘Warrior’, and ‘Omar’ (Platt et al., 1974). Some of these cultivars (Justin, Anza) have breadmaking potential similar t o Scout, Cheyenne, Ponca, and Inia 66, which produce A-gliadins. Although i t is not yet known whether the A-gliadin-deficient, cultivars elicit celiac disease, the absence of these proteins in cultivars having good processing properties suggests that such proteins of low nutritive value and toxic properties can be modified, deleted, or substituted by other component proteins without sacrificing use quality. Studies using stocks nullisomic (deficient) for the group 6 chromosomes should help to further identify the gliadin component (s) responsible for inciting celiac disease (D. D. Kasarda and C. 0. Qualset, personal communication, 1976). More recently, wheat gluten [component(s) not yet identified] has been implicated as a pathogenic factor also in schizophrenia (Singh and Kay, 1976). The same gliadin fraction(s) may prove to be causally associated with the two diseases (D. D. Kasarda et al., personal communication, 1976). AFFECTINGPROCESSING CHARACTERISTICS AND B. MAJORFACTORS NUTRITIONAL VALUEOF WHEATFLOUR I . Milling

Milling to separate the bran from the endosperm and reduce the endosperm to flour is an extremely important factor affecting the amino acid composition and processing properties of the flour (Pomeranz and MacMasters, 1968). Higher amounts of nutritionally better quality proteins are contained in the aleurone layer of the endosperm, but the aleurone is mostly separated with the bran (including also the seed coat and several other outer seed tissues) by the milling process. The bran is generally considered to be undesirable in flour for two reasons: (1) discoloration of the preferred white flour (Ziegler and Greer, 1971 ; Orth and Mander, 1975) and (2) components in the bran and aleurone, i.e., tannins, .and especially the nondigestible sugars raffinose and stachyose (Saunders and Walker, 1969; Stevens, 1970; Saunders et al., 1975a) often have undesirable effects on human digestion (Steggerda, 1968; Hickey et al., 1972; Turner, 1965). In contrast, the aleurone also contains proteins of high nutritional value (see Fig. 2 ) . The higher mineral content of the aleurone layer (see a comparable study on barley in Lin and Pomeranz, 1975)

464

CALVIN F. KONZAK

provides the basis of the method (flour ash content) used to test the efficiency of the milling process and the extraction rate. Even for the best milling wheats, flour ash content increases rapidly as the flour extraction rate exceeds 70% (Ziegler and Greer, 1971; Farrand, 1974a,b). The modern roller milling process itself involves the separation of the bran [and most of the aleurone layer(s) close to it] from the endosperm by a crushing, tearing, and shearing process, achieved by forcing the grains between cross-corrugated rollers moving a t different rates. The separation of the bran from the endosperm is improved by increasing the moisture content of the grain (especially of the bran) so that the bran is toughened and can be separated from the endosperm in relatively large flakes. Most commerical flour is produced from the separated endosperm after several sequential reduction and sieving processes (Ziegler and Greer, 1971). a. Milling Qua1it.y-Flour Yield. Grain hardness lias been associated with improved milling properties, higher flour yield, and high bran cleanup (Wrigley and MOSS,1968; Symes, 1969; Simmonds, 1972b; Simmonds et al., 1973; Mattern et al., 1973; B. Belderok and G. J. Doekes, personal communication, 1974). However, the degree of hardness typical of high quality durum wheats is not considered desirable in bread wheats (F. Fajersson and G. Rubenthaler, personal communication, 1974). The strong-gluten common wheats and durums (often with weak gluten) generally have hard endosperms, while weaker-gluten common wheats tend to have soft endosperms, even though the kernels of either type of wheat may be vitreous or opaque in appearance as the result of environmental influences. The harder wheats tend to mill a t faster rates and give higher flour yields a t any given ash content than do most weak-gluten “soft” wheats (Ziegler and Greer, 1971). The genetic basis for hardness seems to be relatively well established (Symes, 1969), with a t least one major hardness-controlling gene and two or more less effective genes contributing most to the differences in milling performance. One of these factors evidently is genetically associated with factors for higher protein content in Timstein and ‘Cheyenne’, as compared with Chinese Spring (B. Belderok and G. J. Doekes, personal communication, 1974). Grain hardness also has an influence on the susceptibility of the endosperm to granulation, on the particle size distribution, and on the amount of damaged starch (broken starch granules) obtained in milling. The biochemical basis for grain hardness has been investigated. Simmonds et al. (1973) found that composition of the protein matrix which holds the starch granules together in cells was similar for hard and soft isogenic lines, but that higher amounts of soluble material extractable from starch granules were associated with grain hardness. The soluble material was

GENETIC CONTROL O F PROTEINS I N WHEAT

465

30% protein carrying protease and a- and p-amylase activities, while the nonprotein material was mainly carbohydrate which on hydrolysis yielded glucose and traces of xylose, arabinose, and mannose. Hardness was believed to be due to the degree of adhesion between starch and protein. It should be noted, however, that soft endosperm “weak” gluten wheats need not be poor milling, as measured by the flour yield and rate of milling. The T. aestivum ssp. compactum, or “club” wheats, commonly grown in the Pacific Northwest area of the United States produce some of the highest yields of low-ash flour obtainable and also mill more quickly with less energy input than typical hard endosperm wheats (Reitz, 1964; G. L. Rubenthalcr, personal communication, 1974). Many club wheats, like some recent common soft white derivatives, evidently have thinner and less bran, since their bran clean-up is usually less complete than the better-milling hard endosperm wheats (C. F. Konzak and G. L. Rubenthaler, unpublished.) However, Larkin et al. (1951, 1952) found no influence of bran thickness on the millability of seven Pacific Northwest wheats. The two compressed cell layers of the central part of the seed coat on soft white wheats are of cellulose and are not suberized (MacMasters et al., 1971). Whether that difference is sufficient to account for the different milling properties of certain white wheats is not clear, but the “total inner endosperm extraction” technique of Vogel et al. (1976) should permit greater insight into the basis for flour yield differences. b. Effects of Milling on Starch Damage and Processing Characteristics of Flour. One study has associated the pentosan content of the grain with lower milling yield (Weswig et al., 1962). Pentosans have high waterabsorbing capacity and appear to be major constituents of cell walls of cereals (McNeil et al., 1974). Since the aleurone cell walls generally are thicker than those of endosperm cells (Buttrose, 1963), the separation of the aleurone layer with the bran and shorts fractions appears to be important for high milling yields of low-ash flour (Ziegler and Greer, 1971). The alcuroiie layer in different wheats varies in thickness (3256.8 pm) according to studies reviewed by MacMasters et al. (1971) and thus may account for some of the observed differences in milling properties. The thickness (number of cell layers and/or size of cells) of the bran (including aleurone and subaleurone tissues) differs among varieties and may affect flour yield. Recent work (Butcher and Stenvert, 1973a,b; Moss, 1973) also shows that poorer-milling soft-endosperm wheats absorb water more rapidly than do the hard-endosperm wheats and that a higher “detergent fiber” content is associated with lower-milling yield (Stenvert and MOSS,1974). Detergent fiber content may be influenced greatly by damage to aleurone cells in milling, hence comparisons are valid only with iden-

466

CALVIN F. KONZAK

tical conditions of grinding (Butcher, 1975). Differences in rates of water absorption between hard and soft wheats, and between good and poor milling wheats, were not considered in earlier studies (Everson and Seaborg, 1958) and still are not adequately reflected in milling procedures used in many experimental laboratories (G. L. Rubenthaler, personal communication, 1975). Differences in the rate of water uptake by hard and soft wheats appear to be of major importance to the ease and degree of endosperm separation in milling (Moss, 1973 ; Stenvert and Kingswood, 1976). Individual wheat kernels in a bulk sample may differ considerably in moisture contcnt before tempering, and may take up additional water a t different rates unless the grains are equilibrated to a pretempering moisturc of 1276, after which the final adjustment before milling can be made (G. W. Fisher, Technovators, Inc., personal communication, 1976). The degree of fragmcntation of thc bran may bc less in soft wheat than in hard or seinihard wheats, permitting a higher extraction rate to be achieved a t lower flour ash levels (Orth and Mander, 1975). However, bran fragmentation and bran clean-up both appear to be genetically controlled since these properties are variety dependent ( C . F. Konzak and G. L. Rubenthaler, unpublished, 1976). Grain hardness affects the percent starch damage (Symes, 1969; Simmonds, 1972b). Thus, flours milled from soft endosperm wheats have less starch damage and the flour particle size generally is smaller than those from hard endosperm wheats (Williams and McEwin, 1967). The alkaline water and water-retention tests (AWRC, WRC) widely used to predict pastry us breadmaking characteristics of wheat flours are based upon the high water-absorption capacity of damaged starch (Farrand, 1964, 1969, 1972; Yamazaki and Lord, 1971 ; Sollars, 1972; Greenwood, 1976). Flours of higher gluten content tolerate more starch damage and still perform satisfactorily in breadmaking (Pomeranz, 1971), but they are unsuitable for pastry (Yamazaki and Lord, 1971). Small differences in water absorption are apparently due to the water-soluble and starch fractions, but changes in the propcrtirs of the cornponcnts of hard us soft wheat flours occur during separation prior to reciprocal reconstitution (Sollars and Rubenthaler, 1975). Some damaged starch seems desirable for yeast breads since the increased water absorption may be beneficial and the damaged starch is more readily a\-ailable for dcgradation by amylases, providing sugar for fermentation during the dough-handling processes (see Wrigley and Moss, 1968; Pratt, 1971; Pomeranz, 1971; Bloksma, 1971; Kulp, 1972; W. Bushuk, personal communication, 1974). Chemical, biochemical, or physical damage may be necessary before amylolytic enzymes can degrade the starch granules (Greenwood, 1976). However, sugar or malt added

GENETIC CONTROL OF PROTEINS IN WHEAT

467

to the mix fully compensates for damaged starch in the breadmaking process. Recent work on “no-sugar” high-yeast short-time bread formulations also indicates that malt or malted wheat will compensate for sugar used in breadmaking with proof height another critical factor (Finney et al., 1977; Magoffin et nl., 1976). Schlesinger (1964) and Farrand (1969, 1972) showed that starch damage, while increasing water absorption and increasing gassing power, reduces tolerance to mixing, reduces loaf volume, and is generally deleterious to bread quality. If the wheat has been weathered, damage to starch by milling is undesirable because the increased activity of amylase enzymes induced during weathering causes excessive gassing and stickiness in the crumb structure of the baked products (Farrand, 1964, 1969, 1972). Different sizes of starch granules appear to have different contents of amylose us amylopectin types of starch, which may degrade differently during weathering or gelatinize differently in processing. Gelatinization, pasting behavior, and viscosity of starches are related, and are influenced by interactions with organic acids, especially linolenic acid (Goering et al., 1974, 1975). Recent evidence suggests that protease enzyme activity also increases during weathering and may be at least as important as amylase activity on the deterioration of Japanese noodle processing quality (Bean et al., 1974a,b). Protease activity also affects the bread-baking quality of wheat (Pomeranz et al., 1966a). The proportion of damaged starch in flour may be influenced by the control of tempering (moisture adjustment process) and mill grinding practices, such as varying roll-speed ratios and roll pressures in the reduction system (Ziegler and Greer, 1971 ; Farrand, 1972). Farrand (1972) obtained flours with three different levels of starch damage from the same wheat, using the same mill, yet keeping constant the homogeneity of the grist, extraction rate, and flour color. After studying the usual quality parameters, he concluded that the physical state of the starch component in flour has an influence on conventional rheological parameters and sedimentation values for estimating baking quality. The technological capability to effect any prescribed level of starch damage would provide a greater measure of flexibility in flour quality than would normally be encountered in conventional laboratory assessments ( Farrand, 1969). Thus, Farrand (1972) was able to achieve a greater distinction of the parameters controlled by the characteristics and content of the protein in wheat flours. The new short-time baking systems indicate possibilities for obtaining more accurate estimates of the processing properties and potential of different wheats via evaluation of several compensating and interdependent factors (Finney et al., 1977). The bread- or pastry-making properties of wheat flours may be in-

468

CALVIN F. KONZAK

fluenced by the ability of the miller to control the compensating interrelationships between the protein and starch components (Farrand, 1972) or of the baker to control the dough properties by adjustments in the relative proportions of sugar, malt, and a-amylase in the dough (Pomeranz et al., 1966b; K. F. Finney et al., 1972; P. L. Finney et al., 1977; P. L. Finney, personal communication, 1976). The importance of milling t o wheat processing characteristics is suggested from (1) evidence for wide and complex variability in the proteins comprising gluten, (2) the evidence that part of this observed complexity may be an analytically induced artifact, (3) the evidence that major modifications in the physical properties and chemical composition of flours can be made in the milling process and in the final processing into food products, and (4) the evidence that the proportions of solubility fractions change during dough mixing (Kasarda et al., 1971, 1976a; Hoseney and Finney, 1971; Mecham et al., 1972; Farrand, 1972; Tanaka and Bushuk, 1973a,b,c; Orth and Mander, 1975). Better estimates of starch damage than AWRC may be needed (such as particle size distribution) to distinguish differences in the water-absorption capacity of the protein components, and different milling procedures may be needed to reveal the processing potential of future high-protein and high-lysine varieties. Selection for high water absorption properties of wheat gluten (independent of influences by starch damage) should be possible using semiautomatic gluten washing methods (Greenaway and Watson, 1975). The relationship of possible true differences in the relative water absorption capacity of gluten (wet:dry gluten ratio) to pastry or bread processing characteristics is not clear, but genetically controlled (varietal) differences apparently have been identified (Pollhamer, 1967, 1969). c. Eflects of MilZing on the Amino Acid Composition of Flour. White flour has a lower lysine content than the whole grain wheat from which it was milled (Toepfer et al., 1972). The wheat varieties tested by Simmonds (1962) differed with respect to the relative proportion of lysine in the wheat and extracted in the flour. Surprisingly, two varieties, ‘Gabo’ and ‘Broughton’, with slightly lower flour yields retained the highest proportion of lysine in the flour, indicating that the lysine-carrying proteins are better dispersed through the endosperm tissue in some wheats than is the case with others. The subaleurone cells of wheat generally have an appreciably higher protein content than the rest of the endosperm (Kent and Evers, 1969) ; the differences are even greater in high protein varieties (Barlow et al., 1974; Simmonds, 1974). Recently, V. A. Johnson et al. (1975a,b) also demonstrated that varieties and their cross progeny

GENETIC CONTROL OF PROTEINS IN WHEAT

469

may differ with relation to the proportion of lysine and protein present in the endosperm and extractable in white flour. Exploitation of these important genetic differences may improve the nutritional properties of wheat flour. 2. Nutritional Improvement by Supplements-Malt,

Wheat Malt Flour,

or Other Protein Sources

The nutritional value, including lysine content of cereal grains can be greatly enhanced on germination or malting. Cereal foods may be nutritionally improved when significant amounts of ground whole malted grain replaces white flour (P. L. Finney, personal communication, 1976). The nutritional value of bread can be easily and inexpensively increased between 20 and 60% by the inclusion of wheat malt as a large part of the dough. Until recently, it seemed impossible to substitute nutritionally significant amounts of malt in bread doughs. The major limiting factors were believed to involve sugar, low levels of yeast, and the long preparation times in the standard bread dough formulas used by bakers. The discovery of various compensating factors has led to the development of no-sugar, short-time bread formulations in which malts replace sugar. Studies show that a significant portion of the flour (up to 60%) can be replaced by wheat malt, resulting in an enhanced nutritional value and with improved dough processing characteristics. Strong, longer mix time wheats probably can be germinated longer with less loss of processing functionality with increased levels of malt (K. F. Finney, 1975 ; Pomeranz and Finney, 1975; P. L. Finney, personal communication, 1976) than standard all-purpose wheat blends. Soft endosperm wheats may be malted more easily (Pomeranz et al., 1975b), possibly owing to their faster wetting and the genetic association of germination inhibitors with red seed coat color. The “high-yeast,” “no-sugar” bread formulations under investigation also promise to shorten processing time and thus reduce cost. Studies t o achieve even more nutritional malts are in progress (Dalby and Tsai, 1976; P. L. Finney, personal communication, 1976). The development of high-lysine synthesizing yeasts might now be a useful approach to complement the nutritional potential of such formulations, since high-lysine yeasts should be inducible with mutagens. Such new approaches may also be more convenient and economically feasible worldwide than the use of special additives, protein concentrates, synthetic amino acids, or soy flour. Wheat protein concentrates (WPC), prepared by alkaline extraction of protein from the bran by-products of flour milling, may have promise as inexpensive supplements to improve the nutritional value of baked

470

CALVIN F. KONZAK

products (Fellers et al., 1966; Miladi et al., 1972; Saunders et al., 1975a). As with soy flours and other additives, only limited amounts of WPC can be added to bread formulations without significantly affecting the processing characteristics, although the amounts permissible may improve the amino acid balance of the products (Betschart et al., 1975). 3. Rheological Properties of Flour Proteins and Other Components

a. Thiols, Disuljide Linkages, Other Chemical Bonding Mechanisms. The contents of reactive thiol and disulfide groups in wheat flour and the rheological properties (viscosity, elasticity, strength) of dough correlate well with recognized baking quality characteristics (Pomerans, 1971, 1973c; Kasarda et al., 1971, 1976a; Redman and Ewart, 1971). The rheological properties of dough largely determine the volume and crumb structure of the baked loaf of bread (Wrigley and Moss, 1968; Bloksma, 1971; Pomeranz, 1971). It is a common practice to add oxidizing agents, such as iodate or bromate, and other flour improvers (ascorbic acid in Europe and more recently in the United States) to stiffen dough. The stiffening of dough occurs by the oxidative removal of thiol groups rather than by the formation of additional disulfide linkages between polypeptides and is explained by the reduction of thiol-disulfide interchange reactions in dough (Ewart, 1972a,b,c; Bloksma, 1975). An apparent anomaly in this regard is the similarity in effects of ascorbic acid and bromate as dough improvers. Grant (1974) found evidence that ascorbic acid acts via an enzymic mechanism related to a butanol-extracted lipid fraction of flour. Only a small portion of the thiol and disulfide bonds in isolated gluten (about 376, or about 20% of those that are reactive) are rheologically effective, i.e., restrict the elastic deformation (Bloksma, 1971, 1972a,b). The rheologically effective thiol groups are considered to be present in small molecules, such as glutathione and cysteine, since these compounds will diffuse much more rapidly in dough than do proteins and may participate in exchange and transfer reactions with protein disulfide groups, permitting a certain amount of slippage during the dough-mixing process (Archer, 1972; Jones et al., 1974). However, thiols also are chemically sensitive to inactivation by oxidation reactions, possibly yielding disulfide linkages, which may cause the dough t o stiffen (Ewart, 1972a,c; Bloksma, 1971, 1972a,b). Jones et al. (1974) indicate that less than 4% of the total disulfide is involved with determining the development time of dough during mixing; 11-13% of the total disulfide is involved with mixing tolerance. Differences in the rheological properties of doughs were directly related to the content of rheologically effective thiol and disulfide groups. G. Fabriani

GENETIC CONTROL OF PROTEINS I N WHEAT

47 1

(cited by Matsuo and Irvine, 1975) indicates that the ratio of reactive

SH to total SH is related to the cooking quality of spaghetti. The rheo-

logical properties of semolina doughs, measured on a Brabender Farinograph, show that higher dough consistency and strength estimated by farinogram band width are closely correlated with cooked spaghetti firmness or tenderness index (Matsuo and Irvine, 1975). b. Protein and Carbohydrates. The important rheological properties of wheat seem t o be more characteristic of the glutenins than of the gliadins (Hoseney et al., 1970a, 1972; Bietz et al., 1973; Greenwood and Ewart, 1975). Redman and Ewart (1971) suggest that the differences in rheological properties between glutelins of oats and barley from those of wheat may be due to the fact that glutenins of wheat have much less intermolecular disulfide cross-linking (and may have none, Greenwood and Ewart, 1975) but are intramolecularly linked by SS bonds. However, the complete lack of viscoelasticity of maize and oat flours may also be due in part to the influence of different amino acid composition and solubility properties of the proteins in those species. Barley, oats, and maize also lack certain alcohol-soluble proteins (Elton and Ewart, 1962), and the glutelin subunits of oats, maize, and sorghum are of lower MW than those of wheat, rye, and barley (Ewart, 1972b, 1975). The role of the protein in soft-wheat flours used for pastry products is not well understood. The carbohydrate components, particularly starch tailings (fine and damaged starch) and pentosans, are more important than protein (Yamazaki and Lord, 1971 ; Pomeranz, 1971 ; Bloksma, 1971; D’Appolonia et al., 1971; Wade and Elton, 1967; Sollars and Rubenthaler, 1975). However, recent studies on corn and barley starches suggest that pasting viscosity is due not only to the gelatinization and breakdown of starch granules (low viscosity is important to soft-wheat pastry-making potential) , but, rather, is controlled by the breakdown of an amylose-fatty acid (largely linolenic acid) complex (Goering et al., 1975). Many soft wheats have a low protein content, but some soft wheats with high protein, such as Atlas 66, prove to have both pastry and breadmaking properties. The high-protein flours are preferable for bread, whereas the low protein flours are suitable for pastries. The soft wheats generally have weak gluten traits and short-time mixing properties, quite unlike the hard-endospcrm, strong-gluten, bread wheats (Reitz, 1964). Soft wheats studied by Jones et al. (1974) had lower amounts of rheologically effective thiol and disulfide groups. Recent work a t Washington State University indicates that it may be possible to extend the mixing time and tolerance of soft common wheats, retaining the pastry-making properties while improving the bread dough properties (C. F. Konzak and G. L.

472

CALVIN F. KONZAK

Rubenthaler, 1974, 1975, unpublished). Also, it may be notable that some of the softer Australian wheats, ‘Sherpa’, ‘Ford’, and ‘Javelin’, have both pastry and general purpose breadmaking flour characteristics (Moss, 1973; G. L. Rubenthaler, personal communication, 1975). However, those cultivars still lack the mixing strength and stability of the standard hardendosperm bread wheats. The seeming absence of soft-wheat recombinant lines with more stable and stronger protein characteristics in crosses of wheats with weak vs strong protein properties may be due to genetic linkages. However, the selection against these recombinant types by breeders and technologists also may be a factor, due in part to assumptions about quality factors drawn by cereal technologists from their analyses of wheat quality “standard” varieties. Genetics and breeding experiments designed to isolate recombinant soft wheat genotypes with short us long mixing stability and low us high extensibility suggest that the doughmixing properties of wheat protein may not be the critical determinants of loaf volume, crumb characteristics, or pastry-making properties and that it may be possible to develop truly dual-purpose pastry and bread quality types of varieties (C. F. Konzak, G. L. Rubenthaler, and P. L. Finney, unpublished, 1976). Michigan State University recently has released to production a higher protein soft wheat variety, ‘Tecumseh,’ with good pastry-making properties. Tecumseh flours have large particle size even a t higher protein than normal soft wheats, providing positive evidence that even pastry products can contribute protein as well as calories to human nutrition (E. H. Everson, personal communication, 1976). c. Lipids. One other group of compounds, the glycolipids, present as only a small fraction of the total lipids, appear to be important to the rheological properties of bread dough. Synthetic glycolipids have considerable potentiql as dough improvers (Pomeranz, 1973b,c; Pomeranz and Finney, 1973a,b). The glycolipids have been extensively investigated by Pomeranz and colleagues (Pomeranz and Finney, 1969, 1973a; Wehrli and Pomeranz, 1970; Hoseney et al., 1970a,b; Pomeranz, 1971, 1973b,c). Lipids also seem to be important to the cooking properties of pasta made from durum wheat semolina (Irvine, 1971) and to bakery products made from soft wheats (Yamazaki and Lord, 1971). Many of the lipids are bound with protein as lipoprotein, but some are thought to be derived from the embryo and extracted with the flour during milling (Pomeranz, 1971).

4. Relation of Flour Protein Solubility Fractions to

Processing Characteristics Flour component fractionation and reconstitution experiments have established that the modified “Osborne” solubility characteristics of

GENETIC CONTROL O F PROTEINS I N WHEAT

473

wheat proteins relate directly to the functional role of these proteins in breadmaking (Hoseney et al., 1969a-d, 1970a,b ; Hoseney and Finney, 1971 ; Pomeranz et al., 1970). The gluten proteins are primarily responsible for the dough-forming properties of wheat flours, whereas the watersoluble components of wheat flours are important to maximum performance of the glutens in baking for all wheats used for bread (Pomeranz, 1971; Kasarda e t al., 1971, 1976a). However, it should be noted t h a t the albumin and globulin (water- and salt-soluble) fractions comprise a small proportion of the total protein (Bushuk and Wrigley, 1974) and that the percentage of these fractions decreases slightly with increasing protein content (Mitra and Bhatia, 1973). The “soluble” proteins are abundant in rye and triticale (Bushuk, 1972). High dough extensibility, a qualitative characteristic of the gluten proteins, may be preferred for breadmaking but not for the making of pastry or pasta (W. C. Shuey and G. L. Rubenthaler, personal communication, 1974). The quantity of gluten protein required for optimum product quality must be above a certain minimum (9-11%) for pasta, bread, and other leavened foods, but below that range for fine pastries (Reitz, 1964), because the quantity of protein directly relates t o the functional properties of dough. Several reports indicate that the gliadin: glutenin ratio is correlated with the rheological and bread baking properties of wheat flours and of the pasta-making properties of semolinas (Bushuk et al., 1969; Bushuk and Wrigley, 1971; Pomeranz, 1971; Wrigley, 1972a; Orth and Bushuk, 1972; MacRitchie, 1973; Matsuo and Irvine, 1975). The correlations ohtained, however, are not so high as to indicate an invariant relationship. Gliadins as a group contribute to dough stiffness, while glutenins, including high MW “residue” proteins, contribute to elasticity and stability during mixing (Hoseney and Finney, 1971 ; Finney, 1971; Wrigley, 1972a ; Bietz et al., 1973). The qualitative and quantitative composition of the proteins within the general gliadin and glutenin groups may vary within limits determined by their functional interactions. Results of Elton and Ewart (1962), Doekes (1968), Doekes and Hack (1971), and Wrigley and Shepherd (1974) suggest that wheats with similar functional properties may have distinctly different combinations of specific gliadins, and conversely, genetically closely related varieties may have similar gliadin spectra but different breadmaking properties. However, Tanaka and Bushuk (1973a,b,c) found the very strong gluten wheat, ‘Red River 68’, has more high M W proteins than ‘Thatcher’, which has medium-strength gluten, while Thatcher had more high MW proteins than Talbot, a weak gluten soft wheat. Similarly, quantitative and qualitative differences in specific glutenin proteins have been associated with the breadmaking properties of a number of wheat cultivars (Cole et al., 1976; Huebner and Wall, 1976).

474

CALVIN F. XONZAK

Recent work, made possible as a result of the development of methods for solubilizing the “residue” protein of earlier extractions, has confirmed the suggestions that residue proteins were largely glutenins and that the glutenins possess viscoelastic characteristics important to the rheological properties of wheat gluten (Bietz and Wall, 1975; Bietz et al., 1975; Kasarda et al., 1976a). Glutenin proteins are not cross-linked by SS bonds. Different glutenin molecules are comprised of variable numbers of polypeptide chains not necessarily alike, which are joined end to end by SS bonds to form super molecules or concatenations possessing a linear, nonbranched configuration. The viscoelastic properties of glutenin are thought to be due t o complex conformations with water and interand intramolecular entanglements. The complex entanglements of gluten molecules provide the (loose) cross-links responsible for the properties of rubberlike elasticity. Stress causes the unfolding of individual polypeptide chains in a concatenation, and the natural tendency of the molecules to return to their compact or relaxed state of lower free energy accounts for the elastic responses. Intermolecular slip processes account for the viscous flow (Greenwood and Ewart, 1975). Genetic factors controlling important functional (breadmaking) properties of wheat have been identified with specific chromosomes. For example, studies using chromosome substitution lines (one pair of chromosomes from the test variety substituted for the same chromosome of a standard variety by backcrossing) show that dough-mixing strength in the variety ‘Itana’, as compared with ‘Kharkof MC22’, is associated with its chromosome 1D (Welsh and Hehn, 1964). The substitution of chromosome 1D from the variety Hope into the variety Chinese Spring increases loaf volume, indicating that genetic factors concerned also with loaf volume are located on chromosome 1D (B. Belderok and G. J . Doekes, personal communication, 1974). Glutenin subunits associated with breadmaking quality are coded by D genome chromosomes (Orth and Bushuk, 1973c; Bietz et al., 1975). The higher loaf volume capacities of Timstein, ‘Capelle Desprez’, and Cheyenne relative to Chinese Spring are associated with chromosomes 2D, 3B, and 1A but not with 1D (B. Belderok and G. J. Doekes, personal communication, 1974). Also, as indicated earlier, Kasarda et al. (1973) have demonstrated that the A-gliadin fraction of a-gliadins is absent from some wheats, such as Justin and Inia 66, which are known to have strong dough properties, as well as from wheats like Atlas 66 and Omar with weak dough properties. The converse is also true-A-gliadins are present in strong-gluten wheats like Cheyenne, and also in Capelle Desprez which has weaker gluten strength. Using the Cheyenne into Chinese Spring disomic substitution lines of Morris et al. (1966, 1968,

GENETIC CONTROL OF PROTEINS IN WHEAT

475

19731, Kasarda et al. (1973, 1974a,c, 1976a,b), and Platt et al. (1974) have established that the 4-5 aggregatable A-gliadin subfractions of the a gliadins are coded for by chromosome 6A. I n fact, most of the gliadin proteins are produced by chromosomes of groups 1 and 6 (Wrigley and Shepherd, 1973; D. D. Kasarda, personal communication, 1976). Baking quality, e.g., loaf volume, crust appearance and crumb grain and texture, are associated with chromosomes in group 1 of Cheyenne as compared to weak gluten Chinese Spring. Mixing strength is associated with chromosome 1 D in Cheyenne (Morris et al., 1966, 1968; Mattern et al., 1968a, 1973). Dough and baking properties are controlled also by factors on several other chromosomes including groups 2, 6, and 7, the specific relations depending on the variety (see below). The results suggest that certain specific gluten proteins may be relatively more important than others to the processing properties of wheats. The replacement of gliadin with high MW gliadin (MW 100,000) isolated by gel filtration in a synthetic dough system consisting of gliadin, glutenin, and starch caused a slight strengthening of the mixing properties, while the replacement of gliadin by lower MW gliadin (MW 44,000 and 27,000) caused a slight weakening of mixing properties. Replacement of gliadin by acetic acid-soluble glutenin or albumin fractions greatly weakened the mixing curves, suggesting that the quantitative MW distribution of gliadin components may be important in determining the functional properties of wheat flour dough systems. Interestingly, mixing of dough under conditions of accentuated breakdown did not change the proportion or characteristics of water- or salt-soluble proteins but increased the amounts of extractable alcohol-soluble and acetic acidsoluble proteins with a decrease of the “insoluble” fraction. Further analyses showed that the amounts of low MW gliadins and glutenins were increased by mixing (Preston eE al., 1975). Wasik and Rushuk (1975a) found that the quality of spaghetti made from durum wheat semolina was strongly correlated with glutenin :gliadin ratio, with correlations significant to 1% obtained with farinograph mixing tolerance index (-0.661), with gluten strength (0.845), and with tenderness index (-0.681) ; glutenin: gliadin ratio with farinograph mixing tolerance index (-0.666). The differences in sphaghetti-making quality were also found to be associated with the MW distribution and relative concentrations of the six largest glutenin subunits identified by SDS-polyacrylamide electrophoresis of reduced glutenin (see also Matsuo and Irvine, 1975). The proportion of residue protein (high M W glutenin) in gluten of herd endosperm wheats is closely correlated with baking and rheological properties (Tsen, 1967; Huebner 1970; Wall et al., 1971; Orth and Bushuk, 1972,1973b; Bietz et al., 1973; Huebner and Wall, 1975). The

476

CALVIN F. KONZAK

correlation may result (at least in part) because the lower waterabsorbing ability of the high-MW glutenin compensates for increased starch damage caused by milling, with only the correct balance of water absorbing capacities of the gliadins, glutenins, starch, and lipid providing for satisfactory mixing properties. The results of Farrand (1969, 1972) suggest that modifications of the milling procedures to produce lower levels of damaged starch may greatly change estimates of the relative importance attached to several parameters used for processing quality estimation, e.g., water absorption, sedimentation, stiffness, extensibility, farinograph stability, AWRC and protein-loaf volume relationships (see also Pomeranz, 1971 ; Bloksma, 1971 ; Greenwood, 1976). The major function of gluten in making bread and other leavened products is to form a network able to hold gas bubbles formed by fermentation, and to keep starch granules separated, preventing them from growing together as they gelatinize during baking. Gluten is not absolutely essential for crumb structure, since completely protein-free bread with a reasonably good (albeit substandard) volume and crumb structure has been made of starches from wheat, rye, or potatoes using saturated monoglycerides (such as glycerol monostearate), vegetable gums, or gelatinized starch as binders (D’Appolonia et al., 1971), or using the microbial gum xanthan (J. A. Bietz, personal communication, 1975). IX. Genetic Variation for Protein Content and Lysine Composition i n Wheat

Many possible genetic sources of variation for improved protein content and lysine composition in common wheats are listed in reports by Lawrence et al. (1958), McDermott and Pace (1960), Tyuterev et al. (19731, Villegas et al. (1968), V. A. Johnson and Mattern (1972, 19751, and Vogel et al. (1973), but only a small number of these sources have been widely utilized (Table 4). Breeding experiments and other analyses indicate that a small number of major genes contribute to the high protein content in the Brazilian wheats ‘Frondoso’, ‘Frontiera’, and derivatives, especially Atlas 66, which have been used as parents in breeding wheats of higher protein content (Middleton et al., 1954; Davis et al., 1961; V. A. Johnson et al., 1963, 1968b, 1975b; V. A. Johnson and Mattern, 1975; Lofgren et al., 1968; Caepoiu et al., 1975; Balla and Gaspar, 1975). Improved protein mutants also have been induced (Dumanovib et d.,1970; Pepe and Heiner, 1975; Konzak, 1975, unpublished). Heritability estimates for grain protein content in wheat indicate in some cases important and variable influences of environment, with genes

GENETIC CONTROL OF PROTEINS IN WHEAT

477

for protein content showing largely additive genetic effects. Generally, heritability estimates have been sufficiently high to show that selection for higher protein content can be made with relative certainty of success. Davis et al. (1961) reported heritability estimates of 54% to 69% for crosses of soft red winter wheats (Frondoso and Frontiera sources) and Haunold et al. (1962) and Stuber et al. (1962) obtained heritability estimates of 56% and 83% respectively for crosses involving Atlas 66, while Sunderman el al. (1965) obtained heritability estimates of only 15-26% for protein content in the cross Atlas 66/Itana. Heritability estimates also for grain proteins calculated by Lebsock et al. (1964) for a hard red spring wheat cross varied from 37% to 70% based on the regression of F, and F, data, respectively, on F, line values. Flour protein content was inherited bimodally with little or no dominance for the spring wheat cross Selkirk/Gabo (Kaul and Sosulski, 1965). The lowest estimate was 66% for ffour protein content heritability based on analyses of flour from F, and F, plants, while heritability estimates of 76% to 89% were obtained for F, to F, lines, with F,/F,, to F,/F, correlations ranging from 92% to 102.6%. Heritability estimates for DBC value were only 27% in comparisons of the F,/F, and FJF, generations of 51 lines grown in six environments (Sharma et al., 1973). Protein content variation in F,, F,, and F, generations of crosses between Atlas 66 and derivatives with local hard red winter wheat varieties was found to show transgressive segregation for protein content. I n this study, thousand-kernel weight was negatively correlated with protein in one cross combination, but no relation was observed with another (Caepoiu et al., 1975). However, Jain et al. (1976), in a detailed analysis, consider the high heritability of TKW as an advantage in selecting for high protein content and high protein yield per acre. They observed strong to moderate negative correlations for protein content and TKW for four hard spring wheat crosses involving both Frontiera and Nap Hal as high-protein gene sources. The varieties Frondoso, Frontiera, ‘Frontana’, ‘Atlas 50’, Atlas 66, ‘Magnif 41’, ‘Aniversario’, and more modern derivatives of these cultivars probably carry the same gene(s) for high protein content (E. Favret and 0. Klein, personal communication, 1972; V. A. Johnson and Mattern, 1972, 1975; V. A. Johnson and Lay, 1974). Argentine wheats studied by Caepoiu et al. (1975) may have a largely similar gene base. The pedigrees of Frondoso and Frontiera trace to old Brazilian criollo (land) varieties (Silva, 1966). A factor (or factors) controlling the high endosperm protein content of Atlas 66 is genetically linked to leaf rust resistance and is located on chromosome 5D (Morris et at., 1973). Tolerance to acid soils (aluminum toxicity) and high protein content of

478

CALVIN F. KONZAK

TABLE 4 Some Useful or Potentially Useful Genetic Sources of Genes for High Protein and/or Lysine, Basic Amino Acids (BAA) in Common and Durum Wheats

Variety

Sel., P.I. or (3.1. No.

Source or origin

Vernalization response

Useful (high) trait ( 8 )

Common wheats

April Bearded Atlas 50 Atlas 66 Atlas 66-derived lines Kaw/Atlas 50 lines Frondoso Frontiera Aniversario Magnif 41 Magnif 41-ERT 1 Malans Nebr. Male Fert. Restorer Nap Hal Nap Hal Nap Hal Nugaines Mahratta Plainsman V

Hume*2//Nebred+/Agatha Hybrid English Pearl Fultz/Hungarian Fultz/Hungarian/Minturki/ Fultz Sel. H 189-29 Norin lO/Brevor. Sel. 14/ Sel. 27:15/Rioj/Rex, Sel. 53 Norin lO/Brevor, Sel. 14)/ (Brevor Sib, 50-3) Timgalen 22 A Induced mutants Chris Mutant MN6616M Marfed Mutant MJD720175 Rageni 15

England S I USA, N.C. USA, N.C. I D W USA, Neb. c W USA, Kansas Brazil CI 12078 S Brazil CI12019 S Argentina CI12578 S PI344466 Argentina S USA, Wash. WA6096 S S. Afr. CI6127 S NB542437 USA, Neb. W PI176217 India S and w PI 176221 India S PI176223 India S GI13968 USA, Wash. W Australia CIS500 S USA (Seed ReW search Associates, Scott City, Kansas) SD69103 USA, S.D. W CI16225 England W CI3285 S Sweden CI11849 USA, Ind. W CI12756 USA, Wis. W

Protein, Lysineasb Proteine Pr0teina.b Protein0.b Proteinbsc ProteinaJ Proteinaab Pr0teina.b Proteinb Proteinb Proteinnsb ProteinaSb Protein, lysineQLb Protein, BAAb Protein, BAAb Ly sine' Lysine0.b Proteinc

CI13447

USA. Wash.

W

Lysine0Bb

CI13449

USA, Wash.

W

Lysineaeb

CI15400 CI5484

Australia USSR

S S

Proteinb ProteinaJ

CI17241 WA6270 PI383308

USA, Minn. USA, Wash. Pakistan

S S S

Protein, Lysineb Proteinb

CI7337 CI12534 CI12561

Pr0teina.b Proteinneb Lysineanb Proteina Protein'

GENETIC CONTROL O F PROTEINS IN WHEAT

479

TABLE 4 (Continued)

Variety

Sel., P.I. or C.I. No.

Source or origin

Vernalization response

Useful (high) trait(s)

Durum whealsg Coll. F. E. Johnson Coll. If. V. Harlan Abyss. 27 Coll. H. V. Harlan 345 USSR 19284, Dickson 415 fulvo obscurum Obispado Zybicum, HN1 Anafil Claro Alonso Perez Jurado Dabat 119 Coll. ELS 3604-8D, Akaki COIL ELS 6404-2B, Akaki

CI3117 CI7499 CI7799 PI94712 P I 134919 PI136574 PI184542 PI 185760 PI 190992 PI192129 PI297852 PI297855

Tunisia Egypt Ethiopia Ethiopia Portugal Spain Portugal Portugal Spain Ethiopia Ethiopia Ethiopia

S S S S S S S S S S S S

Protein Protein Protein Lysine, BAA Protein Protein Protein Protein Protein Protein, lysine Protein Protein

V. A. Johnson ct al., 1972b; V. A. Johnson and Mattern, 1972, 1975. C. F. Konzak and G. Rubenthaler, 1974, unpublished. E. G. Heyne (personal communication, 1974, Kansas State University) ; Lofgren et al., 1968; C. F. Konsak and G. Rubenthaler, 1975, unpublished. Nap Hal, PI176217, is a mixture of lines having winter, semi-winter, or spring vernalization responses. Also, Bietz el al., (1975) detected variability for the presence versus absence of individual glutenin protein components in a PI176217 population. R. E. Allan and G. Rubenthaler, 197.5, unpublished. f J. F. Pepe and R. E. Heiner, 1975. Evaluations confirmed by cooperative research a t Washington State University and a t North Dakota State University (J. Quick, personal communication, 1976). a

Atlas 66 are genetically associated (Mesdag et al., 1970). At least one important gene for A1 tolerance also appears to be on chromosome 5D (C. F. Konzak and A. M. Prestes, unpublished, 1975). A gene for high lysine content may be present in chromosoine 1D of wheat (Mattern et al., 1968b), which may relate to the producer gene for the high lysine purothionin protein also located on that chromosome (Garcia-Olmedo et al., 1975). Riley and Ewart (1970) obtained evidence that King rye chromosome 11, which is homoeologous to group 5 wheat chromosomes, increases lysine in wheat protein by nearly 9%. Three induced mutants recently identified in advanced cultivars show promise as useful germ plasm sources for increasing protein content or improving protein composition. The male fertility restorer (for cytoplasmic sterile lines used in hybrid wheat breeding) and the Agropyron derivative (SD69103) may be distinct sources of high protein germ

480

CALVIN F. RONZAK

plasm. Atlas 66 appears to carry a different gene(s) for protein content from Nap Hal (PI176217), which also transmits higher lysine composition to its progeny (V. A. Johnson et al., 1973). Other Nap Hal accessions, PI176221 and PI176223, appear to be even more promising than PI176217 as protein and dibasic amino acid (DBAA) gene sources (C. F. Konzak, unpublished, 1975). The gene(s) from Atlas 66 appear(s) to provide capability for 2-4 percentage points more protein than that of ordinary wheats and is effective over a wide range of fertilizer levels. Atlas 66 and derivatives also have higher NRA than normal wheats and their foliage contains more N throughout the growing season. The high protein wheats also translocate more of the N taken up into grain protein (Croy and Hageman, 1970; Duffield e t al., 1972; V. A. Johnson et al., 1973; Schmidt et al., 1974; Dalling et al., 1975; Klepper, 1975), but take up no more soil N (V. A. Johnson et al., 1967). The genetic contribution to higher protein content is inherited independently from grain yield (Middleton e t al., 1954; V . A. Johnson et al., 1967; V. A. Johnson and Mattern, 19751, by way of increased N-use efficiency. The gene(s) for higher lysine in Nap Hal and (313449 also appear (s) to be different (V. A. Johnson e t al., 1975a,b; V. A. Johnson and Mattern, 1975). The variety ‘April Bearded’ (CI7337) appears to carry some of the same genes as Nap Hal for high protein and high lysine (V. A. Johnson and Mattern, 1975). Likewise, additivity of genes for high protein accompanied by increased DBAA values occurs in derivatives of Magnif 41 ert l/April Bearded, and high-protein genes of Magnif 41 ert 1 and CI5484 appear to be independent and additive in action (Nguyen Van Mung and C. F. Konzak, unpublished, 1976). Most important, recombinants with the high milling yield of Magnif 41 ert 1 appear to carry high protein content and DBAA value improvements in the milled white flour. Preliminary results also indicate that the high flour DBAA value of ‘Mahratta’ also is recombinable with the highprotein flour characteristic of Magnif 41 ert 1 (N. V. Mung and C. F. Konzak, unpublished, 1976). Indian wheats appear to be a major source of the high-lysine germ plasm. Nap Hal was obtained from India (possibly Nepal), while Mahratta (CI8500) from Australia, has the pedigree ‘Indian 17’ X ‘Federation’. Federation (CI4734) could also be a source of higher-lysine germ plasm, since it is a parent in the background of (2113447 and CI13449 and is a parent of ‘Marfed’ (CI11919) and numerous other Pacific Northwest wheats. Federation was selected from the cross ‘Purple Straw’ Sel. 14A/‘Yandilla’. Yandilla came from the cross ‘Improved Fife’TEtawah’, while the Etawah parent came from India (MacIndoe and WalkdenBrown, 1968). Some recent analyses of a series of Nepalese wheats sug-

GENETIC CONTROL O F PROTEINS I N WHEAT

481

gest that Nepal is in fact a geographic center of genetic variability for protein and DBAA composition in wheat (Table 5 ) . A. AMINO ACIDCOMPOSITION OF HIGH-LYSINE AND HIGH-PROTEIN WHEATS

No selection or mutant has yet been found in wheat which has lysine composition as high as those identified in barley, oats, or maize (V. A. Johnson et al., 1968a,b, 1972, 1975a; V. A. Johnson and Mattern 1972; Siddiqui and Doll, 1973; Doll, 1973; Munck et al., 1970; Mertz et al., 1974). Lysine values above 4.2 gm per 100 gm of protein have been obtained for Nap Hal, one of the promising sources of high-protein, highlysine germ-plasm (V. A. Johnson and Mattern, 1972). However, the environmental component of variation for lysine appears to be quite large (V. A. Johnson et al., 1973, 1975a,b; V. A. Johnson and Mattern, 1975; Gain et al., 1975). Recent amino acid analyses made a t the IAEA Laboratory a t Seibersdorf, Austria, on whole grain samples of Nap Hal, Mahratta, and several soft white wheat mutants generally confirm data of V. A. .Johnson et al. (1973) and Johnson and Mattern (1972, 1975) that Nap Hal has very high protein content (Table 6 ) . The lysine composition of Nap Hal is 3.17% in 19.7% protein, or about the same as Marfed mutant MJD720175 (3.21% in 13.7% protein grain). Grain from highprotein derivatives of Atlas 66 appears to contain more total lysine, but their lysine/protein ratio is the same as for normal wheats (Mattern el al., 1968b). However, sufficient variability in amino acid composition was present among Atlas 66 derivatives to suggest that selection might improve the balance of lysine and other amino acids (Mattern et al., 1968b, 1975). B. SITE OF THE HIGH-PROTEIN AND HIGH-LYSINE IN THE WHEATGRAIN COMPOSITION The embryo, and the aleurone layer of the endosperm, removed from the kernel by milling to produce white flour, are normally higher in protein and essential amino acids than the inner endosperm parts, but protein appears more concentrated near the aleurone tissues (Kent and Evers, 1969; Mattern et al., 1975), due largely to different size and ages of cells (Evers, 1970). It is not known if the same relations pertain for all wheats, since a larger portion of lysine is recovered in the flour from CI13449 than from Nap Hal, while the bran of both wheats retains proportionately more protein than does that of Atlas 66 (V. A. Johnson and Mattern, 1975). Recombinant derivatives with increased endosperm lysine and/or

TABLE 5 Analysis of Wheat vs Flour Protein Values for New Breeding Materials (1974 Crop, Pullman)

Variety or Sel. No.

Average flour yield

Wheat Udya DBC %

Wheat Kjeldahl

Flour Udya DBC %

Flour Udya protein

Flour Kjeldahl

CI 14995 subbarbarrosa CI14996 turcomanicum CI14997 lutinflalum CI14998 alborubrum CI14999 ferrugineum C115000 glaucolutescens CI15279 subrubruminjlatum C115001 graecum CIl5002 a2bidum CIl5003 lutescens CI15005 pseudoingrediens C115007 nigricolor CIl5008 milturum CIl5011 caesium

73.8 76.8 70.6 70.9 68.8 75.7 70.5 72.6 74.8 73.0 73.1 75.9 64.4 74.8

34.25 38.00 37.00 31.75 36.25 34.25 37.50 37.00 39.25 34.75 39.00 40.00 36.00 33.25

14.2 15.7 15.2 13.6 15.5 15.6 16.3 15.0 16.5 15.7 16.2 17.2 15.2 14.3

36.50 38.75 39.25 33.50 41.25 35.50 43.50 37.00 44.00 39.25 45.50 48.50 38.00 39.25

14.1 15.0 15.2 12.5 16.0 13.6 16.9 14.3 17.1 15.2 17.7 18.9 14.7 15.2

12.8 14.1 13.7 11.7 14.4 13.2 14.8 13.5 15.0 14.5 15.3 16.1 13.5 13.0

Difference U-K flour prot. 0.4 0.9 1.5 0.8 1.6 0.4 2.1 0.8 2.1 0.7 2.4 2.8 1.2 2.2

Udy (U) protein value is based on content of dibasic amino acid composition (DBAA), i.e., a dye-binding capacity (DBC). High deviations from Kjeldahl (K) protein values indicate a higher proportion of DBAA than for standard wheats. Analyses via cooperation of G. L. Rubenthaler, Western Wheat Quality Laboratory, USDA-ARS, Pullman, Washington.

Es

Z r

2 R

TABLE 6 Amino Acid Analyses of Wheats Grown at Pullman, Washington in 1972 and 1973 (Analyses IAEA Seibersdorf Laboratory, May 1974) Amino acid composition (gm/16 gm N)

Protein" content Crop year

Accession No.

1973 1973 1972 1973 1973 1973 1973 1973 1972 1973 1973 1973

MJD720 175 MJD720175 M JD720175 MJD720433 MJD720435 MJD720437 MJD720442 CI11919 CI11919 PI 176217 CI8500 CI4127

Name Marfed mutant Marfed mutant Marfed mutant Marfed mutant Marfed mutant Marfed mutant Marfed mutant Marfed (check) Marfed (check) Nap Hal Mahratta

UdY (DBC) Kjeldahla 17.7

-

16.0 14.4 15.3 16.1 15.4 24.2 17.3 17.3

14.2 14.3 14.0 13.3 13.5

14.3 14.8 14.0 14.3 19.7 13.7 14.3

% Lysa protein

mg Lys/gm sample

Lysine

3.16 3.19 3.19 3.06 2.93 2.87 2.93 3.04 3.00 3.17 3.20 2.87

4.49 4.56 4.47 4.07 3.95 4.11 4.33 4.26 4.28 6.24 4.39 4.11

2.90 2.90 2.90 2.80 2.68 2.63 2.67 2.78 2.74 2.88 2.93 2.62

Glutamic acid Arginine 29.8 28.7 30.6 29.9 30.9 31.0 30.4 30.0 31.2 32.0 28.6 29.8

Dry weight basis; all values other than dye-binding capacity (DBC) are based on Kjeldahl N (protein

=

5.07 5.01 5.03 4.66 4.62 4.46 4.59 4.72 4.96 4.85 5.23 4.92

N X 5.7).

Proline 9.8 9.5 9.9 10.0 10.2 10.2 10.1 9.8 10.2 11.0 9.43 10.1

0 3

w

T1Y

M

2

484

CALVIN F. KONZAK

protein have been recovered from crosses Atlas 66/Nap Hal, and Atlas 66/CI13449 (V. A. Johnson et al., 1975b). Important differences have been observed in milling and protein analyses of several spring wheat sources of high protein and high lysine grown under two Washington State environments in 1974 (Table 7). The crop year rainfall a t Pullman was 719 mm, but only 351 mm a t Lind. The temperature and evapotranspiration conditions a t Lind also were higher than those a t Pullman during the period of grain development and maturation. In the analyses, differences between the Kjeldahl protein and Udy protein values were observed for both whole grain and flour samples of Nap Hal and Mahratta, but not for Magnif 41 ert 1. Magnif 41 is genetiTABLE 7 Protein Content of Selected Wheats (1974 Crop)O

Name Seed coat color Yield Bu/A Pullman Lind

TKWb

Pullman Lind Protein content Pullman: Wheat Kc Wheat U d Flour K Flour U Lind: Wheat K Wheat U Flour K Flour U AWh + F protein Pullman K Lind K AK -+ U protein, flour Pullman Lind

Mahratta CI8500

Nap Hal PI176217

Magnif 41 ert. 1 WA6096

Marfed CI11919

April Bearded CI7337

Wared CI15926

W

R

R

W

R

R

29.4 27.5

23.9 11.2

3.5.1 9.6

32.2 29.8

28.5 17.5

34.8 -

34.5 39.0

21.8 23.0

38.5 35.3

32.5

33.5 31.3

29.5 -

12.3 14.2 10.3 12.0 13.7 14.9 12.3 14.3

15.7 16.7 13.6 14.1 18.0 18.2 15.5 16.5

16.0 15.7 15.8 16.2 20.0 18.4 19.8 20.4

12.1 14.4 10.5 11.7 14.3 15.8 12.8 13.3

1.5.5 16.4 13.7 14.0 17.8 17.7 16.3 17.0

13.8 12.6 13.6 -

2 .Q 1.4

2.1 2.5

0.2 0.2

1.6 1.5

1.8 1.5

-

1.7 2.0

0.5 1.0

0.4 0.6

1.2 0.5

0.3 0.7

1.0 -

1.2

Analyses via cooperation of G. L. Rubenthaler, Technologist in Charge, Western Wheat Quality Laboratory (U.S.Department of Agriculture, Agricultural Research Service), Pullman, Washington. TKW = thousand-kernel weight. < K = Kjeldahl protein (N x 5.7). d U = Udy protein.

GENETIC CONTROL O F PROTEINS I N WHEAT

485

cally related to Atlas 66. Higher Udy or DBC protein values (based on acilane orange dye-binding capacity) , indicate relatively higher amounts of DBAA, including lysine (Mossberg, 1969). The higher protein content grain of Magnif 41 ert 1 demonstrated from two different cultural environments may involve a better genecontrolled distribution of protein throughout the endosperm than is the case for Nap Hal. The flour yield for Nap Hal was low compared with Magnif 41 ert 1 and Wared, but similar to the poorer milling Marfed check. However, a comparison of the Udy vs Kjeldahl protein values for the whole grain vs flour fractions indicates that the grain of Nap Hal has above normal DBAA content and shows that relatively more of the DBAA are extracted with the bran than with the flour in the milling process. I n striking contrast, the grain from Mahratta, while lower in protein than Nap Hal st both Pullman and Lind, loses about the same amount of Kjeldahl as Udy protein to the bran fractions, retaining the highest Udy-Kjeldahl protein in the flour for the group of wheats tested. Among the remaining group, CI3285 has what might be a significant Udy-Kj eldahl protein difference, but only from the Pullman location, whereas CI6127 shows a similar relation, but only from the Lind location. The results suggest that the protein composition of GI3285 and GI6127 has a greater sensitivity to environmental influences than is the case for Magnif 41 ert 1, Mahratta, or Nap Hal. The differences in the UdyKjeldahl values for whole grain and flour suggest that the distribution of protein in the endosperm of wheat may be an important factor affecting the contribution of lysine and protein to the flour. The protein (and lysine) of wheats like Nap Hal may be more concentrated in and near the aleurone region, while that of varieties like Mahratta and Magnif 41 ert 1 may be better distributed in the cells away from the aleurone region. These differences suggest that both the location and distribution of proteins in the wheat endosperm may be genetically independent from the protein content and composition. The milling characteristics (flour yield under comparable conditions, including ash content) may also limit the extraction of the protein and lysine into the flour fraction. However, Nap Hal and Mahratta both have poor milling properties. Some of the observed differences may simply reflect the relative ease by which the high protein cells near the aleurone may be separated with the inner endosperm vs the bran, since Evers (1970) found the same protein content per cell in hard and soft wheats, but more protein was extracted from the hard wheats because of their better endosperm separation. Mattern et al. (1975) has obtained similar results with Nap Hal and derivatives. Preliminary inheritance studies conducted at Pullman, Washington, indicate genetic transmission of the high milling yield by Magnif 41 ert 1 permits the

486

CALVIN F. KONZAK

isolation of recombinant lines with higher flour protein and DBAA from crosses involving CI3285, CI5484, CI6127, CI7337, and CI8500 (N. V. Mung and C. F. Konzak, unpublished, 1976). The higher protein content of grain from Atlas 66 and derivatives is contributed entirely to the endosperm (white flour) fraction, while that of Nap Hal is divided between the bran and flour fractions (V. A. Johnson ot al., 1975a,b). The poor bran cleanup and low flour yield characteristics of Nap Hal may be responsible for the main loss of protein and lysine to the bran fraction. The higher lysine content of Nap Hal separates partly into the bran and endosperm fractions, with the endosperm fraction containing no more than the normal lysine content observed for Atlas 66 and ‘Centurk’. Relatively more protein, as well as more lysine, is extracted in the endosperm fraction from the lower protein soft white winter wheat CI13449 and derivatives, which yield more flour than Nap Hal, but still have relatively poor cleanup compared with wheats like Centurk (V. A. Johnson et al., 1975a). However, some selections from Nap Hal/ Atlas 66 show increased endosperm lysine content (V. A. Johnson and Mattern, 1975). Genetic factors regulating protein content are recombinable by breeding and show high heritability (V. A. Johnson et al., 1972; Middleton et al., 1954; Lofgren et al., 1968). Difficulties were not encountered in recombining high protein factors in high-yielding lines. Lofgren e t aE. (1968) estimated that Kaw and Atlas 50 differed by three to four genes affecting protein content, by a t least two genes affecting mixing time, and by two to four genes controlling flour yield. Satisfactory breadmaking properties were recovered in high-protein derivatives of triparental crosses (V. A. Johnson et al., 1975a,b). One hard red winter wheat selection, named ‘Lancota’ (CI17389, formerly NE701132) from the cross Atlas 66/ ‘Comanche’//’Lancer’ was released in 1975 by the Nebraska and South Dakota Agricultural Experiment Stations. In tests conducted recently at Pullman, Lancota did not have the full protein content potential shown by weaker gluten derivatives from crosses of Atlas 66 parentage (C. F. Konzak and G. L. Rubenthaler, unpublished, 1974) and appeared to show strong effects of environment on protein content (E. Donaldson and G. L. Rubenthaler, personal communication, 1975). However, high-protein hard red spring wheat derivatives with stronger gluten characteristics have been recovered from crosses made at Pullman with other weak-gluten but high-protein Atlas 66/Comanche hard red winter wheat lines from Nebraska (C. F. Konzak, unpublished). Schmidt e t al. (1974) and Mattern et al. (1975) also note that high protein content factors appear to be inherited independently of protein quality for breadmaking.

GENETIC CONTROL OF PROTEINS IN WHEAT

487

X. Chromosomal location of Genes Affecting Protein Content, Composition, and Use Properties

The establishment, by aneuploid techniques, of genetic near-isolines in the wheat Chinese Spring (Sears, 1954, 1969; Morris and Sears, 1967) has permitted a considerable expansion in knowledge of the genetics of wheat proteins. Near-isolines include intervarietal and intergeneric chromosome substitutions, ditelosomics, monosomics, nullisomics, tetrasomics, nullitetrasomics, and synthetic polyploid series. Some protein differences have been identified by monosomic analyses. Induced mutant isolines have figured relatively little in the analyses conducted to date, but can be expected to add to the expansion of knowledge for wheat, as has been the case for other organisms. With the genetic near-isolines, the genotypeenvironment interactions and genotypic differences can be markedly reduced, permitting the identification of genetic differences in a wide range of properties, including protein content and composition, enzyme activity, baking properties, mixing characteristics, etc. The identification of proteins by electrophoresis and electrofocusing techniques has added considerably to the base of information already available, because there is a low or negligible environmental component t o the variation observed, and the differences identified seem to be the products of specific genes or gene interactions. A summary of data on the chromosome locations of genes controlling the synthesis, composition, and properties of proteins and of processing quality-related factors shows that the biochemical genetic analysis of wheat is one of the most rapidly developing areas of modern plant science (Table 8 ) . In many cases the chromosomal location of genes controlling specific proteins have been identified and their homoeologous relationships in the three wheat genomes have been established (Mclntosh, 1973). Genetic associations of quality-related components are common: particular chromosomes often carry concentrations of particular kinds of genes. The evolution of genes as identified by their products, the genetic buffering and intergenomic chromosome compensation proved by cytogenetic and other analyses, and the effects of deleting, adding, or substituting chromosomal units or sets provide new insight into the genetic variability that may yet be revealed by genetic recombination or induced by mutagens. An excellent illustration both of the homoeology and genetic associations of wheat gliadins has been obtained by Wrigley and Shepherd (1973). The chromosomes responsible have been identified by combined electrofocusing and electrophoresis of gliadins from the nullitetrasomic

TABLE 8 Chromosomal Association of Genetic Factors Controlling Proteins, Protein Quality, and Related Characteristics in Wheat Chromosome and arm( ) 1A

Trait Protein content: Total protein (flour, Prolamines

Material0

+ effect)

Gluten (+ effect) or Gliadin

Gliadin Non-gliadin proteolipid (component 21 of RodriguezLoperena et al., 1975) Miscellaneous: &Purothionin (apoprotein producer gene) Processing properties: Loaf volume (+ effect) Loaf volume, crust appearance (+ effect, minor factor) Protein content: Gliadin (3 components)

Gluten (fractions AI, BI, CS, and D, = gliadins?)

Method(s)*

b P

00 00

Reference

TST subst. in CS TC subst. in CS, CS N-T

1, 2 3

CNN subst. in CS X CS (Fi hyb.), CNN, CS CNN subst. in CS CNN subst. in CS CS N-T

4

Siddiqui, 1972 Manghers et al., 1973; Solari and Favret, 1970 Sasaki et al., 1973

36, 3 3 5, 37

Kasarda et al., 1974c Kasarda et al., 1976b Aragoncillo el al., 1975s

d 9

F

s3 r fl

CS N-T

5

Garcia-Olmedo et al., 1975; Fernandez de Caleya el al., 1976

CNN subst. in CS

6, 7, 20

CNN subst. in CS

4

B. Belderok and G. J. Doekes, personal communication, 1974 Morris et al., 1966

CS N-T

3, 5

CS D T

3

Shepherd, 1968; Wrigley and Shepherd, 1973; K. W. S h e p herd (R. Morris, personal communication, 1975) Khrabrova el ul., 1973

z Pi

0

Gliadin (band KI) Enzyme(s): a-Amylase ( effect) Peroxidase (- effect = inhibitor?) Miscellaneous: Nucleolar organizer

+

1B

CS DT, N-T

3

Shepherd, 1968

CS D T CS D T

11 11

Gale-and Spencer, 1974 Gale and Spencer, 1974

CS Mono, Null, D T

8

Nucleolar activity (regulator, RNA synthesis) RNA synthesis (regulator, f effect) Nitrate reduction (+ effect)

CS DT, alien subst., 6X/4X derivatives CS D T

8, 9

8, 9

Crosby, 1957; Crosby, Lmgwell, and Svihla 1960; Sears, 1959b Viegas and Mello-Sampayo, 1975 Jain et al., 1968a

CS DT, N-T

10

Edwards, 1973, 1974

Protein content: Total protein (kernel) Total protein (flour, - effect)

TST subst. in CS, Hope subst.

CS D T

12 1, 2

Nods and Tsunewaki, 1972 Siddiqui, 1972

4

Sasaki et al., 1973

36, 3 3 3

Kasarda et al., 1974c Kasarda et al., 1976b Manghers el al., 1973; Solari and Favret, 1970

3

Garcia-Olmedo el al., 1975; Fernandez de Caleya el al., 1976

13, 4

Welsh and Watson, 1965

4

Morris et al., 1966

Gluten a-Gliadin Gliadins Prolamines (+ effect)

in CS C N N subst. in CS x CS (F1 hyb.), CNN, CS C N N subst. in CS CNN subst. in CS T C subst. in CS, CS N-T

Miscellaneous: a-Purothionin (apoprotein pro- CS N-T ducer gene) Processing properties: Dough properties ( f effect, TST subst. in CS farinograph stability). Loaf volume, crust appearance, CNN subst. in CS crumb grain and texture (+ effect)

(Continued)

lb

TABLE 8 (Continued) Chromosome and arm( ) 1B( S )

Trait

Material'

Protein content: Gluten (fractions AI, BI, Cg, CS DT and D4 = gliadins?) Gliadin (bands K,, K4, and Mz) CS N-T Gliadins CS DT; 6X and 4X RSC, TC and PLD Gliadins (6 components) CS DT, N-T

(0

0

Method (s)*

Reference

3

Khrabrova et al., 1973

3

Shepherd, 1968 Wrigley, 1970a

12, 3

Shepherd, 1968; Wrigley, 1972a; Wrigley and Shepherd, 1973; K. W. Shepherd (R. Morris, personal communication, 1975)

3

Enzyme: Peroxidase (leaf, Per 1BS1) Miscellaneous: Nucleolar organizer

CS DT, N-T

3, 14

May et al., 1973

CS Mono, Null, DT

8

Nucleolar organizer (structural gene)

CS DT, alien subst., 6X/4X derivatives

8, 9

Crosby, 1957; Crosby Longwell and Svihla, 1960; Sears,J1959b Viegas and Mello-Sampayo, 1975

Protein content: Glutenin (subunits 2, 3) CS DT, N-T Enzyme(s): Malate dehydrogenase (Mdh-I) CS DT, N-T, LDN subst. in a-Amylase (+ effect) Miscellaneous: Nitrate reduction (+ effect)

3, 14

Bietz et al., 1975

3, 14

Bergman and Williams, 1972

CS DT

11, 14

Gale and Spencer, 1974

CS DT, N-T

10

Edwards, 1973, 1974; Deckard el al., 1975

cs

c)

2 w

2!

5

1D

Protein content: Total protein (kernel, Total protein (kernel, Gluten (+ effect)

+ effect) + effect)

KKF-MC22 Mono CS Mono X SN64 and X LR CNN subst. in CS X CS (F1 hyb.), CNN, CS T C subst. in CS, CS N-T

2 1 4

Welsh and Watson, 1965 Jha et al., 1971 Sasaki et al., 1973

3

6X and 4X RSC, T C and PLD 6X and 4X C T H and PLD CNN subst. in CS CNN subst. in CS LDN, CNN subst. in CS LDN, SN64, L R

3, 5 3 36, 3 3 3 3

Manghers et al., 1973; Solari and Favret, 1970 Wrigley, 1970a Konarev et al., 1972 Kasarda et al., 1974c Kasarda et al., 1976b Bietz et al., 1975 Joppa et al., 1975

CS Mono X SN64 and X LR CNN subst. in CS

15 15

Jha t-t al., 1971 Mattern et a/., 1968a

CS N-T

3

Garcia-Olmedo et al., 1976; Fernandez de Caleya et al., 1976

CS Mono X SRV 29 and X SRV 210 CNN subst. in CS

23

Maystrenko et a/., 1973b

4

TST subst. in CS

4

Imf volume (+ effect)

Hope subst. in CS

6, 7, 20

Dough properties (- effects, strength)

K K F MC22 Mono, TST subst. in CS

2, 13, 16

R. Morris et a/., personal communicat.ion, 1974 B. Belderok and G. J. Doekes, personal communication, 1974 B. Belderok and G. J. Doekes, personal communication, 1974 Welsh et al., 1968

Gliadins (2 prolamine components) Gliadins Gliadins (2 components) a-Gliadin Gliadins Glutenin (subunits 1, 4) Glutenin (subunits 1, 4) Amino acid: Tryptophan content (+ effect) Lysine content (possible 15 % increase) Miscellaneous: u-Purothionin (apoprotein producer gene) Processing properties: Gluten quality (+ effects, strength) Dough properties (mixing strength, effect) Loaf volume (+ effect)

+

(Continued)

TABLE 8 (Continued) Chromosome and arm( ) 1D

Method(s)b

Reference

Dough properties (mixing tolerance, gas retention, effect) Dough properties (strength)

KKF MC22 Mono X Itana

13, 17, 18

Welsh and Hehn, 1964

LDN, CS 1D addition, D T

3

Joppa el al., 1975

Protein content: Gliadins (4 components)

CS DT, N-T

5, 3

+

WS)

Material-

Trait

Gliadins (bands J 2 and 5 3 ) Gliadins (bands Jf and 5 3 ) Gluten (fractions A,, BI, C,, and D4-gliadins?) Gluten (2 components, urea extract of kernels) Enzyme: Peroxidase (leaf, Per-1 DS-1, Per-1 D S d ) Miscellaneous: Nitrate reduction (+ effect) Peroxidase activity ( - effect = inhibitor) Processing properties: Dough properties (enhancer)

CS DT, N-T Wheat-rye chr. transloc lD(L)-E(S) CS DT

3 3

Wrigley and Shepherd, 1973; K. W. Shepherd (R. Morris, personal communication, 1975) Shepherd, 1968; Wrigley, 1972a Shepherd, 1973

3

Khrabrova et al., 1973

CS DT

3

Boyd and Lee, 1967; Boyd el al., 1969

CS DT, N-T

3, 14

May et al., 1973

CS DT, N-T CS DT

10 11

Edwards, 1973, 1974 Gale and Spencer, 1974

CS DT

19, 13

Maystrenko et al., 1973a

E

2 s

4

0

2* w

Protein content: Glutenin (high M W subunits 1, CS DT, N-T, extracted tetraploids PLD C T H RC, T C 4; 2 lower MW subunits of Orth and Bushuk 1974, not observed) CS DT, N-T, extracted tetraGlutenin (4 subunits; high ploids PLD, CTH, R C MW = subunits 1, 4 of Bietz et al., 1975) Processing properties: Flour quality (dough strength) CS DT, extracted tetraploids, PLD 2A

Protein content: Total protein (kernel, effects)

+

Gluten (+effect) Total protein (kernel,

+ effect)

Gliadins? (joint control with 6-41 Enzyme(s): Phenolase complex activity (+effect, reported as t yrosinaee) Phenolase complex activity

3

Bietz el al., 1976

3

Orth and Bushuk, 1974

20, 13

Kaltsikes el al., 1968b

Rye chr. subst. in CS

21,22

CS Mono X SRV 210 CNN subst. in CS X CS (FI hyb.), CNN, CS CS N-T

23 4

N. Darvey, personal communication, 1974; Jagannathan and Bhatia, 1972 Maystrenko el al., 1973b Sasaki et al., 1973

3

Waines, 1973

Hope and TST subst. in CS

25

Zeven, 1972

p-Glucosidase

25 Koga I1 X subst. of Peko, Hope, and T S T in CS 25 CNN, Hope, KF, MQ, TC, T S T and 2R(2A) subst. in CS 3, 14 2R(2A) subst. in CS

Miscellaneous: Inhibitor (of 6D gliadins)

CS N-T, tetrasomics

Phenolase complex activity

3

Larsen 1974 Wrigley and McIntosh, 1976

C. E. May, personal communication, 1975 Shepherd, 1968 (Continued)

TABLE 8 (Continued) Chromosome and arm( ) 2A

2A(L)

I&

W

lh

Trait

Material"

Processing properties: Baking absorption, loaf volume TC subst. in CS (+ effects) Baking absorption (+ effects) Hope subst. in CS Milling properties (+ effect) CNN subst. in CS Protein content: Total protein (- effect, major CS D T factor) Enzyme (s): Phenolase complex activity CS dimonotelo 2A X TST 2A (kernel phenol reaction; subst. in CS 36.8 5.8% linkage to centromere) Acid phosphatase (+ effect) CS D T a-Amylase (+effect) CS D T Miscellaneous: Regulator? (gluten protein CS D T fractions B, and B, merge, joint control with group 6 chr.) Nucleolus organizer (regulator) CS DT, alien subst. in CS 4X/6X Nitrate reduction (regulator or CS DT, N-T inhibitor) Protein component ratio CS D T (+effect, albumins/globulins) Processing properties: Dough properties (inhibitor) CS D T

Method ( s ) b

Reference

4

Welsh et al., 1968

4 33, 24

Welsh et al., 1968 Mattern et al., 1973

22

Bozzini and Giacomelli, 1973

25

Wrigley and McIntosh, 1975

E2

1:

kl

11 11

Gale and Spencer, 1974 Gale and Spencer, 1974

3

Khrabrova et al., 1973

8, 9 10

Viegas and Mello-Sampayo, 1975 Edwards, 1973, 1974

26

Bozzini and Giacomelli, 1973

19, 13

Maystrenko et al., 197313

z

5

Protein content: Total protein (flour,

+ effect) Total protein (kernel, + effect)

Gluten ( - effect) Gliadin? (joint control with 2 D and 6D) Miscellaneous: “Regulator or inhibitor” of gluten subunits Processing properties: Gluten quality (weak inhibitor)

Dough strength, baking absorption (+ effects, major factor) Loaf volume Enzyme: a-Amylase (+ effect) Miscellaneous: Regulator? (gluten protein fractions B3 and B4,gliadins merge, joint control with group 6 chr.) Processing properties: Dough strength (enhancer)

TST, S61.5, and Hope subst.

in CS Rye chr. subst. in CS

1, 2

Siddiqui, 1972

21, 22

CNN subst. in CS X CS (FI hyb.), CNN, CS CS N-T

4

N. Darvey, personal communication, 1974; Jagannathan and Bhatia, 1972 Sasaki et al., 1973

3

Waines, 1973

CS N-T

3

Orth and Bushuk, 1974

CS Mono X SRV 210

23

Hope subst. in CS

4

Maystrenko et al., 1973b; 0. I. Maystrenko (R. Morris, personal communication, 1975) Welsh el al., 1968

Hope and T S T subst. in CS

4

Welsh et al., 1968

CS D T

11

Gale and Spencer, 1974

CS D T

3

Khrabrova et al., 1973

CS DT

19, 13

Maystrenko et al., 1973a ~

(Continued)

I&

TABLE 8 (Continued) Chromosome and arm( ) 2D

Materiala

Trait Protein eontent: Total protein (kernel, effect) Gluten ( effect) Gliadin (35 mm band, joint control with 2B and 6D) Enzyme (s): Peroxidase Per 5C (leaf blade, stem, root, glume)

+

+

Phenolase complex activity (reported as tyrosinase, effect) Phenolase complex activity (kernel phenol reaction) Processing properties: Gluten properties (+ effect dispersion) Dough properties (strength, effects) Loaf volume ( f effects)

+

+

Enzyme: Phenolase complex activity (kernel phenol reaction) Protein content: Gluten (fractions B, and B4

CD

Q,

Method(s)b

Reference

Rye chr. subst. in CS CS Mono x SRV 29 CS N-T

21,22 27 3

Irani and Bhatia, 1972 Maystrenko et al., 1973b Waines, 1973

CS N-T

3, 14

TST subst. in CS

25

Kamagata and Nishikawa, 1973; Nishikawa and Kamagata, 1974 Zeven, 1972

E

3 4

3

CS N-T; KF, RE, TST subst. in CS

25

Wrigley and McIntosh, 1975

TC subst. in CS

28

Favret et al., 1969

Hope subst. in CS

13, 17

Welsh et al., 1968

TST subst. in CS

7

B. Belderok and G. J. Doekes,

CS N-T, D T

25

Wrigley and McIntosh, 1975

CS D T

3

Khrabrova et al., 1973

personal communication, 1974

z8

$

gliadins merge, joint control with group 6 chr.) Enzyme: Peroxidase ( - effect = inhibitor?) Processing properties: Dough properties ( - strength, = inhibitor) 3A

Protein content: Total protein (kernel, Total protein (flour, Total protein (kernel,

+ effect) + effect) + effect)

Albumins (bands 1, 2, and 6) Amino acid: Tryptophan content (feffect) Enzyme(s): Esterases (3 bands) Esterase (plant development) Peroxidase, Per 5C, root, empty glume Esterases (leaf and root, 3 slow bands) Processing properties: Loaf volume (+ effect, minor factor) Baking absorption (+ effect, minor factor) Kernel hardness, water absorption (+ effects, major factor)

CS DT

11

Gale and Spencer, 1974

CS DT

13, 19

Maystrenko el al., 1973a

CS Mono X SN64 and X L R TST subst. in CS CNN subst. in CS X CS (FI hyb.), parents CS N-T

1 1, 2 4

J h a et al., 1971 Siddiqui, 1972 Sasaki et al., 1973

3, 14

Cubadda, 1975

CS Mono X SN64 and X LR

15

J h a el al., 1971

CS N-T CS aneuploids CS N-T

3, 14 3, 14 3, 14

Boeeini et al., 1973 Nakai and Tsunewaki, 1974 Kamagata and Nishikawa, 1973

CS DT, N-T, D genome subst. L D N

3, 14

Bergman and Williams, 1972

Hope subst. in CS

4

Welsh et al., 1968

TST subst. in CS

4

Welsh et al., 1968

TST subst. in CS

29, 30

B. Belderok and G. J. Doekes, personal communication, 1974 (Continued)

TABLE 8 (Continued) Chromosome and arm( ) 3A(a

=

L)

3A(B = S)

Trait Enzyme(s): Esterases (bands 3Aa-1, 3Aa-2, 3Aa-3) Esterases (bands 1, 2, and 6) Esterase (kernel, band 9) Acid phosphatase (+effect) a-Amylase (+ effect) Glutamate oxaloacetate transaminase Got-AS Miscellaneous: Nitrate reduction (- effect) Protein content: Total protein (kernel) Gluten (fractions B,, B?, Cgand C6 = non-gliadins?) Enzyme (s): Esterases (3 bands) Esterase (fast-moving dimer) Peroxidase ( - effect = inhibitor?) Processing properties: Dough properties (enhancer)

Material.

Method(s)b

Reference

CS DT, N-T

3, 12

CS DT, N-T

CS DT CS DT CS DT, N-T

3, 12, 14 3, 14 11 11 3, 14

C. E. MayJ personal communication, 1975 Cubadda et al., 1973, 1975 Nakai, 1973 Gale and Spencer, 1974 Gale and Spencer, 1974 Hart, 1975

CS DT, N-T

10

Deckard et al., 1975

CS DT CS D T

12 3

Noda and Tsunewaki, 1972 Khrabrova et al., 1973

CS Null, DT CS DT, N-T CS D T

3, 14 3, 14 11

Barber et al., 1968a,b Bergman and Williams, 1972 Gale and Spencer, 1974

CS D T

13, 19

Maystrenko et al., 1973a

CS DT, N-T

3B

Protein content: Total protein (kernel, Gluten ( + effect)

+ effect)

Gluten ( - effect) Albumin (76mm band) Amino acid: Tryptophan content (+ effect) Enzyme (s): Esterase (6 bands, kernel) Esterase (kernel, bands 3, 8, 11, 13) Esterase (plant development) Peroxidase, Per 4C, root, stem Peroxidase (culm, root)

Processing properties: Loaf volume (+ effect) Dough strength, baking absorption (+ effects) Loaf volume (+ effect)

CS Mono X SN64 and X L R CS Mono X SRV 210 CNN subst. in CS X CS (PI hyb.), CNN, CS CS N-T

1 23

Jha et al., 1971 Maystrenko et al., 1973b

4

Sasaki et al., 1973

3

Waines, 1973

CS Mono X SN64 and X LR

15

Jha et al., 1971

CS Null, N-T CS N-T

3, 14 3, 14

Barber el al., 1968s Cubadda et al., 1975

CS aneuploids CS N-T CS N-T

3, 14 3, 14 3, 14

Nakai and Tsunewaki, 1974 Kamagata and Nishikawa, 1973 Nishikawa and Kamagata, 1974; K. Nishikawa (R. Morris, personal communication, 1975)

CD subst. in CS

7

Hope and TST subst. in CS TST subst. in CS

Protein content: Total protein (kernel) CS D T Non-gliadin proteolipid (CM CS DT, N-T components 6, 7 = protein band 4 and components 14 and 15 of Rodriguez-Loperena et al., 1975 = protein band 3 of Noda and Tsunewaki, 1972)

0

8m

2 8

2

r

s hj

g

4

B. Belderok and G. J. Doekes, personal communication, 1974 Welsh et al., 1968

4

Welsh et at., 1968

2

3, 12, 14 5, 37

Noda and Tsenewaki, 1972 Aragoncillo el al., 1975a

L2

ZJ

dF r3

is (Continued)

E

TABLE 8 (Cuntinued) Chromosome and arm( ) 3B (S)

3B (L)

3D

Trait

Materide

Enzyme: Esterase (fsst-moving dimer) Processing properties: Dough propreties (inhibitor) Enzyme(s): Esterase (leaf, band Est-SBL-1) Esterase (kernels, ban& Est S B-6, Es~-SBL-?') Esterases (2 bands) Acid phosphatase a-Amylase (+ effect) Glutamate oxdoacetate transaminase GotBS Miacellaneuus: Nitrate reduction (+effect) Protein content: Total protein (kernel, effect) Albumins Albumin (P.C.S.) Albumins (bands 4,6, 7) Albumins (82,91 and 97 mm bands) Enzyme (s) : Esterases (kernel, 2 bands, regulator?) Esterase (bands 10 and 13) Esterase (kernel, bands 5 and 12)

+

Method(s)b

Reference

CS DT, N-T

3, 14

Bergman and Williams, 1972

CS D T

13, 19

Maystrenko et al., 19738

CS DT, N-T CS DT, N-T

3, 12, 14 3, 12, 14

May et al., 1973;Nakai, 1973 Nakai, 1973

CS DT, N-T CS D T CS DT CS DT, N-T

3, 12, 14 11 11 3, 14

Cubadda et al., 1975 Gale and Spencer, 1974 Gale and Spencer, 1974 Hart, 1975

CS DT, N-T

10

Edwards, 1973, 1974

TC subst. in CS CS Mono X T. durum CS Mono X T . durum CS N-T CS N-T, Ae. squarrosa

2

31, 32 31, 32 3 3

Kuspira and Unrau, 1957 Borzini et al., 1970 Boarini et al., 1971 Cubadda, 1975 Waines, 1973

CS N-T

3, 14

CS Mono X T. durum CS N-T

3, 14 3, 14

,

Bosaini et al., 1973; Cubadda et al., 1975 Nagayoshi et al., 1974 Cubadda et al., 1975

sE

Z

r

t2 3 R

CS aneuploids Esterase (plant development) Esterases (root, 2 slow-moving CS N-T bands) Processing properties: Hope and TST subst. in CS Milling properties (flour yield effects, minor factors) Loaf volume TST subst. in CS Gluten quality (weak inhibitor) CS Mono X SRV 29

+

3, 14 3, 14

Nakai and Tsunewaki, 1974 Bergman and Williams, 1972

24

Welsh et al., 1968

4 23

Welsh et al., 1968 Maystrenko et al., 1973b; 0 . I. Maystrenko (R. Morris, personal communication, 1976)

3 D ( a = L)

Enzyme(s): Esterase (leaf, 1 slow-moving CS DT, N-T band) Esterases (kernels, bands EstCS DT, N-T SDa-1 , Est-3Da-2) Esterases (leaf, EstSDa-1, CS DT, N-T Ed-3Da-2) Glutamate oxaloacetate trans- CS DT, N-T aminase Got-D3 (location approximately 4.3 C.O. units from centromere) Glutamate oxaloacetate trans- CS DT, Trd-Agro. elong aminase Got-AgS transloc. Miscellaneous: Nitrate reduction (leaf extracts, CS DT, N-T in vivo, - effect)

3D(@= S = Left) Protein content: Total protein (kernel) Total protein (kernel)

CS D T Triticum-Agropyron transloc

3, 14

Bergman and Williams, 1972

3, 12, 14

Nakai, 1973

3, 12, 14

May et al., 1973

12

Hart, 1971; Hart et al., 1976

12

Hart et al., 1976

10

Deckard et al., 1973

3, 5 3, 5

Noda and Tsunewaki, 1972 Rodriguez-Loperena et al., 1976 (Continued)

cn

0,

TABLE 8 (Continued) Chromosome and arm( ) 3D(P = S = Left)

Trait

Materiala

Gluten (fractions B,, BI, Ca, and Cg = gliadins?) Non-gliadin proteolipid (CM component 5 = protein of Noda and Tsunewaki, 1972 = 1 compound of Waines, 1973) Enzyme(s): Esterase (fast-moving band) Esterase Peroxidase ( - effect = inhibitor?) Processing properties: Dough properties = enhancer, modifier; effect dough strength, - effect extensibility

+

4A

Protein content: Total protein (flour, effect) Gluten (kernel, effect)

+

+

Method(s)b

Reference

CS DT

3

Khrabrova et al., 1973

CS DT, N-T

5, 37

Aragoncillo et al., 1975a

CS DT, N-T CS N-T CS DT

37 3 11

Bergman and Williams, 1972 Cubadda et al., 1975 Gale and Spencer, 1974

E

9

Z

1

R

0

CS DT

19, 13

Maystrenko et al., 1973a

Hope, and S-615, subst. in CS CNN subst. in CS X CS (F1 hyb.), CNN, CS CS N-T

1, 2 4

Siddiqui, 1972 Sasaki et al., 1973

3

Waines, 1973

3, 14 3

Nagayoshi, 1973 Avivi et al., 1972

Albumins (83 and 69 mm, joint control with 4B) Enzyme(s): 8-Amylase CS Null Alcohol dehydrogenase CS N-T

3 L

Alcohol dehydrogenase

Rye chr. additions in HFT, Triticale CS N-T

Acid phosphatase (reported as alk. phosphatases; see Hart, 1973a; Torres and Hart, 1976) Processing properties: CNN subst. in CS Milling properties ( effect, minor factor) Dough properties (J'alorimeter, TST subst. in CS effect) Doughproperties (+effect, bak- Hope subst. in CS ing absorption, minor factor)

+

+

3, 14

Irani and Bhatia, 1972

3, 14

Brewer et al., 1969

24, 33 13

Mattern et al., 1973; Sasaki et al., 1973 Welsh et al., 1968

4

Welsh et al., 1968

z%

0

Enzyme(s): Alcohol dehydrogenase, Adh-Al, a subunit Lipoxygenase Lpx-A1 (Coleoptile) a-Amylase (+ effect) Protein content: Total protein (kernel) Non-gliadin proteolipid (components 12, 13= part of CM3 proteinof Aragoncillo 1973 and 83mm band of Waines, 1973; component 16 = part of 69mm band of Waines, 1973) Enzyme(s): Acid phosphatase (+ effect) Acid phosphatases (Acph 4, Acph 8) 8-Amylase (component C)

0

CS DT, N-T

3, 14

CS DT, N-T

3, 14

Hart, 1970, 1971, 1973a,b; Tang and Hart, 1975 Hart and Langston, 1975

CS D T

11

Gale and Spencer, 1974

CS D T CS DT, N-T

12 5, 37

Noda and Tsunewaki, 1972 Aragoncillo et al., 1975a

TIr 0

cd

s L-d

3

2

2 8

3> r3

CS D T CS DT, N-T

11 3, 14

CS D T

3, 14

Gale and Spencer, 1974 Hart, 1973a,b; Torres and Hart. 1976 Joudrier and Cauderon, 1976 (Continued)

en

0 W

TABLE 8 (Cmtinued) Chromosome and arm( ) 4-4(8)

4B

Materiala

Trait a-Amylase (+ effect) Processing properties: Dough properties (inhibitor)

Reference

CS DT

11

Gale and Spencer, 1974

CS DT

13, 19

Maystrenko et al., 1973a

4

1, 2

Siddiqui, 1972 Sasaki et al., 1973

23 3

Maystrenko et al., 1973b Waines, 1973

3, 14 3, 14 3, 14

Avivi et al., 1972 Kobrehel and Feillet, 1975 Brewer et al., 1969

4

Welsh et d.,1968

24, 33

Mattern et al.. 1973

4

Morris et al., 1966

23

Maystrenko et al., 1973b; 0. I. Maystrenko (R. Morris, personal communication, 1975)

Protein contend: Total protein (flour, - effect) Gluten (+effect)

S-615 subst. in CS CNN subst. in CS X CS (FI hyb.), CNN, CS Gluten (+ effect) CS Mono X SRV 29 Albumins (83 and 69 mm, joint CS N-T control with 4A) Enzyme(8): Alcohol dehydrogenase CS N-T CS N-T Peroxidase (band C) CS N-T Acid phosphatases (originally reported alk. phosphatases; see Torres and Hart, 1976) Processing properties: Loaf volume, baking absorption TST subst. in CS (+effects, minor factor) CNN subst. in CS Milling properties (+ effect, tentative) CNN subst. in CS Dough propertiea (strength), loaf volume, crust appearance, crumb grain and texture (+ effects, major factors) Gluten quality (weak inhibitor)

Method(@

CS Mono X SRV 210

E

d

:T’

E

5 k

Protein content: Total protein (kernel) Enzyme(s): Acid phosphatases (Acph 2, Acph 3) Acid phosphatase (+ effect) a-Amylase (+ effect) Processing properties: Dough properties (major inhibitor) Enzyme (s): Alcohol dehydrogenase, Adh-B1, 0 subunit Lipoxygenase Lpx-B1 (Coleoptile) 4D

Protein content: Total protein (kernel, effect) Total protein (flour, - effect) Total protein (crude protein, effect) Gluten (+ effect)

+

+

Gluten (+ effect) Albumin (Mb 0.19)

CS D T

12

Nods and Tsunewaki, 1972

CS DT, N-T

3, 14

CS D T CS D T

11 11

Hart, 1973a,b; Torres and Hart, 1976 Gale and Spencer, 1974 Gale and Spencer, 1974

CS D T

13, 19

Maystrenko et al., 1973a

0 H

33 Q

CS DT, N-T

3, 14

CS DT, N-T

3, 14

Hart, 1973a,b; Torres and Hart, 1976 Hart and Langston, 1975; G. E. Hart, personal communication, 1976

81: ;3

0 F

o

r

'd

z5

td

0

TC subst. in CS Hope and S-615 subst. in CS CNN subst. in CS X CS (FI hyb.), CNN, CS CNN subst. in CS X CS (FI hyb.), CNN, CS CS Mono X SRV 29 CS D-genome X durum hybrids

2 1, 2

4

Kuspira and Unrau, 1957 Siddiqui, 1972 Sasaki et al., 1973

4

Sasaki et al., 1973

8

23 32

Maystrenko et al., 1973b Bozrini et aZ., 1971

?i

(Continued)

G

01

%

TABLE 8 (Continued) Chromosome and arm( ) 4D

Trait Albumin (65-mm band) Non-gliadin proteolipid (CM 3 = component 17 of Rodriguez-loperena et al., 1975; = 65 mm band of Waines, 1973) Enzyme (9): Alcohol dehydrogenase Acid phosphatases (reported as alk. phosphatases; see Torres and Hart, 1976) Processing properties: Loaf volume, crumb grain and texture (+ effects, minor factors) Loaf volume (+ effect) Milling properties (flour yield, effect, minor factor)

+

Enzyme(s): Alcohol dehydrogenase, Adh-Dl, 6 subunit @Amylase ( f effect) Lipoxygenask Lpx-Dl (Coleoptile)

Materialm

method(^)^

Reference

CS N-T, Ae. squarrosa CS DT, N-T

3, 14 5, 37

Waines, 1973 Aragoncillo et al., 1975s

CS N-T CS N-T

3, 14 3, 14

Avivi et al., 1972 Brewer et al.. 1969

CNN subst. in CS

4

Morris et al., 1966

TST subst. in CS Hope subst. in CS

4 24

Welsh et al., 1968 Welsh et al., 1968

CS DT, N-T

3, 14

CS DT CS DT, N-T

11 3, 14

Hart, 1973a,b; Tang and Hart, 1975 Gale and Spencer, 1974 Hart and Langston, 1975; G. E. Hart, personal communication, 1976

zT: R

Protein content: Glutenin (high MW subunit 5 ) Enzyme(s): Acid phosphatases (Acph 5, Acph 6) 8-Amylase (2-Ca components)

5A

Protein content: Total protein (kernel, effect) Total protein (flour, - effect) Total protein (kernel, effect) Total protein (kernel, effect)

+ +

+

CS DT, N-T

3, 14

Bietz et al., 1975

CS DT, N-T

3, 14

CS D T

3, 14

Hart, 1973a,b;Torres and Hart, 1976 Joudrier and Cauderon, 1976

TC subst. in CS TST subst. in CS CSMono XSN64 , CNN subst. in CS X CS (FI hyb.), CNN, CS CS Mono X SRV 210

2 1, 2 1

Kuspira and Unrau, 1957 Siddiqui, 1972 Jha et al., 1971

!3 M

4

Sasaki et al., 1973

3 0

23

Maystrenko et at., 1973b

0

15

Jha et al., 1971

5rJ 0

Gluten (+effect) Amino acid: Tryptophan content (+ effect, CS Mono X SN64 major factor) Miscellaneous: Purothionin lipid moiety (least CS N-T effective gene) Processing properties: Baking absorption (+ eff eet, Hope and TST subst. in CS minor factor) Gluten quality (weak inhibitor) CS Mono X SRV 29 Protein content: Gluten protein (fractions BI, B4, and D4) Miscellaneous: Peroxidase activity ( - effect = inhibitor) Nitrate reduction (- effect, leaf ext.)

2

xe r 0

34

4

Garcia-Olmedo et al., 1975;Fernandez de Caleya et al., 1976

'd

4

Welsh et al., 1968

2

23

Maystrenko et al., 197313; 0. I. Maystrenko, (R. Morris, personal communication, 1976)

CS DT

3

Khrabrova et al., 1973

CS DT

11

Gale and Spencer, 1974

CS DT, N-T

10

Deckard et al., 1973

ge

5

4

ie2

01 0

(Continued)

-3

TABLE 8 (Continued) Chromosome and arm( ) 5 W )

5NL)

Trait

Materiala

Processing properties: Dough properties (major inhibitor) Protein content: Albumin Enzyme: Lipoxygenaae Lpx-A& (Coleoptile) Miscellaneous: Nitrate reduction (+effect)

5B

Reference

CS D T

13, 19

Maystrenko el al., 1973a

CS DT, N-T

3

Shepherd, 1971, 1973

CS DT, N-T

3, 14

Hart and Langston, 1975; G. E. Hart, personal communication, 1976

CS DT, N-T

10, 42

Deckard et al., 1975

2 1, 2 4

Kuspira and Unrau, 1957 Siddiqui, 1972 Sasaki et al., 1973

4

Welsh el al., 1968

4

Welsh et al., 1968

34

Garcia-Olmedo et at., 1975

CS D T

3

Khrabrova et al., 1973

CS D T

13, 19

Maystrenko et al., 1973a

Protein contat: Total protein (kernel, effect) TC subst. in CS Total protein (flour effect) TST subst. in CS Gluten ( - effect) CNN subst. in CS X CS (F1 hyb.), CNN, CS Processing properties: Baking absorption (minor Hope subst. in CS factor) Loaf volume (+effect) TST subst. in CS Miscellaneous: Purothionin lipid moiety CS N-T (moderate effectiveness)

+ +

Protein content: Gluten (fractions Bl, Be, and D4) Processing Properties: Dough properties (inhibitor)

method(^)^

Protein content: Albumin Enzyme: Lipoxygenase Lpx-BB (Coleoptile)

5D

Protein content: Total protein (flour,

+ effect)

Total protein (flour, - effect) Total protein (kernel) Total protein (kernel, effect) Gluten (kernel, effect)

+

+ Gluten (kernel, + effect)

Gluten ( - effect) Non-gliadin proteolipid (component 1 of RodriguezLoperena et al., 1975) Miscellaneous: Purothionin lipid moiety (most effective locus) Processing properties: Milling properties (flour yield, effects), kernel hardness

+

CS DT, N-T

3

Shepherd, 1971, 1973

CS DT, N-T

3, 14

Hart and Langston, 1975; G. E. Hart, personal communication, 1976

TST and C D subst. in CS

2

Hope subst. in CS Hope subst. in CS ATL 66 subst. in CS CNN subst. in CS X CS (FI hyb.), parents CS Mono X SRV 29 CS Mono x SVL CS DT, N-T

1, 2 3 2 4

B. Belderok and G. J. Doekes, personal communication, 1974 Siddiqui, 1972 Halloran, 1975 Morris et al., 1973 Sasaki ct al., 1973

23 28 5, 37

Maystrenko et al., 1973b Avila and Favret, 1966 Aragoncillo et al., 1975a

CS N-T

34

Garcia-Olmedo et aZ., 1975; Fernandez de Caleya et al., 1976

CNN subst. in CS

33, 24

B. Belderok and G. J. Doekes, personal communication, 1974; Mattern et al., 1973; M. Sasaki et aZ., personal communication, 1974 (Continued)

z

0

TABLE 8 (Continued) Chromosome and arm( ) -

5D

Reference

Trait

Materida

Method (s)~

Milling properties (flour yield, effect; hardness?)

Hope and TST subst. in CS

24

Baking (water) absorption (+effect) Baking (water) absorption (+effect) Dough strength (major factor) Loaf volume, crumb grain and texture (+ effects, minor factor)

CNN, Hope and TST subst. in CS Hope and TST subst. in CS

4

4

B. Belderok and G. J. Doekes, personal communication, 1974; Welsh et al., 1968 B. Relderok and G. J. Doekes, personal communication, 1974 Welsh et al., 1968

CNN subst. in CS CNN subst. in CS

13, 35 4

Morris et al., 1966 Morris et al., 1966

~~

+

Protein content: Gluten (fractions B,, Bq, and D4) Total protein (genes Prol, Pro2, assoc. with nucleolus organizer region) Miscellaneous: Nucleolus organizer (structural gene) Nitrate reduction (+effect) Processing properties: Dough strength properties (major inhibitor) Milling properties (- effect, flour yield)

? CS D T

3

Khrabrova et al., 1973

Hope subst. in CS, CS D T

8

C. N. Law, personal communication, 1976

CS DT, alien subst. 6X/4X derivatives CS DT N-T

8, 9

Viegas and Mello-Sampayo,

10

Edwards, 1973, 1974

CS DT

13, 19

Mayitrenko et al., 1973s

CS DT

6

Maystrenko et al., 1973a

1975

2 k

Protein content: Albumin Enzyme ( 9 ) : Lipoxygenase Lpx-D8 (Coleoptile) Miscellaneous: Nucleolus organizer (regulator) Nitrate reduction (+effect) 6A

Protein content: Gluten (+effect) Gluten (+ effect)

CS DT, N-T

3

Shepherd, 1971, 1973

CS DT, N-T

3, 14

Hart and Langston, 1975; G. E Hart, persond communication, 1976

CS DT, alien subst. 6X/4X derivatives CS DT, N-T

8, 9 10, 42

Viegas and Mello-Sampayo, 1975 Deckard et al., 1975

CS Mono X SRV 29

23

Maystrenko el al., 197313

4

Sasaki el al., 1973

CNN subst. in CS X CS (FI hyb.), CNN, CS Gliadins (joint control by regu- CS N-T lator in 2A?) Gliadins (5 components) Gliadins (4 components, aggregatable A-gliadins)

Enzyme(s): a-Amylase Esterase (flag leaf) 6A(a = S)

CS N-T CNN subst. in CS

CS Null CS aneuploids

Protein content: A-gliadins (bands 17,19,21,22) CS DT, N-T Enzyme(s): Aminopeptidase ( A m p A I ) CS DT, N-T

n

n

2 3

n n

0

2

!a

0

3

Shepherd, 1968; Waines, 1973

3, 5 3

Wrigley and Shepherd, 1973 Kasarda el al., 1973, 1974a, 1976b

r q

3, 14 3, 14

Nsgayoshi, 1973 Nakai and Tsunewaki, 1974

3

Kasarda et al., 197613

3, 14

G. E. Hart, personal communications, 1973, 1976

(Continued)

+d

m 0 Y

E

5

z d

9Y

z Y

TABLE 8 (Continued) Chromosome andarm( ) 6 A ( a = S)

6A@ =L)

Trait

Material.

Glutamate oxaloacetate transaminase Got-A3 (1st leaf blade) Miscellaneous: Nitrate reduction (+effect) Protein content: Gliadins (5 components)

+

Total protein (kernel, effect) Gluten Gliadins (bands Me, M7) Gluten (fractions Al, D4 = gliadms?, joint control of fractions Be B4, with group 2 chr.) Enzyme (s) : Esterase (band Est-6A-I) a-Amylase (band 2)

*Amylase Glutamate oxaloacetate transaminase Got-Ad (1st leaf blade)

Method(s)b

Reference

CS DT, N-T

3, 14

Hart, 1975

CS DT, N-T

42

Deckard et aE., 1975

CS DT, N-T

3, 4

I(.W. Shepherd (R. Morris,

CS D T CS D T CS DT, N-T CS D T

12 4 3 3

CS DT, N-T CS D T

3, 14 3, 14

CS D T CS DT, N-T

11 3, 14

personal communication, 1975); Wrigley, 1972a; Wrigley and Shepherd, 1973 Noda and Tsunewaki, 1972 Maystrenko et al., 1973a Shepherd, 1968, 1973 Khrabrova et al.. 1973

May et al., 1973 Nishikawa and Nobuhara, 1971; K. Nishikawa and M. Nobuham, personal communication, 1975 Gale and Spencer, 1974 Hart, 1975

Miscellanems: Nitrate reduction (structural gene) Nitrate reduction (+effect) Protein component ratio (+ prolamines) Processing properties: Dough properties (enhancer) 6B

Protein content: a-Gliadin Gliadin L4, “regulator”? Gliadins (10 components)

CS DT, N-T

10

Edwards, 1973, 1974

CS DT, N-T CS D T

10 26

Deckard el al., 1975 Bozzini and Giacomelli, 1973

CS D T

13, 19

Maystrenko et al., 1973a 0 M

CNN subst. in CS CS N-T CS N-T

Total protein (kernel, - effect) CNN subst. in CS X CS @‘I hyb.), CNN, CS Enzyme: Peroxidase CS and transfer from Ae. umbellulata Miscellaneous: “Regulator or inhibitor” (of CS N-T glutenin subunit) Protein component ratio CS D T (+prolamines) Processing properties: Gluten quality (weak inhibitor)

Milling properties (flour yield, effect) Loaf volume ( f effect) Dough strength (+ effect)

+

3, 36 3 3 4

Kasarda et al., 1974a, 1976b Shepherd, 1968 Wrigley, 1972a; Wrigley and Shepherd, 1973 Sasaki et al., 1973

2 3

0 0

0

3

!a

0

r

3

0

Upadhya, 1968

crl +d

3

Orth and Bushuk, 1974

26

Bozzini and Giacomelli, 1973; A. Bozzini, personal communication, 1974

CS Mono X SRV 210

23

TST subst. in CS

24

Maystrenko et al., 1973b; 0. I. Maystrenko (R. Morris, personal communication, 1975) Welsh et al., 1968

TST subst. in CS TST subst. in CS

24 24

Welsh et al., 1968 Welsh el al., 1968 (Continued)

e

E?

5

E

d H

bJ

TABLE 8 (Continued) Chromosome and arm( ) 6B (S)

Trait

Materiala

Protein content: Non-gliadin, proteolipid (CM components 2 and 10 of Rodriguez-Loperena et al., 1975) Gluten (+effect) Enzyme(8): Peroxidase (leaf, band C-2; root, band C-3; "suppressor" peroxidase (root C-9); peroxidase C-2 located distally to root peroxidase C-3 and root peptidase A-3) Acid phosphataee ( effect) Aminopeptidase, Amp-B1

+

a-Amylase (+ effect) Glutamate oxaloacetate transaminase Got-B1 (1st leaf blade) Peptidase (root band A-3)

Reference

CS DT, N-T

5, 37

Aragoncillo et al., 1975a

CS D T

4

Maystrenko et al., 1973a; 0 . I. Maystrenko (R. Morris, personal communication, 1975)

CS DT; Ae. umbelluhta, and chr. addition and translocation lines; T. dicoceum

3, 14

MacDonald and Smith, 1972

11

CS D T CS DT, N-T

3, 14

CS DT CS DT, N-T

3, 14

Gale and Spencer, 1974 Hart, 1973a; G. E. Hart, personal communication, 1976 Gale and Spencer, 1974 Hart, 1975

3, 14

MacDonald and Smith, 1972

CS DT; Ae. umbellulata and chr. addition and translocation lines; T. dimecum

11

Miscellaneous: Nucleolus organizer (structural gene) Nitrate reduction ( +effect) Protein content: Gliadins (10 components) Gluten (fractions A,, Da = gliadins, joint control fractions Ba and B, with group 2 chr.) Enzyme(s): Peroxidase (suppressor, band C-9 root) a-Amylase Amy-GB (19.5 2.7 map units from centromere) a-Amylase (band 3)

6D

+

a-Amylase (+effect) Esterase (leaf, band Est-GBL-I) Esterase (leaf) Glutamate oxaloacetate transaminase Got-Bb Processing properties: Dough strength properties (enhancer) Protein content: Gliadins (joint control by 2B and 2D) Gluten (+ effect, dispersion index)

CS DT, alien subst. 6X/4X derivatives CS DT, N-T

8, 9

Viegas and Mello-Sanipayo,

10

Deckard et al., 1975

CS DT, N-T CS DT

3, 5 3

Wrigley and Shepherd, 1973 Khrabrova et al., 1973

CS DT, addition and translocation lines of CS and Ae. umbellulato; T. dicoccum CS DT 6B(@)X PLD

3, 14

MacDonald and Smith, 1972

3, 14

K. Nishikawa and M. Nobu-

CS DT

3, 14

CS DT CS DT, N-T CS aneuploids CS DT, N-T

11 3, 14 3, 14 3, 5

hara, personal communication, 1975 Nishikawa and Nobuhara, 1971; Nishikawa, 1973 Gale and Spencer, 1974 May et al., 1973 Nakai and Tsunewaki, 1974 Hart, 1975

CS D T

13, 19

Maystrenko et al., 1973a

CS N-T

3

Shepherd, 1968; Waines, 1973

T H subst. in CS

28

Solari and Favret, 1970

1975

~~

-

(Continued)

TABLE 8 (Continued) Chromosome and arm( ) 6D

6D(a

=

S)

Q,

Trait

Material.

method(^)^

Reference

Gliadin (bands 18, 20, part of band 16) Enzyme(s): Amylase

CS N-T

3

Kasarda ei al., 1976b

CS Null, CS Mono X 7'. durum

3

Nagayoshi, 1973

Esterase (flag leaf) Protein content: Gluten

CS ancuploids

3, 14

Nakai and Tsunewaki, 1974

CS D T

4

Khrabrova et al., 1973; 0 . I. Maystrenko (R. Morns, personal communication, 1976)

CS DT, N-T CS DT, N-T

3 3, 14

Hart, 1973a Hart, 1975; Hart et al., 1976 (G. E. Hart, personal communication, 1976)

Enzyme(s): Aminopeptidase, A m p D l Glutamate oxaloacetate transaminase Got-Dl (1st leaf blade) Miscellaneous: Nitrate reduction (+ effect) 6D@ = L)

E!

Protein content: Gliadins (bands M , and La, regulated also by 2A) Gliadins (4 components)

Gliadin (fractions AI, Dd, joint control fractions BJ and B, with group 2 chr.)

CS DT, N-T

10

Deckard et al., 1975

CS DT, N-T

3

Shepherd, 1968

CS N-T, D T

3, 5

CS D T

3

Shepherd, 1968: K. W. Shepherd (R. Morris, personal communication, 1975); Wrigley, 1972a: Wrigley and Shepherd, 1973 Khrabrova et al., 1973

s2 r

0

?w

Enzyme ( s ): a-Amylase (band l ) , Amy 6D-I, CS DT, CS 6D(@ X PLD, 1 1 . 9 k 3 map units from tetra CTH, T . durum, centromere Aegilops spp.

a-Amylase (+ effect) a-Amylase Amy 6D-8 Esterase (leaf; bands Est6DL1, Est-GDL-8) Glutamate oxaloacetate transaminase Got-DI (1st leaf blade) Miscellaneous: PEP carboxylase activity ( - effect) Processing properties: Dough strength properties (inhibitor or regulator) 7A

Protein content: Total protein (kernel, effect) Total protein (kernel, gluten, effect) Total protein (flour, - effect) Gluten Amino acid: Tryptophan content (strong effect) Enzyme(s): Esterase (slow moving band)

+

+

+

3, 14

Nishikawa et al., 1976; K. Nishikawa and M. Nobuhara, personal communication, 1975,

CS DT CS DT CS DT, N-T

11 3, 14 3, 14

CS DT, N-T

3, 14

Gale and Spencer, 1974 Nishikawa et al., 1976 May el al., 1973; C. E. May personal communication, 1975 Hart, 1975

1976

CS DT

38

Fortini el al., 1973

CS D T

13, 19

Maystrenko et al., 1973a

0

2

1 0

0 ?¶

‘d

z

0

CS Mono X SN64 CNN subst. in CS X CS (F, hyb.), CNN, CS TST subst. in CS CS Mono X SRV 210

23

CS Mono X SN64 D chr. subst. in LDN

1

Jha et al., 1971 Sasaki et al., 1973

4

Siddiqui, 1972 Maystrenko et al., 1973b

4

15

Jha et al., 1971

cj

3, 14

Bergman and Williams, 1972

11

2

(Continued)

3>

z -J

TABLE 8 (Continued) Chromosome and arm( )

Trait

Materials ~~

Peroxidase (band d) Miscellaneous: Nucleolus activity (regulator)

~

~~

a-Amylase (+ effect) Endopeptidase Ep-A1 (seedling) Miscellaneous: Protein component ratio Nitrate reduction (inhibitor or regulator)

Method(s)* ~

Reference ~

CS N-T

3, 14

Kobrehel and Feillet, 1975

CS DT, alien subst. 6X/4X derivatives

8, 9

Viegas and MeUo-Sampayo, 1975

11

Gale and Spencer, 1974

10

Edwards, 1973, 1974

13, 19

Maystrenko et al.. 1973a

CS D T

11

Gale and Spencer, 1974

CS DT

3, 14

CS DT CS DT, N-T

3, 14

Nishikawa and Nobuhara, 1971; Nishikawa, 1973; Nishikawa et al., 1976 Gale and Spencer, 1974 Hart and Langston, 1975; G. E. Hart, personal communication, 1976

CS D T

26

CS DT, N-T

10

Enzyme: a-Amylase (+effect) CS D T Miscellaneous: Nitrate reduction (regulator or CS DT, N-T inhibitor) Processing properties: Dough properties (strength, CS D T minor inhibitor) Enzyme ( 8 ): Peroxidase ( - effect = inhibitor?) a-Amylase (band 13), Amy-7A

E 00

11

A. Bozzini, personal communication, 1974 Edwards, 1973, 1974

d

3 ?

w

7B

Protein content: Total protein (kernel, Total protein (kernel, Total protein (kernel,

+ effect) + effect) + effect)

Gluten (+ effect)

T C subst. in CS CS Mono X SN64 CNN subst. in CS X CS (F1 hyb.), parents C N N subst. in CS X CS (F1 hyb.), CNN, CS CS Mono, DT

CM 2 protein (chloroformmethanol extract) Amino acid: Tryptophan content CS Mono X SN64 and X L R Miscellaneous: Inhibitor (of C M 1 component CS N-T 2, structural gene in 6B) Processing properties: Loaf volume (+ effect, minor TST subst. in CS factor) Gluten quality (weak inhibitor) CS Mono X SRV 29 Milling properties (major gene) Dough properties (strength, loaf volume, crust appearance, grain and texture; effect) Protein content: Non-gliadin proteolipid (components 8 and 9 of Rodriguez-Loperena et al., 1975 = albumins of Waines, 1973)

2 1 4

Kuspira and Unrau, 1957 J h a et al., 1971 Sasaki et al., 1973

4

Sasaki el al., 1973

37

Garcia-Olmedo and Carbonero, 1970

15

J h a et al., 1971

5, 37

Aragoncillo et al., 1975a

4

Welsh et al., 1968

23

CNN subst. in CS CNN subst. in CS

4

4

Maystrenko et al., 1973b; 0 . I. Maystrenko (R. Morris, personal communication, 1975) Mattern et al., 1973 Morris et al., 1966

CS DT, N-T

5, 37

Aragoncillo et al., 1975a

+

7WS)

(Continued)

TABLE 8 (Continued) Chromosome and arm( )

7B(S)

Trait

Material"

Enzyme(s): a-Amylase (+ effect) Esterase (intermediate moving band) Processing properties: Milling properties (flour yield, effect)

Reference

CS D T CS DT, N-T

11 3, 14

Gale and Spencer, 1974 Bergman and Williams, 1972

CS DT

6

Maystrenko et al., 1973a

CS D T

22

Fortini et al., 1973

CS D T

3, 14

a-Amylase (+ effect) Endopeptidase, E p B l , Epl (seedling)

CS D T CS DT, N-T

11 3, 14

RuDPCase activity (+ effect) Miscellaneous: Protein component ratio

CS D T

38

Nishikawa and Nobuhara, 1971; Nishikawa et al., 1976 Gale and Spencer, 1974 Hart and Langston, 1975; G. E. Hart, personal communication, 1975 Fortini et al., 1973

CS D T

26

A. Bozzini, personal communication, 1974

3, 39

Favret et al., 1970

1 1, 2

Jha et al., 1971 Siddiqui, 1972

+

Protein content: Total protein (seedling leaf, - effect) Enzyme(s): a-Amylase (band 15), Amy-7Rl

7D

method(^)^

Protein content: Total protein (kernel, effect, TC subst. in CS 4 bands = regulator gene) Total protein (kernel, effect) CS Mono X LR Total protein (flour, - effect) Hope subst. in CS

+ +

Total protein (kernel,

- effect)

CNN subst. in CS X CS (Fj hyb.), parents Albumin CS N-T, Ae. squarrosa Proteolipid C M 3 protein (com- CS N-T ponent 11) Enzyme ( s ): Peroxidase (band a) CS N-T Esterase (intermediate band) CS DT, N-T Esterase (slow band) D chr. subst. in LDN Glutamate oxaloscetate trans- CS DT, N-T aminase Got-I Processing properties: Milling properties (+effect; TST subst. in CS flour yield, minor factor) Kernel hardness (water absorp- TST subst. in CS tion, effects, major factor) Loaf volume, crumb grain and CNN subst.. in CS texture (+effect) Loaf volume (+ effect, minor TST subst. in CS factor)

+

Prolein conlent: Non-gliadin proteolipid (CM 1 CS DT, N-T components 3 and 4 of Rodriguez-Loperena et al., 1975 = albumins of Waines, 1973) Miscellaneous: Nucleolus activity (regulator) CS DT, N-T, 6X/4X derivatives

Esterification of sterols of endosperm

CS DT, N-T

4

Sasaki et al., 1973

3 5, 40

Waines, 1973 Rodriguez-Loperena et al., 1975

3, 3, 3, 3,

Kobrehel and Feillet, 1975 Bergman and Williams, 1972 Bergman and Williams, 1972 Hart, 1975

14 14 14 14

24

Welsh et al., 1968

4, 6

4

B. Belderok and G. J. Doekes, personal communication, 1974 Morris et al., 1966

4

Welsh et al., 1968

5, 37

Aragoncillo et al., 1975a

8, 9

W. S. Viegas and T. MelloSampayo, personal communication, 1974 Torres and Garcia-Olmedo, 1974

41

(Continued)

TABLE 8 (Continwd) Chromosome andarm(

cn

to

Trait Enzyme(s): a-Amylase (band ll), Amy-TD

Materials

Method (sY

Reference

CS DT, Aegilops spp., TetraCTH

3, 14

a-Amylase (+ effect) Acid phosphatase (+ effect) Endopeptidase Ep-D1 (seedling)

CS DT CS D T CS DT, N-T

11 11 3, 14

Peroxidase (+ effect) Miscellaneous: RuDPCase activity (- effect) Processing properties: Dough properties (extensibility, “enhancer”)

CS D T

38

Nishikawa and Nobuhara, 1971; Nishikawa, 1973; Nishikawa et al., 1976 Gale and Spencer, 1974 Gale and Spencer, 1974 Hart and Langston, 1975; G. E. Hart, personal communication, 1976 Gale and Spencer, 1974

CS D T

38

Fortini et al., 1973

CS D T

13, 19

Maystrenko et al., 1973a

a Variety abbreviations: ATL 66 = Atlas 66; CD = Capelle Desprea; CNN = Cheyenne; CS = Chinese Spring: CTH = Canthatch; HFT = Holdfast; KKF MC22 = Kharkof MC22: LDN = Langdon (durum); LR = Lerma Rojo; PLD = Prelude; RE = Red Egyptian: RSC = Rescue; SN64 = Sonora 64;SRV 29 = Saratovskaya 29; SRV 210 = Saratovskaya 210; TC = Thatcher; TST = Timstein. Genetic stock abbreviations: D T = ditelosomics; Mono = monosomics; N-T = nullitetrasomics; Null = nullisomics; subst. = substitutions; chr. = chromosome. * Code for method(s): 1 = dye-binding capacity; 2 = Kjeldahl; 3 = electrophoresis: 4 = AACC; 5 = electrofocusing: 6 = quadrumat Jr. Mill; 7 = experimental baking; 8 = cytology; 9 = autoradiography; 10 = in vivo NRA analyses; 11 = enzyme yield; 12 = isoelectrofocusing; 13 = farinograph; 14 = chemical tests; 15 = amino acid analyses; 16 = sedimentation; 17 = doughball; 18 = fermentation test; 19 = alveograph; 20 = mixing; 21 = micro-Kjeldahl; 22 = Lowry N ; 23 = gluten swelling; 24 = Buhler Mill; 25 = phenol reaction; 26 = solubility fractionation; 27 = flour fractionation; 28 = gluten dispersion index; 29 = starch damage; 30 = water absorption; 31 = immunochemical tests; 32 = immunoelectrophoresis; 33 = quadrumat; 34 = petroleum ether extract; 35 = mixograph; 36 = pH separation, aggregation; 37 = electrophoresis ETOH extract; 38 = in vitro enzyme activity analysis; 39 = Coleman nitrogen analyzer; 40 = electrophoresis of chloroform-methanol extracts; 41 = ether extract, 4 kernels mc, fluorometry; 42 = i n vitro NRA analyses.

to

*F

0

2 J

p

P4

GENETIC CONTROL OF PROTEINS I N WHEAT

523

stocks of the hexaploid wheat variety Chinese Spring (Fig. 6). Even better illustrations of homoeology and genetic relations have been shown in isoenzyme studies (Hart, 1970, 1973a,b, 1975). May et al. (1973) have distinguished four ways by which homoeologous genes of hexaploid wheat may function in accordance with the Jacob and Monod (1961) concept of structural (producer and conformation) genes and regulator genes: (1) The structural and regulatory genes on homoeologous chromosomes are identical. The biosynthetic products of such genes are identical and do not change in concentration in the nullisomictetrasomic lines, indicating complete compensation, i.e., triplicated genes (Morris and Sears, 1967). Duplicated genes controlled in a similar way would produce a constant amount of product independent of the number of gene loci present in the genome. (2) The structural genes differ, but the regulatory genes on homoeologous chromosomes are identical. The products of such genes are distinguishable in various ways, e.g., as doublet or triplet sets of isozymes, and compensation in the nullisomic-tetrasomic lines occurs by increase of an homoeologous gene product. Example: group 6 esterases, and possibly the a-amylases in groups 6 and 7. (3) The structural genes are identical but the regulator genes are different in homoeologous chromosomes. The genetic relations involved are distinguishable by the occurrence of different amounts of identical products and thus only partial compensation occurs in nullisomic, tetraso-

0

FIG.6. Chromosomal control of gliadin synthesis in wheat endosperm as detected using combined electrofocusing and electrophoresis techniques in the analysis of proteins of aneuploid genetic stocks of Chinese Spring wheat. (Reproduced from Wrigley and Shepherd, 1973. Courtesy of the authors and New York Academy of Sciences.)

524

CALVIN F. KONZAK

mic and disomic substitution lines. Minor genes differ from major genes as regards regulatory functions. (4) The structural genes and regulatory genes are in each of the homoeologous chromosomes. In this case the genetic relations involved are distinguishable by the absence of genetic compensation. As for the “diploidized” genes described by Morris and Sears (1967), the absence of a particular homoeologous chromosome or segment results in the absence of the gene product. Biochemical genetic analyses offer to reduce to more simple terms the evident complexity of protein differences among wheat varieties. For example, several prolamine (gliadin) proteins which distinguish the strong us weak gluten varieties, respectively, Thatcher (TH) and Chinese Spring (CS), prove to be controlled by genes located in chromosomes of the A, B, and D genomes of homoeologous group 1 (Fig. 7). Electrophoretic analyses (Manghers et al., 1973) using nullitetrasomic stocks and disomic substitutions of group 1 Thatcher chromosomes into Chinese Spring demonstrate that of the gliadins by which the two varieties differ: (1) Three are controlled by genes present in TH 1A. (2) Another three proteins, which are structurally evolved from those of TH lA, are controlled by the homoeologous chromosome TH 1B. (3) Two other proteins are structurally identical (duplicated) and controlled by genes present in TH 1A and 1B. (4) CS carries the null alleles for all 8 of these proteins. ( 5 ) Two gliadins common to TH and CS are shown to be controlled by chromosome lD, from their absence in nulli 1D tetra 1A and nulli 1D tetra 1B. (6) One protein controlled by chromosome 1B is present in CS;

- - Bzzm

Fzmz&l tzzzzzm r?zzzzm

ezzzza

m

-

b

!m%zEa

B

-

b

-4-44

!&z8zta44

-4-

-4-

EBziza

EZwa?a+a

Ezzz?aa

mBZm

mm2m

EzPzzm

THATCHER

C. SPRING

C.S.+TclA

-*o

-+-e

3-4mZzPA4C.S.+TclB

-

C.S.+ N1O.TlA NID.TlB

FIQ.7. Prolamine patterns for slow-moving bands (buffer pH = 3.2) in Thatcher and Chinese Spring wheats, substitution lines of Thatcher 1A and 1B of Chinese Spring. Arrows on the left show deletion, and those on the right show addition of proteins. Arrows with a circle indicate a probable case of duplicated proteins. (Reproduced from Manghers et a l , 1973.)

GENETIC CONTROL O F PROTEINS IN WHEAT

525

the null allele is in TH 1B. Differences in the genetic regulation of proteins also are evident by the changes in genetic compensation shown in the electrophoretograms : (a) Thatcher 1B carries a regulator gene which increases the amount of one protein common to both varieties. I n this case the homoeologous locus for the structural protein is identical in the two varieties but the regulator genes differ. (b) I n the second case, genetic compensation by TH 1B fails because TH 1B carries the null allele for the structural gene of a homoeologous protein, controlled also by chromosome 1A in both varieties. However, the regulator gene for this protein is present in TH 1B. (c) The third protein is deleted from the TH 1B substitution because TH 1B lacks the structural and regulator genes present in CS 1B. I n similar ways, a considerable number of structural and regulator loci controlling protein synthesis have been identified (Table 8 ) . Certain general conclusions relative to the genetics of protein content, amino acid composition, various processing factors, and specific proteins are now apparent from the experimental results described earlier and those listed in Table 8. 1. Homoeologous, structurally evolved genes which code for proteins are located in several chromosome groups of each of the three genomes. 2. The majority of gliadin proteins (prolamines) are controlled by genes in chromosomes of groups 1 and 6. 3. Specific proteins are absent when arms of chromosomes are deleted. The concentrations of these same proteins are usually increased when the dosage of the chromosomes carrying the genes is increased. The concentrations of specific proteins may depend on whether there are 0, 1, 2, 4,or 6 chromosome doses. 4. Monomer subunits of dimer enzymes, such as alcohol dehydrogenase, are produced in amounts proportional to the dosage of the homoeologous chromosomes carrying the structural genes. 5. Evolutionary changes often have modified the structural and sometimes the functional characteristics of the gene products coded by different homoeologous chromosomes (presumably the gene structure has been altered), but regulation may remain similar in each genome for the homoeologous proteins (e.g., gliadins, alcohol dehydrogenases) . 6. Hybrid enzymes (isozymes of phosphodiesterase) occur in hexaploid wheat that are comprised of components identified in A . squarrosa (D genome) and components present in several tetraploids (probably B genome), but are not present in a number of diploids. A component similar to one in tetraploids occurs in several diploids. New hybrid dimer enzymes (alcohol dehydrogenases) occur in wheat-rye hybrids and rye chromosome substitution lines.

526

CALVIN F. KONZAK

7. Genes controlling the production of unique proteins are present in each of the three wheat genomes. 8. Homoeologous alleles may have different levels of activity or “strength” with relation to other alleles (regulatory genes evolved) ; this relation is exemplified by the different production of the lipid moiety for purothionins by group 5 homoeologs. Some specifics relative to proteins identified in the seven chromosome groups are summarized below :

Chromosome group 1 1. Genomes A, B, and D each may carry homoeologous, evolved, producer (structural) genes for gliadins, purothionins, peroxidases. 2. Chromosome arms 1A(S), l B ( L ) , and 1D(S) are implicated in the process of nitrogen reduction and probably carry structural genes. 3. Chromosome 1D codes for two unique subunits of high MW glutenin, proteins that are important to dough elasticity in hexaploid wheat. 4. Differentiation occurs in gliadins of different varieties, possibly also relating to differences associated with regulation or inhibition by factors in group 2 chromosomes. 5. Chromosome 1D of Cheyenne may carry ( a ) gene(s) increasing lysine over 15%. Chromosome group 2 1. Chromosome 2A and probably chromosomes 2B and 2D carry regulators (or inhibitors) of nitrate reductase activity (NRA). The alien chromosome substitutions with high protein content and the inhibition of 2B and 2D of gliadin synthesis in 6D indicate a homoeologous series of inhibitor loci. 2. Chromosome 2A in ‘Saratovskaya 210’, Hope, Timstein and ‘S-615’, and 2D in Saratovskaya 29 carry genes for high crude protein content, indicating the possibility that the NRA “inhibitor” factors have been modified in these varieties. 3. Group 2 chromosomes apparently carry genes affecting a-amylase enzyme content. 4. Phenolase complex activity is associated with chromosomes 2B and 2D, suggesting that a homoeologous series will be found. Chromosome group 3 1. Genomes A, B, and D may carry homoeologous esterase, peroxidase (Per), a-amylase ( A m y ) , glutamate-oxaloacetate transaminase ( G o t ) , and possibly for acid phosphatase (Acph) loci. 2. Genomes A and D carry a homoeologous esterase seemingly absent from B. 3. Genome A carries a unique esterase locus.

GENETIC CONTROL O F PROTEINS I N WHEAT

527

4. Chromosome arm 3B(L) is implicated in the process of nitrate reduction. 5. Genomes A and D carry loci controlling several “gliadinlike” proteins. 6. Genomes B and D carry loci controlling proteolipids.

Chromosome group 4 1. Genomes A, B, and D may carry evolved homoeologous acid phosphatase, alcohol dehydrogenase (Adh), and lipoxygenase ( L p z ) genes as well as genes affecting a-amylase content. 2. Genomes A, B, and D carry genes affecting milling properties (flour yield). 3. Chromosome 4D controls the formation of a glutenin subunit. Chromosome group 5 1. Genomes A, B, and D in Chinese Spring may carry homoeologous genes controlling the production of the lipid moiety for the purothionins, with the gene on 5 D most effective. 2. Genomes A, B, and D carry homoeologous structural genes for lipoxygenase and for “gliadinlike” proteins. 3. Chromosome 5 D of Atlas 66 carries gene (s) for high protein content. This factor may be concerned with translocation of N into the grain. 4. Chromosomes 5D in Cheyenne, Timstein, and Hope carry a kernel hardness gene affecting milling properties. 5. Chromosomes 5 D in Capelle Desprez, Hope, Timstein, and ‘Sinvalocho’ carry gene (s) for higher protein or gluten content. 6. Chromosome 5 D carries nucleolus organizer, regulator, and structural genes and a factor affecting nitrogen reduction (a single cistron?). Chromosome group 6 1. Genomes A, B, and D may carry homoeologous structural gene loci controlling the synthesis of gliadins, esterases ( E s t ) , Amy, aminopeptidases (Amp),Got, and are of major importance for gliadin production. 2. Chromosome arm 6A(p) carries a structural gene concerned with N reduction. 3. Chromosome 6B carries unique Amy and Per loci. 4. Chromosome 6D carries an inhibitor (or regulator) of PEP carboxylase activity. Chromosome group 7 1. Genomes A, B, and D carry possibly homoeologous, structurally evolved loci controlling a-amylases ( A m y ) ,and endopeptidases ( E p ). 2. Genomes A and B carry possibly homoeologous esterase loci. 3. Genomes B and D carry possibly homoeologous esterase loci.

528

CALVIN F. KONZAK

4. Chromosome arms 7A(S) and 7A(L) carry regulators or inhibitors of NRA. 5. Chromosomes 7D carry gene(s) affecting kernel hardness and milling properties of Timstein, a regulator of nucleolar activity in Chinese Spring, and an inhibitor of RuDPCase activity. 6. Chromosome 7B carries a gene affecting dough mixing characteristics in Cheyenne, and a promotor of RuDPCase activity in Chinese Spring. 7. Chromosome 7A of Cheyenne carries a dominant gene having a negative effect on protein content. XI. General Discussion

Compared with the other main cereals, wheats differ in two important ways that have genetic consequences: (1) All important cultivated wheats are polyploids, i.e., tetraploid or hexaploid, while maize, barley, rice, and sorghum are all diploids. (2) I n wheats, as in barley, corn, rice, and sorghum, the genetically controlled protein components present in highest proportion have the lowest nutritional value. Yet many protein components, mainly prolamines, are evidently vital to the unique properties of wheats. I n all the high-lysine sources of maize, barley, and sorghum identified to date, the proportion of prolamines in the proteins is sharply reduced. The main uses of wheats are now determined by the suitability (quality) of flours (or semolina) milled from them for often rather specialized baked products, e.g., breads, pasta, cakes, and cookies. Through intense selection and breeding over centuries, classes or genetic races of wheats adapted for those end-product uses have been isolated, and in many cases have displaced from cultivation those wheats with other quality properties. The main unique features of wheats are concerned with the doughforming properties of gluten, fundamental to the structure of leavened and unleavened bakery products. Processing technology, constantly changing, can exploit or reduce the importance of protein compositional differences. Current trends are toward stronger gluten wheats, with strength mainly due to high MW glutenins. Wheat gluten (once thought to be simple) has proved t o be comprised of polypeptide chains which are distinct in many ways-molecular size, charge, and solubility in various polar and nonpolar solvents, detergents and other chemical compounds (mercuric chloride, mercaptoethanol, etc.) . The subcomponents of gluten show variability in amino acid composition and in their associations with lipids, thiols, and carbohydrates. Certain

GENETIC CONTROL OF PROTEINS IN WHEAT

529

of the “insoluble” (glutenin) components of wheat gluten are undoubtedly remnants of cellular organelles and the endoplasmic reticulum of the cytoplasm. Protein bodies are observed in developing endosperm, but their identity is virtually lost in the mature endosperm and in flour. The laminar structure of protein bodies observed in the immature endosperm is disturbed in the mature grain, and subjected to considerable further modification by interactions with other cellular components upon wetting and mixing of flour components during the formation of dough. According to a recent hypothesis, many of the characteristics of wheat gluten proteins from flour may be artifacts (Bernardin and Kasarda, 1973a,b; Mecham, 1973; Kasarda, 1970; Bernardin, 1975). Flour proteins released by breaking open endosperm cells during milling or by the crushing forces of dehydration processes during maturation are free to interact with other cellular constituents, such as salts, sulfhydryls (glutathione), etc., and do so instantly upon wetting. The solubility characteristics of the gluten a t high protein concentrations (considered mainly gliadins) change as water absorption (and mixing in dough) proceeds (Kasarda et al., 1976a). There still is some uncertainty about the nature, MW, and properties of the glutenins; hence the very large molecular size and low water-absorbing capacity of some glutenins also may be artifacts reflecting the state of gluten protein components in dough, but not in the intact endosperm cells. However, after reduction, alkylation, and other treatments, a portion of the high-MW proteins remain insoluble in alcohol but soluble in dilute acid. The latter are considered glutenins. Conformational and other alterations can be induced in proteins by changes in pH and ionic strength, and by chemical bonding due to oxidative reactions, once the protein subunits are free (Bernardin and Kasarda, 1973a). The relationships between the individual gliadin components and glutenin subunits are not yet clear, but it should now be possible to investigate them, since virtually all of the protein in wheat can be made soluble without denaturing any one component. The rapid wetting ability of proteins observed by Bernardin and Kasarda (1973a) suggests that the slower water absorbing (long mixing time) ability of certain wheat flours may be due to differences in their composition of hydrophilic us hydrophobic chemical groups, which eventually are modified during dough mixing. The complexity of wheat proteins seems to present a formidable obstacle to the nutritional improvement of wheat proteins via plant breeding, while maintaining the unique processing and product-use characteristics currently important to man. Nevertheless, the prospects for improving the nutritional composition of wheat proteins are not so limited as they may seem.

530

CALVIN F. KONZAK

Essentially all wheat protein components and quality factors are controlled by genes, but genetic linkages may impede recombination, and limit progress in selection for quality improvements. The useful germplasm sources already identified indicate that a basis for significant improvements (although yet smaller than those in other cereals) is available in wheat. If the higher lysine factor on Cheyenne 1D is confirmed, the 15% increase in lysine associated with that chromosome is equal to the advantage of Hiproly over normal barley. I n fact, the gene duplication resulting from the polyploid nature of wheat may endow wheats with greater, rather than less, genetic potential for the desired changes in protein composition: (1) Only minor adjustments in protein composition may be necessary to achieve important nutritional improvements. (2) Those proteins containing the highest concentrations of lysine (especially globulins and albumins) normally are present as a small proportion of the total wheat protein and evidently are of minor importance to doughforming properties. (3) Any negative effects of increases in the soluble proteins on the functional properties of dough might be compensated by introducing genes for greater gluten strength, for which considerable variability is available. Certain components in the glutenin fraction also may have appreciably higher lysine than others, and although the lysine composition of glutenin is lower than in albumins and globulins, the glutenin fraction is a much greater part of the total wheat protein. Furthermore, the evident complexity of wheat proteins may not have the significance that seems apparent: (1) All gliadin proteins (a, p, y , and 0 ) separated by electrophoresis differ only slightly in amino acid composition and amino acid sequence, although the differences evidently are sufficient to alter some physical properties in solutions differing in pH and ionic strength. (2) All gliadins appear to be compactly folded globular proteins with similar functions in dough (Bernardin, 1975; Kasarda et al., 1976a). (3) Certain glutenin proteins may be far more important than others (Bietz et al., 1975; Orth and Bushuk, 1974). Hence, it may be possible to genetically reconstruct other equally effective protein complexes, e.g., increasing or decreasing some gliadins and increasing some glutenins. Some specific proteins a t lower concentrations may be rheologically more effective than others per unit of protein, as is suggested from recent studies on cy-gliadins by Bernardin (1975). The basis for wheat quality may, in fact, be much more simple than might be supposed from detailed analyses (Wrigley and Moss, 1968; Orth et al., 1972; Bushuk, 1972). Only four measurements of grain and flourmixing properties may determine the quality of sound normal wheats-

GENETIC CONTROL O F PROTEINS IN WHEAT

531

kernel hardness, dough stiffness, dough strength, and dough stability. New breadmaking processes, among them the Chorleywood process (highspeed mixing), a new nonfermentation process (Chamberlain e t al., 1962; Kilborn and Tipples, 1972a,b; J . A. Johnson and Sanchez, 1972; Bushuk, 1972), and the short-time baking systems (Finney et al., 1977; Magoffin et ul., 1976) also have a bearing on the importance assigned to particular protein components, especially in technologically advanced countries. However, it appears that wheat flours with stronger gluten properties may become increasingly important because of their use in blends with weaker flours (wheat, rye, oats, etc.) as well as with materials added to bolster the nutritional properties of bakery products, e.g., malt, soy flour. Stronger gluten wheats also appear to retain dough strength longer during malting, possibly permitting greater development of nutritionally improved protein components (P. L. Finney et al., personal communication, 1976). Genetic linkages, as indicated in the studies by Shepherd (1968), can be expected t o limit the facility with which protein composition and characteristics can be manipulated by breeding (Orth and Bushuk, 1972; Manghers et al., 1973; Wrigley and Shepherd, 1973; Bietz et al., 1975). However, genetic variability in proteins coded by individual chromosomes does exist and can be induced (Avila and Favret, 1966), permitting the reconstruction of varieties with newly designed protein complexes. A rather wide genetic variation in molecular composition of gliadins is possible, but is of questionable consequence to rheological and other measured properties of doughs, even though the proportion of gliadin present in the flours often influences the rheological properties of doughs by shortening mix time. Starch damage incurred during milling, especially of hard endosperm wheats, may confound data from sedimentation, mix time, loaf volume, and other measurements, such as water absorption, so that the true effects of some protein differences are obscured (Farrand, 1964, 1969, 1972). In barley, the high lysine content of Hiproly and several mutants may be due in part to a higher-lysine glutelin and in part t o a marked increase in the proportion of a particular saline-soluble fraction that is high in lysine. I n barley, as well as in wheat, the saline-soluble (globulin) purothionins have an exceptionally high lysine content (9.8 to 15 gm of lysine per 100 gm of protein). Purothionins are bound with lipid as lipoproteins, but are separable from the lipid (Nimmo et al., 1968; Garcia-Olmedo et al., personal communication, 1974; 1975). This protein may be one of those increased in the high-lysine barley variants. However, the potential nutritional value of purothionins is dubious owing

532

CALVIN F. KONZAK

to their antibiotic properties and possible toxicity to animals. Recent evidence that the purothionins may be extraction artifacts suggests that the native proteins involved could still have nutritional importance. Perhaps more significant is the evidence that the lysine content of the prolamine fraction in a high-lysine barley mutant, Bomi 1508, has been increased by about 300% (albeit with a reduction in proportion of prolamines from 29 to 9%) , with a lower, but significant, lysine composition increase occurring also in the glutelin fraction. The increased lysine in the glutelin fraction of Bomi 1508, though relatively small, is of significance because of the high proportion (39%) of the glutelin protein. I n some maize mutants, a glutelin fraction is increased both in proportion and in composition of lysine. Considerable variation in lysine composition among different gliadins and lox higher lysine composition for some soluble proteins has been observed by Booth and Ewart (1970). Since the evolutionary relationships generally indicate similar potential for genetic variability among all Gramineae, it should be possible to identify and increase high-lysine components among those glutenins of wheat that are genetically related to the glutelins of barley and maize. The relatively higher lysine composition of some wheat glutenin fractions compared with gliadins suggests that some subcomponents in glutenins may contain above-average amounts of lysine as is the case for rice glutelin. The amino acid composition may also be influenced by the addition (and presumably by substitution) of rye chromosomes to wheat. Riley and Ewart (1970) found the addition of chromosome I of ‘King 11’ rye to ‘Holdfast’ wheat increased the lysine content of the endosperm by 8.7%. Since rye chromosome I is homoeologous to group 5 chromosomes of wheat, they suggested that group 5 wheat chromosomes may be important in the determination of the lysine content of wheat proteins. The King I1 rye chromosome I also increases the cysteine content by 10.70J0,while rye chromosome I1 increases proline by 9.1% and reduces aspartic acid by 8.676. Chromosome IV increases, but VI reduces, arginine by 11.7 and 8%, respectively. Rye chromosome VI also increases proline by 11.8% while chromosome VII reduces threonine by 10.4% (Riley and Ewart, 1970). Resolution of the many protein complexes by combinations of electrophoretic, electrofocusing, and chemical separation techniques using aneuploid stocks is clearly illustrated in the identification of the genetic basis for the synthesis and regulation of the lipoprotein purothionins (GarciaOlmedo and Carbonero, 1970; Garcia-Olmedo et al., 1975). The use of different extraction methods for analyses of a wide variety of wheats and wheat relatives (in addition to the aneuploid material) revealed structural genes coding the two purothionin analogs evolved prior to the for-

GENETIC CONTROL O F PROTEINS IN WHEAT

533

mation of tetraploid wheats. The additive contributions t o the total purothionin content from genes in each of the homoelogous chromosomes has been demonstrated, while differences in apparent concentration of the two purothionin analogs in different wheats is a consequence of the different production of the lipid moiety by genes in different chromosomes of the three genomes. Many others have observed differences in the density of proteins on electrophoretograms which suggest additive contributions of duplicate and triplicate genes coding for the same protein (Shepherd, 1973; Bozzini et al., 1973; B. L. Johnson et al., 1967; Cuhadda et al., 1975; GarciaOlmedo et al., 1975; Hart, 1970, 1975; Tang and Hart, 1975; H a r t et al., 1976). However, additive epistatic and complementary types of gene action are known for many genetic traits; hence the genetic control of proteins can be expected to be similar. In fact, Riley and Ewart (1970) reported that the effects of rye chromosome additions on amino acid content in triticale are independent of the presence of wheat genomes; instead, the wheat genomes appear to be epistatic to individual rye chromosomes in their effect on the content of threonine, glutamic acid, proline, alanine, valine, and lysine. Similarly, the effects of individual rye chromosomes on amino acid composition may be reversed or concealed by additive or interactive effects of other rye chromosomes (Riley and Ewart, 1970). Changes in these genetic relationships may improve nutritional value. Recent work using scanning EM demonstrates genetically controlled differences in the ultrastructure of wheat proteins, and indicates that this new technique can be usefully applied to the resolution of the genetic basis for differences in protein structure. This work has revealed variability in the ultrastructure of protein fractions which was not detected or predicted by previous chemical and physical methods of analysis. Likewise the new work relating the conformational structure of individual proteins to physical-chemical properties and interactions in dough has suggested new hypotheses about t.he nature of native proteins and their reactions to solvents. Sequencing of individual proteins and subunits has only begun, but should answer important questions. New extraction methods have achieved nearly complete dissolution of all wheat proteins. The use of HgC1, and mercaptoethanol, reveals heterogeneity in the subunit composition of the high M W glutenins (Mecham et al., 1972; Bietz et al., 1973; Bietz and Wall, 1975; Huebner and Wall, 1975, 1976; Cole et al., 1976). These glutenins were previously found to be present in a residue insoluble in acetic acid, alcohol, or salt water, but are of greatest importance to the strength and elasticity properties of wheat gluten (Orth and Bushuk, 1972; Hoseney et al., 1970a; Bietz et al., 1973). The new methods of Bietz et al. (1975) almost totally ex-

534

CALVIN F. KONZAK

tract glutenins, and may be more suitable for genetic and breeding studies, since the absence of a band in electrophoretograms must be related to a genetic difference, and not to possible differences in the assembly of subunits into native glutenin molecules. The new evidence revealing the complex genetic regulation of ratelimiting steps in the N reduction process (especially the identification of inhibitors of these processes and the increased protein content resulting from their deletion or substitution by alien chromosomes) has already provided a new insight into ways to increase protein content and enhance N use efficiency of wheat by breeding. Further knowledge about the enzymological and genetic aspects of N reduction, N utilization, and the translocation of amino acid and protein subunits to the developing endosperm should provide the basis for removal by breeding of factors that limit protein accumulation in the endosperm and which affect the chemical and physical characteristics of the proteins stored therein. The existence of genes responsible for inibiting such processes as N reduction is evidence that energetics calculations comparing the ability of wheats to produce protein us carbohydrates cannot provide realistic estimates of the possibilities for improvements by genetic means. Clearly, our existing wheats do not synthesize proteins, or carbohydrates for that matter, as efficiently as they might upon removal of these inhibitors, or by modifying such energy-wasting processes as photorespiration. Genetic variation with respect to most protein traits investigated seems to be detectable if a sufficient search is made using modern methods of extraction and analysis (Doekes, 1968, 1973; Khrabrova e t al., 1973; Wrigley and Shepherd, 1974). This suggests that the potential for recombination breeding to improve protein nutritional value may be enhanced by electrophoretic studies and chemical analysis of specific proteins. Use of these laboratory screening methods may assist the breaking of genetic associations which may be required to achieve the desired goals. Chromosomes of the same or different homoeologous groups share in the genetically controlled synthesis of protein dimers, and may carry genes which regulate the production of proteins structurally determined by loci on other chromosomes in different genomes. The complex interactions possible in the hexaploid wheat system make the analytical work especially difficult, hence several approaches and methods of analyses often must be used. The introduction of alien species chromosomes, induction of mutations, transfer of nuclear material to different cytoplasms, and the resynthesis of new tetraploid and hexaploid combinations from wild diploid wheat relatives and other grass species further expand the opportunities for biochemical genetic analyses and for improvements by

GENETIC CONTROL O F PROTEINS IN WHEAT

535

breeding. The use of the two-dimensional electrofocusing-electrophoresis method may also distinguish more variations than the standard onedimensional procedure. The higher resolution possible with this method has been demonstrated with gliadin proteins by Favret et al. (1970) and by Wrigley and Shepherd (1973). XII. Role for Induced Mutations

Several lines of evidence suggest ways in which induced mutation methods can be employed to improve wheat protein, and may explain some mutations already identified in induced and natural genetic resource materials. A. PRINCIPLES 1. Removal of “Inhibitors” Affecting the Quantity, Composition, and Physical Properties of the Endosperm Proteins

Maystrenko et al. (1973a) have shown that the rheological properties of ditelosomic stocks 5A(L), 7A(L), 4B(L), 2D(L), 5D(L), and SD(L), each lacking one arm of the respective chromosome, were improved over those of the standard euploid Chinese Spring. Whether any of the improvements were due to increased protein content or changed composition has not been determined. They found, however, that several other ditelosomic lines had significantly poorer rheological properties than Chinese Spring. Ditelosomic 5D (L) had a significantly higher flour yield, suggesting that the grain hardness factor, which is responsible for higher starch damage and important to the milling and baking properties of hard wheats like Cheyenne, may be a gene inactivation or deletion in the short arm of chromosome 5D. Edwards (1974) and A. Bozzini and M. Giacomelli (personal communication, 1974) observed that Chinese Spring ditelo 2A(S) has higher NRA than the euploid. Edwards (1974) noted also that similar “inhibitors” or “regulators” identified using the ditelo and nullitetrasomic stocks are present in each arm of chromosome 7A of Chinese Spring, and he observed that several other chromosomes were concerned with N reduction. Obviously, where it can be determined by genetic or other tests that useful wheat varieties carry inhibitors of N metabolism, these inhibitors could be inactivated or deleted by the use of mutagen treatments, resulting in improved genotypes. The variety Cheyenne, for example, appears to carry the 2A inhibitor of protein content, since Cheyenne chromosome 2A substitutions into Chinese Spring have no higher protein

536

CALVIN F. KONZAK

content than Chinese Spring (B. Belderok, personal communication, 1974;

M. Sasaki et al, personal communication, 1975) and the NRA of these lines is similar to Chinese Spring (R. L. Warner, personal communica-

tion, 1974). On the other hand, genetic tests show that Saratovskaya 210 carries a high protein gene on chromosome 2A suggesting that this cultivar may already carry a deletion or inactivation of the 2A inhibitor. Other lines of evidence suggest a possible basis for the inhibitor US enhancer interactions observed. One of these concerns the roles and interaction of nuclear and cytoplasmic organelle “genes” on the various synthetic processes : (a) Nuclear genes and cytoplasmic organelle DNAs may code different subcomponents of a protein polymer, such as RuDPCase, reported by Wildman and associates (see Kawashima and Wildman, 1971, 1972; McFadden and Tabita, 1974). (b) Electrophoretically different acid phosphatases are produced when genomes of wheat-rye hybrids or amphiploids are introduced into rye as compared to wheat cytoplasm (J. Rimpau and G. Robbelen, personal communication, 1974). (c) The number of proteins made on 80 S ribosomes is directly related to chromosome number in tetraploid and hexaploid wheats which have similar cytoplasm (Nagabhushan and Zalik, 1974). These lines of evidence suggest that in certain genotypes various alternative and functionally different (enzyme protein) subunit components may be produced by the action of nuclear genes in each of the three wheat genomes ; or, functionally different regulators which interact with cytoplasmic organelles may be produced by genes in each of the three genomes. Thus, genes of three genomes must interact in a cytoplasm with semiautonomous organelles controlled by, and originally evolved for, interaction with genes of one genome. Sears (1972) has suggested a similar basis for the action of certain nuclear genes that cause chlorophyll deficiency in wheat (see also Khush, 1973). Arnason (1956) has reported a maternally inherited striata chlorophyll deficiency mutation in wheat, presumably due to an induced alteration of plastom DNA. Thus, similar genetic phenomena also can occur as the consequence of evolutionary mutations in the plastom or nuclear counterparts of nuclear gene-cytoplasmic organelle interactive systems. A second line of evidence suggests that the inhibitor us enhancer gene relationship could be due to different intergenomic gene (or gene-product) interactions in the formation of polymers, which may function differentially as enzymes (or as important subunit components of gluten) : (a) New alcohol dehydrogenase (ADH) dimers are produced upon substitution of the homoeologous rye chromosome for wheat chromosomes in group 4; the different isozymes of ADH present in hexaploid wheat prove to be homo- and heterodimers of components produced by

GENETIC CONTROL OF PROTEINS I N WHEAT

537

genes in each of the three genomes (Hart, 1970, 1971, 1973, 1975; Irani and Bhatia, 1972). Similarly, the glutamate oxaloacetate transaminase (Got) isozymes of homoeologous groups 3 and 6 may be homo- or heterodimers of components produced by genes in the different genomes (Hart, 1975; Hart et al., 1976). Evidence from Nishikawa and Nobuhara (1971) suggests that isozymes of a-amylase may be similarly constructed. (b) Two or more chromosomes from the same or different genomes are involved in the production of specific gliadin polypeptides (Shepherd, 1968; Waines, 1973). (c) Different component density (quantitative) changes for specific gliadins are found in different nullisomic-tetrasomic combinations (Shepherd, 1968, 1973 ; Wrigley and Shepherd, 1973). New “gliadinlike” proteins occur in an amphiploid of A . caudata and A . umbellulata (Waines and Johnson, 1971). A third line of evidence suggests that one gene-controlled component may affect the function of another, e.g., the activity of an enzyme, by changing the cell pH, by directly interfering with a regulating system, or by functioning as an inducer: (a) an organic acid produced in maize cells inhibits NRA in vitro (Ku, 1973) ; (b) a low-hlW protein in maize stimulates NRA (Ku et al., 1973) ; (c) an enzyme, possibly a protease, degrades nitrate reductases (Wallace, 1974) possibly a t faster rates in different genotypes. Each of these possibilities seems compatible with the theoretical considerations about gene regulation in higher plants proposed by Britten and Davidson (1969). The various evolutionary processes undoubtedly have modified the nuclear vs cytoplasmic “gene” and gene-gene interrelations associated with the construction of enzymes or other active (functional) components in dough. Evolutionary modification, with selection toward better balance and greater efficiency of biosynthetic systems, is still a basis for improvements by breeding. This evolutionary modification may be akin to diploidization, which has proceeded only a relatively small degree in polyploid wheats (see MacKey, 1968a,b, 1970). In Triticale, an artificially synthesized species, the genetic systems controlling protein synthesis and accumulation appear to be the sum of those from the two parents functioning independently in the cells throughout endosperm development (Dexter and Dronzek, 1975a,b). In some triticales, however, the genetic systems responsible for amylase control appear to be out of phase, resulting in some degradation of starch and, hence, poor endosperm development upon maturation (Simmonds, 1974; Dedio et al., 1975). Some genetic systems in polyploid wheats may become similarly out of phase by evolutionary changes or may be unnecessarily redundant because evolutionary changes have not yet occurred. Important functional and compositional improvements in wheat proteins might be achieved by

538

CALVIN F. KONZAK

adjusting such “gene”-controlled relationships via recombination breeding, induced mutations, and by cytoplasm changes, perhaps followed by mutation induction. 2. Inactivation of Deletion of Genes Controlling the Production of

Antimetabolites, Toxic, or Otherwise Undesirable Components Genetically controlled differences in the content of specific proteins occur among cereal varieties, species or genera. Albumin proteins known to inhibit animal, human, and insect, but not wheat amylases have been identified in different varieties (Silano et al., 1973, 1975; Petrucci et al., 1974). Protease (trypsin) inhibitor(s), more abundant in some rye and triticale varieties than in wheat or other triticales, may function as antimetabolites by lowering feeding efficiency of the grain or flour (Madl and Tsen, 1974a,b), Moreover, in certain instances the antimetabolite components appear to be rather heat stable (Madl and Tsen, 1974b). Certain gliadin components (including a-gliadins) appear t o have a causal relationship to celiac disease in man (Kasarda, 1975; Kasarda et aZ., 1976a,b), but may be present or absent in varieties with similar processing quality characteristics (Kasarda et al., 1973). The fact that quantitative and/or qualitative differences in antimetabolites, etc., occur among wheat varieties and related forms indicates that these components are not essential in modern cultivars. The genetic removal of antimetabolites would likely improve the nutritional value of many cereal varieties (Kakade, 1974). 3. Modifications to the Amino Acid Composition of Storage Protein

Data relating the effects of genetic variations in storage protein composition are yet limited. However, some gliadin fractions and glutenin subunits may contain distinctly more lysine than others (see Kasarda et al., 1976a). Cereal chemists, wheat geneticists, and breeders know that varieties may differ markedly with regard to the stiffness, extensibility, and as well as the water absorption speed and capacity of their proteins. The amino acid components and their order in the protein molecules are important determinants of these and other physical properties. Thus, the properties of these proteins (or protein subunits in the case of glutenins) may be influenced by the interaction of the amino acid components in determining the net molecular charge, balance of hydrophobic us hydrophilic groups, etc. (Ewart, 1975; Kasarda et al., 1976a). An increase in the amount of lysine, for example, might be expected to increase the water solubility and negative charge balance of the protein in which it is located. Such changes could have important effects on the processing characteristics of wheat proteins. Genetic variations of this type un-

GENETIC CONTROL O F PROTEINS IN WHEAT

539

doubtedly occur to some extent already among existing genetic resource stocks. The variety Mahratta, and Nap Hal accessions are possible examples. These wheats have extremely weak, rapid-wetting flours. Similarly, the slightly higher lysine mutant MJD720175 induced in Marfed spring wheat apparently has weaker, more wettable flour traits. However, reductions in protein strength and increased wettability are no serious problem to the breeder since he has available a great diversity of genetic sources for even excessive protein strength and/or with slower flour wetting properties. Most surely he can recombine the various traits to achieve compensation for any effects due to increased lysine by adjustments in other proteins, and thus recover progeny with satisfactory flour processing properties. Consequently, the probability that the processing properties of storage proteins might be altered by changes in amino acid composition need not deter efforts to induce or select genetic variants with significantly improved protein nutritional value. Theoretically, all genes are mutable, possibly differentially so with different mutagenic agents, and the relative frequencies of different types of genetic alterations also can be expected to differ (Nilan e t al., 1976a). The polyploid nature of wheats endows them with considerable genetic buffering capacity to withstand alterations severe enough to be lethal in diploids. But, it is likely that the storage proteins have no vital function except to provide nutrition for the developing seedling during germination. Thus, viability should be unaffected by mutagen-induced structural or regulatory changes in storage proteins. Furthermore, such alterations may not lead to the defective endosperm conditions encountered in high lysine corn, barley, and sorghum. In fact, all higher-lysine wheats identified so far have normal endosperms.

B. MUTATIONS IN PROTEIN STRUCTURE AND

THE

GENETICCODE

The genetic codes for lysine, as an example, as compared with nutritionally poorer amino acids in storage proteins, may also be favorable for improving protein nutritional quality. According to Yanofsky et al. (1964a,b, 1966), Garen (1968) , and Schaap (1971), the RNA codons GAA or GAG code for glutamic acid, which is the most abundant amino acid in wheat proteins, while the codons for lysine, AAA or AAG, can be derived from GAB or GAG by mutation of G to A, which requires one DNA transition mutation from C to T. Gene transitions are among the most common base substitution mutations induced by chemical mutagens. Likewise, RNA arginine codon AGA, can be converted to a lysine codon by a similar base transition. The change from arginine to threonine is also possible, but requires a transversion, which is a less common base

540

CALVIN F. KONZAK

substitution. Transversions evidently may be inducible by mutagens like sodium azide (A. Kleinhofs, personal communication, 1976). Codon changes from GAA or GAG (both of which code for glutamic acid) would require two base substitutions to achieve the AUG codon for methionine, whereas the change from the leucine codons AUU or AUC to that for methionine would require only one transition type of base substitution. In these instances, the cereal storage proteins should be responsive to alterations, especially toward increased lysine composition. Another type of mutation might involve alteration of the regulator or operator site on a gene responsible for the synthesis of lysine or other desired amino acid in such a way that the feedback-inhibition control might be impaired. This type of change would result in a higher pool of the free amino acid, possibly to its incorporation into proteins, but more likely to an increase in free amino acids in the endosperm tissues. Base substitution mutations of the type described are brought about by errors induced by damage to DNA which cause DNA polymerase to incorporate the wrong bases (Saffhill, 1974). However, simple amino acid substitutions in specific storage proteins may have less, if identifiable, impact on the total AA composition than mutations of regulatory genes.

C. EFFICIENT INDUCTION OF IMPROVED PROTEIN MUTANTS Two general approaches offer promise for inducing mutations for improving the content or amino acid composition of wheat protein. These approaches may not be as efficient as might be possible via application of cell culture techniques, but they are practical in view of the fact that methods using cell culture have not been devised for isolating plants carrying gene-controlled changes in storage proteins. The first, or more conventional, approach simply involves mutagen treatments to seed of a selected cultivar, growing the M, generation under conditions to avoid contamination, then screening the M, endosperms or M, endosperms from M, plants for protein content and/or compositional variations. The chemical mutagens, ethyl methanesulfonate (EMS), diethyl sulfate (dES), isopropyl methanesulfonate (iPMS), methyl and ethyl nitroso urea (MNH, ENH), ethyleneimine (EI), and possibly sodium aeide (AZ) are among the more effective and efficient agents for inducing mutations in wheat. It should be noted, however, that EI, MNH, and ENH, are potent carcinogens as well as mutagens, whereas the others except for A 2 are weak carcinogens. Efficient methods for azide treatments to polyploid wheats have not been fully developed; this mutagen, however, is among the most potent agents for barley but is inactive as a chromosome-breaking agent or as a carcinogen (Nilan et al., 1973, 1976a,b; C. F. Konzak, A. Kleinhofs, and R. A. Nilan,

GENETIC CONTROL OF PROTEINS I N WHEAT

541

unpublished). The second method, using monosomic lines, is much more efficient from the standpoint of selection, but has the disadvantage that any mutations isolated will likely be valuable only as gene resources since monosomic series are seldom if ever available in currently successful cultivars. Both approaches require efficient preliminary screening or selection methods, but it should be noted that Doll (1972) isolated several high-lysine barley mutants from analyses of only about 6000 M, lines. The monosomic method as outlined by Tsunewaki and Heyne (1959) involves treatment of seed derived from proved monosomic plants, growing the MI in isolation to avoid cross contamination, then screening seed from M, plants for the desired trait. The advantage is that nullisomics are few and often abnormal and most of the nearnormal progeny derived from an MI-treated monosomic individual will carry the treated chromosome, either in a mono- or disomic condition. Since all individuals will be essentially “homozygous” for an induced change in the critical chromosome, the realized efficiency is about 9 or 10fold, while the probability of recovering a mutation in the noncritical chromosomes of the population is the same as for an euploid cultivar. Mutagen treatments (usually radiation-UV or X- or 7-rays) to pollen can also be made to inactivate an inhibitor, etc. Pollen of a normal plant is treated either in the spike or in collected anthers and applied to emasculated spikes of identified monosomics or nullisomics (when viable and sufficiently female fertile). Seeds grown on the first-generation plants could be individually analyzed or samples of their progeny tested for any induced change. New developments in screening methods may greatly improve the efficiency of mutagen techniques for the production of useful genetic variation. These new developments include near infrared protein analyzers (Pomeranz and Moore, 1975; Williams, 1975; Pomeranz, 1976; Watson et al., 1976), and single-seed test techniques (Brunckhorst e t al., 1974a,b; Mertz et al., 1974). Recent research indicates that the infrared system may be able to simultaneously screen samples for protein and lysine, as well as certain other amino acids, ash (mineral) content, and possibly other components all at one time on the same unweighed sample; the data can be computed and registered electronically, and may require only 11 seconds per sample (K. A. Norris, personal communication, 1976). XIII. Directions for Future Research

Considerable progress already has been made by cereal chemists and geneticists toward an understanding of wheat quality parameters and of the nature, variability, and genetic basis for specific endosperm (protein and other) components of wheat. Until recently, genetic tools have played

542

CALVIN F. KONZAK

a minor part in the analysis of wheat protein and processing quality components. To date induced mutants have had essentially no roIe in the analyses, but offer unique advantages for future investigations. There is a sound basis for the more conventional or empirical approaches commonly practiced in wheat research. The discovery and synthesis of bread flavor by Wiseblatt and Zoumut (1963) is a striking example of the success of an empirical approach by which tests of only a few concoctions of dough components saved perhaps decades of systematic research to the same end. Likewise, the development of the so-called high-yielding wheat and rice varieties exemplifies the success of conventional plant breeding. More systematic approaches often provide the important new leads for exploitation, as evidenced by the discovery of the inhibitors of nitrate reductase and RuDPCase activity in wheat. Also, new analytical methods are continually being developed (see Novacky and Wheeler, 1971; Lein et al., 1973; Concon, 1972; Brunckhorst et al., 1974a,b; Merts et al., 1974; Pomeranz and Moore, 1975; Pomeranz, 1976; Williams, 1975; Dohan et al., 1976) , and more efficient laboratory equipment is now generally available to obtain information for saving valuable time and resources in practical research programs. Of particular significance are new developments in near-infra-red spectroscopy that permit simultaneous rapid screening for protein content and for several specific amino acids on the same unweighed sample (K. A. Norris, personal communication, 1976). Certain improvements in the nutritional value of wheat protein may require genetic engineering, including the induction of mutations. These genetic tools can be used to isolate new germplasm sources and to better define approaches likely to be most successful and exploitable by more conventional techniques. The genetic data already available have great value for both practical application and basic research. These data suggest several lines of investigation that should contribute new knowledge and/or useful resource materials toward the development of wheats with satisfactory processing qualities and with more nutritionally improved protein : 1. Develop caryopsis (kernel) composition ideotypes for the important quality classes of wheats. Although the scientific basis for ideotype concepts of wheat quality types is still relatively weak, enough evidence is now available to suggest several (including alternative) lines of investigation. 2. Increase research into the genetic basis of processing quality differences, preferably by means of reciprocal chromosome substitution series. 3. Identify additional genetic resources for improving protein content and nutritional composition by (a) more intensive screening of available genetic resource collections, including induced mutations, and (b) routine

GENETIC CONTROL OF PROTEINS IN WHEAT

543

screening of segregates from intercrosses among wheat varieties, as well as from crosses between wheat species utilizing as necessary special genetic stocks to enhance interspecific and intergenomic chromosome pairing (see Sears, 1974). 4. Determine the location of high protein and high lysine components in promising genetic stocks by milling and other kernel fractionation studies. 5. Investigate the detailed protein and amino acid composition of the proteins in the higher protein and higher lysine sources, especially of induced mutants, aneuploids, and substitutions (since these genetic stocks may be more nearly isogenic) to estimate the potential for shifts in the amounts of high-lysine components in the albumin-globulin and glutenin proteins of wheat. 6. Conduct classical genetic studies of possible high-protein and highlysine sources to distinguish genetic differences, recover transgressive segregates, and determine the protein composition in endosperms of hybrid genotypes including recombinants, especially to determine whether separate genes control the distribution of protein and lysine fractions contained in the aleurone, subaleurone, and interior endosperm cells. 7. Increase the use by wheat breeders of identified high-protein and higher-lysine resource materials to combine higher protein and lysine composition into varieties with acceptable processing characteristics. 8. Encourage breeding and biochemical genetic studies to increase NRA of wheats, identify sources of genes controlling NRA and relationships t o specific proteins. 9. Investigate further the role of the cytoplasm us nuclear components in the synthesis of wheat endosperm proteins. Several different cytoplasm sources have been incorporated in a t least two varieties of HRS wheat and one of durum, and many lines isocytoplasmic for T. timopheevi L. us T. aestivum L. cytoplasm, have been developed in hybrid wheat breeding programs. Investigations of these materials should include (a) the protein composition, solubility groups, and amino acid distribution, including analyses of components within solubility groups, and (b) the number and characteristics of ribosomal proteins. Several kinds of investigations utilizing the various chromosome substitution series and the Chinese Spring aneuploids should be done to provide further background for mutation and other genetic experiments. Among these are: 1. Analyze the amino acid composition and protein fractions of all available relatively stable aneuploid stocks, including tetrasomics, nullitetrasomics, ditelosomics, chromosome addition and untested substitution series. 2. Study the number of 80 S ribosomal proteins associated with the

544

CALVIN F. KONZAK

above stocks to assess the roles of the nucleus us the cytoplasm in protein synthesis, and to identify specific genetically controlled relationships. 3. Synthesize additional tools for investigating the role of the cytoplasm in the genetic control of biosynthetic processes, e.g., the transfer of Chinese Spring and at least one other contrasting use-quality “base” variety (possibly Cheyenne, because of existing substitutions, etc.) into a different cytoplasm; transfer monosomic series in Cheyenne to this different cytoplasm and into the original Cheyenne cytoplasm (the series now has Chinese Spring cytoplasm). The selection of “foreign” cytoplasm(s) for this purpose might be based on analyses of existing hexaploid and tetraploid materials in those cytoplasms. 4. Synthesize “new” tetraploid and hexaploid combinations from diploid forms (including induced mutants) selected for lysine composition. 5. Induce specific mutations using the “monosomic” approach to expand the utility of aneuploid series, while maintaining desired linkages. For example, (a) the induction of spring habit and short straw traits in Cheyenne would adapt this variety for a wider range of research, (b) the inactivation of “inhibitors” of particular processes, as NRA in chromosome groups 2 and 7, may provide useful genetic testers and possibly useful germ plasm for breeding, (c) induction of mutations affecting the structural and/or regulator functions of genes on chromosomes known to control proteins of nutritional and/or processing quality would greatly assist analyses of these traits, and enhance the scientific basis for breeding developments, (d) isolation of a wider base of genetic sources for improved lysine composition would add greatly to the likelihood of success in breeding nutritionally better wheats. 6. Develop efficient methods for inducing specific gene duplications (possibly via acridines) using the “monosomic” approach to induced mutations. 7. Identify high lysine protein components, especially among gliadin and glutenin, then use the various aneuploid series to identify chromosome (9) responsible for their production and regulation; determine the effect of duplicating the chromosome(s), and of induced duplications or deficiencies, as appropriate. 8. Identify and remove by genetic means antagonistic or toxic factors limiting the nutritional utilization of wheat proteins and carbohydrates. XIV. Summary and Conclusions

1. The genetic basis for the control of protein content and composition appears to be more complex in polyploid wheats than in diploid species,

GENETIC CONTROL OF PROTEINS IN WHEAT

545

but changes in a specific gene may produce large and important effects. Only four main parameters (kernel hardness, dough strength, dough stiffness, dough stability) appear to be concerned with wheat quality. The extent of starch damage incurred during milling is a prime factor in pastry flour quality. However, the possible role of organic acids needs investigation. 2. The number and variety of the proteins in the endosperm of polyploid wheat is essentially the sum of those in their diploid progenitors. This offers opportunities rather than limitations for the selection or induction of genetic variations affecting endosperm protein composition. 3. Gene interactions appear to be especially important in polyploid wheats. The basis for some of the interactions, which are concerned with protein synthesis, evidently also involves relations with cytoplasmic organelles. 4. The biochemica1 systems of current wheat varieties may not be as efficient or harmoniously functioning as those theoretically achievable by genetic means. This is indicated from the discovery of genetically controlled inhibitors of the important biosynthetic processes, NRA, and RuDPCase activity. Removal of these inhibitors by genetic means may increase the biosynthetic efficiency of wheats for protein production and yield. 5. The analytical potentialities of aneuploid and related genetic materials, including induced mutants, are yet relatively unexploited. Exploitation of the potentials of these materials in research on wheat quality and nutrition should lead to important new discoveries. 6. Newly developed isocytoplasmic lines in wheats offer unusual opportunities to investigate the roles of the cytoplasm and nuclear systems controlling endosperm protein composition in wheat. 7. Model induced mutation studies conducted using monosomic lines offer the potential for increased efficiency for detecting mutations in specific chromosomes, without any loss of efficiency for the production of mutations in other chromosomes. The efficiency is, however, dependent on easy (morphological) or rapid biochemical distinction of monosomic individuals or expression of the desired trait in the hemizygous condition. 8. Based on existing evidence, experiments to induce mutations (delete inhibitors) , especially of loci affecting NRA and RuDPCase activity, may be warranted to improve varieties in which monosomic series are available (and where the inhibitors still exist). 9. Given the genetic variability in resources for improving the nutritional composition of wheats, breeders can be expected to develop cultivars which are not only high yielding, agronomically successful and widely adapted, but which also produce more and nutritionally better

546

CALVIN F. KONZAK

protein while retaining or even improving upon those processing characteristics required for making traditional foods.

ACKNOWLEDGMENTS The helpful suggestions generously made by Drs. J. A. Bietz (Cereal Properties Laboratory, USDA-ARS, Peoria, Illinois 61604), J. S. Wall (Cereal Properties Laboratory, USDA-ARS, Peoria, Illinois 616041, Rosalind Morris (Department of Agronomy, University of Nebraska, Lincoln, Nebraska 68503), P. Mattern (Department of Agronomy, University of Nebraska, Lincoln, Nebraska 68503), and D. D. Kasarda (Western Regional Research Center, 800 Buchanan Street, Albany, California 94710) are greatly appreciated. Special thanks are due also to Dr. C. J. Peterson (USDA-ARS Wheat Investigations), and Drs. R. L. Warner and J. A. Vigue (Department of Agronomy and Soils, Washington State University), Dr. P. L. Finney (USDA-AM), Mr. G. L. Rubenthaler (Western Wheat Quality Laboratory, Pullman, Washington), and Dr. W. J. R. Boyd, Visiting Professor in Agronomy and Soils on professional leave from the University of Western Australia, Perth, Australia, for their reviews; and particularly to Mrs. Dorothy Sander for editorial and other suggestions on the manuscript. Sincere appreciation is also expressed to Drs. M. Fried, B. Sigurbjornsson, C. Lamm, A. Micke, and R. Rabson (Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture, Vienna) for stimulating this review and for making much of the work possible. Analytical data provided through the cooperation of Ms. Helga Axmann, Drs. S. C. Hsieh, and H. Perschke (IAEA Laboratory, Seibersdorf, Austria) and through the cooperation of Dr. Stephen Muench and Mr. Kurt Jackson (Department of Agronomy and Soils, Pullman, Washington) and Mr. G. Rubenthaler (Technologist in Charge, Western Wheat Quality Laboratory, USDA-ARS, Pullman, Washington) are greatly appreciated. The dedicated secretarial assistance of Ms. Dorothy Larson, Susan Jones, Catherine Smith (Washington State University), and Marilyn Muller (IAEA) during the development and completion of the manuscript is gratefully acknowledged.

REFERENCES Adam, C. A., and Novellie, L. 1975. Acid hydrolases and autolytic properties of protein bodies and spherosomes isolated from ungerminated seeds of Sorghum bicolor (Linn.) Moench. Plant Physiol. 55, 7-11. Adam, C. A., Novellie, L., and Liebenberg, N. V. W. 1976. Biochemical properties and ultrastructure of protein bodies isolated from selected cereals. Cereal Chem. 53, 1-12. Andrews, T. J., Lorimer, G. H., and Tolbert, N. E. 1973. Ribulose diphosphate oxygenase. I. Synthesis of phosphoglycolate by Fraction I protein of leaves. Biochemistry 12, 11-18. Aragoncillo, C., Rodriquez-Loperena, M. A., Carbonero, P., and Garcia-Olmedo, F. 1975a. Chromosomal control of non-gliadin proteins from the 70% ethanol extract of wheat endosperm. Theor. A p p l . Genet. 45, 322-326. Aragoncillo, C., Rodriquez-Loperena, M. A., Carbonero, P., and Garcia-Olmedo, F.

GENETIC CONTROL OF PROTEINS IN WHEAT

547

1975b. Nigrosine staining of wheat endosperm proteolipid patterns on starch

gels. Anal. Biochem. 63, 603-606.

Aranyi, C., and Hawylewicz, E. J. 1969. Application of scanning electron microscopy to cereal specimens. Cereal Sci. Today 230-233. Archer, M. J. 1972. Relationship between free glutathione content and quality assessment parameters of wheat cultivars (Tm’ticum aestivum L.). J . Sci. Food Agric. 23, 485-491. Amason, T. J. 1956. Maternal inheritance of leaf variegation in hexaploid wheat. Can. J. Bot. 34, 801-804. Aslam, M., Huffaker, R. C., and Travis, R. L. 1973. The interaction of respiration and photosynthesis in induction of nitrate reductase activity. Plant Physiol. 52, 137-141. Auricchio, S., De Viria, B., Carta De Angelis, L., and Silano, V. 1974. Alphaamylase protein inhibitors from wheat in coeliac disease. Lancet 1, 98. Avila, A., and Favret, E. A. 1966. Inheritance of gluten quality in wheat. Proc. Int. Wheat Genet. Symp., Znd, 1963 Hereditas, Suppl. 2,55-61. Avivi, L., Efron, Y., and Feldman, M. 1972. Effect of somatic chromosomal association on the zymograph of alcohol dehydrogenase (ADH) in common wheat. Genetics 71, Suppl. 3, Part 2, 52. Axtell, J. D., Oswalt, D. L., Mertz, E. T., Pickett, R. C., Jambunathan, R., and Srinivasan, G. 1974. Components of nutritional quality in grain sorghum. In “High-Quality Protein Maize,” pp. 374-385. Dowden, Hutchinson and Ross, Stroudsburg, Pennsylvania. Balla, L., and Gaspar, L. 1975. Some results and problems in wheat breeding for higher protein content and improved quality a t Martonvbsbr. Proc. Int. Winter Wheat C o n f , 2nd, 1976 pp. 367-371. Balls, A. K., Hale, W. S., and Harris, T. H. 1942. A crystalline protein extracted from a lipoprotein of wheat flour. Cereal Chem. 19,279. Barber, H. N., Driscoll, C. J., and Vickery, R. S. 1968a. Enzymic markers for wheat and rye chromosomes. Proc. Znt. Wheat Genet. Symp., 3rd, 1968 pp. 116-121. Barber, H. N., Driscoll, C. J., Long, P. M., and Vickery, R. S. 196813. Protein genetics of wheat and homoeologous relationships of chromosomes. Nature (London) 218, 450-452. Barlow, K. K., Simmonds, D. H., and Kenrick, K. G. 1973a. The localization of water soluble proteins in wheat endosperm as revealed by fluorescent antibody techniques. Experientia 29, 229-231. Barlow, K. K., Buttrose, M. S., Simmonds, D. H., and Vesk, M. 1973b. The nature of the starch-protein interface in wheat endosperm. Cereal Chem. 50, 443454. Barlow, K. K., Lee, J. W., and Vesk, M. 1974. Morphological development of storage protein bodies in wheat. In “Mechanisms of Regulation of Plant Growth” (R. L. Bielleski, A. R. Ferguson, and M. M. Cresswell, eds.), pp. 793797. Royal Society of New Zealand, Wellington. Bean, M. M., Keagy, P. M., Fullington, J. G., Jones, F. T., and Mecham, D. K. 1974a. Dried Japanese noodles. I. Properties of laboratory prepared noodle doughs from sound and damaged wheat flours. Cereal Chem. 51, 416-427. Bean, M. M., Nimmo, C. C., Fullington, J. G., Keagy, P. M., and Mecham, D. K. 1974b. Dried Japanese noodles. 11. Effect of amylase, protease, salts and p H on noodle doughs. Cereal Chem. 51,427433. Beck, J. C.,Parker, J. H., Balcavage, W. X., and Matson, J. R. 1971. Mendelian

548

CALVIN F. RONZAK

genes affecting development and function of yeast mitochondria. In “Autonomy and Biogenesis of Mitochondria and Chloroplasts” (N. K. Boardman, A. W. Linnane, and R. M. Smillie, eds.), pp. 194-204. North Holland Publ., Amsterdam. Beckwith, A. C., Nielsen, H. C., Wall, J. S., and Huebner, F. R. 1966. Isolation and characterization of a high-molecular weight protein from wheat gliadin. Cereal Chem. 43, 14-28. Bedetti, C., Bozzini, A., Silano, V., and Vitoazi, L. 1974. Amylase protein inhibitors and the role of AegiEops species in polyploid wheat speciation. Biochim. Biophys. Acta 360, 299307. Bennett, M. D., and Smith, J. B. 1973. Genotypic, nucleotypic and environmental effects on meiotic time in wheat. Proc. Int. Wheat Genet. Symp., 4th, 1979 pp. 637-644. Bergman, J. W., and Maan, S. S. 1973. Genetic control of isozymes in wheat rye addition lines with rye or wheat cytoplasm. Proc. Int. Wheat Genet. Symp., 4th, 1979 pp. 329-335. Bergman, J. W., and Williams, N. D. 1972. Isozyme variants of esterase and malate dehydrogenase among wheat aneuploids. Agron. Abstr. p. 23 (by permission). Bernardin, J. E. 1974. Wheat protein fibrils and their relationship to dough properties. In “Proceedings of the 12th Western Wheat Worker’s Conference” (W.E. Kronstad and F. H. McNeal, eds.), pp. 15-16. Agricultural Research Service, USDA Publ. Boseman, Montana (by permission). Bernardin, J. E. 1975. The rheology of concentrated gliadin solutions. Cereal Chem. 52, 136r-145r. Bernardin, J. E., and Kasarda, D. D. 1973a. Hydrated protein fibrils from wheat endosperm. Cereal Chem. 50,829-536. Bernardin, J. E., and Kasarda, D. D. 197313. The microstructure of wheat protein fibrils. Cereal Chem. 50,735-745. Betschart, A. A., Saunders, R. M., Bean, M. M., and Kohler, G. 0. 1975. Effects of processing on the baking quality of wet alkaline process wheat protein concentrate. Cereal Chem. 52, 812-822. Bhatia, C. R.. 1968. Electrophoresis of analogous enzymes in Triticinae. Proc. Znt. Wheat Genet. Symp., Srd, 1968 pp. 111-115. Bhatia, C. R., and Rabson, R. 1976. Bioenergetic considerations in cereal breeding for protein improvement. Science 194, 1418-1421. Bietz, J. A., and Rothfus, J. A. 1970. Comparison of peptides from wheat gliadin and glutenin. Cereal Chem. 47, 381392. Bietz, J. A., and Rothfus, J. A. 1971. Differences in amino acid sequences of gliadin and glutenin. Cereal Chem. 48,677-690. Bietz, J. A,, and Wall, J. S. 1972. Wheat gluten subunits: Molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cereal Chem. 49, 418-430. Bietr, J. A., and Wall, J. S. 1973a. Glutenin variability among wheats with known genetic differences. Cereal Sci. Today IS, 301 (Abstr. No. 136). Bietr, J. A., and Wall, J. S. 1973b. Isolation and characterization of gliadin-like subunits from glutenin. Cereal Chem. 50, 537447. Bietz, J. A., and Wall, J. S. 1975. The effect of various extractants on the subunit composition and associations of wheat glutenins. Cereal Chem. 52, 145-155. Bietz, J. A., Huebner, R. F., and Wall, J. S. 1973. Glutenin-the strength protein of wheat flour. Baker’s Dig.47, 26-31, 34-35, and 67. Bietz, J. A., Shepherd, K. W., and Wall, J. S. 1975. Single-kernel analysis of glutenin: Use in wheat genetics and breeding. Cereal Chem. 52, 513-532.

GENETIC CONTROL OF PROTEINS I N WHEAT

549

Bloksma, A. H. 1971. Rheology and chemistry of dough. In “WheatChemistry and Technology” (Y. Pomeranz, ed.), 2nd ed., pp. 523-584. Am. Assoc. Cereal Chem. St. Paul, Minnesota. Bloksma, A. H. 1972a. The role of thiol and disulphide groups in dough rheology. Ztz “Working and Discussion Meeting, 7th Meeting of the International Association for Cereal Chemists (ICC), June 8-10, 1972” (ICC Secretariat, ed.), pp. 100-107. Int. Assoc. Cereal Chem., Vienna. Bloksma, A. H. 197213. The relation between the thiol and disulfide contents of dough and its rheological properties. Cereal Chem. 49, 104-118. Bloksma, A. H. 1975. Thiol and disulfide groups in dough rheology. Cereal Chem. 52, 170r-183r. Booth, M. R., and Ewart, J. A. D. 1969. Studies on four components of wheat gliadins. Biochim. Biophys. Acta 181, 226-233. Booth, M. R., and Ewart, J. A. D. 1970. Relationship between wheat proteins. J. Sci. Food Agric. 21, 187-192. Boulter, D., Ellis, R. J., and Yarwood, A. 1972. Biochemistry of protein synthesis in plants. Biol. R e v . Cambridge Philos. SOC.47, 113-175. Bourque, D. P., and Wildman, S. G. 1972. Evidence that nuclear genes code for several chloroplast ribosomal proteins. Biochem. Biophys. Res. Commun. 50, 532-537.

Bowden, W. M. 1959. The taxonomy and nomenclature of the wheats, barley, and ryes and their wild relatives. Can. J. Bol. 37, 657-684. Bowes, G., and Ogren, W. L. 1972. Oxygen inhibition and other properties of soybean ribulose 1,5-diphosphate carboxylase. J. Biol. Chem. 747, 2171-2176. Bowes, G., Ogren, W. L., and Hageman, R. H. 1971. Phosphoglycolate production catalysed by ribulose diphosphate carboxylase. Biochem. Biophys. Res. Commun. 45, 716-722.

Boyd, W. J. R., and Lee, J. W. 1967. The control of wheat gluten synthesis a t the genome and chromosome levels. Experientia 23, 332-333. Boyd, W. J. R., Lee, J. W., and Wrigley, C. W. 1969. The D-genome and the control of wheat gluten synthesis. Erperientiu 25, 317319. Bozzini, A., and Giacomelli, M. 1973. Endosperm composition of Chinese Spring wheat ditelocentrics. Proc. Znt. Cong. Genet., 13th, 19Y3 Genetics 74, Suppl. 2, Part 2, s29. Bozzini, A., Cantagalli, P., Piazzi, S. E., and Sordi, S. 1970. An immunochemical approach to species relationship in Tn‘ticum and some related species. Theor. Appl. Genet. 40, 300-304. Bozzini, A,, Cantagalli, P., and Piazzi, S. E. 1971. Chromosomal location of the genetic control of two proteins specifically attributed to hexaploid wheat by immunochemical methods. E.W.A.C. Newsl. 3, 16-17 (by permission). Bozzini, A., Cubadda, R., and Quattrucci, E. 1973. Esterases in Triticum and some related species. Proc. Int. Wheat Genet. Symp., 4th, l9Y3 pp. 783-787. Brewer, G. J., Sing, C. F., and Sears, E. R. (1969). Studies of isozyme patterns in nullisomic-tetrasomic combinations of hexaploid wheat. Proc. Natl. Acud. Sci. U S A . 64, 1224-1229. Britten, R. J., and Davidson, E. H. 1969. Gene regulation in higher cells: A theory. Science 165, 349-357. Brohult, S., and Sandgren, E. 1954. Seed proteins. In “The Proteins” (H. Neurath and K. Bailey, eds.), 1st ed., Vol. 2, Part A, pp. 487-512. Academic Press, New York. Brown, L. R. 1975. The world food prospect. Science 190, 1053-1059.

550

CALVIN F. KONZAK

Brunckhorst, K., Lein, K. A., and Schon, W. J. 1974a. Bestimmung des Lysingehaltes und Selektion des Hiproly-Merkmales nach RuTkkreuzung bei Gerste. I. Prufung und Entwicklung analytischer Methoden. Z . Pflanzenzuecht. 73, 269-283. Brunckhorst, K., Robbelen, G., and Zosche, M. 1974b. Bestimmung des Lysingehaltes und Selektion des Hiproly-Merkmals nach Ruckkreuzung bei Gerste. 11. Vererbungstudien und Auslese auf Lysin in ‘Asse’ x ‘Hipro1y’-Kreuaungen. 2. Pflanzenzuecht. 73, 1-12. Busch, R. H., and Maan, S. S. 1974. Possible use of cytoplasmic variability in wheat improvement. Wheat Newsl. 20, 163-166 (by permission). Bushuk, W. 1972. Ultrastructure related to wheat processing and products. In “Symposium: Seed Proteins” (G. E. Inglett, ed.), pp. 193-211. Avi Publ., Westport, Connecticut. Bushuk, W., and Wrigley, C. W. 1971. Glutenin in developing wheat grain. Cereal Chem. 48, 448-455. Bushuk, W., and Wrigley, C. W. 1974. Proteins: Composition, structure, and function. In “Wheat: Production and Utilization” (G. E. Inglett, ed.), pp. 119-145. Avi Publ., Westport, Connecticut. Bushuk, W., Briggs, K. G., and Shebeski, L. H. 1969. Protein quantity and quality as factors in the evaluation of bread wheats. Can. J. Plant Sci. 49, 113-122. Butcher, J. 1975. Effect of aleurone cell damages on wheat fibre values as determined by the neutral detergent technique. J. Sci. Food Agric. 26,345-350. Butcher, J., and Stenvert, N. L. 1973a. Conditioning studies on Australian wheat. I. The effect of conditioning on milling behavior. J. Sci. Food Agric. 24, 10551066. Butcher, J., and Stenvert, N. L. 1973b. Conditioning studies on Australian wheat. 111. The role of the rate of water penetration into the wheat grain. J. Sci: Food Agric. 24, 1077-1084. Buttrose, M. S. 1963. Ultrastructure of the developing wheat endosperm. Aust. J. Biol. Sci. 16, 305-316. Caepoiu, N., Euststiu, N., Ittu, Gh., and Shlescu, N. N. 1975. Wheat breeding for improved nutritional quality in Romania. Proc. Int. Winter Wheat Conf., 2nd, 1976 pp. 362-366. Cain, R. F., Schulta, H. W., and Peters, L. J. 1975. “Nutritional Evaluation of Wheat,” 1st Annu. Rep. National Wheat Institute, Department of Food Science and Technology, Oregon State University, Corvallis. Carbonero, P., and Garcia-Olmedo, F. 1969. Purothionins in Aegilops Triticum spp. Ezperientiu 25, 1110. Cataldo, D. A., Schrader, L. E., Peterson, D. M., and Smith, D. 1975. Factors affecting seed protein concentration in oats. I. Metabolism and distribution of N and carbohydrate in two cultivars that differ in groat protein concentration. Crop Sci. 15, 19-23. Chamberlain, N., Collins, T. H.. and Elton, G. A. H. 1962. The chorleywood bread process. Baker‘s Dig. 36, 52-53. Chan, P. H., Sakano, K., Singh, S., and Wildman, S. G. 1972. Crystalline Fraction I protein : Preparation in large yield. Science 176, 1145-1146. Chapman, V., Mittler, T. E., and Riley, R. 1976. Equivalence of the A genome of bread wheat and that of T . urartu. Genet. Res. 27, 69-76. Charbonnier, L. 1973. Studies of the all-soluble proteins of wheat flour. Biochimie 55, 1217-1225.

GENETIC CONTROL OF PROTEINS IN WHEAT

551

Charbonnier, L. 1974. Isolation and characterization of omega-gliadin fractions. Biochim. Biophys. Acta 359, 142-151. Chen, C. H., and Bushuk, W. 1970a. Nature of proteins in Triticale and its parental species. I. Solubility characteristics and amino acid composition of endosperm proteins. Can. J. Plant Sci. 50, 9-14. Chen, C. H., and Bushuk, W. 1970b. Nature of proteins in TriticaZe and its parental species. 11. Gel filtration and disc electrophoresis results. Can. J. Plant Sci. 50, 15-24. Chen, K., Gray, J. C., and Wildman, S. G. 1975. Fraction I protein and the origin of polyploid wheats. Science 190, 1304-1306. Chmeleva, Z. V., and Medredev, A. M. 1973. Effect of fertilizers on the accumulation of protein and lysine in cultivars of spring wheat (In Russian). Bull. Appl. Bot., Genet. Plant Breed. 52,242-248 (Chem. Abstr. 8 1 2 4 ~ ) . Cole, E. W., Ng, H., and Mecham, D. K. 1973. Further studies on the fractionation and characterization of the gel proteins of wheat flour by column chromatography. Cereal Chem. 50, 336345. Cole, E. W., Ng, H., and Mecham, D. K. 1976. Some chemical and physical properties of the mercuric chloride-solubilized gel protein from different wheat varieties. Cereal Chem. 53, 250-257. Concon, J. M. 1972. Determination of lysine in cereal proteins. In “Symposium: Seed Proteins” (G. E. Inglett, ed.), pp. 292-311. Avi Publ., Westport, Connecticut. Crosby, A. R. 1957. Nucleolar activity of lagging chromosomes in wheat. A m . J. Bot. 44, 813-822. Crosby Longwell, A., and Svihla, G. 1960. Specific chromosomal control of the nucleolus and of the cytoplasm in wheat. Exp. Cell Res. 20,294-312. Croy, L. I., and Hageman, R. H. 1970. Relationship of nitrate reductase activity to grain protein production in wheat. Crop Sci. 10, 280-285. Crozet, N., Godon, B., Petit, L., and Guilbot, A. 1974. Submicroscopic structure of wheat flour and gluten lipoprotein components. Cereal Chem. 51, 288-299. Cubadda, R. 1975. Chromosome location of genes controlling the synthesis of some soluble proteins in T. durum and T . aestivum. In “Genetics and Breeding of Durum Wheat” (G. T. Scarascia-Mugnozza, ed.), pp. 653-658. Library of the Faculty of Agriculture, Bari, and National Institute for Nutrition, Rome. Cubadda, R., and Quattrucci, E. 1974. Separation by gel electrofocusing and characterization of wheat esterases. J. Sci. Food Agric. 25, 417-422. Cubadda, R., Quattrucci, E., and Virtuoso, G. 1973. Una nuova tecnica per la separazione de forme multiple di esterasi vegetali mediante gel-focalirzazione ionica (In Italian). Boll. SOC.I t . B i d . Sper. 49, 328-332 (Chem. Abstr. 116382~). Cubadda, R., Bozzini, A,, and Quattrucci, E. 1975. Genetic control of esterases in common wheat. Theor. Appl. Genet. 45,29&293. Dahlberg, A. E., Dingman, C. W., and Peacock, A. C. 1969. Electrophoretic characterization of bacterial polyribosomes on agarose-acrylamide composite gels. J. Mol. Biol. 41, 139-147. Dalby, A., and Tsai, C. Y. 1976. Lysine and tryptophan increases during germination of cereal grains. Cereal Chem. 53, 222-226. Dalling, M. J., Tolbert, N. E., and Hageman, R. H. 1972. Intracellular location of nitrate reductase and nitrite reductase. I. Spinach and tobacco leaves. Biochim. Biophys. Acta 283, 505-512. Dalling, M. J., Halloran, G. M., and Wilson, J. H. 1975. The relation between

552

CALVIN F. KONZAK

nitrate reductase activity and grain nitrogen productivity in wheat. Aust. J. Agric. Res. 26, 1-10. D’Appolonia, B. L., ‘Gilles, K. A., Osman, E. M., and Pomeranz, Y. 1971. Carbohydrates. In ‘LWheat-Chemistry and Technology” (Y. Pomeranz, ed.), pp. 301492. Am. Assoc. Cereal Chem. St. Paul, Minnesota. Davis, W. H., Middleton, G. K., and Hebert, T. T. 1961. Inheritance of protein, texture, and yield in wheat. Crop Sci. 1, 235-238. Deckard, E. L., Williams, N. D., and Hammond, J. J. 1975. Nitrate reductase activity of wheat aneuploids. Agron. Abstr. p. 106 (by permission). Dedio, W., Simmonds, D. H., Hill, R. D., and Shealy, H. 1975. Distribution of aamylase in the triticale kernel during development. Can. J . Plant Sci. 55, 29-36. Deponte, R., Parlamenti, R., Petrucci, T., Silano, V., and Tomasi, M. 1976. Albumin a-amylase inhibitor families from wheat flour. Cereal Chem. 53, 805-820. Dexter, J. E., and Dronzek, B. L. 1975a. Protein synthesis in triticale and its durum wheat and rye parents. Cereal Chem. 52,577-586. Dexter, J. E., and Dronzek, B. L 1975b. Note on the amino acid composition of protein fractions from a developing triticale and its rye and durum wheat parents. Cereal Chem. 52, 587-596. Dhaliwal, H. S. 1976. Cytoplasmic relationship between Triticum boeotrium and Triticum urartu. Wheat In!. Serv. 41-42, 1-2 (by permission). Dhaliwal, H. S., and Johnson, B. L. 1976a. Anther morphology and the origin of the tetraploid wheats. Am. J . Bot. 63, 363368. Dhaliwal, H. S., and Johnson, B. L. 1976b. Cross-compatibility of diploids and tetraploids and the origin of the cytoplasm of the tetraploid wheats. Heredity (in press). Dhaliwal, H. S., and Johnson, B. L. 1976c. Chromosomal rearrangement and the origin of the Timopheevi wheats. Can. J . Genet. Cytol. (in press). Dieckert, J. W., and Dieckert, M. C. 1972. The deposition of vacuolar proteins in oilseeds. In “Symposium: Seed Proteins” (G. E. Inglett, ed.), pp. 52-85. Avi Publ., Westport, Connecticut. Doekes, G. J. 1968. Comparison of wheat varieties by starch gel electrophoresis of their grain proteins. J. Sci. Food Agric. 10, 169-176. Doekes, G. J. 1973. Inheritance of gliadin composition in bread wheat, Triticum aestivum L. Euphytica 22, 28-33. Doekes, G. J., and Hack, A. 1971. Electrophoresis of grain proteins as an aid in wheat breeding. EUCARPIA Bull. pp. 329-345. Dohan, D. A., Standing, K. G., and Bushuk, W. 1976. A new method of analysis of protein content in grain by proton activation. Cereal Chem. 53, 91-100. Doll, H. 1972. Variation in protein quantity and quality induced in barely by EMS treatment. In “Induced Mutations and Plant Improvement’’ (Proceedings of a Latin American Study Group meeting on Induced Mutations and Plant Improvement organized by the Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture held in Buenos Aires, 16-20. November, 19701, STI/ PUB/297. IAEA, Vienna. Doll, H. 1973. Inheritance of the high lysine character of a barley mutant. Hereditas 74, 293-294. Doll, H. 1976. Genetic studies of high lysine barley mutants. In “Barley Genetics 111” (H. Gaul, ed.), pp. 542-546. Karl Thiemig, Munich. Donelson, D. H., and Yamazaki, W. T. 1972. Soft wheat flour particle size analysis by integrated sieve and Coulter counter procedures. Cereal Chem. 49, 641-653. Dronzek, B. L., Kaltsikes, P. J., and Bushuk, W. 1970. Eflect of the D genome on

GENETIC CONTROL OF PROTEINS IN WHEAT

553

the protein of three cultivars of hard red spring wheat. Can. J. Plant Sci. 50, 389400. Duffield, R. D., Croy, L. I., and Smith, E. L. 1972. Inheritance of nitrate reductase activity, grain protein, and straw protein in a hard red winter cross. Agron. J. 64, 249-251. DumanoviE, J., Ehrenberg, L., and Denii., M. 1970. Induced variation of protein content and composition in hexaploid wheat. Improv. P h n t Protein Nucl. Tech., Proc. Symp., 1970 STI/PUB/258, pp. 107-120. Dunstone, R. L., and Evans, L. T. 1974. Role of changes in cell size in the evolution of wheat. Aust. J . Plant Physiol. 1, 157-165. DvoFik, J. 1976. The relationship between the genome of Triticum urartu and the A and B genomes of Triticum aestiuum. Can. J . Genet. Cytol. 18, 371-377. Eastin, J. D., Morris, R., Schmidt, J. W., Mattern, P. J., and Johnson, V. A. 1966. Chromosomal association with gliadin proteins in the wheat variety ‘Cheyenne.’ Crop Sci. 7, 674-676. Edgar, K. F., and Draper, S. R. 1975. Physiological aspects of nitrate utilization in a barley variety exhibiting high seed protein content. J . Sci. Food Agric. 26, 1311-1318. Edwards, I. B. 1973. Physiologic and genetic studies of nitrate reductase activity and nitrogen distribution in spring wheat (Triticum aestivum L.) . Ph.D. Thesis, North Dakota State University, Fargo. Edwards, I. B. 1974. Heritability estimates of nitrate reductase activity in spring wheat and the chromosomal location of genes effecting nitrogen reduction. South A f r . Genet. Congr. 6th, 1974 (Abstr.). Eilrich, G. L., and Hageman, R. H. 1973. Nitrate reductase activity and its relationship to accumulation of vegetative and grain nitrogen in wheat. Crop Sci. 13, 59-66. Elton, G. A. H., and Ewart, J. A. D. 1962. Starch gel electrophoresis of cereal proteins. J. Sci. Food Agric. 13, 62-72 Endo, T. R., and Tsunewaki, K. 1975. Genetic diversity of the cytoplasm in Triticum and Aegilops. I. On the origin of the cytoplasm of Aegilops triuncialis L. Seiken Ziho 25-26, 55-66. Eslick, R . F., and Hockett, E. A. 1976. A second locus for high lysine barley. I n “Barley Genetics 111”(H. Gaul, ed.), p. 634. Karl Thiemig, Munich. Evers, A. D. 1970. Development of the endosperm of wheat. Ann. Bot. (London) CN.S.1 34, 547-555. Everson, E. H., and Seeborg, E. F. 1958. The heritability of milling quality in wheat as measured by the separation of the bran and endosperm. Agron. J. 50, 511513. Ewart, J. A. D. 1966a. Fingerprinting of glutenin and gliadin. J. Sci. Food Agric. 17, 30-33. Ewart, J. A. D. 1966b. Cereal proteins: Immunological studies. J . Sci. Food Agric. 17, 279-284. Ewart, J. A. D. 1967a. Amino acid analyses of glutenins and gliadins. J. Sci. Food Agric. IS, 111-116. Ewart, J. A. D. 196713. Amino acid analyses of cereal flour proteins. J . Sci. Food Agric. 18, 548-552. Ewart, J. A. D. 1968. Fractional extraction of cereal flour proteins. J . Sci. Food Agric. 19, 241-245. Ewart, J. A. D. 1969. Isolation and characterization of a wheat albumin. J. Sci. Food Agric. 20, 730-733. Ewart, J. A. D. 1972a. Recent research and dough viscoelasticity. In “Working

554

CALVIN F. KONZAK

and Discussion Meeting, 7th Meeting of the International Association for Cereal Chemists (ICC), June 8-10, 1972” (ICC Secretariat, ed.), pp. 108-112. Int. Assoc. Cereal Chem., Vienna. Ewart, J. A. D. 1972b. Further studies on SS bonds in cereal glutelins. J . Sci. Food Agric. 23, 567-579. Ewart, J. A. D. 1972c. A modified hypothesis for the structure and rheology of glutelins. J . Sci. Food Agric. 23, 687-699. Ewart, J. A. D. 1973. Sodium dodecyl sulphate electrophoresis of wheat gliadins. J. Sci. Food Agric. 24, 685-689. Ewart, J. A. D. 1975. Isolation of a Cappelle-Desprez gliadin. J . Sci. Food Agric. 26, 1021-1025. Farrand, E. A. 1964. Flour properties in relation to modern bread processes in the United Kingdom, with special reference to alpha-amylase and starch damage. Cereal Chem. 41, 99-111. Farrand, E. A. 1969. Starch damage and alpha-amylase as bases for mathematical model relating to flour water absorption. Cereal Chem. 46, 103-116. Farrand, E. A. 1972. Controlled levels of starch damage in a commerical United Kingdom bread flour and effects on absorption, sedimentation value, and loaf quality. Cereal Chem. 49, 479-488. Farrand, E. A. 1974a. Study of relationships between wheat flour protein contents of two United Kingdom varieties and derived flour protein contents a t varying extraction rates. I. Studies on an experimental commercial mill and a laboratory Buhler mill. Cereal Chem. 51, 56-66. Farrand, E. A. 1974b. Study of relationships between wheat protein contents of two United Kingdom varieties and derived flour protein contents at varying extraction rates. 11. Studies by hand dissection of individual grains. Cereal Chem. 51, 66-74. Favret, E. A., Solari, R., Manghers, L., and Avila, A. 1969. Genetic control of the qualitative and quantitative production of endosperm proteins in wheat and barley. New Approaches Breed. Improved Plant Protein, Proc. Panel Meet., 1968 pp. 87-107. Favret, E. A., Manghers, L., Solari, R., A d a , A., and Monesiglio, J. C. 1970. Gene control of protein production in cereal seeds. Improv. Plant Nucl. Techn., Proc. Sump., 1970. pp. 87-97. Feillet, P., and Bourdet, A . 1967. Composition protkique et caractkristiques gbnktique des blks (In French, Engl. Summary). Bull. SOC.Chim. Biol. 49, 1273-1283. Feillet, P., and Nimmo, C. C. 1970. Soluble proteins in wheat. 111. Isolation and characterization of two albumins, ALB 13A and ALB 13B, from flour. Cereal Chem. 47, 447464, Fellers, D. A., Sinkey, V., Shepherd, A. D., and Pence, J. W. 1966. Solubilization and recovery of protein from wheat millfeeds. Cereal Chem. 43, 1-13. Fernandez de Caleya, R., Gonzalez-Pascual, B., Garcia-Olmedo, F., and Carbonero, P. 1972. Susceptibility of phytopathogenic bacteria to wheat purothionins in vitro Appl. Microbial. 23, 998-1000. Fernandez de Caleya, R., Hernandez-Lucas, C., Carbonero, P., and GarciaOlmedo, F. 1976. Gene expression in allopolyploids. Genetic control of lipopurothionins of wheat. Genetics 83, 687-699. Fincher, G. B., Sawyer, W. H., and Stone, B. A. 1974. Chemical and physical properties of an arahinogalactan-peptide from wheat endosperm. Biochem. J . 139, 535-545.

GENETIC CONTROL OF PROTEINS I N WHEAT

555

Rnney, K. F. 1943. Fractionating and reconstituting techniques as tools in wheat flour research. Cereal Chem. 20, 381-396. Finney, K. F. 1971. Fractionating and reconstituting techniques to relate functional (breadmaking) to biochemical properties of wheat flour components. Cereal Sci. Today 16, 342-356. Finnry, K. F. 1975. A sugar-free formula for regular and high protein breads. Baker’s Dig. 49, 18-22. Finney, K. F., and Fryer, H. C. 1958. Effect on loaf volume of high-temperature during the fruiting period of wheat. Agron. J . 50, 28-34. Finney, K. F., Shogren, M. D., Pomeranz, Y., and Bolte, L. C. 1972. Cereal malts in breadmaking. Baker’s Dig. 46, 36-38 and 55. Finney, P. L., Magoffin, C. D., Hoseney, R. C., and Finney, K. F. 1976. Short-time baking systems. I. Interdependence of yeast concentration, fermentation time, proof time, and oxidation requirement. Cereal Chem. 53, 12b134. Finney, P. L., Rubenthaler, G. L., Jeffers, H. C., and Anderson, P. D. 1977. Shorttime baking systems. 111. Malt independence in a sugar-free formula. Cereal Chem. (in press). Fish, W. W., and Abbott, D. C. 1969. Isolation and characterization of a watersoluble wheat protein. J. Sci. Food Agric. 20, 723-730. Fisher, N., Redman, D. G., and Elton, G. A. H. 1968. Fractionation and characterization of purothionin. Cereal Chem. 45, 48. Flavell, R. B., and Smith, D. B. 1974a. Variation in nucleolar organizer rRNA gene multiplicity in wheat and rye. Chromosoma 47,327334. Flavell, R. B., and Smith D. B. 197413. The role of homoeologous group 1 chromosomes in the control of rRNA genes in wheat. Biochem. Genet. 12, 271-279. Fortini, S., Giorgi, B., Giacomelli, M., Mannino, P., and Cordischi, M. 1973. Modification of RuDP and PEP carboxylase activities, protein content and leaf weight in ditelocentric lines of bread wheat in Chinese Spring. Ann. Ist. Sper. Cerealicolt. 4, 133-144. Gale, M. D., and Spencer, D. 1974. Location of genes controlling enzyme production in endosperm. E.WA.C. Newsl. 4, 16-17 (by permission). Garcia-Olmedo, F., and Carbonero, P. 1970. Homoeologous protein synthesis controlled by homoeologous chromosomes in wheat. Phytochemktry 9, 1495-1497. Garcia-Olmedo, F., Sotelo, I., and Garcia-Faure, R. 1968. Identification of Triticum aestivum products in alimentary paste. IV. Petroleum ether soluble lipoproteins. A n . Inst. Nac. Invest. Agron. (Spain) 17, 433-443. Garcia-Olmedo, F., Carbonero, P., Aragoncillo, C., Fernandez de Caleya, R., and Torres, J. V. 1975. Expression of homoeologous molecular systems in wheat alloploids. In “Heterosis in Plant Breeding. Section I, Principles of Heterosis” Proc. V I I Eucarpia Congress, Budapest, 1974 (in press). Garen, A. 1968. Sense and nonsense in the genetic code. Science 160, 149-159. Gavrilyuk, I. P., Gubareva, N. K., and Konarev, V. G. 1973. Methods for extraction, seperation into fractions and identification of proteins used for genome analysis of cultivated plants. (In Russian, Engl. Summary) Bull. Appl. Bat., Genet. Plant Breed. 52, 249-281 (Chem. Abstr. 40116m). Gibson, R. A., and Paleg, L. G. 1975. Further experiments on the a-amylase containing lysosomes of wheat aleurone cells. Aust. J. Physiol. 2, 4149. Glasziou, K . T. 1969. Control of enzyme formation and inactivation in plants. Annu. R e v . Plant Physiol. 20, 63-88.

556

CALVIN F. KONZAK

Goering, K. J., Fritts, D. H., and Allen, K. G. D. 1974. A comparison of the 10sa of birefrigence with the percent gelatinization and viscosity on potato, wheat, rice, corn, cow cockle, and several barley starches. Cereal Chem. 51, 764-771. Goering, K. J., Jackson, L. L., and Dehaas, B. W. 1975. Effect of some nonstarch components in corn and barley starch granules on the viscosity of heated starch-water suspensions. Cereal Chem. 52, 493-500. Graham, J. S. D., Jennings, A. C., Morton, R. K., Palk, B. A., and Raison, J. K. 1962. Protein bodies and protein synthesis in developing wheat endosperm. Nature (London) 196, 967-969. Graham, J. S. D., Morton, R. K., and Raison, J. K. 1963. Isolation and characterization of protein bodies from developing wheat endosperm. Aust. J. Biol. Sci. 16, 375-384. Grant, D.R. 1974. Studies of the role of ascorbic acid in chemical dough development. I. Reaction of ascorbic acid with flour-water suspensions. Cereal Chem. 51, 684-692. Green, M. R.,and Pastewka, J. V. 1974s. Simultaneous differential staining by a cationic carbocyanine dye of nucleic acids, proteins, and conjugated proteins. I. Phosphoproteins. J . Histochem. Cytochem. 22,767-773. Green, M. R., and Pastewka, J. V. 1974b. Simultaneous differential staining by a cationic carbocyanine dye of nucleic acids, proteins, and conjugated proteins. 11. Carbohydrate and sulfated carbohydrate-containing proteins. J. Histochem. Cytochem. 22, 774-781. Green, M. R.,Pastewka, J. V., and Goerttler, K. L. 1973. Differential staining of phosphoproteins on polyacrylamide gels with a cationic carbocyanine dye. Anal. Biochem. 56, 43. Greenaway, W. T., and Watson, C. A. 1975. The gluto-matic for semiautomatic determination of wet and dry gluten content of wheat flour. Cereal Chem. 52, 367473. Greenwood, C. T. 1976. Starch. I n “Advances in Cereal Science and Technology” (Y. Pomeranz, ed.), pp. 119-157. Am. Assoc. Cereal Chem., St. Paul, Minnesota. Greenwood, C. T., and Ewart, J. A. D. 1975. Hypothesis for the structure of glutenin in relation to rheological properties of gluten and dough. Cereal Chern. 52(3, 11) 146r-153r. Grimes, G. W., Mahler, H. R., and Perlman, P. S. 1974. Nuclear gene dosage effects on mitochondria1 mass and DNA. J . Cell Biol. 61, 565-574. Gunthardt, H.,and McGinnis, J. 1957. Effect of nitrogen fertilization on amino acids in whole wheat. J. Nutr. 61,167-170. Haag, D., Tschahargane, C., and Goerttler, K. L. 1971. Simultaneous differential staining of nucleic acids and proteins in histological tissues by means of J-band effect. Histochemie 26, 190-193. Hall, O., Johnson, B. L., and Olered, R. 1966. Evaluation of genome relationships in wheat from their protein homologies, Proc. Int. Wheat Genet. Symp., Srd, 1963 pp. 47-54. Halloran, G. M. 1975.Genetic analysis of grain protein percentage in wheat. Theor. Appl. Genet. 46,74-86. Hamauzu, Z., Toyomasu, T., and Yonezawa, I?. 1974. Molecular weight determination of gliadin fractions in gel filtration by SDS-polyacrylamide gel electrophoresis and sedimentation equilibrium. Agric. Biol. Chem. 38, 2445-2450. Hammes, G. G., and Wu,C. W. 1971. Regulation of enzyme activity. Science 172, 1205-1211.

GENETIC CONTROL OF PROTEINS IN WHEAT

557

Hanif, M., and Langer, R. H. M. 1972. The vascular system of the spikelet in wheat (Triticum aestivum). Ann. Bat. 36, 721-728. Harlan, J. R. 1970. Evolution of cultivated plants. I n “Genetic Resources in Plants: Their Exploration and Conservation” (0.H. Frankel and E. Bennett, eds.), Int. Biol. Program, pp. 19-32. Davis, Philadelphia, Pennsylvania. Harlan, J. R. 1975. “Crops and Man.” American Society of Agronomy, Crop Science Society of America, Madison, Wisconsin. Harlan, J. R., and Zohary, D. 1966. Distribution of wild wheats and barley. Science 153, 1074-1080.

Harris, D. R. 1967. New light on plant domestication and the origins of agriculture. A review. Geogr. Rev. 57, 90-107. Hart, G. E. 1970. Evidence for triplicate genes for alcohol dehydrogenase in hexaploid wheat. Proc. Natl. Acad. Sci. U S . A . 66, 1136-1141. Hart, G. E. 1971. Alcohol dehydrogenase isozymes of Triticum: Dissociation and recombination of subunits. Mol. Gen. Genet. 111,61-65. Hart, G. E. 1973a. Homoeologous gene evolution in hexaploid wheat. Proc. Int. Wheat Genet. Symp., 4th, f973 pp. 805410. Hart, G. E. 1973b. Acid phosphatase of hexaploid wheat: linkage to homoeologous chromosomes of structural genes for six isozymes. Proc. Int. Cong. Genet. fSth Genet. 74 Suppl. 2, s110. Hart, G. E. 1975. Glutamate oxaloacetate transaminase isozymes of Triticum: Evidence for multiple systems of triplicate structural genes in hexaploid wheat. In “Isozymes, 111. Developmental Biology” ( C . L. Markert, ed.), pp. 637-657. Academic Press, New York. Hart, G . E., and Langston, P. 1975. Evidence for triplicate endopeptidase and lipoxygenase structural gene sets in hexaploid wheat. Isozyme Bull. 8, 12. Hart, G. E., McMillin, D. E., and Sears, E. R. 1976. Determination of the chromosomal location of a glutamate oxaloacetate transaminase structural gene using Triticum-Agropyron translocations. Genetics 83, 49-61. Haunold, A., Johnson, V. A., and Schmidt, J. W. 1962. Genetic measurements of protein in the grain of Triticum aestivum L. Agron. J . 54, 203-206. Hehn, E. R., and Barmore, M. A. 1965. Breeding wheat for quality. Adv. Agron. 17, 85-114.

Henningsen, K. W., Baynton, J. E., von Wettstein, D., and Boardman, N. K. 1973. Nuclear genes controlling chloroplast development in barley. Biochem. Gene Expression Higher Org., Proc. Symp., 1972 pp, 457-478. Hernandez, H. H., Walsh, D. E., and Bauer, A. 1974. Nitrate reductase of wheat: Its relation to nitrogen fertilization. Cereal Chem. 51, 330-336. Hernandez-Lucas, C., Fernandez de Caleya, R., and Carbonero, P. 1974. Inhibition of brewer’s yeasts by wheat purothionins. Appl. Microbial. 28, 165-168. Hertel, W. 1974. Biosynthesis of wheat protcins (In German). Getreide, Mehl, Brat 28, 10-12 (Chem. Ahstr. 47444K). Hickey, C. A., Murphy, E. L., and Calloway, D. H. 1972. Intestinal-gas production following ingestion of commercial wheat cereals and milling fractions. Cereal Chem. 49, 276-282. Hillel, J., and Simchen, G. 1973. Mating systems, population structure and chromosome behavior in Triticum speltoides and T . longissimum. Proc. Int. Wheat Genet. Symp., 4th, 1973 pp. 521-526. Hochstrasser, K., Werle, E., Schwarz, S., and Siegelmann, R. 1969. Uber pflnnzliclie Proteaseinhibitoren, 111. Reindarstellung der Trypsininhibitoren aus Keimen

558

CALVIN F. KONZAK

von Weiaen- und Roggensamen, Lokalisierung der aktiven Zentren (In German, Engl. Summary.) Hoppe-Seyler’s Z. Physiol. Chem. 350, 249-254 (Chem. Abstr.

.

74681G)

Hoseney, R. C., and Finney, K. F. 1971. Functional (breadmaking) and biochemical properties of wheat flour components. XI. Review. Baker’s Dig.45, 30-36, 39-40, and 64-67. Hoseney, R. C., Finney, K. F., Bhorgren, M. D., and Pomeranz, Y . 1969a. Functional (breadmaking) and biochemical properties of wheat flour components. 11. Role of water-solubles. Cereal Chem. 46, 117-125. Hoseney, R. C., Finney, K. F., Shogren, M. D., and Pomeranz, Y . 1969b. Functional (breadmaking) and biochemical properties of wheat flour comopnents. 111. Characterization of gluten protein fractions obtained by ultracentrifugation. Cereal Chem. 48, 126135. Hoseney, R. C., Finney, K. F., Shogren, M. D., and Pomeranz, Y . 1969c. Functional (breadmaking) and biochemical properties of wheat flour components. IV. Gluten protein fractionation by solubilizing in 70% ethyl alcohol and in dilute lactic acid. Cereal Chem. 46, 495-502. Hoseney, R. C., Finney, K. F., Pomeranz, Y., and Shogren, M. D. 1969d. Functional (breadmaking) and biochemical properties of wheat flour components. V. Role of total extractable lipids. Cereal Chem. 46, 606-613. Hoseney, R. C., Finney, K. F., and Pomeranz, Y . 1970a. Functional (breadmaking) and biochemical properties of wheat flour components. VI. Gliadin-lipid- glutenin interaction in wheat gluten. Cereal Chem. 47, 135-140. Hoseney, R. C., Pomeranz, Y., and Finney, K. F. 1970b. Functional (breadmaking) and biochemical properties of wheat flour components. VII. Petroleum ethersoluble lipoproteins of wheat flour. Cereal Chem. 47, 153-160. Hoseney, R. C., Finney, K. F., and Shogren M. D. 1972. Functional (breadmaking) and biochemical properties of wheat flour components. IX. Replacing total free lipid with synthetic lipid. Cereal Chem. 49,366371. Hsieh, S. C., and Hsieh, J. S. 1976. Studies on protein and amino acid improvemenls by chromosome manipulation in wheat (In Chinese, Engl. Summary). Nat. Sci. Council Monthly (Taiwan) 10, 44-52. Huebner, F. R. 1970. Comparative studies on glutenins from different classes of wheat. J . Agric. Food Chem. 18,256-259. Huebner, F. R., and Wall, J. S. 1974. Wheat glutenin subunits. I. preparative separation by gel filtration and ion-exchange chromatography. Cereal Chem. 51, 228-240. Huebner, F. R., and Wall, J. S. 1975. Fractionation and quantitative differences of glutenin from wheat varieties varying in baking quality. Cereal Chem. (in press). Huebner, F. R., and Wall J. S. 1976. Fractionation and quantitative differences of glutenin from wheat varieties varying in baking quality. Cereal Chem. 53, 258469. Huebner, F. R., Donaldson, G. L., and Wall, J. S. 1974. Wheat glutenin subunits. 11. Compositional differences. Cereal Chem. 51, 240-249. Huffaker, R. C., and Peterson, L. W. 1974. Protein turnover in plants and possible means of its regulation. Annu. Rev. Plant Physiol. 25,363392. Inamine, E. S., Noble, E. G., and Mecham, D. K. 1967. Solubilization and fractionation of wheat flour proteins insoluble in dilute acetic acid. Cereal Chem. 44, 143-151. Inglett, G. E., and Anderson, R. A. 1974. Flour milling. In “Wheat: Production

GENETIC CONTROL OF PROTEINS IN WHEAT

559

and Utilization” (G. E. Inglett, ed.), pp. 186-198. Avi Publ., Westport, Connecticut. Ingversen, J. 1975. Structure and composition of protein bodies from wild-type and high-lysine barley endosperm. Hereditas 81, 69-76. Ingversen, J. 1976. Ultrastructural and biochemical differences between the protein bodies from wild type and high lysine barley seeds. In “Barley Genetics 111” (H. Gaul, ed.), p. 547. Karl Thiemig, Munich. Ingversen, J., and KGie, B. 1973. Lysine-rich proteins in high lysine Hordeum uulgare grain. Phytochemistry 12, 1107-1111. Ingversen, J., KZie, B., and Doll, H. 1973. Induced seed protein mutant of barley. Ezperientiu 29, 1151-1152. Irani, B. N., and Bhatia, C. R. 1972. Chromosomal location of alcohol dehydrogenase gene(s) in rye, using wheat-rye addition lines. Genetica 43, 195-200. Irvine, G. N. 1971. Durum wheat and paste products. In “Wheat: Chemistry and Technology” ( Y . Pomeranz, ed.), pp. 777-798. Am. Assoc. Cereal Chem. St. Paul, Minnesota. Islam, A. K. M. R., Shepherd, K. W., and Sparrow, D. H. B. 1976. Addition of individual barley chromosomes to wheat. In “Barley Genetics 111” (H. Gaul, ed.), pp. 260-270. Karl Thiemig, Munich. Jaaska, V. 1974. The origin of tetraploid wheats based on electrophoretic studies of enzymes. Eesti NSV Tead. Akad. Toim., Biol. 23,201-220. Jacob, F., and Monod, J. 1961. Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3, 31tL356. Jagannathan, D. R., and Bhatia, C. R. 1972. Effect of rye chromosome 2 substitution on endosperm proteins of wheat. Theor. Appl. Genet. 42,89-92. Jahn-Deesbach, W., and Jurgens, U. 1973. Der Einfluss variierter Stickstoffgahen auf die Ertragsmorphologie und die Stickstoffeinlagerung bei Sommerweizen in einem Gef&sversuch. IV. Stickstoffeinlagerung in die verschiedenen Proteinfraktionen des Ganzkornes und der einzelnen Mahlfraktionen. (In German, Engl. Summary) 2. Acker- PfEanzenbau 138, 276-286 (Chem. Abstr. 12366e). Jain, H. K., Singh, M. P., and Raut, R. N. 1968a. Genomic differentiation and RNA synthesis in wheat. Proc. Znt. Wheat Genet. Symp., 3rd, 1968 pp. 99-104. Jain, H. K., Raut, R. N., and Nerwal, S. K. 196813. Nuclear organizer as hyperactive locus for RNA synthesis. Heredity 24, 59-67. Jain, H. K., Singhal, N. C., Singh, M. P., and Austin, A. 1975. An approach to breeding for higher protein content in bread wheat. In “Breeding For Seed Protein Improvement Using Nuclear Techniques,” pp. 39-46. Proc. IAEA Symp., Vienna. Jain, H. K., Singhal, N. C., and Austin, A. 1976. Breeding for higher protein yields in bread wheat: Experimental approach and a phenotypic marker. Z. Pflanzenzuecht. 77, 100-111. Jambunathan, R., Mertz, E. T., and Axtell, J. D. 1975. Fractionation of soluble proteins of high lysine and normal sorghum grain. Cereal Chem. 52, 119-121. Jaworski, A., and Key, J. L. 1972. Distribution of ribosomal deoxyribonucleic acid in subcellular fractions of higher plants. Plant Physiol. 53, 366-369. Jenkins, L. D., Loney, D. P., and Meredith, P. 1974. Periodicity of growth and starch deposition in the developing wheat grain. Cereal Chem. 51, 718-733. Jha, M. P., Kaul, A. K., Raghaviah, P., and Swaminathan, M. S. 1971. Identification of chromosomes carrying factors for seed storage proteins. Wheat In!. S e w . 32, 9-13.

Jimenez, J. R. 1968. The effect of the opaque-2 and floury-2 genes on the produc-

560

CALVIN F. KONZAR

tion of protein in maize endosperm. Ph.D. Thesis, Purdue University, Lafayette, Indiana. Johnson, B. L. 1968. Electrophoretic evidence on the origin of Triticum zhukovskyi. Proc. Znt. Wheat Genet. Symp., Srd, 196s pp. 105-110. Johnson, B. L. 1972a. Seed protein profiles and the origin of the hexaploid wheats. Am. J . Bot. 59, 952-960. Johnson, B. L. 1972b. Protein electrophoretic profiles and the origin of the B genome of wheat. Proc. Natl. Acad. Sci. USA. 89, 1398-1402. Johnson, B. L. 1975. Identification of the apparent B genome donor of wheat. Can. J . Genet. Cytol. 17, 21-39. Johnson, B. L. 1976. Polyploid wheats and fraction 1 protein. Science 192, 1252. Johnson, B. L., and Dhaliwal, H. S. 1976. Reproductive isolation of Triticum boeoticum and Triticum urartu and the origin of tetraploid wheats. A m . J . Bot. 83, 1088-1094. Johnson, B. L., and Hall. 0. 1966. Electrophoretic studies of species relationships in Trilicum. Acta Agric. Scand., Suppl. 18, 222224. Johnson, B. L., Barnhart, D., and Hall, 0. 1967. Analysis of genome and species relationships in the polyploid wheats by protein electrophoresis. Am. J . Bot. 54, 1089-1098. Johnson, J. A., and Sanchez, R. S. 1972. New no-fermentation process controls bread flour and costs. Baker’s Dig. 48, 30-32. Johnson, J. A., Khan, M. N. A., and Sanchez, C. R. S. 1972. Wheat cultivars, environment and bread baking quality. Cereal Sci. Today 17, 323-326. Johnson, V. A., and Lay, C. L. 1974. Genetic improvement of plant protein. J . Agric. Food Chem. 22, 558-566. Johnson, V. A., and Mattern, P. J. 1972. “Improvement of the Nutritional Quality of Wheat through Increased Protein Content and Improved Amino Acid Balance,” Summary report of research findings July 1, 1966-Dec. 31, 1972, Contract AID/Csd-1208. Agency for International Development, Department of State, Washington, D.C. Johnson, V. A., and Mattern, P. J. 1975. “Improvement of the Nutritional Quality of Wheat Protein through Increased Protein Content and Improved Amino Acid Balance,” Report of research findings Jan. 1, 1973-March 31, 1975, Contracts AID/Csd-1208 and AID/ta-C-1093. Agency for International Development, Department of State, Washington, D.C. Johnson, V. A., Schmidt, J. W., Mattern, P. J., and Haunold, A. 1963. Agronomic and quality characteristics of high protein Fz-derived families from a soft red winter-hard wheat cross. Crop Sci. 3, 7-10. Johnson, V. A., Mattern, P. J., and Schmidt, J. W. 1967. Nitrogen relations during spring growth in varieties of Triticum aestivum L. differing in grain protein content. Crop. Sci. 7 , 664-667. Johnson, V. A., Whited, D. A., Mattern, P. J., and Schmidt, J. W. 1968a. Nutritional improvement of wheat by breeding. Proc. Znt. Wheat Genet. Sym?., Srd, 1968 pp. 457-461. Johnson, V. A., Schmidt, J. W., and Mattern, P. J. 1968b. Cereal breeding for better protein impact. Econ. Bot. 22, 16-25. Johnson, V. A., Mattern, P. J., and Schmidt, J. W. 1972a. Wheat protein improvement. Int. Rice Res. Inst., Rice Breed. pp. 407418. Johnson, V. A., Mattern, P. J., and Schmidt, J. W. 197213. Genetic studies of wheat protein. In “Symposium: Seed Proteins” (G. E. Inglett, ed.), pp. 126-136. Avi Publ. Go., Westport, Connecticut.

GENETIC CONTROL OF PROTEINS I N WHEAT

561

Johnson, V. A., Dreier, A. F., and Grabouski, P. H. 1973. Yield and protein responses to nitrogen fertilizer of two winter wheat varieties differing in inherent protein content of their grain. Agron. J. 65, 259-263. Johnson, V. A., Mattern, P. J., Stroike, J. E., and Wilhelmi, K. D. 1975a. Breeding for improved nutritional quality in wheat. Proc. Int. Winter Wheat Conf. 2nd, 1975 pp. 316-333. Johnson, V. A., Mattern, P. J., and Vogel, K. P. 1975b. Cultural genetic, and other factors affecting quality of wheat. In “Bread: Social, Nutritional, and Agricultural Aspects of Wheat Bread” (A. Spencer, ed.), pp. 127-140. Applied Science Publ., London. Jones, I. K., Phillips, J. W., and Hird, F. J. R. 1974. The estimation of rheologically important thiol and disulphide groups in dough. J . Sci. Food Agric. 25, 1-10. Jones, R. W., and Cluskey, J. E. 1963. Preparation of aluminum lactate. (Communication to the Editor.) Cereal Chem. 40, 589-591. Jones, R. W., Babcock, G. E., Taylor, N. W., and Senti, F. R. 1961. Molecular weight of wheat gluten fractions. Arch. Biochem. Biophys. 94, 483-488. Jones, R. W., Babcock, G. E., Taylor, N. W., and Dimler, R. J. 1963. Fractionation of wheat gluten by gel filtration. Cereal Chem. 40, 409414. Joppa, L. R. 1973. Development of disomic substitution lines of durum wheat (Tn‘ticum turgidum L.) Proc. Znt. Wheat Genet. Symp., 4th, 1973 pp. 685-690. Joppa, L. R., Bietz, J. A., and McDonald, C. 1975. Development and characteristics of a disomic-1D addition line of durum wheat. Can. J. Genet. Cytol. 17, 355463. Joudrier, M. P., and Cauderon, Y. 1976. Localisation chromosomique de genes contr8lant la synth6se de certains constituants p amylasique du grain de B16 tendre (In French, Engl. Summary). Note. C. R . Hebd. Seances Acad. Sci., Ser. D . 282, 115-118 (Chem.Abstr. 161857j). Juliano, B. 0. 1972. Studies on protein quality and quantity of rice. In “Symposium: Seed Proteins” (G. E. Inglett, ed.), pp. 114-125. Avi Publ., Westport, Connecticut. Kakade, M. L. 1974. Biochemical basis for the differences in plant protein utilization. J. Agric. Food Chem. 22, 550-555. Kaltsikes, P. J., Evans, L. E., and Bushuk, W. 1968a. Durum type wheat with high breadmaking quality. Science 159, 211-213. Kaltsikes, P. J., Evans, L. E., and Larter, E. N. 196813. Identification of a chromosome segment controlling breadmaking quality in common wheat. Can. J. Genet. Cytol. 10, 763. Kamagata, T., and Nishikawa, K. 1973. Genetic variation of peroxidase isozymes in wheat (In Japanese) Jpn. J . Genel. 48, 422423 (abstr.). Kanazana, H., and Yonezawa, D. 1973. Studies on polypeptide composition of “low molecular weight glutenin” (In Japanese, English Summary). Nippon Nogei Kagaku Kaishi 47, 17-22 (Chem. Abstr. 11550g). Kanazana, H., and Yonezawa, D. 1974a. Aggregative polypeptides as component polypeptides of wheat glutenin (In Japanese, English Summary). Nippon Nogei Kngaku Kaishi 48, 113-117 (Chem. Abstr. 2242ln). Kanaaawa, H., and Yonezawa, D. 1974b. Conversions of intra- to interchain disulfide bonds in gluten polypeptides by S H S S exchange reaction (In Japanese, English Summary). Nippon Nogei Kagaku Kaishi 48, 245-253 (Chem. Abstr. f47137j). Kannangara, C. G . 1969. The formation of ribulose diphosphate carboxylase proteins during the chloroplast development in barley. Plant Physiol. 44, 1533-1537.

562

CALVIN F. KONZAK

Kasarda, D. D. 1970. The conformational structure of wheat proteins. Baker’s Dig. 44,20-26, Kasarda, D. D. 1975. Celiac disease: Malabsorption of nutrients induced by a toxic factor in gluten, In “Protein Nutritional Quality of Foods and Feeds” (M. Friedman, ed.), Vol. 1, Part 2,pp. 565-593. Dekker, New York. Kasarda, D. D., Bernardin, J. E., and Thomas, R. S. 1967. Reversible aggregation of alpha-gliadin to fibrils. Science 155, 203-205. Kasarda, D. D., Nimmo, C. C., and Kohler, G. 0. 1971.Proteins and the amino acid composition of wheat fractions. Zn “Wheat-Chemistry and Technology” (Y. Pomerans, ed.), pp. 227-299. Am. Assoc. Cereal Chem., St. Paul, Minnesota. Kasarda, D. D., Qualset, C. O., and Platt, 8. G. 1973. Varietal relationships of the alpha-gliadin proteins in wheat. Proc. Int. Wheat Genet. Symp., 4th, 19Y3 pp. 811-815. Kasarda, D. D., Da Rosa, D. A., and Ohms, J. I. 1974a. N-terminal sequence of cun-gliadin.Biochim. Biophys. Acla 351, 290-294. Kasarda, D. D., Nimmo, C. C., and Bernardin, J. E. 1974b. Structural aspects and genetic relationships of gliadins. In “Coeliac Disease” J. M. Hekkens and A. S. Pena, eds.), pp. 25-36. H. E. Stenfert Kroese, Leiden, Netherlands. Kasarda, D. D., Bernardin, J. E., and Qualset, C. 0. 1974c. Relationship of gliadin protein components to chromosomes through the use of substitution lines. Cereal Sci. Today 19,403. Kasarda, D. D., Bernardin, J. E., and Nimmo, C. C. 1976a. Wheat proteins. In “Advances in Cereal Science and Technology” (Y. Pomeranz, ed.), pp. 158-236. Am. Assoc. Cereal Chem., St. Paul, Minnesota. Kasarda, D. D., Bernardin, J. E., and Qualset, C. 0. 1976b. Relationship of gliadin protein components to chromosomes in hexaploid wheats (Trilicum aestivum L.).Proc. Natl. Acad. Sci. USA. 73, 3646-3650. Kato, M., Hiromi, K., and Morita, Y.1974. Purification and kinetic studies of wheat bran @-amylase. Evaluation of subsite affinities. J . Biochem. (Tokyo) 75, 563576. Kaul, A. K., and Sosulski, F. W. 1965. Inheritance of flour protein content in a Selkirk x Gab0 cross. Can. J . Genet. Cytol. 7, 12-17. Kawashima, N., and Wildman, S. G. 1971. Studies of Fraction I protein. I. Effect of crystallization of Fraction I protein from tobacco leaves on ribulose diphosphate carboxylase activity. Biochim. Biophys. Acta 299,240-249. Kawashima, N., and Wildman, S. G. 1972. Studies on Fraction I protein. IV. Mode of inheritance of primary structure in relation to whether chloroplast or nuclear DNA contains the code for a chloroplast protein. Biochirn. Biophys. Acta 282, 42-49. Kendall, M. J., Schneider, R., Cox, P. S., and Hawkins, C. F. 1972. Gluten subfractions in coeliac disease. Lancet 2, 1065-1067. Kent, N. L. 1966. Subaleurone cells of high protein content. Cereal Chem. 43, 585601. Kent, N. L., and Evers, A. D. 1969. Variation in protein composition within the endosperm of hard wheat. Cereal Chem. 46,293-300. Kerber, E. R. 1964. Wheat: Reconstitution of the tetraploid component (AABB) of hexaploids. Science 143, 253-255. Kerber, E. R., and Tipples, K. H. 1969. Effects of the D genome on milling and baking properties of wheat. Can. J . Plant Sci. 49,225-263.

GENETIC CONTROL OF PROTEINS IN WHEAT

563

Khan, A. H.1972. Improvement of quality and yield of wheat by mutation breeding. Wheat hj. S e w . 36, 7-8. Khrabrova, M. A,, Galai, V. S., and Maystrenko, 0. 1. 1973. Electrophoretic patterns of gluten proteins of some common wheat varieties and of a set of Chinese Spring ditelosomics. In “Cytogenetic Studies on Aneuploids of Common Wheat” (0. I. Maystrenko and V. V. Xhvostova, eds.) (In Russian, English Summary), pp. 218-232. Acad. Sci. U.S.S.R., Siberian Branch, Inst. Cytol. Genet. Khush, G. S. 1973. “Cytogenetics of Aneuploids.” Academic Press, New York. Kies, C., and Fox, H.M. 1970a. Determination of the first-limiting amino acid of wheat and Triticale grain for humans. Cereal Chem. 47, 615-625. Kies, C., and Fox, H.M.1970b. Protein nutritive value of wheat and Triticale grain for humans, studied at two levels of protein intake. Cereal Chem. 47, 671-678. Kihara, H. 1944. Discovery of the DD analyzer, one of the ancestors of Triticum vulgare. Agric. Hort. 19, 889-890. Kihara, H. 1954.Consideration on the evolution and distribution of Aegilops species based on the analyser-method. Cytologk 19, 336357. Kihara, H.1959. Fertility and morphological variation in the substitution and reetoration backcrosses of the hybrids, Triticum vulgare x Aegilops caudata. Proc. Int. Congr. Genet., loth, 1968 Vol. 1, pp. 142-171. Kihara, H. 1966. Factors effecting the evolution of common wheat. Indian J. Genet. Plant Breed. 26a, 14-28. Kihara, H.1968. Cytoplasmic relationships in the Triticinae. Proc. Znt. Wheat Genet. Symp., Srd, 1968 pp. 125-134. Kihara, H. 1974. The history of all organisms is inscribed in the chromosomes (in Japanese). Kromosomo 96-97, 2995-3008. Kihara, H. 1975. Origin of cultivated plants with special reference to wheat. Seiken Ziho 25-26, 1-24. Kihara, H.,and Lilienfeld, F. A. 1949.A new synthesized 6 x wheat. Proc. Znt. Congr. Genet., Sth, 1948 Hereditas, Suppl. 1, 307-319. Kihara, H.,and Tsunewaki, K. 1968. Some fundamental problems underlying the program for hybrid wheat breeding. Seiken Ziho 16,l-14. Kilborn, R. H., and Tipples, K. H.1972a. Factors affecting mechanical dough development. I. Effect of mixing intensity and work input. Cereal Chem. 49, 3447. Kilborn, R. H.,and Tipples, K. H.1972b. Factors affecting mechanical dough development. 11. Implications of mixing a t a constant rate of energy input. Cereal Chem. 49, 48-53. Kimber, G. 1966. Estimate of the number of genes involved in the genetic suppression of the cytological diploidization of wheat Nature (London) 212, 317-318. Kimber, G. 1973. The relationships of the S-genome diploids to polyploid wheats. Proc. Int. Wheat Genet. Symp., 4th. 1973 pp. 81-85. Kimber, G., and Athwal, R. S. 1972. A reassessment of the course of evolution in wheat. Proc. Natl. Acad. Sci. U S A . 69, 912915. Klepper, L. A. 1975. Nitrate assimilation enzymes and seed protein in wheat. Proc. Int. Winter Wheat Conf.,grid. 1976 pp. 334-340. Kneen, E., and Sandstedt, R. M. 1943.An amylase inhibitor from certain cereals. J. A m . Chem. SOC.65, 1247. Kobrehel, K., and Feillet, P. 1975. Identification of genomes and chromosomes involved in peroxidase synthesis of wheat seeds. Can. J. Bot. 53, 2336-2344.

564

CALVIN F. KONZAK

Kobrehel, K., and Gautier, M. F. 1974. Variability in peroxidase isozymes in wheat and related species. Can. J . Bot. 52, 755-759. Koenig, V. L., Ogrins, A., Trimbo, H. B., and Miller, B. S. 1974. The electrophoretic analysis of flour from several varieties of hard red winter wheat grown a t several locations. J . Sci. Food. Agric. 15,492497. Kolderup, F. 1975. Effects of temperature, photoperiod and light quantity on protein production in wheat grains. J . Sci. Food. Agric. 26, 583-592. Konarev, V. G., Gubareva, N. K., Gavrilyuk, I. P., and Bushuk, U'. 1972. Identification of genome D in wheats with respect to gliadin (In Russian, English Summary). Vestn. Skh. Nauki (Moscow) 17,108-114 (Chem. Abstr. 111649~). Kruger, J. E. 1973. Changes in the levels of proteolytic enzymes from hard red spring wheat during growth and maturation. Cerea2 Chem. 50, 122-131. Kruger, J. E. 1976. Changes in the polyphenol oxidases of wheat during kernel growth and maturation. Cereal Chem. 53, 201-213. Kruger, J. E., and LaBerge, D. E. 1974. Changes in peroxidase activity and peroxidase isoayme patterns of wheat during kernel growth and maturation. Cereal Chem. 51, 345-354. Ku, H. S. 1973. Stimulation of nitrate reductase by a protein fraction. Agron. Abstr. p. 35 (by permission). Ku, H., Omata, S., and Tolbert, N. E. 1973. Nitrate reductase inhibitors from leaf tissue. Agron. Abstr. p. 36 (by permission). Kulp, K. 1972. Physicochemical properties of starches of wheats and flours. Cereal Chem. 49, 697-706. Kung, S. D. 1976. Tobacco fraction I protein: A unique genetic marker. Science 191, 429-434.

Kung, S. D., Sakano, K., and Wildman, S. G. 1974a. Multiple peptide composition of the large and small subunits of Nicotiana tabaccum Fraction I protein ascertained by fingerprinting and electrofocusing. Biochem. Biophys. Acta 365, 138-147. Kung, S. D., Sakano, K., and Wildman, S. G. 1974b. Evolution of Fraction I protein. Phnt Physiol. 53, (Suppl.) 63 (Abstr. No. 356). Kuspira, J., and Unrau, J. 1957. Genetic analysis of certain characters in common wheat using whole chromosome substitution lines. Can. J . Plant Sci. 37, 300-326. Kuspira, J., and Unrau, J. 1958. Determination of the number and dominance of genes on substituted chromosomes in common wheat. Can. J . Phnt Sci. 38, 199-205.

Kwanyuen, P., and Wildman, S. G. 1975. Nuclear DNA codes for Nicotiana ferredoxin. Biochim. Biophys. Acta 405, 167-174. La Clerc, J. A,, Bailey, L. H., and Wessling, H. L. 1918. Milling and baking tests of Einkorn, Emmer, Spelt, and Polish wheat. Agron. J. 10, 215-217. Landry, J., and Moureaux. T. 1970. HBtBrogBnBi'tB des glutelines du grain de Mays: Extraction selertive et composition en acides amines des trois fractiones isoldes (In French). Bull. Soc. Chim. Biol. 52, 1021-1037 (Chem. Abstr. 7877Sh). Larkin, R. A., MacMasters, M. M., Wolf, M. J., and Rist, C. E. 1951. Studies on the relation of bran thickness to millability of some Pacific Northwest wheats. Cereal Chem. 28, 247-258. Larkin, R. A., MacMaster, M. M., and Rist, C. E. 1952. Relation of endosperm cell wall thickness t o the milling quality of seven Pacific Northwest wheats. Cereal Chem. 29, 407-413. Larsen, J. 1974. The localization of phenol reaction genes in hexaploid wheat. E.W.A.C. Newslett. No. 4,80 (by permission).

GENETIC CONTROL OF PROTEINS IN WHEAT

565

Larsen, J., and Kimber, G. 1973. The effect of the genotype of Triticum speltoides on the pairing of homologous chromosomes. Cnnnd. J . Genet. Cytol. 15, 233-236. Law, C. N. 1968. Genetic analysis using inter-varietal substitution lines. Proc. Int. Wheat Genet. Symp., Srd, 196s pp. 331-342. Law, C. N., and Worland, A. J. 1972. “Aneuploidy in Wheat and its Uses in Genetic Analysis.” Annu. Rep., 1972, pp. 25-65. Plant Breed. Inst., Cambridge, England. Law, C. N., and Worland, A. J. 1973. Chromosome substitutions and their use in the analysis and prediction of wheat varietal performance. Proc. Int. Wheat Genet. Symp., 4th, 1975 pp. 41-49. Lawrence, J. M., Day, K . M., Huey, E., and Lee, B. 1958. Lysine content of wheat varieties, species, and related genera. Cereal Chem. 35, 169-178. Lawrence, J. M., Lin, S.-C. and Grant, D. R. 1970. Dye-binding capacity and amino acid content of wheat-protein-gel-electrophoresis-bands. Cereal Chem. 47, 110117.

Leaback, D. H., and Wrigley, C. W. 1976. The isoelectric focusing of proteins. I n “Chromatographic and Electrophoretic Techniques” (I. Smith, ed.), 4th ed., Vol. 2. Heinemann, London (in press). Lebsock, K. L., Fifield, C . C., Gurney, G. M., and Greenaway, W. T. 1964. Variation and evaluation of mixing tolerance, protein content, and sedimentation value in early generations of spring wheat, Triticum aestivum L. Crop Sci. 4, 111-174. Lee, J. W., and Wrigley, C. w. 1963, The protein composition of gluten extracted from different wheats. Aust. J. E x p . Agric. Anim. Husb. 3, 85-88. Lein, I<. A., Brunckhorst, X., and Schon, W. J. 1973. Assessment of different techniques for estimation of total nitrogen and lysine in cereals. In “Nuclear Techniques for Seed Protein Improvement,” STI/PUB/320, IAEA, Vienna, pp. 363369.

Lin, D. J., and Pomeranz, Y. 1975. Distribution of minerals in barley at the cellular level by X-ray analysis. Cereal Chem. 52, 620-629. Lofgren, J. R . , Finney, K. F., Heyne, E. G., Bolte, L. C., Hoseney, R. C., and Shogren, M. D. 1968. Heritability estimates of protein content and certain quality and agronomic properties in bread wheats (Triticum aestivum L.). Crop Sci. 8, 563-567. Longwell, J. R., Jr., and Sears, E. R. 1963. Nullisomics in tetraploid wheat. Am. N u t . 97, 401-403. Lorimer, G . H., Andrews, T. J., and Tolbert, N. E. 1973. Ribulose diphosphate oxygenase. 11. Further proof of reaction products and mechanism of action. Biochemistry 12, 18-23. Ma,, Y., and Nelson, 0. E. 1975. Amino acid composition and storage proteins in two new high-lysine mutants in maize. Cereai Chem. 52,412419. Maan, S . S. 1973a. Cytoplasmic and cytogenetic relationships among tetraploid Triticum species. Euphytica 22, 287-300. Maan, S. S. 1973b. Cytoplasmic variability in Triticinae. P I O CZnt. . Wheat Genet. Symp., 4th, lgY5 PD. 367-373. Maan, 5. S. 1975. Cytoplasmic male-sterility and male-fertility systems in wheat. In “Genetics and Breeding of Durum Wheat” (G. T.Scarascia-Mugnozza, ed.), pp. 117-137. Library of the Faculty of Agriculture, Bari, and National Institute for Nutrition, Rome. Maan, S. S.. and Lucken, K. A. 1970. Interaction of Triticum boeoticum cytoplasm and genomes of T . aestivum and T. durum: Restoration of male fertility and plant vigor. Euphytica 19, 498-508.

566

CALVIN F. RONZAK

Maan, S. S., and Lucken, K. A. 1971a. Nucleo-cytoplasmic interactions involving Aegilops cytoplasm and Triticum genomes. J. Hered. 62, 149-152. Mann, S.S., and Lucken, K. A. 1971b. Male sterile wheat with rye cytoplasm: Restoration of male fertility and plant vigor. J. Hered. 62, 353-355. Maan, S. S., and Lucken, K. A. 1972. Interacting male sterility-fertility restoration systems for hybrid wheat research. Crop Sci. 12, 360-364. McDermott, E. E., and Pace, J. 1960. Comparison of the amino acid composition of the protein in flour and endosperm from different types of wheat, with particular reference to variation in lysine content. J. Sci. Food Agric. 11, 109-115. MacDonald, T., and Brewbaker, J. L. 1975. Isoenzyme polymorphism in flowering plants. V. The isoesterases of maize: Tissue and substrate specificities, and responses to chemical inhibitors. Hawaii, Agric. Exp. Stn., Tech. Bull. 89, 1-24. MacDonald, T., and Smith, H. H. 1972. Variation associated with an Aegilops umbellulata chromosome segment incorporated in wheat. 11. Peroxidase and leucine aminopeptidase isorymes. Genetics 72, 77-86. McFadden, B. A. 1973. Autotropic C02 assimilation and the evolution of ribulose diphosphate carboxylase. Bacteriol. Rev. 37, 2%-319. McFadden, B. A., and Tabita, F. R. 1974. n-Ribulose-l,5-diphosphate carboxylase and the evolution of autotrophy. BioSystems 6, 93-112. McFadden, E. S., and Sears, E. R. 1944. The artificial synthesis of Triticum spelta. Rec. Genet. SOC.Am. 13,2627. McFadden, E. S., and Sears, E. R. 1946. The origin of Triticum speltn and its freethreshing relatives. J . Hered. 37, 81-89 and 107-116. McGuire, C. F., and McNeal, F. H. 1974. Quality response of 10 hard red spring wheat cultivars to 25 environments. Crop Sci. 14, 175-178. MacIndoe, S. L., and Walkden-Brown, C. 1968. Wheat breeding and varieties in Australia. N . S. W . , Dep. Agric., Sci. Bull. 76,l-255. Mclntosh, R. A. 1973. A catalogue of gene symbols for wheat. Proc. Znt. Wheat Genet. Symp., $th, 1973 pp. 893-937. MacKey, J. 1966. Species relationship in Triticum. Proc. Znt. Wheat Genet. Symp., 2nd, 1963 Hereditas, Suppl, 2, 237-276. MacKey, J. 1968a. Mutagenesis in wulgare wheat. Hereditas 59, 505-517. MacKey, J. 196813. Relationships in the Triticinae. Proc. Znt. Wheat Genet. Symp., Srd, 1968 pp. 39-50. MacKey, J. 1970. Significance of mating systems for chromosomes and gametes in polyploids. Hereditas 66, 165-176. MacKey, J. 1975. The boundaries and subdivision of the genus Triticum. Znt. Bot. Congr., 12th 1976, Vol. 2, p. 509 (abstr.). MacMasters, M. M., Hinton, J. J. C., and Bradbury, D. 1971. Microscopic structure and composition of the wheat kernel. In “Wheat-Chemistry and Technology” (Y.Pomeranz, ed.), pp. 51-113. Am. Assoc. Cereal Chem., St. Paul, Minnesota. McNeil, M., Albersheim, P., Taiz, L., and Jones, R. 1974. On the structure of the walls of barley aleurone cells. Plant Physiol. 53 (Suppl.) 15 (Abstr. No. 85). MacRitchie, R. 1973. Conversion of a weak flour to a strong one by increasing the proportion of its high molecular weight gluten protein. J . Sci. Food Agric. 24, 1325-1329. Madl, R. L., and Tsen, C. C. 1974a. The proteolytic enzynie system of triticale. In “Triticale: First Man-Made Cereal” (C. C. Tsen, ed.), pp. 157-167. Am. Awoc. Cereal Chem., St. Paul Minnesota. Madl, R. L., and Tsen, C. C. 1974b. Trypsin and chymotrypsin inhibitors of t,riticale.

GENETIC CONTROL OF PROTEINS I N WHEAT

567

In “Triticale: First, Man-Made Cereal” (C. C. Tsen, ed.), p. 168-182. Am. Assoc. Cereal Chem., St. Paul, Minnesota. Maes, E. E. A. 1966. Protein solubility and baking quality. Cereal Sci. Today 11, 200. Magalhaes, A. C., Neyra, C. A., and Hageman, R. H. 1974. Nitrite assimilation and amino nitrogen synthesis in isolated spinach chloroplasts. Plant Physiol. 53, 411-415. Magoffin, C. D., Finney, P. L., and Finney, K. F. 1976. Short-time baking systems. 11. A sugar-free formula for conventional and high protein breads. Cereal Chem. 53, 61. Mak, A., and Jones, B. L. 1974. Studies of the primary structure of wheat purothionin. Cereal Sci. Today 19, 397 (Abstr. No. 86). Manghers, L. E., Solari, R. M., Avila, A., and Favret, E. A. 1973. Implications of the genetics of cereal seed proteins in induced mutagenesis. In “Nuclear Techniques for Seed Protein Improvement,” STI/PUB/320. pp. 305-320. IAEA, Vienna. Mares, D. J., Norstog, K., and Stone, B. A. 1975. Early stages in the development of wheat endosperm. I. The change from free nuclear to cellular endosperm. Aust. J. Bot. 23, 311-326. Matsuo, R. R., and Irvine, G. N. 1975. Rheology of durum wheat products. Cereal Chem. 52, 131r-135r. Mattern, P. J., Schmidt, J. W., Morris, R., and Johnson, V. A. 1968a. A feasibility study of the use of a modified Maes protein extraction process and chromosome substitution lines for bread wheat quality identification. Proc. Int. Wheat Genet. Symp., Srd, 1968 pp. 449456. Mattern, P. J., Salem, A., Johnson, V. A., and Schmidt, J. W. 1968b. Amino acid composition of selected high protein wheats. Cereal Chem. 45, 437444. Mattern, P. J., Morris, R., Schmidt, J. W., and Johnson, V. A. 1973. Locations of genes for kernel properties in the wheat, variety ‘Cheyenne’ using rhromosome substitution lines. Proc. Int. Wheat Genet. Symp., 4th, 1573 pp. 703-707. Mattern, P. J., Vogel, K. P., and Johnson, V. A. 1975. Processing and nutritional quality of high protein wheats. Proe. Int. Winter Wheat Conf., bnd, 1976 pp. 305-31 5. Maugh, T. H., 11. 1975. Ribosomes (11): A complicated structure begins to emerge. Science 190,258-260. May, C. E., Vickery, R. S., and Driscoll, C. J. 1973. Gene control in hexaploid wheat. Proc. I n t . Wheat Genet. Syrnp., 4th, 1973 pp. 843-850. Maystrenko, 0. I., Troshina, A. V., and Ermakova, M. F. 1973a. Chromosomal arm location of genes for flour quality in wheat using ditelosomic lines. Proc. I n t . Wheat Genet. Sump., dth, 1973 pp. 51-56. Maystrenko, 0. I., Ermakova, M. F., and Talyzina, G. G . 1973b. Monosomic analysis of gluten swelling in high quality varieties of common wheat from selected Saratovskaya varieties. In “Cytogenetic Studies on Aneuploids of Common Wheat” (0. I. Maystrenko and V. V. Khvostova, eds.), pp. 246258 (In Russian, English Summary). Acad. Sci. U.S.S.R., Siberian Branch, Inst. Cytol. Genet. Mecham, D. K. 1971. Lipids, In “Wheat-Chemistry and Technology” (Y. Pomeranc, ed.), pp. 343452. Am. Assoc. Cereal Chem., St. Paul, Minnesota. Mecham, D. K. 1973. Wheat and flour proteins: Recent research. Baker’s Dig. 47, 24-26, 30, and 128.

568

CALVIN F. KONZAK

Mecham, D. K., Cole, E. W., and Ng, H. 1972. Solubilizing effect of mercuric chloride on the “gel” protein of wheat flour. Cereal Chem. 49, 52-67. Mello-Sampayo, T. 1971a. Promotion of homoeologous pairing in hybrids of Triticum aestivum and Aegilops longissima. Genet. Iber. 23, 1-9. Mello-Sampayo, T. 1971b. Genetic regulation of chromosome pairing by the chromosome 3D of wheat. Nature (London), New Biol. 230, 22-23. Meredith, 0. B., and Wren, J. J. 1966. Determination of molecular weight distribution of wheat-flour proteins by extraction and gel filtration in a dissociating medium. Cereal Chem. 43, 169-186. Meredith, P. 1965a. On the solubility of gliadinlike proteins. I. Solubility in nonaqueous media. Cereal Chem. 42, 54-63. Meredith, P. 1965b. On the solubility of gliadinlike proteins. 111. Fractionation by solubility. Cereal Chem. 42, 149-160. Meredith, P., and Jenkins, L. D. 1973. Anylases of developing wheat, barley and oat grains. Cereal Chem. 50, 240-254. Mertz, E. T., Misra, P. S., and Jambunathan, R. 1974. Rapid ninhydrin color test for screening high lysine mutants of maize, sorghum and other cereal grains. Cereal Chem 51, 304-307. Mesdag, J., Slootmaker, L. A. J., and Post, J., Jr. 1970. Linkage between tolerance to high soil acidity and genetically high protein content in the kernel of wheat, Triticum aestivum L. and its possible use in breeding. Euphytica 19, 163-174. Middleton, G. K., Bode, C. E., and Bayles, B. B. 1954. A comparison of the quantity and quality of protein in certain varieties of soft wheat. Agron. J . 46, 500-502. Miflin, B. J. 1974. Nitrite reduction in leaves; studies on isolated chloroplasts. Planta 116, 187-196. Mikola, J., and Kirsi, M. 1972. Differences between endospermal and embryonal trypsin inhibitors in barley, wheat and rye. Acta Chem. Scand. 26, 787-795. Miladi, S., Hegsted, D. M., Saunders, R. M., and Kohler, G. 0. 1972. The relative nutritive value, amino acid content, and digestibility of the proteins of wheat mill fractions. Cereal Chem. 49, 119-126. Minetti, M., Petrucci, T., Pocchiari, F., Silano, V., and Avella, R. 1971. Varietal differences in water-soluble gliadin fractions of Triticum aestivum and of Triticum &rum seeds. J . Sci. Food Agric. 22, 72-74. Minetti, M., Petrucci, T., Cattaneo, S., Pocchiari, F., and Silano, V. 1973. Studies of the differential staining of wheat albumins, globulins, and gliadins in polyacrylamide gel by Aniline blue-black. Cereal Chem. 50, 198-209. Minetti, M., Morisi, G., Silano, V., and Accardi, N. 1975. Albumin pools with different extraction behaviors in wheat seed. Cereal Chem. 52, 298-304. Misra, P. S., and Mertz, E. T. 1975a. Studies on corn proteins. VI. Endosperm protein changes in single and double endosperm mutants of maize. Cereal Chem. 52, 161-166. Misra, P. S., and Mertz, E. T. 1975b. Studies on corn proteins. VII. Developmental changes in endosperm proteins of high-lysine mutants. Cereal Chem. 52, 734739. Misra, P. S., Jambunathan, R., Mertz, E. T., Glover, D. V., Barbosa, H. M., and McWhirter, K. S. 1972. Endosperm protein synthesis in maize mutants with increased lysine. Science 176, 1425-1427. Misra, P. S., Merta, E. T., and Glover, D. V. 1974. Characteristics of proteins in single and double endosperm mutants of maize. In “High-Quality Protein Maize,” pp. 291-305. Dowden, Hutchinson and Ross, Stroudsburg, Pennsylvania.

GENETIC CONTROL OF PROTEINS I N WHEAT

569

Misra, P. S., Mertz, E. T., and Glover, D. V. 1975a. Studies on corn proteins. VI. Endosperm protein changes in single and double endosperm mutants of maize. Cereal Chem. 52, 161-166. Misra, P. S., Mertz, E. T., and Glover, D. V. 1975b. Studies on corn protein. VII. Developmental changes in endosperm proteins of high-lysine mutants. Cereat Chem. 52, 734-739. Misra, P. S., Mertz, E. T., and Glover, D. V. 1975c. Studies on corn proteins. VIII. Free amino acid content of opaque-2 double mutants. Cereal Chem. 52, 844-849. Mita, T., and Yonezawa, D. (1971. Separation of component-polypeptides of glutenin by isoelectric focusing. Agric. Biol. Chem. 35, 1972-1978. Mitra, R., and Bhatia, C. R. 1971. Isoenzymes and polyploidy. I. Qualitative and quantitative isoeneyme studies in Triticinae. Genet. Res. 18, 57-69. Mitra, R., and Bhatia, C. R. 1973. Studies on protein biosynthesis in developing wheat kernels. In “Nuclear Techniques for Protein Improvement” (B. Kaufmann and M. Lewis, eds.), STI/PUB/320, pp. 379-389. IAEA, Vienna. Mochizuki, A. 1968. The monosomics of durum wheat. Proc. Int. Wheat Genet. Symp., Srd, 1968 pp. 31&315. Morris, R., and Sears, E. R. 1967. The cytogenetics of wheat and wheat relatives. I n “Wheat and Wheat Improvement” (K. S. Quisenberry and L. P. Reitz, eds.), pp. 19-87. Am. SOC.Agron., Madison, Wisconsin. Morris, R., Schmidt, J. W., Mattern, P. J., and Johnson, V. A. 1966. Chromosomal locations of genes for flour quality in the wheat variety “Cheyenne” using substitution lines. Crop Sci. 6, 119-122. Morris, R., Schmidt, J. W., Mattern, P. J., and Johnson, V. A. 1968. Quality tests for six substitution lines involving ‘Cheyenne’ wheat chromosomes. Crop S C ~ . 8, 121-122. Morris, R., Schmidt, J. W., Mattern, P. J., and Johnson, V. A. 1973. Chromosomal locations of genes for high protein in the wheat cultivar Atlas 66. Proc. I n t . Wheat Genet. Symp., 4th, 1978 pp. 715-718. Moss, R. 1973. Conditioning studies on Australian wheat. 11. Morphology of wheat and its relationship to conditioning. J . Sci. Food Agric. 24, 1067-1076. Mossberg, R. 1969. Evaluation of protein quality and quantity by dye-binding capacity: A tool in plant breeding. New Approaches Breed. Improved Plant Protein, Proc. Panel Meet., 196s pp. 151-160. Mosse, J. 1973. HCtCrogCnBitk et polyrnorphisme des protCines et isoenzymes d g 6 tales: Aspects molCculaires et Bvolutifs (In French, English Summary). Ann. Physiol. Veg. 11, 361384 (Chem. Abstr. 12S676e). Muench, S. R., Lejeune, A. J., Nilan, R. A., and Kleinhofs, A. 1976. Evidence for two independent high lysine genes in barley. Crop Sci. 16, 283-285. Mukai, Y., and Tsunewaki, K. 1975. Genetic diversity of the cytoplasm in Triticum and Aegilops. 11. Comparison of the cytoplasms between four 4>< Aegilops species and their 2>( relatives. Seiken Ziho 25-26, 67-78. Munck, L. 1976. Developing the concept, of quality in barley breeding programs for malt and feed. I n “Barley Genetics 111” (H. Gaul, ed.), pp. 526535. Karl Thiemig, Munich. Munck, L., Karlsson, K. E., Hagberg, A,, and Eggum, B. 0. 1970. Gene for improved nutritional value in barley seed protein. Science 168, 985-987. Mylotte, M., Egan-Mitchell, B., McCarthy, C. F., and McNicholl, B. 1973. Incidence of coeliac disease in the west of Ireland. Br. M e d . J . 1, 703-705. Nagabhushan, N., Gulyas, A., and Zalik, S. 1974. Comparison of plant cytoplasmic

570

CALVIN F. KONZAK

ribosomal proteins by two dimensional polyacrylamide gel electrophoresis. PEant Physiol. 53, 516518. Nagayoshi, T. 1973. Genetical studies on amylase isozymes of D genome chromosomes in common wheat (in Japanese). Jpn. J . Genet. 48, 433 (abstr.). Nagayoshi, T., Kakuda, T., and Yasuhara, A. 1974. Genetical studies on esterase isorymes of D genome chromosomes in common wheat (in Japanese). Jpn. J . Genet. 49, 312 (Abstr.). Nakai, Y. 1973. Isozymes variations in Aegilops and Triticum. 11. Esterase and acid phosphatase isozymes studied by the gel isoelectrofocusing method. Seiken Ziho 24, 45-73. Nakai, Y., and Tsunewaki, K. 1974. Genetic studies on the developmental changes of esterase isozymes in aneuploids of common wheat (in Japanese). Jpn. J . Genet. 49, 313. (Abstr.) National Research Council. Board on Agriculture and Renewable Resources. 1976. “Genetic Improvement of Seed Proteins,” pp. 1-394. Washington D.C. Neyra, C. A., and Hageman, R. H. 1974. Dependence of nitrite reduction on electron transport in chloroplasts. Plant Physiol. 54, 480-483. Nielsen, H. C., Beckwith, A. C., and Wall, J. S. 1968. Effect of disulphide-bond cleavage on wheat gliadin fractions obtained by gel filtration. Cereal Chem. 45, 3747.

Nilan, R. A. 1964. “The Cytology and Genetics of Barley, 1951-1962.” Washington State Univ. Press, Pullman. Nilan, R. A., Sideris, E. G., Kleinhofs, A., Sander, C., and Konzak, C. F. 1973. Azide-a potent mutagen. Mutat. Res. 17, 142-144. Nilan, R. A., Kleinhofs, A., and Konzak, C. F. 1976a. The role of induced mutation in supplementing natural genetic variability. Proc. N . Y . Acad. Sci. (in press). Nilan, R. A., Kleinhofs, A., and Sander, C. 1976b. Azide mutagenesis in barley. In “Barley Genetics 111” (H. Gaul, ed.), pp. 113-122. Karl Thiemig, Munich. Nimmo, C. C., O’Sullivan, M. T., and Bernardin, J. E. 1968. The relation of a globulin of wheat flour to purothionin. Cereal Chem. 45, 28. Nimmo, C. C., Kasarda, D. D., and Lew, E. J.-L. 1974. Physical characterization of the wheat protein purothionin. J . Sci. Food Agric. 25,607-617. Nishikawa, K. 1973. Alpha-amylase isozymes and phylogeny of hexaploid wheat. Proc. Int. Wheat Genet. Symp., 4th, i9Y3 pp. 851-855. Nishikawa, K., and Kamagata, T. 1974. Organ-specific peroxidase isorymes in aneuploids of common wheat (in Japanese). Jpn. J . Genet. 49, 314-315 (abstr.). Nishikawa, K., and Nobuhara, M. 1971. Genetic studies of m-amylase isozymes in wheat. I. Location of genes and variation in tetra- and hexaploid wheat. Jpn. J . Genet. 46, 345-353. Nishikawa, K., Hina, Y., Oonishi, S., and Furuta, Y. 1976. The D genome dependent isozymes of a-amylase in wheat. Wheat Znf. Serv. Nos. 41-42, March, 1976, p. 8 (by permission). Nittler, L. W., and Jensen, H. A. 1974. Cultivar differences among barley plants grown with different nutrient treatments. Agron. J. 66, 397-399. Noda, I<., and Tsunewaki, K . 1972. Analysis of seed proteins in ditelosomes of common wheat. Jpn. J . Genet. 47, 315-318. Noronha-Wagner, M., and Mello-Sampayo, T. 1966. Aneuploids in durum wheat. Proc. Int. Wheat Genet. Symp., Bnd, 1963 Hereditas, Suppl. 2, 382-392. Novacky, A,, and Wheeler, H. 1971. Stimulation of peroxidase isozymes by actinomycin D. A m . J . Bot. 58, 213-224. Ogren, W. L., and Bowes, G. 1970. Ribulose diphosphate carboxylase regulates soybean photorespiration. Nature (London), New Biol. 230, 159-160.

GENETIC CONTROL OF PROTEINS IN WHEAT

571

Olson, R. A., and Sander, D. H. 1975. The effective use of fertilizers in wheat production. Proc. Int. Winter Wheat Conf.2nd, 1976 pp. 256-271. Oram, R. N., Doll, H., and KGie, B. 1975. Genetics of two storage protein variants in barley. Hereditas 80, 53-58. Orr, M. L., and Watt, B. K. 1957. Amino acid content of foods. US.,Dep. Agric., Home Econ. Res. Rep. 4, 1-82. Orth, R. A., and Bushuk, W. 1972. A comparative study of the proteins of wheats of diverse baking qualities. Cereal Chem. 49, 268-275. Orth, R. A., and Bushuk, W. 1973a. Studies of glutenin. I. Comparison of preparative methods. Cereal Chem. 50, 106-114. Orth, R. A , and Bushuk, W. 1973b. Studies of glutenin. 11. Relation of variety, location of growth and baking quality of molecular weight distribution of subunits. Cereal Chem. 50, 191-197. Orth, R. A., and Bushuk, W. 1973c. Studies of Glutenin. 111. Identification of subunits coded by the D-genome and their relation to breadmaking quality. Cereal Chem. 50, 680-687. Orth, R. A., and Bushuk, W. 1974. Studies of glutenin. VI. Chromosomal location of genes coding for subunits of glutenin of common wheat. Cereal Chem. 51, 118-126. Orth, R. A., and Mander, K. C. 1975. Effect of milling yield on flour composition and bread making quality. Cereal Chem. 52, 305-314. Orth, R. A., Baker, R. J., and Bushuk, W. 1972. Statistical evaluation of techniques for predicting baking quality of wheat cultivars. Can. J. Plant Sci. 52, 139-146. Orth, R. A., Dronzek, B. L., and Bushuk, W. 1973. Studies of glutenin. IV. Microscopic structure and its relation to breadmaking quality. Cereal Chem. 50, 688-701. Orth, R. A., Dronzek, B. L., and Bushuk, W. 1974a. Studies of glutenin. VII. Inheritance of its physicochemical factors in Tri‘lieale. Cereal Chem. 51, 281-288. Orth, R. A., Dronzek, B. L., and Bushuk, W. 1974b. Scanning electron microscopy of bread wheat proteins fractionated by gel filtration. Cereal Chem. 51, 696-701. Ory, R. L., and Henningsen, K. W. 1969. Enzymes associated with protein bodies isolated from ungerminated barley seeds. Plant Physiol. 44, 1488-1498. Osborne, T. B. 1907. The proteins of the wheat kernel. Carnegie Inst. Washington Publ. 84, 1-119. Osborne, T. B. 1924. “The Vegetable Proteins,” 2nd ed. Longmans, Green, New York. Pace, J. L., Kemper, D. L., and Ragland, W. L. 1974. The relationship of molecular weight to electrophoretic mobility of ffuorescamine-labeled proteins in polyacrylamide gels. Bwchem. Biophys. Res. Commun. 57, 482-487. Paneque, A., Ramirez, J. M., DelCampo, F. F., and Losada, M. 1964. Light and dark reduction of nitrite in a reconstituted enzymic system. J. Biol. Chem. 239, 1737-1741. Pepe, J. F., and Heiner, R. E. 1975. Plant height, protein percentage, and yield relationships in spring wheat. Crop Sci. 15, 793-797. Petrucci, T., Tomasi, M., Cantagalli, P., and Silano, V. 1974. Comparison of wheat albumin inhibitors of alpha-amylase and trypsin. Phytochemistry 13, 2487-2495. Piano, E., Arcioni, S., and Lorenzetti, F. 1976. Natura dell’effetto materno sul contenuto proteico nel frumento tenero. Genet. Agr. 30, 163-176. Platt, S. G., and Kasarda, D. D. 1971. Separation and characterization of alphagliadin fractions. Biochim. Biophys. Acta 243, 407415. PIatt, S. G., Kasarda, D. D., and Qualset, C. 0. 1974. Varietal relationships of the alpha-gliadin proteins in wheat. J. Sci. Food Agric. 25, 1555-1561.

572

CALVIN F. KONZAK

Pollhamer, E. -n6 1967. The conlplex qualitative index of wheat (In Hungarian, English Summary). Acta Agron. Acad. Sci. Hung. 16, 339-344. Pollhamer, E. -n6 1969. Quality of winter wheats (In Hungarian, English Summary). Acta Agron. Acad. Sci. Hung. 18, 369-376. Polya, G. M., and Jagendorf, A. T. 1971a. Wheat leaf RNA polymerases. I. Partial purification and characterization of nuclear, chloroplast and soluble DNAdependent enzymes. Arch. Biochem. Biophys. 146, 635-648. Polya, G. M., and Jagendorf, A. T. 1971b. Wheat leaf RNA polymerases. 11. Kinetic characterization and template specificities of nuclear, chloroplast and soluble enzymes. Arch. Biochern. Biophys. 146, 649-657. Pomeranz, Y. 1971. Composition and functionality of wheat flour components. I n “Wheat-Chemistry and Technology” (Y. Pomeranz, ed.), 2nd ed., pp. 585-674. Am. Assoc. Cereal Chem., St. Paul, Minnesota. Pomeranz, Y. 1973a. Industrial utilization of cereals. Annotated bibliography 1948-1972. I n “Industrial Uses of Cereals” (Y. Pomeranz, ed.), pp. 418-478. Am. Assoc. Cereal Chem. St. Paul, Minnesota. Pomerans, Y. 1973b. Interaction between glycolipids and wheat flour macromolecules in breadmaking. Adv. Food Res. 20, 153-188. Pomeranz, Y. 1973c. From wheat to bread. Am. Sci. 61,683-691. Pomeranz, Y. 1976. Particle size of wheat in infra-red analysis. TJS. News 2 ( l ) , 4-6. Pomeranz, Y., and Finney, K. F. 1969. Glycolipids: Their distribution, chemistry, and role in breadmaking. Cereal Sci. Today 14, 172-176. Pomeranz, Y., and Finney, K. F. 1973a. Glycolipids: Key to protein-enriched bread. Food Eng. 45, 134-137. Pomeranz, Y., and Finney, K. F, 1973b. Protein-enriched bread, Process Biochem. 8, 16-18. Pomeranz, Y., and Finney, K. F. 1975. Sugars in breadmaking. Baker’s Dig. 49, 20-27. Pomeranz, Y., and MacMasters, M. M. 1968. Structure and composition of the wheat kernel. Baker’s Dig. 42, 24-26, 28-29, and 32. Pomeranz, Y., and Moore, R. B. 1975. Reliability of several methods for protein determination of wheat. Baker’s DG. 49, 44-58. Pomeranz, Y., and Robbins, G. S. 1976. Amino acid composition of isogenic lines in barley. J . Agric. Food Chem. 24, 196-198. Pomeranz, Y., Rubenthaler, G. L., and Finney, K. F. 1966a. Evaluation of the effects on proteolytic enzymes on bread flour properties. Food TechnoE. 20, 95-98. Pomeranz, Y., Rubenthaler, G. L., and Finney, K. F. 1966b. Studies on the mechanism of the bread improving effect of lipids. Food Technol. 20, 105-108. Pomeranz, Y., Finney, K. F., and Hoseney, R. C. 1966c. Amino acid composition of maturing wheat. J. Sci. Food Agric. 17, 485487. Pomeranz, Y., Finney, K. F., and Hoseney, R. C. 1970. Molecular approach t o breadmaking. Science 167, 944-949. Pomeranz, Y., Wesenberg, D. M., Robbins, G. S., and Gilbertson, J. T. 1974. Changes in amino (acid) composition of maturing Hiproly barley. Cereal Chem. 51, 635-640. Pomeranz, Y., Marshall, H. G., Robbins, G . S., and Gilbertson, J. T. 1975a. Protein content and amino acid composition of maturing buckwheat (Fagopyrum esculentum Moench). Cereal Chem. 52, 479-484.

GENETIC CONTROL OF PROTEINS IN WHEAT

573

Pomeranz, Y., Standridge, G. S., Robbins, G. S., and Goplin, E. D. 197513. Malting of new wheat cultivars. Cereal Chem. 52, 485-492. Porceddu, E., Pacucci, G., Perrino, P., Della Gatta, C., and Maellaro, I. 1975. Protein content and seed characteristics in populations of 2”. durum grown a t three different locations. In “Genetics and Breeding of Durum Wheat” (G. T. Scarascia-Mugnozza, ed.), pp. 217-224. Library of the Faculty of Agriculture, Bari, and National Institute for Nutrition, Rome. Pratt, D. B., Jr. 1971. Criteria of flour quality. I n “Wheat-Chemistry and Technology” (Y. Pomeranz, ed.), 2nd ed., pp. 201-226. Am. Assoc. Cereal Chem., St. Paul, Minnesota. Preston, K. R., and Woodbury, W. 1975. Amino acid composition and subunit structure of rye gliadin proteins fractionatcd by gel filtration. Cereal Chem. 52, 719-726. Preston, K . R., and Woodbury, W. 1976. Properties of wheat gliadins separated by gel filtration. Cereal Chem. 53, 181-190. Preston, K. R., Woodbury, W., and Bendelow, V. 1975. The effects of gliadin fractions of varying molecular weight on the mixing properties of a synthetic dough system. Cereal Chem. 52, 427430. Protein-Caloric Advisory Group (PAG) of the United Nations System. 1976. Protein methods for cereal breeders as related to human nutritional requirements. (PAC Guideline No. 16-Cereal Breeders’ Protein Methods.) I n “Advances in Cereal Science and Technology” (Y. Pomeranz, ed.), pp. 378-408. Am. Assoc. Cereal Chem., St. Paul, Minnesota. Puls, W., and Keup, U. 1973. Influence of an alpha-amylase inhibitor (BAYd7791) on blood glucose, serum insulin, and NEFA in starch loading tests in rats, dogs, and man. Diabetologia 9, 97-101. Purvis, A. C., Peters, D. B., and Hageman, R. H. 1974. Effect of carbon dioxide on nitrate accumulation and nitrate reductase induction in corn seedlings. Plant Physiol. 53, 93&941. Redman, D. G., and Ewart, J. A. D. 1971. Measurements on disulphide bonds in cereal glutelins. J . Sci. Food Agric. 22, 19-21. Redman, D. G., and Ewart, J. A. D. 1973. Characterization of three wheat proteins found in chloroform-methanol extracts of flour. J . Sci. Food Agric. 24, 6294536. Redman, D. G., and Fisher, N. 1968. Fractionation and comparison of purothionin and glohulin components of wheat. J . Sci. Food Agric. 19, 651-655. Redman, D. G., and Fisher, N. 1969. Purothionin analogues from barley flour. J . Sci. Food Agric. 20, 427-432, Reitz, L. P. 1964. Wheat quality components responsive to genic control. &uaL Plant. Muter. Veg. 11, 1-16. Rhodcs, A. P., and Jenkins, G. 1975. The effect of varying nitrogen supply on the protein composition of a high lysine mutant of barley. J. Sci. Food Agtic. 26, 705-709.

Rhodes, A. P., and Mathers, J. C. 1974. Varietal differences in thc amino acid composition of barley grain during development under varying nitrogen supply. J . Sci. Food Agric. 25, 963-972. Rijven, A. H. G. C., and Banbury, C. A. 1960. Role of the grain coat in wheat grain development. N a l w e (London) 188, 546-547. Riley, R., and Ewart, J. A. D. 1970. The effect of individual rye chromosomes on the amino acid content of wheat grains. Genet. Res. 15, 209-220.

574

CALVIN F. KONZAR

Riley, R., Kimber, G., and Chapman, V. 1961. The origin of genetic control of diploid-like behavior of polyploid wheat. J . Hered. 52, 22-25. Robbins, G. S., Pomeranz, Y., and Briggle, L. W. 1971. Amino acid composition of oat groats. J . Agric. Food Chem. 19, 53e539. Robertson, D. W. 1971. Recent information of linkage and chromosome mapping. Barley Genetics 2, Proc. Int. Symp., 2nd, 1969 pp. 220-242. Rodriguez-Loperena, M. A., Aragoncillo, C., Carbonero, P., and Garcia-Olmedo, F. 1975. Heterogeneity of wheat endosperm proteolipids (CM proteins). Phytochemistry 14, 1219-1223. Rodriguez-Loperena, M. A., Aragoncillo, C., Torres, J. V., Carbonero, P., and Garcia-Olmedo, F. 1976. Biochemical evidence of chromosome homoeology among related plant genera. Plant Sci. Lett. (in press). Rothfus, J. A., and Crow, M. J. A, 1968. Aminoethylation and fractionation of glutenin-evidence of differences from gliadin. Biochim. Biophys. Acta 160, 404412.

Rothfus, J. A., and Kennel, S. J. 1970. Properties of wheat beta-amylase adsorbed on glutenin. Cereal Ch8m. 47, 140-146. Roy, H., and Jagendorf, A. T. 1974. In vitro synthesis of polypeptides related to the small subunit of ribulose-1,5-diphosphate carboxylase (RuDPCase) . Plant Physiol. 53 (Suppl.), p. 8 (Abstr. No. 37). Ruckman, J. E., Zscheile, F. P., Jr., and Qualset, C. 0. 1973. Protein, lysine and grain yields of triticale and wheat as influenced by genotype and location. Agric. Food Chem. 21, 697-700. Ryan, C. A. 1973. Proteolytic enzymes and their inhibitors in plants. Annu. Rev. Plant. Physiol. 24, 173-193. Saffhill, R. 1974. The incorporation of wrong bases by DNA polymerme I following uv irradiation of DNA-like templates. Biochim. Biophys. Acta 349, 23-31. Sakano, K., Kung, S. D., and Wildman, S. G. 1974a. Change in solubility of crystalline Fraction I proteins correlated with change in the composition of the small subunit. Plant Cell Physiol. 15, 611-617. Sakano, K., Kung, S. D., and Wildman, S. G. 197413. Identification of several chloroplast DNA genes which code for the large subunit of Nicotkna Fraction I proteins. Mol. Gen. Genet. 130, 91-97. Sandstedt, R. M. 1946. Photomicrographic studies of wheat starch I. Development of the starch granules. Cereal Chem. 23, 337-359. Sandstedt, R. M., and Beckord, 0. C. 1946. Photomicrographic studies of wheat starch. 11 Amylolytic enzymes and the amylase inhibitor of the developing wheat kernel. Cereal Chem. 23, 548-559. Sarkar, P., and Stebbins, G. L. 1956. Morphological evidence concerning the origin of the B genome in wheat. Am. J . Bot. 43, 297-304. Sasaki, M., Ogawa, S., Nishiyama, K., and Ito, T. 1973. Chromosomal analysis of biochemical properties of wheat flour in relation to its baking quality. Proc. Int. Congr. Genet., ISth, 19YS Genetics 74, Suppl. 2, Part 2, s239-s240. Saunders, R. M., and Lang, J. M. 1973. Alpha-amylase inhibitors in Triticum aestivum: Purification and physical-chemical properties. Phytochemistry 12, 1237-124 1.

Saunders, R. M., and Walker, H. G., Jr. 1969. The sugars of wheat bran. Cereal Chem. 46, 85-92. Saunders, R. M., Conner, M. A., Edwards, R. H., and Kohler, G. 0. 1975a.

GENETIC CONTROL OF PROTEINS IN WHEAT

575

Preparation of protein concentrates from wheat shorts and wheat millrun by a wet alkaline process. Cereal Chem. 52, 93400. Saunders, R. M., Betschart, A. A., and Lorenz, K. 197513. The sugars of triticale bran. Cereal Chem. 52, 472-478. Scandelios, J. 1969. Genetic control of multiple molecular forms of enzymes in plants. A review. Biochem. Genet. 3, 37-79. Scandelios, J. G. 1974. Isozymes in development and differentiation. Annu. R e v . Plant Physiol. 25, 225-258. Schaap, T. 1971. Dual information in DNA and the evolution of the genetic code. J. Theor. Biol. 32, 293-298. Schlesinger, J. 1964. Results of ball milling Buhler experimentally milled hard winter wheat flour. Cereal Chem. 41, 465474. Schmidt, J. W., Morris, R., Mattern, P. J., and Johnson, V. A. 1966. Comparison of the chromosome substitution and monosomic methods for wheat quality studies. Crop Sci. 6, 370-372. Schmidt, J. W., Mattern, P. J., Johnson, V. A., and Morris, R. L. 1974. Investigations of the genetics of bread wheat baking quality. In “Genetics Lectures 111” (R. Bogart, ed.), pp. 83-101. Genetics Institute, Oregon State Univ. Press, Corvallis. Schrader, L. E., Cataldo, D. A., and Peterson, D. M. 1974. Use of protein in extraction and stabilization of nitrate reductase. Plant Physiol. 53, 688-690. Sears, E. R. 1953. Nullisomic analysis in common wheat. A m . Nut. 87, 245-252. Sears, E. R. 1954. The aneuploids of common wheat. Mo., Agric. E z p . Stn., Res. Bull. 572, 1-58. Sears, E. R. 1959a. The aneuploids of common wheat. Proc. Int. Wheat Genet. Symp., l s t , 1958 pp. 221-229. Sears, E. R. 1959b. The systematics, cytology, and genetics of wheat. In “Handbuch fur Pflanzenzuchtung” (H. Kappert and W. Rudorf, eds.), Vol. 2, pp. 164-187. Parey, Berlin. Sears, E. R. 1966. Chromosome mapping with the aid of telocentrics. Proc. Int. Wheat Genet. Symp., 2nd, 1968 Hereditas, Suppl. 2, 370-381. Sears, E. R. 1969. Wheat cytogenetics. Annu. R e v . Genet. 3, 451468. Sears, E. R. 1972. The nature of mutation in hexaploid wheat. Symp. Biol. Hung. 12, 73-82. Sears, E. R. 1974. Telocentric chromosomes in wheat and their uses. Genetics 77, s59. Sears, E. R. 1975a. The wheats and their relatives. Handb. Genet. 2,59-91. Sears, E. R. 197513. An induced homoeologous-pairing mutant in Triticum aestivum. Genetics 80, s74. Seckinger, H. L., and Wolf, M. J. 1970. Electron microscopy of endosperm proteins from hard and soft wheats. Cereal Chem. 47, 236-243. Seckinger, H. L,, and Wolf, M. J. 1973. Sorghum protein ultrastructure as i t relates to composition. Cereal Chem. 50, 455-465. Sexson, K. R., and Wu, Y. V. 1972. Molecular weights of wheat gamma-1- and gamma-3-gliadins in various solvents. Biochim. Biophys. Acta 263, 651-657. Shainkin, R., and Birk, Y. 1970. Alpha-amylase inhibitors from wheat: Isolation and characterization. Biochim. Biophys. Acta 221, 502513. Shands, H. L., and Kimber, G. 1973. Reallocation of the genomes of Triticum timopheevii Zhuk. Proc. Int. Wheat Genet. Symp., &h, 1.973 pp. 101-108. Sharma, T. R., Gupta, V. P., Gasei, S., and Kaul, A. K. 1973. Estimation of genetic

576

CALVIN F. KONZAK

parameters of some quality characters in wheat (Triticum aestivum L.) and and Bengal gram (Cicer arietinum L.). In “Nuclear Techniques for Seed Protein Improvement” (B. Kaufmann and M. Lewis, eds.), STI/PUB/320, pp. 273-289. IAEA, Vienna. Shealy, H. E., and Simmonds, D. H. 1973. The early developmental morphology of the triticale grain. Proc. Int. Wheat Genet. Symp., 4th, 1973 pp. 265-270. Shepherd, K. W. 1968. Chromosomal control of endosperm proteins in wheat and rye. Proc. Int. Wheat Genet. Symp., 3rd, 1968 pp. 86-96. Shepherd, K. W. 1971. Genetic control of albumins and globulins (fast moving proteins). Aust. Wheat. Cytogenet. Newsl. No. 4, p. 9 (by permission). Shepherd, K. W. 1973. Homeology of wheat and alien chromosomes controlling endosperm protein phenotypes. Proc. Int. Wheat Genet. Symp., 4th, 1973 pp. 745-760. Shogren, M. D., Finney, K. F., and Hoseney, R. C. 1969. Functional (breadmaking) and biochemical properties of wheat flour components. I. Solubilising gluten and flour protein. Cereal Chem. 46, 93-102. Siddiqui, K. A. 1972. Protein content and quality of wheat chromosome substitution lines. Hereditas 71, 157-160. Siddiqui, K. A., and Doll, H. 1973. Screening for improved protein quality mutants in wheat. Z . Pflanzenziicht. 70, 143-147. Silano, V., and Pocchiari, F. 1971. A specific color reaction of albumin and gliadin preparations from wheat. Cereal Chem. 48,445-447. Silano, V., de Cillis, W., and Pocchiari, F. 1969. Varietal differences in albumin and globulin fractions of Triticum aestivum and T . durum. J . Sci. Food A&. 20, 260-261. Silano, V., Pocchiari, F., and Kasarda, D. D. 1973. Physical characterization of alpha-amylase inhibitors from wheat. Bwchim. Biophys. Acta 317, 139-148. Silano, V., Furia, M., Gianfreda, L., Macri, A., Palescandolo, R., Rab, A., Scardi, V., Stella, E., and Valfre, F. 1975. Inhibition of amylases from different origins by albumins from the wheat kernel. Biochim. Biophys. Acta 391, 170-178. Silva, A. R., da 1966. “Melhoramento das Variedades de Trigo Destinadas as Diferented Regioes do Brasil.” Servico de Informacao Agricola Ministerio da Agricultura Rio de Janeiro, GB Brasil (In Portuguese). Estudos Technicos 33, 1-82. Simmonds, D. H. 1962. Variations in the amino acid composition of Australian wheats and flours. Cereal Chem. 39, 445-455. Simmonds, D. H. 1972a. The ultrastructure of the mature wheat endosperm. Cereal Chem. 49, 212-222. Simmonds, D. H. 197213. Wheat grain morphology and its relationship to dough structure. Cereal Chem. 49, 324-335. Simmonds, D. H. 1974. The structure of the developing and mature triticale kernel. In “Triticale: First Man-Made Cereal” (C. C. Tsen, ed.), pp. 105-121. Am. Assoc. Cereal Chem., St. Paul, Minnesota. Simmonds, D. H., and Orth, R. A. 1973. Structure and composition of cereal proteins as related to their potential industrial utilization. In “Industrial Uses of Cereals” (Y. Pomeranz, ed.), pp. 51-120. Am. Aesoc. Cereal Chem., St. Paul, Minnesota. Simmonds, D. H., and Wngley, C. W. 1972. The effect of lipid on the solubility and molecular-weight range of wheat gluten and storage protein. Cereal Chem. 49, 317423.

GENETIC CONTROL O F PROTEINS IN WHEAT

577

Simmonds, D. H., Barlow, K. K,, and Wrigley, C. W. 1973. The biochemical basis of grain hardness in wheat. Cereal Chem. 50, 553-562. Singer, M. 1952. Factors which control the staining of tissue sections with acid and basic dyes. Znt. R e v . Cytol. 1, 211-255. Singh, M. M., and Widholm, J. M. 1974. Measurement of five enzymes which convert chorisimate to tryptophan in wheat plants (Triticum aestivum cv. Kalyansona). Plant Physiol. 32, 240-246. Singh, M. M., and Kay, S. R. 1976. Wheat gluten as a pathogenic factor in Schizophrenia. Scknce 191, 401-402. Singh, R., and Axtell, U. 1973. High lysine mutant gene (hl) that improves protein quality and biological value of grain sorghum. Crop Sci. 13, 535539. Singh, S., and Wildman, S. G. 1973. Chloroplast DNA codes for the rihulose diphosphate carhoxylase catalytic site on Fraction I protein of Nicotiana species. Mol. Gen. Genet. 124, 187-196. Sodini, G., Silano, V., de Agazio, M., Pocchiari, F., Tentori, L., and Vivaldi, G. 1970. Purification and properties of a Triticum aestivum specific albumin. Phytochemitry 9, 1167-1172. Solari, R. M., and Favret, E. A. 1968. Genetic control of protein constitution in wheat endosperm and its implication on induced mutagenesis. Mutat. Plant Breed., Proc. Panel, dnd, i967 STI/PUB/182, pp. 219-231. Solari, R. M., and Favret, E. A. 1970. Chromosome localization of genes for protein synthesis in wheat endosperm. Bol. Genet. Inst. Fitotec. Castelar No. 7, 23-26. Sollars, W. F. 1972. Relation of distilled water retention to alkaline water retention, water absorption, and baking properties of wheat flours. Cereal Chem. 49, 168-172. Sollars, W. F., and Rubenthaler, G. L. 1975. Flour fractions affecting farinograph absorption. Cereal Chem. 52, 420-427. Stehhins, G. L., Jr. 1947. Types of polyploids: Their classification and significance. Adv. Genet. 1, 403-428. Stebbins, G. L., Jr. 1951. Cataclysmic evolution. Sci. Am. 184, 54-59. Steggerda, F. R. 1968. Gastrointestinal gas following food consumption. Ann. N . Y . Acad. Sci. 150, 57-66. Stein, S., Chang, C. H., Bohlen, P., Imai, K., and Udenfriend, S. 1974. Amino acid analysis with fluorescamine of stained protein bands from polyacrylamidc gels. Anal. Biochem. 60, 27Z277. Stenvert, N. L., and Kingswood, K. 1976. An autoradiographic demonstration of the penetration of water into wheat during tempering. Cereal Chem. 53, 141-149. Stenvert, N. L., and Moss, R. 1974. The separation and technological significance of the outer layers of the wheat grain. J. Sci. Food Agric. 25, 62W35. Stevens, D. J. 1970. Free sugars of wheat aleurone cells. J. Sci. Food Agric. 21, 31-34. Stevens, D. J. 1973. Reaction of wheat proteins with sulphite. 111. Measurement of labile and reactive disulphide honds in gliadin and in the protein of aleurone cells. J. Sci. Food Agric. 24, 279-283. Strbac, V. D., Ayers, G. S., and Ries, S. K. 1974. The protein fractions in chemically induced high protein wheat seed. Cereal Chem. 51, 316-323. Stroike, J. E., and Johnson, V. A. 1972. Winter wheat cultivar performance in an

578

CALVIN F. RONZAK

international array of environments, Nebr., Agric. Exp. Stn., Res. Bull. 251, 148. Stuart, L. S., and Harris, T. H. 1942. Bactericidal and fungicidal properties of a crystalline protein isolated from unbleached wheat flour. Cereal Chem. 19, 288-300. Stuber, C. W.,Johnson, V. A., and Schmidt, J. W. 1962. Grain protein content and the relationship to other plant and seed characters in the parents and progeny of a cross of Triticum aestivum L. Crop Sci. 2, 506-508. Suemoto, H. 1968. The origin of the cytoplasm of the tetraploid wheats. Proc. Int. Wheat Genet. Symp., Srd, 1968 pp. 141-152. Suemoto, H. 1973. The origin of the cytoplasm of tetraploid wheats. Proc. Int. Wheat Genet. Symp., dth, 1979 pp. 109-113. Sunderman, D. W., Wise, M., and Sneed, E. M. 1965. Interrelationships of wheat protein content, flour sedimentation value, farinograph peak time, and dough mixing and baking characteristics in the F, and F, generations of winter wheat, Triticum aestivum L. Crop Sci. 5, 537-540. Symes, K. J. 1969. Influence of a gene causing hardness on the milling and baking quality of two wheats. Aust. J . Agric. Res. 20, 971-979. Tanaka, K., and Bushuk, W. 1972. Effect of protein content and wheat variety on solubility and electrophoretic properties of flour proteins. Cereal Chem. 49, 247-257. Tanaka, K., and Bushuk, W.1973a. Changes in flour proteins during dough mixing. I. Solubility results. CereaE Chem. 50, 590-596. Tanaka, K., and Bushuk, W. 1973b. Changes in flour proteins during dough mixing. 11. Gel filtration and electrophoresis results. Cereal Chem. 50, 597-605. Tanaka, K., and Bushuk, W.1973c. Changes in flour proteins during dough mixing. 111. Analytical results and mechanisms. Cereal Chem. 50, 605-612. Tanaka, M.,and Ishii, H. 1973. Cytogenetical evidence on the speciation of wild tetraploid wheats collected in Iraq, Turkey and Iran. Proc. Int. Wheat Genet. Symp., 4th, 1979 pp. 115-121. Taneja, S. R., Abrol, Y. P., and Sachar, R. C. 1974. Modulation of o-diphenolase and monophenolase enzymes during wheat grain development. Cereal Chem. 51, 457-465. Tang, K. S., and Hart, G. E. 1975. Use of isozymes as chromosome markers in wheat-rye addition lines and triticales. Genet. Res. 26, 187-201. Thomas, J. R., and Tewari, K. K. 1974. Ribosomal RNA genes in the chloroplast DNA of pea leaves. Biochim. Bwphys. Acta 361, 73-83. Tkachuk, R. 1975. Competitive affinity chromatography of wheat a-amylase. FEBS Lett. 52, 66-68. Tkachuk, R., and Kruger, J. E. 1974. Wheat a-amylases 11. Physical Characterization. Cereal Chem. 51, 508-529. Toepfer, E. W., Polansky, M. N., Eheart, J. F., Slover, H. T., Morris, E. R., Hepburn, F. N., and Quackenbush, F. W. 1972. Nutrient composition of selected wheats and wheat products. XI. Summary. Cereal Chem. 49, 173-186. Torres, J. V., and Garcia-Olmedo, F. 1974. Chromosomal location of a gene that controls sterol esterification in Triticum aestivum L. Plant Sci. Lett. 3, 213-217. Torres, M. A., and Hart, G. E. 1976. Developmental specificity and evolution of

GENETIC CONTROL OF PROTEINS IN WHEAT

579

the acid phosphatase isozymes of Triticum aestivum and its progenitor species. Biochem. Genet. 14, 595-609. Tronier, B., and Ory, R. L. (1970). Association of bound beta-amylase with protein bodies in barley. Cereal Chem. 47, 464-471. Tsen, C. C. 1967. Changes in flour proteins during dough mixing. Cereal Chem. 44, 308-317. Tsunewaki, K., and Endo, T. 1973. Genetic relatedness among five cytoplasms in Triticum and Aegilops. Proc. Int. Wheat Genet. Symp., 4th, 1973 pp. 391-397. Tsunewaki, K., and Heyne, E. G. 1959. Radiological study of wheat monosomics. 11. Differential sensitivity of 16 monosomics and the disomic to a single dosage of X-irradiation. Genetics 44, 947-954. Turner, D. 1965. “Handbook of Diet Therapy,” 4th ed., Vol. XV. Univ. of Chicago Press, Chicago, Illinois. Tyuterev, S. L., Chmeleva, A. V., Moisa, I. I., and Dorofeev, V. F. 1973. Studies on species of wheat and its wild relatives for protein and uninterchangable amino acids content in relation to specific diversity and genome composition (In Russian). Bull. Appl. Bot., Genet. Plant Breed. 52, 222-241. Udy, D. C. 1971. Improved dye method for estimating protein. J . Am. Oil Chem. SOC.48, 29A33A. Ulmer, R. L. 1973. Relationships of endosperm prptein fractions from normal and high protein wheats to protein content, lysine content, quality, and nitrogen fertilization. Ph.D. Thesis, Nebraska Univ., Lincoln, Nebraska. p. 126. Unrau, J. 1958. Genetic analysis of wheat chromosomes. I. Description of proposed methods. Can. J . Plant Sci. 38,415-418. Upadhya, M. D. 1968. Peroxidase isoenzymes associated with the Aegilops umbellulata chromosome segment transferred to Chinese Spring (Triticum aestivum) . Experientia 24, 613. Upcroft, J. A., and Done, J. 1974. Starch gel electrophoresis of plant NADH-nitrate reductase and nitrite reductase. J . Exp. Bot. 25, 503-508. Vardi, A. 1973. Introgression between different ploidy levels in the wheat group. Proc. Int. Wheat Genet. Symp., 4th, 1978 pp. 131-141. Viegas, W. S., and MelloSampayo, T. 1975. NucleoIar organization in the genus Triticum. Broteria, Ser. Cienc. Nat. 44, 3-4. Villegas, E., McDonald, C. E., and Gilles, K. A. 1968. “Variability in the Lysine Content of Wheat, Rye, and Triticaie Proteins.” Res. Bull. No. 10. Int. Maize and Wheat Improvement Center (CIMMYT), Mexico. Vogel, K. P., Johnson, V. A. and Mattern, P. J. 1973. Results of systematic analyses for protein and lysine composition of common wheats (Triticum aestivum L.) in the USDA World Collection. Nebr., Agric. Exp. Stn. Res. Bull. 258, 27. Vogel, K. P., Mattern, P. J., and Lenser, G. W. 1976. Modified milling procedure for separating endosperm and nonendosperm portions of the wheat kernel for protein and lysine analysis. Agron. J . 68, 686-690. Wade, P., m d Elton, G. A. H. 1967. Flour properties relating to the manufacture of sweet biscuits. Cereal Sci. Today 12, 8-9. Waines, J. G. 1973. Chromosomal location of genes controlling endosperm protein production in Triticum aestivum cv. Chinese Spring. Proc. Int. Wheat Genet. Symp., 4th, 1978 pp. 873-877. Waines, J. G., and Johnson, B. L. 1971. New protein bands in an amphiploid of Aegilops caudata and Ae. umbellulata. Wheat Inf. Serv. 32, 22-24 (by permission).

580

CALVIN F. KONZAK

Wall, J. S., Bietz, J. A., Huebner, F. R., Anderson. R. A., and Stringfellow, A. C. 1971. Effect of variety and processing on wheat and triticale proteins. Proc. Natl. Conj. Wheat Util. Res., 7th, 1971 USDA-ARS nc-1, pp. 119-135 (by permission). Wallace, W. 1974. Purification and properties of a nitrate reductase inactivating enzyme. Biochim. Biophys. Acta 341, 265-276. Warner, R. L., and Kleinhofs, A. 1974. Relationship of reductase, nitrite reductase, and ribulose diphosphate carboxylase activities in chlorophyll-deficient mutants of barley. Crop Sci. 14, 654458. Warner, R. L., and Konzak, C. F. 1975. Nitrate reductase activity in wheat substitution lines. Wheat N e d . 21, 160-161. Wasik, R. J., and Bushuk, W. 1974. Studies of glutenin. V. Note on additional preparative methods. Cereal Chem. 51, 112-118. Wasik, R. J., and Bushuk, W. 1975a. Relation between molecular weight distribution of endosperm proteins and spaghetti-making quality of wheats. Cereal Chem. 52, 322-328. Wasik, R. J., and Bushuk, W. 1975b. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reduced glutenin of durum wheats of different spaghetti-making quality. Cereal Chem. 52,328-334. Watson, C. A., and Welsh, J. R. 1966. Monosomics. Cereal Sci. Today 11, 286-290. Watson, C. A., Carville, D., Dikeman, E., Daigger, G., and Booth, G. D. 1976. Evaluation of two infrared instruments for determining protein content of hard red winter wheat. Cereal Chem. 53, 214-222. Weber, K., and Osbbrn, M. 1969. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J . Biol. Chem. 244, 4 4 0 6 4412. Wehrli, H. P., and Pomeranz, Y. 1970. A note on the interaction between glycolipids and wheat flour macromolecules. Cereal Chem. 47, 160-166. Wells, G. N., and Hageman, R. H. 1974. Specificity for nicotinamide adenine dinucleotide by nitrate reductase from leaves. Plant Physiol. 54, 136-141. Welsh, J. R., and Hehn, E. R. 1964. The effect of chromosome 1D on hexaploid wheat flour quality. Crop Sci. 4 , 320-323. Welsh, J. R., and Watson, C. A. 1965. Flour quality inheritance studies. Wheat Newts. 11, 66 (by permission). Welsh, J. R., Watson, C. A., and Green, C. W. 1968. Chromosomal control of flour properties in three substitution sets of common wheat (Triticum aestivum L.) . Crop Sci. 8, 81232. Weswig, P. H., Foote, W. H., and Drusts, G. R. 1962. The acid-extracted pentosan content of wheat as a measure of milling quality of Pacific Northwest wheats. Cereal Chem. 40, 169-174. Wettstein, D. von, Henningsen, K. W., Boynton, J. E., Kannangara, G. C. and Nielsen. 0. F. 1971. The genic control of chloroplast development in barley. In “Autonomy and Biogenesis of Mitochondria and Chloroplasts” (N. K . Boardman, A. W. Linnane, and R. M. Smillie, eds.), pp. 205-223. North-Holland Publ., Amsterdam. Wettstein, D. von, Kahn, A., Nielsen, 0. F., and Gough, S. 1974. Genetic regulation of chlorophyll synthesis analyzed with mutants in barley. Science 184, 800-802. Whitfeld, P. R., Spencer, D., and Bottomley, W. 1973. Products of chloroplast DNAdirected transcription and translation. Biochem. Gene Expression Higher Org., Proc. Symp., lgr.8 pp. 504-522. Wildman, S. G., Kawashima, N., Bourque, D. P., Wong, F., Singh, S., Chan, P. H.,

GENETIC CONTROL OF PROTEINS IN WHEAT

58 1

Kwok, S. Y., Sakano, K., Kung, S. D., and Thornber, J. P. 1973. Location of DNA’s coding for various kinds of chloroplast proteins. Biochem. Gene Expression Higher Org., Proc. Sump., 1972 pp. 310-329. Wildman, S. G., Jope, C., and Atchison, B. A. 1974. Role of mitochondria in the origin of chloroplast starch grains. Plant Physiol. 54, 231-237. Wildman, S. G., Chen, K., Gray, J. C., Kung, S. D., Kwanyuen, P., and Sakano, K. 1975. Evolution of Ferredoxin and Fraction I protein in the genus Nicotiana. In “Genetics and Biogenesis of Mitochondria and Chloroplasts” (C. W. Birky, Jr., P. S. Perlman, and T. J. Byers, eds.), pp. 310-329. Ohio State Univ. Press, Columbus. Williams, P. C. 1975. Application of near infrared reflectance spectroscopy to analysis of cereal grains and oilseeds. Cereal Chem. 52, 561-576. Williams, P. C., and McEwin, N . M. 1967. A note on the influence of kernel hardn e s on the extractability of wheat proteins. J . Sci. Food Agric. 18, 184-186. Wiseblatt, L., and Zoumut, H. F. 1963. Isolation, origin and synthesis of a bread flavor constituent. Cereal Chem. 40, 162-169. Wong-Staal, F., and Wildman, S. G. 1973. Identification of a mutation in chloroplast DNA correlated with formation of defective chloroplasts in a variegated mutant of Nicotinna tabacum. Planta 113, 313-326. Woodbury, W. 1972. Biochemical genetics and its potential for cereal improvement. Brew. Dig. 47, 70-81. Woodbury, W., Spencer, A. K., and Stahmann, M. A. 1971. An improved procedure using ferricyanide for detecting catalase isozymcs. Anal. Biochem. 44, 301-305. Wrigley, C. W. 1968a. Gel electrofocusing-a technique for analysing multiple protein samples by isoelectric focusing. Sci. Tools 15, 17-23. Wrigley, C. W. 1968b. Analytical fractionation of plant and animal proteins by gel electrofocusing. J . Chromatog. 36, 362. Wrigley, C. W. 1969. Analytical fractionation of proteins according to isoclectric point by gel electrofocusing. Shandon Instrum. Appl. No. 29, p. 1. Wrigley, C. W. 1970a. Protein mapping by combined gel electrofocusing and electrophoresis : Application to the study of genotypic variations in wheat gliadins. Biochem. Genet. 4, 509-516. Wrigley, C. W. 1970b. Sample application for protein fractionation by gel electrofocusing. Proc. Protides Biot. Fluids, 17 Cofloq.,1.969, pp. 417-421. Wrigley, C. W. 1971a. Electrofocusing of proteins. I n “New Techniques in Amino Acid, Peptide and Protein Analysis” (A. Niederwicser and G. Patctki, eds.), pp. 231-339. Ann Arbor Sci. Publ., Ann Arbor, Michigan. Wrigley, C. W. 1971b. Gel electrofocusing. In “Methods in Enzymology” (W. B. Jakoby, ed.), Vol. 22, pp. 559-564. Academic Press, New York. Wrigley, C. W. 1972a. The biochemistry of the wheat protein complex and its genetic control. Cereal Sci. Todag 17, 37CL-375. Wrigley, C. W. 1972b. Protein fractionation according to isoelectric point by gel . 10. elcctrofocusing. A laboratory Experiment. Biochem. E ~ w 1, Wrigley, C. W. 1976a. Isoelectric focusing/electrophoresis in gels. In “Isoelectric Focusing” ( N . Catsimpoolas, ed.), Chapter 8. Academic Press, New York (in press). Wrigley, C. W. 1976b. Isoelcctric focusing of seed proteins. I n “Biological and Biomedical Applications of Isoelectric Focusing” (N. Catsimpoolas and J. W. Drysdale, eds.), Plenum, Yew York (in preparation). Wrigley, C. W., and McIntosh, R. A. 1975. Genetic control of factors regulating the

582

CALVIN F. KONZAK

phenol reaction of wheat and rye grain, Wheat I n f . S e w . No. 40, 6-10 (by permission). Wrigley, C. W., and Moss, H. J. 1968. Selection for grain quality in wheat breeding. Proc. h t . Wheat Genet. Symp., Srd, 196‘s pp. 439-448. Wrigley, C. W., and Shepherd, K. W. 1973. Electrofocusing of grain proteins from wheat genotypes. Ann. N . Y . Acad. Sci. 209, 154-162. Wrigley, C. W., and Shepherd, K. W. 1974. Identification of Australian wheat cultivars by laboratory procedures: Examination of pure samples of grain. Aust. J . Exp. Agric. 14, 796-810. Wu, Y. V., and Cluskey, J. E. 1965. Optical rotatory dispersion studies on wheat gluten proteins: Gluten, glutenin, and gliadin in urea and hydrochloric acid solutions. Arch. Biochem. Biophys. 112, 32-36. Wu, Y. V., Cluskey, J. E., and Sexson, R. 1967. Effect of ionic strength on the molecular weight and conformation of wheat gluten proteins in 3M-urea sohtions. Biochim. Biophys. Acta 133, 83-90. Yamashita, K. 1953. The quality of einkorn wheat. Seiken Ziho 6, 32-35. Yamaeaki, W. T., and Lord, D. D. 1971. Soft wheat products. In “Wheat-Chemistry and Technology” (Y. Pomerana, ed.), 2nd ed., pp. 743-776. Am. Assoc. Cereal Chem., St. Paul, Minnesota. Yampolsky, C. 1957. Wheat. Wnllerstein Lab. Cornmun. 20, 343-358. Yanofsky, C., Carlton, B. C., Guest, J. R., Helinski, D. R., and Henning, U. 1964a. On the colinearity of gene structure and protein structure. Proc. Natl. Acnd. Sci. U S . A . 51, 266272. Yanofsky, C., Horn, V., and Thorpe, D. 1964b. Protein structure relationships revealed by mutational analysis. Science 146, 1593-1594. Yanofsky, C., Ito, J., and Horn, V. 1966. Amino acid replacements and the genetic code. Cold Spring Harbor Symp. Quant. Biol. 31, 151-162. Zak, D. L., and Keeney, P. G. 1974. Disc gel electrophoresis. A technique involving fluorescent staining prior to separation. Agric. Food Chem. 22, 800-802. Zee, S. Y. 1975. Scanning electron microscope observations of the pigment strand in the caryopsis of wheat. Aust. J. Bot 23,107-110. Zce, S. Y., and O’Brien, T. P. 1970a. A special type of trachery element, associated with “xylem discontinuity” in the floral axis of wheat. Aust. J . Biol. Sci. 23, 783-791. Zee, S. Y., and O’Brien, T. P. 1970b. Studies on the ontogeny of the pigment strand in the caryopsis of wheat. Aust. J. Biol. Sci. 23, 1153-1174. Zeven, A. C. 1972. Identification of chromosomes carrying a locus for a gene conditioning the production of tyrosinase in wheat grains. Wheat Inf. S e w . 35, 3-8.

Ziegler, E., and Greer, E. N. 1971. Principles of milling. I n “Wheat-Chemistry and Technology” (Y. Pomeranz, ed.), 2nd ed., pp. 115-200. Am. Assoc. Cereal Chem., St. Paul, Minnesota. Zohary, D. 1966. The evolution of genomes in Aegilops and Triticum. Proc. Int. Wheat Genet. Symp., Ind, 1965 Hereditas, Suppl. 2, 207-217. Zohary, D. 1973. The origin of cultivated cereals and pulses in the Near East. Chromosomes Today 4, 307-320.

SUBJECT INDEX A

organelle DNAs and structure,

Aspergillus aneuploids from controls, and from cromes heterozygous for reciprocal translocations, 43-50 crossing-over in the centromere area of group I and genetic differences between strains, 67-74 genetic mapping of mutants and effects of translocations, 101-107 induced mitotic recombination in standard diploids, 81-84 interchromosomal effects of translocations in meiosis, 56-58 mapping of centromeres and use of translocations for sequencing meiotic fragments, 116-124 meiotic crossing over in standard crosses, 35-40 meiotic nondkjmction frequencies in crosses heterozygous for reciprocal translocations, 50-5fj mitotic crossing-over and nondisjunction in normal diploids, 58-67 mitotic crossing-over in disomics from translocation crosses, especially in “stable” disomics, 97-100 mitotic recombination in translocation heterozygotes, 85-97 mitotic recombination in triploids, 74-80 reduction of crossing-over by chromosomal aberrations, especially reciprocal translocations, 4 M 3 translocations, frequencies, detection and mapping of, 107-115

cell wall mutants, 333-335 is cytoplasmic linkage group located in chloroplast DNA?, 330-331 is sd a mitochondria1 gene?, 331-332 is spc 1-27-3 a chloroplast gene?,

329, 335-336

C

332-333

identification of cytoplasmic genes in criteria for, 289-293 phenotypic classes of cytoplasmic mutants, 293-295 terminology, 289 mechanism of maternal inheritance differential fates of chloroplast DNAs in zygotes, 296-300 the mat mutations, 300-304 UV irradiation and drug pretreatment of gametes, 295-296 methods of genetic analysis, 304-305 liquid-culture kinetic analysis, 309310

pedigree analysis, 305-309 statistical analysis of large undissected zygote colonies, 310-311 results of analysis of chloroplast genome, 311-314 closing the circle, 317-321 genetic diploidy of the chloroplast genome in zoospores, 323-329 mapping by means of cosegregation frequencies, 317 persistent cytoplasmic heterozygotes, 321-323

segregation process in zoospore clones, 314-317

D Drosophila behavioral and structural phylogenies,

CRlamydomonas association of cytoplasmic genes with

10-14 583

584

SUBJECT INDEX

closed and open programs and sexual isolation, 2-6 deviations from random mating within species, 6-10 isofemale strains, 23-24 physiological stresses and behavior boundary conditions, 15-18 nonsexual behaviors within boundary conditions, 18-23

N Neurospora biochemical genetics of hyphal development cell membrane in determination of morphology, 356 cell wall in morphogenesis, 343-356 gene, enzyme and morphology, 357-

sexual development and the matingtype locus, 387-388 normal meiosis and mitosis in the ascus, 142-148 somatic divisions, 148 wild-type genome chromosomes, 152-162 cytoplasmic genes, 177 linkage group-chromosome correlations, 172-176 marker distribution and genetic mapping, 162-172

T Triticum, origin and genetic relationships and closely related species, 417-426

W

380

chromosome rearrangements, 177-178 that do not make viable duplications, 186-193 that generate viable nontandem duplications via meiotic recombination, 193-210 identified : types, frequencies, origins and phenotypes, 183-186 methods of identifying and mapping, 178-183

nontandem duplications and their characteristics, 210-220 in other eukaryotic microorganisms, 220-223

conidiogenesis biochemistry and genetics of, 381-384 circadian rhythm, 385386 ultrastructure and surface architecture of conidia, 380-381 cytogenetics, appendix, 226-2435 genetic variants affecting meiosis or related stages, 148-152 life cycle, incompatibility and ploidy, 138-142

morphogenesis during sexual development, 386-387 genetics and biochemistry of perithecial development, 388-390 genetics of ascus and ascospore development, 390-394

Wheat(s) cultivated, genetic relationships and homoeologies among chromosomes of, 426-427 genetic variation for protein content and lysine content, 476-481 amino acid composition of highlysine and high protein wheats, 48 1

site of high-lysine and high-protein composition in wheat grain, 481486

nuclear gene control of protein content and composition genetic control of 80 S ribosomal proteins, 436-437 nitrate reductase synthesis and genetic control of nitrogen reduction, 437-440 role for induced mutations efficient induction of improved protein mutants, 540-541 principles, 535-539 protein structure and genetic code, 539-540

role of cytoplasm in hereditary control of protein content and composition chloroplast-nuclear gene interactions in nitrogen reduction, 435

SUBJECT INDEX

cytoplasmic organelle-nuclear gene interactions, 432-434 cytoplasm source and protein composition, 435-436 joint chloroplast-nuclear gene control of ribulose-1,5-diphosphate carboxylase, 434-435 Wheat endosperm environmental factors in protein synthesis and accumulation, 427-

428 amino acid composition, 42&430 genetic regulation and control of protein content, composition and processing properties, 430-432 Wheat kernel genetics of endosperm proteins, 417

A 8 7 C 8 0 € F 6

9 0 1 2

ti3

1 4 J 5

585

intracellular sites of protein synthesis and storage, 415-416 origin, development and ontogenetic relationship among tissues, 411-415 structure; 411 Wheat protein chromosomal location of genes affecting content, composition and use properties, 487-528 directions for future research, 541-544 general discussion, 528-535 genetic variability in nature and properties endosperm protein composition,

440-463 major factors affecting processing characteristics and nutritional value of wheat flour, 463-476

GENES, BEHAVIOR, AND EVOLUTIONARY PROCESSES: THE GENUS Drosophila P. A. Parsons Department of Genetics and Human Variation, La Trobe University, Bundoora, Victoria, Australia

Introduction . . . . . . . . . . . . Closed and Open Programs and Sexual Isolation. . Deviations from Random Mating within Species. . Behavioral and Structural Phylogenies . . . . . Physiological Stresses and Behavior . . . . . A. Boundary Conditions. . . . . . . . . B. Nonsexual Behaviors within Boundary Conditions VI. Isofemale Strains . . . . . . . . . . . VII. Discussion and Conclusions . . . . . . . . References . . . . . . . . . . . . I. 11. 111. IV. V.

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

1 2 6

10 15 15 18 23 24 27

1. Introduction

Research in behavior genetics largely began as a by-product of other investigations in a number of organisms including Drosophila. Not until the last 15 years have attempts been made to integrate this information (Fuller and Thompson, 1960; Hirsch, 1967; Parsons, 1967a). More recently, texts have appeared in this hybrid field (De Fries and McClearn, 1973; Ehrman and Parsons, 1976). I n particular, Ehrman and Parsons (1976) argued that the special emphases of behavior genetics compared with other areas of genetics have warranted these recent developments. I n brief, these emphases are (1) difficulty of environmental control, (2) the difficulty of objective measurement, and (3)the importance of the study of learning and reasoning. It must be stressed th a t no unique genetic principles are required when we consider the mechanism of the inheritance of behavioral traits. To date studies on mechanisms have been the predominant approach in behavior genetics, as shown by perusal of Ehrman and Parsons (1976). Even so, in the later chapters of this book evolutionary considerations assume progressively more importance. Indeed prophetically for behavior genetics, Caspari (1967) 1

2

P. A. PARSONS

wrote that “all biological phenomena can be considered from two points of view: mechanism and evolution.” I n agreement, the whole movement of the coverage of Ehrman and Parsons’ book goes from the mechanism of the inheritance of behavioral traits to the evolutionary aspects of behavior. More recently the need to stress evolutionary principles was emphasized by authors such as Wallace (1974). Mayr (1963) wrote that “the shift into a new niche or adaptive zone is almost without exception, initiated by a change in behavior.” That is, there will initially be only minor changes a t the structural level, and the evolution of morphological changes may follow behavioral changes. He returned to this theme again, in a perceptive article (1974) entitled “Behavior Patterns and Evolutionary Strategies,” where he rightly comments that in recent years there has been a growing synthesis between behavioral biology and evolutionary biology. This has led “to the posing of a whole battery of new questions on the evolution of behavior patterns and on the impact of behavior on the course of evolution.” With this comment in mind, it is logical to look a t its application with respect to genetically well-known groups of organisms. This review will be concerned with Drosophila. II. Closed and Open Programs and Sexual Isolation

Some behaviors are regarded as innate-that is they are predominantly determined by the genotype. These behaviors are based on genetic programs not allowing of appreciable modifications during the process of translation into the phenotype, and are called closed programs by Mayr (1974). Other genetic programs are modified during the translation into the phenotype, by input occurring during the life-span of its owner ; they thus have an acquired component. Mayr refers to this as an open program. Closed programs are widespread among so-called lower animals, which to date must include Drosophila. But here we must be careful, since although many genotypes have been tested for a great variety of behaviors and closed programs appear to predominate, this does not mean that the use of appropriate testing procedures over a period of time would not reveal results ascribable in part to open programs-this point will be further considered later in this review. One behavior where the evolutionary significance of a closed program is evident is mating. Male animals preferably display to females of their own species, and females normally respond to the displays of their own males. A female Drosophila, if kept in isolation until ready to mate, and then offered the simultaneous choice of males of several species in-

BEHAVIOR AND EVOLUTION IN

Drosophila

3

cluding her own, will almost unerringly accept the courtship of the male of her own species, as though there is innate knowledge of her own species. Quite clearly, if mating choice were more loose, considerable gamete and other wastage on the part of the female would ensue, so that natural selection for a closed system is to be expected. Even when we go to extremely closely related species, such as sibling species, isolation is normally perfect in the wild. For example, the sibling species D . pseudoobscura and D. persirnilis are sympatric in some regions and are isolated by a complex of ecological and ethological factors (for references see Parsons, 1973) including temperature and food preference differences, activity and photoresponse differences, and sexual isolation associated with differing male courtship songs. I n natural habitats, therefore, it is not surprising that interspecific matings are not found even though hybridization occurs relatively readily in the laboratory. In many laboratory experiments virgins were about 4 days old, but when flies of both sexes were placed together a few hours after emergence the proportion of hybrids was lower-a situation likely to pertain in the wild. Spieth (1958) suggests that the higher level of isolation under the latter situation may be due to individuals of both species maturing together, then acquiring the ability to discriminate between the species before sexual maturity. Furthermore, a D. persirnilis female once having mated with a D.persirnilis male, will not accept a D. pseudoobscura male subsequently. This shows that there may be a learned component in maintaining isolation in the wild. Laboratory experiments indicate other variables-for example, an environmental variable is that isolation is relatively low a t 16.5OC (Mayr and Dobzhansky, 1945). A genetic variable is indicated, since isolation can be increased or decreased by selection (Koopman, 1950; Kessler, 1966). I n conclusion, the program (s) leading to sexual isolation can be assumed to be closed in the wild, and only under the artificial laboratory condition is there any relaxation of this. Therefore efficient premating isolating mechanisms preventing copulation between closely related species are usually the rule. For another pair of sibling species studied in depth ( D . melamgaster and D . simulans), sexual isolation is even more extreme, again based on a complex of ethological (in particular) and ecological factors. Even so, a few hybrids can be obtained in the laboratory, and as for the above pair of sibling species, the level of hybridization is under both environmental and especially genetic control (Parsons, 1972, 1975a). Drosophila paulistorum is a taxon that contains an extraordinary complex of geographic races or incipient species, endemic in Central and South America ; six units are close to the status of reproductively isolated, but morphologically indistinguishable, species. They display pronounced,

4

P. A. PARSONS

in some cases nearly complete, sexual isolation associated with sterility of hybrid males. Observations of the courtship patterns involved show both qualitative and quantitative differences between races. A seventh race, called the Transitional one, occurs exclusively in Colombia. All strains of this race can be crossed and produce fertile hybrids with a t least one of the other races; in fact,, this is regarded by Dobahansky et al. (1969) as a relic of the ancestral population from which the other races have differentiated. Therefore, we have a situation of an extremely dissected and differentiated gene pool, making it a near unique example of a situation where it is difficult to decide whether there are one or several species. Sexual isolation is determined by polygenic differences between the races (Ehrman, 1961). All stages of isolation occur in D. paulistorum, ranging from none to almost complete separation. The variations in sexual isolation have presumably arisen by natural selection occurring as a result of geographic separation and the accumulation of genetic differences in the course of adjustment to different environments. Ehrman (1965) using multiple-choice tests compared given pairs of races that have been found to occur both sympatrically and allopatrically. I n allopatric crosses, the average isolation coefficient was +0.67 and in sympatric crosses was $0.85 (Table 1). Thus pairs of races TABLE 1 Numbers of Matings Observed and Isolation Coefficients Calculated for Sympatric and for Allopatric Crosses. Races

Origin

1. Amazonian X Andean 2. Amazonian X Guianan 3. Amazonian X Orinocan

4. Andean X Guianan 5. Orinocan X Andean 6. Orinocan X Guianan 7. Centro-American X Amazonian 8. Centro-American X Orinocan

(2; {Z; {Z:

E: {E K:

{E

Matings

Coefficient*

108 100 104 109 106 124 109 102 100 111 104 100 102 103 110 103

0 . 8 6 f 0.049 0 . 6 6 f 0.074 0 . 9 4 0.033 0.76 f 0.061 0.75 f 0.065 0 . 6 1 f 0.070 0 . 9 6 f 0.026 0 . 7 4 f 0.066 0 . 9 4 k 0.033 0 . 4 6 f 0.084 0.85 f 0.053 0 . 7 2 f 0.069 0 . 6 8 f 0.072 0.71 f 0.070 0.85 f 0.052 0 . 7 3 & 0.069

From Ehrman (1965). The total number of matings observed was 1695. *Average (sympatric) = 0.85; average (allopatric) = 0.67. a

+

BEHAVIOR AND EVOLUTION IN

Drosophila

5

occurring sympatrically exhibit more sexual isolation than the same pairs occurring allopatrically, or races coexisting geographically tend to be reproductively more isolated than those that do not. This is reasonable as the production of a large number of hybrids would be very inefficient. These differences are presumably due to natural selection within this complex taxon. Because of the variations of sexual isolation from random mating to complete isolation, it would seem also likely that variations may occur according to whether flies of different races are isolated or otherwise before mating. Further, variations may occur for flies once having mated, with respect to subsequent mating. The likely situation is that for a fly of a given strain or race, having mated with that strain or race, subsequent matings would be more likely to be with that given strain or race-that is, isolation would be enhanced and eventually this would be fixed into the genome, thus increasing levels of sexual isolation by natural selection [ L. Ehrman (personal communication) informs me that she has evidence in support of this]. Such variations would indicate a component of sexual isolation controlled by an open program. This would seem reasonable in a situation where levels of sexual isolation themselves are highly fluid, and may represent the situation characteristic of a series of races not yet quite with specific status. A number of experimental possibilities emerge that are testable; such testing may add insight into the processes whereby sexual isolation is enhanced or reduced-in the former case leading to speciation. Within D. pseudoobsclira, Pruzan and Ehrman (1974) found evidence indicating age and previous experience effects with respect to the now well-documented phenomenon of frequency-dependent sexual selection (Petit and Ehrman, 1969). All previous experiments studying this phenomenon had used exclusively young virgins. Pruzan and Ehrman found that 4-day-old virgin females confer mating advantages on all tested rare males as expected, but females who had a previous mating experience when younger award a rare-male advantage only when the rare male is of the same karyotype as their first mate, and matings are random when the first-mate type males are common. Equivalently aged ll-day-old virgin females mate significantly more than expected with minority males if they are of the same karyotype as the females themselves, whereas matings are near random when the males differ. Frequency-dependent mating is therefore both age and experience dependent. Therefore variations in choice may occur within species according to previous experiences. It should be noted that degrees of sexual isolation within species can be modified by artificial selection, as shown by experiments in D. melanogaster where homogamic matings are favored at the expense of heterogamic ones (Wallace, 1954; Knight et al., 1956; Hoenigs-

6

P. A. PARSONS

berg et al., 1966; Crossley, 1974). I n artificially isolated populations of both D . melanogaster and D . pseudoobscura, preferences for homogamic matings tend to develop over time (Koref-Santibaiiez and Waddington, 1958; Ehrman, 1964)-in this case sexual isolation has occurred as a byproduct of genetic divergence. In conclusion, levels of sexual isolation within species, between races within species complexes, and between species are under genetic control. Suggestions occur for previous experience effects. This latter phenomenon needs further exploration, but in conjunction with more experiments on the genetic basis of sexual isolation. Extrapolation to the wild is essential, since laboratory situations may be far from those pertaining in the wild. For example, to what extent do repeat matings occur in the wild, and how do they depend upon previous experience? I n spite of previous experience effects, sexual isolation is mainly under the control of a closed program; in any case, the normal result of previous experience is likely to be toward isolation. Ill. Deviations from Random Mating within Species

Random mating is commonly assumed in natural populations-and indeed the theoretical foundation of population genetics is based on this assumption. It must be admitted that this is partly because of the simplifications leading to simple mathematical modeling. I n practice, random mating rarely occurs (Parsons, 1967b). The increased study of mating patterns within species should be encouraged, coupled with considerations of their evolutionary consequences. Such studies might be both theoretical and observational. The genetic and behavioral study of the benefits that secondary sexual characteristics confer on their bearers also needs more emphasis. I n Drosophila, for example, sexual dimorphism for traits involved in courtship is well developed in some of the Hawaiian species (Carson et al., 1970; Spieth, 1973a,b), but not elsewhere [for example, the endemic Australian species many of which belong to subgenus Scaptodrosophila (Bock and Parsons, 1975)l. There are two main types of benefits of such sexual dimorphism: (1) their bearers may have superior competitive ability against others of the same sex; or (2) they may increase sex appeal. I n the first case, selection is within one sex; and in the second case, selection is made by the opposite sex. While there are difficulties in distinguishing these two components of sexual selection, in Drosophila when flies meet on food masses, each female has the opportunity of choosing among competing males, and because she effectively rejects somc suitors, sexual selection-in this case

BEHAVIOR AND EVOLUTION IN

Drosophila

7

intersexual selection-can be inferred to have occurred. By using mutant flies, Sturtevant (1915) in a seminal paper showed sexual selection in D . melanogaster, as have many other investigators working on this and other species. Using karyotypes polymorphic in natural populations, the importance of heterozygosity on the effectiveness of the males has been clearly shown in species such as D. pseudoobscura (Kaul and Parsons, 1965; Spiess et al., 1966) and D . pavani (Brncic and Koref-Santibaiiez, 1964). As discussed by Parsons (1974), a number of experiments suggest that male mating behavior is an important component of fitness; this agrees with early experiments of Merrell (1953), who found gene frequency changes in experimental populations of D. melanogaster to be predictable from male mating behavior variations. The need for more experiments of the type carried out in D . melanogaster, by Prout (1971a,b), who attempted t o define a small number of fitness components that encompass the entire life cycle and are accessible t o experimental evaluation, is clear-and indeed in his experiments, compared with other components of fitness, relative male mating abilities between genotypes were very important. The evolutionary significance of results of this type is, however, difficult to assess without extrapolation to nature. Emphasis for this comes from the observation that some of the experimental data in D . pseudoobscura show a dependence of relative mating speeds among karyotypes on the environment, such that there may be a tendency for heterokaryotypes to show less variability across environments (usually temperature changes) than homokaryotypes (for references, see Parsons, 1973, 1974). Therefore, we have the situation of possible genotype X environment interactions affecting levels of sexual selection-but it is noteworthy that heterokaryotype advantage is greatest under conditions of environmental stress, which may be rarely effective in nature considering that flies can frequently move away from stressed microhabitats ; specific evidence will be given later for Australian endemic Drosophila. Even so, it appears desirable to study sexual selection in all environments to which the population may be exposed. I n the Hawaiian Islands, in the region where many of the endemic species occur, the environmental extremes t o which many of the cosmopolitan species must be subjected are unlikely to occur. The adults are able to distribute themselves into microhabitats suiting their ecological requirements, the main controlling factors being wind intensity, humidity, temperature, light intensity, food sources, and acceptable courting and oviposition sites. Of these, temperature is the most important factor. I n the Hawaiian Drosophila, a courtship behavior pattern occurs that is not found elsewhere in the genus and has far reaching effects on its biology. In the field, flies of both sexes feed quietly with no male court-

8

P. A. PARSONS

ship activities. After a short period, males leave the food and each selects a territory or lek in the surrounding vegetation (Carson et al., 1970), which is small in size and species specific in character. The lek occupant defends his territory against intruders and advertises his presence, so that sexually receptive females are attracted to advertising males. When she arrives a t the lek she is treated agonistically until the male determines her sex. Then courtship occurs, but the male drives her from the lek if she finds his display unacceptable. Hawaiian lek Drosophila are sexually dimorphic associated with diverse and unique courtship patterns. I n the remainder of the genus, other than male sex combs on the fore tarsi, which are not involved in producing courtship signals, visible dimorphism involved in courtship and mating does not occur. It can be inferred that the Hawaiian lek species have been subjected to intense sexual selection since the male does not court on feeding-ovipositing sites, but rather attracts sexually receptive females to his lek. Spieth (1974b) suggests that the insectivorous behavior of the honey-creepers, the flycatcher, and the predatory muscoid genus Lispocephala, are responsible for powerful selection pressure upon the Hawaiian Drosophila. This selection has resulted in four discrete but interrelated events: 1. The selection of adults that are alert, wary and highly cryptic, both behaviorally and structurally 2. The drastic increase in size of some species 3. The abandonment of courtship on the feeding and ovipositional sites, thus eliminating the incessant courtship activity easily detectable by predators 4. The emergence of lek behavior and a concomitant drastic increase in sexual selection For understanding the relationship between genes, behavior, and evolutionary processes, the comparative study of sexual selection among the various species of Drosophila must continue to be a fruitful area of study-in particular the genetic systems involved, with particular reference to the comparison between the sexually dimorphic and monomorphic species. Further, the complex behavioral patterns involved in achieving a successful mating would seem to indicate the unlikelihood, as can be shown in man (Ehrman and Parsons, 1976), that random mating is the rule as assumed by many population geneticists. It is the job of behavior geneticists interested in evolution to investigate the mechanisms leading to deviations from random mating and asses their effects on populations. Considering the “laboratory” species, probably the most elegant proof of sexual selection is the phenomenon, already referred to, of frequencydependent mating whereby rare males have an advantage compared with when they are common (Petit and Ehrman, 1969). From the population

BEHAVIOR AND EVOLUTION IN

Drosophila

9

point of view, the advantage of the rare male would be expected to lead to an increase in its frequency provided that there are no other selective forces acting against it. As the rare type of male increases, the mating advantage would slowly diminish, and it would appear that a number of gene and chromosomal polymorphisms in Drosophila are maintained by such frequency-balancing selection based on mating behavior. The mechanisms of frequency-dependence are still obscure, but it is clear that the females do discriminate subtle airborne cues from the initiating male-the cue being probably a lipid or steroid (Leonard et al., 1974). Hay (1972) believes that the recognition of minority individuals is due to the existence of a colony order, such that the presence of this volatile male-borne material permits females to recognize when there are two types of males present, and thus modify their receptivity toward the minority males. Whatever the mechanism, the favoring of minority males in mating must ensure heterozygosity in the gene pool of the population, without the need to invoke classical overdominance. I n man, positive assortative mating (the tendency of like phenotypes to mate by choice) is found for numerous physical traits such as stature and arm span, and for many behavioral traits such as intelligence and personality (references in Ehrman and Parsons, 1976). Since these traits are heritable, then as Fisher (1930) argued, assortative mating is an agent important in modifying the genetic constitution of populations. A comparison with populations where matings are arranged would be of considerable interest. Evidence for positive assortative mating occurs between the color phases of the Blue Snow Goose and the Arctic skua (Cooch and Beardmore, 1959; O’Donald, 1959). In Drosophila, few relevant experiments have been done, although Parsons (1965, 1973) found positive assortative mating for sternopleural and abdominal chaeta number in D . melanogaster. This could be a direct effect of fly size, as sternopleural chaeta number and fly size are positively correlated when fly size is altered by environmental means (Parsons, 1961). Alternatively, there may be behavioral differences between flies of different sizes leading to minor modifications in courtship behavior. For example, wing area, related to fly size, is a factor in determining male sexual success (Ewing, 1964). If assortative mating is general, as in man, evolutionary processes will clearly be affected. The possible evolutionary significance of assortative mating was brought home meaningfully as a result of Gibson and Thoday’s (1962) disruptive selection experiments for high and low sternopleural chaeta numbers in D.melanogaster in a single population maintaining gene flow between the high and low components. Within ten generations the population split into two subpopulations, which were characterized by high

10

P. A. PARSONS

and low chaeta numbers. As predicted by Mather (1955) therefore, the population became bimodal and polymorphic under disruptive selection. Maynard Smith (1962) considered on theoretical grounds that these results are difficult to understand unless there is positive assortative mating for sternopleural chaeta number in the base population, or unless there is selection favoring positive assortative mating during the experiment. While the base population was not tested, Thoday (1964) found strong positive assortative mating within the high and low lines. Hence the isolation developed by disruptive selection is associated with strong positive assortative mating within the subpopulations so contructed. I n this way we have a laboratory model of the development of sexual isolation. These results therefore relate to the sexual isolation between races of D. paulistorum, and that developed by artificial selection for homogamy in D. melanogaster referred to a t the end of Section 11. The further study of sexual selection and sexual isolation in the laboratory combined with field observations as carried out in Hawaii, is thus essential to ascertain the processes involved in mating success in diverse species groups and the degree to which they are modifiable by experience and by selection. A combined behavioral and genetic approach is needed for this important segment of the synthesis between behavioral and evolutionary biology. We have gone past the stage of proving that components of mating behavior are heritable-and experiments to do just that alone may well fall into Wallace’s (1974) category of experimental data in behavior genetics no longer worth the effort needed to obtain them. For example, a question to investigate is Spieth’s (1974a) conclusion that, for Drosophila in nature, males are primarily responsible for sexual isolation and females for sexual selection. Another neglected question is the frequency of multiple insemination in the wild-a point emphasized by Anderson’s (1974) report in I). pseudoobscura that a t least half of females in a sample from San Gabriel Canyon, near Riverside, California, carried sperm of more than one male. It is also known that multiple insemination is common in laboratory cultures of the same species (Dobehansky and Spassky, 1967). IV. Behavioral and Structural Phylogenies

If one constructs the phylogeny of a group of animals, such as ducks and pigeons, based on behavioral traits, that phylogeny is exceedingly similar to an independently designed one based upon strictly morphological traits. The interpretation of this parallelism is that both sets of characters are the product of the same genotype-representing a closed

BEHAVIOR AND EVOLUTION I N

Drosophila

11

genetic program (Mayr, 1974). Other examples given by Mayr include gulls and storks. The best insect example is the genus Drosophila (Spieth, 1952). Spieth’s study encompassed 101 species and subspecies representing 21 species groups, and generally the evolution of mating behavior paralleled the morphological evolution of the group. Further, he concluded that divergence of the mating behavior between species occurs first a t the physiological and behavioral levels, and that the visually observable morphological differences arise much later. Brown (1965) quantified differences between 11 species of the obscura group for behavioral and morphological characters utilizing a measure of “Mean Character Differences” based on twenty behavioral and twentyfour morphological traits (Table 2), from which a high correlation between behavioral and morphological divergence emerged. Considering these data and the sibling species D. melanogaster and D . simulans (Bastock, 1956; Crossley and Zuill, 1970; Parsons, 1975a), it is clear that both behavioral and morphological differences between mutants within species are slight, those between sibling species are greater, and those between nonsibling species in the same division greater still. The same applies to the level of subgenera which show the major differences in behavior and morphology. Spieth ( 1974b) reiterated this general view after working on the Hawaiian Drosophilidae. Furthermore, Ewing and Bennet-Clarke (1968) analyzed the male courtship songs of species of the D. melanogaster and D. obscura species groups, and found pairs of sibling species to be more similar than pairs of nonsibling species. It can therefore be presumed that behavioral differences in courtship within species could become, under suitable conditions, the prime traits differentiating closely related species-and eventually associated morphological differences would become apparent. What is needed is t o see how such differences could have arisen during the course of evolution, and how the behavior serves to adapt the animal to its environment. This question in the more general sense-that is, extrapolating to behavior apart from courtship behavior-has been rarely posed, although mutants and evidence of genetic control affecting other behaviors are well known, e.g., affecting the central nervous system, the visual system, taxes such as geotaxis and phototaxis, and activity (for references, see Parsons, 1973). In the case of mutants affecting the central nervous system, studies with gynandromorphs have enabled the morphological localization of genetically controlled defects (Hotta and Benzer, 1972). Another approach is to look a t known morphological mutants with respect to their effects on behavior. Grossfield (1975) tabulated a number of such mutants and showed frequent and diverse behavioral changes, e.g., alterations in flight activity, wing-beat frequency, courtship and

TABLE 2 Mean Character Differences in Behavior Using 20 Traits (Upper Right Half of Table) and in Morphology Using 24 Traits (Lower Left Half of Table in the obscura Group of Drosophik7)a.b pseudo. pseudoobscura persimilis miranda bijasciata tristis obscura silvestris ambigua subobscura helvetia afinis

X 0

9 54 48 74 59 48 83 122 67

per. 10 X

9 50 48 74 59 48 83 122 67

mir . 45 55

x

54 61 74 65 52 83 122 69

bif. 84

80 89 X

58 78 59 48 87 117 106

Iris. 80 80 89 40 X

96 89 46 92 129 119

ob. 120 121 105 100 90 X 33 87 108 146 156

sil. 105 10.5 100 74 55 21 X 67 111 161 140

amb.

sub.

helv.

aff

110 110 95 100 100 70 63

125 130 95 130 130 130 116 60 X 117 119

150 150 150 158 158 150 1.50 92 58 X

129 112 119 106 94 69 75 87 119 58

25

X

X

75 104 87

Drawn up from data from Brown (1965). The correlation coefficient between the fifty-five possible species combinations in pairs between behavioral and morphological traits comes to 0.576 (P < 0.001 for difference from 0 ) . a

b

bd

. ~

:

'

BEHAVIOR AND EVOLUTION IN

Drosophila

13

mating success, phototaxis, and optomotor response. These occurred for a variety of different mutants. Such approaches are slowly making possible the connection between genes, morphology, and behavior to be elucidated-in some cases with associated physiological and electrophysiological changes. A question for the evolutionary biologist must surely be to ascertain which of these behaviors are differentially selected in different populations and species and why. Considering the large literature on phototaxis, studies on natural populations are minimal. However, MCdioni (1962) found variations in natural populations for phototaxis a t different localities in the northern hemisphere, such that positive phototaxis increased going north. It is difficult to speculate on the selective advantage of such a phototactic gradient. Drosophila is sensitive to desiccation and high temperatures (Parsons, 1973), and is likely under conditions of desiccation stress to migrate to damp habitats, which are often shaded. Therefore under conditions of high desiccation/temperature stress, natural selection for negative phototaxis would be greater. In nature such selection pressure would decrease with increased latitude, so that the observed phototactic gradient may be plausible. This hypothesis is testable. But with a trait such as phototaxis care is needed, since, as Rockwell and Seiger (1973) have correctly pointed out, its measurement may be a function of experimental design, so making extrapolation from the laboratory to natural populations difficult. This is because phototaxis is a complex response to light, beginning when the stimulus impinges on the light receptor and culminating with the locomotion or lack of locomotion of the animal. As Rockwell and Seiger further point out, there is no a priori reason for supposing that one particular design provides a measure of phototaxis more closely related to that in nature than any other design. Proceeding to comparisons between the sibling species D. melanogaster and D. simulans, the females of D. simulans are more responsive to the visual aspects of the male’s courtship than D. melanogaster, and furthermore D. simulans shows greater phototaxis, and greater dispersal toward a light source (McDonald and Parsons, 1973; Parsons, 1975b). D. melanogaster belongs to a group (Table 3) of cosmopolitan species that are both light-independent in mating and have a wide geographic distribution (Grossfield, 1971, 1972). A second group of species, which are inhibited by darkness such that only facultative dark mating occurs, are more narrowly distributed with the exception of D. sirnulans, which has a wide distribution. The unique position of D . simulans may reside in its close relationship to D. melanogaster, such that character displacement has occurred. This behavioral divergence reflects a divergence in courtship organization and its underlying genetic architecture. In view of known

14

P. A. PARSONS

TABLE 3 Numbers of Species of Drosophila Occurring in 1 to 6 Geographic Life Zones Plotted with Respect to Three Categories of Light Dependency in Matinga Life zones Dependency category

1

2

3

4

Light independent Facultative dark mating Dark repression

-

1 2 6

-

2 -

9 17

5

6

-

1C -

-

-

5*

The species are listed in Grossfield (1972). D. ananassae, D. funebris, D. hydei, D. immigrans, and D. melanogaster. c D. simulans. 0

latitudinal variations in D.melanogaster for phototaxis, comparisons for flies from a number of habitats paying attention to light-dependency is desirable, comparing too the sibling species when sympatric or allopatric. Finally, there is a third group of species in which mating is blocked by darkness. These species are narrowly distributed. They include the sexually dimorphic “picture wing” Hawaiian drosophilids, where light intensity influences not only the microniche necessary for oviposition but also adult resting positions and leks. In these Hawaiian species therefore, visual cues are of critical importance, such that particular insects orient themselves in spots of specific and consistent light intensity (Grossfield, 1966). In fact, Grossfield suggested that responses to oviposition stimuli and light levels have contributed to speciation in the Hawaiian Drosophila, but, subsequent to his work, little work on phototaxis in Hawaiian Drosophilo has been reported (Carson et al., 1970). Spieth (1974a) commented that in darkness no type of male, mutant or otherwise, orients normally in courtship. Crossley (1970) and Jacobs (1960) noted that in darkness abnormal D.melanogaster courtships are the rule. For the light-independent species, therefore, such courtships still contain sufficient stimuli to lower the female’s threshold to the level a t which she will copulate, whereas in the light-dependent species, the female’s threshold remains high. Spieth considers light dependency to be primitive and that light independence has evolved secondarily in a number of species, but he finds the adaptive significance of light-independence unclear. Experimental work in closely related species, such as D. melanogaster and D . simulans, could provide answers to such questions as: To what extent is light-dependency modifiable by artificial selection? If it is modifiable, what is the genetic architecture of such changes?

BEHAVIOR AND EVOLUTION IN

Drosophila

15

V. Physiological Stresses and Behavior

A. BOUNDARY CONDITIONS As already noted, Carson et al. (1970) observed that Hawaiian Drosophila do not randomly distribute themselves throughout the forest habitat, but select and tend to accumulate in microhabitats meeting their specific requirements. While each species appears to have its own specific requirements, adults of most species avoid even moderate wind currents and light intensities, humidities below 90%, and temperatures above 21OC. So far as the physical stress components are concerned, humidity and temperature are likely boundary conditions delineating the extremes within which a range of response to other environmental variables occurs, for example, light intensity as above. In particular, temperature requirements are relatively exacting in Hawaiian Drosophila. I n rearing these species in the laboratory, it is mandatory that the temperature be kept under 21OC. At one of the richer collecting sites on Maui a t 4000 f t elevation during summer, temperatures a t 5 ft from the ground had a range of 21OC to 12OC, while the surface of the forest floor hovers around 16OC during all times of the day. Winter temperatures are lower but flies are active the year round. At 5 ft from the ground the relative humidity was 95% or higher for 77.5% of the time, and was less than 90% only 2% of the time. Endemic Drosophila in southeastern Australia are found in moist habitats frequently characterized by tree ferns (Grossfield and Parsons, 1975). The endemics mainly belong to the subgenus Scaptodrosophila, in contrast with the Hawaiian species, which belong to the subgenus Drosophila. I n the genus Scaptodrosophila, rare elsewhere in the world, there are a t least 45 Australian species, suggestive of an Australian adaptive radiation (Bock and Parsons, 1975). The main studies have been carried out in southeastern Australia, where the climate is much more variable than in the Hawaiian forests. Even so, fern gullies form refuges, usually isolated from each other, which offer temperature/humidity conditions compatible with the survival of species of the genus (Parsons and Bock, 1976; Parsons et al., 1976). Below 12OC, flies are inactive and are rarely found in the wild. I n parallel, Australian strains of the cosmopolitan species D.melanogaster and D . simulans do not mate or oviposit below about 12O-13OC in the winter. Indeed when flies of either species are placed between 12OC and 2OoC, both mating and oviposition increase almost linearly (Fig. 1) with temperature increase (McKenzie, 1975; J. A. McKenzie and P. A. Par-

16

P. A. PARSONS

0 Trm(rr.tun ('C)

Temperature (OC)

FIQ.1. Mating percentages of three strains (V,A, 0 : strains 1-3) of Drosophila melanogaster after 48 hours at seven experimental temperatures between 8" and 2O"C, and percentage of eggs deposited relative to 20°C controls at six experimental temperatures between 8" and 18°C. (After McKenzie, 1975.)

sons, unpublished). In an ectothermic organism the temperature-energy relation is critical. At lower temperatures the physiological capabilities of the organism will be limited. It seems reasonable that in winter energy wastage will be selected against: oviposition during this period represents such a wastage because of the low probability of the development of larvae. Field data agree, since in the cellar of the "Chateau Tahbilk" winery in Victoria, larvae and pupae are not found when the winter temperature falls below 13OC, although adults can be collected (McKenzie, 1975). This suggests that 12OC or thereabouts may be a lower boundary condition for the activity of flies of the genus Drosophila, although they will survive considerable periods of time a t much lower temperatures (Anoxalabehere and Periquet, 1970). While no detailed surveys of overwintering have been done in Australia, only rarely would temperatures fall below zero for long periods of time in habitats where the endemics are found. In any case it can be surmised that 12OC represents a lower boundary condition for the reproduction of some Drosophila species as assessed by mating and activity. Quite possibly this limit may be genus-specific (Parsons, 1 9 7 5 ~ ) . Turning to the upper boundary temperature, it is known that those in the 25°-300C range are stressful for the above cosmopolitan species (Parsons, 1973), although D. melanogaster can tolerate higher temperatures than D. simulans. Field and laboratory experiments are in agreement. Not unexpectedly, D . simulans is more sensitive to the associated stress of desiccation than is D. melanogaster (Parsons, 1975a). Adaptation to high temperatures and desiccation is a complex of physiological acclimation, genetic variation, and behavior, the relative importance of

BEHAVIOR AND EVOLUTION IN

Drosophila

17

these three components varying among species (Levins, 1969). Laboratory studies on environmental stresses pay little attention to the mobility of drosophilids. Behavioral adaptations add a new dimension to the capabilities of an animal, for when stressed, it can avoid stress by judicious and alterable microhabitat selection. The Australian endemic species are collected in the wild by sweeping them from their actual microhabitats. Because of this, it is possible to characterize the temperature/humidity relationships of these habitats. Collection data show quite clearly that flies adjust to microhabitats based on temperature/humidity relationships. In brief, then (and including the lower boundary conditions for completeness) : ( 1) temperatures below 1l0C-usually no flies; (2) temperatures between 12' and 15OC with high relative humidities-flies widely scattered and difficult to find ; (3) temperatures 16OC and up with high relative humidities-flies on tree ferns 1-4 m above water or permanent moisture; (4) temperatures 16O21OC with relative humidities 80% and below-flies on tree ferns up to 3 m above water or permanent moisture; (5) temperatures 2 l o C and up with relative humidities 80% and below-flies very close to water on tree ferns. Figure 2 gives schematically the situation for 15O-24OC, showing the effect of humidity on the position of flies for ambient temperatures in this range. Quite clearly flies adjust to microhabitats based on temperature/humidity relationships. Within habitats, temperature gradients occur such that temperatures a t the fern frond, where flies are collected, were generally in the range 15O-2OoC irrespective of site (Parsons, 1 9 7 5 ~ )The .

D i d ance from permanent moistwe (m)

Ambient

temperature

'C

FIG.2. A schematic diagram showing the distance of endemic Australian flies in tree fern habitats from permanent moisture for ambient temperatures between 15" and 24"C, for high relative humidity ( 2 9 0 % ) implying little or no deeiccation stress, and for humidities <SO% implying a much greater desiccation stress.

18

P.

A.

PARSONS

inferred behavioral responses to temperature and humidity are not surprising, since small insects have an extremely high ratio of surface area to volume, so that the amount of water that can be lost by evaporation is large compared with the amount that can be stored. Under conditions of extreme stress, genetic differences in resistance to temperature extremes and desiccation may become critical in determining survival. It is not surprising therefore that genetically resistant D. melanogaster individuals tend to be large and to have high wet and dry weights (Parsons, 1970). Therefore between the lower (12OC) and upper ( 20°-30°C depending on relative humidity) boundary conditions, flies can be found a t varying distances from permanent moisture ; i.e., there is adaptation bp behavioral means. I n other words, there is a favorable range of components in the environment (see Andrewartha and Birch, 1954). Clearly the range may vary from species to species, but the broadest constraint must be regarded as genus specific. This is followed by species-specific constraints as shown by comparisons between D. melanogaster and D. simulans (Parsons, 1975a), and between population-specific constraints as found for desiccation resistance in D. simulans (McKenzie and Parsons, 1974a). More detailed exploration of behavioral responses to temperature and moisture variations is obviously necessary throughout the genus, having been largely neglected so far. BEHAVIORS WITHIN BOUNDARY CONDITIONS B. NONSEXUAL Given that species and populations are within the genus-specific boundary conditions, normal behaviors in relation to variations in the environment can be studied: i.e., mating, feeding, ovipositing etc. (for a discussion of what constitutes the generalized environment, see Andrewartha and Birch, 1954). Apart from behaviors discussed so far, feeding and oviposition are the main ones to be considered further. They are of special importance since the family Drosophilidae apparently gained ascendancy through specializing on organisms causing fermentation. This then gave them a great variety of substrates in a variety of habitats, including presumably a great variety of fermenters in addition. Throckmorton (1976) regards the history of the family as one of mastering this niche complex over the last fifty million years or so over the major continents of the globe. Drosophila adults seek fermenting and/or decomposing masses of plant material from which they selectively consume the yeasts and/or bacteria abundantly growing in such substrates (for references, see Carson, 1971; Parsons, 1973) according to species. The main types of substrates are (1) freshly decaying plant parts, e.g., fallen flowers, fruits, leaves, bark wounds, rotting bark on broken twigs, limbs, or dying trees;

BEHAVIOR AND EVOLUTION IN

Drosophila

19

(2) deliquescing or rotting fungi; (3) slime fluxes; and (4) (more rarely) living attached flowers. The food sites are generally small in size, discrete, and periodic in occurrence. Apart from constraints of the physical environment, their periodicity of occurrence leads to great variations in population numbers during the year, often on a cyclical basis (McKenzie and Parsons, 1974b). Attraction t o feeding sites occurs via odor. A considerable number of flies may be collected from a given food mass, suggesting opportunism in feeding behavior. Throckmorton ( 1975) considers that originally the Drosophilidae were associated with slowly fermenting leaves and other fleshy plant parts on the humid forest floor, and probably also with sap and broken and damaged parts of living plants themselves. This would have been a relatively austere existence, but i t provided a step toward the exploitation of fleshy fungi, and much richer fermentation sites, such as fruits. Not a great deal of detailed experimental work has been done on feeding sites, in spite of their central importance in the evolutionary biology of Drosophila. However, there has been some niche-breadth work based on food selectivity of several species using several kinds of bait, for example, banana, tomato, potato, and orange in Puerto Rico (Levins, 1968). A species such as D. curdini was attracted to each bait with almost equal frequency and so can be said to have a broad niche for the baits used compared with D. repleta and D. hydei, which had a strong preference for potato. Comparing the sibling species D. melanogaster and D . simulans, adults of the former species show a slight preference for media containing alcohol, and the latter a substantial aversion (P. A. Parsons, unpublished), and furthermore, oviposition differences between the two species occur in a similar way (McKenzie and Parsons, 1972). Isono and Kikuchi (1974) found an autosomal recessive mutation for lowered response to the presence of the sugars glucose, sucrose, and maltose in D. melanogaster involving receptor sites in labellar chemosensory hairs. Researches of this type within and between closely related species ought t o be steps in ascertaining the precise resources utilized. This would be especially true for D . melanogaster and D. simulans, about which much evolutionary biology is known, but the exact resources utilized by the species are not (Parsons, 1975a). Kikuchi (1973) has found an olfactory mutant in D. melanogaster attracted to 18 chemicals repellant to a parental strain. Kikuchi’s studies are far distant from relating behavior and evolutionary processes to date. However, ethyl alcohol has been shown, by release-recapture experiments during vintage a t the Chateau Tahbilk winery in Victoria, to encourage migration toward the fermentation tank in D. melanogaster and away from it in D. simulans. I n other words, in adequately strong concentrations of alcohol, the two species are

20

P. A. PARSONS

separated completely, and indeed in the cellar of the winery, only D. melanogaster is found a t all stages of the life cycle-eggs, larvae, pupae, and adults-provided that the temperature is 13OC or higher (McKenzie and Parsons, 1972; McKenzie, 1975). The study of fermentation products would seem an obvious step in trying to define more precisely the feeding sites of these two species. Turning to oviposition sites, Carson (1971) and others have pointed out that while most species are opportunistic generalists in their feeding behavior, many species are selective in their choice of ovipositional substrates. That is, they will oviposit in only one of several substrates on which they regularly feed. Spieth (1974a) further comments that he knows of no instance of flies ovipositing on a substance on which they do not feed. Considering host plants, for example, oviposition ranges from species with only one host plant (monophagous) to those with a multiplicity (polyphagous). For example, D. buzzattii is associated only with the cactus Opuntia (Carson and Wassermann, 1965), and D.pachea only with the cactus Lophocereus schottii. In the latter case D . pachea utilizes a unique sterol provided by the cactus, and furthermore, there is an alkaloid pilocereine in the cactus that kills other species, but not D . pachea (Kircher et al., 1967). The South American species D. flavopilosa lays eggs only in the flowers of Cestrum parcqui (Solanaceae), and development takes place in these flowers (Brncic, 1966). Two Hawaiian sibling species, which are homosequential and monomorphic, D. heedi and D . silvarentis, coexist in a sparsely vegetated area on the island of Hawaii (Kaneshiro et al. 1973). Of the two major trees in the area, Myoporum sandwicense alone appears to be able to support both species. Adults of both species feed on Myoporum flux, but most remarkably, D. silvarentis oviposits only on fluxes that wet the trunk well above the ground surface, whereas D . heedi larvae are exclusively found in caked soil moistened by flux dripping from above (Fig. 3 ) . Therefore, the only facet of the environment in which significant separation of the species occurs is in the choice of oviposition sites. This is in accord with Carson (1971), who argues that many species are highly specific for oviposition site, but less so for larval or adult feeding sites. Indeed, the above pair of species suggest that a shift in oviposition site can be one of the most crucial phases of adaptive separation of two Drosophila species -a point worthy of much more study. Monophagous species are often difficult to culture in the laboratory, whereas polyphagous species, such as the cosmopolitans, having a variety of hosts, are easier to culture, presumably because of less specialized requirements compared with monophagous species. Many of the Hawaiian species must fall into the category of specialists (Carson et al., 1970).

BEHAVIOR AND EVOLUTION IN

Drosophila

21

FIG.3. Breeding sites of Drosophila heedi and D . silvarentis. Myoporum tree (A) has a trunk flux 7 feet above the ground (B) that wets the trunk for about 1 foot below it (C). Horizontal branch (D) has fluxing point (E) resulting in free-falling flux (F) which forms flux puddle (G) in soil (H). Fluxing point close to ground (I) wets trunk (J) and results in flux puddle close to the base of t.he tree (K). Only D. silvarentis larvae would be found in C. E. and J. Drosophila heedi larvae will be found at G and K. The latter may contain a fen. D . silvarentis larvae reaching it from J. (From Kaneshiro et al., 1973.)

One of the most bizarre examples of specialism concerns three species of drosophilid flies that have evolved an obligatory commensal association with three different species of gecarcinid land crabs on different islands (Carson, 1974). Other than these three crab flies, Drosophila has little use for animals or animal protein. All three crab flies utilize the exterior nephric nerve and pad. These are basically cases of parallel evolution, in each case the fly belonging to a distinctly different phyletic line of the family Drosophilidae (one species, in fact, is in genus Lissocephala) . Returning to the laboratory experiments, when offered a series of apparently similar oviposition sites, several species of Drosophila do not distribute their eggs a t random. The main factor bringing about the

22

P. A. PARSONS

aggregation of the eggs is the preference of female flies for ovipositing near sites previously used for oviposition by other females. Flies will go to a given site simply by placing eggs on one of several sites offered to them in D. pseudoobscura (del Solar and Palomino, 1966). I n D. melanogaster, Mainardi (1968) showed that females preferred to lay eggs where males had previously been present, as compared with sites where males had not previously been present, when given the choice. That is, female flies can recognize the previous presence of males a t a site. I n D. pseudoobscura, the degree of aggregation can be changed by selection and so is genetically controlled (del Solar, 1968). Presumably there is a compromise between a single oviposition site, leaving all others unexploited, and the situation of having many sites with so few eggs deposited that the food is not conditioned by the larvae. Natural selection strikes the compromise between some oviposition sites receiving larger numbers of eggs than others, and others remaining unoccupied. Extrapolation to the wild has not been done, and additional laboratory work on a number of species seems desirable. Comparing the sibling species D. melanogaster and D . simulans, D. simulans is better able to oviposit in the center of food cups and on food having a surface crust-that is, desiccation makes the medium less favorable for D. melanogaster (for references, see Parsons, 1975a). Pupation sites have been little studied in the laboratory or the wild. However, variations in pupation site are known in different strains of D. melanogaster (Wallace, 1968). Of more significance are the experiments in D. willistoni (de Souza et al., 1970) revealing that there is a pair of major genes a t a locus controlling pupation site-namely, on the food of a population cage or on the bottom of the cage. Polymorphic populations with both types of flies reach larger population sizes and have a greater biomass than monomorphic populations. That the polymorphism depends on the multiniche environment is shown by the rapid elimination of genes of preference for pupation outside the food in populations kept in 250-cm3 bottles. Therefore, there are a number of behaviors occurring throughout the life cycle that have been shown to be under partial or complete genetic control based on comparisons within and between species, and can be studied within the boundary conditions of temperature and humidity already discussed. However, these conditions refer to adults, and extrapolation is needed to all phases of the life cycle (and species). Considering survival in a Colorado population of D. pseudoobscura (Crumpacker and Marinkovic, 1967), in order of decreasing cold resistance gives: adults > pupae, larvae > eggs. The eggs are substantially affected by temperatures of 5°C and below. Genetic studies of cold resistance, and

BEHAVIOR

AND EVOLUTION IN

Drosophila

23

comparisons with other species, are minimal, but would seem to be necessary for explaining seasonal distributions and overwintering mechanisms. Indeed, such studies are needed to define the environments under which all stages of the life cycle exhibit normal behavior patterns, considering any stress to which a population is likely to be exposed. VI. Isofernale Strains

I n the study of natural populations, isofemale strains (those set up from single inseminated females in the wild) have been used especially in Drosophila. Initially, variation between isofemale strains derived from natural populations, known for essentially all measurable traits tested-behavioral, morphological, and physiological-tended to be put into the nuisance category. However, since the variation between strains persists consistently over a number of generations, this directly implies polymorphic differences in natural populations originating from the founder females. Recently McKenzie and Parsons (1974~)and Parsons (1975d) considered the isofemale strain approach with particular reference to directional selection, genetic architecture studies, population variability, and microdiff erentiation in response to differential selection pressures. As pointed out, a major advantage of the approach is that inferences about natural populations can be made directly and quickly. In particular, this is important for species for which there is little genetic information, but that can be cultured in the laboratory. I n this way comparisons, already referred to, between natural populations of D . melanogaster and D . simulans for phototaxis and dispersal toward a light source and levels of hybridization have been made. An obvious example where the isofemale strain approach is likely t o be invaluable is in comparative studies of populations of such endemic species as can be cultured. This would enable rapid assessments of the genetic architecture of some of the traits characterizing these populations and could aid, for example, in finding out more genetically about the high levels of sexual dimorphism and lek behavior in the Hawaiian species. The technique of diallel crossing between isofemale strains is a one-generation approach, which will provide estimates of additive and dominance components of a trait. For example, in D . melanogaster, isofemale strain heterogeneity has been found for duration of copulation and mating speed. Carrying out diallel crosses between strains, for the former trait male-controlled additive differences were found, whereas for mating speed both additive and nonadditive effects occured, the male being more important than the female. The nonadditive effects were mainly in the direction of rapid mating-

24

P. A. PARSONS

agreeing with Parsons (19741, who argued that mating speed is under directional selection for rapid mating. While these are simple behaviors, they illustrate an approach that could be applied to more complex problems. Isofemale strain studies can also be used to assay differences among natural populations within species. An example where the approach would be of use is MBdioni’s (1962) finding of variations between wild strains of D. melanogaster collected a t different localities in the northern hemisphere with greater phototaxis in the more northern flies. Indeed, the approach has been successfully used by McKenzie and Parsons (1974~) in D. melanogaster for sensitivity to alcohol within the wine cellar of Chateau Tahbilk winery, immediately outside the cellar and distant from the cellar, so demonstrating genetic heterogeneity due to selection over a remarkably short distance. Hence isofemale strain studies permit conclusions on microdifferentiation of populations as the result of ecological heterogeneity (some behavioral aspects of this system were considered in Section V ) . I n addition, McKenzie and Parsons (1974a) found differing genetic architectures for resistance to desiccation for two populations of D. simulans (Melbourne and Brisbane) using diallel crosses from isofemale strains. It is hoped that these examples will illustrate the pointto be referred to again in the next section when learning is discussedthat the isofemale strain approach is of great value if rapid and quick inferences about natural populations are required for any species that can be cultured in the laboratory. This applies for any measurable trait, including behavioral ones, over a multiplicity of environments. VII. Discussion and Conclusions

The behaviors discussed in Drosophila lean heavily toward those under closed genetic programs, since the shorter the life-span of an individual, the smaller the opportunity to learn from experience. I n species with a more or less extended period of parental care, the situation may be radically different, since the longer the period of parental care, the more time will be available for learning, and so for a shift toward an open genetic program. The only mention of this so far in Drosophila involves mating choice as influenced by previous experience. The selective advantage of the open program is that far more detailed experiences about the environment can be stored than can be transmitted across generations via the genetic code. Given that, in Drosophila, we are mainly dealing with closed genetic programs, the task of relating genes, behavior, and evolutionary processes is simplified.

BEHAVIOR AND EVOLUTION IN

Drosophila

25

However, there is some recent evidence for maze-learning ability in D . melanogaster (Hay, 1975) based on the mazes used to study taxes in Drosophila (e.g., Hirsch, 1963 ; Hadler, 1964 ; Dobzhansky and Spassky, 1969). The procedure entailed modification of the 11-unit mazes where flies initially turn left or right, so that the subsequent tendency of flies to turn left (or right) is followed. Isofemale strain heterogeneity was found indicating more successfully than previously that (a) learning does occur and (b) it is under genetic control. This is a system difficult to relate to the wild, but it indicates that, apart from sexual isolation, learning may occur in Drosophila. Of more obvious significance for evolutionary processes is Manning’s (1967) observation that Drosophila reared on a medium containing geraniol ( a peppermint smell) show reduced aversion to its odor when adult. Manning considers this to be a form of habituation. However, it is reasonable to postulate that over a number of generations there could be genetic assimilation for such behavior as suggested by Moray and Connolly (1963). Extrapolating to the wild, if a niche is entered characterized by some sort of metabolic change that can be assimilated genetically, the possibility of evolutionary change, perhaps to the level of speciation, occurs. Studies to separate clearly learning from habituation are of obvious significance in this regard, as shown by Hershberger and Smith (1967). Indeed Quinn e t al. (1974) claim to have established memory persisting for 24 hours in populations of D . melanogaster trained by alternately exposing them to two odorants, 3-octanol and 4-methylcyclohexanol, one coupled with electric shock. They eliminate pseudoconditioning, excitatory states, odor preference, sensitization, habituation, and subjective bias as explanations. From the point of view of population continuity, the genus Drosophila illustrates well the idea that species have a tolerable range for components of the environment beyond which they cannot survive and reproduce. It has been shown that temperature/humidity relationships provide such boundaries in Drosophila, with some variation among species of the genus, and even among populations within species. The local population is therefore the unit for identifying exact tolerable ranges within a broader species range and an even broader genus range. Tolerable ranges for the components of the environment for existence have been little explored either by ecologists or evolutionary biologists. For behaviors such as movement, mating, feeding and oviposition, the boundary conditions must be met, otherwise resource utilization is minimal. Particularly noteworthy are the precise microhabitats occupied by Australian endemic species as dependent upon temperature/humidity relationships. Here the program must be regarded as closed, and the microhabitats occupied by flies depends upon the physical features of the environment.

26

P. A. PARSONS

Behavioral variation is subject to natural selection, but being more difficult to study than morphological variation, has been little studied, except under very artificial situations in Drosophila, as this review and the literature in general indicate. Furthermore, the relevance of the individual behaviors studied to survival in nature is frequently not assessable. Including behavior, there is a large unknown field of endeavor in the determination of the selective advantages of various possible options in different organisms under different environmental conditions. The detailed focusing of attention on this must surely provide, with time, insight into the relationships between behavior genetics and evolutionary processes. On the other hand, the complete answer is not just to concentrate on behavior but upon all factors determining the distribution and abundance of animals. Major reasons for stressing behavior include its relative past neglect in the study of evolutionary processes, and its now clear interaction with all parameters determining survival. It has been shown that the behavior geneticist interested in evolutionary processes should rely more on natural populations. The concept of studying species in a tolerable range of environments for population continuity must again be stressed. For example, in comparative studies of components of fitness in the sibling species D. melanogaster and D.simulans, the majority of experiments have been carried out a t 25OC only, an environment relatively unfavorable for D. simulans. Hence genuine interspecific comparisons are impossible. Furthermore, many of the studies were carried out with very few and often special strains, many having been in the laboratory for generations. Extreme caution is therefore necessary in extrapolating from the strain to the species level in view of the genetic heterogeneity found in both natural and even laboratory populations for almost any measurable trait (Parsons, 1975a). Although these two species are easy to culture in the laboratory, how can the heterogeneity of the microhabitat in the wild be replicated? And furthermore, how do physiological and behavioral mechanisms help to adapt to this heterogeneity? Even though these two species have been extensively studied for many decades, their value in helping to link genetics, behavior and ecology is only just beginning to be realized. Studies of this multidisciplinary nature, including behavioral work, should contribute much to evolutionary biology. The emphasis should be on those behavioral traits determining population continuity under the multiplicity of environments to which populations are normally subjected. As already shown, a common approach in behavior genetics, i.e., to study only the heritable components of traits, does not contribute greatly to the elucidation of the role of behavior in evolutionary processes. Such studies may well be part of a larger investigation in the future, but it

BEHAVIOR AND EVOLUTION IN

Drosophila

27

would seem that solo studies of this nature, being a major feature of behavior genetic work in the 1960s, is likely, as already suggested, to slip from the prominence they formerly possessed. Another reason for this is that the evolutionary significance of some of the behaviors studied during this period-often under highly artificial laboratory conditions-is most obscure. That is, the extent to which the traits are relevant in determining the population continuity of genotypes controlling them is unknown. Behaviors relevant in population continuity need t o be isolated and studied under a multiplicity of environments. I n other words the question of the connection of a trait to fitness in nature must be posed. As shown by Parsons (1974) when making an assessment of the importance of male mating speed as a component of fitness, this will not easily be done. The answering of questions of these types for Drosophila, with its presumed largely closed genetic program, will surely aid in the study of the more complex paths between genes, behavior, and evolutionary processes, which must occur in organisms higher in the phylogenetic series, such as mice and men, where genetic programs are certainly more open.

ACKNOWLEDGMENTS I am grateful to Dr. Lee Ehrman for a critical assessment of an earlier version of the manuscript, to Mrs. Cheryl Wynd for typing the various versions, and to Miss Michele Jones for drawing the diagrams. The work has been supported in part by the Australian Research Grants Committee.

REFERENCES Anderson, W. W. 1974. Frequent multiple insemination in a natural population of Drosophila pseudoobscura. A m . Nat. 108, 709-711. Andrewartha, H. G., and Birch, L. C. 1954. “The Distribution and Abundance of Animals.” Univ. of Chicago Press, Chicago Illinois. Anoxalabrhere, D., and Periquet, G. 1970. RCsistance des imagos aux basses temp6ratures chez Drosophila melanogaster. Bull. Soc. 2001.Fr. 95,61-70. Bastock, M. 1956. A gene mutation which changes a behavior pattern. Evolution 10, 421439.

Bock, I. R., and Parsons, P. A. 1975. Australian Drosophila-an adaptive radiation in the subgenus Scaptodrosophila. Nature (London) 258,602. Brncic, D. 1966. Ecological and cytogenetic studies of Drosophilia flavopilosa, a neotropical species living in Cestrum flowers. Evolution 20, 1629. Brncic, D., and Koref-Santibafiez, S. 1964. Mating activity of homo- and heterokaryotypes in Drosophiln pavani. Genetics 49, 585-591. Brown, R. G. B. 1965. Courtship behaviour in the Drosophila obscura group. Part 11. Comparative studies. Behaviour 25, 281-323. Carson, H. L. 1971. “The Ecology of Drosophila Breeding Sites,” Harold L. Lyon Arboretum Lecture Number Two. University of Hawaii, Honolulu.

28

P. A. PARSONS

Carson, H. L. 1974. Three flies and three islands: Parallel evolution in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 71, 3517-3521. Carson, H. L., and Wasserman, M. 1965. A widespread chromosomal polymorphism in a widespread species, Drosophila buzzatii. A m . Nat. 99, 111-115. Carson, H. L., Hardy, D. E., Spieth, H. T., and Stone, W. S. 1970. The evolutionary biology of the Hawaiian Drosophilidae. I n “Essays in Evolution and Genetics in Honor of Theodosius Dobzhansky” (M. K. Hecht and W. C. Steere, eds.), pp. 437-543. Appleton, New York. Caspari, E. 1967. I n “Behavior-Genetic Analysis” (J. Hirsch ed.), pp. 3-9. McGrawHill, New York. Cooch, F. G., and Beardmore, J. A. 1959. Assortative mating and reciprocal difference in the Blue Snow Goose complex. Nature (London) 183, 1833-1834. Crossley, S. 1970. Mating reactions of certain mutants. Drosophila lnf. Serv. 45, 170. Crossley, S. 1974. Changes in mating behavior produced by selection for ethological isolation between ebony and vestigial mutants of Drosophila melanogaster. Evolution 28, 631-647. Crossley, S., Zuill, E. 1970. Courtship behaviour of some Drosophila melanogaster mutants. Nature (London) 225, 1061-1065. Crumpacker, D. W., and Marinkovic, D. 1967. Preliminary evidence of cold temperature resistance in Drosophiln pseudoobscura. A m . Nat. 101,505-514. De Fries, J. C., and McClearn, J. C. 1973. “Introduction to Behavioral Genetics.” Freeman, San Francisco, California. Del Solar, E. 1968. Selection for and against gregariousness in the choice of oviposition sites by Drosophih pseudoobscura. Genetics 58,275-282. Del Solar, E., and Palomino, H. 1966. Choice of oviposition sites in Drosophila melanogaster. A m . Nat. 100, 127-133. De Souza, H. M. L., da Cunha, A. B., and Dos Santos, E. P. 1970. Adaptive polymorphism of behavior evolved in laboratory populations of Drosophila willistoni. A m . Nat. 104, 175-189. Dobzhansky, T. and Spassky, B. 1967. Repeated mating and sperm mixing in Drosophila pseudoobscura. A m . Nat. 101,527-533. Dobzhansky, T., and Spassky, B. 1969. Artificial and natural selection for two behavioral traits in Drosophila pseudoobscura. Proc. Natl. Acad. Sci. U S A . 62, 75-80.

Dobzhansky, T. Pavlovsky, O., and Ehrman, L. 1969. Transitional populations of Drosophila paulistorum. Evolution 23, 482-492. Ehrman, L. 1961. The genetics of sexual isolation in Drosophila paulistorum. Genetics 46, 102.5-1038.

Ehrman, L. 1964. Genetic divergence in M. Vetukhiv’s experimental populations of Drosophila pseudoobscura. I. Rudiments of sexual isolation. Genet. Res. 5, 150-157.

Ehrman, L. 1965. Direct observation of sexual isolation between allopatric and between sympatric strains of the different Drosophila paulistorum races. Evolution 19, 459-464.

Ehrman, L., and Parsons, P. A. 1976. “The Genetics of Behavior.” Sinauer Assoc., Sunderland, Massachusetts. Ewing, A. W. 1964. The influence of wing area on the courtship behaviour of Drosophila mehnogaster. Anim. Behav. 12, 316-320. Ewing, A. W., and Bennet-Clark, H. C. 1968. The courtship songs of Drosophila. Behaviour 31, 288-301.

BEHAVIOR AND EVOLCTION IN

Drosophila

29

Fisher, R. A. 1930. “The Genetical Theory of Natural Selection.” Oxford Univ. Press (Clarendon), London and New York. Fuller, J. L., and Thompson, W. R. 1960. “Behavior Genetics.” Wiley, New York. Gibson, J. B., and Thoday, J. M. 1962. Effects of disruptive selection. VI. A second chromosome polymorphism. Heredity 17, 1-26. Grossfield, J. 1966. VI. The influence of light on the mating behavior of Drosophila. Univ. Tex. Publ. 6615, 147-176. Grossfield, J. 1971. Geographic distribution and light dependent behavior in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 68, 2669-2673. Grossfield, J. 1972. The use of behavioral mutants in biological control. Behav. Genet. 2, 311-319. Grossfield, J. 1975. Behavioral mutants of Drosophila. In “Handbook of Genetics” (R. C. King, ed.), Vol. 3, pp. 679-702. Plenum, New York. Grossfield, J., and Parsons, P. A. 1975. Locality records for some endemic Australian Drosophilidae based on winter collections. Proc. R . SOC.Victoria 87, 235-238. Hadler, N. M. 1964. Heritability and phototaxis in Drosophila melnnogaster. Genetics 50, 1269-1277. Hay, D. A. 1972. Recognition by Drosophiln melanogaster of individuals from other strains or cultures: Support for the role of olfactory cues in selective mating? Evolution 26, 171-176. Hay, D. A. 1975. Strain differences in the maze-learning abilities of D. melanogaster. Nature (Lolldon) 257, 44-46. Hershberger, W. A., and Smith, M. P. 1967. Conditioning in Drosophila melanogaster. Anim. Behav. 15, 259462. Hirsch, J. 1963. Behavior genetics and individuality understood. Science 142, 1436-1442. Hirsch, J., ed. 1967. “Behavior-Genetic Analysis.” McGraw-Hill, New York. Hoenigsberg, H. F., Chejne, A. J., and Hortobagji-German, E . 1966. Preliminary report on artificial selection towards sexual isolation in Drosophila. Z. Tierpsychol. 23, 129-135. Hotta, Y.,and Benzer, S. 1972. Mapping of behaviour in Drosophila mosaics. Nature (London) 240, 527-535. Isono, K., and Kikuchi, T. 1974. Autosomal recessive mutation in sugar response of Drosophila. Nature (London) 248, 243-244. Jacobs, M. E . 1960. The influence of light on mating of D . melanogaster. Ecology 41, 182-188. Kaneshiro, K. Y., Carson, H. I,., Clayton, F. E., and Heed, W. B. 1973. Nirhe separation in a pair of homosequential Drosophila species from the island of Hawaii. A m . Nat. 107, 766-774. Kaul, D., and Parsons, P. A. 1965. The genotypic control of mating speed and duration of copulation in Drosophila pseudoobscura. Heredity 20, 381-392. Kessler, S. 1966. Selection for and against ethological isolation between Drosophila pseudoobscura and Drosophila persimilis. Evolution 20, 634645. Kikuchi, T. 1973. Genetic alteration of olfactory functions in Drosophila melanogaster. J p n . J . Genet. 48, 105-118. Kircher, H. W., Heed, W. B., Russell, J. S., and Grove, J . 1967. Senita cactus alkaloids : their significance to Sonoran desert Drosophila ecology. J. Insect. Physiol. 13, 1869-1874. Knight, G. R., Robertson, A., and Waddington, C. H. 1956. Selection for sexual isolation within a species. Evolution 10, 14-22.

30

P. A. PARSONS

Koopman, K. F. 1950. Natural selection for reproductive isolation between Drosophila pseudoobscura and Drosophila persimilis. Evolution 4, 135-148. Koref-Santibaiiez, S., and Waddington, C. H. 1958. The origin of sexual isolation between different lines within a species. Evolution 12, 485-493. Leonard, J. E., Ehrman, L., and Pruzan, A. 1974. Pheromones as a means of genetic control of behavior. Annu. Rev. Genet. 8, 179-193. Levins, R. 1968. “Evolution in Changing Environments: Some Theoretical Explorations.” Princeton Univ. Press, Princeton, New Jersey. Levins, R. 1969. Thermal acclimation and heat resistance in Drosophih species. Am. Nat. 103, 483-499. McDonald, J., and Parsons, P. A. 1973. Dispersal activities of the sibling species Drosophila melanogaster and Drosophila simulans. Behav. Genet. 3, 293-301. McKenzie, J. A. 1975. The influence of low temperature on survival and reproduction in populations of Drosophila melanogaster. Aust. J . Zool. 23, 237-247. McKenzie, J. A., and Parsons, P. A. 1972. Alcohol tolerance: An ecological parameter in the relative success of Drosophila melanogaster and Drosophila simulans. Oecologia 10, 373-388. McKenzie, J. A,, and Parsons, P. A. 1974a. The genetic architecture of desiccation resistance in populations of Drosophila melanogaster and Drosophila simulans. Aust. J . Biol. Sci. 27, 441-450. McKenzie, J. A., and Parsons, P. A. 1974b. Numerical changes and environmental utilization in natural populations of Drosophila. Aust. J . Zool. 22, 175-187. McKenzie, J. A,, and Parsons, P. A. 1974c. Microdifferentiation in a natural population of Drosophila melanogaster to alcohol in the environment. Genetics 77, 385-394.

Mainardi, M. 1968. Gregarious oviposition and pheromones in Drosophila melanogaster. Boll. Zool. 35, 135-136. Manning, A. 1967. Pre-imaginal conditioning in Drosophila. Nature (London) 216, 338-340.

Mather, K. 1955. Polymorphism as an outcome of disruptive selection. Evolution 9, 52-61. Maynard Smith, J. 1962. Disruptive selection, polymorphism and sympatric speciation. Nature (London) 195, 60-62. Mayr, E. 1963. “Animal Species and Evolution.” Harvard Univ. Press, Cambridge, Massachusetts. Mayr, E. 1974. Behavior programs and evolutionary strategies. Am. Sci. 62, 650-659. Mayr, E., and Dobzhansky, T. 1945. Experiments on sexual isolation in Drosophila. IV. Modification of the degree of isolation between Drosophila pseudoobscura and Drosophila persimilis and of sexual preferences in DrosophiEa prosaltans. Proc. Natl. Acad. Sci. U8.A. 31, 75-82. MBdioni, J. 1962. Contribution Zt 1’6tude psycho-physiologique et g6nBtique du phototropisme d’un insecte Drosophila melanogaster Meigen. Bull. PsychoE., Paris 16, (2) 8. Merrell, D. J. 1953. Selective mating as a cause of gene frequency changes in laboratory populations of Drosophila melanogaster. Evolution 7, 287-296. Moray, N., and Connolly, K. 1963. A possible case of genetic assimilation of behaviour. Nature (London) 199, 35g360. O’Donald, P. 1959. Possibility of assortive mating in the Arctic Skua. Nature (London) 183, 1210-1211.

BEHAVIOR AND EVOLUTION IN

Drosophila

31

Parsons, P. A. 1961. Fly size, emergence time and sternopleural chaeta number in Drosophila. Heredity 16, 455-473. Parsons, P. A. 1965. Assortative mating for a metrical characteristic in Drosophila. Heredity 20, 161-167. Parsons, P. A. 1967a. “The Genetic Analysis of Behaviour.” Methuen, London. Parsons, P. A. 1967b. Behaviour and random mating. Experienlia 23,585. Parsons, P. A. 1970. Genetic heterogeneity in natural populations of Drosophila melanogaster for ability to withstand desiccation. Theor. Appl. Genet. 40, 261-266. Parsons, P. A. 1972. Variations between strains of Drosophila melanogaster and D . simulans in giving offspring in interspecific crosses. Can. J . Genet. Cytol. 14, 77-80. Parsons, P. A. 1973. “Behavioural and Ecological Genetics: A Study in Drosophila.” Oxford Univ. Press (Clarendon), London and New York. Parsons, P. A. 1974. Male mating speed as a component of fitness in Drosophila. Behav. Genet. 4, 395-404. Parsons, P. A. 1975a. The comparative evolutionary biology of the sibling species, Drosophila melanogaster and D . simulans. Q. R e v . Biol. 50, 151-169. Parsons, P. A. 1975b. Phototactic responses along a gradient of light intensities for the sibling species Drosophila melanogaster and Drosophila simulans. Behav. Genet. 5, 17-25. Parsons, P. A. 1975c. The effect of temperature and humidity on the distribution patterns of Drosophila inornata in Victoria, Australia. Environ. Entomol. 4, 961-964. Parsons, P. A. 1975d. Quantitative variation in natural populations. Genetics 79, 127-136. Parsons, P. A., and Bock, I. R. 1976. Drosophilidae (Diptera: Insecta) in the Otway Region of Victoria: Species diversity. Proc. R . Sac. Victoria (in press). Parsons, P. A,, Bock, I. R., Hay, D. A., and Grossfield, J. 1976. Endemic Drosophila species in Victoria, Australia : Habitats, ecological factors and evolutionary implications. Drosophila Inf. Serv. (in press). Petit, C., and Ehrman, L. 1969. Sexual selection in Drosophila. Evol. Biol. 3, 177-223. Prout, T. 1971a. The relation between fitness components and population prediction in Drosophila. I. The estimate of fitness components. Genetics 68, 127-149. Prout, T. 1971b. The relation between fitness components and population prediction in Drosophila. 11. Population prediction. Genetics 68, 151-167. Pruzan, A., and Ehrman, L. 1974. Age, experience, and rare-male mating advantages in Drosophila pseudoobscura. Behav. Genet. 4, 159-164. Quinn, W. G., Harris, W. A., and Benzer, S. 1974. Conditioned behavior in Drosophila melanogaster. Proc. Natl. Acad. Sci. U S . A . 71, 708-712. Rockwell, R. F., and Seiger, M. B. 1973. Phototaxis in Drosophila: A critical evaluation. Am. Sci. 61, 339-345. Spiess, E. B., Langer, B., and Spiess, L. D. 1966. Mating control by gene arrangements in Drosophila pseudoobscura. Genetics 54, 1139-1149. Spieth, H. T. 1952. Mating behavior within the genus Drosophila (Diptera). Bull. Am. Mus. Nut. Hist. 99, 401-474. Spieth, H. T. 1958. Behavior and isolating mechanisms. In “Behavior and Evolution” (A. Roe and G. G. Simpson, eds.), pp. 363-389 Yale Univ. Press, New Haven, Connecticut. Spkth, H. T. 1974a. Courtship behavior in Drosophila. Annu. R e v . Entomol. 19, 385-405.

32

P. A. PARSONS

Spieth, H. T. 1974b. Mating behavior and evolution of the Hawaiian Drosophila. I n “Genetic Mechanisms of Speciation in Insects” (M. J. D. White, ed.), pp. 94101. A.N.Z. Book Company, Sydney. Sturtevant, A. H. 1915. Experiments on sex recognition and the problem of sexual selection in Drosophila. J . Anim. Behav. 5, 351-366. Thoday, J. M. 1964. Genetics and the integration of reproductive systems. Symp. R . Entomol. SOC.London 2, 108-119. Throckmorton, L. 1975. The phylogeny, ecology, and geography of Drosophila. In “Handbook of Genetics” (R. C. King, ed.), Vol. 3, pp. 421-469. Plenum, New York. Wallace, B. 1954. Genetic divergence of isolated populations of Drosophila melanogaster. Caryologk 6, Suppl., 761-764. Wallace, B. 1968. “Topics in Population Genetics,” Norton, New York. Wallace, B. 1974. In a review of “Behavioural and Ecological Genetics: A Study in Drosophila” by P. A. Parsons, Oxford University Press, New York. Bioscience 24, 467.

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

Recommend Documents