Advances in Genetics Volume 14

ADVANCES IN GENETICS VOLUME 14 Edited by

E. W. CASPARI Department of Biology University of Rochester Rochester, N e w York

1968

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0

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CONTRIBUTORS TO VOLUME 14 R. W. ALLARD, Departments of Agronomy and Genetics, University of California, Davis, California WILLIAMK. BAKER,Department of Biology, University of Chicago, Chicago, Illinois

OSWALDHESS,* Max Plan&-Znstitut fur Biologie, Tubingen, Germany S. K. JAIN, Department of Agronomy, University of California, Davis, Calif orniu GUNTHERF. MEYER,Max Planclc-Znstitut fur Bwlogie, Tubingen, Germany

ANIL SADGOPAL? Division of Biology, California Institute of Technology, Pasadena, California J. SCHWEMMLE, Botanical Institute, University of Erlangen-Nurnberg, Germany

HAROLD H. SMITH,Biology Department, Brookhaven National Laboratory, Upton, New York

P. L. WORKMAN, Department of Agronomy, University of California, Davis, California

* Present address : Zoologisches Institut der Universitat, Freiburg. Germany.

t Present address: Molecular Biology Laboratory, Tata Institute of Fundamental Research, Colaba, Bombay, India. V

RECENT CYTOGENETIC STUDIES IN THE GENUS Nicotiana Harold H. Smith Biology Department, Brookhaven National Laboratory, Upton, New York

I. Introduction 11. The Species 111. Euploidy .

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

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

Pairing in Interspecific Hybrids . . . . . . . V. Allopolyploids . . . . . . . . . . . . . . . . VI. Aneuploidy and Multiple Genomes . . . . . . . . . . VII. Interspecific Recombination, Introgressive Hybridization, and Disease Resistance . . . . . . . . . . . . . . . . . VIII. Genetic Tumors . . . . . . . . . . . . . . . . IX. Cytogenetic Instability in Species Hybrids . . . . . . . . X. Cytoplasmic Inheritance: Male Sterility and Plastids . . . . . XI. Inheritance of Alkaloids . . . . . . . . . . . . . XII. Biometrical Studies . . . . . . . . . . . . . . . XIII. Miscellaneous . . . . . . . . . . . . . . . . XW.Summary . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

IV.Chromosome

. .

.

.

. . . . . . . .

1 2 2 5 6 8

15 22

27 30 34

38 41 42 43

1. Introduction

The genus Nicotiuna has been a favored material for studies on inheritance and evolution in higher plants since the days of the preMendelian hybridizers. Two features of the genus have had a profound influence on the type of investigations undertaken: (1) The species have evolved into a broad spectrum of different degrees of divergence in cytogenetic and morphological systems and therefore provide rich material for studying general problems on the origin and interrelationships among plant species as well as certain specific phenomena of frequent occurrence in interspecific Nicotiana hybrids such as cytogenetic instabilities and tumor formation. (2) The genus contains the cultivated species N . tubacum, which, because of its commercial value, has been the object of extensive studies on cytogenetic relationships with progenitor species, introgressive hybridization to incorporate disease resistance, and studies on the inheritance of alkaloids, quantitative traits, and cytoplasmic effects. 1

2

HAROLD H. SMITH

Cytogenetic studies on Nicotiana have been reviewed in the past by East (1928), Kostoff (1943), and Goodspeed (1954). The present review will deal mainly with work published in the late 1950’s and early 1960’s including reference to earlier publications where needed for background and clarity. II. The Species

A taxonomic monograph of the genus Nicotiana by Goodspeed, Wheeler, and Hutchison is included in Goodspeed’s book (1954). The genus is classified into three subgenera, fourteen sections, and sixty species in this definitive work. Forty-five of the species are indigenous to North or South America and fifteen, comprising the Suaveolentes section, to Australasia. I n a subsequent revision, Burbidge (1960) added five new species to the Australian group and changed N . stenoCarpa to N . rosulata. A minor revision has been suggested by Wells (1960), who found a continuous intergradation of N . palmeri with N . trigonophylla and hence questioned the validity of assigning species rank to N. palmeri. The 64 presently recognized species of Nicotiana are listed in Table 1 with the chromosome number of each. Nicotianu sanderae is not included in the table, since it is a horticultural species that originated as a hybrid between N . forgetiana and N . alata. The origins and evolution of species of the genus have been presented in detail by Goodspeed (1954) and Goodspeed and Thompson (1959), and these relationships have been summarized diagrammatically in the form of three phylogenetic arcs. In the first two arcs the genus is envisaged as derived from a pregeneric reservoir of related forms with six pairs of chromosomes and evolving into three complexes, at the 12-paired level, that are hypothetical precursors of the three modern subgenera. The third arc contains the present day species, represented with their various degrees of interconnection, a t the 12- and 24-paired chromosome level. I n summary, the evidence shows that interspecific hybridization, with subsequent amphiploidy as well as genetic recombination, has played an important role in the evolution of the genus Nico tiana . 111. Euploidy

Haploids, triploids, and tetraploids of a number of species of Nicotiana have been reported in the literature, and their incidence and meiotic behavior summarieed by Goodspeed (1954). Haploids are phenotypic replicas of the diploid parent on a reduced scale and are

CYTOGENETICS OF

Nicotiana

3

TABLE 1 Classification of the Genus Nicotiana*

Subgenus Rustica

Tabacum

Pelunioides

Section Paniculatae

Species

Authority

Graham Linnaeus Goodspeed Walpers Goodspeed Philippi Macbride Bitter ex GoodThyrsiflorae speed Rustics 9. rustica Linnaeus Ruiz and Pavon Tomentosae 10. lomenha 11. tomentosiformis Goodspeed Grisebach 12. oiophora 13. sefchellii Goodspeed Linnaeus 14. glutinosa Linnaeus Genuinae 16. tabacum Ruiz and Pavon Undulatae 16. undulata 17. arentsii Goodspeed Koch and Fintel18. wigandivides mann Dona1 Trigonophyllae 19. trigonnphylla Spegaazini and Alatae 20. sylvestris Comes Weinmann 21, langsdorfii Link and Otto 22. alata Hort. ex Hemsley 23. jorgetiana Lehmann 24. bonariensis Cavanilles 25. longiJEora 26. plumbaginifolia Viviani Willdenow ex Repandae 27. repanda Lehmann Brandegee 28. stocktonii Johnston 29. nesophila Noctiflorae 30. noctiftma Hooker (Grisebach) 31. petimioides Milltin Spegazzini 32. acaulis Spegeazini 33. ameghinoi Acuminatae 34. acuminata (Graham) Hooker 35. pauci$ora Rerny Torrey ex Watson 36. altenuata Philippi 37. longibracteata 38. miersii Remy 1. glauca 2. paniculata 3. knightiana 4. solanijolia 5. benavidesii 6 . cordijolia 7. r a i m d i i 8. thyrsiflora

Somatic chromosome number 24 24 24 24 24 24 24 24 48 24 24 24 24 24 48 24 48 24 24 24 18 18 18

18

20 20 48 48 48 24

24

24

?

24 24 24 1 24

4

HAROLD H. SMITH

TABLE 1 (Continued)

Subgenus

Section

Species 39. wrymbosa

Bigelovianae Nudicaulea Sauveolentea

40. linearis 41. spegazzini 42. higelovii 43. clevelandii 44. n d i m u l i s 45. benlhamiana 46. umbratica 47. cavicola 48. dehneyi

49. gossei 50. amplexicaulis 51. mam'tima 52. velutina 53. hespes-is 54. oecidentalis 55. simulans 56. megalosiphon 57. rotundifolia 58. excelsior 59. suaveolens 60. in gulba 61. exigua 62. goodspeedii 63. rosulata 64. fragrans

Authority Remy Philippi hfilkn (Torrey) Watson Gray Watson Domin Burbidge Burbidge Domin Domin Burbidge Wheeler Wheeler Burbidge Wheeler Burbidge Heurck and Muell. Lindley J. M. Black Lehmann J. M. Black Wheeler Wheeler (S. Moore) Domin Hooker

Somatic chromosome number 24 24 24 48 48 48 38 46 46

48

36 36 32 32 42 42 40 40 44 38 32 40 32

40

40 48

*After Goodspeed (1954).

almost completely sterile. A number of haploids of N. tubacum have been reported recently (Takenaka and Takenaka, 1956 ; de Nettancourt and Stokes, 1960; Burk, 1962; Dulieu, 1964); and also a subhaploid with 22 chromosomes, two less than a complete haploid set (Burk and Gerstel, 1961). Burk (1962) devised a method for detecting haploids through crossing recessive yg yg plants with normal green ones and picking out yg haploids as light yellowish-green seedlings. I n this way he recovered 0.09% maternal haploids rand 0.04% paternal. Haploids have also been selected on the basis of reaction to tobacco mosaic virus, and these are being doubled in chromosome number by use of colchicine to produce homozygous lines of tobacco varieties (Stokes, 1963). An F1 generation of 334 plants from a cross between an N. tabucum haploid, obtained from a twin seedling, and a diploid was examined

CYTOGENETICS OF

Nicotiana

5

cytologically by Rao and Stokes (1963). Meiosis in the haploid female was irregular, showed a pairing range of 0 to 5 bivalents, and produced female gametes of varying chromosome numbers (24 4 5). Four major categories of chromosomal types were recognized in the F1 ( n X 2n) ; trisomics, disomics (diploids), monosomics, and monotrisomics. Nearly half of the F1 progeny were monosomics.

IV. Chromosome

Pairing in Interspecific Hybrids

More than 300 interspecific hybrids have been reported in the genus Nicotiana (East, 1928; Kostoff, 1943; Goodspeed, 1954). Those that develop normally show, with few exceptions and regardless of the parental chromosome numbers, intermediate characteristics of leaf and flower (East, 1935; Kehr and Smith, 1952). Of these, 215 were analyzed cytologically by Goodspeed (1954) for meiotic behavior in the F1.The pairing relationships have been useful in analyzing phylogenetic relationships. Five categories of pairing were distinguished, based on the amount of conjugation a t first meiotic metaphase (MI): (1) “complete or almost complete” pairing (30 F1 hybrids) ; (2) “drosera scheme” pairing (50 F1 hybrids) ; (3) “highly variable” pairing (35 F1 hybrids), where the range is wide, from 1 or 2 pairs to almost complete pairing, and the mode is half the number of possible pairs; (4) “low variable” pairing, also with a wide range (M), but with a mode of no more than 2 or 3 pairs; (5) “minimum” pairing (70 F1 hybrids), in which the pairing range is 0-3 and the mode is commonly zero. Takenaka (1962a,b,c,d, 1963) has more recently reported pairing relationships in nine interspecific hybrids not included in Goodspeed’s studies. These are N . debneyi X N . glutinosa and N . otophora; N . knightzizna X N . rustica; N . panicukzta x N . longiflora and N . plumbaginifolia; and N . tabacum x N . jorgetiana, N . gossei, N . knightiana, and N . megalosiphon. The pairing of chromosomes in interspecific hybrids is of interest in studies of the evolution of the genus as well as in problems of transferring genes or chromosome segments from one species to another. In the former, the degree of pairing of chromosomes can be considered a measure of chromosome homology, and hence, as evidence of the degree of species relationships and their ancestral origin. I n the latter, the success of transferring desirable hereditary traits from wild t o cultivated species depends largely on pairing relationships of their chromosomes, The pairing relationships are difficult to analyze in hybrids, which show a low amount of variable pairing and in which the chromosomes cannot be distinguished cytologically. Sficas (1962) has devel-

6

HAROLD H. SMITH

oped probability distributions for the number of bivalents to be expected a t meiotic metaphase of interpsecific hybrids under the following conditions: all chromosomes vs. only certain ones pairing; specific vs. random associations; and with the same or different probabilities of pairing for the different chromosomes. These statistical tests were applied by Sficas and Gerstel (1962) to the bivalent frequency distribution observed at MI in the F1 hybrids N . g2utinosa X N . syZvestris, N . glutinosa X N . otophora, and N . tabacum X N . glutinosa, and in two haploids of N . tabacum. In general they concluded that, although the number of bivalents formed in the hybrids rarely exceeded 3, the number of possible metaphase associations was not limited to just a few; but rather that all 12 chromosomes are potentially capable of forming biparental pairs, but with unequal frequency. Sficas and Gerstel pointed out that this statistical technique may be used to investigate phylogenetic relationships in Nicotiana and be applied to the problem of the basic chromosome number of the genus. Since pairing of chromosomes assures their distribution to the poles in equal numbers, in species hybrids or other organisms where a variable number of chromosomes remain unpaired a t MI, their distribution to the poles is variably unequal. Sficas (1963) developed a probability distribution for testing the randomness of movement of univalents where a variable amount of meiotic pairing occurs. Applying it t o the three Nicotiana hybrids listed in the preceding paragraph, he found a tendency toward an equal, rather than a random, distribution of unpaired chromosomes to the poles. A possible explanation for this is that some chromosomes tend to come together without forming chiasmata; then move to opposite poles. V. Allopolyploids

Allopolyploidy, and particularly amphiploidy, has played an irnportant role in the evolution of the genus Nicotiana. Eleven species with 24 pairs of chromosomes, and hence of probable amphiploid origin, are known today. I n addition 18 of the 20 Australian species represent probable aneuploid derivations of the 24-paired polyploid level (Goodspeed, 1954). A large number of amphiploids have been produced in experiments with Nicotiana, either spontaneously following interspecific hybridization or by artificial means, notably with colchicine. The frequency of multi- vs. bivalent chromosome associations in amphiploids varies with the degree of homology between parental chromosomes, and is reflected in the fertility and variability among progeny.

CYTOGENETICS OF

Nicotiana

7

The main tobacco species of commerce, N . tabacum, is of amphiploid origin and much interest and research has centered on the relationships of the present day tobacco to its putative wild progenitors. The original evidence of Goodspeed and Clausen (1928) was interpreted to indicate that N . tabacum arose from chromosome doubling following hybridization between a progenitor of N . sylvestris (S’ genome, n = 12) and a member of the Tomentosae section, either N . otophora, N . tomentosiformis or, more likely, an ancestral type similar to, but not identical with, either of these present day species (T’ genome, n = 12). I n order to test genetically whether the chromosomes of N . tomentosiformis or those of N . otophora are more nearly homologous with the T genome of N . tabacum Gerstel (1960) performed the following experiments. Several N. tabacum (S S T T ) stocks with altogether six recessive genes marking five chromosomes were crossed to each of the two diploid species carrying corresponding dominants. The Fl’s ( 3 X ) chromosome numbers were doubled to form 6 X polyploids (T’T’TTSS) and these in turn were backcrossed to recessive tester stocks of N . tabacum. The observed segregation frequencies, therefore, reflect directly the gametic output of the duplex (ZZzz) amphiploid (T’T’TT) genomes. Few or no recessive segregants would be expected if the parental T’ and T genomes behaved as typical allopolyploids, whereas ratios of 5 : 1 (or 3.7 : 1 as a limiting value with chromatid segregation) would be expected if homologies between chromosomes of the T’ and T genomes caused multivalent associations. The observed results were that 6X ( N . tabacum X N . tomentosiformis) gave consistently smaller ratios than 6 X ( N . tabacum X N . otophora). This is an indication that N . tomentosiformis is the more closely related to N . tabacum in chromosome homology. However, additional complications occurred in that the ratios were generally even smaller than 3.7 : 1 (indicating elimination of the dominant gene). Yang (1964) found that there was not a sufficient amount of chromosome elimination to explain the small ratios and concluded that still other factors must be involved. Similar tests, using the amphiploid segregation technique, were carried out by Gerstel (1963, 1966) to explore homologies between chromosomes of the S genome of N . tabacum and those of N . sylvestris (S’ genome), the only extant relative of the form from which the S genome of tobacco is derived. Pronounced differences were observed in the segregation ratios among the characters used to mark eight of the chromosomes. From these results it was concluded that some chromosomes of the two species have remained completely homologous while others have become differentiated to some degree during evolution.

8

HAROLD H. SMITH

In order to test if preferential pairing is determined by the homologies of the individual chromosomes, the amphiploid segregation technique was used by Gerstel (1961) with an N . tabacum parent in which one of the pairs of chromosomes was substituted from the taxonomically distant N . glutinosa. The other parent in the synthetic amphiploid was N . tomentosiformis. Characteristically, segregates for duplex loci in N . tabacum X N . tomentosiformis amphiploids give a gametic output of about 3 : 1, but for the factor located on the substituted glutinosa chromosome, the output was found to be 59 : 1. This result suggests that preferential pairing is a property of the individual chromosome and not of the genotype as a whole. VI. Aneuploidy and Multiple Genomes

The monosomics of Nicotiana tabacum are aneuploid types of particular current interest since they provide material for a rapid method of locating genes on specific chromosomes. These monosomics have arisen spontaneously (Clausen and Goodspeed, 1926a), as derivatives from hybridization of N . tabacum and N . sylvestris (Clausen and Goodspeed, 1926b), and by use of a genetically controlled asynaptic condition (Clausen and Cameron, 1944). The 24 monosomic lines, which were assembled largely through the efforts of the late Prof. R. E. Clausen, have been characterized on the basis of their most readily identifiable features by Cameron (195913). They are listed in Table 2 and described according to their appearance on the genetic background of Red Russian tobacco. One or two of the lines may not be primary types (Cameron, 1952). The listing of genes associated with the monosomic chromosomes was kindly supplied in correspondence by Dr. D. R. Cameron. All primary trisomic types have been identified in only one species of NicoCiana, N . sylvestris, n= 12 (Goodspeed and Avery, 1939, 1941). Eight of the nine possible trisomics have been identified in N . Zangsdorfii (Abraham, 1947; Lee, 1950; Smith, 1943, unpublished. In both of these species the trisomic types are readily distinguishable from the diploid in morphological features of plant, leaf, and flower. Aneuploidy, as well as amphiploidy, has played a part in the evolution of the genus Nicotiana as shown by the occurrence of 9- and 10paired species in the Alatae section and 16- to 23-paired species in the Suaveolentes section (Table 1). The fornier are considered to have resulted from chromosomal loss from the 12-paired level and the latter from the 24-paired level. The importance of aneuploidy in plant evolution in general may be judged by the great diversity in chromosome

CYTOGENETICS OF

Nicotiana

9

numbers in the kingdom that are not readily interpretable as due to polyploidy alone. Aneuploidy may offer an opportunity for finer adjustment in evolution through loss of certain chromosomes, a t least a t higher levels of ploidy, rather than simply by addition. An example is afforded by the cross N . bigelovii ( n E 24) x N . glutinosa ( n = 12) in which the F1 and amphiploid show heterosis of the flower (Kehr and Smith, 1952). When the amphiploid was backcrossed to N . bigelovii, and the interspecific triploid self-pollinated, segregants were produced which exceeded the amphiploid in flower size. I n an effort t o stabilize the transgressive character of these exceptional segregants, they were crossed with the amphiploid to establish disomes of N . glutinosa. Relatively uniform populations were established (Smith, 1964) which exceeded by as much as 30% the flower size of the heterotic amphiploid. These populations had 62-64 chromosomes; that is, 48 from N . bigelovii plus 7 or 8 extra pairs from the 12 pairs of N . glutinosa chromosomes originally added in the amphiploid. Trispecies combinations in Nicotiana have been made by crossing an amphiploid with a third species. Twelve different hybrids in which three species were combined were reported by various workers between 1931 and 1945 (Krishnamurty et al., 1960), 18 more in 1952 by Kehr and Smith, seven by Krishnamurty et al. (1960), and an addit,ional six by Smith and Abashian (1963). Hybrids in which the genomes of four different species are combined have also been reported (Kehr and Smith, 1952; Appa Rao and Krishnamurty, 1963; Smith and Abashian, 1963) . The trispecies hybrid N . tabacirm X ( N . glutinosa-trigonophy lla) and its reciprocal showed the expected somatic chromosome number of 48, but morphological differences were noted between the reciprocal crosses (Krishnamurty et al., 1960). This trispecific hybrid was treated with colchicine and although most of the affected plants had the full doubled chromosome number (96), three out of 15 studied showed chromosomal elimination of varying numbers (Appa Rao and Krishnamurty, 1963). I n the combination of N . glutinosa and N . glutinosutrigonophylla with N . megalosiphon chromososmes were frequently eliminated in hybrid plants (Krishnamurty and Satyanarayana, 1962 ; Satyanarayana and Subhashini, 1964) and this general phenomenon of cytogenetic instability in species hybrids of Nicotkna will be discussed in a subsequent section. An early effort to explore the limits and consequences of multiple aIlopolyploidy showed chromosome crowding and loss with consequent production of aneuploid types. An allopolyploid was produced (Kehr and Smith, 1952) which combined the genomes of three distantly related amphiploid species: N . bigelovii (n= 24, North America), N .

TABLE 2 The Monosomic Types of Niwtkzna tabacum* Designatior

c1

0

Distinguishing charscteristics -.

Plant height

Associated genes

Pollen

Monosome

Essentially Smaller,basal constrio 48.643.1 ,somewhat normal paler in color, fading tion more proearlier nounced

Medium small

hfl, hairy filaments; pa, asynaptic

Very small

MI, many leaves; Pp, purpleplant; yb,, yellow burley; Pb, purple buds, N. otophoraS

Leaves

1. Haplo-A

Somewhat below normal

2. Haplo-B

Subnormal, sparsely Smaller,narrow, basal constriction less branched abrupt, auricles strongly reduced

'lowers (length in mm)j

53.6-41 .O, more strongly Essentially

bent, color darker

normal

cd,crinkled dwarf; Zf, light filaments; wh, white flower; Wh-P, pale; wc,white center, N. dophora$

3. Haplo-C

Often taller than normal, longer internodes

Narrow, basal eon5 8 . M . 7 , longer and broader, color paler striction less abrupt in tube and throat

Marked abortion

Medium small

4. Haplo-C

Normal but maturity delayed

Brighter green in young plants, leaf base semibroad

Essentially normal

Incorporated in a fsl, fasciated trivalent in about 50% PMC

50.6-39.7, slightly

reduced in size

x

a,

E

2 IZ

5. Haplo-E

Subnormal

Smaller, constriction less abrupt

51.3-41.1, calyx inflatec

Essentially normal

Very small

6. Haplc-F

Subnormal, shorter internodes

Small, more erect

44.5-37.5, distinctly

Moderate abortion

Large with char- co, coral flower; acteristic medimml, maman constriction moth; snl, spontaneous necrosis

7. Haplo-G

Subnormal, meager Small with rounded tips, basal constricinflorescence, mation pronounced turity delayed

55.6-42.3, tapering

Variable aa to cy- Large toplasmic rontent but few grains completely aborted

tg, tinged; vb,

Smll, narrow, basal Normal but stems constriction less and branches den, der, reduced pronounced branching

51.8-38.3, narrow tube,

High abortion bui Medium large variable as to contents

Nc, necrotic.

8. Haplo-H

shorter limb fluted

gradually to limb, style short. Capsules small and poorly filled

limb reduced, calyx lobes pointed

veinbanding; wsl, white seedling

N . glutinosaz; td, toadskin

9. Haplo-I

Normal slender branches, delayed maturity

Small, more sharply pointed

53.1-41.2, corolla lobes

Low abortion but Very small dimorphic

ccl, cata-

10. Haplo-J

Subnormal, maturity delayed, leaves small, narrow

Small and narrow

53.2-42.6, limb charac-

High abortion, sharp distinction between stainable and aborted grains

cy, calycine); Zc, larerates

pointed. Capsules long, narrow, poorly filled, calyx inflated

teristically wavy a t maturity, color less intense. Capsules small, poorly filled

Medium

corolla); rd, red modifier

(Continued)

38

E i 6 02

$

3:

s. %

ij.

TABLE 2 Designation

(Con+nued)

c. N

Distinguishing charrtcteristies Plant height

11. Hap1o-E

Subnormal, maturity delayed

12. Haplo-I

Above normal, stem heavy, maturity somewhat delayed

Leaves Semibroad at base

'lowers (length in mm).

Pollen

Monosome

Associated genes

48.3-36.7, tube short, infundibulum propor tionately longer, anthem small with delayed dehiscence

Low abortion, dimorphic

Very large, medianly constrictec (cf. Clausen anc Cameron, 1944:

8.6-37.1, tube shorter and broader, color distinctly paler

High abortion, variable in size

Large with promi. at, Ambalema nent constrictdl;gb, green buds; Tr, tube tion retarder, N. setchellii$ Ap, apetalous; Rf,ruffled

SubnormalJ branching a t the base

Large, basal constric- 53.9-40.7, color fades to a purplish hue at tion less pronounce( maturity, calyx conspicuously longer

Medium large, High abortion, characteristivariable in concally ovoid tent

14.Haplo-P

Distinctly subnormal, short internodes, compact inflorescence

Small, erect

43.9-34.0, visibly smaller, color darker red

Low abortion, dimorphic

Large with median constriction (cf. Haplo-F)

mm2, mammoth; sn,, spontaneous necrosis

15. Haplo4

Close to normal

Slightly smaller, basal constriction more pronounced

49.2-39.5, size reduced, paler in color, stamen and pistils slightly ex

Low abortion

Medium large

hf,, hairy fila-

13.Haplo-k

ments; gbr, yellow burley

5

8 x

m

g

3

X

serted, pollen shedding delayed, capsuli small and poorly fillec 16. Haplo-P

Normal, maturity delayed

Small, tips rounded, semibroad at base

49.3-37.5, limb narrow, corolla lobes leas pro nounced. Capsules small and poorly filled 54.8-38.8, tube longer, limb spread reduced. Capsules pointed, small and poorly filled

Marked abortion subnormal grains variable in size

Medium large Br, broad; Fsz, with characterfasciated; pk, istic subtermpink flower; inal constrictior sg, stigmatoid

Very high abortion, sharply divided into two classes

Medium

17. Haplo-Q Reduced, little branching, maturity delayed

Narrow, basal constriction pronounced, auricles strongly reduced, ruffled

18. Haplo-R Subnormal, thick stems, profusely branched

Small, darker green, auricles reduced

49.2-41.9, enlarged infundibulum, wide throat, color paler

High abortion, bu completely empty grains rare

Very large

mi2, mosaic tol-

19. HapleS Normal, maturity usually retarded

Lighter green surface smooth

47.3-41.7, color more vivid, stamens and pistils exserted, poller shedding delayed

Low abortion, grains variable in size

Large, frequently associatedwith a bivalent

cl, chimeral; yg, yellowish green

20. Haplo-'I Subnormal, matu-

Small, darker green, basal constriction elongated

56.e41.7, tube longer, High abortion merging gradually into t h e infundibulum, stamens and pistils relatively short. Capsules small poorly filled

rity delayed

Large, usually with a welldefined constriction

-

erant; Pd, petioloid

white seedling

WSa,

(COntinUed)

TABLE 2 (Continued) Designation

w

Distinguishing characteristics

Pollen

Monosome

Plant height

Leaves

21. Haplo-T

Subnormal, bushy

Large, frequently with a pronounced petiole

High abortion, 8.5-40.9, corolla lobes aborted grains acutely pointed, tub1 variable in size pale, l i b and throai strongly colored

Medium large

22. Haplo-\

Subnormal

Small, basal constrio tion less abrupt, auricles reduced

47.S39.8, tube stout

High abortion, visibly so in freshly opened %owem

Medium large

Long, narrow, sharply pointed, auricles reduced

52.8-40.8, color lighter, High abortion,

23.Haplo-m Subnormal, elonga-

ted internodes, sparsely branched maturity delayed

'lowers (length in mm)'

pollen scanty, sometimes lacking in earlj flowers

aborted grains small

Associated genes

Large, but PMC frequently unobtainable during early flowering

24. Haplo-Z Normal, maturity conspicuously delayed

Small, basal constric- 52.W.3, style tends to tion less pronounced be curved, limb auriclesless ruffled frequently fails to open fully

Abortion very Large high, sharp dir, tinction between normal and aborted grains

*After Cameron (1959b). t The flower measurements are averages of 10 representative flowers and show tube length-limb spread, both meaaured in millimeters. These are to be compared with normal values of about 53-43. $ Transfers to N.labacum from other species. 8 Not clear-cut distinct characters.

A

x

CYTOGENETICS OF

Nicotiana

15

debneyi ( n = 24, Australia), and N . tabaeum ( n = 24, South America),

In an individual which contained the full doubled complement of 144 chromosomes only bivalents were formed, but laggards were observed in metaphase and anaphase stages of meiosis. The progeny were variable in appearance and fertility and were continued through 10 generations by self-pollination accompanied by selection for three distinctly different complexes of morphological characteristics (Smith et al., 1958). By the tenth inbred generation each selected race had become relatively uniform in appearance and the pollen was over 90% fertile in typical plants. I n each race there had been a loss of different chromosomes from the original 144 to 108 -+ 6. It may be of some significance that the genomes became relatively stabilized a t approximately the “9-fold” level (9 x 12) of ploidy. The phenomenon observed in this multiple allopolyploid would seem to offer opportunities for exploitation in the evolution of some plant groups in that there is wide variability in early generations without the serious loss in fertility usually associated with species hybridization a t the diploid level. VII. lnterspecific Recombination, lntrogressive Hybridization, and Disease Resistance

The foregoing sections have dealt mainly with problems of altered chromosome number, and have also served to introduce the subject of interspecific recombination through discussion of pairing relationships among chromosomes in species hybrids and allopolyploids. This section will consider the problem of the transfer of genes between species, and subsequent establishment of new types with a parental chromosome number and containing genes introgressed from an alien species. The urgency to develop disease-resistant commercial tobacco by utilizing resistance found mainly or exclusively in wild species of the genus has been an impetus to studies of introgression. The problem of effecting recombination varies with the degree of genetic and cytological differentiation in the genomes of the parental species (Stephens, 1961). Surmounting barriers to crossability and fertility are initial steps to gene transfer. Sterility barriers may be chromosomal, genic (Ar-Rushdi, 1956; Cameron and Moav, 1957), cytoplasmic (Clayton, 1950; Burk, 1960; Smith, 1962b; Cameron, 1965; Hart, 1965), or due to a combination of causes. Some problems in the relative ease of gene trander and chromosomal substitution into N . t a b a c m from other Nicotiana species have been summarized by Chaplin and Mann (1961). The species that have provided sources of disease resistance are

16

HAROLD H. SMITH

sdhiently remote from N . tabacum so that in these intersectional hybrids the parental genomes show partial or complete failure of chromosome pairing. Such hybrids are highly sterile as a result of the various unbalanced chromosomal products of meiosis. The opportunity for interspecific recombination is slight. Doubled chromosome complements, before or after hybridization, can be obtained by using colchicine, or they may occur spontaneously, and a fertile amphiploid or sesquidiploid is produced. This is used as the starting material for a series of backcrosses to N . tabacum accompanied by selection for disease resistance. Ultimate success of such a program of controlled introgression requires that a chromosomal exchange take place and the gene block governing the inheritance of the desired characteristic of the nonrecurrent parent become incorporated in a N . tabacum chromosome (Mann et al., 1963). The first use of this method was in an effort to transfer a gene, which confers resistance t o tobacco mosaic virus by causing lesions of localized necrotic leaf tissue, from N . glutinosa to N . tabacum (Holmes, 1938). In the F1 N . tabacum ( n = 24) X N . glutinosa ( n = 12) pairing is variable with a modal number of three bivalents (Goodspeed, 1954). The amphiploid N . dig2uta (n = 36) shows preferential pairing, giving 36 bivalents and little distributional irregularity. The cross of tetreploid N . tabacum with diploid N . glutinosa yields an F1 with 24 pairs of N . tabacum chromosomes and 12 N . glutinosa chromosomes, with some limited trivalent production (Clausen and Cameron, 1957; K. A. Patel and Gerstel, 1961). Backcrosses t o N . tabacum lead to rapid elimination of N . glutinosa chromosomes, except for the one carrying the gene for necrotic resistance, which is maintained by selection. Continued self-fertilization of such strains led to the formation of a type of tobacco that bred true for the local-lesion type of mosaic resistance (Holmes, 1938). It was later shown that when a plant of this type was crossed with N . tabacum there was a failure of the chromosome carrying the resistance factor to pair (2311and 21) with a tobacco chromosome (Gerstel, 1943, 1945). An entire N . glutinosa chromosome had been substituted for the H chromosome of N . tabacum to produce a n “alien substitution” race. With continued backcrossing and self-fertilization of such strains, however, plants were eventually obtained which either bred true for mosaic resistance or segregated in typical Mendelian fashion: 3 necrotic resistant : 1 systemic susceptible. The necrosis factor had been transferred from the N . glutinosa chromosome to a chromosome that now contained a sector from N . tabacum of sufficient length to permit conjugation in a majority of cells with the homologue shown to be the N . tabacum H chromo-

CYTOGENETICS OF

Nicotiana

17

some (Gerstel, 1948). The mechanism of this controlled introgression through formation of a segmental chromosome following chromosomal exchange in an alien substitution heteroeygote has been discussed in publications of Gerstel and Burk (1960), K. A. Pate1 and Gerstel (1961), and Mann et al. (1963). It has been shown that even after many backcrosses to N . tabacum the factor for mosaic resistance or closely linked N . glutinosa genes cause significant effects on certain agronomic characters, including reduced yields (Chaplin et al., 1961). The success in transferring the N . glutinosa mosaic resistance factor t o commercial tobacco has now been realized with other diseases whose source of resistance is also found in species of Nicotkna remotely related to N . tabacum. Resistance t o a bacterial disease wildfire [Pseudomonas tabaci (Wolf and Foster) F. L. Stevens] has been reported in a number of species and N . Zongiflora was chosen by Clayton (1947) t o hybridize vith N . tabacum ( 4 n X 4n). Homoeygous wildfire-resistant lines were established and were stabilized after a series of backcrosses t o White Burley tobacco and selfing for five generations (Valleau, 1959; Clayton, 1958). A variety, called Burley 21, which is resistant to both wildfire and tobacco mosaic virus was released in 1955 (Heggestad et al., 1960). Resistance to each of these diseases showed completely dominant monogenic inheritance. Satisfactory resistance to blue mold (Clayton, 1958) has been found in N . debneyi but the mode of inheritance is neither monogenic nor completely dominant. After a number of backcrosses of an N . debneyitabacum allopolyploid t o N . tabacum the blue mold-resistant plants contained 24 N . tabacum bivalents plus a number of (+9) N . debneyi univalents. Apparently a sufficient number of segments of N . debneyi chromosomes have now been translocated to the N . tabacum genome to produce an acceptable commercial type resistant to blue mold (Lea, 1961). With regard t o three other diseases of tobacco (Table 3) the resistant varieties in current use derive their resistance from N . tabacum, but favorable sources have been found in remote species of the genus and the programs of transfer are in various stages of development. These diseases are black root rot [Thieluvia basicola (Berk. and Br.) Ferr], black shank [Phytophthora parasitica Dast. var. nicotianae (Breda de Haan) Tucker], and root knot nematodes (Meloidogyne spp.) (cf. Burk and Heggested, 1966). The transfer of black root rot resistance was delayed by the complete male sterility of the resistant lines in the third backcross when N . tabacum chromosomes were accumulated in a cytoplasm of N . debneyi (Clayton, 1950). Cytoplasmic effects will be discussed in a later section. The sterility was overcome by reversing the

TABLE 3 Examples of Genetically Controlled Disease Resistance Transferred into or Found in Ndiana tabacum Disease

Pathogen

Primary source of resistance

Inheritance

1. Resistance source mainly in

wild species Tobacco mosaic virus (TMV) Wildfire

Blue mold 2. Resistance source in wild species and N. tabacum Black root rot

Marmor taban’ Holmes PsaLdomonas tabaci (Wolf and Foster) F. L. Stevens Peronospora tabacina Adam Thielaviapsis basieola (Berk. and Br.) Ferr.

Black shank

Phytophthora parasitica (Dast.) var. nicdianae (Breda de Haan) Tucker

Root knot nematodes

Meloiclogyne sp.

3. Resistance source mainly in N. tabmm Granville wilt Fusarium wilt Powdery mildew

N . glutinosa N . longiflora

Monogenic, dominant Monogenic, dominant

N . debneyl

oligc- or polygenic, intermediate

E

N . debmyi

Monogenic, dominant

F

N . tabacum, T . I. 87,88,89 N . longi$ora and N. plumbqiniforia N . labacum, Florida 301 N . repanda N . t a b m m , T . I. 706

Polygenic, intermediate Monogenic, dominant

Pseudomanas solanoeearum (E. F. Smith) N . tabacum, T . I. 448A Fusarium oxysporium Wr. var. N . tabacum, cigar and aromatic types nicotianae Johnson Erysiphe cichoracearum D. C . N. tabmsm, kuo-fan

Oligogenic, intermediate

‘‘Bridged,’through N . sylvestris Monogenic, dominant

Polygenic, intermediate Polygenic, intermediate Digenic, recessive

8 P

8

CYTOGENETICS OF

Nicotiana

19

direction of the cross and the transfer of root rot resistance progressed so that homozygous resistant lines of White Burley were obtained (Clayton, 1958) and are currently being evaluated. Among the various species of Nicotiana found to be resistant to black shank, N . longiflot-awas selected by Valleau et al. (1960) and N . plumbaganifoh by Clayton (1958), Chaplin (1962), and Apple (1962) for a backcrossing and gene transfer breeding program. In the work initiated in 1951 in South Carolina, N . plumbaginifolia was used to pollinate a tetraploid of N . tabacum, this sesquidiploid was backcrossed three times t o N . tabacum, then selfed three times, each step accompanied by selection for black shank resistance. A highly resistant variety with favorable agronomic characteristics was established and is being used as source material for further plant breeding (Chaplin, 1962). Experiments by Cameron (1959a), Cameron and Moav (1957), and Ar-Rushdi (1957), began with the same sesquidiploid, N . tabacumtabacum-plumbaginifoliu and were carried out to study the mode of transference of genes for black shank resistance and other characters from N . plumbaginifoliu ( p ) to N . habacum. Backcrossing to diploid N. tabacum (t t ) accompanied by selection for immunity led rapidly to the establishment of a 24 ( t t ) bivalent plus one ( p ) univalent condition. I n one line all bivalents plus one trivalent were formed, indicating that an exchange had occurred between N . tabacum and N . plumbaginifoliu chromosomes. Subsequently, Moav (1958) studied 14 segmental substitution lines in which another factor from N . plumbaginifolia had been introduced into a chromosome of N . tabacum to determine which N . tabacum chromosomes were involved. He found that eight of the transfers were incorporated into the same N . tabacum chromosome, and the remaining six were equally distributed among three other N . tabacum chromosomes. Since this distribution is nonrandom i t supports the hypothesis that residual homology exists between the two species and was responsible for the interspecific exchanges. In this cross it is evidently possible t o have interspecific exchanges with an alien addition chromosome (trivalent formation). Some peculiarities and difficulties for transmission of genes between N . plumbaginifolia and N . tabacum are, however, indicated by ( 1 ) a gene located in the same chromosome as the black shank resistance factor that kills pollen with 24 N . tabacum chromosomes in the same quartet (Cameron and Moav, 1957) ; (2) variegation and cytological instability of introgressed N . plumbaginifolia chromosomes (Ar-Rushdi, 1957) ; and (3) low female transmission and aberrant segregation of the chromosome carrying black shank resistance (Cameron, 1959). Moav (1962) has suggested the use of autotriploidy as a means for increasing the rate of interspecific gene

20

HAROLD H. SMITH

transfer because a greater length of unpaired chromosome would be available for pairing and segmental exchange with an alien chromosome. More difficulty in gene transfer to N . tabacum is encountered when the wild species fails either to hybridize or t o produce a cross fertile combination with the commercial species. This problem is being met in an effort to transfer resistance to root knot nematodes from N . repanda by using N . sylvestris as a “bridging” species (Burk and Dropkin, 1961). The amphiploid N . repanda-sylvestris shows a high degree of resistance. Although it could not be hybridized directly with N . tabacum, steps are now being taken to backcross the amphiploid to N . sylvestds, to retain by selection those N . repanda chromosomes carrying nematode resistance, and to then cross this introgressive genotype to N . tabacum. For the most part success in experimental introgression of disease resistance has been with the transfer of single dominant genes or individual chromosome segments accompanied by restricted intrachromosoma1 recombination. Disease resistance among varieties within the cultivated species usually exhibits polygenic control; that is, a series of modifiers has been accumulated in the hereditary system (Table 3 ) . The plant breeding programs which have been followed to incorporate resistance to Granville wilt, fusarium wilt, and powdery mildew (Wan, 1962) into various commercial varieties from sources within N . tabacum (Table 3) are outside the scope of this review. In the foregoing discussion the transfer of disease resistance has provided a number of examples of introgressive hybridization in Nicotiana. Other studies on the mechanism and effects of incorporating alien genetic material into the genome of N . tabacum have been under study a t the University of California for some years, particularly with reference to identifying the N . tabacum chromosome into which an introduced locus was transferred (Cameron, 1962). Eight introgressed lines were investigated in which marker loci from two related species ( N . sylvestris and N . setchellii) and three unrelated ones ( N . glutinosa, N . plumbaginifolia, and N . paniculata) were introduced into N . tabacum chromosomes. The primary purpose was to determine whether chromosome exchange regularly involved the N . tabacum chromosome known to bear the recessive locus corresponding to the one introduced. Selected monosomic types were used for the analysis. Introgressed loci from the related species, N . sylvestris and N . setchellii, were identified with the homologous chromosome in the former, and with a nonhomologue in the latter. Results were inconsistent where N . glutinosa was the donor species. This is not surprising in view of the small amount of homology which might be expected between N . glutinosa and N . tubacum chromosomes. With the two species least closely related to N . tabacum, N .

CYTOGENETICS OF

Nicotiana

21

plumbaginifolia and N . paniculata, the data were too limited to indicate an association of the marker loci with either N . sylvestris or N . tomentosa genomes. Records of hybridization between the two cultivated species, N . rustics and N . tabacum, extend back to the pre-Mendelian plant hybridizers (East, 1928). The cross is usually made, N . rustica ( 9 ) X N . tabacum (d),but the reciprocal has been successful using X-irradiated pollen of N . rmstiea (Swaminathan and Murty, 1959). Although the introduction of genetic material from N . rustica into N . tabacum does not appear to be a useful way to improve quantitatively inherited traits in the commercial tobacco (Legg and Mann, 1961), novel geniccytoplasmic interactions have been observed (Hart, 1965). Introgressive hybridization in which the mammoth character of N . tabacum was transferred to N . rustica yielded types with altered requirements for flowering. This program is now in the eighth backcross generation to N rustica and all plants have 24 bivalents; however, some cytological anomalies and unselected characteristics of N . tabacum were observed to persist in the sixth backcross (Murty and Swaminathan, 1957). Early in the backcrossing series, some of the mammoth segregants failed to flower under short days (Smith, 1950), though a shortday response is characteristic of this genotype in N . tabacum. Some of these segregants were brought to flower with low temperatures (Steinberg, 1953), but in the fourth and subsequent backcrosses N . TUStica-like mammoths were produced which consistently failed to flower under any conditions tried, including grafting to flowering plants. A clone from such a nonflowering segregant has now been induced to flower under prolonged treatment with gibberellic acid (GAS), low temperatures ( l l o to 16OC), and short photoperiod (Hillman and Smith, 1965). These results are of genetic significance as a demonstration of the profound changes that may occur in requirements for gene expression when an allele is transferred from one residual genotype to that of another species. The induced flowers are abnormal and are similar to those of a related mammoth N . rustica introgressant, which reverts briefly to flowering (Murty, 1960). This flower-revertant character is of incomplete penetrance, is controlled by a gene linked to a mammoth locus, and is characterized by a “turning-on” of flowering under early season conditions, followed shortly by a “turning-off” or reversion to the mammoth, nonflowering condition. Hybrid derivatives of a cross between N . langsdorfii and N . sanderae have been used to study the effect of selection, following interspecific recombination, on morphological divergence and genetic isolation as a problem in evolution under controlled conditions a t two levels of ploidy

22

HAROLD H. SMITH

(Smith and Daly, 1959). Analyses of diploid and amphiploid lines each selected on the basis of small, intermediate, and large corolla size showed that by the Fa generation the diploid lines were as uniform as the parental species and that the large selection eventually exceeded the larger parent in flower size-an introgressive transgressant population. Investigation of reproductive isolation based on crossability, meiotic abberation frequency, and pollen abortion indicated that each selected line was isolated from its parents on the basis of one or more of these criteria. Lines selected from the amphiploid remained highly variable throughout the experiment and showed relatively less response to selection, but all were partially isolated from the parental species by pollen sterility. Populations satisfying two of the formal criteria of speciation, that is, morphological divergence and genetic isolation, were developed from one hybridization, followed by single plant selection and self-pollination, within six generations. VIII. Genetic Tumors

The occurrence of spontaneous tumors in interspecific hybrids of Nicotiana was first reported by Kostoff (1930). These abnormal growths, which constitute striking example of hybrid instability, usually appear after the flowering period and are similar to, but generally more differentiated than, crown gall tumors (Braun and Stonier, 1958; Kupila and Therman, 1962). The following lines of evidence support the conclusion that the basic cause of tumor formation in these hybrids rests in particulate genes located in the chromosomes. (1) Of more than 300 different interspecific hybrids reported among species of Nicotiuna only about 34 produce spontaneous tumors throughout the whole plant and in entire populations; a n additional 25 Fl’s produce tumors sporadically (Kehr and Smith, 1954; Takenaka and Yoneda, 1962). Naf (1958) proposed that species involved in tumorous combinations may be divided into two groups; and the contribution of each group is envisaged as differing in some biochemical or physiological manner under genetic control so that products conducive to tumorous growth are formed only in intergroup hybrids. The restriction of the occurrence of tumors to only certain hybrid combinations can be considered as general evidence of their genetic basis. (2) No pathogenic causative agent has been isolated from Nicotiana hybrid tumors in spite of attempts by workers over a period of years to do so (Kehr, 1951). Tumor induction is not transmitted across a graft union; the abnormal growths arise only from cells of the plant parts that are so constituted genetically as to form them (Kehr and Smith, 1954).

CYTOGENETICS OF

Nicotiana

23

(3) Tumor formation is the same in reciprocal hybrids, indicating that the cause is determined not by cytoplasmic elements present in only one of the parental species, but rather by nuclear elements contributed equally by male and female parents. (4) With the hybrid Nicotiana glauca x N . langsdorfii it was possible to obtain various genomic combinations ranging from two N . langsdorfii genomes and one of N . glauca ( L L G ) , to one of N . langsdorfii and three of N . glauca (LGGG). All these combinations are tumorous, indicating that the tumor-forming potential of the hybrid N . glauca X N . langsdorfii remains qualitatively the same regardless of the ratio of genomes (Kehr and Smith, 1954). (5) When all chromosomes of N . glauca are added to diploid N . langsdorfii the resultant triploid plants develop tumors ; but tumor formation was not observed by Kehr and Smith (1954) on a variety of plants with only one or a few N . glauca chromosomes added to N . langsdorfii. Evidence has not yet been obtained on just which, or how many, N . glauca chromosomes must be combined with those of N . langsdorfii in order to produce the tumorous condition. Ahuja (1965) reported that tumors may occur in plants with fewer than the full complement of N . glauca chromosomes added to diploid N . langsdorfii. These results have provided preliminary evidence that the hereditary capacity for tumor formation is governed by factors residing in the chromosomes and, in N . glauca x N . langsdorfii hybrids a t least, the genetic situation is complex. A simpler genetic control has been found in crosses between N . longiflora and the amphiploid involving N . tabacum and N . debneyi. By a program of repeated backcrossing, it was possible to obtain on the background of the amphiploid a single N . longiflora chromosome or even fragment of chromosome associated with tumor formation (Ahuja, 1962). (6) The two rather closely related species N . langsdorfii and N . sanderae, which give a partially fertile nontumorous hybrid, have genotypes with markedly different effects on tumor formation (Brieger and Forster, 1942). The N . langsdorfii complement contains genes that enhance tumor expression, whereas N . sanderae may inhibit formation of tumors, at least in plant parts observable above the ground line, in appropriate crosses. When the F1, N . langsdorfii x N . sanderae was hybridi~edin a number of interspecific combinations with a third species, selected as conducive to tumor formation, evidence was obtained for segregation of genes that enhance or inhibit expression of tumors (Smith and Stevenson, 1961). (7) Further evidence of genetic control is afforded by correlations: ( a ) between N . langsdorfii genes governing small corolla size and tumor enhancement, and ( b ) between N . sanderae genes governing large

24

HAROLD H. SMITH

corolla size and tumor inhibition (Smith and Stevenson, 1961). These associations are best interpreted as resulting from genetic linkage and indicate that the genes affecting tumor formation are located in the chromosomes interspersed with genes that govern the normal morphology of plant parts, such as flower sise. (8) In order to establish populations that show evidence of recombination of tumor-controlling genes, different F2 segregants of N . langsdorfii X N . sanderae were inbred to the F4 generation and were then crossed with other species, in combinations conducive to tumor formation. This gave progenies that were characterized by marked differences in tumor expression as a result of the recombination of tumorcontrolling genes from N . langsdorfii and N . sanderae (Smith, 1962a). (9) A final point of evidence indicating that genes control tumor formation in Nicotiana hybrids is afforded by the induction with X-rays (Izard, 1957) of a nontumorous fertile mutant from the characteristically tumorous amphiploid, N . glauca-Zangsdorfii. This mutant has the same chromosome number as the parent, is dominant t o the tumorous condition, and segregates in the FB and first backcross generation (Smith and Stevenson, 1961). Abnormal mitoses and some consequent alterations in chromosome number are characteristic of genetic tumor tissue. Kostoff (1930, 1943) found that aberrant chromosome numbers in somatic tumor cells of N . glauca X N . lungsdorfii were relatively rare (1 in 50) and involved primarily additions (22, 23, 28, and 42) to the normal complement of 21. Burk and Tso (1960) have reported that cells in tumor tissue of this same hybrid characteristically involve reduction to 18, 19, and possibly 38 (19 doubled) chromosomes. Although variation in chromosome number is characteristic of a number of plant and animal tumor tissues it may well be coincidental rather than causal t o the tumorous condition. Cytological studies of tissue cultures reveal interrelationships between genotype and components of the nutrient medium (Yoneda and Takenaka, 1963) in influencing chromosomal abnormalities. A fundamental question with regard to the Nicotiana hybrid tumors is whether the change to abnormal growth involves a change in gene structure, that is, a somatic mutation, or whether i t is explained better as due t o differences in the regulation of gene activity. The following lines of evidence tend to support the latter interpretation. (1) Braun (1959, 1965) was able to demonstrate that a single tobacco cell, made tumorous by crown gall infection, could recover and develop into a normal plant. (2) Stemlike protuberances originating from partially differentiated genetic tumors on Nicotiana hybrid plants may occasionally yield flow-

CYTOGENETICS OF

Nicotiana

25

ers, seeds, and plants that differ in no observable way, including tumor formation, from progeny derived from seed produced on normal branches of the same hybrid, N . snuveolens-langsdorffii (Smith, 1962a). (3) Direct observations were made on the relationship between somatic mutation and tumor initiation in hybrids synthesized so that both phenomena could be scored quantitatively on the same individuals. Exposure to chronic 7-irradiation a t dosage levels that gave more than a 100-fold increase in “mutation” rate (loss of a dominant gene for anthocyanin pigmentation) failed to elicit a significant increase in tumor initiation (Smith, 1957). There was no simple relation between the two phenomena. (4) Different Nicotiana genotypes respond differently to irradiation (Smith and Stevenson, 1961). Some show enhanced tumor formation, particularly a t levels of chronic y-irradiation above 200 r/day (Sparrow and Schairer, 1958) or 2000 r of acute X-irradiation of seedlings (Ahuja and Cameron, 1963). The effect could result from radiation-induced somatic mutation or, alternatively, be a response in gene activity t o stress conditions imposed on the cellular metabolism. The latter interpretation is favored as the more influential since similar effects may be obtained under nonmutagenic stress conditions, such as merely crowding of seedlings during growth (Smith, 1962a). The effects of ionizing radiation in causing or accentuating morphological and physiological changes in plants and leaves of N . glauca, N . langsdorfii, and their tumorous hybrid have been studied in detail by Gunckel (1957), Hagen and Gunckel (1958, 1962), and Hagen et al. (1961). Investigations utilizing tissue cultures have contributed significantly to an understanding of differences in growth requirements of normal and tumorous cells. Braun (1958, 1965) has shown that plant cells altered to tumor formation under the influence of crown gall bacteria gradually acquire the ability to grow rapidly on a minimal culture medium, whereas normal cells require the addition of certain organic compounds as well as the growth-promoting substances cytokinin and auxin. Tissue cultures of normal types of cells and those of genetically tumorous Nicotiana hybrids show much the same kinds of difference in growth factor requirements (Schaeffer and Smith, 1963; Schaeffer et al., 1963). Tissues of the parental species N . langsdorfii and N . suaveolens require both auxin and kinetin for rapid growth whereas tissues of tumorous hybrids fail to respond to additions of auxin and kinetin to the culture medium. Not only the growth rate, but also the morphology of the cultured tumor, can be a function of the metabolites available. The degree of differentiation, from highly organized to amorphous, can

26

HAROLD H. SMITH

be induced by manipulation of the medium on which the tumor is grown (Hagen, 1962) ; and also by the inducing agent (Kupila and Therman, 1962). The evidence from tissue cultures, of higher effective levels of growthpromoting substances produced in the genetically tumorous tissue, indicates that the reason for spontaneous tumor formation in intact plants is the production or accumulation of greater than regulatory amounts of growth-promoting substances as the plant matures. Application of a drop of growth factor solution containing indoleacetic acid and kinetin to the stem growing point of young seedlings of tumorous hybrids will induce the plants to tumor formation a t this early stage (Schaeffer, 1962). A number of differences in chemical composition between tumorous and nontumorous Nicotiana tissues have been reported (Tso et al., 1962). The content of free amino acids (Anders and Vester, 1960; Vester and Anders, 1960; Steitz, 1963) and the rate of scopoline production are higher in N . langsdorfii-glauca than in the parent species (Tso et al., 1964). Leaf tissue of this hybrid not only contains more free auxin but is more efficient in converting tryptophan into auxin and has more free tryptophan to start with than either of the parents from which it was derived (Kehr and Smith, 1954). The high level of auxin in the hybrid seems to be regulated by a variety of inhibitors with different specificities in the growth-regulating process (Bayer and Hagen, 1964). When auxins and auxin inhibitors were extracted from tissue of N . glauca-langsdorfii and the parent species, and separated by paper chromatography, it was found that N . langsdorfii and the hybrid contain three different auxin inhibitors while N . glauca contains only two of them (Bayer, 1965). The addition of either auxin or kinetin to tissue cultures of N . glauca and of N . suaveolens-langsdorfii decreased the activities of enzymes of the hexose monophosphate shunt (Scott et al., 1964). The mechanism by which phytohormones affect growth is not known with certainty, but experimental evidence is accumulating with a variety of plant materials that regulation of DNAdependent RNA synthesis is primarily concerned; and this is reflected in the rate of synthesis of proteins including enzymes of systems that control physiological processes culminating in cell division and expansion. Genetic control of tumor formation in Nicotiana hybrids is envisaged as depending primarily on the accumulation of greater than regulatory amounts of growth-promoting substances, which affects the regulation of gene activity and DNA-dependent RNA synthesis, thus activating enzyme systems limiting to growth and differentiation, Hypothetical

CYTOGENETICS OF

Nicotiana

27

gene regulation and activation circuits which relate regulator-operon systems to phytohormone production and cell division have been suggested by Smith (1966). IX. Cytogenetic Instability in Species Hybrids

I n addition to tumor formation, other manifestations of instability occur in hybrids among species of Nicotiana. These include variegation in pigment of flower or leaf, variations in morphology and habit during plant development, and variability in growth among hybrid plants. Kostoff (1935, 1943) cited several examples of instability in interspecific Nicotiana hybrids, and in all these combinations one parent species was a member of the Alatae section. Plants with variegated anthocyanin pigmentation in the flower have appeared sporadically in the Fz and subsequent generations of the cross N . 1angsdorfi.i x N . sanderae (Smith and Sand, 1957). One of these variegated types has been analyzed in detail and is attributed to a mutable locus, v. Two alleles at this locus are necessary and sufficient to account genetically for the different modal breeding behavior of the three major variegated phenotypes: speckled (v,/v,) , sectorial (vB/v8),rare sectorial (vs/vs).Both the alleles are unstable and somatic mutations occur in both directions, so that a chromosomal loss is apparently not involved. Differences in frequency and developmental timing of these reversible mutations have both heritable and environmental components (Sand, 1957). Higher temperatures decrease somatic speckling (v, + v g ) but increase sectoring (va+ v,) . Another environmental variable, the node position of the flower on the plant, was shown by Sand (1961a) to affect both the unstable v locus and a stable R locus in this hybrid material. The effects were different, however; stable and unstable loci may exhibit differential sensitivity to heritable change in varying metabolic or cellular environments (Sand, 1962a). This differential effect is strikingly evident under exposure to low levels of yirradiation. The slope of the somatic response curve for the mutable gene is about 10 times greater than for the stable gene a t dosage levels below 12 r/day (Sand et al., 1960). The unstable v locus is located in chromosome 2, whereas another more extreme instability that occurs in segregating generations of the same N . langsdorfii x N . sanderae hybrid is associated with chromosome 7 (Urata, 1959). The latter type of segregant is completely sterile, is small and slow growing, has mottled leaves and flowers, and forms frequent chromosomal abnormalities. The interspecific hybrids Nicotiana bigelovii X N . glauca and N . glutinosa X N . glauca exhibit aberrant phenotypes characterized by

28

HAROLD H. SMITH

leaves that are asymmetrical and variegated in color and texture, and by variability in growth habit among F1 plants (McCray, 1932; East, 1935; Kehr and Smith, 1952). These abnormalities are peculiar t o the F1 in that amphiploids produced from them are somatically stable and yield uniform progeny (Latterell, 1958). Anaphase stages in somatic mitoses of both the F1 and the amphiploid show a relatively high frequency of chromosome bridges, indicating that chromosomal instability is unlikely to be the sole cause of the somatic instability. The F1hybrid, Nicotiuna bigelovii x N . glauca, in which spontaneous growth abnormalities occur, was studied by Meiselman et al. (1961a,b) with particular reference to the effects of irradiation. Grown at levels of chronic y-irradiation of 300-375 r/day for one month, the hybrid was more adversely affected than either parent species. Bridges and fragments occur in 38% of the hybrid cells under control conditions, and the increased radiosensitivity of the hybrid was considered to be due to these inherent cytological as well as physiological instabilities. All hybrid combinations of Nicotiana tabacum (tbc) and N . plumbaginifolia (pbg) that were studied by Ar-Rushdi (1957), Moav and Cameron (1960), and Moav (1961) showed somatic variegation of dominant characters carried on the pbg genome. It was established by various direct and indirect procedures that the variegation in five pbg markers was due to somatic elimination of whole chromosomes. Most of the quantitative work was done with W s (pbg), a dominant locus which permits chlorophyll production on a background of albino N . tabacum and the transfer of which to N . tabacum can apparently be induced by radiation (Niwa, 1965). Backcrosses of the sesquidiploid hybrid (24 tt 10 p ) t o diploid tbc (24 t t ) resulted in a marked increase of the variegation intensity, which was attributed to the resolution of the pbg genome into individual chromosomes. The great majority of 24 tt 1 p plants obtained by recurrent backcrossing was highly unstable, but about 1% became completely or relatively more stable. Some of the spontaneously stabilized Ws (pbg) loci were found to have been transferred to a tbc chromosome. Mitotic bridgelike structures in the tbc-pbg and other related hybrids have been observed ; however, the exact cytological nature of the chromosomal elimination is still to be found. Genetic instability has also been found repeatedly in hybrids and hybrid derivatives between N . tabacum and diploid species which, as presentday representatives of putative ancestral forms, are closely related to the cultivated allopolyploid. In hexaploids synthesiaed from N . tabacum X N . otophora abnormal segregation ratios, variegation, and chromosomal aberrations were encountered (Gerstel, 1960).

+

+

CYTOGENETICS OF

Nicotiana

29

Chromosomes of extraordinary size, which were called “megachromosomes” (Gerstel and Burns, 1966a), were found in scattered cells of derivatives from N . tabacum X N . otophora which showed variegation, It may be of some significance, in connection with the instabilities observed, that the patterns of distribution of heterochromatin in N . tabacum and N . otophora are greatly different and the total amount of heterochromatin appears to be much larger in N . otophora (Burns, 1966). Carmine-coral variegation of corollas, one of the several disorders found following hybridization between N . tabacum and N . otophora, occurs as coral spots on carmine background of plants heterozygous (Co” GO), homozygous (Cov Co”), and hemizygous (Cov -) for the carmine (Co”) allele derived from N . otophora (Gerstel and Burns, 1966b). The inheritance of “carmine-coral variegation” is essentially Mendelian, shows a dosage effect, and increases from F1 to advanced backcross generations. The latter trend parallels that of the decrease in amount of chromatin, especially heterochromatin, from N . otophora with repeated backcrossing. The switches from carmine to coral apparently do not entail chromosome losses or involve separate controlling elements. The phenomenon may be governed by a V-type position effect (Gerstel and Burns, 1966b), but the authors are also considering other explanations. Yang reported both interplant (1964) and intraplant variation (1965) in chromosome numbers in hexaploid and pentaploid derivatives of N . tabacum crossed with N . sylvestris, N . otophora, and N . tmnentosiformis. The hexaploids were produced by colchicine treatment of interspecific triploids, and the pentaploids (2N= 60) by backcrossing to N . tabacum (n=24) hexaploids with a confirmed number of 7t = 36. The hexaploids not only produced balanced triploid microspores, but their chromosome numbers varied from 33 to 39 with modes a t the expected number of 36. Similarly, the somatic chromosome numbers in root tip cells of pentaploids ranged from 56 to 64 with a mode a t the expected number of 60. The variations in chromosome number appeared to have arisen from meiotic irregularities in the hexaploid, and chromosomal deficiency was found more frequently than hyperploidy in both the basically 3X microspores and the pentaploid offspring. I n addition to irregular chromosome numbers between pentaploid plants, Yang (1965) observed that different cells in the same plant varied in number of chromosomes. Hybridity, high chromosome numbers, and the unbalance of aneuploidy all added to the degree of chromosomal instability. An interesting example of chromosome elimination coupled with

30

HAROLD H. SMITH

plant to plant morphological variation was observed by Krishnamurty and Satyanarayana (1962) in the F1 generation of a cross between the stable amphiploid, N . glutinosa-trigonophylla (2n = 48) and N . megalosiphon (2n = 40). Cytological examination of 35 trispecific hybrids showed that from one to eleven chromosomes were eliminated in different plants from the expected number of 44. The elimination apparently took place in the early stages of embryo development, and loss of different chromosomes was responsible for the morphological variation observed in the trispecific progeny. Phenotypic variation and chromosomal elimination of similar nature and probably of similar origin was reported in the F1 and amphiploid, N . megalosiphon X N . glutinosa (Satyanarayana and Subhashini, 1964). Aneuploidy, resulting from elimination of chromosomes, has played a role in the evolution of the Suaveolentes section (Satyanarayana and Subhashini, 1964) and is of potential importance in the future evolution of the genus (Smith et al., 1958; Smith, 1964). Developmental instability, defined as the intragenotype variability of quantitative characters, has been investigated in N . rustica (Jinks and Mather, 1955; Paxman, 1956) and in N . tabacum (Sakai and Shimamoto, 1965). Insofar as these species are of allopolyploid origin the experiments may appropriately be discsussed under the general heading of cytogenetic instability in species hybrids. In N . rmstica, developmental instabilities of various characters were found to have a genetic component. Developmental instabilities in foliar and floral organs vary among varieties of N . tabacum (Sakai and Shimamoto, 1965), suggesting that they are governed by genetic factors. Furthermore, there was evidence that the genes responsible for developmental instabilities of leaves may be different from those affecting flowers; and, based on differences in stability a t different developmental stages, that these genes may be more active a t a certain period of growth than a t others. X. Cytoplasmic Inheritance: Male Sterility and Plartids

Evidence for extrachromosomal or cytoplasmic inheritance has been found for two characters in Nicotiuna, male sterility and chlorophyll variegation, East (1932) was the first to report experimental results in Nicotiana showing that a factor in the cytoplasm, combined with specific nuclear genes, governed the expression of male sterility. I n the presence of N . langsdorfii cytoplasm [ms], plank homozygous for a gene from N . sanderae are male sterile. East concluded that the N . sanderae genes in-

CYTOGENETICS OF

Nicotiana

31

volved were alleles a t the S locus which control self-incompatibility ; however, it could be considered as the recessive gene (rfl) a t a locus for restoring pollen fertility (Rfl), Nicotiana langsdorfii would then be [ms] Rfl Rfl, N . sanderae [MF] rfl rfl, and male sterile segregants are those with cytoplasm from N . langsdorfii [ms] and no restorer gene (rfl rfl). Additional complexities to this pattern of inheritance were reported by Smith (196213) in another cross of N . langsdorfii N . sanderae which was similar except that the N . sanderae (male) parent was self-compatible. The experimental data could be explained by the presence of a second independent restorer of pollen fertility (Rf2). These parental genotypes are N . Zangsdorfii [ms] Rfl Rfl rf2 rf2 and N . sanderae [MF] rfl rfl R f 2 Rf2; and only [ms] rfl rfl rfz rf2 segregants are male sterile. That is, the complete genetic information for ensuring normal microgametogenesis is present a t each of two independent gene loci in the nucleus as well as in a factor in the cytoplasm. Certain anomalies to regular Mendelian inheritance of the pollen restorer genes were attributable to a disadvantage of pollen carrying the Rfl allele when in competition with rfl pollen (Smith, 1962b). A number of other examples of cytoplasmic and genic-cytoplasmic control of male sterility hqve appeared in the Nicotiana literature. I n fact, it now appears to be a widespread phenomenon in the genus; that is, the cytoplasm of one species (A) combined with the partial or complete genome of another species (B) will often produce male sterility, and furthermore, genetic restorers to pollen fertility will be found in certain chromosomes of species A. An early example reported by Clayton (1950) appeared in pedigreed cultures begun in the 1930’s in an effort to transfer resistance to blue mold from N . debneyi to N . tabacum. The amphiploid N . debneyitabacum was backcrossed to N . tabacum, used as the male parent, through a number of generations. In the first backcross generation (BC1) many of the plants were male sterile and by the BC3 generation all were male sterile and exhibited a characteristic morphological anomaly of split corollas. At this stage there were still 4 t o 7 univalent N . debneyi chromosomes present, but by the BClo generation these were apparently completely eliminated and the male sterility persisted. Fertility can be restored in earIy stages of the backcrossing program by using N . tabacum as the female parent. It is thus evident that male sterility is caused by a combination of N . debneyi cytoplasm with genes from N . tabacum, tbc ( d b n ) . Negative results were obtained by Sand (1960) in experiments designed to detect transmission of this cytoplasmic male sterility to progeny by grafting. Derivatives having a genome of N . tabacum in the cytoplasm of another Australian spe-

x

32

HAROLD H. SMITH

cies, N . megalosiphon, tbc (mgl) also gave male sterility (Clayton, 1950). Burk (1960) reported that male sterility and characteristic flower anomalies gradually develop through backcrosses of N. bigelovii as female (cytoplasmic donor) parent hybridized with N . tabacum as the male parent, tbc (bgl). As above, pollen fertility is restored in the reciprocal cross which introduces cytoplasm of N . tabacum. The extent of flower modification is apparently a function of the Nicotiana species involved as the cytoplasmic donor parent (Cameron, 1965; Chaplin, 1964). I n two other male-sterile types reported by Burk (1960), which originated in single plant derivatives of N . t a b a m m X N . plumbaginif o l k and N. tabacum x N . gbtinosa, both the cytoplasm and the recurrent parent in the backcrossing program were N . tabacum. Male sterility was maintained undimished through a number of generations even though the procedure should have restored N . tabacum genomes in an N . tabacum cytoplasm, tbc ( t b c ) . An unusual case of male sterility was described by Hart (1965) in which the chromosomes of N . tabacum were combined with the cytoplasm (plasmon) of N . rwtica t o produce ,pollen that essentially failed to germinate, and was thus nonfunctional, even though i t was indistinguishable morphologically from functional pollen. Partial male fertility was restored by the action of one or more restoring genes on specific N . m t i c a chromosomes. Genetic tests firmly established that the fertility restoration induced by N . rustica chromosomes occurs in the sporophyte. This suggests that the male sterility of plants with N. tabacum chromosomes and N. rustica plasmon is due to the failure of a genome-plasmon interaction which normally occurs in the sporophyte. Reciprocal hybrids between N . tabacum, N . sylvestrie, and members of the Tomentosae section have been carried out over many years by Cameron (1965). He concluded that since combinations of N . tabacum with the Tomentosae section are highly aberrant there seems little doubt that the N. sylvestria parent provided the cytoplasm in the original hybrid from which N . tabacum was derived. The potential use of cytoplasmic male sterility in the commercial production of flue-cured tobacco has been investigated by Mann et al. (1962), Aycock et al. (1963), and Chaplin et al. (1963). In these investigations flue-cured derivatives of Clayton’s 402 male-sterile line ( N . megalosiphon cytoplasm-N. tabacum genome) were used. The general conclusion reached was that commercial advantages, if any, of F1 hybrids over available pure line varieties appear to be very slight.

CYTOGENETICS OF

Nicotiana

33

T h e growth retardation associated with the N . megalosiphon source of cytoplasmic male sterility essentially obliterates any superiority of F1 hybrids. However, these hybrids may be used as temporary measures to combine desirable traits of two varieties, though the best method in the long run for developing improved tobacco varieties is still the accumulation of desirable factors in a homozygous condition (Smith, 1952; Matzinger and Mann, 1962; Matzinger et al., 1962). Eight different sources of cytoplasmic-inherited male sterility were investigated by Chaplin (1964) to determine their relative use in producing hybrid tobacco seed. The eight male sterile lines and a norma1 flowered (Type 1 ) tobacco were arranged into the following morphological types. In Type 2 the flowers had normal corollas with modified anthers as featherlike structures ( N . bigelovii cytoplasm). Flowers on Type 3 had normal corollas but the anthers were stigmatoid on shortened filaments ( N . megalosiphon and N . suaveolens cytoplasm). Type 4 corollas were shortened with normal-appearing anthers borne on shortened filaments and stigmas protruding ( N . plurrtbaginifolia cytoplasm). Type 5 had corollas shortened, with modified anthers petaloid, and protruding stigmas ( N . undulata and N . tabacum cytoplasm). In Type 6 the corollas were split and the stigmatoid anthers were borne on shortened filaments ( N . debneyi cytoplasm). Chaplin (1964) concluded that Type 5 flowers would probably have the most economic value in production of hybrid tobacco seed, but that hand pollination would be required. Cytoplasmic inheritance of chlorophyll variegation in tobacco has been reported by Dermen (1960), Wolf (1959), Burk and Grosso (1963), Burk et al. (1964), Burk (1965), and Edwardson (1965). Defective plastid inheritance is transmitted only through the female and the progeny of variegated plants have varied from segregation into all seedlings either white or green (Dermen, 1960; Burk and Grosso, 1963), or into white, variegated, and green seedlings (Wolf, 1959). An explanation for somatic patterns observed and for differences in heritabilities of plastid-controlled chlorophyll variegation in tobacco has been advanced by Burk et al. (1964). Patterns of variegation in which the sporogenous tissue was derived from the second histogenic layer (L-11) containing only one type of plastid produced only green or only white offspring. When L-I1 is a mosaic of both normal and deficient mutant plastids the inheritance pattern depends on the inclusion or loss of the mutant type in mature cells of sporogenous tissue and successive cell generations in sexually produced seedlings. The genetic determiner of the defective plastid type studied by Burk et al. (1964) is located in the plastid itself. It is dominant in the sense that when

34

HAROLD H. SMITH

present in sufficient numbers i t suppresses chlorophyll formation by genetically normal plastids in the same cell. Evidence of the genetic autonomy of plastids was afforded by the observation in single cells of young leaves both normal and mutant types of plastid. Defective plastids can be used to detect migration of cells between histogenic layers. Evidence for this phenomenon was based on the occurrence of green spots in cell layers that are otherwise albino, as well as the rare occurrence of green seedlings from a gamete-producing histogen (L-11) known to contain only chlorophyll-defective plastids (Burk, 1965). I n studies on a leaf variegation that appeared in N . tabacum var. Hicks Broadleaf, and that was inherited in non-Mendelian fashion only through egg cells, Edwardson (1965) found that in crosses with two Turkish varieties the variegation was completely eliminated. The data were interpreted t o indicate that these varieties possess nuclear genes which block both the expression and transmission of variegation. The interaction of these variegation “eliminator” genes with cytoplasmic factors is not analogous to the interaction of fertility restorer genes, discussed above, with cytoplasmic male sterility factors, since variegation does not recur even in the presence of presumably homozygous “permitter)’ genes. XI. Inheritance of Alkaloids

Recent work on the inheritance of alkaloids can conveniently be discussed under three headings : alkaloid content among Nicotiana species and interspecific hybrids, inheritance of conversion of nicotine to nornicotine, and associations between nicotine content and yield in commercial tobacco. Alkaloid content was recently analyzed by paper-partition chromatography in 52 species of Nicotiana representing all taxonomic sections and centers of geographical distribution (Smith and Abashian, 1963). Most of the species contained predominantly three identified alkaloids : nicotine, nornicotine, and anabasine. In addition, a t least six other alkaloids, which are separable chromatographically but were not identified chemically, are characteristic of the genus and are present in varied but characteristic patterns in each species. Marion (1950) summarized the work on 36 species analy~edby other methods to that date and these results can be compared with the above more recent ones. I n 25 of these species the same alkaloid was found. I n 10 other species the alkaloid reported to be the main one in earlier work was secondary in the analyses of Smith and Abashian (1963) or vice versa. Alkaloid composition of some Nicotiana species has been determined

CYTOGENETICS OF

Nicotiana

35

by means of chromatography by Jeffrey (1959) and his results can be compared with both earlier and later efforts. Twenty-three species have now been analyzed by a t least three independent investigators. There is complete agreement as to the main alkaloid in eleven of these species : predominantly nicotine producing are N . attenuata, N . benavidesii, N . bigelovii, N . gossei, and N . rustica; predominantly nornicotine producing are N . ghctinosa, N . megalosiphon, N . otophora, and N . palmeri; and predominantly anabasine producing are N . debneyi and N . glauca. I n seven of the 23 species there is a disagreement as to whether nicotine or nornicotine is the main alkaloid, both having usually been detected. These species are: N . acuminata, N . longijlora, N . nesophila, N . paniculata, N . repanda, N . sylvestris, and N . undulata. In three other species that have been analyzed by a t least three independent investigators, namely, N. benthamima, N . clevelandii, and N . nudicaulis, Smith and Abashian (1963) found anabasine to be the main alkaloid, whereas previously nicotine or nornicotine had been so reported. The unidentified alkaloids are rarely found in as great amounts as the three identified alkaloids. No clear-cut relationships were observed between types of alkaloid and phylogenetic position (Imai, 1959), geographical distribution, habitat, or habit of growth. However, since all species of the genus contain one or more alkaloids, their presence may have had an adaptive significance early in the evolution of the genus; and the biochemistry of their formation been fixed with a significant role in metabolism a t the cellular level in present day Nicotiana plants. The role of alkaloids in the overall development of the tobacco plant is, however, not clearly understood. Tso (1962) and Tso and Jeffrey (1961) demonstrated that the alkaloids take an active part in the metabolism of the plant and thus cannot be considered as simple waste products. Dawson (1960) relates the pathway of alkaloid synthesis and growth of the plant as progressing through some common intermediate steps, thus associating alkaloid formation and growth. In the work of Smith and Abashian (1963) , 35 two-species combinations, 14 three-species combinations, and 2 four-species combinations were analyzed for alkaloid composition. When a predominantly “anabasine species” is crossed with an anabasine, nicotine, or nornicotine species, the main alkaloid in the combination is most frequently anabasine (Smith and Smith, 1942). The biosynthesis of this alkaloid is an essentially dominant genetic characteristic. The alkaloid produced in crosses between predominantly nicotine and nornicotine species is most frequently nornicotine. The genetic factors controlling nornicotine formation are usually partly dominant over those producing nicotine,

36

HAROLD H. SMITH

but the relationship is clearIy not simple (Mann and Weybrew, 1958; Burk and Jeffrey, 1958). The arrays of unidentified alkaloids in interspecific Fl’s and amphiploids were compared with those in the two corresponding parental species. The most frequent result is the disappearance of one or more of the parental unidentified alkaloids in the hybrid and amphiploid. No generalizations about the inheritance of unidentified alkaloids seem justified a t this time except that they appear to be governed largely by genes that behave as recessives (Smith and Abashian, 1963). While some features of alkaloid inheritance were evident from the work on interspecific F1 hybrids and multiple genome combinations, it is clear that more simple genotypic differences need to be studied for further genetic analysis. One method, used by Smith (1965), was t o produce a hybrid between N . langsdorfli and N . sanderae, each of which has relatively simple alkaloid contents, then to develop reciprocal introgressive hybrids by transferring marker genes controlling color characters through a series of backcrosses, and ultimately to relate differences in alkaloid content t o the introgressed markers. Nicotianu sanderae synthesizes only nicotine, nornicotine, and unidentified alkaloid 2 (U2) . Nicotiana langsdorfii synthesizes, in addition, anabasine, and unidentified alkaloids U1, U4, U5, and U6. I n the F1, anabasine and U6 behave as dominants, U4 and U5 as recessives. None of the N . sanderae types, introgressed with marker genes from N . langsdorfii, synthesized anabasine, U1, or U6; one, with seven introgressed genes of N . langsdorfii, synthesized U4 and U5. With this exception, and a similar one on the background of N . Zangsdorffii, all other introgressed genes apparently marked regions of the genotype different from those closely linked with genes governing alkaloid synthesis. Pronounced correlations of anabasine with U6 (possibly N-methylanabasine), U4 with U5, and nicotine with nornicotine were observed in species, hybrids, amphiploids, and introgressive types. These paired associations may be taken to indicate genetic linkage, pleiotropism, and (or) biochemical relationships, that is, an inherent consequence of the biochemical pathways followed. Some 12 or more different alkaloids have been identified in the cultivated species, N. tubacum, mainly by Spath and his students using classical extraction and isolation procedures (for summaries, see Henry, 1949; Marion, 1950, 1960). Little is known about the inheritance of these alkaloids. A great deal of work has been done on the biosynthesis of tobacco alkaloids which is beyond the scope of this review; however, in brief with regard to nicotine, it is synthesized mostly in the roots and translocated to the leaves (Dawson and Solt, 1959). The concen-

CYTOGENETICS OF

Nicotiana

37

tration of nicotine in the leaves is thus a function of rate and amount of synthesis in the roots, followed by the rate of translocation, and h a l l y by the storage facilities in the leaves. Variations in genotypes and cultural or environmental effects may operate on any of these phenomena. I n certain low alkaloid strains of burley tobacco a conversion of nicotine to nornicotine during air curing was found to be controlled by a single dominant factor (Valleau, 1949; Griflith et al., 1955). This conversion has been described as a demethylation of nicotine to nornicotine (Dawson, 1952). I n crosses between a Maryland “converter” variety and three “nonconverter” varieties, the conversion of nicotine to nornicotine was found by Burk and Jeffrey (1958) to be controlled by a major gene with modifiers. From a series of studies on the genetics of conversion in N . tabacum (nonconverting, recessive) and related (converter, dominant) new allopolyploids (Mann and Weybrew, 1958; Gerstel and Mann, 1964; Mann et al., 1964), the following conclusions were reached. The ability t o convert nicotine to nornicotine, which is not a characteristic of most varieties in N . tabacum, is governed mainly by two pairs of dominant and independent genes. One locus, C1,common to eleven selected nornicotine-containing tobacco varieties, is located in the Tomentosae genome ; the other is in the N . sylvestris genome. The former locus, when recessive (clcl), as in most tobacco varieties, appears to be unstable and gene changes are thought to represent back-mutations (cl + Cl) or reversions to the primitive form with the ability to convert nicotine to nornicotine. Conversion has been investigated by using reciprocal grafts (Hall et al., 1965) of “red-free” nonconverter (c1c1czc2) tobacco, “cherry-red” (C,C,czcz) tobacco, and crosses with N . sylvestris (C2C2). Grafting per se did not influence the level of alkaloid production in these materials. The degree of conversion and alkaloid production in a scion or host was not influenced by the other graft component. Conversion capability is associated with the genotype: those plants with two genes for conversion exhibited a greater degree of conversion than those with only one. Matainger and Mann (1963) conducted experiments to assess the relationship between yield and percentage of nicotine relative to its genetic and environmental components. Through estimates of genetic, environmental, and phenotypic correlations, it was demonstrated that the relationship between yield and alkaloid production is governed by genetic as well as other factors. In a number of populations studied, the genetic correlation ranged from -0.21 to -0.85. The primary implication from this information is that selection for yield alone will be expected to lead to varieties which, on the average, are reduced in alkaloids.

38

HAROLD H. SMITH

XII. Biometrical Studies

Biometrical methods have been used in genetic studies on Nicotiana in order to analyze the inheritance of continuous variation in quantitative characters in terms of the nature of the polygenic systems responsible. An analysis of means and variance components applied to data from a series of generations following prescribed breeding programs has made possible interpretations about kinds of gene action, that is, additive, dominance, and various nonallelic interactions. The total phenotypic variance for quantitative characters can be partitioned by appropriate methods into genetic and environmental components, and the genetic component further partitioned into the proportionate contribution due to additive, dominance , and various epistatic gene effects. Information gained from such genetical analyses can be utilized in designing breeding methods to give maximum expectations for achieving desired practical goals in plant improvement. Early studies used data on plant height and other quantitative characters in N . rustica to demonstrate the applicability of these biometrical methods to appropriate data and to gain insight into the genetic system governing polygenic inheritance in a highly inbreeding species (Mather, 1949; Smith, 1952; Mather and Vines, 1952). In these studies only additive and dominance (allelic interaction) effects were included and nonallelic (epistatic) interactions were considered to be negligible. In general, additive effects ( d ) were found t o contribute more to the E ) than dominance effects ( h ). Consequently total variance (D H heritability was high and the expected efficacy of selection great. Subsequently, F1 and derived generations from crosses among varieties of N . rustica have been used for the analysis of diallel crosses (Jinks, 1954, 1956; Hayman, 1954), the separation of epistatic from additive and dominance variation in generation means (Hayman, 1960), and the study of the effects of reciprocal differences (Gilbert and Jinks, 1964). A recent experiment with N . rustica by Hill (1905) has provided evidence for a rarely reported result, namely, that changes produced by different fertilizer treatments in an inbred variety apparently are heritable since they have now been transmitted through two generations without any further treatments being applied. Studies on the biometrical analysis of quantitative inheritance in the commercial species, N . tabacum, have been carried out in the United States by a group of investigators a t North Carolina State University. I n an early publication by Robinson et al. (1954) estimates of sizable amounts of additive genetic variance but little dominance variance were obtained under the assumption of no epistasis. They concluded

+ +

CYTOGENETICS OF

Nicotiuna

39

that, if these results are generally applicable for N . tabacum, a breeding program designed to accumulate the maximum number of favorable genes in homozygous genotypes would offer more promise for tobacco improvement than attempting to utilize F1 hybrids on a commercial scale. Applying the same gene model to two populations of different flue-cured tobaccos, G. J. Pate1 (1959) found evidence in one of them for an appreciable dominance effect in gene action controlling yield and quality of the cured leaf. Estimates of additive X additive epistatic variances were obtained by Matzinger et al. (1960), under the assumption of no other types of epistasis, from a comparison of variances of cross-bred and selfed progeny of Hicks Broadleaf X Coker 139, and from the covariance of F2 plants in crosses vs. selfed progeny. A diallel analysis of F1 and F2 generations from crosses among eight flue-cured varieties of N . tabacum (Matzinger et al., 1962) again showed the predominance of additive genetic variance and only small amounts of heterosis and inbreeding depression. Most recently the North Carolina group (Legg et al., 1965) has described results from the first selfed generation of a synthetic variety combining randomly intermixed germ plasm from eight fluecured varieties. Estimates of additive genetic variance were significant for both the agronomic and chemical traits evaluated; and two advantages were noted, namely, that the number of desirable genes to select from is increased and that there is more opportunity for recombination than when only two lines a t a time are hybridized. In contrast to the results with N . tabacum discussed above, Murty et al. (1962) and Murty (1965) in India found evidence for the presence of considerable nonallelic interaction among genes controlling flowering time, number of curable leaves, percentage bright grades, and leaf burn in crosses among widely divergent flue-cured varieties. Associations between heterosis and nonallelic interactions were observed and the degree of heterosis could be related to the magnitude of genetic divergence between the parental varieties, which was greater than in the North Carolina experiments. Povilaitis (1964) has also found evidence for epistatic effects in crosses among flue-cured varieties grown in Canada. He noted that “The portion of epistatic effects due to additive X dominance interactions was small, but the estimated additive x additive effects were comparable to the additive effects but not significant for days to flower and height measurements; so were also the dominance X dominance effects for days to flower, number of leaves and height measurements.” Most workers have reported, for most characters in crosses among varieties of N . tabacum, a preponderance of additive gene effects and

40

HAROLD H. SMITH

high heritability (Oka, 1959; Wittmer and Scossiroli, 1961 ; Luthra, 1964). However, it should be noted that Lamprecht (1964), from a n analysis of diallel crosses among flue-cured varieties grown in South Africa, found significant general and specific combining ability as well as reciprocal effects for all characters, and the estimated heritability for plant height and for leaf color was low. Proposals for the utilization of heterosis in F1 hybrids in commercial production of tobacco continue to appear in the world literature (Ahmad and Termazi, 1960; Bawolska et al., 1960; Chen and Chu, 1960; Sikka and Batra, 1960; Chaudry and Munshi, 1964). Irradiation of tobacco seeds causea in segregating progeny an increase over controls in the genetic component of phenotypic variability for various quantitative traits (Wittmer, 1960, 1961; K. A. Pate1 and Swaminathan, 1961 ; Dalebroux, 1962). It was shown in biometrical analyses performed by Bsgnara et al. (1964) that not only does radiation increase genetic variability but differences in response occur depending on the genetic background of different tobacco varieties. The inheritance of leaf shape in N . tabacum is controlled by three pairs of genes-Br, Pd, and Pt-which show little or no dominance, and P t has about twice the effect of Pd (Van der Veen, 1957; Humphrey et al., 1964) as well as producing multiple effects throughout the leaf (Van der Veen and Bink, 1961). The dimensions of flower parts in crosses between species of Nicotiana were used in early experiments on the inheritance of quantitative characters and these studies have been continued utilizing methods of biometrical analysis to assess gene effects (Smith and Robson, 1959). Measurements of corolla tube length and lobe length were made over the period 1933 to 1955 on N . langsdorfii and N . sanderae and their derived generations through the F4 and second backcross. On a logarithmic scale, environmental variances as measured in the nonsegregating generations were essentially constant through time and independent of genotype. First-degree genetic parameters, including epistatic effects of triplets of nonallelic genes, were estimated by a regression analysis of t h e weighted generation means. The results of the analysis suggested that epistasis occurs in this hereditary system; nevertheless, the simple nonepistatic , three-parameter model fitted remarkably well to the means of 13 generations. By selecting for large corolla size in selfed generations derived from the hybrid between N . langsdorfii and N . sanderae an inbred line was produced with a mean flower size that exceeded that of the largerflowered parent, N . sanderae. To gain information on the nature of gene action controlling the transgressive phenotype an analysis of means and

CYTOGENETICS OF

Nicotiuna

41

variance components was carried out by Daly (1958). Three epistatic parameters for digenic interactions, additive X additive, additive X dominance, and dominance X dominance, were estimated in addition to additive and dominance effects. The additive genic effect exceeded the dominance effect for each flower dimension measured. Variance component estimates were consistent with results for gene effects in indicating the presence of nonallelic gene interactions. The major portion of the increase in size of the selected line over its larger parent could be attributed to additive gene action and the remaining smaller portion to additive X additive interaction. Estimates made of the minimum number of effective factors responsible for the difference in corolla tube length were as follows: (1) between N . langsdorfii and N . sanderae, 11; (2) between N . langsdorfii and the large-flowered transgressive selection, 9 ; and ( 3 ) between N . sanderae and the large-flowered selection, 3. The seeming anomaly, that smallflowered N . langsdorfii differs by fewer genes for flower size from the transgressively large-flowered selection than from N . sanderae, is in complete agreement with the known genetics of the three types. That is, the large selection is larger than N . sanderae because it contains additional genes for large flower size from N . langsdorfii. Experiments programmed to isolate the nine chromosomes of N . sanderae on a background of N . longiflora as alien addition races are being carried out by Sand (1961b, 196213). The purpose of these investigations is to plot the “topography” of the genome with respect to the location in chromosomes of genes affecting quantitative characters, rather than only to assess gene action and partition variance components for the genome as a whole. X111. Miscellaneous

New spontaneous mutations continue t o be uncovered in N . tabacum (Wolf, 1965). Of those reported during the past 10 years, some behave as dominants, aurea (Burk and Menser, 1964) and wrinkled-leaf (Mann and Matzinger, 1965); some are partially dominant, variegation D (Valleau, 1958b), corrugated dwarf (Dean, 1964), and leaf curl (Silber and Burk, 1965) ; and still others are recessive, yellow Crittenden (Valleau, 1957) , virescent (Valleau and Stokes, 1957), veinbanding (Valleau, 1958a), and ivory (Gwynn and Mann, 1965). Although a few of the mutants are governed in their inheritance by two genetic factors, most segregate in monogenic ratios. This may, however, reflect a “functional diploidization” through mutation a t one of duplicate loci, originally present because of the amphiploid origin of N . tabacum (Clausen and

42

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Cameron, 1950; Stines and Mann, 1960; Povilaitis and Cameron, 1963). The 5 gene, that controls self-incompatibility reactions in plants, has been studied by Pandey (1962, 1964, 1965) in order (1) to elucidate the structure of the locus in terms of allelic components, and (2) to distinguish between true radiation-induced pollen-part mutants and those requiring an extra chromosome fragment to effect self -compatibility in N . alata. Radiation has been used to treat different species of Nicotiana to investigate differences in radiation sensitivity among species (Scarascia, 1960) and in gametes vs. zygotes vs. proembryos (Devreux and Scarascia Mugnozza, 1962, 1964; Tramvalidis and Devreux, 1964). Various mutants affecting pollen sterility, chlorophyll production, morphology of leaf, and chromosomal aberrations have been produced in N . tabacum with thermal neutrons (Murty et aZ., 1963). XIV. Summary

The genus Nicotiana, which comprises 64 presently recognized species, has been studied extensively cytogenetically because of the different degrees of evolutionary divergence and their consequences that can be observed in hybrids, and because of problems associated with the commercial production of N . tabacum. Studies on pairing of chromosomes in species hybrids have been helpful in clarifying phylogenetic relationships, and determining the progenitors of N . tabacum and other species of amphiploid origin. Limits and consequences of multiple allopolyploidy have been explored, and the role of aneuploidy assessed. The use of introgressive hybridization to transfer resistance to various diseases into commercial tobacco from wild species has been an important part of recent tobacco breeding efforts. Certain Nicotiana interspecific hybrids exhibit a striking somatic instability in forming spontaneous tumors. These are controlled by genes and appear to result from production of more than regulatory amounts of growth-promoting substances. Other manifestations of hybrid instability are variegations in pigment and variations in growth pattern, some of which have been analyzed in terms of the cytogenetic phenomena involved. I n certain interspecific combinations evidence for a hereditary component in the cytoplasm has been found. This usually affects either pollen sterility or plastid-controlled chlorophyll variegation. All species of Nicotiana synthesize alkaloids, of which three-nicotine, nornicotine, and anabasine-are particularly prevalent throughout the genus. Most

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varieties of N . tabacum lack the genes for converting nicotine to nornicotine which are found in progenitor species. Biornetrical analyses of quantitative character inheritance in N . tabucum and in species crosses have shown preponderant additive gene effects, generally high heritability, and consequent effective response to selection. ACKNOWLEDGMENTS Grateful acknowledgment and thanks are expressed to Dr. L. G. Burk, Dr. D. R. Cameron, Dr. D. U.Gerstel, and Dr. T. J. Mann for help extended in many ways, from making available unpublished material to critical reading of the manuscript. The Tobacco Literature Service of North Carolina State University, through publication of Tobacco Abstracts, aided greatly in the literature search.

REFERENCES Abraham, A. 1947.A cytogenetical study of trisomic types in Nicotiana langsdorfii. Ph.D. Thesis, Cornell University. Ahmad, N., and Termazi, S. A. 1960.Heterosis in tobacco. Pakistan J. Sci. 12, 74-82. Ahuja, M. R. 1962. A cytogenetic study of heritable tumors in Nicotiana species hybrids. Genetics 47, 865-880. Ahuja, M. R. 1965. A cytogenetic study of the tumor-bearing hybrid Nicotiana glauca x N . langsdorjii. Proc. 11th Intern. Congr. Genet., The Hague, 1963 Vol. 1, p. 118.Pergamon Press, Oxford. Ahuja, M. R., and Cameron, D. R. 1963. The effects of X-irradiation on seedling tumor production in Nicotwlnn species and hybrids. Radiation, Botany 3, 55-57. Anders, F.,and Vester, F. 1960. Genetisch bedingte Tumoren und der Gehalt an freien Aminosauren bei Nicotiana. Experientia 16, 65-67. Appa Rao, K.,and Krishnamurty, K. V. 1963. Studies on multiple polyploids in Nicotiana. Cenetica 34, 66-78. Apple, J. L. 1962. Transfer of resistance to black shank (Phytophthora parasitica var. nicotianae) from Nicotiana plumbaginifolia to N . tabacum. Phytopathology 52, 1. Ar-Rushdi, A. H. 1956. Inheritance in Nicotkana tabacum. XXVI. Sterility genes from Tomentosae species. J. Genet. 54, 9-22. Ar-Rushdi, A. H. 1957. The cytogenetics of variegation in a species hybrid in Nicotiana. Genetic8 42, 312-325. Aycock, M. K. Jr., Mann, T. J., and Matzinger, D. F. 1963. Investigations with a form of cytoplasmic male-sterility in flue-cured tobacco. Tobacco Sei. 7 , 130-136. Bagnara, D.,Palenzona, D. L., and Wittmer, G.1964.Valuation in R, of the effect of ionizing radiations on the genetic variability of some leaf characters in N . tabacum. Genet. Agrar. 18, 389-394. Bawolska, M.,BerbeE, J., and Konczerewicz, A. 1960. Effect of heterosis in tobacco hybrids ( N . tabacum L . ) .Roczniki Nauk Rolniczych A81, 845-878. Bayer, M. H. 1965.Paper chromatography of auxins and inhibitors in two Nicotiana species and their hybrid. Am. J. Botany 52, 883-890.

44

HAROLD H. SMITH

Bayer, M. H.,and Hagen, G. L. 1964. The extractable and difiusible auxin-auxin inhibitor level in Nicotianii glazrecr, Nicotknu lawsdorfii and their amphidiploid hybrid. Am. J. Botany 51, 643-648. Braun, A. C. 1968. A physiological basis for autonomous growth of the crown-gall tumor cell. Proc. Natl. Acad. Sci. US.44, 344-349. Braun, A. C. 1959. A demonstration of the recovery of the crown-gall tumor cell with the use of complex tumors of single cell origin. Proc. Natl. Acad. Sci. U S .

45,932-938.

Braun, A. C. 1965.The reversal of tumor growth. Sci. Am. 213, 75-83. Braun, A. C.,and Stonier, T. 1958. Morphology and physiology of plant tumors. Protoplasmatologia 10, 1-93. Brieger, F.G., and Forater, R. 1942.Tumores en certos hibridos do gsnero Nicotiana. Bragantia 2, 259-274. Burbridge, N.T.1960. The Australian species of Nicotiana L. (Solanaceae). Australian J. Botany 8,342-380. Burk, L. G. 1960. Male-sterile flower anomalies in interspecific tobacco hybrids. J. Heredity 51,27-31. Burk, L. G. 1962. Haploids in genetically marked progenies of tobacco. J. Heredity

53,222-226.

Burk, L.G. 1965. Inter-histogenic cell migration measured by a plastid defect of tobacco. Am. J. Botany 52, 616-617. Burk, L. G., and Dropkin, V. H. 1961.Response of Nicotiana repanda, N . sylvestris, and their amphidiploid hybrid to root-knot nematodes. Plant Disease Reptr. 45,

734-735.

Burk, L. G.,and Gerstel, D. U. 1961. Haploid Nicotiana deficient for two chromosomes. J. Heredity 52, 203-206. Burk, L. G., and Grosso, J. J. 1963. Plasmagenes in variegated tobacco. J. Heredity

54, 23-26.

Burk, L. G., and Heggestad, H. E. 1966.The genus Nicotiana: A source of resistance to diseases of cultivated tobacco. Econ. Botany 20,7648. Burk, L. G., and Jeffrey, R. N. 1968. A study of the inheritance of alkaloid quality in tobacco. Tobacco Sci. 2, 139-141. Burk, L. G., and Menser, H. A. 1964.Dominant aurea mutation in tobacco. Tobacco Sci. 8,101-104 Burk, L. G., and Tso, T. C. 1960. Genetic tumors of Nicotiana associated with chromosome loas. J. Heredity 51, 184-187. Burk, L.G., Stewart, R. N., and Dermen, H. 1964. Histogenesis and genetics of a plastid-controlled chlorophyll variegation in tobacco. Am. J. Botany 51, 713424. Burns, J. A. 1966. The heterochromatin of two species of Nicotiana: cytological observations. J. Heredity 57, 43-47. Cameron, D.R. 1962. Inheritance in Nicotiana tabacum XXIV. Intraspecific difTerencea in chromosome structure. Genetics 37, 288-296. Cameron, D. R. 1969a. Alien substitution of a locus effecting immunity to black shank in Nicotiana tabacum. Proc. l o t h Intern. Congr. Genet. Montreal, 1958 Vol. 2, p. 41. Univ. of Toronto Press, Toronto. Cameron, D. R. 1959b. The monosomics of Nicotiana tabacum. Tobacco Sci. 3,

164166.

Cameron, D.R. 1962. Studies of introgressed loci in N . tabacum. Tobacco Sci. 6,

137-139.

CYTOGENETICS OF

Nicotiana

45

Cameron, D. R. 1965. Cytoplasmic effects in Nicotiana. Proc. 11th Intern. Congr. Genet. The Hague, 1968 Vol. 1,pp. 203-204. Pergamon Press, Oxford. Cameron, D. R. and Moav, R. 1957. Inheritance in Nicotiana tabacum XXVII. Pollen killer, an alien genetic locus inducing abortion of microspores not carrying it. Genetics 42, 326335. Chaplin, J. F. 1962. Transfer of black shank resistance from Nicotiana plumbaginif o l k to flue cured N . tabacum. Tobacco Sci. 6, 184-189. Chaplin, J. F. 1964. Use of male-sterile tobaccos in the production of hybrid seed. Tobacco Sci. 8, 105-109. Chaplin, J. F., and Mann, T. J. 1961. Interspecific hybridization, gene transfer and chromosome substitution in Nicotiana. N . Carolina State Coll, Agr. Expt. Sta., Tech. Bull. 145, 1-31. Chaplin, J. F.,Mann, T. J., and Apple, J. L. 1961. Some effects of the Nicotiana glutinosa type of mosaic resistance on agronomic characters of flue cured tobacco. Tobacco Sci. 5 , 80-83. Chaplin, J. F., Ford, Z. T., and Jones, C. W. 1963. Use of male-sterile tobacco in relation to topping and suckering practices. Tobacco Sci. 7 , 158-162. Chaudry, A. H.,and Munshi, A. 1964.Heterosis studies in inter-varietal crosses of Nicotiana tabacum. West Pakistan J . Agr. Res. 2, 96105. Chen, S. T., and Chu, J. M. 1960.Utilization of hybrid vigor of tobacco during the last ten years in China. Chinese J . Agr. Sci. (In Chinese.) 4, 23-27. Clausen, R. E., and Cameron, D. R. 1944.Inheritance in Nicotiana tabacum XVIII. Monosomic analysis. Genetics 29, 447-477. Clausen, R.E., and Cameron, D. R. 1950.Inheritance in Nicotiana tabacum. XXIII. Duplicate factors for chlorophyll production. Genetics 35, 4-10. Clausen, R. E., and Cameron, D. R. 1957. Inheritance in Nicotiana tabacum XXVIII. The cytogenetics of introgression. Proc. Natl. Acad. Sci. U S . 43, 908913. Clausen, R.E., and Goodspeed, T. H.1926a.Inheritance in Nicotianu tabacum. VII. The monosomic character “fluted.” Uniu. Calif. (Berkeley) Publ. Botany 11, 61-82. Clausen, R. E., and Goodspeed, T. H.1926h.Interspecific hybridization in Nicotiana. 111. The monosomic Tabacum derivative, “corrugated,” from the sylwestristabacum hybrid. Univ. Calif. (Berkeley) Publ. Botany 11, 83-101. Clayton, E. E. 1947. A wildfire resistant tobacco. J . Heredity 38, 35-40. Clayton, E.E.1950. Male sterile tobacco. J . Heredity 41, 171-175. Clayton, E.E.1958.The genetics and breeding progress in tobacco during the last 50 years. Agron. J . 352-356. Dalebroux, M. 1962. Biometrical study of radiation-induced genetic variability in a pure diploid selfing line. Ann. Amelioration Plantes 12, 96-115. Daly, K.1958. Genetic studies of biotypes derived from hybridization in Nicotiana. Ph.D. Thesis, Cornell University. Dawson, R. F. 1952. Alkaloid biogenesis : Nicotine demethylation in excised leaves of Nicotiana glutinosa. Am. J . Botany 39, 250-253. Dawson, R. F.1960.Biosynthesis of the Nicotiana alkaloids. Am Sci. 48, 321340, Dawson, R. F., and Solt, M. L. 1959. Estimated contributions of root and shoot to the nicotine content of the tobacco plant. Plant Physiol. 34, 656661. Dean, C. E. 1964. Corrugated dwarf tobacco: A semidominant lethal mutation in Nicotiana tabacum L.J . Heredity 55, 104-106.

46

HAROLD H. SMITH

de Nettancourt, D., and Stokes, G. W. 1960.Haploidy in tobacco. J. Heredity 51, 102-104. Dermen, H.1960. Nature of plant sports. Am. Hort. Mag. 39, 123-173. Devreux, M., and Scarascia Mugnozza, G. T. 1962.Action des rayons gamma sur les premiers studes de dkveloppement de l’embryon de Nicotiana rustica L. CaryoZogia 15,279-291. Devreux, M.,and Scarascia Mugnozza, G. T. 1964. Effects of gamma radiation of the gametes, zygote and proembryo in Nicotiana tabacum L. Radiation Botany 4, 373-386. Dulieu, H. 1964. Detection of haploid plants among the progeny Nicotiana tabacum L. x N . sanderae Hort. after pollen irradiation. Compt. Rend. 259, 4126-4129. East, E. M. 1928.The genetics of the genus Nicotiana. Bibliog. Genet. 4, 243-318. East, E. M. 1932. Studies on self-sterility. IX. The behavior of crosses between self-sterile and self-fertile plants. Genetics 17, 175-202. East, E.M.1935. Genetic reactions in Nicotiana. 11. Phenotypic reaction patterns. Genetics 20, 414-442. Edwardson, J. R. 1965. Gene control of non-Mendelian variegation in Nicotiana tabacum. Genetics 52, 365-370. Gerstel, D. U. 1943.Inheritance in Nicotiana tabacum. XVII. Cytogenetical analysis of glutinosa-type resistance to mosaic disease. Genetics 28, 533-536. Gerstel, D. U. 1945.Inheritance in Nicotiana tabacum. XX. The addition of Nicotiana glutinosa chromosomes to tobacco. J . Heredity 36, 197-206. Gerstel, D. U. 1948. Transfer of the mosaic-resistance factor between H-chromosomes of Nicotiana glutinosa and N . tabacum. J . Agr. Res. 76, 219-223. Gerstel, D. U. 1960.Segregation in new allopolyploids of Nicotiana. I. Comparison of 6 X ( N . tabacum X tomentosiforml) and 6 x ( N . tabacum x otophora). Genetics 45,1723-1734. Gerstel, D. U. 1961. Chromosomal control of preferential pairing in Nicotiana. Science 133, 579-680. Gerstel, D.U. 1963. Segregation in new allopolyploids of Nicotiana. 11. Discordant ratios from individual loci in 6 x ( N . tabacum x N . sylvestris). Genetics 48, 677-689. Gerstel, D.U.1966.Evolutionary problems in some polyploid crop plants. Hereditas S ~ p p l2,481-504. . Gerstel, D. U., and Burk, L. G. 1960. Controlled introgression in Nicotiana. A cytological study. Tobacco Sci. 4, 146150. Gerstel, D. U., and Burns, J. A. 1966a. Chromosomes of unusual length in hybrids between two species of Nicotiana. Chromosomes Today 1,41-56. Gerstel, D. U., and Burns, J. A. 1966b.Flower variegation in hybrids between NiCotiana tabacum and N . otophora. Genetics 53, 551-567. Gerstel, D. U., and Mann, T. J. 1964.Segregation in new allopolyploids in Nicotiana. 111. Nicotine-converter genes in allopolyploids from N . tomentosiformis, N . sylvestris and N . tabacum. Crop Sci. 4, 387-388. Gilbert, N.,and Jinks, J. L. 1964.Non-additive combining abilities. 11. Reciprocals and backcrosses. Heredity 19, 97-103. Goodspeed, T. H. 1954. “The Genus Nicotiana.” Chronica Botanica, Waltham, Massachusetts. Goodspeed, T. H., and Avery, P. 1939. Trisomic and other types in Nicotkna sylvestris J . Genet. 38, 381-458.

CYTOGENETICS OF

Nicotiana

47

Goodspeed, T. H., and Avery, P. 1941. The twelfth primary trisomic type in Nicotiana sylvestris. Proc. Natl. Acad. Sci. U S . 27, 13-14. Goodspeed, T. H., and Clausen, R. E. 1928. Interspecific hybridiaation in Nicotiana. VIII. The sylvestris-tomentosa-tabacum hybrid triangle and its bearing on the origin of tabacum. Univ. Calif. (Berkeley) Publ. Botany 11, 245-256. Goodspeed, T. H., and Thompson, M. C. 1959. Cytotaxonomy of Nicotidna. 11. Botan. Rev. 25, 385415. Griffith, R. B., Valleau, W. D., and Stokes, G. W. 1956. Determination and inheritance of nicotine to nornicotine conversion in tobacco. Science 121, 343-344. Gunokel, J. E. 1957. IV The effects of ionizing radiation on plants: Morphological effects. Quart. Rev. Biol. 32, 46-56. Gwynn, G. R.,and Mann, T. J. 1965. Ivory-a virescent character in tobacco. Tobacco Sci. 9,17-20. Hagen, G. L. 1962.Morphogenesis in a tobacco hybrid tumor. Develop. Biol. 4, 569579. Hagen, G. L., and Gunckel, J. E. 1958. Free amino acids in Nicotiana glauca, N . langsdorfii and their interspecific hybrid following gamma irradiation. Plant Physiol. 33, 439443. Hagen, G. L.,and Gunckel, J. E. 1962.Tumor formation on irradiated leaves of a tobacco hybrid. Radiation Botany 1, 247-249. Hagen, G. L.,Gunckel, J. E., and Sparrow, A. H. 1961. Morphology and histology of tumor types induced by X, gamma, and beta irradiation of a tobacco hybrid. Am. J. Botany 48, 691-699. Hall, J. L.,Weybrew, J. A., and Mann, T. J. 1965. Conversion of nicotine to nornicotine in grafts between cherry-red tobacco and related materials. Plant Physiol. 40, 45-48. Hart, G. E. 1965. Studies on extrachromosomal male sterility in Nicotidna. Ph.D. Thesis, University of California, Berkeley, California. Hayman, B. I. 1954.The theory and analysis of diallel crosses. Genetics 39, 789-809. Hayman, B. I, 1960. Separation of epistatic from additive and dominance variation in generation means. 11. Genetica 31, 133-146. Heggestad, H, E., Clayton, E. E., Neas, M. O., and Skoog, H. A. 1960.Development of Burley 21 the first wildfire-resistant tobacco variety, including results of variety trials. Tenn. Univ. Agr. Expt. Sta., Bull. 321, 1-51. Henry, T. A 1949. “The Plant Alkaloids,” 4th ed. Blakiston, Philadelphia, Pennsylvania. Hill, J. 1966. Environmental induction of heritable changes in Nicotiana rustica. Nature, 207, 732-734. Hillman, W.S., and Smith, H. H. 1965. Induced flowering in a vegetative tobacco hybrid. J . Heredity 56, 2-6. Holmes, F.0.1938.Inheritance of resistance to tobacco mosaic disease in tobacco. PhytopathoEogy 28, 553-561. Humphrey, A. B., Matzinger, D. F., and Mann, T. J. 1964. Inheritance of leaf shape in flue-cured tobacco (Nicotiana tabacum LJ.Heredity 19, 615-628. Imai, S. 1959. Alkaloids in Nicotiana. Hatano Tobacco Expt. Sta., Bull. (In Japanese with English summary) 44,129-136. Izard, C. 1957. Obtention et fixation des lign6es tumorales e t non tumorales B partir de mutations exp6rimentales de l’hybride N . glauca x N . Eangsdorfii. Compt. Rend. Acad. Agr. France 43, 325-327.

48

HAROLD H. SMITH

Jeffrey, R. N. 1959. Alkaloid composition of species of Nicotiana. Tobacco Sci. 3, 89-93. Jinks, J. L. 1964.The analysis of continuous variation in a diallel cross of Nicotiana rustica varieties. Genetics 39, 767-788. Jinks, J. L. 1956. The Fp and backcross generations from a set of diallel crosses. Heredity 10, 1-30. Jinks, J. L., and Mather, K. 1955. Stability in development of heterozygotes and homosygotes. Proc. R o y . Soc. B143, 561-578. Kehr, A. E. 1951. Genetic tumors in Nicotiana. A m . Naturalist 85, 51-64. Kehr, A. E., and Smith, H. H. 1952. Multiple genome relationships in Nicotiana. Cornell Univ., Agr. Expt. Sta. Mem. 311,l-19. Kehr, A. E.,and Smith, H. H.1964. Genetic tumors in Nicotiuna hybrids. Brookhaven Symp. Biol. 6, 65-78. Kostoff, D. 1930. Tumors and other malformations on certain Nicotiana hybrids. Zentr. Bakteriol., Parasitenk., A b t . IZ 81, 244-260. Kostoff, D. 1935. On the increase of mutation frequency following interspecific hybridization. Current Sci. (India) 3, 302-304. Kostoff, D. 1943. “Cytogenetics of the Genus Nicotiana,” 1071 pp. State Printing House, Sofia, Bulgaria. Krishnamurty, K. V., and Satyanarayana, K.V. 1962.Chromosome elimination and phenotypic variability in a trispecific Nicotiana hybrid. Japan. J . Genet. 31, 485-497. Krishnamurty, K. V., Murty, G. S., and Appa Rao, K. 1960. Cytogenetics of the trispecific hybrid Nicotiana tabacum x ( N . glutinosa x N . trigonophylla) and its reciprocal. Euphytica 9, 111-121. Kupila, S., and Therman, E. 1962. Anatomical observations on genetic tumors and crown gall in amphidiploid Nicotiana glauca x langsdorfii. Ann. Botan. SOC. Zool. Botan. Fennicae “Vanamo” 32, 1-21. Lamprecht, M. P. 1964. Diallel crosses for yield improvement with flue cured tobacco cultivars. S. African J . Agr. 81%.7, 717-729. Latterell, R. L. 1968. Morphogenetic investigations on somatic instability in the F1hybrid of Nicotiana glutinosa by N . glauca. Ph.D. Thesis, Cornell University, Ithaca, New York. Lea, H. W. 1961.A review of two commerical flue cured blue mold resistant tobacco varieties. Coresta, Park Znf. Bull. 2, 21-27. Lee, R.E. 1950.A cytogenetic study of extra chromosomes in Nicotiana langsdorfii and in crosses with N . sanderae. Ph.D. thesis, Cornell University. Legg, P. D., and Mann, T. J. 1961. Study of introgression of N . rwtica germplasm into flue cured varieties of N . tabacum. Tobacco Sd.5, 136-139. Legg, P. D., Matzinger, D. F., and Mann, T. J. 1965. Genetic variation and covariation in a Nicotiana tabacum L. synthetic two generations after synthesis. Crop Sci. 5 , 30-33. Luthra, J. K. 1964. Inheritance of quantitative characters in Nicotiana tabacum. Indian J . Genet. Plant Breeding 24, 276-279. McCray, F. A. 1932. Compatibility of certain Nicotiana species. Genetics 17, 621636. Maw, T. J., and Matzinger, D. F. 1965. Wrinkled-leafdominant mutant in flue cured tobacco. Tobacco Sci. 9, 44-77. Mann, T.J., and Weybrew, J. A. 1968. Inheritance of alkaloids in hybrids between flue cured tobacco and related amphidiploids. Tobacco Sci. 2, 29-34.

CYTOGENETICS OF

Nicotiana

49

Mann, T. J., Jones, G. L., and Matzinger, D. F. 1962. The use of cytoplasmic male sterility in flue cured tobacco hybrids. Crop Sci. 2, 407410. Mann, T. J., Gerstel, D. U., and Apple, J. L. 1963. The role of interspecific hybridization in tobacco disease control. Proc. 3rd World Tobacco Sci. Congr., Salisbury, S. Africa, 1988 pp. 201-205. Mardon Printers, Salisbury, Southern Rhodesia. Mann, T. J., Weybrew, J. A., Manringer, D. F., and Hall, J. L. 1964. Inheritance of the conversion of mcotine to nornicotine in varieties of Nicotiana tabacum L. and related amphiploids. Crop Sci. 4, 349-353. Marion, L.1950. The pyridine alkaloids. In “The Alkaloids” (R. H. F. Manske and H. L. Holmes, eds.), Vol. 1, pp. 228-253. Academic Press, New York. Marion, L. 1960.The pyridine alkaloids. In “The Alkaloids” (R. H. F. Manske, ed.), Vol. 6,pp. 128-132. Academic Press, New York. Mather, K. 1949. The genetical theory of continuous variation. Proc. 8th Intern. C w r . Genet. Stockholm, 1949. Hereditas Suppl. pp. 376-401. Mendelian SOC., Lund, Sweden. Mather, K., and Vines, A. 1952. The inheritance of height and flowering time in a cross of Nicotiana m t i c a . In “Quantitative Inheritance” (Agr. Res. Council Colloq.), pp. 49-79. H. M. Stationery Office, London. Matzinger, D. F., and Mann, T. J. 1962. Hybrids among flue-cured varieties of Nicotiana tabacum in the F, and Fp generations. Tobacco Sci. 6, 125-132. Matzinger, D.F.,and Mann, T. J. 1963. Genetic studies on associations between nicotine content and yield of due-cured tobacco. Proc. 3rd World Tobacco Sci. Congr., Sulkbury, S. Africa, 1963, pp. 357-365. Mardon Printers, Salisbury, Southern Rhodesia. Matzinger, D. F., Mann, T. J., and Robinson, H. F. 1960. Genetic variability in flue-cured varieties of Nicotiana tahacum. I. Hicks Broadleaf x Coker 139. Agron. J. 52, 8-11. Matzinger, D.F.,Mann, T. J., and Cockerham, C. C. 1962. Diallel crosses in Nicotiana tabacum. crop Sci. 2, 383-386. Meiselman, N.,Gunckel, J. E., and Sparrow, A. H. 1961a. The general morphology and growth responses of two species of Nicotiana and their interspecific hybrid after chronic gamma irradiation. Radiation Botany 1, 69-79. Meiselman, N., Sparrow, A. H., and Gunckel, J. E. 1961b. The Radiosensitivity of two species of Nicotiana and of their interspecific hybrid. Bull. Torrey Botan. Club. 88, 30-38. Moav, R. 1958. Inheritance in Nicotiana tabacum. XXIX. The relationship of residual chromosome homology to interspecific gene transfer. Am. Natural& 92, 267-278. Moav, R. 1961. Genetic instability in Nirotiana hybrids. 11. Studies of the Ws (pbg) locus of N . plumbaginifolia in N . tabacum nuclei. Genetics 46, 1069-1087. Moav, R. 1962. Inheritance in Nicotiana tabacum. XXX. Autotriploidy, a possible means of increasing the rate of interspecific gene transfer. Heredity 17, 373-379. Moav, R., and Cameron, D.R. 1960. Genetic instability in Nicotiana hybrids. I. The expression of instability in N . tabncum x N . plumbaginifolia. Am. J. Botany 47, 87-93. Murty, B. R. 1960. Genetic studies of some hybrid derivatives of Nicotiana rustica and Nicotiana tabacum. PhD. Thesis, Cornell University, Ithaca, New York. Murty, B. R. 1965. Heterosis and combining ability in relation to genetic divergence in flue-cured tobacco. Indian J. Genet. Plant Breeding 25, 46-56.

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Murty, B. R., and Swaminathan, M. S. 1967. Cytogenetic studies in derivatives of Nicotiana rustica x N . tabacum. Euphytica 6, 227-236. Murty, B. R.,Murty, G. S., and Pavate, M. V. 1962. Studies on quantitative inheritance in Nicotiana tabncum L. 11. Components of genetic variation for flowering time, leaf number, grade performance and leaf burn. Ziichter 32, 361-369. Murty, G. S., Krishnamurty, K. V., and Appa Rao, K. 1963. Cytogenetics of neutronic mutants in Nicotianu tabacum. Euphytica 12, 67-68. Naf, U.1968. Studies on tumor formation in Nicotiana hybrids. I. The classification of the parents into two etiologically significant groups. Growth 22, 167-180. Niwa, M.1965. Radiation induced interspecific transfer of Ws gene from Nicotiana plumbaginifolia to N . tabacum. 111. Differential frequencies of the interspecific transfer during gametogenesis. Japan. J. Breeding (In Japanese.) 15, 64. Oka, M.1959.The analysis of inheritance of quantitative characters with flue-cured tobacco varieties in diallel cross. Japan. J . Breeding (In Japanese). 9, 87-92. Pandey K. K. 1962. A theory of &gene structure. Nature 196, 236-238. Pandey, K.K.1964.Elements of the S-gene complex. Genet. Res. 5, 397-409. Pandey, K. K. 1966. Centric chromosome fragments and pollen-part mutation of the incompatibility gene in Nicotiana alata. Nature 206, 792-795. Patel, G. J. 1959. Estimates of genotypic and phenotypic variances and covariaances in a high and low yielding population of flue-cured tobacco and their implications in selection. Dissertation Abstr. 20, 1649-1650. Patel, K. A., and Gerstel, D.U. 1961. Additional information on the mechanism of chromosome substitution in Nicotiana. Tobacco 8ci. 5, 18-20. Patel, K. A., and Swaminathan, M. S. 1961. Mutation breeding in tobacco. Tobacco Sci. 5, 67-69. Paxman, G.J. 1966. Differentiation and stability in the development of Nicotiana wstica. Ann. Botany (London) [N.S.] 20, 331-347. Povilaitis, B. 1964. Inheritance of certain quantitative characters in tobacco. Can. J. Genet. Cgtol. 6, 472-479. Povilaitis, B.,and Cameron, D. R. 1963. A mutation causing chlorophyll deficiency in Nicotiana tabacum. Can. J . Genet. Cytol. 5,233-238. Rao, P.N.,and Stokes, G. W. 1963. Cytogenetic analysis of the F1 of haploid x diploid tobacco. Genetics 48, 1423-1433. Robinson, H.F.,Mann, T. J., and Comstock, R. E. 1954. An analysis of quantitative variability in Nicotiana tabacum. Hereditv 8, 366-376. Sakai, K. I., and Shimamoto, Y. 1965. Developmental instability in leaves and flowers of Nicotiana tabacum. Genetics 51, 801-813. Sand, S. A. 1957. Phenotypic variability and the influence of temperature on somatic instability in cultures derived from hybrids between Nicotiana Zangsclorjjii and N . sanderae. Genetics 42,686-703. Sand, 8. A. 1960. Autonomy of cytoplasmic male sterility in grafted scions of tobacco. Science 131, 665. Sand, S. A. 1961a. Effects of flower node position on the mutable V and stable R loci in a clone of Nicotiana. Genetics 46, 569-574. Sand, 5. A. 1961b. Alien chromosome addition races in Nicotiana. Genetics 46, 895. Sand, S. A. 1962a. Temperature response of the genetically stable R locus in comparison with the mutable V locus in a clone of Nicotiann. Nature 196, 91-92.

CYTOGENETICS OF

Nicotiana

51

Sand, S. A. 1962b. A model for the cytogenetic partitioning of quantitative genetic expression. Genetics 47, 982. Sand, S. A,, Sparrow, A. H., and Smith, H. H. 1960. Chronic gamma irradiation effects on the mutable V and stable R loci in a clone of Nicotiana. Genetics 45, 289408. Satyanarayana, K. V., and Subhashini, L. 1964. Interspecific hybridization and aneuploidy in the genus Nicotiana. Indian J. Genet. Plant Breeding 24, 264-271. Scarascia, G. T. 1960. Studies on the effect,s of radiation in Nicotiana. 111. Aspects of radioresistance in the genus Nicotiana. Genet. Agrar. 13, 123-156. Schaeffer, G. W. 1962. Tumour induction by an indolyl-3-acetic acid-kinetin interaction in a Nicotiana hybrid. Nature 196,1326-1327. Schaeffer, G . W., and Smith, H. H. 1963. Auxin-kinetin interaction in tissue cultures of Nicotiana species and tumor-conditioned hybrids. Plant Physiol. 38, 29 1-29 7. Schaeffer, G. W., Smith, H. H., and Perkus, M. P. 1963. Growth factor interactions in the tissue culture of tumorous and nontumorous Nicotiana glaucalangsdorfii. Am. J . Botany 50, 766-771. Scott, K. J., Daly, J., and Smith, H. El. 1964. Effects of indoleacetic acid and kinetin on activities of enzymes of the hexose monophosphate shunt in tissue cultures of Nicotiana. Plant Physiol. 39, 709-711. Sficas, A. G. 1962. Statistical analysis of chromosome pairing in interspecific hybrids. I. The probability distributions. Genetics 47, 1163-1170. Sficas, A. G . 1963. Statistical analysis of chromosome distribution to the poles in interspecific hybrids with variable chromosome pairing. Genet. Res. 4, 2 6 6 275. Sficas, A. G., and Gerstel, D. U. 1962. Statistical analysis of chromosome pairing in interspecific hybrids. 11. Applications to some Nicotkna hybrids. Genetics 47, 1171-1185. Sikka, L. C., and Batra, B. M. 1960. Studies on commercial feasibility of artificially produced hybrid vigour in tobacco (Nicotiana Rustica). Indian Tobacco 10, 43-53. Silber, G., and Burk, L. G. 1966. A genetic leaf curl of tobacco. J. Heredity 56, 2 15-21 8. Smith, H. H. 1943. Effects of genome balance, polyploidy and single extra chromosomes on size in Nicotiana. Genetics 28, 227-236. Smith, H. H. 1950. Differential photoperiod response from a n interspecific gene transfer. J . Heredity 41, 199-203. Smith, H. H. 1952. Fixing transgressive vigor in Nicotiana ruatica. In “Heterosis” (J. Gowen, ed.), Chapter 10, pp. 161-174. Iowa State Univ. Press, Ames, Iowa. Smith, H. H. 1957. Genetic plant tumors in Nicotiana. Ann. N . Y . Acad. Sci. 71, 1163-1178. Smith, H. H. 1962a. Genetic control of Nicotinnn plant tumors. Trans. N . Y . Acad. Sci. [2] 24, 741-746. Smith, H. H. 1962b. Studies on the origin, inheritance and mutation of geniccytoplasmic male sterility in NicoLiana. Genetics 47, 985-986. Smith, H. H. 1964. Stabilized transgression through alien disome addition. Genetics 50, 287. Smith, H. H. 1965. Inheritance of alkaloids in introgressive hybrids of Nicotiana. A m . Naturalist 99, 73-79.

52

HAROLD H. SMITH

Smith, H. H. 1966. Genetic tobacco tumors and the problem of difTerentiation. Brookhaven Lecture Ser. 52,l-8. Smith, H. H., and Abashian, D. V. 1963. Chromatographic investigations on the alkaloid content of Nicotiana species and interspecific combinations. Am. J. Botany 50, 435-447. Smith, H. H., and Daly, K. 1959. Discrete populations derived by interspecific hybridization and selection in Nicotiana. Evolution 13, 476487. Smith, H. H., and Robson, D. 8. 1959. A quantitative inheritance study of dimensions of flower parts in tobacco. Biometries 15, 147. Smith, H. H., and Sand, S. A. 1957. Genetic studies on somatic instability in cultures derived from hybrids between Nicotiana langsdorfii and N . sanderae. Genetics 42, 560-582. Smith, H. H., and Smith, C. R. 1942. Alkaloids in certain species and interspecific hybrids of Nicotiana. J. Agr. Res. 65, 347-359. Smith, H. H., and Stevenson, H. Q. 1961. Genetic control and radiation effects in Nicotiana tumors. 2.Vererbungslehre 92, 100-118. Smith, H. H., Stevenson, H. Q., and Kehr, A. E. 1968. Limits and consequences of multiple allopolyploidy in Nicotkna. Nucleus (Calcutta) 1, 205-222. Sparrow, A. H., and Schairer, L. A. 1958. Some factors influencing radio-resistance and tumor induction in plants. Proc. U.N. Ind Intern. Conj. Peaceful Uses A t . Energy Geneva, 1958 Vol. 27, 335-340. United Nations, New York. Steinberg, R. A. 1953. Low temperature induction of flowering in a Nicotiana rustica x N . tabacum hybrid. Plant Physiol. 28, 131-134. Steitz, E. 1963. Untemchungen uber die Tumorbildung bei Bastarden von Nicotiana glauca und N . langsdorfii. Ph.D. Thesis, University of Saarland, Saarbriiken, Germany. Stephens, 8.G. 1961. Species differentiation in relation to crop improvement. Crop S C ~1., 1-5. Stines, B. J., and Mann, T. J. 1960. Diploidiaation of Nicotiana tabacum. A study of the yellow burley character. J . Heredity 51, 222-227. Stokes, G. W. 1963. Development of complete homozygotes of tobacco. Science 141, 1185-1186. Swaminathan, M. S., and Murty, B. R. 1959. Effect of x-radiation on pollen tube growth and seed setting in crosses between Nicotiana tabacum and N . rustica. 2. Vererbungslehre 90, 393-399. Takenaka, Y. 1962a. Cytogenetic studies in Nicotiana. XV. Reduction divisions in five interspecific hybrids between section Alatae and Suaveolentes. Japan. J. Genet. 37, 80-85. Takenaka, Y. 1962b. Cytogenetic studies in Nicotiana. XVI. Reduction divisions in six interspecific hybrids between N. tabacum and six other species. Botan. Mag. 75, 237-241. Takenaka, Y. 1962~.Cytogenetic studies in Nkotiana. XVII. Reduction divisions in five interspecific hybrids. Japan. J . Genet. 37, 343-347. Takenaka, Y. 1962d. Cytogenetic studies in Nicotiana. XIX. The reduction division in four hybrids between N. paniculata and other species of section Alatae. Japan. J. Breeding 12, 278-280. Takenaka, Y. 1963. Cytogenetic studies in Nicotiana. XX. Reduction divisions in three interspecific hybrids and one amphidiploid. Japan. J . Genet. 38, 135-140. Takenaka, Y., and Takenaka, M. 1956. Cytogenetic studies in Nicotiana. XIII. Haploid plant of Nicotiana tabacum. Botan. Mag. 69, 193-198.

CYTOGENETICS OF " k O k k ? t U

53

Takenaka, Y., and Yoneda, Y. 1962. Tumorous hybrids in Nicotiana. Natl. Znst. Genet. Japan. Ann. Rept. 13, 63-64. Tramvalidis, C.,and Devreux, M. 1964. Fertilitk pollinique de Nicotiana tabacum L. a p r k irradiation gamma aux stctdes gambtes, zygote e t proembryon. Caryologia 17, 453457. Tso, T. C. 1962. Some novel concepts on the biosynthesis and biogenesis of tobacco alkaIoids. Botan. Bull. Acad. Sinica (Taiwan) Znst. Botany rN.S.1 3, 61-71. Tso, T. C., and Jeffrey, R. N. 1961. Biochemical studies on tobacco alkaloids. IV. The dynamic state of nicotine supplied to N . rustica. Arch. Biochem. Biophys. 92, 253-256. Tso, T. C., Burk, L. G., Sorokin, T. P., and Engelhaupt, M. E. 1962. Genetic tumors of Nicotiana. I. Chemical composition of N . glauca, N . Zangdorfii, and their F1 hybrid. Plant Physiol. 37, 257-260. Tso, T. C.,Burk, L. G., Dieterman, L. J., and Wender, S. H. 1964. Scopoletin, scopolin and cholorogenic acid in tumours of interspecific Nicotiana hybrids. Nature 204, 779-780. Urata, U. 1959.A cytogenetic study of the dwarf-variegated phenotype in Nicotkna. W.D. Thesis, Cornell University. Valleau, W.D. 1949.Breeding low nicotine tobacco. J . Agr. Res. 78, 171-181. Valleau, W. D.1957. Yellow Crittenden-a mutant in dark tobacco. Tobacco Sci. 1, 91-92. Valleau, W. D. 195%. Genetic veinbanding-a white-flowered tobacco mutant. Tobacco Sci. 2, 20-22. Valleau, W. D. 195813. Variegation D-a dominant mutation in burley tobacco. Tobacco Sci. 2, 77-79. Valleau, W. D.1959.Tabak. 111.Variability and genetics, special methods and general breeding methods. Handbuch PfEanzenziiecht 5, 135-152. Valleau, W. D., and Stokes, G. W. 1957. V i r e s c e n t a chlorophyll deficiency in tobacco. Tobacco Sci. 1, 175-176. Valleau, W. D., Stokes, G. W., and Johnson, E. M. 1960. Nine years experience with the Nicotiuna 1ongifEora factor for resistance to Phytophthora parasitica var. nicotianae in the control of black shank. Tobacco Sci. 4, 9294. Van der Veen, J. H. 1957. Studies on the inheritance of leaf shape in Nicotiana tabacum L. Thesis, Agric. Univ. Wageningen, The Netherlands. Van der Veen, J. H., and Bink, J. P. M. 1961. Multiple effects of the leaf shape allele Pt in Nicotiana tabacum L. Genetica 32, 33-50, Vester, F., and Anders, F. 1960. Der Gehalt an freien Aminosiiuren des spontan tumorbildenden Artbastards von Nicotiana glauca und N . langsdorfii. Biochem. 2.332, 396-402. Wan, H. 1962. Inheritance of resistance to powdery mildrew in Nicotianu tabacum L. Tobacco Sci. 6, 178-181. Wells, P. V. 1960. Variation in section Trigonophyllae of Nicotiana. Madrono 15, 148-151. Wittmer, G. 1960. Influence of treatments with X rays applied to seeds on the variability of some flower characters in Nicotiana tabacum. Genet. Agrar. 13, 157-169. Wittmer, G. 1961. Polygenic mutability induced by X rays in Nicotiana tabacum. Atti Assoc. Genet. Ital. 6, 333-342. Wittmer, G., and Scossiroli, R.E. 1961. Estimation of genetic variability for several quantitative traits in two tobacco varieties. Genet. Agrar. 14, 223-233.

54

HAROLD H. SMITH

Wolf, F. A. 1959. Cytoplasmic inheritance of albinism in tobacco. Tobacco Sci. 3,

39-43.

Wolf, F.A. 1965. Hereditary abnormalities in tobacco. J. Elisher Mitchell Sci. SOC.

81,144-1'72.

Yang, S.J. 1964.Numerical chromosome instability in Nicotiana hybrids. I. Interplant variation among offspring of amphiploids. Genetics 50, 746-756. Yang, S.-J. 1965.Numerical chromosome instability of Nicotiana hybrids. 11. Intraplant variation. Can. J . Genet. Cytol. 7 , 112-119. Yoneda, Y., and Takenaka, Y. 1963. Cytological studies on a Nicotiana hybrid and its parents. Natl. Inst. Genet. Japan. Ann. R e p t . 14, 78-79.

.. .

THE GENETICS OF INBREEDING POPULATIONS

. .

. L. Workman?

R W Allard. S K Jain. f and P

University of California. Davis. California

I. Introduction . . . . . . . . . I1. Theoretical Analysis of Genetic Models

. . A. General Considerations . . . . . . B. Single-Locus Models . . . . . . .

.

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

C Multilocus Population Models: Effects of Linkage and Epistasis under Inbreeding D . Complex Models . . . . . . . . . . . . . . . E Conclusions from Theoretical Analyses . . . . . . . . 111. Experimental Analyses of Polymorphisms . . . . . . . . A. Estimation of Parameters for Single-Locus Polymorphisms . . . B Complex Polymorphisms . . . . . . . . . . . . . IV Genetic Variability in Quantitative Characters . . . . . . . A Geographical Variability B. Variability within Populations . . . . . . . . . . . C . Variability within Families . . . . . . . . . . . . V Responses to Selection . . . . . . . . . . . . . . A . Competition in Mixtures of Pure Lines B. Responses to Natural Selection . . . . . . . . . . . C . Responses to Artificial Selection . . . . . . . . . . VI Effect of Altering the Mating System . . . . . . . . . . A . Enforced Self-Fertilization . . . . . . . . . . . . B. Increased Outbreeding by Introduction of Male Sterility . . . VII . Population Structure under Extreme Inbreeding : The Festuca microstachya Complex . . . . . . . . . . . . . . VIII. Concluding Remarks . . . . . . . . . . . . . . . IX Summary . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

. . . .

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

.

. . .

.

. .

. .

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

.

.

. .

. . . . . . . . .

.

.

.

.

55 57 57 58

68 81 85 86 86 92 94 95

97 99 103 103 105

. 110 . 113 . 113 .

.

.

115

117 120 123

. . 125

1 Introduction

The term inbreeding is applied when the individuals which mate together are more closely related to each other than are random members of an indefinitely large population . The notion of the closeness of the relationship between two individuals is most easily visualized in simple cases and much of inbreeding theory has been based on the analyses of regular systems of inbreeding involving repeated sib, cousin,

* Departments of Agronomy and Genetics. t Department of Agronomy.

55

56

R. W. ALLARD, S. I(. J A I N , AND P. L. WORKMAN

or other types of matings between close relatives. From a population standpoint, however, these systems of inbreeding are interesting only in a formal sense because, aside from special experimental situations, matings in populations do not usually follow regular patterns. I n natural populations there are two general causes of inbreeding: restriction of population size and variations in the mating system. In a population of bisexual organisms each individual has two parents (uniparental, in case of selfing), four grandparents, and so on, so that n generations back an individual has 2n ancestors. The number of individuals required to provide separate ancestors for each present individual obviously becomes very large within a few generations so that any pair of individuals within a population must have common ancestors in the not too remote past. The smaller the population the less remote are the common ancestors and the higher the level of inbreeding. Inbreeding due to restricted population size is not confined to populations consisting of a few individuals isolated from others of their kind. Many factors can lead to effective restriction of population size, even though the total number of individuals in a population may be large (Wright, 1943, 1946). Isolation by distance is a n example: there is a tendency for close neighbors t o mate and proximity in time or space increases the probability that these close neighbors will be relatives, The amount of inbreeding that results from the mating of close neighbors, even in a panmictic population, is largely a function of the relative mobility of the organisms or their gametes or propagules. The other main source of inbreeding in natural populations derives from various mechanisms in both plants and animals that affect the mating system (reviews in Grant, 1956, 1958; Mayr, 1963). An example in animals is the brother-sister mating observed in parasitic hymenopterans that mate within the host (Dreyfus and Breuer, 1944). I n plants, modifications of floral structure which encourage self-fertilization or assortative mating are widespread and in many instances they are remarkably effective, reducing the amount of outcrossing to 1% or less. Preferential mating between similar genotypes can also be an important source of inbreeding. It should be noted that these two main causes of inbreeding, restriction of population size and control of mating system, can occur simultaneously and reinforce one another in their effects. Inbreeding increases the probability that individuals which mate carry alleles that are alike by virtue of descent from a common ancestor. The essential consequences, as shown theoretically by Wahlund (1928) and Wright (1951), are a reduction in genetic variability within

GENETICS OF INBREEDING POPULATIONS

57

families (kinships of related individuals) and an increase in genetic variability between families, which become genetically differentiated from one another under inbreeding. The magnitude of these effects was well illustrated by Johannsen’s population of Princess beans which was differentiated into a large number of strikingly different, true breeding families (Johannsen, 1926). There have been many deductions regarding the genetic structure of inbreeding populations since Johannsen’s work. It has usually been argued that inbreeding, by increasing homozygosity, also increases genetic uniformity and hence that i t allows populations to achieve “closer adaptation’’ (or adaptedness) to the immediate environment. It is also often argued that the gain in adaptation (fitness in the current environment) conferred by inbreeding is achieved largely through loss in adaptability (flexibility or capacity for change in adaptation). Thus, the mating system is commonly considered to be the chief factor determining the genetic structure and evolutionary potential of populations and a sharp distinction is usually drawn between inbreeders and outbreeders (see discussions by Darlington and Mather, 1949; Stebbins, 1950). Such considerations have also been the basis for discussions of evolutionary changes in breeding systems (Mather, 1943; Stebbins, 1957; Grant, 1958). I n this review we shall consider, in a population context, the effects of inbreeding in terms of genetic models of steadily increasing complexity, We will then correlate theoretical effects with observations and measurements which show that inbreeding populations contain large amounts of genetic variability and that this variability is organized into highly integrated and flexible systems. The thesis that we shall develop is that the observed structure of inbreeding populations results from an appropriate integration of inbreeding into the constellation of genetic and ecological factors which are involved in the regulation of variability and maintenance of flexibility. II. Theoretical Analysis of Genetic Models

A. GENERALCONSIDERATIONS An understanding of the genetic structure of inbreeding species derives from combining analyses of theoretical models with studies on experimental and natural populations. By varying the restrictions on the genetic parameters which define the theoretical models (e.g., those describing population size, the mating system, the selective forces) i t is possible to explore the relations between these various parameters.

58

R. W. ALLARD, S. K. J A I N , AND P. L. WORKMAN

Generally this entails a comparison of gene and genotypic frequencies, both a t equilibrium and during the approach t o equilibrium, for models with different specifications. One of the important discoveries of population genetics is that all populations, including inbreeding populations, contain large stores of genetic variability. It is, therefore, of particular interest to ascertain which combinations of values of the genetic parameters result in a population structure in which genetic variability is retained permanently. We shall consider first models describing the distribution of genotypes at a single diallelic locus in an indefinitely large diploid population. An examination of the simple models of complete random mating and complete selfing will be followed by a treatment of models pertaining to partial inbreeding and partial inbreeding with selection. Next we shall consider two locus models with particular attention directed to the joint effects of inbreeding, linkage, and epistasis on population structure. The aspect of epistasis that will be emphasized is the manifestation of interactions between loci as they are expressed on the fitness scale. Finally we shall discuss the view of population structure provided by analyses of several more complex models such as those including multiple loci, stochastic variation of the genetic parameters, and restriction of population size. All of the models to be considered assume that generations are discrete (i,e., nonoverlapping). However, it can be shown that corresponding continuous time models, that is, those assuming overlapping generations, lead to the same conclusions as those derived from the discrete models.

B. SINGLE-LOCUS MODELS I n the discussion of single-locus models we shall restrict the treatment to a locus with two alleles, say A1 and A2. We shall denote the genotypic proportions of (AlAI, AlA2, A2A2) in generation n by (fl(ll), fz("), fa(")) and the allelic proportions of (A1? A,) by ( p @ ) ,q(")),where ptn) q(") = 1.

+

1. Complete Random Mating and Complete Inbreeding

The most elementary model is that of random mating in a n indefinitely large population, described by the Hardy-Weinberg theorem. If the frequencies of alleles Al and A%,at any generation, n, are given by p and q = 1 - p , then the genotypic proportions in the next and all subsequent generations are given by (&A1, A1A2, A2A2) = ( p 2 , 2pq, q z ) .For multi-

GENETICS OF INBREEDlNG POPULATIONS

59

ple alleles the genotypic proportions are given by A d , = pi2, and A& = 2 p & p j ( i # j ) . For the diallelic model, the heterozygous genotype, A1A2, has frequency fz = 2 p q and consequently the maximum possible heterozygosity is determined from the solution of d(fz)/dq = 0, for which q = $/2. Thus, under random mating without selection the maximum heterozygosity possible is 50% and the range of possible values for f2 is clearly 0 < f 2 5 1/2, depending only on p and q . I n contrast to the case of random mating, consider population structure under a system of complete inbreeding. For plant popul8tions this could result from complete self-fertilization; for both plant and animal populations it might occur if the only matings permitted were those between like genotypes (genotypic positive assortative mating) or like phenotypes (phenotypic positive assortative mating). Complete self-fertilization and genotypic assortative mating are formally identical systems and by the technique of generation matrices (Haldane and Waddington, 1931 ; Fisher, 1949; Kempthorne, 1957) it is possible to derive n-step recurrence formulas for these mating systems, T h e recurrences give the genotypic proportions a t any generation, n, in and n. For terms of the initial genotypic distribution (fl@), f 2 @ ) , complete selfing these can be shown t o be fi(") = fi'o) ( 1 / 2 ) [ 1 - (1/2)n]f2(D) A1A1: AIAz: fiCn) = (1/2)" fz'"' (1) fa(") = fa(') ( 1 / 2 ) [ 1 - (1/2)*]fi(O) A2Az: Equations (1) show that the amount of heterozygosity in a population is halved every generation. I n the limit (limn+m f(@) the genotypic distribution (fl", f z m l f a m ) is given by ( f ~ @ ) ( 1 / 2 ) * f z ( O ) , 0, f a @ ) (1/2)f3(0)), or ( p @ ) ,0, q ( O ) ) . Thus, under this model, the limiting state consists solely of homozygous genotypes in the same proportion as the original gene frequencies and no heterozygosity is retained. For phenotypic assortative mating, in the case of dominance, i t can be shown (e.g., Hogben, 1946) that the limiting distribution is identical to that for genotypic assortative mating. However, the rate of approach t o equilibrium is different. The heterozygosity a t generation n is given by

+ +

+

+

and the rate of decrease in heterozygosity is much slower than in the case .of genotypic assortative mating. For both cases, gene frequencies remain constant over all generations (pi(")= p P for all n, m ) ; only the genotypic proportions change. These models of complete random mating and complete inbreeding represent extremes that rarely, if ever, would occur in real populations.

60

R. W. ALLARD, S. K . JAIN, AND P. L. WORKMAN

A more realistic appraisal of population structure, therefore, requires consideration of mating systems in which the actual amount of inbreeding lies somewhere between the extreme values of 0 and 100%. 2. Partial Inbreeding

The mating system of a population may be one of partial inbreeding for many different reasons. Dioecious plants (those producing both male and female gametes) may mate according t o some mixed system of random mating and self-fertilization. Genetic control of variation in flowering time might result in assortative mating among those plants flowering concurrently. Genetic variation in flower color or in the structure of the reproductive organs may lead to assortative mating in insect-pollinated species when the pollinators have preferences for particular colors or shapes. Positive sssortative mating arising from mating preference between similar types has also been observed in a number of animal populations (e.g., O'Donald, 1959; Cooch and Beardmore, 1959). Pearson and Lee (1903) and others have noted its occurrence in human populations with respect to various physical characteristics, Mating among members of the same family either as a regular system (e.g., sib mating in parasitic hymenopterans; review in Mayr, 1963) or as some proportion of the total matings (uncle-niece or cousin matings in man) is another common cause of partial inbreeding. The first systematic study of models of inbreeding systems was that of Wright (1921), reported in a classic series of papers entitled "Systems of Mating." Since then, numerous workers, employing a variety of techniques, have considered the problem, both generally and in terms of specific systems. We shall consider here only the most general models of mixed random mating and selfing, or equivalently, partial positive assortative mating, these two systems being formally identical. References to more specific models (e.g., regular systems such as those involving uncle-niece, full sib, or parent-offspring mating) and descriptions of the various techniques used for their analysis are found in Kempthorne (1957). Consider a population in which there is a constant probability, s, for self-fertilisation and a corresponding constant probability, t = 1 s, for random outcrossing. Recurrence equations relating genotypic proportions in successive generations are given by

-

=

fz("+l)

=

f3("+')

+ + +

+

+

s[fiCn) (1/4) f2(")] t[fi(") (1/2) f2'"'12 ~[(1/2) fz'"'] 2t[fl'"' (1/2) fz'"'][f3'"' = ~[f3("' (1/4) fz'"'] t[f3("' (1/2) fz""Ia

fi(n+l'

+

+

+

+ (1/2) f~'"']

(3)

GENETICS OF INBREEDING POPULATIONS

61

It can be shown from these equations that the gene frequencies remain

constant in all generations, but the genotypic frequencies change until an equilibrium state is attained. The general n-step formulas for this system, given as Eqs. (4),can be obtained by matrix methods (Workman and Allard, 1962) or by the methods appropriate to the solution of finite difference equations (Haldane, 1924) :

fi'")

= S"

f3(4

= s= .f3'")

fl'"'

+ (1/2) (S")[l - (1/2)"1

+ (1/2)(9)"[1 - (1/2)"]fP

where p and q are and

f2'"

[fl'"'

If3'"'

+ (1/2) + (1/2)

fi'"'1

f2'")I

respertively. In the limit (as n + co ) the equilibrium state is given by

It is of particular interest to know how much heterozygosity can be maintained indefinitely, given various assumptions about the initial population and the amount of outcrossing. Table 1 gives the amount of heterozygosity expected a t equilibrium for different values of p and t. By the symmetry of the model, the tabular value of p is that of q when p > 0.5. An alternative derivation of the above results can be obtained by the use of the coefficient of inbreeding, F (Wright, 1921), which was first defined as "the correlation between homologous genes of uniting gametes under a given mating pattern relative to the total array of those in random derivations of the foundation stock" (Wright, 1965a). Other interpretations of F in terms of probabilities of identity of uniting gametes (MalBcot, 1948) or as a function of the relative amount of heterozygosity are reviewed by Wright (1965a). The use of F statistics has proven to be one of the most successful methods employed in the study of inbreeding systems (see Kempthorne, 1957, for examples).

62

R. W. ALLARD, S. K. J A I N , AND P. L. WORKMAN

TABLE

1

Expected Equilibrium Proportions of Heteroeygotes under Mixed Random Mating and Selfing (without Selection) for Various Assumptions about t and p (Initial Frequency of Allele At)

P t

0.05

0.10

0.20

0.30

0.40

0.50

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

0.0173 0.0317 0.0439 0.0543 0.0633 0.0713 0.0783 0.0844 0.0900 0.0950

0.0327 0.0600 0.0831 0.1029 0.1200 0.1350 0.1482 0.1600 0.1705 0.1800

0.0582 0.1067 0.1477 0.1829 0.2133 0.2400 0.2636 0.2844 0,3022 0.3200

0.0764 0.1400 0,1939 0.2610 0.2800 0.3150 0.3459 0.3733 0.3969

0.0783 0.1600 0.2216 0.2743 0.3200 0.3600 0.3953 0.4266 0.4548 0.4800

0.0909 0.1667 0.2308 0.2857 0.3333 0.3750 0.4118 0.4444 0.4737

-

0.4200

0.5oOo

At equilibrium, any inbreeding population can be written as (AIA1, A I A z , A z A ~= ) ( p a pqP, 2 p q ( l - F ) , q2 p q F ) where F is the equilibrium coefficient of inbreeding, and both p and F are constant (e.g., Wright, 1942). For the case of mixed random mating and selfing, an n-step equation for F can be shown to be (Kempthorne, 1957)

+

+

where F(O) is the inbreeding coefficient in the original population. The = s/(2 - s) and the equilibrium limiting formula ( n +=00) gives Frn) hetero~ygosity,given by 2 p q ( l - F ) , is f 2 = 2pq{2t/ (2 - s) }, which is equal to the expression ( 5 ) obtained by matrix or finite difference methods. It is possible that the probabilities for selfing versus outcrossing differ for each of the genotypes. For this situation let a,/3, y be the probabilities for selfing of AIA1, A1A2, A2A2, respectively. Then (1- a), (1 - p ) , (1- 7) are the corresponding probabilities for outcrossing of these three genotypes. Note that when a = p = y = s , this model reduces to that of mixed random mating and selfing already considered. The case when ff, p, and y are different and operate on both sexes has been considered by O’Donald (1960b), who obtained the recurrence equation for t h e hetero~ygote,fz, and an exact equation for the equilibrium state. He also considered the case of dominance (partial phenotypic assortative mating). His results showed that when the tendency for assortative mating is

GENETICS OF INBREEDING POPULATIONS

63

small, the equilibrium distribution is very close to that expected under random mating. A variant of the above is the model in which only the female parent has different probabilities for random mating versus selfing (or assortative mating). This could apply to plant populations in which there is an excess of pollen disseminated a t random, or to animal populations in which mating is determined by female preference. The relevance of models based on 0, 8, and y , as opposed to s alone, is indicated, for example, by Harding and Tucker (1964), who showed that estimates of the amount of outcrossing a t a locus varied with the particular genotype used in the estimation. Mating preference leading to assortat.ive mating might also be determined in part by the genotype of one or both of the parents. The effect of this imprinting on genotypic proportions has been considered by O’Donald (1960a) and Mainardi et al. (1965). Such a system was suggested as the basis for the assortative mating in the Blue Goose (Huxley, 1955; Cooch and Beardmore, 1959). The models thus far considered vary only with respect to the mating system and represent, as such, extremely idealized representations of natural populations. In the following section we shall introduce variation due to differential selective forces in order to determine the kinds of interactions between the mating system and the selective forces which result in the maintenance of genetic heterogeneity. 3. Inbreeding and Selection

There are many different ways in which selective forces can act to alter gene and genotype frequencies, in terms of both their possible equilibrium distributions and the approach to equilibrium. Selection pressure can be assumed to be constant over time or regularly cyclic within generations or between generations [e.g., this has been suggested as the explanation for a chromosomal polymorphism in the mantid, Ameles heldreichi (Wahrman, 1965)l. The intensity of selection may vary over an environment (clinal selection, habitat, or disruptive selection), differ for different sexes or morphs, vary according to the frequencies of the genotypes in the population (Clarke and O’Donald, 1964) , or vary with population density. Selection can act directly on the gametes or on zygotes a t different stages in the life cycle (differential viability, differential fecundity), Combinations of these selective forces acting a t several different times may account for the differential reproductive capacities of genotypes a t a given locus. I n practice, however, it is usually necessary to consider only simple models in which selection, operating a t a specified stage, is ascribable to a

64

R. W. ALLARD, S. K. JAIN, AND P. L. WORKMAN

single specified cause. We shall consider only two general models in detail: selfing with zygotic selection, and mixed random mating and selfing with zygotic selection. The conclusions drawn from analyses of these models regarding the relation between inbreeding and selection are applicable to a wide range of more specific models. a. Selfing and Zygotic Selection. The most general solution is that given by Hayman and Mather (1953, 1956)' who incorporated into their model selection between lines, seIection within lines, and seed selection. They obtained the n-step relations,

a

wncfs(")

- vfi(0))

1 +sf2(0) 2"

where (z,1, y) are the relative survival rates of (fl, fi, fa) in segregating families, z and w are the relative survival rates of fl and f 3 in truebreeding families, and u = x / [ 2 (1 - 22)] and v = y/[2 ( 1 - 2w)l are compound survival parameters. When z = w = (%)x (%)y 1/2 selection occurs only within lines; when z = 'y = 1 selection occurs only between lines; and when z = x and w = y only seed selection operates. The equilibrium distributions are as follows. The population will be homozygous f l (or fa) if z > 1/2, w (w > 1/2, 2 ) . For z = w > 1/2 there will be a mixture of homozygotes fl and fa. In particular, if 1/2 > z, w, heterosygosity persists and the equilibrium genotypic frequencies are

+

+

Thus, for the case of full selfing, the heterozygotes must have a marked superiority over the homozygotes (z, y 5 1/2) in order to maintain variability in the population. Hayman and Mather (1953) also treated particular cases of heterozygote advantage for several regular systems of inbreeding (e.g., sib mating, parent-offspring mating) and made comparisons among these systems and the case of complete selfing. b. Mixed Random Mating and Selfing with Zygotic Selection. Models involving selection give rise to a series of nonlinear recurrence equations for which, except for the case of full selfing, it has not been possible t o obtain general n-step formulas. The analysis of such models involves the examination of the distribution of genotypic frequencies at equilibrium either directly, or in terms of gene frequency and Wright's F. At equilibrium ft("+l) = f i @ ) = f 6 and the solution is given by the simultaneous

GENETICS OF INBREEDING POPULATIONS

65

solution of the equations in f4. The values of the relevant parameters which result in stable nontrivial equilibria can be determined by testing the stability by whichever of the available methods is most appropriate (e.g., Owen, 1953; Lewontin, 1958; and general methods of Saaty and Bram, 1964). Suppose that the relative viabilities of (Il, f2, f 3 ) are in the ratio (2, 1, y) and assume constant probabilities for selfing (s) and random mating ( t = 1 - s) , If the genotypic frequencies are assumed to be scored just before mating and after all of the zygotic selection has occurred, the model is that described by Hayman (1953). The recurrence relations are fi(n+l) a

f2(n+l)

a

f~(~+') a

+ (1/4) + + (1/2) + 2t(jI(n) + (1/2) fz(n))(ja(n) + (1/2) j ~ ' ~ )(9))

X( s(fi(") (1/2) sj2'")

y(S(fs'"'

f2(n))

t(fi(")

(1/4) fz'"')

t(f3(n)

fi'n')2)

(1/2) ~Z(~)'I

The most direct approach to a solution of these proportionalities is that described by Workman and Jain (1966) and Jain and Workman (1966), If we define F = 1 - f n / 2 p q , when p = ( f l + ( 1 / 2 ) f 2 ) , then the genotypic distribution a t any generation, n, can be written as

If the only force causing genotypic frequencies to depart from HardyWeinberg expectations is that of inbreeding, then F(n)is the coefficient of inbreeding a t generation n. If selection is also involved, then F(*) denotes the joint effects of inbreeding and selection and, in Wright's (1965s) terminology, F(n)is the fixation index. At equilibrium both Ap = 0 and AF= 0 (or dp/dt = dF/dt = 0 ) . Using this transform, the above equations (9) at equilibrium can be written as fi: f2:

s3:

+

pZ p q F 2Pdl - F ) !I2 P q F

+

a

a a

+

+

x ( p 2 (1/2)s p q ( 1 F)) (2PP - pq(1 F)I Y{q2 (1/2)s P d l F)1

+

+

+

(11)

where F is the fixation index a t equilibrium and p is the equilibrium gene frequency. From the relations (11) Workman and Jain (1966) showed that both of the following equations must be satisfied a t equilibrium:

- (1/2)s(l - X)(1 + F ) - y) [ l - (1/2)8(1 + F ) ] sF2(1 - X) + F ( 2 ( ~ y- 1) + ~ ( 2 z - g)} - 2(1 - ~ ) ( 1- y) + ~ ( -l ZY) = 0 (1 - y)

= (2

-x

(13)

66

R. W. ALLARD, S. K. J A I N , A N D P. L. WORKMAN

I n considering equilibrium populations it is useful to examine phase diagrams (Figs, l, 2) as described by Hayman (1953) and Workman and Jain (1966). These diagrams show types of equilibria for different magnitudes of the relative viabilities (z, y) of the two homozygotes, given some particular amount of selfing ( 5 ) . The areas marked A and B cor""I

/

3

x

FIG.1. Phase dingrrtm for s = 1.00. See text for description of areas A, B, C, D.

0.4 1

\

I

a

FIG.2. Phase diagram for s = 0.95. See text for description of areas A, B, C, D.

respond to populations homozygous for AIAl (fl) and AzAz (fs) , respectively; in C,heterozygotes are present but in a frequency lower than in a population mating a t random without selection ( F > 0) ; in D there are more heterozygotes than in a random mating population without selection ( F < 0). It can be seen that the amount of heterosygote ad-

GENETICS OF INBREEDING POPULATIONS

67

vantage needed to maintain variability is a function of the amount of selfing. As the amount of selfing increases, increasing heterozygote advantage is required for the maintenance of a nontrivial equilibrium. Two variations of this model have been considered by Workman and Jain (1966). First, suppose that genotypic frequencies are scored just after mating. The genotypic distribution a t equilibrium will be equal to that obtained by imposing one generation of mixed random mating and selfing upon the genotypic proportions obtained in the above model (11) in which the genotype proportions are determined prior to mating. Consequently, the value of p will be the same since gene frequencies remain constant under mixed mating; but F, which depends on both genotypic and gene frequencies, will be different. As can be shown by setting n = 1 in (6) above, the appropriate value of F , say F‘, will be equal to ( x ) s (1 F ) , Thus, for this model, the equilibrium is described by

+

(1 = (2

- y) - F’(1 - z) - z - y)(l - F’)

2F’2(1 -X)(1 - y > + F ’ ( ( z + y - 2 )

-t(z+y-2xy)}

+ (X + 2/ - 2zy)(l - t ) = 0

(13’)

The phase diagrams for this model, which differs only operationally from the previous one [see Eqs. ( l l ) ] , are the same as given by Figs. 1 and 2. If selection acts only on one sex, the model is one of differential fecundity, rather than one of differential viability as above. If genotypes are counted after mating, the equilibrium conditions are found to be identical to those in the above model of differential viability (selection in both sexes) when genotypes are scored after mating [Eqs. (12‘, 13’)]. Thus, for each of the above three models of selection and inbreeding the equilibrium values of p and F are identical a t the same stage of the life cycle. However, the approach to equilibrium differs among the three models. Figures 3 and 4 show changes in p and F plotted against time for x = 0.50, y = 0.75 and ( P o , Ro, Qo) = (0.25, 0.50, 0.25). Note that p goes monotonically to its equilibrium value, but F can approach its equilibrium value by complex paths. The models so far discussed show that the permanent maintenance of variability under inbreeding requires some net heterozygote advantage, the degree of advantage required varying directly with the intensity of inbreeding. It should be noted that net heterozygote advantage does not necessarily imply that the heterozygote is adaptively superior, per se, to both homozygotes since various mechanisms such as frequency-dependent selection or disruptive selection can create a net heterozygote advantage (see Dempster, 1955). Other kinds of interactions that result in the

68

11. W. ALLARD, S. K. J A I N , AND P. L. WORKMAN

5

10 15 Generation

20

25

FIG.3. Changes in p and F for three different models during the approach to equilibrium for z = 0.50, y = 0.75; s = 0.0. p,FI, selection on both sexes, census prior to mating, pI,FII, selection on both sexes, census after mating. pIIIFIII,selection on one sex only, census after mating. 1.0 I

5

FIG.4. Changes in p and F for three different models during the approach to equilibrium for z = 0.50, y = 0.75; s = 0.50. See Fig. 3 for key.

maintenance of genetic variability will be considered next in the discussion of two-locus and more complex genetic models.

C. MULTILOCUS POPULATION MODELS : EFFECTS OF LINKAGE AND EPISTASIS UNDER INBREEDING The simplest model that permits the effects of linkage to be considered is one of two linked loci with two alleles each. The notation that we shall

69

GENETICS OF INBREEDING POPULATIONS

use in the discussion of two-locus models is given in the accompanying tabulation. Loci and allelic designation Gene frequencies Gametes Gametic frequencies

A q1

PI

=

a I

- Pl

B

b

pz

qz = 1

AB

Ab

aB

91

Ba

93

Genotypes

A B AB

Genotypic frequencies

fl

- pz

ab

94 =

1

- 81 - Yz - 93

AB Ab AB A B Ab - Ab - -aB_ aB_ ab Ab

A6

aB

f?

f3

f4

ab

f6

aB

ab

aB

ab

ab

f6

f7

fS

fB

f10

I n order to discuss the effects of linkage and epistasis on genetic changes a t two or more loci jointly, it is necessary to define the terms “linkage equilibrium” and “linkage disequilibrium.” Linkage equilibrium is defined as the state in which gametic frequencies correspond to the products of the appropriate gene frequencies : 91 = PIP2 92 = m q 2

93 =

QlPZ

g4 = QlQZ

It can be shown (Geiringer, 1944, 1945) that gametes in populations in linkage disequilibrium have the frequencies

+

gi = P ~ P Z D gz = piqz - D

93 = pzqi y4

=

~

1

-D

+D ~

2

where D = glg4 - g2g3. These relationships also hold for inbreeding. The parameter D is consequently a useful general measure of unbalance in the proportion of coupling and repulsion gametes. As early as 1909 Weinberg suggested that linkage equilibrium (D = 0) is, in general, not reached in a single generation of random mating, even without linkage. Jennings (1917) and Robbins (1918) provided explicit expressions for the rate a t which any initial linkage disequilibrium in a population, such as that in a population derived from the cross of two homozygous parents, disappears under random mating. The recursion for change in D is simply D(n) = (1 - c)DCn-I) or (14) D(n) = (1

- c)nD(o)

where D(0) and D(”) measure the amount of gametic array unbalance in the initial and nth generations and c is the recombination value between the two loci. I n the absence of selection limn+mD ( n )= 0, so that after

70

R. W. ALLARD,

S.

K.

J A I N , AND P. L. WORKMAN

a suf6cient number of generations in any random mating population the loci will come into linkage equilibrium (D= 0). When the mating system is one of complete selfing the recursion for the gametic frequency of AB(gl) is (Bennett and Binet, 1956)

so that D does not become zero at genotypic frequency equilibrium. Thus a correlated gene distribution, once created by any factor causing fti # j e t persists to genotypic frequency equilibrium even in the absence of linkage. As we shall see in the next section this result does not hold for partial inbreeding. Consequently the case of complete selfing cannot be treated simply as the limiting case of partial inbreeding systems.

I. Partial Inbreeding Systems of partial inbreeding have been treated analytically by three main methods. The generation matrix method was developed by Haldane and Waddington (1931), who used it to analyze rates of approach t o equilibrium for certain regular systems of inbreeding (e.g., parentoffspring and full sib mating). Wright (1933) approached the problem through the shorter method of path coefficients, using the correlation coefficient ( r ) between gene distributions a t two linked loci, where r is given by r =

D (PlqlPzqz)lIa

His analysis of a wide variety of mating systems led to the conclusion that “different pairs of allelomorphs, even in the same chromosome, come to be combined practically a t random in any freely interbreeding population of long standing” and so also “within different subgroups of a population unless these subgroups are very small or linkage is extremely close.” Bennett and Binet (1956) and Binet et al. (1959) developed n-step recursions for determining changes in the quantities D and v = (f6 - fe) for the case of mixed selfing and random mating. Using methods of matrix algebra they showed that for s < 1, both these quantities eventually become zero in the absence of selection. Thus, mating systems involving mixed selfing and random mating also lead t o linkage equilibrium. Haldane (1949) used the probability method of Malecot (1948) to investigate the joint effects of inbreeding on two linked loci. He established that excesses or deficiencies of doubly homozygous and doubly heterozygous gene combinations occur under partial inbreeding, even in populations which are in linkage equilibrium (D= 0). Such zygotic as-

GENETICS OF INBREEDING POPULATIONS

71

sociations become increasingly pronounced as linkage intensity increases.

It was shown later by Schnell (1961) and Kimura (1963) that rates of

approach to equilibrium are also affected by Haldane’s joint inbreeding function, defined as a measure of identity of allelic pairs by descent. The role of this “mating system effect” under mixed selfing and random mating has been investigated by Bennett and Binet (1956). They showed that the excess of double heterozygotes under free recombination ( c = 0.50) is given by

or generally, for any c, w =

(4

-

1 6 ~ ( l- S ) [2 - s 4 ~ ( 1- ~ ) ( l - ~ ) (2 s ) ~ 4 - 2s f 4 ~ ( l C)

-

‘’1

~ 1 ~ 2 4 1 ~ (18) 2

where the parameter w provides a measure of zygotic associations independent of the gametic unbalance parameter D. Changes that occur in D and o as an arbitrary population progresses toward equilibrium are plotted in Figs. 5 and 6. Figure 7 gives the magnitude of o measuring the zygotic associations in equilibrium populations for various specifications regarding s and c. The effect of w when there is selection will be considered later. These results lead to two main conclusions. First, the description of genotypic frequencies is complicated under partial inbreeding since 0.25 I

-1

I

5

10

15 Genera tion

20

25

Fra. 5. Changes in D and w under random mating for c=0.10 and c =OM.The genotypes AABB and aabb were equally frequent in the original populations 80 that D and LO had their maximum values of 0.25. Notice that the value of o decreases slowly when linkage is tight.

72

R. W. ALLARD, S. K. JAIN, AND P. L. WORKMAN

---___ -----__ -. ..\.

0.25p,

C

'\ 0.20- \ \ 0.15

~

=0.01 D

.\\

%\<:\ ;0, OD

\ '\

\

/ A \

Proportion of selfing, S

FIG.7. Equilibrium valuw of w under varying proportions of self-fertilization and different recombination values (c).

(8)

gametic associations ( D ),zygotic associations (a),and the joint inbreeding effect must all be taken into account. Second, linkage has greater effects on both gametic and zygotic associations under partial inbreeding than under random mating. Before turning to models in which selection is incorporated, note should

73

GENETICS OF INBREEDING POPULATIONS

be taken of investigations of multilocus systems in terms of the distribution in succeeding generations of complete linkage blocks. Fisher (1948, 1949) Bennett (1954) and Hanson (1959), using the concept of average recombination rates, have shown that linkage has characteristically different effects under different mating systems and that these effects have a bearing on population change under selection. )

)

6. Random Mating: Selection and Linkage

It is tempting to conclude from studies of models of random mating without selection that linkage effects are eventually dissipated and that they have no significant influence on the structure of the equilibrium population. Indeed, as emphasized by Lewontin (1964a)) most of the formulations of multiple gene theory have been extensions of single gene models that ignore linkage and epistasis. Wright, however, pointed out in 1942 that selection “tends to bring about departures from random combination among different series of alleles. The effects are unimportant in most cases, especially if all relative selective differences are slight.’’ Later, in investigations of an optimum model, Wright (1952) demonstrated that epistasis expressed on the fitness scale can lead to nonrandom gene combinations (D# 0) in equilibrium populations. The role of linkage in the quantitative theory of selection has also been discussed by Griffing (1960) and Gibson and Thoday (1962). Information on conditions which permit permanent linkage disequilibrium and on the magnitude of the resulting gametic and zygotic associations is therefore clearly necessary to an understanding of population structure. Consider again the two-locus model presented earlier in which the fls are the ten genotypic frequencies. Further assume that the selective values, wi,of the various genotypes represent relative probabilities that a zygote in one generation will leave a zygote in the following generation. Numerical values can be assigned to the w;s so as to produce selection models without epistasis, or with any kind of epistasis. Given the AB A6 aB ab

Marginal means

AR

Ab

aB

ab

w1

W2

wb

WAB

WS

Wa

W7

WAb

w4

ws

w4 wa

?UQ

WaB

W5

w7

WD

WlO

Wab

wa

accompanying matrix of selective values, the average selective value of gametic type AB in all its combinations under random mating is w.48 = wlgl f w2gz 4- wag3 i w&74 The average selective values of the other gametic types,

W A b ) W,B,

and

74

R. W. ALLARD, 8. K. J A I N , AND P. L. WORKMAN

wab, can be obtained similarly from appropriate marginal means. Since the genotypic frequencies, ji, are obtained directly as the products g4g5, the population is a t equilibrium when Agi = 0 (Lewontin and Kojima, 1960) :

+

+

+

where W = glWAB g2wAb g3waR g4Wab denotes mean population fitness. The expressions (19) are cubic equations in the gi)s and they are consequently difficult to solve for general wls. However, analytical solutions are possible for w1 = w3 = w8 = wlo = u, w4 = w7 = v, w2 = w9 = w, WE = we = x, where values of u, v, w, x can be taken which will produce overdominance a t one or both loci, and epistasis results when ( u 2 w - v) # 0. By virtue of the symmetry of the model, Eqs. (19) reduce to a single cubic equation

+

&(ZJ

f W

- X - U)(4gi2 - 3gi -k

+

1/2)

- W(g1 - 114)

=

0

(20)

When there is no epistasis (u z - w - v = 0) the only nontrivial solution of Eq. (20) is G1 = & = = J4 = 1/4. Hence a t equilibrium D = 0. However, when the loci interact ( u x - v - w # 0) Eq. (20) has three solutions :

+

and

61

-

=

114

=

1/4 f

cx

where GI= = 1/2 G2 = 1/2 - i3.Thus for recombination values c < (u x - v -w) /4 there will be permanent gametic phase unbalance (D # 0) a t gene frequency equilibrium and the linkage value required to yield D # 0 a t gene frequency equilibrium depends on the type and extent of epistasis. Lewontin and Kojima concluded from studies of an heterotic model that “as a general rule joint effects of linkage and epistasis do not produce serious changes in population structure except under special circumstances. These circumstances are the simultaneous existence of marked epistasis and tight linkage.”

+

75

GENETICS OF INBREEDING POPULATIONS

More recent,ly, i t has been possible t o extend studies of the effects of linkage and epistasis to a wider variety of two-locus models, including asymmetrical selection models, through simulation on digital computers (Lewontin, 1964a,b; Kojima, 1965; Jain and Allard, 1966). Some of the conclusions are (a) When there is no epistasis, linkage affects the rate of progress toward equilibrium but not the final equilibrium state of a population. (b) Under certain conditions of epistasis permanent gametic phase unbalance is maintained even for genes that are unlinked. (0) The epistasis which results in gametic phase unbalance can be generated by certain multiplicative processes and also those which characterize optimising selection. (d) The initial composition of a population respecting gametic phase can affect the final gene frequency equilibrium which is attained. Thus populations which have identical gene frequencies but differ respecting gametic phase may reach different gene frequency equilibrium states. (e) Gametic unbalance enhances mean populational fitness a t gene frequency equilibrium. These points are illustrated by the following two genetic models (Table 2). The first model (Model 1) involves asymmetrical selection T.4BLE 2 Selective Values for Models 1 and 2 Model 1

BB

Bb bb

Model 2

AA

AlZ

aa

0.8 0.6 0.7

0.5

0.8 0.5 0.6

1.0 0.6

BB Bb bb

AA

Aa

aa

0.64 1.00 0.94

1 .oo 1.05 1 .oo

0.96 1.00 0.64

with mixed under- and overdominance a t the two loci (Jain and Allard, 1966). Gene frequency equilibria and the trajectories of change in gene frequency leading to these equilibria for four different linkage values are superimposed on Wrightian adaptive topographies in Fig. 8. These topographies were drawn under the assumption that D = 0, an assumption that is not correct since genotypic frequencies are functions of both D and gene frequencies and this model produces permanent gametic phase unbalance a t gene frequency equilibrium even for c = 0.50. The peak at A and the saddles a t S are expected equilibrium points if D is ignored. Note that the equilibria actually reached do not correspond to these points. For c = 0.50, D takes the value 0.0046 a t gene frequency

76

R. \V. ALLARD, S. K . J A W , A S D 1’. L . WORKMAN

FIG.8. Adaptive topographies for Model 1 with (a) c = 0.50, ( b ) c = 0.25, (c) c = 0.10, and ((1) c = 0.001. Tlir isoiidi~~)ts (tlottcd cwntoiir lines) shown in all four topographies were computed assuming 1) = 0. Numbers along the trajectories give the generation a t which the population point reached the position indicated. Initial grnc frcquency sets were: I, pl = p z = 0.6, D = 0.25; 11, pl = 0.6, p 2 = 0.4, D = - 0 . 2 5 ; 111, pi=pn=0.5, D = O ; IV, p i = p n = 0 . 2 , D = O ; V, pi=p:!=OS, D = 0 ; VI, p , = 0.9, p 2 = 0.1, D = 0 ; and VII, pl = 0.1, p~ = 0.9, D = 0. The peak at A and saddlc~at S are given by the topography drawn assuming D = 0, whereas the nctual peitks obtained by simiilation are nenr A in Fig. 8a and a t B-F in Figs. Sb,c.tl. After Jain and Allard (1966). equilibrium. In this case the dcpurture of D from zero is small and the actual equilibrium lies near the peak ( A ) which is expected when D is ignored. With c = 0.25, all trajectories lead to fixation of AABB. With tight linkage ( c = O.OOl), however, there are two stable equilibrium points, a t E and F, for which Zl = 0.2214 and D = -0.2393, respectively. These points itre locatcd a t some distance from the single peak ( A )

77

GENETICS OF INBREEDING POPULATIONS

expected when D is ignored, and they represent equilibria for which mean = 0.866 for E and 0.879 for F) than for population fitness is higher 0.712). An interesting feature revealed by these the peak a t A (p= figures is the crossing of trajectories of gene frequency change. This indicates that populations which have identical gene frequencies may have very different evolutionary futures owing to differences in their evolutionary histories. The second model (Model 2) is one of optimizing selection due to Wright (1959). I n common with other models of optimiaing selection this model leads to permanent gametic phase unbalance, with D < 0, a t gene frequency equilibrium (Table 6). If D is ignored three equilibrium points are indicated for c = 0.50: (a) 61 = $2 = 0.746; (b) = fj2 = 0.158; (c) GI = lj2 = 0.548. This corresponds to an adaptive topography with stable equilibria with peaks a t (a) and (b) and an unstable equilibrium a t the saddle point (c). When D is taken into account there is found to be only a single equilibrium point a t = jj2 = 0.1764 with D = -0.0041 (Jain and Allard, 1965). Thus D cannot be ignored, even when it takes small values. Notice from Table 3 that gene frequencies

(w

TABLE 3 Stable Equilibria under Model 2 for Random Mating (t

=

1 .O)*

Recombination value (e) Item?

0.50

0.25

0.10

0.01

=

0.1764 0.9713 -0.0041

0.212Y 0.9725 -0.0121

0.3995 0.9792 -0.0599

0.4534 0.9962 -0.1664

$1

w

D

(i2

* After Jain - and Allard (1965). t f,, i2,r,and D represent equilibrium gene frequencies, mean population fitness,

and linkage disequilibrium values, respectively.

tend to be intermediate and that I D I and are larger with tighter linkage. Another feature of linkage which should be stressed is its general effect of reducing the rate of approach to equilibrium, or fixation. This can lead to the maintenance of variability over large numbers of generations, producing quasi-stable equilibrium populations as discussed by Lewontin (1964b) and Kimura (1965). Wright (1965b) has shown that overdominance tends to reduce the number of peaks (equilibrium points) in multigenic systems whereas increasing linkage, epistasis, and asymmetry of selection tend to in-

78

R. W. ALLARD, S. K. JAIN, AND P. L. WORKMAN

crease the number of peaks. Numerical examples indicate that overdominance on marginal means a t individual loci is a necessary but not a sufficient condition for stable equilibrium (Lewontin and Kojima, 1960; Jain and Allard, 1966). The above analytical and numerical studies of multilocus models make i t clear that linkage and epistasis can have significant effects on population structure under random mating. I n addition, Kimura (1956) and Bodmer and Parsons (1962) have discussed models which illustrate the critical role of linkage in the initial stages of survival of newly arising mutations and their maintenance in stable polymorphisms. An experiment on changes in the recombination system produced by selection was reported by Allard (1963). These studies suggest that evolutionary adjustments in the recombination system are an integral feature of the population structure. Next, we shall consider the effects of linkage and epistasis on population changes under inbreeding. 3. Selection and Linkage under Inbreeding The case of complete selfing, with selection, for two-locus models has been treated by Reeve (1955) and Shikata (1963) and the case of sib mating, with selection, has been treated by Reeve and Gower (1957). In general, however, partial inbreeding with selection leads to systems of nonlinear equations that are difficult to handle analytically and it has consequently been necessary to resort to numerical methods to gain insight into the effects of linkage and epistasis under inbreeding. This approach was used by Jain and Allard (1965, 1966) in studies of a wide variety of selection models under mixed selfing and random outcrossing. The general pattern of the results indicated that increasing the level of inbreeding has much the same effect on D as tightening linkage, The amount of gametic phase unbalance ( D # 0) is relatively greater with inbreeding than with random mating and it rises more or less directly as the amount of selfing increases. This effect of inbreeding is particularly marked with loose linkage. As a result, permanent gametic unbalance is attained under a wider range of conditions with inbreeding than with random mating. I n fact, under certain conditions of selection, permanent gametic unbalance is possible a t gene frequency equilibrium under intense inbreeding even without epistasis. These results are illustrated by the models given in Table 4, which represent four main types of selection (intermediate optimum, mixed

GENETICS OF INBREEDING POPULATIONS

79

TABLE 4 Selective Values for Models 3 to 6 3. Intermediate Optimum

4. Mixed Optimum-Heterotic 0.50 0.70 0.85 0.70 1.0 0.70 0.85 0.70 0.50

0 . 6 0 . 8 1.0 0 . 8 1.0 0 . 8 1.0 0 . 8 0.6

5. Het,erotio (epistatic) 0.5 0.7 0.5 0 . 7 1.0 0 . 7 0.5 0.7 0.5

6. Mixed Under-, Overdominance 0.9 0.2 0.9 0 . 2 1.0 0 . 2 0.9 0.2 0.9

optimum-heterotic, heterotic with epistasis, and mixed under- and overdominance). Values of F , D, and a t gene frequency equilibrium, for various specifications regarding mating system ( t ) and linkage (c) are given in Table 5. It can be seen that values of F, the fixation index (see Wright, 1965a; Jain and Workman, 1966), were small and negative for t = 1 (random mating) and that they increased steadily as t decreased. Thus, inbreeding had its usual effect of increasing homozygosity. It can also be seen that inbreeding led to larger values of D. The effect was small for c = 0.01 (tight linkage) but it became increasingly pronounced as linkage became looser. Mean population fitness is correlated with 1 D 1 and, with the intermediate optimum model (Model 3), inbreeding led to small inThe relationships among the variables t, c, F , and D creases in are, however, not simple and their interactions with each other and with selection as stipulated in the model influenced in complex ways. I n addition to gametic unbalance (D) inbreeding also affects zy) [AaBb](")gotic associations as measured by the parameter o f a= [Aa](")* [Bb](").As noted earlier, inbreeding leads to an excess of multiple homozygotes or heterozygotes even without selection. Table 6 gives values of w for various values of c and t for the case of no selection and for Models 3-6. It can be seen that w takes its maximum value for different combinations of values of c and t for the different selection models, indicating that the interrelationships between linkage and selection are complex under inbreeding. Moreover, the effects are often large and take forms that can, in theory, significantly influence the genetic organization of populations. Clearly, gametic and zygotic associations as influenced by linkage and inbreeding may be important factors in the maintenance of both stable and quasi-stable gene frequency equilibria.

w,

v.

Values of D, Model 3 t

1.0 0.90 0.30 0.10

C

F

0 0.01 0.10 -0.0114 0.50 -0.0460 0.01 0.0524 0.10 0.0440 0.0094 0.50 0.01 0.5396 0.10 0.5464 0.5484 0.50 0.01 0.8190 0.8248 0.10 0.8326 0.50

D

Model 4 -

W

-0.2375 -0.1359 -0.0274 -0.2387 -0.1469 -0.0339 -0.2457 -0.2123 -0.1389

0.9901 0.9192 0.9616 0.9910 0.9256 0.8621 0.9966 0.9710 0.9210 -0.2484 0.9988 -0.2366 0.9895 -0.2094 0.9702

* Adapted from Jain and Allard (1966).

Fd

TABLE 5

w,and F at Gene Frequenry Equilibria (Models 3-6)* F

D

-0.2388 -0.0688 -0.1408 -0.0942 -0.0251 -0.2396 -0.0102 -0,0190 -0,1496 -0.04% -0.0307 0.4446 -0.2451 0.4658 -0.2066 0.4872 -0.1265 0.7616 -0.2480 0.7774 -0.2329 0.7978 -0.2014 -0.0784

9

Model 5

i7

F

0.9151 -0,2702 0.8398 -0.1724 0.7782 -0,1724 0.9122 -0.2412 0.8423 -0.1366 0.7755 -0.1328 0.0790 0.8830 0.2340 0.8516 0.2752 0.8081 0.3762 0.8639 0.5528 0.8516 0.6106 0.8293

AD

Model 6

F

fD

- 1 %-

W

*

0.1936 0.7400 0.7250 0 0.7250 0 0.1970 0.7315 0.7152 0 0.7146 0 0.2143 0.6519 0.6274 0 0.6212 0 0.2201 0.5934 0.5674 0 0.5592 0

-0.0524 -0.0510 -0.0434

-0.0020

0.0048 0.0396 0.4822 0.5234 0.6868 0.7862 0.8160 0.9022

0.2466 0.2141 0 0.2468 0.2154 0 0.2479 0.2268 0 0.2486 0.2331 0

0.9400 0.8500 0.5750 0.9380 0.8528 0.5888 0.9191 0.8780 0.7594 0.9078 0.8916 0.8502

m

.

5

Q

”

*

‘d

8 0

$

E

81

GENETICS O F INBREEDING POPULATIONS

TABLE 6 Equilibrium Values of w for the Case of No Selection and for Models 3 to 6 t

0.1

0. 3

0.9

1.o

0.01 0.25

0.50

0.0365 0.0265 0.0239

0.0614 0.0427 0.0376

0.0104 0.0079 0.0064

0 0 0

3

0.01 0.25 0.50

0.0809 0.0571 0.0501

0.1726 0.1032 0.0810

0.2321 0.0468 0.0284

0.2303 0.0338 0.0201

4

0.01 0.25 0.50

0.1031 0.0675 0.0581

0.1953 0.1110 0.0860

0.2347 0.0508 0.0329

0,2317 0,0385 0.0252

5

0.01 0.25

0.1940 0.0480 0.0369

0.2092 0.0491 0.0368

0.1492 0.0075 0.0054

0.1371 0.0012 0.0012

0.01

0.0951 0.0629 0.0376

0.1912 0.1475* 0.1038

0.2486 0.2062* 0.1684

0.2479 0.2018* 0,1626

Selection model

No selection

C

0.50

6

0.25

0.50

* Metastable equilibria. See Jain and Allard (1966). D. COMPLEX MODELS Deterministic one- or two-locus models can be extended by introducing stochastic genetic parameters or by considering populations of finite size. Stochastic models involving the process of gene frequency change in small random mating populations with nonoverlapping generations assume that the process can be described by a finite Markov chain and solutions are obtained from the diffusion equation or by simulation on digital computers (for reviews, see Kimura, 1964; Wright, 1964). The effects of random drift arising from sampling errors in small populations and the interactions of random drift with various directed processes have been formulated in terms of effective population size N and the distribution of gene frequencies (Wright, 1948). The effects on the distribution of gene frequencies of fluctuations in the coefficients of the parameters describing the directed processes have also been investigated. Both fluctuations due to sampling in small populations and those due to varying selective values affect the distribution of gene frequencies. While the former

82

R. W. ALLARD, S. K. JAIN, AND P. L. WORKMAN

can bring about fixation in small populations, the latter type of fluctuation, theoretically, leads to gene frequencies in the neighborhood of fixation but never a t fixation (quasi-fixation; Kimura, 1954). The joint effect of small size and inbreeding on the relative rates of gene dispersion is of interest in that variance in gene frequency changes ( U q 2 ) can in theory be expressed in terms of the effective population number (Kimura and Crow, 1963), or alternatively, in terms of the fixation index, F, and its variance, oF2.Using a single locus model, with size N = 10, 20, 100, and partial selfing of varying degrees, Allard and Hansche (1964) studied the gene dispersion process due to drift with and without selection. When N is as small as 10 the drift effects are so large that even heavy inbreeding only slightly increases the rate of dispersion (Figs. 9 and 10). However, as N increases, selection and mating system have an increasingly greater effect on the rate of dispersion until, in populations of modest size (say N = 100 to lOOO), drift effects tend to become small relative to the effects of the directed processes. Similar analyses of rates of fixation in models involving six linked loci, with and without selection, showed that rate of approach to fixation is initially greater in small populations under inbreeding ( t < 1) than under random mating. This is particularly the case when inbreeding is heavy and linkages are tight. However, when popula-

5

0 2.

018016-

Random mating (51% lost) 95% Selfing (65% lost)

3 Allele frequency

FIQ.9. Random dispersion of allelic frequencies in the Bth generation among populations of size N = 10 (no selection, initial allelic frequencies= 0.1). Dispersion among random mating families was determined using generation matrix techniques. Diaperdon among inbreeding populations (s = 0.95) reflects the results of 300 Monte Carlo simulations. After Allard and Hansche (1964).

GENETICS OF INBREEDING POPULATIONS

83

-

5 0.12 .%n a0 2 ace

m mating (41% lost)

95 % Selfing (59 %lost

0.06

ao4 0.E

'0

0.1 0.2 0.3 0.4 0.50.6 0.7 0.8 0.9 0 Allele frequency

FIG.10. Random dispersion of allelic frequencies in the fifth generation among population of size N = 10 (z= 1, y = 0.9; initial allelic frequencies = 0.1). Dispersion was determined as in Fig, 9. After Allard and Hansche (1964).

tion size is as small as 20 (Fig. l l ) , and there is no selection, fixation is rapid whether mating occurs a t random or there is heavy inbreeding. Perhaps the most interesting point revealed by these studies is that tighter linkage tends t o reduce rates of gene dispersion, especially under inbreeding, when the genetic model is one which leads to gametic phase unbalance (e.g., optimum models). 100 80 0

2

G.60

.0

0

-2 40

-

0

z

-- c -c

20

'0

20

40

60

Generation

= 0.01 = 0.50

80

FIG.11. Random drift for 6 linked loci in populations of size N = 20. All gene frequencies were initially 0.5 and no selection was assumed. The percentage of loci fixed was computed from 20 runs for each pair of values of s (percent self-fertilization) and c (recombination value) (Jain, 1968a).

84

R. W. ALLARD, S. K. J A I N , AND P. L. WORKMAN

With increasingly larger population sizes, the pattern of the effects of linkage and inbreeding for six-locus optimum, heterotic, or mixed models becomes much the same as observed with two-locus deterministic models. As population size increased, drift effects diminished relative to the effects of selection and mating system until, for populations of size N = 1000, the distribution of gene frequencies approached those for deterministic cases (Jain, 1968). An additional feature revealed by studies of six-locus models is a cumulative effect of linkage along the chromosome such that genes far apart on the chromosome can be held in gametic phase unbalance by genes located between them, particularly under inbreeding. This linkage effect was observed by Lewontin (1964a) in his study of five-locus heterotic models, and as pointed out by him, such a property of linked gene complexes could play a significant role in the storage of variability under conditions leading t o permanent gametic unbalance. Studies of six-locus models also reveal that factors such as pleiotropic gene action and fluctuating positions of the optimum provide for an extensive range of conditions favoring the maintenance of unfixed loci in permanent gametic unbalance. Stochastic processes treating the mating system ( t ) and the selection parameters (x, y) as normal random variables have also been investigated by simulation, As an example, E(E) = 0.10 and E ( o t ) = 0.05 or 0.09 were taken as expected means and variances over 100 generations, with 20 replicate runs. The resulting variation among replicates is illustrated in Fig. 12 in terms of changes in the fixation index, F. Similar 1.0

0.8 0.6

0;

20

40

60

Generation

80

Ib

FIG.12. Effect of fluctuation in t (percent of outcrossing) on the fixation index, F. Three replicates are plotted for t = 0.10, me = 0.0025 and for t = 0.10, utp = 0.0080. No selection was assumed. Note that for this model F is relatively insensitive to differences in the magnitude of the variance in t .

GENETICS OF INBREEDING POPULATIONS

85

results were obtained by taking z, y as normal random variables with known variances us2, ow2and plotting the resulting fluctuations in the trajectories around the expected deterministic path of changes in allelic frequency, p , and fixation index F (Jain, 1968). The average of all replicates was found to approach the deterministic curve, and the variation in F, or up2,was as expected found to be proportional to the input variances of the parameters t, z, y. The important point is that fluctuations in the parameters x: and y cause the bounds of the areas describing stable equilibria (e.g., Figs. 1 and 2) to become larger such that a wider range of conditions allowed stable polymorphisms under a model with stochastic z and y (Jain and Marshall, 1968).

E. CONCLUSIONS FROM THEORETICAL ANALYSES In this section the theory of inbreeding and selection was introduced in terms of simple one-locus, deterministic models and then developed by reference to genetic models of steadily increasing complexity. One-gene models of mixed random mating and positive assortative mating (e.g., selfing) in which selection confers net heterozygote advantage provide a simple theoretical basis for explaining the maintenance of balanced polymorphisms in inbreeding populations. I n such models the net superiority of heterozygotes need not result from superiority of heterozygotes per se but might be associated with frequency-dependent selection, cyclic selection, or other causes. Variations in the mating system, such as the occurrence of some proportion of negative assortative mating, can also provide a basis for maintenance of stable polymorphisms under otherwise predominant inbreeding. Thus, the a priori assumption that inbreeding leads to homozygosity and hence to genetic uniformity does not find support in theoretical models which take both selection and mating system into account. Investigations of two-locus models in which the effects of linkage and epistasis are incorporated show for many models that one of the effects of inbreeding is to reinforce the effect of linkage in the development and maintenance of nonrandom gametic or zygotic associations. Heterozygote advantage on marginal means is a necessary condition for stable nontrivial equilibrium in multilocus situations and the maintenance of genetic variability in general involves the net selective advantage of heterozygous segments in conjunction with interlocus interactions. There are, however, a wide range of biologically reasonable values for the relevant parameters which allow for the maintenance of unfixed loci under inbreeding. Investigations of mul-

86

R. W. ALLARD, S. K. J A I N , AND P.

L.

WORKMAN

tilocus models involving stochastic variation in the genetic parameters and restricted population size lead to essentially the same conclusions as the simple models, that is, dispersive processes reinforce the tendency toward reduction in the within-family genetic variability and concomitant increase in between-family genetic variability without much reduction in the total populational variability. Migration between subdivisions of a population is a factor that also promotes maintenance of genetic variability in the total population. Theoretical studies suggest that the many factors which affect population structure are interrelated and that there are a multiplicity of pathways by which inbreeding populations might evolve a coadaptive gene pool featuring a stable high level of genetic variability. 111. Experimental Analyses of Polyrnorphisrns

A. ESTIMATION OF PARAMETERS FOR SINGLE-LOCUS POLYMORPHISMS A body of evidence is now in existence to indicate that polymorphisms are a commonplace in both natural and agricultural species of inbreeding plants (review in Allard, 1965). The genetic models discussed in the previous section identify the parameters that are believed to be relevant to the development of stable polyrnorphisms but it is obvious that the models can be little more than abstractions in the absence of quantitative estimates of the parameters obtained from actual populations. In particular, it is important t o determine whether the parameters describing the mating system and the relative viabilities take numerical values that will explain the polymorphisms which are observed in natural and experimental populations. In this section we shall consider methods for estimating the genetic parameters and some of the problems involved in estimation and interpretation of the resulting statistics. The discussion will be restricted to plant populations in which there is predominant selffertilization because adequate data do not appear to be available in other species. 1. Analyses of the Mating System

Census data on genotypic frequencies in two successive generations, (fl("), f ~ ( ~fg(")) ) , and ( f l ( n + l ) , f 2 ( # + l )f3(nf1)) , provide two degrees of freedom and allow only two parameters to be estimated in the recurrence equations which relate genotypic proportions in successive generations. Consequently, before relative viabilities can be estimated it is necessary to obtain an independent estimate of the amount of outcrossing ( t ) .

GENETICS OF INBREEDING POPULATIONS

87

Methods of estimating t , assuming no selection and equilibrium gene frequencies, have been given by Fyfe and Bailey (1951) and Nei and Syaktdo (1958). I n general, however, these assumptions will not be satisfied. Allard and Workman (1963) and Harding and Tucker (1964) have given methods for estimating t when selection is present and (or) equilibrium cannot be assumed. These methods are based on estimates of gene frequency in the population and estimates of the frequency of dominant individuals appearing in the progeny of recessive individuals taken a t random from the population. It has been assumed in the formulation of the above models that outcrossing occurs a t random. A number of studies suggest that this assumption may not be justified (Guitierrez and Sprague, 1959; Barnes and Cleveland, 1963; Harding and Tucker, 1964). If the outcrossing that occurs tends to be between unlike individuals, that is, the outcrossing involves negative assortative mating, then, as shown by Workman (1964), lower levels of heteroxygote advantage suffice to maintain polymorphisms than is the case with random outcrossing. Conversely, if part of the outcrossing involves positive assortative mating, greater heteroxygote advantage is required. Some of the difficulties pertaining to a correct assessment of the mating systems are made less critical when an “effective” amount of random outcrossing rather than the actual amount of outcrossing is estimated (Allard and Workman, 1963). The estimate of t used in the estimation of the relative viabilities should, if possible, be the average of a series of estimates. Studies in a wide variety of organisms (e.g., the French bean, Bateman, 1952; safflower, Claassen, 1950; flax, Dillman, 1938) show the actual frequency of outcrossing is often quite variable. It can differ between years or locations within a year for a given marker locus, and i t can differ from locus to locus. Some of the results of Harding and Tucker (1964) showing variation in the estimate of t in experimental lima bean populations are summarized in Table 7. The amount of outcrossing can also vary with respect to sex. Smeltzer analyzed outcrossing in a population made up of five varieties of sorghum. Each line was considered in terms of its contribution to the outcrossing both as a pollen source and an ovule source. Significant differences between lines with respect to sex were noted as shown in Table 8, 2.. Estimation of Relative Viabilities

As noted earlier, selection models vary according to the time and mode of action of the selection and the stage of the life cycle a t which the genotypic frequencies are determined. In estimating relative via-

88

R. W. ALLARD, S. K. JAIN, AND P. L. WORKMAN

TABLE 7 Estimates of Outorossing in Three Experimental Lima Bean Populations* Year Populationf

Locus

1957

1958

I

D/d

-

-

Wlw

I1

Did

0.0806

0.0254 0.0478 0.0632

Vlv

c/c

Srlsr ss

ss

-

-

-

1959

1960

0.0892 0.1792

0.0730 0.2979

-

0.0479 0.0293 0.1710 0.0924 0.1173 0.1667

0.0180 0.0722 0.19291

-

0.5247$

Location and year Population I11

Locus CIC 85

88

(Davis)

(Irvine)

(Davis)

(Irvine)

0.0539

0.0615

0.0712 0.1718 0.0834

0.0053 0.0295 0.0292

1961

-

1961

-

1962

1962

* Adapted from Hardmg and Tucker (1964).

t Populations I and I1 grown at Davis, California;population I11 grown at Davis and Irvine, California. $ Standard error of estimates > O . 05. bilities it is therefore necessary to choose the model which most closely fits the biological situation. Estimators of 2 and y, and their variances, can then be obtained from the recurrence equations by standard methods (e.g., by the method of maximum likelihood, or least-squares analysis). These estimates of 5 and y are assumed t o represent so-called net Darwinian fitnesses defined in terms of the relative number of progeny left by different genotypes, These estimates will be biased if the selection has not been completed by the time of the genotypic census, as shown by Prout (1965). For the case of predominantly self-fertilixing populations, with which we are concerned here, Workman and Jain (1966) have shown that the errors which attend the estimation of selective values at a partially selected stage are too small to be of any practical significance. Examples of estimators for differing modes of selection and time of genotypic census are give11 by Allard and Workman (1963) and

89

GENETICS OF INBREEDING POPULATIONS

TABLE 8 Estimates of Outcrossing in Mixtures of Grain Sorghum Varietiea at Davis, California* Outcrossing as estimated from female parentst

Proportional contribution to outcrossing aa male parentst

Variety

1959

1963

1959

1963

DD38 CK60 DWD DW39 EH BDS

0.1321 0.0279 0.0508 0.0506 0.0721

0.0721 0.0258 0.0447 0.0246 0.0355 -

0.0362 0.1335 0.1014 0.1733 0.1563 0.3993

0.0857 0.2200 0.0859 0.1667 0.4417

~

* Smelteer (1965).

-

~~

-

~~

?All estimates have been adjusted to compensate for differences in the relative proportions of the linea in the mixture. $ Accurate clas~ificationwas found to be difficult in the 1959 experiment. This genotype waa therefore not included in 1963.

Workman and Jain (1966). Table 9 gives numerical values of the selection parameters obtained for various marker loci in some experimental populations of liina beans and barley and in some natural populations of wild oats, These data indicate that heterozygotes nearly always produce more viable progeny than homozygotes and reference to the phase diagrams for t = 0.05 (Fig. 2) shows that the observed level of heterozygote advantage is sufficient to account for permanent maintenance of the polymorphisms. The data of Table 9 can be considered in another way. Inbreeding species have usually been described in terms of the amount or intensity of inbreeding without consideration of the effects of selection. Allard and Workman (1963) defined a parameter, t*, the effective amount of outcrossing, which permits one to equate the observed population with parameters x, y, and t, t o a population in which x = y = 1 and there is outcrossing in the proportion t*. Although the actual t was of the order of 3 to 5% for the lima bean populations, t" was approximately 13 to 14%. Thus, heterozygote advantage had the effect of substantially reducing the amount of homozygosity that would have resulted if inbreeding had been the major factor in determining variability. Some other aspects of the problems involved in the estimation of the genetic parameters should be mentioned. Allard and Workman (1963) studied fluctuations in estimates of x and y over several generations in experimental lima bean populations (Fig. 13). Their results show that year-to-year fluctuations in environment lead to sharp

90

R. W. ALLARD, S. K. JAIN, AND P. L. WORKMAN

TABLE 9 Estimated Relative Viabdities for the Two Homozygotea and the Heterozygotea for Chromosome Segments Marked by Major Genes

Locus or population

Mean percentage of outorwing

Lima bean'

s/s

Dld SIs Barleyt Blb Sb Wlg

Ele

Bllhl Rlr

Bt/bt Sh./sh Wild oatst (lemma color locus) Population 1 (1960) Population 1 (1960) Population 5 (1960) Population 7 (1961)

4 5 3

Relative viabilities of

AIAI

AiAt

0.53 0.58 0.66

1 .oo 1 .oo 1 .oo

1.06

0.81

1.04 0.47

0.61 0.82

0.96 0.71 0.50

0.64 0.41 0.57

1.00 1 .oo 1.00 1.00 1 .oo 1 .oo 1 .oo 1.00 1 .oo 1.00 1.00 1 .oo

A 4 2

0.35

0.47 0.66 1.31

0.96 0.82 0.59

0.54 0.68

1.06 0.63 0.30

0.56

0.44 0.54

*After Allard and Hansche (1964). t After Jain and Allard (1960). 2 After Imam and Allard (1965).

shifts in the relative viabilities of different genotypes suggesting that caution must be exercised in interpreting results based on data from a limited sample of generations. Sampling errors, particularly if one of the genotypes is in low frequency, can also be an important source of fluctuations in the estimates of the viabilities (Workman and Allard, 1964). Frequency-dependent selection may be involved but go unrecognired. Harding et al. (1966) submitted one of the lima bean populations studied by Allard and Workman (1963) to a more detailed experimental analysis and established that the fitness of heterozygotes increased strikingly as their frequency decreased in the population (see Fig. 14). Consequently, it seems likely that some of the observed fluctuations in x and 9 reported by Allard and Workman may be ascribed to the effect of frequency-dependent selection. Such selection can be a potent force in retaining variability in popu-

GENETICS O F INBREEDING POPULATIONS

91

x, y 0.8

0.4

,:-:\' '

Pdpuiknk '

L i ----

.

Y

.,as[

0.4 0.0

k ,ik2iAiMizi Year

k 7imi m / !

FIG.13. Year-to-year fluctuations in estimates of selective values for the S/s locus in three experimental lima bean populations. After Allard and Workman (1963).

% 4.0 -

0

03

c

m

=.

N

E 3.0-

All populations

26

016

f

c

r

-F v)

._ r

2.0-

s QCT 1.0-

200

._

L

Y e = 3.38

- 16.77):

s b = 2.74

FIG.14. Relationship between frequency of heterozygotes and their fitness relative to homozygotes for the S/s locus in lima bean populations. Homozygotes were assigned a relative fitness of 1.0. After Harding et al. (1966).

lations when the selective value of any genotype increases as its frequency decreases. Workman and Jain (1966) have shown that restriction of selection to one sex affects rate of approach to equilibrium but that the ultimate equilibrium reached is the same, for given 2, y, and t, as when

92

R. W. ALLARD, S. K. J A I N , AND P. L. WORKMAN

selection affects both sexes equally. Thus, it is possible that different models may appear to fit a given set of experimental data, particularly for populations near equilibrium. This can lead to serious problems of interpretation, particularly if the data available span only a limited number of generations. The genetic basis for the selfing can vary from control by a single locus to control by a large number of loci (e.g., East, 1919; Clarke, 1935; Holden and Bond, 1960). When a single locus or very few loci control selfing there will be little or no outcrossing a t all in many families and the distribution of outcrossing among families may be satisfied approximately by a Poisson or negative binomial distribution. Control by a large number of loci is expected to lead to an approximately normal distribution of amount of outcrossing in different families. Differences in the genetic control of selfing lead to considerable differences in the observed variation in the estimate of t , especially in experiments in which only a few families a r e studied. Linkage is another factor which affects estimates of the relevant parameters. Suppose that a marker locus under study (say A ) is linked to a locus ( B ) a t which variability is retained due to heterozygote advantage. The estimates of x and y for locus A then depend not only on the relative viabilities of the genotypes a t the A locus but also on the intensity of linkage, the viabilities a t the locus B, and the presence or absence of linkage disequilibrium between loci A and B. This problem has been considered indirectly by Kimura (1956) and by Parsons (1957) for the case of full selfing. Despite the caution that must be observed in analyzing estimates of genetic parameters, certain conclusions can be drawn with confidence. The general pattern is that heterozygotes usually show considerable selective advantage over the corresponding homozygotes for marker loci and the net heterozygote advantage observed is often at levels which permit permanent retention of polymorphisms, even in very heavily inbreeding populations.

B. COMPLEX POLYMORPHISMS The models discussed in Sections 11, C and 11, D indicate some of the possibilities regarding equilibrium states and progress toward equilibrium in populations t.hat are polymorphic for more than a single Mendelian unit. However, a number of difficulties are encountered when attempts are made to obtain numerical estimates of the relevant parameters in actual populations. Not the least of these diffi-

GENETICS OF INBREEDING POPULATIONS

93

culties is the sampling problem associated with the necessity of estimating the frequencies of nine genotypes when two polymorphisms are studied simultaneously, 27 genotypes with three-unit polymorphisms and discouragingly large numbers of genotypes with more complex cases. It is not surprising that experiment has lagged behind the development of theory. Despite the difficulties, there have been several studies of complex polymorphisms in experimental populations and also a few studies in natural populations. Examples are wing characters of the grasshopper Pazatettix by Fisher (1939),shell color of Cepaea nemoralis studied by Lamotte (1951) and Gain and Sheppard (1952),the inversion polymorphisms of Drosophilia robusta reported by Levitan (1955, 1958), the complex mimicry patterns in certain butterflies (Sheppard, 1959), mutant markers in D . melunogaster studied by Cannon (1963) and further analyzed by Lewontin (1964a),marked chromosomes of D . pseudoobscura studied by Spassky et al. (1965), and lethals in D. uri2listoni studied by Magahaes et al. (1964). The general pattern of results with these presumably random mating species indicates that different Mendelian units are often not independent in their fates in populations. Instead interactions between different units frequently affect selective values and hence the organization of the population genotype. Experimental data on complex polymorphisms are also scanty in inbreeding populations. One of the most thoroughly analyzed cases occurs in the Australian grasshopper, Moraba scurra. White (1957) and Lewontin and White (1960) presented evidence that pericentric inversions carried on two different chromosome pairs in this species are not combined a t random in adult male individuals. Their analysis of the deviations from the expected random combinations provided evidence for genetic interaction between the two systems of cytological polymorphism as they affect viability. However, these wingless grasshoppers have limited mobility and if it is assumed that there is a tendency for close neighbors to mate and that close neighbors are likely to be relatives, i t is expected that some equilibrium level of inbreeding will develop in populations of these sedentary insects. As discussed by Allard and Wehrhahn (1964) the assumption of a low level of inbreeding (0.10< F < 0.15) gave a good fit t o the data. Thus epistatic interactions can be a feature of an apparently stable nontrivial gene frequency equilibrium in populations in which inbreeding arises as a result of isolation by distance. The polymorphisms in experimental populations of lima beans discussed previously are favorable materials for the study of interactions

94

R. W. ALLARD, S. K. JAIN, AND P. L. WORKMAN

between different Mendelian units under heavy inbreeding and they have been used for that purpose by Harding and Allard (in preparation). In the population chosen for detailed study it was possible to score seven marker loci individually and 21 possible two-locus combinations. In 16 of the 21 two-locus cases studied, certain genotypes were in significant excess or deficiency. Two of the loci are located on the same chromosome and the type of gametic unbalance observed for these two loci ( D > 0, loci in coupling in the initial population) suggested that the unbalance represented undissipated coupling associations due to the linkage. However, the remaining 15 cases of zygotic association cannot be attributed to linkage because the loci involved are known to be located on different chromosomes. The basis of these associations is therefore presumably either the mating system effect of Bennett and Binet (see Section 11), or epistatic interactions between the marked linkage blocks. More extensive and more precise numerical estimates are clearly necessary before the relative importance of the parameters identified in the two-locus models of the previous section can be assessed. Results obtained thus far point, however, to the possibility, indeed the probability, that the destiny of genes in populations depends not only on the effects of the genes themselves but also on their interactions with all of the factors that affect the organization of the gene pool. The mating system appears t o be only one among a number of interdependent factors that affect the organization of populations, and its effects, no less than those of the other factors, are integrated in the whole. As an example, the effects of inbreeding due to restricted population size in Cepaea nemoralis appear to be mitigated by multiple matings and sperm storage mechanisms (Murray, 1964). The discovery that complex interactions are a feature of two-locus systems thus provides an experimental basis for extrapolation to the more complicated systems of interaction which are presumably involved in the control of continuously varying characters and which in total lead presumably to the “coadapted” population genotype. IV. Genetic Variability in Quantitative Characters

Populations of inbreeding species have commonly been supposed to be highly uniform respecting measurement characters. However, it has been known since ancient times that selection is effective in improving performance and modifying various other characteristics in populations of wheat, barley, oats, and other predominantly selfpollinated crop plants (Roberts, 1929). The first recorded accounts showing that selection is effective in changing inbreeding populations

GENETICS OF INBREEDING POPULATIONS

95

appeared in the eighteenth century as a result of methodical attempts at plant breeding. Van Mons in Belgium, Knight in England, and Cooper in America demonstrated that selection can lead to worthwhile improvement in agricultural performance. More fully documented evidence followed in the early nineteenth century. John Le Couteur, a farmer of the Isle of Jersey, described the diversity of plant types in his wheat field and established that there were differences in the agricultural value of various selections. B y the late nineteenth century selection in “land varieties” had become an established method of plant breeding. Land varieties still exist in some of the less advanced agricultural areas and it is easily demonstrated by progeny tests that such populations contain many different genotypes. Harlan (1957), in discussing variability in barley, states that in each local area “there has evolved a type peculiarly fitted for conditions as they exist there. Slight changes in altitude are accompanied by corresponding changes in the barleys, The barleys from each tiny area are made up of large numbers of strains that look much alike, but that may differ greatly in ways useful to the plant breeder. Even out on the plains where the superficial appearance of the crop may be the same over a large area, these constituents are present in endless variety and shift as one goes to drier or colder sections.” Thus, it has been recognized since the earliest days of cultivation that populations of predominantly self-pollinating agricultural plants are repositories of great amounts of genetic variability. It is only recently that detailed quantitative studies of variability in measurement characters have been undertaken in inbreeding populations and that adequate estimates have been made of the components of this variability under population conditions. I n these studies the basic procedure has been to collect seeds from random plants in a number of populations. These seeds were then sown to establish families of 20 or more plants in replicated experiments in a common nursery environment and each individual in each family was measured for various morphological and physiological attributes. Data from such studies permit quantification of three aspects of genetic variability, that associated with broad geographical regions, that associated with specific sites within regions , and variability occurring within sites. The general pattern of variability that exists in inbreeding species can be established by considering a few examples. A. GEOGRAPHICAL VARIABILITY

Knowles (1943) made collections of Soft Chess (Bromus mollis), a highly self-pollinated annual grass, along a transect from the cold,

96

R. W. ALLARD, 6 . K. JAIN, AND P. L. WORKMAN

humid coastal regions of northern California t o the warmer semiarid Sacramento Valley to the interior. Data from progenies grown in a common environment (Yolo County) revealed a clinal pattern of variation (Table 10). Size of plant, as measured by height, and time required to reach maturity, as measured by days to flowering, decreased progressively but tillering capacity increased with increasing aridity. Similar clines have been found in all other inbreeding species for which adequate measurement data are available. Thus, for example, seed size in Bur Clover (Medicago hispidu) decreases progressively with increasing altitude, petal length of Erodium &uturiurn increases with increasing rainfall, leaf length in Foxtail barley (Hordeurn nodosum) decreases from north to south in California,

-

TABLE 10 Genetic Variability between and within Populations of Bromus mollis' ~

Location of populationt

Number of progenies

Del Norte Humboldt Contra Costa Solano Yolo Sutter

16 23 18 44 9

6

~~

Daya t o heading

Plant height

Tiller number

Mean

Range

Mean

Range

Mean

Range

186 174 164 153 154 151

174-201 163-183 155-169 151-164 151-160 151

88

5&102 49-103 72-89 55-92 57-86 58-63

31 27 29 33 40 35

22-45 17-43 2043 23-62 32-49 30-40

77

80 67 71 60

* After Knowles 11943).

t Listed in increaeing order of aridity. and wild oats (Avena fatua) from deep fertile soils are more robust and tiller more profusely than wild oats from infertile soils (Allard, 1956). Superimposed on this clinal variability is another and much more striking pattern of variation. This is a patchwork or mosaic pattern which is reflected in sharp differentiations between different sites (populations) within regions. Such variation is illustrated by a sample of data on wild oats (Avena jatua) from the studies of Imam and Allard (1965) and Jain and Marshall (1967). Comparisons of collections taken from a number of sites within one of the regions studied (the Coast Range of California) indicated sharp differentiations between sites (Table ll). For example, flowering was earlier for site 4 than for the other sites. The habitat in this case was a west-facing arid hillside which might be expected to be unfavorable for late maturing genotypes in a Mediterranean climate.

97

GENETICS OF INBREEDING POPULATIONS

TABLE 11 Estimated Means for Different Natural Populations of Wild Oats within a Geographical Region

Populationt

Spikelet number per panicle

Panicle length (cm)

112

El

7.7

9.9

103

E2

6.3

8.2

14.0

15.2

Population*

Flowering time (date in April)

Height (cm)

4

11.5

5

16.5

6

25.4

107

FI

* After Imam and Allard (1965).

t After Jain and Marshall (1966). Differentiations are often striking over very short distances. Within one site heading time changed more than 15 days in a distance of 5 meters, apparently in association with local topography. Progeny of planta taken from a flat area immediately above a short steep slope flowered on April 18 on the average, those from the steep slope flowered on April 11, and those from the well-watered area a t the base of the slope flowered on April 26. Results for many different species follow a pattern that can be summarized as follows, Inbreeding species of plants are differentiated geographically and the differentiations observed appear to be those which provide each population with the adaptive properties needed to meet the requirements of the local environment. The predominant pattern is one of striking local differentiations, often involving areas of a few square meters or less, but clinal gradients also occur in association with progressive changes in rainfall, temperature, and other factors of the physical or biotic environment.

B. VARIABILITY WITHIN POPULATIONS Perhaps the first quantitative study of genetic variability arising from differences among families whose seed parents were single plants taken from the same population was Johannsen’s (see Johannsen, 1926) classic experiment with the Princess variety of garden bean (Phaseolus vulgaris). Johannsen observed that a great deal of variation occurred in the unselected population with respect to seed size and other characters and, by dividing the seed into classes according to weight, he was able to show that smaller seeds in general produced small-seeded and large seeds produced large-seeded progeny. B u t he also observed that seeds in a given class produced progeny with

It. W. ALLARD, 6. K. JAIN, AND P. L. WORKMAN

98

rather widely different weights and this observation led him to compare mean seed weights of families. Johannsen established families by growing separately the progenies of 19 plants from the original population and he found that each family had a characteristic seed weight forming a continuous series from 35.1 centigrams for the smallest family to 64.2 centigrams for the largest family. It is not clear whether these 19 families represented a random or selected sample from the original population. However, the nearly twofold range in seed size from smallest to largest family established that the original population was highly variable respecting this character. Knowles’ (1943) study of Soft Chess demonstrated that similar variability occurs in natural populations of inbreeding species. It can be seen from Table 10 that families from the same population varied over very wide ranges respecting flowering time, height, and number of tillers. The difference between earliest and latest family was as much as 25 days in flowering time, the range from shortest to tallest was more than 50 inches, and there was more than twofold difference in tillering capacity. Knowles concluded from his measurements and observations on several characters that each family is genetically distinct from each other family, implying that few if any individuals in the original populations had exactly the same genotype. Knowles’ conclusion regarding the genetic distinctiveness of each individual has been placed on a firmer basis by subsequent studies in other species. In wild oats, for example, very wide differences occur among progenies from the same population with respect to flowering time, height, germination percentage, number of tillers, length of panicle, seed size, and other characters. When the means of families are compared for any single character, using a Duncan multiple range test, it is found that the families from a single population fall into a t least two but usually more groups which differ significantly from one another ( P 0.05). Families in different groups clearly have different genotypes. When two characters are considered simultaneously i t is found that families which fall in the same group for one character (say flowering time) often fall into different groups with respect t o the second character (say height); such families also do not have the same genotype. Extension of this sort of analysis over additional characters ultimately separates the families into as many groups as there are families. This measure of intrapopulation variability therefore leads to the conclusion that each population includes plants of many different genotypes respecting measurement characters. No single genotype is represented by more than a few individuals and perhaps

<

GENETICS O F INBREEDING POPULATIONS

99

every individual in t,he population differs genotypically from every other individual.

C. VARIABILITY WITHIN FAMILIES Again, the first precise quantitative study appears to have been Johannsen’s study of the Princess variety of the garden bean. This study is too well known to require detailed description. It is sufficient to say that six generations of selection within families failed to establish a significant difference between lines selected for light seeds and lines selected for heavy seeds. This result led t o the formulation of the pure-line theory and it is also the basis for many of the deductions that have been made concerning the genetic structure of inbreeding populations. More recently, evidence has been accumulating that Johannsen’s population of garden beans represented a special and apparently very rare situation, that of a population in which selective pressures have been low and reproduction has been exclusively by self-fertilization for many generations. There have been many experiments to indicate that continued propagation of selfed families under conditions of low competition leads to high levels of homozygosity and these experiments provide a basis for the result observed by Johannsen. In the population studied by Johannsen a history of propagation under garden conditions may have provided for high survival and the absence of outcrossing might have been due t o lack of appropriate insect pollinators in Northern Europe. However, the garden bean is typically not completely self- fertilized in the more southerly areas where this species is grown on a large scale. Instead, a low rate of outcrossing (less than 1%)is usual and in some years there may be as much as 5% of outcrossing between certain genotypes. Furthqrmore, plantings are usually dense, which suggests that competition may be intense. The effect of this low order of outcrossing and presumed competition on within-family variability was determined by Allard and Golden (1954), who conducted an experiment analogous to that of Johannsen. A lot of foundation seed of the Red Kidney bean ( P . vulgaris) provided by the California Crop Improvement Association was sown and seed weight index (grams/100 seeds) was determined for each of 100 plants. The range in seed weight index varied from 49.1 to 54.3. This represents a smaller range of variability than Johannsen found in the Princess variety and i t no doubt reflects the stringent selection for conformity to “type” that characterizes present day seed practices.

100

R. W. ALLARD, S. K. JAIN, AND P. L. WORKMAN

Twenty of these plants were then chosen by random methods and seeds from each plant were sown to establish 20 families each containing approximately 25 individuals. Seed-weight indexes were determined a t maturity and seeds from the plant with the smallest seed-weight index and from the plant with the largest seed-weight index were sown the next year to establish minus and plus selection lines. This process was repeated for four generations. The results of selection in the families are presented in Table 12. I n contrast to Johannsen’s result a significant response to selection occurred in each of the 20 families. TABLE 12 Effect of Four Generations of Selection for Seed Weight within Families 7 and 16 of the Red Kidney Bean* Family 7, seed weight index (grams/lOO seeds)

Family 16, seed weight index (grams/100 seeds)

Generation

Small

Large

Difference

Small

Large

Difference

0 1 2 3 4

52.2 50.1 50.8 50.3 49.2

52.2 52.3 52.9 52.6 52.9

0 2.2t 2.lt 2.3$ 3.7$

51.1 50.0 51.2 49.0 48.1

51.1 53.1 55.5 54.1 56.1

0 3.1$

4.3f

5.1$

6.0$

* Among 20 families studied, response to selection was smallest in family 7 and greatest in family 16. From Allard and Golden (1954). t Difference significant P 5 0.05. 3 Difference signifirant P 5 0.01. The greatest progress occurred in family 16. I n that family the final difference between the small and large selection lines was 6.0 g/100 seeds, which represents a spread of nearly 12% in terms of the base weight of line 16 (51.1 g/100 seeds). The smallest response was in family 7 but even in that family the final difference between small and large was 3.7 g/100 seeds, or about 7% of the base weight of the family (52.2 g/100 seeds). Similar responses to plus and minus selection within families were obtained in a companion experiment with the Henderson variety of the lima bean (Phaseolus lunatus). In this case the base population was a highly selected elite seed lot obtained from a commercial seedsman. The seed weight index of the base population was 43.8 and the mean observed seed weights for progenies of plants derived from 100 random seeds taken from the base population varied from 39.8 to 46.1 g/100

101

GENETICS OF INBREEDING POPULATIONS

seeds. Selection for low and high seed-weight index in 20 families led t o statistically significant responses in each case. The smallest response observed after four generations of selection was 4.1 and the largest response was 8.4 g/lOO seeds (Table 13). It can be inferred from studies of intervarietal rates of outcrossing that a very high proportion, probably more than 99%) of the fertilizations result from selfing in both the Red Kidney and Henderson varieties. However, the precise rate of outcrossing which occurs within these varieties under population conditions cannot be determined because TABLE 13 Data Showing Response to within Family Selection in the Henderson Variety of the Lima Bean (Pha-seoluslunatus)* Family 11, seed weight. index (gramsj100 seeds)

Generation

Family 8, seed weight index (grarrm/100 seeds)

Small

Large

Difference

Small

Large

Difference

43.6 41.8 41.9 41.5 41.7 41.6

43.6 43.8 43.9 45.1 45.1 45.7

0 2.0t 2.0t 3.6$ 3.4$ 4.1$

41.5 41.3 40.8 39.1 37.9 37.1

41.5 42.6 42.6 43.4 43.3 45.5

0 1.3 1.8t 4.3t 5.4t 8.4$

* Family 11 showed the smallest response and family 8 the largest response among 20 families studied. From Allard and Golden (1954). t Difference significant, p 5 0.05. 1Difference significant, p 5 0.01. both varieties are monomorphic. It is therefore of particular interest t o examine response to selection within families in populations in which precise quantitative data on outcrossing rates have been obtained from studies of polymorphisms. I n their study of variability in wild oats, Imam and Allard (1965) noted that, in addition to the differences between families that were considered in the previous section, there was also distinct variability within the great majority of families. A selection experiment was conducted to determine whether at least part of the variability for measurement characters could be ascribed t o heterozygosity of the plants taken from the natural population. The selection experiment was based on 10 plants chosen by random methods from a natural population in which the outcrossing rate varied from 1.49 to 6.68%) depending on

102

R. W. ALLARD, S . K. JAIN, AND P. L. WORKMAN

the year and the marker gene used to determine rate of outcrossing. A progeny of 20 individuals was grown from the seeds harvested from each of these 10 plants and within each progeny the individual with the lowest score and the individual with the highest score were selected respecting the following three characters : prostrate vs. erect growth habit; early vs. late flowering time; short vs. tall. The next season paired plots were grown, one plot of each pair containing 10 plants derived from the extreme low individual and the other containing 10 plants derived from the extreme high individual for each character. The 30 paired plots for each character were arranged in a randomized design, replicated twice. Each replication also included a plot derived from reserve seed of each of the ten original plants. The results of measurements taken on an individual plant basis in these 180 plots are summarized in Table 14. TABLE 14 Effects of a Single Generation of High and Low Selection on Three Quantitative Characters in Lines Derived from 10 Wild Oat Plants Taken from a Natural Population* Unselected lines Character

Range

Mean

Low selected lines

Range

Mean

High selected lines Range

Growth habitt 2.70-3.71 3.26 1.89-2.46 2.2418 3.34-4.11 Days to 10.63-16.99 15.62 4 . 6 5 1 5 . 5 1 11.1918 16.16-19.18 flowering Height 93.1-115.2 105.6 82.5-106.3 89.3$0 107.3-139.4

Mean 3.68 17.72 116.2

* After Imam and Allard (1965). t Scored on an arbritrary scale; 1 = prostrate; 5 = erect.

1Significantly different (P< 0.05) from the mean of the unselected lines. Q Significantly different (P < 0.01) from the mean of the high selectians.

Two aspects of the results can be considered. First the data given in Table 14 show that the mean of the 10 low selected lines was significantly lower ( P < 0.05) than the mean of the 10 high selected lines for all three characters, Also the means of both the low and high selected lines were significantly different from the means of the unselected lines for each character. The second aspect of the results is the divergence within individual families produced by the selection. For growth habit and flowering time the difference between the high and low selections was significant in each of the 10 families and the difference between the selected and unselected lines was also significant in all comparisons. The response to selection for height was only slightly

103 less clear-cut. For this character one generation of selection failed to produce a significant difference between high and low selection lines in only two families and the difference between unselected and selected lines failed to reach significance in only 8 of the 20 comparisons that could be made. The results of various experiments involving selection within families in several different heavily inbreeding species can be summarized as follows. Individuals within a population are often heterozygous for many genes governing quantitative characters. The variability that is commonly observed within individual families is therefore not exclusively environmental or developmental but much of it can be ascribed to segregation. It is clear that the genetic organization of a population cannot be deduced solely from the amount of outcrossing that occurs in the population. GENETICS OF INBREEDING POPULATIONS

V. Responses to Selection

The responses of inbreeding populations to mass selection, both artificial and natural, have been studied in many different species. I n this section we shall consider some representative experiments which illustrate the main patterns of response.

A. COMPETITION IN MIXTURES OF PURELINES There have been a number of studies of natural selection in populations synthesized by mechanical mixing of two or more pure lines (e.g., Harlan and Martini, 1938; Suneson, 1949; Allard e t al., 1966). The results of these experiments have been remarkably consistent; almost without exception one pure line rapidly became predominant and all other pure lines were drastically reduced in frequency. A typical result is given in Fig. 15. I n this experiment the four competing genotypes were fully vigorous and well adapted commercial varieties. Nevertheless, relative selective values (Fig. 15) show that the poorest competitor produced 23% fewer progeny on the average than the best competitor (Workman and Allard, 1964). I n some other experiments selective differences were much greater. In certain cases genotypes that were not obviously defective left only of the order of 5% as many progeny as the best competitor. It is obvious that selection of very great intensity occurs under population conditions. Results such as these lead to the expectation that local populations of highly inbreeding populations should soon be reduced t o a single highly competitive genotype. However, we have already seen that both

104

R. W. ALLARD, S. K. JAIN, AND P. L. WORKMAN

natural and agricultural populations of inbreeders are variable and that, in fact, no single genotype makes up any substantial proportion of the population. One line of evidence that provides a partial answer to this seeming paradox is a tendency noted in several populations for the selective values of certain genotypes t o increase as these genotypes become increasingly rare in the population. For example, Workman and Allard (1964) found evidence to suggest that the selective values of Club Mariout, Vaughn, and Hero were higher when they were in low than when they were in high frequency. This type of frequency-dependent selection could be a powerful force in maintaining many genotypes in a population, each in low frequency. 100,

.

Selected Values

._

._______--*'.

-.

220

\--.

10 0

I

2 3 4

5

6

8 9 10 I I Generation

7

f2 13 14 15 16

ha. 15. Effect of natural selection on genot,ypic frequencies in a mixture of four pure lines of barley. Data of Suneson (1949).

Another type of experiment provides additional information which is helpful in understanding the large number of genotypes which occur in natural populations. In the experiments that have just been considered, the integrity of the small number of competing pure lines was protected by removing interline hybrids. If, however, the occasional hybrids that occur were to be left in the population, the situation would correspond more closely to that in natural populations, or in unselected agricultural populations. An experiment with a population of lima beans which was synthesized by mechanical mixing of two pure lines illustrates the type of result which is obtained. The two homozygous lines that were mixed were differentiated by five major genes affecting conspicuous morphological characters so that the great majority of individuals derived from hybrids between the original homozygotes could be identifled easily. Consequently it was possible t o classify the population, generation by generation, into parental and

GENETICS OF INBRREDING POPULATIONS

105

nonparental types, as shown in Fig. 16. Within a few generations the original pure lines had been swamped by a hybrid swarm, even though the proportion of outcrossing in the population averaged only about 5 % . Moreover, the population had become enormously variable for quantitative characters by the fifth or sixth generation. Observations and measurements on progenies derived from single random plants taken from the population in the ninth and tenth generations indicated that each progeny was characterized by a constellation of characteristics, such as time to maturity, leaflet color (yellow green to dark green), leaflet size, seed size, and other more subtle differences,

.-c m0

I

I

560- I I

Parental line L 121

" O I 2 3 4 5 6 7 8 9 1 0 1 1 Generation

FIG.16. Proportions of parental and nonparental types in a lima bean population which was synthesized by the mechanical mixing of two pure lines (L21 and L121). The average amount of outcrossing in this population was 5%. After Jans and

Allard (in preparation).

which distinguished it from all other progenies. It is clear that competition among homozygotes rarely if ever occurs in pure form in natural populations because even low levels of outcrossing set the stage for conversion of simple mixtures of limited numbers of homozygotes into a complex and dynamic populational system within a few generations. B. RESPONSES TO NATURAL SELECTION

There have been a number of studies of both short- and long-term responses to natural selection in populations that were developed from intercrosses between different genotypes. An early experiment which provided quantitative data on two aspects of response to natural

106

R. W. ALLARD, S. K. JAIN, AND P. L. WORKMAN

selection was reported by Adair and Jones (1946). These investigators blended F2 seed of 20 different rice hybrids and grew the resulting population without conscious selection at Biggs, California, Stuttgart, Arkansas, and Beaumont, Texas, for eight generations. A random sample of 1000 seeds was then drawn from the population a t each location, grown a t Stuttgart, Arkansas, and the plants in each sample measured or classified for various characteristics. The average number of days from seeding to heading was shortest for the California grown and longest for the Texas grown materials, as shown in Table 15. This is in accord with differences in the length of growing season a t the three locations and it indicates that rather intense directional selection occurred for this character. Intense directional selection also occurred for grain type, awn length, and pigmentation as evidenced by the differing proportions of plants with varying expression of these characters a t the three locations. However, mean height was about the same a t the three locations and it was also about equal to the mean of the original parents. Apparently, therefore, the height of the original hybrid population was close to that which is most desirable for each of the environments sampled. These results make i t clear that one of the major patterns of change in response to natural selection is rather rapid directional adjustment of measurement characters toward optimum values and that the optimum is often not the same for different environments. Table 15 also reports the frequency with which plants with various phenotypes survived a t the three locations. Plants with a wide range of heading dates and with a wide range of height survived at all locations. The three populations also remained highly variable with respect to grain type, degree of awning, and color. Among the surviving plants these characters occurred in all combinations. The second major effect of natural selection is therefore the preservation of many different genotypes and not reduction to uniformity as has commonly been supposed. The experiments of Akemine and Kikuchi (1958) and Allard and Jain (1962) reveal that stabilising selection, as well as directional selection, occura for measurement characters. Akemine and Kikuchi grew hybrid populations of rice a t 20 experiment stations located from 31" to 43" north latitude in Japan. Each generation, random samples of seed were drawn from the population a t each location, grown in a common environment in central Japan, and measured for various characters. The data showed that strong directional selection occurred for earliness and for lateness a t northern and southern locations, respectively, whereas there was little change in mean heading time a t intermediate latitudes. The progressive elimination of late or early

TABLE 15 Effect of Natural Selection in Three Environments on the Composition of a Population of Rice* Days from seeding to heading

Source of seed Mean California 102 Arkansas 110 Texas 120

Height.

Range

Mean (in.)

R.ange (in.)

83-127 94-127

38.8 39.9

20-48

105-151

40.1

*Data of Adair and Jones (1946).

20-55 20-55

Awn classes (percent)

Grain types (percent) Short 46 24

50

Medium 48

41 44

Long

6 35 6

Awnless

53 77

52

Tip

awDs 24 16 22

Patially awned

17

6 21

i

B

Color of hull apex (percent)

Awned 6 1 5

Purple

2

46 11

Red 16 27 9

g

2

Green LJ

82 28 80

cd 0

35

108

R. W. ALLARD, S. K. JAIN, AND P. L. WORKMAN

heading genotypes which occurred a t the extreme latitudes led to skewed distributions in early generations and to some reduction in variability, whereas a t central locations selection led t o less drastic elimination of individuals from both tails of the distribution curve. Nevertheless, the amount of variation remained large at all locations showing that stabilizing as well as directional selection occurred for heading time. Similar concurrent directional and stabilizing selection responses have also been observed for various metrical characters in experimental populations of barley, wheat, and lima beans. For example, in a study of a barley population (Composite Cross V) synthesized by mixing intercrosses among 31 varieties of barley, Allard and Jain (1962) observed that mean heading date of the population shifted rapidly in the direction of earliness in the first 4 or 5 generations after synthesis and then more slowly during the next 15 generations. Frequency distributions for various generations showed that there was steady elimination of individuals from both tails of the curve, indicating that stabilizing selection was also occurring for this character. The effect of this combined directional and stabilizing selection was a small change in the mean heading time of the population and a moderate decrease in variance over 20 generations. To determine the basis of these changes in mean and variance, random samples of plants were taken from Composite Cross V in various generations and their progeny grown in replicated experiments. Measurements on heading date and other metrical characters allowed estimates to be made of the variance of family means and also of within-family variances. The between-family variance was high in early generations and it decreased steadily generation by generation. The distribution of family means indicated that genotypes with intermediate heading dates were favored and that selection operated against both early and late heading genotypes. However, between-family variance remained high in the Flg generation, which was the most advanced generation available for study. Measurements on the Fan generation of a similar barley population (Composite Cross 11) indicated that 20 additionaI generations of stabilizing selection led to little if any further decrease in between-family variance. Apparently, by the Fl9 generation, a balance had been struck between factors that increase and factors that decrease variability. Within-family variances for heading date and other metrical characters were high in the Fr generation of Composite Cross V, as expected in materials recently derived from hybrids. During the next 15 generations the mean within-family variance decreased steadily.

GENETICS OF INBREEDING POPULATIONS

109

However, in the FIQgeneration it remained larger than the average within-family variance of the original homozygous parents of the population. One possible explanation for this excess of variability over that of the parents is that selection favored genotypes which, owing to poor buffering, were more variable than equally homozygous but well-buffered genotypes. However, in a study of seed size, tiller height, and similar characters for which it is possible to make multiple measurements on single plants, Kikuchi et al. (1967) found that withinplant variances were higher in early than in late generations. Selection thus appears to favor well-buffered individuals and this reinforces the argument that the high within-family variability observed in the Fle generation is due to the segregation of genes governing metrical characters. Studies of the FSQgeneration of barley Composite Cross I1 indicate that little if any further decrease in within-family variability OCcurred after the FIQgeneration. This result provides further evidence that the individuals of which inbreeding populations are comprised are not fully homozygous but that they provide new variability each generation through segregation and recombination of genes governing quantitative characters. This result also suggests that populations derived from hybrids rather rapidly reach an equilibrium in which loss of variability due to the combination of directional and stabilizing selection is balanced by steady release of new variability resulting from intercrosses between individuals within the population. Studies of experimental populations derived from hybrids have also provided information about the effect of natural selection on the mean fitness of populations, as measured by seed yield. Changes in seed yield of several representative populations are given in Fig. 17. I n the F z to Fa generations yields were conspicuously inferior t o those of standard locally adapted varieties. This inferiority is not unexpected because many of the genotypes included as parents were unadapted types. I n the Fs to F16 generations there was rapid improvement in seed yield until the level of performance of each population approached or even exceeded that of the standard commercial variety with which it was compared. Jain and Allard (1960) studied the number of seeds produced per individual in various generations. There was a high proportion of individuals of very low fecundity in early generations. The proportion of these inferior genotypes declined sharply in the generations when seed yield was improving rapidly and there was a corresponding rapid increase in the proportion of individuals of mediocre fecundity, I n late generations there was some increase in the proportion of individuals which produced large numbers of seeds

110

R. W. ALLARD, S. K . J A I N , AND P. L. WORKMAN 120

I

I10 -

-.--.

7-. I

,I

?'

100

I

I

L

I

501

' F4

I

F,

I

6,

I

1

56

40

I

64

I

58

Generation

FIG.17. Changes in seed yield in four representative populations synthesized from hybrids between homozygous varieties. The yield of each population is given as a percentage of the yield of a standard commercial variety. After Allard and Hansche (1965).

and interestingly, a few individuals with very high seed-producing capacity appeared. These results indicate that the mean fitness of population derived recently from crosses between a set of arbitrary parents is low owing to the production, by segregation and recombination, of a swarm of unbalanced genotypes. The initial stage of the evolution of such populations is characterized by elimination of these inferior genotypes, leading to rapid improvement in mean population fitness. Continued evolution of the system appears to involve the slow sorting out and incorporation into the populational system of the occasional superior genotype produced by continued segregation and recombination.

C . RESPONSES TO ABTIFICIAL SELECTION The question variability that grated systems. tion for various

which must now be considered is whether the genetic occurs in inbreeding populations is organized into inteA number of experiments involving directional selecsingle characters help to answer this question.

111

GENETICS OF INBREEDING POPULATIONS

I n one such experiment selection for intense green seed coat color has been practiced in an experimental population of lima beans. In each of 15 successive generations the planting seed was chosen from among the seeds which fell in the most green 10% of the harvest (Allard and Sanchez, 1965). There was steady response to selection but the increase in greenness of seed coat color was accompanied by a series of correlated responses in other characters, such as maturity date and seed size. Most important there was severe deterioration in seed yield and general vigor of the population. Upon relaxation of selection, seed coat color reverted within two or three generations to neatthat of the unselected population and there was an accompanying improvement in general thriftiness and productivity. Parallel results have been obtained in several other experimental populations of lima beans (Allard, 1965a) and in populations of barley (Suneson, 1965) which were selected for large or for small seed size. A selection experiment with the Yellow Double Dwarf 38 variety of grain sorghum (Sorghum vulgare) is interesting because the base population was a highly purified seed lot obtained from the California Crop Improvement Association. Six mass selection populations were established in which upward or downward selection was practiced for seed size, height, or flowering date (Sanchez et al., 1963). I n each generation 500 individuals were measured for these characters and the planting seed for the next generation was taken from the 50 most extreme individuals. As shown in Table 16 all three characters showed TABLE 16 Effect of Six Successive Generations of Mass Selection on Three Measurement Characters in a Highly Purified Seed Lot of the Yellow Double Dwarf 38 Variety of Grain Sorghum*

Seed size (gr-/lW kernels) Generation

B&W

Flowering date (days after seeding)

Height (inches)

Small

Large

Short

Tall

Early

Late

2.46 2.35 2.34 2.25 2.00 1.96 1.83

2.46 2.53 2.82 3.07 3.06 3.32 3.38

53.3 49.7 50.7

53.3 55.0 54.0

44.3 48.7 47.3

63.7 66.7 72.3

71.7 71.7 71.0 71.0 70.7 69.0 68.7

71.7 72.3 71.3 72.0 75.3 75.0

1 2 3 4 5 6

* Benjasil (1968).

-

-

-

TABLE 17 Mean Seed Yield of Selected Populations of Grain Sorghum Expressed as a Percentage of Mean of the Urnelected Base Population* Character under dection ~~

Tdl

Short I

Location

0

I I1

I11 IV V VL

100.0 96.5 79.3 68.9 63.9 51.0 65.0

100.0 100.0 98.8 99.4 105.4 106.1 88.4 85.8 71.7 81.1 62.2 71.2 70.5 75.6

100.0 100.0 100.0 99.0 95.3 108.4 103.5 100.6 97.9 Q7.9 105.7 94.7 99.1 98.4 96.2 102.4 90.4 101.6 115.4 90.6 95.4

11

100.0 100.0 97.5 96.5 83.8 97.6

_

87.0 82.9 78.2

-

94.6 88.1 80.7

I11

I

100.0 92.2 91.9

100.0 89.9 92.8

-

88.2 97.9 93.7

rA

Anthesis date

Eeight

-

I1

Early 111

100.0 100.0 108.0 97.7 101.4 92.9

-

104.6 107.0 116.3 100.9 127.1 109.8

-

90.5 98.6 100.8

I

I1

100.0 100.0 97.3 90.1 86.4 93.8 87.3 101.3 81.7 104.8 93.5 99.3 99.8 104.1

w

Late

I11

I

I1

100.0 100.4 99.1 100.9 93.2 99.3 100.4

100.0 96.8 101.1 104.5 106.7 104.9

100.0 92.5 98.6

-

I11

100.0 100.0 96.8 108.0 95.4 97.5 102.4 106.2 93.3

-

-

i

4

3 R2

GENETICS OF INBREEDING POPULATIONS

113

steady response to selection in both plus and minus directions over six successive generations. This response was accompanied by correlated responses in characters other than those under selection. In the sixth generation the performance of each selected population was compared with that of the base population in a replicated yield trial. The results, which are shown in Table 17, show that the general effect of directional selection for single characters was to reduce performance, as measured by seed yield. In general, directional selection for any one character changes the mean of the character in the direction of the selection. At the same time, changes occur in various other characters that are seemingly unrelated to the character under selection, and the totality of the population phenotype is also altered, usually adversely; upon relaxation of selection the population reverts toward its unselected state. Such responses are not consistent with the idea that the population is a simple mixture of highly adapted genotypes. They indicate instead that every component of the system depends on every other component and hence that the population genotype is an integrated and cohesive system (Allard, 1966). VI. Effect of Altering the Mating System

A. ENFORCED SELF-FERTILIZATION Estimates of selective values given in Section 111 suggest that much of the variability observed in inbreeding populations may be associated with loci held heterozygous as a result of some net advantage of heterozygotes over homozygotes. If this hypothesis is correct, forced self-fertilization should lead to a reduction in reproductive capacity. However, it is virtually impossible t o design experiments which give meaningful measures of reproductive capacity under natural conditions and it has consequently been necessary to use characters such as plant height, leaf number, survival after emergence, and flowering time, measured under nursery conditions, as criteria of the fitness of inbred materials. I n an inbreeding experiment with wild oats, Imam and Allard (1965) bagged panicles from 60 randomly chosen plants. A t maturity seeds were harvested from these selfed panicles and also from comparable open-pollinated panicles on the same plants. The next season these seeds were used to establish paired plots in a replicated experiment conducted under nursery conditions. The data of Table 18 indicate that plants from bagged and open-pollinated panicles were equal

114

R. W. ALLARD, S. K . JAIN, AND P . L. WORKMAN

in height but that plants from bagged panicles were significantly later in flowering, tillered less profusely, and were less likely to survive from seedling stage to maturity. Wild oats outcross at a rate of approximately 5%. Consequently 95% of the plants in plots from openpollinated panicles are expected to be selfs, that is, only 5% fewer than in plots from bagged panicles. The 5% of outcrossed plants in plots from open-pollinated panicles must have been substantially more vigorous than the selfed plants since plots containing these few outcrosses were significantly different from plots containing only selfs. There was, however, some indication that the kernels produced under TABLE 18 Effect of a Single Generation of Enforced Self-Fertilization OD Four QuantitativeCharacters in Wild Oats* Character

Selfed Open-pollinated

Height (cm)

Tiller number

Percentage survival after emergence

100.3 101.2

40.6t 45.8

90. lt 98.1

Heading time (day8 after April 1)

17.lt 13.3

* From Imam and Allard (1965). t Significantly different (P < 0.05) from progenies derived from open-pollinated seed. bags were smaller than kernels produced on unbagged panicles on the same plant and i t is possible that the effects seen in Table 18 trace to adverse effects of bagging, rather than to inbreeding. Hence this experiment did not fully resolve the issue of inbreeding depression in wild oats. I n an inbreeding experiment with grain sorghums, Sanchez et al. (1963) bagged 100 random plants in a foundation seed lot of the variety Yellow Double Dwarf 38. For the next 5 generations each family was perpetuated from a single selfed plant chosen a t random. These families gradually diverged from each other in height, flowering time, seed size, and various other characters but there was no observable deterioration in general vitality or robustness. I n the Sa generation the seed yields of the 100 families and the base population were determined in a replicated field experiment. The majority of the inbred families were lower in productivity than the base population but differences were generally small and nonsignificant. Thus increasing the level of inbreeding does not appear t o have very drastic effects on general vigor in this already heavily inbred population of sorghums.

GENETICS OF INBREEDING POPULATIONS

115

B. INCREASED OUTBREEDING BY INTRODUCTION OF MALESTERILITY Jain and Suneson (1964, 1966) have made use of a recessive malesterility allele (ms) to study the effect of increasing the level of outbreeding on variability and fitness in barley. Their experimental material was Composite Cross XIV, which was developed by intercrossing eight barley varieties of nearly common origin by means of the malesterility gene. Two levels of outcrossing were obtained by manipulation of the male sterility gene. One level was obtained by propagating the population in mass for 21 successive generations, ignoring the male-sterility gene. I n this population the proportion of male-sterile plants decreased from 26% in F2 to 5% in Flo, 2% in F14, and to 1% in F22.I n a second series the population was propagated exclusively from seeds produced on malesterile plants. After 7 and 15 generations of enforced outcrossing samples were taken from the population and propagated thereafter by the natural mating system of self-fertilization, The generations studied in detail in the selfing series were the F,, FI5, and FZ1and in the outcrossing series the 07F4 (outcross 7, F4), O7Fl2,and O15F12 generations were studied in detail. Progeny tests established that the proportion of heterozygotes a t various marker loci was higher in the 07F4,07F12, and OlaF12 generations than in the selfing series. However, in both series i t was necessary to invoke heterozygote advantage to explain the observed level of heterozygosity a t marker loci. Variability in quantitative characters was determined from measurements made on the progeny of 64 randomly chosen individuals from each of the six generations indicated above. These progenies were grown in a replicated experiment in which the eight original parents of the population were also induced. The Fl, F15, F21, 0,F4, O15F4,and O.rF12 families grown along with parents were scored for plant height (in centimeters) heading date (in days after March 31), and seed size on a single plant basis. Yields were taken on a plot (row) basis and seed number per family was determined by the yield to seed size ratio. The range in height and seed size among the parents was rather small but they differed from one another rather substantially in heading date, seed number per plot, and yield per plot (Table 19). Table 19 also gives generation means, the range in family means, and estimates of betweenand within-family variances (sa2, sW2) for various generations of the selfing and outcrossing series. General features that should be noted are (1) Large amounts of variability are present in all generations and in general the range of types in the population exceeds the range exhibited by the parents.

TAl3LE 19 Estimates of Mean, Between-, and Within-Family Variances in Composite Cross XIV Populations* Character Plant height (cm)

18.4 9.86 12.7-27.3 9.18

18.7 6.50 12.9-24.4 10.11

19.4 5.87 12.4-23.6 8.48

19.8 15.95 14.9-2.82 7.81

F

33.51

35.93 16.32 24.4-46.3

32.32 9.98 28.2-37.0

P

21.0-46.4

33.56 18.15 24.2-44.5

222.1 3528.72 127-380

240.9 3354.72 105-411

204.9 2566.53 97-339

195.8 2769.78 125313

102.14 45.08 88.4-118.8 64.47

102.4 77.23 82.4-122.4 101.13

19.1 9.21 15.4-27.5 9.04

19.4 10.80 11.8-29.4 6.99

18.2 5.72 13.2-24.9 7.03

33.16 18.50 19.1-40.6

33.76 13.48 32.0-41.6

35.13 10.71 24.2-42.7

228.0 4567.78 84418

264.7 3274.67 95-389

3306.37 159-454

Mean 83

Mean Sb'

Range Mean Sb'

Range Seed no. per family

99.3 14.62 92.7-104.5 34.72

100.5 56.38 83.3-116.4 73.86

Range sw=

Yield/plot (grams)

105.8 71.28 88.8-125.3 101.02

98.7 96.94 75.8-138.8 89.19

s WZ

(decigrams)

100.7 74.68 77.6-118.6 126.95

Mean Range

Seed size

Parents

F4

Sb'

Heading date (days after March 31)

OBii

Statistic

Mean 8'b

Range

*After Jain and Suneson (1966).

7088 3838306

-

Fis

7539 4062509

-

Fzi

290.9

8329 3694965

-

O7F4

27.40

6729 3456909

-

olsF4

7282 3379905

-

5635

2864554

-

6035 2374271

-

4

2

Z

3

GENETICS OF INBREEDING POPULATIONS

117

(2) The within-family variance was large in all generations, including the FS1, which suggests that many loci affecting quantitative characters remain heterozygous, even under intense inbreeding. (3) The generation means are in the intermediate range and they remain virtually unchanged from early to late generations. This is expected because the parents were largely adapted types with heading dates appropriate to the local environment. (4) Between-family variance decreased in later generations (except for height in the outcrossing series), which suggests that optimizing selection occurred in the population, that is, that genotypes producing extreme phenotypes are at a disadvantage. These results are parallel to and corroborate those discussed in Section IV.

The main point of this experiment, however, is the comparison between the selfing and outcrossing series. Variability, as estimated by the between- and within-family components, tends to be a little larger in the outcrossing series as would be expected from greater heterozygosis and higher recombination potential than in the selfing series. measured by average number Yet, the mean population fitness of seeds produced per family and the yielding ability of total population were higher in the selfing than in the higher outbreeding series. The rates of change, A V , computed from the yield trial results over the period 1954-1965, are compared in Table 20. It can be seen that the selfing series of two composite crosses, CC V, and CC XIV, and the outcrossing series of CC XIV improved a t nearly the same rate. Thus, it is apparent that the low levels of outcrossing (1-2%) that occur normally in these barley populations are integrated into a system which released sufficient variability so that increasing the recombination potential by altering the mating system did not increase the rate of change in fitness.

(w)

VII. Population Structure under Extreme Inbreeding: The Festuca microstachys Complex

The Festuca microstachys complex provides particularly favorable material for the study of population structure under extreme inbreeding because the cleistogamous habit of these slender annual grasses virtually ensures self-fertiliaation (Kannenberg and Allard, 1966). The stamens and stigma are tightly enclosed by the lemma and palea and pollination occurs before the panicle is exserted from the “boot.” Moreover, it is usual for only one of the three anthers in a flower to develop to anthesis and this anther, which is tightly

TABLE 20 Average Yields (lb/Acre) and Yearly Rate of Change in Composite Cross V and XIV Populations*

(AF)

CC V, Selfing series Period 1954-1957 1958-1961 1962-1965

Generation Fu-Fi, FlrFzo Fu-Fu

Average yield

A w = 31.4

* Afhr Jein and Suneson (1966).

2415 3474 3740

CC XIV, Selfing series Generation Fii-Fu F16-Fli FirFa

Average yield

AF = 31.6

2572 3860 3972

CC XIV, Outcrossing seriea Generation

Average yield

O?Fz*rFa

OEsOBe

07Fl&7Fla-

AW = 32.2

2390 3675 3808

4

2

z

GENETICS OF INBREEDING POPULATIONS

119

enclosed in the stigmatic hairs, is usually very small and contains only a half dozen or so pollen grains, Under favorable environmental conditions an occasional floret is chasmogamous, thus providing opportunity for outcrossing. However, when Kannenberg and Allard examined more than 20,000 progeny of plants taken from nature they found no recognizable hybrids. They concluded that the rate of outcrossing is not likely to be as high as one per 1000 fertilizations and it is probably lower than one per 10,000 fertilizations. An extensive quantitative study of genetic variability between and within populations revealed that these grasses exhibit an intricate and complex pattern of variation in the hundreds of habitats they occupy. Distinct clinal trends were observed but differentiations between local populations were even more striking. Measurements made on the progeny of single plants revealed that any single microniche contains individuals which are genotypically very different from one another with respect to height, maturity date, and other continuously varying characters. For any single measurement character the genotypes within a local population represent a graded series such that there is a normal distribution between wide extremes. Consideration of several measurement characters simultaneously showed that each natural population consists of a very large number of genotypes, no one of which makes up more than a small fraction of the total. However, each population has a characteristic pattern of variability for measurement characters which distinguishes it from other local populations. The population phenotype appears to result from interactions among many different genotypes, giving the population a coordinated and cohesive structure. Because of the low level of intrapopulational gene flow, these interactions are presumably between individual plants and result from factors such as frequency and density dependence. The following inferences were made concerning breeding behavior. The progeny of seeds collected from single plants are quite uniform, giving the impression that the great majority of individuals in the population are rather highly homozygous. Although it could not be demonstrated it was presumed that outcrossing occasionally occurs between different families and that numerous novel genotypes segregate from such hybrids. If heterozygotes are at an advantage, or advantage attends rarity in the population, as observed in other inbreeding species, a burst of segregation initiated by even a single outcross could have considerable persistence and result in the release of large amounts of genetic variability. The population into which these new genotypes are infused is the product of many generations of natural selection. It is prosperous in

120

R. W. ALLARD, S. K. J A I N , AND P. L. WORKMAN

the habitat it occupies owing to harmonious balances among the many genotypes of which it is composed and it has cohesive properties due to interactions and interdependencies among the constituent genotypes. When new genotypes are introduced into the population as a result of hybridization or migration, the ones that interact disharmoniously with the other genotypes in the population are eliminated and the ones which contribute to a better adapted population genotype are incorporated into the system. Although most individuals in the population are presumably rather highly homozygous, and hence capable of producing progeny only like themselves, each such potentially uniform family appears to be represented in the population by only a single individual, or a t most a few individuals. The population owes its variability to the genetic uniqueness of each of many different relatively homozygous individuals of which it is composed. Kannenberg and Allard compared measures of within-population variability of the fescues with measures on other annual grasses obtained in similar experiments. The results indicated that the fescues were no less variable respecting comparable quantitative characters than either wild oats (see Section I V ) , a species in which the extent of outcrossing is many times higher, or Lolium nvultiflorum (Schulke, 1963), an outcrossing species. Measurement data therefore do not support the idea that there is any general or consistent relationship between mating system and total populational variability.

VIII. Concluding Remarks

If recent experiments conducted to quantify the extent of genetic variability in inbreeding species point to any single conclusion, it is that there is remarkable genetic diversity within natural and domestic populations of such species. The demonstration that inbreeding species are highly variable genetically calls for explanations of the persistence of genetic variability under inbreeding and it also points t o the desirability of a reassessment of deductions concerning the evolutionary role of inbreeding which have been made on the assumption that inbreeding popuIations are uniform genetically. Quantitative studies of inbreeding species reveal that any given population contains individuals of many different genotypes. Individuals within populations often differ from one another with respect to single- and multiple-unit polymorphisms and also for continuously varying characters. The extent of variation for continuously varying characters is striking. For example, the range in height from the

GENETICS OF INBREEDING POPULATIONS

121

genetically shortest to the genetically tallest individual within a population is often twofold or greater and between-family variation for other measurement characters is usually equally large. Another feature of variability in inbreeding populations is that the individuals within a population are frequently heteroeygous a t many loci, that is, there is a substantial component of within-family variability. The extent of the response to selection within families is often large, which suggests that segregational variability is an important feature of the variability system under inbreeding. Still another feature of variability in inbreeding species is genetic differentiation between different populations. Clinal variation is frequently observed in association with progressive changes in rainfall, temperature, and other factors of the physical environment. Superimposed on such broad geographical variations is a patchwork or mosaic pattern of variation which reflects adaptations to the local environment. The pattern is often a very fine one and since the numbers of niches included in areas as small as a few square meters may be considerable, such local differentiation appears to provide for massive storage of genetic variability. It is apparent that population structure in inbreeding species is much more complicated than has been commonly supposed and that it probably does not take the same form in all inbreeding species or even in different populations of the same species. Population structure in inbreeding species cannot be explained by focusing attention on any single factor among the complex of interacting genetic and ecological factors that are involved. It is clearly inadequate, for example, to make predictions concerning population structure or evolutionary potential on the basis of simple genetic models which take only within-family variability into account. Such models predict that heavy inbreeding leads to homozygosity, a prediction which is a t variance with the observed facts. The incorporation of selection (heterozygote advantage) into simple single-locus models helps to explain the existence of stable polymorphisms under heavy inbreeding. There is, however, experimental evideiice to indicate that different polymorphisms interact with one another and theory shows that the effects of such epistatic interactions on population structure vary with the type of epistasis and the intensity of inbreeding. One of the discoveries from theoretical investigations of multilocus models is that inbreeding can have the same effect as linkage and that with some types of epistasis inbreeding can reinforce or substitute for linkage in the maintenance of balanced gene complexes. It seems clear that simple genetic models are inadequate and that analyses of complex multilocus models are

122

R. W. ALLARD, S. K. JAIN, AND P. L. WORKMAN

prerequisite to an understanding of the genetic aspects of the structure of inbreeding populations. The situation becomes even more complex when various factors which are more ecological than genetic are taken into account. One such factor is frequency-dependent selection in which the pattern of interactions between individuals is a function of the frequency of various genotypes in the population. In one case which has been analyzed in detail, the S/s locus in the lima bean, heterozygotes and homozygotes are equal in fitness when all three genotypes are frequent in the population. However, when heterozygotes are rare in the population their fitness increases relative to the homozygotes. In this case it is clear that maintenance of a stable nontrivial polymorphism depends on a complex set of interactions between genetic factors, mating system, and ecological factors. The maintenance of numerous relatively homozygous genotypes in populations of the heavily inbreeding grasses of the Festuca microslachys complex may depend in part on a similar complex pattern of interactions between individuals in the population. Other items of a similar nature which are believed to affect the extent of genetic variability include niche arrangement, local subdivision, mobility in animals, pollen and seed dispersal mechanisms in plants, and interactions of these factors with each other and with the genetic determinants of population structure. Many aspects of genetic systems and of ecology have been studied individually in different species but rarely if ever have several factors been studied simultaneously in a species as components of a single integrated system for the regulation of variability. The discovery that remarkable genetic variability exists within populations of inbreeding species suggests that high variability is essential to the survival of populations. It also suggests that change of any component of the variability system which leads t o undue restriction of variability must be mediated by compensatory changes in other components if the population is to survive. For example, should adoption of self-fertilization as a device to ensure fertilization in unfavorable weather have the correlated effect of reducing genetic variability in the population to critical levels, survival of the population would presumably depend on compensating adjustments in other components of population structure which affect the variability. Viewed in this way the mating system is seen t o be only one component of the variability system and what is needed are comprehensive studies to determine how it is integrated into the totality of factors which determine population structure and net evolutionary potential. Broad generalizations about the evolution-

GENETICS OF INBREEDING POPULATIONS

123

ary implications of mating systems do not appear t o be warranted until this type of information becomes available. IX. Summary

Inbreeding can arise in populations either as a result of various mechanisms which affect the mating system, or from restrictions in actual or effective population size. In this review the effects of inbreeding have been considered, in a populational context, with respect to theory and also in terms of studies conducted with natural and experimental populations. The theoretical effects of inbreeding were introduced in terms of single-locus population models in which population size was assumed sufficiently large to avoid sampling effects and in which i t was assumed that selective values, mating system parameters, and other population parameters were constant in all environments. Examination of equilibrium values and rate of approach to equilibrium in terms of simplified population models show that net heterozygote advantage is a necessary condition for stable nontrivial equilibrium when the mating system imposes some degree of inbreeding. The extent of heterozygote advantage required to avoid fixation is least when homozygotes have equal selective values and there is little inbreeding; i t increases with increasing asymmetry of selection and increasing levels of inbreeding. Under the most intense inbreeding possible with diploid organisms (complete assortative mating) the selective value of the heterozygote must be a t least double the selective values of the homozygotes to maintain stable nontrivial equilibrium. The effects on population structure of linkage and epistatic interactions between different polymorphisms were then considered in terms of multilocus genetic models involving deviations from a fixed optimum, heterotic models, and mixed optimum-heterotic models. The basis for this choice is the general experience that the types of selection implied by these models are widespread in actual populations. The results were expressed in terms of the extent of linkage disequilibrium (gametic phase unbalance), mean population fitness, and the number and nature of gene frequency equilibria. Complex interactions occur among linkage, epistasis, and inbreeding, and different combinations of these factors can lead to retention of variability in populations. Finally, the models were extended to include stochastic variation, which appears to have the effect of producing quasi-stable equilibria a t critical values of the relevant parameters.

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Estimates of selective values for the simple polymorphic characters commonly found in natural and experimental populations of predominantly self-pollinated plants show that linkage blocks marked b y major genes are often subject to very strong selection. I n a high proportion of cases the estimated selective values show that such linkage block heterozygotes have striking net selective advantage over homozygotes. The selective values of heterozygotes often fall within ranges which permit stable equilibrium a t intermediate gene frequencies. Inbreeding populations also frequently carry several t o many polymorphisms whose distributions in the population are interdependent. Such, interactions extended over the totality of the gene pool may be the basis of the cohesive properties and “coadapted” nature of the population genotype. There is a substantial body of measurement data which shows that natural populations of inbreeding species contain great stores of genetic variability respecting continuously varying characters. Part of this variability takes the form of geographical differentiations, including a mosaic pattern of differentiations associated with adaptation to microniches. There is also extensive intrapopulational variability both between and within families. Between-family variability is shown by significant differences among the means of the progenies of individuals taken from nature, Within-family variability is demonstrated by responses to selection within single kinships, indicating that the individuals within natural populations are heterozygous for numerous genes affecting quantitative characters. Theory and experimental studies lead to the following conception of population structure under inbreeding. The natural population contains a very large number of different genotypes none of which makes up more than s small proportion of the total. For any single measurement character the genotypes in a population represent a graded series such that there is a more or less normal distribution from one extreme to the other with all degrees of intermediacy represented. When many characters are considered simultaneously, each local population is found t o have a characteristic pattern of variability which distinguishes it from other local populations. The mating system is only one of many factors which affect the amount and kinds of variability in populations. The observed structure of inbreeding populations is seen to result from an integration of inbreeding into the totality of genetic and ecological factors affecting population structure. The structure of inbreeding species provides both for storage of large amounts of genetic variability and for retention of sufficient flexibility t o meet the requirements imposed by heterogeneity of the

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local environment, including season-to-season or year-to-year fluctuations. Species which meet these requirements appear t o possess ample stores of variability to satisfy the challenges imposed by long-term ‘changes in environment. ACKNOWLEDGMENTS This study was supported in part by grants from the National Science Foundation (GB-3246) and the National Institutes of Health (GM 10476).

REFERENCES Adair, C. R., and Jones, J. W. 1946. Effect of environment on the characteristics of plants surviving in a bulk hybrid population of rice. J . Am. Soe. Agron. 38, 708-716. Akemine, H., and Kikuchi, F. 1958. Genetic variability among hybrid populations of rice plants grown under various environments. (In Japanese.) In “Studies on the Bulk Method of Plant Breeding” (K. Sakai, R. Takahashi and H. Akemine, eds.), pp. 89-105. Allard, R. W. 1956. Unpublished results. Allard, R. W. 1963. Evidence for genetic restriction of recombination in the lima bean. Genetics 48,1389-1395. Allard, R. W. 1965. Genetic systems associated with colonizing ability in predominantly self-pollinated species. In “The Genetics of Colonizing Species” (H. G. Baker and G. L. Stebbins, eds.), pp. 50-76. Academic Press, New York. Allard, R. W. 1965. Unpublished results. Allard, R. W. 1966. Population structure and performance in crop plants. Ciencia Cult. Sao Paulo 19,145-150. Allard, R. W., and Golden, W. G. 1954. Unpublished results. Allard, R. W., and Hrtnsche, P. E. 1964. Some parameters of population variability and their implications in plant breeding. Advan. Agron. 16, 281-325. Allard, R. W., and Hansche, P. E. 1965. Population and biometrical genetics in plant breeding. Proc. 11th Intern. Congr. Genet., The Hague, 1963 Vol. 3, pp. 665-679. Pergamon Press,Oxford. Allard, R. W., and Jain, S. K . 1962. Population studies in predominantly selfpollinated species. 11. Analysis of quantitative changes in a bulk hybrid population of barley. Evolution 16, 90-101. .Allard, R. W., and Sanchez, R. L. 1965. Unpublished results. Allard, R. W., and Wehrhahn, C. 1964. A theory which predicts stable equilibrium for inversion polymorphisms in the grasshopper, Moraba scurra. Evolution 18, 129-130. Allard, R. W., and Workman, P. L. 1963. Population studies in predominantly self-pollinated species. IV. Seasonal fluctuations in estimated values of genetic parameters in lima bean populations. Evolution 18, 470-480. Allard, R. W., Harding, J., and Wehrhahn, C. 1966. The estimation of selective values and their use in predicting population change. Heredity 21, 547-564. Barnes, D. I<., m d Cleveland, R. W. 1963. Pollen tube growth o f diploid alfalfa in vitro. Crop Sci. 3, 291-297.

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R. W. ALLARD, S. K. JAIN, AND P. L. WORKMAN

Bartlett, M. S., and Haldane, J. B. S. 1936. The theory of inbreeding with forced heterozygosis. J. Genet. 31, 327-340. Bateman, A. J. 1962. Variation within French bean varieties. Ann. Appl. Biol. 39,

129-138.

Benjasil, V . 1968. IMecta of ma= selection for quantitative traits in sorghum. Ph.D. Dissertation. Univ. California, Davis, California. Bennett, J. H. 1964. The distribution of heterogeneity upon inbreeding. J . Roy. Statist. SOC. B16, 88-99. Bennett, J. H.,and Binet, F. E. 1956. Association between Mendelian factors with mixed selfing and random mating. Heredity 10, 51-55. Binet, F. E.,Clark, A. M., and Clifford, H. T. 1969. Correlation due t o linkage in certain wild plants. Genetics 44,613. Bodmer, W.F.,and Parsons, P. A. 1962. Linkage and recombination in evolution. Advan. Genet. 11,147. Cain, A. S.,and Sheppard, P. M. 1952. The effects of natural selection on body color in the land snail Cepaea nemoralis. Heredity 6,217-231. Cannon, G. B. 1963.The effects of natural selection on linkage disequilibrium and relative fitness in experimental populations of Drosophila melanogaster. Genetics 48,1201-1216. Claassen, C. E. 1950. Natural and controlled crossing in safflower, Carthamus tknctorius L. Agron. J . 42,381484. Clarke, A. E. 1935. Inheritance of annual habit and mode of pollination in an annual White Sweet Clover. Agron. J. 27, 492-496. Clarke, B., and O’DonaId, P. 1964. Frequency-dependent selection. Heredity 19,

201-206.

Cooch, F. G.,and Beardmore, J. A. 1959. Assortative mating and reciprocal difference in the Blue-Snow goose complex. Nature 183, 1833-1834. Darlington, C. D., and Mather, I(. 1949. “Elements of Genetics.” Allen & Unwin, London. Dempster, E. R. 1956. Maintenance of genetic heterogeneity. Cold Spring Harbor Symp. Quant. Biol. 20, 25-32. Dillman, A. C. 1938.Natural crossing in flax. Agron. J. 30, 279-286. Dreyfus, A., and Breuer, M. E. 1944. Chromosome and sex determination in the parasitic hymenopteron Telenomus furiai (Lima). Genetics 29, 75-82. East, E. M. 1919. Studies on self-sterility. 111. The relation between self-fertile and self-sterile plants. Genetics 4, 341-346. Fisher, R. A. 1939. Selective forces in wild populations of Paratettix tezunus. Ann Eugen. (London) 9,109-122. Fisher, R . A. 1948. A quantitative theory of genetic recombination and chiasma formation. Biometldcs 4,l-13. Fisher, R. A. 1949. “The Theory of Inbreeding.” Oliver & Boyd, Edinburgh and London. Fyfe, J. L., and Bailey, N. T. J. 1961. Plant breeding studies in leguminous forage crops. I. Natural crossing in winter beans. J . Agr. Sci. 41, 371-378. Geiringer, H.1944. On the probability theory of linkage in Mendelian heredity. Ann. Math. Statist. 15, 26-57. Geiringer, H.1945.Further remarks on linkage theory in Mendelian heredity. Ann. Math. Statist. 15,26-67. Gibson, J. B., and Thoday, J. M. 1962. Effects of disruptive selection. VI. A second chromosome polymorphism. Heredity 17,l-26.

GENETICS OF INBREEDING POPULATIONS

127

Grant, V. 1956. The influence of breeding habit on the outcome of natural hybridization in plants. Am. Naturalist 90, 319-322. Grant, V. 1958. The regulation of recombination in plants. Cold Spring Harbor Symp. Quant. Biol.23, 337-363. Griffing, B. 1960. Accommodation of linkage in mass selection theory. Australian J. Biol.Sci. 13, 501-526. Guitierrez, M. G., and Sprague, G. F. 1959. Randomness of mating in isolated polycross phntings of maize. Genetics 44, 1075-1082. Haldane, J. B. S. 1924. A mathematical theory of natural and artificial selection. Part 11. Proc. Cambridge Phil. SOC.1, 168-163. Haldane, J. B. S. 1949. The association of characters as a result of inbreeding and linkage. Ann. Eugen. (London) 15,16-23. Haldane, J. B. S., and Waddington, C. H. 1931. Inbreeding and linkage. Genetics 16,357-374. Hanson, W. D. 1959. The breakup of initial linkage blocks under selected mating systems. Genetics 44, 857-868. Harding, J., and Allard, R. W. Population studies in predominantly self-pollinated species. XI1 Zygotic ‘and gametic interactions in a population of Phaseolus Zunatus (in preparation). Harding, J., and Tucker, C. L. 1964. Quantitative studies on mating systems. I. Evidence for the non-randomness of outcrossing in Phaseolus lunatus. Heredity 19,369-381. Harding, J., Allard, R. W., and Smeltzer, D. G. 1966. Population studies in predominantly self-pollinated species. IX. Frequency dependent selection in Phaseolus lunatus. Proc. Natl. Acad. Sci. U.S. 56, 99-104. Harlan, H. V. 1957. “One Man’s Life with Barley.” Exposition Press, New York. Harlan, H. V., and Martini, M. L. 1938. The effect of natural selection in a mixture of barley varieties. J. Agr. Res. 67, 189-199. Hayman, B. I. 1953. Mixed selfing and random mating when homozygotes are a t a disadvantage. Heredity 7, 185-192. Hayman, B. I., and Mather, K. 1953.The progress of inbreeding when homozygotes are at a disadvantage. Heredity 7, 166-183. Hayman, B. I., and Mather, K. 1956. Inbreeding when homozygotes are a t a disadvantage : A reply. Heredity 10, 271-274. Hogben, L. 1946. “An Introduction to Mathematical Genetics.” Horton, New York. Holden, J. H. W., and Bond, D. A. 1960. Studies on the breeding system of the field bean, Vicia labia (L.). Heredity 15, 175-192. Huxley, J. S. 1965. Morphism in birds. Acta 11th Congr. Intern. Omithol. Basel, 1954 pp, 309-328. Imam, A. G., and Allard, R. W. 1965. Population studies in predominantly selfpollinated species. VI. Genetic variability between and within natural populations of wild oats from differing habitats in California. Genetics 61, 49-62. Jain, S.K.1968.Unpublished results. J a b , S. K. 1968. Simulation of models involving mixed selfing and linkage in finite populations. Theoret. Appl. Genet. 38 (in press). Jain, S. K., and Allard, R. W. 1960. Population studies in predominantly selfpollinated species. I. Evidence for heterozygote advantage in a closed population of barley. Proc. Natl. Acad. Sci. U B . 46, 1371-1377. Jain, S. K.,and Allard, R. W. 1966. The nature and stability of equilibria under optimizing selection. Proc. Natl. Acad. Sci. U.S. 54, 1436-1443.

128

R. W. ALLARD, S. K. JAIN, AND P. L. WORKMAN

Jain, S. K., and Allard, R. W. 1966. The effects of linkage, epistasis, and inbreeding on population changes under selection. Genetics 53, 633-659. Jain, S. K.,and Marshall, D. R. 1967. Population studies in predominantly selfpollinated species. X. Variation in natural populations of Awena f a t m and A . barbata. Am. Naturalist 101,19-33. Jain, 8. K.,and Marshall, D. G.1968.Simulation of models involving mixed selfing and random mating. I. Stochastic variation in outcrossing and selection parameters. Heredity (in press). Jain, S. K., and Suneson, C. A. 1964. Population studies in predominantly selfpollinated species. VII. Survival of a male-sterility gene in relation to heterozygosis in barley populations. Genetics 50, 905-913. Jain, S. K., and Suneson, C. A. 1966. Increased recombination and selection in barley populations carrying a male sterility factor. I. Quantitative variability. Genetics 54,1215-1224. Jain, S . K.,and Workman, P. L. 1966. The use of generalized F-statistic in t h e theory of inbreeding and selection, Nature 214, 674-678. Jana, S., and Allard, R. W. Population studies in predominantly self-pollinating species. XIII. Genetic variability in populations of lirna beans composed originally of a mixture of two pure lines (in preparation). Jennings, H. S. 1917. The numerical results of diverse systems of breeding, with respeot to two pairs of characters, linked or independent, with special relation to the effects of linkage. Genetics 2, 97-154. Johannsen, W. 1926. “Elemmte der exakten Erblichkeitslehre,” 3rd ed. Fischer, Jena. Kannenberg, L. W., and Allard, R. W. 1967. Population studies in predominantly self-pollinated species. VIII. Genetic variability in the Festuca microstachys complex. Ewolution 21, 227-240. Kempthorne, 0. 1957. “An Introduction to Genetic Statistics,” Wiley, New York. Kikuchi, F., Allard, R. W., and Chapman, S. R. The effect of natural selection on phenotypic plasticity and genetic polymorphism in a barley population. In “Food for the Man of Tomorrow” Symp. Am. Univ. Beirut, 1967 (in press). Kimura, M. 1954. Process leading to quasi-fixation of genes in natural populations due to random fluctuation of selection intensities. Genetics 39, 280-295. Kimura, M. 1956. A model of a genetic system which leads to closer linkage b y natural selection. Evolution 10, 278-287. Kimura, M. 1963. A probability method for treating inbreeding systems, especially with linked genes. Biometries 19, 1-17. Kimura, M. 1964. “Diffusion Models in Population Genetics,]’ p. 57. Methuen, London. Kimura, M. 1966. Attainment of quasi-linkage equilibrium when gene frequencies are changing by natural selection. Genetics 52, 876-890. Kimura, M., and Crow, J. F. 1963. The measurement of effective population number. Evolution 17, 279-288. Knowles, P.F. 1943. Improving an annual bromegrass, Bromus moUk L., for range purposes. J. Am. SOC.Agron. 35, 584-694. Kojima, K. 1959. Role of epistasis and overdominance in stability of equilibria with selection. Proc. Natl. Acad. Xci. U.S. 45, 984-989. Kojima, K. 1966. The evolutionary dynamics of two-gene systems. In “Computers in Biomedical Research” (R. W. Stacy and B. D. Waxman, eds.), Vol. 1, Chapter 9,pp. 197-220. Academic Press, New York.

GENETICS OF INBREEDING POPULATIONS

129

Lamotte, M. 1961. Recherches sur la structure genetique des populations naturelles de Cepaea nemoralis L. Bull. Biol. France Belg. Suppl. 35, 1-239. Levitan, M. 1965. Studies of linkage in populations. I. Association of second chromosome linkages in Drosophila robusta. Evolution 9, 2741. Levitan, M. 1958. Nonrandom association of inversions. Cold Spring Harbor Symp. Quant. Biol. 23,251-268. Lewontin, R. C. 1958. A general method for investigating the equilibrium of gene frequency in a population. Genetics 43,419434. Lewontin, R. C. 1964a. The interaction of selection and linkage. I. General considerations: Heterotic models. Genetics 49, 49-67. Lewontin, R. C. 1964b. The interaction of selection and linkage. 11. Optimum models. Genetics 50, 757-782. Lewontin, R. C., and Hojima, K. 1960. The evolutionary dynamics of complex polymorphisms. Evolution 14, 458-472. Lewontin, R. C., and White, M. J. D. 1960. Interaction between inversion polymorphisms of two chromosome pairs in the grasshopper Moraba scurra. Evolution 14, 116129. Magahaes, L. E.,Brito Da Cunha, A., De Toledo, J. S., Toledo, S. A., De Soura, H. L., Targa, H. J., Setzer, V., and Pavan, A. 1964. On lethals and their suppressors in experimental populations of Drosophila willistoni. Mutation Res. 5 45-54. Mainardi, D., Scudo, F. M., and Barbieri, D. 1965. Assortative mating based on early learning: population genetics. Ateneo Parmense 36, 583-606. MalBcot, G. 1948. “Les math6matiques de l’hCr6ditk.” Masson, Paris. Mather, K. 1943.Polygenic inheritance and natural selection. Biol. Rev. 18, 32-64. Mayr, E. 1963. “Animal Species and Evolution.” Harvard Univ. Press, Cambridge, Massachusetts. Murray, J. 1964.Multiple mating and effective population size in Cepaea nemoralis. Evolution 18, 283-291. Nei, M., and Syaktdo, K. 1958.The estimation of outcrossing in natural populations. Japan. J . Genet. 33,46-51. O’Donald, P.1959.Possibility of assortatire mating in the Arctic Skua. Nature 183, 1210-1211. O’Donald, P. 1960a. Inbreeding as a result of imprinting. Heredity 15, 79-85. O’Donald, P. 1960b. Assortative mating in a population in which two alleles are segregating. Heredity 15, 389-396. Owen, A. R. G.1953. A genetical system admitting of two distinct stable equilibria under natural selection. Heredity 7 , 97-102. Parsons, P. A. 1957. Selfing under conditions favoring heterozygosity. Heredity 11, 411-421. Pearson, K.,and Lee, A. 1903. On the laws of inheritance in man. I. Inheritanre of physical characters. Biometrilia 2, 357-462. Prout, T. 1965. The estimation of fitness from genotypic frequencies. Evolution 19, 546551. Reeve, E. C. R. 1956. Inbreeding with the homozygotes a t a disadvantage. Ann. Human Genet. 19, 332-346. Reeve, E. C . R., and Gower, J. C. 1957. Inbreeding with selection and linkage. 11. Sib mating. Ann. Human Genet. 23, 36-49. Robbins, R. B. 1918.Applications of mathematics to breeding problems. 11. Genetics 3, 73-92.

130

R. W. ALLARD, S. K. JAIN, AND P. L. WORKMAN

Roberts, H.F. 1929. “Plant Hybridization Before Mendel.” Princeton Univ. Press, Princeton, New Jersey. Saaty, T. L., and Bram, J. 1964. “Nonlinear Mathematics.” McGraw-Hill, New York. Sanchez, R. L. Smeltzer, D. G., and Allard, R. W. 1963. Unpublished data. Schnell, F. W. 1961. Some general formulations of linkage effects in inbreeding. Genetics 46, 947-967. Schulke, J. D. 1963. Genetic variability in natural populations of Lolium multifioruna. Ph.D. Dissertation, University of California, Davis, California. Sheppard, P. M.1959.The evolution of mimicry; a problem in ecology and genetics. Cold Spring Harbor Symp. Quant. Bwl. 24, 131-140. Shikata, M. 1963. Representation and calculation of selfed population by group ring. J. Theoret. Biol. 5, 142-160. Smeltzer, D. G. 1965.Unpublished data. Spassky, B., Dobzhansky, T., and Anderson, W. W. 1965. The genetica of natural populations. XXXVI. Epistatic interactions of genetic load in Drosophila pseudoobscura. Genetics 52, 653-664. Stebbins, G. L. 1950. “Variation and Evolution in Plants.” Columbia Univ. Press, New York. Stebbins, G. L. 1967. Self-fertilization and population variability in the higher plants. Am. Naturalist 41, 337-353. Suneson, C. A. 1949. Survival of four barley varieties in a mixture. Agron. J. 41,

459461.

Suneson, C.A. 1966.Personal communication. Tucker, C. L., and Harding, J. 1965. Quantitative studies on mating systems. 11. Estimation of fitness parameters in a population of PhaseoZus Zunatus. Heredity 20, 393-402. Wahlund, S. 1928. Zusammensetzung von Populationen und Korrelationserscheinungen vom Standpunkt der Vererbungslehre aus betrachtet. Hereditas 11, 65-106. Wahrman, J. 1965.Permnal communication. Weinberg, W. 1909. Ober Vererbungsgesetae beim Menschen. 2. Znduktive Abstammungs- Vererbungslehre 1, 277-330. White, M. J. D. 1957. Cytogenetics of the grasshopper, Moraba scurra. 11. Heterotic systems and their interactions. Australian J . 2001.5, 305-337. Workman, P. L. 1964. The maintenance of heterozygosity by partial negative assortative mating. Genetics 50, 1369-1382. Workman, P. L., and Allard, R. W. 1962. Population studies in predominantly self-pollinated species. 111. A matrix model for mixed selfing and random outcrossing. Proc. Natl. Acad. Sci. US. 48, 1318-1325. Workman, P. L., and Allard, R. W. 1964. Population studies in predominantly self-pollinated species. V. Analysis of differential and random viabilities in mixtures of competing pure lines. Heredity 19, 181-189. Workman, P. L., and Jain, S. K. 1966. Zygotic selection under mixed random mating and self-fertilization : Theory and problems of estimation. Genetics

54,159-171.

Wright, 8. 1921. Systems of mating, I-V. Genetics 6, 111-1’78. Wright, 6. 1933. Inbreeding and recombination. Proc. Natl. Acad. Sci. U S . 19,

4201433.

Wright, S. 1940. Breeding structure of populations in relation to speciation. Am. Naturalist 74, 232-248. Wright, S. 1942.Statistical genetics and evolution. Bull. Am. Math. SOC.48, 223-246.

GENETICS OF INBREEDING POPULATIONS

131

Wright, S. 1943. Isolation by distance. Genetics 28, 114-138. Wright, S. 1946. Isolation by distance under diverse systems of mating. Genetics 31, 39-59. Wright, S. 1948. On the roles of directed and random changes in gene frequency in the genetics of populations. Evolution 2, 279-294. Wright, S. 1951. The genetic structure of populations. Ann. Eugen. (London) 15, 323-354. Wright, S. 1952. Quantitative inheritance. In “The Genetics of Quantitative Variability” (E. C. R. Reeve and C. H. Waddington, eds.), pp. S-41. Agr. Res. Council, London. Wright, S. 1959. Physiological genetics, ecology of populations, and natural selection. Perspectives Biol. Med. 3, 107-161. Wright, S. 1964. Stochastic processes in evolution. In “Stochastic Models in Medicine and Biology” (J. Gurland, ed.), pp. 199-244. Univ. of Wisconsin Press, Madison, Wisconsin. Wright, S. 1965a. The interpretation of population structure by F-statistics with special regard to systems of mating. Evolution 19, 395-420. Wright, S. 1965b. Factor interaction and linkage in evolution. Proc. Roy. SOC. B162, 80-104.

POSITION-EFFECT VARIEGATION"

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William K Baker Department of Biology. University of Chicago. Chicago. Illinois

I. Introduction . . . I1. Cytogenetic Aspects

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. . . . . . . . . . . . . . . A . The cis-trans Relation . . . . . . . . . . . . . . B . Proofs of Position-Effect Nature . . . . . . . . . . . C. Variegation of Genes Normally in a Heterochromatic Region . . D . Y Chromosome Suppression and the It Exception . . . . . E. The Polarized Spreading Effect . . . . . . . . . . .

I11. Position-Effect Variegation in the Mouse

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A Cytogenetics . . . . . . . . . . . . . . . . B Relation between Normal X Suppression and Position-Effect Variegation . . . . . . . . . . . . . . . . IV. Parental Modification of Variegated Gene Expression . . . . . A Discovery of These Effects . . . . . . . . . . . . B . Parental Homozygosity for Rearrangement . . . . . . . C. Effect of Parental Y Chromosomes . . . . . . . . . . D Parental Source of Rearrangement . . . . . . . . . . E. Comments on Parental Effects . . . . . . . . . . . V . Variegation. a Model System for Simplified Studies in Developmental Biology . . . . . . . . . . . . . . . . . A . A Process of Regulation of Gene Activity . . . . . . . . B Clonal Nature . . . . . . . . . . . . . . . . C Implications for Development . . . . . . . . . . . VI . Thoughts, Perhaps Themselves Too Variegated . . . . . . . A Determinative and Ditrerentiative Events . . . . . . . . B Tissue Specificity of Variegation . . . . . . . . . . C Possibility of Studying Variegated Transcription . . . . . . References . . . . . . . . . . . . . . . . . .

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1 Introduction

The cytogenetic aspects of position-effect variegation were thoughtfully and ably reviewed by Lewis (1950). and any reader interested in the historical development of the subject or in the details of experiments performed until that date should become familiar with his re-

* The research of the author and his students reported herein has been supported by the U.S. Public Health Service. Grant No . GM 07428 . 133

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view. A brief summary of our current cytogenetical knowledge of the phenomenon will be undertaken; however, the main emphasis of this review will be on aspects of variegation that have been discovered or have been exploited within the past decade or two. Because of space limitation, variegated phenotypes observed, for example, in tobacco and tomatoes, will not be discussed ; their position-effect nature is uncertain. The “rules” for the association between the affected gene and the heterochromatic regions of chromosomes have only been slightly modified during this period. Perhaps the greatest advances concern (1) the generality of variegation, having been discovered and extensively studied in mice and Drosophila virilis, and shown to apply t o practically any gene in D. melanogaster; (2) extensive analyses of the effects of the parental genotypes on expression of variegation in the offspring; and (3) the use of position-effect variegation as a tool in studies of developmental biology. The greatest disappointment of research in this area over the past 15 years is that we have learned so little about the underlying cytochemical basis of variegation, and nothing about its molecular cause. Explanations of the reason a gene is not producing its product (or at least, a normal one) in a given region of the variegated tissue are still put in terms of “heterochromatiaation” or “compaction,” terms that, in reality, expose our ignorance rather than our understanding. Some speculations will be made as to possible reasons for the refractivity of this problem. A fresh approach to the subject is badly needed. II. Cytogenetic Aspects

A. THEcis-trans RELATION Position-effect variegation is the name given to the phenomenon of suppression of gene action caused by a chromosomal rearrangement (R) located cis to the allele of the gene affected. For example, consider a gene g whose wild-type allele is dominant over the mutant allele. If a chromosomal rearrangement has a break close to this locus, and if this rearrangement fulfills certain other criteria discussed subsequently, then the heteroaygote R ( g + ) / g will often show a mosaic of wild-type and mutant expression within the tissue affected by this gene, whereas the R(g)/g+will most often be wild type. The break in the rearrangement that is responsible for this gene suppression is invariably found within the heterochromatic regions close to a centromere in Drosophila, and within the X chromosome of the mouse. One of the two X chro-

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mosomes of a female mouse also becomes heteropycnotic. Therefore, if position-effect variegation of a particular gene is to be observed, there are two requirements of the rearrangement: one break must occur in a heterochromatic region, and a second break must be such that the “heterochromatic” breakpoint is brought close to the gene whose action is being studied. It is not sufficient t o have the gene brought close to a heterochromatic region; rather it is necessary that the gene be brought into proximity with a heterochromatic region that has itself been broken. Therefore, if we let the symbol b p indicate the “heterochromatic” breakpoint, and bp+ denote the normal chromosomal arrangement in the heterochromatic region, a more adequate description of the genotypes pertaining to variegation is R(g+ bp)/g bp+ (variegated) vs. R ( g bp)/g+ bp+ (wild type). You will note in the first genotype that the mutant condition of the gene and the chromosomal arrangement are trans to each other and a mutant gene expression is observed, whereas in the second genotype, they are Cis t o each other and this produces wild-type expression. Thus this type of variegation should be considered as a special case of the usual cis-trans position effect. This more complete notation of the genotypes involved emphasizes the dual nature of the interesting questions raised by this gene suppression: How is the gene action suppressed in particular cells of the affected tissue? Why is a break in the linear sequence of a heterochromatic region necessary for any gene suppression to occur? The latter question has only seldom been raised, and no experiments have been directed toward it-a rather surprising fact since it implies a higher level of organization of the heterochromatic regions as compared with the euchromatic regions of chromosomes.

B. PROOFS OF POSITION-EFFECT NATURE 1. Trans to cis Proof

This notation also makes clear the experimental conditions that must be met for a direct proof that an observed variegation is caused by position effect: one observes whether the phenotype changes from variegated t o wild type upon a change from the trans to the cis genotype caused by crossing over between the gene and the breakpoint. Such direct proofs had been obtained in D. melanogaster by Dubinin and Sidorov (1935) for the hairy gene, and for the curled gene by Panshin (1935). More recently, direct proofs have been obtained for the white gene of D. melanogaster by Judd (1955), the peach locus in D. virilis by Baker (1954), and for the brown locus in the mouse by Russell

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and Bangham (1961). The last case will be discussed further in Section 111. 9. Reversion of Phenotype with Reversion of Rewrangement

A less critical type of proof has been obtained by irradiating R(g+) chromosomes and then studying induced chromosomal changes that revert the variegation to wild type. Gruneberg (1937) found that a reversion of the rsts phenotype in D. melanogaster was accompanied by a complete reinversion of the original In(l)rstS inversion. Novitski (1961) was able to obtain 11 phenotypic reversions of the rstS phenotype by irradiating I n (l)rstS/+ heterozygotes. Six were accompanied by reinversions of the chromosome that were indistinguishable from the normal gene order either by analysis of the salivary gland chromosomes or by a sensitive genetic test. (Novitski argues convincingly that these reversions are not induced multiple crossovers which switch the markers between the rstS and the wild-type chromosome.) The test consisted of recovering the two complementary crossover products between each reinversion and a normal chromosome. If the breaks of a reinversion were not a t precisely the same breakpoints as the originaI inversion, then the crossovers would be duplication-deficiency products and should show phenotypic effects, None was observed. The breakpoints could have differed by a small heterochromatic region which remained undetected by the cytological and genetic tests. Perhaps a recombinations1 fine structure analysis in the rst region of the reinversions might be revealing. Since it would be highly unlikely that the two breaks in the reinversion occurred a t the same molecular spots as the original inversion, one concludes that there remains in the revertants a cytologically invisible piece of the originally basal heterochromatic region now located distally near the rst locus, and that the new basal heterochromatic region has the corresponding deficiency. This condition does not produce variegation, implying that the heterochromatic regions are discrete insofar as their variegation, as well as other, properties are concerned, a conclusion to which attention will be given later. Hinton and Goodsmith (1950) obtained similar complete phenotypic and cytological revertants of the bwD phenotype in D. melanogaster, which was shown conclusively by Slatis (1955a) to be a case of variegation at the brown locus caused by the insertion of a few bands of heterochromatic material next t o this locus. Even when the induced change in expression of the variegated phenotype may not revert completely to wild type, this change is often accompanied by a new chromosomal

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rearrangement with one or both breakpoints close to the original ones as Hinton (1950) found with the In (2LR)40d arrangement.

3. One Break in a Heterochromatic Region

It is obvious that the latter proof of the position-effect nature of a variegated phenotype is difficult and sometimes impossible to execute. Most cases of variegation in Drosophila are assigned a position-effect cause (even if only the trans genotype is observed) if one of the breaks in the accompanying rearrangement is in a heterochromatic region and another break close t o the gene whose action is seen to variegate, and (or) if the expression of variegation is more toward wild type with the addition of extra Y chromosomes and more toward the mutant phenotype upon removal of Y chromosomes. Either of these criteria appears to be adequate. In the new "Mutants of Drosophila melanogaster" (1968) Lindsley and Grell list 72 different genes whose expression has been shown to variegate when involved with rearrangements. In all 312 cases, there is one break in the proximity of the locus affected, and in all but five cases another break is in a recognized heterochromatic region near a centromere or in the Y chromosome. It is believed that most of the exceptions are the result of inaccurate cytological analysis.

4. Suppression by Y Chromosomes Just as striking confirmation of the adequacy of these criteria are the sex-linked, X-ray induced, recessive loci discovered by Lindsley et al. (1960) which are viable as XY males but lethal as XO. If lethals in the nucleolus-organizing region of the X are excluded, each of these lethals was accompanied, without exception, by a chromosomal rearrangement with one break in the euchroniatic region of the S and another break in a heterochromatic region. Thus, although the phenotype of these lethal genes cannot give a variegated appearance in somatic tissue, these are genes whose actions are being suppressed by the same process that is responsible for position-eff ect variegation. Ratty (1954) has also provided evidence for variegation of lethal genes. Lindsley et al., noted that with a dosage of 3 or 4 kr about 20% of all X-ray induced sex-linked recessive lethals were of the position-effect type. This points to the ubiquity of the phenomenon. I n fact, there is no evidence against the assumption that every gene is subject t o this type of suppression. The importance of this generality should not be overlooked because it implies that whatever the nature of this suppression, it affects a process common to the action of all genes. I n terms of molecular biology, i t means that the suppression is acting probably

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WILLIAM K. BAKER

a t the levels of transcription or translation rather than at the level of e n ~ y m e sand substrates.

c. VARIEGATION OF GENESNORM.4LLY IN A HETEROCHROMATIC REGION As we have seen, the requirements of a rearrangement necessary to evoke variegation are one break in a heterochromatic region, and another break close t o the affected gene. These are sufficient to cause variegation of the expression of genes normally located in euchromatic chromosome sections; however, there appears to be a further requirement for genes normally located in heterochromatic regions. This was first pointed out by Khvostova (1939) in studies with D. melanogaster dealing with position effects of the cubitus interruptus (ci) gene located on chromosome 4. Position effects at this locus are detected by comparing the length in the gap of the fourth longitudinal vein between individuals of the R(+)/ci and +/ci genotypes, and between R (ci)/ci and ci/ci. Khvostova found that X-ray induced position-effect rearrangements in R(+)/ci flies always had one break in the centromeric region of chromosome 4; however, the other break was not located a t random in the other chromosomes, but was primarily limited to the distal portions of euchromatic regions, although in a relatively few cases the second break was within another heterochromatic region near a centromere. Practically none of the breaks associated with variegation were found in the proximal euchromatic regions although other rearrangements that did not produce variegation had breaks induced in these regions. It appeared, therefore, that suppression of ci+ by a rearrangement did not occur unless this allele was removed from the centromere of chromosome 4 and placed either a t the distal end of another chromosome or into another disrupted heterochroniatic region. Stern and Kodani (1955) confirmed these observations on the R(+)/ci position effects, and found they also apply to R(ci)/ci position effects. The same restrictions on variegation of genes normally located in heterochromatic regions were found by Baker (1953) in studies of 30 rearrangements causing variegation of the peach gene of D. virilis which is located in the distal end of the heterochromatic region of chromosome 5 next to the centromere (Baker, 1954). Finally, Hessler (1958) reported the same breakage distribution among 35 rearrangements responsible for variegation of the light gene (located in basal heterochromatic region of 2L) in D.melanogaster, with the exception that no Y-2 translocations were observed nor were any breaks reported in other heterochromatic regions. The latter exception may be an artifact since

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the cytology of the rearrangements was analyzed in salivary gland chromosomes where translocations between two heterochromatic regions would not be detected. In fact, she mentioned two additional cases of variegation which were not associated with observable rearrangements; however, no genetic analyses were made. I n view of the apparent generality of this “centromere distance” effect on variegated expression of genes normally located in heterochromatic regions, its significance as an indication of higher levels of chromosome organization should not be overlooked. One way of redescribing

b

uu

g-variegation

FIG.1. The centromere-distance effect on the variegated expression of genes normally located in a heterochromatic region. Three illustrative translocations, A, B, and C are shown. The arrows indicate the extent of spread of the hypothetical repair process from an undisturbed heterochromatic region linearly along the chromosome.

the effect is pictured in Fig. 1. The wild-type allele of these “heterochromatic” genes acts normally only when moved (case B) t o a euchromatic region that is close to a n undisturbed heterochromatic region, whereas if they are placed in a distal euchromatic region (case A) or in another disrupt,ed heterochromatic region (case C) , then variegation results. It is as if only the products of an undisturbed heterochromatic region can repair the damage, and then only if the affected gene is close by; that is, the repair mechanisms spread linearly along the chromosome. This spreading is indicated in Fig. 1 by the black arrow.

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WILLIAM R. BAKER

The centromere-distance effect is subject to experimental verification and study. I n theory, this could be accomplished by obtaining a crossover between the gene and the breakpoint of the rearranged chromosome causing variegation with another rearranged chromosome that has this interstitial section located close to a normal heterochromatic region -in other words, to go from a genotype like A to B in Fig. 1 by crossing over and then see if the phenotype changed from variegated t o wild type. I have made several large-scale attempts to do this in D . virilis, but have been thwarted by technical problems. In order to obtain a euploid gamete of the desired type from the heterozygote between two translocations, it is not only necessary to recover the interstitial crossover, but this must occur in an egg which resulted in the proper type of segregation; furthermore, a second crossover is required. The simultaneous occurrence of these three events must be quite rare. Further study of the centromere-distance effect would be rewarding.

D. Y CHROMOSOME SUPPRESSION AND

THE

It EXCEPTION

One of the diagnostic characteristics of position-eff ect variegation, at least in Drosophila, is the suppression of the variegation toward wild type upon the addition of a Y chromosome and, conversely, the enhancement of the variegation toward the mutant phenotype upon the deletion of this element. This effect was first discovered by Gowen and Gay (1933a) and has since been observed in almost all cases in which a critical study has been made. Exceptions have been reported, however, in D. melanogaster (the rolled locus, Morgan and Schultz, 1942; the white mottled of Cicak, Oster, 1957) and in D. viriZis (the yellow locus, Girvin, 1949). There is one long-known exception to this general rule, the light (It) locus of D . melanogaster. As pointed out by Schultz (1936), the effect of Y chromosomes is diametrically opposed to the usual situation: addition of Y’s enhances the mutant tissue in the light-variegated eyes, whereas the deletion of Y’s suppresses the mutant tissue. It has been critically shown (Baker and Rein, 1962) that this is indeed an opposite effect. One and the same Y chromosome, or fragments thereof, was introduced by single individual males into a genotype which would show It variegation and then into two genotypes which would exhibit white variegation. Quantitative measurements were made of the pteridines responsible for the drosopterin pigmenta in the variegated eyes. The results were unequivocal: the same Y fragments that were most effective in suppressing the mutant tissue in the white variegated genotypes were the ones most effective in enhancing

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the mutant phenotype in the light system. I n fact, i t was suggested from the data that Y chromosomes are having opposite effects in the It and w variegated systems in spite of the fact that apparently the same biosynthetic pathway leading to the drosopterin pigments is involved. Since Et is located within the heterochromatic region of 2L, it has been suggested (Schultz, 1941; Lewis, 1950) that this antagonistic effect of added Y chromosomes is characteristic of genes normally located in heterochromatic regions. This has proved not to be the case. In a careful study, Grell (1959) found that in R(ci+)/ci heterozygous females the addition of a Y chromosome definitely shortens the gap in the wing vein. The presence of two Y’s made the vein more normal than one Y, which was more normal than a female with no Y. Altorfer (1952) also reports Y suppression of the wing-vein gap in R(ci)/ci heterozygotes. The peach gene in D . uirilis is the other gene normally located in a heterochromatic region that has been extensively studied from the standpoint of variegation, and Schneider (1962) likewise found that an extra Y suppresses the mutant tissue in the variegated eye. It appears that It variegation is a unique case in this respect, although it is similar to other “heterochromatic” loci with regard to the centromere-distance effect. The suppression of variegation is not limited to the heterochromatic Y chromosome but may be observed with other chromosome elements containing heterochromatic regions. Grell (1958) reported a particularly interesting gynandromorph in D. melanogaster that was exhibiting variegated expression of the autosomal brown locus. The left side of the head on this individual was female and XXY and showed wild-type expression of brown, whereas the right side was male and XY and showed the mutant expression of brown over almost the entire eye. The somatic loss of an X chromosome enhanced the mutant expression and thus had the same effect as the loss of a Y would have had. These studies, along with the commonly held belief that the heterochromatic regions are relatively inert insofar as their number of discrete genes, have been responsible for the widespread use of the term “heterochromatin” to describe the material in these chromosomal regions. If this term is used, i t should not imply genetic homogeneity or inertness. This has been emphatically pointed out and fully documented by Cooper (1959) in his elegant study of the heterochromatic regions of the X and Y chromosomes of D . melanogaster. Evidence of the inhomogeneity of the Y chromosome with regard to its suppression of variegation was studied by Baker and Spofford (1959), under rather rigidly

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WILLIAM K. BAKER

controlled genetic backgrounds, by investigating the effect of 15 different fragments of Y chromosomes on white variegation in D. melanogaster. The drosopterin pigments and some of their presumed precursors in the variegated eyes were separated chromatographically and their relative amounts determined photometrically. The Y fragments differed remarkably in their suppressive ability, some were even more effective than complete Y chromosomes, and this ability was not necessarily correlated with the cytological length of the fragment since some of the shorter ones were the most effective. Brosseau (1960b, 1964) utilized his series of Y fragments containing various numbers of the five kl and the two lcs fertility factors (Brosseau, 1960a) t o localise within the Y chromosome the factors which suppress position-effect variegation at the Bar loci, Variegation for Bar was observed among some derivatives of the Bs.Y chromosome that had been irradiated. In these BsV*Y derivatives, the eye was normal in males instead of being Bar-shaped. Since the Bar phenotype is caused by a duplication of the 16A region in the salivary gland chromosomes, apparently in these derivatives one of the loci had been inactivated. If this is correct, then addition of another Y to the genome should reactivate this locus (suppress the variegation) with the result that the eye would once again appear as Bar. This was found to be the case. By use of this system, Brosseau was able to show (1964) the presence of one suppressor of variegation to be located near the lcl-2 fertility factor on YL,and another was on Ys proximal to lcs-1. We conclude, therefore, that there are discrete loci which suppress variegation located within the Y chromosome and that most Y chromosomes contain alleles of these genes that are active. It would be illuminating t o learn the molecular mechanism by which this type of suppressor acts. E. THE POLARIZED SPREADING EFFECT Three facts have been mentioned which suggest that position-effect variegation is operating a t the transcriptional, or possibly the translational, level of gene expression: (1) the ubiquity of genes affected, (2) the &-trans nature of the effect, and possibly (3) the centromeredistance effect. The second and third of these illustrate a linear effect that extends along one homologue, but not between homologues. There is a fourth fact which also supports this argument. The suppression of gene activity spreads from the "heterochromatic" breakpoint t o the nearest gene, and if its activity is blocked, then the next most distal

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gene may be affected. This was illustrated quite clearly by Demerec and Slizynska (1937) and by Schultz (1941) in their studies of the association between variegation of roughest and split and variegation a t the white locus. In both of these cases, rst+ and spl+ (genes that produce a ((rough” arrangement of the eye facets) were located closer to the breakpoint than was w+. I n a variegated eye, the white areas were invariably rough, whereas a normally pigmented area could be either rough or not. Cohen (1962) showed that polarization of this spreading effect was the result of the location of the breakpoint in the heterochromatic region and not to the orientation of the centromere. It has previously been noted (Baker, 1963) that the spreading effect observed by Cohen cannot be explained as a sequential limitation of pigment differentiative abilities imposed on disarranged ommatidia; we are indeed dealing with a polarization along the chromosome. It is quite remarkable that this suppressive action of the “heterochromatic” break can extend for appreciable distances along the chromosome. In the euchromatic regions it has been shown to extend from white t o bifid in D. melanogaster, a distance of about 50 salivary chromosome bands, or in terms of crossing over, a distance of about 6% (Demerec, 1941). In D. virilis the breakpoint may be removed from the affected peach gene by almost the complete basal heterochromatic region, a distance of about one-third the length of the mitotic chromosome 5 (Baker, 1954). Russell and Montgomery (1965) reported that in the mouse a gene may be 25 crossover units removed from the breakpoint and still a detectable phenotypic effect is observed. The mouse results will be discussed in detail later. It need hardly be emphasized that these linear (intrahomologue) interactions extend over distances of entirely different magnitudes than operons in microorganisms. The vast majority of cases of position-effect variegation are like those previously described in which it is necessary to have the (‘heterochromatic” break and the affected gene in the same homologue. Slatis (195513) described some interesting cases of variegation of the brown locus in D. melanogaster in which R ( b w ) / b w + flies show variegation, whereas bw/bw+ individuals are wild type. Although this variegation may be ascribed t o an interhomologue effect since the bw allele behaves as an amorph, completely blocking the production of the red drosopterin pigments, the possibility should be considered that bw is a complex locus and that rearrangements of the locus may restore activity, producing a dominant type of variegation. Certainly the phenomenon of position-effect variegation is overly blessed with the number of interesting observations that have been

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WILLIAM K. BAKER

made. It is difficult to decide which are relevant to the underlying cause of variegation and which are not. 111. Position-Effect Variegation in the Mouse

Until 1959 some biologists viewed position-effect variegation as of rather limited interest since its occurrence was restricted t o Drosophih and to a single case in Oenothera (Catcheside, 1947). But like other genetic discoveries, its generality did not remain hidden. Russell and Bangham (1959) reported the first case in the mouse and have subsequently shown that variegation in mice and in Drosophila share many common characteristics. In fact, the mouse work has added important information to our general knowledge. A. CYTOCENETICS Variegation has been observed by utilizing primarily the following autosomal genes which affect coat color: b = brown, genophore 8 ; cch= the chinchilla allele of the albino gene, genophore 1; p = pink eye, genophore 1. A t the present time, eight different rearrangements which evoke variegation have been found, and seven extensively studied: one translocation between the X chromosome and genophore 8 which exhibits brown variegation, and six translocations between X and genophore 1 which show variegation for c or p or both, All the rearrangements occurred in mice bearing the wild-type alleles of these genes. Five were radiation induced, one induced by a chemical mutagen, and one was spontaneous. It is important to note that all are X-autosome translocations. Although translocations between autosomes would have been produced and were detected, none showed variegation for the autosomal genes. The variegated phenotype is produced only in XX females, the XY males have a wild-type coat (Russell and Bangham, 1961; Cattanach, 1961). That this is due to the X chromosome constitution and not t o the sex of the individual is demonstrated by the fact that XO females carrying the translocation are wild type (Russell and Bangham, 1961), whereas XXY males are variegated (Cattanach, 1961). It is clear that one possible basis of variegation may be the well-known “inactivation” of one of the X chromosomes in XX somatic tissue (Lyon, 1961). Therefore, in a R ( g + ) / g heterozygote, if the part of the translocated X bearing the wild-type alleles of the autosornal genes is inactive in particular cells of the variegated tissue, then those cells may express the mutant

POSITION-EFFECT VARIEGATION

145

phenotype; if the normal X is inactivated, then the wild-type phenotype will be exhibited in these cells. It is obvious, of course, that in a female heteroeygous for a reciprocal translocation involving the X and an autosome there are three elements that have X-chromosome material: the normal X, and each of the two translocated chromosomes. Single-X inactivation, or suppression as I prefer to call it, will be discussed more fully subsequently. If the reasonable assumption is made that it is the association of the autosomal loci with the X chromosome that causes variegation, then SUPRESSION OF C+

R2

R 3 R 4

R5 R6

P+

+

0 +tt

(t1

t ttt tt?

t+t? +tt?

O?

Fro. 2. The breakpoints of translocations between the X chromosome and genophore I which cause suppression of genes c+ and p+ in the mouse. The table at the top of the figure indicates the extent of suppression in each of the five rearrangements (Russell, 1963). The breakpoints and their recombination values are taken from Russell and Montgomery (1965).

translocations between the X and genophore 1 containing c and p can provide information relative to breakpoints and affected loci, Figure 2 summarises the data on the location of the autosomal breaks in these translocations as reported by Russell and Montgomery (1965), along with the data on the relative suppression of c+ and p + in these rearranged chromosomes (Russell, 1963). Notice that the crossover distance may be quite large between the breakpoint and the affected locus; for example, R5 and R6 cause suppression of cf although removed from

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WILLIAM K. BAKER

this locus by approximately 20 crossover units. On the other hand, c+ is not suppressed by R.2 although it is only 0.3 units from the break. The simplest and most logical interpretation of this finding is that the part of the X chromosome attached to c+ in R2 has no suppressive activity. You will note from this figure that in general the gene closer to the breakpoint is suppressed to a greater extent than the more distant gene, Cattanach (1961) reported a similar spreading effect in a transposition in which c+ and p + have been inserted into the X. Now this spreading effect and the lack of c+ suppression in R2 are suggestive; perhaps there is a site within the X chromosome from which the suppression spreads (Russell e t aZ., 1962). It has been possible to place some limits on the location of this site of suppression (Russell and Montgomery, 1965). At the top of Fig. 2 are shown the X breakpoints of R2,R3, R5, and R6 relative to the location of the Tabby ( T a ) gene on the X chromosome. I n these four translocations, R (+)/ T a has the same phenotype as +/Ta, indicating that this locus is under the influence of the suppression site. Therefore, the autosomal loci that show suppression must be on the same member of the translocated pair as the Ta+ allele. This fact, in conjunction with the frequency of exchange between T a and the breakpoint for each of the translocations, allows one to place the breaks in the positions shown in this figure. The centromere location is unknown for either chromosome, but in this figure the chromosomes have been so oriented relative to one another that both centromeres are either to the left or to the right. The suppression site is delimited by the breaks in R2 and R5. The reason for this is apparent upon examination of Fig. 3, which portrays the translocation heterozygotes of R2,R3, R5,and R6. Since T a + , c+, and p + may be suppressed in R5, the site must be between the R5 break and the “distal” end of the X shown at the top of the figure. The “distal” limit of the site location is given by R2. I n this translocation c+ is not suppressed; therefore, the portion of the X “distal” to the R2 break does not contain the site. These limits for the site of suppression are indicated in Fig. 3 for R3 and R6. It is not known on which side of Ta the site lies within this limited region. A spreading effect from the site is shown (Russell and Montgomery, 1965) by the relation between the Ta-p crossover distance and the extent of the recessive phenotype shown in the variegated coat. For example, with R5 where the crossover distance is 9 to 12 units, 50% of the coat is phenotypioally p and 38% c, whereas with R6 the distance is 14 t o 23 units and only 24% of the coat is p and 12% c. Therefore, the findings are similar to those known in Drosophila: the closer the affected gene is to the “hetero-

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POSITION-EFFECT VARIEGATION

chromatic” breakpoint, the more extreme the variegation (Schultz, 1941). The position-effect nature of the variegation in the mouse was definitively demonstrated by Russell and Bangham (1961) by use of a translocation between the X chromosome and genophore 8 which produced brown variegation. Approximately 15% crossing over was observed between b and the breakpoint, enabling the following genotypes to be constructed in sequence: R ( b + ) / b (variegated) to R ( b ) / b + (no variegation) to R ( b + ) / b (variegated). The cis-trans nature of the relation between breakpoint and affected gene is obvious.

R2

I

To

I

6

T“ R3

i

Ta

FIQ.3. Location of the suppression site on the X chromosome of the mouse. The shaded area indicates the limits within which the site resides, based on rearrangements R5 and R2. Dats, from Russell and Montgomery (1965).

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WILLIAM K. BAKER

One of the unique features of the mouse research on variegation is the finding reported by Ohno and Cattanach (1962) of the correlation ' between phenotypic expression and cytologically observed heteropycnosis in T p ( X ; l )Ct. This rearrangement discovered by Cattanach (1961) is a transpositional insertion of a region of genophore 1 into the X chromosome, and reciprocally there appears t o be a small piece of the X inserted into the autosome (Sliaynski, 1967). The autosomal insertion contains c+ and p + , the left breakpoint being between Hbb and sh-1 and the right between p and pu (Eicher, 1967; Wolfe, 1967). The location of the insertion within the X chromosome has been shown (Cattanach, 1966) to be Gy-Insertion-Mobr-Tu-Bn-spf. Both C + and p + variegate in their expression, producing colorless patches on the fur. This insertion makes an appreciable increase in cytological length of the X chromosome and it can be distinguished from a normal X. Ohno and Cattanach took advantage of this situation to ask the question whether the X with the insertion was heteropycnotic (suppressed) in the skin cells in which the phenotype was white (both c+ and p + were suppressed), and whether the normal X was the heteropycnotic element in the pigmented patches. They report a complete correlation between the cytological picture and the phenotypic expression, a remarkable finding indeed. It remains t o be shown that the pigment granules, or their absence, were derived from the same cells as the skin cells which were examined cytologically. BETWEEN NORMAL S SUPPRESSION AND B. RELATION POSITION-EFFECT VARIEGATION

There is considerable debate on the question as to the relevance of the facts learned about gene suppression in the X-autosome translocations to the normal suppression of genes on one X chromosome in an XX individual (Russell, 1964; Lyon, 1966). The data previously discussed clearly point to the existence of a suppression site on the X near Ta whose action can spread t o attached autosomal genes. The question raised by Lyon (1966) is whether there is evidence for such a site in normal X chromosomes, or whether, alternatively, all genes on a normal X are suppressed to the same degree, namely completely inactivated. In support of the alternative explanation, Lyon (1966) studied expression of X chromosomal genes in an X-autosome translocation, T(X;?)16H, in which the translocated part of the X containing !Pa+ was never suppressed, while the normal X bearing T a was always suppressed (Lyon et al., 1964). In other words the R ( T a + ) / T a heterozygote was wild type. There is no evidence on the activity of genes on the

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other part of the translocation that does not contain the Tabby locus. The experiment was designed around the X chromosome gene sparse fur, spj, which shows no mosaicism when heterozygous with ita wildtype allele when in normal chromosomes. Russell (1963, 1964) raised the possibility that genes of this type on the X are never suppressed, presumably because of their distant location relative t o the suppression site. Lyon (1966) was able to show that in Rl6H(spf+)/spf hetero~ygotes there is never any indication of sparse fur, whereas in R16H (spf)/spf+ individuals the sparse fur phenotype is invariably expressed, and in some spf+/spf heterozygotes there is a mild expression. Since these three heterozygotes differ in expression, Lyon considers this as evidence that suppression is actually occurring (but is not often phenotypically expressed) in spj/+ individuals. Viewed in a different light, these experiments might support a localized site of suppression. Why are the Ta+ and spf+ genes always fully expressed on the translocated chromosomes, but never on the normal X? One possibility is that the translocation break has occurred between the suppression site and the loci of Ta and spf, placing the suppression site on the other member of the translocated pair. If the breakpoint was a t or close to the suppression site, perhaps the site could be inactivated, allowing the site on the normal X to be continually active in suppression. To obtain evidence on this possibility it would be necessary to study the suppression of autosomal and (or) X-linked genes located on the complementary member of the translocated pair, that is, the member not containing Ta+ and spff. Position-effect variegation in the mouse is always associated with rearrangements of the X, an element that is known t o become heteropycnotic in one of the X’s of an XX cell. This correlates well with the heterochromatic regions responsible for variegation in Drosophila. One clear distinction between this heterochromatic suppression in the two organisms must be kept in mind: the suppression does not spread in a normal X chromosome of Drosophila, only if the heterochromatic region has become interrupted by rearrangement can it spread to contiguous euchromatic regions. IV. Parental Modification of Variegated Gene Expression

A. DISCOVERY OF THESEEFFECTS The expression of variegation is not only the product of the genomeenvironment interactions in the individuals being studied, but it can also be modulated by the parental genotype. This was first reported in

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WILLIAM K. BAKER

some detail by Noujdin (1944). His results have received little attention in spite of their striking and unexpected nature. Perhaps, among other things, this is due to the fact that the measure of expression of variegation used was solely its penetrance (that is, percentage of variegated flies), with no measure of the extent of variegation within an individual. The main body of work is a study of achaete variegation caused by In(l)sc8 and yellow variegation evoked by In(l)ysP, although similar results were reported for brown variegation caused by In(2r)PmD1. A small portion of his data has been abstracted in Table 1, but this will suEce to illustrate his three main observations. The TABLE 1

A Sample of Noujdin’s (1944) Data showing Effect of Parental Genomea on Expression of Variegation

Cross No.

Mother

Father

Offspring

Percentage variegated

Maternal Homozygosity for Rearrangement 1 2 3 4

scs/scs sc8/ sce/scs

scs/Y

Sc“/

SCs/Y

3 5

scyscs

SC0/8C8

4.8 40.0 13.9 40.6

Effect of Maternal X Fragment SCyY sCs/scs scyy scs/sca scs/scs/XR

13.9 7.0

+ +

+/y +/Y

S8/+

s8/+ scs/sca

Effect of Paternal Y Material 6

F/Pp/XR

8

Y”/@~/XR

7

y”/@P/XR

laply

X .Ys/Y X*YL/Y

%p/XRrY 2/”/XR/Y 1/3’/XR/Y

16.4 3.2 2.6

first four crosses clearly show that mothers which are homozygous for the rearrangement responsible for variegation produce fewer variegated offspring than heterozygous mothers. His second parental effect can be seen by comparing the frequency of variegation in the sca/sc8 offspring of crosses three and five: extra heterochromatic elements in the mother lowers the penetrance of variegation even though these elements are not in the genome of the offspring examined. (XR presumably is the element of T(1;4)A1 which carries the proximal portion of the X inserted into 4.) The third finding is a similar lowering of penetrance when the male parent carries extra heterochromatic regions (in this

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case parts of the Y are attached to the X) as illustrated in the last three crosses. Note that the males which differed in the amount of Y chromosome attached to the X did not carry the rearrangement causing variegation. Luning (1954) also briefly reported a maternal effect on yellow variegation. A similar set of parental effects was rediscovered by Spofford (1958) and Baker and Spofford (1959), and have been studied rather extensively in D. melanogaster (Spofford, 1959, 1961, 1962, 1965; Hessler, 1961; Cohen, 1962) and in D. wirilis (Schneider, 1962). The studies in D . melanogaster dealt with white-eyed variegation caused by Dp (wm)264-58a, a 20-band insertion of the X chromosome containing the w+ locus into the heterochroniatic region a t the base of 3L. The D . virilis work concerned peach variegation caused by a series of translocations involving the fifth chromosome which normally contains, in its basal heterochromatic region, the peach ( p e ) locus (Baker, 1953). B. PARENTAL HOMOZYGOSITY FOR REARRANGEMENT One of the most consistent and general of the parental effects on variegation concerns the homozygosity of the rearrangement in the parents of the individuals being studied. If the mother is hoinozygous for the rearrangement, there is more wild-type tissue in the variegatcd eye than if she is heterozygous. This has been conclusively demonstrated not only by the penetrance of variegation but by visual estimates of the eye pigmentation and by quantitative measurements of the amount of drosopterin pigments, using chromatographic techniques in both D . melanogaster (Spofford, 1959; Hessler, 1961) and D. virilis (Schneider, 1962). It should be noted that in both white and peach variegation the testis sheath as well as the compound eye shows vnricgation and the homozygosity effect was also observed in this tissue (Hessler, 1961; Schneider, 1962). Spofford (1966) suggests that a great deal of this maternal effect in D. inelanogaster may be caused by the maternal effect of a suppressor gene (Su-V) closely linked to the dupli~) in D. naelanogaster cation (see Section IV, d). The D p ( ~ 1 utilized usually does not survive homozygously in males SO that this effect could not be tested in fathers. However, Schneider (1962) observed some suppression of variegation with the peach-variegated rearrangements in heterozygous offspring of homozygous, as compared with heterozygous, fathers. This observation, therefore, would not support as an explanation of the suppression any simple scheme of cytoplasniic inheritance.

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C. EFFECT OF PARENTAL Y CHROMOSOMES Although the suppressive action on variegation of extra Y chromosomes in the genome has long been known, i t was not realized that a similar suppression is observed if a Y chromosome is present in the genome of the mother although not present in the genome of the offspring whose variegation is being studied. Quite striking effects were observed by Spofford (1959) not only with the presence and absence of Y’s in the mother, but also with Y fragments, one of which was even more effective than a complete Y in this maternal suppression. The suppression was more complete if Dp(wm) was in the maternal genome, but this was not necessary for an effect. Schneider (1962) showed that variegation of the p e gene in D . virilis (normally located in a heterochromatic region) was also subject to suppression by the presence of a Y in the mother. Although this suppression may be a case of maternal inheritance, Spofford reported (1959) that in certain experiments, but not in others, the Y chromosome constitution of the father affected the extent of offspring variegation; in this case, only if the Dp(wm) was maternal in origin and thus not in the same genome as the Y chromosome causing suppression. It will be recalled that Noujdin reported such a paternal effect of extra Y material.

D. PARENTAL SOURCE OF REARRANGEMENT

It has been shown (Spofford, 1959, 1962; Hessler, 1961; Schneider, 1962) that the parent contributing the rearrangement to the offspring

is influential in determining the extent of variegation in these offspring. In D . virilis and in one of the ‘Lstates” (Dp‘) of the duplication in D . rnelanogaster, the variegated offspring have more wild-type tissue if the rearrangement comes from the mother as compared with a paternal origin. One could interpret this as an extension of the maternal homozygous vs. heterozygous effect of the rearrangement if it were not for the fact that another state of the duplication (Dpa) behaved strikingly in the opposite manner, that is, much more pigment was present in the eye of variegated offspring if the duplication was paternal in origin. This was in no way connected with a maternal effect per se since Baker (1963) found that this greater paternal suppression was observed among sibs from crosses in which identical mothers were used. Two groups of sibs from the same mothers were compared: one group received Dp” from the father, the other from the mother. The variegation was greatly suppressed in the first group.

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The basis for the two different states of the duplication is now known. Spofford (1Qf32,1967) has shown convincingly the presence of a semidominant suppressor of variegation, Su-V, which is rather closely linked (about 6%) with the duplication on the third chromosome. Variegation is suppressed more in Su-V/Su-V individuals than in Su-V/+ heterozygotes. In addition t o this direct effect of Su-V in the genome of the variegating individual, this suppressor also acts maternally. As much pigment is formed in the eye of a fly by the presence of Su-V in its mother as by its presence in the genome. Spofford has demonstrated that the difference between Dpf and Dpa is solely due to the presence of Su-V in the former state; that is, Dpf = Su-V Dp(w") and Dpa = D p (wm). We conclude that the primary parent.al-source effect is that exhibited by Dp", suppression caused by passage of Dp(zo*) through the sperm as compared with passage through the egg.

E. COMMENTS ON PARENTAL EFFECTS I n contrast to the claim of Noujdin (1946), none of the investigations described above gives any indication that the parental effects last more than one generation; there is no evidence of grandparental effects (Spofford, 1959; Hessler, 1961). There. still remains an intriguing puaele: How is the parental information transmitted to the offspring, and how is this information retained during ontogeny until the time the tissue exhibiting variegation is differentiated? There would appear to be two types of information transmitted: one maternal and, therefore, possibly cytoplasmic; the other presumably nuclear. The maternal suppression of Su-V and the action of Y chromosomes in maternal genomes that do not contain the variegation-evoking rearrangement are presumably cases of cytoplasmic modification acting through the materials poured into the Drosophila oocyte by nurse and follicle cells during its formation. On the other hand, the paternal suppression evidenced by the parental-source effect of Dpa is a nuclear determination, probably acting a t the chromosome level on the duplication itself. It is not too difficult to imagine how a chromosomal change might be somatically inherited through the mitotic divisions of the developing tissue until the time when pigment differentiation, for example, takes place, It is somewhat more difficult t o envisage how cytoplasmic components would be perpetuated unless one wishes to propose replicating cytoplasmic particles, a concept which, in my opinion, has not been fruitful in explaining development in higher organisms. Another possibility remains. Perhaps the variegation pattern is determined very

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early in the anlage of the compound eye, a t a much earlier time than the actual pigmentation, and thus i t is not necessary for the cytoplasmic suppressors to persist throughout larval development. In any event, a study of the developmental biology of variegating tissue is necessary for our understanding of parental effects. V. Variegation, a Model System for Simplified Studies in Developmental Biology

A. A PROCESS OF RECULATION OF GENE ACTIVITY The term “variegation” implies a tissue composed of at least two different phenotypes. I n certain cases of white variegation in D. melanogaster, the pigmented areas of the eye appear visually to be composed primarily of wild-type pigmentation and colorless mutant areas, although small amounts of pigment can be seen in some sectors. These alternative states are evident even at the level of the pigment granules in the individual pigment cells. Electron microscope studies (Shoup, 1966) show that in a white variegated eye all the pigment cells within B given ommatidium either do or do not have pigment granules. This raised the question as t o whether the alternative phenotypes have their basis in somatic mutation, in the sense that the informational DNA has been altered, or in the suppression of genic activity in a portion of the tissue. There are five lines of evidence against the somatic mutation hypothesis (Baker, 1963) : 1. The antagonistic effect of Y chromosomes on the expression of variegation of the light and white genes of D . melanogaster militate against the hypothesis. As previously discussed, a given Y chromosome would have to be at the same time a mutagenic agent in the one system and an antimutagen in the other if it is to increase the amount of mutant tissue in the light-variegated eye and to decrease the amount in a white-variegated eye. 2. Raising Drosophila showing eye-pigment variegation a t a low temperature often increases the amount of mutant tissue (e.g., Gowen and Gay, 193313). The period a t which the low temperature is effective is during pupation (Chen, 1948; Becker, 1961) when pigment is being deposited and not during the end of the first larval instar when the pigment potentialities of the eye anlagen are being determined (see Section V, B ) . Furthermore, such a hypothetical mutation process would have a negative Ql0. 3. No germinal mutations are observed in individuals that show position-effect variegation. If somatic mutation were the cause of var-

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iegation, these mutations would have to be limited strictly to somatic cells, even excluding gonial cells. 4. Chromatographic studies of the pteridines in white-variegated DrosophiEa (Baker and Spofford, 1959; Hessler, 1961; Baker and Rein, 1962) show the accumulation of certain pteridine precursors of the drosopterin pigments in amounts in excess over that observed in wild type. These precursors are virtually absent in the eyes of white-eyed adults. No such accumulation is seen in any flies homozygous for the tested mutant alleles a t this locus (Hadorn and Mitchell, 1951). It is not known whether these intermediates are accumulating in the colorless areas of the tissue or in the pigmented areas, but such accumulation is not characteristic of known mutants. 5. The effect of chromosome elements of the parent on the expression of variegation in the offspring even though they do not enter the offspring’s genome can hardly be accommodated by any reasonable mutation hypothesis. When taken together, these lines of evidence seem overwhelmingposition-effect variegation is not based on somatic mutation. Therefore, variegation and certain differentiative processes taking place during development share the common property of differential genic activity within a tissue. €3. CLONAL NATURE

One of the conspicuous features of certain cases of eye-pigment variegation in Drosophila is that large areas of the eye may be either mutant or wild type. As examples, the ventral portion of a series of eyes of D . melanogaster with the D p (w”)26&58a duplication are presented in Fig. 4. Aside from the large size of the mosaic areas, two other facts should be noted. First, there appear t o be repeated patterns of pigmentation; for example, the horizontal sector across the middle of the eye (eyes b, c, f , and g) , or the vertical division in the middle of the eye (eyes f and g). The variegation is occurring within definite sectors. Second, the members of each pair of eyes shown in the figure have been picked so that one is the “negative” image of the other b and c, d and e, f and g ) , insofar as these were represented in a sample of eyes from which the ones in this figure were picked. This observation means that within a given sector, pigment either may, or may not, be formed. These three features of variegated eyes raise the interesting possibility that the pattern may be based on the cell lineage of the ommatidia.

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This lineage has been determined by the pattern of twin spots produced in the eye by somatic crossing over when such exchanges are X-ray induced in the developing eye anlagen of larvae of particular genotypes in D. meEanogaster (Becker, 1957) and in D. virilis (Baker, 1967). Such studies also allow one to put a time scale on the eye anlage development: the smaller the number of cells in the anlage, the larger the twin spots in the eye of the adult. This is true because the earlier the crossover is induced, the larger the number of cell divisions that will ensue to form the adult eye. By making a comparative study of the induced twin spots and the patterns of position-effect variegation, one can answer two questions: Are similar sectors of the eye affected in both cases? If so, i t means that position-effect variegation has a cell-lineage basis. If the first question is answered affirmatively, then a second question can be framed. At what developmental time does an induced somatic crossover occur such that the twin spots formed will have the same shape as the sectors

a.

FIQ.4. Patterns of white variegation in the eyes of Drosophila melanogmter. Eyes b and c, d and e, f and g are pairs of positive-negative images. Eye a indicates diagrammatically the areas filled by the descendants of the eight cells present at the end of the first larval instar which will form the ventral half of the eye. (From Baker, 1963.)

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in the variegated eyes? This time will then coincide with the time that the gene suppression is determined within the anlagen of variegated eyes. Conclusive answers to these questions have been given by studies with D . melanogaster by Becker (1961, 1966) utilizing the T(1;4) ~"258-18 rearrangement, and by Baker (1963) using the Dp(wm)26458a duplication to induce variegation; and in D. virilis by Baker (1967) using t.he T (Y ;5)pemnrtranslocation. The variegated pattern in these systems was compared with the twin spots produced in w/wco heteroup heterozygotes in D. zygotes in D . melanogaster, and in w t&ilis. A sample of the twin spots and variegation patterns in D. virilis from which this comparison was made is shown in Fig. 5. The somatic exchange which formed the twin spots was induced a t the end of the

+/+

w

9P

+

FIG.5. Patterns of position-effect variegation (eyes F to K) in peach-mottled eyes and patterns of twin spots (eyes A to E, and M to Q ) caused by sometir crossing over induced at the end of the first larval instar in Drosophih vidis. A comparison of the patterns from the two event,s indicate the cell-lineage nature of posit.ion-effect variegation. (From Baker, 1967.)

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first larval instar. Therefore, we conclude that position-effect variegation in these systems is based on cell lineage, and the pattern of variegation is determined in the eye anlagen a t the end of the first larval instar a t which time there are from 16 to 20 presumptive eye cells present. Some of the sectors formed from the descendants of these cells are diagrammed in Fig. 4a. This early determination of pattern leads to the suggestion that the maternal effects on variegation expression discussed previously may operate on this determinative event.

C. IMPLICATIONS FOR DEVELOPMENT The implications of these findings are of some importance. The determination of gene suppression in a variegated eye is made very early in development of the eye anlage, many days and many cell divisions before the actual differentiation of pigment (during the mid-pupal stage). The determinative and differentiative events of variegation are widely separated in time. The same time separation is also seen in normal Drosophila development. For example, the cells in the larval genital disc are determined as to the structures (anal plates, claspers, and so on) they will form although they do not become differentiated and form these structures until pupation (Hadorn, 1965). The second important implication is that this determination of gene suppression in the case of variegation is clonal; that is, it is somatically inherited within a cell lineage. This, also, is a property of normal Drosophila development. Hadorn (1965) has shown that determined, but undifferentiated, genital discs may be transplanted almost indefinitely in aduIts where the cells multiply but remain undifferentiated as long as they are in adult hosts. However, even after many such adult transfers, the tissue usually differentiates into one or more genital structures if it is transplanted into a larval host which is allowed to pupate. These implications raise two fundamental questions : What is the molecular basis of this determination, and how is this determination of gene regulation inherited from one cell generation to the next? It is doubtful if the second question will be answered before information is obtained on the first. The pattern of variegation as seen in the adult eye apparently remains unaltered when actinomycin D is administered to larvae during a period including the end of the first larval instar when this pattern is determined (Baker, 1967). This is in spite of the fact that cell di-

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vision within the eye anlage is completely blocked by this inhibitor from this stage on through the second instar (Perez-DBvila and Baker, 1967). At the end of the first instar, there are about 140 cells present in a head anlage in both the treated and control larvae, whereas, a t the end of the second instar, there are almost 1200 cells present in controls but still only 140 in the actinomycin-treated larvae. I n order to learn whether the 16 t o 20 presumptive eye cells within the late first-instar head anlage exhibited the same lack of cell division upon actinomycin treatment as did the remaining 120 or so other cells in the anlage, somatic crossing over was induced in treated and control larvae at the end of the first and a t the end of the second instar, and the twin spots scored. Contrary to expectation, it was found (Perez-Dhvila and Baker, 1967) that more, but smaller, twin spots were observed in both control and treated series when crossing over was induced a t the end of the second instar as compared with induction a t the end of the first. This was in spite of the fact that no increase in cell number within an anlage was observed during this period if the larvae were administered actinomycin D. Two possible explanations can be proffered: either only the presumptive eye cells divided and the other cells in the anlage did not, or the chromosomes replicated within the treated anlage cells but there was no cell division. One would have to assume as a corollary to the latter explanation that upon removal of the larvae from the actinomycin treatment, the replicated chromosomes segregated out in the normal manner, as in colchicine-treated cells, for example. The first explanation does not seem likely since two cell divisions of the 20 presumptive eye cells would make a detectable rise in the total number of cells in the anlage, and this was not observed. No obvious cell necrosis was seen in either series. The second explanation is directly testable by making measurements of the DNA content in the nuclei. These were accomplished on Feulgen-stained squashes of head anlagen using microspectrophotometry (Baker and Swift, 1967). The preliminary results are equivocal. The actinomycin-treated anlagen when squashed a t the end of the second instar sometimes contain a larger number of cells with poly -C values than do the control cells, but this may be an artifact of dissection. If this preliminary finding is verified, an interesting conclusion can be reached concerning the determinative event that sets the pattern of position-effect variegation: the determination is made at the level of the chromosome, and not made by other nuclear or cytoplasmic

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components. This is so because the variegated patterns were unaffected by actinomycin treatment which, however, produced a polytene( 1 ) condition. The only known component of a polytene cell that can later segregate out in a normal mitotic fashion is a chromosome. Such a conclusion would be of some importance in framing our concepts of regulative processes during development, but it is unfortunate that oiir present preliminary experiments are inconclusive.

VI. Thoughts, Perhaps Themselves Too Variegated

The reader may now feel overwhelmed by the myriads of facts known about position-effect variegation which have yet to fall within a simple, rational, biological framework. If this review has not saturated his storage capacity for a confusing array of information, he is referred to the recent reviews of Schultz (1965) and Brown (1966) or the older ones of Hannah (1951) and Cooper (1956) which cover broader aspects of the role of heterochromatic chromosomes or chromosmal regions in addition to their role in position-effect variegation. After such a survey one could readily come to the conclusion that the basis of the phenomenon is much too complex for profitable continuation of its investigation a t this time. Justification for such a view could be based on the fact that we are no closer to a chemical or molecular interpretation than we were 30 years ago. Is the situation really as complex a s it appears? It seems to me that the process of gene suppression accompanying position-effect variegation is so ubiquitous, so subject to the stringent cis-trans rules of action, and so polarized in its suppression, that it cannot be complex a t the molecular level. The basic simplicity of the process remains hidden among the great diversity of experimental observation, and I wish to suggest that the primary reason for this shroud is that many of these observations are really irrelevant to the basic mcchanism involved. The reasons for this irrelevancy are threefold: (1) There has been a failure t o diatinguish between the determinative event which underlies the pattern of variegation and the differentiative event which exposes the variegated gene expression; (2) the cytology and cytochemistry is often done on tissues other than those known to be expressing variegation; (3) the variegating phenotypes usually studied are so far removed from the molecular level of the suppression that they are relatively uninformative insofar as the basic process is concerned. Each of these points will be developed more fully.

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A. DETERMINATIVE AND DIFFERENTIATIVE EVENTS The evidence is quite clear in several Drosophila cases that the pattern of eye-pigment variegation is determined in the anlage a t the end of the first larval instar. It is a t this time that the cells are programmed for their eventual activity in eye pigmentation or, to be more explicit, it is a t this time that the program of gene suppression associated with the position-effect aspects of variegation is laid down. It seems valid, therefore, to assume as a working hypothesis that the molecular action lying a t the basis of the variegation is occurring a t this stage. As has been previously discussed, i t is likely that this basis lies in the transcription or, possibly, the translation process. In any event, this is the stage of the developing eye anlage when one should be looking for the presence of new molecules, possibly attached to the sites of affected genes which have the potentiality of blocking transcription when it eventually occurs. The evocation of this early determined program only becomes a morphological reality after the tissue, or the process expressing variegation, becomes differentiated. Certainly the predetermined program can be modulated during the differentiative process, and certainly different morphological structures differentiate a t different developmental times. It would seem logical to attempt to divide the confusing array of facts a t our disposal concerning position-effect variegation into two groups: those most likely dealing with the determinative event and thus a t the basis of variegation, and those dealing with the secondary differentiative process. It is fortunate that in Drosophila the determination of the pattern of eye pigmentation and the differentiation of pigment are so widely separated in time that experimental distinction between the processes is possible. Let us first consider two modifying effects of variegation which almost certainly act on the differentiative process: temperature and chromosomes added to the genome (Becker, 1961). Experiments by Becker (1961) and Chen (1948) show that the long-known enhancement of the mutant area in a variegated eye caused by decreased temperature is observed solely for a lowering of the temperature during pupation when pigment differentiation is occurring; a lowering during the larval instars is ineffective. From a study of the relative number of eye sectors expressing the mutant phenotype upon combining temperature shifts and added Y chromosome material to the genome, Becker (1961) argues that Y suppression cannot be acting by shifting the time of the determinative event that affects the pattern. Although the ~

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evidence in this case is circumstantial instead of being direct, his conclusion seems reasonable. Therefore, temperature and perhaps Y suppression are only modulating a previously determined pattern, as if they were effective by changing a threshold for pigment production, conceivably by acting a t the enzyme-substrate level. It is doubtful if the reported (Schultz, 1956, 1965) effects of Y chromosome on RNA metabolism, while interesting in themselves, will tell us much about the basis of variegation. The time separation of the determination and the differentiation of variegation allows for a simpler understanding of many spreading effects in Drosophila (see Section 11, E). Schultz (1965) assumes that the reason the roughest areas in an eye may or may not show white facets when both w+ and rst+ are expressing variegation and the break is closer to rst is that “, . . the effect on the arrangement of the ommatidia occurs prior in development to that on the eye pigment, so that one has within a roughest area both normally colored and changed facets.” Of course, as he states, it would be necessary to assume the reverse developinental timing if the break in a rearrangement was closer to w than rst, and the roughest areas were invariably white. This precise test, however, has not been made to my knowledge. Cohen’s data (1962) suggest that there is no reversal of developmental timing. It is much simpler to assume that a polarized suppression of potential gene activity occurs a t the pattern-determining stage and spreads from the breakpoint. If rst+ is closer t o the breakpoint and it is suppressed, then a more distal gene like W + may be suppressed in some cells and not in others. The embryological fact that the differentiation of facet arrangement is evident a t the end of the third instar whereas pigmentation occurs in the mid-pupal period is irrelevant to the spreading effect. The fact that position effect may suppress genes whose morphological expression may become differentiated a t different developmental periods is no evidence that the initial suppression occurred a t different times. I do not wish to leave the impression that these differentiative events are unimportant or uninteresting, but it does not seem likely that we can understand their modulating effects on variegation until the basis of the suppression pattern determined a t a much earlier developmental stage is known.

B. TISSUESPECIFICITY OF VARIEGATION

If one sets out to use the tools of cytology and cytochemistry to investigate Drosophila chromosomes in variegated tissue, the almost

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invariable result is that the tissue exhibiting variegation has chromosomes that are unsuitable for analysis. One cannot observe, for example, if there is “heterochromatization” of the rearranged chromosome carrying the wild-type alleles of the affected genes within the mutant part of the variegated eye tissue and not in the wild-type part. Therefore as a substitute, the cytologically beautiful salivary gland chromosomes are examined but, as a matter of fact, there is no evidence that variegated suppression of the genes under investigation is actually occurring in this tissue. We just do not know if there if, any tissue specificity involved in the initial determination of suppression. Even if not, we do not know if the determination occurs a t the end of the first instar in wing or leg anlagen as it does in the eye anlage. Furthermore, even though the determinative event may occur in all tissues, it seems unlikely that this determination woiild become differentiated into a structure with the same cytological appearance in all tissues. This is why the cytological study of Ohno and Cattanach (1962) on the chromosome appearance within the nuclei of cells of the mutant and wild-type areas of the variegated mouse coat was so potentially important. It is unfortunate that in vertebrates the pigment granules which determine the coat color may migrate into the skin cells from areas quite distantly removed; therefore, evaluation of their results is difficult. Perhaps the picture is not quite so dismal. Schultz (1965) discussed and Rudkin (1965) pictured the salivary gland chromosomes in a case of white variegation in which the region (including the white locus) close t o the break is sometimes compacted in the translocated chromosome as compared with the normal X in the same cell. A band between the break and the white locus that normally puffs does not do so in the compacted member, an indication of the suppression of gene activity. In view of these circumstances, it might be profitable to look for position-eff ect variegation of chromosome morphology in cells on which localized cytochemistry can be accomplished ; for example, variegation in the puffing pattern of a particular band in salivary gland chromosomes caused by a rearrangement involving a heterochromatic region, If such variegation could be found, and if i t obeyed the criteria set out for variegations of the position-effect type, then cytochemical analysis could be done on cells with both wild-type and mutant puffing within the same gland. It should be recalled, however, that even if all this could be accomplished, one would still have to determine if this variegated puffing were the differentiative event or the determinative event. It is conceivable that the pattern of cells within the gland which will show chromosomal puffs a t a part,icular time has already been

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determined during the cell divisions of gland anlage, and that the cytochemical analysis would be relevant primarily to the differentiative process. Cell lineage studies would be informative for this question (Baker, 1967). It is important to establish cases of variegation in the puffing patterns of salivary gland chromosomes per se because Rudkin (1965) has established the fact that in these chromosomes, in contrast to neuroblast cells, the DNA in the heterochromatic regions replicates very little, or possibly very late. Since he and Schultz attribute the variegated phenotype to this abnormal replication, known positioneffect variegation within the salivary gland is essential for support of their hypothesis.

C. POSSIBILITY OF STUDYING VARIEGATED TRANSCRIPTION One of the advantages of studying variegation of a process like chromosome puffing over eye-pigment variegation is that the phenotype is much closer to the initial gene suppression: a long series of biosynthetic pathways does not intervene. An even more revealing phenotype would be a study of the variegation of the transcribed RNA itself. Such a product is available for study in Drosophila: the ribosomal RNA which is transcribed from the genes in the nucleolusorganizer region of the X and Y chromosomes (Ritossa and Spiegelman, 1965). Since these genes are normally located in a heterochromatic region, one would expect them to show variegated expression if there were a rearrangement with a break between their loci on the X chromosome and the centromere such that they would be moved to a distal euchromatic region. Inversions sc8 and scsl have appropriate breakpoints, and interestingly, sca/O and scsl/O males are usually inviable. Is this lethality caused by position-eff ect variegation of the genes in the euchromatic tip of the X whose lethality is expressed in the absence of the Y chromosome (an element that retards mutant expression in a variegated phenotype), or is the lethality caused by variegated expression of the ribosomal RNA genes in the absence of a normal set of these genes in the Y chromosome? The first alternative may be unlikely since the addition of a duplication of the tip of the X down to sc (scJ4, a translocation of the X tip to the tip of 3L) does not allow either the sca/O or the scS1/0 males to survive (Baker, 1968). Although this rules out variegation of lethal genes located on the X to the left of sc as being the lethal agent, there are known lethal genes close to the

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right of sc, and duplications covering these are being tested. If the latter duplications do not cover the lethality, i t will be interesting to study the r-RNA content of the scH/O and scsl/O larvae before their death. I n this connection one recalls the interesting case described by Green (1960) in which alleles of the hairy gene (chromosome 3) showed enhanced expression when present with X inversions in which the break was between the nucleolus organizer and the centromere, but no such enhancement when an X inversion (sc4) with breaks distal to the nucleolus organizer was used, Although this is probably a position effect on the Hw locus a t the tip of the X chromosome, it might possibly be an expression of position-eff ect Variegation of the r-RNA genes. REFERENCES Altorfer, N. 1952. Influence du chromosome Y sur I’expression de la mutation cubitus interrupts ches Drosophda melanogaster. Ann. SOC. Roy. 2001.Belg. 82, 447-462. Baker, W. K. 1953. V-type position effects of a gene normally located in heterochromatin. Genetics 38,328-344. Baker, W. K. 1954. Data on the physical distance breakage point and affected locus in a V-type position effect. J. Heredity 45, 65-68. Baker, W. K. 1963. Genetic control of pigment differentiation in somatic cells. Am. Zoologist 3, 57-69. Baker, W. K. 1967. A clonal Rystem of differential gene activity in Drosophila. Develop. Biol. 16, 1-17. Baker, W. K. 1968.Unpublished data. Baker, W. K., and Rein, A. 1962. The dichotomous action of Y chromosomes on the expression of position-effect variegation. Genetics 47, 1399-1407. Baker, W. K., and Spofford, J. B. 1959. Heterochromatic control of position-effect, variegation in Drosophila. Texas Univ. Publ. 5914, 135-154. Baker, W. K., and Swift, H. 1967.Unpublished data. Becker, H . J. 1957.Uber Rongenmosaikflecken und Defektmutationen am Auge von Drosophila und die Entwicklungsphysiologie des Auges. Z . Znduktive Abstammungs- Vererbungslehre 88, 333-373. Becker, H. J. 1961. Untersuchungen sur Wirkung des Heterochromatins auf die Genmanifestierung bei Drosophila melanogaster. Verhandl. Deut. Zool. Ges. Bonn-Rhein Suppl. 24, pp. 283-291. Becker, H. J. 1966. Genetic and variegation mosaics in the eye of Drosophila. In “Current Topics in Developmental Biology” (A. A. Moscona and A. Monroy, eds.), Vol. 1,pp. 155-171. Academic Press, New Pork. Brosseau, G. E., Jr. 1960a. Genetic analysis of the male fertility factors on the Y chromosomes of Drosophila melanogaster. Genetics 45, 257-274. Brosseau, G. E.,Jr. 1960b. V-type effects influencing the action of the Bar locus in Drosophila. Genetics 45, 979.

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Broeseau, G. E.,Jr. 1964. Evidence that heterochromatin does not suppress V-type position effect. Genetics 50, 237. Brown, S. W. 1966. Heterochromatin. Science 151, 417-425. Catcheside, D. G. 1947.The P-locus position effect in Oenothera. J. Genet. 48, 3142. Cattanach, B. M.1961. A chemically-induced variegated-type position effect in the mouse. 2. Vererbungslehre 92, 165-182. Cattrtnach, B. M. 1966. The location of Cattanach’s translocation in the Xchromosome linkage of the mouse. Genet. Res. 8, 253-256. Chen, S. Y. 1948. Action de la temperature sur trois mutants iL panachure de Drosophila melanogaster: wpss-18,wm6, et z. Bull. Biol. France Belg. 82, 114-129. Cohen, J. 1962. Position-effect variegation at several closely linked loci in Drosophila melanogasler. Genetics 47, 647-659. Cooper, K. W. 1956.Phenotypic effects of Y chromosome hyperploidy in Drosophila melanogaster and their relation to Variegation. Genetics 41, 242-654. Cooper, K. W. 1959. Cytogenetics analysis of major heterochromatic elements (especially X h and Y) in Drosophda mdanogaster and the theory of “heterochromatin.” Chromosoma 10,535488. Demerec, M. 1941. The nature of changes in the white-Notch region of the Xchromosome of Drosophila melanogaster. Proc. 7th Intern. Congr. Genet., Edinburgh, 1939. pp. 99-103. Demerec, M., Slizynska, H. 1937.Mottled white 258-18 of Drosophila melanogastor. Genetics 22,641-649. Dubinin, N. P.,and Sidorov, B. N. 1935. The position effect of the Hairy gene. Biol. Zh. 4,555-568. Eicher, E. M. 1967. The genetic extent of the insertion involved in the flecked translocation in the mouse. Genetics 55, 203-212. Girvin, E.C. 1949. X-ray produced mutations, deletions and mosaics in Drosophila virilia. Texas Univ. Publ. 4920, 42-56. Gowen, J. W., and Gay, E. H. 1933a. Eversporting as a function of the Ychromosome in Drosophila melanogaster. Pmc. Natl. Acad. Sci. U.S. 19,122-126. Gowen, J. W.,and Gay, E. H. 1933b. Effect of temperature on ever-sporting eye color in Drosophila melanogaster. Science 77, 312. Green, EM. M. 1960.A new heterochromatin effect in Drosophila melanogaster. Proc. Natl. Acad. Sci. U S . 46, 524-528. Grell, R. F. 1958. The effect of X-chromosome loss on variegation. Drosophila Inform. Serv. 32, 124. Grell, R. F. 1959.The Dubinin effect and the Y chromosome. Genetics 44, 911-922. Griineberg, H. 1937. The position effect proved by a spontaneous reinversion of the X-chromosome in Drosophila melanogaster. J. Genet. 34, 169-189. Hadorn, E. 1965. Problems of determination and transdetermination. Brookhaven Symp. Biol. 18, 148-161. Hadorn, E., and Mitchell, H. K. 1951. Properties of mutants of Drosophila melanogaster and changes during development as revealed by paper chromatography. Proc. Natl. Acad. Sci. U S . 37, 650-665. Hannah, A. 1951. Localization and function of heterochromatin in Drosophila melanogaster. Advan. Genet. 4, 87-125. Healer, A. Y. 1958. V-type position effects a t the light locus in Drosophila melanogaster. Genetics 43. 395-403. Hessler, A. Y. 1961. A study of parental modification of variegated position effects. Genetics 46, 463-484.

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Hinton, T. 1950. A correlation of phenotypic changes and chromosomal rearrangements at the two ends of an inversion. Ge,rretics 35, 188-205. Hinton, T., and Goodsmith, W. 1950. An analysis of phenotypic reversions a t the brown locus in Drosophila. J . E x p t l . Zool.114,103-114. Judd, B. H. 1955. Direct proof of a varicgated-t>ype posit,ion effect a t the white locus in Drosophila melrrrroga.ster.Grnetics 40, 739-744. Khvostova, V. V. 1939. The role played by the inert chromosome regions in the position effect. of the cuhitus intwruptus gene in Drosophila melanogasler. Bull. Acad. Sci. USSR, Ser. Biol.pp. 541-574. Lewis, E. B. 1960. The phenomenon of position effect. Advan. Genet. 3, 73-116. Lindsley, D. L., and Grell. E. H. 1968. “Mutants of Drosophiln melanogaster” (in preparation). Lindsley, D. L., Edington, C. W.,ant1 von Halle, E. 8. 1960. Sex-linked recessive lethals in Drosophila whose expression is siippressed by the Y chromosome. Genetics 45,1649-1670. Liining, K. C. 1954. Maternal influence on ii crise of variegation in Drosophila melanogaster. Hercditas 40, 538-541. Lyon, M. F. 1961. Gene a h o n in the X-chromosome of the mouse ( M u s r n u s c z h ) . Nature 190, 372-373. Lyon, M. F. 1966. Lack of evidence that inactivat,ion of t.he mouse X-chromosome is incomplete. Genet. Res. 8, 197-204. Lyon, M. F., Searle, A. G . , Ford, C. E., and Ohno, 8. 1964. A mouse translocation suppreseing sex-linked variegation. Cytogenelics (Basel) 3, 306323. Morgan, T. H., and Schultz, J. 1942. Invrstigat,ions on the constitution of t,he germinal material in relation to heredity. C’amrgie Znst. Wash. Year Book 41, 242-245. Noujdin, N . I. 1944. The regularities of hrterochromatin influence on mosaicism. J . Gen. Biol. (USSR) 5, 357-388. Noujdin, N. I. 1946. The role of hybridization in variability. I. Influence of heterozygous structure upon variegat,ion of mosaic characters. 11. Influence of an extra heterochromatin upon variation of the wild type allelomorph for bobbed Drosophdu melanogaster. J . Gen. Biol. (C’SSR) 7, 176-208. Novit,ski, E. 1961. The regular reinversion of the roughest, inversion. Genetics 46, 711-717. Ohno, S., and Cattanach, B. M. 1962. Cytological study of an X-aut,osome translocation in M m musculus. rytogenelics (Basel) 1, 129-140. Oster, I. I. 1957. Two unusual cases of whit,e-variegat,ion. Drosophila Inform. Serv. 31, 150. Panshin, I. B. 1935. New evidence for the posit,ion effect hypothesis C m p t . Rend. Acad. Sci. USSR [ N S . ] 4 , 8 5 4 8 . Perez-Dftvila, Y., and Baker, W. K. 1967. EKect of actinomycin D on the development of the early imaginal eye disks of Drosophila melariogasfer. Develop. B i d . 16, 18-35. R,atty, F. J., Jr. 1964. Gene itction and posit,ion effect in duplications in Drosophila melanogaster. Genetics 39, 513-528. Ritossa, F. M., and SpiegeIman, S. 1966. Localization of DNA complementary to ribosomal RNA in the nucleolus organizer region of Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S. 53, 737-745.

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Rudkin, G. T. 1966. The structure and function of heterochromatin. Proc. 11th Intern. Congr. Genet., The Hague, 1963 Vol. 2, pp. 369-374. Pergamon Press, Oxford. Russell, L. B. 1963. Mammalian X-chromosome action : Inactivation limited in spread and in region of origin. Science 140, 976-978. Russell, L. B. 1964. Another look at the single-active-X hypothesis. Trans. N.Y. Acad. SCi. 26, 726-736. Russell, L. B., and Bangham, J. W. 1959. Variegated-type position effects in the mouse. Genetics 44, 632. Russell, L. B.,and Bangham, J. W. 1961. Variegated type position effects in the mouse. Genetics 46, 609-626. Russell, L. B., and Montgomery, C.S. 1966. The use of X-autosome translocations in locating the 1-chromosome inactivation center. Genetics 52, 470-471. Russell, L. B., Bangham, J. W., and Saylars, C. L. 1962. Delimitation of chromosomal regions involved in V-type position effects from X-autosome translocations in the mouse. Genetics 47,981-982. Schneider, I. 1962. Modification of V-type position effects in Drosophila virilk. Genetics 47, 26-44. Schultz, J. 1936. Variegation in Drosophila and the inert chromosome regions. Proc. Natl. Acad. Sci. U.S. 22, 27-35. Schultz, J. 1941.The function of heterochromatin. Proc. 7th Intern. Congr. Genet. Edinburgh, 1939 pp. 267-262. Schultz, J. 1956. The relation of the heterochromatic chromosome regions to the nucleic acids of the cell. Cold Spring Harbor Symp. Quant. Biol, 21, 307-32&, Schultz, J. 1966.Genes, differentiation, and animal development. Brookhaven Sump. Biol. 18,116-147. Shoup, J. R. 1966. The development of pigment granules in the eyes of wild type and mutant Drosophila melanogaster. J . Cell. Biol. 29, 223-249. Slatis, H. M. 1956a. A reconsideration of the brown-dominant position effect. Genetics 40,246-261. Slatis, H.M.1965b.Position effects at the brown locus in Drosophih melanogaster. Genetics 40, 5-23. Slizynski, B. M. 1967. Oocyte pachytene analysis of Cattanach's fd translocation. Genet. Res. 9, 17-22. Spofford, J. B. 1968. Parental control of position-effect variegation. Proc. 10th Intern. Congr. Genet., Montreal, 1968 Vol. 2,p. 270. Spofford J. B. 1959. Parental control of position-effect variegation. I. Parental heterochromatin and expression of the white locus in compound-X Drosophila melanogaster. Proc. Natl. Acad. Ed. U S . 45, 1003-1007. Spofford, J. B. 1961. Parental control of position-effect variegation. 11. Effect of parent contributing white-mottled rearrangement in Drosophila melanogaster. Genetics 46,1161-1167. Spofford, J. B. 1962. Direct and parental phenotypic effects of a euchromatic variegation-suppressor locus in Drosophila melanogaster. Genetke 47, 986987. Spofford, J. B. 1966. Non-specific genetic control of variegation in Drosophila melanogaster. Proc. 11th Intern. Congr. Genet., The Hague, 1965 Vol. 1, p. 41. Pergamon Press, Oxford. Spofford, J. B. 1966.Variegation in progeny of mothers homozygous or heterozygous for rearrangement. Drosophila Inform. Serv. 41, 82-83.

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Spofford, J. B. 1967. Single-locus modificat~ionof position-effect variegation in Drosophila melanogaster. I. White variegation. Genetics 57, 751-766. Stern, C., and Kodani, M. 1966. Studies on the position effect of the cubitus interruptus locus of Drosophila melanogaster. Genetics 40, 343-373. Wolfe, H. G. 1967. Mapping the hemoglobin locus in mice transmitting the flecked translocation. Genetics 55,213-218.

GENETIC ACTIVITIES OF THE Y CHROMOSOME IN Drosophila DURING SPERMATOGENESIS Oswald Hess* and Gunther

F.

Meyer

Max Pbnck-lnrtitut &r Biologie, Tubingen, Germany

I. Genetic Studies of the Actions of the Y Chromosome on Male Fertilit,y . . 171 11. Cytogenetic Studies of the Y Chromosome . . . . . . . . . . 176 A. Morphological Appearance of Spermatorytc Structure6 . . . . . . 176 B. The Role of the Y Chromosome in the Formation of the Spermatocyte Structures . . . . . . . . . . . . . . . . 183 C. The Functional Nature of the Y-Chromosomal Loops . . . . . . 187 D. The Determination of the Shape of thc Y-Chromosomal Loops . . . 196 111. Morphogenetic Effects of the Y-Chromosomal Loops in Spermiogrnesis . 199 A. Normal Spermatogenesis in D . melanogaster and D . hydei . . . . 200 B. Spermatogenesis in XO Males and Y-Deficient Males of D . melanogaster . . . . . . . . . . . . . . . . . . 208 C . Spermatogenesis in XO Males and Y-Deficient Males of D . hgdei . . 209 D. Thr Influence of the Y-Chromosomal Idoopson Growth of Spermatozoa . 211 IV. Conclusions . . . . . . . . . . . . . . . . . . . . . 212 V. Summary . . . . . . . . . . . . . . . . . . . . . 216 References . . . . . . . . . . . . . . . . . . . . . 218

1. Genetic Studies of the Actions of the Y Chromosome on Male Fertility

Bridges (1916) was the first to demonstrate that in Drosophila rnelanogaster the processes of spermiogenesis are under the control of genetic factors. He found exceptional patroclinous males which lacked a Y chromosome. Viability and phenotype of such XO males, as they are usually called, are as a rule not changed. However, they are completely sterile. Thus, factors located in the Y chromosome are involved in the formation of normal functioning sperm. Stern (1929) found that males of D . melanogaster with deficiencies for only a part of the Y chromosome-for instance, either the long or the short arm-are also sterile. He concluded that each of the two arms of the Y chromosome carries a fertility factor or a complex of such factors. The two com-

* Present

address : Zoologisches Institut der Universitiit, Freihurg, Germany. 171

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plexes, K1 in the long arm and K2 in the short arm, are not complementary. Therefore, a defect in the complex K1 cannot be compensated by duplications of complex K2. Neuhaus (1939) tried to obtain more detailed information about the number, sequence, and localization of the fertility genes in the Y chromosome of D . melanogaster. According to a hypothesis of position effects, which in those days was widely accepted, he based his experiments on the assumption that any break of the Y chromosome would cause a defect or mutation in the fertility gene lying near the breakage point. Therefore, reciprocal translocations in which the Y chromosome was involved were expected necessarily to result in male sterility. Moreover, in different translocations with different breakage points in the Y chromosome different genes should be affected. Therefore, complementation could be expected if a proximal translocated fragment was combined with the distal Y fragment of another translocation in which the breakage point in the Y chromosome was located proximally to that of the first translocation. The reciprocal combination should be sterile, under these conditions. One would also expect that combinations of Y fragments of other pairs of translocations would not be complementary, namely, in those cases in which the proximal Y fragment was derived from a translocation with a breakage point proximally to that of the second translocation from which the distal Y fragment was derived. Indeed, Neuhaus found complementation and noncomplementation in a large series of combination experiments. The resultg were consistent with the interpretation that the long arm of the Y chromosome has a t least five fertility genes, one of which presumably has an allele in the X chromosome near the locus of bobbed. In addition, the short arm seemed to have a minimum also of five fertility genes. However, our present knowledge indicakes several errors that may have occurred in these experiments (see also the criticism of Brosseau, 1960, p. 269). Translocations were recovered only by genetic methods, namely, by searching for newly induced mutations of ci after irradiation of ci+ males. These new mutations were considered to be position effects caused by translocations. Of all “translocations” found in this way 85% turned out to cause sterility in males under special conditions. (The new mutations were kept together with X.YL or X.Y8 compound chromosomes; in this system the X-YL chromosome would cover induced defects of fertility factors in the long Y arm, and X*YS would cover defects in the short Y arm; in this way “sterile” reciprocal translocations could be maintained and, if wanted, sterile males for experiments could be obtained by replacing the compound chromosome by a normal X chromosome.) Those translocations that

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caused sterility were considered to be Y-IV, the rest, which did not affect sterility, were considered to be II-IV and III-IV translocations. There was no further attempt to prove that Y-IV translocations had really occurred. In fact, Neuhaus was not able to see cytologically translocation chromosomes in any of his translocation stocks. To explain this observation, the author assumed that the translocated Y fragments were always of a size close to, or even smaller than, the dotted chromosome IV and were, therefore, invisible in cytological preparations. Furthermore, from this assumption he concluded that both groups of fertility factors were located in small clusters a t the very tip of each arm of the Y chromosome. More recently, the fertility factors in the Y chromosome of D. mehogaster have been investigated by Brosseau (1960). Males possessing a marked Y chromosome (sc8.Y which carries a small X duplication including y f)were irradiated and mated with females homozygous for an attached X - Y compound chromosome. As this compound chromosome contained all fertility factors of the Y chromosome, FI males were fertile even in cases where the irradiation had caused defects in the free Y chromosome. In order to detect the cases with sterile Y chromosomes, F1 males were pair-mated with females of the constitution In(1)dl-49, ywm/X.Y, yB (the first X chromosome carries two inversions and, therefore, inhibits crossing-over nearly completely ; the second is the above-mentioned compound chromosome, marked with y and B ) . If the irradiation had caused sterility in the free Y chromosome, one class of males in the F B progeny, namely, the In(1)dl-49 males, would be sterile. The second class would be fertile, because they contain all necessary fertility factors in the compound chromosome. In this way i t was possible to detect defects in the Y chromosome and, a t the same time, to maintain the sterile Y chromosomes. A number of such sterile Y chromosomes have been recovered. Whether factors on the long or on the short Y arm had been changed was determined in the next step of the investigations. This was done by combination of each defective Y chromosome with either an ;Y.Y1. or X.Ys compound chromosome in order to check which of the two was complementary. Second, pairs of mutated Y chromosomes defective in the same complex were combined and tested for complementation. As a result of a great number of such combination crosses it was found that there are at least five fertility factors in the long arm and two in the short arm of the Y chromosome. Some information of the loci of these seven factors has also been obtained. Sterile Y’s were combined with different X-Y translocations

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of known cytogenetic constitution. These translocations had been induced by detachments in XX/Y females (“capped” and “captured” X chromosomes; see Kau’ffnsnn, 1933; Abrahamson et al., 1956). The findings of these experiments are consistent with the view that the factors are arranged in a linear sequence in the Y chromosome. Moreover, the proximal-distal direction of each group of fertility factors has been revealed. The fertility factors are not all located at the tips of the two arms of the Y chromosome. There is also some evidence that the complexes of fertility factors in both Y arms are not located in clusters, as Neuhaus had formerly claimed. It is clear from these investi,gations that both arms of the Y chromoEome contain several fertility factors each of which has its own specific function. Therefore, they all are essential, and the absence or mutation of one of these factors cannot be compensated by an excess of other regions of the Y chromosome. However, the X and the Y chromosomes segregate during meiosis and only one half of the spermatids possess a Y chromosome. In spite of this, both X- and Y-bearing spermatids differentiate normally. On the other hand, X spermatids of XO males which have exactly the same genetic constitution as the X spermatids of normal males are not able to differentiate into normally functioning sperm. In addition, spermatids lacking both the X and Y chromosome are well able to form completely normal sperm if they occur in normal males, for instance, after nondisjunction of the X and Y chromosomes (Kuhn, 1930) or in stocks with attached X.Y compound chromosomes. In contrast to this, spermatids of the same genetic constitution in XO males cannot differentiate into normal spermatozoa. Thus, spermiogenesis appears to depend on the genetic constitution of the male as a whole, but not on the genetic constitution of the differentiating spermatids themselves. Experiments designed to clarify the mechanisms involved here have been carried out by Stern and Hadorn (1938). Testes of sterile males with the constitution X.YL were transplanted into fertile hosts, and testes of fertile donors into sterile hosts. In both types of experiments the transplanted testes could occasionally fuse with the testes of the hosts. Because one of the partners in the experiment was always marked with white which causes also colorless testis sheets, the fusion of the transplanted and host testes could be directly checked in the microscope. With the aid of this marker gene it was also possible to demonstrate that whenever a fusion was fertile, the progeny was only derived from the supplier of germ material containing a complete Y chromosome. There was no inhibition of sperm differentiation in transplanted genetically fertile material by the surrounding steriIe host tissue, and also no stimula-

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tion of sperm differentiation in sterile transplants by the surrounding tissue of a fertile host. Two possible explanations for these results have been discussed. First, it seems possible that the genetic constitution of a certain big nutritive cell is decisive. During one stage of spermiogenesis the differentiating spermatids of a bundle have their heads regularly inserted into such cells. There could be some specific local influence of the nutritive cell on the attached spermatids which determines their fate. The second and more likely explanation assumes that the genetic constitution of the male germ cells in the diploid phase before meiotic reduction is itself decisive. This would mean that the actions of the fertility factors in the Y chromosome are cell autonomous and t.hat the capacity for normal spermatid differentiation is predetermined. I n other words, the differentiation processes in spermatids are directed by preformed and stored key substances (messenger RNA?). This view is in accordance with the finding that spermatid nuclei are synthetically inactive (Henderson, 1964; Das et a!., 1965; Olivieri and Olivieri, 1965; and many others). In addition to the effects 011 fertility other genetic effects of the Y chroinosonie have been found. For instance, chromosome rearrangements in which the Y chromosome is involved may show position effects and position effects of the variegation type may be strongly modified by changes in the Y constitution (Lewis, 1950; Gowen and Gay, 1933. 1934; Dubinin and Heptner, 1935; Schultz, 1936; Baker and Spofford, 1959). Phenotypic effects of quantitative characters such as changes of number and size of chaetae or wing hairs may also be influenced by Y chromosomes (Mather, 1944; Bnrigozzi, 1948, 1951 ; Barigozzi and DiPasquale, 1953). Special types of lethal factors have been found which are suppressed by the Y chromosome (Lindsley e t al., 1960; Hess, 1963). Supernumerary Y cliromosonies may cause mottling of the eyes (Cooper, 1956), and sterility of males and females (Morgan e t al., 1934; Cooper, 1949; Grell, 1959). It is also known that the Y clironiosome contains an allele of bobbed and a nucleolus organizer (Stern, 1927, 1929; Heitz, 1934). Their exact loci have not been established with certainty. I n conclusion, the studies described here have shown that the Y chromosome of Drosophila does not contain genetic information that would be indispensable for somatic development. However, i t carries factors that are essential in spermiogenesis. Whether or not these fertility factors are conventional genes is not yet clear. Cytologically, the Y chromosome is seen to be entirely “heterochromatic,” and it is now widely accepted that the genetic material of the heterochromatin is

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in a state that is not competent to react on the normal gene activation processes; in other words, heterochromatin is thought to be genetically inactive. However, there is experimental evidence that heterochromatization of a chromosome or a part thereof is not an irreversible inactivation of genetic material. Thus, the possibility suggests itself that the fertility factors which are located in the Y chromosome may be genes functioning exclusively during spermiogenesis. A new approach to solve these problems experimentally has recently been found in cytogenetic studies of spermatocyte nuclei, the results of which will now he discussed. II. Cytogenic Studies of the Y Chromosome

A. MORPHOLOGICAL APPEARANCE OF SPERMATOCYTE STRUCTURES In the primary spermatocytes of D.melanogaster during their growth stage intranuclear structures are found that are characteristic of this stage of the male germ line cells and which have never been found in nuclei of other tissues. As will be shown later in this review, these structures are formed by the Y chromosome. They consist, as seen in the electron microscope, of tubuli with a diameter of 300 to 400 A, of reticular elements with a diameter of 100 to 200 A, and of strongly basophilic granules with a diameter of 400 t o 700 A. During diakinesis the structures are separated from the chromosomal fibrils and are decomposed (Meyer et al., 1961). So far, more than 50 different species of the genus Drosophila have been examined (Table 1). The spermatocyte nuclei of all these species contain characteristic structures. Although organized structures (tubuli, for instance) may be absent in some species, the spermatocyte nuclei always contain some striking formations that are not found in other nuclei of the organism. In general, the morphological appearance of all these structures varies strongly between different species. However, it is highly constant within the same species. It is, therefore, oftcn possible to use the different appearance of the spermatocyte structures as a reliable criterion for taxonomic classification. Even very closely related species, such as D. rnelanogaster and D. simu2ans, or D. hydei and D. neohydei, can be easily distinguished (Meyer, 1965). A more detailed and illustrated description of the specific spermatocyte structures of 54 Drosophila species has been given by Hess (196713). Especially large spermatocyte structures with a complicated morphology have been found in D . hydei and some other species belonging t o the D. hydei subgroup (Hess and Meyer, 1963b) (Fig. 1). In these

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TABLE 1 SPEEIMATOCYTE STRUCTURES IN DIFFERENT Drosonhila SPECIES Subgenus: Hirtodrosophila D . duncani: Spermatocyte nuclei comparatively small, with masses of material that cannot be identified in the light microscope, some accumulated granules. Subgenus: Pholadoris D . victoria: Spermatocyte nuclei small and spherical, with a field of homogeneous diffuse material and with granules which are arranged in rows so that a twin loop is formed. Subgenus : Dorsilopha D . busckii: Field of homogeneous material ; accumulation of granules ; many spheres of nucleolus-like material which are distributed over the entire nuclear space. Subgenus: Sophophora Species group : willistoni D . paulistomm: Large field of grana. D . willistoni: Field of homogeneous material which is composed of tubular ribbons (as seen in the electron microscope) ; globular masses of nucleolus-like material. Species group : melanogaster D . ananassae: Spermatocyte nuclei relatively small ; nucleolus spherical and not attached to the nuclear membrane; field of granular material around the nucleolus. D . melanogaster: Masses of tubular ribbons; aggregations of reticular elements ; accumulations of granules. D , sirnulam: A field of tubular masses; one pair of thin and short compact threads ; aggregations of granules ; nucleolus surrounded by a hollow sphere of a dense network of strongly osmiophilic material. Species group : obscura D.azteca: Small field of homogeneOUE diffuse material; very small granules which are often accumulated into two distinct groups.

Species group: obscura (Cont.) D . athabasca: Very small field of diffuse material; some groups of very small granules which are nearly invisible in the light microscope. D . miranda: Aggregations of grainula; some homogeneous diffuse material. D . persimilis: Very small granules of nucleolus-like material; diffuse material which appears in the electron microscope to be composed of granular material. D . pseudoobscura: Many spheres of nucleolus-like material ; masses of granular and tubular material. D. afink: Field of diffuse granular material ; nucleolus relatively large Subgenus : Drosophila Species group : virilk D . montana: Spermatocyte nuclri spherical, of medium size; nucleolus loosely attached to the nuclear membrane; large field of completely homogeneous material which occupies nearly the whole nuclear space. D . borealis: Spermatocyte nuclei spherical ; nucleolus relatively large, spherical, with eccentrically located vacuoles; aggregation of highly refractive grana which are attached to a ground substance possibly of the shape of a loop. D. americana: Spermatocyte nuclei large and spherical; nucleolus spherical and hollow with only a thin wall; large field of homogeneous material which appears light gray in phase contrast, D . virilis: Large accumulations of grana; masses of diffuse material which in the electron microscope seems to be composed of tubuli. D. novamexicana: Field of coarse reticular material.

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TABLE 1 (Continued) Species group : virilis (Cont.) D . texana: Spermatocyte nuclei large and spherical; nucleolus large and eccentrically located ; small field of rather coarse and refractive reticular material; one body of irregular shape and a spongy ground substance (similar to pseudonucleolus of D . hydei). Species group : funebris D . funebris: Big spheres of nucleolus-like material; field of homogeneous diffuse material. Species group: repleta. Subgroup : hydei D . bifurca: Spcrmatocyte nuclei peardaped; nucleolus attached to the nuclear membrane; one pair of threads with compact and diffuse sections; one pair of completely diffuse threads which originate from a field of tubular material; one pair of diffuw threads to which are attached many granules; many large and highly refractive grana which are distributed all over the nuclear space (Fig. 1). D. eohydei: Spermatocyte nucleus pear-shaped ; nucleolus attached to the nuclear membrane; large but, thin plate of tubular material; pseudonucleolus ; one pair of clubs (Fig. 1). D. hydei: Spermatocyte nucleus pear-shaped ; nucleolus attached to the nuclear membrane; one pair of threads with highly compact and diffuse sections; one pair of clubs; pseudonucleolus with one pair of rones; one or two loop pairs of tubular ribbons; one or t8wopairs of nooses (Fig. 1). D. neohydei: One pair of threads with highly refractive and diffuse sections; one pair of long and thin clubs; one pair of dense knots of tubular ribbons; diffuse field of tubular material; big grana distrib-

Sp. repleta. Subgroup : hydei (Cont.) uted over the whole nuclear space (Fig. I ) . D . nigrohydei: One pair of diffuse threads ; accumulation of grana of different sizes; small group of tubular material near the nucleolus; several small fields of homogeneous diffuse material (Fig. 11, Species group : repleta. Subgroup : melano palpa D . canapdpa: Spermatocyte nuclei irregularly dumbbell-shaped ; large field of homogeneous material; many large hollow spheres; one clump of coarse reticular material. D . fulvirnacula: Spermatocyte nucleus dumbbell-shaped ; nucleolus with large vacuoles; paired pseudonucleolus body attached to nucleolus; large field of homogeneous material in nucleolus-near half of the nuclear space, with ribbon-shaped protrusions into the upper half of the nucleus ; inclusions of coarse reticular material. D . fulvimaculoides : Spermatocyte nucleus pearahaped with constriction ; nucleolus vacuolated ; near the constriction one large pseudonucleolus body; in the larger part of the nuclear space a large field of coarse reticular material. D . fkvorepleta: Spermatocyte nucleus pear-shaped; large field of homogeneous diffuse material with included grana of different sizes and accumulations of reticular elements. D. limensk: Spermatocyte nucleus dumbbell-shaped ; opposite to the nucleolus a large number of big hollow grana which are often arranged in several lines; near the nucleolus a triangular field of homogeneous diffuse material. D . melanopalpa: Spermatocyte nucleus very large, elongated, with ronstriction; many very large hol-

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TABLE 1 (Continued) Sp. repleta. Subgroup : melampalrs (Cont.) low grana which are distributed over the whole nuclear space, but arc more frequent in the part opposite to the nucleolus, often arranged in lines; grana sometimes contain a smaller granule eccentrically. D. repleta: Spermatocyte nucleus dumbbell-shaped ; one very large field of diffuse homogeneous material in which is included R small field of tubuli near the nucleolus; sonic compact globules of nucleoluslike material (accessory nucleoli) opposite to the nucleolus (Fig. 1). Species group : repleta. Subgroup : mulleri D . anceps: Spermatocyte nucleus miall and dumbbell-shaped ; small and spherical nurleolus ; large pseudonucleolus with protrusions. D. owonensis: Spermatocyte nuc h i s small and dumbbell-shaped ; large field of homogeneous diffuhe material; one very large and srveral smaller hollow grana opposite to tlic nucleolus. D. hamatojih: Nucleolus in the shape of a narrow crescent which is attached to the nuclear membrane ; small field of homogeneous material with inclusions of coarse granular material. D. hoeckeri: Spermatocyte nucleus small; field of homogeneous material ; two knots of small c~ompac-t threads. D. longicornis: Spermatocyte nucleus small with constriction ; field of homogeneous material which fills nearly the entire nuclear space; one triangular body with a reticular matrix to which are attached masses of granules located opposite to the nucleolus. D . mojavensis: Spermatocyte nucleus small and dumbbell-shaped ;

Sp. w p l e f t r . Subgroup: mislleri (Cont.) field of homogeneous material which, however, sometimes shows a separation into knots of distinct diffuse threads ; opposite to the nucleolus one large pseudonueleolus with a matrix of c’oarse filamentous material. I). pc riz~uulork; Spermatocyte nucleus of medium size and beanrhaped ; very m a l l nucleolus ; some rows of coarse reticular material to which are attached rows of granules D. rtigricrurin: Field of homogeneous diffuse material; inclusions of small unidentified structures of rompact material. D. buzzatiit Spermatocyte nucleus dumbbellshaped, with a deep constriction whic,li divides the nuclear spave into two equal parts; nucleolus often in the shape of a narrow cws,*rnt which is attached to the nuclear membrane ; several acrumulations of fine granular material which is arrungrd in the form of knotted loops. Species group : ropletn. Subgroup : mercnfonim D . rnercatorzmz: Syermatocyte nucaleus dumbbell-shaped ; field of hotnogrneous material near the nucleolus; opposite to the nucleolus n group of three grana, one in the middle very large, at its sides two smaller ones ; grana are vacuolated with it wall of densp, high refractive material and are embedded in a matrix of coarse granular material. D . pnrnnaensi?: Spermatocyte nucleus dumbbell-shaped : nucleolus large and with vacuoles; near the nucleolus a field of homogeneous material and a number of grana; the middle of the nuclear space remains empty; opposite to the nucl~olusabout 10 large grana, of a material which appears dark gray

(Continued)

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OSWALD HESS AND GUNTHER F. MEYER

TABLE 1 (Continued) Sp. repleta. Subgroup : mercatorum (Cont.) in phase contrast, and with a small hollow each. D. pararepleta: Spermatocyte nucleus dumbbell-shaped with a deep constriction; small field of homogeneous material in the middle of the nuclear space near the constriction; opposite to the nucleolus three large grana of nucleolus-like material which are embedded in a matrix. D. mercatorum: Spermatocyte nucleus dumbbell-shaped ; two fields of slightly granular material which differ in optical density; opposite to the nucleolus several groups of coarse granular material. Species group : repleta. Unclassified D . moju-like: Spermatocyte nucleus spherical with small lobelike protrusions in which the nucleolus is located; large field of homogeneous material which fills nearly the entire nuclear space; one large knot of compact threads. Species group : robusta D . robusta: Spermatocyte nucleus large and spherical; large field of homogeneous material in which are embedded one body of coarse reticular material and a number of evenly distributed large grana. Species group : cardini D. cardini: Spermatocyte nucleus spherical ; nuclear space evenly filled with homogeneous diffuse material. D. dunni: Spermatocyte nucleus very small; one small crescent-

Species group: cardini (Cont.) shaped field of homogeneous material. D. neomorpha: Spermatocyte nucleus small and spherical; nucleolus attached to the nuclear membrane, usually with two big vacuoles; near the nucleolus h o t s of diffuse thread-shaped loops. Species group : guarani D . guarani: One twin loop of thin diffuse threads which originate from the nucleolus. Species group : pallidipennis D . pallidipennis: Nucleolus in the shape of a narrow crescent which is attached to the nuclear membrane; several knotted loops of granular material originate near the nucleolus, Species group : calloptera D . omatipennis: Spermatocyte nucleus small; nucleolus in the shape of a narrow crescent which is attached to the nuclear membrane ; small field of homogeneous material near the nucleolus. Species group : rubrijrons D. parachrogaster: Spermatocyte nucleus very small and spherical; some granular material which could not be identified in the light microscope. Unclassified D . lacertosa: Spermatocyte nucleus small and spherical ; nucleolus large, spherical, and eccentrically located; several roundish bodies; one structure with reticular material to which are attached many granules.

species the structures occur as several pairs of loops, each pair having its own characteristic form. The most striking structure in the spermatocyte nuclei of D . hydei, for instance, is a pair of compact and highly refractive threads, which arise in the neighborhood of the nucleolus and become diffuse distally (Figs. 1 and 2a). The diffuse sections are continuoup with each other, so that the two compact threads are con-

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THE Y CHROMOSOME IN I ) V O S O p h i h

nected in a single loop. The threads are surrounded also in their compact sections by diffuse material, which appears in the electron microscope to be formed of tubuli. A pair of club-shaped bodies is also found in these nuclei. These structures are seen in the electron microscope to be composed of a reticular ground substance to which are attached large and usually hollow granules which proliferate many smaller granules from their surface. A third structure in the spermatocyte nuclei of D. hydei has been termed (‘pse~don~cleol~s.” In the electron microscope its fine structure is clearly distinct from that of the nucleolus, which in spermatocyte nuclei is always attached t o the nuclear membrane. The pseudonucleolus consists of a loose spongy ground substance which is penetrated by canals. These are filled with less electron-dense filamentous and reticular material. On the two opposite sides of the pseudonucleolus two dark protrusions can be recognized in phase contrast. I n many cases the pseudonucleolus consists of

Th

D.repleta

D.bifurca

0. nigmhydei

D.neohydei

D.eohydei

D. hydei

FIG.1. Diagrams of the looplike formations in sperrnatocyte nuclei of D. repleta and five species of the D. hydei subgroup. In each case on the right-hand side, a mitotic metaphase plate and an indication of the species-specific sperm length (twice the actual size) is shown. Explanation: aN, accessory nucleoli; C, clubs; N, nucleolus; P, pseudonucleolus; T, tubular ribbons; Th, threads (with c, compact, cp, compact proximal, d, diffuse, dd, distal diffuse sections).

182 OSWALD HEW AND GUNTHER I!>. MEYER

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two separate parts, which then have one protrusion each. Fourth, tubuli are found in the nuclei. They are formed into ribbons and the ribbons are wound in knots which lie in the central region of the nucleus and appear in phase contrast as a large gray area (Figs. 1 and 2a). A more detailed description of these spermatocyte structures has been given by Meyer (1963). In other species of the D. hydei subgroup, for instance, in D . neohydei, D . bifurca, D . eohydei, and D . nigroh ydei, similar intranuclear structures are found (Fig. 1). The different formations may be classified on the basis of their morphological appearance into compact threads, diffuse threads, clubs, pseudonucleoli, and bands of tubular ribbons. Not all of these different types are found in every species, and there are typical morphological properties and characteristic topographical arrangements for each particular species (Fig. 1) (Hess and Meyer, 1963b).

B. THEROLEOF

Y CHROMOSOME IN THE FORMATIOX SPERMATOCYTE STRUCTURES

THE

OF THE

I n several species, especially clearly in D. hydei, it can be observed that the spermatocyte structures in question originate from the chromocenter by some form of outgrowth or unfolding during the transition from the spermatogonia to the young primary spermatoeytes. The chromocenter is formed by the heterochromatin of both the X and Y chromosomes. Its position is marked by the nucleolus, because, as was already mentioned, these two chromosomes carry nucleolus organizers. Moreover, i t is expected that the Y chromosome is involved in the metabolism of spermatocytes since factors located in this chromosome are essential for the formation of normal sperm (see Section I ) . Therefore, a correlation between the Y chromosome and the spennatocyte structures seems likely. In fact, in D.melanogaster the spermatocyte nuclei of XO males do not contain tubuli and reticular elements. Thus, the Y chromosome must play a decisive role in the formation of these structures. The examination of males possessing only a part of the Y chromosome (only the long or the short arm, for example) showed that a t least the tubuli are not dependent on the activity of a single locus in the Y chromosome, This follows from the fact that both the X/YL and X/Ys spermatocytes contain tubuli and the other intranuclear formations. Moreover, a quantitative correlation is found: The X/YL spermatocyte nuclei possess tubuli more frequently and in larger quantities than the X/Ys spermatocytes.

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Long needle-shaped protein crystals which occur a t the same time as do the tubuli in normal spermatocytes are regularly found in XO spermatocytes of D . melanogaster. The crystals always appear when the formation of tubuli is completely blocked or seriously inhibited. A reasonable explanation is that the crystals are intermediate products in a chain of synthesis, and that they are normally used up by the activity of the Y chromosome, or in other words, by the tubuli. However, it could also be the case that the crystals are composed, a t least partially, of material which in normal spermatocytes is used for the formation of the tubuli and the other intranuclear structures and which in the absence of the Y chromosome is not organized and, therefore, crystallizes in a different form. In D . hydei, too, spermatocyte nuclei of XO males do not contain any of the looplike structures (Fig. 2b) (Hess and Meyer, 1963a). In the light microscope the nuclei appear empty except for the nucleolus and some very small autosomal loops. This confirms the conclusion that the Y chromosome is involved in the formation of the spermatocyte structures also in this species. However, the specific mode of the Y-chromosomal action is not clear from these experiments. The structures could be built either by the Y chromosome itself, or under the inductive influence of the Y chromosome by some other chromosome. This question could be cleared up by observations of males of D . hydei with two Y chromosomes. Such males were prepared by crossing homozygous white light (w It) females with wild-type males, Among the F1 exceptional matroclinous females were recovered. These females arose by nondisjunction of the maternal X chromosomes and, at the same time, possessed an additional Y chromosome. Therefore, one half of the male progeny of such nondisjunction females possessed two Y chromosomes. Indeed, spermatocyte nuclei of the males with two Y chromosomes contain a duplicated set of intranuclear structures (Fig. 2c). The observed numerical duplication clearly indicates that the structures in the spermatocytes develop exclusively from the Y chromosome. Theoretically, the entire Y chromosome could be transformed into the visible structures. If, on the other hand, the structures represent structural modifications of small segments of the Y chromosome, then large segments could exist between them which do not transform into visible structures in spermatocytes. These questions could be solved by an analysis of Y translocations (Hess, 1965~). A Y-autosome translocation has been found which has its first break in the long arm of the Y chromosome and the second immediately next to the kinetochore of one of the rod-shaped autosomes (Fig. 3). Thus, the two translocation chromosomes can segregate independently

FIG.3. Y-Autosome translocation of D. hydei. Diagram of spermatocyte nuelei with Y chromosome loops and the corresponding metaphase plates of larval neuroblast mitoses of six different male classes. Explanations : A, YLd,translocation chromosome containing a proximal fragment of the autosome A1 with only heterochromatin and the kinetochore and a distal fragment of the long Y arm ; Ys’Lp* A,, translocation chromosome containing a proximal fragment of the Y chromosome with the short Y arm, the Y kinetochore, and a proximal fragment of the long Y arm, and a distal fragmeut of the autosame At with the complete euchromatic section; A, unchanged homolog of A,; C , clubs; N, nucleolus; P, pseudonucleolus; T , tubular ribbons; Thdd, Thpc, distal diffuse, and proximal compact sections of the threads.

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OYWALD HESS AND CiUNTHER F. MEYER

without causing autosomal duplications or deficiencies which would be cell-lethal. A stock was built which does not contain a normal Y chromosome, but rather contains the two complementary translocated Y fragments. In this stock different classes of males are found (Fig. 3 ) . Besides males with a normal set of Y-chromosomal structures in their spermatocyte nuclei, others are regularly found which show partial deficiencies, partial or complete duplications of the Y-chromosomal structures. By an analysis of metaphase plates in larval neuroblast mitoses it was found that each such class of males has its specific chromosomal constitution. The two translocation chromosomes turned out to be present either once or in duplicate, or to be absent. These data are consistent with the conclusion that the sites of formstion of the thread-shaped loop and the pseudonucleolus are located in the distal part of the long arm of the Y chromosome, and that the sites for the clubs and tubular ribbons are on the remaining part of the Y chromosome, that is to say, either in the short arm or in the proximal fragment of the long arm. The localization analysis was further clarified with the aid of X-Y translocations. The genetic technique used in these experiments involves the selection of exceptional matroclinous males from the progeny of X-irracfiated, attached-X females carrying recessive markers in both Zt/Y). Most their X chromosomes and an additional Y chromosome (w __ of these “detachment males” proved to carry an X-Y translocation, in addition to their normal Y chromosome. From each case of detachment a stock was built by crossing the exceptional males in pair matings with attached-X females. In this way each translocation can be kept permanently. All males from such a stock possess a normal autosomal set, a translocation chromosome containing the euchromatic part of the X chromosome and a translocated fragment of the Y chromosome, and a free Y chromosome. The extent of the translocated Y fragment was determined in metaphases of larval neuroblasts. Which loops were formed by these fragments was established in males having no free Y chromosome. Such males were recovered in special crosses as rare cases of patroclinous exceptions occurring after meiotic nondisjunction of the X chromosomes in their mothers (Figs. 4, 5 ) . As a result of these experiments a sequence of the various loopforming sites in the Y chromosome could be established. The location and the approximate linear extension of these sites could also be defined, The threads represent a very short segment a t the very tip of the long arm, less than one-tenth the length of that arm. The site of the threads is followed immediately by that of the pseudonucleolus. The tubular ribbons and the clubs are formed by a proximal portion

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of the long arm, which is not longer than two or three tenths of the long arm. The site forming the clubs is located more proximally to the site which forms the tubular ribbons. There have been found loops of a new type, called the ‘(nooses,” which usually cannot be seen in spermatocyte nuclei because of their faint diffuse morphology and their tendency t o fuse with the other structures of the Y chromosome. However, this loop is clearly visible in nuclei where it is the only present loop (Fig. 5a). The nooses are developed by a locus in the short arm of the Y chromosome. I n summary, it seems that relatively short segments of the Y chromosome transform into the giant loops during the spermatocyte stage and that the rest of the Y chromosome either forms no loops a t all, or loops that are too small to be seen. There is also some indication that the loop-forming sites are not evenly distributed over the entire Y chromosome, but occur in groups. For instance, one such group is formed by the threads and the pseudonucleolus a t the tip of the long arm (Fig. 6). Both these structures are perhaps complex in nature, the threads being composed of compact and diffuse regions, and the pseudonucleolus being composed of a fused body with a spongy ground substance and of a pair of “cones.” Each of these subunits might be formed by its own specific site. A second group of structures is formed by the tubular ribbons, the clubs, and the nooses. Again, there is experimental evidence that the tubular ribbons and the nooses are subdivided and their subunits can be separated in translocations.

C. THEFUNCTIONAL NATURE OF

THE

Y-CHROMOSOMAL LOOPS

The observed structures formed by the Y chromosome in the spermatocyte nuclei of D . hydei and related species are paired and polarized formations. They are thus organized in a way that is reminiscent of many of the typical features of “lampbrush chromosomes,” which are found in the oocytes of many vertebrate and some invertebrate animal species, especially in the urodele amphibia (Callan, 1963). It was, therefore, not unreasonable t o homologize the intranuclear formations in Drosophila spermatocytes with the loops of lampbrush chromosomes. In other words, the Y chromosome would assume the state of a lampbrush chromosome during the growth stage of the primary spermatocytes. The loop-forming sites would correspond, then, to chromomeres which during certain phases of high synthetic activity unfold (a part of) their DNA into pairs of lateral loops (Fig. 6). However, in contrast to the conditions realized in amphibian oocytes, Drosophila displays only a limited number of very large loops. During the spermatocyte

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OSWALD HESS AND GUNTHER F. MEYER

FIa. 4. Diagram of spermatocyte nuclei of six different X-Y translocations induced by detachments of attached-X chromosomes. On top, normal X and Y chromosome, with indications of the breakage points. The translocated Y fragments carry loop-

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stage all chromosomes are in a highly uncoiled condition. Therefore, they are invisible as distinctive structures. This holds also for the Y chromosome with the exception of the loop-shaped formations, which are the only structures that can be identified individually in these nuclei. Furthermore, in those cases where no closed paired loops are seen, as, for instance, in the clubs and in the pseudonucleolus of D. hydei, one has to suppose that invisible submicroscopic connections to the-also invisible-chromomere are existing. I n such a model the clubs and pseudonucleoli would be organized in a manner similar to the so-called “giant granular” and “giant fused loops” found in oocytes of Triturus (Callan, 1963). In other spermatocyte formations, for example, the threads of D . hydei, axial breakages within chromomeres similar to those obtained experimentally in amphibian lampbrush chromosomes seem to occur regularly. Even a complete “open chain” model with (regional) separation of half-chromatids might be realized. The results of histochemical investigations of the Y-chromosomal loops in spermatocyte nuclei of D. hydei are in good agreement with the lampbrush hypothesis. In sections or total mounts of spermatocytes fixed during the growth stage all chromosomal structures are stained with toluidine blue. The staining reaction increases as the loop formation proceeds. The reaction disappears completely after a treatment with RNase (Meyer et al., 1961; Meyer, 1963). These findings indicate that the loops are rich in nucleoproteins. The basophilic material is removed from the loops during the condensation of the chromosomes in the late meiotic prophase I (Fig. 9f). The remnants can still be recognized during the first meiotic division as clumps of highly basophilic material which are then transferred through the secondary spermatocytes into the spermatids. These clumps seem to be identical with the “chromatoid bodies” as described by Zujitin (1929). As was already mentioned, in late primary spermatocytes of Drosophila all chromosomes are invisible in the living state in phase contrast because of their highly extended state. They also remain invisible after staining with Feulgen. The looplike structures in spermatocyte nuclei of D. hydei are Feulgen-negative as well. Only in young spermatocyte stages four slightly Feulgen-positive bodies are forming sites of (al, as) nooses, clubs, and tubular ribbons; (b) nooses and clubs; (0) nooses only; (d) threads only; (el threads and pseudonucleolus; (f) threads, pseudonucleolus, and tubular ribbons. Explanations: solid, heterochromatin of the Y chromosome; dotted, heterochromatin of the X chromosome (Xh); white, euchromatin of the X chromosome (Pa) ; YL,Ye, long and short Y arm ; C, clubs; N, nucleolus ; Noo, nooses; P, pseudonucleolus; T, tubular ribbons; Thdd, Thcp, diffuse distal and compact proximal sections of the threads.

190 OSWALD HESS AND GUNTHER F. MEYEB

THE Y CHROMOSOME IN Drosophila

191

FIG.5. Males of D. hydei with three different deficiencies of the Y chromosome. Phase contrast photographs of living spermatocyte nuclei ( x 1200) with (a) nooses only, (b) nooses, clubs, and tubular ribbons, (c) threads. al-cl corresponding metaphase plates of larval neuroblast mitoses, diagrams and phase contrast photographs of lactic-orcein preparations ( x 2500). Explanations: ArA5, the five pairs of autosomes; C, clubs; MTh, matrix of tlireads; N, nucleolus; Noo, nooses; T, tubular ribbons; Thpc, proximal rompart sections of the threads ; T(XY), translocation chromosome.

found. These can also be observed in phase contrast as four diffuse spheres (Fig. 9b,c). They obviously represent the four pairs of somatically paired big rod-shaped autosomes. For comparison, i t may be mentioned here that the lateral loops of lampbrush chromosomes in oocytes of amphibia are also Feulgen-negative. Although the loop-shaped structures are highly sensitive to changes of ion concentrations in the surrounding medium, it has been possible by treatment with hypotonic solutions or by ultrasonic irradiation to break up the nuclear membrane and release the loop structures from the spermatocyte nuclei. If the isolated loops are treated with RNase or proteases, their form but not their structural continuity is changed. In contrast to this finding, the loops are fragmented by DNase (Hennig, 1967). This observation demonstrates that the loops contain an axis composed of DNA and provides further support for the lampbrush hypothesis. The identification of the spermatocyte nuclear structures as lampbrush loops leads to the conclusion that these structures must represent highly activated segments, possibly genes, of the Y chromosome. Direct experimental evidence for this has been found in the fact that the looplike structures appear labeled in autoradiographs after in vivo incubation with tritiated uridine. Such investigations have only just

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FIQ.6. Diagram of the Y chroniosonie of D. hytlei in the state of an univalent lampbrush chromosome in spermatocytes. (a) Very young spermatocyte (+ chromomerio structure in detail; (b) early growth stage of a primary spermatocyte nucleus, unfolding of the Y loops; ( c ) full grown spermatocyte nucleus with fully developed loops. Explanations : Chr, chromomore; C, clubs; Kin, kinetochore ; N, nucleolus; NO, nucleolus organizer; Noo, nooses; P, pseudonucleolus; T, tubular ribbons; Thdd, Thpc, diffuse distal, and proxinial compact sections of the threads ; YL,YE,long and short Y arm. Exact locus of nucleolus organizer is not yet known; the thin connections between pscudonuc*lcolusand dubs and the chromomere are not visible and are hypothetical.

become possible because for a long time it was not possible to preserve the loops after fixation in total mounts. Recently, a deep-freezing method that gives good results has been worked out (Hennig, 1967). Studies of such a kind are now in progress on a larger scale. Experiments with actinomycin likewise support the lampbrush hypothesis. Actinomycin is known to block DNA-dependent RNA synthesis by interacting with DNA (Kersten and Kersten, 1962; Goldberg et al., 1962, 1963; Reich et al., 1962; Acs et al., 1963). After injection of 0.01 pg of actinomycin into larvae or imagoes of D. hydei, a disintegration of all spermatocyte loops has been observed (Meyer and Hess, 1965). The first visible changes occur as early as 6 hours after injection (Fig. 7s). These changes can most easily be followed in the thread-shaped loop. The compact proximal sections of this loop become shorter and thicker. Moreover, the material puffs and forms

THE Y CHROMOSOME IN

Drosophila FIG.7. Alterations of the Y-ehromosomal loops of D . hydei (a) 7 hours, (b) 15% hours, (c) 60 hours after injection of 0.01 actinomycin per animal. Explanations: C, clubs; N, nucleolus; P. pseudonucleolus; Thpc, proximal compact sections of the threads. Phase contrast, photographs of living nuclei, x 1200.

pg

193

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OSWALD HESS AND GUNTHER F. MEYER

small cavities. Five to seven hours after injection, the threads have t-he appearance of a string of hollow beads. The number of these beads or spheres decreases gradually. Twenty-four hours after injection, one usually finds only one pair of rather big spheres located near the nucleolus (Fig. 7b). Still some hours later, the threads have completely disappeared (Fig. 7c). The other pairs of loops also show striking alterations. The matrix of the so-called clubs swells, and the normally attached granules are spread gradually over the entire nuclear area. Around the tubular ribbons, many new granules are formed which are never found in normal nuclei. The material of the tubular ribbons is clotted together. The pseudonucleolus seems to lose material. Its structure appears much lighter than under normal conditions and sometimes it becomes nearly invisible. These changes reach a maximum at about 2440 hours after injection. A t this time all spermatocyte structures are disintegrated. In addition, also the nucleolus is changed under the influence of actinomycin. It becomes spherical in shape and often detaches itself from the nuclear membrane where i t is normally located (Fig. 7c). A similar behavior has been observed in the puffs and Balbiani rings of polytene chromosomes in Diptera (Beermann, 1964), and in the loops of amphibian lampbrush chromosomes (Izawa et al., 1963). A regression of lateral loops under the influence of actinomycin , therefore, seems to be a general reaction of functional structures in chromosomes. In the spermatocytes of Drosophila the alterations caused by actinomycin are reversible. Two to three days after the injection a11 loops have returned to normal. Again, this process can most easily be followed in the thread-shaped pair of loops. At first, two very short pieces of compact threads which are connected by a diffuse distal section, appear near the nucleolus. These extend gradually until they attain the dimension and appearance of the original threads in untreated nuclei. The other types of structures exhibit similar processes of outgrowing. It has not been possibIe, so far, to follow these processes of outgrowing or unfolding in uilro. Therefore, the sequence of changes caused by actinomycin in the spermatocyte loops can only be followed in testes of different animals killed at various times after injection o t the antibiotic. However, the possibility could be excluded that the regeneration of the loops is simulated by the proliferation of spermatogonia which might have developed into new spermatocytes with normal loops during the course of the experiment. First, third-instar larvae contain some growing spermatocytes, but no spermatids. The oldest spermatocytes are located in a few cysts a t one side of the testis. The spermatocytes

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Y CHROMOSOME IN L)'rOSOphdU

195

can be seen to contain fully regenerated loops 3 days after an injection of actinomycin. Degenerated cells and nuclei with disintegrated loops are not detectable a t this stage. Second, young spermatocyte nuclei, in which the first (normal) formation of the loops occurs, are less than half the size of full grown spermatocyte nuclei. I n contrast to this, the regeneration of loops after treatment with actinomycin, which can be observed in several gradual stages, occurs only in full grown spermatocyte nuclei. Thus, the normal outgrowth of loops in young spermatocytes and the regeneration of the loops after treatment with actinomycin are two processes that are clearly distinguishable. It could be shown in autoradiographs that soon after injection of actinomycin the rate of RNA synthesis is reduced to about 10% of the normal value. About 5 hours after injection, however, the rate of RNA synthesis is increasing again. At the same time the morphological disintegration of the loop structures is in full progress and continues for a t least 18 hours. Twenty-four hours after injection, when the morphological alterations of the loops reach their maximum effect, the rate of RNA synthesis is still not higher than 50% of the normal value (Hennig, 1967). The results of the experiments with actinomycin clearly indicate a correlation between the existence of spermatocyte structures and the continuation of DNA-dependent RNA synthesis. These data, therefore, provide further substantiation of the assumption that the loops formed by the Y chromosome in Drosophila spermatocytes are modifications of the chromosome structure a t sites of active genes. The morphological changes produced by actinomycin can similarly be induced also by X-irradiation with doses ranging from loo0 to l0,OOO r (Hess, 1965b). The structural alterations caused by irradiation are strictly analogous to the changes observed after injection of actinomycin, and they occur a t the same time after treatment and in the same sequence. With doses between 2500 and 10,000 r, the maximum effect attained and the speed with which i t develops are the same. I n other words, the effects are independent of the irradiation dose. However, with lower doses, for example 1000 r, only some of the spermatocyte nuclei are affected. Many nuclei do not show any alterations. As in the experiments using actinomycin it was found that the structural alterations caused by X-irradiation are also reversible. A gradual but complete regeneration of the loops occurs 80-100 hours after irradiation, Unlike actinomycin, X-irradiation may also cause some irreversible changes in the chromosomal loops. In some nuclei, for instance, the regeneration of the threads is blocked either on both sides,

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or on only one side. Breaks within the loops can also be seen occasionally. I n contrast to the lampbrush chromosomes in amphibian oocytes in which breaks of the loops have been observed immediately after the irradiation (von Borstel et al., 1966; Miller et al., 1965), no such direct breaks have so far been detected in the loops in spermatocytes of D . hydei. One may raise the question as to whether the primary effects of both actinomycin and X-irradiation are on the same target, namely the priming activity of the DNA in RNA synthesis. Whereas the synthesis of DNA is well known to be radiosensitive, there is no evidence that X-rays inhibit the RNA polymerase system in vivo. However, it was recently shown that the ability of Escherichia coli (Harrington, 1964, 1966) and of calf thymus (Zimmermann et al., 1964, 1965) DNA’s to prime in vitro RNA synthesis is severely depressed by X-irradiations. The doses used in these experiments were the same as in the irradiations of D . hydei spermatocytes. Thus, our findings are not inconsistent with the suggestion that in Drosophila spermatooytes the DNAdirected synthesis of RNA can be reversibly inhibited by X-irradiation. Preliminary data obtained after incubation of X-irradiated males with tritiated uridine support this assumption. During the first hours after irradiation the rate of incorporation of uridine is considerably reduced as compared with untreated males (Hennig, 1967).

D. THEDETERMINATION OF THE SHAPE OF

THE

Y-CHROMOSOMAL LOOPS

1. Species Hybrids between D . hydei and D. neohydei

The described observations are in favor of the assumption that each of the loops has an axis formed of DNA which in normal chromasomes, that is to say, in the inactive condition without structural modifications, is folded tightly within chromomeres. Concomitant with, or even as a result of loop formation, the DNA is directed to synthesize RNA. In addition, nucleoproteins are attached to the enfolded DNA axis. Each loop may have its own specific nucleoproteins, and it may be that in this way the characteristic form of each loop is determined. There are not only morphological differences between the different loops of one species, but there are also differences between homologous loops in different species. One may assume, therefore, that the shape-promoting nucleoproteins are also species specific. The available data throw some light on the mechanisms by which the characteristic form of each loop is determined. Drosophila hydei and D . neohydei are two closely related species ths t possess spermatocyte structures with clearly distinctive form (Fig.

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1 ) . They can be crossed in either direction. The hybrids are viable and fertile. The spermatocyte nuclei of hybrid males never contain Y-chromosomal loops of an intermediate type, but contain always the typical structures of that parental species from which the Y chromosome was derived (Hew and Meyer, 1963a). The hybrid males were back-crossed several times with females of the maternal species in which all autosomes were marked with recessive genes. In this way all autosomes which were derived from the same species as the Y chromosome were gradually eliminated, and hybrid males were produced which had a Y chromosome from D. neohydei, whereas all other chromosomes, X and autosomes, were from D. hydei. The spermatocyte loops of such males were still of pure D . neohydei type. It is likely, then, that factors in the X chromosome or in the autosomes do not participate in the determination of the characteristic morphology of the loops, but that genetic information situated in the Y chromosome itself is responsible for the shape and appearance of the spermatocyte loops. On the other hand, in our translocation stocks rather small fragments of the Y chromosome are able to give rise to loops of quite normal shape, provided that the corresponding loop-forming site is located within that fragment (Hess, 196%) (Figs. 4, 5 ) . This shows that the information for the shape-promoting proteins must be located very near to the loop-forming site. It may even be located within this loop. 2. Mutunts Changing the Shape of the Loops

There are other findings that support this view (Hess, 1965a, 1966). The thread-shaped loop of D. hydei is a twin structure consisting of a pair of highly refractive threads t.hat originate near the nucleolus, and of distal segments that are composed of diffuse material. I n the progeny of a male which was X-irradiated with 5000 r, sons were detected in which the proximal compact threads were changed into wide tubes. The distal diffuse sections were not changed (Fig. 8a). I n another irradiation experiment, a second case of alteration of the threads was found. This time the proximal sections of the threads remained unchanged, but the distal diffuse parts were altered into two knots of narrow tubes (Fig. 8b). Both alterations were found to be heritable and linked t o the Y chromosome. From both mutations, which are called “tube-proximal” and “tube-distal,” pure stocks have been successfully built up. In males carrying a “tube” mutation no deviations from normal viability and fertility have been found so far. I n mitotic metaphase plates of ‘%ube” males the Y chromosomes do not show

198 OSWALD HESS AND GUNTHER F. MEYER

Fic. 8. Mutations of the thread-shaped loop of the Y chromosome of D . hydei. (a) Mutant tube-proximal, (b) mutant tubedistal, ( c ) male with a normal and a second tube-proximal Y chromosome; autonomous development of different types of threads. Explanations: C, clubs ; N, nucleolus; P, pseudonucleolus; T, tubular ribbons; Thdd, distal diffuse, Thdt, distal tube-shaped, Thpc, proximal compact, Thpt, proximal tube-shaped sections of the threads. Phase contrast photographs of living spermatocyte nuclei, x 1200.

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any visible changes. Moreover, it has been established that both mutations are not accompanied by alterations in the sequence and loci of the loop-forming sites on the Y chromosome. In addition, the mutanttype loops undergo the same reversible alterations following injection of actinomycin or irradiation with X-rays as do the normal loops. Thus, one may conclude that point mutations or very small deletions or duplications have occurred, most probably within the limits of the changed loop itself. The normal as well as the mutant phenotypes of the threads are completely autonomous. If one produces males with two Y chromosomes one of which is normal and the second mutated, then each of the two Y chromosomes gives rise autonomously to threads of its own characteristic form (Fig. 8c). If actinomycin is injected into males with two different Y chromosomes, then 3 days after the injection each pair of threads is regenerated in its original form; that is, i t corresponds to the genetic constitution of its Y chromosome. Thus, the factors determining the shape of the loops act in cis relationship only. This finding is difficult t o explain. Some of the conclusions which have to be drawn from this observation will be discussed later in this review. 111. Morphogenetic Effects of the Y-Chromosomal Loops in Spermiogenesis

A system like the one which is realized in the Y chromosome of Drosophila may help to gain further insight into those mechanisms by which the chromosomes and their subunits control growth and differentiation. Several properties are united here, and, together, offer unique possibilities to tackle such questions: Only a few, giant loop pairs, each with distinctive qualities, are found in an otherwise “silent” chromosome, and in a nucleus where no other chromosome develops giant loops. There is further a perfect correlation between the presence of these loops and a defined type of cellular differentiation, that of spermiogenesis, which in itself shows several unique features. And, last but not least, i t is possible to manipulate the cytogenetic situation of the animals in experiments within wide limits. Therefore, in order to elucidate the physiological significance of those long sections of the DNA which are unfolded from chromomeres during loop formation, the morphogenetic effects of a number of Y deficiencies have been studied, mostly with the aid of the electron microscope. A brief outline of normal spermiogenesis will be given first. [A detailed investigation on the spermiogenesis of both D . inelanogaster

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and D.hydei as revealed by light and electron microscopy has recently been published (Meyer, 1968).I

A. NORMAL SPERMATOGENESIS IN D . melanogaster

AND

D. hydei

Spermatogenesis in Drosophila has been the subject of several investigations (Metz, 1927; G u y h o t and Naville, 1929; Huettner, 1930; Woskressensky and Scheremetjewa, 1930; Dobzhansky, 1934; Cooper, 1950; Meyer et al., 1961; Meyer, 1963). Electron microscopical investigations dealt mainly with special problems of spermiogenesis (Yasuzumi et al., 1958; Daems et al., 1963; Kaufmann and Gay, 1963; Bacetti and Bairati, 1964; Meyer, 1964; Bairati and Bacetti, 19%). The synthesis of nucleic acids during spermatogenesis in D . melanogaster has also been studied (Olivieri and Olivieri, 1965; Hennig, 1967). Of the spermatogonia which represent the first stage of spermatogenesis, three types can be distinguished (Hannah-Alava, 1966). The predefinitive spermatogonia consist of a few cells near the tip of the gonad. They are followed by the “indefinitive spermatogonia” (= primary spermatogonia, according t o Cooper) and finally by the “definitive spermatogonia” (= secondary spermatogonia according to Cooper) which show a remarkable increase of their cell size (Fig. 9a). With the onset of the meiotic prophase the coarse chromatin reticulum of the definitive spermatogonia is replaced by thread-shaped chromosomes which clearly are paired somatically (Cooper, 1950). Characteristic for the youngest stages of primary spermatocytes in D. hydeei is the attachment of the nucleolus to the nuclear membrane (Fig. 9b) and a gradual dispersion of the chromocenter (Fig. 9b-e). The latter is in the process of formation of loops by the Y chromosome. The autosomes are only visible in young spermatocytes as four spherical diffuse bodies which are slightly Feulgen positive (Fig. 8c). In later prophase or early diakinesis the spermatocyte nuclei become spherical again. A multiple layer of membranes parallel to the nuclear membrane is formed. It originates from small vesicles which possibly are derived from Golgi material (dictyosomes) (Meyer, 1963). During the condensation of the chromosomes the material of the Y chromosomal loops is released from the chromosomal axis. It clumps and forms bodies of dense material which often hide the chromosomes (Fig. 9g). The stage of secondary spermatocytes is very short, because the two meiotic divisions follow each other very quickly. In D. hydei, therefore, interphase stages of secondary spermatocytes have so far never been seen. Nuclei of secondary spermatocytes are much smaller than nuclei of primary spermatocytes and are always observed to lie within a fully developed spindle apparatus (Fig. 9i).

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In young spermatids the size of the nuclei increases rapidly. At the same time a nebenkern is formed (Fig. 9j) by an accumulation of mitochondria. Also an acroblast is formed by dictyosomes. The remnants of the Y-chromosomal loops are still visible in the cytoplasm (Fig. 9k). A little later, in the stage of the initial elongation of spermatids the acroblast has moved to an apical position, the “chromatoid body” is now dispersed, and the centriol divided. The flagellum starts t o grow in a caudal direction between the two nebenkern derivatives which have been formed in the meantime by a division of the nebenkern (Fig. lob). The next stage is characterized by an increased elongation of the nebenkern derivatives (Fig. 1Oc). A very small acrosome has been formed. The dictyosomes have disappeared. Still later the nucleus becomes spindle-shaped (Fig. 10d) while the nebenkern derivatives transform into paracrystalline material (Fig. 10d, Cry). I n the final stages of spermiogenesis the nebenkern derivatives are completely transformed. I n the anterior parts of the spermatids or the spermatozoa the flagellum is located in an invagination of the nucleus (Fig. 10e,f). In principle, spermatogenesis is similar in D. melanogaster and D . hydei. However, there are two main differences. First, in D . melunogaster only one nebenkern derivative is transformed into a paracrystalline body, while in D . hydei both undergo this transformation. Second, in D. melanogaster the number of sperm in each cyst is usually 64. In contrast to this, in D . hydei no more than 32 spermatozoa per cyst have ever been found. In order to facilitate the studies of the morphogenetic effects of Y deficiencies spermiogenesis has been subdivided into eight stages. However, this subdivision is to some extent arbitrary and there are no sharp demarcations between the stages. FIG.9. (See pp. 202 and 203.) Spermatogenesis in D.hyrlei. Phase contrast photographs of living cells, approx. x 900. ( a ) Spermatogonia and very young primary spermatocytes; (b) primary spermatocyte at the beginning of the growth phase; (c) growth stage; loops begin to appear; (d) later growth stage; different loops now distinguishable ; (e) full grown spermatocyte nucleus ; loops completely unfolded ; (f) late prophase I ; matrix of loops condensed; (g) early metaphase I ; ( h ) tnetaphase I ; ( i ) secondary spermatocyte in prophase 11; ( j ) young spermatids; forniation of nebenkern; (k) young spermatid; nebenkern fully differentiated; (1) later sperniatid ; elongation of nucleus and nebenkern derivatives starts. Explanations : A, autosomes; Ab, acroblast ; C, clubs; Ch, chromosomes ; Chb, chromatoid bodies; Chc, chromocenter; FS, functional structures of the Y chromosome; N, nucleolus; ND,, ND1, nebenkern derivatives ; NK, nebenkern ; NU,nucleus ; P, pseudonucleolus; Spc. spermatocytes; Spg, spermatogoniu ; T, tubular ribbons; Th, threads.

202 OSWALD HESS AKD GUNTHER F. MEYER

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FIG.9. For legend see p. 201.

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FIG.10. Diagram of spermiogenesis in D. hydei. (a) Young spermatid; (b) beginning of elongation, formation of nebenkern derivatives; (c, d) later stages of elongation; nucleus spindleshaped ; transformation of nebenkern derivatives into

paracrystalline bodies; (e) late spermatid; almost completed elongation of nucleus and transformation of nebenkern derivatives; (f) mature sperm; (g) cross sections through stages (el and (f). Explanations: Ab, acroblast; As, acrosome; Ce, centriol; Ced, Cep, distal and proximal daughter centrioles; Chb, chromatoid body; Cry, paracrystalline bodies ; F, flagellum ; N, nucleus ; Nd, nebenkern derivative; NK, nebenkern; T, tail of sperm.

Stage 1: Early spermatid, single cells tightly packed in cysts, nucleus spherical, acroblast and nebenkern formation completed, flagellum established.

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FIG.11. Diagram of a cross section through the flagellum of a mature sperm of Drosophila. Diameter of whole flagellum approx. 220 mp. Explanations: CF, central dense fibers; is, intersatellite space; pF, peripheral fibers; Sa, satellites; sF, sccondary fibers; Sp, spokes. After Persijn, Daems, and Tates, modified.

Stage 2: Beginning of spermatid elongation, but nucleus still spherical, growth of flagellum in caudal direction, formation of two roundish nebenkern derivatives. Stage 3: Further elongation of spermatids which are separated by wide intercellular spaces, nucleus spindle-shaped, formation of central flagellar fibers, transformation of the nebenkern derivatives into paracrystalline bodies. Stage 4: Spermatids of each cyst fuse again to form a syncytium, continuation of the transformation of nebenkern derivatives, further differentiation of the flagellum with formation of satellites and secondary fibers (Figs. 11, 12). Stage 5 : Spermatids still syncytially connected, continued linear elongation of the cells and continued transformation of nebenkern derivatives (in D . melanogaster degeneration of the second nebenkern derivative). Stage 6: Second separation of spermatids, which remain, however, connected by thin cytoplasmic bridges, tight package of the flagellum and the paracrystalline bodies, completion of the paracrystalline organization of the nebenkern derivatives (Fig. 13). Stage 7: End of syncytial stage, complete separation of sperm, which are, however, still immotile. Stage 8 : Motile sperm, characteristic modifications of the pattern in paracrystalline organization as compared to stage 7 (Fig. 13); the mature axial fiber complex of the flagellum now contains satellites, fibers, spokes, secondary fibers, and dense central fibers (Fig. 11).

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FIG.12. Cross section through a part of a spermatid cyst of D.hydei. Spermatids still syncytial. Explanations : Cryl, Cry?, crystals in nebenkern derivatives ; F, flagellum ; Nd, nebenkern derivative. Electron micrograph of ultrathin section, x 21,000.

FIG.13. Paracrystallme bodies (transformed nebenliem derivatives) in sperm of D. hytlei. (a) Immotile. young sperm, x 200,000; (b) mature sperm, x 200,000;

( c ) paracrystalline bodies a t higher magnification, showing details of macromolecular organization: the longitudinal units (200 A long) are composed of %-A subunits which seem to be part of a continuous cross-structure; thin 13-A elements connect the subunits in transverse direction, x 1,160,000. Electron micrographs of whole mounts after negative staining with phosphotungstic acid.

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B. SPERMATOGENESIS IN XO MALES AND Y-DEFICIENT MALESOF D . melanogaster Spermatogenesis in XO males has been the subject of several investigations, which had, however, somewhat contradictory results. Shen (1932) claimed meiosis and sperm differentiation to be normal in XO males. He observed only immotility of sperm. In contrast to this, Morgan et al. (1925) and Safir (1920) have demonstrated that spermatids of XO males do not develop into sperm of normal morphology. All spermatids remain connected syncytially. Schultz (1947) observed that spermatid nuclei of XO males were only incompletely elongated. Similar deviations from normal have also been found in genetically sterile testes (of the constitution X.YL) which were transplanted into fertile hosts (Stern and Hadorn, 1938). Our own examinations confirm the results of Morgan et al. (1925) and of Schultz (1947). XO spermatids do not develop beyond stages 4 or 5 (Meyer, 1968). Early XO spermatids usually show a normal organization of the acroblast and nebenkern. There are, however, other cases in which the formation of the acroblast is disturbed. The shape of the nuclei may become irregular during elongation. Particularly striking are irregularities which are found on the submicroscopical level in the organization of the flagellar subcomponents and the nebenkern derivatives. Normally, both nebenkern derivatives are formed, grow in contact with the axial fiber complex, and thus develop a regular geometric figure. This process can be disturbed a t an early stage of differentiation in XO males. Either both of the nebenkern derivatives or only one are located free in the cytoplasm and, therefore, none or only one instead of two is in contact with the flagellum. Spermatids with nebenkern derivatives but without a flagellum are also found. The formation of groups of flagella without nebenkern derivatives is possible, but very rare. In other cysts morphogenesis may even be more severely disturbed. There are regions which seem to be made of the remains of a few spermatids bounded by a common membrane. In each such region many paracrystalline bodies are found which are formed within numerous fragments of the nebenkern. Furthermore, in contrast to normal males in which the formation of the paracrystalline bodies always starts at the point of contact of the nebenkern derivative with the axial fiber complex (Fig. lo), the transformation in XO males may begin in any region of the nebenkern derivatives. The formation of two and sometimes three separated paracrystalline bodies in the same nebenkern derivative has also been observed.

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The differentiation of the flagellar axial fiber complex may also be remarkably disturbed in XO males. Each component of the complex, that is, the. central and peripheral fibers, secondary fibers, satellites, and spokes (Fig. l l ) , may show irregularities in shape and topographical relations. A distortion of the circular arrangement of the fibers is frequent. The central fibers of the axial complex may be completely absent, or they may not be transformed into the dense compact form which is found in mature sperm of normal flies. Some spermatids have been found in which all flagellar fibers are lacking. Cysts of XO males contain often only 40 or 30 spermatids instead of the normal number of 64. Males with deficiencies of the Y chromosome, lacking, for instance, either the long or the short Y arm, show similar defects (Meyer et al., 1961). It seems possible that spermiogenesis might. proceed beyond stage 4; however, this can not yet be clarified with certainty.

C. SPERMATOGENESIS IN XO MALESAND Y-DEFICIENT MALESOF D . hydei I n contrast to D. melanogaster, development of germ line cells in XO males of D. hydei does not proceed beyond the stage of the first

spermatocyte (Hess and Meyer, 1963a). Spermatogenesis is always blocked a t the spermatocyte stage. The crystalline needles that are characteristic of XO males in D. melanogaster are also not found in testes of XO males of D. hydei. As soon as a small fragment of the Y chromosome is present, spermatogenesis always proceeds further than in XO males. At least in a few cells the meiotic divisions are completed and spermatids are formed. The differentiation of the spermatids continues to various stages depending on the character of the Y fragment present. Generally, spermiogenesis in Y-deficient males is considerably slowed down, and maximum development of spermatids is often not reached sooner than 20 days after the emergence of male flies. [A brief outline of the spermiogenesis in Y-deficient males of D . hydei has been given by Hess (1967a), and a comprehensive description by Meyer (1968)l. The effects of the various Y-chromosome deficiencies which have been studied may be roughly classified into those that interfere with spermatid differentiation immediately following meiosis (“early effects”) , and those that permit the development of nearly complete sperm, at least in some spermatids, and in some cysts of the testis (“late effects”).

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Early effects are found with deficiencies of the entire long arm of the Y chromosonie (including the sites for threads, pseudonucleolus, tubular ribbons, and clubs), and also with deficiencies of the short Y arm (including only the nooses) , and further with deficiencies involving the short arm plus various proximal portions of the long arm. The generai aspect of the testes in these different cases is not uniform. For instance, in the case of 8 deficiency for the entire long arm including the kinetochore and possibly a small piece of the short arm, the normal number of spermatogonia and spermatocytes seems to be contained in the testes. However, the majority of the spermatids appears to be completely degenerated. Only a few spermatids are found in the intermediate region between the spermatocytes and the degenerated cells. These spermatids may possess slightly elongated nebenkern derivatives and a short piece of flagellum. On the other hand, with deficiencies for the short arm, including the nooses and the kinetochore region, one finds early effects of a less drastic kind. The testes contain a relatively small number of spermatids. In some cysts all spermatids show identical irregularities, such as failure of mitochondria to form a unitary nebenkern and malformations of the nuclei. In other cysts some spermatids proceed to the formation of a nebenkern and its derivatives. These cells also elongate. Flagella may also be formed in such cases. It is not yet known whether or not the nebenkern derivatives can also be transformed into paracrystalline bodies. In some cases the mitochondria of which the nebenkern is composed do not form a regular roundish aggregate a t stage 1. Then, the two nebenkern derivatives are not formed. In such cases the development of the flagellum is blocked, whereas spermatids with a normal nebenkern possess a flagellum. One may assume from these observations that in D . hydei, in contrast to the situation realized in D. melanogaster, the differentiation of the flagellum is dependent on a regular formation of the nebenkern derivatives. Comparable effects are caused by deficiencies for the short arm (nooses) plus various proximal portions of the long arm, including the clubs, or clubs and tubular ribbons, or these two together with the pseudonucleolus. Late effects are found in males with deficiencies for only the threads, the threads and the pseudonucleolus, or these two together with the tubular ribbons, that is, in deficiencies for various lengths of the distal part of the long arm. The kinetochore region of the Y chromosome is always present in these cases. Here, many cysts are found

THE Y CHROMOSOME I N

21 1

Drosophila

with spermatids developing almost normally up to the end of the syncytial phase. Sometimes, there are produced sperm, which, however, are not yet fully elongated and do not move. Their fine structure is not deficient in any element recognizable by electron microscopy. In many cysts spermatids may differentiate as far as stage 6, but in other cysts only aberrant forms are found. Here the flagellum may be completely absent, or the arrangement of the flagellar fibers is disordered. The transformation of the nebenkern derivatives into paracrystalline bodies may also be inhibited. Summarizing the results of the investigations on the morphogenetic effects of Y-chromosome deficiencies, one can draw the following conclusions: None of the deficiencies studied results in inability to form any of the major structural components of spermatozoa. All observed defects are organizational, or regulatory in kind. Furthermore, all observed effects involve, in addition to defects of organization, more or less severe inhibitions of growth (see also p. 212). The individual contributions of the various loop pairs to sperm differentiation and growth are hard to assess a t present. This is partly due t o the uncertainty with respect to the role played by the kinetochore region of the Y chromosome and the still undetermined location of the nucleolus-organizing region. Certainly, the absence of one specific loop pair can not be balanced by the presence of another one in duplicate. Therefore, the effects of the various Y-chromosomal loci are not just cumulative. This is especially clearly demonstrated in experiments in which different proximal and distal fragments of the Y chromosome are combined and tested for complementation. If the combined pairs of Y fragments have overlapping regions, fertility is regularly reestablished. If , however, by the combination of two Y fragments the genetic constitution remains still deficient for an interstitial region that carries the site for one of the loops, males are sterile and the differentiation of their spermatids shows the characteristic malformations, such as described above (Hess, 1967~).

D. THEINFLUENCE OF ON

THE Y-CHROMOSOMAL LOOPS GROWTHOF SPERMATOZOA

Not only the morphology and motility of sperm, lengths seem to be dependent on the loops of the in spermatocytes. Species-specific differences of total within the genus Drosophila appear to be correlated

but also their

Y chromosome sperm lengths with both the

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degree of differentiation and the number of loop-organizing sites present in the Y chromosome. For instance, the species-specific sperm length within the D . hydei subgroup varies from 1.2 mm for D . nigrohydei, which has only a single pair of Y chromosomal loops, up to 6.6 mm for D. hydei, which possesses a t least five different loop pairs of relatively complicated structure (Fig. 1 ; Hess and Meyer, 1963b). Similar relationships are found in other species 8s well, indicating that sperm length increases with increasing number and complexity of lampbrush loops of the Y chromosome. Also intraspecific effects of the Y chromosome on sperm length are found. For instance, in D. melanogaster the immotile sperm of XO males are on the average 1.2 mm long, sperm of XYs males are 1.3 mm, sperm of XYL males are 1.5 mm, sperm of normal males are 1.8 mm, and sperm of XYY males are up to 3.5 mm long. The fact that hyperploidy of the Y chromosome reduces fertility, or may even cause sterility in males of D . meEanogaster (Cooper, 1956), is comprehensible in the light of these observations. Similarly, males of D . hydei with two Y chromosomes have sperm of 13-14 mm as compared to 6.6 mm for sperm of normal males. Males carrying partial duplications of the Y chromosome produce motile sperm of intermediate lengths, with differences in the growth-promoting capacities of Y segments carrying the sites for different loops, On the other hand, the different types of defective sperm formed by males with Y deficiencies are always shorter than normal. IV. Conclusions

At present, nothing is known about the function of the genes located in the loops of the Y chromosome. If one assumes that they are identical with the fertility factors (as first described by Stern, 1929), there still remains the problem of why such genes form complex giant loops in spermatocyte nuclei, and what might be the physiological significance of loop formation. In thinking of possible explanations one has to consider the special situation that is generally realized in gametogenesis. As has been demonstrated, spermiogenesis is not directed by the genetic constitution of the differentiating spermatids themselves, but rather by the constitution of the diploid cells of the testis. Therefore, either key substances of spermiogenesis which are under the control of Y-chromosomal factors have to be synthesized before the meiotic divisions, or their messages have to be transcribed, stabilised, and stored during earlier stages of spermatogenesis.

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On the other hand, it is perhaps not by accident that in homologous stages of female germ line cells, namely, in the oocytes, the formation of lampbrush loops by the chromosomes is also observed in a great number of different animal species. Indeed, a very similar situation is realized in oogenesis. As has been demonstrated in recent years, there is onIy very little de nouo RNA synthesis during the early embryological stages, and the process of development does not depend on newly synthesized RNA. However, there is protein synthesis during these stages, and the newly synthesized proteins turned out to be essential for normal development. Therefore, development is not impaired by actinomycin, but immediately blocked by puromycin. Thus, all processes are under the direction of messengers which have been preformed during oogenesis and stored in the egg in a temporarily blocked and stabilized form (Brown and Gurdon, 1964; Brown and Littna, 1964a,b; Gross and Cousineau, 1964; Gross et al., 1964; Nemer and Infante, 1965; Monroy and Gross, 1967). It seems, therefore, that in both male and female germ cells a phase of relative synthetic inactivity of the nucleus is preceded by a stage in which the nucleus is highly active and special mechanisms are developed which enable the cell to store and stabilize the gene products for use during later stages. It seems likely, then, that the formation of lampbrush loops with their complicated morphology is involved in such processes. It has in fact been found that unfertilized sea urchin eggs contain particles with messenger RNA in an inactive state which are activated to start translation into proteins upon fertilization (Monroy et al., 1965). These findings lead us back to a general consideration of the organization of lampbrush loops. Beermann (1965) proposes that chromosomes of higher organisms have generally a chromomeric organization. This is partially based on the fact that the chromosomes of nearly all species of higher organisms show a t certain stages, usually before meiosis, a subdivision into chromomeres. Moreover, the number of chromomeres visible is of the same order of magnitude in all species, namely, about 108-104. The same number is found for the bands in polytene chromosomes. Thus i t is possible that the bands are nothing but chromomeres in polyteny. There is, on the other hand, good evidence that chromomeres act as functional units during both transcription (Pelling, 1964, 1966; Gall and Callan, 1962; Beermann, 1965) and DNA replication (Plaut, 1963; Keyl and Pelling, 1963; Hsu, 1964; Hinegardner et al., 1964; Keyl, 1965a,b). However, the giant loops in amphibian lampbrush chromosomes and the loops of

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the Y chromosome of D . hydei in spermatocytes are 50-100 p long. I n terms’of DNA, this is enough to code for several dozens of proteins. Thus, one may expect that the DNA of such loops is subdivided. Gall and Callan (1962) have indeed found that the so-called “giant granular loops” of Triturus seem to be composed of several independent transcriptional units. This was concluded from the observation that in these loops whose lengths correspond to a t least lo5 pairs of nucleotides, incorporation of tritiated uridine begins always a t a small initial section situated a t one end of the loop. As the incubation time is increased the border of labeling moves gradually around the loop until after 2 weeks of incubation the loop is labeled along its entire length. As has been mentioned earlier in this review, in D. hydei the individual shape of each giant loop is dependent on factors that are located in the Y chromosome and, more specifically, must lie very near to or even within the regions of the loop-forming sites themselves. If the shape of each loop is determined by special proteins, it therefore seems likely that regions within each loop code for these proteins. However, the fact that mutations exist by which the shape of a loop is changed on its entire length indicates that only one cistron of a loop codes for a shape-determining protein. All the rest of the DNA in a loop should have, then, other functions. Moreover, one could assume an affinity to exist between the entire DNA axis of a loop and its specific shape-determining protein. However, as in males with two genetically different Y chromosomes homologous loops with different shapes are formed autonomously within the same spermatocyte nucleus, this assumption would necessitate the assumption that the affinity changes simultaneously with the occurrence of a mutation in the cistron coding for the shape-determining protein. This seems unlikely. A possible explanation would be that RNA and proteins which are combined in the loop matrix remain attached in situ a t the site of the RNA synthesis, but are not taken up by the loop axis from the surrounding. Beermann (1965) in his working hypothesis assumed that only an initial “master” segment of the chromomeric DNA in each loop is active in synthesizing messengers coding for protein synthesis. The completed messenger RNA molecules could be sequentially attached to the rest of the chromomeric DNA. This (larger) portion of the DNA in a chromomere would then have the principal function of binding messenger molecules and providing a structural substrate to package them into ribonucleoprotein particles. These special functions of the “posterior portions” of the DNA in the I

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loop could be made possible in the following way: These posterior portions could be partially redundant copies of the initial segment and base sequences which all subunits in a chromomere have in common could enable certain portions of the DNA in a loop t o specifically bind messengers synthesized by the master segment. This model does not seem unlikely since Key1 (1965a,b) could demonstrate the existence of DNA duplications in homologous bands of polytene chromosomes. Besides the fact that this model is in accordance with all experimental data from our studies on the function of the Y chromosome in spermatocytes, it might also be more generally applicable. For instance, the sequential labeling of the giant granular loops in Triturms oocytes as described by Gall and Callan (1962) could easily be explained with the aid of such a model. Moreover, it is a fact that in higher organisms the haploid chromosome set contains much more DNA than is needed to code for all necessary information. I n addition, closely related species often have DNA contents per haploid chromosome set which differ by one or two orders of magnitude. For instance, in Amphibia, newts may have about 20 times more DNA than toads (Mirsky and Ris, 1951). These problems have recently been discussed in detail by Ullerich (1966). As this can not be explained by the assumption of a similar difference in information content, and as a variety of experimental data has recently appeared which speaks against a multistranded organization of the DNA in the chromosomes, these observations have been especially difficult to explain. These difficulties could be overcome with the aid of the newly proposed model: According to this model a great part of the DNA may be noninformational, and it seems well understandable that in different species the relative proportions of DNA with only auxiliary functions might be different. A very similar model for the chromomeric organization has recently been proposed by Callan (1967) and Whitehouse (1967). The most important point, however, is the fact that this model suggests that the synthesis of (messenger) RNA is not automatically followed by removal of the RNA copies from the chromomere. It seems that the loops are elaborate devices not only t o keep the RNA attached to the chromosome, but also to facilitate its association with specific nuclear proteins. This, as was mentioned in the beginning of this review, seems to be a requirement for a system like the spermatocyte, which is followed by stages with an inactive (and genetically incomplete) nucleus and, therefore, needs to be directed by preformed, stored, and stabilized messengers. Our studies on the morphogenetic effects of various Y-chromosomal

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deficiencies on spermatid differentiation have clearly demonstrated that the factors in the Y chromosome do not code for proteins necessary for the formation of sperm organelles a s far as these are visible in the light and the electron microscope, This finding has a parallel in the long known and well-established fact that the Y chromosome does not carry any information essential for normal somatic differentiation. Thus, one may doubt whether or not the stored RNA in the loops is a true messenger, It seems also possible that specific transfer RNA’s, or their precursors, might be stored in this fashion. If certain transfer RNA’s had a key function in the translation of some messages, the release of such molecules from a storage form could be essential for certain phases of spermatid differentiation. Also, an increase in the available amount of the key compound would be expected to lead to a proportional stimulation of growth. This is indeed found in the case of Y-chromosomal duplications. It could be demonstrated with the aid of DNA-RNA hybridization techniques that in male germ line cellp specific species of RNA molecules are synthesized which are not found in somatic tissues. Moreover, the sequences complementary to these testis-specific RNA’s seem to be located in the Y chromosome. Thus, it is very probable that sites in the Y chromosome are active only in male germ line cells and are synthesizing highly specific RNA’s. In the moment, no further information about the nature of these specific RNA’s is available (Hennig, unpublished). It was the aim of this review to demonstrate not only that the investigations of the loops formed by the Y chromosome in spermatocyte nuclei of Drosophila may reveal further insight into those processes by which the fertility factors postulated long ago may act, but also that such studies may contribute to our knowledge about the genetic regulation of cell differentiation processes and the structural organization of chromosomes in higher organisms. V. Summary

1. The Y chromosome of Drosophila contains a number of fertility factors which are all essential for the formation of normally functioning sperm. These factors are cell autonomous and act even during the diploid phase, so that X-bearing spermatids that do not possess a Y chromosome are able in normal males to differentiate into normal spermatozoa. 2. In spermatocyte nuclei of Drosophila species special structures occur which are not found in the nuclei of other tissues. Moreover,

THE Y CHROMOSOME I N

Drosophila

217

they are restricted to the growth phase of primary spermatocytes. 3. These structures develop by an unfolding of DNA from chromomeres in the Y chromosome in a way similar to the formation of lateral loops in lampbrush chromosomes. In the Y chromosome of D . hydei a t least five different loop pairs of distinctive morphology are formed by relatively small sites. Sequence and loci of these loop-forming sites have been determined. 4. The functional nature of the Y-chromosomal loops has been demonstrated in two independent ways: First, in autoradiographs the loops appear labeled after incubation with tritiated uridine. Second, if the DNA-dependent synthesis of RNA is blocked by actinomycin, the loops disintegrate within a few hours. The disintegration is reversible. Similar effects are caused by X-irradiation. Irreversible damage, such as inhibition of loop regeneration and breaks within loops, are induced by X-rays. Histochemical studies reveal the presence of proteins, RNA, and DNA in the loops. 5. The morphology of the loops of the Y chromosome is species specific. Studies of species hybrids demonstrate that the form of the loops depends only on factors located in the Y chromosome. Analyses of Y-chromosome translocations show that these factors must be located very near or within the loop-forming sites. Moreover, point mutations have been isolated by which the shape of single loops is altered. In males with two genetically different Y chromosomes these mutations act autonomously within the same spermatocyte nucleus. 6. The morphogenetic effects of Y deficiencies on spermiogenesis have been studied. If a segment of the Y chromosome containing any of the loop-forming sites is lacking, males are invariably sterile. The observations of spermiogenesis in males with deficient Y chromosomes are consistent with the idea that the DNA within the loops does not code for proteins necessary for the formation of any sperm organelle. However, growth of sperm depends on the number of Y chromosomes present. Males with two Y chromosomes form sperm that are about twice as long as normal sperm. Partial duplications of the Y chromosome provoke an intermediate sperm elongation with differences in the growth-promoting capacities of Y segments carrying different loops. 7. A working hypothesis has been offered to explain the possible physiological significance of the lampbrush loops. It is assumed that only a relatively short segment of the DNA in each chromomere is transcribed whereas the rest of the DNA is not informative, but has some auxiliary functions. These could be to provide a structural substrate for packaging and storing messenger RNA molecules or to ac-

218

OSWALD HESS AND GUNTHER F. M E Y W

cumulate especially large amounts of key substances for spermiogenesis -for instance, specific transfer RNA’s which are synthesized during the spermatocyte stage, but used only during spermiogenesis, in which stage the nucleus is genetically incomplete and physiologically inactive. ACKNOWLEDGMENT The authors’ thanks are due to the able technical assistance of Miss R. Bromberg, Miss T. Fleischmann, Mrs. C. Hess, Miss I. Hi&, Miss M. Hinrichs, Miss C. Runk, and Miss G. Thies. Mr. E. Freiberg has drawn the figures. We want to thank especially Prof. W. Beermann for many discussions and his interest in our work. We are also grateful to Dr. D. R. Wolstenholme and Dr. J. Boyd for correcting the English text. Oswald Hess receives support from the Deutsche Forschungsgemeinschaft.

REFERENCES Abrahamson, S., Herskowits, I. H., and MuIler, H. J. 1966. Identification of halftranslocations produced by X-rays in detaching attached-X chromosomes of Drosophila melanogaster. Genetics 41,410-420. Acs, G., Reich, E., and Valanju, S. 1963. RNA metabolism of B . subtilk. Effects of actinomycin. Biochim. Biophys. Acta 76,68-79. Baccetti, B., and Bairati, A. 1964. Indagini comparative sull’ultrastruttura delle cellule g e r m i d i manchili in Dacus oleae ed in Drosophila melanogaster. Re& [2] 49, 1-29.

Bairati, A., and Baccetti, B. 1965. Indagini comparative sull’ultrastruttura delle cellule germinali maschili in D a m okae ed in Drosophila melanogaster. 11. Nuovo reperti ultrastrutturali sul filament0 aside degli spermatosoi. Redia [2]49, 81-86. Baker, W. K., and Spofford, J. B. 1969. Heterochromatic control of positioneffect variegation in Drosophila. Texas Univ. Publ. 5914, 136-154. Barigorsi, C. 1948. Role of the Y chromosome in the determination of cell sire in Drosophila melanogaster. Nature 162, 30. Barigozri, C. 1961. The influence of the Y chromosome on quantitative characters of Drosophila melanogaster. Heredity 5 , 416-432. Barigozsi, C., and DiPasquale, A. 1963. Heterochromatic and euchromatic genes acting on quantitative characters in Drosophila melanogaster. Hereditu 7 ,

389399.

Beermann, W. 1964. Control of differentiation at the chromosomal level. J . Ezptl.

200l.lS7,49-62.

Beermann, W . 1966. Operative Gliederung der Chromosomen. Natwwksemchajtem

52,3664‘75.

Bridges, C. B. 1916. Non-disjunction as a proof of the chromosome theory of heredity. Genetics 1,142 and 107-163. Brosseau, G. 1960. Genetic analysis of the male fertility factors on the Y chromosome of Drosophila melanogaster. Genetics 45, 267-274. Brown, D. D.,and Gurdon, J. B. 1864. Absence of ribosomal RNA synthesis in the anucleolate mutant of Xenopus laevis. Proc. Natl. Aoad. Sci. U S . 51,

139-146.

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D~OSOphilU

219

Brown, D. D., and Littna, E. 1964a. RNA synthesis during the development of Xenopus laevds, the South African clawed toad. J . Mol. Biol. 8, 669-687. Brown, D. D., and Littna, E. 196413. Variations in the synthesis of stable RNA’s during oogenesis and development of Xenopus laevis. J . Mol. Bwl. 8, 688-695. ,Callan, H.G. 1963. The nature of lampbrush chromosomes. Intern. Rev. Cytol. 15, 1-34. Callan, H. G. 1967. The organization of genetic units in chromosomes. J . Cell Sci. 2.1-7. Cooper, K. W. 1949. The cytogenetics of meiosis in Drosophila. Mitotic and meiotic autosomal chiasmata without crossing over in the male. J . Morphol. 84,81-122. Cooper, K. W.1950. Normal spermatogenesis in Drosophaa. I n “Biology of Drosophila” (M. Demerec, ed.), pp. 1-61. Wiley, New York. Cooper, K. W. 1956. Phenotypic effect of the Y chromosome hyperploidy in Drosophila melanogaster, and their relation to variegation. Genetics 41, 242-264. Cooper, K. W. 1969. Cytogenetic analysis of major heterochromatic elements (especially Xh and Y) in Drosophila melanogaster and the theory of “heterochromatin.” Chromosoma 10,535388. Daems, W. T., Persijn, J. P., and Tates, A. D. 1963. Fine structural localization of ATPase activity in mature sperm of Drosophila melanogaster. Exptl. Cell Res. 32,163-167. Das, N.K.,Siegel, E. P., and Alfert, M. 1965. Synthetic activities during spermatogenesis in the locust. J . CeU Biol. 25, No. 2,Part 1,387495. Dobzhansky, T. 1934. Studies on hybrid sterility. I. Spermatogenesis in pure and hybrid Drosophila pseudoobscura. 2. Zelljorsch. Milcroskop. Anat. 21, 169223. Dubinin, N. P., and Heptner, M. A. 1935. A new phenotypic effect of the Y chromosome in Drosophila melanogaster. J . Genet. 30, 423-446. Gall, J. G.,and Callan, H. G. 1962. HS uridine incorporation in lampbrush chromosomes. Proc. Natl. Acad. Sci. U S . 48,562-670. Goldberg, I. H., Rabinowitz, M., and Reich, E. 1962. Basis of actinomycin action. I. DNA binding and inhibition of RNA-polymerase synthetic reactions by actinomycin. Proc. Natl. Acad. Sci. US.48.2094-2101. Goldberg, I. H.,Rabinowitz, M., and Reich, E. 1963. Basis of actinomycin action. 11. Effect of actinomycin on the nucleoside triphasphate-inorganic pyrophosphate exchange. Proc. Natl. Acad. Sci. U.S.49, 22C229. Gowen, J. W.,and Gay, E. H. 1933.Eversporting as a function of the Y chromosome in Drosophila melanogaster. Proe. Natl. Acad. Sei. U S . 19,122-126. Gowen, J. W., and Gay, E. H. 1934. Chromosome constitution and behaviour in eversporting and mottling in Drosophila melanogaster. Genetics 19, 189-208. Grell, R. F. 1969. The Dubinin effect and the Y chromosome. Genetics 44, 911922. Gross, P. R., and Cousineau, G. H.1964. Macromolecule synthesis and the influence of actinomycin on early development. Exptl. Cell Res. 33, 368-395. Gross, P. R., Malkin, L. I., and Moyer, W. A. 1964. Templates for the first proteins of embryonic development. Proc. Natl. Acad. Sci. UB. 51, 407-414. Guyknot, E.,and Naville, A. 1929. Les chromosomes et la rkduction chromatique chez Drosophila melanogaster. Cellule 39, 25-82. Hannah-Alava, A. 1966.The premeiotic stages. Advan. Genet. 13, 157-226.

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OSWALD HESS AND CUNTHER F. MEYER

Harrington, H. 1964. Effect of X irradiation on the priming activity of DNA. Proc. Natl. Acad. Sci. U S . 51, 69-66. Harrington, H. 1966. Effects of various agents on the priming activity of DNA for Escherichia coli polymerase. J. Mol. Biol. 15, 162-169. Heitz, E. 1934. Uber a- und 8-Heterochromatin sowie Konstanz und Bau der Chromomeren bei Drosophila. Biol.Zentr. 54, 688-609. Henderson, 8. A. 1964. RNA synthesis during male meiosis and spermatogenesis. Chromosoma 15, 346-366. Hennig, W. 1967. Untersuchungen zur Struktur und Funktion des LampenburstenY-Chromosoms in der Spermatogenese von Drosophila. Chromosoma 22, 2 9 6 367. Hem, 0. 1963. Die Mutation scute-8 von Drosophila melanogaster als ein Letalfaktor, dessen Penetranz vom Heterochromatin abhangt. VerhandZ. Deut. Zool. Ges., Zool. Anz. Suppl. 26, pp. 87-92. Hess, 0. 1966a. Strukturdifferenzierngen im Y-Chromosom von Drosophda hydei und ihre Beziehungen zu Gen-Aktivitaten. I. Mutanten der Funktionsstrukturen. Verhandl. Deut. 2001.Ges., Zool. Anz. Suppl. 28, pp. 166163. Hem, 0. 1965b. The effect of X-rays on the functional structures of the Y chromosome in spermatocytes of Drosophila hydei. J. Cell Biol. 25, 169-173. Hess, 0. 1 9 6 6 ~ .Strukturdifferenzierungen im Y-Chromosom von Drosophila hydei und ihre Beziehungen zu Gen-Aktivitaten. 111. Sequenz und Lokalisation der SchIeifen-Bildungsorte. Chromosoma 16, 222-248. Heas, 0. 1966. Structural modifications of the Y chromosome in Drosophila hydei and their relations to gene activity. I n “Chromosomes Today” (C. D. Darlington and K. R. Lewis, eds.), Vol. 1, pp. 167-173. Oliver & Boyd, Edinburgh and London. EewJ 0. 1967a. Genetic control of differentiation in male germ line cells of Drosophila. Exptl. Biol. Med. 1, 90-109. Hem, 0. 196713. Morphologische Variabilitiit der chromosomalen Funktionsstrukturen in den Spermatocytenkernen von Drosophda-Arten. Chromosoma 21, 429-446. . of genetic activity in translocated fragments of Hess, 0. 1 9 6 7 ~ Complementation the Y chromosome in Drosophila hydei. Genetics 56,283-296. Hess, O., and Meyer, G. F. 1963s. Chromosomal differentiations of the lampbrush type formed by the Y chromosome in Drosophila hydei and D. neohydei. J. Cell Biol. 16, 527439. Hess, O., and Meyer, G. F. 1963b. Artspezifische funktionelle Differenzierungen des Y-Heterochromatins bei Drosophila-Arten der D. hydei-Subgruppe. Port. Acta Biol. A7, 2 9 4 6 (E. Heitz-Festschrift). Hinegardner, R. T., Rao, B., and Feldman, D. E. 1964. The DNA synthetic period during early development of the sea urchin egg. Exptl. Cell Res. 36, 63-61. Hsu, T. C. 1964. Mammalian chromosomes in vitro. XVIII. DNA replication sequence in the Chinese hamster. J. Cell Biol. 23, 53-62. Huettner, A. F. 1930. The spermatogenesis of Drosophila melanogaster. Z. Zellforsch. Mikroskop. Amt. 11, 616-637. Iaawa, M., Allfrey, V. G., and Mirsky, A. E. 1963. The relationship between RNA synthesis and loop structure in lampbrush chromosomes. Proc. Natl. Acad. S& US. 49,644-651. Kaufmann, B. P. 1933. Interchange between X- and Y-chromosomes in attached-

THE Y CHROMOSOME IN

Drosophila

22 1

X females of Drosophila melanogaster. Proc. Natl. Acad. Sci. US. 19, 830838. Kaufmann, B. P., and Gay, H. 1963. Cytological evaluation of differential radiosensitivity in spermatogenous cells of Drosophila. In “Repair from Genetic Radiation Damage and Differential Radiosensitivity in Germ Cells” (F. H. Sobels, ed.), p. 398. Pergamon Press, Oxford. Kernten, W., and Kersten, H. 1962. Zrir Wirkungsweise von Actinomycinen. 11. Bildung uberschikiger Desoxyribonucleinsaure in Bacillus subtdis. 111. Bindung von Actinomycin C an Nucleinsauren und Nucleotide. 2. Physiol. Chem. 327.234-242 ; 330,2130. Keyl, H. G. 1965a. A demonstrable local and geometric increase in the chromosomal DNA of Chironomus. Ezperientia 21,191-193. Keyl, H. G. 196513. Duplikation von Untereinheiten der chromosomalen DNS wiihrend der Evolution von Chironomus thummi. Chromosoma 17, 139-180. Keyl, H. G., and Pelling, C. 1963. Differentielle DNS-Replikation in den Speicheldrusenchromosomen von Chironomus thummi. Chromosoma 14, 347-359. Kuhn, E. 1930. Ein Beweis fur die Lebensfahigkeit von Spermatoeoen ohne Xund Y-Chromosom bei Drosophila mclnnogaster. Z . Znduktive Abstamrnungs Vererbungslehre 53, 2 6 3 7 . Lewis, E. B. 1950. The phenomenon of position effect. Advan. Genet. 3, 73-115. Lindsley, D. L., Edington, C. W., and von Halle, E. S. 1960. Sex-linked recesssive lethals in Drosophila whose expression is suppressed by the Y-chromosome. Genetics 45, 1649-1670. Mather, K. 1944. The genetical activity of heterochromatin. Proc. Roy. SOC. B132,308-332. Mete, C. W. 1927. Observations on spermatogenesis in Drosophila. 2. Zellforsch. Mikroskop. Anat. 4,l-28. Meyer, G. F. 1963. Die Funktionsstrukturen des Y-Chromosoms in den Spermatocyten-Kernen von Drosophila hydei, D . neohydei, D . repleta und einigen anderen Drosophila-Arten. Chromosoma 14, 207-255. Meyer, G. F. 1964. Die parakristallinen Korper in den Spermienschwanzen von Drosophila. 2.Zellfarsch. Mikroskop. Anat. 62, 762-784. Meyer, G. F. 1965. A reliable cytological method for classification of Drosophila species. Drosophila Inform.Serv. 40, 80. Meyer, G. F. 1968. Spermiogenese in normalen und Y-defizienten Miinnchen von Drosophila melanogaster und D . hydei. 2. Zellforsch. Mikroskop. Anat. 84, 141-175. Meyer, G. F., and Hess, 0. 1965. Strukturdifferenzierungen im Y-Chromosom von Drosophda hyded und ihre Beziehungen zu Gen-Aktivitiiten. 11. Effekt der RNS-Synthese-Hemmung durch Actinomycin. Chromosoma 16, 249-270. Meyer, G. F., Hess, O., and Beermann, W. 1961. Phasenspeeihhe Funktionsstrukturen in den Spermatocytenkernen von Drosophila melanogaster und ihre Abhhgigkeit vom Y-Chromosom. Chromosoma 12, 676716. Miller, 0. L., Carrier, R. F., and von Borstel, R. C. 1966. In situ and in vitro breakage of lampbrush chromosomes by X-irradiation. Nature 206, 905-908. Mirsky, A. E., and Ris, H. 1951. The desoxyribonucleic acid content of animal cells and its evolutionary significance. J . Gen. Physiol. 34, 451-462. Monroy, A., and Gross, P. R. 1967. The control of gene action during echinoderm embryogenesis. Morphological and biochemical aspects of cytodifferentiation. Ezpptl. Biol. M e d . 1,37-51.

222

OSWALD HESS AND G ~ N T H E RF. MEYER

Monroy, A., Maggio, R., and Rinaldi, A. M. 1965. Experimentally induced activation of the ribosomes of the unfertilized sea urchin egg. Proc. Natl. Acad. ScL U S . 54,107-111. Morgan, T. H.,Bridges, C. B., and Sturtevant, A. H. 1925. The genetics of Drosophila. Bibliogr. Genet. 2,l-262. Morgan, T. H.,Bridges, C. P., and Schultz, J. 1934. Report of investigations on the constitution of the germinal material in relation to heredity. Carnegie Inst. Wash. Publ. 33, 274-280. Nemer, M., and Infante, A. A. 1965. Messenger RNA in early sea urchin embryos: Size classes. Science 150, 217-221. Neuhaus, M. J. 1939. A cytogenetic study of the Y chromosome of Drosophila melanogaster. J. Genet. 37, 229-254. Olivieri, G., and Olivieri, A. 1965. Autoradiogmphic study of nucleic acid synthesis during spermatogenesis in Drosophila melanogaster. Mutation Res. 2,

366-380.

Pelling, C. 1964. Ribonukleinsaure-Sythese der Riesenchromosomen. Autoradiografische Untersuchungen an Chironomus tentans. Chromosoma 15, 71-122. Pelling, C. 1966. A replicative and synthetic chromosomal unit-the modern concept of the chromomere. Proc. Roy. Soc. B164,279-289. Plaut, W. 1963. On the replicative organization of DNA in the polytene chromosome of Drosophila melanogaster. J. Mol. Biol. 7 , 632-635. Reich, E., Franklin, R. M., Shatkin, A. T., and Tatum, E. L. 1962. Action of actinomycin on animal cells and viruses. Proc. Natl. Acad. Sci. 77.8. 48, 1238-

1245. Safir, S. R. 1920. Genetic and cytological examination of the phenomena of primary non-disjunction in Drosophila melanogaster. Genetics 5, 459-487. Schults, J. 1936. Variegation in Drosophila and the inert chromosome regions. Proc. Natl. Acad. Bci. UB. 22, 27-33. Schultz, J. 1947. The nature of heterochromatin. Cold Spring Harb. Symp. Quant. BWl. 12,179-191. Shen, T. H.1932. Cytologische Untersuchungen uber Sterilitiit bei Mannchen von Drosophila melanogaster und bei Fl-Miinnchen der Kreuzung swischen D . simulans-Weibchen und D . melanogaster-Mannchen. Z. Zelljorsch. Mikroskop. Anat. 15,547-580. Stern, C. 1927. Ein genetischer und cytologischer Beweis fiir Vererbung im YChromosom von Drosophila melanogaster. Z. Induktive Abstammungs- Vererbungslehre 44, 188-231. Stern, C. 1929. Untersuchungen uber Aberrationen des Y-Chromosoms von Drosophila melanogaster. Z . Induktive Abstammungs- Vererbungslehre 51, 253-353. Stern, C., and Hadorn, E. 1938. The determination of sterility in Drosophila. males without a complete Y chromosome. Am. Naturalist 72, 42-52. Ullerich, F. H. 1966.Karyotyp und DNS-Gehalt von Bujo bufo, B . viridis, B. bufo x B. viridis und B. calamita (Amphibia, Anura). Chromosoma 18, 316342. von Borstel, R. C., Miller, 0. L., and Carrier, R. F. 1966. X-irradiation-induced breakage of lampbrush chromosomes. In “Chromosomes Today” (C.D. Darlington and H. R. Lewis, eds.), Vol. 1, pp. 141-144. Oliver & Boyd, Edinburgh and London. Whitehouse, H.L. I(. 1967. A cycloid model for the chromosome. J . Cell Sci. 2, 9-22.

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223

Woskressensky, N. M., and Scheremetjewa, E. A. 1930. Die Spermiogenese bei Drosophila melanogaster. 2.Zellfmch. Mikroskrop. Anat. 10, 411-426. Yssusumi, G.,Jujimura, W., and Ishida, H. 1958. Spermatogenesis in animals aa revealed by electron microscopy. V. Spermatid differentiation of Drosophila and grasshopper. Exptl. Cell Res. 14, 268-285. Zimmermann, F., Kroger, H., Hagen, U., and Keck, K. 1964. The effect of Xirradiation on the priming ability of the D N A in the RNA polymerase system. Biochim. Biophys. Acta 87, 160-162. Zimmermann, F., Kroger, H., and Lucking, T. 1965. Interaction of RNA polymerase with irradiated DNA. Biochem. 2.342, 115-119. Zirjtin, A. I. 1929. On the the peculiarities of spermatogenesis in Drosophdu melanogaster. B i d . Bur. Genet., Leningrad 7 , 97-107.

SELECTIVE FERTILIZATION

IN Oenothera"

J . Schwemmle Botanical Institute. University of ErlangemNurnberg. Germany

I. Selective Frrtilization in the Crosses Oe . berlerinna x Oe . odorata . . A . Reciprocal Crosses B.1 x v.1 . . . . . . . . . . B . Proof of Selective Fertilization . . . . . . . . . C . Definition of the Concept of Affinity . . . . . . . . D . Determination of the Affinity v-B . . . . . . . . E . Supplementary Crosses . . . . . . . . . . . F. Additional Examples of Lark of Affinity . . . . . . . I1. Selective Fertilization in Selfing of Oe . herferiana (B.I) . . . . A . Question of the Fate of the Homozygotrs B.B and 1.1 . . . . B . Anatomical Investigations . . . . . . . . . . C . Proof of Selective Fertilization hy Breeding . . . . . . I11. Selective Fertilization in Srlfing of Oe . odorata (v.1) . . . . . A . Question of the Fate of the Homozygotes v.v and 1.1 . . . . B . Anatomical Investigations . . . . . . . . . . C . Proof of Selective Fertilization by Breeding . . . . . . and of Oe . IV. Selective Fertilization in Selfings of Oe . X ~ J ~ C(Istr.vstr) ~ U selowii (2se.vse) . . . . . . . . . . . . . . V . Selective Fertilization in Selfing of Or . cnmpylocalyz (cek.cl) . . . VI . Changes in Affinities . . . . . . . . . . . . . A . Influence of Environmental Factors . . . . . . . . B . Condition of Maturity of the Ovules . . . . . . . . C. Influence of Cytoplasm and Plnstids on the Affinities . . . . D . Changes of Afkitirs by Changes of the Genome . Influence of the Maternal Diplonts . . . . . . . . . . . . VII . Affinities in Trisomic Mutants . . . . . . . . . . VIII . Chemotropism of the Pollen Tubes . . . . . . . . . Appendix . Survey of the Strains Used in the Crosses . . . . . Rrfrrences . . . . . . . . . . . . . . .

226 226 230 232 233 235 237 239 239 240 243 254 254 255 256 268 270 278 279 280 298 306 313 316 321 321

In 1929 the analysis of Oenothera berteriana and Oe . odorata. section Raimannia. was started . Both are complex heterozygotes as defined by Renner . As in many of the Oenothera the 14 chromosomes are connected *Editor's note . The manuscript has been translated from the German orginal by

Miss Heide Ingenohl. to whom I want to express my gratitude for her competent

and thorough work .

226

226

J. SCHWEMMLE

to form a ring or a chain in diakinesis of meiosis. As a result of the zig-zag arrangement in metaphase, the chromosomes with their genes move alternatively to one or the other pole of the spindle. In this way the complexes are formed. They are designated B and 1 in Oenothera berteriana, and v and I in Oenothera odorata (Schwemmle, 1938a). 1. Selective Fertilization in Crosses Oe. berteriana X Oe. odorata

A. RECIPROCAL CROSSES B.Z X v.1 When Oe. berteriana (B.1) with the gametes B and 1 is crossed to Oe. odoruttz (v.1) with gametes v and I, the following new complex heterozygotes would be expected to occur with equal frequencies, assuming equal probabilities for the possible combinations, B.v, B.1, l.v, and 2.1. It was striking that in the 46 crosses of P B.1 X 6 v.1 which were grown up to 1942, the B.v was almost completely missing. Only 2 B.v were found out of a total of 7,484 plants (Table 1). 3,379 B and 4,088 Z ova were fertilized. That means that the B ovules were almost exclusively fertilized by I-pollen tubes. Since 1.v were more frequent than 1.1, it has to be assumed that the v-pollen tubes grow faster than the I-pollen tubes, and therefore compete more successfully for the 1 ovules. I n the 33 crosses of o v.1 X 8 B.1 145 ( = 5.7%) v.B were found (Table 1). They were normally green. The B.1 were mostly, and the Z.v always completely yellowish, since in both of them the Oe. odorata plastids were not able to become green and the 1 pollen tubes never transfer Oe. berteriana plastids (Haustein, 1938). When they are carried by . do not apB-pollen tubes the resulting B.1 become variegated. The Z1 pear a t all; these embryos die when they get Oe. odorata plastids through the ova (Binder, 1938). Meanwhile it has been confirmed in other crosses that proplastids may be able to inhibit the development of embryos (Kistner, 1955). It was not possible to establish the number of 1-pollen tubes which fertilized ova since the 1.1 did not appear in the o v.1 )( 8 B.Z crosses. Therefore the v.1 with Oe. berteriana cytoplasm and plastids was crossed with B.Z. These plantdl are obtained from the cross o B.1 ( 0 B.1 X 6 v.I), F1X 8 v.1. I n this experiment B.v were also found but somewhat more rarely, only 1.5% (see Table 1). They look exactly like the B.v from the cross 9 v.1 X 8 B.Z. Altogether 338 B-pollen tubes (14 B.v 324 B.1) and 595 Z-pollen tubes (304 Z.v+!291 1.1) accomplished fertilization. That means that the Z-pollen tubes have grown faster than the B-pollen tubes. Beeide the

+

UJ

H

P

E

3

TABLE 1 Progeny Obtained from Crosses B.1 x v.I* Crosses B.1 X v.1 v.1 X B.Z v.1 [Eert. Pl.] X B.1

Number 46

33 9

B.v 2

145 14

*In all crows, the female parent is given first.

3La

B.1

l.V

z.1

Dev.

Total seeds

3,377 1,135 324

2,755 1,242

1,333

7,484

304

291

17 3

-

2

2,545

935

2

E ?-

2

r,

2

F $

?r

Y

P

228

J. SCHWEMMLE

expected types some aberrant plants were found (“dev.” in Table 1 ) ; these were mostly trisomic mutants. Other crosses were made in which the complex B was introduced through the ovum and the complex v through the pollen (e.g., 0 B.2 X 8 B.v or o B.I. X 8 v.1) and vice versa (e.g., P B . v X 8 B.1 or ? 1.v X 8 B.1). Altogether out of 128 crosses (15,224 plants) in which B was transmitted through the ovum only 24 B.v (= 0.15%) were counted. But in the 128 crosses (10,093 plants) in which the complex v was introduced through the ovum 953 B.v = 9.4% were found. Since not all seedlings in the above crosses had been planted it is quite possible that, for example, in the crosses o B.1 X 8 v.1 the late germinating seeds with B.v embryos were lost. Therefore in 1943 and 1944 a larger number of crosses was set up. Before opening, the capsules were removed and their contents counted under a dissecting microscope. The following types of seeds were found: (1) Large seeds (L.S.) mostly with healthy embryos; (2) Shrunken seeds (sh. s.) which had developed from seeds with defective embryos, according to Schutz (1934) and Weidner-Rauh (1939) ; (3) the coarse powder (co. PO.) that develops out of ovules which have received pollen tubes ; (4) the fine powder (f. PO.) which develops out of unfertilized ovules and sterile ovules with decaying tetrads. The proportions were related t o the whole content of the capsules as percentages. Seeds from those capsules which contained the largest number of seeds were used. The seedlings were planted out until germination was completely finished. Their number was again related to the total content of the capsules. The same was done for the later appearing types and the other values included in Table 2. In the column “germinated” there is added in parentheses the percentage of the germinated and planted seeds related to the seeds which were laid out as loo%, that is, the percentage germinating. It could not be avoided that some of the seedlings and plants were lost. Their proportion in percentage is included in Table 2. The results of these crosses in which all seedlings were counted are the same as those described earlier. Again the B.v are missing in the crosses ? B.1 x 8 v.1. Since in these crosses the 1.v were more frequent than the 1.1 the v-pollen tubes must have grown faster than the I-pollen tubes. I n the crosses ? v.1 x 8 B.1 the B.v were found in every case. They were again more frequent when the ova had Oe. odoruta cytoplasm and plastids. In this case, 2.1 was missing. Since the amount of the coarse powder was increased considerably the dead 1.1

v,

m

TABLE 2 Types of Seeds and Genotypes of Progeny Obtained in Crosses B.1 X v.T, in Percentage* Crosses B.2 X v.1 (1943)

B.1 X v.1 (1944)

r

n

d

2 c

Cultures

L.S.

sh.8.

co.po.

Germinated

B.v

1.v

B.1

1.1

Dev.

Total see&

Imt

4

36.2

0.1

0.3

3.20 (90.7) 32.3 (80.0) 30.8 (75.0) 35.6 (89.3)

-

15.3

9.2

6.6

0.2

31.3

0.7

4

40.7

0.1

0.8

v.1 X B.I

3

41.3

0.4

10.1

v.1 [bert. PI.] X B.Z

4

40.4

0.2

0.1

-

13.1 12.4

5.1

0.4

31.0

M

1.3

2.7

13.6 12.8

-

-

29.1

1.7

0.2

11.7 1 0 . 7

9.9

0.5

33.0

2.6

* Abbreviations:L.S., large seeds; sh. a., shrunkenseeds; co. PO., coarse powder; dev., aberrant forms.

3

sE N

8

;P

Z

2 3

2

ir

'1

P

h3 N

W

230

J. SCHWEMMLE

embryos must have been contained in this powder. In the crosses P v.1. [bert]” X 8 B.Z, 21.6% Z-pollen tubes (11.7% Z.v 9.9% 1.1) have fertilized ova, but only 10.9% B-pollen tubes (0.2% B.v 10.7% B.1). This means that the Z-pollen tubes again must have grown faster than the B-pollen tubes. All these crosses o B.1 x 8 v.1 show unambiguously that the B ovules are not fertilized by the v-pollen tubes; they are fertilized only by the slower growing I-pollen tubes. The v-pollen tubes compete with the I-pollen tubes only for the Z ovules. These experiments demonstrate for the first time the actual occurrence of selective fertilization.

+

+

OF SELECTIVE FERTILIZATION B. PROOF

The question remains why the B ovules are not fertilined by the v-pollen tubes. It may be assumed that the v-pollen tubes do not react to chemotropic substances which are released by the B ovules and therefore fertilize almost without exception the 2 ovules. If this assumption is correct then pollination with v pollen only should result in a much lower number of seeds than in the cross o B.ZX 8 v.1. Fortunately only the v complex is transmitted by the pollen of the trisomic mutant “type A” which was derived from Oe. odorata (v.1) (Schwemmle, 1956a). When 2 lobes of the stigma of a castrated flower of B.1 are pollinated by the pollen of type A and 18-20 hours later both of the other two lobes with homozygous 1.1pollen, then the pollen tubes of the postpollination will fertilize all the B ovules which were not fertilized earlier by the v-pollen tubes, and possibly in addition also some Z ovules. A t all events the number of seeds should be much higher than in crosses 0 B.2 X 8 type A (v). For the experiments involving pre- and postpollination other homozygotes besides type A(v) were used. One of them was 1.1. This is a homozygote of the I complex of Oe. odorata (v.1) which is viable with Oe. berteriana plastids. It produces only I pollen. Seven of each of the crosses mentioned in Table 3 were made in 1944. For culturing *Editor‘s note. Following the suggestions of the International Committee on Genetic Nomenclature, cytoplasmic constitutions are indicated in square brackets. fiert.] and [bert. Pl.] means that the plant has the cytoplasm, including plastids, derived from Oe. berteriana; [od.] and [od. Pl.], the cytoplasm derived from Oe. odorata. Where the cytoplasmic constitution has been separated from the plastid constitution, the plastid constitution is indicated by Plstd., the remainder of the cytoplasm by Pl., P1. always preceding plstd. For example, v.1 [od. P1. bert. Plstd.] indicates a plant with the chromosomal complexes v and I, plastids derived from Oe. berteriann, and the remainder of the cytoplasm derived from Oe. odorata.

TABLE 3 Types of Seeds and Genotypes of Progeny Obtained in Crosses of 0 B.1 with v- and I-Pollen Tubes Alone, Simultaneously, and in Pre- and Postpollination*t Crosses

Cultures

L.S.

sh.s.

co.po.

4

40.7

0.1

0.8

B.I X v.1 B.1 X type A(v)

B.1

X 1.1

B.Z X type A(v)+ x 1.1

* Abbreviations: See Table 2.

3

20.4

0.1

1.8

2

48.4

0.1

0.9

4

49.9

0.5

2.5

Germinated

B.v

32.2

Dev.

Total seeds

Lost

i

5.1

0.4

31.0

1.3

=!

0.8

-

0.1

14.6

1.3

-

21.1

18.9

0.4

40.5

1.5

7.3

18.6

11.5

0.5

37.9

2.3

B.1

Z.1

-

13.1 12.4

15.9

-

13.7

42.0

-

(80.0) (77.5) (86.0) 40.2

(80.7)

0

1.v

t In the latter experiment, the genotype of the maternal parent is given first, those of the pre- and postpollinating pollen parents in this order.

2

' r2

8 3 0

c)

3

232

J. SCHWEMMLE

those capsules were used which possessed the most seeds. The values in Table 3 are mean values. I n crosses o B.1 x 8 v.1 with 40.7% large seeds and only few shrunken seeds and coarse powder the B.v did not appear. The B ovules were only fertilized by the I-pollen tubes. 12.4% B.1 were counted. Because of the faster growth rate of v-pollen tubes the number of Z.v (13.1%) was again higher than 1.1 (5.1%).Altogether 18.2% 1 ovules were fertilized. In o B.1 X 8 type A(v) the B.v did not appear; the B ovules were not fertilized by the v-pollen tubes. Therefore the number of seeds produced was much reduced. The few appearing B.1 plants show that sometimes the supernumerary chromosome of the altered I complex can be lost. The number of 1.v (13.7%) was the same as in the crosses o B.1 x 8 v.1. That means that in the latter cross the v-pollen tubes had such an advantage that they could fertilize the 1 ovules with maximum success. In the crosses o B.1 X 8 1.1the number of B.1 (21.1%) was higher than that of 1.1 (18.9%). The crosses of o B.1 X 8 type A o x 8 1.1 are very informative. With 49.9% large seeds they are even somewhat more successful than the 9 B.ZX 8 1.1crosses, which is not easily understandable. B.v were again missing. The B ovules which had not been fertilized by the v-pollen tubes of the prepollination were only fertilized by the I-pollen tubes of the postpollination. 18.6% B.1 were counted. They were somewhat less frequent than in the crosses of o B.1 x 8 1.1, with 21.1% B.1, because germination was lower and the losses larger. The number of 1.v (7.3%) was now lower than that of 1.1 from the postpollination (11.5%) even though the v-pollen tubes had a large advantage in time. This striking result can only be explained by the assumption that the I-pollen tubes with Oe. berteriana cytoplasm and plastids grow faster and could thus overtake the v-pollen tubes.

+

C. DEFINITION OF THE CONCEPT OF AFFINITY The investigations of Schutz and Weidner-Rauh have shown that the ovules in the ovary of B.1 are usually 40-50% sterile through decay of the tetrads after meiosis. Accordingly, 6 0 4 0 % of the ovules are fertile and can be fertilized. Of these 25430% are B or I ovules, respectively. However, only 13.7% 1.v have appeared in the crosses 0 B.1 X 8 type A (v). Therefore not all 1 ovules were fertilized by the v-pollen tubes, just as not all 1 ovules were fertilized by the I-pollen tubes in the crosses 0 B.1 and 8 1.1, since only 18.9% 1.1 were counted in these crosses. Even in the crosses o B.1 X 8 1.1,with 21.1% B.1, a con-

SELECTIVE FERTILIZATION IN

Oenothera

233

siderable number of B ovules were not fertilized. Apparently the frequency with which complex heteroeygotes are obtained in crosses depends on the genetic constitution of the ova and synergids in the embryo sac of the ovules and on that of the pollen tubes. That means that certain particular ovules have an affinity to certain pollen tubes which depends on the genetic constitutions of both. A measure of this affinity is the frequency in percentage of the possible complex heterozygotes relative to the total content of the capsule. That means that the affinity B to v, in short, B-v = 0, Gv = 13.7, Z-I = 18.9, B-I = 21.1. I n the crosses o B.2 X 8 v.1 and o B.1 X 8 type A(v) no more 1.v could be formed than corresponds to the affinity 1-v. Since the affinity Z-I = 18.9 is larger than Z-v in the crosses ? B.2 x 8 v.1, the I-pollen tubes could only fertilize the 2 ovules up to this maximal amount. Altogether 13.1 5.1 = 18.2% I ovules were fertilized. Surely many 2 ovules must have remained unfertilized, although there was still a large number of I-pollen tubes available. The same is true for the crosses ? B.2 X 8 type A ( v ) + X 8 1.1. Here only 7.3 11.5 = 18.8% 2 ovules were fertilized, the same amount as in crosses ? B.2 X 6 1.1. We shall become acquainted with further examples of this generalization.

+

+

D. DETERMINATION OF THE AFFINITYv-B B.v appeared only in the crosses 9 v.1 x 8 B.2 in which the B- and 2-pollen tubes compete for the v ovules. Therefore the affinity v-B could not be evaluated from this cross. The affinity could be found best if a type was used which transmits only the complex B through the pollen. Fortunately, the semiheterogamous type B .I1 produces only active B-pollen grains. B y crossing 9 Z1 . F1 [bert.] X 8 B.Z the B.11 is obtained. In 1.1 the complex I1 arises from the I complex by exchange of two I chromosomes with two 1-chromosomes (Haustein, 1952). In the crosses ? v.1 X 8 B.11 (Table 4) with only 27.4% large seeds, 7.2% B.v and 15.9% B.1 were counted. As the female parent, v.1 with Oe. berteriana cytoplasm and plastids (called from now on only [bert. Pl.] was chosen in order to avoid complication of the results due to the weakness of the B.1 from the crosses with the original v.1 female. Since there was no competition of the pollen tubes, B.v were more frequent than in the crosses ? v.1 [bert. Pl.] X 8 B.1 with only 0.2% B.v (see Table 2). The affinity v-B = 7.2 is not large. Even though this result proves that B-pollen tubes not infrequently fertilhe v ovules, nevertheless crosses with pre- and postpollination were carried out. Oenothera argentinea (ha.ha) was used as the homo-

TABLE 4 Results of Crosses for the Determination of the AfEnity v-B* Crosses

Cultures

L.S.

8h.s.

v.1 [bert Pl.] X B.IJ

4

27.4

0.1

v.1 [bert. PI.] X ha.ha

3

24.7

0.1

v.1 X B.II+ X ha.ha

* Abbreviations: See Table 2.

4

29.5

-

co.po.

-

Germinated

B.v

B.1

v.ha

I.ha

Dev.

Total seeds

Lost 1.7 0.4

1.8

7.2

15.9

-

(92.4)

-

-

7.7

15.9

-

23.3 23.6

5.0

18.3

1.0

0.8

-

25.1

24.0

243.9

(94.2)

8 kc

25.0

-

(98.2)

4

o.2

3K E

SELECTIVE FERTILIZATION

IN

Oenothera

235

zygote. The genome ha is transmitted through the pollen (Schwemmle and Zintl, 1939). From the crosses o v.1 x 8 ha.ha the affinities v-ha = 7.7 and I-ha = 15.9 could be obtained (Table 4). Apparently the ha-pollen tubes are less attracted chemotropically by the v ovules than by the I ovules. In the crosses o v.1 x 8 B.II+ x 8 ha-ha 5.0 B.v were found. Since part of the v ovules had already been fertilized by the B-pollen tubes, the ha-pollen tubes from the postpollination could fertilize only very few v ovules; 1.0% v.ha were counted. Altogether 6.0% v ovules were fertilized; that means somewhat less than in the other two crosses. The 18.3% B.1 were derived from the prepollination, the 0.8% I.ha from the postpollination. Altogether 19.1% I ovules were fertilized, that is, more than would have been expected according to the affinity I-B = 15.9. These small deviations are to be expected since the conditions of pollination and fertilization will never be exactly the same. At all events, a large part of both the v and the I ovules remain unfertilized. The number which will be fertilized depends on the highest affinities.

E. SUPPLEIMENTARY CROSSES I n order to confirm unambiguously that the v-pollen tubes never fertilize the B ovules whereas the B-pollen tubes fertilize the v ovules, crosses were set up with B.1 and l.v, both derived from o B.2 X 8 v.1, as female parents. In the crosses o B.I. x 8 type A(v) (Table 5 ) the B.v did not appear. 16.7% 1.v were counted. The affinities B-ha = 4.5 and I-ha = 14.2 were obtained from the crosses o B.1 X 8 ha.ha. In the crosses Q B.1 X 8 type A(v)+ X 8 ha.ha the faster growing v-pollen tubes of the prepollination fertilized 14.2% I ovules but not a single B ovule. The 1.7% I.ha were derived from the postpollination. They are so low in number since most of the I ovules had already been fertilized by the v-pollen tubes. Altogether 14.2 1.7 = 15.9% I ovules were fertilized, that is nearly the same number as in the crosses o B.1 X 8 type A (16.7%). Surely a large number of I ovules must have remained unfertilized. The B ovules which had not been fertilized by the v-pollen tubes were fertilized by the ha-pollen tubes to the extent of 5.5% , that is, somewhat more frequently than in crosses P B.1 X 8 ha.ha. Thus a sufficient number of ha-pollen tubes grew out which could have fertilized the unfertilized I ovules. But how many of these ovules are actually fertilized is limited by the highest affinities, that is I-v A6.7. I n the crosses o 2.v X 8 B.11 (Table 5 ) the B.v were found as ex-

+

M

Q,

TABLE 5 Itesults of Crosses for the Determination of Affinities B-v and v-B* Total

Crosses

Cultures

L.S.

sh.s.

co.po.

Germinated

B.v

1.v

B.ha

I.ha

Dev.

seeds

Lost

B.1 [bert. PI.] X Type A(v)

2

18.1

-

0.2

-

16.7

-

-

0.3

17.0

0.4

B.1 [hert. Pl.] X ha.ha

3

19.1

3.9

0.1

17.4 (97.3) 18.7

-

-

4.5

14.2

-

18.7

-

1.7

-

21.4

Lost

B.1 X Type A + X ha.ha

4

23.6

6.2

0.3

(99.6)

22.2 (96.0)

-

14.2

5.5

0.8

3z

Cultures

L.S.

sh.s.

co.pn.

Germinated

I.B

v.B

Z.ha

v.ha

Dev.

Total seeds

1.v X B.11

3

27.4

0.1

-

22.2

3.7

-

-

-

25.9

1.2

2.v X haha

2

22.6

0.1

-

-

-

13.6

7.7

-

27.3

0.2

1.v X B.II+ X ha.ha

2

23.7

-

0.2

27.1 (98.9) 21.5 (97.1) 22.4 (96.0)

15.9

4.8

1.7

-

-

22.4

-

Crosses

* Abbreviations: See Table 2.

4 u)

E

SELECTIVE FERTILIZATION IN

Oenothera

237

pected. But there was only 3.7% B.v as opposed to 7.2% in the crosses 0 v.1 X 8 B.11 (see Table 4). At all events the affinity v-B is not large. The affinities 2-ha = 13.6 and v-ha = 7.7 were obtained in the crosses ? 2.v X 8 ha-ha, the latter value being exactly the same as in the crosses P v.1 X 8 ha.ha (see Table 4). In the crosses 9 2.v )( 8 B.II+ X 8 ha.ha 15.9% 1 ovules were fertilized by B-pollen tubes from the prepollination. There could have been more since the affinity Z-B was 22.2 in the crosses P 1.v x 8 B.11. But in this case all four lobes of the stigma had been heavily pollinated by pollen of B.11 while in the crosses ? Z.v X 8 B.II+ X 8 ha.ha only two lobes were pollinated. Probably the number of B-pollen tubes was not sufficient. 1.7% E.ha were counted; the ha-pollen tubes must have caught up with the B-pollen tubes and thus competed with them. For if already 15.9% Z ovules had been fertilized earlier by B-pollen tubes the hapollen tubes would not have been able to fertilize any more Z ovules since the a.ffinity &ha was only 13.6. Unexpectedly, the B.v amounted t o 4.8%, more frequent than in crosses P Z.v X 8 B.11. But this difference is not too large. It is not understandable in view of the affinity Vha=7.2 why the v.ha did not appear. These examples, selected from a variety of crosses, confirm again that the B ovules are not fertilized by the v-pollen tubes but the v ovules are fertilized by the B-pollen tubes. This confirms unambiguously the existence of selective fertilization.

F. ADDITIONAL EXAMPLES OF LACKOF AFFINITY There exist other examples showing that some types of ovules are not fertilized by certain pollen tubes. Ruhl analyzed Oe. campylocalyx of the section Renneria recently established by Fischer (1962). It is a semiheterogamous complex heterozygote. The complexes ck and cl are transmitted through the eggs, but only ck through the pollen. (In the appendix, all species and complexes used in the crosses are mentioned. The compilation indicates also which complexes are transmitted through the ova and through the pollen.). I n the crosses P v.1 x 8 ck.cl, v.ck and I.ck only appeared with nearly equal frequencies (see Table 6). I n the reciprocal crosses 0 ck.cl X 8 v.1 the ck.v was missing. This means that the affinity ck-v = 0. The c1.v are not too frequent, representing 18.0% of all plants raised. There were only 9.0% ck.1 but 72.9% cl.1. Apparently the Ipollen tubes are less strongly attracted chemotropically by the ck ovules than by the cl ovules. That only very few cl ovules were

238

J. SCHWEMMLE

TABLE 6 Progeny of Croesea of Oe. campylocalyx to Oe. odorata, Oe. stricta, and Oe. selowii

Crosses

% ck.v

%

%

%

C1.V

ck.1 9.0 52,l

Cl.1 72.9 cl.vstr

ck.cl X v.1 v.1 X ck.cl

-

47.9

18.0

ck.cl X Zatr.vstr Istr.vstr X ck.cl

ck.ktr 12.1 52.6

cl.lstr 27.9

ck.vstr

ck.ke 21.4

cl.Zae 57.1

ck.we 0.7 36.4

ck.cl X Zac.vse kc.vee X ck.cl

63.6

-

60.0

47.6

cl.vse 20.7

Number of plant8 ( = 100%) 222 338 215 491 285 286

fertilized by v-pollen tubes cannot be due to competition of the pollen tubes since the v-pollen tubes grow faster than the I-pollen tubes. The isogamous complex heterozygotes Oe. stricta (1str.vstr) and Oe. selowii (1se.vse) which had been analyzed by Hsbryka (1951) were also crossed to ck.cl. These have 1 and v complexes designated correspondingly which are very similar to the 1 complex of Oe. berteriana (B.2) and the v complex of Oe. odorata (v.1). I n the crosses Q ck.cl x 8 Zstr.vstr the ck.vstr were missing although a large number of vstr pollen tubes had grown out as shown by the 60% cl.vstr. In the crosses P ck.cl X 8 lse.vse, the ck.vse was not entirely missing but very rare, 0.7%. Just as in the crosses o B.1 x 8 v.1 the B ovules are never or only very rarely fertilized by the,v-pollen tubes, the ck ovules are never fertilized by the v- and vstr-pollen tubes and only rarely by the vsepollen tubes. In the crosses 9 ck.cl X 8 Oe. hoolceri (hhook.hhook) neither the ck nor the cl ovules were fertilized by the hhmk-pollen tubes, which grew, however, through the style as was demonstrated directly. In the crosses P Oe. mollissima (mk.ml) x 8 Oe. hookeri the mk ovules were not fertilized a t all ; the ml ovules only in 9.0-6.0%. On the other hand, the crosses o Oe. hoolceri X 8 Oe. argentinea (ha.ha) did not give any progeny. In this case it was not investigated whether the ha-pollen tubes grow to the ovules but were not attracted by them chemotropically.

SELECTIVE FERTILIZATION IN

Oenothera

239

II. Selective Fertilization in Selfing of Oe. berteriana (8.1)

A. QUESTION OF THE FATE OF THE HOMOZYGOTES B.B AND 1.1 I n the isogamous complex heterozygote Oe. berteriana (B.1) the complexes B and 1 are inherited through the pollen as well as through the ova. If the gametes should combine with equal probabilities as is usually assumed then the following would be formed: 25% B.B

25% 1.B

25% B.1

25% 1.1

(the female gametes are written first). But during all the years in which plants were grown by cross-pollination, self-pollination, and crosses B.1X B.Z the homozygotes B.B and 1.1 never appeared. It was obvious t o assume that they are contained in the sterile seeds. But the germination experiments did not confirm this. Of the 1300 seeds of the 1927 harvest 97.3% germinated, of the 4300 seeds of the 1928 harvest 93.0%, of the 865 seeds of the 1937 harvest 98.1%. I n the last only 39 seeds = 4.5% were sterile. But there are found in the ripe capsules of the Oenothera in addition to the large seeds, the so-called powder. It was therefore possible that the homosygotes were contained in it. Ten capsules each from harvests 1927 and 1928 were counted out and the following mean values were found : 1927: 51.6% large seeds, 48.4% powder (lowest value 40.9%) ; 1928: 55.0% large seeds, 45.0% powder (lowest value 37.6%).

If the homozygotes B.B and 1.1 were contained in the powder then the proportion of powder should be lower in the crosses B.2 )( v.1. Therefore 8 capsules of the 1928 harvest were counted out. There were 2308 large seeds = 54.4%. The preportion of the powder was 45.6% (lowest value 39.5%). This was exactly the same frequency as in the selfings from the same year. Since in the crosses B.1 X 1.1 only B.1 and 1.1 were formed it was expected that with the aid of this cross it would be possible t o find out how many of the ovules can be fertilized. But the harvest of the year 1939 gave a lower yield with 55.4-31.9% large seeds, an average of 41.4% large seeds. The question could be raised now whether in selfings of B.1 the homozygotes are formed at all. Schutz has answered this question.

240

J. SCHWEMMLE

B. ANATOMICAL INVESTIGATIONS I n a capsule of B.1 selfed 286 (48.6%) large seeds and 302 (51.4%) powder were found. They were measured individually. It was found that the large seeds were distinctly separated from the powder without intermediates. I n the powder, a distinction was possible between coarse and fine powder; but between these there existed all kinds of intermediates so that a sharp distinction between the two types of powder was not possible. To observe the development especially of the powder the contents of two pistils were fixed and sectioned with a microtome 12 and 16 days after pollination of the stigma of B.2 with its own pollen. The results were : 470 ovules with embryo with two cotyledons 20 ovules with head-shaped (dying) embryo 14 ovules with entering pollen tubes (ovum not divided)

( 93.2%) ( 4.0%) ( 2.8%)

504 ovules

(100 %)

152 fertile ovules remained unfertilized. So altogether there had been 656 fertile ovules of which 504 = 76.8% had been fertilized. Furthermore there were 572 sterile ovules, in which the 4 gametocytes formed by meiosis had degenerated for unknown reasons (Weidner-Rauh, 1939). Relative to the complete contents of both capsules 46.6% of all ovules were sterile. It was important to know how large a percentage of the ovules in B.2 was sterile. Weidner-Rauh and Schutz investigated the proportion of sterile seeds in 16 pistils. It varies between 29.6% and 50.3%. Most frequent are values between 40 and 50% as seen in the folIowing compilation : % sterile ovules: No. of pistils investigated:

29.6 - 30 - 35 - 40 - 45 - 50 - 55 2 2 6 5 1

~~

The results of Huber (1955) and Leuchtmann (1955) are in good agreement. The occurrence of sterile ovules and the differences in their frequencies waa disturbing for the investigations on the physiology of fertilization. Intensive investigations demonstrated that the ovules with normal embryos develop into the germinating fertile seeds, and the ovules with the head-shaped embryos into sterile and shrunken seeds; the ovules with the entering pollen tubes and unfertilised ovules become coarse powder. The fine powder develops from the sterile ovules. In 4 pistils with 655 fertilized ovules 31 head-shaped embryos = 5.4%

SELECTIVE FERTILIZATION IN

Oenothera

241

were counted. Out of a total of 1124 5 8 ~ 5 . 2 %seeds did not germinate; they were sterile. This confirms unambiguously that the ovules with head-shaped dying embryos develop into nongenninating sterile seeds. If the dying embryos were B.B and 1.1 homozygotes, which is by no means proved yet, their proportions would be much too small. The proportion is too small even if we add the ovules with entering pollen tubes. This means that in the selfing of B.1 the B-pollen tubes fertilize the 1 ovules, the l-pollen tubes the B ovules. This would therefore constitute a selective fertilization. Whether and how frequently the B.B and 1.1 homozygotes are formed would be seen best in crosses p €3.1 X 8 B.11 (only B pollen) and 0 B.1 X 8 1.11 (only 1 pollen). I n the first cross BB and 1.B are formed, in the second B.1 and 1.1. Schutz made sections of 6 pistils of the cross 0 B.Z X 8 B.11. Besides ovules with normal 1.B embryos many ovules with dying embryos were indeed formed. Apparently the B.B ovules are those with the headshaped embryos; they show irregular cell wall formation, a constricted embryo sac, and vesicularly enlarged nuclei in the endosperm. I n addition, there occur ovules with dying embryos which are different from the described ones. These can only be 1.B embryos which die for nutritional reasons. I n Table 7 are summarized the results from 4 pistils which were fixed 10-17 days after pollination. The pollination was heavy in capsules 1-3, light in capsule 4. The percentages are related to the whole content of the capsule = 100. In capsule 1 in which only 6.7% of the ovules remained unfertilized the B.B were equally frequent (27.9%) as the 1.B (25.1 1.9 = 27.0%). The ratio 1.B : B.B is 1 : 1. That means that the 1 and B ovules were fertilized equally frequently at heavy pollination. It is the more surprising that in selfing of B.1 no B.B embryos are found, for the dying head-shaped embryos do not show the typical characteristics of B.B embryos. We see in the column “unfertilized ovules” that the number of unfertilized ovules increases in capsules 2 4 . The ratio l.B : B.B is shifted in a parallel fashion in favor of 1.B until the B.B do not appear a t all in capsule 4 with light pollination. Schutz interpreted this important observation to mean that the B-pollen tubes a t first preferentially fertilize the 1 ovules and fertilize the B ovules only hestitatingly, although the affinity B-B is apparently as large as 1-B. We shall become acquainted later with other examples which confirm the correctness of the results of Schiitz. Consequently, the frequency with which new complex heterozygotes are formed depends on (a) the affinities of the two kinds of ovules to the

+

TABLE 7 Contents of Four capsules from a Croes 9 B.Z X

Capsule 1 2 3 4

Head-shaped embryw

Total content (100%)

Normal 1.B

B.B

1.B

463 584 616 519

25.1 29.5 17.5 20.6

27.9 27.4 12.8

1.9 0.8 2.6

-

0.8

8 B.11, in Percentage 4

Undivided ova

Unfertilized ovules

Sterile

-

6.7 12.7 23.1 29.5

38.4 29.6 42.9 49.1

1.1

-

OVUleS

Ratio 1.B :B.B 1:l 1.1 : 1 1.6 : 1

-

3E

g

SELECTIVE FERTILIZATION

IN

Oenothera

243

one type of pollen tubes, and (b) the speed with which the pollen tubes react on one or the other kind of ovules, that is, probably on the chemotropic substances released by them, as defined by Haustein. The crosses 0 B.1 X 8 1.11 produce mostly a lower seed set than o B.1 X 8 B.11. The shrunken seeds are less frequent but the coarse powder has increased in amount. The content of 6 capsules was investigated anatomically. They were fixed again 10-17 days after pollination (Table 8). Besides ovules with normal B.1 embryos there were also some with head-shaped embryos which were often poor in cytoplasm; the embryo sac was developed normally; the nuclei of the endosperm often vesicularly enlarged. Even if all of these had been 1.1 embryos, they would still be much less frequent than the B.1 embryos even a t optimal seed set (capsule 1). That means the affinity Z-Z is much lower than B-1. Schiitz came to the same conclusion on the basis of the higher percentage of ovules with entering pollen tubes but with undivided ova which do not appear in o B.1 X 8 B.11 crosses. According to him a high proportion of the head-shaped embryos represent B.Z embryos which were disturbed in their developmentbut they cannot be distinguished from the 1.1 embryos. Therefore it cannot be decided whether ti difference in the speed of reaction is also involved in the crosses o B.l X 8 1.11. I n selfings of B.1 there occurs a low percentage of dying head-shaped embryos. According to their appearance, they could be 1.1. But this is improbable because of the low affinity 1-1. They are rather defective B.l embryos. That means that in selfings of B.1, B.B. and 1.1 embryos are not formed at all. The anatomical investigations of Schiitz also suggest the existence of selective fertilization in selfings of B.1, just a r ~the earlier observations mentioned above.

C. PROOF OF SELECTIVE FERTILIZATION BY BREEDINQ To prove the occurrence of selective fertilization definitely, many crosses were carried out in 1949. Some of these will be reported in the following. Ten to twelve capsules from each cross were counted, resulting in a value for mean seed set. For each experiment the 3 capsules with the highest number of large seeds were used for the cultures (see Table 9). Open pollination of B.1 resulted in a good yield with 52.2% large .seeds. Shrunken seeds were rare; the coarse powder more frequent (6.8%). Germination was good (94.8%); 1.8% sterile seeds were counted. I n crosses with Oe. scabra male, a homozygote of the section Renneria producing only hsc pollen, B.hsc and Lhsc were found. Al-

TABLE 8 Contents of Six Capsules from a Cross Q B.I X

Capsule 1

2 3 4 5 6

Total content (100%) 569 446 498 536 410 499

Ovules with embryos with two cotyledons 32.5 26.2 7.6 15.7 5.1 8.2

Ovules with head-haped embryos 8.8 2.0 5.0 0.2 9.3 1.2

8 LIT, in Perrentage Undivided ova 2.5 0.2 0.6 0.7 3.4 0.4

Unfertilized ovules 15.5 24.7 43.2 33.4 36.3 42.1

Sterile Ovules

40.7 46.9 43.6 50.0

45.9

48.1

4

u,

2

z4

$

TABLE 9 Progeny of Crosses of Oe. bwleriana (B.1) Selfed and as Maternal Parent to Oe. scabra (bsc-hsc) and 6.11, in Percentage* Crosses B.1 open

co.

Cultures

L.S.

sh.s

PO.

3

52.2

0.9

6.8

Germinated

Sterile seeds

B.B

B.Z

B.hsc

Lhsc

Dev.

49.6 (94.8)

1.8

-

49.6

-

-

-

41.8 (96.7) 54.5 (97.1) 50.5

1.5

-

-

16.3

23.9

0.3

40.5

13

1.4

-

-

22.9

29.5

0.4

52.8

1.7

1.1

-

-

21.0

27.7

0.4

49.1

1.4

3

43.5

-

4.7

3

56.3

-

3.7

3

51.8

-

2.5

B.1 X B J I (4 and 3

6

45.0

0.6

6.9

20.8 (44.6)

21.5

(21.5)

20.8

-

-

-

3

35.1

0.2

6.6

1’7.0

15.7

(15.7)

17.0

-

-

-

B.I X B.11 (3 loEes)+ x hsc.hsc B.2 X B.11 (2 lobes)+ X hsc.hsc

49.6

Lost

-

(97.8)

2

3

s

zE N

P

20.8

-

G 17.0

-

(48.9)

3 0

rc

3

46.2

0.9

4.6

28.4 (61 .5)

17.3

(17.3)

15.1

4.3

7.8

0.3

27.5

0.9

3

47.1

0.2

4.8

28.5 (60 .6)

18.2

(18.2)

12.7

5.4

9.3

0.2

27.6

0.9

* Abbreviations: See Table 2.

E M

B.1 X hsc.hsc (4 lohee) B.1 X hsc.hsc (2 lobes) B.1 X hsc.hsc (1 lobe)

lobes) B.2 X B.11 (2 lobes)

Total seeds

$-

N I@ cn

246

J. SCHWEMMLE

though in this case no lethal homozygotes are formed the coarse powder is nearly as frequent as in open pollination of B.Z. Therefore, B.B and 1.1 embryos are formed only rarely if a t all. The crosses 9 B.Z X 6 hsc.hsc in which two or one lobes of the stigma were heavily pollinated with hsc pollen produced nearly the same good yield. Also in this case the coarse powder was present. Germination was good. The sterile seeds (1.4 and 1.1%) were nearly as frequent as in open pollination of B.1. That means that they also appear where no lethal embryos are expected. The proportions of B.hsc and Z.hsc are seen in the table. Since the crosses o B.1 1N(N = lobes of the stigma) X 8 hsc.hsc produce such a good yield, pollination of only one lobe of the stigma is apparently sufficient. I n 6 crosses the mean values were 21.9% B.hsc and 28.6% 1.hsc. Only very few I ovules remained unfertilized. Consequently the affinities are B-hsc = 21.9 and Z-hsc = 28.6. Apparently the hsc-pollen tubes are attracted better by the 1 ovules. It was striking that the crosses o B.Z X 8 hsc.hsc which were carried out a t the same time and in which all 4 lobes of the stigma were heavily pollinated all produced a lower seed set. It seems as though with heavy pollination the pollen tubes inhibit each other in growing and that therefore not as many ovules were fertilized aa would have been possible. Schneider (1956) could confirm this assumption through her investigations. I n the 6 crosses 9 B.1 X 8 B.11 in which either 4 or 3 lobes of the stigma were pollinated with B.11 pollen 45.0% large seeds were found. Shrunken seeds and coarse powder were not more frequent than in open pollinated B.1, although B.B. homosygotes were formed in large numbers. Therefore only 44.6% of the large seeds germinated. 21.5% were sterile, which probably contained mostly the dead B.B. Consequently, the affinity B-B is estimated as 21.5. It is true that the value is somewhat too high since a small proportion of sterile seeds is always found even where they are not expected. Thus, also in the present crosses some 1.B may have died, the ovules developing into sterile seeds. 20.8% 1.B were counted. Considering the nongerminating full seeds, which certainly carried some 1.B embryos, the affinity 1-B is 23.4. The crosses o B.1 X 6 B.11 in which two lobes of the stigma were pollinated all produced a reduced seed set. It has to be concluded that a prepollination of two lobes of the stigma is not sufficient. In the halfheterogamous B.11 only the B pollen grew out. I n comparison to 9 B.1 X 6 B.11 (4and 3 stigma lobes pollinated) the B.B (relative to 21.5 BB = 100%) decreased by 27.0% ; the 1.B (relative t o 20.8% 1.B = 100% ) decreased by 18.3%-bscause the B-pollen tubes preferentially

SELECTIVE FERTILIZATION

IN

Oenotheru

247

fertilized the 1 ovules even though the affinity B-B is not much smaller than LB. I n the crosses o B.Z X 8 B.11 (3 stigma lobes = 3N)+ X 8 hsc.hsc the one unpollinated lobe of t.he stigma was postpollinated by pollen from hsc.hsc 17 hours after prepollination. The seed set was generally better than in the crosses 9 B.1 X 8 B.11 but lower than in the crosses 9 B.1 X 8 hsc.hsc (1N). I n the 3 capsules used for the raising of progeny, 46.2% large seeds were found. Of these 61.5% germinated; 17.3% of the seeds were sterile; they contained the B.B. The 15.1% B.1 were formed by the prepollination. Both are less frequent than in the crosses o B.Z X 8 B.11 (4 and 3 N ) . I n spite of their large advantage the outgrowing B-pollen tubes did not have enough time to fertilize a number of ovules which would correspond to their affinity. They were partly overtaken by the apparently faster growing hsc-pollen tubes. Schneider has shown that the hsc-pollen tubes grow much faster than the Z- and the still slower B-pollen tubes. Therefore 4.3% B.hsc and 7.8% 1.hsc were counted. Altogether 21.6% B ovules were fertilized (17.3% B.B. 4.3% B.hsc) ,that is, as many as correspond to the equa1 values of the f f i i t i e s B-hsc and B-B. Certainly many additional B ovules remained unfertilized. It was astonishing that only 22.9% I ovules were fertilized (15.1% B.l+ 7.8% Lhsc). Since the affinity is 1-hsc = 28.6 more 1 ovules should have been fertilized. It might be suspected that the 1 ovules lost their chemotropic attraction earlier than the B ovules when they are aging. But, as will be shown later, this was not the case. In the o B.Z X 8 B.11 (2N)+ X 8 hsc.hsc, of course, more hscpollen tubes competed with the B-pollen tubes. Therefore there are now fewer B.2 but more Z.hsc. The B.B., a t 18.2%, are more frequent than in the previously discussed crosses. Since in addition 5.4% B.hsc were counted, altogether 23.6% B ovules were fertilized, that is, slightly more than expected from the affinity B-hsc = 21.9. But such deviations are to be expected. Again too few 1 ovules were fertilized (22.0%). I n parallel crosses B.l stigmata were postpollinated by pollen from 1.1. Since the results were the same as in the previous crosses they will not be described. In any case, all crosses demonstrate unambiguously that the B.B. are often formed in suitable crosses even though they do not appear in selfings. I n order to find out whether and with what frequency the 1.2 homoaygotes are formed B.1 can be crossed with the half-heterogamous 1.11 male, which produces only active 1 pollen. Only crosses which have been raised will be discussed (see Table 10).

+

248 J. SCHWEMMLE

SELECTIVE FERTILIZATION

IN

Oeraothera

249

The 12 crosses o B.1 X 8 1.11 in which 4, 3, and 2 stigmatic lobes were pollinated with equal success produced with 30.0% large seeds a much lower seed set than the open pollinations of B.1, apparently because the 1.1 homozygotes are formed only rarely. Therefore the Z-pollen tubes are sufficient if only two stigmatic lobes are pollinated. Shrunken seeds were very scarce with 0.2%, but the coarse powder was strikingly frequent with 11.2%. It might be assumed that the 1.1 homozygotes are partly contained in the powder, but this is not the case. I n 1944, 11 crosses o B.l x 8 1.11 were carried out. I n 4 of these capsules the coarse powder was missing while in the others it ranged between 0.7 and 0.1%. Also in the open pollinated B.1 from the year 1949 the amount of coarse powder was quite high, 6.8% (Table 10). I n the 10 selfings of B.1 from the year 1944 there had been only 3.3% just as in the crosses B.1 X B.Z. Schutz has demonstrated that sometimes embryos may die which are normally able to develop. The hot summer of the year 1949 in which the crosses were made may have contributed to it. The germination was quite good, 87.7%. 3.6% sterile seeds were found; that means more than in free pollination of B.1. I n these there may have been part of the 1.1. Since even in the crosses o B.1 X 8 hac. hsc which had the best viable seed set sterile seeds occurred, the assumption appears justified that there were about 2.6% 1.1. The affinity l-l= 2.6 is very low. 26.2% B.2 were counted. Including the nongerminatecl large seeds in the affinity B-1 is 27.4. In the crosses o B.1 X 8 1.11 X 8 hsc.hsc sterile seeds were very rare. This indicates that the 1-pollen tubes fertilize the 1 ovules only hesitatingly, but preferentially the B ovules. The 15.1% B.1 were derived from the prepollination, the 7.6% B.hsc from the postpollination. Altogether 22.7% B ovules were fertilized, less than expected since the affinity B-1 is 27.4. The 18.1% Lhsc are derived from the postpollination. There are so many because the 1 ovules had not been fertilized by the Z-pollen tubes. But their proportion was still much lower than in the crosses o B.l x 8 hsc.hsc; possibly the aged 1 ovules did not sufficiently attract the hsc-pollen tubes. The crosses o B.1 X 8 1.1 (Table 11) produced a good yield with 53.5% large seeds. The proportion of coarse powder was strikingly high. This is found every time in crosses involving 1.1 males. The affinity LI = 25.3 was larger than B-I = 21.0. I n the crosses o B.1 X 8 1.11 ( 3 N ) + x 8 1.1the first outgrowing 1-pollen tubes had time enough to fertilize 20.0% B ovules. 2.4% B.1 were formed in the postpollination. Altogether 22.4 B ovules were fertilized, less than would have been possible according to the affinity B-l = 27.4. But it cannot be expected that

+

TABLE 11 3 1.1 and X $ 1-11, in Percentage*

Progeny of Crosses of 0 B.1 X CrorcseS

B.1 X 1.1 (4, 2, and 1N) B.2 X 2.11 (4, 3, and 2N) B.1 X 2.11 (3N)S

B.Z X Z.11

x 1.1

(2ivI-E x 1.1

L.S.

8h.s.

co. po.

Germi-

9

53.3

0.3

5.3

12

30.0

0.2

11.2

3

47.5

0.2

6.9

4

53.9

0.8

5.6

48.6 (91.1) 26.2 (87.7) 44.0 (93.2) 50.5 (93.3)

Culturea

* Abbreviations: See Table 2.

nated

Sterile seeds

B.1

1.1

B.1

1.1

Dev.

Total seeds

Loat

4.1

-

-

21.0

25.3

0.5

46.8

1.8

3.6

26.2

(2.6)

-

-

-

26.2

-

0.3

20.0

-

2.4

19.5

0.5

42.4

1.6

0.5

15.1

-

8.8

25.1

0.7

47.7

2.8

Q

m

2

3iz K

SELECTIVE FERTILIZATION IN

Oenothera

251

the Z-pollen tubes of the prepollination fertilize as many B ovules as they do in the crosses ? B.Z X 8 Z1 .1 with the highest yields. The high number of 1.1 (19.5%) can again be interpreted to mean that Z ovules remained available because they had not been fertilized by 1-pollen . was still tubes. Only very few seeds were sterile. The number of Z1 lower than in the crosses P B.Z X 8 1.1; but this was only the case because the Z-pollen tubes from the pollination of only one lobe of the stigma were not sufficient. But when in the crosses o B.Z X 8 Z1 .1 (2N)+X 8 1.12 lobes were postpollinated, the I-pollen tubes were more successful in the competition with the Z-pollen tubes. More I-pollen tubes could overtake the earlier outgrowing Z-pollen tubes. Therefore less B.Z and more B.1 were found than in the former crosses. The 1.1 (25.1%) were now as frequent as in the o B.Z X 8 1.1 crosses (25.3%). This shows again unambiguously that the Z ovules were avoided by the Z-pollen tubes, which fertilized, however, 15.1% B ovules, but could still be fertilized by the I-pollen tubes. The above crosses show (a) that the B.B homozygotes are formed plentifully as long as only B-pollen tubes fertilize without competition with other types; (b) that the 1.1 homozygotes are formed a t the most in small proportion even if only Z-pollen tubes are used for fertilization since the affinity L Z is only low. But in selfings of B.1 also the B.B are missing even though the affinity B-B is high. In this case the Z-pollen tubes should fertilize only the B ovules, the B-pollen tubes only the Z ovules. This would mean that there exists in this cross a distinct selective fertilization. In the earlier crosses (see page 230) it had been found that the Z-pollen tubes grow faster than the B-pollen tubes, since it could be shown that the former exclusively fertilized the B ovules. If in addition the B-pollen tubes fertilize first the unfertilized Z ovules left over by the Z-pollen tubes and only hesitatingly the B ovules, which in the meantime have been fertilized completely by the l-pollen tubes, the lack of homozygotes would be explained. This assumption was confirmed by the experiments of Table 12. I n 6 selfings of B.11 the seed set (27.8% large seeds) was not high. The seeds germinated only to 47.1%. 9.7% sterile seeds were counted. They contained, if not exclusively, a t least mostly the dead B.B homozygotes. There were 12.8% B.11, giving a ratio of B.B : B.11 = 1 : 1.3. The B.B therefore were nearly as frequent as the B.11. In the crosses Q B.11 x 8 1.11 17.0% B.I and only 1.3% 1.11were counted. The afinity 11-1 is apparently low. In the crosses o B.11 X 8 B.II+ X 8 1.11 the

TABLE 12 Progeny of Crosses B.11 Selfed, and 9 B.11 X # Z.11, in Percentage*

Cultures

L.S.

sh.s.

PO.

B.11 X B.11

5

27.8

1.3

0.8

B.11 X I.11

5

22.8

0.3

1.4

Crosses

B.11 X B.II+ X I.11

co.

1

40.4

2.5

0.9

1

37.1

1.4

1.4

1

30.5

2.0

4.5

3

20.8

4.4

7.9

* Abbreviations: See Table 2,

Germinated

Sterile seeds

B.11

B.1

1.11

12.8 (47.1) 19.2

9.7

12.8

-

2.5

-

16.4

(84.1)

23.8 (58.9) 25.5 (67.5) 23.6

(77.2) 17.2 (84.9)

seeds

Lost

Ratio B.B :B.II

-

12.8

-

1 :1.3

17.0

1 3

18.3

09

-

20.1

3.2

-

23.3

0.5

1 :1.2

12.1

23.2

1.7

-

24.9

0.6

1 :1.9

6.9

18.8

3.0

-

21.8

1.8

1:2.7

2.2

15.6

1.2

0.1

16.9

0.3

1 :7.1

Total

y ix d

E

3 z

SELECTIVE FERTILIZATION IN

Oenothera

253

postpollination had only little effect since there were only 3.2-1.2% B.E. The seed set was variable. It dropped from 40.4% to only 20.8% in an average of 3 lumped crosses. Concomitantly, the proportion of B.11 decreased. The number of sterile seeds, that is, B.B, decreased even more sharply. Therefore, when many B-pollen tubes achieve fertilization the ratio B.B : B.11 is 1 : 1.2, as in the selfings of B.11. But when only a small number of B-pollen tubes grow out these fertiliae preferentially the I1 ovules and only rarely the B ovules, since the ratio is now B.B : B.11 = 1 : 7.1. I n accordance with Schutz’s results this indicates a difference in reaction speed. This confirms the correctness of our second assumption, made to explain why B.B do not appear in selfings of B.1, namely that the B-pollen tubes fertilize predominantly the Z ovules and only hesitatingly the B ovules. I n addition the following observation shows that in selfings of B.1 the B-pollen tubes fertilize the 1 ovules and the 1-pollen tubes the B ovules. In 1944, the affinities B-Z = 24.2 and Z-B = 16.8 were established in suitable crosses. This would result in a viable seed set in selfings of B.1 of 41%. Actually, 41.3% were found.

16.8

24.2

41.0

Furthermore, in 1949 the sum of the affinities (B-Z=27.4 and GB = 23.4) was 50.8, as large as the value found in open pollination of B.2, namely 50.2% large seeds. The two affinities obtained in different years are not equal. But in both cases B-Z is larger than Z-B. Consequently in selfings of B.1 the B.1 should be more frequent than the 1.B (the complex derived from the ovum written first). Of course the two cannot be distinguished except by marking of the 1 complex of either the male or the female parent. By exchanging the chromosome 9 (Z2)10 containing the factor T (T = dotted petals) of the 1 complex of B&, the original Oe. berteriuna, against the chromosome 9(12)10 of the I complex of v.1, a B.Zt with self-colored petals is obtained. I n the same I chromosome of the v.1 the dominant allele Ri arose by mutation. Ri is expressed by a peculiarly grooved formation of the leaves. From suitable crosses B .ZIji were obtained.

254

J . SCHWEMMLE

By means of cross o B.ZT X 6 B.Zt it should be immediately possible to establish whether B.2, is more frequent than ZT.B (the complex derived from the ovum written first) since the affinity B-1 is larger than 2-B. Furthermore, it can be established whether the B-pollen tubes fertilize only the 1 ovules and the Z-pollen tubes only the B ovules. In 1951 a large number of reciprocal crosses were set up. Every time, the seeds from the 5 capsules with the highest number of seeds were raised. Surprisingly, in the crosses o B . h X 8 B.2t the B.Zt (20.3%) were equally frequent as the ZT.B (21.0%). In an earlier cross, o B.1p X 8 B&, the B.Zt (102plants) had been more frequent than ZT.B (88 plants) , a result which could not be explained at the time. In the other crosses the B.1 were more frequent than the Z.B. B.Zt X B.ZT B.1p X B.lnr B.2~4X B.Zt

: : :

21.7% B.Zp, 29.1% B.ZR(, 26.1% B.ZT,

18.5% 2t.B 16.4% 2p.B 19.3% 2~4.B

Since, for example, in crosses o B.2, X 6 B.Zp sterile seeds were not more frequent than in open pollinations of B.Z raised a t the same time, or in crosses in which no dying embryos are expected, it is established that the faster growing Z-pollen tubes fertilize exclusively B ovules, to which they are strongly attracted chemotropically as shown by their high affinity. The slower growing B-pollen tubes fertilize only the unfertilized Z ovules, even though the affinity B-B is quite high. But because of the difference in reaction speed there are no B ovules left over for the B-pollen tubes. B.B and 1.1 homozygotes are completely lacking. These results confirm unambiguously the occurrence of selective fertilization in selfings of Oe. berteriana (B.1). 111. Selective Fertilization in Selfing of Oe. odorafa (v.1)

A. QUESTIONOF

THE

FATEOF

THE

HOMOZYGOTES v.v AND 1.1

For the Oe. odoruta (v.1) with v and I pollen as well as v- and I-egg cells the question has to be asked where the homozygotes remain. If v.v and 1.1were t o be formed according to the probabilities of the combination, 25% v.v, 50% v.1 and I.v, and 25% 1.1 would be found. The v.v plants are viable even with Oe. odoruta plastids as characteristically dwarfed plants. This is shown by experiments with the mutant type A derived from Oe. odoratu (v.1). Type A transmits only complex v through the pollen. In 11 selfings of type A, involving 465 plants, 6.7% v.v were formed. But in 22 selfings of v.1 in the years 19271948 containing 1716 plants, 99.4% v.1, 0.1% v.v, and 0.5% 1.1 were

SELECTIVE FERTILIZATION IN

Oenothera

255

,counted. The 1.1 appeared only in the year 1927. They were yellowish seedlings which died early. Therefore it could be assumed that the 1.1 with Oe. odorat,a plastids are not viable. But sterile seeds are not frequent. In 1927 34 out of 3800 seeds = 0.9% were sterile; in 1944 65 out of 1788 seeds = 3.6%. About 81.3% of the seeds germinated. The shrunken seeds and the coarse powder also were infrequent, with 2.8%. I n 20 selfings of v.1 with Oe. berteriana cytoplasm and plastids in which 81.6% of the seeds had germinated out of 1455 plants, 89.9% v.1, 9.5% 1.1, and only 0.6% v.v were counted. The 1.1, which are viable in combination with Oe. berteriam plastids, are not too rare, but the v.v are very rare. But it has t o be mentioned that not all seedlings were planted. It was thus possible that the v.v were contained in the late germinating seeds. It has to be examined whether also in the selfings of v.1 the homozygotes do not appear in expected frequencies because of selective fertilization. Since 1944 many experiments have been carried out, only some of which will be described. The resulk of the others were the same.

B. ANATOMICAL INVESTIGATIONS First it is necessary to investigate at what time the 1.1 with Oe. odorata plastids die (von Zitek, 1954). v.1 with Oe. odoruta plastids were crossed to 1.1 male parents since many 1.1 should be formed in this cross. The whole contents of a pistil were fixed, embedded, and sectioned with the microtome at different days after pollination. Table 13 shows the results from one pistil 18 days after pollination. TABLE 13 Contents of One Pistil from a Cross 9 v.1 [od. Plstd.] X after Pollination

3 1.1, 18 Days

106 = 24.4% ovules with large normal embryos 23 = 5.3% ovules with small normal embryos 35 = 8.0% ovules with bilobed normal embryos 58 = 13.4% ovules with head-shaped embryos 14 = 3.2% ovules without embryos

130 = 29.9%

236 = 54.3% fertilized ovules 199 = 45.7% unfertilized fertile and sterile ovules 435

-

loo % total ovules

256

J. SCHWEMMLE

Apparently the 29.9% 1.1 die at different stages. Sometimes they can develop even so far that germinating seeds are formed. In order to determine to what stage the ovules with 1.1 embryos develop the structure of the seed coat had to be taken into account (Table 14). In one capsule of a cross o v.1 x 8 1.1the following structures were counted (Table 14): 40.3% large seeds of which 15.7% did not germinate, 7.9% shrunken seeds, and 6.6% coarse powder. If in the anatomically investigated capsule, the percentage of presumably nongerminating seeds was lower (10.3%) than expected but the coarse powder was more frequent (10.8%) than in the capsule which was raised, this may be explained by assuming that unfavorable conditions inhibited the development of the 1.1embryos at an earlier stage. In 8 pistil of the cross v.1 X v.1 selfed, which was also fixed 18 days after pollination, the counts in Table 15 were obtained. Beside the percentages obtained in the anatomical investigations are indicated the phenotypes to which different types of ovules probably would have developed and the percentages formed in a capsule whose seeds were raised. The agreement is good. We can thus evaluate the proportion of usually lethal 1.1 with Oe. odorata plastids appearing in selfings and crosses by adding the appropriate percentages of nongerminating seeds, shrunken seeds, and coarse powder. The values obtained will be somewhat too high since even in crosses in which no lethal embryos are expected not all seeds germinate and shrunken seeds and coarse powder occur. The v.1 with Oe. berteriana cytoplasm and plastids was also investigated anatomically. Here fertile ovules are more frequent. Besides they contain more ovules in a pistil than the original Oe. odorata v.1. When the capsules with the best rate of seed set are considered, the following proportions of fertile ovules are obtained: %fertile ovules 50 55 60 65 70 v.1 [od. Pl.1 21.1 68.4 10.5 (19 pistils = 100%) v. I [bert. Pl.] - 36.4 45.4 19.2 (11 pistils = 100%)

C. PROOF OF SELECTIVE FERTILIZATION BY BRE~DINCI Three out of 20 open pollinations of the year 1949 were raised. 85.5% of all ovules were fertilized. This means, assuming equal probabilities of the possible combinations, that 1/4 each (i.e., 14.6%) v.v and 1.1 and 2/4 (i.e., 29.2%) v.1 should be formed. 8.8% v.v, 8.4% 1.1, and 27.1% v.1 were counted. Contrary to the selfings of B.Z in this

TABLE 14 Developmental Fate of 1.1 Embryos: Seeds from One Capsule of a Cross of One Sectioned Pistil Seed coat

Embryos in ovule

0

v.1 X cjll 1.1, Compared to the Contents

% in

pistil

Developmental fate

%in

capsule

i3

2 E

N

8 a

Normal Slightly indented Strongly deformed

Small, two cotyledons Small, two cotyledons Bilobed, head-shaped, in part missing

Nongerminating seeds Shrunken seeds Coarse powder

10.3 8.8 10.8

Total

29.9

15.7

7.9 6.6

2

5

TABLE 15 Developmental Fate of Embryos from v.1 Selfed: Comparison of Embryos from Sectioned Pistils and the Progeny from One Capsule

% in

Seed coat

Embryos in ovule

Developmental fate

pistil

%in capsule

Normal Normal Slightly indented Strongly deformed

Large,two cotyledons

Germinating seeds Nongerminating seeds Shrunken seeds Coarse powder

48.6 2.9 3.2 0.8

46.6 3.1 2.5 3.3

Small, two cotyledons Small, two cotyledons Bilohed and head-shaped

4 m

ii

3

SELECTIVE FERTILIZATION IN

Oenothera

259

cross the homozygotes are formed, but their number is too low and that of v.1 too high. This means that also in the selfing of v.1 selective fertilization occurs. In addition capsules with lower seed sets were raised. The result wa.s that the number of v.v decreased more than that of v.1. This indicates a different reaction speed of the v-pollen tubes. In order to establish how many v.v and 1.1 can be formed a t all, v.1 were crossed with type A(v) males and with 1.1 males. In the 24 crosses with type A males producing a mean of 44.7% large seeds, only 0.4% shrunken seeds and 0.9% coarse powder were found. Four capsule contents were raised (Table 16). The affinity v-v = 22.2 was lower than the d n i t y I-v=28.9. The v.v decreased more than 1.v in the seeds from 5 capsules which had reduced seed sets. The ratio v.v : 1.v was 1 : 1.9 as opposed to 1 : 1.3 in the progenies discussed earlier. The same result was found in selfings of v.1. The seed set from 24 crosses 9 v.1 x 8 1.1 waa much lower with 26.1% large seeds; shrunken seeds (5.8%) and coarse powder (11.4%) were more frequent than in the crosses with type A males. They contained the dead seedlings 1.1as did some of the nongerminating seeds. For this reason germination was much lower. In 4 cultures from the seeds of capsules with the highest seed set, considering the nongerminating seeds, the shrunken seeds, and the coarse powder, the ailhities v-I = 27.2,I-I = 25.6 were obtained (Table 17). They are not very different. When the crosses produced a lower seed set the v.1 decreased more than the 1.1. The ratio was shifted in favor of 1.1 from 1 : 1.1 to 1 : 0.7. This is exactly reverse of the v.v, which decrease relatively as the seed set becomes lower. The same experiments were carried out with v.1 [bert. Pl.]. Since in this cross the 1.1 are viable it could have been assumed that their proportion could be most easily established. However, shrunken seeds and coarse powder were always frequent when 1.1 should have appeared. I n 2 capsules of selfings v.1 [bert. Pl.] 1.5% shrunken seeds and 11.7% coarse powder were counted. In the raised cultures only 0.1% 1.1 appeared. Therefore even here the proportions have to be estimated as described above. Only a few examples out of the large number of experiments will be presented. The selfings and crosses produced generally a better seed set than the original v.1. Also the comparable affiities were quite different. This indicates an influence of the cytoplasm, including the plastids, which will be discussed separately. Table 18 shows that too few homozygotes are formed as in the experiments with v.1 [od. P1.1. The deviation from the expected values is lowest in 1.1. Also in this case the ratio 1.1 : v.1 is shifted at lower seed sets in favor of 1.1. In two

260 T. SCHWEMMLE

TABLE 17 Progeny from Four Culturea of the Cross 9 v.1 X

Crosses

v.1

x 1.1

co.

r

8 1.1, in Percentage*

M

Cultures

L.S.

8h.s.

PO.

Germinated

v.1

1.1

4 4

36.4 14.6

7.2 3.2

9.2 14.3

27.2 (75.3) 13.1 (88 7)

27.2 13 1

125.6) (19 0 )

* Abbreviations: See Table 2.

Ratio

1.1 :v.1 1 :1.1 1~0.7

!E E $

g

U

c

Z

8 Y 0

cc

3-

CB

2

TABLE 18 Progeny from Crosses v.1 X v.1 and v.1 [bert. Pl.] X v.1 [bert. PI.], in Percentage, Compared to Expectation

%

Crosses

Cultures

v.1 [bert. Pl.] open v.1 [bert. Pl.]self v.1 x v.r

3 2

1

Expected

4 m

Observed

d

Fertilized ovules

% v.1

% v.v

% 1.1

% v.1

% v.v

% 1.1

63.5 61.5 58.3

31.8 30.6 29.2

15.9 15.3 14.6

15.9 15.3 14.6

38.9 34.5 38.7

5.4 6.3 7.8

12.0 13.3 8.4

w

9E E

SELECTIVE FERTILIZATION IN

Oenothera

263

selfings with 48.0% large seeds, the ratio was 1 : 1.6, but in two selfings with only 24.4% large seeds 1 : 1.4. In 20 crosses of o v.1 [bert. Pl.] x 8 type A there were on the average 48.9% large seeds. Shrunken seeds (0.4%) and coarse powder (0.2%) were very rare, just as in the crosses with v.1 [od. Pl.]. The v.v were less frequent than the 1.v in the raised cultures (Table 19). The v-pollen tubes were attracted less by the v ovules than by I ovules. The ratio v.v : 1.v is shifted again in favor of 1.v a t low seed sets. The same was found by Koepchen in his crosses P v.1 X 8 v.11. The v.11 is a half-heterogamous complex heterozygote which transfers only v through the pollen. As can be seen from Table 20, the v.v decrease the more strongly the lower the seed set. I n parentheses, the values from the crosses with o v.1 [bert. Pl.] are given. In 15 crosses o v.1 [bert. Pl.] X 8 1.1 with 31.7% large seeds, the shrunken seeds (4.3%) and the coarse powder (7.3%) were strikingly frequent since the 1.1 with Oe. berteriana plastids are viable. Von Zitek could prove that the 1.1 embryos die and that these ovules develop into nongerminating seeds, shrunken seeds, and coarse powder if the temperature drops strongly in the days after pollination. The v.v and 1.v embryos are not damaged by this drop in temperature since they are less sensitive. Therefore the proportions of 1.1 had to be evaluated in these experiments. As shown in Table 21 the affinity vI z 17.3 is strikingly low, but the affinity 1-1 = 27.7 very high. The 1.1 are relatively more frequent a t low seed sets than at high yields of seeds, as in the earlier experiments. We conclude therefore that a t low seed set the ratio v.v : v.1 is shifted in favor of v.1, but the ratio v.1 : 1.1 in favor of 1.1. This can be explained in the following way. In crosses o v.1 X 8 type A(v) the v-pollen tubes preferentially fertilize the I ovules and only hesitatingly the v ovules. When at plentiful pollination many pollen tubes grow down the style, this will actually be found; but there are sufficient v-pollen tubes available to fertilize also the v ovules which a t first had been avoided up t o the maximum value given by the affinity v-v. But when a t low yield less v-pollen tubes grow out the advantage of the I ovules can be recognized in a stronger decrease of V.V. Again there are different reaction velocities. If only the affinities were to determine the frequencies of fertilization of v and I ovules by v-pollen tubes the ratio v.v : 1.v should be the same in all cases. As was shown in the crosses 0 v.1 x 8 1.1the ratio v.1 : 1.1 is shifted in favor of 1.1 a t lower seed set. This means that the I-pollen tubes preferentially fertilize the I ovules and only hesitatingly the v ovules.

TABLE 19 Progeny from Cultures of the Cross 9 v.1 [bert. PI.] X

3 Type A(v), in Percentage

?

Crosses

Cultures

L.S.*

Germinated

v.v

1.v

Ratio v.v : 1.v

v.1 (bert. P1.J X type A(v)

4 1

55.1 28.5

51.9 (94.5) 26.3 (92.9)

19.8

31.9 20.5

1:1.6 1:3.8

* L.S. = large seeds.

5.4

8 u1

9Y a:

2

u,

Im?

TABLE 20

Progeny from Cultures of the Cross 9 v.1 X

Crosses

v.1 x v.11

Cultures

f+

L.S.

sh.8 and

co.po.

3 v.11, at Different Seed Sets, in Percentage*

G~minated

v.v

1.v

Dev.

Seeds

Lost

8

0

Ratio v.v : 1.1

cl

m

a +i +I

8(6)

52.5

1.2

+

8(9)

36.0

1.1

-

6(7)

21.5

0.6

--

6(5)

6.8

0.5

* Abbreviations: + + = high; + = slightly high; -

49.1 (94.5) 34.6 (96.8) 20.3 (95.4) 6.3 (95.1) = slightly low;

E

16.4

27.7

0.4

44.5

4.6

1 : 1 . 7 (1 : 2 . 5 )

g

9.4

22.3

0.4

32.1

2.5

1 ~ 2 . 4(1 :3.3)

1:

3.7

15.1

0.1

18.9

1.4

1 : 4 . 1 (1:5.8)

1.1

4.9

-

6.0

0.3

1 : 4 . 5 (1 :9.8)

CI

0

m

g

c)

23-

--

m

= low.

see a h Table 2.

3

F)

TABLE 21 Progeny from Cultures of the C r w 9 v.1 [bert. PI.] X 3 1.1in Percentage at Different Seed Sets* co.

Crosses

Cultures

L.S.

sh.s.

PO.

v.1 pert. PI.]X 1.1

4 3

47.8 26.2

3.8 4.1

5.1 13.1

* Abbreviations: See Table 2.

.

Germinated

v.1

1.1

Ratio v.1 :I.I

44.3 (92.7) 26.2 (100)

17.3 12.5

27.7 24.5

1 :1.6 1 :2.0

W

3m

0

zz E

SELECTIVE FERTILIZATION IN

Oenothera

267

But it also has to be mentioned that the difference between the reaction

speeds is not as large as it is for the v-pollen tubes. At all events it is the more surprising that the 1.1 occur too rarely in selfings of V.I.

Lechner (1964) could also demonstrate the difference in reaction speeds of the B- and v-pollen tubes in his extensive investigations. However, in crosses o B.Z x 8 hsc.hsc the ratio B.hsc : Z.hsc is the same a t plentiful and a t sparse pollination. There is no evidence for differences in reaction speeds. The same result was obtained in crosses 9 B.Z x 8 1.1. The difference in reaction speed of the v-pollen tubes offers the first possibility of explaining the fact that in selfings of v.1 the homosygotes do not appear with the expected frequency. The second possibility is to assume that the v-pollen tubes grow faster than the I-pollen tubes as suggested by the crosses 9 B.1 )( 8 v.1. Loertzer (1954) has demonstrated this fact directly. She found that v-pollen tubes grew through a sectioned style 2.5 cm in length in Oe. longiflora in 5 hours 40 minutes, whereas I-pollen tubes needed 6 hours 35 minutes, that is, about 1 hour more. Schneider confirmed these results. The faster growth of v-pollen tubes was demonstrated in a different way by von Zitek. When the faster growing v-pollen tubes reach the lower part of the ovary, competition with the I-pollen tubes is low. Therefore many v.v and 1.v are formed but only few 1.1. The competition with the I-pollen tubes is stronger in the upper regions of the ovary. The v.v will become less frequent whereas the 1.1 will increase considerably. Five capsules were divided into 3 pieces and the proportions of v.1, v.v, and 1.1in the upper, middle, and lower pieces determined and related to the total number of planted seeds in each third. The mean values are found in Table 22. It can be seen that the proportion of v.v is considerably reduced from the bottom to the top, while the 1.1 increase in the same direction. We can now explain the results obtained in the selfings of v.1 in the following way: The faster growing v-pollen tubes fertilize first the I ovules to which they have a high affinity and only hesitatingly the v ovules. These are preferentially fertilized by the more slowly growing I-pollen tubes. Since many I ovules had been fertilized earlier by v-pollen tubes not as many 1.1 appear as should be formed according to the high a%hity 1-1. But the difference in growth rate of v- and I-pollen tubes will not always be the same, depending on environmental conditions. When the v-pollen tubes grow faster than the I-pollen tubes there will appear

268

J . SCHWEMMLE

TABLE 22 Relative Frequencies of v.1, v.v, and 1.1 in the Upper, Middle, and Lower Regions of Cappsules from Crosses v.1 x v.1, in Percentage: Meana from Five Capsules Third of the capsule

v.1

v.v r.1

Upper

Middle

Lower

63.1 1.6 24.9

63.2 6.7 14.8

68.7 14.4 6.9

more v.v and less 1.1. When the difference is reduced the number of v.v tends to decrease, while the number of 1.1 will increase since now

the I-pollen tubes compete more successfully with the v-pollen tubes for the I ovules. Since the affinity I-v is larger than v-I the 1.v must be more frequent than the v.1 in selfings of v.1. Of course the two cannot be distinguished unless the I complex of one of the partners is marked by the genes T (dotted petals) or Ri (grooved leaves). The v.1, v.IT, and v . 1 ~were crossed with each other. Each cross was carried out 20 times. Each time the 5 capsules with the highest yields were raised. It is sufficient t o use the mean values obtained in the crosses 0 v.IT X 8 v.1 as an example, The results of the 5 other crosses are in principle the same.

4.5% v.v and 6.5% IT.Iwere obtained. Therefore the homozygotes did &s frequently as would be expected under equal frequencies of the possible combinations (9.5%). The I T ovules were fertilized in preference by the faster growing v-pollen tubes, the v ovules by the I-pollen tubes. That means that also in the selfings of v.1 selective fertilization occurs. The effect is, however, not as pronounced as in the selfings of B.1.

not appear

IV. Selective Fertilization in Selfings of Oe. sfricfu (Istr.vstr) and of Oe. selowii (Ise.vse)

x

I n the crosses Oe. berteriuna (B.1) Oe. odoorata (v.1) Z.v can be obtained. Already two species have been kept for a long time in culture; they are quite similar to 1.v (Oe. stricta and Oe. selowii).

SELECTIVE FERTILIZATION IN

Oenothera

269

They were analyzed by Habryka. Both are complex heterozygotes with complexes similar to the 1 and v complex. They were designated accordingly. In selfings of Zstr.vstr, the 1str.Zstr are missing. They are contained in the sterile seeds. Since these appear, sometimes in even higher frequencies, in crosses in which sterile seeds are not expected, their proportion as estimated (3.7%) is certainly too high. Besides 35.9% lstr.vstr., 2.8% vstr.vstr. were counted. Since the homoeygotes are so rare it could be assumed that in selfing of Zstr.vstr. selective fertilization occurs, and the same applies t o Oe. selowii. I n the latter the sterile seeds are somewhat more frequent; the vse.vse did not appear. I n order to demonstrate the occurrence of selective fertilization in both species it is sufficient to establish the occurrence of selective fertilization in Z.V. 46.9% large seeds 46.3% large seeds 45.3% large seeds 82.1% germination 80.4% germination 87.4% germination

29.1

35.9

1 ~ i . vself

2str.vstr self

When 1Rk.v are selfed (see diagram) the seed set is as high as in Oe. stricta selfed. But the seeds did not germinate as well and the loss of seedlings was higher so that only 29.1% 1Ri.V were obtained. The ~RI.IR( were very rare since in the 1.3% sterile seeds constitutions other than 1 ~ & must have been present. The v.v (7.2%) were more frequent than the vstr.vstr. It could be assumed that in selfings of lBI.v the faster growing v-pollen tubes fertilize preferentially the lRi ovules and only hesitatingly the v ovules, not many of which remain unfertilized for the slower growing ZRi-pollen tubes. Crosses o ZR6.v x 8 Zpv show that this explanation is correct. The seed set was the same as in 1Ri.V selfed and 1str.vstr. selfed. The v-pollen tubes fertilized 22.4% 1 ~ ovules t and in addition 7.5% v ovules. The slower growing ZT-pollen tubes found only it is assumed that all sterile seeds are 2.4% ZRi ovules available-if R l&a-nd in addition 5.2% v ovules. Apparently the affinity v-IT is lower than &$Cv. Since selective fertilization could be demonstrated for ZR6.v by this model experiment the same can be assumed for Zstr.vstr. I n this species, Zstr.lstr are more frequent, and the vstr.vstr less frequent than in Zm.v selfed and in crosses 0 ZRI.V X 8 14.v. Accordingly the lstr-pollen tubes

270

J. SCHWEMMLE

compete more successfully with the vstr-pollen tubes for the Zstr and vstr ovules since they grow faster. The experiments demonstrating selective fertilization in selfings of 2se.vse will not be discussed. I n every case it had been assumed that the v-pollen tubes grow faster than the I-pollen tubes. This was proved by crosses with 1.1 females. 3 1.1 X c? ke.vse: 7.1% I.lse (19.8%) 28.9% Lvse seeds: 35.9% 9 1.1 X c? 2str.vstr: 10.2% I.lstr (31.4%) 22.3% I.vstr seeds: 32.6%

The I.vstr and I.vse are more frequent than the I.lstr and I h e . Furthermore it can be seen that the proportion of I.Zse, that is, 19.8%, relative to the 35.9% plants counted = loo%, is lower than the 1.Zstr with a corresponding proportion of 31.4%. The Zstr-pollen tubes are faster than the Zse-pollen tubes. Also in the present examples the speed of growth of the pollen tubes is very important. In this connection the investigations of Schneider may be mentioned. She measured how fast pollen tubes of different genetic constitutions grow through a 3 cm long style of B.Z and of v.1. The pollen used for pollination was derived only from paternal plants which transmit only one type of active pollen grains. Pollen Style of

V

B.1

4h29’ 4W’

v.r

I

hso

1

B

+ 4h37’ + 5h19‘ + 6h0’ + 6h16’ 4b40’ + 5h35’ + 5 W ’ + 6h28’

The plus signs indicate that the differences in growth rate are statistically significant. The data show also that the genetic constitution of the tissues of the style through which the pollen tubes grow has an effect. Schneider’s experiments confirmed almost in every case the conclusions concerning the relative growth rates of geneticalIy different pollen tubes which had been first derived from the crosses involving pre- and postpollination. V. Selective Fertilization in Selfings of Oe. campylocalyx (ck.cl)

Oenothera campy ZocaZyz, which was analyzed by Ruhl (1952) belongs together with Oe. scabra t o the section R e n n e k established by Fischer. The plants belonging to this section possess capsules like those of the Eu-Oenothera (biennis group) and oval seeds like the species of the

SELECTIVE FERTILIZATION IN

Oenothera

271

section Raimannia; they are annuals. Oenothera campylocalyx (ck.cl) is half-heterogamous. The complexes ck and cl are both transmitted through the ovum, but only ck through the pollen. In crosses to species which transmit only one kind of pollen, the hybrids with the ck complex and with the cl complex occur with different frequencies. This is shown in Table 23. The crosses produced different yields. The c1 ovules are always fertilized more frequently than the ck ovules. I n the extreme case the latter were fertilized only very rarely by the vTABLE 23 Progeny Obtained in Crosses of Oe. campylocalyz (ck.cl) of Pollen

0 to Different Genotypes

3

Pollen

0 ck.cl

Ratio ck :cl

hsc.hsc ha.ha h1.hl B.11 1.11 v.11 1.1

hsc ha hl B 1

152 ck.hm 274 cl.hsc 1 ck.ha 21 cl.ha 6 ck.hl 58 cl,hl 75 ck.B 150 c1.B 38 ck.2 104 c1.l 1 ck.v 113 c1.v 74 ck.T 133 cl.1

1 :1.8 1 :21 1 :9.7 1 :2 1 :2.7 1 :113 1 :3.1

V

I

pollen tubes. The afhities of the ck and cl ovules to the different pollen tubes are very different. On the other hand, ck-pollen tubes are attracted chemotropically to different degrees by ovules with genetically different egg cells (Table 24). These crosses will not be discussed further. Only one remarkable fact should be mentioned. While the ck ovules are fertilized only very rarely by v-pollen tubes the v ovules attract the ck-pollen tubes as well as the I ovules. The crosses of Riihl led to the assumption that selective fertilization occurs also in Oe. campylocalyx. First it had to be investigated whether in selfings of ck.cl the ck.ck homozygotes appear and what becomes of these ovules. Lohmeyer demTABLE 24 Progeny of Different Complex Eeterozygotea Pollinated as Female Parents by ck Pollen

9 Zstr.vstr B.1 v.1 mk.ml Zse.vse

Ova lstr B

Pollen: ck

vstr l

V

I

mk lse

ml vse

258 Zstr.ck 481 B.ck 162 v.ck 78 mkxk 182 be.ck

233 vstr.ck 456 1.ck 176 I.ck 165 ml.ck 104 vse.ck

Ratio 1 :0 . 9 1 :0.9 1 :1.1 1 :2.1 1 :0 . 6

272

J . SCHWEMMLE

onstrated by means of anatomical investigations that the ck.ck die in different stages of development and the ovules give rise to sterile and shrunken seeds and to coarse powder. He found also that the cl ovules are fertilized more frequently by the ck-pollen tubes than the ck ovules. The ratio ck.ck to cl.ck was 1 : 1.9. From 52.3 to 39.9% (mean 45.9%) of all ovules and ovaries were sterile. These are values of a magnitude found in other crosses. Thus it was possible to evaluate the proportion of ck.ck in selfings. It was only necessary to add the sterile and shrunken seeds and the coarse powder together and to calculate the percentage relative to the total content of the capsule. Since some sterile seeds and so forth occur also in crosses in which they are not expected, the calculated proportions of ck.ck are somewhat too high. In counting capsules from selfings with different seed sets (Table 25) TABLE 25 Seed Set and Occurrence of cl.ck and ckxk in Selfings of Oe. campylocalyx (ck.cl)

%

%

%

Ratio

L.S.*

cl.ck

ck.ck

cl :ck

42.6 39.6 35.7 35.1 29.1

25.4 28.5 25.6 25.8 27.1

21.1 16.0 11.7 10.4 4.2

1.2 : 1 1.8 : 1 2 . 2 :1 2.5 : 1 6.5 :1

* L.8.= large seeds.

it was found that the proportion of ck.ck was lower, the lower the seed set of the selfings. However the cl.ck were about equally frequent at all seed sets. Probably few of the cl ovules remained unfertilized,

but certainly many ck ovules. These results can be interpreted only by assuming that the ck-pollen tubes fertilize preferentially the cl ovules and only hesitatingly the ck ovules. If many ck-pollen tubes grow out the ck ovules are fertilized up to the maximum determined by the affinity ck-ck. As less ck-pollen tubes grow out ck.ck is formed more rarely. This represents evidence for a difference in reaction velocities just as in the B- and v-pollen tubes. I n order t o find out whether the ck and cl ovules show differences in chemotropic attraction, Q ck.cl were crossed t o Oe. embra (hsc pollen), 1.1. (I pollen), and B.11 (B pollen) as male parents. The afEnities found in 3 or 4 cultures each are found in Table 26. I n every case the cl ovules are fertilized more frequently than the ck ovules,

SELECTIVE FERTILIZATION I N

Oenothera

273

TABLE 26 Progeny in Percentage from Pollination of 9 ck.cl with Pollen of Different Genotypes, Establishing the Different Affinities ~

Pollen Ova ck cl

ck :(*I

ck

11.7 25.6 1 :2.2

hsc 11.1 34.0 1 :3.1

I 6.8 16.5

1 :2.4

B 1.8 22.0 1 : 12.2

which have the strongest attraction for ck-pollen tubes and the lowest for B-pollen tubes. The hsc-pollen tubes fertilize the cl ovules most frequently, the I-pollen tubes most rarely. If the assumption is correct that the ck-pollen tubes preferentially fertilize the cl ovules and only hesitatingly the ck ovules, then the ratio cl : ck should be shifted against ck in prepollination with ck pollen and postpollinstion with, for example, hsc pollen if the pollen tubes of the postpollination can still compete with the ck-pollen tubes of the prepollination. In crosses ? ck.cl (2N)X 6 ck.cl two lobes of the stigma were pollinated with ck.cl pollen (Table 27). I n the 2 capsules raised, 35.1% large seeds were found, giving rise to 25.8% cl.ck and 10.4% ck.ck. The ratio cl : ck is therefore 2.5 : 1. Altogether 36.2% ck-pollen tubes had accomplished fertilization. The fact that the sum cl.ck ck.ck = 36.2% is higher than the percentage of large seeds (35.1%) is due t o the fact that the shrunken seeds and the coarse powder were included in ck.ck. The 4 crosses o ck.cl x 8 hsc.hsc gave a much better yield with 47.8%. 34.0% cl.hsc and 11.1% ck.hsc were counted. I n crosses o ck.cl x 8 ck.cl+ X 8 hsc.hsc two lobes of the stigma were prepollinated with pollen from ck.cl and 8-12 hours later the two other lobes were postpollinated with pollen from hsc.hsc. The 16.5% cl.ck and 4.6% ck.ck were derived from the prepollination. Altogether 21.1% ck-pollen tubes had accomplished fertilization as opposed to 36.7% in the cross o ck.cl X 8 ck.cl. That means that they were overtaken by the hsc-pollen tubes from the postpollination. Since fewer ck-pollen tubes had accomplished fertilization, the ratio was shifted from 2.5 : 1 to 3.6 : 1 in favor of ck.cl. Therefore ck-pollen tubes indeed fertilize the cl ovules preferentially and only the ck ovules hesitatingly. If this was not the case the ratio cl.ck should remain a t 2.5 : 1. The 14.5% cl.hsc and 6.9% ck.hsc were formed by the postpollination. Altogether 4.6 6.9= 11.5% ck ovules were fertilized, not more than corresponds

+

+

TABLE 27 Progeny Obtained from ck.cl aa Female Parents Pollinated by ck Pollen in Prepollination, and by hsc Pollen in Postpollination, in Percentage Crosses

Cultures

% L.S.'

ck.cl X ck.cl (2N) ck.cl X hsc.hsc ( 2 N ) ck.cl X ck.cl+ X hsc.hsc

2 4 4

35.1 47.8 43.4

~~

* L.S.

= large seeds.

4

ck.ck

el :ck

cl.hsc

%

rn

cl.ck

ck.hsc

25.8

10.4

2.5 : 1

-

-

16.5

4.6

3 . 6 :1

E

%

-

%

-

Ratio

-

%

34.0 14.5

11.1 6.9

g

33:

SELECTIVE FERTILIZATION IN

Oenothera

275

to their maximum affinity c k - c k = 11.7 (see Table 26) even though there were sufficient pollen tubes available. 16.5 14.5 = 31.0% cl ovules were fertilized, 3% less than in the crosses P ck.cl x 8 hsc.hsc. But the latter had a higher seed set with 47.8% large seeds; and of these the capsules with the highest seed sets were raised. In the crosses P ck.cl X 8 ck.cl+ X 8 hsc.hsc it is not likely that just those crosses will be examined in which the highest number of hsc-pollen tubes accomplished fertilization. The crosses in which ck.cl plants were postpollinated with the B pollen of B.11 gave in principle the same results (see Table 28). The two crosses ? c k d (2N)X 8 ck.cl with 35.1% large seeds have slready been discussed above. I n parentheses are added the values from two crosses with lower seed sets in which because of the different reaction velocities the ratio cl : ck is shifted from 2.5 : 1 to 6.5 : 1. In both crosses P ck.ck (2N)X 8 ck.cl+ X 8 B.11 with 38.4% large seeds the clxk are more frequent than they are in crosses P ck.cl X ck.cl. This is quite possible since in selfings up to 32.8% cl.ck were found. But the ck.ck are rarer than in the crosses 9 ck.cl X 8 ck.cl since the ck-pollen tubes fertilize ck ovules only hesitatingly. The ratio in this experiment is cl : ck = 3.8 : 1. The B-pollen tubes compete less successfully with the ck-pollen tubes from the prepollination for the cl ovules since only 1.8% c1.B are formed in the postpollination. Completely different results are obtained in the competition for the ck ovules since the ck.B from the postpollination are, a t 1.4%, nearly as frequent as in the crosses P ck.cl X 8 B.11-again indicating differences in reaction velocities. This is also shown in the two crosses with lower seed set. The values from these crosses are given in parentheses. The ck.ck decreased relatively more strongly than the cl.ck. The ratio cl : ck therefore becomes 4.9 : 1. Since fewer ck-pollen tubes grew down to the ovules the B-pollen tubes could compete more successfully with the ck-pollen tubes for the cl ovules; 2.9% c1.B were formed. The B-pollen tubes are still more successful in competition for the ck ovules since the ck-pollen tubes fertilize them only hesitatingly. The fact that the cl.hsc and ck.hsc are more frequent in the crosses with hsc.hsc pollen than the c1.B and ck.B are in the crosses with B.11 pollen indicates that the hsc-pollen tubes grow faster than the B-pollen tubes (see p. 270). Thus our experiments with Oe. campylocalyx from the section Renneria gave the same results as the experiments with species of the section Raimannia. Probably selective fertilization occurs also in species of the section Eu-Oenothera (biennis group) *

+

TABLE 28 Progeny from ck.rl 9 Pollinated by ck Pollen in Prepollination, and by B Pollen in Postpollination, in Percentage

%

%

Ratio cl:ck

10.4

-

( 4.2)

2.5 : 1 (6.5 : 1)

27.4 (24.5)

7.2 ( 5.0)

3.8 :1 (4.9 .1)

%

Cultures

L.S.*

dck

ck.ck

rk.c.1 X ck.rl (2N)

2C2)

ck.cl X B.11 ( 2 N ) ck.cl X ck.cl+ X B.11

3

35.1 (29.8) 25.7 38.4

25.8 (27.1)

Crosses

* L.S.

= large seeds.

W)

(33 5)

-

-

%

c1.B

% ck.B

m

-

-

M

22.0 1.8 (2.9)

1.8 1.4 (2.2)

4

8

z E

s

SELECTIVE FERTILIZATION IN

Oenothera

277

The above discussed experiments indicate that the frequencies with which new complex heterozygotes are formed in crosses depend on the genetic constitution of the egg cells (synergids) in the ovules and of the fertilizing pollen tubes. I n order t o show this once more the affinities obtained in reciprocal crosses of different species of the section Raimannia with Oe. hookeri are summarized in Table 29 (Schwemmle and Simon, 1956). The frequencies of the possible combinations in the crosses were not related to the whole contents of the capsule as percentages but calculated, for example, for the cross 2 B.Z X 6 Oe. hookeri, how many B and 1 ovules were fertilized by hhmk-pollen TABLE 29 Affinities of Oe. hookeri (hhook)Ovules and Pollen with Different Complexes* 1

Complex for affinities hhaok, via. -hhook hsc

ck I koak

1 ml hl

B V

ha

2

% Fertilized

fertile ovules Oe. hoolceri 9 99.4-93.5 66. Y-62.9 35.2-33.2 32.1-30.2 17 .O-16.0 10.9-10.2 9.6- 9 . 1 9.3- 8 . 7 5.2- 4 . 9

0

3

% Fertilized

fertile ovqles

Oe. hookeri 8 94.0-82.6 0 40.5-30.4 50.5-33.7 29.6-24.7 9.0- 6 . 0 21.1-18.5 17.2-14.3 49.641.3 27 3-14.1

*After Schwemmle and Simon (1956).

tubes. For this purpose i t had to be established how many fertile ovules were available in the plants which were used as female parents. This has been determined by anatomical investigations and by the use of crosses with highest seed set. The calculated values vary within certain limits since the proportions of fertile ovules also varied. I n column 1 is indicated the genetic constitution of the pollen tubes in the crosses used with Oe. hookeri as females or of the ovules (egg cells) in the reciprocal crosses with Oe. hookeri pollen. In column 2 is found how many ovules of Oe hookeri were fertilized by different types of pollen tubes. The highest number was formed in the cross ? Oe. hookeri X 6 Oe. scabra (hsc.hsc), a very low number in the cross 0 Oe. hookeri X 8 type A(v). The crosses ? Oe. hookeri X 8

278

J. SCHWEMMLE

Oe. urgentinea (ha.ha) did not produce seed. The summary shows that the same ovules, that is, those of Oe. hookeri, are fertilized with different frequencies by different types of pollen tubes. The pollen tubes, depending on their genetic constitutions, react differently to the chemotropically active substances secreted by the Oe. hookeri ovules. Column 3 shows how often ovules (egg cells) of different genetic constitutions are fertilized by Oe. hookeri pollen tubes. The same pollen tubes are apparently attracted chemotropically to a different degree by different types of ovules. Usually the afbities of Oe hookeri ovules to the different types of pollen tubes are lower than the corresponding affinities obtained in the reciprocal crosses. Normally the different affinities do not determine by themselves the occurrence of selective fertilization. In addition, competition between pollen tubes and sometimes different reaction velocities play a decisive role. VI. Changes in Afinitier

Very early i t became obvious that the same crosses produced different seed sets in different years. The crosses o B.Z )( 8 1.1 will be used as an example. The seed sets were 1943, 48.8%; 1944, 55.6%; 1949, 58.9%. Accordingly different proportions of B.1 and 1.1 were obtained. In Table 30 the afkities obtained in crosses with BJ pistillate TABLE 30 Affinities of B and 1 Ovules from B.1 to Five Genotypes of Pollen Tubes: Obtained in 1944 (in parentheses) and in 1949

Q B 91

8 hsc

8 1

C3B

81

8 ha

(25.8) 21.9 (20.9) 28.6

(21.1) 21.0 (19.0) 25.3

(15.2) 21.5 (16.8) 23.4

(24.2) 27.4 ( 1.2) 2.6

(12.4) 12.1 (20.3) 16.8

parents are given. The values in parentheses refer to crosses made in the year 1944. In most cases, the affinities were higher in 1949 than in 1944. The affinities B-I and B-ha were equal in the two years. The affinities B-hsc and &ha were lower in 1949 than in 1944. It was therefore assumed that the environmental conditions at the time the crosses were made might have an influence on the seed set and on the values of the affinities.

SELECTIVE FERTILIZATION IN

Oenothera

279

A. INFLUENCE OF ENVIRONMENTAL FACTORS Richter (1956) could demonstrate in her investigations that the seed set depends on the exact time of pollination. For example, the crosses set up on 8/19/1953 produced in part a lower seed set than those of 8/11/1953, as can be seen from the summary:

B ~ TX 1.1 B.11 X 1.1 B.1~6X 1.1 ~~

~

~~

811111953

8/19/1953

53. 1% large seeds 52.9% large seeds 49.8% large seeds

40,8% large seeds

~

~

~~

49.4% large seeds

49.2% large seeds ~

~

It is striking that the reduction in seed set is different for different

crosses. The largest and statistically most significant reduction is found in the crosses o B.Zt X 8 1.1. It is noteworthy that the crosses o B.1~4X 8 1.1had equal seed sets on both days. It seems therefore that the effect depends on the genetic constitution of the egg cells in the ovules and possibly also of the maternal diplonts. Possibly genetically identical pollen tubes may react differently on different days. Richter (1956) compared cultures of four crosses made during the period 7/177/25/1953 and on 8/14/1955 with each other. I n the cross P B.ZT x 8 B.11, the ZTB had decreased slightly in comparison to 7/17-7/25 while the B.B had increased. I n P B.ZT X 8 B.11 the higher seed set was due exclusiv,ely to the B.B which had increased by 3.5%. On the other hand, in 0 B.lRl X d B.11 the B.B had increased by 2.9% whereas lRi. B had decreased by 3.7%. I n spite of all precautions the same crosses gave different seed sets on different days, certainly because of external conditions which can never be kept constant. This made the investigations difficult, because experiments from various years could not be used for comparison but always had to be repeated. Crosses which were to be compared were therefore set up on the same day if a t all possible. The obtained results could then be regarded as established since differences could not be caused by changes in environmental conditions. But i t could also be imagined that the time of pollination, as counted from the day of castration, may be of importance. Possibly the B and 1 ovules of B.1 mature and age at different rates in the pistil, that is, they may reach the maximum chemotropic attraction with different speeds and not a t the same time and they may also lose their ability to attract a t different rates. This would explain why in crosses 0 B.Z x 8 1.1 sometimes the B.1 are more frequent than the 1.1 while a t other times exactly the opposite occurs.

280

J . SCHWEMMLE

Therefore in 1952 many crosses with early and late pollination were carried out. The results will be discussed in the following sections.

B. CONDITION OF MATURITY OF

THE

OVULES

1. Maturation and Aging of the B and 1 Ovules of Oe. berteriana ( B J )

The crosses o B.2 X 8 hsc.hsc in which pollination was carried out 10-12 hours after castration produced a good seed set with a mean of 50.4% large seeds in capsules 1-10. For the late pollination the flowers to be used had been castrated a t the same time as the flowers used for early pollination-but they were pollinated after 48, 80, and sometimes after 96 hours. The seed set was much lower (43.2% large seeds in capsules 1-10). The difference is statistically significant. The seeds from 16 capsules of the crosses with early pollination and 14 capsules of crosses with late pollination were raised. In Fig. 1

---__ Early

FIG.1. Seed set and progeny from P B.1 x 8 hsc.hsc. Left, early pollination; right, late pollination. Abscissa: capsules, arranged in order of increasing seed set at the left, in order of decreasing seed set at the right. -, % large seeds; , % B.hsc; , % Lhsc. From Schaemmle (1957a).

--------

the percentages of large seeds and the proportions of B.hsc and 1.hsc are arranged according to rising and falling values. I n crosses with early pollination sometimes the B.hsc were more frequent and sometimes the Lhsc. When the seed set increased both complexes increased in the same direction. Therefore it may be concluded that in B and 1 ovules the ability to attract pollen tubes chemotropically increases gradually and reaches a maximum a t 25.9% B.hsc and 27.5% l.hsc. I n crosses with late pollination the 1.hsc were always more frequent than the B.hsc. This can only mean that the B ovules lose their ability to attract pollen tubes faster than the I ovules; in other words, they age faster. Crosses with Oe. berteriana (BJ,) (no dots on their petals) which were made up and grown at the same time show that the B and I t ovules T dotted petals. mature more slowly than the B and 1~ ovules of B ~ with

SELECTIVE FERTILIZATION IN

Oenothera

281

I n crosses with late pollination the It ovules seem to age slightly faster than the ZF ovules. However, the B ovules of B.Zt age more slowly than the B ovules of B.ZT. It might be assumed that crosses with late pollination produce generally a lower seed set than early pollinations. But in the crosses trisomic mutant of Oe. odorata with v.1 tenella as pollen parent-a producing only I pollen-which were carried out a t the same time as those involving hsc.hsc males this was not found to be the case (Fig. 2). I n capsules 1-10 of these crosses with early pollination there were in the mean 33.9% large seeds while in the late pollinations the mean was 54.4%. The latter thus had had a higher seed set. This is also shown in Fig. 2 concerning the proportions of B.1 and 1.1 which were

FIQ.2. Seed set and progeny from 9 R.2 x 8 v.1. var. tenella (I). Left, early pollination; right, late pollination. Abscissa: capsules, arranged in order of increasing seed set at the left, in order of decreasing seed set at the right. ,% large seeds; - - - - - - - -, % B.1.; -._._._._ , % 1.1. From Schwemmle (1957a).

-

found in the cultures. I n crosses with early pollination the B.1 were always less frequent than the 1.1. Apparently the B ovules mature more slowly than the 1 ovules. Both reach their maximum chemotropic attraction relatively late; in this case the B.1 and the 1.1 are formed with equal frequencies. With decreasing yield which indicates aging of the ovules, the B.1 become again less frequent than the 1.1. The B ovules age faster than the Z ovules. The same result had been obtained in the crosses with hsc.hsc pollen parents. Since the crosses to be compared were made up and grown at the same time the results show unambiguously the difference in reaction of the hsc- and the I-pollen tubes to the chemotropically active substances secreted by the ovules. The experiments are to be interpreted in the following way: I n the crosses with early pollination, the concentration of the substances se-

282

J . SCHWEMMLE

creted by the B and 1 ovules is sufficient over a relatively wide range to attract the more strongly reacting hsc-pollen tubes equally well. Therefore the B.hsc are almost as frequent as the 1.hsc. But especially in the beginning the concentration is still too low for the less strongly reacting I-pollen tubes. Therefore the P B.1 X 8 v.1 tenella crosses produce a lower seed set in early pollination. Furthermore, the I-pollen tubes are more strongly attracted by the faster maturing 1 ovules than by the B ovules. The attraction reaches its maximum relatively late and is then equally strong for both types of ovules. Later on the B ovules age faster than the 1 ovules. In comparable crosses involving B.Zt pistillate parents it became clear that the lt ovules mature more slowly and age faster than the IT ovules. The same result was obtained in crosses with hsc.hsc pollen parents. The difference in maturation and aging between the B and Z ovules was found also in other crosses. These show especially distinctly how differently the pollen tubes react depending on their genetic constitutions. If the B ovules age faster than the I ovules, the same might be the case for the B pollen as compared to the 2-pollen. Therefore on the same day 1.1 stigmata were pollinated by B.Z pollen taken directly out of the anthers whereas other 1.1 stigmata were pollinated with B.Z pollen obtained from anthers which had been kept for 6 days in an open Petri dish near a northern window. The seed set was very good in pollinations with fresh pollen (maximum 56.3% large seeds), but poor when old pollen was used (maximum 17.8% large seeds). I n the six cultures 0 1.1X 8 B.Z (fresh pollen) in the mean 12.4% 1.B and 33.8% 1.1 were obtained. Apparently the l-pollen tubes grow much faster than the B-pollen tubes. I n the six crosses P 1.1X 8 B.1 (old pollen) the 1.B were absent and the 1.1 amounted to only 9.3%. It is therefore certain that the B-pollen grains age and die faster than the l-pollen grains, just as the B ovules age faster than the 1 ovules. In the crosses in which fertilization occurred 47 hours and more after castration, there were less seeds in the lower parts of the capsules than at the top. This finding suggests the assumption that the ovules do not age uniformly throughout the ovary but age earlier in the lower regions than at the top. Similarly the ovules will probably mature earlier in the lower parts of the ovary than in the upper parts. To examine this assumption crosses of the following types were made in 1953: o B.1 X 8 type A ( v ) , o B.1 X 8 1.1, and P B.1 X 8 type A(v)+ X 8 1.1, 20 crosses each. The flowers were castrated at the same time. They were pollinated with type A(v) pollen 22 and

SELECTIVE FERTILIZATION IN

Oenothera

283

26 hours after castration and with 1.1. pollen 57 and 53 hours after castration. The capsules were divided into three pieces and the proportions of large seeds were expressed as percentages relative to the whole contents of the upper, middle, and lower third. The seeds were grown separately. In the crosses P B.Z X 8 type A(v) in which only 1.v are formed the proportions were nearly the same in all three parts in the capsules with the highest seed sets. But in the others it becomes increasingly clear that the percentage of large seeds decreases from below to the top. This result should be examined with reference to Fig. 3. In capsule 15 19.4% large seeds = 1.v in the lower piece, 13.5% in the middle piece, and 9.9% in the upper piece %

301 1 15

I

16

I

I

I

1

I

13

8

7

5

3

I

I

I

4

I

2 Ka

FIQ.3. Percentage of large seeds in capsules from crosses 0 B.2 x 8 type Atv). Upper third; - - - -, middle third; -.-.-+--, lower third. Abscissa: Ka=capsules, arranged according to % large seeds in the upper third, i.e., ac-

-,

-- --

cording to age of ovary at pollination. The numbers of the capsules are arbitrary. From Schwemmle (195%).

were counted. The chemotropic attraction decreases from below to the top. At the time of the pollination the 1 ovules in the lower third were not yet in a condition in which they could have attracted the v-pollen tubes optimally. Nevertheless a large part of them were fertilized. In the middle third the ovules were still less mature and the immaturity was even more pronounced in the upper third. Therefore the seed set decreased toward the top. I n capsules 16-5 the 1 ovules of the lower third approach increasingly more closely the condition of optimal attraction for the v-pollen tubes. This is also the case for the middle and upper third since the curves rise steadily; but the differences in the condition of maturity are maintained. I n capsule 3, 28.4% large seeds were counted in the lower third. In this case its 1 ovules have reached the condition of optimal attraction for the v-pollen tubes. Most ovules will have become fertilized. The ovules of

284

J . SCHWEMMLE

the middle third with 32.8% large seeds and of the upper piece with 19.6% large seeds are not yet mature enough. I n capsule 1 the seed set is reduced to 24.8% in the lower third. The ovules have started to age and therefore their chemotropic attraction becomes again lower. But the ovules of both the other two pieces continued to mature; the seed sets rose to 25.0% in the middle and t o 21.0% in the upper third. I n capsules 4 and 2 the period of optimum attraction had already passed even in the middle third; the seed sets dropped to 22.3% and 21.7% respectively. Since the 1 ovules age slowly the differences between the middle and the lower third become smaller. The curve for the upper third continues to rise up to 25.9%. Its 1 ovules approach the condition in which they attract the v-pollen tubes optimally. I n the P B.1 x 8 1.1crosses, which were pollinated 47 and 53 hours after castration, most seeds were found in the upper third. The mean seed sets and the corresponding proportions of B.1 and 1.1 in 10 cultures each are given in Table 31. Apparently the ovules of the upper TABLE 31 Seed Set and B.1 and 1.1 Progeny in the Upper, Middle, and Lower Thirds from 10 Capsules of Crosses 9 B.Z X 8 1.1: Pollination 47 and 53 Hours after Castration

Upper third Middle third Lower third

* L.S.

=

Ratio

% L.S.*

% B.1

% 1.1

B.1 : 1.1

62.1 38.6 14.8

25.7

28.9 21.1 9.9

1 :1.1 1 :1.5 1 :2.6

14.2

3.8

large seeds.

third have aged only little or not a t all. This is indicated by the high relative values for B.1 and 1.1. I n the middle and still more in the lower piece the ovules have aged so that they do not attract the I-pollen tubes very strongly. The ovules which are in the lower part of the ovary mature and age earlier than those in the middle third. The same is true for the middle third in comparison t o the upper part. Already in the experiments in which the seeds from whole capsules were grown, faster aging of the B ovules could be established. The same is found again in the present experiment. The B.1 decrease more strongly from the top to the bottom than the 1.1, as indicated by the ratio B.1 : 2.1. The crosses P B.1 X 8 type A ( v ) were carried out in order to demonstrate definitely that the B ovules are not fertilized by the v-pollen

SELECTIVE FERTILIZATION IN

Oenothera

285

tubes from the prepollination with type A. Surprisingly, the seed set of the 10 crosses ? B.2 X 8 type A ( v ) + X 6 1.1 with 42.4% large seeds in the whole capsules was better by about 5.9% than that of the 10 crosses ? B.2 X 8 1.1with 36.3% large seeds. From the summary (Table 32) it can be concluded that in crosses o B.1 x 8 type A+ X 8 1.1the seed set was lower in the upper third but higher in the middle and especially in the lower piece. Therefore the seed set of the whole capsules was higher. Corresponding results are obtained after postpollination of B.1 with pollen from 1.1. There are two possibilities to explain this peculiar result: (1) The tissues of the style and of the ovary become softened by the growth of the v-pollen tubes from the prepollination favoring the I-pollen tubes of the postpollination. Therefore the ovules become more reactive than they are in the crosses 9 B.1 X 8 1.1. (2) The B ovules age more slowly in the crosses 0 B.Z X 8 type A(v)+ X 6 1.1 than in the crosses o B.1 X 6 1.1 since the 2 ovules had been fertilized by the v-pollen tubes of the prepollination. If the proportion of B.1 in the crosses o B.1 x 8 1.1 in the upper third is taken as loo%, then B.1 decreases to 55.2% in the middle and to 14.8% in the lower third. I n the crosses ? B.2 X 8 type A(v)+ X 8 1.1 the rate of decrease is much lower. Similar results are found for the 2 ovules. In ? B.2 X 8 type A+ X s 1.1 fewer 1 ovules were fertilized in the upper third, slightly more in the middle piece, but considerably more in the lower piece in comparison with 0 B.2 x 8 1.1 (Table 33). That means that the 1 ovules age also more slowly in o B.Z x 8 type A (v) X 8 1.1than in ? B.1 X 8 1.1. If in the former cross about 3.4% less 1 ovules were fertilized in the upper part (28.9%-25.4%), the reason may be that just as the 1 ovules age more slowly in the middle and lower thirds, the 2 ovules of the upper piece mature more slowly and have not yet reached their optimal chemotropic attraction. In 1953, crosses 0 B.Z x 8 2.11; o B.Z x 8 hsc.hsc, and ? B.2 X 8 HI+ x 8 hsc.hsc were also carried out. It was intended t o show again that the 1.1 are formed only rarely. Already here it should be mentioned that in these crosses the 1 ovules were not fertilized a t all by the Z-pollen tubes. B.Z x 8 I.11 in which pollination was carried I n the 10 crosses out 24 and 28 hours after castration, 13.6% large seeds with B.1 embryos were counted in the upper third, 12.4% in the middle third, and 15.5% in the lower third. I n this cross, no difference between the top and the bottom is visible. Thus by the use of Z-pollen tubes it could

+

TABLE 32

Seed Set and Percentage of Fertilized B Ovules in the Upper, Middle, and Lower Third of Capsules from Crosaea 0 B.2 X 31.1 and X 8 Type A(v)+ X 81.1 Upper third

Middle third

?

Lower third

u1

% L.S.*

% €3.1

% L.S.

% B.1

% L.S.

%B.I

B.1 X 1.1

62.1

38.6

54.6

14.2 (55.2) 16.6 (68.0)

14.8

B.1 X type A+ X 1.1

25.7 (=100%) 24.4 ( = 100%)

3.8 (14.8)

Crosses

* L.S.

= large seeds.

42.8

31.6

8.0

(32.8)

2

g

SELECTIVE FERTILIZATION IN

Oenothera 287

288

J. SCHWEMMLE

not be established that the B ovules mature earlier in the lower part of the ovary than in the middle part or a t the top. I n the 10 crosses 0 B.2 X 8 hsc.hsc in which pollination was carried out 48 and 52 hours after castration the seed set decreased strongly from the top to the bottom, since the ovules were too old and therefore showed reduced chemotropic attraction for the hsc-pollen tubes (Table 34). But the faster aging of the B ovules as compared to the 2 ovules could not be demonstrated. The reduction in percentage from the top to the bottom is the same for both genotypes. This can be seen easily from the table. Thus not all types of pollen tubes are suitable for the demonstration of differences in maturation and aging between the ovules in different parts of the ovary of B.Z. Landspersky (1963) could demonstrate only in crosses o B.1 X 8 ha.ha that the ovules mature earlier a t the bottom TABLE 34 Seed Set and B.hsc and l . b c Progeny in the Upper, Middle, and Lower Thirds from 10 Capsules of Crosses 9 B.Z X 8 hsc.hsc

Upper third Middle third Lower third

* L.S.

=

69.9 31.3 10.1

31.9 (=loo%) 14.2 (44.5) 4.7 (14.7)

34.1 (~100%) 16.1 (47.0) 4.7 (13.8)

large seeds.

of the ovary than a t the top, But all his crosses showed that they age correspondingly faster-the B ovules earlier than the I ovules. I n the crosses with B.Z as pistillate parents, in which the capsules were divided into three pieces, it could be established that sterile ovules are less frequent in the upper third than in the lower parts. Weidner had found that in Oe. longiflora sterile ovules are distributed evenly through the whole ovary. It had been assumed previously that the same would apply in Oe. berteriana. It was important to find out whether the pollen tubes start t o fertilize ovules a t the top or a t the bottom of the ovary. Therefore in 19564958, crosses with sparse pollination were set up. The results from 5 crosses 9 B.Z X 8 v.II+ X 8 1.1 are found in Table 35. The v-pollen tubes, the only ones transmitted in the prepollination, do not fertilize the B ovules. The Z.v, which were counted in the cultures, indicate how many v-pollen tubes have accomplished fertilization in the prepollination; the counted B.1 and Z.1, how many I-pollen tubes have fertilized ovules in the postpollination. Of all the v-pollen tubes

SELECTIVE FERTILIZATION IN

Oenothern

289

TABLE 35 Progeny from 5 Crosses 9 B.1 X 3 v.II+ X 3 1.1, at Different Levels of the Capsules

Upper third Middle third Lower Third

Seeds Number of pollen tubes

% v tubes

% I tubes

5.7 28.4 65.9

48.6 35.2 16.2

100 33 1

100 976

(331) not less than 65.9% had fertilized ovules in the lower third of the ovary; but only 5.7% had fertilized ovules in the upper third. This shows as clearly as anything that the I ovules a t the lower part of the ovary attract the v-pollen tubes much more strongly than those in the middle or particularly a t the top, where the ovules are not yet mature enough to assert their attraction. The I-pollen tubes from the postpollination, which was made 22 hours later, fertilized mostly the B and 1 ovules in the upper third since the ovules a t the bottom of the ovary were already too old and therefore did not strongly attract the I-pollen tubes. The question arises why especially in these crosses the difference in rate of maturation of the ovules in the lower and upper parts of the ovary could be shown so unambiguously. I n the crosses 0 B.1 X 8 type A(v) discussed earlier the differential frequencies of 1.v in the three parts of the capsules were the more striking the lower the seed set. It can be concluded that the v-pollen tubes pass the not yet completely mature ovules a t the top of the ovary in sparse pollinations, only few v-pollen tubes being attracted chemotropically. The 1.v are somewhat more frequent in the middle third since there the I ovules are already better able to attract the v-pollen tubes. The 1.v occur most frequently a t the bottom of the ovary since there the 1 ovules are mature enough to attract the v-pollen tubes well, maybe even optimally. We know through the investigations of Schneider that the pollen tubes grow through the styles in a few hours. Then i t still takes considerable time until the ovules of the ovary are fertilized. It would be possible with plentiful pollination for the v-pollen tubes to grow through the pistil at equal rates and wait at the top of the ovary until the ovules are sufficiently mature to attract the pollen tubes. In this way, the difference in seed set between the upper, middle, and lower

290

J. SCHWEMMLE

thirds would disappear and the differences in maturation could not be recognized. The I-pollen tubes from the postpollination are attracted very well by the ovules a t the top of the ovary, since they have become sufficiently mature in the meantime. I n the middle third they are attracted less well since the ovules are already aging. This is still more pronounced in the lower third where only 159 pollen tubes = 16.2% have accomplished fertilization. Also other experiments have shown that the pollen tubes start fertilizing ovules a t the top. If only few ovules in this position are fertilized, the reason is that they are not yet sufficiently mature. The five crosses o B.1 X 8 Z.II+ x 8 hsc.hsc with scarce pollination show the same result (Table 36). The 1-pollen tubes from the prepollination fertilized most frequently ovules in the lower part of the ovary, TABLE 36 Progeny from 5 Crosses 0 B.2 X 3 LII+ X c? hsc.hsc, at Different Levels of the Capsules

% I tubes Upper third Middle third Lower third

Seeds Number of pollen tubes

14.5 32.7 52.8

100 715

% hsc tubes 58.5 40.1 1.4

100 577

bypassing in their growth the immature ovules a t the top. It is noteworthy that the differences in seed set depending on the different states of maturity were less pronounced as more 2-pollen tubes accomplished fertilization. It is thus understandable that in the earlier mentioned crosses 0 B.2 X s 1.11no difference was found a t all. Landspersky (1963) could prove the differential maturation of the ovules only in the poorly yielding crosses o B.l X 8 ha.ha. The B.hsc and l.hsc seeds from the postpollination were most frequent in the upper third since meanwhile the ovules in this position had reached their optimal attraction. I n the middle piece they are starting to age. I n the lower piece in two out of five crosses only 8 hsc pollen tubes accomplished fertilization. This shows that there were not sufticient pollen tubes left t o fertilize more ovules. If the hsc-pollen tubes had started fertililization in the lower part of the ovary, the B.hsc and Lhsc would have been more frequent. The ovules cannot have been so old that only 8 of them could be fertilized by hsc-pollen tubes.

SELECTIVE FERTILIZATION IN

Oenothera

29 1

The other experiments from the years 1955 and 1958 support these conclusions very well. Again i t could be shown that the B ovules age faster than the I ovules but that the hsc-pollen tubes are not as suitable for the demonstration as the I-pollen tubes.

2.Maturation and Aging of the v and I Ovules of Oe. odorata (v.I)

It was investigated whether also in the v.1 the v and I ovules mature and age at different rates. Therefore they were crossed in 1953

with different types of pollen parents. Care was taken that the crosses were set up at the same time since only under this condition are they comparable. I n crosses of the original Oe. odorata v.1 with Oe. odorata cytoplasm and plastids to type A f v ) as pollen parent the seed sets obtained with early pollination ( 8 hours after castration) were not large (Table 37). 35.3% large seeds were in capsules 1-10, Starting with the 11th capsule the yield became increasingly lower. The seeds from seven capsules with different seed sets were raised. The results are shown in Fig. 4. I n this figure the proportions of 1.v and v.v are indicated according to rising values. The v.v were always less frequent than the I.v. The crosses of o v.1 [od. Pl.] X 8 type A(v) with late pollination produced a better seed set. In capsules 1-10 there were 51.7% large seeds. This is surprising since the plants were pollinated 30 and 54 hours after castration. The petals were already changing their colors and the stigmatic lobes did not look as if pollen grains could still germinate on them. The seed sets of the eight crosses raised are shown in Fig. 4, as are the proportions of the 1.v and v.v arranged according

zE ?4

r

6 5o0 1 Early

20 10

0 13

_----

(_--c

--12

10

8

3

2

I

1

2

7

9

I2

17

18

I 9 KO

FIG.4. Percentage of large seeds, and of 1.v and v.v progeny in capsules from cromes 0 v.1 x 8 type A(v). Left, early pollination; right, late pollination. Abscissa: K a Icapsules, arranged according to increasing values at left, according to de, % Large seeds; -, yo v.1; - - - - _ - - creasing values a t right. % v.v. After Schwemmle (1958a).

TABLE 37 Seed Set after Early and Late Pollination (the Latter in Italics) after Pollination of v.1 with Different Cytoplasmic Constitutions by Pollen of Different Genotypes 8

%

L.S.

+

Ca

1-10

9

v

9I

v.1 [od. Pl.] [od. Plstd.] v.1 [bert. Pl.] [od. Plstd.] v.1 [bert. Pl.] [bert. Plstd.] [od. PI.] [od. Plstd.] [bert. PI.] [od. Plstd.] [bert. PI.] [Eert. Plstd.] [od.PI.] [od. Plstd.] [bert. PI. I [od. Plstd.] [bert. PI.] [bert. Plstd.]

V

81

8 ha

8 ck

8 B

14.9

38.7 18.6

30.0 l Y . 4

8h c

35.3 51.7

53.0 57.7

20.4

43.1 51.Y

47.5 55.1

24.8 25.7,

28.5 90.0

48.1 39.1

32.7 98.9

33.4 55.0

46.5 54.4

24.5 29.6

34.3 15.3

40.1 95.6

36.4

2.8.2

13.3 81.8

24.5 26.9

11.3

14.4

22.3

16.2

8.4

17.3 21.3

23.9 26.9

14.3 13.5

18.7 11.3

13.4 21.Y

24.4 26.1

12.6 15.5

18.5

21.7 88.8

30.4 30.9

12.1 10.Y

13.0

27.8 51.5

23.2 88.7

25.3 52.4

24.9 99.6

18.0

6.4

22.3

9.9

10.4

23.2 17.4

19.1 13.4

9.3

20.9

13.1

18.9

9.9

8.7

21.0

12.6

17.3

9.Y

16.1 17.5

15.8 18.5

24.7

18.9

16.2 15.2

14.9 19.4

18.9

23.2 16.3

13.1

19.7

16.1

B

3

s

@

SELECTIVE FERTILIZATION IN

Oenothera

293

to decreasing values. Also here the v.v are less frequent than the I.v. But it is clear that the difference in the crosses with the highest seed sets is smaller. This finding can be explained by assuming that the ovules a t first exerted only a weak chemotropic attraction. Both types of ovules matured gradually and were fertilized more and more frequently. The differences in the affinities v-v and I-v were preserved. When the I ovules have reached their optimal chemotropic attraction the v ovules have not yet reached it. Therefore the difference becomes reduced. Maybe there exists a time when the v and I ovules attract the v-pollen tubes equally well. This time was not observed in the present experiments. Later on, chemotropic attraction decreases. The ovules age but apparently at different rates. Whereas the I.v. decrease from the highest values found, 29.3%, to 24.3%, a value which is still as high as the highest values obtained in the crosses with early pollination (23.5%), the corresponding limiting values for v.v are 25.2% and only 6.6%. I n cultures of seeds from capsules with still lower yields, probably only 1.v would have appeared, as well as in the crosses with the lowest seed sets from early pollinations. In order t o eliminate the differences in the affinities the proportions (in percent) of 1.v and v.v were related to their respective highest values = 100%. In this way the values of Fig. 5 were obtained. This curve shows the increasing %

100

-

90 -

-

r''*4 /

/

/

/

/

0

FIG.5. Maturation (left) and aging (right) of the I ovules (heavy dashed and solid lines) and the v ovules (light dashed and solid lines) of v.1 [od.Pl. od.Plstd.1 tested by v-pollen tubes (drawn curves) and I-pollen tubes ( h e a t e d curves). After Schwemmle (1958a).

294

J . SCHWEMMLE

maturation and the differential aging of the v and I ovules. It can also be seen that the v ovules get a later start in their maturation, that is, in their ability to attract pollen tubes chemotropically, and that this difference is preserved through a long time. Afterward, the v ovules age faster and more precipitously than the I ovules. Also in the crosses with 1.1pollen parents cultures produced by late pollination showed a better seed set (57.7% large seeds) than those with early pollination (53.0% large seeds). In Table 37 the values obtained in crosses with late pollination are printed in italics. In every case, the 1.1 are more frequent than the v.1. Apparently the I-pollen tubes react differently than the v-pollen tubes since in crosses with early as well as with late pollination the v.1 are always more frequent than the v.v, and the 1.1 more frequent than the I.v. The I-pollen tubes are attracted better than the v-pollen tubes by both the v and the I ovules. It is important t o state that the comparable crosses were made a t the same time, so that the ovules of the v.1 parent will have been in nearly the same condition. Also for the crosses with 1.1 the curves for maturation and aging were drawn and included in Fig. 5. The values for maturation and aging of the ovules turn out to be completely different when the more strongly reacting I-pollen tubes are used rather than the v-pollen tubes. A more detailed discussion is not necessary. In 1952 v.1 with Oe. berteriana cytoplasm and Oe. odorata plastids [bert. P1. od. Plstd.] and v.1 [bert. P1. bert. Plstd.] were made up by suitable crosses. In 1953 these were crossed at the same time as v.1 [od. P1. od. Plstd.] with the same pollen parents and the crosses also raised in 1954. In this way a possible influence of the cytoplasm and of the plastids could be detected. In the upper part of Table 37 the mean values for large seeds found in capsules 1-10 are given for the different crosses, and for the crosses with 1.1 pollen the percentage of fertilized ovules. The values obtained in crosses with late pollination are printed in italics. The same applies to the affinities which can easily be derived from the table. When the seed sets in crosses with early and late pollination are compared it is seen that the seed set with type A(v) as male parents and 1.1 males is always higher with late pollination: accordingly the afbities v-v and I-v are increased. In the crosses with hsc.hsc, ck.cl, and B.11 as pollen parents, completely different results are obtained. In these combinations the crosses with early pollination always produced a higher yield, resulting in higher affinities. The results in the crosses with ha-ha male parents are peculiar. In the crosses with v.1 [od. P1. od. Plstd.] those involving late pollination gave a lower yield;

SELECTIVE FERTILIZATIONIN

Oenotheru

295

the affinities were accordingly reduced. They agreed thus with the crosses t o hsc.hsc, ck.cl, and B.11 males. However, in the crosses o v.1 [bert. P1. bert. Plstd.] x 8 ha.ha the crosses with late pollination produced higher seed sets, just as in the crosses with type A males and 1.1 males. The affinity v-ha is increased only slightly, that is, from 12.6 to 13.5, but the a n i t y I-ha is increased from 14.9 to 19.4. The crosses 0 v.1 [bert. P1. od. Plstd.] X 8 ha.ha produced equally good seed sets in early and late pollinations; accordingly, the differences in affinity are small. It is easy to see from the table that the chemotropic attraction exerted by the v and I ovules for the different pollen tubes is different. Table 37 also gives information on the influence of cytoplasm and plastids. First the crosses involving early pollination will be considered. I n the crosses of o v.1 [bert. P1. od. Plstd.] with different males the v ovules attract the different pollen tubes better than the v ovules of the v.1 [od. P1. od. Plstd.] with the exception of the I-pollen tubes. That means that the Oe. berteriuna cytoplasm increases the affinities v-v, v-ha, etc. But depending on the type of pollen tubes used the amount of the increase may be different. In the crosses with type A(v) males and hsc.hsc males it is largest; in the crosses with ck.cl males, lowest. Contrary to the other crosses, in the crosses with 1.1 male the affinity v[bert. P1. od. Plstd.1-I = 23.9 is lower than the affinity v[od. P1. od. Plstd.1-1~24.5. This difference is only small. The genetically different pollen tubes react completely differently to the changes in chemotropic attraction by the v ovules, induced by the Oe. berteriana cytoplasm. I n most of the crosses involving v.1 [bert. P1. bed. Plstd.] generally the chemotropic attraction exerted by the v ovules is again reduced. This is indicated only slightly in the crosses with hsc.hsc and B.11 males but is much more pronounced in the crosses with type A(v) and ha-ha males. The affinity v[bert. P1. bert. Plstd.1-ha = 12.6 is still higher than the affinity v[od. P1. od. Plstd.1-ha = 11.3. The v-pollen tubes were attracted equally well by both of these types of v ovules. The effect of Oe. berteriuna cytoplasm appears in this case only in the decrease in comparison to the crosses with o v.1 [bert. P1. od. Plstd.]. I n the crosses with ck.cl males the ck-pollen tubes are less strongly attracted by the v [bert. P1. bert. Plstd.] ovules. In the crosses with 1.1 males the different types of v.1 are equally frequent. The I-pollen tubes are thus not very suitable to test for changes caused by the Oe. berterianu cytoplasm or plastids in the chemotropic attraction of the v ovules. Similar results are obtained for the crosses with late pollination. The

296

J . SCHWEMMLE

Oe. berteriana cytoplasm again increases the affinities v-ha, v-hsc, v-ck, and v-B, but to a much higher degree than in the crosses with early pollination. I n those with type A male and 1.1 male an influence of the Oe. berteriana cytoplasm cannot be observed. The reduction of the affinities caused by the Oe. berteriana plastids in the crosses of P v.1 [bert. P1. bert. Plstd.] as compared to P v.1 [bert. H. od. Plstd.] can be easily demonstzated in the crosses with hsc.hSC, ck.cl, and B.11 males but not in those with type A ( v ) , 1.1,and ha.ha males. With I ovules in principle the same results are obtained. In the crosses with early pollination the I ovules of the v.1 [bert, P1. od. Plstd.] attract the v-, ha-, hsc-, and ck-pollen tubes more strongly than the I ovules of v.1 [od. P1. od. Plstd.]. But in the crosses with 1.1 males and B.11 males Oe. berteriana cytoplasm causes a reduction of the affinities. In P v.1 [bert. P1. bert. Plstd.] the Oe. berteriana plastids decrease the affinities in comparison to the crosses with 4 v.1 [bert. P1. od. Plstd.] when type A ( v ) , ha.ha, and ck.cl are used as males, But an increase of different degrees is found in the crosses with 1.1, hsc.hsc, and B.11 males. Also in the crosses with late pollination the influence of the cytoplasm and the plastids is seen, in most cases even more distinctly. Thus the Oe. berteriana cytoplasm increases the affinities of the I ovules for the v-, ha-, hsc-, ck-, and B-pollen tubes. Only in the crosses with 1.1 male the affinity 1-1 decreases, though not as much as in the crosses with early pollination. Another increase, different in size according t o the type of pollen tubes used, is found in the crosses of P v.1 [bert. P1. bert. Plstd.] with type A(v), 1.1, ha.ha, hsc.hsc, and B.11 males. Only in the crosses with 8 ck.cl, affinity decreases as in the crosses with early pollination. Obviously the different types of pollen tubes are not equally suitable to demonstrate the influence of the cytoplasm and plastids on the affinities. I n addition, the chemotropic attraction of the v and I ovules does not change by the same amount. I n order to find out whether cytoplasm and plastids also influence the maturation and aging of the ovules the curves were drawn (Fig. 6 ) . It should be mentioned that the difference in the affinities is irrelevant here. As an example, the curves of the crosses with 8 ha.ha are given. It should be noted that they are comparable with each other. Of the 20 crosses each which were arranged according to decreasing values of the seed set, capsules with equal numbers were raised. Clearly the v and I ovules of the v.1 [od. P1. od. Plstd.] mature faster than those of both the other v.1 types which do not show large differences with each other. The differences are more distinct in the curves for aging

SELECTIVE FERTILIZATION IN

Oenothera

297

of the ovules. The faster maturing v and I ovules of v.1 [od. P1. od. Plstd.] also age earlier. In the ovules of v.1 [bert. P1. od. Plstd.] aging is retarded, and it is still more retarded in the ovules of v.1 [bert. P1. bert. Plstd]. That means that both cytoplasm and plastids have an influence on the maturation and aging of the ovules. It is again important which types of pollen tubes are used for the demonstration. In the crosses with 8 ck.cl, for instance, the I ovules of the v.1 [bert. P1. od. Plstd.] mature faster than those of v.1 [od. PI. od. Plstd.]. It might be assumed that the differences in affinities caused by the cytoplasm and the plastids are only due to the differences in matura-

t

/ /

4030 =-Id 20 c

10

/

-

-

0.

*

'

a

I

j

a

\

298

J. BCHWEMMLE

tion and aging of the ovules. But this is not the case. The v ovules of v.1 [od. P1. od. Plstd.] mature faster than the v ovules of v.1 [bert. P1. od. Plstd.]. Nevertheless, the affinity v[od. P1. od. Plstd.1-v = 13.3 is lower than the affinity v[bert. pl. od. Plstd.1-v = 17.3. The affinity of the most slowly aging v ovules of v.1 [bert. P1. bert. Plstd.] to the hapollen tubes (13.5) is just as large as the affinity of the much faster aging v ovules of v.1 [bert. P1. od. Plstd.] to the ha-pollen tubes (13.3). It appears therefore that the ovules of different v.1 in the same condition of maturity, which unfortunately cannot be exactly observed, attract a particular type of pollen tubes with different strengths. Also in other cases an influence of cytoplasm and plastids on the affinities was found. I n the next section such cases will be reported. It appears simple t o compare the different investigations and to demonstrate contradictory results; but knowing how much the affinities depend on external conditions and the state of maturity of the ovules, crosses obtained in different years can be compared only with difficulty. Particularly crosses with early and late pollination cannot be compared to those in which the stigmata of the plants used as females were in their best condition. It had been planned to cross the different types of v.1 with different types of males a t intervals of 0, 8, 16, 24, etc. hours from the time of castration, to obtain a deeper insight into the processes of fertilization and to define unambiguously the influence of cytoplasm and plastids. Unfortunately it has not been possible to carry out these plans.

C. INFLUENCE OF CYTOPLASM AND PLASTIDS ON THE AFFINITIES The cytoplasm and plastids of Oe. odorata (v.1) and Oe. berteriana (B.1) are different. The B.1 from o v.1 x 6 B.Z possess longer hypanthia than the B.1 from the reciprocal cross o B.1 x 8 v.1. It could be shown that the Oe. odorata cytoplasm causes this lengthening of the hypanthia. I n the B.1 and 1.v from the ? v.1 )( 6 B.1 the Oe. odorata plastids are not able to become green; if Oe. odorata proplastids only are present in 1.1, they even cause death of embryos. I n the cross o B.Z X 8 v.1 the B.1, 1.1, and 1.v are normally green if no Oe. odorata plastids were transmitted. In the latter case they are variegated. The Oe. berteriana plastids are therefore different from the Oe. odorata plastids. Because B.2 and v.1 are compl.ex heterozygotes it was possible, in addition to the original v.1, to obtain three more types of v.1 which differ in their cytoplasms and plastids from the original form. Their constitutions are listed in Table 38. It is not possible here to describe their pedigrees; the reader is referred to Schwemmle (1957~).It will also not be discussed whether the different

SELECTIVE FERTILIZATION IN

299

Oenothera

v.1 reaIly have the constitutions assigned to them in Table 38. Many arguments favor the assumption (Hagemann, 1964). The different v.1 were crossed with type A, a trisomic mutant derived from the original v.1. It has Oe. odorata cytoplasm and plastids [od. Pl.]. Only the v comp1,ex is transmitted through the pollen (v. [od. Pl.]). Type A derived from v.1 [bert. P1. bert. Plstd.] contains, like the pollen grains, Oe. berteriana cytoplasm and plastids (v [bert. PI.]). The homozygote 1.1which was also used as male parent has Oe. berteriana cytoplasm and plastids, also in its I pollen (I [bert. Pl.]). In the trisomic mutant v.1 var. tenella derived from the original v.1 the I pollen which alone is active carries Oe. odorata plastids and cytoplasm (I [od. Pl.]). TABLE 38 Seed Sets Produced by v.1 Pistillate Parents with Different Constitutions of the Cytoplasm and Plsstids, Crossed to v Pollen with od. and bert. Cytoplasm and Plastids*

3 v [bert. Pl.]

3 v [od. PI.] Cal-10 ~

Cal-5

Cal-10

Ca 1-5

~

9 v.1 [od.P1. od. Plstd.]

45.9

49.9

41.6

44.0

9 v.1 [od. P1. bert. Plstd.]

-

38.3

41.1

38.9

41.6

9 v.1 [bert. P1. od. Plstd.]

42.4

44.2

44.0

45.9

9 v.1 [bert. P1. bert. Plstd.]

(+I

-

+ 51 .O

53.8

(+I

(+)

50.8

54.8

+,

*Abbreviations: difference significant at 0.01 level; (+),difference significant at 0.05 level; -, difference not significant.

In 1950, 20 crosses each were made and the seed set was determined by counting the contents of the capsules. In Table 38 and 40 only the 10 highest yielding capsules were taken and in addition capsules 1-5 whose seeds were raised. The seed set of the crosses 9 v.1 [od. P1. bert. Plstd.] X 8 v [od. PI.] was lower than that from o v.1 [od. PI. od. Plstd.]. The Oe. berteriana plastids reduce the affinities v-v as well as I-v (Table 39). I n the crosses involving o v.1 [bert. P1. od. Plstd.] more large seeds are produced than in v.1 [lsert. PI od. Plstd.], but still less than in the crosses with o v.1 [od. H.od. Plstd.]. Compared to the latter with affinity I-v=29.8, the same affinity was reduced in the former by about 5.7 to 24.1 due to the influence of the Oe. berteriana cytoplasm. The seed set decreases by about the same amount in capsules 1-5. However the affinity v--v was not affected by the Oe. berteriana

300

J. SCHWESIMLE

cytoplasm. It is true that the v.v are less frequent in the crosses with P v.1 [bert. P1. od. Plstd.] (16.9%) than in the crosses with 0 v.1 [od. P1. od. Plstd.] (17.7%) but this reduction is due to lower germination of seeds and lower viability. We established thus that the Oe. berteriana plastids lower the affinities v-v and I-v but the Oe. berteriana cytoplasm only the affinity I-v. It is very surprising that the cross with ? v.1 [bert. P1. bert. Plstd.] produces the highest yield. The v.v are here most frequent even though the differences are not large in comparison to crosses with o v.1 [od. P1. od. Plstd.]. Therefore it is understandable that in the crosses of Gress in the year 1953 whose values are added in parentheses in Table 39 no differences were found. At all events, Oe. berteriana cytoplasm and plastids together do not low,er the affinities I-v and v-v but may even increase them. This is seen distinctly in the crosses TABLE 39 Influence of the Cytoplasmic and Plastid Constitutions of Pistillate v-I and of v-Pollen Tubes on the Affinities v-v and I-v

8 v [od. Pl.]

’

[od. PI. od. Plstd.] [od. P1. bert. Plstd.] [bert. PI. od. Plstd.] [bert. P1. bert. Plstd.] [od. P1. od. Plstd.] I [od. P1. hert. Plstd.] [bert. PI. od. Plstd.]

[bert. P1. bert. Plstd.]

d’ v Ibert. Pl.]

17.7 (17.9) 12.9 16.9 18.6 (18.1)

14.7 (12.4) 13.7 18.3 18.0 (16.5)

29.8 (26.8) 24.7 24.1 31.8 (26.5)

27.2 (25.3)(27.7; 25.1) 25.1 24.4 33.1 (28.4)(31.3;29.3)

with 8 v. [bert. PI] which give the same results in principle as the crosses with d v [od. Pl.]. Therefore they will not be discussed separately. Gress obtained in her crosses the same results (values in the first parentheses). Also the values from the crosses with 8 v.11 (values in the second parentheses) by Koepchen are in agreement. While apparently the v[od. P1.1- and the v[bert. PI.]-pollen tubes react in the same manner in crosses involving synthetic v.1 as already shown by their seed sets, they seem to behave differently in crosses with ? v.1 [od. P1. od. Plstd.]. If this is crossed to 8 v[bert. Pl.] the seed act is lower: the v.v as well a s the 1.v are less frequent than they are in the crosses with 8 v [od. Pl.]. This means that in this case the v[bert. P1.1-pollen tubes do not react as well as the v[od. H.1-pollen tubes. But the difference in yields is not statistically significant; nevertheless, Gress obtained th,e same result (values in parentheses). At all events it is important which kind of

SELECTIVE FERTILIZATION IN

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301

TABLE 40 Influence of the Cytoplasmic and Plastid Constitutions of Pistillate v.1 and of I-Pollen Tubes on Seed Set*

8 I [od. PI.] Ca1-10 ~

~~

~

~

9 v.1 [od. PI. od. Plstd ]

~~

8 I [bert. Pl.]

Ca 1-5

Ca1-10

Ca 1-5

53.2

56.6

~~

51.0

51.8

9 v.1 [od. PI. bert. Phtd.]

+ 43.1

46.0

+ 46.6

48.9

9 v.1 [bert. P1. od. Plstd.]

-

44.5

47.9

49.2

51.1

9 v.1 [bert. PI. bert. Plstd.]

58.0

60.4

-

+

+ 62.1

63.1

* Abbreviations: +, statistically significant; -, not significant. cytoplasm and plastids the egg cells (synergids) of the ovules possess. The same result is found in the crosses with 6 I [od. Pl.] which will be discussed first (Tables 40 and 41). The seed set in the crosses with 0 v.1 [od. PI. bert. Plstd.] was much lower than in the crosses with 9 v.1 [od. P1. od. Plstd.]. The v.1 were reduced from 25.0% to 20.0%. The difference is much smaller in 1.1 (26.8%-25.4%). That means that the Oe. berteriana plastids cause a strong reduction of the affinity v-I but only a slight reduction of 1-1. In the crosses with 8 v.1 [bert. P1. od. Plstd.] the number of large seeds increased slightly. The v.1 (21.6%) and 1.1 (26.3%) are more frequent than in the former crosses. In order to establish the influence of the cytoplasm, crosses to o v.1 [bert. P1. od. TABLE 41 Influence of the Cytoplasmic and Plastid Constitutions of Pistillate v.1 and of I-Pollen Tubes on the Affinities v-I and 1-1

Q v

I

81

8 1 [od. PI.]

[bert. PI.]

[od. PI. od. Plstd.] [od. PI. bert. Plstd.] [bert. PI. od. PLstd.1 [bert. PI. bert. Plstd.]

25.0 20.0 21.6 25.4

29.5 21.6 24.1 28.0

[od. PI. od. Plstd.] [od. PI. hert. Plstd.] [bert. P1. od. Plstd.] [bert. PI. bert. Plstd.]

26.8 25.4 26.3 35.0

27.1 27.3 27.0 35.2

302

J . SCHWEMMLE

Plstd.] are compared with crosses to o v.1 [od. P1. od. Plstd.]. The difference in seed set is statistically significant a t the 0.05 level. The v.1 (21.6%) are rarer in the crosses with 0 v.1 [bert. P1. od. Plstd.] than they are in the crosses with 0 v.1 [od. PI. od. Plstd.] (25.0%). In the 1.1the decrease (from 26.8% t o 26.3%) is only slightly indicated. Thus the Oe. berteriana cytoplasm lowers especially the a w i t y v-I. The v.1 and also the 1.1are rarest in the crosses with Q v.1 [od. PI. bert. Plstd.]. These results indicate that the effect of the Oe. berteriana plastids is larger than that of the Oe. berteriana cytoplasm. The best yield is produced in the crosses with ? v.1 [bert. P1. bert. Plstd.]. The differences with all the other crosses are statistically significant a t the 0.01 level. Especially the 1.1 are considerably more frequent; therefore Oe. berteriana cytoplasm and plastids in combination increase the affinities which are reduced by each one alone. The yield from crosses with 8 I [bert. P1.1 is always higher; in the crosses with 8 v.1 [bert. P1. bert. Plstd.] the difference is statistically significant as compared to the crosses involving 8 I Cod. Pl.]. The I [bert. PI.]-pollen tubes react more strongly than the I [od. PI.]-pollen tubes. Especially the v.1 are more frequent in crosses with 8 I [bert. Pl.]. The change of the affinities v-I caused by the cytoplasms and plastids of the pistillate parents can be measured in the same way, only they are much more pronounced. The 1 [bert. P1.1-pollen tubes indicate this difference more distinctly than the I rod. P1.1-pollen tubes. The increase is only small in 1.1.Furthermore, the 1.1are equally frequent in all crosses, with the exception of the crosses with 0 v.1 [bert. Pl. bert. Plstd.]. A change of the affinity 1-1 caused by cytoplasm and plastids is not apparent. Even in the crosses with 8 I [od. Pl.] only a slight indication of such a change was found. It was therefore advantageous that the different types of v.1 had also been crossed with both plasmonic constitutions of type A (v). In these crosses it could be shown that the attraction of the v ovules just as well as that of the I ovules is influenced by the cytoplasm and plastids. The investigations show therefore that the v and I ovules with Oe. berteriana cytoplasm and plastids show a stronger chemotropic attraction for the v- and I-pollen tubes than the v and I ovules with Oe. odorata cytoplasm and plastids. The same is true for the B- and Zpollen tubes. The following affinities were obtained:

I[od. Pl.1- B I[bert. PI.]-B

=

=

18.4 25.2

1949

1950

I[od. P1.1- I = 14.7 I[bert. P11-l = 1 7 . 1

8.0 12.0

SELECTIVE FERTILIZATION IN

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303

Comparable crosses were also carried out with B.1 [bert. PI. bert. Plstd.], that is, with the original Oe. berterianu, and with o B.Z [od. PI. bert. Plstd.]. This constitution was obtained from the cross o Z.v [od. P1. od Plstd.] 8 B.Z.If the B.1 possess only Oe. odozata plastids they are not viable. When Oe. berteriunu plastids are transmitted by the B-pollen tubes chimeras are obtained. The yellowish sections have Oe. odorata plastids which are unable to become green while the green sectors contain Oe. berteriana plastids. By selfing a branch in the green sector, B.1 [od. Pl.bert. Plstd.] plants are obtained. These differ only in their cytoplasm from the original Oe. berteriana. That they possess Oe. odorata cytoplasm can be seen from the fact that the hypanthia of their flowers are longer than those of the original B.Z. In the crosses with 8 type A(v) the B.v are missing. When the large seeds, the shrunken seeds, and the coarse powder are added together it is known how many 1 ovules had been fertilized by the v-pollen tubes. I n the crosses o B.1 [od. Pl.] 8 type A(v) [od. Pl] more Z.v were found than in the crosses with o B.Z [bert. PI.] (Table 42). Thus the Oe. odorata TABLE 42 Influence of the Cytoplasmic Constitution of B.1 Pistillate Parent and of v-Polle R Tubes from Type A(v) on Number of Fertilized I Ovules (in Percentage) Crosses in 1952 B.Z [bert. PI.] X type A(v) [ad. PI.] B.Z [bert. Pl.] X type A(v) [bert. PI.] B.Z [ad. PI.] X type A h ) [od. PI.] B.1 [ad. Pl.] X type A(v) [bert. PI.]

Fertilized ovules = 1.v 31.1 (26.7) 31.4 (26.1) 34.8 30.8

cytoplasm of the mother increases the affinity Z-v. The crosses with the different cytoplasmic conditions of v.1 had giv,en corresponding results (see Tables 38 and 39). But if type A(v) [bert. PI.] is used as the pollen parent the influence of the maternal cytoplasm cannot be seen. We conclude that the cytoplasmic constitutions of v-pollen tubes react differently only when the egg cells possess Oe. odorata cytoplasm. The same result had been found earlier in the experiments with v.1. As can be seen in Table 38 the differ,ence in seed set in the crosses with 8 type A(v) [od. Pl.] and 8 type A(v) [bert. Pl.] are most pronounced when v.1 [od. P1. od. Plstd.] were used as the pistillate parents. The two constitutions of B.Z had also been crossed with 8 1.1 (I [bert. PI.]) and 8 v.1 var. tenella (I [od. Pl.]). I n 1950 and again in 1952 the yield from the crosses to 8 I [bat. Pl.] was better than that

304

J . SCHWEhIMLE

from the crosses to 8 I [od. Pl.]. The differences are statistically significant. As shown in Table 43, the B.1 as well as the Z.1 are increased in crosses to 8 I [bert. PI.]. Therefore the I [bert. P1.1-pollen tubes are attracted more strongly by the B and I ovules than the I [od. P1.1pollen tubes. The same had been found in the crosses with the different constitutions of v.1 (see Table 40). In 1950 th.e affinities of the B and Z ovules for the I [od. P1.1-pollen tubes were lower than in 1952. The affinities for the I [bert P1.1-pollen tubes w.ere, however, equal in both years. This is another indication that the I-pollen tubes with different cytoplasms exhibit different resctivities. TABLE 43 Influence of the Cytoplasmic Constitution of Pistillate Parent B.Z and of I-Pollen Tubes on the Affinities B-I and Z-I

8 I [od. PI.]

9 9 9 9

B [bert. Pl.] B [ad. Pl.] 1 [bert. Pl.] 2 [od. Pl.]

8 I [bert. PI.]

1950

1952

1950

1952

19.3

23.6 25.0 26.3 24.2

28.4

28.6 30.8 27.3 28.4

23.5

27.0

The influence of the maternal cytoplasm is not as obvious as it is in the crosses with 8 type A. The B.1 were more frequent when the B.1 Cod. Pl.] were used as pistillate parent. The same applies to the Z.1 from the crosses 0 B.Z [od. Pl.] X 8 I [bert. Pl.]. But the difference is not large. Thus it may be due to chance that in the crosses with 8 I [od. Pl.] the 1. I appear less frequently in the cross with Q B.Z [od. Pl.] than in the cross with 0 B.2 [bert. Pl.]. The cytoplasm of the egg cells (synergids) and, as far as they can be separated, the plastids, as well as the cytoplasm of the pollen tubes, including the plastids, influence the affinity between the two. The cytoplasm, including the plastids, affects not only the reactivity of the pollen tubes but also their growth rates. In the crosses Q B.1 x 8 v.1 of the year 1953 the Z.v (14.7%) were more frequent than the 1.1 (6.6%). That means that the v-pollen tubes grow faster than the Ipollen tubes, and therefore compete more successfully with the I-pollen tubes for the 1 ovules. This is also seen in the crosses Q B.Z X 8 v.1 from the year 1944 (Table 44) in.which the Z.v were as frequent as in the crosses 0 B.Z X 6 type A(v) [od. Pl.] where the v-pollen tubes did not have t o compete with any other type of pollen tube. I n the crosses

SELECTIVE FERTILIZATION IN

Oenothera

305

o B.1 x 6 type A ( v ) [od. Pl.]+ x 8 1.1 in which the pistillate parents were prepollinated with v pollen and 26 hours later post pollinated with I pol1,en it could be expected that the Z.v would be a t least as frequent as in the crosses O B.1 X 8 v.1. Surprisingly, the 1.v decreased considerably, whereas the 1.1 increased very much. The I-pollen tubes of the post pollination overtook th,e v-pollen tubes, and must in this cross have grown faster than the v-pollen tubes. This can only be due to an effect of their Oe. berteriana cytoplasm. TABLE 44 Influence of the Cytoplasmic Constitutions on the Growth Rate of I-Pollen Tubes

B.Z X type A(v) [od. Pl.] B.1 X v.1 [od. PI.] B.1 X type A(v) [od. Pl.]+ X 1.1

% 1.v

% 1.1

13.7 13.1 7.3

-

3.4 11.5

The same is true for the v-pollen tubes. In the two crosses of Table o v.1 [bert. Pl.] were prepollinated with v [od. Pl.] pollen of type A the v.v and 1.v derived from it are not frequent. The Z-pollen tubes have grown very quickly and have overtaken the v[od. P1.1-pollen tubes considerably. Therefore many v.1 and 1.1 were formed. But when ? v.1 [bert. Pl] was prepollinated with v [bert. Pl.] pollen from v.11, the v.v 45 postpollination was carried out with 1 pollen from 1.11. When

TABLE 45 Influence of the Cytoplasmic Constitution on the Growth Rate of v-Pollen Tubes

v.1 [bert. Pl.] X type A(v) [od. Pl.]+ X 1.11 v.1 [bert. PI.] X v.11 [bert. PI.]+ X Z.11

% v.v

% 1.v

% v.1

% 1.1

0.3

7.1

18.7

15.2

9.9

23.8

7.6

0.8

and 1.v derived from it were formed much more frequently. Only 7.6% v.1 were formed by the postpollination since already 9.9% v ovules had been fertilized by the v[bert. P1.1-pollen tubes. The 1.1 decreased even more, since the v[bert. PI.]-pollen tubes had already fertilized most of the I ovules. Therefore the v-pollen tubes must have grown much faster than the Z-pollen tubes due t o the Oe. berteriana cytoplasm. I n the crosses o B.Z x 8 v.1 [bert. Pl.] the 1.v again are more frequent than

306

J . SCHWEMMLE

the 1.1 since again the v[bert. P1.1-pollen tubes are faster than the I [bert. P1.1-pollen tubes. For the same reason, in selfings of v.1 [bert. P1.1 the v.v are as frequent as in v.1 [od. Pl.] selfed. If in these cases we speak of growth rates we must consider that this expression is not quite correct. The pollen tubes grow rather fast through the style as was shown by Schneider. But then it takes a certain amount of time until fertilization is achieved. Otherwise it would not be possible that the pollen tubes of the postpollination overtake the pollen tubes of the prepollination even though many hours elapse between them. It is the time from pollination t o fertilization which is shorter for the vCbert.Pl.1and I [bert. P1.1-pollen tubes in comparison to the v[od. P1.1- and I [od. P1.1-pollen tubes.

D. CHANGESOF AFFINITIESBY CHANGES OF THE

THE GENOME. INFLUENCE OF MATERNAL DIPLONTS

The experiments discussed above show that the frequency with which certain complex heterozygotes are formed depends on the genetic constitution of the egg cells (synergids) in the ovules as well as on that of the pollen tubes. Therefore there is a possibility that changes of the genetic constitution may also induce changes of the affinities. As mentioned earlier, the complex I1 arose from 1.1 with the chromosome constitution 8 4 1.2 in the following way: in complex 11, the chromosomes I4 and I1 were replaced by chromosomes 16 and 11 from complex 1, so that complex I1 contains 5 I and 2 1 chromosomes. If the complex I1 also transmits the gene T (dotted petals) (IIT), the chromosome I2 is in addition replaced by 12. Thus we can investigate the afbities of the ovules with the complexes I, 11, and I in their egg cells toward the different types of pollen tubes. I n Table 46 the affinities obtained are listed. The affinity dI=23.5 is much larger than the affinity 1-1 = 17.0. The affinity 11-1 = 20 is intermediate. These differences are caused by the 2 or 3 1 chromosomes of the altered I complex. The affinity 1-1 = 2 is small but I-Z = 22.7 is high.

+ +

TABLE 46 AfFinities of Complexes 1, IT, and I in the Ovules t o

91

9

11

9 1

23 5 20 17

2

6

22.7

I-, Z-, and B-Pollen Tubes

23 21 20.8

SELECTIVE FERTILIZATION IN

Oenothera

307

The 2 or 3 1 chromosomes of the altered I complex, that is, complex 11, are sufficient to reduce the affinity 11-1. The affinity ZB - = 23 is larger than I-B = 20.8. The affinity II-B = 21 is correspondingly reduced by the effect of the I chromosomes, A differential effect of the complexes IIT with 3 and I I t with 2 1 chromosomes could not be established with certainty. Now the results of the crosses P B.11 X 8 B.Z and P Z1 .1 x 8 B.Z can be understood. I n 9 B.11 X 8 B.1 the following were expected: B.B, B.11, B.1, and Z.11. I n seven crosses, 10.2% B.11 and 18.0% B.Z .1 were missing, as were apparently the B.B since were obtained. The Z1 there were only few sterile seeds (1.9%). Therefore the l-pollen tubes fertilizing exclusively the B ovules, the B-pollen tubes only the I1 ovules. I n 3 crosses P 1.11 X 8 B.Z with 3.7% sterile seeds which in part contained the 1.1, 11.4% B.1, 8.7% B.11, and 3.5% 1.11 were counted. The latter are not very frequent but a t least they appeared. The results of this cross can be explained in the following way: The faster growing 1 pollen tubes had enough time to fertilize the 1 and I1 ovules. But since the affinities 1-1 and 11-1 are not large only few 1.1 and 1.11 were formed. The slower growing B-pollen tubes fertilized plentifully the 1 and I1 ovules to which they have a much higher affinity. So the unexpected results in the crosses o B.11 X 8 B.1 could be explained by means of selective fertilization. Richter has carried out extensive experiments involving B.lT, B.lt, and B.lR6. B.lT is the original Oe. berteriana with gene T (dotted petals) on the 1 2 chromosome. In the B.Zt with undotted petals this chromosome is replaced by the I 2 chromosome from the I complex. The B.lRd with grooved leaves also has the I 2 chromosome but with the gene Ri which originated by mutation. By comparing these crosses it would be possible to find out whether a single chromosome or even a single gene can affect the atbities. I n the crosses with 8 type A(v) and 8 Z1 .1 the expected forms occurred with equal frequencies. The affinities toward the v- and 1-pollen tubes were not changed by the I 2 chromosome and the gene Ri. The results in the crosses with 8 hsc.hsc (Table 47) were different. Eight cultures of each cross were raised. The seed sets from crosses with B.1T were highest, with B.bc lowest, the difference being statistically significant. Apparently the B.hsc were equally frequent in all crosses, but not the 1.hsc. The ZT ovules attract the hsc pollen tubes best since 26.6% L h s c were counted. The affinity lrhsc 23.7 is lower than lphsc. This difference is due to the I 2 chromosome. The gene Ri lowers the affinity Ghsc still further, since in the cross P B . l ~X i 8 hsc.hsc only 22.7% lR$.hsc were counted. I n the crosses with ? B.lt and o B.L, the difference in

308

J. SCHWEMMLE

the seed sets is larger than the difference in the 1t.hsc and Zsr.hsc. The seeds had germinated differentially and the viabilities were different. The lower seed set in the crosses with 9 B.lR4 is certainly caused by the lower frequency of L4.hsc. The crosses with 6 1.1 and 6 ha.ha gave different results (Table 48). In the crosses with 8 1.1 the yield of 9 B.bi X 8 1.1 was the TABLE 47 Effect of the Chromosome I 2 (in 21) and of the Mutant Gene Ri on Affinity of the Ovules to hsc-Pollen Tubes

%

B.ZT X hsc.hsc B.lt X h8C.hsc B.Zzi X hsc.hsc

* L.S.

%

%

L.S.*

B.hsc

l.hc

56.6 53.6 49.7

24.5 24.0 23.5

26.6 23.7 22.7

= large seeds.

lowest. It might have been expected t.hat the Im.1 progeny would be less frequent than the ZTI and It1 in the comparable crosses, But apparently the 1.1 are equally frequent. But in the crosses o B.lR+X 6 1.1the B.1 are less frequent than in both of the others. Therefore their seed set was lower. TABLE 48 Effect of Chromosome I 2 (in I t ) and of the Mutant Gene Ri on the AffiSlities 1-1 and h a 4 ha.ha 3

1.1 $

9 B.IT 9 BJr 9 BJR~

* L.S.

% L.S.*

7 0 B.1

70 I.1

% L.S.*

% B.ha

%/.ha

54.2 54.6 51.9

21.9

24.4 25.2 25.7

39.3 43.4 44.2

15.6 18.3 18.6

21.5 22.2 23.3

23.5 20.7

= large seeds.

I n the crosses with male ha.ha the B.ha were less frequent in the crosses with B.lT than in both of the others. On the other hand the different types of 1 ovules were fertilized with equal frequencies. These results are surprising since the B complexes of B.lT, B.Zt, and B.lR4are identical, and the B.Z were equally frequent in the crosses with 6 1.11. Apparently the maternal diplont influences in some way

SELECTIVE FERTILIZATION IN

Oenotkera

309

the affinity to the pollen tubes used which react in completely different ways. Thus the B.1 are least frequent in the crosses with P B.lR+ whereas the B.ha are least frequent in the crosses with P B.Z,. Since the affinities are also determined by the reactivity of the pollen tubes i t was investigated whether this is different for the lT-, It-, ZBi-pollen tubes. Therefore they were crossed to female ha.ha and female 1.1 (Table 49). The seed set of crosses P ha.ha X 8 B.1, was lowest, that of P ha.ha 8 B.Zt better, and that of P ha.ha X 8 B.lR4 highest. Accordingly, the Lha increased. The effect of the I 2 chromosome and of the factor Ri is very obvious. But the B.ha also increased. Since

x

TABLE 49 Influence of Chromosome I 2 (in I t ) and of the Mutant Gene Ri in 2-Pollen Tubes on Their Reactions to ha and I Ovules ha.ha 0

8 B.IT 8 B.lt 3 BJRi * L.H.

=

1.1 Q

% L.S.*

% B.ha

% 1.ha

% L.S.*

% B.1

% /.I

18.7 26.2 28.5

8.0 9.0 10.2

7.4 9.0 10.2

55.6 43.2 58.4

9.9 10.9 5.5

42.7 24.7 46.4

large seeds.

in ha.ha no sterile ovules occur and altogether relatively very few ovules were fertilized, competition between pollen tubes may be excluded. Maybe the more strongly reacting It- and ZRi-pollen tubes influence the B-pollen tubes in the same direction. Also in the crosses with 9 1.1 the ZRi-pollen tubes were attracted more strongly. But the yield from crosses P 1.1X 8 B.Zt was now the lowest since the I 2 chromosome reduces the affinity I-&. Also the B.1 occur with different frequencies. This is certainly due t o competition of pollen tubes. The less 1.1 are found, the more frequent are the B.I. This means that the lRi-pollen tubes grow fastest. In parallel experiments Gress investigated the influence of the I 2 chromosome and of the gene Ri on the affinities in crosses with three different v.1. These contained Oe. berteriana cytoplasm and plastids. I n the v.IT the I 2 chromosome is replaced by the I 2 chromosome with the I 2 chromosome posthe gene T (= dotted petals) ; in the sesses the gene Ri. As can be seen in Table 50 the seed set from crosses P v . I T x 8 type A(v) [bert. Pl.] is much reduced. It is noticeable that the IT.v are nearly as frequent as I.v, but the v.v have

310

J. SCHWEMMLE

decreased considerably, even though the v complexes of the three different v.1 are identical. Thus, also in this cross the maternal diplont influences the affinity v-v. In the crosses with v.IB the v.v are slightly more frequent but the IR6.v less frequent than in comparable crosses. The affinity Irv is only insignificantly lowered in comparison to I-v, the afbity I R much ~ more strongly. Also in the crosses with male TABLE 50 Influence of Chromosome I 2 (in IT) and of the Gene Ri on the Affiities of I Ovules to v-Pollen Tubes with bert. and od. Cytoplasms v [bert. Pl.] 9

9 v.1 9 v.1~

9 v.IR<

* L.S.

v [od. Pl.] 3

% L.S.*

% v.v

% 1.v

% LA.*

% v.v

% 1.v

51.9 42.1 40.3

16.5 9.5 10.6

28.4 27.6 23.5

51.6 43.6 40.2

18.1 12.3 10.2

26.5 24.5 23.2

= large seeds.

type A(v) [od. Pl.] the influence of the I 2 chromosome and the gene Ri can be recognized, as well as the peculiar and inexplicable effect of the maternal diplont. How the reactivities of IT- and IRi-pollen tubes are changed can be seen from the affinities obtained in crosses listed in Table 51. The IT-pollen tubes were less strongly attracted by the v ovules than the I-pollen tubes, and the IRi-pollen tubes still less. The effect of the 1 2 chromosome and of the gene Ri is unmistakable. TABLE 51 Influence of Chromosome 2 2 (in IT) and of the Gene Ri on the Reaction of I-Pollen Tubes to v Ovules

Crosses v.1 [bert. PI.] x 1.1 v.1~1[bert. PI.] X v . 1 ~ v . 1 ~[bert. Pl.] X v.1~6

Affinities

- I = 19.3 - I T = 12.2 V - I R I = 10.8

v v

The I-pollen tubes of the three different types v.1 also grow with different speeds. I n the crosses o v.1 X 8 v . 1 ~the v.v were with 12.4% especially frequent. The v-pollen tubes could fertilize many v ovules since they had a large advantage over the slower growing IT-pollen tubes. As calculated from the frequency of v.v, the I-pollen

SELECTIVE FERTILIZATION IN

Oenothera

311

tubes grow fastest, the IB4-pollentubes were slower, and the IT-pollen tubes slowest. It is noteworthy that in the crosses with male B.ZT, B.Zt, and B.lRr the results were completely different. The gene Ri increased the affinity I& (see Table 49) and the ZRa-pollentubes showed the highest growth rate. It is therefore important in which complex the 1 2 chromosome and the gene Ri are contained. The I d T are missing in the crosses o v.111.X 8 v.1~4 and 9 v.1~1 8 v.IT. The seeds germinated as well as those of the comparable crosses. Besides, there were no more sterile seeds and the mortality was normal. But the seed set in these crosses was quite low. Thus it can be stated with certainty that the IT ovules were neither fertilized by the IBr-pollen tubes nor the IBi ovules by the IT-pollen tubes; the affinities I T - I R I = 0 and I R & = 0. I n 1942 three B.1 with light green leaves appeared beside twelve normal B.2 in a culture from B.1 selfed. Haustein proved that the abnormal color of the leaves was caused by a recessive gene h. This gene is transferred very easily from one complex to the other. In this way it is possible to obtain all genetic types with light green leaves. Thus it could be investigated whether this gene h is able to affect the affinities between ovules and pollen tubes. Therefore numerous comparable crosses with normal and light green forms were set up. The seed set from crosses 0 vb.Ih X 8 hsc.hsc was about 14.9% lower compared to that of crosses o v.1 X 8 hsc.hsc put equal to 100%. Calculated in the same way, there were about 17.1% less Ih.hsc than I.hsc and about 11.8% less Vh.hSC than v.hsc. The gene h reduces the affinities I-hsc and v-hsc, and its effect is more pronounced in the I complex than in the v complex. X 8 v.11 than The seed set w a ~again lower in the cross ? in the comparable cross 9 v.1 X 8 v.11. But this time the 1h.V decreased only by about 2.8% ; the vh.v, however, by about 32.4%. The gene h has a much larger effect in the v complex than in the I complex, contrary t o the above discussed crosses. The result depends therefore on the type of pollen tubes used to demonstrate the effect of the gene h. Unexpectedly the yield of crosses 9 v.1 X 8 V h I I h (with only Vh pollen) was much better than that of crosses 0 v.1 X 8 v.11. The 1.v and I.Vh progeny were equally frequent but there were 59.0% more v.vh than V.V. The reactivity of the vh-pollen tubes to the chemotropically effective substances secreted by the v ovules is considerably increased. Comparative crosses with o B.2 and 8 B h l h were also made. These also showed how important the type of pollen tubes is. Thus the B.1

x

312

J. SCHWEMMLE

are as frequent as the Bh.Z and the 1.1 as frequent as the 1h.l in the crosses 0 €3.1 X 8 1.11 and O BhlhX 8 1.11. This means the factor h has no influence on the affinities. But in the crosses ? B.1 X 8 B.11 and 0 Bh.lhX 8 B.11 there were about 18.1% less ZhB than 1.B and about 5.7% less BhB than B.B. Here the effect of the gene h is shown unambiguously; it is stronger in the 1 complex than in the B complex. In the crosses with 8 hsc.hsc it is exactly the reverse; the Bh.hsc decreased by about 9.4%, the l,.hsc by only about 3.6%. I n the crosses with 8 hl.hl there is a surprisingly large reduction by 47.9% and 46.7% in the Bh.hl and 1h.hl compared to B.hl and Lhl, respectively. It is noteworthy that a single gene can influence so strongly the complicated processes which occur during fertilization. On page 308 i t has already been pointed out that the maternal diplont can influence the affinities. This phenomenon will be discussed again very briefly. It should be expected that the affinity, for example, of 8 B ovule to a particular type of pollen tube is always the some in crosses set up at the same time, independently of the complex heterozygotes in which the B complex is present. Furthermore a certain type of pollen tube should be attracted equally well by the same type of ovule independently of the pollen parent from which the pollen is derived. Indeed, the affinities hhook-B and hhoor-l are the same, independently of whether the B or 1 pollen is taken from B.1 or from B.11 and 1.11. Also the affinity d B obtained in the cross o B.2 x 8 B.11 is, at 16.8, the same as that obtained in the cross o Z.v )( 8 B.11, which is 16.5. Still more examples could be given. But not infrequently different values are found for the same affinities from different crosses. This can be easily seen in Table 62. Apparently it is not unimportant whether the ovules containing egg cells of equal constitutions are present in the ovary of this diplont or that; no explanation can be given. Maybe experiments with the low yielding B.v from P v.1 X 8 B.1 would have given some indication. TABLE 52 Differences in Affinities of Ovules with Identical Complexes in Different Maternal Diplonts ~

_

_

~

_

~

Crosses

Affiaities

Crosses

B.1 X hhook-hhmk V.1 x hhook.hhook B.1 X ha.ha B.1 X ha.ha

Z-hlrwk = 7 . 4 V-hhook = 3 . 5 B-ha = 12.4 Z-ha = 20.3

2.V x hhookhhook l.V x hhookahhaok B.1 X ha.ha I.v X ha.ha

Affinities 2-hb-k V-hhook B-ha 1-ha

= 14.9 = 18.7 = 7.3 = 13.6

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313

Arnold investigated a peculiar case of influence by the maternal diplont on the affinities. Simon had observed that the crosses 0 Oe. campylocalyx (ck.cl) X 6 Oe. hookeri (hhook.hhook) did not produce seed in repeated attempts, although the hhook-pollentubes grew through the style. Therefore it had to be assumed that the ovules of ck.cl do not attract the hhmk-pollen tubes chemotropically. Six hundred experiments in which ovules were placed separately on a suitable nutrient medium surrounded by a circle of pollen grains from Oe. hookeri (distance to ovules about 1 mm.) confirm this assumption. The hhookpollen tubes grew out without direction while the ck-pollen tubes in the control experiments grew unambiguously toward the ovules. Arnold repeated the crosses with the same negative results. But in 1956 the same crosses produced seeds. The seeds were raised in the year 1957 and 3 ck.hhmkand 52 cl.hhmkwere obtained. Also the crosses from the year 1957 produced seed. I n 1958 ck.cl seeds from the harvests of 1950 (ck.cl 50) and 1957 (ck.cl 57) were sown and the seedlings raised. I n the crosses of these plants with Oe. hookeri males i t was found that the crosses from o ck.cl 50 did not produce seed but those with Q ck.cl 57 did. The two samples of ck.cl were not genetically identical, although an exact comparison did not show any visible differences. From a number of crosses Arnold came to the following explanation: in ck.cl 50 a gene “H50” is present in t.he complex cl which inhibits the ovules of ckxl from being fertilized. But the gene H50 has this effect only in the presence of the ck complex since the crosses with cl 50.v females obtained through suitable crosses produced seed. The gene H50 in the ck.cl 50 causes the ck and cl ovules t o produce substances by which the Oe. hookeri pollen tubes are not attracted while other pollen tubes are. The gene H57 which originated by mutation in the ck.cl 57 permits the formation of substances to which also the Oe. hookeri pollen tubes react. The decision concerning this character in both cases has already occurred in the diploid megaspore mother cell. VII. Affinities in Trisornic Mutants

I n the cross 0 B.Z X 6 B.11 grown in 1951 several aberrant plants appeared which resembled B.Z but had small characteristically serrated leaves. They were investigated by Arnold, who found that these aberrant plants were trisomic mutants. He called them B.Z angusta. The unchanged complex B from B.Z is transmitted through pollen and egg cells, while an altered Z complex with 8 chromosomes ( I 8) is transmitted only through the egg cells. The chromosome 7 ( 1 4)14 has been lost through nondisjunction and has been replaced by the chromosomes

314

J . SCHWEMMLE

13(B4)14 and 4(B5)7. It was possible that the affinity of the ovules with the Z 8 complex in their egg cells to a particular type of pollen tube, e.g., to hsc, may be different than the affinity 1-hsc. I n order t o test this assumption many comparable crosses with B.l females and B.1 8 females were made. By counting out the capsules it was found first that the crosses with B.l 8 produced a much lower seed set, and second that the B.1 8 had much fewer ovules in the ovary than the B.1. Therefore it was not possible to compare directly the seed sets and the genetic types which appeared in the cultures. First it had to be found how many ovules are fertile. A renewed anatomical investigation of B.Z demonstrated that 65.2% and 62.1% of all ovules were fertile, in agreement with the result of earlier determinations. Therefore there were 3 3 4 1 % B ovules and the same number of 1 ovules. In the B.2 8 the fertile ovules were much rarer (30.2 and 26.6%). ConTABLE 53 Progeny Produced by B.Z and the Trisomic Complex €3.1 8 Pollinated by Different Types of Pollen ~~

~

c? 1.1 TypeA hsc.hsc ha.ha

~

Pollen

I

v hsc ha

Q B.1 B

9 B.18 B

$2 B.1

1

79.4-84.7 (111.6) 76.4-81.3 87.0-92.6 72.7-77.4 68.1-72.7 (93.9) 71.8-76.4 55.4-59.0 85.0-90.7 (163.4) 67.3-71.6

71.2-75.8

-

0 B.18 18

53.7-57.3 (70.3) 39.4-42.0 (46.5) 34.4-36.7 (4'7.9) 50.0-53.5 (74.3)

sidering the results from the cultured progeny of the crosses it may be assumed that there were 16-15% B ovules and a corresponding number of Z 8 ovules among the ovules of B.Z 8. Now i t is possible to calculate the percentage of fertile B and 1 ovules and of B and 1 8 ovules, respectively (each taken as loo%), which were fertilized. Table 53 shows that the 1 ovules were fertilized with different frequencies by different pollen tubes. The reactivity of the pollen tubes depends on their genetic constitution. Furthermore, it can be seen that the Z 8 ovules were fertilized more rarely than the 1 ovules. Their chemotropic attraction was reduced, the extent of the difference depending on the type of pollen tubes used. It is largest in the crosses with type A(v) as male, smaller in the crosses with ha.ha as male parent. This is shown best by the values given in parenthesis. They show how many I 8 ovules were fertilized in comparison to the 1 ovules = 100%. The lower the calculated value, the larger is the difference. It could have been expected that the B ovules of B.1 and B.1 8 mhich have the same con-

SELECTIVE FERTILIZATION IN

315

Oenothera

stitution would be fertilized equally frequently. But this is not the case. I n the crosses with 8 hsc.hsc the B ovules of B.2 were fertilized somewhat less frequently than the B ovules of B.1 8; but the difference is small. Possibly the comparable crosses with 8 hsc.hsc correspond to the expectation. But in the crosses with 8 1.1 and 8 ha.ha the B ovules of B.2 8 were fertilized more frequently than the B ovules of B.Z. The difference is largest in the crosses with 8 ha.ha. There exist several possibilities to explain this striking phenomenon. But since none has been tested they will not be discussed further. Also in these crosses the B ovules were not fertilized by the v-pollen tubes. Corresponding experiments were carried out with Oe. odorata var. tenella. This is a trisomic mutant of v.1 as was found by Arnold. Of the TABLE 54

Progeny Produced by v.1 and the Trisomic Complex v 8.1 Pollinated by Different Types of Pollen _

_

_

_

_

~

~~

9

8 Type A

2.11 1.1 B.11 hsc.hsc ha.ha

Pollen V

I

I B hsc ha

9

9

9

V

v 8.1 v8

v.1 I

v 8.1 I

66.1 79.4 82.7 49.1 43.6 34.2

17.7 (26.8) 31.9 (40.2) 69.6 (84.2) 38.1 (77.6) 28.5 (66.4) 37.3 (108.7)

86.4 42.7 77.9 47.1 39.7 36.7

72.3 (83.7) 5 6 . 5 (132.3) 8 9 . 2 (114.6) 78.8 (167.7) 94.2 (23r.3) 86.9 (969.2)

v.1

7 v chromosomes the 10(v3)14 chromosome has been replaced by two I chromosomes, to wit 13(13)14 and 9(12)10. Both the unaltered I complex and the altered v complex called v8 are transmitted through the egg cells but only the I complex through the pollen. Also in this v8.I the number of ovules is smaller than in v.1 and it also has fewer fertile ovules, only 52% as compared to 66% in v.1. Thus the v.1 has 33% v and 33% I ovules, the v8.1 only 26% v8 and 26% I ovules. It was calculated how many of the fertile ovules of v.1 and v8.I were fertilized. This can be seen in Table 54. We see again that the v and I ovules of the v.1 are fertilized with different frequencies depending on the type of pollen tubes used. But how about the v8 ovules in comparison to the v ovules? Obviously the v8 ovules of v8.I were fertilized less frequently than the v ovules of v.1. Only the crosses o v8.I X 8 ha.ha do not show this difference. Furthermore, it can be seen that the differences in the comparable crosses are of different sizes. This is

316

J . SCHWEMMLE

shown by the calculated values given in parentheses as mentioned above. The difference is largest in the crosses with type A(v) male. I n other crosses, the difference becomes smaller and smaller, until surprisingly the v8 ovules were fertilized more frequently than the v ovules in the crosses with 8 ha-ha. It is now noteworthy that also in the crosses with B.Z and B.Z8 female (Table 53) the differences decrease in the same succession depending on the type of pollen tubes used, that is, in the order v, hsc, I, ha. Already in the crosses involving B.Z and B.Z 8 it had been found that B ovules with equal constitutions from B.1 8 were in part more frequently fertilized than the B ovules from B.Z.This occurs still more strikingly in the crosses now under discussion. 86.4% of the I ovules of v.1 were fertilized by the v-pollen tubes, but only 72.3% I ovules of the v8.1, in other words less. But in the other crosses, the I ovules of v8.I were fertilized more frequently than the I ovules of v.1. The calculated values in parentheses show clearly how large the differences are, depending on the type of pollen tubes used. An explanation for these striking results cannot be given. Since the experiments have shown that not all ovules containing egg cells with 8 chromosomes are fertilized] their frequency in a diploid mother plant cannot be exactly determined. V111. Chemotropirm of the Pollen Tubes

The experiments discussed above could be explained by assuming that the ovules produce certain substances depending on the genetic constitution of the egg cells (synergids) to which the pollen tubes react differently depending on their own genetic constitution. It was necessary t o demonstrate this chemotropism of the pollen tubes. Koepchen carried out the first experiments. First a suitable nutrient medium had to be found on which the pollen grains could germinate and form sufficiently long pollen tubes. Ovules were laid out separately on slides with a thin layer of this substrate. After 20 hours pollen grains were sown circularly around the ovules a t a distance of about 2 mm. After another 10 hours the results were scored. Only those experiments were listed as positive (+)in which the pollen tubes grew unambiguously toward the ovules. Then the percentage of positive experiments, relative t o all experiments = loo%, was calculated. From this value a certain percentage of positive experiments was subtracted, namely the percentage of positive experiments obtained when a thoroughly cleaned grain of sand was substituted for an ovule. The values obtained by this method are found in Table 55. Many experiments were

SELECTIVE FERTILIZATION IN

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317

carried out blind, the observer not knowing from which plant the ovules or pollen grains were derived. Each series of experiments consisted of 629-718 single experiments. I n the experiments with ovules of B.2 and pollen from 1.1 there were 41.2% positive experiments. 38.7% large seeds were found in the crosses P B.1 X 8 1.1. I n the experiments with the v pollen of v.11 18.4% were positive in the crosses 0 B.Z X 8 type A (also only with v pollen) the seed set was 16.1% since the B ovules are not fertilized by the vpollen tubes. I n the experiments with the ovules of Oe. odorata (v,I), the fewer+- experiments were obtained, the lower the seed set in the comparable crosses. TABLE 55 Chemotropic Attraction of Ovules from B.2 to v.1 for Pollen Tubes of Different Constitutions, Compared to the Seed Set of the Corresponding C r m Ovules from

Ova

Pollen

% exps. pos.

B.E B.1 v.1 v.1 v.1 v.1

Band2 B and 1 v and I vandI v and I v and 1 .

I

41.2 18.4 41 .O 33.1 18.0 18.7

* L.S.

V

I B hl I

% L.S.'

Cross

B.1 X 1.1 38.2 B.Z X type A(v) 16.1 v.1 x 1.1 38.6 v.1 X B.11 25.7 v.1 X hl.hl 1.4 pollen sown immediately

= large seeds.

If the ovules secrete certain chemotropically effective substances, their concentration will probably increase during the 20 hours after the ovules are layed out. Thus there should be fewer experiments when the pollen grains are sown directly after laying out the ovules. This was found to be the case. In the experiments where the pollen grains were sown at once there were 18.7% positives; in the original experiments, 41.0%. Loertzer continued the experiments using the same method. Only pollen of Oe. ZongifEora (hl.hl) was used. She also found that the perexperiments increased parallel to the improvement in centage of the seed set in the comparable crosses (Table 56). Schneider devised a method which permits the demonstration of the chemotropic attraction of the pollen tubes by the ovules in a way which is suitable for statistical treatment. H e determined the percentage of pollen tubes which grew unambiguously in the direction of ovules which had been arranged in a row on the nutrient medium. The pollen

+-

+-

318

J. SCHWEMMLE

grains were sown out parallel to the ovules. The compilation of data in Table 57 shows that the better the pollen tubes were attracted chemotropically the higher the seed set was in comparable crosses; furthermore, the pollen tubes with genetically different constitutions reacted differently to the chemotropically active substances which were secreted by the B and 2 ovules. TABLE 56 Chemotropic Attraction of Ovules from Different Homozygotes for Pollen Tubes from Oe. Zongi$ma (hLhl), Compared to the Seed Set of the Corresponding Crosses ~~

Ovules from

% exps. pos.

ha.ha v.v hsc.hsc 1.1 hl.hl

* L.S.

9.8

16.1 26.0

43.1 50.2

% L.S.* in crosses

2.7 4.6 13.7 26.3 75.7

= large seeds.

Table 58 shows that the same pollen tubes were attracted with different success by Ovule6 with different constitutions with regard to their egg cells. It should be mentioned again that the differences in the chemotropic attractions are statistically significant with a probability of 0.0027. TABLE 57 Percentage Pollen Tubes of Different Constitutions Reacting Positively to Ovules from B.2, Compared to the Seed Set of the Corresponding Crosses ~

Ovules from

B.1

Pollen hsc

I

V

* L.S.

% Pos. pollen tubes

Crosses

% L.S.*

45.3 39.4 30.1

B.Z X hsc.hac B.E X 1.1 B.1 X type A(v)

47.3 38.2

16.1

= large seeds.

Glenk (1964) showed by his anatomical investigations that the pollen tubes are indeed attracted chemotropically by the ovules as long as the affinity is high enough. In the upper part of the ovary the socalled conducting tissue fills the whole center of the cross section. I n the middle and lower parts this tissue is restricted to four elliptical areas beneath the placental groove which Glenk designated as “pla-

SELECTIVE FERTILIZATION IN

Oenothera

319

TABLE 58 Percentage ha-Pollen Tubes Reacting Positively to Ovules of Different Genetic Constitutions, Compared to the Seed Set of the Corresponding Crosses ~

Ovules

Pollen

pollen tubes ~

I ha

ha

V

hsc

* L.S.

= large

~

% Pos.

% L.8.*

Crosses _______

~~

45.2 44.8 39.8 36.2

~

~

1.1 X ha.ha ha.ha X ha.ha v.v X hs.ha hsc.hsc X h a h a

70.3

100 48.9

30.7

seeds.

cental pockets.” The pollen tubes grow through this conducting tissue which is especially loose. I n the crosses o Oe. berteriana x 6 Oe. hoolceri, which produce poor seed sets, the pollen tubes grow in the conducting tissue straight to the lower end of the ovary. Not a single pollen tube grew toward the ovules (Figs. 7 and 8 ) . I n the crosses o Oe. berteriana x 8 Oe. scabra, however, which produce a good yield, the pollen tubes grew obIiqueIy upward through the placental grooves

Rh. -

A

Plt.

SA

Plri. I

PI.

Fr.W

Fr. W

SA

Plri.

Plt. A

I

PI

FIG.7. Left. Pollen tubes growing &might down in a placental pocket in a case of missing affinity ( 0 BZ. x 8 hhook. hhmk). Semidiagrammatic. Lines indicate direction of the tissue. Abbreviations: Fr. W, ovarial wall; SA, ovules; Pl., placenta; Phi., placental groove; Plt., placental pocket; A, axial cone; Rh., cells of raphe. After Glenk (1964). Fro. 8. Right. Pollen tubes penetrating the placental grooves obliquely and branching on the surface of the placenta, in a case of positive affinity ( 9 B.1 x 6 hsc.hsc.). For abbreviations, see Fig. 7. After Glenk (1964).

320

J. SCHWEMMLE

which, however, did not determine their direction of growth in any way, to the surface of the placenta. There they branched and a branch of the pollen tube penetrated into the micropyle of the ovule. Thus the pollen tubes are already deflected from their original growth direction in the conducting tissue by the chemotropically active substances of the ovules. It is noteworthy that in crosses with a good seed set, pollen tubes which had lost their way in the tissue of the stigma changed their wrong growth direction and grew toward the base of the style. It seems as if a certain chemotropism already acts a t this stage. Seufert’s experiments (1965) lead to the same conclusion. He irradiated pollen of tetraploid Oe. berteriana and used it for pollination. At a dose of 32,000 r the pollen grains still germinated and pollen tubes grew to the ovaries; but only very few ovules were fertilized since the reactivity of the pollen tubes was strongly reduced. At still higher dosages no fertilization occurred. The pollen tubes grew without any direction on the stigma and were not able t o penetrate into the style. Schildknecht and Benoni (1963) tried to identify the chemotropically active substances which are secreted by the ovules. Ovules from five different Oenothera were extracted. Schneider’s method was used to test whether the pollen tubes were attracted chemotropically by the extracts. This proved to be the case. The pollen tubes of Oe. scabra were attracted most strongly by the extracts from ovules of the same species. The selfings of Oe. scabra also produced the highest seed set. By means of paper chromatography the following substances were demonstrated: amino acids, five peptides, one primary aliphatic amine, one indol compound, two ninhydrin-negative amines, and five sugars. Most of the compounds could be further identified. The identified substances were tested for their chemotropic effects, especially with pollen of Oe. Zongiflora, using eluates from cut-out parts of the paper chromatograms, as well as with the corresponding synthetic compounds. At different dilutions characteristic for each compound the pollen tubes were attracted optimally. The chemotropic activity can be increased by addition of K+ ions or of mixtures of amino acids and sugars, which were also found in the extracts. The extracts of ovules from genetically different Oenothera did not differ qualitatively. The fact that the same pollen tubes are attracted chemotropically to different degrees by ovules with different constitutions with respect to their egg cells (synergids) can probably be explained by quantitative differences in the composition of the mixture which is secreted by the ovules. In this summary only the results of experiments with Oenothem have been discussed. It can be regarded as firmly established that here selective fertilization occurs. The question of how far selective fertiliza-

SELECTIVE FERTILIZATION IN

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321

tion has been demonstrated in other plants will not be discussed in this paper. In 1958 Arnold reviewed all cases known at that time. The reader is referred to his review. Finally I would like to express my sincere gratitude to all my collaborators for their help and also t o the Deutschen Forschungsgemeinschaft whose support made these investigations possible. Appendix. Survey of the Strains Used in the Crosses Jsoeamous complex heterozygotes Y

Oe. berteriana Oe. odvrata Oe. stricta Oe. selowii Oe. mollissima Half heterogamous complex heterozygotes Oe. campylocalyx Oe. oakesiana Homorygotes Oe. urgenlinea Oe. Zon&ba Oe. scabra Oe. hookwi Trisomic mutants (a) Oe. odorata type A var. tenella (b) Oe. berterianu var angusta Forms obtained from crosses Homozygote 1.1 Half-heterogamous B.11 1.11 v.11

Genetic constitution

B.1 v.1 1str.vstr 1se.vse mk.ml ck.cl koak.loak

Gametes

-

9

B

8 1

B

l

2str lse mk

I vstr vse ml

V

ck koak

cl loak

ck koak

V

ha.ha hl.hl hsc.hsc hhook. hhmk

I

vstr

ktr ke mk

ha hl hsc hhaok

vse ml

-

-

ha hl hsc hook

v.18 v8.I

V

I8 I

V

-

v8

B.18

B

18

B

-

1.1 B.11

1.11

v.11

- I

I

I B 1 V

I1 I1 I1

B 1

V

-

-

REFERENCES Arnold, C.-G. 1965. Genetische und rytologische Untersuchungen an trisomen Mutanten der Oenothera berteriuna. Z . Vererbungslehre 86, 622-662. Arnold, C.-G. 1967. Uber 15-chromosomige Mutanten der Oenothera odorata. Flora (Jena) 144, 637-661.

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Arnold, C.-G. 1958.Selektive Befruchtung. Ergeb. Biol. 20, 67-96. Arnold, C.-G. 1962.Zum Problem der selektiven Befruchtung. Z . Botan. 50, 113-126. Binder, M.1938.Die Eliminierung der Zl-Embryonen mit Plastiden der Oenothera odorata. 2.Vererbungslehre 75, 739-796. Fischer, H.P. 1962.Drei Oenotheren aus dem neugeschaffenen Subgenus Renneria. Feddes Rept. 64,233-240. Glenk, H. 0. 1964. Untersuchungen uber die sexuelle Affinitat bei Oenotheren. “Pollen Physiology and Fertilization,” pp. 170-181. Gress, M.1959.Der Einfluss des Plasmas und einer geringen Anderung des Genoms auf die Affinitat rwischen Samenanlagen und Pollenschlauchen bei der Oenothera odorata. Biol. Zentr. 78, 871-888. Gross, H. 1959. Untersuchungen zum Problem der “Vegetativen Annaherung” bei Oenotheren. Zuechter 29, 6-20. Habryka, K. 1951. Analyse der Oenothera stricta und Oenothera selowii. Dissertation, Erlangen. Hagemann, R. 1964. “Plasmatische Vererbung.” G. Fischer, Jena. Haustein, E. 1938. Die Plastidenvererbung bei Oenothera berteriana und Oenothera odorata. Z . Vererbungslehre 75, 661-689. Haustein, E. 1962.Die Endenbezifferung der Chromosomen einiger Oenotheren aus dem Subgenus Raimannia. 2. Vererbungslehre 84, 417-453. Haustein, E. 1955. Neuere Arbeiten zur Befruchtungsphysiologie. Z . Botan. 43,

263-261.

Haustein, E. 1957. “Selektive Befruchtung” im Pflanzenreich. Umschau 57, 432-435. Haustein, E. 1962. Die chlorina-Mutante der Oenothera berteriana. 2. Vererbungslehre 93,249-253. Heymer, T . 1946. Die Analyse der Oenothera oakesiana. Dissertation, Erlangen. Huber, H.1955. Die Eliminierung von Austauschgonen bei Oenotheren. Z . Vererbungslehre 86, 469-479. Kistner, G. 1955. Uber plastidenbedingtes Absterben wahrend der Embryonalentwicklung einiger Oenotherenbastsrde. 2.Vererbungslehre 86, 521-644. Landspersky, N. 1963. Das unterschiedliche Reifen und Altern der Samenanlagen in den verschiedenen Ahschnitten des Fruchtknotens der Oenothera Berteriana. Biol.Zentr. 82, 9-29. Lechner, K. 1964. Selektive Befruchtung auf Grund unterschiedlicher Reaktionsgeschwindigkeiten zwischen Pollenschlauch und Eizellsorten. Biol. Zentr. 83,

641-660.

Leuchtmann, G. 1955. Die Eliminierung von Austauschgonen bei Oenotheren 11. 2.Vererbungslehre 86, 480-497. Loertrer, B. 1954.Weitere Untersuchungen zur selektiven Befruchtung. 11. Dissertation, Erlangen. Richter, C.-M. 1956. Untersuchungen zur Physiologie der Befruchtung bei Oenotheren. 2.Botan. 44, 377-407. Riihl, F. 1952. Die genetische Analyse der Oenothera campylocalyz. Dissertation, Erlangen. Schildknecht, H., and Benoni, H. 1963. Uber die Chemie der Anziehung von Pollenschlauchen durch die Samenadagen von Oenotheren. Z . Naturforsch. 18b,

46-64.

Schneider, G. 1956. Wachstum und Chemotropismus von Pollenschlauchen. 2. Botan. 44, 175-205.

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Oenothera

323

Schiitz, G. 1939. Genetische und aytologische Untersuchungen an Eu-Oenotheren. Selektive Befruchtung bei der Komplexheteroaygote Oenothera Berteriana. Z . Botan. 33,481-525. Schwemmle, J. 1938a. Die Analyse der Oenothera Berteriana und Oenothera odorata. Z . Vererbungslehre 75,361-468. Schwemmle, J. 1938b. Untersuchungen iiber das Zusammenwirken von Kern, Plasma und Plastiden. Z . Vererbungslehre 75, 486660. Schwemmle, J. 1943. Plastiden und Genmanifestation. Flora (Jena) 37, 61-72. Schwemmle, J. 1949. Gibt es eine selektive Befruchtung? Bio2. Zentr. 68, 195-231. Schwemmle, J. 1951. Gibt es eine selektive Befruchtung? 11. Biol. Zentr. 70, 193252. Schwemmle, J. 1952a. Gibt es eine selektive Befruchtiig? 111. Biol. Zentr. 71, 152183. Schwemmle, J. 1952b. Selektive Befruchtung als Erklarung unerwarteter Kreuzungsergebnisse. Biol. Zentr. 71,353-384. Schwemmle, J. 195%. Der Einfluss des Plasmas auf die Affinitat zwischen Samenanlagen und Pollenschlauchen. Biol. Zentr. 71, 487-499. Schwemmle, J. 1953a. Selektive Befruchtung bei der Oenothera berteriana. Biol. Zentr. 72,129-146. Schwemmle, J. 1953b. Selektive Befruchtung bei der Oenothera odorata. Biol. Zentr. 72,405-424. Schwemmle, J. 1956a. Trisome Mutanten der Oenothera odorata. Flora (Jena) 143, 356-384. Schwemmle, J. 1956b. Selektive Befruchtung hei Oenothera selowii und Oenothera stricta. Biol. Zentr. 75, 563-575. Schwemmle, J. 1956c. Uber Vergleichskrcuzungen mit ahnlich koustituierten Oenotheren. Biol. Zentr. 75, 643-664. Schwemmle, J. 1957a. Der Samenansatz bei Kreuzungen mit der Oenothera Berteriana 0 nach Friih- und Spatbestiiubung. Planta 49, 135-167. Schwemmle, J. 1957b. Das unterschiedliche Reifen und Altern der Samenanlagen in den verschiedenen Abschnitten des Fruchtknotens der Oenothera Berteriana. Planta 49,168-207. Schwemmle, J. 1957~.Der Einfluss des Plasmas auf die Affinitat zwischen Samenanlagen und Pollenschlauchen. Biol. Zentr. 76, 443-453. Schwemmle, J. 1957d. Der Eintluss des Plasmas und der Plastiden auf die Affinitat zwischen Samenanlagen und Pollenschliiuchen. Biol. Zentr. 76, 529-549. Schwemmle, J. 1958a. Uber Kreuzungen mit verschiedenen Formen der Oenothera odorata bei Friih- und Spatbestaubung. I. Planta 51, 223-248. Schwemmle, J. 1958b. Uber Kreuzungen mit verschiedenen Formen der Oenothera odorata bei Friih- und Spiitbestaubung. 11. Planta 51, 249-287. Schwemmle, J. 195th. Der Samenansatz bei Kreuzungen mit der trisomen Oenothera Berteriana var. angusta. BioZ. Zentr. 77, 153-165. Schwemmle, J. 1958d. Uber Kreuzungen niit der trisomen Ocnothera odorata var. tenella. Biol. Zentr. 77, 329-347. Schwemmle, J. 1963a. Der Einfiuss des Faktors h auf die Affiitat zwischcn Samenanlagen und Pollenschliiuchen. I. Z . Vererbungslehre 94, 133-162. Schwemmle, J. 196313. Der Einfiuss des Faktors h auf die A h i t a t zwischen Samenanlagen und Pollenschlauchen. 11. Z . Botan. 51, 217-232. Schwemmle, J. 1964. Weitere Untersuchungen zur Physiologie der Befruchtung hei Oenothera berteriana. Biol. Zentr. 83, 409-425.

324

J. SCHWEMMLE

Schwemmle, J. and Koepchen, W, 1953. Weitere Untersuchungen zur selektiven Befruchtung. 2. Vererbungslehre 85, 307446. Schwemmle, J., and Lohmeyer, H. 1963.Uber Versuche mit Oenothera campylocalyx. 2. Vererbungslehre 94,412-426. Schwemmle, J., and Simon, R. 1956. Der Samenansata bei Oenotherenkreuzungen. Planta 46, 552-568. Schwemmle, J., and Zintl, M. 1939. Genetische und eytologische Untemchungen an Eu-Oenotheren. Die Analyae der Oenothera argentinea. 2. Verebungslehre 76,

353-410.

Seufert, R. 1965. Die Strahlenempfindlichkeit verschiedener Entwicklungsstadien der tetraploiden Oenothera berteriana. Radiation Botany 5 , 163-169. von Schlenk-Barnsdorf, M. 1951. Analyse der Oenothera scabra. Dissertation, Er1a.ngen. von Zitek, R. 1954. Der Nachweis der v.v- und 1.1-Homosygoten der Oenothera odorata. Dissertation, Erlangen. Weidner-Rauh, E. 1939, Untersuchungen uber die partielle Sterilitat der Oenotheren: Das Pulver bei Eu-Oenotheren. 2. Vererbungslehre 76, 422486. Wensauer, H.1952.Die Analyse der Oenothera mollissirnu. Dissertation, Erlangen.

THE GENETIC CODE AFTER THE EXCITEMENTii2 Anil Sadgopa12’ Division of Biology, California Institute of Technology, Pasadena, California

.

I. Introduction . . . . . . . . . . . . . . , . , 11. The General Nature and Historical Background of the Genetic Code . 111. Codon Assignments . . . . . . . . . . . . . . . A. Genetic Studies . . . . . . . . . . . . . . . . B. Biochemical Studies . . . . . . . . . . . . . . . C. Amino Acid Replacement Studies . . . . . . . . . . . D. The Pattern of Degeneracy . . . . . . . . . . . . . IV. Chain Initiation and Initiator Codons . . . . . . . . . . A. N-Formylmet-tRNA and Its Codons . . . . . . . . . . B. Involvement of AUG in Initiating and Phasing the Reading . . C. The Role of Formylation and the Mechanism of Initiation . . D. Initiator Codons in Polycistronic Messengers . . . . . . . E. Requirement for Magnesium Ions in the Absence of an Initiator Codon . . . . . , . . . . . . . . . . . . . F. Are Initiator Codons Universal? . . . . . . . . . . V. Chain Termination and Nonsense Codons . . . . . . . . . A. The Need for a Mechanism of Chain Termination . . . . . B. The Nonsense Codons . . . . . . . . . . . . . . C. Amber and Ochre Mutants and Their Suppressors . . . . . D. UAG, UAA, and Chain Termination . . . . . . . . . . E. UGA and Its Nonsense . . . . . . . . . . . . . .

.

.

. . .

. . .

. .

326 327 329 329 331 337 344 345 346

. . 348

. .

349 350

. .

352 354 . 355 .355 .356 . 357 . 359 . 360

’The survey of literature for this review was concluded in J u n d u l y , 1967. isoleucine, asparagine, and glutamine are abbreviated 89 N-formylmet (or formylmet), ilu, asN, and glN, respectively; all other amino acids are abbreviated with the first three letters of their names. Other abbreviations are as follows: A (adenine), C (cytosine), G (guanine), T (thymine), U (uracil), I (hypoxanthine), and 9 (pseudouracil) ; Sm (streptomycin) and DSm (dihydrostreptomycin) ; Me (methyl) ; TMV (tobacco mosaic virus) ; E. coli (E8chekhk coli) ; tRNA (transfer or soluble RNA), and mRNA (messenger RNA). Poly-, oligo-, and trinucleotides are written with the 5’-OHto the left and the 3’-OH to the right; phosphate groups are usually omitted for brevity. Polypeptides are written with the amino terminal end to the left and the carboxy terminal end to the right. ** Present Address: Molecular Biology Laboratory, Tata Institute of Fundamental Research, Colaba, Bombay, India.

’The amino acids N-formylmethionine,

326

326

ANIL SADGOPAL

VI. Ambiguity in the Genetic Code . . . . . . . . . . . . . A. Coding Environment . . . . . . . . . . . . . . . . B. The Pattern of Ambiguity . . . . . . . . . . . . . . C . Suppression and tRNA . . . . . . . . . . . . . . . D. Streptomycin-Activated Suppression and Ribosomes . . . . . . VII. tRNA and Its Synthetase in Codon Recognition . . . . . . . . A. The Relationship between Anticodons and Synthetnse-Recognition Sites. . . . . . . . . . . . . . . . . . . . . B. Is the Anticode Degenerate? . . . . . . . . . . . . . C. The Experimentally Observed Patterns of Multiple Codon Recognition . . . . . . . . . . . . . . . . . . . D. Crick’s Wobble Hypothesis and the Nature of the Anticodon . . . E. Predictions and Tests of Crick’s Wobble Hypothesis . . . . . . F. The Degeneracy of tRNA Synthetases . . . . . . . . . . VIII. The Universality and Evolution of the Genetic Code . . . . . . A. Evaluation of the Universality of Codon Assignments and Anticodons B. The Possibility of Structural Relationships between Amino Acids and Codons or Anticodons . . . . . . . . . . . . . . . C. Ideas on the Evolution of the Code . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

361 362 364 365 367 370 371 374 377 378 380 383

384 384 386

388 390

I. introduction

In 1963, F. H. C. Crick reviewed the field of genetic coding in an article entitled “The Recent Excitement in the Coding Problem.” His title reflected the tremendous activity then being directed toward deciphering the genetic code. In this article, Crick laid down the basic definitions and concepts involved in the effort to “crack the code.” Now that the excitement generated by the deciphering of the code is almost over, it is an appropriate time to summarize the numerous advances made in this field in the past few years. This review discusses the various approaches used to decipher the code and evaluates the validity of present codon assignments both in vitro and in vivo. Special attention is devoted to the recent progress made in elucidating the nature of the signals for initiation and termination of protein synthesis. It has become increasingly clear that the coding ambiguity observed in vitro may have a significant in vivo role during protein synthesis. Therefore, the effect of the various components involved in protein synthesis on the fidelity of the coding process will be examined. The most challenging problem after the deciphering of the genetic code has been presented by the discovery that a transfer RNA (tRNA) species can recognize more than one codon for the same amino acid. Various experiments designed to test the validity and implications of a recently proposed mechanism for multiple codon recognition are discussed, as are investigations explaining the anticodon and its role in

THE GENETIC CODE

327

synthetase recognition. Finally, the concepts of universality and evolution of the genetic code will be reevaluated in the light of these developments. For current ideas on protein synthesis, see the reviews by Campbell (1965) and Schweet and Heintz (1966). II. The General Nature and Historical Background of the Genetic Code

The essential problem of genetic coding was the determination of the exact number and sequence of the nucleotide bases within each code word, or codon. The development of this coding problem can be looked a t in quite simple terms. If only one base were assigned t o an amino acid, no more than four amino acids could be coded. Two bases taken together for a code word (doublet) would define 16 sets, leaving 4 of the 20 amino acids without codons. The combination of three bases to define a code word (triplet) provides 64 unique sequences, with the possibility of having more than one codon per amino acid. If the resulting genetic language is such that more than one triplet code for any given amino acid, the code is said to be degenerate. The possibility also exists that up to 44 of these triplets may be nonsense codons, designating no amino acid. The genetic code is now well known to be highly degenerate, with a very limited number of nonsense codons. I n 1954, Gamow proposed a “diamond” code based on a concept of stereospecificity between the bases in the DNA double helix and the coded amino acid. This proposal drew attention because only 20 diiferent stereospecific diamond-shaped holes specified by three bases could be formed from 64 possibilities. This code was completely degenerate and was further characterized by its fully overlapping nature, that is, each code letter was used in more than one codon. Such an overlapping code imposes constraints on the possible sequences of amino acids, and it was soon shown by Gamow et al. (1956) that this was incompatible with the known sequence of amino acids in insulin. Since that time the existence of a nonoverlapping triplet code has been well demonstrated. Brenner (1957) showed the absence of any restrictions on neighboring amino acids in the sequences of a number of proteins. The fully overlapping code predicts that a mutation in one base would result in changes in three neighboring amino acids in a protein, but a large number of single amino acid substitutions have been found in mutant proteins of tobacco mosaic virus (TMV) coat protein (Tsugita, 1962; Wittmann and Wittmann-Liebold, 1963) , human hemoglobins (Baglioni, 1963) , and tryptophan synthetase A of E. coli (Yanofsky, 1963). I n addition, synthesis of homopolypeptides

328

ANIL SADGOPAL

under the direction of ribopolynucleotides of repeating triplet sequences (such as poly AAG) in cell-free systems also indicates the nonoverlapping nature of the code (cf. Table 1 and Section IIIJ3). The need for establishing a direct stereochemical relationship between an amino acid and its coding triplet was obviated by the formulation of the adaptor hypothesis by Crick (1957). Although interest in finding such a relationship has been recently revived (see Section VIII,B), Crick observed that it was difficult t o fit amino acids directly on the bases of DNA or RNA in such a way as to obtain the specificity required for such interactions. The adaptor hypothesis suggested a role for a molecule with which an amino acid could be enzymically combined and which would have a hydrogen-bonding surface to specifically interact with a nucleic acid template. Hoagland et al. (1958) identified tRNA as this adaptor. An alternate model for coding, concerned with the problem of punctuation or spacing of the individual code words in a nonoverlapping code, was proposed by Crick e t al. (1957). They developed a “commafree” triplet code by composing a list of 20 unique triplets such that no codon had the same composition as any possible overlap segment. Thus when any two “sense” triplets were put side by side, the overlapping triplets generated by them were designated nonsense. Such a nondegenerate code implied the presence of many nonsense codons, but Crick e t al. (1961) showed that “addition” and “deletion” mutations in the rII region of the Ta bacteriophage did not generate much nonsense. I n addition, the subsequent demonstration that randomly linked nucleotide copolymers could be used as messengers to direct polypeptide synthesis in vitro made it very unlikely that the code possesses many nonsense codons (see Section II1,B). On the basis of their studies of “addition” and “deletion” mutations in T4, Crick et al. (1961) proposed that the genetic message is read from one end to the other and that protein synthesis begins a t a fixed point on the mRNA. The direction of reading of the polynucleotide message was investigated in bacterial (Salas e t al., 1965) and rabbit reticulocyte (Lamfrom et al., 1966) cell-free systems in which the amino acid incorporation directed by the oligonucleotide (A) ,,C was shown to produce polylys with an asN a t the carboxy terminal end (AAA and AAC are known to code for lys and asN, respectively). Similarly, i t was demonstrated (Thach et al., 1965) that the oligonucleotide A3U3 directed synthesis of the dipeptide lys-phe (UUU-phe codon) but never of phe-lys. Hence, if the polypeptide is indeed assembled from the amino to the carboxy terminal end, as is currently believed, then the message must be read in the 5‘ to 3’ direction. An

THE GENETIC CODE

329

unequivocal evidence in favor of the 5' to 3' direction of translation of the messenger was provided by the comparison of the repeating tetrapeptide sequences in polypeptides, synthesized in cell-free systems, with the repeating tetranucleotide sequences in the ribopolynucleotide templates (Kossel e t al., 1967; cf. Table 1 and Section IIIJ3). Furthermore, the results of acridine-induced mutations in the phage lysozyme system (described in Section II1,A) can best be interpreted if the direction of translation of the mRNA is taken to be 5' to 3'. The need for commas was eliminated by the discovery of Nformylmet-tRNA and its codon AUG, which are involved in initiating protein synthesis a t a fixed point in E . coli (see Section IV). These findings clearly indicate how a precise chain-initiating mechanism coupled with a fixed direction of translation of the mRNA can fix the frame of reading of the message, eliminating the need for punctuation in the genetic code. 111. Codon Assignments

The most direct way of discovering the size of a code word might well have been by comparing the size of a piece of DNA (presumably a gene) or mRNA with the size of the protein for which it codes and calculating the ratio of the number of nucleotide residues to the number of amino acids. However, currently available techniques are not precise enough for such a study. The data supporting a triplet code and assigning the codons to their amino acids have been developed through three major lines of investigation to be discussed below: genetic studies, biochemical studies, and amino acid replacement data. A. GENETICSTUDIES From studies on the interaction of acridine and related compounds with DNA, Lerman (1961, 1963) proposed a model in which the acridine molecule was intercalated between the base pairs in a DNA strand. Crick et al. (1961) suggested that this resulted in addition or deletion of a base (or bases) during replication of DNA. This in turn would lead to shifts in the reading frame of the mRNA a t that point, producing an incorrect reading from there on. Such a shift would be expected to produce a nonfunctional protein. On the basis of their investigations of such frameshift mutations in T4 lysozyme synthesiaed in E . coli, Streisinger et al. (1966) proposed a mechanism for the mutagenic action of acridines which involved the presence of an acri-

330

ANIL SADGOPAL

dine-stabilized gap in one of the two chains of a D N A molecule a t or near a region of a repeating set of bases (e.g., AAAA or GUGU). The synthesis of D N A to filI the gap formed by the mispairing of bases a t the repeating sequence results in the addition or deletion of a base (or bases), The earliest evidence concerning the size of the codon came from the work of Crick et al. (1961) on frameshift mutations in the B cistron of the rII region of Tq. They showed that a mutation of a given type, arbitrarily designated “deletion” (or -) , can be compensated for by introducing a second mutation of a different type, arbitrarily designated “addition” (or +), a t a nearby point. This second mutation shifted the reading frame back to its original form and restored the function of the B cistron. Pairs of mutants from the same class did not compensate for one another, but the combination of three mutants from the same class resulted in wild or pseudo-wild type activity. This indicated that three additions or three deletions shifted the reading frame three units and allowed correct reading of the message from that point on. A coding ratio of 3 therefore seemed possible. However, Wall (1962) pointed out that these data imply only the coding ratio and not the word size (for a discussion of this point, see Crick, 1963). These experiments also predicted the presence in double mutants of a region of altered amino acid sequence between the two points where addition or deletion had occurred. The confirmation of code word size and altered amino acid sequence had to await the development of the phage lysozyme system involving acridine mutants and sequence analysis of the protein product (Terzaghi et al., 1966). When a particular deletion mutant was crossed with a particular addition mutant, the lysozyme produced had a block of five amino acids different from wild type lysozyme. Utilizing our knowledge of codon assignments (Table 2) from the in vitro experiments to be described below, a unique sequence of bases can be written which will code for the amino acids of the wild type protein and will also code for the five amino acids of the double mutant protein by a deletion of one base, and a shift in reading frame followed by insertion of one base (Fig. 1 ) . Recently, Streisinger et al. (1966) have reported in phage lysozyme and a - mutation and additional double mutant strains carrying a a triple mutant carrying three mutations of the same class, all of which produced active proteins containing a short stretch of amino acids that differed from the wild type. For all of these mutants it was possible to assign a base sequence as was done in the case presented in Fig. 1. The sequences can be related to each other only if the 5’ to 3’

+

331

THE GENETIC CODE

direction of the codons is made to parallel the amino terminal t o carboxy terminal polarity of the protein. These studies provide further evidence for a triplet code and verify that several of the codons assigned by in vitro experiments occur in vivo in &NA (Streisinger et al., 1966). Degeneracy in the code used by phage T4 can be observed in Fig. 1.

...

1. Wild type:

...

I. rn ANA- wild type:

2. mRNA-double mutant:

2. Double mutant:

Pro

ser

I

leu asN ala

...

CCA UCA CUU A A U a C .

I

'7

... AC. AAA GUC CAU CAC UUA A U T C . ... ... thr lys val his his leu met ala ... ,

'I

FIG.1. Frameshift mutations in phage lysoayme and codon assignments (from Table

21, Adapted from Terzaghi et al. (1960).

Genetic studies have also aided in the elucidation of the nature of nonsense codons and chain termination, as will be discussed separately in Section V.

B. BIOCHEMICAL STUDIES Early efforts to relate various code words to their amino acids utiliaed the in vitro E. coli system for amino acid incorporation developed by Nirenberg and Matthaei (1961). The use of synthetic polynucleotides t o decipher the genetic code was suggested by the discovery that poly U directed the synthesis of polyphenylalanine (Nirenberg and Matthaei, 1961). Nirenberg and co-workers (1963) and Ochoa and co-workers (see Speyer e t al., 1963) used randomly linked synthetic ribopolynucleotides of varying base ratios containing two or three of the four bases. The code words were deduced by comparing the frequencies of incorporation of various radioactive amino acids with the theoretical code word frequencies calculated for each of the polymers. Interpretation of these results involved three major assumptions (Crick, 1963) : (a) the code word is a triplet, (b) the composition of the polynucleotide is the same as that of the mixture of bases used to produce it, and (c) the sequence of bases in the polymer is random. Various criticisms of the techniques used and the methods of interpretation have been discussed in detail by Crick (1963). Although this approach was limited in that it provided only the composition but not the order of the bases in each triplet, a large number of triplets could be allotted to a group of several amino acids, pro-

332

ANIL SADGOPAL

viding tentative evidence for a degenerate code. For example, random poly AC directed the incorporation of pro, his, thr, asN, glN, and lys into polypeptides (Nirenberg et al., 1963). The percent incorporation of lys, asN, glN, and his agreed well with the triplet code word frequencies, but not with doublet frequencies, whereas the percent incorporation of both thr and pro could be explained by either one doublet TABIX 1 Summary of Amino Acid Incorporations (in Cell-Free Systems from E. coli) Directed by Ribopolynucleotides with Repeating Nucleotide Sequences and Codon Assignments (inParentheses)* Ribopolynucleotide

Amino acids incorporated and codon assignment

Ribopolynucleotides with Repeating Trinucleotide Sequencest Poly UAC tyr (UAC), thr (ACU), leu (CUA) Poly GUA Val (GUA), ser (AGU), 1 Poly AUC ilu (AUC), ser (UCA), his (CAU) Poly GAU asp (GAU), met (AUG), 1 Poly UUG leu (UUG), cys (UGU), Val (GUU) Poly CAA glN (CAA), thr (ACA), asN (AAC) Poly wc phe (UUC), ser (UCU), leu (CUU) Poly AAG lys (AAG), glu (GAA), arg (AGA) Ribopolynucleotides with Repeating DinucIeotide Sequences0 Poly uc ser (UCU), leu (CUC) Poly AG arg (AGA), glu (GAG) Poly UG val (GUG), CJW (UGU) Poly AC thr (ACA), his (CAC) Ribopolynucleotides with Repeating Tetranucleotide Sequences11 Poly UAUC leu (CUA), ser (UCU), ilu (AUC), tyr (UAU) Poly UUAC thr (ACU), tyr (UAC), leu (UUA), leu (CUU) Poly GUAA # Poly GAUA # *Adapted and modified from Morgan et a2. (1966). t Morgan et al. (1966) and Nishimura d al. (196610). 1Although polymers with repeating trinucleotide sequences would be expected to direct the incorporation of three amino acids, only two were incorporated in these cmea. This result is consistent with the conclusion that codons UAG in poly GUA and UGA in poly GAU are nonsense. Nishimura et al. (1966a) and Jones et al. (1966). 11 Kbsel et al. (1967). The sequence of four amino acids in repeating tetrapeptide sequences of the polypeptide products was the same in which they are listed here. #No acid-insoluble polypeptides were made by theae template. Thia result is consistent with the conclusion that codons UAA in poly GUAA and UAG in poly GAUA, occurring at every fourth place in the polymers, are nonsense codons.

333

THE GENETIC CODE

(AC-thr; CC-pro) or two triplets (ACA, ACC-thr; CCC, CCA-pro) . By using poly UC, it was shown that phe, ser, pro, and leu were also coded for either by two triplets or by one doublet. It could be concluded from these studies that the genetic code could not consist of only doublets, although a mixed doublet-triplet code was not excluded. A different approach t o the investigation of the amino acid-code word TABLE 2 Present Codon Assignments*t 2nd letter 1st letter

U

C

A

G

U Phe Phe

c

~~

~~

~~

A

G

3rd letter

U

Leu$

Ser Ser Ser# SerS

TYr TYr Ochre$§ Amber f i

CYd CYS Nonsense#§ Try $

Leu$ Leu Leu Leu

Pro Pro Pro$ Pro

Hisf Hisf GINS GINS

Arg Arg -4% Arg

U

Ilu Ilu Ilu$ Met$

Thr Thr Thr$ Thr#

AsNf

U

Lysf Lys$

ser $ Ser ArO# Arg

Val

Ala Ale Ala$ Ala

ASP Glu$ Glu 1

Lac$

Val$

Val$ Val

AsN

ASP

* Adapted and modified from Morgan ef al. (1966).

GIY G 1y GlY t GlY

c

A G

c A

G C

-4

G

U

c

A G

The assignments in regular type are on the basis of binding experiments only. Boldface assignments are from binding data and have been confirmed by cell-free polypeptide synthesis using completely defined polymers. Italicized assignments are deduced from incorporation experiments with defined polymem but gave essentially no binding. "'hem results are based on studies with bacterial cell-free systems conducted independently by three groups of workers (see Section 111, B). $ These assignments are consistent with the blocks of changed amino acid sequencea in frameshift mutations of phage lysozyme (see Section III,A), amino acid replacement data (see Section HI$), or substitutions of amino acids in revertants of the umber and ochre mutants (see Section V,C and E). The sequences of bases in the first two positiom of many of the remaining assignments are also consistent with the amino acid replacement data (see Fig. 2b, Tables 3 and 4), but these cases, in which the third position cannot be deduced uniquely because of degeneracy, have not been included in this table. 4 Deduced by genetic arguments (see Section V,C and E).

334

ANIL SADGOPAL

relationship was developed by Nirenberg and Leder (1964). I n this method, ribotrinucleotidesS of defined base sequences were used to stimulate the binding of specific aminoacyl-tRNA’s to ribosomes in vitro. Nirenberg and co-workers (see Trupin et al., 1965; Brimacornbe et al., 1965) and Khorana and co-workers (see Soll et al., 1965) studied the binding properties of all 64 trinucleotide templates in cell-free systems from E . coli and were able to allocate a large number of codons to their respective amino acids. The data obtained by the two groups independently have shown good agreement and their results are summarized in Table 2. The results of binding studies, however, are not always interpretable in an unambiguous way and i t is appropriate here to discuss the difficulties encountered by workers in this area. While many trinucleotides caused relatively specific stimulation of the binding of only one aminoacyl-tRNA to ribosomes, allowing codon assignments to be made with considerable certainty, other trinucleotides induced the binding of more than one aminoacyl-tRNA. In the latter cases, some of these trinucleotides stimulated the binding of one aminoacyl-tRNA t o a greater extent than that of others and could still be assigned (Soll et al., 1965). I n addition, many cases of ambiguity arose due to the in vitro conditions (e.g., temperature and Mg++ concentration) used in the assays. Often ambiguity could not be resolved by altering the conditions. For example, GGA stimulated the binding of both gly- and glu-tRNA’s, the stimulation in the latter case being comparable to that caused by GAG, a codon already assigned to glu (Soll et al., 1965). Although the assignment of GGA to gly was consistent with the amino acid replacement data of Yanofsky (1965) for the tryptophan synthetase A protein of E . coli (see Fig. 2a and Section III,C),the ambiguity shown by the GGA binding of glutRNA could not be decreased by changing temperature and Mg++ concentration. The above complications were further augmented by the lack or weakness of stimulation of binding by about 20% of all the triplets (Brimacombe et al., 1965; So11 et al., 1965). The lack of binding by two of these, UAA and UAG, is consistent with the conclusion from genetic studies that they are nonsense codons (see Section V,C). The inactivity of other nonbinding codons is, however, hard to explain, since many of these have been assigned to various amino acids on the ‘The ribotrinucleotides used were of two general classes: pXpYpZ and XpYpZ. However, most of the later work utilired trinucleotides of the latter type only. For brevity, such trinucleotide diphosphates will be referred to as trinucleotides only and are represented by the XYZ notation.

T H E GENETIC CODE

335

basis of amino acid replacement data and amino acid incorporation directed by polymers of random or defined sequences. For example, the trinucleotides containing only U and C failed to stimulate the binding of E . coli leu-tRNA to ribosomes (Brimacombe et al., 1965; Sol1 et al., 1965) whereas random poly UC stimulated such binding (Bernfield and Nirenberg, 1965) as well as leu incorporation into proteins (Nirenberg et al., 1963; Speyer et d.,1963). A possible explanation for this type of discrepancy could involve a tRNA forming an unstable tRNA-ribosome-codon complex (Brimacombe et al., 1965). Each triplet may occur in three structural forms in RNA, as 5'terminal, 3'-terminal, and internal codon, with the trinucleotides of the XpYpZ class representing a fourth form. Each of these forms may differ in its template activity in binding assays (Nirenberg and Leder, 1964; Brimacornbe et al., 1965; Rottman and Nirenberg, 1966). Indeed, it is conceivable that the mechanism of trinucleotide-induced binding of aminoacyl-tRNA to ribosomes is only partially analogous to the binding stimulated by internal triplets in RNA-like long polynucleotides during protein synthesis (Brimacombe et aZ., 1965; Rottman and Nirenberg, 1966). Thus the codon assignments made on the basis of binding data can be fully accepted only in the presence of supplementary evidence obtained by other approaches. The codon assignments shown in Table 2 were further verified, and sometimes unambiguously made, by the use of a third approach in which ribopolynucleotides containing completely defined nucleotide sequences were used to direct amino a u d incorporation in cell-free systems (Khorana, 1965). Alternating copolymers such as poly UC, poly UG, poly AC, and poly AG were found t o direct the synthesis of copolypeptides of ser and Ieu, Val and cys, thr and his, and arg and glu, respectively (Nishimura et al., 1965a; Jones et al., 1966). Subsequently a variety of ribopolynucleotides containing repeating tri- and tetranucleotide sequences were prepared by a combination of chemical and enzymic methods by Khorana and co-workers (Nishimura et al., 196513; Morgan et al., 1966; Kossel et al., 1967). One such polymer, poly AAGP directed the synthesis of three homopolypeptides, polylys, polyglu, and polyarg (Nishimura et al., 1965b). This undoubtedly occurred because only a single triplet sequence (AAG, GAA, or AGA) could be translated depending on the position a t which the reading of the message started. The point of chain initiation in this polynucleotide was not fixed, presumably owing to lack of an initiating codon. It is

'The terminal nucleotides

of these repeating polymers are not known. Therefore,

poly AAG can equally well be written as poly AGA or poly GAA.

336

ANIL SADGOPAL

interesting to note that poly AAG-directed synthesis of homopolypeptides provides independent evidence in favor of a nonoverlapping triplet code (Nishimura et al., 196513.) Ribopolynucleotides containing repeating tetranucleotide sequences were demonstrated to direct the synthesis of polypeptides containing repeating tetrapeptide sequences, and identification of certain specific repeating tetrapeptide sequences in the polypeptide products proved the direction of translation of mRNA to be 5’ to 3’ (Kossel et al., 1967). The results of such studies are summarized in Table 1. These methods opened up a reliable approach for deduction and confirmation of codon assignments. For example, AGA can be assigned to arg on the basis of arg incorporation by repeating poly AG and poly AAG, although it gave no stimulation of binding of arg-tRNA to ribosomes. One major complication encountered in the studies with polymers of repeating trinucleotide sequences is the great variation in the extent of incorporation of the three amino acids into homopolypeptides by the same ribopolynucleotide (Morgan et al., 1966). Two possible reasons for this discrepancy have been considered by Morgan e t al. (1966). The use of oligonucleotides of defined sequences has shown that the first triplet from the 5’-end of synthetic oligonucleotides is usually not translated and reading begins frequently from the second triplet (Salas et al., 1965; Stanley et ul., 1966). Therefore, the synthesis of three homopolypeptides under the direction of repeating trinucleotide polymers requires polynucleotide chains which possess the three possible codons as the second triplet from the 5’-end. The variations in the incorporation of three amino acids could then be partially due to the presence of different proportions of polymers containing the three different codons a t this position (see footnote 4). A second cause of this variation may be differences in the concentrations of tRNA’s specific for the three codons of each polymer. Recently, Morgan et al. (1967) have demonstrated the synthesis of polypeptides by direct translation of single-stranded DNA-like polymers with repeating nucleotide sequences in the presence of neomycin B; in addition, specific binding of appropriate tRNA’s by such deoxyribopolynucleotides is also described. The amino acids incorporated and the tRNA’s bound were found to be the same as expected from the RNA code. Although the biological significance of these findings is not clear, the above authors discuss several practical uses of deoxypolymers in the study of the genetic code. The use of certain “block” oligonucleotides of specified base sequence in cell-free protein-synthesizing systems has provided independent sup-

THE GENETIC CODE

337

port for several codons assigned by the methods described above (Stanley e t al., 1966). I n particular, these studies gave evidence for AUA as a codon for ilu, since the oligonucleotide AAAAU(A),, directed the incorporation of lys and ilu only (the other triplets in this oligonucleotide, AAA, AAU, and UAA, represent lys, asN, and nonsense codons, respectively). The assignment of AUA on the basis of binding studies with the E . coli system had been inconclusive (Brimacombe et al., 1965; Sol1 et al., 1965). It may be noted that AUA has recently been shown to stimulate the binding to E. coli ribosomes of ilu-tRNA isolated from the livers of guinea pig and Xenopus Zaevis (Marshall et al., 1967).

C. AMINOACID REPLACEMENT STUDIES

It has been proposed that mutational changes involving a single amino acid residue in proteins of related species or in mutant proteins originate predominantly from single-base changes in the DNA (Wittmann, 1961 ; Tsugita and Fraenkel-Conrat, 1960; E. L. Smith, 1962; Jukes, 1962). The base change leads to alteration in the codon in the mRNA, which results in the substitution of a different amino acid in the polypeptide chain. More complicated explanations may be needed, however, for comparison of polypeptide chains distantly related in the evolutionary scale, the a- and P-chains of hemoglobin, for example. Even single amino acid replacements among the mutant hemoglobins may sometimes be explained by the occurrence of genetic hot spots (Benzer, 1961). Changes induced by some chemical mutagens, however, particularly when the mutational mechanism is known, can be explained as a result of single-base alterations with greater confidence. A large number of mutations resulting in the replacement of one amino acid by another have been described in various proteins, such as hemoglobin, TMV coat protein, alkaline phosphatase and tryptophan synthetase A of E. coli, and a few others (Tsugita, 1962; Wittmann and Wittmann-Liebold, 1963; Baglioni, 1963; Yanofsky, 1963; Beale and Lehmann, 1965; Jukes, 1965). The correlation between the amino acid substitutions in various abnormal hemoglobins and the alterations in their codons (assigned from in vitro studies on bacterial systems), shown in Table 3, presents a convincing case for the occurrence of these codon assignments in mammals. Confidence in these assignments is increased by consideration of the amino acid alteration which was reported for the a16 position of hemoglobin I as lys + asp (Murayama, 1962). This could not be explained on the basis of single-base alteration in the codons, but subsequent reinvestigation showed i t to actually possess a lys + glu substitution at the a16 position, easily explainable

w w

00

TABLE 3 Correlation between Amino Acid Replacements*in the Variants of Hemoglobin and Codon Assignmentst Abnormal hemoglobin

Hikari I

Abnormal chain

Position

Amino acid substitution

61

lys + asN

16

D Ibadan

87

G Philadelphia

68

0 Indonesia

G Chinese San Jose

G Gdveston

S

116 30

lye +glu thr

asN +lys glu

+ lys

-

glu -glN

7

glu

43

gIu

6

+ lys

Codon alteration AAG AAG ACA ACG AAC

-+

AAC

GAG + AAA -+ AAG} -+

Shimonoseki

54

glN -+ arg

-+

Transversion

GAG + AAG

GAG -+CAG

Transversion Transition Transversion Transversion

glu -Val glN

Transition

t

AAG

+ ala

54

'Jkansvemion

3

gly

Mexico

Mutational mechanism

Transversion

glu CAG +CGG

Transition

$

K Ibadan

P

Norfolk

ff

Seattle

M Milwaukee

46 or 56

glY

-

glu

57

glY -'asp

(3

70 or 76

ala --f glu

P

67

val

-+

glu

L Ferrara

(Y

47

asp

G Acrra

B

79

asp +asN

+

GGG

--f

GAG

GGC + GAC GCG -+ GAG GUG

-+

GAG

Trami ti on Transition Transversion Transversion Transition

dY

-+

GAC AAC GAU CAU -P UAU\ CAC +UAC(

Transition

Ivt Boston

Q!

58

Kenwood

B

143

Zurich

P

63

his

+

tyr

his -+ asp his

-+

arg

CAU CAC UUU -+ UUC

-+

--f

Horse Hemoglobin

ff

24

phe

-+

tyr

-+

* Amino acid replacement data from Beale and Lehrnann

t From Table 2.

CGU\ CGCj UAU\ UAC)

I3

Transition

H

Transversion

2

--f

Transition Transversion

0

m

d 0

8

(19851, except for horse hemoglobin, from Kilmartin and Clegg (1967).

w w

CD

340

ANIL SADGOPAL

as a consequence of single-base change (Beale and Lehmann, 1965). Furthermore, the amino acid replacements shown in Table 3 present in vivo evidence supporting degeneracy in the code. For example, gly + glu substitution suggests that the codon for gly a t this position can be either GGA or GGG, while a gly + asp substitution would lead us to assign either GGU or GGC for gly. Many of the earlier efforts to study the correlation between codon assignments and amino acid replacements in the nitrous acid-induced mutants of TMV coat protein were made before the final picture of codon assignments emerged (Tsugita, 1962; Wittmann and WittmannLiebold, 1963). Therefore, a fresh examination of these replacements now seems appropriate (see Table 4). We find that 16 out of 21 different kinds of amino acid replacements (or 56 of the 65 observed mutaTABLE 4 Correlation between Amino Acid Replacements0 in the Nitrous Acid Mutants of TMV Coat Protein and Codon Assignments from Table 2 Times observed Amino acid replacements

Tubingen

Berkeley

Codon alterationb

A G ~ } GG;}

glY

4

arg + lys

1

1

-

*

-+

asp -+ ala

4

dY

2

WP

+

glN +arg

1

-+

CA.)

-+

*

asp

1

-

glY

1

1

Val

2

-

met

1

-

+ vaI

3

2

AUE] A

leu +phe

1

-

CUE}

pro leu pro +ser

4 4

2 -

cc. cc.

glu glu glu ilu ilu

-.+

.+ -+

-+

-+

G G '}:

* ~

AUA -+AUG +G {/

4

-+ --f

UUZ}

cu. uc.

(Continued)

341

THE GENETIC CODE

TABLE 4 (Continued) ser + leu

3

ser

phe

4

3

thr +&la

-

2

thr +ilu

9

-

thr +met

3

-

Val ~~

--+

-+

met ~~

-

AC. + GC. Ul U A") + AUZ] A ACG + A U G

1 ~

~

~

~ D a t afrom the laboratories a t Tubingen, Germany (Wittmann and WittmannLiebold, 1963 ,1966)and Berkeley, Calif., U.S.A. (Tsugita, 1962;Funatsu and FraenkelConrat, 1964). b The bases involved in mutation are indicated by boldface letters. A dot at the third place of triplets indicates that this place in the synonym codons can be occupied by any one of the four bases. *$ These amino acid exchangea are not explained by A G and C + U transitions, but can be explained by single-base changes involving either transversions of various types (*) or transitions of the type G -+A (I). --+

tional instances) can be explained on the basis of single-base changes induced by the predicted mechanism of the nitrous acid mutagen (A + G; C + U) ; those remaining are not explainable this way, although they too involve only single-base changes. It may be noted that three of the amino acid replacements can be explained only by singlebase changes involving transversions while two would require transitions of the type G + A (see Table 4 and its footnotes). Some of these inconsistent amino acid replacements may have resulted from spontaneous mutation, but such a high frequency of spontaneous mutation seems unlikely. A more probable explanation is that nitrous acid may not always act as predicted on the basis of its action on isolated TMV RNA and intact viruses. Indeed, Tessman et al. (1964) reported that nitrous acid mutates all four bases to varying degrees in single-stranded DNAcontaining phages, and established a G + A mutation. It has been reported that guanine is also converted into some unidentified sideproducts in addition to its more favored conversion to xanthine when TMV RNA is treated with nitrous acid after isolation (Schuster and Wilhelm, 1963). The effect of these unidentified products of guanine as well as of xanthine on the coding properties of the triplets is not yet understood. Since a large number of the mutants reported were ob-

342

ANIL SADGOPAL

tained by nitrous acid treatment of isolated TMV RNA (Wittmann and Wittmann-Liebold, 1963; Tsugita and Fraenkel-Conrat, 1962), it is conceivable that the transformed products of guanine might cause some of the unexpected base changes in the coding triplets. Furthermore, nitrous acid is known to produce cross linkages (Geiduschek, 1961) and large deletions (Tessman, 1962) in DNA. If similar changes occur in TMV RNA, alterations other than A + G and C + U type transitions might well be expected. Despite these possibilities, the high degree of correlation observed between the amino acid replacements in TMV mutants and codon alterations on the basis of the predicted mechanism of mutation by nitrous acid is worth noting. Amino acid replacement data from nitrous acid-induced mutants of TMV coat protein also demonstrate degeneracy in vivo (see Table 4). An interesting exchange observed in these studies is ilu +met, which c a n be explained on the basis of the predicted mechanism of nitrous acid mutagenicity only if AUA is assigned t o ilu (and not to met, as would be expected from the general pattern of degeneracy discussed in Section 111,D). Since the assignment of AUA to ilu is based only on a limited amount of in vitro data (cf. Section III,B), this particular amino acid exchange assumes added significance. The correlation between codon assignments and single amino acid changes is well illustrated by the findings of Yanofsky (1965) and Guest and Yanofsky (1966) of several amino acid changes a t a single site in tryptophan synthetase A of E. coli. The serial changes of amino acids and code words are shown in Figs. 2a and 2b for the two sites reported. All of the amino acid exchanges observed in mutant and revertant bacteria a t each of the two sites can be explained by singlebase changes in the coding triplets, confirming the code word assignments of Table 2. I n addition, these studies provide an in vivo proof of degeneracy in the same gene by showing that gly is coded at one site by GGA and at the other by GGU or GGC. Once again, these studies give additional evidence in favor of the assignment of AUA to ilu, since the arg+ ilu change (Fig. 2s) can be explained only if AUA is assigned to ilu and not to met. The excellent agreement between the code word assignments and amino acid replacement data can be further emphasized in the following manner (see Table 5 ) . Of the 170 single amino acid replacements predicted, possible as a result of single-base changes in the codons of Table 2 (including those between amino acids and gaps due to nonsense codons), 70 have already been reported to occur. In contrast, only one exchange reported, a spontaneous mutation in TMV coat protein leading

OlY GGA

OlY GGU (GGC)

-

A :;B

ilu AUA

2-aminopurine ser AGU

1

arg AGA

gly GGA

thr ACA

/\

glu

i?

ala GCA

GGA 9lY GGA

Gr glu GAA

asp GAU (GAC)

favored by 2-aminopurine ala GCU (GCC)

glY GGU (GGC)

9lY GGU (GGC)

FIG.2. (a) Correlation between codon assignments from Table 2 and serial single amino acid replacements (data from Yanofsky, 1965) in the pedtide CP2 of tryptophan synthetase A from E . coli. (b) Correlation between codon assignments from Table 2 and serial single-amino acid replacements (data from Guest and Yanofsky, 1966) in the peptidr TP6 of tryptophan synthetase A from E . coli. Codons in parentheses indicate an alternate series which also fits the data.

344

ANIL SADGOPAL

TABLE 5 Amino Acid Replacements* aa the Result of Smgle-Base Changes in Codons from Table 2

~~

Amino acid

Possible exchangest

try tYr Val

asp g b gZy pro ser thr val cys glN gly his ilu leu lys met nonl pro ser thr try asp his ilu lys ser thr tyr ala asN glu gly his tyr V a l arg gly non phe ser try tyr arg glu his leu lys non pro ala asp glN gly lys non val ala arg asp cys glu non ser try val arg asN asp glN leu pro tyr arg aeN leu lys met phe ser thr Val arg glN his ilu met non phe pro ser try val arg asN glN glu ilu met non thr arg ilu leu lys thr Val arg cys glN glu gly leu lys ser try tyr cys ilu Zeu ser tyr V a l ala arg glN his leu ser thr ala arg asN cys gly ilu leu uon phe pro thr try tyr ala arg asN ilu lys met pro ser arg cye gly leu non ser asN asp cys his non phe ser ala asp glu g l y ilu leu met phe

~

* From Tsugita

~

(1962), Wittmann and Wittmann-Liebold (1963, 1966), Yanofsky (1965), Beale and Lehmann (1965), Jukes (1965), Guest and Yanofsky (1966), Weigert and Garen (1965b), Kilmartin and Clegq (1967), and Funatsu and Fraenkel-Conrat (1964). t Italicized amino acids have been observed as replacements in mutations. 1G a p m R result of nonsense codons.

to asN + arg substitution (Wittmann and Wittmann-Liebold, 1963), cannot be explained in this manner.

D. THEPATTERN OF DEGENERACY Well before the amino acid-code word relationships had been completely worked out, several investigators speculated whether a pattern of degeneracy existed among the limited number of known codon assignments. Eck (1963) arranged 64 codons in 32 pairs in which both members of a pair were identical except for a purine in one being replaced by a different purine in the other, or a pyrimidine by a pyrimidine. Each pair, according to this suggestion, codes for one amino acid, U being equivalent to C and A to G a t an unspecified position in a

THE GENETIC CODE

345

triplet. Woese (1962, 1965a) postulated that the genetic code has specific degeneracy confined to a particular position in the codon. The above ideas have been proven to be generally correct (see Table 2). It is now established that the synonym codons (codons that code for the same amino acid) share a common sequence of bases a t the first two positions in the triplet, with the exception of the codons for leu and arg, whose first bases may also vary, and for ser, all of whose bases may vary. It is possible to divide all the amino acids into three major groups on the basis of the pattern of degeneracy at the third position of their codons. This position may be occupied by either (a) U or C,5 (b) A or G, or (c) U, C, A, or G. I n the special case of ilu, the third position may be occupied by either U, C, or A, while for met and try, it is occupied by only one base, G. This pattern of degeneracy a t the third position has interesting implications for the problem of codon recognition in the light of Crick’s wobble hypothesis, which will be discussed in Section VI1,D. IV. Chain Initiation and Initiator Codons

Protein synthesis can be initiated randomly a t different points along artificial messengers (Sundararajan and Thach, 1966; Stanley e t al., 1966). However, in the case of translation of natural messengers, random initiation would lead to synthesis of not only incomplete protein fragments but, often, also of proteins translated out-of-phase. This suggests the existence of a specific chain initiation site a t the 5’-end of each natural messenger. The existence of such sites in wivo has also been implied by electron micrographic studies on polysomes (Rich et al., 1963) as well as by work on sequential or simultaneous translation of various cistrons in polycistronic messengers (e.g., see Ames and Hartman, 1963; Alpers and Tomkins, 1966; Berberich et al., 1966, 1967). I n particular, the studies on the effect of polar mutations on the translation of polycistronic messengers suggested the presence of initiation sites at the beginning of each cistron (Newton et al., 1965; Whitfield e t al., 1966; Martin et al., 1966; Yanofsky and Ito, 1966; see Section IV,D) . ‘Such equivalence of bases at the third position of codons is usually represented &S

G

346

ANIL SADGOPAL

An early clue regarding the nature of initiation sites came from the finding that the amino terminal position of most of the soluble proteins in E . coli is occupied by either met, ala, ser, or thr (Waller, 1963). Two groups of investigators have now shown that in a cell-free system from E. coli directed by either phage fz or R17 RNA, the amino terminal amino acid of newly synthesized phage coat protein is Nformylmet (J. M. Adams and Capecchi, 1966; Webster et al., 1966). However, the phage coat protein made in uiuo has ala a t its amino terminal end, followed by -ser-asN-phe-. J. M. Adams and Capecchi (1966) demonstrated that coat protein synthesized in vitro also contained the amino terminal sequence N-formylmet-ala. Both groups of researchers conclude that the phage coat protein is made with the amino terminal fragment N-formylmet-ala-ser-asN-phe-, the N-formylmet probably being split off enzymically in vivo. Many E. coli proteins may be made in this way. Sometimes, only the formyl group will be removed from the nascent proteins, leaving met a t the amino terminal end; an enzyme having this function has been reported in E . coli extracts (Gussin et al., 1966). Evidence suggesting that all E . coli proteins are initiated with N-formylmet is available (Gussin et al., 1966). A. N-FORMYLMET-tRNA

AND

ITSCODONS

The role of N-formylmet in protein biosynthesis is emphasized by the demonstration of the formylation of methionyl-tRNA t o N-formylmethionyl-tRNA in extracts of E. coli (Marcker and Sanger, 1964). A cell-free system can be established a t a low Mg++ concentration (0.007-0.009 M ) in which amino acid incorporation directed by either random poly AGU or RNA from phages or a plant virus is dependent upon the addition of N-formylmethionyl-tRNA or N6-formyltetrahydrofolate (Nakamoto and Kolakofsky, 1966; Kolakofsky and Nakamoto, 1966). A specific requirement for N-formylmet and its tRNA in protein synthesis is also shown by the use of extracts of E. coli treated with trimethoprim (an inhibitor of dihydrofolate reductase) so that the extracts lack a source of formyl groups (Eisenstadt and Lengyel, 1966). At a low M g f + concentration, the amino acid incorporation in these extracts under the direction of phage fa RNA has been shown to depend on the addition of N-formylmethionyl-tRNA or N6-formyltetrahydrofolate. The specific nature of the participation by N-formylmet in the synthesis of proteins was clarified by investigations on the properties of N-formylmethionyl-tRNA. Only a part of the total methionyl-tRNA can be formylated in the cell-free system, suggesting that different

THE QENETIC CODE

347

tRNA molecules are used for the formation of methionyl- and N-formylmethionyl-tRNA’s (Marcker, 1965). In confirmation of this, the mettRNA has been fractionated into two species, one of which, mettRNAF, can be formylated, while the other, met-tRNAM, cannot be (Clark and Marcker, 1966a; Kellogg et al., 1966). A specific difference in the coding properties of these two species is that in cell-free amino acid-incorporating systems, met-tRNAM responded only to the random heteropolynucleotide, poly UAG, but met-tRNAF responded to both random poly UG and poly UAG (Clark and Marcker, 1965, 1966a). These results are in agreement with the data obtained from the tRNAribosome binding assays, which showed that the binding of met-tRNAM was stimulated by the trinucleotide AUG alone while that of mettRNAF was stimulated by AUG, UUG, and GUG and to a lesser degree by CUG (Clark and Marcker, 1966a; Kellogg et al., 1966; Ghosh et al., 1967). This shows a lack of specificity for recognition of the first nucleotide a t the 5’-end of the codon by met-tRNAF. The response of mettRNAF to UUG and GUG, codons assigned to leu and Val, respectively, presents a situation of apparent ambiguity. It is likely that UUG and GUG are recognized by leu- and val-tRNA’s, respectively, whenever they occur in an internal position in the mRNA but are recognized by met-tRNAF a t the 5’-terminal position. This would be possible in view of the special structural and functional characteristics of met-tRNAp as discussed in Section IV,C. Ghosh et at?. (1967) have recently examined the initiating properties of the above-mentioned codons in cell-free protein-synthesizing systems directed by polynucleotides with repeating di- or trinucleotide sequences. They demonstrated that at low Mg++ concentrations poly UGdirected synthesis of the val-cys copolypeptide was dependent on the addition of N-formylmethionyl-tRNAF and the amino terminal sequence of the polypeptide product was formylmet-cys-Val. The above finding led the authors t o conclude that GUG acts both as an initiator and a Val codon at the 5’-end and internal positions, respectively. Similarly, a t low Mg+ + concentrations, poly AUG-directed synthesis of polymet and poly GUA-directed incorporation of Val were dependent on added N-formylmethionyl-tRNAF, while poly UUG failed to direct any polypeptide synthesis and formylmet was unable to restore amino acid incorporation. These results failed to demonstrate a capacity for UUG to initiate protein synthesis in spite of its demonstrated stimulation of the binding of met-tRNAF to ribosomes. I n contrast, these results established GUA as an initiator (although a weak one) in addition to AUG and GUG and indicated the possibility of a degeneracy among the initiator codons a t the first as well as the third position.

348

ANIL SADGOPAL

B. INVOLVEMENT OF AUG IN INITIATING AND PHASING THE READING The results described in the preceding paragraphs suggest that mettRNAF is directly involved in the mechanism of initiation of polypeptide chains and that the codons AUG, GUG, GUA, and possibly UUG a t or near the 5’-terminus act as chain initiators in bacteria by directing the incorporation of N-formylmet a t the amino terminal positions of proteins. This proposal is consistent with the results of Stanley et al. (1966) , who assigned a role in the initiation of protein synthesis to two partially purified factors (F1 and Fz) which are usually removed from ribosomes during their purification, It was reported that natural messengers such as MS2, &a, or TMV RNA’s (each of which presumably possesses a chain-initiating codon) were quite inefficient in promoting protein synthesis on the purified ribosomes but showed a several-fold stimulation of amino acid incorporation upon the addition of factors F1 and Fa. However, poly A, which lacks a chain-initiating codon, does not respond to these factors. By using synthetic oligonucleotides of specified base sequences, the effect of these factors was correlated with the presence of the AUG codon at or near the 5’-terminus, whereas the reading of the oligonucleotide of the type A,AUG was not affected by the absence or presence of initiation factors (Salas et al., 1967a). It was further demonstrated that the transfer of met from met-tRNAF into peptide linkage took place in the presence of either AUGA, or ArUGA, messenger; in contrast, A A U G transferred met from met-tRNAM, but not from met-tRNAF (Salas e t al., 1967a). The initiation factors were found to stimulate the transfer of met from mettRNAF, but not from met-tRNAM in the above experiments (Stanley et al., 1966; Salas et al., 1967a). These factors also accelerate the binding of met-tRNAF t o purified ribosomes in the presence of AUG, but are without effect on the binding of gly- or lys-tRNA’s in the presence of GGU or AAA, respectively (Salas et al., 1967a). Thach et al. (1966) have shown that synthetic oligonucleotides containing the sequence AUG a t or near the 5’-end stimulate incorporation of N-formylmet but not of free met, and that AUG is more active as a chain-initiating codon than UUG. The role of AUG in the initiation of polypeptide synthesis is also supported by the observation that the translation of polymers is greatly accelerated by AUG and other initiator codons (such as GUG) in the first or second position (Stanley et al., 1966; Ghosh et al., 1967). B y using oligonucleotides of known sequences, containing specific triplets a t their 5’-ends, it has been shown that with the exception of AUG, the initial triplet is infrequently translated (Stanley et al., 1966).

THE GENETIC CODE

349

The importance of AUG in phasing the reading of mRNA as well as in initiating it has been brought out by Sundararajan and Thach (1966), who demonstrated that when AUG is incorporated into longer polynucleotide chains, it suppresses the reading of codons which partially overlap its sequence and promotes the reading of the adjacent 3’codon. For example, AUG(U);; stimulated the binding of met- and phe-tRNA’s but not of Val-tRNA to ribosomes, whereas AUGG (U), stimulated the binding of met- and Val-tRNA’s, but reduced the binding of phe-tRNA to a low level. Similarly, AAAUG stimulated binding of met-tRNA but not of lys-tRNA, as compared to AAACG, which coded for lys- but not for thr-tRNA. The triplets ACG, UAG, and AAG did not show this phasing activity.

C. THEROLEOF FORMYLATION AND THE MECHANISM OF INITIATION An interesting feature of met-tRNAF is the absence of a significant difference between the binding of formylated and unformylated methionyl-tRNAF to ribosomes under the direction of AUG, UUG, and GUG (Clark and Marcker, 1966a). I n a cell-free system free of formylation activity, both random poly UG and poly UAG directed the incorporation of most of the met attached to met-tRNAF into the amino terminal positions, while met attached to met-tRNAM was transferred by poly UAG to the internal positions of the newly synthesized protein chains (Clark and Marcker, 1966a). This result agrees with the finding that methionyl-tRNAF, formylated or unformylated, directed the incorporation of met into the amino terminal position of protein synthesized in the presence of mu 2 phage RNA in a nonformylating cell-free system (Clark and Marcker, 1966b). On the other hand, met attached to met-tRNA, was transferred to internal positions. Similar conclusions were reached by Leder and Bursztyn (1966), who showed that met-tRNAF, prior to formylation, recognized the AUG codon in phase and that formylation actually reduced the efficiency of this process, possibly by changing the secondary structure of the tRNA. They also demonstrated that formylation of met-tRNA could occur following its binding to the messenger-ribosome complex. However, when N formylmethionyl-tRNA or means of formylation is added to a cell-free system lacking transformylase activity, the rate of protein synthesis is stimulated under the direction of either synthetic messengers (e.g., random poly UG and poly UAG) or natural messengers, such as mu 2 phage RNA (Clark and Marcker, 1965, 1966a,b). The formyl group probably affects the efficiency of formation of the initiaI peptide bond, which may be the rate-limiting step in the initiation of protein synthesis.

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It has been proposed that N-formylmethionyl-tRNAr enters the pep-

tidyl-tRNA site on the ribosome directly because of the similarity to a peptidyl-tRNA conferred on it by formylation (Nakamoto and Kolakofsky, 1966; Nijll, 1966). However, we have seen the lack of effect of formylation on the coding characteristics of methionyl-tRNAF, leading u9 to t,he contrary conclusion that the direction of met into the amino terminal position is determined by the nature of met-tRNAF itself (Clark and Marcker, 1966a,b; Leder and Bursztyn, 1966). According to this theory, met-tRNAF, formylated or not, has a unique structure which allows it to select the initiator codon and to fit directly into the peptidyl-tRNA site on the ribosome. This is supported by the experiments of Bretscher and Marcker (1966) involving the use of puromycin, which terminates protein synthesis by substituting for an aminoacyl-tRNA on the “amino acid site” of the ribosome and displacing the polypeptide bound to the “peptide site” as polypeptidylpuromycin. Both formylated and unformylated methionyl-tRNAp bound to ribosomes with the triplet AUG reacted with puromycin to release formylmethionyl- and methionyl-puromycin, respectively. In contrast, methionyl-tRNA= complexed with ribosomes in the presence of AUG was unaffected by puromycin. Similar results have also been reported by Zamir e t al. (1966). We are still left with the dilemma of how a single codon, AUG, can distinguish between met-tRNAF and met-tRNAM..A possible explanation lies in the presence of the two tRNA-binding sites on the ribosomes (Warner and Rich, 1964; Arlinghaus et at., 1964) which may have different specificities of attachment for peptidyl- and aminoacyl-tRNA’s. Thus if AUG appears at the peptidyl-tRNA site, mettRNA, carrying N-formylmet would bind to the ribosome but AUG a t the aminoacyl-tRNA site would bind met-tRNAM carrying met. Once AUG has established the reading frame by phasing the message at the peptidyl-tRNA site, sequential reading of the message ensures that an internal AUG codon always appears at the aminoacyl-tRNA site and thus codes for met carried by met-tRNAM. The results of Ghosh et d. (1967), demonstrating the requirement for both met-tRNAv and met-tRNAM during polymet synthesis directed by repeating poly AUG, provide support for this idea.

D. INITIATOR CODONS IN POLYCISTRONIC MESSENGERS The above model for the mechanism of chain initiation by mettRNA, in the presence of initiator triplets has a special bearing on the nature of punctuation in the translation of polycistronic messengers.

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This model implies the presence of a chain terminating codon preceding an intercistronic initiator site so that the peptidyl-tRNA site on the advancing ribosome is vacated before encountering an initiator during the sequential translation of a polycistronic messenger (see Section V on nonsense codons). The peptidyl-tRNA site thus vacated could be occupied by met-tRNAF in the presence of an initiator triplet a t the beginning of an internal cistron, allowing the reading to be phased. This concept is supported by the studies of Sarabhai and Brenner (196713)) who generated a mutation in the rII B cistron of T4 which allows reinitiation of protein synthesis after the chain has been terminated by a nonsense mutation preceding it in the same cistron. These authors presented evidence showing that chain termination is obligatory for the functioning of this artifically generated initiator site and that reinitiation is independent of frameshifts inserted between the terminator and the initiator. The mechanism is also consistent with a model which has been proposed to explain the polarity effects of nonsense and frameshift mutations (thought to act by generating nonsense codons) observed in lactose (Newton et al., 1965), tryptophan (Yanofsky and Ito, 1966), and histidine (Whitfield et al., 1966) operons in bacteria. According to this model (Martin et al., 1966), which provides a molecular basis for the phenomenon of polarity in polycistronic messengers, ribosomes become associated only at the beginning of the mRNA. On encountering a nonsense codon, the peptide chain is terminated and the ribosome continues t o move along the messenger with no defined phase. During this phaseless wandering the ribosome has a probability of becoming dissociated from the messenger. Protein synthesis is initiated in the proper phase when the ribosome encounters a chain-initiating triplet. I n the above context, Martin et al. (1966) point out that the degree of polarity due t o dissociation of the ribosome from the mRNA is not entirely a function of the physical distance between the nonsense mutation and the subsequent chain-initiating triplet. Instead, they suggest that it is also dependent on the relative efficiency of the particular initiator signal in appropriately phasing the ribosomes, thus permitting protein synthesis to be reinitiated subsequent to the cistron containing the nonsense codon. One may speculate regarding the biological significance of the different in vitro efficiencies of the various initiator triplets (AUG, GUG, GUA, or UUG) in initiating and phasing translation, as discussed in previous sections. It is conceivable that these triplets are employed as intercistronic initiators according to the above suggestion of Martin

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et al. (1966). Suggestive evidence for the use of these triplets as intercistronic initiators comes from the demonstration that derepression of various cistrons of the histidine operon in Salmonella may proceed by two modes depending upon the availability of one of the metabolites produced as a by-product of the histidine biosynthetic pathway (Berberich et al., 1966). Derepression of the enzymes occurs simultaneously in mutants of the histidine operon incapable of producing this metabolite, but it proceeds sequentially along the positional sequence of genes in the mutants which produce this metabolite. It has now been demonstrated that this metabolite allows only sequential derepression by limiting the availability of formyl groups (Berberich et al., 1967). Accordingly, the translation of the polycistronic histidine message can be initiated a t multiple sites (at the beginning of each cistron) only when formyl groups are not limiting; when the formylation capacity of the cell is limiting, the translation is preferentially initiated at the operator end. This finding suggests that formyl groups are utilized in the chain initiation process (presumably through N-formylmethionyl-tRNA) a t the beginning of each cistron in a polycistronic mRNA. More directly, i t has been shown that N-formylmethionyl-tRNA is required for amino acid incorporation into all proteins programed by the polycistronic fz RNA in cell-free systems (Eisenstadt and Lengyel, 1966). The existence of certain codons coding for N-formylmet a t or near the beginning of various internal cistrons is indicated by the demonstration that each of the polypeptide chains (viral RNA synthetase and coat protein) synthesized in a cell-free E. coli system directed by MS2 polycistronic phage RNA contains formylmet at its amino terminal end (Viiiuela e t al., 1967). Similar evidence exists in the case of proteins directed by R17 phage RNA (Gussin et al., 1966).

E. REQUIREMENT FOR MAGNESIUM IONS IN OF AN INITIATOR CODON

THE

ABSENCE

We shall now consider the in vitro requirement of relatively high Mg+ + concentrations (0.015-0.02 M ) for optimal amino acid incorporation under the direction of synthetic polynucleotides and for trinucleotide-induced binding of tRNA to ribosomes. Revel and Hiatt (1965) have pointed out that the optimum Mg++ concentration required for amino acid incorporation by synthetic messengers, such as poly U, is about twice that for native mRNA. In a system directed by

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poly U, the step requiring the higher Mg++ concentration (0.0150.02 M ) was shown to be the binding of the messenger to ribosomes with subsequent protein synthesis occurring optimally at the lower concentration (0.007-0.01 M).I n contrast, it is known that several viral messengers are capable of carrying on protein synthesis a t the lower Mg+ + concentration. These differences may be understood in light of the observations (Clark and Marcker, 1966a) that the concentration of Mg++ required for the optimum binding of met-tRNAF to ribosomes, under the stimulation of AUG, is lower (0.01 M ) than that required for met-tRNAM (0.02-0.03 M ) . In addition, the phasing activity of AUG is a t a maximum at about 0.009 M Mg++, decreasing sharply at higher concentrations (Sundararajan and Thach, 1966). The oligonucleotide AUGA, directs amino acid incorporation efficiently at lower Mg++ concentration than that required for the translation of A,AUG (Salas et al., 1967a). Using polymers containing repeating dinucleotide sequences, Ghosh et al. (1967) demonstrated that poly UG (containing the initiator codon, GUG) has an optimum Mg++ requirement lower than either poly AC, poly UC, or poly AG for directing the synthesis of copolypeptides in cell-free systems. It has also been shown that the dependence on the initiation factors (discussed in Section IV,B) as well as on the formylation of met-tRNAF of the ribosomal binding of met-tRNAF in the presence of AUG and of the polypeptide synthesis directed by the oligonucleotide AUGA, is pronounced a t low Mg++ concentrations (0.005-0.01 M ) and decreases at higher Mg++ concentrations (Salas et d.,1967a,b). Similar dependence on the initiation factors and N-formylmethionyl-tRNAF a t low Mg+ + concentrations has been demonstrated for the amino acid incorporation by repeating polynucleotides containing initiator codons (poly UG, poly AUG, poly GUA) while polynucleotides lacking initiator codons do not show this dependence (Ghosh et al., 1967). Consistent with this is the observation discussed earlier showing that an amino acid incorporating system directed by a natural messenger can be made dependent on added N-formylmethionyl-tRNA if low concentrations of Mg++ are used. It would seem then that protein synthesis can take place at the lower Mg++ concentration if an initiator signal is present a t the beginning of the message. Higher Mg++ probably modifies the interaction between the messenger and transfer RNA’s on the ribosome such that chain initiation takes place in the absence of a specific initiator. The tendency for t,he reading to begin at the second triplet in systems directed by synthetic oligonucleotides, as observed by Stanley et al. (1966), may be related to this.

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F. ARE INITIATOR CODONS UNIVERSAL?

It is not known a t this time if the AUG triplet acts as an initiator of protein synthesis in organisms other than E. coli, but N-formylmethionyl-tRNA has been found in growing yeast cells (Marcker and Sanger, 1964). The efforts to formylate methionyl-tRNA’s from rat liver and hen oviduct have been unsuccessful to date (Marcker and Sanger, 1964; Clark and Marcker, 1966b). On the other hand, Nformylmethionyl-tRNA has been isolated from rabbit reticulocytes (NO11, 1966), and a specific met-tRNA fraction from guinea pig liver was recently shown to accept formyl groups in the presence of a formylase preparation from E . coli (Caskey et al., 1967). Further, Schwartz et al. (1967) have provided evidence in favor of the involvement of N-formylmethionine during the synthesis of viral coat protein in extract% of Euglena gracilis directed by RNA from phage f p . It is conceivable that amino acids other than formylmet are involved in the initiation of protein synthesis. Horikoshi and Doi (1967) found a very high ala : met (8 : 1) ratio at the amino terminal positions of soluble and ribosomal proteins of Bacillus subtilis. They also demonstrated that in several in vivo and in vitro pulse-labeling experiments, the ratio of newly incorporated ala to met a t amino terminal positions was high (6 : 1) and decreased from 25 : 1 at early times of synthesis to a more constant ratio of 5 : 1. The above findings suggest that ala is involved in the initiation of protein synthesis in Bacillus subtilis. Reichmann et al. (1966) have shown that the initial triplet on the 5’terminus of satellite tobacco necrosis virus RNA is the serine codon AG:] and the in vitru translation of this RNA produces viral coat protein with an amino terminal blocked product that does not contain N-formylmet. Binding studies performed on a yeast system have shown that lys-tRNA as well as met-tRNA binds to ribosomes optimally at lower Mg++ concentration than does tyr-tRNA (Tanner, 1966). In addition, lys-tRNA of yeast recognized AAA, AAG, UAG, GAG, and weakly CAG in a binding pattern similar to that of formylmet-tRNA of both E . coli and yeast. If the degeneracy of the first base in a triplet or the use of ambiguous codons is a common pattern of all initiator triplets, as suggested by NO11 (1966), the binding pattern of lys-tRNA in yeast may be worth noting. NO11 (1966) has compiled a list of the known amino terminal amino acids of a large number of proteins from a variety of organisms. This tabulation shows that ala, asp, glu, gly, ser, Val, ilu, and leu occur with high frequency a t the amino terminal positions while met and thr, found frequently a t these positions in E . coli, are much less frequent

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or completely absent. Whether the amino acids encountered frequently a t the amino terminal positions of proteins of higher organisms play an important role in the initiation mechanism or occupy this position as a result of formylmet being removed from the amino terminal end of the nascent proteins is not known. In mammalian and other proteins, a large number of amino terminal amino acids have been found whose a-amino groups are blocked by acetylation (e.g., N-acetylgly and N-acetylser) , by the presence of aromatic rings (e.g., pyrrolidone), or by other means. In particular, the earlier findings of amino terminal acetyl groups in several proteins prompted the suggestion that the synthesis of proteins is initiated with an N-acetylamino acid or an amino acid with a similarly blocked amino group (Narita, 1963; Pearlman and Bloch, 1963). Repeated attempts to demonstrate the activation of N-acetylamino acids which could esterify tRNA and be subsequently transferred to proteins have given either negative or contradictory results (Pearlman and Bloch, 1963; Karasek, 1963; Narita et al., 1965; Marchis-Mouren and Lipmann, 1965; Haenni and Chapeville, 1966). In contrast, it has been shown that acetylation of human fetal and chicken hemoglobins (Marchis-Mouren and Lipmann, 1965) and of histones (Allfrey et al., 1964) occurs independently of protein synthesis and that these proteins are probably acetylated after their synthesis. The possible involvement of N-acetylamino acids in the initiation of protein synthesis, however, has not been completely eliminated as yet. Indeed, the physiological significance of the recent reports (Haenni and Chapeville, 1966 ; LucasLenard and Lipmann, 1967) demonstrating the participation of chemically synthesized N-acetylphenylalanyl-tRNA in the initiation of polyphe synthesis in poly U-directed bacterial cell-free systems remains unclear. Furthermore, tentative evidence has been presented which suggests that the in vitro translation of satellite tobacco necrosis virus RNA is initiated with N-acetylserine (Reichmann et al., 1966). V. Chain Termination and Nonsense Codons

A. THENEEDFOR

A

MECHANISM OF CHAIN TERMINATION

According to the currently accepted model of protein biosynthesis, the growing polypeptide chain remains attached t o the surface of the ribosome by tRNA. Gilbert (1963) showed that polyphenylalanine synthesized in vitro under the direction of poly U was bound to the 50 S subunit of the ribosome and the growing chain remained linked to a tRNA molecule by a covalent bond similar to that in aminoacyl-

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tRNA. I n contrast, when endogenous native RNA or RNA from phage fa was used as a source of messenger in cell-free E . coli systems, free polypeptides were released to the supernatant (Zinder, 1963; Ganosa and Nakamoto, 1966). The above evidence suggests that natural messengers are endowed with a chain-terminating mechanism to separate the finished polypeptide from tRNA by cleavage of the ester linkage. Although such a mechanism for spontaneous chain termination is apparently lacking in many synthetic polynucleotides used, certain polynucleotides composed of A’s and U’slead to the production of a significant proportion of free polypeptides (Ganoza and Nakamoto, 1966; Bretscher et al., 1965), suggesting that the signal for chain termination may consist of certain specified nucleotide sequences. Increasing the frequency of triplets containing two A and one U on the poly UA messenger led to a corresponding increase in the release of free polypeptides. These observations, along with the evidence for nonsense codons (see next section), suggest that the chain terminating signal is probably a triplet composed of U and A which does not code for any amino acid. B. THENONSENSE CODONS During their investigations of the acridine-induced frameshift mutations in the r I I region of T d , Crick et al. (1961) noticed that the construction of double mutants containing an addition and a deletion mutation did not always restore the activity. The frameshift in such cases was interpreted as having produced nonsense code words which led to an interruption in the reading of the mRNA (Champe and Benser, 1962; Benser and Champe, 1962). This has recently been supported by the identification of these suggested nonsense codons as amber and ochre codons (Brenner et al., 1967). Benzer and Champe (1962) provided an operative definition of the nonsense mutation by the use of a special kind of deletion mutant called r1589, in which the portion joining the A and B cistrons of the r I I region in T4 was missing. Only the A function was lost, but due to the deletion of the cistrondividing component, the B fragment was not translated independently of A. It was shown that certain base changes in the A cistron would block the B function. Such base substitutions were interpreted as having produced a nonsense word. Many of these nonsense mutants in r1589 have subsequently been identified as amber and ochre codons (Brenner et al., 1965; Brenner and Beckwith, 1965). Garen and Siddiqi (1962) also showed the presence of nonsense codons in the alkaline phosphatase gene of E . coli.

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C. Amber

AND

357

Ochre MUTANTSAND THEIRSUPPBESSOBS

Further understanding of the role of nonsense codons in protein synthesis is derived from the study of a special set of mutants called ambers. An amber mutation does not allow the gene in which it has occurred t o be expressed in a particular host cell, called the nonpermissive or restrictive host, but allows it to be expressed in a permissive or suppressor host. Amber-type mutations have been reported in a variety of systems, such as the rII region (Benzer and Champe, 1962; Brenner et al., 1967) and the head protein gene (R. H. Epstein et al., 1963; Brenner et al., 1965) of the phage T4, the RNA-containing phage f2 (Zinder and Cooper, 1964), several genes of the singlestranded DNA-containing phage 4x174 (Hutchison and Sinsheimer, 1966; Sinsheimer et al., 1967), and the alkaline phosphatase gene (Garen and Siddiqi, 1962), lactose (Newton et al., 1965), tryptophan (Yanofsky and Ito, 1966), and histidine (Whitfield et al., 1966) operons, and pyruvate dehydrogenase complex (Henning et al., 1965) of E. coli. Evidence suggesting that amber mutations lead to the production of 8 chain-terminating codon came from studies (Sarabhai et al., 1964; Stretton and Brenner, 1965) on the amber mutants of the Tq head protein gene. The mutants produced only incomplete fragments of the head protein in a nonpermissive host (su-) but made complete proteins in the permissive host (CR 63 s ~ + ) . The ~ mutant polypeptide chains were shown to be the amino terminal fragments proportional in length to the position of each mutation mapped on the gene, indicating that the chain reading was prematurely interrupted a t t.he site of mutation. Similar results were obtained from a study of several amber mutants of the p-galactosidase gene of E . coli (Fowler and Zabin, 1966). In vitro translation of phage f2 RNA containing an amber mutation in the coat protein gene also leads to chain termination at the site of mutation and results in release of amino terminal coat protein fragments free of tRNA (Zinder et al., 1966). I n the proteins made by various amber mutants of T4 head, f 2 coat and E. coli alkaline phosphatase genes in the permissive host SUI+ (or SU-I +) , a glN or try in the wild type is replaced by a ser a t the site of "This strain of E . coli has been shown t o carry a suppressor gene, SU T +. which appears to be indistinguishable from the Su-1' (Weigert and Garen, 1965a) used to suppress the amber type mutants of the alkaline phosphatase gene. The other suppressor genes, S W ~and + Sui?+,utilized as suppressors in the latter system, also seem to be similar to SUII+ and SUIII+, respectively, which were found to suppress the umber mutanks of the head protein and rII genes of T4.

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mutation in the suppressed protein (Stretton and Brenner, 1965; Weigert and Garen, 1965a; Notani et ul., 1965). It seems that the codon for glN or try in the wild strain is mutated to a nonsense codon which is read as ser in the permissive host suI+, leading to suppression of the umber mutation. The remarkable feature of this suppression is that irrespective of the structural gene, the position of the mutation, and the original amino acid affected by the mutation, the amino acid inserted by the suI+ suppressor gene is always ser. Similarly the suppressor genes, suII+ and suIII+,were shown to insert glN and tyr, respectively, a t the site of the umber mutation in both T4 head protein and alkaline phosphatase (Kaplan et al., 1965; Weigert et al., 1965). An additional suppressor gene, sun+, has been shown to insert a certain basic amino acid at the site of umber mutation in T4 head protein (Stretton et al., 1966). The nucleotide sequence of the umber codon was identified by comparing the amino acids replaced a t the mutated site in various revertants of an amber mutant of alkaline phosphatase in E . coli (Weigert and Garen, 1965b). The revertants of the umber mutant substituted glN, glu, tyr, ser, try, leu, and lys (or arg) in the place of the original try. From the known codon assignments for these amino acids, the umber codon was uniquely identified as UAG. In the r I I region of T4also, by using mutagens whose mutational mechanism is known and by analyzing the pattern of base substitutions in the umber and ochre mutants, the umber codon was confirmed to be UAG (Brenner et al., 1965; Stretton et al., 1966). Another class of nonsense mutations, suppressible by a new class of suppressor hosts, has been found in the alkaline phosphatase system of E . coli (Gallucci and Garen, 1966), the rII region and head protein gene of T4 (Brenner et ul., 1965; Brenner and Beckwith, 1965), and a few other systems. The nonsense codon of this class of mutants, called ochre,7 has been identified as UAA (Brenner et al., 1965; Weigert et uE., 1967a). A triplet with a nucleotide composition like that of the ochre triplet has been shown to cause release of the growing polypeptide from its linkage to tRNA (discussed in Section V,A). The role of UAA in chain termination is unequivocally demonstrated by the following finding. Last et al. (1967) have shown that the oligonucleotide AUGA, (lacking a UAA triplet) directs thc incorporation of met and ‘The N1and Na classes of nonsense mutations in the alkaline phosphatase gene

are analogous in most respects to the amber and ochre of the r I I and head genes in T4, respectively (Gallucci and Garen, 1966; Brenner et al., 1966). The fact that the suppressors of NS class do not suppress ochre mutants in the rII gene has been ascribed to a strain difference unrelated to suppression (Brenner et al., 1966). For simplicity, the terms amber and ochre have been used in this account for all of these systems.

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lys into polypeptides, and AUGU2A, (lacking a UAA triplet in phase) directs the incorporation of met, leu, and lys; on the other hand, the oligonucleotides AUGUA, and AUGU4A, (both containing a UAA triplet in phase) stimulate the incorporation of only small amounts of lys, with little incorporation of met and phe. Furthermore, AUGU4A, directs the synthesis and release of formylmet-phe as a free dipeptide (not as a peptidyl-tRNA) . Fan (cited by Stretton et al., 1966) has presented additional evidence suggesting that ochre mutants of the p-galactosidase gene in E . coli produce fragments of the enzyme terminating a t the site of mutation. An interesting difference between the amber and ochre suppressors is the ability of the latter t o suppress both amber and ochre mutations, while the amber suppressors are specific for amber mutants only (Brenner et al., 1965; Brenner and Beckwith, 1965; Gallucci and Garen, 1966). Since all the ochre suppressors are weak, suppressing by only 1 to 15% (Brenner et al., 1965; Gallucci and Garen, 1966), in contrast to amber suppressors with efficiencies ranging from 15 to 65% (Kaplan et al., 1965; Garen et al., 1965), the isolation of proteins of ochre mutants has proven to be difficult. Despite this, there is some evidence that various suppressors of the ochre mutants of alkaline phosphatase, like those of amber, insert different amino acids a t the site of mutation (Gallucci and Garen, 1966). Recently, the Su-4+ suppressor gene, which acts on both UAG and UAA, has been shown to cause both of the nonsense codons to specify tyrosine (Weigert et al., 1967b).

D. UAG, UAA,

AND

CHAINTERMINATION

The question may now be posed whether UAG and UAA, the amber and ochre codons, respectively, are chain terminating signals of natural messengers in E . coli. Although the amber suppressors suppress the UAG codon very strongly (up to 65%), the growth rates of su+ and su- strains are similar. If the normal mechanism of chain termination can be suppressed so efficiently in a cell, the growth rate would be expected to be seriously reduced. Since this was not found to be the case, one would expect the UAG codon to be rarely, if ever, present in the wild type strains of E . coli and Tq (Garen et al., 1965; Stretton et al., 1966). The possibility that UAA or some other type of signal may act as a natural chain terminator a t the end of cistrons in E. coli remains likely. Since several tRNA’s from mammalian and amphibian sources failed t o recognize UAA and UAG in binding assays, these codons may play a role in chain termination in higher organisms also (Marshall et al., 1967). Although a tRNA specific for UAA, UAG, or any other chain ter-

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minator has not been demonstrated, the involvement of tRNA in the mechanism of chain termination and polypeptide release is assumed. Such a tRNA may be analogous to puromycin (which breaks the ester linkage between polypeptide and tRNA) ; it may carry an amino acid-like molecule which forms a labile bond with the released polypeptide to be cleaved off later. A possible candidate for this molecule is an amino acid bearing a tertiary amino function. This would form a quaternary bond with the growing chain which would be hydrolyzed because of its instability in aqueous conditions (Hawtrey and Biedron, 1966).

E. UGA

AND

ITSNONSENSE

It has not been possible to assign UGA conclusively to any amino acid on the basis of binding data, although it causes a slight stimulation of binding to ribosomes of cys-tRNA from E. coli and a relatively more positive stimulation of cys-tRNA from guinea pig liver (Sol1 et al., 1965; Marshall et aZ., 1967). However, the triplet UGA failed to direct detectable incorporation of any amino acid, including cys and try, when present in the repeating copolymer poly GAU in an E. coli cell-free system (Morgan et al., 1966; see Table 1 ) . Furthermore, Weigert et al. (1967a) found that while amber mutants (UAG) in the alkaline phosphatase gene of E . coli reverted to seven different amino acids including try (UGG) , the ochre mutants (UAA) reverted t o six of these, but not to try. Similarly, Sarabhai and Brenner (1967a) reported complete absence of phenotypes (temperature resistant) expected to contain try among the base analog-induced revertants of an ochre mutant in the r I I A cistron of Tq. These findings suggest that UGA does not code for try. The above evidence indicates that UGA may be an additional nonsense codon in E. coli. This suggestion is supported by the genetic analysis of certain mutants of the rII region in Ta by Brenner et d . (1967). These authors have isolated a mutant, X655, in the left-hand end of the B cistron in the r I I region, and have shown it to be UGA by converting i t to an ochre (UAA) with the use of mutagens of known behavior. By using selected frameshifts in the same region which do not restore activity of the B cistron, they have also shown in three cases that the shifted frame led to the production of the UGA triplet, because these barriers could be induced to revert to ochres and then t o ambers at the same site. The mutant X655 and these frameshift-induced barriers occur in a region which is nonessential for the function of the B cistron, since i t can accept extensive frameshifts and can be re-

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placed by varying lengths of the A cistron through deletions joining the two genes. Therefore, the unacceptability of UGA in this region is unlikely to be the result of a missense mutation. The inability of UGA to code for any amino acid is further confirmed by studying the effect of UGA in the A cistron in combination with the deletion r1589 on the activity of the B cistron. In accordance with the test for nonsense proposed by Benzer and Champe (1962), the mutant X665 in the A cistron, also identified as UGA, abolished the activity of the B cistron. Further evidence in favor of UGA as nonsense is derived from its effect in restoration of part of the B activity (i.e., production of minute plaques on the restrictive host E . coli K12) in certain combinations of the type (++) in the B cistron. This effect has been interpreted to be the result of some sort of phase error in reading dependent on the presence of UGA (Brenner et ul., 1967). Whether UGA acts as a chain terminator in E . coli or has some other role is still uncertain. UGA has been suggested as a “spacer” to separate cistrons in a polycistronic message in E . coli (Brenner et al., 1967). It is possible that some of the alkaline phosphatase-negative mutants of E. coli, exhibiting phenotypic characteristics of nonsense mutants but not suppressible by any of the suppressor genes for umber (or N1) and ochre (or N2)mutants, may possess UGA codon a t the site of mutation (Gallucci and Garen, 1966; Weigert et ul., 19674. VI. Ambiguity in the Genetic Code

Ambiguity in the genetic code refers to the situation of one triplet coding for more than one amino acid, suggesting that a given codon can be recognized by more than one species of tRNA. It is possible to identify three distinct types of ambiguities. A codon can be misread owing to environmental conditions existing around the tRNA-mRNAribosome complex which interfere with the codon recognition process (see Section V1,A). Alternatively, a codon can be misread because of a specific change in the primary, secondary, or tertiary structure of either the tRNA (see Section V1,C) or the ribosome (see Section VI,D) such that it affects the codon-anticodon interaction. A third type of ambiguity occurs when a tRNA synthetase misrecognizes either an amino acid or a tRNA, leading to the formation of the wrong aminoacyl-tRNA. This type of ambiguity will not be considered here since the codon-anticodon interaction remains undisturbed. Ambiguity in genetic coding became obvious when it was revealed that poly U, containing the UUU codon for phe, also codes for small

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ANIL SADGOPAL

but significant amounts of leu. Such a leu-phe8 ambiguity under poly U stimulation was reported in cell-free systems derived from E. coli (Matthaei et al., 1962; Bretscher and Grunberg-Manago, 1962; Weisblum et al., 1962), Bacillus stearothermophilus (Friedman and Weinstein, 1964), Chlamydomonas (Sager et al., 1963), mouse leukemia cells (Ochoa and Weinstein, 1964), mouse plasma cell tumor (Weinstein and Schechter, 1962), rat liver and rabbit reticulocytes (Weinstein et al., 1966), and sea urchin eggs (Nemer, 1962). An ilu-phe ambiguity, also under poly U stimulation, was reported in the Chlamydomonas system (Sager and Toback, 1965). A basis for leu-phe ambiguity was established by the finding of a fraction of leu-tRNA from E. coli which preferentially responded to poly U (von Ehrenstein and Dais, 1963). An in vivo example of coding ambiguity appears to be the situation in which each of six positions in the a-chain of hemoglobin from an individual rabbit may be occupied by more than one amino acid (von Ehrenstein, 1966). Thus 50% leu and 50% phe was found in position 48 in the a-chain from a single rabbit. Although alternative explanations are not completely eliminated, these observations have been explained on the basis of an ambiguous translation of certain triplets. The occurrence of ser and thr in position 68 of the a-chain of hemoglobin from two highly inbred and related strains of mice (Rifkin et al., 1966; Popp, 1967) as well as similar findings in hemoglobins of horse (Kilmartin and Clegg, 1967) and man (von Ehrenstein, 1966) can also be interpreted in this manner. In vivo ambiguity may mean that a gene can, in certain cases, serve as a template for a group of related polypeptide chains instead of only one as currently accepted. The evolutionary significance of in vivo ambiguity is difficult to fully understand at this time. A. CODINGENVIRONMENT

A variety of chemical and physical environmental factors, both individually and in combination, have been shown to influence the degree of leu-phe, ilu-phe, ser-phe, and other types of ambiguities in vitro. Some of these factors include p H (Thach et al., 1966; GrunbergManago and Dondon, 1965), temperature (Soll et al., 1965; Friedman and Weinstein, 1964; Szer and Ochoa, 1964), alcohol (So et al., 1964), ammonium ions (So e t al., 1964), Mg++ (Soll et al., 1965; Friedman and Weinstein, 1964; Sager and Toback, 1965; So et al., 1964; Szer In this notation for ambiguity, the amino acid not primarily coded for by the triplet being considered is written first.

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and Ochoa, 1964) , polycations such as spermine and spermidine (Friedman and Weinstein, 1964)) aminoglycoside antibiotics such as Sm and DSm (Friedman and Weinstein, 1964; So e t al., 1964; Pestka e t al., 1965; J. Davies et al., 1964, 1966)) urea (So et al., 1964)) base analogs in mRNA (Grunberg-Manago and Michelson, 1964), and concentration of tRNA (Grunberg-Manago and Dondon, 1965; Pestka e t al., 1965). Many of the factors cause ambiguity in polynucleotide-directed amino acid incorporation as well as in trinucleotide-stimulated binding of tRNA to ribosomes. In general , increasing concentrations of Mg+ + inhibit incorporation of phe and stimulate that of leu and ilu in the presence of poly U (So e t al., 1964). An interaction between temperature and Mg++ has been illustrated in a thermophile system from Bacillus stearothermophilus, in which low temperature and high Mg++ concentration enhanced leu-phe ambiguity in response to poly U (Friedman and Weinstein, 1964). I n addition, random poly UG coded for phe and gly with only slight incorporation of arg (poly UG lacks arg codons) a t 65°C and low Mg++ concentration (0.01 M ) , whereas a t 37°C and higher Mg+ + concentration (0.018 M ) , arg incorporation exceeded that of gly (Friedman and Weinstein, 1964). Similarly, in a study of interaction of cations, polar and nonpolar compounds, and Sm on amino acid incorporation directed by polynucleotides, So et d. (1964) reported that the ratio of various agents to Mg++ was an important factor in stimulation or inhibition of poly U-directed phe incorporation. The effects of Mg++ and organic compounds on the incorporation of different amino acids by poly U were closely interrelated, alcohol lowering the optimum Mg++ concentration and urea increasing it. These authors proposed that some miscoding may be due to modification of hydrophobic interactions and hydrogen bonding, altering the type of complex formed between tRNA , mRNA, and ribosomes. A possible explanation for the miscoding effect of low temperature and high Mg++ was forwarded by Szer and Ochoa (1964)) who compared the influence of poly r T and poly U on leu-phe ambiguity in the E . coli system. Decreasing temperature and increasing M g + + concentration led to an increase in leu-phe ambiguity with both polymers; however, this was much more pronounced with poly rT than with poly U. Since poly r T has a higher complexing ability than poly U, it was suggested that the higher ambiguity shown by poly rT was the result of its better interaction with tRNA. For several reasons (see GrunbergManago and Dondon, 1965) this explanation of ambiguity seems inadequate to cover the miscoding effect of several factors. A more probable explanation is an environmentally induced modification of the conforma-

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ANIL SADGOPAL

tion of either the ribosome or the tRNA or both, which allows two of the three bases in a triplet to form sufficiently stable complexes with tRNA on the ribosome. This is supported by studies to be described in Section VI,D on the influence of Sm on ambiguity through ribosomal modifications. Binding assays have also been used to reveal the miscoding effect of several factors. At 25OC and 0.02 M Mg++, GCG (codon for ala) and CCG (codon for pro) both stimulated the binding of arg-tRNA with a template activity higher than that of CGG (codon for arg), but at 37OC and 0.01 M Mg++, the stimulation of arg-tRNA binding by GCG and CCG was depressed relative to CGG ($611 et al., 1965). Interference by environmental factors at the level of codon recognition was also shown by the influence of tRNA concentration (Pestka et al., 1965) and Sm (Pestka et al., 1965) on ambiguity in binding assays. Clearly, the above results would raise questions regarding the validity of codon assignments made from in vitro data if these had not been confirmed by a variety of evidence from in vivo studies. As we have seen, a very high correlation exists between codon assignments and single amino acid substitutions in various proteins, and these assignments are also consistent with the frameshift mutations of phage lyso~yme(see Section II1,A and C) ,

B. THEPATTEIRN OF AMBIGUITY By the use of repeating copolymers, Davies e t al. (1966)have demonstrated that misreading induced by Sm during polynucleotidedependent amino acid incorporation is much more specific than that suspected during earlier studies with homopolymers. They reported the following results on incorporation of amino acids by various repeating copolymers: poly UG

arg and some ser incorporatedin addition to the normal val and cys incorporation poly UC %phe, pro, and some his incorporated in addition to the normal leu and ser incorporation poly AG

no misreading observed

On the basis of codon assignments, it was concluded that Sm induces misreading of the following kinds: (1) only the two pyrimidine bases are misread; (2) pyrimidine bases in the 5'-terminal position of a codon are misread as pyrimidines; (3) internal pyrimidines can be misread as either pyrimidines or purines; (4) misreading is influenced considerably by the nature of the neighboring base.

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Pestka et al. (1965) have also pointed out that ambiguous codon recognitions are not random errors, but involve aminoacyl-tRNA species which respond to similar codons. For example, ilu, leu, ser, Val, and tyr, which show ambiguity with phe, have codons that differ from phe codons by one base only. On the basis of ambiguity produced by various in vitro conditions as well as that produced by the presence of unnatural base analogs in the synthetic messengers, Woese (1965b) proposed that there is a pattern that characterizes translation errors. This pattern for UUU is Position on the codon Base mistaken for U

I C,A

I1

c-low (A-very low)

111

C,A,G

Under “normal” conditions for the in vitro system, that is, 0.015 M Mg++and 37”C, the error rate in the I11 position of the codon is about 100 times that for the I position, which in turn is 10-fold greater than that for the I1 position. In contrast, the errors involving the purinerich codons lack a distinct pattern and the overall error rate involved is considerably lower (Woese, 1965b).

C. SUPPRESSION AND tRNA The properties of amber suppressor strains of E. coli have already been discussed in Section V,C. It is clear that the nonsense codon is read differently in the permissive and restrictive hosts. This indicates a situation of coding ambiguity, which can result from a modification of any component concerned with the mechanism of accurate reading of a codon. Capecchi and Gussin (1965) pointed out that these components could be: (1) an altered aminoacyl-tRNA synthetase which can transfer the amino acid to a tRNA species which recognizes the nonsense codon but normally does not carry any amino acid, (2) an erratic RNA polymerase which alters the nonsense codon during transcription, (3) mutated ribosomes which distort the natural mRNA-tRNA complex and permit the recognition of a nonsense codon by an aminoacyltRNA species (cf. the next section on Sm-activated suppression), (4) a modified species of tRNA, specific for a particular amino acid, yet capable of recognizing the nonsense codon a t least part of the time. Capecchi and Gussin (1965) and Engelhardt et al. (1965), using an in vitro assay for suppression of an amber mutation in the ooat protein cistron of the RNA-bacteriophage R17 (or fi), identified the com-

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ANIL SADGOPAL

ponent active in suppression in a permissive host carrying the Su-l+ gene as a ser-accepting tRNA species not present in the nonpermissive isogenic host. It was shown that no functional coat protein was synthesized in vitro unless this species of tRNA waa provided. Engelhardt et al. (1965) also demonstrated that the ser-tRNA from the suppressor strain inserted a ser residue into the coat protein a t the position specified by the nonsense triplet, and that in the absence of such a suppression a fragment of the coat protein was produced in vitro which terminated a t the same position. Since this suppressor activity cochromatographed both by salt and temperature gradient elutions from DEAE-Sephadex with one of the major ser-tRNA's, it was suggested that the suppressor tRNA could arise from a preexisting ser-tRNA (Bergquist and Capecchi, 1966). It has been further reported that strains carrying the amber suppressor genes Su-d+ and 8%-J+and ochre suppressor genes Su-4+ and Su-6+ contain tRNA species capable of translating nonsense codons in vitro (Wilhelm, 1966). J. D. Smith et al. (1966) have demonstrated that the amber suppressor gene SUIII+ produces a tyr-accepting tRNA which recognizes the amber codon UAG and appears not to recognize either the two tyr codons UAU and UAC or the ochre codon UAA. Recently, Carbon et al. (1966a) identified an ambiguous tRNA from a suppressor mutant (suss+) known to suppress a gly+ arg mutation in tryptophan synthetase A of E . coli. By the use of the alternating copolymer poly AG, which stimulates the incorporation of arg and glu, the above authors showed that C14-gly was incorporated in the presence of tRNA from su36+ but not in the presence of tRNA from su36- strain. The finding of C1*-labeled gly-glu and glu-gly sequences in the polypeptide demonstrated that gly was incorporated in place of arg. It was further shown (Carbon et al., 1966b) that the 6uQ6+tRNA suppressor activity chromatographed with gly-tRNA and not with argtRNA, indicating that a gly-tRNA species from suss+ can recognize an arg codon (AGA) and part of the time insert a gly in place of arg in the suppressed gly + arg mutant of tryptophan synthetase A, Parallel results have been reported by Gupta and Khorana (1966), who utilized a suppressor mutant (su7s+) for a gly 4cys mutation in the tryptophan synthetase A protein. It was shown that poly UG, an alternating copolymer which normally synthesizes a val-cys copolypeptide, directed the synthesis of a val-gly copolypeptide when the cell-free amino acid incorporating system was supplemented with su78+ tRNA. Although the origin of the altered tRNA in this system is not certain, i t appears from countercurrent distribution studies on SU78' tRNA that a very minor species of gly-tRNA is involved (Gupta et al., 1966).

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A modification of the overall structure of the suppressor tRNA may permit i t to recognize the nonsense codon in addition to its normal codon. Thus the amber suppressor mutation could be a mutation in a particular region of the tRNA cistron such that a major secondary or tertiary structural feature is affected. A similar alteration could also result from a mutation in a gene which codes for an enzyme involved in tRNA production, such as one producing the unusual bases in tRNA (Capecchi and Gussin, 1965). I n this connection, the altered coding properties of the methyl-deficient phe-tRNA (Revel and Littauer, 1966) and leu-tRNA (Capra and Peterkofsky, 1966) of E . coli may be noted. However, such a model is somewhat difficult to accept in view of observations showing that the suppressor tRNA may have the same chromatographic mobility as a t least a fraction of the particular tRNA species from which it is assumed to originate. A change in overall structure would be expected to alter its chromatographic properties also. A more likely model for amber suppression envisages a point mutation in the anticodon of tRNA so that i t specifically recognizes the nonsense codon although still aminoacylated by the same synthetase. A crucial corollary of this model is that the codon whose tRNA is altered by a suppressor mutation must be recognized by more than one species of tRNA in the wild-type strains ; otherwise the suppressor mutation would be lethal. This model also places restrictions on the type of mutations allowed in the anticodon. These will be considered in light of the wobble hypothesis in Section VI1,E (cf. Table 9). Consistent with this model and its restrictions is the recent report showing that the anticodon GUA in the su- tyr-tRNA is changed to CUA in the su+ tyr-tRNA isolated from the suppressor strain which inserts tyr a t the site of amber codon, UAG (Crick, 1 9 6 7 ~ )The . weak suppression by ochre suppressors makes it difficult to investigate if they operate by a similar mechanism.

D. STREPTOMYCIN-ACTIVATED SUPPRESSION AND RIBOSOMES Streptomycin is known to inhibit poly U-directed incorporation of phe in cell-free systems derived from Sm-sensitive strains of E. coli (Flaks et al., 1962; Speyer et al., 1962); resistant strains are able to withstand much greater concentrations of Sm. The difference in the response of the two strains was attributable to the ribosomal fraction. Similarly, the stimulatory or inhibitory effects of Sm on the synthesis of polyphe in poly U-primed systems were shown to be correlated with the ribosomes isolated from Sm-dependent, Sm-dependence-suppressed, Sm-sensitive, or Sm-resistant strains (Brownstein and Lewandowski, 1967; Likover and Kurland, 1967). B y reaggregation of 30 S

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and 50 S ribosomal subunits prepared from Sm-sensitive and -resistant strains, it was found that only the ribosomes containing the 30 S component from the Sm-sensitive strain were sensitive to the action of the drug, showing that the inhibitory action of Sm was correlated with the 30 S subunit (Davies, 1964; Cox et al., 1964). The requirement for Sm for the maximum poly U-directed polyphe synthesis by ribosomes from a Sm-dependent strain has also been demonstrated to be a property of the 30 S subunit (Likover and Kurland, 1967). The site of Sm-sensitivity (enhancement of poly U-directed ilu-phe ambiguity) has been further localized to the 23 S ribonucleoprotein core of the 30 S subunit (Staehelin and Meselson, 1966; Traub et al., 1966). A basis for this sensitivity in bacteria is found in the stimulation by Sm of coding ambiguity in E. coli extracts (So et al., 1964; J. Davies et al., 1964, 1966). For example, Sm inhibited poly U-directed incorporation of phe but stimulated incorporation of ilu, ser, and leu (So et al., 1964; Davies et al., 1964); ribosomes from Sm-resistant strains did not show this miscoding. The specific pattern of the enhancement in ambiguity in the presence of Sm has already been discussed in Section V1,B. Stimulation of ambiguity by Sm in binding assays showed that it acts at the level of codon recognition (Pestka et al., 1965; Kaji and Kaji, 1965). Sm also promotes misreading of natural messengers (Schwartz, 1965), resulting in the formation of nonfunctional protein (Bissell, 1965). The fact that an external agent acting on a specific ribosomal component can cause coding ambiguity implies that the fidelity of the translational mechanism depends not only on the particular codon and anticodon but also on the conformation of the ribosomal site holding the tRNA to the messenger. A modification of this site could allow a “wrong” tRNA to fit so well against the messenger that a “wrong” amino acid is incorporated into the polypeptide chain. A mutation to Sm resistance could modify this site so that the correct tRNA is paired whether or not Sm is present, All the studies reported above on the miscoding action of Sm were performed in extracts from microorganisms. No similar miscoding action is found in cell-free systems derived from higher organisms. For example, poly U-directed leu-phe and ilu-phe ambiguity exhibited in extracts of Chlumydomonas in the absence of Sm was actually repressed in the presence of the drug (Sager and Toback, 1965). I n the same system, no appreciable incorporation of ser or tyr occurred either in the presence or absence of Sm. A lack of Sm-induced ambiguity in a poly U-dependent system was also reported in extracts of rat liver (Weinstein et al., 1966), rabbit reticulocytes (Weinstein e t al., 1966)

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and chick embryos and fibroblasts (Soeiro, cited by Weinstein et at., 1966). An in vivo confirmation came from studies on Euglena in which Scher (1966) showed that Sm-induced mutations in the photosynthetic apparatus of the organism were not caused by translational errors. Ribosomes of higher organisms may therefore be inherently different in the structure of one of their components (e.g., the 23 S core particle). Indeed, these studies with higher organisms reflect a parallel with the resistance to Sm-induced miscoding exhibited by ribosomes from Smresistant bacterial strains. Further understanding of the action of Sm on the codon recognition process comes from the discovery of Sm-activated suppression and its interaction with genetic suppressors. Gorini and Kataj a (1964) demonstrated that a variety of E . coli mutants deficient in different metabolic functions were able to grown in the absence of their specific growth factors provided that Sm was present in the culture medium. This new class of mutants was defined as “conditionally Sm dependent (CSD) .” In a particular CSD mutant requiring arg it was shown (Gorini et al., 1961) that active ornithine transcarbamylase (OTC) was found only in cells grown in the presence of Sm. It was thus established that informational suppression may be achieved not only through mutations in the genome (e.g., a mutation leading to a new tRNA found in amber suppressors), but also through alteration of the cytoplasmic conditions (e.g., by addition of Sm). Other examples of Sm-suppressible mutants have been found in various genes of bacteria and phages (Lederberg et al., 1964; Whitfield et al., 1966; Orias and Gartner, 1965; Valentine and Zinder, 1964). It has also been shown (Gorini and Kataja, 1964) that from a Smsensitive (Sms) strain carrying an OTC defect suppressible by Sm (SSu) , two classes of Sm-resistant (SmR) mutants may be derived which differ in their competence for permitting suppression by Sm: one is responsive to the suppressive action of Sm (i.e., competent), while the other does not respond to any concentration of Sm and requires arg under all conditions (i.e., incompetent). Since i t is known that the Sm locus in the genome is also one of the determinants of the 23 S ribonucleoprotein particle, it may be concluded that the various alleles of the Sm locus control their suppressive action through the ribosomes by influencing the codon recognition process. Anderson et al. (1965) pointed out that a SmR mutant was resistant to the suppressible action of Sm only in relation to the particular defect being suppressed and could act as a competent strain for another defect. Within each class of competent and incompetent strains for the OTCssU defect the imposition of further auxotrophic mutations yielded strains

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ANIL SADGOPAL

that were competent for the Sm-activated suppression of some of these additional mutations. This specific effect of various alleles a t the Sm locus on Sm-activated suppression was further emphasized by the studies on the interaction of these alleles with genetic suppression. For example, it is found that a mutation from Sms to SmR can impose a restriction on the efficiency with which a genetic suppressor acts in a given host (Gorini et al., 1966). In some cases this restriction can be so severe that the effect of suppressor mutation disappears in the phenotype of a Su+SmR mutant derived from a Su+Sma strain. Addition of Sm may or may not lead to the suppressed phenotype again. Gorini et al. (1966) studied the effect of different Sm alleles, including the Sms wild type, on a given suppression system which consisted of two independent suppressible mutations (OTC!,S" and leussu). They demonstrated that the Sm allele controlled the suppression of each mutation independently, and moreover that the "restriction" of suppression is not necessarily associated with SmR alleles since suppression of leusBu can be positive in a SmR cell and negative in the corresponding Sms one. Gorini et al. (1966) have argued that the different suppression patterns of OTC and leu in strains T19-32 (arg' leu- Sms) and T23-61 (arg* leu' SmR) suggest that different ribosomal mutations lead to different types, rather than simply different amounts, of ambiguities. The above studies clearly bring out the important distinction between two types of suppressions: suppression through the ribosome and suppression through adaptors. Gorini et al. (1966) propose that only mutations leading to altered properties of the adaptors can provide new alternative versions of a given codon (as in amber suppressors), while a change in the ribosomal structure can only affect the fidelity with which the alternative versions already present are translated (as in the interaction of Sm alleles with different SSu defects). Because of the relationship between ribosomes and adaptors in the codon recognition process, suppression via adaptors can be defined only in terms of a given form of ribosomes allowing the use of alternative versions of codons, and conversely, suppression via the ribosome is only definable in relation to a particular set of adaptors providing alternative codon versions. VII. tRNA and Its Synthetase in Codon Recognition

The existence of a tRNA and an aminoacyl-tRNA synthetase (activating enzyme) specific for each of the 20 amino acids has been demonstrated (Berg, 1961; Brown, 1963). For tRNA to find its specific tRNA synthetase, and to recognize its specific codon in the subsequent

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interaction with mRNA requires three separate functional sites on the tRNA molecule. One of these, the synthetase-recognition site, interacts with the specific tRNA synthetase which attaches the amino acid to the second site, now well identified as the 2'- or 3'-hydroxyl group of the adenosinyl residue of the terminal -CCA sequence. The third site, the specific nucleotide sequence (anticodon) , recognizes the codon defining the particular amino acid. BETWEEN ANTICODONS AND A. THERELATIONSHIP SYNTHETASE-RECOGNITION SITES

The precise nature of the interrelationship of anticodon and synthetase-recognition site is not yet clear. It is conceivable that the synthetase-recognition site on the tRNA may partially or completely overlap9 the anticodon site. If these sites are identical, the reaction between tRNA from one species and synthetase from another species would lead to more-or-less successful aminoacylation of tRNA, assuming a universal anticode.1° The studies on the cross-reaction between tRNA and synthetase from different species, reviewed by Brown (1963) , reveal considerable species specificity. Although several reports have demonstrated fairly good cross-reaction for certain amino acids among the mammalian species tested, very little cross-reaction is observed between these components derived from mammalian, fungal, and bacterial species. Since the anticodon site was assumed to be universal, these results indicated that the synthetase-recognition site varies from species to species and thus must differ from the anticodon site. However, part of the synthetase-recognition site may overlap the anticodon, with only the nonoverlapping region changing from species to species during evolution. Furthermore, Loftfield and Eigner (1963) found the Michaelis-Menten constants t o be similar for the aminoacylation of tRNA by heterologous and homologous enzymes with only the reaction rates differing. They concluded that the synthetase-recognition site for a particular tRNA may be identical in all species making it unnecessary to distinguish it from the anticodon site. @Overlapof sites, in this context, refers t.o regions coincident with or adjacent to each other on the primary structure of tRNA as well as to regions spatially separated on the primary structure but physically or functionally involved with each other due t o the particular folding pattern of tRNA. loThe original assumption in these considerations regarding the universality of the anticode is most likely not true. This is shown by the difference in the pattern of codon recognition at the third position in the case of ala-tRNA of E . coli and yeast in Table 6, and also explained in Section VII,C.

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ANIL SADGOPAL

Another approach has been to study the effects of modifioations of tRNA by various physical and chemical agents on its acceptor and subsequent transfer activities. Yu and Zamecnik (1963a) showed that the treatment of tRNA with bromine (which reacts mainly with pyrimidines) inactived aminoacylation of phe-, Val-, and lys-tRNA’s from yeast at different rates than of those from E. coli. They concluded that the pyrimidine composition of synthetase-recognition sites in the yeast tRNA’s tested was different from those of the corresponding E . coli tRNA’s. At a bromination level of 1 bromine per 40 nucleotide residues of tRNA, the formation of phenylalanyl-tRNA was significantly inhibited (Yu and Zamecnik, 1963a,b), leading the authors to conclude that the synthetase-recognition site of phe-tRNA consisted of at least one bromine-sensitive residue, probably a pyrimidine, and thus differed from the suggested anticodon sequence (AAA) for phetRNA. Similar conclusions were derived from studies on the effect of modification of tRNA by treatment with nitrous acid (Carbon, 1964) and methylation and brornination (Weil et al., 1964). However, the above results do not eliminate the possibility of the anticodon forming just a part of the synthetase-recognition site. A more serious criticism of the above studies is that the chemical treatments may inactivate tRNA by changes in the secondary or tertiary structure essential for synthetase recognition. Indeed such a possibility is suggested by various experiments on the inactivation of aminoacylation of tRNA by semicarbazide modification of the cytosine residues (Muto et al., 1966) and by ribonuclease treatment (Nishimura and Novelli, 1964). Some other experiments on the modification of tRNA have suggested that the synthetase-recognition and anticodon sites overlap each other. Penniston et al. (1964) showed that the inactivation rates of the aminoacylation and of transfer activity for formaldehyde-treated tRNA were similar. Gottschling and Zachau (1965) found that the acceptor and transfer activities had the same sensitivity to ultraviolet (W) irradiation for both phe- and lys-tRNA’s. Their observation that the acceptor activity of lys-tRNA, presumably containing the anticodon UUU, is destroyed much more by UV irradiation (which dimerizes uracil residues) than that of phe-tRNA, presumably containing the anticodon AAA, is also consistent with the possibility of anticodon being involved in synthetase recognition. Harriman and Zachau (1966) further demonstrated that the target for W inactivation of the acceptor and transfer functions of tRNA was very small with a high overlap between the targets in phe-tRNA and a low overlap between those of lys-tRNA. However, the above studies with W irradiation, suggesting that the synthetase-recognition site may include

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the anticodon, are subject t o the same criticism as the previous studies with chemical agents which suggested otherwise. The possibility that the cause of W inactivation of tRNA may be a change in its secondary structure as a result of the formation of uracil dimers has been considered by the latter authors themselves (Harriman and Zachau, 1966). In a novel approach, Hayashi and Miura (1966) studied the inhibitory effects of various kinds of oligonucleotides (average chain length 5-6 nucleotides) on the aminoacylation of tRNA by yeast synthetases. They assumed that inhibition would be observed when the added oligonucleotide possessed a specific structure corresponding to the site in tRNA which recognizes the aminoacyl-tRNA synthetase. The formation of phenylalanyl-tRNA (suggested anticodon, AAA) was competitively inhibited by oligo-A but only slightly by oligo-U, while oligo-C caused no inhibition. Similarly, the aminoacylation of lystRNA (suggested anticodon, UUU) and pro-tRNA (suggested anticodon, GGG) was specifically inhibited by oligo-U and oligo-G, respectively, and not by other oligonucleotides. These experiments suggest that the anticodon is either identical to or a t least a part of the synthetase-recognition site in tRNA. However, other workers using bacterial synthetases have failed to relate the inhibitory effects of oiigo- and polynucleotides on aminoacylation of tRNA to any specific competition with the anticodon site (Letendre et al., 1966; Stulberg and Isham, 1967). A somewhat similar approach consists of using periodate- or exonuclease-treated tRNA’s as inhibitors of aminoacylation reactions but has so far not given any definite clues regarding the question of involvement of the anticodon in the synthetase-recognition site (Torres-Gallardo and Kern, 1965; Stulberg and Isham, 1967). B y using “denatured” and “renatured” forms of tRNA (interconvertible by altering certain i n witro conditions) , the importance of secondary and tertiary configuration of native tRNA in synthetase recognition has been established (Lindahl et al., 1967; A. Adams et al., 1967; Sueoka et al., 1966). The interpretation of these results is difficult since the precise nature of the change caused by “denaturation” is unknown. However, it is conceivable that the synthetase-recognition site may include the anticodon along with some specific secondary or tertiary feature, such as the loop on the right-hand part of the “clover-leaf” model of tRNA which varies in structure in ser-, tyr-, and ala-tRNA’s (see the models proposed in Zachau et al., 1966; Madison et al., 1966; Holley et al., 1965). As discussed in Section VI,C, the amber suppression may result from a mutation in the anticodon such that the recognition of the mutated tRNA by its specific synthetase remains unaffected. I n view of this, the degree t o which the

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anticodon may determine the specificity of synthetase recognition remains an open question.

B. Is

THE

ANTICODE DEGENERATE?

A highly degenerate genetic code suggests the possibility of more than one kind of tRNA for each amino acid. This was indicated by DNA-tRNA hybridization experiments which demonstrated the presence of at least 40 tRNA-complementary sites in the E . coli genome (Goodman and Rich, 1962; Giacomoni and Spiegelman, 1962) and of a 13-fold multiplicity of the 60 tRNA species in the Drosophila genome (Ritossa et al., 1966). Several authors have investigated heterogeneity in various tRNA’s by the use of such techniques as countercurrent distribution (Holley et d.,1963), and chromatography on columns of methylated albuminkieselguhr (Sueoka and Yamane, 1962), DEAE-cellulose and DEAESephadex (Cherayil and Bock, 1965)) Sephadex G-25 (Muench and Berg, 1966a), and hydroxylapatite (Muench and Berg, 196613). The heterogeneity of several tRNA’s in E . coli, yeast, and mammalian systems has been described (Clark and Marcker, 1966s; Weisblum et al., 1962; Muench and Berg, 1966a; Doctor et al., 1961; Apgar et al., 1961; Karau and Zachau, 1964; Hoskinson and Khorana, 1965; Sol1 et al., 1966; Apgar and Holley, 1962; Berg et al., 1961; Bennett et al., 1965; Goldstein et al., 1964). It should be noted that the physicochemical heterogeneity of tRNA may be an indication not of degeneracy, but of redundancy in tRNA. We shall consider a tRNA for a given amino acid as degenerate if its multiple fractions differ a t least in their specificities of codon recognition and consequently also in their anticodons; degenerate tRNA species may have differences in their primary structure in addition to those in the anticodon. On the other hand, redundancy shall refer to those multiple species of tRNA for an amino acid that do not differ in their coding attributes but possess differences in regions other than the anticodon. It is pertinent to point out that small changes in the anticodon only, leading t o degeneracy, may not be sufficient to render a particular tRNA physicochemically separable into its various species. A possible example of redundancy appears to be the case of sertRNA I and I1 described by Zachau et al. (1966) ; the base sequence of these two species differs in regions outside the predicted anticodon site. In binding assays, peak I of E . coli lys-tRNA responded to poly A in preference to the repeating polymer poly AAG while peak I1 responded preferentially to poly AAG (Nishimura et al., 1966b). How-

THE GENETIC CODE

3 75

ever, the magnitude of difference in the responses shown by the two peaks was not such as to indicate a specificity for either triplet. The use of trinucleotides in binding assays failed to show even this difference since peak I1 of lys-tRNA responded to the triplet AAA in preference to AAG. The two peaks of lys-tRNA in E. coli may constitute an example of redundancy in tRNA. Redundancy can have great biological importance. It could affect the functioning of genetic suppressors (to be discussed in Section VI1,E) and the expression of certain mutations affecting the regulation of protein synthesis, e.g., in the case of “his R” regulatory mutants of Salmonella histidine biosynthetic enzymes (Silbert et al., 1966). I n the past few years several experiments have been reported which indicate an involvement of tRNA in various cellular developmental and regulatory processes (e.g., see Sueoka et al., 1966; Subak-Sharpe et al., 1966; Kaneko and Doi, 1966; Silbert et al., 1966; Waters and Novelli, 1967; Taylor et al., 1967). This question, although of growing importance, will not be discussed further in the present review. It suffices to mention in this connection that redundancy (as well as degeneracy) of tRNA may play an important role in control mechanisms through modifications in the concentrations of various tRNA species in a cell (cf. the codon restriction hypothesis of cellular differentiation proposed by Strehler et al., 1967). Furthermore, it is conceivable that the various redundant tRNA species may be confined to different parts of a cell or to different tissues of an organism. I n this context, one may note the findings of Barnett and Brown (1967) showing that the mitochondria of Neurospora contain a full complement of tRNA’s and that the two (asp- and phe-tRNA’s) which were examined in detail are distinct from their cytoplasmic counterparts in aminoacylation specificities (cf. the work of Barnett et al., 1967, on mitochondrial-specific synthetases). A degeneracy specifically located in the anticodon was first demonstrated by Weisblum et a2. (1962) and von Ehrenstein and Dais (1963), who reported that the three peaks of leu-tRNA of E. coli responded to random polynucleotides of different compositions in polynucleotidedirected amino acid incorporation experiments (peak I to poly UC, peak I I a to poly U, and peak I I b to poly UG). The above results were generally confirmed by Sol1 et al. (1966), who showed that CUG and UUG specifically stimulated the binding of peak I and peak I11 to ribosomes, respectively; however, some discrepancy in these studies is pointed out in the footnote * of Table 6. On the basis of degeneracy of the anticodon sites of tRNA, it was predicted that the different leu-tRNA’s would incorporate leu into different positions of a protein molecule. Weisblum et a2. (1965) showed

376

ANIL SADGOPAL

TABLE 6 Summary of Experimentally Observed Patterns of Codon Recognition by tRNA Source of tRNA

tRNA

Codons recognized

References

1. Specificity for First Letter of Codons

arg-tRNA-I arg-tRNA-I1 leu-tRNA-I* leu-tRN A-I11 ser-tRNA-I ser-tRNA-111

Yeast Yeast E. coli E. coli E. coli E. coli

CGX (X = U, C, A) AGA, AGG CUG UUG

ucu, ucc

AGU, AGC

1

1 1 1 1 1

2. Multiple Recognition of First Letter of Codons

mebtRNAF

B. coli

Yeast E. coli E. coli E . coli

E. coli

E. coli Yeast Yeast Yeast 2.coli E. coli B. coli

UUG?, GUG, AUG

3. Multiple Recognition of Third Letter of Codons (a) Recognition of U and C phe-tRNA ZJUU, UUC Val-tRNA-1111 GUU. GUC val-tRNA-III1J GUU, GUC ucu, ucc ser-tRNA-1 ser-tRNA-111 AGU, AGC AUU, AUC ilu-tRNA-IJ and 111 gly-tRNA-I1 and IV GGU, GGC (b) Recognition of A and G mg-tRNA-JI AGA, AGG gly-tRNA-I GGA, GGG ser-tRNA-IIt UCA, UCG ala-tRNA-I $ GCA, GCG Val-tRNA-I 11 GUA, GUG Recognition of IT C, and,A ala-tRNA GCX (X = U, C, A) arg-tRNA-I CGX (X = U, C, A) Val-tRNA-I GUX (X = TJ, C, A) (d) Specific recognition of G gly-tRNA-III$ GGC leu-tRNA-l* CUG leu-tRNA-I11 UUG mebtRNAF, y AUG tv-tRNA UGG (e) Recognition of A, G, and U Val-tRNA-I tind 1111 GUA, GUG, GUU Val-tRNA-I1 GUA, GUG, GUU (f) Recognition of A, G, U, and C ala-tRNA-I1 GCU, GCC, GCA, GCG

2,3

11 4

1 2 1

1 1

1 1 1 1 2 1

((2)

Ye& Yeast Yerrst Yeast E. coli E. coli E. wli E . coli E . coli Yeast

E. coli

1 1 1

1 1 1

2,3 1 2

1 2

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377

that peak I of E. coli leu-tRNA placed leu in six peptides of the a-chain of rabbit reticulocyte hemoglobin but leu from peak I I b of leu-tRNA appeared almost exclusively in a single peptide, indicating to them that leu has a t least two tRNA species with different coding specificities for reading a natural genetic message. However, the interpretation by the above authors can be accepted only with reservation since the C14-leu from peak I I b of leu-tRNA was found in a single peptide which appeared to be related but not identical to the normal hemoglobin peptide chromatographing a t that position. Degeneracy for codon recognition has now also been demonstrated by binding or incorporation experiments, or both, for ser-tRNA (So11 et al., 1966; Bennett et al., 1965), met-tRNA (Clark and Marcker, l966a; Kellogg et al., 1966), Val-tRNA (Kellogg et al., 1966), ala-tRNA (Kellogg et al., 1966), arg-tRNA (Soll et al., 1966), and gly-tRNA (So11 et al., 1966).

C. THEEXPERIMENTALLY OBSERVED PATTERNS OF MULTIPLECODONRECOGNITION A remarkable feature of several of the individual fractions of various tRNA's is their capacity to recognize more than one synonym codon in accordance with a distinct overall pattern summarized in Table 6. For example, t'he single peak of yeast phe-tRNA can recognize two phe codons, UUU and UUC (Bernfield and Nirenberg, 1965; Sol1 et al., 1966), a.nd a highly purified species of yeast ala-tRNA can recognize References: (1) Sol1 et al. (1966); (2) Kellogg et al. (1966); (3) Clark and Marcker (1966a); (4) Bernfield and Nirenberg (1965). * Although leu-tRNA-I responded t o CUG alone in the binding assays, it responded well t o randomly linked poly UC in amino acid incorporation systems (Weisblum et al., 1962; von Ehrenstein and Dais, 1963). The codon assignment of this species therefore may be considered provisional. t The data of Still et al. (1966) show that ser-tRNA-I1 responded appreciably to UCU but much less than to UCA and UCG. This waa interpreted aa an example of multiple recognition of A and G alone at the third place. Kellogg et al. (1966) interpreted the same data as showing multiple recognition of A, G, and U a t the third place. J Ala-tRNA-I is being cited as an example of multiple recognition of A and G a t the third place on the hasis of binding assays in 0.01 M Mg++. However, in 0.02 M Mg++ this species responded to GCU almost as well, making this an example of multiple recognition of A, G, and U a t the third place (Kelloggetal., 1966). $ The data of Soll et al. (1966) show that ply-tRNA-I11 responded very strongly to GGG, but noticeably t o GGU and GGC also. /I Notice the unresolved differences between the results of Soll et al. (1966) and Kellogg et a2. (1966) for the codon recognition patterns of various Val-tRNA species of E. coli.

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ANIL SADGOPAL

three ala codons, GCU, GCC, and GCA (Still et al., 1966). A possible mechanism for this pattern of multiple codon recognition at the third place is discussed in the next section. Using these observed classes of the anticode in Table 6, we can specify the minimum number of tRNA’s needed to code for a given amino acid. This is illustrated by considering the example of an amino acid, such as gly, which is assigned a set of four codons sharing a common base sequence a t the first two positions, the third position being occupied by any one of the four bases. One tRNA, belonging to the type 3f, may suffice for all gly codons by recognining all four bases at the third place equivalently. Alternatively, two types of tRNA’s may be used by an organism in the following three different combinations: (i) One tRNA species recognizes both U and C (type 3a); the second species recognizes both A and G (type 3b). (ii) One tRNA species recognizes U, C, and A (type 3c); the second species recognizes either both A and G (type 3b) or G alone (type 3d). (iii) One tRNA species recognizes A, G, and U (type 3e) ; the second species recognizes either both U and C (type 3a) or U, C, and A (type 3 0 ) . One implication of the property of multiple codon recognition by individual tRNA species is clear ; several different combinations of anticodons may be used by various organisms or by different cell types in one organism to code for the same amino acid. It may also be noted that some organisms may have more species of tRNA than are minimally required, as seems to be the case with gly-tRNA in yeast (see Table 6 ) ; gly-tRNA-I recognized GGG and GGA, while GGG was also recognized by gly-tRNA-111. Similarly, both ilu-tRNA-I1 and -111 in E . coli recognize AUU and AUC.

D. CRICK’S WOBBLEHYPOTHESIS AND NATURE OF

THE

ANTICODON

THE

The experimentally observed multiple codon recognition properties of tRNA described in the previous section raise questions concerning the mechanism of such recognition at the third position of a coding triplet. The “wobble hypothesis” proposed by Crick (1965, 1966) provides an explanationll for this phenomenon. Essentially, the wobble hypothesis l1 Bock (1967a,b) has recently proposed “assisted tautomerization” as an alternative to the wobble hypothesis. This proposal also leads to the same pattern of multiple base recognition a t the third place as the one shown in Table 7 on the basis of the wobble.

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THE GENETIC CODE

states that while the standard base pairs (G = C ; A = U; I = C ) are formed at the first two positions of a codon during its interaction with the anticodon, there can be a certain amount of “wobble” or play in the base pairing a t the third position, making the formation of more than one type of base pair possible. On the basis of a study of molecular models, stereochemical considerations and the nature of the code, Crick suggests that the wobble would permit the following three types of base pairs in addition to the standard base pairs a t the third position of the codon: I = U, G = U, and I = A. The relevant base of an anticodon can, therefore, recognize more than one base a t the third position of the codon according to the rules of base pairing shown in Table 7. I n addition to the base-pairing patterns shown in Table 7, Crick (1965) suggested that I can code €or all four bases a t the third TABLE 7 Pairing at the Third Position of the Codon as Predicted by Crick’s Wobble Hypothesis Base on the anticodon

Bases recognized on the codon

U

c A

G

u

G

I

position of codons by not pairing a t all, that is, by being “tucked” out of the way. Crick’s wobble hypothesis can aid in the recognition of the anticodons which occur a t comparable positions on the primary sequence (at or very near the positions 36, 37, and 38) and the proposed “cloverleaf” secondary structure of the tRNA’s. The anticodons identified to date are presented in Table 8. Although the wobble hypothesis has explained the mechanism of multiple codon recognition by tRNA quite satisfactorily for most the observed cases, i t does not seem to explain the pattern of codon recognition by certain tRNA species (listed under 3e in Table 6 ) , which recognize A, G, and U equivalently a t the third place. This could be due to a failure t o separate two or more types of tRNA’s or due to environmentally induced conformational changes in the tRNA or ribo-

380

ANIL SADGOPAL

TABLE 8 Identified Anticodon Sequences Amino acid

Anticodon*

ah Val

IG C JAC

8er

IGA

tYr Phe

G$A 2’0MeGAA

Codonst GC. GU.

uc.

UA

ULT

z} g}

Reference Holley et al. (1965) Ingram and Sjaquist (1963) Zachau et al. (1966) Madison et aZ. (1966) RajBhandary ef al. (1967)

* Note that codon-anticodonpairing is antiparallel.

t Adot at the third place of tiplets indicatm that this place in the synonym codons can he occupied by any one of the four hases. somes affecting the wobble in such a way that A, G , and U are recognixed at the third place by a single tRNA species (see footnotes 1 and Ij of Table 6). It might then be postulated that certain environmental conditions make it possible to loosen the specifications of Crick’s wobble and allow freer base pairings at the third place of the codon, which may be the very basis of coding ambiguity.

E.

PREDICTIONS AND

TESTSOF CRICK’S WOBBLE HYPOTHESIS

The wobble hypothesis makes very precise predictions about the nature of anticodons, thus rendering itself amenable to several tests. Some of these are discussed below. (a) At least one fraction of ilu-tRNA must contain inosine a t the 5’-terminal position of the anticodon. The presence of inosine in this position is the only way, according to the wobble hypothesis, for a tRNA to recognize A at the third place of a codon equivalently with U and C and yet not recognize G. (b) The wobble hypothesis predicts that no inosine can occur at the 5’-terminus of the anticodon of any tRNA which codes for only or only&} at the third place of synonym codons (types 3a and 3b in Table 6 ) . This prediction would apply to tRNA’s for amino acids such as asN, asp, glN, glu, his, lys, phe, and tyr. Tyr- and phe-tRNA’s, the only tRNA’s of this class whose base sequences have been worked out so far, do not violate this prediction (see Table 8 ) . Of course, this prediction would also apply to tRNA’s of types 3a and 3b in Table 6, which might code for amino acids whose codons use all four bases equivalently at the third place. Possibly few or none of the tRNA’s of E . coli belong to type 3c, listed in Table 6 as recog-

X}

T H I QENETIC CODE

381

nizing U, C, and A equivalently a t the third place of triplets (this type of tRNA is predicted to contain inosine and has so far been found only in yeast), since little inosine is reported t o occur in the total E . coli tRNA (B. F. C. Clark, cited by Tanner, 1966). Thus, amino acids like pro or gly in E . coli could be coded by tRNA’s of types 3a and 3b lacking inosine. (c) According to the wobble hypothesis, a tRNA cannot recognize C alone or A alone a t the third place of codons. However, G can be exclusively recognized at this position (note the single assignments of UGG to try and AUG to met). Thus UGA could code for either cys or try (if the anticodons of cys- and try-tRNA’s contain I and U, respectively, a t the 5’-end) and for no other amino acid. Since evidence is accumulating for UGA being a nonsense codon, the wobble hypothesis predicts that UGA cannot have a tRNA. By preparing radioactively labeled uncharged tRNA it may be possible t o test whether UGA stimulates the binding of any particular tRNA to ribosomes in cell-free systems. The observation that ochre (UAA) suppressors can also suppress amber (UAG) mutations, although suppressors specific for umber mutants exist, is consistent with this prediction. A corollary of this prediction allows only C a t the 5’-terminal position of the anticodon of the suppressor tRNA specific for amber mutants only. The identification of CUA in the anticodon of amber suppressor tyr-tRNA is consistent with this. It is likely that an additional class of suppressors would be found; these suppressors will be able to suppress the nonsense codon UAA exclusively without suppressing UAG. The mutant tRNA’s of this class12 of suppressors are predicted to contain an inosine in their anticodons. (d) An analysis of the nature of the amber suppressor mutation can be made in light of the wobble hypothesis. The simplest case is that of SUIII+ tyr-tRNA which inserts t y r at the site of the UAG codon (see Table 9 and its footnotes). The wobble hyothesis predicts that the suppressor mutation in this case alters either the anticodon AUA or GUA t o CUA. It is further implied that unless the wild-type strain of E . coli contains multiple species of tyr-tRNA to code for UAU and UAC, the suppressor mutation affecting the anticodon GUA would be lethal. In the case of the suI+ suppressor gene, a ser-tRNA recognizes the UAG codon. Of the five possible anticodons for ser-tRNA listed in Table 9, only the species containing the anticodon CGA can give rise to an amber suppressor ser-tRNA by a point mutation. If a species with the antila

The name Topaa has been suggested for this class of suppressors (Bock, 19f37a).

382

ANIL SADGOPAL

TABLE 9 Possible Nature of Mutation in the Anticodon of Amber* Suppressor tRNA Suppressor gene

Aminoacid inserted

Codom assigned U

SUi+

ser1

WlI+

glN

CA

$}

tYr

UA

}:

8U111+

Anticodonst of wild-type tRNA

Mutation in anticodon

(i) AGA

(v) CGA

(iii) GGA (iv) UGA (v) CGA (i) UUG (ii) CUG

CUA

(ii) IGA

(i) AUA (ii) GUA

1

(ii) CUG

1

CUA (i) AUA

1

CUA (ii) GUA

1

CUA *Amber codon UAG; predicted amber anticodon CUA. t The posaible anticodom predicted by the wobble hypothesis are listed in this column. Codons AGU and AGC for ser are eliminated from consideration since a CUA anticodon cannot be formed by a point mutation in their tRNA.

codon UGA exists in the wild-type strain, the mutation to 8uI+,in contrast to the S U ~ I I + mutation, does not require the ser-tRNA species containing the anticodon CGA to be redundant. A similar situation exists in the case of the SUII+ gene. It may be possible to test the proposed mechanism of amber suppression as well as this prediction of the wobble hypothesis by fractionating su- ser-tRNA into its various species. The binding properties of each species may be determined by the binding assay of Nirenberg and Leder (1964). Similarly the 8Ur+ ser-tRNA may be fractionated and the distribution of suppressor activity determined using the in vitro assay (Capecchi and Gussin, 1965; Engelhardt et al., 1965) discussed in Section V1,C. The suppressor activity should cochromatograph with the species of su- ser-tRNA which codes exclusively for the ser codon UCG. If the suppressor activity co-chromatographed with the species of su- ser-tRNA coding for the other three ser codons, the predictions of the wobble hypothesis would be violated. The most direct test of the wobble hypothesis involves a comparison of the binding properties and the anticodon sequences of the various fractions of each tRNA. The results obtained so far, as shown in Tables 6 and 8, generally confirm this hypothesis,

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383

F. THEDEGENERACY OF tRNA SYNTHETASES It is difficult at this time to make an intelligent judgment regarding the degeneracy of the tRNA synthetases. A specific synthetase has been established for each of the 20 amino acids, and a large number of these have been purified (Brown, 1963; Bergmann et al., 1961). If the synthetase recognition of tRNA involves its anticodon site (as discussed in Section VII,A), some kind of degeneracy in the synthetases would be expected. Many previous studies with aminoacyl-tRNA synthetases in E . coli have failed to implicate more than a single synthetase for any amino acid (e.g., see Yamane and Sueoka, 1964; Baldwin and Berg, 1966). However, Yu (1966) has been able to demonstrate the presence of multiple forms of leucyl-tRNA synthetase, each of which appears to aminoacylate different species of leu-tRNA. In relatively more complex organisms, several examples of multiplicity of synthetases have been reported. Barnett and Epler (1966) separated two phenylalanyl-tRNA synthetases from Neurospora crassa, each aminoacylating a different phe-tRNA. Neurospora also seems to contain two separable aspartyltRNA synthetases functionally distinct with respect to their specificity for aminoacylation (Barnett and Epler, 1966). Although it has not been shown that these tRNA’s of Neurospora also differ in their coding specificities, the above experiments point to the possibility of some degeneracy among synthetases. This degeneracy might be based on the specificity of the synthetases toward the anticodons or may simply be due to an overall stereospecificity between other regions of the tRNA’s and their respective synthetases or be due to a combination of both. In this context, it is interesting to note the observations of Imamoto et al. (1965) on the patterns of cross-reactions of the two Neurospora phenylalanyl-tRNA synthetases with tRNA’s from various organisms. These studies indicated that one of the two enzymes is similar to its bacterial counterpart while the other is closer to the phenylalanyltRNA synthetase of higher organisms. The authors suggested that the enzyme similar to the bacterial enzyme might be an evolutionary remnant, or might well be confined to some organelle in the cytoplasm. Indeed, recent experiments indicate that Neurospora mitochondria contain a full complement of synthetases and that at least three of these (namely, aspartyl-, phenylalanyl-, and leucyl-tRNA synthetases) , along with their corresponding tRNA’s, are exclusively associated with mitochondria (Barnett et al., 1967). The aminoacylation specificities of mitochondria1 and cytoplasmic synthetases have been shown to be different by the above authors.

384

ANIL SADGOPAL

In the case of mammals, Strehler e t al. (1967) have demonstrated the existence of chromatographically separable multiple leucyl-tRNA synthetases in rabbit heart muscle; two of these synthetases preferentially attach leu to different species of tRNA. Interest in studies on multiplicities of synthetases is further heightened by experiments suggesting that different cell types from the same organism may contain different synthetases for an amino acid. For instance, while one of the ala-tRNA’s in rabbit is aminoacylated by enzymes from kidney, reticulocytes, and liver, the second ala-tRNA species is charged by enzymes present in liver and reticulocytes but not by enzymes present in kidney (Strehler et al., 1967). Based on this finding, the above authors have proposed a model of differentiation which envisages selective codon usage in different tissues. VIII. The Universality and Evolution of the Genetic Code

A. EVALUATION OF THE UNIVERSALITY OF CODON ASSIGNMENTS AND ANTICODONS Considerable evidence has been collected to support the universality of the codon assignments derived from in vitro studies on E. coli cell-free systems. (1) Hemoglobin synthesized in a cell-free system from rabbit reticulocytes by the use of amino acids bound to E . coli tRNA’s was found to be related t o the normal rabbit hemoglobin (von Ehrenstein and Lipmann, 1961). (2) RNA isolated from the f z coliphage, known to direct the synthesis of its coat protein in extracts of E. coli, also leads to synthesis of f 2 coat protein in extracts of a chloroplast-containing flagellate, Euglena gracilis (Schwartz et al., 1965). (3) Satellite tobacco necrosis virus RNA has been shown to lead to synthesis of viral-specific coat protein in a cell-free system of E. coli (Reichmann et al., 1966). (4) Serratia marcescens synthesized E . coli alkaline phosphatase under the direction of an E . coli episome (Signer et al., 1961). The properties of this enzyme were similar to those of the E. coli enzyme rather than the enzyme normally made by S e r r a t k (5) Bacillus subtilis infected with DNA from animal viruses, vaccinia or polyoma, produced complete particles of these viruses (Abel and Trautner, 1964; Bayreuther and Romig, 1964). The viruses obtained were biologically identical to conventionally grown viruses. (6) Good agreement exists between mutational amino acid replace-

THE GENETIC CODE

385

ment data from human hemoglobin and TMV coat protein and the codon assignments obtained from E. coli in vitro systems (see Section II1,C). (7) Several of the codon assignments have been confirmed in T4 by the study of frameshift mutants of phage lysozyme (see Section 111,A). (8) Binding or incorporation studies, or both, with synthetic polymers and trinucleotides have shown coding relationships similar to those of E . coli for the few amino acids tested in a variety of systems, such as rat liver and other mammalian systems (Maxwell, 1962; Arnstein et al., 1962; Weinstein et al., 1963), Chlamydomonas (Weinstein et al., 1963), yeast (Tanner, 1966; S611 et al., 1966; Marcus and Halvorson, 1963), wheat germ (Basilio et al., 1966), castor bean embryos (Parisi and Ciferri, 1966), and in several microorganisms, such as Bacillus (Friedman and Weinstein, 1964) and Staphylococcus (Mao, 1967). Speyer et al. (1963) showed that 22 triplets specified in a cell-free system from Alcaligenes faecal& (G C content = 66%) are the same as those in E. coli ( G + C content= 52%). (9) One of the most convincing arguments for a universal code comes from the work of Marshall et al. (1967), who examined the binding responses of tRNA’s from the livers of guinea pig and Xenopus laevis (an amphibian) to E. coli ribosomes under the direction of approximately 50 trinucleotides. By comparing these results with their previous results with E. coli tRNA’s, the authors concluded that mammalian, amphibian, and bacterial species have identical codon-amino acid relationships. However, it was demonstrated that guinea pig and Xenopus tRNA’s differ strikingly from the corresponding E. coli tRNA’s in their relative response to certain trinucleotides (Marshall et al., 1967). For example, amphibian and mammalian arg-tRNA responded well to AGG while E. coli arg-tRNA did not; a particular fraction of yeast argtRNA has been shown to respond well to AGA and AGG (see Table 6). Several other such differences in the relative response of tRNA’s from different species were also reported (Marshall et al., 1967). This evidence indicates that different synonym codons for an amino acid are used with varying frequencies in different organisms. Therefore, certain synonym codons with their appropriate tRNA’s may occur more frequently than others in a given organism. A different kind of variation in the tRNA’s is predicted by the wobble hypothesis. Section VI1,C has shown how the same set of codons for an amino acid may be recognized by different sets of tRNA’s in different species. It is tempting to make the generalization that while the codon assignments are essentially universal, the anticodons may

+

386

ANIL SADGOPAL

vary from species to species. Clearly, the wobble hypothesis permits the tRNA for an amino acid to mutate a t the 5'4erminal position of the anticodon in a certain pattern and yet code for the same amino acid, but certain mutations a t this position of the anticodon not allowed by the rules of the wobble hypothesis would lead t o deviations from the universality of the code. Obviously, any mutation in the other two positions of the anticodon will certainly alter codon-amino acid relationships. The altered coding properties of the amber suppressor tRNA's result from such mutations in the anticodon, constituting examples of minor deviations from universality.

B. THEPOSSIBILITY OF STRUCTURAL RELATIONSHIPS BETWEEN AMINOACIDSAND CODONS OR ANTICODONS Universality of the genetic code poses a major question regarding the nature of the ubiquitous evolutionary mechanism which produced it. Woese (1965a) speculated why a particular code word is assigned to its amino acid and on whether codon assignments would be different from the present ones if the genetic code had to evolve again. One way to approach this question is to explore the possibility of any kind of structural relationship existing directly between amino acids and their codons or anticodons. It has been argued by Weinstein (1963) that since the tRNA's and tRNA synthetases are themselves under genetic control, their specificity would be subject to genetic mutation which would change the genetic code in different species. However, a stereochemical relationship between amino acids and their codons or anticodons would ensure maintenance of the code in the face of numerous random mutations (Weinstein, 1963). Pelc (1965) has pointed out the following structural correlations between codons and amino acids: amino acids with hydrocarbon residues have U or C as the second base; those with branched methyl groups have U as the second base; and the basic and acidic amino acids have A or G as the second base. Similar structural correlations have been described by Woese et al. (1966) also, In addition, there are several structurally related amino acids which can change from one to another by single-base changes in their codons (Pelc, 1965; Epstein, 1966). Phe, leu, ilu, and Val can be interconverted by altering the first base of their codons; shifts in the second base can lead t o such interchanges as phe and tyr, ala and val, ala and gly, lys and arg; changes in the third base can accomplish shifts from asp to glu and asN to lys. It was argued (Pelc, 1965) that such a logical code arrangement

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could not have arisen by chance alone and a structural relationship between codons and amino acids seemed likely. Despite the above-mentioned correlations, not much convincing evidence in favor of such stereochemical relationships has been presented so far. Woese e t al. (1966) have presented data showing that amino acids which have been assigned similar codons behave similarly in certain chromatographic systems. Although these data imply “relatedness” among amino acids assigned similar codons, the relevance of such correlations t o the genetic code is not obvious. Felc and Welton (1966) attempted to fit the Courtaulds space-filling models of amino acids into those of nucleotide triplets by means of hydrogen and covalent bonds and stacking interactions. They reported that successful fits of 90% of the codon-amino acid complexes required by the genetic code could be achieved with a high degree of specificity without introducing undue strain (Pelc and Welton, 1966; Welton and Pelc, 1966). A drawback of this work is the use of chemically unusual covalent bonds between the methyl groups of amino acids with hydrocarbon side chains and some part of the bases. Recently, Crick (1967b) pointed out three major reasons for the unacceptability of these models on stereochemical grounds, and further observed that the polynucleotide sequences in these models ran backwards (see Woese et al., 1966, for further comments on the validity of these models). Two mechanisms have been proposed (Welton and Pelc, 1966) to reconcile such a stereochemical relationship, if it exists a t all, with our present model of protein synthesis; however, both can be eliminated from consideration. The first suggests the presence of a codon sequence, identical to the mRNA codon for that amino acid, a t a particular position on the tRNA. This codon on the tRNA would select the specific amino acid for attachment by the synthetase to the -CCA end. However, the nucleotide sequence of yeast tyr-tRNA (Madison et al., 1966) lacks both t y r codons. The second possibility, that the amino acid attached to the tRNA may help in recognizing the codon on the mRNA by stereochemical interaction, is eliminated by experiments demonstrating that chemical conversion of one amino acid to another after it is on the tRNA does not alter the coding properties of the tRNA (Chapeville et al., 1962; von Ehrenstein et al., 1963). A structural relationship between nucleotides and amino acids may involve the anticodon of tRNA, rather than the codon, which would provide a cage to bind amino acids as their acyladenosine monophosphates in a position favorable for establishing the bond to the -CCA end with the aid of synthetase (Weinstein, 1963; Dunnill, 1966). The use of the same site on the tRNA for recognizing the amino acid as

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well as the codon would greatly restrict the changes in the genetic code due to mutations during evolution. Although no direct evidence for such a relationship is available, the suggested model in no way seems to violate the established mechanism for protein synthesis. An anticodon-amino acid relationship would be difficult to eliminate were it not for the proposition that the amber suppressor mutation alters one of the three bases of an anticodon and yet allows the affected tRNA to carry the same amino acid. Experiments on the effects of specific mutations of tRNA synthetases also cast serious doubts on the existence of a role for such stereochemical relationships and further demonstrate that the synthetase specifically recognizes an amino acid without the apparent aid of a nucleotide sequence on tRNA. In an ethionine-resistant strain of the basidiomycete fungus Coprinus lagopus, differing from the wild type in its inability to incorporate ethionine (an analog of met) into proteins, the met-tRNA synthetase did not activate ethionine (Lewis, 1963). Similarly, in a mutant of E . coli resistant to p-fluorophenylalanine, a phe analog, the resistance was found to be due t o the inability of the phetRNA synthetase to activate this analog (Fangman and Neidhardt, 1964). It seems then that if any structural relationship does exist between nucleotides and amino acids, it could only have played a role in the evolution of the code before the adaptor system was completely evolved.

C. IDEAS ON THE EVOLUTION OF THE CODE Two outstanding features arise from an examination of the genetic code : (I) the code is highIy degenerate with a minimum number of nonsense codons, probably only those essential for punctuation of the message; (2) amino acids having similar structural and functional properties are assigned related codons, ensuring that one-step mutations would often replace an amino acid with a similar one. Sonneborn (1965) proposed that a highly ordered and degenerate code could arise by minimizing the lethal effects of mutations during evolution. Selection pressures would lead to (a) the elimination of nonsense codons, (b) the optimization of the frequency of mutations which involve a change in the codon not producing a change in the amino acid (i.e., synonymous or “silent” mutations), and (c) the optimization of the frequency of mutations causing an amino acid to be replaced

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by one functionalIy related to it. Such a code, once developed, would become frozen and universal since any change would be likely to result in lethality. I n an alternate scheme, Woese (1965b) suggested that in a primitive cell with an inefficient translational mechanism the frequency of ambiguous codons was so high that the probability of translating a genetic message correctly was almost zero. In such cells the proteins produced by any given gene were “statistical proteins,” deviating in primary structure from some theoretical mean. Woese proposed that such cells recognized only groups of related amino acids, such as a “nonfunctional” group and a “functional” group. The early proteins were probably very dependent on the position and kind of the “functional” amino acids, using the “nonfunctional” ones to provide a hydrophobic environment. The primitive cell, it was proposed, evolved by accepting the least error-prone purine-rich codons (cf. Section VI,B on the pattern of ambiguity) for the “functional” amino acids, leaving the more errorprone pyrimidine-rich codons for the others. Thus, this scheme envisages that a primitive cell can evolve the highly ordered code by minimizing the effects of translational errors. As Woese noted, the present code maximizes the probability that (a) an error in reading the more errorprone terminal base of a codon would lead to no change in the amino acid, and (b) if a mistake in reading a codon does lead to the introduction of an incorrect amino acid, it will often be functionally and structurally related to the correct one. A similar scheme has also been forwarded by Goldberg and Wittes (1966). A scheme proposed by Jukes (1965) assumes that the proteins in a primitive cell consisted of only 15 amino acids coded by an archetypal doublet code. The code consisted of 15 doublets coding for 15 amino acids and the 16th acting as a gap between genes. According to this suggestion, five of the amino acids are of later origin and the triplet code evolved by adding a third base to each doublet codon. According to an alternate possibility suggested by Crick (1967a), in a primitive code a small number of amino acids were coded for by a small number of triplets. At a more advanced stage the early amino acids became related t o most of the triplets in order to reduce nonsense triplets to a minimum. It was pointed out that a new amino acid was incorporated into the code only if its introduction conferred a selective advantage. Crick also noted that the new amino acid was likely to have been related to the one previously coded by the triplets being shared. The code was frozen when the number and structure of proteins became Sophistic a k d enough SO that no possible new amino acid could add a selective advantage.

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ACKNOWLEDGMENTS I thank Prof. James Bonner for his continuous encouragement and numerous helpful suggestions; John Penswick, whose stimulating comments led to deeper probing of several problems; my co-workers Keiji Marushige, Michael Dahmus, Martin Pall, David Kabat, and Roger Chalkley for many constructive criticisms of the manuscript, and especially m y friend and colleague Sandra Winicur for ruthless and painful overhauling of the original draft. Also, I am indebted to Mary Dakin, who not only cheerfully shared many difficult momenta during the development of this review, but also willingly went through extended periods of preparing the bibliography and proofreading the manuscript. This work was supported by the Lucy Mason Clark Fund.

REFERENCES Abel, P., and Trautner, T. A. 1964.Formation of an animal virus within a bacterium. 2. Vererbungslehre 95, 6672. Adams, A.,Lindahl, T., and Fresco, J. R. 1967. Conformational differences between the biologically active and inactive forms of a transfer ribonucleic acid. Proc. Natl. Acad. Sci. U S . 57,1684-1691. Adams, J. M.,and Capecchi, M. R. 1966.N-formylmethionyl-sRNA as the initiator of protein synthesis. Proc. Natl. Acad. Sci. US. 55, 147-165. Allfrey, V. G., Faulkner, R., and Mirsky, A. E. 1964. Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc. Natl. Acad. Sci. U S . 51, 786-794. Alpers, D. H.,and Tomkins, G. M. 1966. Sequential transcription of the genes of the lactose operon and its regulation by protein synthesis. J. BioE. Chem. 241, 4434-4443. Ames, B. N.,and Hartman, P. E. 1963. The histidine operon. Cold Spring Harbor Symp. Quant. Biol. 28, 349356. Anderson, W. F., Gorini, L., and Breckenridge, L. 1966. Role of ribosomes in streptomycin-activated suppression. Proc. Natl. Acad. Sci. U S . 54, 10761083. Apgar, J., and Holley, R. W. 1962. Differences between yeast and rat-liver amino acid-specific “soluble” ribonucleic acids shown by countercurrent distribution. Biochem. Biophys. Res. Commun. 8, 391-393. Apgar, J., Holley, R. W., and Merrill, S. H. 1961. Countercurrent distribution of yeast “soluble” ribonucleic acids in a modification of the Kirby system. Biochim. Biophys. Aeta 53, 220-221. Arlinghaus, R., Schaeffer, J., and Schweet, R. 1964. Mechanism of peptide bond formation in polypeptide synthesis. Proc. Natl. Acad. Sci. US. 51, 1291-1299. Arnstein, H. R. V., Cox, R. A., and Hunt, J. A. 1962. Function of polyuridylic acid and ribonucleic acid in protein biosynthesis by ribosomes from mammalian reticulocytes. Nature 194, 1042-1044. Baglioni, C. 1963. Correlations between genetics and chemistry of human hemoglobins. In “Molecular Genetics” (J. H. Taylor, ed.), Part 1, pp. 405476. Academic Presa, New York. Baldwin, A. N., and Berg, P. 1966. Purification and properties of isoleucyl ribonucleic acid synthetase from Escherichk coli. J. Biol. Chem. 241, 831-838. Barnett, W. E., and Brown, D. H. 1967. Mitochondria1 transfer ribonucleic acids. Proc. Natl. Acad. Sci. US.57, 462-468.

THE GENETIC CODE

391

Barnett, W. E., and Epler, J. L. 1966.Fractionation and specificities of two aspartylribonucleic acid and two phenylalanyl-ribonucleic acid synthetases. Proc. Natl. Acad. Sci. U.S. 55, 184-189. Barnett, W. E., Brown, D. H., and Epler, J. L. 1967. Mitochondrial-specific aminoawl-RNA synthetases. Proc. Natl. Acad. Sci. U S . 57, 1776-1781. Basilio, C., Bravo, M., and Allende, J. E. 1966. Ribonucleic acid code words in wheat germ. J. Biol. Chem. 241,1917-1919. Bayreuther, K.E.,and Romig, W. R. 1964. Polyoma virus: Production in Bacillus subtilis. Science 146,7784'79. Beale, D., and Lehmann, H.1965. Abnormal haemoglobins and the genetic code. Nature 207,259-261. Bennett, T. P., Goldstein, J., and Lipmann, F. 1965. Coding and charging specificities of sRNA's isolated by countercurrent distribution. Proc. Natl. Acad. Sci. U5.53,385392. Benzer, S. 1961. On the topography of the genetic fine structure. Proc. Natl. Acad. Sd. US.47,403-416. Benzer, S., and Champe, S. P. 1962. A change from nonsense to sense in the genetic code. Proc. Natl. Acad. Sci. UB.48,1114-1121. Berberich, M. A., Venetianer, P., and Goldberger, R. F. 1966. Alternative modes of derepression of the histidine operon observed in Salmonella typhimurium. J. Biol. Chem. 241,4426-4433. Berberich, M. A., Kovach, J. S., and Goldberger, R. F. 1967. Chain initiation in a polycistronic message : sequential versus simultaneous derepression of the enzymes for histidine biosynthesis in Salmonella typhimurium. Proc. Natl. Acad. Sci. U.S. 57, 1857-1864. Berg, P. 1961. Specificity in protein synthesis. Ann. Rev. Biochem. 30, 293-324. Berg, P., Bergmann, F. H., Ofengand, E. J., and Dieckmann, M. 1961. The enzymic synthesis of aminoacyl derivatives of ribonucleic acid. I. The mechanism of leucyl-, valyl-, isoleucyl-, and methionyl ribonucleic acid formation. J . Biol. Chem. 236,1726-1734. Bergmann, F. H.,Berg, P., and Dieckmann, M. 1961. The enzymic synthesis of aminoacyl derivatives of ribonucleic acid. 11. The preparation of leucyl-, valyl-, isoleucyl-, and methionyl ribonucleic acid synthetases from Escherichia coli. J . Biol. Chem. 236, 1736-1740. Bergquist, P. L., and Capecchi, M. R. 1966. Fractionation of a suppressor sRNA. J . Mol. Biol. 19, 202-206. Bernfield, M. R.,and Nirenberg, M. W. 1965. RNA codewords and protein synthesis. The nucleotide sequences of multiple codewords for phenylalanine, serine, leucine, and proline. Science 147, 479484. Biasell, D. M. 1966. Formation of an altered enzyme by Escherichia coli in the presence of neomycin. J. Mol. Biol. 14, 619-622. Bock, R. M. 1967a. Prediction of a topaz suppressor ribonucleic acid. Circulated as memo No. 783 in Information Exchange Group No. 7, Natl. Inst. Health, Bethesda, Maryland. Bock, R. M. 196%. Personal communication. Brenner, S. 1957. On the impossibility of all overlapping triplet codes in information transfer from nucleic acid to proteins. Proc. Natl. Acad. Sci. U.S. 43, 687-694. Brenner, S., and Beckwith, J. R. 1965. Ochre mutants, a new class of suppressible nonsense mutants. J. Mol. Biol. 13, 629-637.

392

ANIL SADGOPAL

Bremer, s., Stretton, A. 0.W., and Kaplan, s. 1966. Genetic Code: The ‘ ~ O I I S ~ triplets for chain termination and their suppression. Nature 206, 994-998. Brenner, s., Kaplan, S., and Stretton, A. 0. 1966. Identity of N2 and ochre nomewe mutants. J. Mol. BioZ. 19,674476. Bremer, s., Barnett, L., Katz, E. R., and Crick, 3’. H. c. 1967. UGA: A third nonsense triplet in the genetic code. Nature 21% 449-460Bretwher, M. S., and Grunberg-Manago, M. 1962. Polyribonucleotide-directed protein synthesis using an E. coli cell-free system. Nature 195, 283-284. Bretscher, M. S., and Marcker, K. A. 1966. Polypeptidyl-s-ribonucleic acid and amino-acylsribonucleic acid binding sites on ribosomes. Nature 211, 380-384. Bretscher, M. S.,Goodman, H. M., Menninger, J. R., and Smith, J. D. 1965. Polypeptide chain termination using synthetic polynucleotides. J. Mol. Biol. 14,

w.

634-639.

Brimacombe, R., ”mpin, J., Nirenberg, M., Leder, P., Bernfield, M.,and Jaouni, T. 1966. RNA codewords and protein synthesis, VIII. Nucleotide sequences of synonym codone for arginine, valine, cysteine, and alanine. Proc. Natl. Acad. Sci. U S . 54,964-960. Brown, G. L. 1963.Preparation, fractionation, and properties of sRNA. Progr. Nucleic Acid Res. 2, 269-310. Brownstein, B. L., and Lewandowski, L. J. 1967. A mutation suppressing atreptomycin dependence. I. An effect on ribosome function. J, Mol. Biol. 26, 99-109. Campbell, P.N. 1966.The biosynthesis of proteins. Progr. Biophys. Mol. Biol. 15,

1-38.

Capecchi, M. R., and Gussin, G. N. 1966. Suppression in vitro: Identification of a serine-sRNA as a “nonsense” suppressor. Science 149, 417-422. Capra, J. D., and Peterkofsky, A. 1966. A difference between ‘Lnormal” and “methyl-deficient” E. coli sRNA in polynucleotide-directed binding of leucylsRNA to ribosomes. Federation Proc. 25, 403. Carbon, J. 1964. The amino acid recognition and ribosome combining sites of E. coli transfer RNA. Biochem. Biophys. Res. Commun. 15, 1-7. Carbon, J., Berg, P., and Yanofsky, C. 1966a. Studies of missense supprmion of the tryptophan wthetase A-protein mutant A36. Proc. Natl. Acad. Sci. U.S.

56,764-771.

Carbon, J., Berg, P., and Yanofsky, C. 1966b. Missense SUppreSsiOD due to a genetically altered tRNA. Cold Spring Harbor Symp. Quant. Biol. 3,487-497. Caskey, C. T., Redfield, B., and Weissbach, H. 1967. Formylation o guinea pig liver methionyl-sRNA. Arch. Biochem. Biophys. 120, 119-12s Champe, S. P.,and Benser, S. 1962. An active cistron fragment. J:MoZ. Biol. 4,

288-292.

ChaPville, F., LiPmann, F.,von Ehrenstein, G., Weisblum, B,,Rv, W. J., Jr., and Benser, s. 1962. On the role of soluble ribonucleic acid in coding for amino acids. Proc. Natl. Acad. Sci. U.S. 48, 1086-1092. Cherayil, J. D., and Bock, R. M. 1966. A column chromatograp procedure for the fractionation of sRNA. Biochemistry 4, 1174-1183. Clark, €3. c.,and Marcker, K. A. 1966.Coding response pf’ N-formyl-nethionyIsRNA to UUG. Nature 207, 1038-1039. Clark €3. F-c.,and Marcker, K. A. 1966a.The role of N-formy1-methionJ-&NA in protein biosynthesis. J. Mol. Biol. 17, 394-406. Clark, €4. F. c., and Marcker, K. A. 1966b. N-formyl-methionyl-s-ribonuckic acid

,

~ ’

THE QENETIC CODE

393

and chain initiation in protein biosynthesis : Polypeptide synthesis directed by a bacteriophage ribonucleic acid in a cell-free system. Nature 211, 378-379. Cox, E.C., White, J. R., and Flaks, J. G. 1964. Streptomycin action and the ribosome. Proc. Natl. Acad. Sci. U S . 51, 703-709. Crick, F. H. C. 1957. Discussion. I n “The Structure of Nucleic Acids and Their Role in Protein Synthesis” (E. M. Crook, ed.), pp. 25-26. Cambridge Univ. Press, London and New York. Crick, F. H. C. 1963. The recent excitement in the coding problem. Progr. Nucleic Acid Res. 1, 163-217. Crick, F. H. C. 1965. Codon-anticodon pairing : The wobble hypothesis. Circulated as Memo No. 14 in Information Exchange Group No. 7, Natl. Inst. Health, Bethesda, Maryland. Crick, F. H. C. 1966.Codon-anticodon pairing: The wobble hypothesis. J . Mol. Biol.

19,548-555.

Crick, F.H. C. 1967a.Origin of the genetic code (correspondence). Nature 213, 119 Crick, F. H. C. 196’7b.An error in model building. Nature 213, 798. Crick, F. H. C. 1967c. Personal communication. Crick, F. H. C., Griffith, J. S., and Orgel, L. E. 1967. Codes without commas. Proc. Natl. Acad. Sci. US. 43, 416-421. Crick, F. H. C., Barnett, L., Brenner, S., and Watts-Tobin, R. J. 1961. General nature of the genetic code for proteins. Nature 192, 1227-1232. Davies, J. 1964. Studies on the ribosomes of streptomycin-sensitive and resistant strains of Escherichia coli. Proc. Natl. Acad. Sci. US. 51, 659-664. Davies, J., Gilbert, W., and Gorini, L. 1964. Streptomycin, suppression and the code. Proc. Natl. Acad. Sci. US.51, 883-890. Davies, J., Jones, D. S., and Khorana, H. G. 1966. A further study of misreading of codons induced by streptomycin and neomycin using ribopolynucleotides containing two nucleotides in alternating sequence as templates. J. Mol. Biol.

18, 48-67.

Doctor, B. P., Apgar, J., and Holley, R. W. 1961. Fractionation of yeast amino acid-acceptor ribonucleic acids by countercurrent distribution. 3. Biol. Chem.

236,1117-1120.

Dunnill, P. 1966. Triplet nucleotide-amino-acid pairing; a stereochemical basis for the division between protein and non-protein amino-acids. Nature 210, 1267-

1268.

Eck, R. V. 1963. Genetic code: Emergence of a symmetrical pattern. Science 140,

477480.

Eisenstadt, J., and Lengyel, P. 1966. Formylmethionyl-tRNA dependence of amino acid incorporation in extracts of trimethoprim-treated Eschen’chia coli. Science

154,524-527.

Engelhardt, D. L.,Webster, R. E., Wilhelm, R. C., and Zinder, N. D. 1965. In vitro studies on the mechanism of suppression of a nonsense mutation. Proc. Natl. Acad. Sci. U.S. 54, 1791-1797. Epstein, C. J. 1966.Role of the amino-acid ‘code’ and of selection for conformation in the evolution of proteins. Nature 210, 25-28. Epstein, R. H.,Bolle, A,, Steinberg, C. M., Kellenberger, E., Boy de la Tour, E., Chevalley, R., Edgar, R. S., Susman, M., Denhardt, G. H., and Lielausis, A. 1963. Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28, 375-392.

394

ANIL SADGOPAL

Fangman, W. L., and Neidhardt, F. C. 1964.Demonstration of an altered aminoacyl ribonucleic acid synthetase in a mutant of Escherichia coli. J . Biol. Chem.

239,1839-1843.

Flab, J. G., Cox, E. C., Witting, M. L., and White, J. R. 1962. Polypeptide synthesis with ribosomes from streptomycin-resistant and dependent E . coli. Biochem. Bwphys. Res. Commun. 7,390-393. Fowler, A. V., and Zabin, I. 1966. Co-linearity of p-galactosidase with its gene by immunological detection of incomplete polypeptide chains. Science 154, 1027-

1029.

Friedman, 8. M., and Weinstein, I. B. 1964. Lack of fidelity in the translation of synthetic polyribonucleotides. Proc. Natl. Acad. Sci. US. 52, 988-996. Funatsu, G., and Fraenkel-Conrat, H. 1964. Location of amino acid exchanges in chemically evoked mutants of tobacco mosaic virus. Biochemistry 3, 1356-1362. Gallucci, E.,and Garen, A. 1966.Suppressor genes for nonsense mutations. 11. The Su-4 and Su-6 suppressor genes of Esherichia coli. J . Mol. Bwl. 15, 193-200. Gamow, G. 1954. Possible relation between deoxyribonucleic acid and protein structures. Nature 173, 318. Gamow, G., Rich, A., and YEW, M. 1956. The problem of information transfer from the nucleic acids to proteins. Advan. Biol. Med. Phys. 4, 23-68. Ganoza, M. C., and Nakamoto, T. 1966. Studies on the mechanism of polypeptide chain termination in cell-free extracts of E . coli. Proc. Natl. Acad. Sci. U S .

55, 162-169.

Garen, A., and Siddiqi, 0. 1962. Suppression of mutations in the alkaline phosphatase structural cistron of E. cold. Proc. Natl. Acad. Sci. U S . 48, 1121-1127. Garen, A., Garen, S., and WiIhelm, R. C. 1965. Suppressor genes for nonsense mutations. I. The Su-1, Su-2 and SuJ genes of Eschenchia coli. J . Mol. Biol.

14,167-178.

Geiduschek, E. P. 1961. “Reversible” DNA. PTOC.Natl. Acad. Sci. U S . 47, 950-955. Ghosh, H. P., 5811, D., and Khorana, H. G. 1967. Studies on polynucleotides. LXVII. Initiation of protein synthesis in vitro as studied by using ribopolynucleotides with repeating nucleotide sequences as messengers. J. Mol. Biol. 25,

275-298.

Giacomoni, D.,and Spiegelman, 8. 1962. Origin and biologic individuality of the genetic dictionary. Science 138, 1328-1331. Gilbert, W. 1963. Polypeptide synthesis in Escherichia coli. 11. The polypeptide chain and s-RNA. J . Mol. Biol. 6,389-403. Goldberg, A. L.,and Wittes, R. E. 1966. Genetic code: Aspects of organization. Science 153,420-424. Goldstein, J., Bennett, T. P., and Craig, L. C. 1964. Countercurrent distribution studies of E. coli B sRNA. Proc. Natl. Acad. Sci. US.51, 119-125. Goodman, € M., I.and Rich, A. 1962. Formation of a DNA-soluble RNA hybrid and its relation to the origin, evolution, and degeneracy of soluble RNA. Proc. Natl. Acad. 815.US.48,2101-2109. Gorini, L., and Kataja, E. 1964. Phenotypic repair by streptomycin of defective genotypes in E . coli. Proc. Natl. Acad. Sci. UB.51, 487-493. Gorini, L.,Gunderson, W., and Burger, M. 1961. Genetics of regulation of enzyme synthesis in the arginine biosynthetic pathway of Escherichia coli. Cold Spring Harbor Symp. Quant. Biol. 26, 173-182. Gorini, L., Jacoby, G. A., and Breckenridge, L. 1966. Ribosomal ambiguity. Cold Spring Harbor Symp. Quant. Biol. 31, 657-664.

THE GENETIC CODE

395

rGottschling, H., and Zachau, H. G. 1965. Ultraviolett-insktivierung der phenylalanin- und lysinspezifischen t r a n s f e r - r i b o n u c l e i . Biochim. Biophys. Acta

103,418-430.

Grunberg-Manago, M., and Dondon, J. 1965. Influence of pH and s-RNA concentration on coding ambiguities. Biochem. Biophys. Res. Commun. 18, 517-622. Grunberg-Manago, M.,and Michelson, A. M. 1964. Polynucleotide analogues. 11. Stimulation of amino acid incorporation by polynucleotide analogues. Biochim. Biophys. Acta 80, 431-440. ,Guest, J. R., and Yanofsky, C. 1966. Relative orientation of gene, messenger and polypeptide chain. Nature 210,799-802. ,Gupta, N. K., and Khorana, R. G. 1966. Missense suppression of the tryptophan synthetase A-protein mutant A78. Proc. Natl. Acad. Sci. US.56, 772-779. Gupta, N. K.,RajBhandary, U. L., and Khorana, H. G. 1966. Mimense suppression in tryptophan synthetase. Cold Spring Harbor Symp. Quant. Biol. 31, 499-500. Gussin, G. N., Capecchi, M. R., Adams, J. M., Argetsinger, J. E., Tooze, J., Weber, K., and Watson, J. D. 1966. Protein synthesis directed by RNA phage messengers. Cold Spring Harbor Symp. Quant. Bdol. 31, 267-271. Haenui, A.-L., and Chapeville, F. 1966. The behavior of acetylphenylalanyl soluble ribonucleic acid in polyphenylalanine synthesis. Bwchim. Biophys. Acta 114,

135-148.

Harriman, P. D., and Zachau, H. G. 1966. Ultraviolet inactivation of transfer ribonucleic acid functions. J. Mol. Biol. 16, 387-403. Hawtrey, A. O., and Biedron, S. I. 1966. Release of peptide chains from rat-liver polysomes by the N,N-dimethyl-p-methoxy-1-phenylalanylanalogue of puromycin. Nature 211, 187-189. Hayashi, H., and Miura, K. 1966. Functional sites in transfer ribonucleic acid. Nature 209,376-378. Henning, U.,Dennerb, G., Szolyvay, K., and Deppe, G. 1966. Amber mutants in the pyruvate dehydrogenase system of Escherichia coli K12. J . Mot. Biol. 13,

692-595.

Hoagland, M. B., Stephenson, M. L., Scott, J. F., Hecht, L. I., and Zamecnik, P. C. 1968. A soluble ribonucleic acid intermediate in protein synthesis. J. Biol. Chem. 231,241-257. Holley, R. W , Apgar, J., Everett, G. A., Madison, J. T., Merrill, S. H., and Zamir, A. 1963. Chemistry of amino acid-specific ribonucleic acids. Cold Spring Harbor Symp. Quant. Bwl. 28,117-121. Holley, R. W.,Apgar, J., Everett, G. A., Madison, J. T., Marquisee, M., Merrill, S. H., Penswick, J. R., and Zamir, A. 1966. Structure of a ribonucleic acid. Science 147,1462-1466. Borikoshi, K., and Doi, R. H. 1967.The N-terminal amino acids of Bacillus subtilis proteins. Federation Proc. 26, 457. Hoskinson, R. M., and Khorana, H. G. 1965. Studies on polynucleotides. XLI. Purification of phenylalanine-specific transfer ribonucleic acid from yeast by countercurrent distribution. J. BioZ. Chem. 240, 2129-2134. Hutchison, C. A., 111, and Sinsheimer, R. L. 1966. The procew of infection with bacteriophage *X174. X. Mutations in a *X lysis gene. J . Mol. B i d . 18,

429-447.

Imamoto, F., Yamane, T., and Sueoka, N. 1966. Existence of two phenylalanylsRNA synthetases in Neurospora crassa. Proc. Natl. Acad. Sci. US.53, 1456-

1462.

396

ANIL SADGOPAL

Ingram, V. M., and SjGquist, J. A. 1963. Studies on the structure of purified alanine and valine transfer RNA from yeast. Cold Spring Harbor Symp. Quant. Biol.

28,183-138.

Jones, D. S., Nishimura, S., and Khorana, H. G. 1966. Studies on polynucleotides. LVI. Further syntheses, in vitro, of copolypeptides containing two amino acids in alternating sequence dependent upon DNA-like polymers containing two nucleotides in alternating sequence. J . Mol. Biol. 16, 464-472. Jukes, T. H. 1962. Possible base sequences in the amino acid code. Biochem. Biophys. Res. Commun. 7, 497-602. Jukes, T. H. 1966. Coding tripleta and their possible evolutionary implications. Biochem. Biophys. Res. Commun. 19,391-396. Kaji, H.,and Kaji, A. 1966. Specifio binding of sRNA to ribosomes: Effect of streptomycin. Proc. Natl. Acad. Sci. U S . 54, 213-219. Kaneko, I., and Doi, R. H. 1966. Alteration of valyl-sRNA during sporulation of Bacillus subtilis. Proc. Natl. Acad. Sci. U S . 55,664-671. Kaplan, S., Stretton, A. 0. W., and Brenner, 5. 1966. Amber suppressors: EXiciency of chain propagation and suppressor specific amino acids. J . Mol. Biol. 14,

628433.

Karasek, M. 1963. Changes in free and acetylated amino acids during tobacco mosaic virus multiplication. Biochim. Biophys. Acta 78. 644448. Karau, W., and Zachau, H. G. 1964. Isolierung von serinspeaifischen transferribonucleinsaure-fraktionen.Biochim. Biophys. Acta 91, 649468. Kellogg, D. A., Doctor, B. P., Loebel, J. E., and Nirenberg, M. W. 1966. RNA codons and protein synthesis, IX. Synonym codon recognition by multiple species of valine-, almine-, and methionine-sRNA. Proc. Natl. Acad. Sci. US.

55,912-919.

Khorana, H.G. 1966. Polynucleotide synthesis and the genetic code. Federation Proc. 24,1473-1487. Kilmartin, J. V.,and Clegg, J. B. 196'7.Amino-acid replacements in horse haemoglobin. Nature 213, 269-271. Kossel, H., Morgan, A. R., and Khorana, H. G. 1967. Studies on polynucleotides. LXXIII. Synthesis in vitro of polypeptides containing repeating tetrapeptide sequences dependent upon DNA-like polymers containing repeating tetranucleotide sequences: Direction of reading of messenger RNA. J . Mol. Biol.

26,449-475.

Kolakofsky, D., and Nakamoto, T. 1966. The initiation of viral protein synthesis in E. coli extracts. Proc. Natl. Acad. Sci. US.56, 1786-1793. Lamfrom, H., McLaughlin, C. S., and Sarabhai, A. 1966. Direction of reading the genetic message in reticulocytes. J . Mol. Biol. 22, 366-368. Last, J. A., Stanley, W. M., Jr., Salas, M., Hille, M. B., Wahba, A. J., and Ochoa, S. 1967. Translation of the genetic message, IV. UAA as a chain termination codon. Proc. Natl. Acad. Sci. US. 57, 1062-1067. Leder, P.,and Bursztyn, H. 1966.Initiation of protein synthesis, I. Effect of formylation of methionyl-tRNA on codon recognition. Proc. Natl. Acad. Sci. US. 56,

1679-1686.

Lederberg, E. M., Cavalli-Sforza, L., and Lederberg, J. 1964. Interaction of streptomycin and a suppressor for galactose fermentation in E . coli K-12. Proc. Natl. Acad. Sci. US. 51,678-682. Lerman, L. 8. 1961. Structural considerations in the interaction of DNA and acridines. J. Mol. Biol. 3, 18-30.

THE GENETIC CODE

397

Lerman, L. S. 1963. The structure of the DNA-acridine complex. Proc. Natl. A d . Sci. U S . 49,94-102. Letendre, C., Michelson, A. M., and Grunberg-Manago, M. 1966. Oligonucleotide inhibition of amino acid attachment. Cold Spring Harbor Sump. Quant. Biol.

31371-76.

Lewis, D. 1963. Structural gene for the methionine-activating enzyme and its mutation as a cam of resistance to ethionine. Nature 200, 161-164. Likover, T.E.,and Kurland, C.G. 1967. Ribosomes from a streptomycin-dependent strain of Escherichia coli. J . Mol. Biol. 25, 497-604. Lindahl, T., Adams, A., Geroch, M., and Fresco, J. R. 1967. Selective recognition of the native conformation of transfer ribonucleic acids by enzymes. Proc. Natl. Acad. Sci. U S . 57,178-186. Loftfield, R. B., and Eigner, E. A. 1963. Species specificity of transfer RNA. Acta Chem. Scand. 17, Suppl., 117-122. Zucas-Lenard, J., and Lipmann, F. 1967. Initiation of polyphenylalanine synthesis by N-acetylphenylalanyl-sRNA. Proc. Natl. Acad. Scd. US. 57, 1060-1067. Madison, J. T.,Everett, G. A,, and Kung, H. 1966. Nucleotide sequence of a yeast tyrosine transfer RNA. Science 153,631-634. Mao, J. C.-H. 1967. Protein synthesis in a cell-free extract from Staphylococcus aureua. J . Bacterial. 94, 00-86. Marchis-Mouren, G., and Lipmann, F. 1966. On the mechanism of acetylation of fetal and chicken hemoglobins. Proc. Natl. Acad. Sci. US.53, 1147-1164. Marcker, K. 1966. The formation of N-formylmethionyl-sRNA. J. Mol. Bwl. 14,

63-70.

Marcker, K.,annd Sanger, F. 1964. N-formylmethionyl-sRNA. J. Mol. Bwl. 8,

036-040.

Marcus, L., and Halvorson, H. 0. 1963. An active cell-free amino acid incorporating system from yeast. Federation Proc. 22, 302. Marshall, R. E., Caskey, C. T., annd Nirenberg, M. 1967. Fine structure of RNA codewords recognized by bacterial, amphibian, and mammalian transfer RNA. Science 155,820-826. Martin, R. G.,Silbert, D. F., Smith, D. W. E., and Whitfield, H. J., Jr. 1966. Polarity in the histidine operon. J . Mol. Biol. 21, 367-369. Matthaei, J. H.,Jones, 0. W., Martin, R. G., and Nirenberg, M. W. 1962. Characteristics and composition of RNA coding units. Proc. Natl. Acad. Sci. US.48,

666-677.

Maxwell, E. S. 1962. Stimulation of amino acid incorporation into protein by natural and synthetic polyribonucleotides in a mammalian cell-free system. Proc. Natl. Acad. Sci. US. 48,1639-1643. Morgan, A. R., Wells, R. D., and Khorana, H. G. 1966. Studies on polynucleotides, LIX. Further codon assignments from amino acid incorporations directed by ribopolynucleotides containing repeating trinucleotide sequences. Proc. Natl. Acad. Sci. U S . 56,1899-1906. Morgan, A. R., Wells, R. D., and Khorana, H. G. 1967. Studies on polynucleotides. LXXIV. Direct translation in vitro of single-stranded DNA-like polymers with repeating nucleotide sequences in the presence of neomycin B. J. Mol. Biol. 26,

477-497.

Muench, K. H.,and Berg, P. 1966a. Fractionation of t r a d e r ribonucleic acid by gradient partition chromatography on sephadex columns. Biochemistry 5, 970-

981.

398

ANIL SADGOPAL

Muench, K. H., and Berg, P. 1966b. Resolution of aminoacyl transfer ribonucleic acid by hydroxylapatite chromatography. Biochemistry 5 , 982-987. Murayama, M. 1962. Chemical difference between normal human haemoglobin and haemoglobin-I. Nature 196, 276-277. Muto, A,, Miura, K., Hayatsu, H., and Ukita, T. 1966. Effect of semicarbazide modification on the amindacylation of transfer ribonucleic acid. Biochim. Biophys. Acta 95, 669-671. Nakamoto, T., and Koldcofsky, D. 1966. A possible mechanism for initiation of protein synthesis. Proc. Natl. Acad. S&. U S . 55,606-613. Narita, K. 1963. “Collection of Lectures Commemorating the Inauguration of the New Shionogi Research Laboratory Building.” p. 106. Narita, K., Sato, N., and Ogata, K. 1966.Rat liver enzyme does not activate acetylamino acids. J. Biochem. (Tokyo) 57,176-183. Nemer, M. 1962. Interrelation of messenger polyribonucleotides and ribosomes in the sea urchin egg during embryonic development. Biochem. Biophys. Res.. Commun. 8,611-516. Newton, W. A., Beckwith, J. R., Zipser, D., and Brenner, S. 1966. Nonsense mutanisl and polarity in the lac operon of Escherichia coli. J . Mol. Biol. 14, 290-296. Nirenberg, M. W.,and Leder, P. 1964. RNA codewords and protein synthesis. The effect of trinucleotides upon the binding of sRNA to ribosomes. Science

145,1399-1407.

Nirenberg, M. W., and Matthaei, J. H. 1961. The dependence of cell-free protein synthesis in E . coli upon naturally occurring or synthetic polyribonucleotides. Proc. Natl. Acad. Sci. U S . 47, 1688-1602. Nirenberg, M. W.,Jones, 0. W., Leder, P., Clark, B. F. C., Sly, W. S., and Pestka, S. 1963. On the coding of genetic information. Cold Spring Harbor Xymp. Q w n t . Biol. 28,549-657. Nishimura, S., and Novelli, G. D. 1964. Species differences in soluble ribonucleic acids detected by differing ribonuclease sensitivity. Biochim. Biophys. Acta 80,

340-342.

Nishimura, S., Jones, D. S., and Khorana, H. G. 1965a. Studies on polynucleotides. XLVIII. The in vitro synthesis of a co-polypeptide containing two amino acids in alternating sequence dependent upon a DNA-like polymer containing two nucleotides in alternating sequence. J . Mol. Biol. 13, 302-324. Nishimure, S., Jones, D. S., Ohtsuka, E., Hayatsu, H., Jacob, T. M., and Khorana, H. G. 1966b. Studies on polynucleotides. XLVII. The in vitro synthesis of homopeptides aa directed by a ribopolynucleotide containing a repeating trinucleotide sequence. New codon sequences for lysine, glutamic acid and arginine. J. Mol. Biol. 13, 283-301. Noll, H.1966.Chain initiation and control of protein synthesis. Science 151, 1241-

1246.

Notani, G. W.,Engelhardt, D. L., Konigsberg, W., and Zinder, N. D. 1966. Suppression of a coat protein mutant of the bacteriophage f2. J. Mol. Biol. 12,

439-447.

Ochoa, M., Jr., and Weinstein, I. B. 1964. Amino acid coding in a subcellular system derived from the L1210 mouse ascites leukemia. Proc. Natl. Acud. Sci. US.52, 470-477. Orias, E., and Gartner, T. K. 1966. Effects of a streptomycin resistance allele and of streptomycin on suppressible mutants of phage T7. Bacteriol. Proc. p. 24.

THE GENETIC CODE

399

Parisi, B., and Ciferri, 0. 1966. Protein synthesis by cell-free extracts from castor bean seedlings. I. Preparation and characteristics of the amino acid incorporating system. Biochemistry 5,1638-1645. Pearlman, R., and Bloch, K. 1963. N-acetylamino acids and protein synthesis. Proc. Natl. Acad. Sci. U.S. 50, 533-537. Pelc, S. R. 1965.Correlation between coding-triplets and amino-acids. Nature 207, 697499. Pelc, S. R.,and Welton, M. G. E. 1966.Stereochemical relationship between coding triplets and amino-acids. Nature 209, 868-870. Penniston, J. T., Steward, M., and Tucker, M. D. 1964. Inactivation of transfer RNA with formaldehyde-a test of the triplet pairing model. Biochem. Biophys. Res. Commun. 15,358-366. Pestka, S., Marshall, R., and Nirenberg, M. 1965. RNA codewords and protein synthesis, V. Effect of streptomycin on the formation of ribosome-sRNA complexes. Proc. Natl. Acad. Sci. US.53, 639-646. Popp, R. A. 1967. Hemoglobins of mice: Sequence and possible ambiguity a t one position. of the alpha chain. J . Mol. Biol. 27, 9-16. RajBhandary, U.L.,Chang, S. H., Stuart, A., Faulkner, R. D., Hoskinson, R. M., and Khorana, H. G. 1967. Studies on polynucleotides, LXVIII. The primary structure of yeast phenylalanine transfer RNA. Proc. Natl. Acad. Sci. U S . 57, 751-758. Reichmann, M. E., Chang, A. Y., Faiman, L., and Clark, J. M., Jr., 1966. The satellite tobacco necrosis virus in studies of genetic coding. Cold Spring Harbor Symp. Quant. Biol. 31, 139-144. Revel, M., and Hiatt, H. H. 1965. Magnesium requirement for the formation of an active messenger RNA-ribosome-s-RNA complex. J . Mol. Biol. 11, 467-475. Revel, M.,and Littauer, U. Z. 1966. The coding properties of methyl-deficient phenylalanine transfer RNA from Escherichia coli. J . Mol. Biol. 15, 389-394. Rich, A., Penman, S., Becker, Y., Darnell, J., and Hall, C. 1963. Polyribosomes: Size in normal and polio-infected HeLa cells. Science 142, 1658-1663. Rifkin, D. B.,Rifkin, M. R., and Konigsberg, W.1966. The presence of two major hemoglobin components in an inbred strain of mice. Proc. Natl. Acad. Sci. US. 55, 586-692. Ritossa, F. M., Atwood, K. C., and Spiegelman, S. 1966. On the redundancy of DNA complementary to amino acid transfer RNA and its absence from the nucleolar organizer region of Drosophila melanogaster. Genetics 54, 663-676. Rottman, F., and Nirenberg, M. 1966. RNA codons and protein synthesis. XI. Template activity of modified RNA codons. J . Mol. Bwl. 21, 555-570. Sager, R., and Toback, F. G. 1965. Coding fidelity of ribosomes from streptomycin sensitive and resistant strains of Chlamydomonas. Circulated as Memo No. 93 in Information Exchange Group No. 7, Natl. Inst. Health, Bethesda, Maryland. Sager, R., Weinstein, I. B., and Ashkenazi, Y. 1963. Coding ambiguity in cell-free extracts of Chlamydomonus. Science 140, 304-306. Salas, M., Smith, M. A., Stanley, W. M., Wahba, A. J., and Ochoa, S. 1965. Direction of reading of the genetic message. J . Biol. Chem. 240, 3988-3995. Salas, M., Hille, M. B., Last, J. A., Wahba, A. J., and Ochoa, S. 1967a. Translation of the genetic message, 11. Effect of initiation factors on the binding of formylmethionyl-tRNA to ribosomes. Proc. Natl. Acad. Sci. US. 57, 387-394.

400

ANIL SADGOPAL

Salas, M., Miller, M. J., Wahba, A. J., and Ochoa, S. 196713. Translation of the genetic measage, V. Effect of Mg++and formylation of methionine in protein synthesis. Proc. Natl. Acad. Scd. US.57, 1865-1869. Sarabhai, A., and Brenner, S. 1967a. Further evidence that UGA does not code for tryptophan. J . M o t . Biol. 26, 141-142. Sarabhai, A,, and Brenner, S. 1967b. A mutant which reinitiates the polypeptide chain after chain termination. J . Mol. Biol. 27, 145-162. Sarabhai, A. S., Stretton, A. 0. W., Brenner, S., Bolle, A. 1964. Co-linearity of the gene with the polypeptide chain. Nature 201, 13-17. Scher, S. 1966. Evidence for the non-essentiality of translation errors (misreading) in mutagenesis by aminoglycoside antibiotics. Biochem. Biophys. Res. Commun.

22,672-678.

Schuster, H.,and Wilhelm, R. C. 1963. Reaction differences between tobacco mosaic virus and its free ribonucleic acid with nitrous acid. Biochim. Biophys, Acta 68, 664-560. Schwarts, J. H.1965. An effect of streptomycin on the biosynthesis of the coat protein of coliphage f2 by extracts of E . coli. Proc. Natl. Acad. See’. US. 53,

1133-1 140.

Schwartz, J. H.,Eisenstadt, J. M., Brawerman, G., and Zinder, N. D. 1966. Biosynthesis of the coat protein of coliphage f2 by extracts of Euglena gracilis. Proc. Natl. Acad. Sci. U S . 53,195-200. Schwartz, J. H., Meyer, R., Eisenstadt, J. M., and Brawerman, G. 1967. Involvement of N-formylmethionine in initiation of protein synthesis in cell-free extracts of Euglena gracilis. J . Mol. Biol. 25, 671474. Schweet, R., and Heintz, R. 1966. Protein synthesis. Ann. Rev. Biochem. 35, 723-

758.

Signer, E.R., Torriani, A., and Levinthal, C. 1961. Gene expression in intergeneric merozygotes. Cold Spring Harbor Symp. Quant. Bwl. 26, 31-34. Silbert, D. F., Fink, G. R., and Ames, B. N. 1966. Histidine regulatory mutants in Salmonella typhimurium. 111. A class of regulatory mutants deficient in tRNA for hietidine. J . Mol. Biol. 22, 335347. Sinsheimer, R.L.,Hutchinson, C. A., 111, and Lindqvist, B. H. 1967. Bacteriophage 0x174: Viral functions. In “The Molecular Biology of Viruses” (J. S. Colter, ed.), pp. 176-192. Academic Press, New York. Smith, E. L. 1962. Nucleotide base coding and amino acid replacements in proteins, 11. Proc. Natl. Acad. Sci. U.S. 48,869-864. Smith, J. D., Abelson, J. N., Clark, B. F. C., Goodman, H. M., and Brenner, S. 1966. Studies on amber suppressor tRNA. Cold Spring Harbor Symp. Quant. Biol. 31,479-486. So, A. G.,Bodley, J. W., and Davie, E. W. 1964. The influence of environment on the specificity of polynucleotide-dependent amino acid incorporation into polypeptide. Bwchemistrg 3,1977-1982. Soll, D.,Ohtsuka, E., Jones, D. S., Lohrmann, R., Hayatsu, H., Nishimura, S., and Khorana, H. G. 1966. Studies on polynucleotides, XLIX. Stimulation of the binding of aminoacyl-sRNA’s to ribosomes by ribotrinucleotides and a survey of codon assignments for 20 amino acids. Proc. Natl. Acad. Sci. US.54, 1378-

1386.

SOU, D., Jones, D. S., Ohtsuka, E., Faulkner, R. D., Lohrmann, R., Hayatsu, H., Khorana, H.G., Cherayil, J. D., Hempel, A., and Bock, R. M.1966. Specificity of sRNA for recognition of codons as studied by the ribosomal binding

THE GENETIC CODE

401

technique. J. Mol. Biol. 19, 556-573; Cold Spring Harbor Symp. Quant. Biol. 31, 51-61. Sonneborn, T. M. 1965. Degeneracy of the genetic code: Extent, nature, and genetic implications. I n “Evolving Genes and Proteins” (V. Bryson and H. J. Vogel, eds.), pp. 377-397. Academic Press, New York. Speyer, J. F., Lengyel, P., and Basilio, C. 1962. Ribosomal localization of streptomycin sensitivity. Proc. Natl. Acad. Sci. U.S. 48, 684-686. Speyer, J. F., Lengyel, P., Basilio, C., Wahba, A. J., Gardner, R. S., and Ochoa, S. 1963. Synthetic polynucleotides and the amino acid code. Cold Spring Harbor Symp. Quant. Biol. 28, 559-567. Staehelin, T., and Meselson, M. 1966. Determination of streptomycin sensitivity by a subunit of the 305 ribosome of Eschen’chia coli. J . Mol. Biol. 19, 207-210. Stanley, W. M., Jr., Salas, M., Wahba, A. J., and Ochoa, S. 1966. Translation of the genetic message : Factors involved in the initiation of protein synthesis. Proc. Natl. Acad. Sci. U S . 56, 290-295. Strehler, B. L., Hendley, D. D., and Hirsch, G. P. 1967. Evidence on a codon restriction hypothesis of cellular differentriation: Multiplicity of mammalian leucyl-sRNA-specific synthetases and tissue-specific deficiency in an alanylsRNA synthetase. Proc. Natl. Acad. Sci. U S . 57, 1751-1758. Streisinger, G., Okada, Y., Emrich, J., Newton, J., Tsugita, A., Terzaghi, E., and Inouye, M. 1966. Frameshift mutations and the genetic code. Cold Spring Harbor Symp. Quant. Biol. 31, 77-84. Stretton, A. 0. W., and Brenner, S. 1965. Molecular consequences of the amber mutation and its suppression. J. Mol. Biol. 12, 456-465. Stretton, A. 0. W., Kaplan, S., and Brenner, S. 1966. Nonsense codons. Cold Spring Harbor Symp. Quant. Biol. 31, 173-179. Stulberg, M. P., and Isham, K. R. 1967. Studies on the locus of the enzyme recognition site in phenylalanine transfer RNA. Proc. Natl. Acad. Sci. U.S. 57, 1310-1316. Subak-Sharpe, H., Shepherd, W. M., and Hay, J. 1966. Studies on sRNA coded by herpes virus. Cold Spring Harbor Symp. Quant. Biol. 31, 583-594. Sueoka, N., and Yamane, T. 1962. Fractionation of aminoacyl-acceptor RNA on a methylated albumin column. Proc. Natl. Acad. Sci. U S . 48, 1454-1461. Sueoka, N., Kano-Sueoka, T., and Gartland, W. J. 1966. Modification of sRNA and regulation of protein synthesis. Cold Spring Harbor Symp. Quant. Biol. 31, 571-580. Sundararajan, T. A., and Thach, R. E. 1966. Role of the formylmethionine codon AUG in phasing translation of synthetic messenger RNA. J . Mol. Biol. 19, 74-90. Szer, W., and Ochoa, S. 1964. Complexing ability and coding properties of synthetic polynucleotides. J. Mol. Biol. 8, 823-834. Tanner, M. J. A. 1966. Studies on amino acid transfer ribonucleic acid. Ph.D. Thesis, University of Cambridge, Cambridge, England. Taylor, M. W., Granger, G. A., Buck, C. A., and Holland, J. J. 1967. Similarities and differences among specific tRNA’s in mammalian tissues. Proc. Natl. Acad. Sci. U S . 57,1712-1719. Terzaghi, E., Okada, Y., Streisinger, G., Emrich, J., Inouye, M., and Tsugita, A. 1966. Change of a sequence of amino acids in phage T4 lysoryme by acridineinduced mutations. Proc. Natl. Acad. Sci. U S . 56, 600-507.

402

ANIL SADGOPAL

Tessman, I. 1962. The induction of large deletions by nitrous acid. J. Mol. Biol. 5,.

442-446.

Tessman, I., Poddar, R, K., and Kumar, S. 1964. Identification of the altered bases in mutated single-stranded DNA. I. I n vitro mutagenesis by hydroxylamine, ethyl methmesulfonate and nitrous acid. J. MoZ. Biol. 9, 362-363. Thach, R. E.,Cecere, M. A., Sundmarajan, T. A., and Doty, P. 1966. The polarity of messenger translation in protein synthesis. Proc. Natl. Acad. Sci. U S . 54,

1167-1173.

Thach, R. E., Dewey, K. F., Brown, J. C., and Doty, P. 1966. Formylmethionine codon AUG as an initiator of polypeptide synthesis. S&me 153, 416-418. Torres-Gallardo, J., and Kern, M. 1966. The specific inhibition of the enzymatic aminoacylation of valyl- and tyrosyl-sRNA by periodate-oxidized sRNA. Proc. Natl. Acad. Sci. US.63, 91-96. Traub, P.,Hosokawa, K., and Nomura, M. 1966. Streptomycin sensitivity and the structural components of the 30s ribosomes of Escherichia coli. J. Mol. Biol. 19,

211-214.

Trupin, J. S., Rottman, F. M., Brimacombe, R. L. C., Leder, P., Bernfield, M. R., and Nirenberg, M. W. 1965. RNA codewords and protein synthesis, VI. On the nucleotide sequences of degenerate codeword sets for isoleucine, tyrosine, asparagine, and lysine. Proc. Natl. Acad. Sci. US.53, 807-811. Tsugita, A. 1962. The proteins of mutants of TMV: Composition and structure of chemically evoked mutants of TMV RNA. J . MoZ. Biol. 5, 284-292. Tsugita, A., and Fraenkel-Conrat, H. 1960. The amino acid composition and Cterminal sequence of a chemically evoked mutant of TMV. Proc. Natl. Acad. Sci. U S . 46,636-642. Tmgita, A,, and Fraenkel-Conrat, H.1962. The composition of proteins of chemically evoked mutants of TMV RNA. J . Mol. Bwl. 4, 73-82. Valentine, R. C., and Zinder, N. D.1964. Phenotypic repair of RNA-bacteriophage mutants by streptomycin. Science 144,1468-1469. Viiluela, E., Salas, M., and Ochoa, S. 1967. Translation of the genetic message, 111. Formylmethionine as initiator of proteins programed by polycistronic messenger RNA. Proc. Natl. Acad. Sci. US. 57, 729-734. von Ehrenstein, G. 1966. Translational variations in the amino acid sequence of the a-chain of rabbit hemoglobin. Cold Spring Harbor Symp. Quant. Biol. 31,

705-714.

von Ehrenstein, G., and Dais, D. 1963. A leucine acceptor sRNA with ambiguous coding properties in polynucleotide-stimulated polypeptide synthesis. Proc. Natl. Acad. Sci. U.S. 50, 81-86. von Ehrenstein, G.,and Lipmann, F. 1961. Experiments on hemoglobin biosynthesis. Proc. Natl. Acad. Sci. U.S. 47, 941-950. von Ehrenstein, G., Weisblum, B., and Benzer, S. 1963. The function of sRNA as amino acid adaptor in the synthesis of hemoglobin. Proc. Natl. Acad. Sci. U.S. 49, 669-676. Wall, R. 1962. Overlapping genetic codes. Nature 193, 1268-1270. Waller, J.-P. 1963. The NH.-terminal residues of the proteins from cell-free extracts of E . coli. J. Mol. B i d . 7,483-496. Warner, J. R., and Rich, A. 1964. The number of soluble RNA molecules on reticulocyte polyribosomes. Proc. Natl. Acad. Sci. U S . 51, 1134-1141. Waters, L. C., and Novelli, G. D. 1967. A new change in leucine transfer RNA

THE GENETIC CODE

403

observed in Eschenchia coli infected with bacteriophage T2. Proc. Natl. Acad. Sei. UB. 67,979-985. Webster, R. E., Engelhardt, D. L., and Zinder, N. D. 1966. In evitro protein SWthesis: Chain initiation. Proe. Natl. Acad. Sci. US.55, 155-161. Weigert, M. G., and Garen, A. 1965a. Amino acid substitutions resulting from suppression of nonsense mutations, I. Serine insertion by the Su-1 suppressor gene. J . Mol. Bwl. 12, 448-455. Weigert, M. G., and Garen, A. 1965b. Base composition of nonsense codons in E . coli: Evidence from amino-acid substitutions a t a tryptophan site in alkaline phosphatase. Nature 206, 992994. Weigert, M. G., Lanka, E., and Garen, A. 1965. Amino acid substitutions resulting from suppression of nonsense mutations. 11. Glutamine insertion by the S u d gene; tyrosine insertion by the Su-3 gene. J . Mol. Biol. 14, 622-527. Weigert, M. G., Lanka, E., and Garen, A. 1967a. Base composition of nonsense codons in Escheriehia coli. 11. The N 2 codon UAA. J . Mol. Biol. 23, 391400. Weigert, M. G., Lanka, E., and Garen, A. 1967b. Amino acid substitutions resulting from suppression of nonsense mutations. 111. Tyrosine insertion by the Su-4 gene. J . Mol. Biol. 23, 401404. Weil, J.-H., Befort, N., Rether, B., and Ebel, J.-P. 1964. Effects of chemical modifications of the biological properties of s-RNA. Biochem. Biophys. Res. Commun. 15,447452. Weinstein, I. B. 1963.Comparative studies on the genetic code. Cold Spring Harbor Symp. @.ant. Bwl. 28, 579-580. Weinstein, I. B., and Schechter, A. N. 1962. Polyuridylic acid stimulation of phenylalanine incorporation in animal cell extracts. Proc. Natl. Acad. Sci. US. 48,1686-1691. Weinstein, I. B., Sager, R., and Fresco, J. R. 1963. “Coding potential” of synthetic polyribonucleotides in non-bacterial systems. Federation Proc. 22, 644. Weinstein, I. B.,Ochoa, M., Jr., and Friedman, S. M. 1966. Fidelity in the translation of messenger ribonucleic acids in mammalian subcellular systems. Biochemistry 5,3332-3339. Weisblum, B., Benzer, S., and Holley, R. W. 1962. A physical basis for degeneracy in the amino acid code. Proc. Natl. Acad. Sci. US. 48, 1449-1454. Weisblum, B., Gonano, F., von Ehrenstein, G., and Benzer, S. 1965. A demonstration of coding degeneracy for leucine in the synthesis of protein. Proc. Natl. Acad. Sci. US.53,328-334. Welton, M. G.E., and Pelc, S. R. 1966. Specificity of the stereochemica1 relationship between ribonucleic acid-triplets and amino-acids. Nature 209, 870-872. Whitfield, H. J., Jr., Martin, R. G., and Ames, B. N. 1966. Classification of amino transferase (C gene) mutants in the histidine operon. J. Mol. Biol. 21, 335-355. Wilhelm, R. C. 1966. Discussion. Cold Spring Harbor Symp. Quant. Biol. 31, 496497. Wittmann, H. G. 1961. Ansatee eur Entschlusselung des genetischen Codes. Naturuksenschajten 48, 729-734. Wittmanu, H. G., and Wittmann-Liebold, B. 1963. Tobacco mosaic virus mutanta and the genetic coding problem. Cold Spring Harbor Symp. Quant. Biol. 28, 589-595. Wittmann, H. G.,and Wittmann-Liebold, B. 1966. Protein chemical studies of two RNA viruses and their mutants. Cold Spring Harbor Symp. Quant. Biol. 31,163-172.

404

ANIL SADGOPAL

Woese, C. R. 1962. Nature of the biological code. Nature 194, 1114-1116. Woese, C. R. 1965a. Order in the genetic code. Proc. Natl. Acad. Sci. U.S.54, 71-75. Woese, C. R. 1966b. On the evolution of the genetic code, Proc. Natl. Acad. Sci. U S . 54,1546-1552. Woese, C. R., Dugre, D. H., Dugre, S. A., Kondo, M., and Saxinger, W. C. 1966. On the fundamental nature and evolution of the genetic code. Cold Spring Harbor Symp. Quant.Biol. 31,723-736. Yamane, T., and Sueoka, N. 1964. Enzymic exchange of leucine between Merent components of leucine acceptor RNA in Escherichia coli. Proc. Natl. Acad. Sci. US.51,1178-1184. Yanofsky, C. 1963. Amino acid replacements associated with mutation and recombination in the A gene and their relationship to in vitro coding data. Cold Spring Harbor Symp. Quant. Biol. 28, 581-688. Yanofsky, C. 1965. Possible RNA codewords for the eight amino acids that can occupy one position in the tryptophan aynthetase A protein. Bwchem. Biophys. Res. Commun. 18, 898-909. Yanofsky, C., and Ito, J. 1966. Nonsense codons and polarity in the tryptophan operon. J. Mol. Biol. 21, 313-334. Yu, C. T. 1966. Multiple forms of leucyl sRNA synthetase of E. coli. Cold Spsng Harbor Symp. Quant. Biol. 31, 565-570. Yu, C. T., and Zamecnik, P. C. 1963a. On the aminoacyl-RNA synthetase recognition sites of yeast and E. coli. transfer RNA. Biochem. Biophys. Res. Commun. 12,457-463. Yu, C . T., and Zamecnik, P. C. 1963b. Effect of bromination on the amino acidaccepting activities of transfer ribonucleic acids. Biochim. Biophys. Acta 76, 209-222. Zachau, H. G., Dutting, D., Feldman, H., Melchers, F., and Karau, W. 1966. Serine specific transfer ribonucleic acids, XIV. Comparison of nucleotide sequences and secondary structure models. Cold Spring Harbor Symp. Quant. BWl. 31,417-424. Zamir, A., Leder, P., and Elson, D. 1966. A ribosome-catalyzed reaction between N-formylmethionyl-tRNA and puromycin. Proc. Natl. Acad. Sci. U.S. 56, 1794-1801. Zinder, N. D. 1963. The information content of an RNA-containing bacteriophage. I n “Informational Macromolecules” (H. J. Vogel, V. Bryson, and J. 0. Lampen, eds.), pp. 229-237. Academic Press, New York. Zinder, N. D., and Cooper, S. 1964. Host-dependent mutants of the bacteriophage f2. I. Isolation and preliminary classification. Virology 23, 162-168. Zinder, N. D., Engelhardt, D. L., and Webster, R. E. 1966. Punctuation in the genetic code, Cold Spring Harbor Symp. Quunt. Biol. 31, 251-256.

AUTHOR INDEX Numbers in italics refer to pages on which the complete references are listed.

A Abashian, D. V., 9, 34, 35, 36, 62 Abel, P.,384, 390 Abelson, J. N., 366,400 Abraham, A., 8, 43 Abrahamson, S.,174, 218 ACS,G.,192, 218 Adair, C. R., 106, 107, 126 Adams, A., 373, 390, 39Y Adams, J. M., 346, 352, 390, 996 Ahmad, N., 40, 4.3 Ahuja, M. R., 23, 25, 43 Akemine, H., 106, 1% Alfert, M.,175, 219 Allard, R. W.,61, 75, 76, 77, 78, 80, 81, 82, 83, 86, 87, 88, 89, 90, 91, 93, 96, 97, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 110, 111, 113, 114, 117, 196, I#, 128, 130 Allende, J. E., 385, 3.91 Allfrey, V. G., 194, 2.2’0, 355, 390 Alpers, D. H., 345, 390 Altorfer, N.,141, 166 Ames, B. N., 345, 351, 357, 369, 375, 390, 400, 403 Anders, F., 26, 43, 63 Anderson, W .F., 369, 390 Anderson, W. W., 93, 130 Apgar, J., 373, 374, 380, 390, 393, 396 Appa Rao, K., 9, 42, 43, 48, 60 Apple, J. L., 16, 17, 19, 43, 46, @ Argetsinger, J. E., 346, 352, 996 Arlinghaus, R., 350, 390 Arnold, C.-G., 321, 322 Arnstein, H. R. V., 385, 990 Ar-Rush&, A. H., 16, 19, 28, 4.9 Ashkenazi, Y., 362, 399 Atwood, K. C., 374, 399

Avew, P., 8, 46, 47 Aycock, M. K., Jr., 32, 43

B Baccetti, B., 200, 218 Baglioni, C., 327, 337, 390 Bagnara, D.,40, 43 Bailey, N. T. J., 87, 126 Bairati, A,, 200, 218 Baker, W. K., 135, 138, 140, 141, 143, 151, 162, 154, 155, 156, 157, 158, 159, 164, 166, 167, 175, 218 Baldwin, A. N.,383, 390 Bangham, J. W., 136, 144, 146, 147, 168 Barbieri, D.,63, 129 Barigozzi, C.,175, 218 Barnes, D.K., 87, 126 Barnett, L.,328, 329, 330, 356, 357, 360, 361, 392, 393 Barnett, W. E., 375, 383, 390, 391 Bartlett, M . S., 126 Basilo, C., 331, 335, 367, 385, 991, 4Ol Bateman, A. J., 87, 126 Batra, B. M., 40, 61 Bawolska, M.,40, 43 Bayreuther, K. E., 384, 391 Bayer, M.H.,26, 43, 44 Beale, D., 337, 339, 340, 344, 391 Beardmore, J. A., 60, 63, 126 Becker, H. J., 154, 156, 157, 161, 166 Becker, Y.,345, 399 Beckwith, J. R., 345, 351, 356, 357, 358, 359, 391, 398 Beennann, W., 176, 189, 194, 200, 209, 213, 214, $18, 221 Befort, N., 372, 403 Benjaail, V., 111, 112, 126 Bennett, J. H., 70, 71, 73, 126

405

406

AUTHOR INDEX

Bennett, T. P., 374, 377, 391, 394 Benoni, H., 320, 322 Benaer, S., 337, 356, 367, 361, 362, 374, 375, 377, 387, 391, 402, 403 Berbek, J., 40, Berberich, M. A., 345, 352, 8 1 Berg, P., 366, 370, 374, 383, 390, 391, 392, 397, 398 Bergmann, F. H., 374, 383, 391 Bergquist, P. L., 366, 391 Bernfield, M.,334, 335, 337, 392 Bernfield, M. R., 334, 335, 376(4), 377, 391, 402 Biedron, S. I., 360, 506 Binder, M.,226, 322 Binet, F. E., 70, 71, 126 Bink, J. P. M.,40,63 Biasell, D. M., 368, 391 Bloch, K.,355, 399 Bock, R. M., 374, 375, 376(1), 377, 378, ai, mi, 391, 392, 400 Bodley, J. W., 362, 363, 368, 400 Bodmer, W. F., 78, 126 Bolle, A., 357, 393, 400 Bond, D. A., 92, 197 Boy de la Tour, E., 357, 593 Bram, J., 65, 130 Bravo, M.,385, 391 Braum, A. C., 22, 24, 25,44 Brawerman, G.,354, 384, 400 Breckenridge, L.,369, 370, 390, 394 Brenner, S., 327, 328, 329, 330, 345, 351, 356, 357, 358, 359, 360, 361, 391, 392, 393, 396, 398, 400, 401 Bretscher, M. S., 350, 356, 362, 392 Breuer, M. E., 56, 126 Bridges, C. B., 171,175, 208,218,222 Brieger, F. G.,23, 44 Brimacombe, R.,334, 335, 337, 392, 402 Brito Da Cunha, A., 93, 129 Brosseau, G.,142, 166, 166, 172, 173, 218 Brown, D.D., 213, 218, 219 Brown, D. H., 375,383,390, 391 Brown, G. L., 370, 371, 383, 392 Brown, J. C., 348, 362, 402 Brown, S. W., 160, 166 Brownstein, B. L., 367, 392 Buck, C. A., 375, 401

Burbidge, N. T., 2, 44 Burger, M.,369, 394 Burk, L. G., 4, 15, 17, 20, 24, 26, 32, 33, 34, 36, 37, 41, 44, 46, 61, 63 Burns, J. A., 29, 4, 46 Rursstyn, H., 349, 350,396 C

Gain, A. S., 93, 126 Callan, H.G., 187, 189, 213, 214, 215, 119 Cameron, D.R., 8, 14, 15, 16, 19, 20, 25, 28, 32, 42, 43, 44, 46, 49, 60 Campbell, P. N., 327, 592 Cannon, G. B., 93, 186 Capecchi, M.R., 346, 352, 365, 366, 367, 382, 390, 391, 392, 396 Capra, J . D., 367, 398 Carbon, J., 366, 372, 392 Carrier, R. F., 196, 221, 222 Caskey, C. T., 337, 354, 359, 360, 385,

392, 397 Catcheside, D.G., 144, 166 Cattanach, B. M., 144, 146, 148, 163, 166, 167 Cavalli-Sforsa, L., 369, 396 Cecere, M. A., 328, 402 Champe, S. P., 356, 357, 361, 391, 992 Chang, A. Y., 354, 355, 384, 399 Chang, S. H., 380, 399 Chapeville, F.,355, 387, 392, 396 Chaplin, J . F., 15, 17, 19, 32, 33, 46 Chapman, S. R., 109, 128 Chaudry, A. H., 40, .@ Chen, S. T., 40, 46 Chen, S. Y., 154, 161, 166 Cherayil, J. D., 374, 375, 376(1), 377, 378, 385, 392,400 Chevalley, R., 357, 393 Chu, J. M., 40, @ Ciferri, O.,385, 9 9 Claassen, C. E., 87, 126 Clark, A. M., 70, 186 Clark, B. F. C., 331, 332, 335, 347, 349, 350, 353, 354, 360, 374, 376(3), 377, 392, 398, 400 Clark, J. M., Jr., 354, 355, 384, 399 Clarke, A. E., 92,126 Clarke, B., 63, 126 Clausen, R. E., 7, 8, 16, 42, 46, 47

407

AUTHOR INDEX

Clayton, E. E., 15, 17, 19, 31, 32, 46, 47 Clegg, J. B., 339, 344, 362, 396 Cleveland, R. W., 87, 186 Clifford, H. T., 70, 126 Cockerham, C. C., 33, 39, 49 Cohen, J., 143, 151, 162, 166 Comstock, R. E., 38, 60 Cooch, F. G., 60, 63, 1,96 Cooper, K. W., 141, 160, 166, 175, 200, 212, 219 Cooper, S., 357, 404 Cousineau, G. H., 213, ,919 Cox, E. C., 367, 368, 393, 394 Cox, R. A., 385, 390 Craig, L. C., 374, 304 Crick, F. H. C., 326, 328, 329, 330, 331, 356, 357, 360, 361, 367, 378, 379, 387, 389, 392, 393 Crow, J. F., 82, 128 D Daems, W. T., 200, ,919 Dais, D., 362, 375, 377, 402 Dalebroux, M., 40, 46 Daly, J., 26, 61 Daly, K., 22, 41, 46, 61 Darlington, C. D., 57, 196 Darnell, J., 345, 399 Das, N. K., 175, 219 Davie, E. W., 362, 363, 368, 400 Davies, J., 363, 364, 368, 393 Dawson, R. F., 35, 36, 37, 46 Dean, C. E., 41, 46 Demerec, M., 143, 166 Dempster, E. R., 67, 116 de Nettancourt, D., 4, 46 Denhardt, G. H., 357, 395 Dennert, G., 357, 396 Deppe, G., 357, 396 Dermen, H., 33, 4, 46 De Souza, H. L., 93, 199 De Toledo, J. S., 93, 1.99 Devreux, M., 42, 46, 63 Dewey, K. F., 348, 362, 402 Diecbmann, M., 374, 383, 391 Dieterman, L. J., 26, 63 Dillman, A. C., 87, 126 Di Pasquale, A., 175, 218 Dobzhansky, T., 93, 130, 200, 819

Doctor, B. P., 347, 374, 376(2), 377, 393, 396

Doi, R. H., 354, 375, 396, 396 Dondon, J., 362, 363, 396 Doty, P., 328, 348, 362, 409 Dreyfus, A., 56, 126 Dropkin, V. H., 20, 44 Dubinin, N. P., 135, 166, 175, 219 Dugre, D. H., 386, 387, 404 Dugre, S. A., 386, 387, 404 Dulicu, H., 4, 46 Dunnill, P., 387, 393 Diitting, D., 373, 374, 380, 404

E East, E. M., 2, 5, 21, 28, 30, 46, 92, 126 Ebel, J. P., 372, 403 Eck, R. V., 344, 393 Edgar, R. S., 357, 393 Edington, C. W., 137, 167, 175, ,991 Edwardson, J. R., 33, 34, 46 Eicher, E. M., 148, 166 Eigner, E. A., 371, 397 Eisenstadt, J., 346, 352, 393 Eisenstadt, J. M., 354, 384, 400 Elson, D., 350, 404 Emrich, J., 329, 330, 331, 401 Engelhardt, D. L., 346, 357, 358, 365, 366, 382, 393, 398, 403, 404 Engelhaupt, M. E., 26,63 Epler, J. L., 375, 383, 391 Epstein, C. J., 386, 393 Epstein, R. H., 357, 393 Everett, G . A,, 373, 374, 380, 387, 396, 397

F Faiman, L., 354, 355, 384, 399 Fangma.n, W. L., 388, 394 Faulker, R., 355, 390 Faulkner, R. D., 374, 375, 376(1), 377, 378, 380, 385, 399, 400 Feldman, D. E., 213, 220 Feldman, H., 373, 374, 380, 404 Fink, G . R., 375, 400 Fischer, H. P., 237, 38.2 Fisher, R. A., 59, 73, 93, 1%' Flaks, J. G., 367, 368, 393, $04

408

AUTHOR INDEX

Ford, C. E., 148, 167 Gorini, L., 363, 368, 369, 370, 390, 393, Ford, 2. T., 32, 46 394 Forster, R.,23, 44 Gottschling, H., 372, 396 Fowler, A. V., 357, 394 Gowen, J. W., 140, 154, 166, 175, 819 Fraenkel-Conrat, a,337, 341, 342, 344, Gower, J. C., 78, 129 Granger, G. A., 375, 401 894, 408 Grant, V., 56, 57, 1.97 Franklin, R. M.,192, E2.9 Green, M. M., 165, 166 Fresco, J. R., 373, 386, 890, 897, 403 Friedman, S. M., 362, 363, 368, 369, 385, Grell, E. H., 137,7'61 Grell, R. F., 141, 166, 175, U 9 394, 403 Funatsu, G., 341, 344, ,994 Gress, M.,322 Griibg, B., 73, 1.97 Fyfe, J. L., 87, 126 Griffith, J. S., 328, 393 Griffith, R. B., 37, 47 G Gross, H.,388 Gross, P.R., 213, 819, 881 Gall, J. G.,213, 214, 215, 219 Grosso, J. J., 33, 4 Gdlueci, E.,358, 359, 361, 39.6 Griineberg, H., 136, 166 Gamow, G., 327, 394 Grunberg-Manago, M.,362, 363, 373,392, Ganosa, M.C., 356, 394 396, 397 Gardner, R. S., 331, 335, 385, 4.01 Garen, A.,344, 356, 357, 358, 359, 360, Guest, J. R., 342, 343, 344, 396 Guitierres, M. G., 87, 1.97 361, 39.4 403 Gunckel, J. E., 25, 28, 47, 49 Garen, S., 359, 894 Gunderson, W., 369, ,994 Gartland, W.J., 373, 375, 401 Gupta, N. K.,366, 396 Gartner, T.K., 369, 398 Gurdon, J. B., 213, 218 Gay, E.R.,140, 154,166, 175, 219 Gus& G. N., 346, 352, 365, 367, 382, Gay, H.,200, 8.91 398, 396 Geiduschek, E. P.,342, 394 Guyhot, E., 200, 219 Geiringer, H., 69, 126 Gwynn, G. R.,41, 47 Geroch, M.,373, 397 Gerstel, D.U., 4, 6, 7, 8, 16, 17, 28, 29, 37s 44, 46, 49, 60, 51 H Ghosh, H. P.,347, 348, 350, 353, 394 Giacomoni, D.,374, 394 Habrykrt, K., 238, 322 Hadorn, E,,155, 158, 166, 174, 208, 89% Gibson, J. B.,73, 1.96 Gilbert, N.,38, 46 Haenni, A. L.,355, 396 Gilbert, W.,355, 363, 368, 393, 394 Hagemann, R., 299, 328 Girvin, E. C., 140, 166 Hagen, G. L., 25, 26, 44, 47 Glenk, H.O., 318, 319, 322 Hagen, U., 196, $83 Goldberg, A. L.,389, 394 Haldane, J. B. S., 59, 61, 70, 126, i5?' Goldberg, I. H., 192, 819 Hall, C., 345, 399 Goldberger, R. F., 345, 352, 391 Hall, J. L., 37, 47, 49 Golden, W.G., 99, 100, 101, 126 Halvorson, H. O., 385, 397 Goldstein, J., 374, 377, 391, 394 Hannah-Alava, A., 160, 166, 200, g19 Gonano, F.,375, 403 Goodman, H. M.,356, 366, 374, 398, Hansche, P .E., 82, 83, 90, 110, 126 Hanson, W.D., 73, 127 394, PJ Harding, J., 63, 87, 88, 90, 91, 103, 186, Goodsmith, W,, 136, 167 187, 130 Goodspeed, T. H., 2, 4, 5, 6, 7, 8, 16, Harlan, H.V., 95, 103, 1%' 44 46, 47

409

AUTHOR INDEX

Harriman, P. D., 372, 373, 396 Harrington, H., 196, ,920 Hart, G. E., 15, 21, 32, 47 Hartman, P. E., 345, 390 Haustein, E., 226, 233, 322 Hawtrey, A. O., 360, 396 Hay, J., 375, 401 Hayatsu, H., 332, 334, 335, 336, 337, 360,

362, 364, 372, 374, 375, 376(1), 377, 378, 385, 398, 400 Hayashi, H., 373, 396 Hayman, B. I., 38, 47, 64, 65, 66, I# Hecht, L. I., 328, 3995 Heggestad, H. E., 17, 44, 47 Heintz, R., 327, 400 Heitz, E., 175, ,920 HempeI, A., 374, 375, 376(1), 377, 378

385,400 Henderson, S. A., 175, 2.80 Hendley, D. D., 375, 384, 401 Henning, U., 357, 396 Hennig, W., 191, 192, 195, 196, 200, 8.80 Henry, T. A., 36, 47 Heptner, M. A., 175, 219 Herskowitz, I. H., 174, 218 Heas, O., 175, 176, 183, 184, 189, 192, 195, 197, 200, 209, 211, 212, 220, 2221 Hessler, A. Y., 138, 151, 152, 153, 155, 166 Heymer, T., 3.82 Hiatt, H. H., 352, 399 Hill,J., 38, 47 Hille, M. B., 348, 353, 358, 396, 399 Hillman, W. S., 21, 47 Hinegardner, R. T., 213, 220 Hinton, T., 136, 137, 167 Himh, G. P., 375, 384, 401 Hoagland, M. B., 328, 596 Hogben, L., 59, l# Holden, 3. H. W., 92, 1% Holland, J. J., 375, 401 Holley, R. W., 362, 373, 374, 375, 377, 380,390,393,396,403 Holmes, F. O., 16, 47 Horikoshi, K., 354, 396 Hoskinson, R. M., 374, 380, 396] 599 Hosokawa, K., 368, 402 Hm, T. C., 213, 220 Huber, H., 240, 32.2

Euettner, A. F., 200, 990

Humphrey, A. B., 40, 47 Hunt, J. A., 385, 390 Hutchison, C. A. 111, 357, 396, 400 Huxley, J. S., 63, 187

I Imai, S., 35, 47 Imam, A. G., 90, 96, 97, 101, 102, 113, 114, 187 Imamoto, F., 383, 396 Infante, A. A., 213, 82.2 Ingram, V. M., 380, 596 Inouye, M., 329, 330, 331, 401 Isham, K. R., 373, 401 Ishida, H., 200, 223 Ito, J., 345, 351, 357, 404 Izard, C., 24, 47 Izawa, M., 194, 280

J Jacob, T. M., 332, 335, 336, 374, 398 Jacoby, G. A., 370, 394 Jain, S. K., 65, 66, 67, 75, 76, 77, 78, 79,

106, 108, 109, 115, 116, 118, 126, 137, 128, 130 Jana, S., 105, 128 Jaouni, T., 334, 335, 337, 392 Jeffrey, R. N., 35, 36, 37, .& 48, 63 Jennings, H. S., 69, 128 Jinks, J. L., 30, 38, 46, 48 Johannsen, W., 67, 97, 128 Johnson, E. M., 19, 63 Jones, C. W., 32, @ , Jones, D. S., 332, 334, 335, 336, 337, 360, 362, 363, 364, 368, 374, 375, 376(1), 377, 378, 385, 393, 396, 398, 400 Jones, G. L., 32, 49 Jones, J. W., 106, 107, 1% Jones, 0. W., 331, 332, 338, 362, 397, 398 Judd, B. H., 135, 167 Jujimura, W., 200, 223 Jukes, T. H., 337, 344, 389, 396

K Kaji, A,, 368, 396 Kaji, H., 368, 396

410

AUTHOR INDEX

Kaneko, I., 375, 396 Kannenberg, L. W., 117, 1988 Kano-Sueoka, T., 373, 375, 401 Kaplan, S., 356, 357, 358, 359, 3998, 396, 401 Karasek, M., 355, 396 Karau, W., 373, 374, 380, 396, 404 Kataja, E., 369, 394 Katz, E. R., 356, 357, 360, 361, 3998 Kaufmann, B. P., 174, 200, 220, 221 Keck, K., 196, 2983 Kehr, A. E., 5, 9, 15, 22, 23, 26, 28, 30,

@, 698

Kellenberger, E., 357, 393 Kellogg, D. A,, 347, 376(2), 377, 396 Kempthorne, O., 59, 60, 61, 62, 128 Kern, M., 373, 4098 Kersten, H., 192, 821 Kersten, W., 192, 9821 Keyl, H. G., 213, 215, 9.21 Khorana, H. G., 329, 332, 333, 335, 336, 337, 347, 348, 350, 353, 360, 362, 363, 364, 366, 368, 374, 376(1), 377, 378, 380, 385, 393, 394, 396, 396, 397, 398, 399, 400 Khvostova, V. V., 138, 167 Kikuchi, F., 106, 109, 126, 128 Kilmartin, J. V., 339, 344, 362, 396 Kimura, M., 71, 77, 78, 81, 82, 92, 118 Kistner, G., 226, 3298 Knowles, P. F., 95, 96, 98, 1.28 Kodani, M.,138,169 Kenigaberg, W., 358, 398 Koepchen, W., 3984 Kiiaeel, H., 329, 332, 335, 336, 396 Kojima, K., 74, 75, 78, 1988, 1.29 Kolakofaky, D., 346, 350, 396, 398 Konczerewicz, A., 40, 43 Kondo, M., 386, 387, 404 Konigsberg, W., 362, 399 Kostoff, D., 2, 5, 22, 24, 27, 48 Kovach, J. S., 345, 352, 391 Krishnamurty, K. V., 9, 30, 42, @, 48, 60 Kroger, H., 196, 9868 Kuhn, E., 174, 221 Kumar, S., 341, 402 Kung, H., 373, 380, 387, 397 Kupila, S., 22, 26, 48 Kurland, C. G., 367, 368,397

L Lamfrom, H., 328, 396 Lamotte, M., 93, 129 Lamprecht, M. P., 40, 48 Landspersky, N., 288, 290, 322 Lanka, E., 358, 359, 360, 361, 403 Last, J. A., 348, 353, 358, 396, 399 Latterell, R. L., 28, 48 Lea, H. W., 17, 48 Lechner, K., 267, 322 Leder, P., 331, 332, 334, 335, 337, 349, 350, 382, 392, 396, 398, @Z, 404 Lederberg, E. M., 369, 396 Lederberg, J., 369, 396 Lee, A., 60, 1.99 Lee, R. E., 8, 48 Legg, P. D., 21, 39, 48 Lehmann, H., 337, 339, 340, 344, Sgl Lengyel, P., 331, 335, M,352, 367, 385. 393, 401 Lerman, L. S., 329, 396, 397 Letendre, C., 373, 397 Leuchtmann, G., 240, 322 Levinthal, C., 384, 400 Levitan, M., 93, 129 Lewandowski, L. J., 367, 8098 Lewis, D., 388, 397 Lewis, E. B., 133, 141,167,176,221 Lewontin, R. C., 65, 73, 74, 75, 77, 78, 93, 129 Lielauais, A., 357, 393 Likover, T. E., 367, 368, 897 Lindahl, T., 373, 390, 397 Lindqvist, B. H., 357, 400 Lindsley, D. L., 137, 167, 176, 221 Lipmann, F., 355, 374, 377, 384, 387, 391, 398, 397, 402 Littauer, U. Z., 367, 399 Littna, E., 213, 619 Loebel, J. E., 347, 376(2), 377, 396 Loert~er,B., 267, 322 Loftfield, R. B., 371, 397 Lohmeyer, H., 884 Lohrmann, R., 334, 335, 337, 360, 362, 364, 374, 375, 376(1), 377, 378, 385, .boo Lucaa-Lenard, J., 355, 397 Lucking, T., 196, 223 Liining, K. c.,151, IST

AUTHOR INDEX

Luthra, J. K., 40, & Lyon, M. F., 144, 148, 149, 167

411

Miller, M. J., 353, 400 Miller, 0.L.,196, 221, 222 Mirsky, A. E.,194, 215, 220, 221,366,390 Mitchell, H.K., 155, I66 M Miura, K.,372, 373, 396, 398 Mow, R.,15, 19, 28, 46, 49 McCray, F. A., 28, 48 Monroy, A,, 213, 221, ,922 McLaughlin, C. S., 328, 396 Madison, J. T., 373, 374, 380, 387, 396, Montgomery, C. S., 143, 145, 146, 147, 168 397 Morgan, A. R., 329, 332, 333, 335, 336, Magahaes, L. E., 93, 129 360, 396, 897 Maggio, R., 213, 228 Morgan., T. H.,140,167,176, 208, 222 Mainardi, D.,63, 1?29 Moyer, W. A., 213, 219 MalBcot, G.,61, 70, 129 Muench, K.H.,374, 397, 398 Malkin, L. I., 213, 219 Mann, T.J., 15, 16, 17, 21, 32, 33, 36, 37, Muller, H. J., 174, 218 38, 39, 40, 41, 42, 43, 46, 46, 47, 48, 49, Munshi, A,, 40, 46 Murayama, M., 337, 398 60, 62 Mantzinger, D. F., 32, 33, 37, 39, 40, 41, Murray, J., 94, 129 Murty, B. R.,21, 39, 49, 60, 62 43, 47, 48, 4g Murty, G. S., 9, 39, 42, 48, 60 Mao, J. C.-H., 385, 397 Muto, A., 372, 598 Marchis-Mouren, G.,355, 89" Marcker, K.,346, 347, 354, 397 Marcker, K.A.,347, 349, 350, 353, 354, N 374, 376(3), 377, 392 Marcus, L., 385, $97 Nilf, U., 22, 60 Marion, L.,34, 36, 49 Nakamoto, T.,346, 350, 356, 394, 396, 398 Marquisee, M., 373, 380, 396 Marshall, D.G.,85, 128 Narita, K., 355, 398 Marshall, D.R.,96, 97, 128 Naville, A., 200, 819 Marshall, R.,363, 364, 365, 368, 399 Neas, M. O.,17,47 Marshall, R. E.,337, 359, 360, 385, 397 Nei, M., 87, 129 Martin, R. G., 345, 351, 352, 357, 362, Neidhardt, F-C.,388, 394 Nemer, M., 213, 282, 362, 398 369, 397, 403 Martini, M. L.,103, 1fl Neuhaus, M. J., 172, 222 Mather, K., 30, 38, 48, 49, 67, 64, 126, Newton, J., 329, 330, 331, 401 Newton, W.A.,345, 351, 357, 398 1.27, 129, 176, 221 Matthaei, J. H., 331, 362, 397, 398 Nirenberg, M. W., 331, 332, 334, 335,337, Maxwell, E. S., 385, 397 347, 359, 360, 363, 365, 367, 368, 376(2, Mayr, E., 56, 60,129 4), 377, 382, 385, 391, 398, 396, 397, Meiselman, N., 28, 49 398, 399, 402. Melchen, F., 373, 374, 380, 404 Nishimura, S., 332, 334, 335, 336, 337, Menninger, J. R., 356, 392 360, 362, 364, 372, 374, 396, 398, 400 Menser, H.A., 41, 44 Niwa, M., 28, 60 Merrill, S. H., 373, 374, 380, 390, 396 NO11, H., 350, 354, 398 Meselson, M., 368, 4Ol Nomura, M., 368, 402 Metz, C. W., 200, 2.21 Notani, G. W.,358, 398 Meyer, G.F., 176, 183, 184, 189, 192, 197, Noujdin, N. I., 150, 153, 167 200, 208, 209, 212, 820, 2S1 Novelli, G. D., 372, 375, 398, 408 Meyer, R., 354, 400 Michelson, A. M., 363, 373, 396, 397 Novitski, E., 136, 167

412

AUTHOR INDEX

0

Ochoa, M., Jr., 362, 368, 369, 398, 403 Ochoa, S., 358, 328, 331, 335, 336, 337, 345, 348, 352, 353, 362, 363, 385, 396, 39, .boo, 401, 402 O'Donald, P., 60,62, 63, 126,, 189 Ofengand, E. J., 374, 391 Ogata, K.,355, 398 Ohno, S., 148, 163, 16'7 Ohtsuka, E.,332, 334, 336, 336, 337, 360, 362, 364, 374, 375, 376(1), 377, 378, 385, 398, 400 Oka, M., 40,60 Okada, Y., 329, 330, 331, 4Ol Olivieri, A., 175, 200, $98 Olivieri, G., 175, 200, 889 Orgel, L. E., 328, 393 Orias, E.,369, 398 Oster, I. I., 140, 167 Owen, A. R. G.,65, 189

P Palenzona, D. L., 40, 43 Pandey, K.K.,42, 60 Panshin, I. B., 135, 167 Parisi, B.,385, 399 Parsons, P. A.,78, 92, 196, 199 Patel, G. J., 39, 60 Patel, K.A,, 16, 17, 40, 60 Pavan, A., 93, 129 Pavate, M. V., 39, 60 Paxmm, G. J., 30, 60 Pearlman, R.,355,399 Pearaon, K.,60, I89 Pelc, S. R.,386, 387, 399, 403 Pelling, C., 213, 981, 882 Penman, S., 345, 309 Penniston, J. T.,372, 399 Penswick, J. R., 373, 380, 396 Perez-DBvila, Y.,159,167 Perkus, M.P., 25,61 Persijn, J. P., 200, 91Q Pestka, S., 331, 332, 335, 363, 364, 365, 368, 398, 399 Petekofsky, A., 367, 398 Plaut, W.,213, 9.28 Poddar, R. K., 341, 402 Popp, R. A., 362, 399

Povilaitis, B., 39, 42,60 Prout, T.,88, 129

R Rabinowits, M.,192, $29 Rao, B., 213, 880 h, P. N., 5, 60 Ratty, F. J., Jr., 137,167 Ray, W. J., Jr., 387, 398 RajBhandary, U. L., 366, 380, 396, 399 Redfield, B.,354, 399 Reeve, E. C. R., 78, 199 Reich, E.,192, 918, 919, g.99 Reichmann, M. E., 354, 355, 384, 399 Rein, A., 140, 155, 166 Rether, B., 372, 403 Revel, M.,352, 367, 399 Rich, A., 327, 345, 350, 374, 394, 399, 408

Richter, C. M.,279, 322 Rifkim, D.B.,362, 399 Rifkin, M. R., 362, 399 Rinaldi, A. M.,213, 928 Ris, H.,215, 891 Ritossa, F. M.,164, 167, 374, 399 Robbins, R. B., 69, 1.99 Roberts, H.F.,94, 130 Robinson, H.F.,38, 39, 49, 60 Robson, D. S., 40, 68 Romig, W. R., 384, 391 Rottman, F.,335, 300 Rottman, F. M., 334, 409 Rudkin, G. T.,163, 164, 168 Riihl, F., 270, 388 Russell, L. B., 136, 143, 144, 145, 147, 148, 149, 168 S

Saaty, T. L., 65, 130 Safir, 8. R., 208, 999 Sager, R., 362,368,385,8988,403 Sakai, K. I., 30, 60 Salas, M.,328, 336, 337, 345, 348, 352, 3 5 3 , 3 5 8 , 3 9 ~ , ~ 9 ~ , 4 0 0 , 440.9 01, Sanchez, R. L., 111, 114,126,190 Sand, S. A., 27, 31, 41, 60, 61, 69 Sanger, F., 346, 364,397

AUTHOR INDEX

Sarabhai, A. S., 328, 351, 357, 360, 396, 400 Sato, N., 355, 598 Satyanarayana, K. V., 9, 30, 48, 61 Saxinger, W. C., 386, 387, 404 Saylars, C . L.,146, 168 Scarascia, G. T., 42, 61 Scarascia Mugnozza, G. T., 42, 46 Schaeffer, G. W., 25, 26, 61 Schaeffer, J., 350, 390 Schirer, L. A., 25,62 Schechter, A. N., 362,403 Scher, S., 400 Scheremetjiwa, E. A., 200, 923 Schildknecht, H., 320, 322 Schneider, G., 246, 322 Schneider, I., 141, 151, 152, 168 Schnell, F. W., 71, 130 Schiitz, G., 228,393 Schulke, J. D., 120, 130 Schultz, J., 140, 141, 143, 147, 160, 162, 163,167,168, 175, 208, 888 Schuster, H., 341, 400 Schwartz, J. H., 354, 368, 384, 400 Schweet, R., 327, 350, 390, 400 Schwemmle, J., 226, 230, 235, 277, 280, 281, 283, 291, 293, 297, 298,393, 384 Scoasiroli, R. E., 40,63 Scott, J. F., 328, 396 Scott, K. J., 26,61 Scudo, F. M., 63, 129 Searle, A. G., 148, 167 Setrer, V.,93, 129 Seufert, R., 320, 32.4 Sficas, A. G., 5, 6, 61 Shatkin, A. T., 192, 222 Shen, T. H., 208, 182 Shepherd, W. M., 375, 401 Sheppard, P. M., 93, 126, 130 Shikata, M., 78, 130 Shimamoto, Y., 30, 60 Shoup, J. R., 154,168 Siddiqi, O., 356, 357, 394 Sidorov, B. N., 135, 166 Siegel, E. P., 175,219 Signer, E. R., 384, 400 Sikka, L. C . , 40, 61 Silber, G., 41, 61 Silbert, D. E., 345, 351, 352, 375, 397, 400 Simon, R., 277, 32.4

413

Sinsheimer, R. L., 357, 396,400 Sjoquist, J. A,, 380, 396 Skoog, H. A., 17, 47 Slatis, H. M., 136, 143, 168 Slizynska, H., 143, 166 Slizynski, B. M., 148,168 sly, W. S., 331, 332, 335, 398 Smeltzer, D. G., 89, 90,91, 111, 114, l%', 130

Smith, C. R., 35, 68 Smith, D. W. E., 345, 351, 352, 397 Smith, E. L., 337,400 Smith, H. H., 5, 8, 9, 15, 21, 22, 23, Za, 25, 26, n,28, 3,31, 33, 3,35, 36, 38, 40, 47, 48, 61, 62 Smith, J. D., 356, 366, 392, 400 Smith, M. A,, 328, 336, 399 So, A. G., 362, 363, 368, 400 So11, D., 334, 335, 337, 347, 348, 353, 360, 362, 364, 374, 375, 376(1), 377, 378, 385, 394, 400

Solt, M. L., 36, 46 Sonneborn, T.M., 388, 401 Sorokin, T. P., 26, 63 Sparrow, A. H., 25, 27, 28, 47, 49, 61, 62 Spassky, B., 93, 130 Speyer, J. F., 331, 335, 367, 385, 401 Spiegelman, S., 164, 167, 374, 394, 399 Spofford, J. B., 141, 151, 152, 153, 155, 166, 168, 169, 175, 218 Sprague, G. F., 8 7 , l R Staehelin, T., 368, 4 O l Stanley, W. M., 328, 336, 399 Stanley, W. M., Jr., 336, 337,345,348,353, 358,396,401 Stebbins, G. L., 57, 130 Steinberg, C. M., 357, 393 Steinberg, R. A., 21, 62 Steitz, E., 26, 68 Stephens, S. G., 15, 62 Stephenson, M. L., 328, 396 Stern, C., 138, 169, 171, 174, 175, 208, 212, 922

Stevenson, H. Q., 15, 23, 24, 25, 30,62 Steward, M., 372, 399 Stewart, R. N., 33,.# Stines, B. J., 42, 62 Stokes, G. W., 4, 5, 19, 37, 41, 46, 47, 60, 69,63 Stonier, T., 22, 4.4

414

AUTHOR INDEX

Strehler, B. L., 375, 384, 401 Streisinger, G., 329, 330, 331, 401 Stretton, A. 0. W., 356, 357, 358, 359, 398, 396, 400, 401 Stuart, A., 380, 399 Stulberg, M. P., 373, 401 Sturtevant, A. H., %IS, 222 Subak-Sharpe, H., 375, 401 Subhaahini, L., 9, 30, 61 Sueoka, N., 373, 374, 375, 383, 396, 401, 404 Sundararajan, T. A., 328, 345, 349, 353,

401,409

Suneson, C. A., 103, 104, 111, 115, 116, 118, 128, 130 Susmm, M., 367, 393 Swift, H., 159, 166 Syaktdo, K., 87, 129 Swaminathan, M. S., 21, 40, 60, 62 Szer, W., 362, 4 1 Szolyvay, K., 357, 396

T Takenaka, M., 4, 62 Takenaka, Y. 4, 5, 22, 24, 62, 63, 64 Tanner, M. J. A , 354, 381, 385, 4Ol Targa, H. J., 93, 189 Tates, A. D., 200, 219 Tatum, E. L., 192, 228 Taylor, M. W., 375, 40f Termazi, S. A., 40, 43 Terzaghi, E., 329, 330, 331,401 Tessmm, I., 341, 342, 402 Thach, R. E., 328, 345, 348, 349, 353, 362, 401, 402 Therman, E., 22, 26, 48 Thoday, J. M., 73, 126 Thompson, M. C., 2, 47 Toback, F. G., 362, 368, 399 Toledo, S. A., 93, 129 Tomkins, G. M., 345, 890 Tooae, J., 346, 352, 396 Torres-Gallardo, J., 373, 408 Torriani, A., 384, 400 Tramvalidis, C., 42, 63 Traub, P., 368, 402 Trautner, T. A,, 384, 390 Trupin, J., 334, 335, 337, 399 Trupin, J. S., 334, 408

Tso, T.C., 24, 26, 35, 44, 63 Tsugita, A., 3!.27, 329, 330, 331, 337, 340, 341, 342, 344, 401, 402 Tucker, C. L., 63, 87, 88, 187, 130 Tucker, M. D., 372, 399

U Ukita, T., 372, 398 Ullerich, F. H., 215, 922 Urata, U.,27, 63

V Valanju, S., 192, 218 Valentine, R. C., 369 40.8 Valleau, W. D., 17, 19, 37, 41, 47, 63 Van der Veen, J. H., 40,63 Venetianer, P., 345, 352, 391 Vester, F., 26, 43, 63 Vines, A., 38,49 Vifiuela, E., 352, 408 von Borstel, R. C., 196, 281, 222 Von Ehremtein, G., 362, 375, 377, 384, 387, 3Q8, 40.2, 403 von Halle, E. S., 137, 167, 175, 221 Von Schlenk-Barnsdorf, M., 324 Von Zitek, R., 255,324

W Waddington, C. H., 59, 70, 137 Wahba, A. J., 328, 331, 335, 336, 337, 345, 348, 353, 358, 385, 396, 399, 400, 401 Wahlund, S., 56, 130 Wahrman, J., 63, 130 Wall, R., 330, 408 Waller, J.-P., 346, 408 Wan, H., 20, 63 Warner, J. R., 350, 409 Waters, L. C., 375, 408 Watson, J. D., 346, 352, 396 Watts-Tobin, R. J., 328, 329, 330, 356, 398 Weber, K., 346, 352, 396 Webster, R. E., 346, 357, 365, 366, 382, 393, 403, 404 Wehrhahn, C., 93, 103, 186 Weidner-Rauh, E., 228, 240, 384 Weigert, M. G., 344, 357, 358, 359, 360, 361, 403

415

AUTHOR INDEX

Weil, J-H., 372, 403 Weinberg, W., 130 Weinstein, I. B., 362, 363, 368, 369, 385, 386, 387, 394, 398, 399, 403 Weisblum, B., 362, 374, 375, 377, 387, 399, 4OB, 403 Weissbach, H., 354, 392 Wells, P. V., 2, 63 Wells, R. D., 332, 333, 335, 336, 360, 397 Welton, M. G. E., 387, 399, 403 Wender, S. H., 26, 63 Wensauer, H., 394 Weybrew, J. A., 36, 37, 4Y, 48, 49 White, J. R., 367, 368, 393, 394 White, M. J. D., 93, 129, 130 Whitehouse, H. L. K., 215,% 2!.9 Whitfield, H. J., Jr., 345, 351, 352, 367, 369, sgr, 403 Wilhelm, R. C., 341, 359, 365, 366, 382, 393, 394, 400, 403 Wittes, R. E., 389, 394 Witting, M. L., 367, 394 Wittmann, H. G., 327, 337, 340, 341, 342, 344, 408 Wittmann-Liebold, B., 327, 337, 340, 341, 342, 344, 403 Wittmer, G., 40, 43, 63 Woese, C. R., 345, 365, 386, 387, 389, 404 Wolf, F. A,, 33, 41, 64 Wolfe, H. G., 148, 169

Workman, P. L., 61, 65, 66, 67, 79, 87, 88, 89, 90,91, 103, 104, 196, 198, 130 Woskressensky, N. M., 200, 9% Wright, S., 56, 60,61, 62, 65, 70, 73, 77, 79, 81, 180, 131

Y Yamane T., 374, 383, 396, 401, 404 Yang, SJ.,7, 29, 64 Yanofsky, C., 327, 334, 337, 342, 343, 344, 345, 351, 357, 366, 399, 396, 404 Yasurumi, G., 200, 223 YFas, M., 327, 394 Yoneda, Y., 22, 24, 63, 64 Yu, C. T., 372, 383, 404 Z

Zabin, I., 357, 394 Zachau, H. G., 372, 373, 374, 380, 396, 396, 404 Zamecnik, P. C., 328, 372, 396, 404 Zamir, A., 350, 373, 374, 380, 396, 404 Zimmermann, F., 196, 2B3 Zinder, N. D., 346, 356, 357, 358, 365, 366, 369,382, 384,393,398,4OO,4Oi?, 408, 404 Zintl, M., 235, 3B4 Zipser, D., 345, 351, 357, 398 Zujtin, A. I., 189, 923

SUBJECT INDEX A Alkaline phosphatase, in genetic code BtUdiM, 337 Alkaloids, of Nicotiana, inheritance of, 34-37 Amber mutants, 357459 Amino acids, in proteins, replacement studies on, 337-344 Aminoacyl-tRNA synthetase, in codon recognition, 370384 Anabasine, in Nicotiana spp., 35 Anticodons, degeneracy of, 374-377 sequences of, 380 synthetase-recognition site and, 371374 wobble hypothesis of, 380

B Bacteriophage T 4 in genetic code studies, 331 Biometrical methods, in Nicotiam cytogenetic studies, 38-41

C

in polycistronic mesaengers, 350-352 universality of, 354 nonsense type, chain termination and, 355-361 recognition, tRNA and, 370384 multiple, 377478 Cytogenetics of Nicotiann, 1-54

D Developmental biology, simplified studies by variegation experiments, 154-160 DNA, in Y chromosome loop, 213-215 Drosophila, position-effect variegation studies on, 133-169 spermatocyte structures in, 177-180 spermatogenesis, Y chromosome in, 171-223 Drosophila hydei, spermatogenesis in, 200-207

abnormal, 209-211 Drosophila melanogasler,spermatogenesis abnormal, 208-209

E Euploidy, in Nicotiana, 25

F Cis-tram relation in position-effect variegation, 134-135 Festuca microstachys complex, in inCodom, 327 breeding population studies, 117-120 amino acid structure and, 386-388 N-Formylmethionine, in protein bioanti-, see Anticodom synthesis, 346-348 assignments of, 32-45, 384-386 amino acid replacement studies of, G 337444 biochemical studies of, 331-337 Genetic code, 325-404 genetic studies, 329-331 ambiguity in, 361470 pattern of degeneracy, 344345 coding environment and, 362-364 of N-formylmet-tRNA, 346-348 pattern of, 364-365 initiator type, 345-355 suppression and, 365-367 416

417

SUBJECT INDEX

chain initiation in, 345-355 chain termination and, 355-361 formylation and, 349-350 general nature of, 327-329 historical background of, 327-329 universality and evolution of, 384 Genetic tumors, of Nicotiana, 2 2 3 0 Genetics of inbreeding populations, 55131

H Hemoglobins, in genetic code studies, 327, 337, 338-340

I Inbreeding populations, genetics of, 55131 effect of altering the mating system, 113-117 enforced self-fertilization, 113-114 male sterility, 115-117 experimental analyses of polymorphisms, 86-94 extreme inbreeding, 117-120 genetic models, theoretical analysis, 57-86 complex types, 81-85 multilocus population types 68-81 single-locus types, 58-68 genetic variability in quantitative characters, 94-103 geographic variability, 95-97 within families, 99-103 within populations, 97-99 responses to selection, 103-113 artificial selection, 110-113 competition in mixtures of pure lines, 103-105 natural selection, 105-110

M Magnesium ions, in genetic coding, 3 5 2 353 Mouse, position-variegation effect in, 144-149

N Nicotiuna, alkaloids of, inheritance, 3 4 3 7 allopolyploids of, 6-8 aneuploidy and multiple genomes of, 8-15 chromosome pairing in interspecific hybrids of, 5-6 classification of, 3-4 cytogenetic studies on, 1-54 biometrical methods in, 38-41 cytoplasmic inheritance, 30-34 disease resistance of, 15-22 euploidy in, 2-5 genetic tumors of, 22-30 species of, 2 Nicotiana tabacum, alkaloids of, 36-37 disease resistance of, 18 monosomic types of, 10-14 Nicotine, in Nicotiana spp., 35 Nornicotine, in Nicotiuna spp., 35

0

Ochre mutants, 357459 Oenothera spp. selective fertilization in, 225-324 a f i i t i e s in trisomic mutants, 313-316 changes in a f i i t i e s of, 278313 cytoplasm and plastid influences 298306 environmental factors in, 279-280 genome changes, 306-313 ovule maturity, 280-298 pollen-tube chemotropism, 316-321 Oenothera berteriana, crosses of, fertilization in 226-238 selfing of, selective fertilization in, 239 Oenothera campylocalyx, selfings of, selective fertilization of, 270-278 Oenothera odorata, crosses of, fertilization in, 226238 selfing of, selective fertilization in 254268 Oenothera selowii, selfings of, selective fertilization of, 268-270 Oenothera stricta, selfing of, selective fertilization in, 268-270

418

SUBJECT INDEX

P Pollen tubes, of Oenothera spp., chemotropism in, 316-321 Polymorphisms, experimental analyses of, 8694 Polyphenylalanine, biosynthesis of, 331 Position-effect variegation, 133-169 cytogenetic aspects, 134-144 cis-tram relation in, 134-135 of genes normally in heterochromatic region, 138-140 as model system for developmental biology studies, 154460 in the m o m , 144-149 cytogenetics of, 144-148 normal X suppression and, 148-149 parental homozygosity for rearrangement in, 151 parental modification and, 149-154 parental source of rearrangement, 152153 parental Y chromosomes and, 152 polarhed spreading effect in, 142144 proofs of, 135-138 Y chromosome suppression in, 140-142

R RNA, in Y chromosome loops, 216-216 transfer type, see Transfer RNA 8 Spermatocytes, of Drosophsla spp., morphology of, 176183 Spermatogenesis, Y chromosome in, 171223

Streptomycin, in codon suppresaion, 367370

T Tobacco mosaic virus coat proteins, in genetic code studies, 327, 337, 340 Topaz mutants, 381 Transfer RNA, in codon recognition, 370384 nonsense codon and, 36S67 Transfer RNA synthetases, degeneracy of, 383-384 Tryptophan synthetase A, in genetic code studies, 327, 337

V Variegation, position-effect type, aee Pwition-effect variegation, 133-169

W Wobble hypothesis, anticodone and, 380382

Y Y chromosome, in spermatogenesis, 171223 cytogenetic studies of, 176-199 morphology of spermatocytes, 176 183 in formation of spermatocyte structures, 183-187 loops of function of, 187-196 shape of, 196199 in spermiogenesis, 199-213