No title

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME43

ADVISORY EDITORS H. W. BEAMS

DONALD G MURPHY

HOWARD A. BERN

ROBERT G. E. MURRAY

W. BERNHARD

ANDREAS OKSCHE

GARY G . BORISY

VLADIMIR R. PANTIC

ROBERT W. BRIGGS

D. C. REANNEY

R. COUTEAUX N. 13. EVERETT DON FAWCETT MICHAEL FELDMAN

LIONEL I. REBHUN JEAN PAUL REVEL WILFRED STEIN ELTON STUBBLEFIELD

CHARLES J. FLICKINGER

H. SWIFT

WINFRID KRONE

DENNIS L. TAYLOR

K. KUROSUMI M A R I A N 0 LA VIA GIU S EPPE M ILLON IG MONTROSE J. MOSES

J. B. THOMAS TADASHI UTAKOJI ROY WIDDUS A. L. YUDIN

111 connection with Dr. James F. Danielli’s editorial responsibilities. please note that, effective May 1, 1975, tlie postal address for

INTERNATIONAL REVIEW OF CYTOLOGY will be: Worcester Polytechnic Institute, Worcester, Massachusetts 01609 for ull new manuscripts and correspondence pertaining thereto.

INTERNATIONAL

Review of Cytology EDITED BY

G . H. ‘BOURNE

J. F. DANIELLI

Yerkes Regional Primate Research Center Emory University Atlanta, Georgia

Worcester Polytechnic Institute Worcester, Massachusetts

ASSISTANT EDITOR K. W. JEON Department of zoology University of Tennessee Knoxville, Tennessee

VOLUME43

ACADEMIC PRESS New York San Francisco London 1975 A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHI 0 1975, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED I N ANY FORM OR rw A N Y MEANS, E I ECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION I N WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC. 1 1 1 Fifth Avenue, New

York. New York 10003

United K i w d o m Edition vublished bv ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London N W l

LIBRARY OF CONGRESS CATALOG CARD NUMBER:52-5203

ISBN 0-12-364343-0 PRINTED IN THE UNITED STATES OF AMERICA

Contents LIST 01: <:ONTRIHUTOWS

,

,

.

.

.

.

ix

The Evolutionary Origin of the Mitochondrion: A Nonsymbiotic Model HENRYH. MAIILEHA N D RUDOLF A. RAFF I. 11. 111. IV. V. VI. VII. VIII. IX.

Introdnction . . . . . . . . . Enkaryotic Origins and the Fossil Record , . . The Sym1)iotic Theory and Its Consequences . . . Aerobic Adaptations of the Erikaryotic Cell Cytoplasm and Primitive Functions . . . . , , . . Mitochondria1 Genes and Their Transcription. . . Mitochondrial Gene Expression . , . . . Mitochondria1 Functions . . . . , . . A Model for the Nonsymbiotic Origin of the Mitochondrion Conclusions and Predictions . . . , . , Note Added in Proof . . . . , , . . References . . . . . . . . . .

. . .

. . .

. .

.

2 3 9

Survival of

.

.

.

. .

. .

. . .

. . .

.

.

.

. ,

. . . .

.

.

11 33 46 74 83 98 11 1 112

Biochemical Studies of Mitochondrial Transcription and Translation C:. SA(’CONEAM)

I. Introduction . . . 11. Mitochondrial Transcription I I I, Mitochondria1 Translation . Note Added in Proof . . References . . . .

E. QUAGLIARIELLO

.

. . . . .

. . .

.

. .

.

. .

.

.

. . . .

. . . .

. . . . .

. . . . .

. . . . .

. . . . .

125 127 152 160 160

The Evolution of the Mitotic Spindle

I. Introduction . . . . . . . . . . . . 11. Some Evolutionary Considerations . . . . . . . . 111. The Possible Involvement of the Nuclear Envelope in Chromosome Movement . . . . . . . . . . . . . IV. Tlie Participation of Microtubules in Typical Mitosis . . . . V. Tlie Possibility of an Unconventional Role for Microtuldes in Chromo. . . . . . . . . . . some Movement . VI. Nuclear Divisions with Microtu1)ule-Mediated Chromosome Movements . . . . . . . . . . . . . . VII. Final Remarks . . . . . . . . . . . . References . . . . . . . . . . . . . V

167 168 171 194 197 205 220 22 1

vi

CONTENTS

Germ Plasm and the Differentiation of the Germ Cell Line E. M. EDDY I. Historical Background . . . . . , . 11. Animal Reported to Have Germ Plasm . . , . 111. Polar Granules and Insect Development . . . . IV. Genninal Determinants and Chaetognath Development. V. Genninal Plasm and Amphibian Development . . V1. The Ntiuge and Germ Cells . . . . . . VII. Coldusions . . . . . . . . , References . . . . . . . . . ,

. . . ,

. .

. .

,

.

.

,

. . . .

. . . . .

,

,

.

229 230 23 1 240 24 1 250 27 1 275

Gene Expression in Cultured Mammalian Cells RODY

I. 11. 111. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV.

xv.

XVI.

P. COX

AN11 JAMES

c. h N C ;

General Introduction , . . , . , , . . . . The Discovery of Gene Regulation in Bacteria . . . . . . Difficulties in Applying the Bacterial Model to Higher Forms . Differences between Prokaryotes and Eukaryotes , . . . . Regulation of Specific Protein Synthesis i n Mammalian Cells . . Differentiated Functions in Mammaliarr Cell Culture . . . . Hormonal Effects on Gene Expression in Cultured Cells . , . Regulation of the Activity of Metalloenzymes by Agents Other than . . . . . . . . . . . . Hormones . Regulation of the Rate of Protein Degradation. . , . . . Protein Modification Altering Gene Expression . , . . , Control of Enzyme Activity in Intact Cells- Role of Substrates and . , . . , . , . . , . . , Cofactors Genetic Coiitrol of the Intracellular Localization of Enzymes . , Gene Expression in Heterokaryons of Mammalian Cells , . , Gene Expression in Synkaryons of Mammalian Cells . . . . Heconstruction of Mamrnalian Cells . . . , , . , . Conclusions , . . . . . . , . . . . References . . . . . . . . . . . , .

282 283 284 285 293 308 313 329 03 1 333 334 335 336 339 343 343 344

Morphology and Cytology of the Accessory Sex Glands in Invertebrates

I. 11. 111. IV.

Introduction . . General Survey . . Evolutionary Aspects . Miitiirtitioii . . ,

. .

.

.

. .

.

.

. . . .

. . . .

. . . .

. .

.

.

.

.

,

.

,

.

. .

. .

. ,

.

.

,

,

353 354 388 390

vii

CONTENTS

V . Differentiation and Secretion: The Role of Hormones VI . Concluding Remarks . . . . . . . References .

.

.

.

SUBJECTINDEX . . . . CONTENTSOF PREVIOUSVOLUMES

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

392 393 396

. .

. .

. .

. .

. .

. .

. .

. .

. .

399 403

This Page Intentionally Left Blank

List of Contributors Numbers in parentheses indicate the pages on which the authors' contributions begin.

K. G. ADIYODI (353),Department of Zoology, Calicut Uniuersity, Kerala, India R. G. ADIYODI (353),Department of Zoology, Calicut Uniuersity, Kerala, India

RODY P. COX (281), Division of Human Genetics, Departments of Medicine and Pharmacology, New York University Medical Center, New York, New York E. M. EDDY(229),Department of Biological Structure, University of Washington, Seattle, Washington C. KING (281), Division of Human Genetics, Department of Microbiology, New York University Medical Center, New York, New York

JAMES

DONNAF . KUBAI" (167), Department of Zoology, University of Wisconsin, Madison, Wisconsin HENRY R. MAHLER (l), Department of Chemisty, Indiana University, Bloomington, Indiana RUDOLF A. RAFF (l),Department of Zoology, Indiana Uniuersity, Bloomington, Indiana C . SACCONE(125), Institute of Biological Chemisty, University of Bari, Bari, Italy

E. QUAGLIARIELLO(125), Institute of Biological Chemistry, University of Bari, Bari, Italy

O

Present address: Deparbnent of Zoology, Duke Universb-Durham, North Carolina. ix

This Page Intentionally Left Blank

The Evolutionary Origin of the Mitochondrion: A Nonsymbiotic Model HENRY R . MAHLERAND RUDOLF A . RAFF Department of Chemistry and Department of Zoology. Indiana University. Bloomington. Indiana I . Introduction . . . . . . . . . . I1. Eukaryotic Origins and the Fossil Record . . . . A . Conditions on the Precambrian Earth . . . . B. The Precambrian Fossil Record . . . . . C . Time of Prokaryote-Eukaryote Divergence Estimated from Rates of Molecular Evolution . . . . 111. The Symbiotic Theory and Its Consequences . . . IV. Aerobic Adaptation of the Eukaryotic Cell Cytoplasm and . . . . . Survival ofprimitive Functions . A . Enzymic Protection from Oxygen Toxicity . . . B. Metabolic Requirements of Anaerobic Eukaryotes . C . Oxygen and Biosynthetic Patterns . . . . . D . Heme Biosynthesis . . . . . . . . E . ElectronTransport Sequences . . . . . F. Energy Coupling and ATPases . . . . . V. Mitochondria] Genes and Their Transcription . . . A . The Mitochondria] Genome . . . . . . B. DNA-DependentRNAPolymerases . . . . VI . Mitochondrial Gene Expression . . . . . . A . Mitochondrial rRNA . . . . . . . B. Mitochondria] mRNA . . . . . . . C . Mitochondria] Protein Synthesis . . . . . VII . Mitochondrial Functions . . . . . . . A . Mitochondrial Topography . . . . . . B. Mitochondria] Lipids . . . . . . . C . Matrix Functions . . . . . . . . D . Functions of the Inner Membrane . . . . E . Autonomy in Biogenesis . . . . . . VIII . A Model for the Nonsymbiotic Origin of the Mitochondrion A . TheModel . . . . . . . . . B. Elements of the Model . . . . . . . IX . Conclusions and Predictions . . . . . . A . The Two Theories: Contrast and Outlook . . . B. Validity of the Molecular Approach . . . . C . Mitochondria as Supramolecular Assemblies . . D. A Speculation: Protoeukaryotes as Living Fossils . Note Added in Proof . . . . . . . . References . . . . . . . . . .

2 3 3 4 7 9 11 12 17 18 20 22 30 33 34 40 46 46 60 65 74 74 75 78 78 83 83 83 86 98 98 104 108 110 111 112

2

HENRY R. MAHLER AND RUDOLF A. RAFF

I. Introduction “All right,” said the Cat; and this time it vanished quite slowly beginning with the end of the tail, and ending with the grin, which remained some time after the rest of it had gone. Lewis Carroll Alice’s Adventures in Wonderland

Those of us concerned with the origin of the mitochondrion find ourselves, like Alice, forced to deal with a very frustrating entity. Essentially, all we have before us is the grin-from which we must attempt to reconstruct the Cheshire Cat. Regardless of the mode of origin of the mitochondrion, and whatever the model selected, both the nuclear-cytoplasmic and nuclear-mitochondria1 systems of the eukaryotic cell have undergone over a billion years of coevolution to their present forms. The almost complete lack of a fossil record bearing on these events requires us to find our clues by unraveling the nature of the contemporary systems available to us. The greatest evolutionary discontinuity between living organisms lies between prokaryotic and eukaryotic cells. Biochemical evidence clearly demonstrates that these two major groups of cells did not arise independently. Nevertheless, the extent of the gap separating them, and the apparent lack of any intermediate forms, has made it difficult to reconstruct the evolutionary transition between them. One of the most puzzling aspects of eukaryotic cellular organization is the existence of semiautonomous cytoplasmic genomes in certain organelles, the mitochondria and chloroplasts. The existence of these self-replicating organellar genomes, and the resemblance of the associated organellar systems of protein synthesis to those of bacteria, has led to the wide acceptance of the hypothesis that these organelles had their origins in symbiotic associations of bacteria and blue-green algae with ancestral protoeukaryotic cells (Sagan, 1967; Nass, 1969; Margulis, 1970, 1971; Stanier, 1970; Cohen, 1970; Raven, 1970; Schnepf and Brown, 1971). This theory requires that several symbiotic events cooperated in the establishment of the various organelles. In our previous article (Raff and Mahler, 1972), we reexamined the data used in support of the symbiotic hypothesis and concluded that at least for the mitochondrion the case for the symbiotic theory is far from totally convincing. We put forward a nonsymbiotic (plasmid) hypothesis which we felt accounted equally well or better for the available data. Since the time of our last writing, much new experimental data have appeared which, as we hope to demonstrate,

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

3

strengthen the case for a nonsymbiotic origin of the mitochondrion. We have previously ignored the chloroplast, and we shall have to do so in this article as well. This is done because we feel that this organelle merits separate consideration, both because of the mass and complexity of the data and because it is quite conceivable that organelles were acquired independently by both symbiotic and nonsymbiotic mechanisms. The case for a symbiotic origin of the chloroplast (Cohen, 1973) is certainly much better than for the mitochondrion. We hope that our effort will help to provide a perspective which we consider vital, since dogmatic adherence to the symbiotic theory is premature and may result in an unfortunate narrowing of experimental approach and interpretation in the study of organelles and their origins. 11. Eukaryotic Origins and the Fossil Record

A. CONDITIONS

ON THE PRECAMBRIAN

EARTH

Most prokaryotes and essentially all eukaryotes are adapted to life in the presence of free oxygen. The two fundamental biochemical adaptations for such life are the possession of two enzymes, superoxide dismutase and catalase, which protect cellular components from autooxidation, and an electron transport system that allows cells to use atmospheric oxygen as a terminal electron acceptor for energyyielding biochemical oxidations. Nevertheless, the original atmosphere of the earth in which life arose and early cellular evolution took place was almost certainly anoxygenic, and may have resembled that of the Jovian planets. Experiments on the abiogenic synthesis of amino acids and other cellular components under hypothetical primitive earth conditions have been found to require a reducing environment and generally are performed with starting materials such as methane, ammonia, water, and hydrogen. Details of such experiments and of the evidence for a reducing atmosphere on the primitive earth are presented by Kenyon and Steinman (1969). Geological evidence for a shift from an anoxygenic atmosphere to an oxygenic one is discussed by Rutten (1971), by Cloud (1968a,b, 1974), and by Cloud and Gibor (1970). Detrital uranite deposits and banded iron formations which are interpreted to have formed under oxygen-poor conditions are confined to deposits 1.8 billion years old or older. However, red beds, which indicate surface oxidation and thus free atmospheric oxygen, occur in deposits of 1.8 billion years

4

HENRY R. MAHLER AND RUDOLF A. RAFF

old or younger. Rutten (1971) concludes that an oxygenated atmosphere became established between 1.4 and 1.8 billion years ago. The first organisms were anaerobic heterotrophs. These gave rise to a variety of anaerobic prokaryotic heterotrophs and photosynthetic organisms. Fossil evidence indicates that blue-green algae (which produce oxygen as a photosynthetic by-product) may have been in existence more than 2.5 billion years ago (Echelin, 1970a,b; Schopf et al., 1971; Schopf, 1970). Photosynthetic production of oxygen was probably the major cause of the replacement of the ancient atmosphere of the earth by one containing free oxygen (Cloud, 1968b). Cloud (1968a,b, 1974), and Cloud and Gibor (1970) suggest that the banded iron formations resulted from the production of oxygen b y primitive photosynthetic organisms that had not yet evolved the enzymes required for oxygen tolerance. Thus these organisms grew in balance with available soluble Fez+. Ferric or ferrous-ferric iron was precipitated on combination of Fez+with photosynthetic oxygen. According to this hypothesis, once oxygen and enzymes capable of detoxifying peroxides had evolved, the photosynthesizers would no longer be sensitive to free oxygen and thus no longer dependent on a balance with the availability of Fez+.These organisms would have increased in number, and oxygen production would have increased eventually to the point of accumulation in excess. An ozone layer providing protection from ultraviolet radiation would have appeared and opened further environments to life (Ratner and Walker, 1972). A timetable of Precambrian events and fossil occurrences is presented in Table I.

B. THE PRECAMBRIANFOSSILRECORD The most prominent macrofossils of the Precambrian are the layered structures known as stromatolites, which formed large reefs in Precambrian times (Glaessner, 1962, 1966, 1968; Howell, 1971). Stromatolites are not confined to the Precambrian, in fact they currently occiir in some tropical regions, which gives us a glimpse of part of the Precambrian environment and allows some paleoecological speculations to be made. Stromatolites are not single organisms, but rather are formed by multispecific mats of filamentous blue-green algae growing in the intertidal zone (Logan, 1961; Logan et al., 1964; Garrett, 1970; Awramik, 1971; Golubic, 1973). During the Precambrian, stromatolites may well have grown in the subtidal zone from which they are now excluded because of competition and grazing (Garrett, 1970; Awramik, 1971). Precambrian stromatolites indicate that oxygen-producing, blue-green algae, photosynthetic

5

MITOCHONDRIA AND T H E ORIGIN O F EUKARYOTES TABLE I A TIMETABLE OF PRECAMBRIAN EVENTSAND FOSSIL OCCURRENCES Age (years x lo8)

3.7 3.4 2.7-3.1 3.0 2.2-2.3 -2.0 -1.8 -1.2-1.5 0.9 0.6-0.7 0.6

Event or occurrence Oldest dated terrestrial rocks; Greenland Oldest dated sedimentary rocks containing globular microstructures of possible biogenic origin; Onverwacht series, South Africa Oldest known stromatolite; Bulawaya, Rhodesia Oldest fossil rod-shaped bacteria; Fig Tree series, South Africa Oldest fossil blue-green algae showing cell diversification; Transvaal sequence, South Africa Diversified prokaryotic microbiota; Gunflint iron formation, Canada First free oxygen in atmosphere Oldest fossil cells resembling eukaryotes; Beck Springs formation, California; Bungle-Bungle dolomite, Australia Oldest large assemblage ofeukaryotic cells and evidence for meiosis; Bitter Springs formation, Australia Oldest fossil multicellular animals End of Precambrian

References"

1 2,3 4, 5 6, 7 8

9 3,lO-12 13-15 15 16 17

~

(1) Moorbath et al. (1973); (2) Hurley et al. (1972); (3) Rutten (1971); (4) Schopf et al. (1971); (5) Vail and Dodson (1970); (6) Barghoorn and Schopf (1966); (7) Sinha (1972); (8) Nagy (1974); (9) Schopf (1970); (10, 11) Cloud (1968a,b); (12) Cloud and Gibor (1970); (13) Cloud et al. (1969); (14) Diver (1974); (15) Schopf and Blacic (1971); (16) Glaessner (1971); (17) Dott and Batten (1971).

organisms, already existed 2.6 x lo9 years ago (Schopf et al., 1971). Throughout the Precambrian such blue-green algal mats would have provided a relatively oxygen-rich environment, shielded from high levels of ultraviolet light (Fischer, 1965). Precambrian microfossils, often found in association with stromatolites, have begun to provide us with the glimmerings of a record of cellular evolution (the literature prior to 1970 is well reviewed by Schopf, 1970). A few bacterialike forms are known from the early Precambrian (more than 2.5 x lo9 years ago) (Table I), but the middle Precambrian (2.5 to 1.7 x lo9 years ago) has yielded a relatively rich set of microfossils from several localities. These provide a record of increasing prokaryotic diversity. The oldest known fossils showing cellular differentiation are those of filamentous blue-green algae from the 2.2 x 109-year-old Transvaal sequence of South Africa

6

HENRY R. MAHLER AND RUDOLF A. RAFF

(Nagy, 1974). The best known middle Precambrian microbiota come from the Gunflint chert of Canada (approximately 1.9 x log years old) (Schopf, 1970; Rutten, 1971). The cellular fossils from this formation consist mainly of several unicellular spheroids and septate filaments preserved in amorphous silica which has hardened to cryptocrystalline quartz. Most of these are assignable to the blue-green algae, although there are other apparently prokaryotic organisms not readily assignable to modern taxa. Eukaryotes are apparently absent from the Gunflint and other middle Precambrian formations. Late Precambrian (approximately 1.7 to 0.6 x log years ago) formations have yielded various microbiota which begin to provide an insight into the origin of eukaryotic cells. Licari and Cloud (1972) studied 1.6 x 1Og-year-old microfossils from stromatolites from Queensland, Australia, which seem to be entirely the remains of bluegreen algae. Other formations of similar age, for example, the Amelia dolomite of Australia (Croxford et al., 1973; Muir, 1974a,b), apparently contain prokaryotic assemblages similar to that of the Gunflint chert. However, some more advanced forms are found in the Amelia dolomite, notably multicellular organisms which superficially resemble colonies of myxobacteria (Stanier et al., 1970), and Muir (1974a) proposes that some of the early (some as old as 1.8 x log years) problematic fossil cells actually represent primitive “protoeukaryotes.” The oldest microfossils definitely assigned to the eukaryotes are from stromatolites from the Beck Springs dolomite dated at about 1.3 x logyears old (Cloud et al., 1969),and from the Bungle-Bungle dolomite of Australia which is assigned a similar age (Diver, 1974). This assignment is based on the presence of spherical cells containing a dark spot interpreted as the possible remains of a nucleus or pyrenoids. Possible eukaryotic cell fossils have also been described from formations of about 1.0-1.1 x log years old (Schopf, 1970; Schopf and Fairchild, 1973). However, the question of the validity of the criteria used to distinguish prokaryotic from eukaryotic fossil cells is of vital importance, particularly since peculiar fossils such as those found in the Gunflint chert and the Amelia dolomite are known. Thus fossils representing either extinct side branches or protoeukaryotes may either b e assigned to prokaryotic genera (Rutten, 1971) or remain enigmatic. Further, there appears to be an overlap of cell size in preserved prokaryotic and eukaryotic fossil cells, and such criteria as fossil “mitosis” and “ nuclei” are not completely convincing. This is of course unfortunate, since the time of origin of eukaryotes is of considerable interest.

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

7

I n summary then, it appears (Schopf, 1970; Licari and Cloud, 1972) that eukaryotes are lacking from microfaunas 1.6-1.9 x lo9 years in age, but made their appearance somewhere between 1.4 and 0.9 x lo9 years ago. They are almost certainly present in the 0.9 x 109-year-old Bitter Springs (Australia) assemblage which in large part appears to be a blue-green algal composite similar to that of the Gunflint chert (Schopf, 1970). However, in it have been noted several apparently eukaryotic cells, as well as spore tetrads, indicating the existence of meiosis and therefore of eukaryotic sexuality b y that time. Thus, according to the fossil record, eukaryotic cells arose at a time when free oxygen had already been accumulating in the atmosphere for several hundred million years. The oldest metazoan fossils are 0.6 x lo6 years old (Glaessner, 1971). Various aspects of eukaryotic cell evolution in the period between 0.9 and 0.6 x lo9 years ago have been discussed by several authors (Cloud, 1968a; Towe, 1970; Raff and Raff, 1970; Rhoads and Morse, 1971; Stanley, 1973).

c.

TIMEOF PROKARYOTE-EUKARYOTE DIVERGENCEESTIMATED FROM RATES OF MOLECULAR EVOLUTION Evolutionary rates of amino acid or nucleotide substitution have been computed for several macromolecules. Such rates are generally quite constant for any particular molecular phylogeny (Dickerson, 1971; Dayhoff, 1972). This has been very nicely illustrated by Dickerson (1971), who correlated sequence changes in cytochromes from diverse organisms with absolute times of phylogenetic divergence obtained from classic paleontological data. Once such a rate is computed, one ought to b e able to apply it to dating divergences not preserved in the fossil record, such as those that may have occurred in the Precambrian (e.g., metazoa from protozoa and plants, eukaryotes from prokaryotes). Using this extrapolation for cytochrome c, Dickerson (1971) estimates the age of divergence of animals and plants at 1.2 x log years. As an argument in favor of this value, h e points out that mitochondria1 electron transport had become established prior to the separation of animals and plants. We have applied his method of calculation to the divergence of eukaryotic from prokaryotic cytochrome (using Rhodospirillum cytochrome c2) and found an apparent divergence date of 2.4 x lo9 years ago, which seems high (see below). McLaughlin and Dayhoff (1970) used sequence data for cytochrome c and tRNA species to estimate the degrees of divergence between eukaryotes and prokaryotes and of eukaryotic kingdoms

8

HENRY R. MAHLER AND RUDOLF A. RAFF

from each other. They conclude that eukaryote-prokaryote divergence was 2.6 times more remote in evolution than the divergence of eukaryotic kingdoms. Jukes (quoted in McLaughlin and Dayhoff, 1970) and Kimura and Ohta (1973) estimated from tRNA data that the eukaryotic-prokaryotic divergence age was twice as long as that between eukaryotic kingdoms. Our only available bench marks for the age of the eukaryotic kingdonis are Dickerson’s estimate of 1.2 x lo9 years derived from cytochrome c data and the interpretation of Schopf et al. (1973) that the 0.9 x 109-year-old Bitter Springs eukaryotes lie near in time to the origin of eukaryotic sexuality. Applying these dates to the estimates of McLaughlin and Dayhoff, Jukes, Kimura, and Ohta yields a divergence age for eukaryotes and prokaryotes in excess of 1.8 x lo9 years. Kimura and Ohta (1973) calculated that 5s RNAs of eukaryotes are 1.5 times more different from those of prokaryotes than from those of other eukaryotic kingdoms. They then applied Dickerson’s estimate of the age of the eukaryotic kingdoms (based on the rate of cytochrome c evolution) to their data, and estimated the time of eukaryote-prokaryote divergence as 1.8 x lo9 years ago. Their estimate of divergence making use of Schopf’s 0.9 x 109-years datum point (first appearance of‘ well-defined eukaryotes in the fossil record) yields a divergence time of 1.4 x lo9 years. All these estimates are shaky, since relevant data are still scarce. Furthermore, extrapolations of rates of molecular evolution may be risky. Simpson (1953)has pointed out that evolution may occur at an. accelerated pace during evolutionary transitions between major adaptive zones. The origin of eukaryotic cells (and to a lesser extent that of multicellularity) clearly involved such a major evolutionary step. The transition may have required a relatively short span of time. Since the innovations were subcellular and biochemical, this transition may have had as a concomitant a high rate of molecular evolution followed b y a slowing of rates to those calculated for Phanerozoic eukaryotes. Such a phenomenon (as recognized by Dickerson, 1971) would cause significant overestimates of the age of eukaryoteprokaryote divergence. Evidence that variations in rate of sequence change in proteins d o occur is provided by an apparently accelerated rate of cytochrome c evolution in snakes (Jukes and Holmquist, 1972a,b), and of a-lactoglobulin subsequent to its divergence from lysozyme (Dickerson and Geis, 1969).

MITOCHONDRIA AND T H E ORIGIN OF EUKARYOTES

9

Nevertheless, as more comparative sequence data become available, rates of molecular evolution may have a great potential for dating Precambrian evolutionary events. Such data may also provide considerable insight into relationships of groups that lack a fossil record for their divergence.

111. The Symbiotic Theory and Its Consequences According to the symbiotic theory of the origin of the eukaryotic cell, as the primitive anoxygenic atmosphere of the earth began to acquire free oxygen as a result of photosynthesis, prokaryotes which had utilized a wide variety of pathways for anaerobic energy metabolism were forced either to adapt to aerobic conditions or to become restricted to the limited anaerobic environments remaining. Prokaryotes exhibit a variety of anaerobic energy-generating pathways, and there is a diversity of anaerobic prokaryotes. Eukaryotes, however, possess only glycolysis as a pathway of anaerobic energy metabolism (Stanier, 1970). Since eukaryotes are thus restricted, the ancestral protoeukaryote likewise utilized glycolysis. This protoeukaryote evolved several adaptations that allowed it to escape from the selective pressure of free oxygen which was the determinant driving the evolution of advanced oxidative metabolic pathways in contemporaneous prokaryotes. By such innovations as larger cell size, intracellular translocation, advanced mechanisms of cell motility, and the ability to phagocytize, the protoeukaryote became able to ingest prokaryotes as prey to provide substrates for glycolysis. Dependent on and subsequent to these advances was the establishment of stable intracellular symbiotic relationships between the protoeukaryote and certain ingested aerobic prokaryotes. Thus the final step in the origin of the eukaryotic cell was the acquisition of oxygen-mediation mechanisms (photosynthesis and respiration) by quantum steps. Symbiotic relationships involving prokaryotic symbionts housed in a eukaryotic cytoplasm exist among present-day organisms (Stanier, 1970; Preer, 1971). Interestingly, however, endosymbiotic relationships in which the host is a prokaryote have apparently not been observed (Stanier, 1970).The symbiotic theory further requires that in the course of time the symbiotic association has become intimate to the point that most of the genetic information required for assembly of the organelle-symbiont has been transferred to the nuclear genome. Concomitantly, the informational content of the organellar genome has been greatly reduced. Two aspects of this hypothesis seem awkward. The model suggests

10

HENRY R. MAHLER AND RUDOLF A. RAFF

that the protoeukaryote possessed many advanced cellular adaptations, yet was primitive and inefficient metabolically. In the face of competition from other prokaryotic organisms possessing more efficient aerobic energy-yielding pathways foreshadowing present-day patterns, such an organism should have found itself at a considerable disadvantage. Second, the integration of the symbiont to the extent of the restricted autonomy observed for the mitochondrion would have required a wholesale transfer of genes from the endosymbiont genome to the unrelated nuclear genome of the host. N o mechanism has been put forward to account for such a transfer. Unfortunately, these aspects of the theory are not amenable to experimental investigation. However, other consequences emerge from this model, for which pertinent experimental data do exist. These are outlined below, and will be discussed in detail in subsequent sections of this article.

1. According to the symbiotic model, eukaryotic cytoplasm should show evidence of a fundamentally anaerobic nature, since the anaerobic protoeukaryote acquired its oxygen-mediating systems from the aerobic symbiont. With the exception of some secondarily evolved anaerobic eukaryotic organisms which are ultimately dependent on molecular oxygen, all eukaryotes are aerobes and utilize the efficient energyyielding pathways of the mitochondrion. Further, as substantiated in Section IV, all eukaryotes appear to b e fundamentally aerobic not only in the possession of oxygen-detoxifying enzymes, and mitochondria, but also in many details of biosynthesis in which several important cellular components synthesized by anaerobic pathways in bacteria are synthesized by reactions utilizing molecular oxygen (Bloch, 1962; Goldfine and Bloch; 1963; Cohen, 1970, 1973). Thus eukaryotic cells probably did not arise from their prokaryotic progenitors until after the atmosphere contained appreciable free oxygen. These ancestors were probably already adapted to the use of oxygen. 2. According to the symbiotic model, the limited organellar genomes of contemporary organisms, as well as their protein synthetic systems, are evolutionary relicts. We shall show that in fact these supposedly relict systems are of vital importance to organellar organization and function, and that the origin of organellar genetic systems probably lies in the necessity for in situ protein synthesis on the inner membrane of a topologically closed organelle. 3. Several properties of present-day organelles are similar to those

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

11

of prokaryotes; such characteristics are interpreted as being simply relicts in nature and have been taken to provide strong support for a symbiotic origin. We shall show that, while many aspects of the mitochondrion are conservative, others are not, so that the degree of homology between prokaryotes and mitochondria is easily overstated. Finally, relict or conservative attributes of these organelles only give evidence for a prokaryotic ancestry (Uzzel and Spolsky, 1974) not for symbiosis, since both genetic systems in the eukaryotic cell have been derived from an ancestral prokaryotic ancestor. During their long coexistence in the eukaryotic cell, these systems have been components of two different, although interdependent, units of selection (Lewontin, 1970), and have diverged both from the ancestral prokaryotic pattern and from each other. 4. There is no evidence for the postulated decrease in mitochondrial complexity as a function of evolution, either with respect to the extent of autonomous informational content or its expression.

IV. Aerobic Adaptations of the Eukaryotic Cell Cytoplasm and Survival of Primitive Functions Several lines of evidence indicate that the eukaryotic cell cytoplasm is not simply an anaerobic system containing an aerobic respiratory organelle, but that in fact it possesses ancient and fundamental adaptations to an aerobic environment. Clearly, the most definitive evidence concerning possible evolutionary relationships is provided by structural information on key enzymes and other proteins inferred as arising early in the postulated series of events described in Fig. 1 and Table 11. These provide, in summary and schematic fashion, our version of a possible phylogenetic tree-and the accompanying biochemical events-which incorporates many of the features proposed and discussed in several recent publications (Uzzell and Spolsky, 1974; Klein and Cronquist,

1967). The most convincing, and so far least accessible, evidence is derived from comparisons of primary structures (amino acid sequences). This of course is the approach taken, among others, by the authors of the Atlas of Protein Structure (Dayhoff, 1972), with the results summarized in that compilation. Lacking this essential information one is forced to have recourse to much less reliable, and occasionally misleading, data on correspondence of molecular weights, subunit composition, and functional analogies.

12

HENRY R. MAHLER AND RUDOLF A. RAFF Other Eubocteria

(e.g. Rhodoparudomonas)

Green aulfur bacteria

(0.g. Deaulfovibrio)

II

Anaerobic heterotropha (0.g. Cloatridia)

I

FIG. 1. A hypothetical phylogeny ofthe eukaryotes. For a fuller description see the text and Tahle 11, which also provides the biochemical parameters used in the definition of the four stages ( I to IV) of prokaryotic evolution. PROTECTION FROM OXYGEN TOXICITY A. ENZYMIC Some extremely interesting recent studies have dealt with the mechanism by which contemporary cells, both pro- and eukaryotic, protect themselves against the potential degradations of [02.1-, the superoxide radical, generated in a variety of enzymic and nonenzymic reactions that utilize molecular oxygen. The reaction catalyzed by the protective enzyme, superoxide dismutase, is

2[0J

+ 2Hf + H202 + 0,

This scavenging of the extremely reactive radical appears to constitute an essential defense mechanism evolved to permit survival of the aerobic cell in its intrinsically toxic environment (McCord et al., 1971; Fridovich, 1972; Lavelle et al., 1973). The dismutase, and catalase, which destroys HO, are apparently

TABLE I1 STAGES IN EVOLUTION^

Novel feature of Stage

I

Transition

I1

Transition 111

Metabolism Glycolysis, “elastic” reactions; ethanol oxidation; N, fixation

Substrate level phosphorylation

Complete citric acid cycle

Acyl

Heme synthesis

Chlorophyll synthesis Photosystem I; CO, fixation (Calvin cycle)

Transition Photosystem 11; 0, evolution

IV

>IV

Energy transduction

0, as electron acceptor Hydroxylase systems 0, protective systems Sequestration and loss of functions

- enzyme

Primitive transducing device;1 ATP/2 e

Integration into membranes Increasing specialization of membrane vesicles + chromatophore; >1 ATP/2 e-; cyclic photophosphorylation Noncyclic phosphorylation

Oxidative phosphorylation; 3 ATP,,/2 e-

Requirementsb Proteins; coenzymes (CoA, TPP, NAD, FH,); fd (type I, MW 6 x 103);primitive ISPs, including P D Clostridial fd, MW 6 x 103; [(IS),& fds; PD complex Low potential cytochrome c; multiplicity of ISPs specialized for reduction of acceptors Multiple cytochromes High potential cytochrome c; high potential ISP with electron transport function b-Type cytochromes; multiplicity of quinones; differentiation of chlorophylls; plant-type fd, MW 1.2 x 104; [(IS),]; cytochrome oxidase of protoheme type (c’ or bSM= o ) Cytochrome aa, fd (or rd) plus cytochrome (e.g., P450) SOD, catalase -

See also Fig. 2. ISP, iron-sulfur protein; PD, pyruvate dehydrogenase; CoA, coenzyme A; TPP, thiamine pyrophosphate; fd, ferredoxin; rd, rubredoxin; fp, flavoprotein; cyt, cytochrome; SOD, superoxide dismutase.

k

z

U

c 0

14

HENRY R. MAHLER AND RUDOLF A. RAFF

ubiquitous in aerobes, and appear to b e vital to the existence of organisms living in the presence of oxygen. This view is supported by the data of McCord et al. (1971), who assayed several species of prokaryotic aerobes, strict anaerobes, and aerotolerant anaerobes for superoxide dismutase and catalase. All aerobes contained both enzymes, strict anaerobes contained neither, while aerotolerant anaerobes contained superoxide dismutase but lacked catalase. Yeasts (an eukaryote) were found to contain both enzymes. Although the patterns of induction of the two enzymes in response to oxygen were not the same in different prokaryotic and eukaryotic organisms, they did support the inference that their dismutase represents the primary-and catalase a secondary-defense mechanism against oxygen toxicity (Gregory and Fridovich, 1973; Gregory et al., 1974). Superoxide dismutase has been purified from several eukaryotes including wheat germ, squash, peas, Neurospora, and various animal cells (Fridovich, 1972; Weser, 1973). There is some direct evidence for a protective role for superoxide dismutase (Fee and Teitelbaum, 1972). Hemolysis of vitamin E-deficient rat erythrocytes induced by dialuric acid apparently involves peroxidation of membrane lipids. Erythrocytes are protected by catalase alone, and to an even greater extent by catalase plus superoxide dismutase. On the basis of the evidence just cited, the speculation that the dismutase must have arisen quite early during evolutionary development, coincident perhaps with the first organisms capable of producing oxygen, appears therefore well founded. The properties of some representative enzymes are described in Table 111. Bacteria, such as Escherichia coli, contain two types of enzymes; one, with iron at the active site, is localized in the periplasmic space and serves primarily as a defense against superoxide in the environment; the other, a manganozinc enzyme localized in the intracellular matrix, functions in the protection of the contents of this compartment against the superoxide generated within (Gregory et al., 1973). Eukaryotic cells also contain two forms of the enzyme. One, a cuprozinc enzyme (two atoms each per 33,000 daltons) is localized in the cell sap and in the intermembrane space of mitochondria (Weisiger and Fridovich, 1973a). It was found to b e identical to the copper protein, erythrocuprein, long known as a constituent of bovine erythrocytes, but of hitherto unknown function (Fridovich, 1972; Weser, 1973). The other enzyme resembles one of the bacterial enzymes in its metal cofactor and subunit mass (one manganese atom per 20,000 daltons), although not in the molecular weight (tetramers of MW

TABLE 111 SUPEROXIDEDISMUTASES IN PRO- AND EUKARYOTES"

Escherichia coli Saccliaromyces cereoisiue

Ref. 1

Refs. 2 and3

Bovine erythrocytesb

-

Chicken liver ~

Ref. 4

Ref. 5

Ref. 5

Ref. 6

Ref. 2

Ref. 2

~~~

Location

Molecular weight ( x Enzyme Subunitsd Metal contente

Periplasmic space

Matrix

Cytosol

Cytosol

Mitochondria] matrixC

Cytosol

Cytosol mitochondria1 intermembrane space

Mitochondria] matrix

38.7 18 (2) 2 Fe

39.5 20 (2) 2Mn

32 16.0 Cu,Zn

32.7 16.0 -

Mn

33.0 16.5 (2) 2 Cu, 2 Zn

31.0 16.8 (2) 2 Cu, 2 Zn

79.6 19.4 (4) 2 Mn

a References: (1) Yost and Fridovich (1973); (2) Weisiger and Fridovich (1973a); (3) Keele et al. (1970); (4) Weser (1973); (5) Weisiger and Fridovich (1973b); Goscin and Fridovich (1972); (6) Keele et al. (1971). * An enzyme from bovine heart has an almost identical amino acid composition. Present in cells of a mutant devoid of mtDNA; hence specified in nucleus and synthesized on cytoribosomes. Number of subunits is given in parentheses. ' Per mol.

16

HENRY R. MAHLER AND RUDOLF A. RAFF

80,000 versus dimers of MW 40,000).Its localization is within the mitochondrial matrix and, like all other proteins found in this compartment, it is specified by nuclear genes and synthesized outside the mitochondria (Weisiger and Fridovich, 1973b). The N-terminal sequences of four of these proteins have recently been obtained by Steinman and Hill (1973). These results, summarized in Fig. 2, show a striking sequence homology between the two bacterial enzymes and that of chick liver mitochondria, and a complete lack thereof between the latter and the protein from beef erythrocytes. Therefore the first three show a remarkably low degree of evolutionary divergence in this region of their protein structure, as a result, presumably, of constraints inherent in their function and/or localization. Whether similar constraints are operative for the remainder of the sequence in these molecules and for those of the other (cuprozinc) class of dismutases can only be revealed by future studies. In any event the close homology of the bacterial and mitochondrial classes of proteins argues in favor of a common ancestry in an early aerobic protoeukaryote-but, other interpretations to the contrary (Weisiger and Fridovich, 1973a; Fridovich, 1974), cannot be used to test the validity of the two alternate theories. It represents the survival of an essential primitive function (Uzzell and Spolsky, 1974) subject to ponderous evolutionary constraints. In this respect, as well as in its small size and lack of membrane attachment, and its

~ ~ :AcAla-Thr-Lys" ( ~ Ala -Val~ - Cys -Val -

~ ~ : : e ( ~ ~ ~Cl~y f Thr o-

-

g

- His - Phe -=U-

-

Leu- Lys-Cly - A s p - m - P r o - V a l

Ala - Lys - Gly

-

Asp - Thr - Val

-

-Gln

Val - Val

FIG. 2. Comparative N-terminal amino acid sequences of fonr different forms of superoxide dismutase: two prokaryotic, from E . coli; two eukaryotic, one from the cytosol (bovine erythrocytes) and one from mitochondria (chick liver). Boxes enclose identical residues in the sequences of the two E . coli and the mitochondria1 dismutases. Underlined capital letters indicate identities between any one or more of these three dismutases and bovine erythrocyte superoxide dismutase. (From Steinman and Hill, 1973, with permission.)

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

17

continued specification b y the principal genome of contemporary eukaryotes, it bears a great deal of resemblance to other essential entities required for aerobic function, such as cytochrome c. The cytosolic dismutases evidently have had a long, divergent evolutionary history from both the prokaryotic and the highly conservative mitochondrial species. Isolated from such diverse eukaryotes as fungi, plants, and mammals, they are in all cases cuprozinc proteins. Unfortunately, not enough sequence data exist on these similar, although apparently not identical, enzymes (Table 111) to trace their evolutionary relationships to each other and to the prokaryotic mangano enzyme (Steinman and Hill, 1973). Nevertheless, the evidence on hand strongly suggests that the basic cuprozinc enzyme of the eukaryotic cell cytoplasm arose earlier in the history of the eukaryotic cell than the origin of the eukaryotic kingdoms, and thus probably represents an ancient adaptation of early eukaryotes to the presence of oxygen in the Precambrian atmosphere. Catalase, the other major oxygen-detoxifying enzyme of eukaryotes, and various oxidases are packaged in specialized organelles called peroxysoines (DeDuve and Baudhuin, 1966; DeDuve, 1969; Avers, 1971). The obvious role of the catalase in these particles is in the disposal of hydrogen peroxide. The oxidases and catalase of the peroxysome may also represent a primitive respiratory pathway allowing the cell to regenerate NAD+ from NADH + H+ and thus utilize nonfermentable substrates such as glycerol. Peroxysomes are found throughout the eukaryotic kingdoms in such evolutionary diverse organisms as Tetrahymena, yeasts, plants, and mammalian lines, and thus may, as originally pointed out by DeDuve and Baudhuin (1966), represent an organelle evolved by primitive eukaryotes for protection from oxygen. B.

METABOLIC REQUIREMENTS OF ANAEROBIC EUKARYOTES

Anaerobic prokaryotes exist in considerable diversity, but there are very few anaerobic eukaryotes. The largest group among them consists of the flagellated protozoa inhabiting the anaerobic environments of the intestinal tracts of animals. Examples of these are the ruinen protozoa of cattle (Hungate, 1966) and trichomonads which lack mitochondria (e.g., see Nielsen et al., 1966). There are also freeliving examples such as the facultative anaerobic fungus Aqualinderella which is an obligate fermenter living on submerged fruit in stagnant water (Emerson and Held, 1969). Stanier (1970) suggests that these organisms are not primitively anaerobic, but represent secondary adaptations to specialized niches. This is certainly the case

18

HENRY R. MAHLER AND RUDOLF A. RAFF

for facultatively anaerobic animals, some of which are capable of surviving and producing ATP for prolonged periods under anaerobic conditions, Examples of such forms are found among the nematodes, platyhelminthes, annelids, and mollusks (Hochachka and Mustafa, 1973). These organisms possess modifications of the glycolytic pathways and the Krebs cycle, which allow efficient anaerobic ATP production by simultaneous catabolism of carbohydrates and amino acids to produce end products such as succinate, alanine, and propionate, with the maintenance of a redox balance between NAD+ and NADH + H + (Hochachka and Mustafa, 1973). An example of a well-known facultative eukaryotic anaerobe is bakers’ yeast, which can be cultured indefinitely under anaerobic conditions. But this is so only if the cells in anaerobic culture are provided with oleate and a steroid (Andreason and Stier, 1953, 1954). Oleate and steroids require the presence of oxygen for their biosynthesis, and yeast cells cultured aerobically are capable of producing these compounds (Yuan and Bloch, 1961). Thus yeasts, primitive eukaryotes capable of anaerobic growth, ultimately exhibit an absolute requirement for oxygen (Keith et al., 1972). The various other anaerobic eukaryotes discussed above probably have similar requirements, which are met b y their close association with aerobic eukaryotes. For example, Aqualinderella, like yeasts, requires an unsaturated fatty acid and a sterol when cultured on artificial medium (Held, 1970).

c.

OXYGEN AND BIOSYNTHETIC PATTERNS

Bloch (1962) and Goldfine and Bloch (1963) have drawn two significant generalizations from their detailed discussions of the relationship of molecular oxygen to biosynthetic patterns. First, universal cellular components are not invariably synthesized by the same pathways in all cells. Many are synthesized by anaerobic pathways in some organisms, and by aerobic pathways in others. Second, there are compounds for which there exist only aerobic pathways; such compounds are consequently confined to aerobic organisms. Thus metabolic specializations have been superimposed on the ancient anaerobic metabolic schemes evolved during the Precambrian prior to the existence of free oxygen in the atmosphere. Alternate aerobic and anaerobic pathways exist for monounsaturated fatty acids, nicotinic acid, tyrosine, carotenoids, and porphyrins. There are no anaerobic pathways at all for steroids and polyunsaturated fatty acids. The mechanisms of synthesis of unsaturated fatty acids and

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

19

steroids are of particular significance to our postulate that the eukaryotic cell is primitively adapted to the utilization of molecular oxygen. Eukaryotes and some advanced prokaryotes use the aerobic pathway for the synthesis of monounsaturated fatty acids. Eubacteria, whether aerobic or anaerobic, utilize an entirely different, anaerobic pathway (Bloch et al., 1961; Bloomfield and Bloch, 1960). Th'is anaerobic pathway for unsaturated fatty acids in eubacteria involves a modification of the elongation pathway as outlined in the accompanying reactions for the synthesis of vaccenic acid (Bloch, 1969). CH,-(CH2),-CH2-COSR

3

0

CH3-(CH2)5-CHz-

II

C-CHZ-

nt.dttct l c r l l

COSR '

OH

I I

CH3- (CHZ)S- CH2-C -CHZ-

COSR

Dc h\
H

CH,-(CH2),-CH=CH-CH2-COSR CH, -(CH&-

Furthrr <.lollgatla,,

>

C H= CH -(CH2)9- COOH

By contrast, the aerobic pathway for the production of unsaturated fatty acids does not involve any modification of the elongation reactions, but begins with a saturated long-chain fatty acid. The essential reactions may be summarized as shown for the synthesis of oleic acid. CH,-(CH2),-CHZ-CH,-

-

(CH2),- COSR

[oxy derivative (exact structure unknown)]

0: \IADPH

CH,3- (CH,),- CH=CH --(CH,),-COSR

In eukaryotes this reaction occurs in the endoplasmic reticulum. Bloch (1962) proposed that the change in pathway occurred in the course of evolution of advanced prokaryotes and was retained by the ancestral eukaryote. The proposed selective advantage was that aerobically synthesized fatty acids such as oleic acid serve as substrates for the production of certain polyunsaturated fatty acids such as linoleic and more highly unsaturated species. Polyunsaturated fatty acids are absent from both aerobic and anaerobic bacteria, but are universal in eukaryotes. The final stage of steroid synthesis, the cyclization of squalene to lanosterol, similarly requires molecular oxygen and is localized in

20

HENRY R. MAHLER AND RUDOLF A. RAFF

the microsonies of eukaryotic cells (Willett et al., 1967; Scaller et nl., 1968; Tchen and Bloch, 1957; Hayaishi and Nozaki, 1969).The reaction sequence is shown in Fig. 3. Steroids, which are ubiquitous in eukaryotes, have been recently reported in prokaryotes as well (summarized b y Bird et al., 1971). In most of these instances, they have been found in minute amounts, so that contamination may account for some of the reports. However, Methylococcus has been shown to contain amounts of steroids coinparable to those found in eukaryotes (Bird et al., 1971).The lack of an anaerobic pathway for steroid synthesis, as well as their universal occurrence in eukaryotes, suggests that this pathway evolved early in eukaryotic cellular evolution.

D. HEME BIOSYNTHESIS In contemporary eukaryotes the enzymes of the biosynthetic pathway for this compound exhibit an interesting and characteristic

Squalene

+ Squalene 2,3-oxide

1

HO

Lanosterol

FIG.3. The postulated intermediate and reaction in the biogenesis of' lanosterol from squalene, catalyzed b y a mixed-function oxidase. (After Hayaishi and Nozaki, 1969.)

21

MITOCHONDRIA AND T H E ORIGIN OF EUKARYOTES

pattern of localization, the first and last enzyme being localized in the mitochondria glycine Succinyl- CoA

svnthetase

6-Aminolevulinate

-

dehydra(ta)se

Protoporphyrin IX

Porphobilinogen

ferrochelatase-

Fez+

-

Heme

and the remainder in the cytosol. Furthermore, it is generally believed that, while the 8-aminolevul(in)ate synthetase is found in the matrix, the ferrochelatase (protoheme ferrolyase) is attached to the inner membrane. As shown in Table IV, heme biosynthesis is required for the functioning of certain autotrophic bacterial forms, such as Desulfovibrio, which, because of their relatively simple metabolic pattern, probably correspond to, or are descendants of, evolutionarily extremely primitive forms. In these, as in bacteria in general, heme synthesis is not associated with plasma or cytomembranes. Unfortunately, not much is known about the constituent enzymes in these anaerobes, but the

TABLE IV ENZYMES OF HEME BIOSYNTHESIS

COMPARISON OF FIRST TWO

Synthetase Property Organism Localization Molecular weight ( x 10-3) Number of subunits Inhibition by heme

Dehydratase

Prokaryotic

Eukaryotic

Prokaryotic

Rhodopseudomonus splieroides Solublea

Rat or guinea pig liver Mitochondria1 matrixb

Rhodopseudomonas spheroides Soluble

Beef liver

57"

77

250 (-+150)c

2.40 (-140)'

1

+

1

+

8 x 36.5d

+

Eu karyotic

Soluble

8

X

42.56

+

" A more recent report (Franica-Gaignier aird Clement-Metl.al, 1973a.b) describes two dillereut enzymes (both of MW 1.0 X lW),one localized in the cytoplasm and the other in the chromatoplrores of this organism under a particular set of growth conditions. Etizyme may also be present i n cytosol which may represent a precursor of'the mitochondria1 enzyme. ' Under dissociating conditions. Symmetry on electron micrographs appears identical.

22

HENRY R. MAHLER AND RUDOLF A. RAFF

somewhat more advanced nonsulfur purple bacteria have provided a fertile source of materials. Table IV summarizes the information available on the two enzymes studied most extensively, the synthetase and dehydratase for S-aminolevulinic acid. It is evident that at this nonsophisticated level of resolution both the mitochondria1 and cytosolic enzymes resemble their bacterial counterparts. It will be particularly interesting to compare the properties of the synthetase enzymes in cases in which they have been reported to exhibit a biphasic localization in both a pro- and a eukaryote: in the cytoplasm and chromatophores of Rhodopseudomonos spheroides (FanicaGaignier and Clement-Metral, 1973a,b) and in the cytoplasm and mitochondria of yeasts (Porra et d . , 1972).

E. ELECTRONTRANSPORT SEQUENCES 1. Electron Transport in Bacteria As outlined in Fig. 4, the key feature of contemporary mitochondria whatever their source-hence presumably the most distinguishing characteristic of their ancestor-is the electron transport function of their inner membrane. In bacteria membrane-associated electron transport appears to have evolved quite early and thus may well represent an exceedingly primitive, perhaps even primordial, function already present in quite early anaerobic forms. In Table 11, further elaborated in Fig. 5, we have indicated what appear to be plausible patterns of evolutionary development with respect to electron transport. In common with other reviewers (Klein and Cronquist, 1967; Olson, 1970; Uzzell and Spolsky, 1974), we consider it likely not only that photosynthetic functions evolved before the development of respiration, but also that at first the latter simply constituted a modification of the former, utilizing many of its components. This close link between the members of the photosynthetic and respiratory electron transport chains and their respective modes of energy generation is still evident today, even in the nonsulfur purple photosynthetic bacteria, such as Rhodospirillum and Rhodopseudomonas, which, according to the phylogenetic tree shown in Fig. 1, represent a stage of evolution beyond the postulated branch point that led to the development of mitochondria. These organisms have already succeeded in sequestering the two types of electron transport into two separate membranous compartments [photosynthesis into chromatophores (Fig. 4),which eventually assume the thylakoid structure characteristic of chloroplasts, and respiration into the plasma

MITOCHONDFUA AND T H E ORIGIN OF EUKARYOTES

23

Coupllng device

ADPerl

ATPexr

FIG. 4. An idealized, "general" mitochondria1 electron transport chain and its link to the generation of biochemical energy in the form ofATP (oxydative phosphorylation). Appropriate partial reactions and their characteristic inhibitors are also indicated, where known. Although there is some variability in details hetween species, and between different cells of the same species, most of the features shown are common to all mitochondria from the simplest unicellular to the most complex multicellular form. Q, Coenzyme Q, ubiquinone; a , b, c , cytochroines a, b, c, etc.; ISC, iron-sulfur centers (nonheme iron-sulfur proteins); fp, flavoprotein. Mitochondria1 complexes (I to V), see text and Table XVI, are in brackets. NADH~,l,,r,,l and ATP~,t,,,,,l, nucleotides internal or external to the inner membrane; TH, transhydrogenase.

membrane and its invaginations proper] (Oelze and Drews, 1972; Niederman, 1974). The possibility that the mesosomes-the most prominent forms of these invaginations-are the actual sites of respiratory activity is considered remote on the basis of the best currently available evidence (Reusch and Burger, 1973). Still they retain evidence of the postulated link on several levels: actual identity or overlap of components such as ubiquinone (coenzyme Q) and cytochromes of the c type (Oelze and Drews, 1972), as well as of key components in energy transduction (Melandri et al., 1971; Lien and Gest, 1973). Particularly striking is a finely tuned interplay of regulatory devices by means of which control of photosynthetic capacity is exercised in response to, among others, oxygen-linked respiratory activity. One of its key components appears to be a regulatory factor which is inactivated by oxygen and reactivated by electron flux from the respiratory system (Marrs et al., 1972; Marrs and Gest, 1973). A consideration

ADP

Closfridia

ATP

Ethanol

NAD(P)H

i

acetate ISP H,

TPP.ISP

ATP

Pyruvate Fumarate

+

N,

ti* Desulfovibrro

,

S,Ob- (trlthlonate)

Chlorobium

Chromnlium

hv

I

1

[Common precursor]

Flerlbaeterlaeeae (respiratory chain In blue green algae probably almllar)

Substrates --+ NAD

-

succtnate

Mycsbocferiurn phlei (other eubaelerla)

- -K

fpD-

fp*

--

Substrates-NAD

Isc-K,

IpD

-"p

NAD(P)H

_c

fp

(ISP)

fp

- 1. - -J

Cyt b-558

Isc? cyt E-551

-'t 3

-eyt

X?

cyt c-550

(CYt c,)

ISC

-- Malate

Also all aeroblr ORGANISMS

cyt b-562

b-563

eyt "a,

Dn

ISC

cytb-559

cyt

C,

-" P

0,

f _

cyt

cyt c

(10.

--*P

0: (Ylelds H,O')

"cyt b"

AH, (Ytelds AH 6 H )

FIG. 5. Evolution ofbacterial electron transport toward the "mitochondrial" pattern outlined in Fig. 4. For the significance of the examples chosen for purposes ofphylogeny and evolution, see Fig. 1 and Table 11. ISP, iron-sulfur protein (e.g., ferredoxin); for all other abbreviations see legend to Fig. 4.

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

25

of Figs. 4 and 5 indicates that certain contemporary, obligately aerobic eubacteria such as Mycobacterium phlei can lay claim to an electron transport system that in all relevant particulars is a virtual copy of that found in mitochondria (Brodie and Gutnick, 1971; Harold, 1972). The only significant difference is the substitution of a naphthoquinone (K,) for the benzoquinone (ubiquinone, coenzyme Q) characteristic of mitochondria. However, other bacteria can either utilize both types of quinone (e.g., E . coli) or are restricted to ubiquinone (e.g., Acetobacter rylinum). An even closer analogy to the mitochondrial pattern is provided by the constitutive portion of the electron transport chain in Paracoccus denitrificans (John and Whatley, 1975). This includes not only all the essential catalytic components, especially an oxidase containing cytochromes aa3, but even such sophisticated features as multiple forms of cytochrome b and a variety of branch points. The tightly membrane-integrated cytochromes, which in mitochondria contain known gene products of the organelle, therefore appear to be the most appropriate objects for further studies of possible evolutionary relationships. Unless we entertain the possibility of parallel evolution, the facts just presented argue for the likelihood that similar electron transport chains might already have been present in the postulated common ancestor of these bacteria and of mitochondria. Since among present-day organisms blue-green algae, and especially their nonphotosynthetic homologs, the Flexibacteriaceae, are frequently regarded as occupying a position closest to, but somewhat beyond, this point, the nature of their respiratory electron transport systems becomes of considerable significance. The best current opinion (Tang and Krogmann, 1972; Wolk, 1973) suggests that these organisms do contain a complete, membrane-integrated respiratory system composed of the canonical triad of cytochromes (b, c, and oxidase). In at least some forms of flexibacteria (e.g., Saprospira) there is good evidence for a truly “mitochondrial” system consisting of two b types, one or more c types, and a CN--sensitive oxidase analogous to cytochrome aa3 (Dietrich and Biggins, 1971). Isolation and analysis of this protein should prove most rewarding, particularly with respect to its prosthetic groups (heme a and copper).

2. Extramitochondrial Electron Transport Systems of Eukaryotes: Mixed-Function Oxidases The most commonly considered electron transport chains are those of the respiratory organelle-the mitochondrion of eukaryotes and the cell membrane of prokaryotes. Nearly all present-day organisms

26

HENRY R. MAHLER AND RUDOLF A. R A F F

(with the exception of Clostridia and Lactobacillus) contain cytochromes (Horio and Kamen, 1970). Aerobic bacteria possess cytochrome respiratory chains similar in function to those of the mitochondrion (i.e., cytochrome types b, c, and a) (Fig. 5 ) . However, it is also significant that cytochromes are widely distributed in anaerobic bacteria, in which they function in electron transport between organic substrates or molecular hydrogen and a variety of inorganic oxidants (Newton and Kamen, 1961; Horio and Kamen, 1970). This suggests that rather than being the exception among Precambrian prokaryotes, cytochrome electron transport chains were probably the rule. When oxygen began to become available as an electron sink, many organisms modified their electron transport systems to utilize oxygen as a terminal acceptor. We assume this process occurred in the cells ancestral to eukaryotes, as well as in other phylogenies (Table I1 and Fig. 1). Eukaryotes and prokaryotes, however, are not limited to the cytochrome chain outlined above for electron transfer processes terminating with molecular oxygen. Both also utilize mixed-function oxidases; these are largely microsomal in their location in the eukaryotic cell. Their reactions are summarized as follows (Ullrich, 1972; LU and Levin, 1974): RH

+ DoHz + 0,--* ROH + DO + HZO

RH is the substrate, and DoHz is the hydrogen donor, generally reduced NADPH. Oxygenases serve to introduce hydroxyl groups into hydrophobic compounds, and function in steroid and fatty acid metabolism, as well as in the synthesis of such amino acids as tyrosine and hydroxyproline, and in the degradation of hydrocarbons. The electron transport chains associated with such reactions vary in complexity, but all serve to carry electrons from NADPH or NADH via a flavoprotein to cytochrome P450which constitutes the terminal oxidase interacting with molecular oxygen. The most elaborate known sequence is that found in the outer mitochondria1 membranes of mammalian adrenals (Omura et al., 1966) (Fig. 6). Another system, involving a somewhat different set of components is involved in fatty-acid desaturation (Oshino et al., 1971; Holloway and Wakil, 1970). This chain is illustrated in Fig. 7. Cytochrome b5, while part of the desaturation scheme, apparently does not function in most mixed-function oxidase chains (Hrycay and O’Brien, 1974; Levin et al., 1974). Two of the components of the microsomal electron transport chains are cytochromes (cytochromes P450 and b5) and thus are of con-

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

iso-

27

,02

RCH2CH@H

FIG.6. A nonmitochondrial, cytoplasmic eukaryotic electron transport chain: a mixed-function oxidase involving cytochrome P4,0. Electrons pass from NADPH via a flavoprotein (F,) and a nonheme iron protein (NHI) to reduce the ferri form of cytochrome P,,o. This carrier interacts with oxygen and a hydrocarbon with the ultimate production of an alcohol, water (not shown), and the ferri state of P450.

siderable evolutionary interest, since all other eukaryotic cytochromes are located in the mitochondria1 inner membrane. Cytochrome P450has been recently purified and crystallized from Pseudomonas putida, a bacterium (Yu et al., 1974), and highly purified from rat liver microsomes (van der Hoeven and Coon, quoted in Dus et al., 1974; Levin et al., 1974). Cytochromes Pds0from the first source has a molecular weight of 45,000; that from the second is <49,000. In spite of their differences in substrate specificity, intracellular localization and requirement for coreactants (an iron sulfur protein in the bacterial, a phospholipid in the mammalian case) the two proteins exhibit remarkable conservative features; their sizes, aminoacid composition, and cyanogen bromide-induced peptides are all similar, and they are capable of immunological crossreaction. Cytochrome b, has been purified from mammalian liver microsomes (Ozols and Strittmatter, 1967; Nobrega and Ozols, 1971; Ozols, 1974), and the complete sequence of the calf liver protein has

NADH

+

Fp

+

Cyt b5

+

DESATURASE

n

Stroryl CoA

Olryl CoA

FIG. 7. A nonmitochondrial, cytoplasmic eukaryotic electron transport chain: a desaturase linked to cytochrome b,. Electrons pass from NADH via a flavoprotein (Fp) and cytochrome b, to the desaturase. This enzyme then passes these electrons together with a pair abstracted from stearyl CoA to oxygen, leading to the simultaneous desaturation of the former to oleyl CoA and reduction of the latter to water.

28

HENRY R. MAHLER AND RUDOLF A. RAFF

been determined. The sequence bears no resemblance to that of cytochrome c, but does show some homology to those of the a and p chains of hemoglobin, as well as to that of cytochrome b2, a component of a mitochondrial enzyme (L-lactate dehydrogenase) in yeasts. The sequence of a tryptic peptide, 80 amino acids long, of this cytochrome b, bearing the heme binding site has been reported recently (Guiard and Lederer, 1973). It shows a striking homology to the same region of microsomal cytochrome b,, and the folding of the polypeptide backbone around the heme in these two cytochromes appears identical. Thus microsomal cytochrome b,, which functions in reactions that appear to be primitively fundamental to eukaryotes, is apparently evolutionarily related to other nonmitochondrial proteins, the globins, and to a mitochondrial cytochrome, cytochrome b,. We feel that such a relationship is best explained by assuming that the protoeukaryote carried the gene for a cytochrome ancestral to both the contemporary cytochrome b, and b, species. AS both mitochondrial and nonmitochondrial electron transport systems evolved, the original gene may have been duplicated and given rise to a pair of related genes, which were subject to divergent evolution to produce the specialized mitochondrial and microsomal species. The most significant inference to be drawn from the available data is that these complex electron transport systems, which allow the cell to use molecular oxygen as a direct reactant in several metabolic schemes, are all derived from the eukaryotic membrane system isolated in the microsomal fraction. In some cases they originate in the outer mitochondrial membrane, which is closely related to and may also have originated from the membranes of the endoplasmic reticulum. Furthermore, these systems for electron transfer are not confined to eukaryotes. We therefore propose that they were also part of the biochemical armamentarium of the ancestral protoeukaryote, which had thus both a respiratory chain (destined for enclosure in the mitochondrion) and a system for mixed-function oxidase reactions (which remained external to the organelle).

3. Ferredoxins and ZronSulfur Centers Ferredoxins are members of a group of soluble, or easily solubilized, proteins of relatively modest size, active in electron transport by virtue of iron atoms coordinated to an unusual constellation of four cysteine residues by means of inorganic sulfur. They therefore constitute a subclass of a larger class of redox carriers, all of which contain similar active centers (iron-sulfur centers, ISCs) but which differ widely in structure, membrane association, and the presence of other pros-

MITOCHONDRIA AND T H E ORIGIN OF EUKARYOTES

29

thetic groups and resultant function. Collectively, they are referred to as iron-sulfur proteins (ISPs) or nonheme iron proteins (NHIs). The ferredoxins themselves are of two main types, frequently called the bacterial (or clostridial) and the plant type. The former have molecular weights near 6000, and contain two clusters each consisting of four iron atoms, four labile sulfur atoms, and four cysteine residues. In their distribution they appear largely restricted to anaerobic bacteria. More interesting for our purposes is the second type, members of which have been reported to be present in sources as diverse as Clostridium pasteurianum, Rhodospirillum rubrum, P . putida, bluegreen algae, and the chloroplasts of all plants examined. Similar entities are present as well in the microsomal fraction of most animal cells and in the mitochondria of adrenal glands. Their molecular weights are 10,000 to 12,000 (occasionally 24,000, probably as the consequence of gene duplication), and they contain clusters with two iron and two labile sulfur atoms coordinated to four cysteine residues, resulting in highly characteristic spectroscopic and magnetic properties. Figure 8 compares the amino acid sequences of two ferredoxins of this class-putidaredoxin, from the aerobic eubacteriuin P . putida, and adrenodoxin, from adrenal mitochondria. It is likely that this last Ad (1)

Ser Ser Ser Gln Asp Lys

Pu (1)

FIG.8. Homology between adrenodoxin (Ad) and putidaredoxin (Pu). The squared off sequences indicated identical residues, while the lines indicate gaps inserted for better alignment of the residues in the two proteins. There was 37% homology in the sequence of the two proteins. (From Tanaka et al., 1974, with permission.)

30

HENRY R. MAHLER AND RUDOLF A. RAFF

species, which is probably localized in the outer mitochondrial membrane and constitutes an essential component of the hydroxylation (mixed-function oxidase) system in this tissue, is a representative example of other ISPs involved in analogous reactions by other metazoan cells. As mentioned earlier, generally these systems are found associated with the endoplasmic reticulum and are concentrated in the microsomal fraction after cell fractionation. The homologies between the bacterial and the mammalian ISPs (37%)are at least as striking and significant as those previously established between bacteria and plants, and even between photosynthetic bacteria and bluegreen algae (see also Table 10.5 of Dayhoff, 1972).

F. ENERGYCOUPLING

AND ATPASES

Another area of possible homology involves the membrane proteins concerned with energy transduction, in particular, the terminal members of the sequence, usually assayed as Mg2+-dependent ATPases (Fig. 4 and Table V). Indeed, at first glance, the apparent analogies between mitochondrial and several bacterial enzymes [those from Streptococcus faecalis, E . coli, and Micrococcus luteus (Zysodeikticus) have been studied most thoroughly] appear quite striking (Razin, 1972; Harold, 1972; Senior, 1973; Nelson et aZ., 1974). When attached to the membrane, the enzymes are organized into knoblike particles with a diameter of -100 A. They are susceptible to several characteristic inhibitors [dicyclohexylcarbodiimide (DCCD) and the antibiotic Dio 91; once detached and solubilized, they become cold-labile (this applies to enzymes from mitochondria and S. faecalis, but not to those from M . luteus). Their particle weights are in the range of 3 X lo5 daltons; their amino acid compositions appear similar (Table VI); they are composed of multiple nonidentical subunits, and they require a separate and distinct protein for reattachment to the membrane. However, these pleasing similarities must be viewed with some caution. As shown in Tables V and VI, they differ in detailed inhibitor specificity (most strikingly to the mitochondrial inhibitors rutamycin and oligomycin) and exhibit a different subunit structure with regard to the number and kind of constituent polypeptides, their molecular weight and, most significantly their amino acid composition. In particular there does not appear to be a close analogy in the bacterial case to the mitochondrial F, (also called CFJ portion (Senior, 1973) and especially to its small polypeptide (subunit 9), most directly concerned with membrane attachment (Sierra and Tzagoloff, 1973; Tzagoloff et ul., 1974). The data in Table VI also provide a cautionary example of the extreme danger of

TABLE V SUBUNIT STRUCTURE AND OTHER PROPERTIES OF MgZf-DEPENDENT ATPASES PRESUMED TO PARTICIPATE IN ENERGYTRANS DUCT ION^ Mitochondria

Property Sensitivity to oligomycin Form 1 Form 2 Sensitivity to cold Form 1 Form 2

Saccharom yces cerevisiaeb

-

-

+ +

+ +

-

Sensitivity of form 1 to Dio 9 + DCCD Molecular weight ( x Form 1 340 Form 2 460 (lipoprotein) Subunit composition, molecular weight (X

10-3) 1 2 3 4 5 6 7 8 9 10

Beef heart?

58.5 54.0 38.5 31.0 [29.0p [22.0]. (18.5) 12.0, [12.0P L7.57 -

-

+ -

360 284’

Membranes

Rat liveld

Streptococcus faecalise

-

+ (on membrane ) -

+ + 386 -

[73.0]? 53.0; three 50.0; three 33.0 [29.0] [20.0] (19.0) 12.5 or 17 [ 1o.oy

8.0

a Form 1 is the solubilized ATPase proper (FJ, incapable of attaching to or forming membranes; form 2 contains additional polypeptides required for this purpose (CF,); these are shown in brackets; one of them (OSCP) can also be obtained separately, and is shown in parentheses. A membrane attachment protein called nectin has also been isolated from S. faecalis. The enzyme from E . coli (MW = 3.8 x lo5) has been reported to consist of either four or five nonidentical subunits [of molecular weight 56.8, 51.8, 30.5, 21.0 and 11.5 x 103 (Bragg and Hou, 1972)l; only the two largest appear essential for activity (Nelson et al., 1974). Tzagoloff et al. (1973). Knowles and Penefsky (1972); Senior (1973); Kozlov and Mikelsaar (1974). Senior (1973). Schnebli et 01. (1970).Two nonidentical subunits of similar MW. Capaldi (1973). Known products of mitochondrial synthesis and-probably-of mitochondrial specification. Site of binding of the potent covalent inhibitor DCCD; a second polypeptide of this size, itself fulfilling an inhibitory function, has also been reported to be present in some preparations of soluble F,.

’

AMINO

TABLE VI ACID COMPOSITION OF POLYPEPTIDES KNOWN TO BE SYNTHESIZED IN MITOCHONDU

AND

PARENT ENZYMES

Cytochrome oxidase MgP+-activated ATPase Neurospora crassab

Residues

Beef heart, total"

Asp + Glu Lys + Arg His Ser + Thr Gly + Ala Pro Phe + Tyr Met Val + Ile Leu

14.92 16.14 6.98 6.75 2.79 3.05 15.76 12.61 16.07 16.42 6.90 6.97 10.5 11.0 2.85 2.05 12.40 13.68 11.37 10.65

a

'

Total

Subunit 1'

10.73 4.32 2.56 14.89 17.73 6.84 12.43 1.27 15.86 13.37

Kuboyama et al. (1972). Sebald et al. (1973) Mitochondria1 products. Tzagoloff et al. (1974). Farron (1970). Catterall and Pedersen (1971). Schnebli et al. (1970).

Subunit 2'

18.35 5.57 2.64 13.51 11.21 7.33 16.57 2.05 16.87 11.68

Subunit 3'

Subunits 4 to 7

13.34 3.80 4.62 14.06 17.02 4.61 12.80 1.28 15.79 12.59

20.03 10.58 2.82 9.73 18.25 6.86 13.04 4.91 9.60 7.47

Beef Rat Streptococcus Yeast, Yeast, heart, liver, faecalis, subunit lcd subunit 9 c * d total' total' total'

14.8 10.0 4.8 15.2 10.7 5.6 6.8 0.0 15.4 12.6

7.8 4.9 0.0 11.3 17.3 3.1 8.2 3.1 18.4 15.5

20.33 20.8 13.06 10.67 1.72 1.67 10.79 11.29 20.0 14.04 4.51 4.72 6.05 5.79 1.96 2.04 15.04 15.81 9.07 8.49

23.0 9.6 1.7 13.0 16.9 3.9 6.4 2.3 13.0 9.3

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

33

basing any arguments on amino acid composition data of multisubunit enzymes. In fact, the most interesting subunits, in the sense of their relevance to the intramitochondrial genetic system (hence to presumptive survival of prokaryotic features), deviate strikingly from the bulkand the bacterial analog-in their content of apolar residues. What then is the significance of the similarities so evident on cursory examination alluded to earlier? Perhaps they simply reflect fundamental constrains on evolutionary development of the properties required either for energy transduction by vectorially oriented membranes (Mitchell, 1973a,b, 1974; Harold, 1972; Brodie and Gutnick, 1971; Brodie et al., 1972), or for extensive one- or two-dimensional aggregation. In that context it may be appropriate to point out that S. faecalis ATPase exhibits a much more highly significant correlation of amino acid composition with tubulin (the protein constituent of' microtubu1es)-considered an unique attribute of eukaryotic cells-than with mitochondrial (or chloroplast) ATPase (Weltman and Dowben, 1973).

V. Mitochondria1 Genes and Their Transcription One of the more attractive features of the endosymbiont theory, and indeed the one that propelled it into the recent scientific consciousness and compelled its almost universal acceptance, is directly related to the discovery that all mitochondria contain their own genetic system. This is composed of a specific mitochondrial DNA (mtDNA), as well as the means for its replication and, at least potentially, expression. Thus the organelles possess a DNA-dependent RNA polymerase and a system for protein synthesis, consisting of ribosomes, mRNA(s), tRNAs, aminoacyl-tRNA ligases, and the three classes of protein factors (initiation, elongation, and termination factors) required for its function. The case for the genetic autonomy of mitochondria was strengthened by the conclusive demonstration that at least in organisms such as Saccharomyces cerevisiae and Neurospora crassa, for which a pattern of extrachromosomal, nonMendelian inheritance had already been established, mtDNA could b e identified as the genophore responsible. Related to this was the discovery that mitochondrial genomes are capable of undergoing not only mutation but also repair and mutual exchanges (recombination). However, the strongest inferences were based on the contention that all these mitochondrial entities, events, and processes appear not only to be conserved throughout the whole eukaryotic realm, from the most primitive to the most complex species, but also to be essen-

34

HENRY R. MAHLER AND RUDOLF A. RAFF

tially bacterialike in their properties. However, the recent rapid expansion of factual material has made possible a more critical reevaluation of these two propositions. As shown in Sections V and VI, neither of them appears tenable without modifications so profound as to make an alternative explanation, that is, divergent evolution of the two genetic systems of the eukaryotic cell, the nuclear-cytosolic and the mitochondrial, at least equally plausible.

A. THE MITOCHONDRIAL GENOME

1. mtDNA As is clearly evident from the data of Table VII, mtDNA has been subject to considerable evolution, not only as concerns its size, but also its base composition. The implication is therefore that there have been corresponding evolutionary changes in the encoded sequence of proximal (RNA) and distal (protein) gene products. Three observations stand out: (1) The buoyant densities assumed by mtDNA encompass a range of 1.684-1.715 gm/ml, equivalent to the total span-between about 20 and 80% G C-found in prokaryotes. (2) But, while bacterial genomes vary in molecular weight between 2 x lo8 (3 x lo5 nucleotide pairs) for the smallest, such as Mycoplasma gallosepticurn to 26 X lo8 (40 X lo5 nucleotide pairs) for the largest, such as E . coli, even the largest mitochondrial genome probably does not exceed a value of 6 x lo7 (or 7.5 x lo4 nucleotide pairs). However, this is a size quite consistent with that of many of the larger bacterial plasmids (Table VIII). Furthermore, there exists a class of small plasmids virtually identical in size to the mtDNA of animals (5 x lo6). Related to these observations is the apparent complete lack of redundancy and gene duplication in “normal” mitochondrial genomes. (3)At least for metazoa, nuclear and mitochondrial genomes appear to have become evolutionarily stabilized coordinately and convergently at a value of their buoyant density close to 1.700 (40%G C) for either, and a mass of the order of 10 x lo8 daltons (1.2 x lo4 nucleotide pairs) for the mitochondrial component.

+

+

2. Homologies It may of course be argued that the apparent divergence evident from Table VII does not reflect a true evolutionary alteration in actual stable mitochondrial gene products, but instead is due to the loss of regions required as punctuation marks, spacers, or promoters for transcription, as elements for processing, as ribosome attachment

35

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES TABLE VII PROPERTIESOF MITOCHONDRIAL DNAaeb Nuclear DNA

-

mtDNA f i

6 (mg/ml)

Contour length (pmY

A (pm)

PB

PB

Species

(gm/ml)

(gm/ml)

Chlamydomonas Euglena gracilis Tetrahymena pyriformis

1.724 1.707 1.688

1.719 1.690 1.684

6 17 4

6

Leishmania enriettii Chlorella Higher plants Physarium polycephalum Neurospora crassa Saccharomyces cerevisiae

1.721 1.716 1.692 1.700 1.713 1.698

1.699 1.712 1.706 1.686 1.702 1.684

22 4 - 14 14 11 14

-

Sea urchin oocyte Housefly Carp liver Xenopus laevis oocyte

1.694 1.696 1.697 1.700

1.704 1.689 1.703 1.702

10 7

5 -

-6

-

-2

13

Chick liver Rat liver Mouse L cells Human leukocytes Human HeLa cells

1.700 1.701 1.701 1.700 1.700

1.708 1.701 1.701 1.701 1.706

-8 0 0 -1 -6

42 31

-

-

40

16, 17.6 (30 x los)

-

-

19

-

20d, 26 25d, (50 X lo6) 4.6d, 4.gd 5.2d 5.4d 5.7d (11.7 X loa) 5.5d 4.96d 4.74d 5.w 5.0d (9.6 x lo6)

All data from the compilations of Mahler (19734 and Meyer (1973). The buoyant density ps is a function of the base composition of DNA which for the species shown is reasonably well represented by X (G + C) = (pB - 1.660)/0.098, where X (G + C ) equals the mole fraction of those bases in the DNA and it is assumed that the DNA of E. coli with X (G + C) = 0.50 bands with pB = 1.710. T h e contour length of a linear, or relaxed circular DNA is directly related to its mass by the relation 0.52 pm = 1.0 x lo6daltons. 8, Difference in density between nDNA and mtDNA; A, difference of separated strands of mtDNA in alkaline cesium chloride, a measure of the predominance of pyrimidines in one of the two strands. When more than one value is shown, this is due to differences in the subspecies examined. Values in parentheses are molecular weight. Molecule has been isolated as a covalently closed circle. a

sites for mRNA, or in other as yet unexplored regulatory functions. Aside from the rather arbitrary distinction this proposition appears to make between structural and regulatory or quasiregulatory genetic elements, it also fails to take into account the very real differences in base composition and the resultant lack in sequence homologies

36

HENRY R. MAHLER AND RUDOLF A. RAFF

SIZE

AND

TABLE VIII OTHER PROPERTIES O F SOME BACTERIALPLASMIDS COMPARED MITOCHONDRIAL DNAs OF S. cereoisiae"

Type of DNA Fertility factor F' Fz F'Lac F'Gal Colicinogenic factor El E2 E3 Ib-P9 E l (Ps)

R1 223lR3 (Ps) Defective phage P1 Mitochondria1 DNA, S. cereuisiae Wild type p- mutant (C"ER)

Molecular weight (x

75 45 81 74 51 4 5 5

61.5 4.2 8.5 12.7 10.6 65 12 54,64 60 50

Circularity

+ + + + + + + + + + + + + + + + + + + + + +

TO

Contour length (/.mi)*

ReferenceC

nd nd nd nd nd

1 2 2 2 2

2.3 1 nd nd nd nd nd nd 5.5 33 -

3 4 4 5 4 4 4 6 6 7 7

31.2

8

26 1.23 2.5 3.7 5.0

9 10 10 10 10

-

a All from E. coli except those marked (Ps), which were isolated from Pseudomonas ntirubilis. nd, Not done. (1) Bazaral and Helinski (1970b); (2) Freifelder (1968); (3) Inselburg and Fuke (1971); (4) Bazaral and Helinski (1970a); ( 5 ) Clewell and Helinski (1970); (6) Cohen and Miller (1969); (7) Nisioka et ul. (1969); ( 8 ) Ikeda and Tomizawa (1968); (9) Hollenberg et d.(1970); (10) Faye et d.(1973).

between at least one stable and well-characterized set of gene products, namely, the mitochondria1 rRNAs. As will be shown (Table XII), the latter reflect the base composition of the DNA from which they are transcribed. In confirmation, more direct comparisons of rRNA, including hybridization (Dawid, 1972a,b; Freeman et al., 1973), indicate an even smaller degree of sequence conservation between

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

37

evolutionarily distant species than holds true for the cytosolic rRNAs of the same organisms (Bendich and McCarthy, 1970; Sinclair and Brown, 1971). A similar lack of homology, even between quite closely related species or even subspecies, has become apparent from studies by Dawid and his colleagues (Dawid, 1972a,b; Dawid and Horak, 1972; Dawid and Blackler, 1972), using a comparison of the transcripts from mtDNA obtained in vitro. These observations are in marked contrast to the situation in bacteria where, in spite of base and sequence divergence of DNAs, the ribosomal cistrons appear to be quite similar in their base composition and exhibit considerable homology (Sogin et al., 1972).

3. Disposition, Replication, and Elimination The number of copies of mtDNA found-not per cell, a number that is not particularly revealing in light of the great variability of the number of mitochondria in different cells, even of the same organism-but per average or unit mitochondrion (Grimes et al., 1974), appears to b e significantly greater than unity in all species examined. It can vary from the order of 5 (in fully derepressed S. cerevisiae or rodent cells in culture) to 100 or more (in fully repressed S. cerevisiae, or with the same or other organisms under conditions of respiratory limitation) (Hoffman and Avers, 1973; Bleeg et al., 1972; Calvayrac et al., 1971).The replication of this mtDNA is integrated into the cell cycle, at least in some species, but can become rather easily dissociated under a variety of conditions. Finally, its replication can be inhibited specifically, and the mtDNA eliminated selectively. This can occur when the mitochondrial system is blocked in the course of repeated and continued cell division-which requires the presence or induction of an alternate pathway for energy generation-or by sequential fragmentation under the influence of certain heteroaromatic polycyclic dyes such as acridinium and phenanthidinium derivatives (Mahler, 1973a,b; Mahler and Bastos, 1974a,b).

4. Mechanism of Genetic Exchanges The discovery of mitochondrial alleles conferring resistance (or sensitivity) to various antibiotics in S. cerevisiae-about eight being known so far (Linnane et al., 1972)-was soon followed by the demonstration of recombination between them (Bolotin et al., 1971; Gingold et al., 1969). These findings appeared to strengthen the analogy between mitochondrial and bacterial genomes. Particularly important was the demonstration of polarity in some mitochondrial

38

HENRY R. MAHLER AND RUDOLF A. RAFF

crosses (Bolotin et al., 1971), apparently conforming to the nonreciprocal transfer of an E . coZi genome from a donor (F+)to a recipient (F-) cell. However, on closer examination (Dujon et al., 1974), this analogy also appears to break down on the following grounds: (1) two parental genomes can exhibit recombination not only in this heteropolar (heterosexual) manner, when they are said to be of opposite polarity (w+ x m-), but also in a homopolar (homosexual) one, when they are of identical polarity (w+ x w+ or w- x w-). Nonreciprocity, defined by a predominance of one or the other recombinant genotype, is therefore not obligatory as in E . coli. It is characteristic only of the first (heteropolar), not of the second (homopolar), type of event. (2) The w allele appears always to be part of the genome itself, and there is no evidence for its potential independence and separate transfer as there is for the bacterial sex factor, the F plasmid (Table VIII). (3) Polarity, where it does exist, appears to be restricted only to three mitochondrial alleles (RI, RII and RllI also called C , S , and R ) , all probably specifying regions in mitochondrial rRNA and closely linked to w. (4) Mitochondria1 polarity and genetic recombination in general appear to obey rules quite foreign to those demonstrated for E . coli (Dujon et al., 1974; Peelman and Birky, 1974). They do not appear to require the process of transfer replication (Curtiss, 1969; Sarathy and Siddiqui, 1973a,b). There is no evidence for the transitory existence of partial heterogenotes, and the pattern of emergence of recombinant genotypes (Lukins et al., 1973; Wilkie and Thomas, 1973) is reminiscent of the events characteristic not so much of the bacterial case but instead of those that describe the genetic pool of replicating bacteriophages (Visconti and Delbriick, 1953). Temperate bacteriophages are of course to be regarded as plasmids, and replication of plasmids, as does that of circular mtDNA, produces certain quite characteristic intermediates (Borst, 1972; Mahler, 1973a; Goebel, 1973). These include “D-loops” and oligomeric, interlinked (catenated) circles (Robberson et al., 1972; Flavell et aZ., 1972; Novick et d . , 1973; Kupersztoch and Helinski, 1973; Koike and Kobayashi, 1973; Berk and Clayton, 1974; Matsumoto et al., 1974).

5. Nuclear Copies of mtDNA Obviously, a demonstration of the presence of partial or complete copies of the mitochondrial genome integrated into one of the nuclear chromosomes would provide a strong impact on any model of mitochondrial evolution. Such nuclear “master copies” have been postulated periodically on the basis of not entirely compelling

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

39

genetic experiments in S. cerevisiae (Wilkie, 1963; Whittaker et a1., 1972) and of morphological studies of oogenesis in Pteridium aquih u m (Bell and Muhlethaler, 1962). However, in the absence of some additional ad hoc assumptions and constraints, the weight of the evidence from mitochondria1 genetics appears to vitiate this hypothesis. Yet its potential impact is sufficiently great to have impelled several recent critical studies specifically and explicitly designed to test it by means of a variety of nucleic acid hybridization techniques. Such studies in S. cerevisiae (Fukuhara, 1970), chick liver (Tabak and Borst, 1970), and Tetrahymena pyriformis (Flavell and Trampe, 1973) all led to the conclusion that such nuclear copies probably do not exist. Horak and Dawid (1972) have drawn a similar inference from their investigations on the maternal inheritance of mtDNA in Xenopus. Still the exercise of some residual caution appears advisable; based on very carefully hybridization studies with S. cerevisiae, Storti and Sinclair (1974) have reached the opposite conclusion.

6. Similarities of mtDNA and Plasmids Let us briefly review and expand on the properties shared b y mitochondrial and plasmid DNA (Tables VII and VIII). These are: (1) size (from -5 to -50 x lo6); (2) configuration (doubly covalently linked, supercoiled circles, easily transformed from this into relaxed circles covalently linked in only one strand); (3) arrest of replication and resultant elimination (curing) b y specific intercalating dyes in the acridinium and phenanthridinium series; (4)ready accumulation of replicative forms, some, such as multiple catenanes, potentially of an aberrant variety; ( 5 ) replication, requiring tight integration with, and potential regulation by, an attachment site on the appropriate membrane; (6) the presence of ribonucleotide tracts in the mature, vegetative form of the genophore (Williams et al., 1973; WongStaal et al., 1973; Grossman et al., 1973); (7) actual (plasmids) or potential (mtDNA) integration into the principal cellular genophore; (8) partial or complete dissociation of replication of plasmid and mtDNA from the DNA of the principal genophore by the application of inhibitors of protein synthesis (Blamire et al., 1972; Goebel, 1973; Kline, 1974) or temperature-sensitive mutations (Cottrell et d.,1973; Goebel and Schrenipf, 1973); (9) the ability to confer antibiotic resistance-albeit by different mechanisms-on cells harboring an antibiotic-resistant form of the subsidiary genome.

40

HENRY R. MAHLER AND RUDOLF A. RAFF

B. DNA-DEPENDENTRNA POLMYERASES All gene products-whether eventually translated into polypeptides or not-are, in the first instance, transcripts of DNA. We therefore turn our attention first to enzymes and processes responsible for transcription in general and in mitochondria in particular.

1. Bacterial RNA Polymeruses The RNA polymerases of bacteria are large, multisubunit enzymes with molecular weights in the range of 400,000 to 500,000. RNA polymerase has been purified from E . coli, as well as other bacteria, and from the blue-green alga Anacystis nidulans. The holoenzyme consists of four major subunits (az, of, p, and u),and frequently a fifth ( 0 ) (Burgess, 1971; Chamberlin, 1974). Molecular weights and stoichiometries of subunits of the E . coli enzyme are presented in Table IX. The subunit structures of other bacterial and of Anacystis RNA polymerases are similar. Some variation in molecular weight has been reported; several gram-negative bacteria have u and a subunits of somewhat higher molecular weight than those of E . coli, while Bacillus appears to have u subunits of lower molecular weight (55,000). Sporulating cells of Bacillus contain an RNA polymerase with few cr subunits and a p subunit of low molecular weight (110,000) (reviewed by Chamberlin, 1974). Bacterial enzymes are strongly inhibited b y the antibiotics streptolydigin, streptovaricin, and rifampicin (or the related compound rifamycin SV-(rifampin) at concentrations of about 1-2 pg/ml or less (Hartmann et al., 1967; Mizumo et al., 1967).These compounds bind to the p' subunit and block the initiation of transcription, but not elongation (Hinkle et al., 1972). 2. Eukaryotic Transcriptional Systems Eukaryotic cells contain several transcriptional systems which can be discriminated in vivo by their differential sensitivities to various inhibitors. The nucleolar system, which produces rRNA, is in most cells sensitive to low concentrations ( < 1 pg/ml) of actinomycin D (Penman, 1968). The nucleoplasmic system, which produces heterogeneous, nuclear RNA (HnRNA), and mRNA, is sensitive to camptothecin (Perlman et al., 1973), while the mitochondria1 system is sensitive to ethidium bromide (Penman, 1968; South and Mahler, 1968; Zylber et al., 1969; Mahler and Dawidowicz, 1973; Meyer et al., 1972) and euflavine (Fukuhara and Kujawa, 1970).

MITOCHONDFUA AND T H E ORIGIN OF EUKARYOTES

41

The basis for these differential sensitivities has not yet been completely elucidated, but it has already become clear that each system has its own species of RNA polymerase. 3. Eukaryotic Nuclear Polymerases Eukaryotic nuclei (of animals, plants, fungi, and protozoa) contain three or more RNA polymerase species (Lindell et al., 1970; Kedinger et al., 1970, 1972; Jacob et al., 1970; Roeder and Rutter, 1969, 1970a,b; Mullinix et al., 1973; Ponta et al., 1972; Roeder, 1974; also reviewed by Chambon, 1974). The major species are enzyme I or A, which is nucleolar in localization and functions in the synthesis of rRNA, and polymerase I1 or B, which is located in the nucleoplasm and apparently synthesizes mRNA. This enzyme, but not the nucleolar species, is inhibited by a-amanitin (Lindell et al., 1970; Kedinger et al., 1972). None of these enzymes is inhibited by rifampicin, although derivatives of rifamycin bearing more complex substituents d o act as inhibitors in high concentrations (Adman et al., 1972; DiMauro et al., 1972; Meilhac et al., 1972). These derivatives also inhibit E . coli RNA polymerase, Some, such as AF/013, inhibit at very low concentrations (65% inhibition at a concentration of 0.4 pg/ml). Comparable inhibition of animal RNA polymerase by AF/013 requires a concentration of greater than 12 pg/ml (Meilhac et al., 1972). As in the case of rifampicin inhibition of bacterial RNA polymerase, inhibition of animal polymerase by AF/013 involves initiation but not elongation. However, the details of the inhibitory action may not be identical, since AF/013 inhibits primary binding of animal but not E . coli RNA polymerase to DNA (Meilhac et al., 1972). Nuclear polymerases resemble prokaryotic polymerases, in being high-molecular-weight (380,000 to 450,000) multisubunit enzymes (Gissinger and Chambon, 1972; Kedinger and Chambon, 1972; Weaver et al., 1971; Ponta et al., 1972; Mullinix et al., 1973; Dezelee and Sentenac, 1973). The number and size of the subunits has not yet been uniquovacally determined, although there appear to be two large subunits present in a 1 : l molar ratio, and two to four small subunits. The subunit properties of the relatively well-characterized calf thymus enzymes are presented in Table IX. This subunit distribution is very similar to that of bacterial polymerase with respect to size, although there is no information whether or not the subunits are functionally homologous. Bacterial polymerase subunit cr has been found to be important in the determination of transcriptional specificity (Burgess, 1971; Roberts, 1969). It

E

z s rc s

F

TABLE IX SVSUNIT PROPERTIES OF PURIFIEDDNA-DEPENDENTRNA POLYMERASES Subunit

Bacterial

Escherichia coli

Molar ratio

8’: 150,000-160,000 8: 145,000-150,000

1 1 1 2 2

U:85,000-90,000

a: 40,000 0:

Eukaryotic Type 1 (A) (calf thymus nucleolar)

Inhibitor

Molecular weight

Polymerase

9000-12,000 200,000

126,000 51,000 44,000 25,000 16,000

3

M

1 1 1 1 2 2

Sensitive to (>5oo/o inhibition at given concentration) Rifampicin, <0.5j@ml Rifamycin SV, C0.5 pg/ml Streptovaricin, <0.5 pglml

73

U

Insensitive to a-Amanitin

s crl

* s

AFl013, <0.5 pglml

AFl013, 12 pglml

s

%crl Rifampicin Rifamycin SV Streptovaricin a-Amanitin

Type I1 (B) (calf thymus nucleoplasmic)

Mitochondria1 Neurosporn Yeast

-200,000 140,000 34,000 25,000 16,000

1 1 1-2 -2 3-4

a-Amanitin, 0.04 pg/ml AF/013, 12 pg/ml

Rifampicin Rifamycin SV Streptovaricin

z

w

cl

Xenopus

46,000

Rifampicin, <6 pg/ml Rifampicin (crude enzyme), 38 pg/ml AF/013,20 pg/ml

Rat Chloroplast Maize

65,000

Rifampicin, < 10 p d m l

65,000 61,000

220,000 150,000 Several smaller proteins

a-Amanitin Rifampicin (purified enzyme) Rifampicin Rifamycin SV a-Amanitin

8z 0 z U

F

a-Amanitin

t?U

Rifamycin SV

4

8

44

HENRY R. MAHLER AND RUDOLF A. RAFF

is not yet known whether or not a similar factor is present in nuclear polymerases, although evidence for a factor that stimulates initiation has been reported (Stein and Hausen, 1970; Mondal et al., 1972; DiMauro et al., 1972; Hall, 1973). 4. Mitochondria1 Polymerase Mitochondria purified from a variety of organisms have been found to contain RNA polymerase activity (Mahler, 1973a). Highly purified polymerases have been prepared from the mitochondria of Neurospora (Kiintzel and Schgfer, 1971) and Xenopus (Wu and Dawid, 1972). These enzymes have only about 5% the activity of purified E . coli polymerase. Both are single polypeptides of similar molecular weight (64,000 for the Neurospora polymerase and 46,000 for the Xenopus enzyme) which aggregate readily at low salt concentrations. Poly d(AT) is the best template for both enzymes, while mtDNA is about one-half as active. Wu and Dawid found the native, covalently closed circular form of mtDNA to b e a better template than its nicked form. The Xenopus enzyme is active with calf thymus DNA, while the Neurospora enzyme is not. Both polymerases are insensitive to a-amanitin. However, there are significant differences in the sensitivities of these enzymes to rifampicin. The Neurospora polymerase is inhibited by this compound (6 pg/ml), while the Xenopus enzyme is unaffected by it at concentrations as high as 100 pg/ml but is completely inhibited by more complex derivatives such as AF/013 at concentrations of about 30 pg/ml. Wu and Dawid have also reported that the Xenopus enzyme is not stimulated by Mn2+ions and is inhibited by salt concentrations above 0.05 M. These characteristics set this polymerase well apart from either Xenopus nuclear polymerases or bacterial polymerases. Less pure RNA polymerase preparations from other organisms have also been studied. Scragg (1971) studied crude RNA polymerase from yeast mitochondria. Molecular-weight estimates based on Sephadex sieving suggest that the main peak of activity has a molecular weight of 200,000. This apparently represents aggregates of a lower-molecular-weight entity (MW 61,000) (Scragg, 1974; see also Rogall and Wintersberger, 1974). High ( > 38 pg/ml) concentrations of rifampicin are required for significant inhibition of the crude enzyme; the purified enzyme appears sensitive to much lower concentrations (10 pg/ml). Tsai et al. (1971) and Eccleshall and Criddle (1974) also studied yeast mitochondria1 polymerase which they found to b e resistant to rifampin, streptovaricin, and a-amanitin. Reid and Parsons (1971)

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

45

have reported that rat liver mitochondrial RNA polymerase has a molecular weight of about 65,000 and is sensitive to 10 pg/ml of rifampicin. Thus the various mitochondrial RNA polymerases that have been studied to date are easily distinguished from both nuclear and bacterial polymerases. Mitochondrial polymerases, in contrast to all other enzymes, are apparently composed of single subunits with molecular weights ranging from 46,000 to 65,000, and differ in catalytic properties and ion requirements as well. A major criterion used to distinguish prokaryotic from eukaryotic nuclear polymerases is the striking difference in sensitivity of these two classes to rifampicin and rifamycin; at low concentrations these compounds completely inhibit bacterial but not nuclear enzymes. Mitochondrial polymerases differ in reported sensitivity to these drugs; the Neurospora enzyme is sensitive, while the yeast enzyme is apparently only inhibited by high doses, and the Xenopus enzyme not at all. The rat liver enzyme appears to b e sensitive. The closest equivalent in the prokaryotic realm is provided by the polymerase encoded in the DNA of bacteriophage T7; it too is a single polypeptide, but with a molecular weight of 70,000, resistant to the action of rifampicin. Mitochondrial polymerases are also strikingly different from chloroplast polymerase, which has been reported to have a molecular weight of about 500,000 and to contain two large subunits of 220,000 and 150,000 molecular weight together with several smaller polypeptides (Bottomley et al., 1971; Smith and Bogorad, quoted in Mullinix et al., 1973).

5. Site of Synthesis of Mitochondrial RNA Polymerase The genetic locus for mitochondrial polymerase has been investigated with petite strains of yeast which either altogether lack or have aberrant mtDNA. Mitochondria from such strains appear to lack the ability to synthesize proteins (Mahler, 1973a). Although the issue has not been completely resolved, evidence does exist to indicate that the mitochondrial RNA polymerase of yeast is specified b y a nuclear gene. RNA polymerase activity has been found in petite strains (South, cited in Mahler, 1973a; Wintersberger, 1970; Tsai et al., 1971). However, Tsai et al. (1971) and Scragg (1971) have reported finding no polymerase activity in petite strains completely lacking in mtDNA. Since the inner membranes of these mitochondria are aberrant, the lack of polymerase activity may have resulted from an inability to stimulate the synthesis of the enzyme or

46

HENRY R. MAHLER AND RUDOLF A. RAFF

to retain the enzyme in the organelle. The experiments of Barath and Kuntzel (1972), which demonstrate the stimulation of mitochondrial polymerase synthesis in the presence of ethidium bromide or chloramphenicol which, respectively, inhibit mitochondrial transcription and translation, provide further support for a nuclear locus for the mitochondrial polymerase gene.

VI. Mitochondria1 Gene Expression A.

MITOCHONDRIALrRNA

1. General Properties of Ribosomes As mentioned, one of the mainstays of the symbiotic hypothesis for the origin of mitochondria and chloroplasts is their possession not only of DNA, but also of the machinery for its transcription and translation. Translation in these organelles is performed by ribosomes analogous to those of prokaryotes and of the eukaryotic cytoplasm. The function of mitochondrial ribosomes (mitoribosomes) in protein synthesis is discussed in Section V1,C. The general physical properties of organellar ribosomes from three diverse groups of organisms are compared to the corresponding cytoplasmic ribosomes (cytoribosomes) and to bacterial ribosomes in Table X. The sedimentation properties of the mitoribosomes differ from species to species and from those of bacteria. The mitoribosomes of fungi have somewhat higher sedimentation coefficients than those of bacteria, while those from the ciliate Tetruhymena, are much higher and those of animals lower. Bacterial ribosomes are composed of about 60% RNA and 40% protein. The proportion of these two constituents in cytoribosomes has been claimed to be approximately equal, but may in fact be similar to that of bacteria (McConkey, 1974). However, the mitoribosomes of Tetrahymena and of higher animals have been reported to contain over 60% protein. The sedimentation coefficients of animal mitoribosomes are low, but the mitoribosomes of Tetruhymena are clearly in a class by themselves. These particles not only exhibit a very high sedimentation coefficient, but appear to b e composed of two subunits with equal sedimentation coefficients, rather than a large and small one as is customary in all other cases. However, the fact that two rRNAs of unequal size can be obtained from these mitoribosomes indicate that the two subunits are probably functionally distinct. A detailed comparative discussion of rRNAs is presented in

TABLE X PROPERTIESOF BACTERIAL, MITOCHONDFUAL, AND EUKARYOTIC CYTOPLASMICRIBOSOMES~ Ascomycetesb Parameter

Bacteriab

Dimensions (A) 200 X 160 Sedimentation coefficient (S) Ribosome 70 Large subunit 50 Small subunit 30 Density (gm/mJ) Ribosome 1.63 Large subunit 1.67 Small subunit 1.63 Average molecular weight ( x Ribosome 2.7 Large subunit 1.8 Small subunit 0.85 Large-subunit rRNA 1.09 0.56 Small-subunit rRNA Peptides of the 0.60 (35) Large subunitb 0.30 (20) Small subunitb Yes Presence of 5s RNA in ribosome Methylation of rRNA Yes Subunit exchange with Yes bacterial ribosomes (various bacteria) Initiation and elongation factors active Yes with bacterial ribosomes

Mitochondria

Cytoplasm

Animalsb

Terrahymena

Mitochondria

265 x 210

Cytoplasm

275 x 230

Mitochondria

Cytoplasm

370 x 240

-320 x 250 80 60 40

72-78 50-58 35-40

80 60 40

80 55 55

80 60 40

50-60 33-45 25-35

1.46-1.48 -

1.52 -

-

1.56 1.57 1.53

1.40-1.43

-

1.46 1.52 1.46

-

1.50 1.60-1.64 1.55

4.16 2.47 1.69 1.30 0.70

4.49

-

-

1.30 0.73

0.82 0.52

1.30 0.69

2.5 1.7 0.80 0.53 0.33

1.4-1.7 -

(30) (23) No Yes, low No

Yes Yes -

-

-

-

-

-

-

-

-

-

-

See text for references. Number of distinct proteins detected in a given subunit is given in parentheses.

-

-

Yes

-

-

1.08 (-30) 0.56 (-20) No Yes, low

0.90 (-35) 0.70 (-20) Yes Yes -

-

48

HENRY R. MAHLER AND RUDOLF A. RAFF

Section VI,A,2, particularly with respect to the observation that mitoribosomes may differ from other ribosomes in their lack of a small RNA with a sedimentation coefficient equal to 5s and in possessing apparently poorly methylated rRNAs. Mitoribosomal proteins have not been sufficiently characterized to make discussion of these entities profitable at this juncture, although certain aspects are discussed where appropriate in other sections of this article. 2. Base Composition of Mitochondria1 rRNAs The DNA base compositions of organisms vary within the rather broad limits of about 22 to -75% G C (Woese and Bleyman, 1972). These values may define the limits beyond which the overall DNA base composition of an organism may not evolve without losing its ability to perform a coding function. There may be two types of genetic code limit organisms-those with as extreme a G + C content as is possible without generating atypical amino acid compositions in their proteins, and ones the proteins of which have evolved such extremes in amino acid composition. I t is unknown whether the latter type exists (Woese and Bleyman, 1972). Ribosomes must exhibit some fundamental similarities. All carry out essentially the same reactions. All consist of two subunits-one large and one small-except for Tetruhymena mitoribosomes. Each subunit contains a distinct major species of rRNA, as well as a set of proteins characteristic of that subunit. rRNAs contain double helical regions which may control their conformation, and appropriate sequences are apparently required for proper ribosome assembly (reviewed by Monier, 1972). The rRNA of the small subunit may also play a direct functional role in protein synthesis. When about 50 nucleotides from the 3’-end of the rRNA have been removed from E . coli 30s subunits by certain colicins, the particles become inactive in protein synthesis (Senior and Holland, 1971; Bowman et al., 1971). The 3’-terminal hexanucleotides of 18s rRNAs from yeasts, Drosophilu and rabbits have an identical sequence, -UCAUUAoH, which is complementary to the known eukaryotic terminator codons and therefore capable of playing a role in polypeptide chain termination b y recognizing the terminator codons of the mRNA under translation (Dalgarno and Shine, 1973). Since the properties of rRNAs from a wide variety of species have been established, it is possible to ask whether there are any constraints on their base compositions. Several generalizations have been noted. The rRNAs of the large and small ribosomal subunits are

+

MITOCHONDRIA AND T H E ORIGIN OF EUKARYOTES

49

similar in base composition (Amaldi, 1969; Lava-Sanchez et al., 1972), but not in nucleotide sequence (reviewed by Monier, 1972). Bacterial rRNAs are quite uniform in base composition (50-55% G C), while eukaryotic cytoribosomal RNAs and mitoribosomal RNAs show considerable variation. These are plotted in Fig. 9 against the total DNA base compositions of the cell or organelle from which they were derived. The constancy of rRNA composition in bacteria regardless of their DNA base composition is in accord with the hypothesis that in these organisms there exist functional con-

+

70

-

60

-

0

250-

rs

9 E40-

6 c

.P

20

30

40 50 60 Conpo8ition of DNA (% G*C)

70

80

FIG. 9. Composition of rRNAs as a function of the corresponding DNA. Bacteria (triangles), eukaryotic nucleocytoplasm (squares), mitochondria (circles). T h e numbers in each symbol represent a particular species of that class. Bacteria: 1, Clostridium perfringens; 2, Staphylococcus pyrogenes; 3, Bacillus cereus; 4, Proteus vulgaris; 5, Diplococcus pneumoniae; 6, Bacillus subtilis; 7, Escherichia coli; 8, Aerobacter aerogenes; 9, Serrutiu marcescens; 10, Pseudomonas aeruginosa; 11, Alkaligenes faecalis; 12, Sarcina lutea; 13, Micrococcus lysodeikticus; 14, Streptomyces griseus. Eukaryotic nucleocytoplasm: 1, Dictyostelium discoides; 2, Tetrahymena pyriformis; 3, Sacchoromyces cerevisiae; 4, Pisum sativum (pea); 5, Lytechinus (sea urchin); 6, Xenopus laeuis (frog); 7, several birds and mammals; 8, Urechis caupo (marine worm); 9, Drosophila melunogaster; 10, Triticum aestivum (wheat); 11, Neurospora crassa. Mitochondria: 1, Saccharomyces sp.; 2, Tetrahymena pyriformis; 3, Aspergillus nidulans; 4 , Euglena gracilis; 5 , Neurospora crassa; 6, mouse; 7, hamster (BHK cells); 8, Xenopus laevis; 9, rat; 10, human (HeLa cells). References: Belozersky and Spirin (1958); Miura (1962); Midgley (1962); Woese (1961); Firtel et al. (1972); Sinclair and Brown (1971); Borst (1972); Borst and Grivell (1971); Mahler (1973); Edelman et al. (1970, 1971); Kroon et al. (1972); Dawid and Chase (1972); Attardi and Attardi (1971); Spirin (1964).

50

HENRY R. MAHLER AND RUDOLF A. RAFF

straints on rRNA, which in eukaryotes are either less severe or are subject to overrides of a different sort. An evolutionary trend is apparent in the base compositions of these eukaryotic rRNAs (Fig. 10). Both the cytoplasmic and mitochondrial rRNAs of lower eukaryotes are generally lower in G C content than those of animals-particularly mammals. Attardi and Amaldi (1970) suggest that this may indicate an evolution toward greater secondary structure in animal rRNAs. If this hypothesis is correct, it would also predict an evolution toward a more compact structure for these RNAs. Since the rDNA of bacteria and eukaryotes occupies only 0.04 to 0.4% of the total genome (Attardi and Amaldi, 1970), one would not expect any correlation between the base compositions of rRNA and the total DNA of either the bacterial cell or the eukaryotic nucleus. However, the compositions of eukaryotic cytoribosomal RNAs vary from 43 to 65% G C and correlate roughly with the compositions of nuclear DNAs. Mitoribosomal RNAs vary from 26%G C to 47% G + C and correlate strongly with the compositions of mtDNAs (Fig. 9), a not unexpected result, since rDNA constitutes 2.5 to 7% of the total mtDNA (Mahler, 1973a).

+

+

+

"I a

f

40

I I I I 20 1 40 45 50 55 60 65 Composition of Cytoplaamic rRNA (XQ C)

I

70

FIG. 10. Composition of mitochondria1 rRNAs as a function of the corresponding cytoplasmic rRNAs. 1, Tetrahymena pyriformis; 2, Saccharomyces cerevisiae; 3, Aspergillus nidulans; 4, Euglena gracilis; 5, Trichoderma oiridis; 6, Neurospora crassa; 7 , mouse (L cells); 8, Xenopus laevis; 9, rat; 10, human (HeLa cells). References: Mahler (1973a); Freeman et al. (1973).

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

51

Thus among mitoribosomal RNAs is found the lowest mole fraction C in any known rRNA, and this constitutes a significant difference between these mitochondria1 rRNAs and bacterial rRNAs. Furthermore, there exists a correlation of base composition of mitochondrial RNAs not only with that of mtDNA but also with those of cytoribosomal RNAs from the same species (Freeman et al., 1973) (Fig. 10). These results suggest that, whatever their nature, the rRNAs and perhaps the ribosomes in the mitochondria and the cytosol of the same cell are subject to similar functional or regulatory constraints. And as already mentioned, this type of constraint on mtRNA is quite dissimilar to that governing the composition of the analogous entity in bacteria.

of G

+

3. Evolution of Size of rRNA Polyacrylamide gel electrophoresis has been used for determination of the molecular weight of a wide variety of rRNAs. The major rRNAs of the large and small subunits of prokaryotic ribosomes are not only nearly identical in composition, but also in size. The molecular weight of the rRNA of the large subunit is 1.07-1.11 x lo6; that of the rRNA of the small subunit is 0.56 x lo6 (Loening, 1968; Monier, 1972). The cytoribosomal RNAs of eukaryotes, however, show distinct, phylogenetically correlated trends in molecular weight. The rRNA of the small subunit appears to be uniformly of a molecular weight of 0.7 X lo6 (Loening, 1968; Monier, 1972), although the rRNAs derived from Euglena and Amoeba, and from the platyhelminth Dugesia, exhibit significantly lower mobilities on polyacrylamide gel electrophoresis (Loening, 1968; Raff, 1970) and that of Crithidia may be larger (0.83 x 106-Reijnders et al., 1973a). The molecular weights of large-subunit rRNAs are about 1.3 x lo6 in fungi, protozoa, and plants (Loening, 1968). The large-subunit rRNAs of metazoans are distinctly larger than this. Invertebrates contain rRNAs with molecular weights of 1.40-1.44 x lo6 (Loening, 1968; Sy and McCarthy, 1968; Raff, 1970). The values for lower vertebrates are about 1.5-1.6 x lo6 (Loening, 1968), while mammals yield values of 1.7-1.9 x lo6 by gel electrophoresis and equilibrium centrifugation (Loening, 1968; McConkey and Hopkins, 1969). Molecular weights of large-subunit rRNAs derived from agar gel electrophoresis are lower than those obtained with acrylamide gels, but show the same trend of increasing size from lower to higher vertebrates (Kokileva et al., 1971). The evaluation of molecular weights of mitoribosomal RNAs has been more difficult than for rRNAs from other sources, because the

52

HENRY R. MAHLER AND RUDOLF A. RAFF

peculiar conformational properties of the former cause them to behave anomalously on electrophoresis or sedimentation (Forrester et al., 1970; Groot et al., 1970; Edelman et al., 1971; Reijnders e t al., 1973a). However, reliable measurements of molecular weight-based on sedimentation and electrophoresis-have recently been obtained under conditions that totally denature the RNA, so as to eliminate all conforinational effects. Direct length measurements have also been made for various species of rRNA by electron microscopy. These determinations are presented in Table XI. It is clear that mitochondrial rRNAs of fungi are longer than the corresponding RNAs from E . coli, while mammalian mitochondrial rRNAs are shorter. As shown in Table XII, molecular-weight values calculated from such measurements agree with those determined by transport methods. The data show clearly that mitochondrial rRNAs are not the same size as those of bacteria, nor are their molecular weights positively correlated with those of the corresponding cytoplasmic rRNAs-in fact the contrary is true. The values of molecular weights indicate a divergent evolutionary trend in these RNAs quite unlike the apparent strong conservativeness of bacterial rRNAs. It is of particular interest to note that these values for the molecular weights of mitochondrial RNAs indicate two opposing trends; molecules larger than bacterial molecules occur in fungi, and smaller ones are found in animals. In neither case can they be considered typically prokaryotic. In fact, the sizes of animal mitochondrial rRNAs are the smallest known.

LENGTH

OF

T A B L E XI rRNAS DETERMINED B Y ELECTRON MICROSCOPY" Cytoplasmic

M itochondrial

Species

Large

Small

Large

Small

Escherichia coli Saccharom yces carlsbergenesis Aspergillus nidulans Human (HeLa)

0.72-0.85

0.38-0.43

-

-

nd

nd

0.92

0.46

1.10 1.16

0.52 0.55-0.59

0.9 1 0.42-0.46

0.47 0.26-0.27

ReferencesL

1-3 4 3 1, 5, 6

nd, Not determined. A l l values are in micrometers. (1) Granboulan and Scherrer (1969); (2) Nanninga et al. (1972); (3) Verma et al. (1970); Reijnders et al. (1973b); (5) Wu et al. (1972); (6) Robberson et al. (1971).

MITOCHONDRIA AND T H E ORIGIN OF EUKARYOTES

53

TABLE XI1 MOLECULARWEIGHTS OF rRNAs"

Species Bacteria Saccharom yces Aspergillus Neurospora crassa Tetralzymena pyriformis Xenopus laeois Human (HeLa)

Cytoplasmic ( x 10-6)

Mitochondria1 ( x 10-8)"

1.07-1.11, 0.56 1.30, 0.72 1.30, 0.73

1.30, 0.70 1.30, 0.70 1.30, 0.70 1.29,0.72 0.82, 0.52 0.53,0.30 0.54, 0.35

nd

1.30, 0.69 1.54, 0.69 1.75, 0.70

Referencesb

1 1, 2 1, 3 , 4 4, 5 1, 4 1, 6 1, 7

a The first value corresponds to the rRNA of the large subunit and the second to the rRNA of the small subunit. * (1) Loening (1968); (2) Reijnders et al. (1973); (3) Edelman et al. (1970); (4) Borst and Grivell (1971); (5) Kuriyama and Luck (1973a); (6) Dawid and Chase (1972); (7) Robberson et al. (1971).

4. Degree of Methylation of rRNA Methylated nucleotides have been isolated from the rRNAs of bacteria and eukaryotes. They represent about 0.6 mole % of E. coli 23s RNA and 0.95 mole % of the 16s RNA. The level of methylation of animal and plant rRNAs is reported to be about 1.2-1.7 mole %; the degree of methylation of the small rRNA is 2 0 3 0 % greater than that of the corresponding large rRNA (Attardi and Amaldi, 1970; Monier, 1972). The study of rRNA methylation has been impeded by the fact that methylation of nucleotides can occur on both the base and on the 2'-OH of the ribose, and by the lability of some methylated bases (Attardi and Amaldi, 1970; Monier, 1972). Methylation of rRNA is apparently important in ribosome assembly, and possibly in function. Vaughan et al. (1967) found that 45 and 32s rRNA precursor RNAs were synthesized by HeLa cells starved for methionine, but that under these conditions the submethylated (approximately 20% the methyl group content of controls) rRNAs could not be utilized for the formation of ribosomal subunits. In contrast, valine starvation allowed continued production of ribosomal subunits. Submethylated rRNAs have also been found to b e incapable of participating in the assembly of bacterial ribosomes both in vivo and in vitro (Beaud and Hayes, 1971; Lowry and Nomura, cited in Lowry and Dahlberg, 1971). Beaud and Hayes produced submethylated rRNAs by culturing cells in ethionine. Ribosomal subunits isolated from these cells did not associate to produce 70s ribosomes. Lowry and Nomura found

54

HENRY R. MAHLER AND RUDOLF A. RAFF

that ribosomal 305 subunits assembled in vitro from the submethylated precursor p16 are inactive in protein synthesis. The nucleotide sequence, mZ6AmzaACUG,which contains methylated adenosine (N6-dimethyladenine, mz6A) moieties has been isolated from the small rRNA of mammals, yeasts, and E . coli (Salim and Maden, 1973). Fellner et al. (1972) have reported about 70% of the sequence of E . coli 16s rRNA; most of the methyl groups are in the 3'-terminal 25% of the molecule. Fellner et al. propose that methylated residues play a role in subunit assembly. Resistance to certain antibiotics in bacterial ribosomes has been shown to be related to changes in rRNA methylation (Helser et d., 1971; Lai and Weisblum, 1971). Such observations may be pertinent to the nature of antibiotic-resistant mutants in mitochondria (Section V,A). Unfortunately, the nature and extent of methylation of mitochondrial rRNAs is still unclear, although recent studies have improved this situation. No11 (1970) has reported that a substantial portion (5.7%) of the phosphate in alkali digests of mitochondrial rRNA from Neurospora resides in a species identified as a 2'-O-methyl dinucleotide. Kuriyama and Luck (1974a,b) have determined the levels of methylation in mitochondrial and cytoplasmic rRNAs from N. C M S S U . Mitochondria1 rRNA has 1.4 methyl groups per 100 nucleotides, while cytoplasmic rRNA has 2.4. However, Vesco and Penman (1969) have reported very low levels of methylation of HeLa mitochondrial rRNA, as have Dubin and Montenecourt (1970), who found that mitochondrial rRNA contains 0.28 methyl groups per 100 nucleotides as compared to 1.4 for cytoplasmic 28s rRNA. Similarly, Dubin and Friend (1972) reported this parameter for baby hamster kidney cells to be low ( < 0.5). Dubin (1974), in a more precise study of cultured hamster (BHK-21) cell mitochondrial rRNA, has found that the 17s rRNA contains 0.13 methyl groups per 100 nucleotides, and the 13s rRNA 0.37. Dubin draws the substantive conclusion that hamster cell mitochondrial rRNA is significantly methylated, although to a considerably lesser extent than all other rRNAs. Methylation even at this low level may well play a functional role. The 13s mitochondrial rRNA, but not the 17s rRNA, contains N6-dimethyladenine which appears to be restricted to or enriched in the smallsubunit rRNAs of bacteria and mammals (Klagsbrun, 1973). Evidence that it plays a role in ribosomal function or maturation has been presented above. The presence of mz6Ap in the 13s mitochondrial rRNA may be homologous to the mZ6Apmz6Aregions of bacterial and eukaryotic cytoplasmic rRNAs previously discussed.

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

55

Mitochondria1 rRNA differs greatly in size and composition from both prokaryotic and eukaryotic cytoplasmic rRNAs. Thus, as Dubin points out, homology in a small region would indicate a remarkable degree of conservation in the face of extensive evolutionary changes in the rest of the molecule. Attardi and Attardi (1971) reported the higher values of 0.8 to 1.0 for HeLa mitochondrial rRNA versus 1.4 for cytoplasmic 28s rRNA. The most convincing evidence for the occurrence and importance of methylation of mitochondrial rRNA is provided b y some recent observations b y Kuriyama and Luck (1974a,b),who found that the deficiency of small ribosomal subunits in mitochondria of the poky mutant of N . crassa is due to abnormal processing of the 32s rRNA precursor. I n poky the precursor and its resultant products are undermethylated, suggesting, in line with data on other systems, that abnormal processing in this mutant is due to faulty methylation.

5 . 5s rRNA The large ribosomal subunit of both prokaryotes and eukaryotes contains a small RNA (about 125 residues in length) corresponding to a molecular weight of 4 x lo4 and a sedimentation coefficient of 5s. This RNA has been isolated from ribosomes of such diverse sources as bacteria, lower eukaryotes, plants, and animals, and appears to be a universal component of cytoribosomes (see Monier, 1972, for references). This RNA plays a structural role in the assembly of the large ribosomal subunits (Erdmann et al., 1971; Gray and Monier, 1971; Gray et al., 1973). Erdmann et al. (1971) have performed studies on the assembly of the 50s subunit of Bacillus stearothermophilus from its RNAs and proteins in vitru and found that absence of the 5s RNA results in reduced binding of at least four proteins to the reassembled particles. Large subunits assembled in the absence of 5 s RNA are much less active in polypeptide synthesis directed by either natural or synthetic templates, as well as in several model reactions, than are subunits containing 5s RNA. A direct functional role in protein synthesis for 5 s RNA is suggested by the results of Horne and Erdmann (1973), who isolated a specific complex composed of 5s RNA and two main proteins of the large ribosomal subunit. This complex exhibits both ATPase and GTPase activity. Fusidic acid and thiostrepton, inhibitors of the ribosomal GTPase activity associated with translocation, inhibit ATP and GTP hydrolysis by the complex. Complete nucleotide sequences have been determined for 5s RNAs from E . coli, Pseudomonas fluorescens, yeast, Xenopus, mouse, rabbit, rat, and human cells (Brownlee et al., 1968, 1972;

56

HENRY R. MAHLER AND RUDOLF A. RAFF

Forget and Weissman, 1969; DuBuy and Weissman, 1971; Hindley and Page, 1972). These sequences, except for that of Xenopus, which is almost identical to that of mammals, aligned for maximum homology (Kimura and Ohta, 1973), are presented below (Fig. 11).The observed differences between these 5s RNA sequences are presented in Table XIII. The rate of sequence divergence has apparently been slow enough for relationships to become discernible (Sankoff et al., 1973) that suggest that, while the sequences of human and yeast RNA are more similar to each other than to bacterial sequences, and vice versa, considerable sequence homology has nevertheless been retained between the eukaryotic and bacterial 5s RNAs. As discussed above, Kimura and Ohta (1973) used these data to estimate the time of divergence of eukaryotes from prokaryotes. Curiously, while mitoribosomes share with all other ribosomes a structure composed of a large and a small subunit, each containing a characteristic high-molecular-weight rRNA, they apparently lack 5s RNA. At least no molecule with such a sedimentation coefficient or corresponding electrophoretic mobility has been found in mitoribosomes of fungi or animals (Mahler, 1973a). Since 5s RNAs play a vital role in the assembly of ribosomes of all other types, it seems a priori peculiar that this function should have become dispensable for mitoribosomes. This is particularly so since chloroplast ribosomes contain 5s RNA (see Section VI,A,7). Two major possibilities are that a 5s analog exists but is concealed under the 4s tRNA peak (Lizardi and Luck, 1971; Gray and Attardi, 1973), or that the role of 5s RNA is performed by a segment of the large rRNA (Lizardi and Luck, 1971). Gray and Attardi (1973) examined the low-molecular-weight RNAs of HeLa mitoribosomes by gel electrophoresis, and partially resolved two components in the 4s region of the gel. The species possessing the higher mobility is 10 to 15 nucleotides shorter than mitochondrial 4s RNA, that is, it would be expected to b e about 60

MBUUUlS

1 10 20 30 40 50 60 -G-UCUACGGCC-AUACCACCCUGMCGCGCCCGAUCUCGU~GAU-CUCGG~GCUMGCAG

Yeast

-G-GUUGCGGCC-AUACCAUCUAGAAACCACCG~~CCGUCCGAUMCCUGUAG~MGCUG

E. ooli

UGCCUGGCGGCC-GUAGCGCGGUGGUCCCACCUGACCCCAUGCCGM~CAG~UG~CGC

P. fluoresoens

UGUUCUUUGACGAGUAGUGGCAUUCGAACACCUGAUCCCAUCCCGM~C~AGGUG~CGA 70 80 90 100 110 120 GGUCGGGCCUG-GUUAGUACUUGGAUCGGAU~AGACCGC~GGG~UACCGGGUGCUGUAG~OUU

GUMGAGCCUGACCGAGUAGUGUAGUGGGUGACCAUACGCG~CCUAGGUGCUGCA--AUCU CGUAGCGCC---GAUGGUAGUGUG--GGGUCUCCCCAUGCGAGAGUA~MCUGCCA~CAU UGCAUCGCC---CAUGGUAGUGUG--GGGUUUCCCCAUGUCMGAUCUCG-ACCAUAGAGCAU

FIG. 11. Comparison of5S RNA sequences.

57

MITOCHONDRIA AND T H E ORIGIN OF EUKARYOTES

TABLE XI11 FRACTIONAL DIFFERENCES IN 5 s RNA SEQUENCES' Pseudomonas

Human Yeast

Escherichia coli Pseudomonas jluorescens

Human

Yeast

Escherichia coli

Jluorescens

0

0.40

0.45 0.47 0

0.50 0.57 0.31 0

0

Data from Dayhoff (1972) and Kimura and Ohta (1973).

nucleotides long. There is one molecule of this RNA in each large ribosomal (45s) subunit. Labeling of the RNA is inhibited by ethidium bromide, indicating that it is a product of mitochondrial transcription. Dubin et al. (1974) studied an RNA of similar low molecular weight (less than 100 nucleotides long) from hamster mitochondria. This RNA is present in approximately a 1: 1 molar ratio with mitochondrial rRNA, is unmethylated, and has a relatively low G C content. Its synthesis is also sensitive to ethidium bromide. The function of these RNAs has not yet been determined. However, since the mitochondrial rRNAs in mammalian cells are considerably smaller than their prokaryotic or cytoplasmic counterparts and yet capable of functioning, it is not unreasonable that such a small RNA molecule could perform the function of the 5s RNAs of other ribosomes. Since prokaryotic and eukaryotic 5 s RNAs exhibit sequence homology, and since it is relatively easy to isolate 5 s RNA genes (Brown and Weber, 1968; Brown et al., 1972), it may well b e possible to test for 5s sequences in the small mitochondrial RNAs isolated by Gray and Attardi and by Dubin et al. or in mitochondrial rRNA from other sources by molecular hybridization techniques. In summary, while mitochondrial rRNAs have diverged from prokaryotic rRNAs, they have also diverged from one another in such properties as base composition and molecular weight. Furthermore, the ordinary (120-nucleotide) small RNA of the large subunit is absent in the mitochondrial particle. Thus the loss or modification of this RNA may have occurred early in the evolutionary history of the mitochondrion and prior to the divergence of fungi and animals. This evidence supports, but of course by no means proves, the hypothesis that mitochondria had a common origin and that the protein synthetic machinery of the ancestral mitochondrion underwent radical modification early in the evolutionary history of the organelle (Table XIII).

+

sa

HENRY R. MAHLER AND RUDOLF A. RAFF

6. Modes i f rRNA Processing The RNA for eukaryotic cytoribosomes is synthesized in the nucleolus, which contains reiternated copies of the genes for large- and small-subunit rRNAs linked in tandem in an alternating fashion (Brown and Weber, 1968; Brown et ul., 1972). These tandem repeats contain spacer regions that d o not appear in the final rRNA product, although the initial product of transcription of the nucleolar rRNA genes is a large precursor species containing the sequences for the large- and small-subunit RNAs, as well as some of the spacer sequences. Similar precursors have been found in yeasts, plants, amphibians, and mammals (reviewed by Loening, 1970). The precursor species is subject to processing by phosphodiesterase cleavage to yield large- and small-subunit rRNAs, as well as other products that apparently are degraded by the cell. Schemes for the processing of rRNAs in mammals and yeasts are presented in Fig. 12. The large polycistronic precursor containing both rRNAs is cleaved to yield two fragments. One of these, the precursor for the rRNA of the small subunit is again rapidly cleaved and the rRNA rapidly transported into the cytoplasm. The precursors of the rRNA destined for the large subunit are then processed more slowly in the nucleus. The processing of these precursors also produces a so-called 7s species about 0.04-0.06 x los in molecular weight. This species is linked to the large-subunit rRNA by hydrogen bonds and is retained in the mature eukaryotic ribosomal particle. No comparable molecule is present in bacterial ribosomes. While a significant amount of excess RNA is discarded during processing in all cases, mammals are apparently peculiar in producing a precursor containing such a great proportion of nonribosoma1 RNA. Methylation and processing are coordinated so that nucleotides in sequences of the precursor RNA not destined to become rRNA are nonmethylated, whereas those conserved in rRNA are. Processing of mitochondrial rRNA has been studied in Neurosporu by Kuriyama and Luck (1973a). These workers detected a 32s RNA by pulse-chase labeling. This species first gives rise to two other short-lived species and ultimately to rRNAs. Both rRNAs compete with the 32s species in molecular hybridization experiments. The scheme of processing for mitochondrial rRNA based on these observations proposed by Kuriyama and Luck is also presented in Fig. 12. It is evident that this processing pattern is strongly reminiscent of those observed for the eukaryotic nuclear-cytoplasmic system, particularly the one for yeasts.

MITOCHONDRIA AND T H E ORIGIN OF EUKARYOTES

59

Prowsting of Eucaryotic Cytoplasmic rRNAs Mammals 0.65 x lo6MW rRNA (18s)

0.9;;;p6-~) Initial Transcript 4.1 x lo6 MW (45S)

) 3.1 x lo6 1.0~106 (41s)

\ 2.1 x I06

1.65 x 106MW rRNA

(28s)

(32 S)

0.45 x lo6

Yeast

106 MW 1 ’

0.8 x I06 (20s)

0.7 x

(18s)

rRNA

ai x 106 Initial

/

Transcript

) 1.6 x

2.5 x lo6 MW (35s)

T)

lo6

1.3 x ( d 4 W rRNA

(plua 0.06 x lo6) 0.3~10~

(7s1

Rocassing of NeurocDom Mitochondria1 rRNA

/

Tmnscript Initial )-

2.4 x lo6 (325)

~ 0 . x9 106.-)0.72 -1.6 x

x lo6 MW rRNA

lo6 v . 1 . 2 8

x

1 8

MW rRNA

J.

-0.4x lo6

FIG. 12. Processing of eukaryotic rRNAs. Cytoplasmic rRNAs are transcribed in the nucleolus. The initial transcript is specifically cleaved to produce precursors of the large and small ribosomal subunit RNAs. Further processing yields the final products. Sizable segments of the original transcript are discarded. Processing is slow enough to permit the detection of measurable pools of the various precursor species. Neurospora mitochondria1 rRNAs are transcribed within the mitochondrion. Processing is apparently similar to that observed for cytoplasmic rRNAs. But, see note added i n proof.

Bacterial rRNA is transcribed from rDNA arranged in repeat units each consisting of a gene for 16,23, and 5s rRNA (reviewed by Pace, 1973). The only precursor species demonstrable in most bacterial cells, the so-called p23 and p16 species, are molecules only slightly larger than the mature rRNA (Fig. 13). This gave rise to the belief (Pace, 1973) that bacteria might lack a polycistronic precursor as the initial transcript of the cluster of ribosomal genes. However, recent studies in two laboratories (Nikolaev et al., 1973, 1974; Dunn and

60

HENRY R. MAHLER AND RUDOLF A. RAFF Processing of Bacterial rRNA

<

1.20x 106 (21s)

MW rRNA

7’ (23 zo.11 1.01-1.11x106

S)

x 106

Initial Tranacrlpt 2.1 x lo6 MW RNA

(30s)

0 56 x lo6 MW rRNA o*?i.i$6’-’ (16 S) co.10 x ‘

106

FIG. 1 3 . Processing of bacterial rRNAs. Processing of rRNA in bacteria is fundamentally similar to that observed in eukaryotic systems. Detection of an initial transcript has been difficult, because the rate of processing is rapid and may normally occur while transcription is still in progress.

Studier, 1973), using a mutant deficient in RNase I11 (and perhaps some other enzymes as well), have identified such a precursor as well as its mode of processing (Fig. 13).

7. A Note on Chloroplast Ribosomes and rRNA While chloroplasts are generally beyond the scope of this article, it is interesting to note that the properties of chloroplast rRNAs are very unlike those of mitochondria, and similar to those of prokaryotes. The large- and small-subunit rRNAs have molecular weights of 1.07-1.11 x los and 0.56 x los, respectively (Loening and Ingle, 1967; Loening, 1968; Scott and Smillie, 1969). The base composition of Euglena and higher-plant rRNAs is restricted to the range 51.554%G C (reviewed by G o o n et al., 1972). rRNA from Euglena chloroplasts shows a significant degree of hybridization with blue-green algal DNA (Pigott and Carr, 1972). Chloroplast ribosomes resemble both cytoplasmic and prokaryotic ribosomes in containing 5s RNA (Payne and Dyer, 1971; Bourque et al., 1971). Finally, ribosomal subunits of both lower and higher plants are capable of forming active, hybrid particles with subunits of E . coli ribosomes (Lee and Evans, 1971; Grivell and Walg, 1972) while subunits from mitochondria are incapable of doing so (Grivell and Walg, 1972).

+

B. MITOCHONDRIAL mRNA

1. Site of Specification Mitochondria of all eukaryotic species examined-as long as they retain an unmodified mtDNA-appear to contain their own system

MITOCHONDRIA AND T H E ORIGIN O F EUKARYOTES

61

for protein synthesis, distinct from that found in the extramitochondrial cytoplasm of the same cell and designed to produce a discrete set of polypeptides (Table XIV). In addition to the class of mitoribosomes described in Section VI,A (and polysomes to be discussed in Section VI,C,2) with their unique RNAs, and a set of separate tRNAs (all of which appear to be specifically encoded in and transcribed from mtDNA), such a translational system must of necessity contain another (set of) essential RNA(s). This is the mitochondrial mRNA, the polynucleotide sequence of which specifies the amino acid sequences to be synthesized. There is therefore no longer any question regarding the mitochondrial location of such an RNA, but until recently there has been some residual doubt concerning its specification. Why should this doubt have arisen? There are several reasons, none of them compelling alone but quite disturbing in the aggregate:

1. Most mitochondrial polypeptides, including those of the inner membrane, are synthesized outside and have to be imported (Section VI1,E); those localized in the inner membrane or the matrix PRODUCTS OF

TABLE XIV MITOCHONDRIALPROTEIN Molecular weight ( x low5)

Complex

Description

Total

I I1

IV

NADH-CoQ reductase Succinate-CoQ reductase CoQH,-cytochrome c reductase Cytochrome-c oxidase

7.0 2.0 2.3 2 x 1.33

V

ATPase complex

111

3.6

Subunits synthesized in mitochondria

2(?) x 2 X 2x 2x

0.3 0.4 0.3 0.2 0.3 2 x 0.2

4(?) x 0.075

Total Fraction of inner membrane

17.6 50%

3.4 (19%) 9.5%

Data adapted from Mahler (1973a) and Schatz and Mason (1974). T h e assumptions made are that (1) all complexes are present in equal mol ratios, (2) the molecular weight of cytochrome oxidase in the membrane is equal to that in solution (MW = 2.5 x lo5)and therefore consists of two sets of subunits, and (3)these complexes account for 50% of the membranes. If these are incorrect, as appears to be the case in heart muscle (Harmon et al., 1974), the fraction synthesized internally may be as high as twice the amount shown. ”

62

HENRY R. MAHLER AND RUDOLF A. RAFF

have to cross one double-membrane barrier and penetrate into or cross a second one as well. The same consideration probably also applies to some tRNAs, particularly of animal mitochondria. Thus there is no fundamental inconsistency in postulating a mRNA synthesized in the nucleus and destined for the mitochondria. 2. In most eukaryotic cells there is a division of labor between two cytosolic systems of protein synthesis consisting of free and membrane-bound polyribosomes (see, e.g., Rolleston, 1972; Lowe and Hallinan, 1973). The nature and destination of the class of polypeptides formed b y these two systems are quite unique, and therefore there must exist a mechanism for the designation and partition of two distinct classes of mRNAs between them. Similar principles might be operative for the postulated third (mitochondrial) class of mRNAs (Kellems et al., 1974a,b). 3. Isolated mitochondria of Xenopus have been shown to be capable of absorbing added polyribonucleotides and to utilize them as templates for protein synthesis (Swanson, 1971; Grivell and Metz, 1973). 4. Dawid (1972a,b) has argued persuasively that the isolated mitochondrial RNAs of Xenopus do not contain any significant amount of a species that can qualify as mRNA; all such RNAs are accounted for in terms of the known stable rRNAs and tRNAs. Furthermore, in comparing the mtRNAs, and DNAs of Xenopus laevis with those of X. mulleri, a closely related species, he finds no evidence for sequences complementary to any species of RNA other than rRNAs and tRNAs. These considerations have engendered a variety of novel experiments and a more critical evaluation of existing ones. As a result, the balance appears to b e tipping strongly against import and in favor of intrumitochondrial specification of its mRNA. The relevant lines of evidence are:

1. The supply and function of mitochondrial mRNA, as defined by its appearance and association with mitoribosomes and its ability to program them for polypeptide synthesis, exhibiting characteristic attributes (Section VI,C), can b e shown to remain unaffected by treatments that completely disrupt the analogous events in cytoplasmic mRNA. The most convincing experiments used a temperature-sensitive mutant of S. cerevisiue deficient in the supply of ull cytoplasmic RNAs of nuclear origin at the nonpermissive temperature (Mahler and Dawidowicz, 1973). Very similar experiments with HeLa cells

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

63

have used the selective inhibitor comptothecin for the same purpose (Perlman et al., 1973). Conversely, mitochondrial mRNA becomes ineffective in both these systems when its generation is interrupted by ethidium bromide, a specific inhibitor of mitochondrial transcription-and, unfortunately, translation. However, the detailed patterns of the polysomal decay engendered by ethidium bromide and chloramphenicol, an authentic inhibitor of translation in the mitochondria of S. cerevisiae, appear to differ. 2. Scragg (1974) has provided some evidence that RNA transcribed by mitochondrial mRNA polymerase from mtDNA in yeasts in vitro can be used as a messenger in a cell-free system from E. coli to produce polypeptides capable of immunological cross-reactions with inner membrane proteins from yeast mitochondria, and Kuntzel and Blossey (1974) have performed analogous experiments with Neurospora. 3. Particles that resemble polymorphic or polyhedral viruses in morphology have been identified in several species of fungi. These viruslike particles” were isolated from u b n l , a cytoplasmic (presumably mitochondrial) respiration-deficient mutant of N . crassa by Kiintzel and his collaborators (Kuntzel et al., 1973a,b, 1974). They were found to contain single-stranded RNA which was complementary to 4.5% of the base sequences of mtDNA from both the wild type and a b n l , but which did not show any homology to either mitochondrial or cytoplasmic rRNA. This RNA could b e used to program a protein-synthesizing system for the production of a protein of molecular weight 11,000, which resembled one of the two protein constituents of the viruslike particle and which is known, furthermore, to be synthesized inside the mitochondria. It is therefore probable that this RNA represents a stable form of mitochondrial mRNA which is overproduced in consequence of the ubnl mutation and its \ resultant lesion in mitochondrial protein synthesis. 4. The capability for using extrinsic polyribonucleotides as artificial messenger appears to be a peculiarity of mitochondria from Xenopus eggs. Mitochondria from other sources such as rat or chick liver, Tetrahymena, and yeasts cannot perform the same reaction (Grivell and Metz, 1973). 5. At least some of the known mitochondrial mutations in S. cerevisiae (those conferring resistance to oligomycin, and perhaps others responsible for resistance to different inhibitors and uncouplers of respiration-Fig. 4) have now been shown to produce altered proteins responsible for these functions (Shannon et al., 1973; Griffiths and Houghton, 1974); these proteins are known to be synthesized in“

64

HENRY R. MAHLER AND RUDOLF A. RAFF

side the mitochondria (Tzagoloff et al., 1973). Such combinations of genetic and biochemical evidence provide the strongest proof for mitochondria] specification of mitochondrially produced polypeptides. 6. Mitochondrial mRNAs appear to exhibit the distinguishing feature of 3’-terminal adenylation (see Section VI,B,2). This has now been demonstrated for HeLa cells (Perlman et al., 1973; Attardi et al., 1975; Ojala and Attardi, 1974a,b,c), insects (Hirsch et al., 1974) and, perhaps, S. cereuisiae (Cooper and Avers, 1974; but see Groot et al., 1974). In the case of HeLa cells this species was not only isolated from mitochondria1 polysomes, but was also be shown to be complementary specifically to mtDNA. Its specification has been reported to be by the H strand either exclusively (Hirsch and Penman, 1973) or predominantly (Ojala and Attardi, 1974a,b). (The seven larger species varying in molecular weight between 5.3 and 2.6 x 105 can be hybridized with the H strand, and the smallest, with a molecular weight of 9.0 x lo4, with the L strand. If the whole length of these molecules is used to code for amino acids, the corresponding molecular weights are approximately 50, 25, and 8 x lo3.) This group of RNAs has been resolved by means of gel electrophoresis and shown to be composed of eight discrete species (Ojala and Attardi, 1974~). This number coincides rather neatly with the one now commonly accepted (Schatz and Mason, 1974; Mahler, 1973a) for the number of polypeptides synthesized inside mitochondria from many species in uiuo and in uitro (e.g., Lederman and Attardi, 1973). Even more striking is the fact that the size distribution of these presumptive mRNA molecules is completely consistent with that expected from the molecular weights of these polypeptides (Section VI,C,6). When added to the other known transcripts, these poly-A-containing species account for the majority (-70%) of the potential informational content encoded in a single strand of mtDNA from this source. 2. Polyadenylation of Mitochondrial mRNA Recent investigations (Perry, 1973; Darnell et al., 1973) have disclosed a striking difference between well-characterized mRNAs of bacterial and eukaryotic origin. Most of the latter (the mRNAs for histones providing the one glaring exception so far) carry at their 3’-OH a sequence of polyriboadenylic acid residues. These are synthesized by a separate enzyme not requiring a template and are attached to the mRNA after the completion of its transcription. Two different classes of such posttranscriptionally polyadenylated RNA have been identified in metazoan cells (for references, see Hirsch and Penman, 1973). One, carrying poly-A stretches 150 to 200 nucleotides long, is

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

65

composed of mRNAs of nuclear origin destined for the cytoplasm and of the RNA of viruses of nuclear localization such as RNA tumor viruses, SV40, and adenoviruses. The second class, with approximately 80 to 110 adenylate residues, consists of the RNA constituent of positive-strand RNA viruses (arbo- and picornaviruses) and of the RNA transcripts formed from negative-strand RNA viruses (paramyxo- and rhabdoviruses) or from the DNA of vaccinia virus. As described in Section VI,B,l similar poly-A-containing RNAs, likely candidates for a messenger function, have now been identified in HeLa, insect, and yeast mitochondria. The enzymic reaction in mitochondria responsible for the formation of this poly A has been characterized (Jacob et al., 1972), and it has been demonstrated that, as in the nucleus, the attachment of poly A to mRNA takes place after the transcription of the latter has been completed (Ojala and Attardi, 197413; Hirsch and Penman, 1973). The length of the poly-A stretch in question is of the order of 55 nucleotides, and thus mitochondrial mRNA appears to be unique in this respect, and clearly distinct from all other mRNAs whether of pro- or eukaryotic origin. The apparent absence of poly-A-containing mRNAs in some mitochondrial mRNAs (Groot et al., 1974; Avadhani et al., 1973) may be d u e to rapid deadenylation (Avadhani et al., 1974). Also, see note added in proof.

C. MITOCHONDRIALPROTEINSYNTHESIS

1. Components of the System That isolated mitochondria contain an intrinsic protein-synthesizing system has now been known for more than 15 years. Before the significance of this fact could be placed in its proper perspective several advances, both technical and conceptual were required: (1) The reaction had to b e shown to be due to mitochondria and not to possible bacterial contaminants; the most convincing demonstration of this fact was provided by the observation that mitochondrial and bacterial protein synthesis differ in several important details at the molecular level (see Section VI,A,B). (2) The reaction had to be demonstrated as occurring and being of importance to cellular function in vivo. This became possible with the advent of pulselabeling techniques, particularly in conjunction with the use of sitespecific inhibitors of protein synthesis (see Section VI,C,5), most convincingly with cell cultures of single celled eukaryotes or of isolated cells from metazoan animals. (3) The products of the reaction had to be identified, and their importance and relevance to mitochondrial autonomy and biogenesis established. This required the

66

HENRY R. MAHLER AND RUDOLF A. RAFF

development of a methodology for the solubilization, separation, purification, and analysis of highly insoluble hydrophobic membrane proteins and their constituent subunits, and a demonstration that mtDNA did indeed carry the information for discrete mitochondrial entities. With this impetus rapid strides have recently been made toward a complete characterization of the mitochondrial protein-synthesizing system and its evolution. All mitochondrial systems studied-as well as all protein-synthesizing systems of pro- and eukaryotes-consist of the following classes of essential components:

1. Ribosomes, that is, ribonucleoproteins, consisting of two nonidentical subunits and each composed of a distinct species of RNA (rRNA)-all already discussed-and a group of associated proteins; one of these, localized in the large subunit, is responsible for catalyzing the peptide bond-forming, chain-elongating step in polypeptide synthesis. 2. mRNA which carries the polynucleotide sequence to be translated into an amino acid sequence. 3. A set of at least 21 aminoacid tRNAs (at least one for each of the 20 amino acids and one for chain initiation), which represents the entries in the genetic dictionary, since they combine the sequence information for the specific attachment of their cognate amino acids, in one part of the molecule, with the ability to recognize the relevant coding triplet on mRNA, in another. 4. At least 20 different enzymes (amino acid tRNA ligases or synthetases, one for each of the 20 amino acids) capable of forming an aminoacyl tRNA bound at the 3’(2’)-OH group of the terminal adenosine residue of the cognate tRNA. 5 . A set of protein factors essential for catalysis of the individual steps required for polypeptide synthesis which, like any other polymerization process, can be subdivided as follows: (a) Initiation factors ( 3 2 ) concerned with the recognition of the proper initiation codon (AUG), the attachment of the small and large ribosomal subunit to an appropriate mRNA in sequential fashion, and the apposition of the appropriate charged aminoacyl tRNA and its localization in a configuration permitting the formation of the first peptide bond. (b) Elongation factors ( 2 2 ) ,concerned with the apposition of the nth aminoacyl tRNA (ah-tRNA,) and the nth codon of the mRNA in the appropriate (recognition or A) site on the ribosome, and with the translocation of the ribosomes relative to the mRNA. The latter term defines a sequence of events that occurs subsequent to the formation

MITOCHONDFUA AND THE ORIGIN OF EUKARYOTES

67

of the nth peptide bond. This, the elongation step proper, involves a transpeptidation of the growing chain, originally attached to the ribosome at a second (peptidyl or P) site as [aa,

. . . aa,-,C-OtRNA,-J[P] II 0

to yield [aa,

. . . aa,-,C-NHaa,tRNAJ[A] II 0

that is, chain elongation involves transfer to the A site. Translocation then consists of transfer of the newly formed molecule back into the P site, subsequent to or coincident with the expulsion of the freed tRNA,-,, and opening the A site for the potential binding of the (n + 1) st aa-tRNA. This transfer is accompanied by a movement ofthe mRNA relative to the ribosome so as to bring the (n + 1) st codon into apposition to the A site, thus permitting the next turn of the cycle to take place. (c) Termination factors (about three), which are concerned with the recognition of one (or more) of the nonsense codons (UAA, UAG, and UGA) as chain terminators, the removal of the completed chain from the last tRNA by hydrolysis, and the detachment of the ribosome from the mRNA, possibly coincident with the dissociation of the former into its subunits. 6. A set of substrates (the 20 amino acids), cosubstrates (ATP and GTP), and cofactors (divalent and monovalent ions, -etc.).

2. mtRNAs and Protein Synthesis The properties of the various RNA species, their changes during evolution, and their site of specification in mtDNA have already been discussed in earlier sections. One observation appears germane at this point, however. This deals with the tRNA involved in chain initiation. It now appears highly probable that the same basic mechanism for chain initiation is operative in both pro- and eukaryotic systems. This mechanism involves the recognition of an AUG codon in the 5'-proximal portion of each message by a special, initiating species of charged methionyl tRNA called Met-tRNAFet,distinct from the second species of methionyl tRNA, Met-tRNAiet, which recognizes the same codon in an internal position. The difference between the two forms of methionyl tRNA and the function of tRNA,M"'in chain initiation are discussed in Section VI,C,3. The important consideration here is that at least in some eukaryotes there is no evidence for more than one form of this molecule (tRNA,), which

68

HENRY R. MAHLER A N D RUDOLF A. FMFF

therefore must act as initiator for both the mitochondrial and cytosolic system of protein synthesis. However in yeast, Halbreich and Rabinowitz (1971) demonstrated that this tRNA is homologous with a base sequence of mtDNA. Therefore either all the cellular tRNAF is provided b y mitochondrial transcription, which seems unlikely, or identical or closely related genes for this tRNA reside in both a nuclear and the mitochondrial chromosome.

3. Mitochondria1 Ribosomes and Polysomes Protein synthesis in both prokaryotic and extramitochondrial eukaryotic systems is known to involve arrays of ribosomes arranged in series along the length of the mRNA. Growing polypeptide chains are attached to the array, so as to increase in size as we progress from the N-terminal to the C-terminal part of the message. Does mitochondrial protein synthesis involve similar structures, that is, is there evidence for mitochondrial polyribosomes or polysomes? The consensus appears to be that there is, although the picture is complicated b y the highly hydrophobic nature of the nascent polypeptides and the resultant tendency to cause aggregation of the ribonucleoprotein particles to which they are still attached, a process not dependent on the presence of intact mRNA (Ledermann and Attardi, 1973; Michel and Neupert, 1974). Polysomes that exhibit the expected properties, including their conversion to monoribosomes or subunits on transfer of the nascent polypeptides to puromycin, and the destruction or removal of mRNA, have been isolated from mitochondria of yeast (Mahler and Dawidowicz, 1973), Neurospora (Agsteribbe et al., 1974), Euglena (Avadhani and Buetow, 1972), and HeLa cells (Ojala and Attardi, 1972).

4. Chain Initiation and Elongation Factors Mitochondrial chain initiation utilizes Met-tRNA!" of the formylated variety, that is, met-tRNAp'. In this it resembles the pattern established for prokaryotes rather than for the eukaryotic cytoplasm. However, as already mentioned, chain initiation by the latter system makes use of the same species of tRNA. What is lacking in the cytoplasm and present in the mitochondria is the transformylase, the enzyme responsible for converting Met-tRNA into met-tRNA with N-lO-formyltetrahydrofolate as the formate donor. In agreement with the postulated basic conservation of the chain-initiating mechanism throughout evolution is the observation that fMet-tRNAFand initiation factors from E . coli and reticulocyte cytosol appear to be interchangeable (Berthelot et al., 1973).

MITOCHONDRIA AND T H E ORIGIN OF EUKARYOTES

69

The ability of supernatant factors (usually elongation factors G and T) isolated from mitochondria of various species, the homologous cell sap, and from bacteria ( E . coli) to support protein synthesis (usually poly Phe formation with poly rU as artificial mRNA) on ribosomes isolated from the same three sources has been investigated with ascomycetes (Kroon et al., 1972; Kuntzel, 1971) and X. laeuis (Swanson, 1971). The results are in reasonable agreement. Protein factors (whether crude mixtures or purified elongation factors) of mitochondria and E . coli are mutually interchangeable, at least qualitatively. but cannot interact with cell sap ribosomes; conversely, elongation factors isolated from the cell sap are specific only for ribosomes from the same cell fraction (regardless of species) and do not interact with ribosomes of mitochondria or E . coli. Similar results were obtained also with a more complex system consisting of ribosomes from yeast mitochondria and elongation factors from this source or E . coli, programmed with mRNA synthesized b y Neurospora mtRNA polymerase transcribing the DNA from E . coli bacteriophage T3 or T7 (Richter et al., 1972).

5 . lnhibition Patterns Many recent reviews and articles have served to expand and qualify the initial postulate by Linnane and his collaborators (ClarkWalker and Linnane, 1966; Huang et al., 1966) that mitochondrial ribosomes were of the “prokaryotic, 70s type” and thus differed qualitatively in terms of possible inhibitors from those of the cell sap (from the same organism) which are of the “eukaryotic, 80s type.” The current status of the problem has been reviewed by one of us (Mahler, 1973a) and by Linnane et al. (1973), and the available data (Table XV) can be summarized as follows:

1. The inhibitors of eukaryotic cell sap ribosomes (80stype), in particular the glutarimides (such as cycloheximide), emetine, and anisomycin-all of which bind to the large subunit-appear specific for this class and do not block mitochondrial or bacterial protein synthesis. Some caution in the interpretation of these and most other inhibition data must be exercised, however, since these agents are known to be capable of affecting other processes as well. Some of these, such as ion and amino acid transport (TimberlakewandGriffin, 1973; Reilly et al., 1970) or RNA synthesis (Cihik and Cerna, 1972; Timberlake et al., 1972; Farber and Farmar, 1973), are not only of particular importance to the cellular economy in general, but may also affect protein synthesis in a quite direct manner.

70

HENRY R. MAHLER AND RUDOLF A. RAFF TABLE XV INHIBITORS OF PROTEIN SYNTHESIS

Acting on ribosomal systems of the 80s type Anisomycina Diphtheria toxin Emetine" Endomycin Glutarimide group Actiphenol Cycloheximide" Streptimidone Streptovitacin A P e d er i n Phenom ycin Tenuazonic acid Tylophora alkaloids' Cryptopleurine Tylocrebrine Tylophorine

Acting on ribosomal systems o f t h e 70 and 80s types Actinobolin Aurintricarboxylic acid Blasticidin S Bottromycin A* Edeine Fusidic acid Gougerotin Nucleocidin Pactamycin Poly-dextran sulfate Puromycin Sparsom ycin Tetracycline group Chlortetracycline Deoxycycline Oxytetracycline Tetracycline

Acting on ribosomal systems of the 70s type

Acting on mitochondria

Aminoglycosides Chloramphenicol Lincomycin Macrolides Carbom ycin Erythromycin Spirainycin Mikamycin (vernamycin)

Aminoglycosides ( ? ) b Chloramphenicol" Lincomycind Macrolides Carbom ycin Erythromycind Spiramycin Mikamycin (vernamycin)

Siomycin Thiostrepton (Bryamycin)

Siomycin Thiostrepton" (Bryamycin)

" Most generally useful inhibitors. Incompletely studied; in yeast neomycin and paromomycin appear to be relatively effective and discriminating mitochondria] inhibitors in uitro, but are ineffective with animal mitochondria; other common members of this group (e.g.,streptomycin, kanamycin) are ineffective even with unicellular eukaryotes. Have also been reported to be effective against yeast mitochondria. Ineffective with mitochondria from animals.

2. Similarly, the group of bacterial (70s type) inhibitors that bind to their large (50s) subunits, consisting of chloramphenicol, mikamycin (vernamycin), carbomycin and, usually, spiramycin, ineffective with HeLa cells or their mitochondria (Dixon et al., 1971), but active with BHK cells (de Vries et aZ., 1973), appear to be effective and specific in their appropriate concentration ranges in blocking mitochondrial protein synthesis in vivo and are also effective against isolated mitochondria from all sources. With all these inhibitors care must be exercised to distinguish a direct mode of inhibition of protein synthesis from an indirect one on respiration exerted at a somewhat higher but frequently overlapping concentration range. Other macrolide antibiotics such as erythromycin and lincomycin, which

MITOCHONDRIA AND T H E ORIGIN OF EUKARYOTES

71

are highly effective with bacteria, are potent inhibitors with certain cells and their mitochondria (e.g., yeasts) but not with others (e.g., Neurospora, HeLa, rat liver), even though at least in some systems they are capable of penetrating the cell and its mitochondria and binding to their ribosomes (Towers et al., 1973; d e Vries et al., 1973; Denslow and O’Brien, 1974). 3. The mode of action of the inhibitors under item 2, although reasonably well established for bacterial systems (Pestka, 1971a,b; Benveniste and Davies, 1973), appears much less certain for mitochondria. Even where inhibition by, or binding of, these compounds can be demonstrated with isolated mitochondria i n uitro, the inference that the mitochondrial ribosome or one of its proteins is the susceptible target is not always justified. In principle, it should be possible to obtain an answer by studies on mitochondrial mutants conferring resistance to the appropriate antibiotic. However, there are several difficulties with this approach; the phenotypic patterns produced b y different mutations in the same gene are very complex when analyzed at the cellular and mitochondrial level, particularly with regard to cross-resistance; the mutations reported from different laboratories are difficult to correlate in a meaningful fashion, and some of the results reported appear contradictory (e.g., Grivell et al., 1973; Malloy et al., 1973; Trembath et al., 1973).Only recently has it been possible to show that mitochondrial mutations to antibiotic resistance at some of these independent, but linked, genetic loci actually lead to alterations in mitochondrial ribosomes. However, the mutational changes probably result in an altered rRNA rather than a ribosomal protein (Grivell et al., 1973). The latter change appears to be the usual basis for erythromycin resistance in most bacteria, but an altered RNA, methylated abnormally as a result of novel information carried by a plasmid, has been observed in Staphylococcus aureus. There is a precedent in a mitochondrial mutation producing an altered pattern of mitochondrial rRNA methylation. Recent results by Kuriyama and Luck (1974a,b) argue strongly that this is at least one of the consequences of the m i l (poky) mutation in Neurospora; mitochondrial rRNA but not tRNA is specifically undermethylated in this strain (Section VI,A,4). 4. The inhibitor fusidic acid, which in bacteria is believed to bind to elongation factor G and to act by fixing the usually transient complex between 50s ribosomes, this elongation factor, and a guanine nucleoside di- or triphosphate (Bodley et al., 1970; Pestka, 1971a,b; Otaka and Kaji, 1973; Haselkorn and Rothman-Denes, 1973), is usually also effective with eukaryotic cell sap systems,

72

HENRY R. MAHLER AND RUDOLF A. RAFF

including those of ascomycetes (Richter and Lipmann, 1970), presumably with a similar mode of action. One would therefore expect it to be effective against mitochondria also, and that is the conclusion reached by Richter et al. (1971, 1972) using ribosomes and elongation factors from yeast. In contrast, Grandi et al. (1971), working with an analogous system from Neurospora, and Dawidowicz (1972), who used the formation of fMet-puromycin by yeast spheroplasts in uiuo, found fusidic acid to be ineffective. Additional complications may arise because of the reported interference of the inhibitor with mitochondrial ATPase (Kroon et al., 1974). No such ambiguity has so far been reported for the inhibitors thiostrepton and siomycin, which are capable of blocking peptide bond formation on bacterial 50s ribosomes through two of their intrinsic proteins called L7 and L12, both acidic, helical proteins lacking cysteine, tryptophan, and histidine, but with a high content in alanine (Haselkorn and RothmanDenes, 1973) b y interacting with both elongation factor G and Tu. Their specificity and mode of action appears similar in bacterial and mitochondrial protein synthesis (Richter and Lipmann, 1970; Modolell et al., 1971a,b; Highland et al., 1971; Murray and Linnane, 1972; Haselkorn and Rothman-Denes, 1973). In any event, the general mechanism and the molecular events of protein synthesis in pro- and eukaryotes (both in the cytosol and mitochondria) exhibit so many fundamental similarities (Haselkorn and Rothman-Denes, 1973) that it is probably extremely risky to ascribe a great deal of significance to the few remaining, rather idiosyncratic, differences. Among the latter is the apparent greater propensity ofbacterial and chick embryo mitoribosomal systems, programed with artificial mRNA, to translational errors in response to aminoglycoside antibiotics, ethanol, and low temperature, as compared to cytoribosomes.

6 . Polypeptides Synthesized b y Mitochondria and (Probably)

Specified by mtDNA Several techniques have been used to determine the nature of the polypeptides synthesized by the mitochondrial system of protein synthesis. Since these results have been reviewed extensively in several recent articles (Mahler, 1973a; Tzagoloff et al., 1973; Schatz and Mason, 1974), we only summarize the most relevant conclusions:

1. The products of mitochondrial protein synthesis appear to be localized exclusively in the inner mitochondrial membrane and probably contribute no more than 15% of its total mass.

MITOCHONDFUA AND THE ORIGIN OF EUKARYOTES

73

2. The total number of these polypeptides synthesized by mitochondria and identified by electrophoresis on acrylamide gels (in the presence of SDS) appears to be of the order of 8 to 10, regardless of the species examined. 3. All these polypeptides, again regardless of species, appear to fall into only a few molecular-weight classes, with masses of -40, -35, -28, -25, -20, -15, -10, and -7.5 x lo3 daltons. All of them also appear to be formed in roughly equimolar amounts. We first need to inquire into the bearing these findings have on the heading of this section. The reason for believing that at least the majority (and therefore by Occam’s razor the totality) of these translational products are encoded in mtDNA has been critically reviewed by Borst (1972, 1974), by Schatz and Mason (1974), and by one of us (Mahler, 1973a). The arguments in favor of the hypothesis have already been summarized in Section VI,B. We therefore assume a mitochondrial specification for these polypeptides and next ask whether any of them have been assigned a definite function in the mitochondrial economy. Again, recent studies in several systems appear to permit a fairly decisive answer (see Table XIV); three (two in metazoan animals) appear to be associated with cytochrome oxidase (cytochrome aaJ, four form part of the membrane attachment site of oligomycin-sensitive ATPase (Tables VI and XIV), and one, with a molecular weight of 30,000 is a part of-or may account for the total proteins of-at least one form of cytochrome b (Weiss and Ziganke, 1974a,b). Finally, there is some indirect evidence that one or more components of the transporter for ATP may also be formed inside the mitochondria (Linnane et al., 1973). The location and function of these components is summarized in Figs. 3 and 17 and is discussed in Section VI1,C. What proportion of the total coding capacity of the mitochondrial genome is represented by these and the other identified gene products? Or, as a coroliary, how many other proteins specified by it remain to be identified and isolated? So far as animal mitochondria are concerned, these known entities come close to but do not exceed their coding capacity (Schatz and Mason, 1974; Attardi et al., 1975). This conclusion can b e arrived at either by calculating the proportion of the H strand-responsible for the bulk of the encoded information-already accounted for in terms of known gene products, or by direct hybridization experiments (see Section VI,A,B). Very few, if any, additional gene products remain therefore to be identified in these instances. Primitive, unicellular eukaryotes have much larger DNAs (Table VII), and therefore the possibility of their coding for

74

HENRY R. MAHLER AND RUDOLF A. RAFF

additional gene products remains open. That at least one, and possibly more than one, such product is involved in DNA replication or repair is indicated by two types of observations: (1) studies with the extrachromosomal, probably mitochondrial, mutant uus p72 (Moustacchi, 1971; Mahler and Perlman, 1973; Mahler and Bastos, 1974a,b; Perlman, personal communication) which exhibits mitochondrial hypermutability to respiratory deficiency b y a variety of mutagens; (2) the induction of such mutations under conditions in which the mitochondrial system of gene expression is shut down by specific inhibitors (Williamson et al., 1971; Carnevali et al., 1971). However, if we assume that in exponentially growing cells with a full complement of competent mitochondria all mitochondrial polypeptide gene products are synthesized roughly to the same extent, the quantitative data summarized in Table XIV preclude the existence of a substantial number (>5 or so) of additional unidentified products. One of the corollaries to this inference is the statement, which has already been verified experimentally, using a variety of independent criteria, that the bulk, and perhaps all, of the proteins required for mitochondrial duplication itself, that is, those concerned in DNA replication, RNA synthesis, and protein synthesis (ribosomal proteins, amino acid tRNA synthetases, and initiation, elongation, and termination factors) all must be extrinsic to the organelle. They are specified by nuclear genes, synthesized on cell-sap ribosomes, and imported into the mitochondrion.

VII. Mitochondrial Functions A.

MITOCHONDRIAL TOPOGRAPHY

Mitochondria, like gram-negative bacteria, but unlike all other organelles, contain two sets of membranes. In contrast to the bacterial case, however, both their inner and outer membranes are bilaminate, and both are essentially devoid of components such as polysaccharides which can contribute to structural rigidity. In any event this kind of topography defines four mitochondrial spaces or compartments usually designated: (1) outer membrane, (2) intermembrane space, (3) inner membrane, and (4) matrix. Among these items 3 and &and particularly item L a r e usually considered most characteristically mitochondrial. This assertion is based not only on the structure, composition, and functions of the entities found there (see Section VII,D), but also on the fact that only the inner membrane itself is functionally, or vectorially, active. It alone is respon-

75

M I T O C ~ O N ~AND ~ A THE ORIGIN OF EWKARYOTES

sive to alterations in osmotic pressure, presents a permeability barrier not only to mitochondria1 substrates and cosubstrates (cytochrome c, ADP and ATP, nicotinamide nucleotides, salts of di- and tricarboxylic acids, etc.), but also to H+ and OH- ions; and it contains devices for an energy-dependent, selective accumulation (active transport) of many of them (Figs. 3 and 14).

B. MITOCHONDRLQL LIPIDS Like other bilaminate unit membranes, both the outer and inner membranes contain a large proportion of lipids, particularly phospholipids, as essential components. However, the two membranes are quite distinct, both in their lipidlprotein ratio-and the resultant buoyant density-and in the details of their composition. Outer membranes are generally much richer in lipids and, in the case of guinea pig liver mitochondria (Parsons and Yano, 1967), may consist of 50% lipid as compared to values of 25 to 33% for the inner membrane. On isopycnic sedimentation they are therefore found to NADP+ ATP*H@

NAY

NADPH

'/,

,NAD+

AHZ car28-1

t

t

6"

ADP+Pi

Inlride fmtrix)

~

t-

H+-

At2H'

u

+ 2s'

(28 1.20-t 2H')

~Divclent+ (plus onion1 i

~

~

~

Monovalent (plus ionophorrs ) Anlonlr (organic wide, amino acids) Nucieotidelr(incl. adenorme di and triphasphate) -2M)mV, inaide negative 1

FIG. 14. Various interconvertible and reversible modes of energy transduction performed by ~iitochondria.Ordinarily-as shown in Fig. 4-oxidation of substrate (right) is rised for the generation of ATP from ADP (left). Alternatively, it may be employed for transhydrogenation (top) or to transport any or all of the entities shown across the membrane, against a concentration gradient, or for the generation of a membrane potential. Conversely, any of these latter processes may be driven by external ATP, used to either generate ATP, or or-when operating in the reverse direction-be reduce the oxidized form of the substrates. All this is made possible by the participain all of them; although its tion of one and the same intermediate, symbolized by precise nature is still unknown, the vectorial organization of the membrane and its maintenance constitutes an essential prerequisite.

-

TABLE XVI PHOSPHOLIPIDCOMPOSITIONOF REPRESENTATIVEMEMBRANES".~

Rat liver Lipid=

1

2

3

4

5

a b

61.4 3.2 22.7 8.6 3.6 nd 3.4 1.3 nd nd

60.9 3.7 18.6 8.9 3.3 nd nd nd 4.7 0

45.3 12.3 17.9 8.7 4.2 nd nd nd 5.9 6.3 30.2d

34.9 17.7 18.5

33.5 32.9 17.9

7'3 9.0 4.8 t 4.4 3.3 nd -

8.9

C

d e f g h i k Sterol

-

Escherichia coli

Yeast mitochondria

nd

6.8 0 nd -

6 49.7 5.0

23.2 12.6 2.2 2.5 3.4 1.3 nd nd 30.1d

7 45.4 2.5 25.3 5.9 0.9 2.1 17.4 0.7 nd nd 5.06d

8

9

38.5 nd 30.6 8.1 4.2 nd 10.9 nd nd nd

34.3 nd 17.9 26.0 3.9 nd 8.9 nd nd nd 7.3'

10 47.5 nd 19.3 12.6 10.0 nd 6.2 nd nd nd 250"

11

12

32.5 nd 22.0 10.7 3.3 2.0 15.6 nd nd nd

35.8 nd 20.5 16.7 7.2 0.8 9.1 nd nd nd -

13

14

63.4 10.6 10.6

78.0 11.0 6.3

9.4 -

<1.0 -

-

-

-

All values as percent of total. nd, Not done or reported; 0, below limit of detection; t, trace, not quantitated. 1, Nuclear (Kleinig, 1970); 2, rough endoplasmic reticulum (Keenan and Morre, 1970); 3, Golgi (Keenan and Morre, 1970); 4, plasma (Ray et d., 1969); 5, lysosomal (Henning et al., 1970); 6, mitochondrial, outer (McMurray and Dawson, 1969); 7, mitochondrial, inner (McMurray and Dawson, 1969); 8, aerobic (Paltauf and Schatz, 1969);9, anaerobic, unsupplemented (Paltauf and Schatz, 1969); 10, anaerobic, supplemented (Paltauf and Schatz, 1969); 11, aerobic, wild type (Jakovcic et QZ., 1971); 12, aerobic, respiration-deficient mutant (Jakovcic et d.,1971); 13, wall (White et d., 1972); 14, plasma (White et al., 1972). ' Lipid: a, Phosphatidylcholine; b, sphingomyelin; c, phosphatidylethanolamine; d, phosphatidylinositol; e, phosphatidylserine; f, phosphatidylglycerol; g, diphosphatidylglycerol; h, phosphatidic acid; i, lysophosphatidyl choline; k, lysophosphatidylethanolamine. Micrograms of cholesterol per milligrartr of protein (Parsons and Yano, 1967). e Micrograms of cholesterol per milligram of protein (Cobon and Haslam, 1973). a

* Membrane:

77

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

be lower in density by about 80 mg/ml (e.g., 1.13 versus 1.21 for rat liver in sucrose gradients). The main compositional differences are (Table XVI) a preponderance of sterols (cholesterol in animals, ergosterol in yeasts) and inositol phosphatides in the outer membrane, and of glycerol diphosphatides (cardiolipin) in the inner membrane. However, as is also shown in the table, cardiolipin cannot be considered a specific mitochondrial component, since it is present also in other membranes of highly specialized tissues such as heart muscle. Another, frequently emphasized, feature of mitochondrial lipids is their apparent high degree of unsaturation. However, as shown in Table XVII, some care must be exercised in applying this property as a diagnostic characteristic: (1) Other membranes in the same cell may exhibit a very similar pattern, and (2) provided that membrane fluidity is preserved, the maintenance of a particular pattern is probably not required for mitochondrial function, since it can be manipulated almost at will and without any apparent ill effects by altering the lipid supplement for certain yeast auxotrophs (i.e., strains deficient in the enzymes responsible for the desaturation of fatty acids). TABLE XVII UNSATURATION PATTERNS OF VARIOUS MEMBRANE LIPIDS"~~ Fatty acid <16 16:O 16:l 18:O 18:l 18:2 18~3 20:4 Others

1 21.3 3.6 10.3 14.7 31.8 18.1

-

2 22.6 2.2 5.5 13.5 36.7 19.2

-

3

4

5

6

-

-

20.4 nd 22.3 11.6 16.3 24.7 -

26 nd 7.5 38 16

0.6 17.9 43.7 3.6 34.2 -

-

-

-

7

8

4.5 20.5 6.5 3.9 61.5 -

1.2 16.1 43.1 3.6 36.0 -

-

-

-

6

-

-

_

-

9

1

0

5.1 8.6 58.4 57.8 12.0 13.9 1.1 19.2 23.4 0.5 -

-

-

-

-

1

1

1.6 2.0 1.2 6.6 47.2 29.4 11.9

2.6 29.0 22 2.0 23.1 -

_

-

-

11.8'

All values as percent of total. 1, Rat microsomes, lecithins (Crane and Sun, 1971); 2, rat mitochondria, lecithins (Crane and Sun, 1971); 3, rat mitochondria, total lipids (Chapman and Leslie, 1970); 4, chick mitochondria, total lipids (Chapman and Leslie, 1970); 5, yeast mitochondria, aerobic, phospholipids (Paltauf and Schatz, 1969); 6, yeast mitochondria, anaerobic, phospholipids (Paltauf and Schatz, 1969); 7, yeast mitochondria, aerobic, neutral lipids (Paltauf and Schatz, 1969); 8, yeast mitochondria, aerobic, fatty acid desaturase mutant, supplemented with 18: 1 (Proudlock e t al., 1971); 9, yeast mitochondria, aerobic, fatty acid desaturase mutant, supplemented with 16: 1 (Proudlock et al., 1971); 10, Neurospora mitochondria (Keith et al., 1968); 11, Escherichia coli membranes (White et al., 1972). 17-Cyclopropane, 8.2; 19-cycIopropane, 3.6.

78

HENRY R. MAHLER AND RUDOLF A. RAFF

C. MATRIXFUNCTIONS The enzymic and other functional characteristics of the different compartments are summarized in Table XVIII. Although matrix proteins constitute indispensable and specific catalysts for key steps in cellular and mitochondrial metabolism, such as the tricarboxylic acid cycle, amino acid metabolism, and heme biosynthesis, several considerations suggest that they are probably no more suitable as indicators of direct evolutionary relationships than are soluble or easily solubilized proteins localized in other cellular compartments. Although they may or may not represent surviving primitive functions, retained ever since they first arose in the cytoplasm of the prokaryotic ancestors of the eukaryotic cell, localization in the matrix simply indicates that they have since become sequestered inside the mitochondria. Such proteins as cytochrome c and s u peroxide dismutase (see Section IV,A) are therefore subject to the same genetic, structural, and functional constraints governing the evolution of their primary sequences that all other proteins are, whether found in the same (e.g., L-malate dehydrogenase) or a different subcellular compartment. Conversely, in this view the conservation (or lack thereof) of their primary structure can shed no light on the evolutionary origin of the mitochondria. This point of view rests on the fact that matrix proteins are soluble; that their localization inside the mitochondrion is not invariant in all species (e.g., in s. cerevisiae, the “typical” mitochondrial enzymes L-glutamate dehydrogenase and aspartate transaminase are found in the cytosol-Hollenberg et al., 1970; Perlman and Mahler, 1970); and that, whenever this question has been analyzed, they have been found to be specified by nuclear genes, synthesized on ribosomes in the cytosol, and imported by the mitochondria (Table XVIII).

D. FUNCTIONS OF THE INNE R MEMBRANE The inner mitochondrial membrane, folded into its typical invaginations called cristae, and with its attached knoblike projections, provides the nonphotosynthetic eukaryotic cell with machinery for generation, conservation, and transduction of its respirationgenerated metabolic energy. I n its topology, and the attendant structural and functional organization, this membrane appears to be invariant and conserved from the simplest unicellular to the most highly differentiated forms. It is here therefore that evolutionary relationships and homologies between mitochondria and their presumed prokaryotic ancestors should become manifest. As shown in Fig. 4, inner membrane functions may be conve-

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

79

niently separated into two main classes: those belonging to (1) the respiratory or electron transport system proper, and ( 2 ) those concerned with the energy generation and transduction device. Although ordinarily tightly coupled, the two systems may b e structurally dissociated or functionally decoupled by appropriate manipulations (Racker, 1972; Kagawa, 1972; Harold, 1972). The respiratory system in turn may be further subdivided by analogous means into four segments or complexes (Hatefi et uZ., 1973): (1) the segment NADH + CoQ (ubiquinone); ( 2 ) the segment succinate (or other substrates oxidized by appropriate flavoproteins such as fatty acyl CoA’s, a-glycerophosphate, choline, etc.) + CoQ; (3) the segment CoQHz + cytochrome c; (4) the segment reduced cytochrome c + 0,. Of these, segments 1,3, and 4 are generally plugged into the energy-generating device and provide the three classic “sites of oxidative phosphorylation.” Segment 1 consists of a flavoprotein dehydrogenase, itself containing (nonheme) iron-sulfur centers, and an iron-sulfur protein portion (and perhaps a second flavoprotein). Segment 3 consists of cytochrome b, a nonheme iron-sulfur protein portion, and cytochrome cl, with the cytochrome b in this segment probably composed of two spectroscopically and functionally distinct entities, b,,, and b562, which may differ in their involvement in electron transport and energy generation and are themselves tightly associated with iron-sulfur centers. Segment 4, also known as cytochrome oxidase, consists of the two functionally and spectroscopically distinguishable cytochromes a and a3, as well as protein-bound copper ions in amounts stoichiometrically equivalent to the total heme present. The energy-transducing device consists of at least three operationally distinguishable parts (Senior, 1973; Mitchell, 1974):

1. The energy-generating device proper, composed of some of the proteins of the inner membrane in proper spatial juxtaposition. 2. A relatively easily detachable segment, morphologically equivalent to the knoblike projections, itself composed of three proteins. The first is concerned with the terminal step in energy generation, that is, ATP synthetase, catalyzing the strongly endergonic dehydration of ADP plus inorganic phosphate to yield ATP; this activity is usually and conveniently assayed in the reverse direction as mitochondrial, Mg2+-dependent ATPase, also called F, (for soluble coupling factor 1). F, when isolated in this soluble form is cold-labile and resistant to the characteristic antibiotic inhibitors oligomycin and rutamycin. It, like some of its bacterial counterparts (S. fuecalis,

TABLE XVIII DISTRIBUTIONOF MITOCHONDFUAL ACTIVITIES AMONG THE FOURCOMPARTMENTS Inner membrane-matrix

m 0

Outer membrane NADH-cytochrome b, reductaseb Monoamine oxidase

Kynurenine hydroxylase Fatty acid-activating enzymes (thiokinase)

Intermembrane space Adenylate kinaseb

Process Citric acid cycle

Citrate synthasec (M); fumarase, aconitase (M) Isocitrate dehydrogenase' (M); L-malate dehydrogenase' (M)

Nucleoside diphosphckinase Superoxide dismutase (cuprozinc type)

Succinate dehydrogenase (I) a-Ketoacid dehydrogenase system (M) Accessory systems: Amino acid metabolism Fatty acid degradation

Enzymes of (phospho) lipid metabolism

Marker"

Heme synthesis

L-Glutamate dehydrogenaseb (M); aspartate transaminases (M) Fatty acid oxidation systemsb (M); P-hydroxybutyrate dehydrogenase (I) 8-Aminolevulinate synthetase (M); ferrochelatase (I)

Electron transport (respiratory chain)

Energy transduction

Oxygen protection Specific phospholipids Semiautonomous replication; DNA replication and repair; RNA synthesis (transcription)

co

NADH, succinate dehydrogenases and cytochrome c reductase (I) (inhibited by antimycin A); cytochrome-c oxidase (I); FP;d CoQ;' ISC;' cytochrome b; cytochrome c,; cytochrome c; cytochrome aa3; Cu (all I) ATPase (F;); inhibited by oligomycin (I); OSCP (I); attachment site for F; (I); NADPH-NAD kanshydrogenase (I); transport system for cations and anions (I) Superoxide dismutase (M) (Mn type) Cardiolipin (I) CTP-phosphatidic acid cytidyl transferase (M) mtDNA, DNA polymerase(s); mtRNAs (see below); RNA polymerase; mitochondrial RNAs and mitochondrial ribosomes; mitochondrial tRNAs and amino acid ligases; transformylase and protein factors (IFs, EFs, TFs);m e t - t R N A and fMet-puromycin

Entities synthesized by mitochondrial genetic system are shown in italics. I, Inner membrane; M, matrix.

* May be absent from mitochondria of S. cereoisiae.

f

May also be present as a separate form-an Flavoprotein. Coenzyme Q, ubiquinone. Iron-sulfur centers.

iso(en)zyme-in

extramitochondrial compartments.

82

HENRY R. MAHLER AND RUDOLF A. RAFF

certain strains or preparations from E . coli-cf. Nieuwenhuis et al., 1973) is sensitive to the highly specific inhibitor Dio 9 (Guillory, 1964). The second is a polypeptide required for the attachment of F, to its appropriate site on the membrane, and thereby makes the enzyme sensitive to these inhibitors (oligomycin-sensitivity conferring protein, OSCP). The third is an inhibitor of the ATPase, itself a polypeptide of low molecular weight ( -lo4). 3. A group of proteins required for F1 attachment; they are sometimes referred to as membrane factors, the membrane sector, Fo, or CFo. Of them, one in particular, subunit 9 from yeast, has been implicated as a primary regulatory and attachment site (Sierra and Tzagoloff, 1973; Tzagoloff e t ul., 1974). It is this protein subunit that probably becomes inactivated by DCCD, an inhibitor of the complete ATPase complex of both mitochondria and certain bacteria such as S. fuecalis (Table V). In some instances, for example, S. cereuisiae or beef heart, the ATPase can be isolated and purified as a complex consisting of F,, OSCP, and attachment proteins (F1-CFo) (Table V). In such a complex, just as in its original membrane-bound version, the ATPase is cold-stable and oligomycin-sensitive. The precise relationship or overlap among the constituent proteins in segments 2 and 3 therefore still remains to be established, as does the actual molecular mechanism of the link between respiration and energy storage in the membrane. Whatever form this mechanism may take, it is clear that it must be accommodated within the framework of the scheme in Fig. 14, which diagrammatically summarizes contemporary knowledge concerning these processes. This scheme states: (1) Oxidation of a substrate at any one of the three sites can be used to drive a variety of endergonic processes, the catalysts or carriers of which are not only tightly membranelinked, but also require integrity and vectorial organization of the membrane for their proper function. Of these the generation of ATP is only one, albeit frequently the most important, manifestation of the transducing ability of the membrane. Alternative uses of the free energy liberated as a consequence of substrate oxidation involving active transport, that is, the accumulation of a variety of ionic species against a concentration gradient, are shown as well. (2) All these reactions are reversible in principle; this means that the various concentration gradients can be maintained at the expense of ATP hydrolysis, and that this hydrolysis or even transfer of ions down the gradient can b e used to drive electron transport in the reverse direction, that is, lead to the reduction of oxidized substrates, the so-

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

83

called reversed electron flow. (3) It is not certain whether the proton gradient shown, like the other gradients, is a product of the transducing device or-as demanded by the chemiosmotic hypothesis for energy generation-a prerequisite for its function (Harold, 1972; Mitchell, 1973a,b, 1974). (4) The directionalities indicated are those for intact mitochondria or for submitochondrial particles derived from them without topological inversion. Particles or vesicles exhibiting such an inversion with an “inside-out’’ orientation may also be obtained, for example, by sonication or mechanical breakage (Kagawa, 1972; Harmon et al., 1974). They exhibit a potential with the outside (the side exposed to the medium) negative, transport protons in and cations out, carry their dehydrogenases and ATPase projections on the outside, and are readily accessible to NADH but relatively inaccessible to cytochrome c added externally.

E. AUTONOMY IN BIOGENESIS Of the various functional segments of the membrane, only three are now believed to depend on polypeptides synthesized in (and probably specified by) the intramitochondrial system (Table XIV). Three polypeptides of cytochrome oxidase, possibly as many as four polypeptides of the attachment site for OSCP-F, ATPase, and at least one of the polypeptides in the cytochrome b part of the CoQ + c segment share in this property. However, except possibly for the last-mentioned protein, none of them appear to be concerned with the primary function of the enzyme complex in question; in cytochronie oxidase activity, heme and copper binding sites are carried b y the four smaller, imported polypeptides of this complex; and all the polypeptides of F, and OSCP are imported. The polypeptides synthesized locally appear to be principally involved in an integrative and regulatory function.

VIII. A Model for the Nonsymbiotic Origin of the Mitochondrion A. THE MODEL Contrary to the symbiotic model which assumes an anaerobic protoeukaryote that acquired a respiratory endosymbiont, we propose that the protoeukaryote was an advanced, aerobic cell rather larger in size than is typical for prokaryotes. Among the changes in cellular organization concomitant with the trend toward larger size (Stanier, 1970) was a large increase in respiratory membrane surface. This was

84

HENRY R. MAHLER AND RUDOLF A. RAFF

initially accomplished by invagination of the inner cell membrane (Fig. lSa), and later by formation of membrane-bound vesicles generated from the inner cell membrane (Fig. 15b and c). The respiratory organelles thus generated were topologically closed objects surrounded by a membrane providing a selective permeability barrier between the respiratory elements and the cytoplasm. This presuinably proved to be evolutionarily advantageous, and provided the basis for a more sophisticated regulation of respiratory metabolism (Hughes et al., 1970). However, the resulting segregation of the respiratory elements from the cytoplasm posed a problem to the cell, since certain constituents of the respiratory chain (e.g., subunits of cytochrome oxidase) apparently require synthesis in situ. While the membrane surrounding the respiratory elements was (like the present-day mitochondria1 membrane) permeable to many proteins, including cytochrome c and protein components of the respiratory and phosphorylation systems, it was impermeable to ribosomes or rRNA. Thus these respiratory organelles required constant de no00 replacement from the cell membrane. This continual turnover of the complex organelle would have been somewhat uneconomical. Thus a device for organelle maintenance based on the presence of a system for protein synthesis intrinsic to it, that was on the inside of the organelle, would have been selectively advantageous. The implantation of such an organellar genome seems at first sight to present a formidable problem. However, genetic systems exist in contemporary eukaryotic and prokaryotic organisms that suggest that this should have been entirely feasible. Our model proposes that the protoeukaryotic cell implanted a protein synthesis system in the respiratory organelle by the incorporation of a stable plasmid containing the appropriate genes for ribosomal components (Fig. 15). A particularly well-studied analogous process occurs in the generation of multiple nucleoli during amphibian oogenesis. In that process, tandem multiple replicates of the chromosomal rRNA genes are released as circles of DNA. These are packaged into free nucleoli which produce the large amounts of rRNA required b y the egg (Brown and Dawid, 1968; Brown and Blackler, 1972). The hypothetical respiratory organellar genome of the protoeukaryote may not of course have been generated by the same mechanism; nevertheless, it serves as a useful example. As pointed out in Section V,A,6, contemporary prokaryotes contain extrachromosomal genomes called plasmids, with characteristics that make them eminently suitable candidates to serve as the evolutionary progenitors of the mitochondrial genome. This aspect is discussed in Section VIII,B,2.

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

85

FIG. 15. A schematic representation of the origin of mitochondria from a simple prokaryotic respiratory organelle. The drawings present cross sections of hypothetical cells representing various evolutionary stages. Blocks on the membrane represent respiratory assemblies. (a) Section of protoeukaryote showing invaginated cell membrane possessing respiratory function. (b) As the protoeukaryote becomes large, a more extensive respiratory surface becomes necessary and is provided by the blebbing off of respiratory membranes from the cell membrane. (c) Topologically closed respiratory organelles generated by blebbing. (d) Establishment of a stable plasmid (schematically represented by a circle) containing genes for ribosomal components and some elements of the respiratory membrane. (e) The final step in the evolution of the mitochondrion is the later acquisition of an outer membrane. (From Raff and Mahler, 1972. Copyright 1972 by the American Association for the Advancement of Science.)

86

HENRY R. MAHLER AND RUDOLF A. RAFF

B. ELEMENTSOF

THE

MODEL

1. Organelles of Prokaryotes While the organelles of eukaryotic cells are more complex (and more thoroughly studied) than the organelles of prokaryotic cells, prokaryotes nonetheless contain several types of membrane-bound organelles. Among these are the chlorobium vesicles and thylakoids of photosynthetic bacteria and blue-green algae, the cristaelike membranes of some bacteria (Figs. 16 and 17), the gas vesicles of photo-

I_ 7

FIG. 16. Arrangements of intracytoplasmic menibrane structures (chromatophores or thylakoids) of photosynthetic bacteria. (1)Chlorobium vesicles (30 x 120 nm) in the green siilfiir bacteria Chlorobirim limicola, Chlorobium thiosrilfatophilum, Chlorobiuin phaeobacteroides, Chlorobium phaeovibrioides, Pelodictyon clothratiforme, Pelodictyon aggregatum, Chloropseudomoiias ethylicum, and Prosthecochloris aestuarii. ( 2 ) Vesicular membrane systems (diameter 30-80 nm) in cells of Rhodospirilluin rubrum, Rhodopseudomonas spheroides, Rhodopseudomonas cupszilatu, Chromatiurn (strain D ) , Chromatium okenii, Chromatium ioeissei, Thiospirillum jenense, and Thiocqisa roseopersicina. ( 3 ) Tubular membrane systems in cells o f Thiocapsa pfennigii (Thiococczis sp.). (4) Single, small, and irregular membrane invaginations in cells of Rhodopseudomonas gelatinosa and Rhodospirillum tenue. ( 5 ) Stacks of short double membranes bound to the cytoplasmic membrane of Rhodospirillum molischiunum. Rhodospirillum~rlvuni,Ectothiorhodospira, and Rhodospirillum photoinetricum. The membranes in the stacks form a sharp angle with the cytoplasmic membrane in Rhodospirihm molischianum and Rhodospirillum fulvirm, but not i n Ectothiorhodospira. (6) Parallel lamellae underlying and continuous with the cytoplasmic membrane, a partly branched and irregularly arranged structure found in

Rhodopseudomonas paliistris and Rhodopseudomonas acidophila. (7) Concentric layers of double membranes in cells of Rhodopseudomonas oannielii. (8) Granalike stacks of double membranes, partly bent parallel to the cytoplasmic membrane, found in cells of Rhodopseudomonas viridis. (From Oelze and Drews, 1972, with permission.)

MITOCHONDRIA AND T H E ORIGIN OF EUKARYOTES

87

FIG. 17. Complex internal membranes of bacteria. (a)Nitrosolohus multiformis, (b) Nitrococcus nobilis, (c) Nitrosocossus oceunus. (From Raff and Mahler, 1972. Copyright 1972 by the American Association for the Advancement of Science.) Original electron micrographs provided by Dr. S. Watson of the Woods Hole Oceanographic Institution.

synthetic bacteria, the carboxysomes of some chemiautotrophs, and the mesosomes of bacilli and other bacteria (Stanier, 1970; Hughes et al., 1970; Echlin, 1970a,b; Walsby, 1972; Shively et al., 1973). The functions of some of these membranous systems are problematical. However, the cell membrane of all bacteria contain the electron transport-oxidative phosphorylation systems of these organisms, and membrane-bound replication sites for the cell’s DNA. Thus the basic membranous equipment of the prokaryotic cell includes elements capable of evolutionary modification in the manner outlined in our model. An early step in this model is the production of internal membranes possessing stnictural and functional specialization different from that of the outer cell membrane (Figs. 16 and 17). The existence of such prokaryotic membrane differentiation is best exemplified by the membranes of photosynthetic bacteria (reviewed by Oelze and Drews, 1972). The bacterial photosynthetic apparatus is an integral part of the intracytoplasmic membrane system (Fig. 17). These membranes form under anaerobic light conditions and disappear when the cultures are made aerobic. Intracytoplasmic membranes probably originate as modified extensions of the cell membrane. Under conditions that induce this formation of intracytoplasmic membranes, there is an increase in their lipids and in the bacteriochlorophyll content. The resultant internal

88

HENRY R. MAHLER AND RUDOLF A. RAFF

membranes possess many of the components of their parental surface membrane but differ from it in phospholipid ratio and in certain proteins and in enzymic activities. The hypothetical scheme of Oelze and Drews for the reversible formation of intracytoplasmic membranes is presented in Fig. 18. By this scheme an accelerated rate of membrane synthesis relative to that of the cell wall causes an invagination of the membrane. This membrane becomes differentiated from the cell membrane by an active process of incorporation of the photosynthetic components into the developing membranes. The complex internal membranes of these bacteria possess knoblike structures like those found in the cristae of mitochondria (Fig. 19) (Bachop et al., 1972). In the reverse process, under conditions in which the intracytoplasmic membranes are not needed, synthesis of photosynthetic units ceases, although synthesis of other membrane components continues. This process results in the dilution of pho-

FIG. 18. Hypothetical scheme for the development of the photosynthetic active intracytoplasmic membrane system and its reversion in Rhodospirillum rubrum. n , represeiitative for all components essential for the formation of the photosynthetic a p p a r a t i i s ; l I , representative for the components of the cytoplasmic membrane of aerobically dark-grown cells without any photosynthetic activity. CW, Cell wall; CM, cytoplasmic membrane; ICM, intracytoplasmic membrane. (From Oelze and Drews, 1972, with permission.)

MITOCHONDRIA AND T H E ORIGIN OF EUKARYOTES

89

FIG.19. Characteristic knoblike projections of the interior (matrix) side of the mitochondrial inner membrane. These particles are now known to be composed of F,, the mitochondrial ATPase, and the stalks are probably composed of a single additional polypeptide, the OSCP (see Table V). Electron micrograph kindly provided by Dr. William Bachop, Clemson University, Clemson, South Carolina.

tosynthetic components and the disappearance of the intracytoplasmic membrane. Photosynthetic membranes are not the only specialized intracytoplasmic membranes in prokaryotes. Some of the most complex membranes in nonphotosynthetic bacteria are found in certain ammonia- and nitrite-oxidizing organisms such as those presented in Fig. 17. Two of these, Nitrosocystis and Nitrosolobus, oxidize animonia to nitrite and fix carbon dioxide as a carbon source (Watson et al., 1971; Watson and Remsen, 1970). Both have extensive internal membranes. Those of Nitrosolobus seem to indicate physiological as well as morphological partitioning, since glycogenlike material is confined to the outer compartments of the cell. Absorption specka indicate the presence of cytochromes b, c, and a,. The nitrite oxidizer Nitrococcus has intracytoplasmic membranes in the form of tubular invaginations of the cell membrane (Watson and Waterbury, 1971). These membranes appear, like those of photosynthetic bacteria, to have approximately 100-A particles similar to those of mitochondrial cristae (Fig. 19) (Remsen e t al., 1967). It is tempting to speculate that these complex membranous systems represent adaptations to the metabolic specializations of these organisms. They certainly imply a great evolutionary flexibility for prokaryotic membranes. It would be

90

HENRY R. MAHLER AND RUDOLF A. RAFF

of interest to find examples of prokaryotes with more advanced organelles such as diagramed in Fig. 16.

2. Plasmids A detailed comparison of plasmid and mtDNAs has been made in Section V,A,6. We thus summarize here only the properties of plasmids that support their evolutionary role as ancestors of the mitochondrial genome (Table XIX). Plasmids are similar in size to mtDNAs, and resemble the latter in being supercoiled circles subject to elimination by acridines and ethidium. Like the primary chromosomes, plasmids are capable of autonomous self-replication in the bacterial cell. This process involves both plasmid-linked and chromosome-linked genes, and may involve specific replication sites. Plasmids, however, contain not only genes required for their replication, but also a considerable variety of other genes as well. Of particular significance to our model is the capability of plasmids for direct genetic interaction with the principal chromosomes by means of physical integration into the latter. The integration process seems to involve insertion of plasmid DNA, by utilization of the cell's recombination enzymes, into a region of the chromosome possessing some homology with a region of the plasmid. Integration is TABLE XIX CHARACTERISTICS OF PLASMIDS" Characteristic Molecular weight Conforniation Distribution Amount of plasmid DNA per cell

Replication

Genoinic content of various plasmids

Effects of acriflavine and other acridines, ethidium bromide "

Observations

1.5 x 10s to 1.0 x lo8daltons Circular duplex, supercoiled Eubacteria, photosynthetic bacteria Varies with plasmid and host; may be 1 to 30 copies per cell, or up to 40% of total cell DNA Probable specific replication points on membrane; plasmid stability mutants linked with both plasmid and chromosome Replication genes, sex factor, colicinogenic Factors, variety of genes for antibiotic resistance, viwious genetic markers excised from bacterial chromosome (lac, gal, trp, cysB) Plasinid eliminated from host

See Raff and Mahler (1972) for references.

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

91

often reversible, and excision of the plasmid in some cases entails a concomitant excision of chromosomal genes, resulting in the generation of a novel plasmid (Fig. 20). These events are not particularly rare and in natural populations subject to particular selection pressures, plasmids bearing advantageous genes very quickly become evident. This property of plasmids has become medically significant, since bacteria carrying plasmids bearing different genes for resis-

0

FIG.20. Model for the reversible integration of a plasmid into a bacterial chromosome [model of Campbell, redrawn from Richmond (1970)l. (a) Plasmid (heavy line) and chromosome (thin line) surviving independently in the same cell. (b) Apposition of homologous regions of plasmids and chromosome followed by single crossover. (c) Integrated plasmid. (d) Apposition of homologous regions again followed by crossingover. (e) Plasmid excised with incorporation of part of the chromosome-carrying marker to yield a novel plasmid. By the proposed model such events occurred in the protoeukaryote, and the marker may have been, for example, a gene for rRNA. (From Raff and Mahler, 1972. Copyrighted 1972 by the American Association for the Advancement of Science.)

92

HENRY R. MAHLER AND RUDOLF A. RAFF

tance to a variety of antibiotics have been found. Since they can be transmitted from one bacterium to another, plasmids provide an evolutionary mechanism of great flexibility. Thus there is a continuous flow of genetic information between chromosomes and plasmids-even among different organisms. The incorporation of chromosomal genes into plasmids is not restricted to genes for antibiotic resistance or for certain metabolic enzymes, but also includes genes for rRNA and tRNA (Russell et al., 1970; Yu et al., 1970). Given a selective pressure on the protoeukaryote for the incorporation of a protein synthesis system into the primitive respiratory organelle, generation of a plasmid with the appropriate genes would have been an efficient way to do so by exploiting the cell’s already well-established plasmid-chromosome genetic interactions.

3. Role of Membrane-Bound Ribosomes An integral part of our model is the postulate that certain elements of the respiratory machinery of the protoeukaryote required synthesis in situ and thus, once a closed protomitochondrion evolved, an internal protein synthetic system was required. There are several lines of evidence which support this proposal. The strongest supporting data for our contention that in situ synthesis of certain components played a key role in mitochondrial origination comes from the nature of the products synthesized on mitochondrial ribosomes. As pointed out in Section VI,C,5, the proteins encoded by the mitochondrial genome and synthesized in the mitochondrion are few in number (< 10) and are restricted in their localization. Neupert and Ludwig (1971) and Bandlow (1972) showed that the components of the outer mitochondrial membrane are synthesized on cytoplasmic polyribosomes, and that the mitochondrial system contributes only to the inner membrane. These mitochondrial contributions are found in oligomycin-sensitive ATPase, cytochrome oxidase, and ubiquinone-cytochrome c reductase. All these products of mitochondrial protein synthesis are extremely hydrophobic in their solubility properties. Beattie et al. (1970)and Coote and Work (1971) had already shown that the products of mitochondrial protein synthesis in mammalian cells were highly insoluble and were associated with the cristae. Kadenbach and Hadvary (1973), working with rat liver mitochondria, found that the products of endogenous protein synthesis (i-e., sensitive to chloramphenicol and ethidium bromide, but insensitive to cycloheximide) were generally insoluble, but that about one-third of the products were soluble in chloroform-methanol (2: 1). Similar

MITOCHONDRIA AND THE ORIGIN OF EUJSARYOTES

93

results have also been reported by several other groups (Burke and Beattie, 1973, 1974; Kuzela et al., 1973). Ledermann and Attardi (1973) and Costantino and Attardi (1973) investigated the incorporation of various amino acids into the protein products of HeLa cell mitochondria and found them to be hydrophobic, several of them being soluble in chloroform-methanol. These properties were found to correlate with high levels of incorporation of hydrophobic amino acids. The proteins synthesized by the mitochondria of yeasts (Tzagoloff and Akai, 1972; Murray and Linnane, 1972; Mitchell and Rogers, 1974; Burke and Beattie, 1974) and Neurosporu (Lowe and Hallinan, 1973; Michel and Neupert, 1973; Kiintzel and Blossey, 1974) are likewise hydrophobic, and many of them are soluble in chloroform-methanol. The components of cytochrome oxidase synthesized inside the mitochondria of both these organisms are higher in apolar amino acids than the ones synthesized externally (Sebald et nl., 1973; Poyton and Schatz, 1975). The similarity of these results obtained with mitochondria of mammals and ascomycetes, organisms evolutionarily distant from each other, indicates that the peculiar properties of the proteins synthesized in the mitochondria are probably ancient, and critical to mitochondrial biogenesis and function. Further evidence for the evolutionary necessity of an internal mitochondrial protein synthetic system comes from the intimate association of mitochondrial ribosomes with the inner mitochondrial membrane. Cytoplasmic ribosomes occur in two forms, free and bound to membranes of the endoplasmic reticulum (see Rolleston, 1974, for a recent review). Membrane-bound ribosomes are particularly prevalent in cells that synthesize proteins for export (Goldberg and Green, 1964; Campbell, 1965; Redman et al., 1966; Redman and Sabatini, 1966; Palade, 1966; Redman, 1967, 1969; Takagi and Ogata, 1968; Kimmel, 1969). Free and membrane-bound ribosomes appear to b e engaged in the synthesis of different proteins. Thus in liver, serum albumin is synthesized on bound ribosomes, while ferritin is mainly a product of free polysomes (Ganoza and Williams, 1969; Hicks et al., 1969; Redman, 1969; Peters, 1962). Membrane-bound ribosomes are also present in nonsecretory cells (Rosbash and Penman, 1971; Andrews and Tata, 1971). Tata (1971) proposed that membrane-bound ribosomes may play a role in the topographical segregation within a cell of different groups of ribosomes synthesizing specific proteins. Tata (1971) also made the interesting suggestion that ribosomes on rough endoplasmic reticulum associated with mitochondria might be

94

HENRY R. MAHLER AND RUDOLF A. RAFF

synthesizing mitochondrial proteins not synthesized by the mitochondrial protein synthetic system. A mechanism whereby the in situ synthesis of membrane components might be accomplished by membrane-bound ribosomes has been suggested by Adelman et al. (1973). This group found that membrane-bound ribosomes can b e removed from microsomal membranes, without disrupting the membrane, by incubation with puromycin and high salt concentrations. Puromycin alone became covalently linked to nascent chains, but only in combination with high salt did it lead to their release, combined with detachment of ribosomes from the membrane. Incorporation studies with p ~ r o m y c i n - ~ H showed that the released nascent chains still remained bound to the membrane. These observations suggest that ribosomes are bound to membranes b y nascent chains and b y a salt-labile bond, and that proteins synthesized by these ribosomes are vectorially released into the membrane complex. Kellems et al. (1974a,b) found that about half of the puromycin-discharged nascent chains from cytoplasmic ribosomes attached to the outer surface of mitochondria similarly remain associated with the mitochondria. For this reason these investigators suggest that in fact both cytoplasmic ribosomes on the outer mitochondrial membrane and mitochondrial ribosomes attached to the inner membrane are involved in the synthesis of mitochondrial proteins in a bivectorial manner. Several lines of evidence show that mitochondrial ribosomes are bound to the inner membrane. Release of ribosomes from mitochondria requires the use of high salt concentrations and a detergent, which suggests membrane attachment (Linnane et al., 1972; H. 0. Halvorson, personal communication). Bunn et al. (1970) studied a cytoplasmically inherited yeast mutation which involves resistance to mikamycin and chloramphenicol in uiuo. Protein synthesis was, however, sensitive in in vitro preparations. Bunn et al. interpret these results to indicate that this was a mutation affecting membrane protein rather than a ribosomal protein. Towers et al. (1973) have reported that Arrhenius plots of protein synthesis by rat liver mitochondria show discontinuities at 23°C. This effect persists in sonicated mitochondria, but is abolished by treatment with digitonin or 2,4-dinitrophenol. Similar Arrhenius plots were obtained from chillsensitive plants, but no break was observed with chill-resistant plant mitochondria in which there was no lipid-phase change. Kuriyama and Luck (1973b), using the poky mutant of Neurosporu, have very convincingly demonstrated that functional mitochondrial ribosomes are membrane-bound. These mitochondria carry out protein synthe-

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

95

sis, albeit at a lower rate than those of wild type. The mitochondria of this mutant contain a mass ratio of large to small ribosomal subunits of 1O:l. Functional ribosomes, however, should have a 2 : l mass ratio of large to small subunits. Kuriyama and Luck sonicated mitochondria from the poky mutant, and found that most of the mitoribosomes were associated with the resultant membrane-bound vesicles. When these were subjected to puromycin plus high salt, the released ribosomal subunits were found to have a ratio of 2 : 1, as predicted for active ribosomes. Ribosomal subunits retained b y vesicles after this treatment are present in a ratio of 10: 1 (large to small), which probably represents adventitious entrapment. These results provide strong evidence for the direct attachment of active mitochondrial ribosomes to the inner membrane. Since nascent chains are involved in the binding of mitochondrial ribosomes to the membrane, it is very likely that they represent in situ synthesis of inner membrane components. Chloroplast ribosomes are also apparently bound to the thylakoids. Chua et al. (1973) have reported that membrane-bound chloroplast ribosomes are released by puromycin and high salt. The nature of the products of mitochondrial protein synthesis and the membrane-bound nature of mitochondrial ribosomes strongly suggest a requirement for in situ synthesis. The exact nature of this requirement is not yet known, but it is of interest, to note that specific protein-protein interactions between membranes and nascent chains of membrane-bound ribosomes have been described. A striking example is provided by Diegelmann et al. (1973), who prepared microsomes from chick embryo connective tissue. These membrane-bound ribosomes were found to carry out the cell-free synthesis of collagen, including hydroxylation of proline in the nascent chains. Polyribosomes released by detergent treatment of such preparations were still competent to synthesize collagen nascent chains, but proline in these chains was not hydroxylated. These results suggest that procollagen chains synthesized on membrane-bound polyribosomes enter the membrane in such a fashion as to interact with prolyl hydroxylase in the membrane. We know that the outer mitochondrial membrane is synthesized by cytoplasmic ribosomes translating mRNAs transcribed in the nucleus. The inner membrane is composed partly of nuclear-encoded proteins which are synthesized by ribosomes on the outer surface of the outer membrane and are then discharged into the membrane system, perhaps at sites of close apposition or fusion of its two (outer and inner) components (Kellems et nl., 1975). In addition, some proteins of the inner membrane are highly insoluble and must

96

HENRY R. MAHLER AND RUDOLF A. RAFF

be synthesized in situ simultaneously with their imported partners. Thus this small group of proteins required the evolution of an organellar genome and a system of protein synthesis. This system has apparently been in many respects evolutionarily conservative and retains many primitive (i.e,, prokaryotic) traits. However, its origin did not require a symbiotic event, since all the necessary genetic and biochemical mechanisms for the production of two divergent protein synthesis systems were already in existence in the ancestral protoeukaryote. 4. Genes f o r Expression of the Mitochondria1 Genome Mitochondrial systems for DNA replication, and RNA and protein synthesis are quite distinct from the corresponding nuclear-cytoplasmic systems. Yet with the exception of rRNAs and tRNAs, the components of these systems are almost exclusively encoded’ by nuclear genes (Table XX). This closely resembles the situation for most mitochondrial enzymes, including the highly conservative superoxide dismutase. According to the symbiotic model, this state of affairs arose through a transfer of genes from the endosymbiont to the nucleus. We, however, propose that the genes involved resided in the nucleus from the first, and that the plasmid contained only the minimal number of genes needed for its replication and function after its sequestration into a closed organelle. The most suggestive evidence comes from the peculiarly divergent properties of gene expression in mitochondria. As shown in Table IX, the transcriptive system of mitochondria has several features not found in either nuclear or bacterial transcription. Most strikingly, mitochondrial RNA polymerases are composed of a single polypeptide chain (MW 45,000-68,000) instead of being a large complex composed of several polypeptide chains, as are the polymerases of both the nucleus and bacteria. At least in fungi this unique polymerase is known to be encoded in the nucleus. The components of the mitochondrial protein synthesis system exhibit a mixture of conservative and divergent properties (Tables X to XI1 and XV, Figs. 9 and 10). Ribosome size and properties of the mitochondrial rRNAs are highly divergent from the prokaryotic pattern; significantly these divergent rRNAs are encoded in the mtDNA. However, ribosomal proteins and elongation factors are encoded in nuclear genes, yet the elongation factors are bacterialike and can substitute for bacterial factors with bacterial ribosomes. We suggest that this peculiar melange of prokaryotelike and uniquely mitochondrial properties is an evolutionary product of two

97

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

TABLE XX INTRACELLULAR LOCATIONOF GENES FOR PROTEINS OF MITOCHONDRIALDNA REPLICATIVE,RNA SYNTHETIC, AND PROTEIN SYNTHETIC SYSTEMS Protein DNA polymerase, mammalian, ascomycetes RNA polymerase Neurospora

Yeast HeLa Ribosomal proteins Neurospora Erythromycin resistance factor, yeast" Polypeptide chain-initiation factors, Neurosporu Polypeptide chain-elongation factors, yeast

Function and properties

Gene location

Polymerase distinct from nuclear enzyme

Nuclear

Rifamicin-sensitive and amanitininsensitive; therefore bacterialike; single polypeptide, 64,000 daltons (smallest known polymerasey Sensitive to ethidium bromide Symmetric transcription sensitive to ethidi um bromide' 30 in large subunit; 23 in small subunit; all distinct from cytoplasmic ribosome proteins Probably a modified RNA

Probably nuclealh

Bacteriali ke Factors required for mitochondrial protein synthesis can substitute for bacterial elongation factors with bacterial ribosomes

Nuclear

? Nuclear

Mitochondria1 Probably nuclear Nuclear

Bacterial polymerase consists of several polypeptide chains. stimulated by ethidium bromide (Barath an d Kiintzel, 1972). See Rafr and Mahler (1972) and text for references. This is unique; bacterial and nuclear transcription are asymmetric. " Also genes conferring resistance to other inhihitors of proteiil synthesis.

* Transcription

different inputs. First, both nuclear-cytoplasmic and nuclear-mitochondrial systems of interactions arose in an advanced prokaryotic cell, thus the divergent evolution of these two systems utilized the same starting material. In several respects the nuclear-mitochondria1 system has been the more conservative. Second, the regulatory requirements of the two systems are very likely different, and the simplest way to manage these controls is to utilize separate components. Thus there are different mitochondrial and nuclear polymerases, although the genes for both are probably nuclear. Precedent for this hypothesis comes from the situation in the nucleus in which there are separate nucleoplasmic and nucleolar polymerases with different transcriptional functions. Further, metabolic pathways in which synthetic and degradative pathways (e.g., for fatty acids) utilize similar reactions employ completely different sets of enzyme s.

98

HENRY R. MAHLER AND RUDOLF A. RAFF

IX. Conclusions and Predictions A. THE TWO THEORIES:CONTRASTAND OUTLOOK

The principal exponent of the endosymbiont hypothesis has been Margulis (1970), whose propositions we have criticized in this and in preceding articles (Raff and Mahler, 1972, 1975). However, our approach differs from the one espoused b y her in quite a fundamental fashion. It exhibits differences in philosophy, paradigm, and methodology, which have not really been discussed so far. This we now proceed to do. An analogous exposition from the point of view of the endosymbiotic theory has recently been published by Taylor (1974). We refer the reader to his article for the details of his presentation, but in brief his arguments for what he refers to as the serial endosymbiosis theory (SET) and against the model presented by us are: 1. The coherent and systematic view of the lower levels of biological organization and their interrelations during evolution provided by the SET. The three types of postulated prokaryotic ancestors-the first for the cell as a whole (host), the second for the protomitochondria, and the third for the protochloroplast-are considered basic units or “monads.” Fusion of the first two produces a “dyad,” and addition of the third a “triad,” exemplifying the unicellular eukaryotes; their polymerization into a multicellular state results in “polydyads” (metazoans) and “polytriads” (metaphytes), respectively. 2. The functional continuity of the endomembrane system of the eukaryotic cell, including the mitochondria1 outer membrane, results in the topological consequence that the inner membrane-matrix spaces of mitochondria (and chloroplasts) are really “outside” the cell proper. They are only “embraced” by it and thus can be regarded as retaining their ancestral status as permanent invaders or symbionts. 3. The existence of obligatory symbionts, some apparently intermediate in type between free-living forms and organelles, in contemporary eukaryotes. 4. The absence of precision and detail concerning some steps and entities in our model: the nature of the postulated prokaryotic ancestor, the “fortuity” of the capture of the right kind of plasmid b y the respiratory assembly, the uncertainty concerning the selective advantage conferred b y first segregating respiratory assemblies and then enclosing them in a second (outer) membrane and attaching them through it to the cellular cytomembrane systems, and so on.

MITOCHONDRIA A N D THE ORIGIN OF EUKARYOTES

99

5. the inadvisability of using biochemical criteria as phylogenetic markers, 6. Our unwillingness to provide a comprehensive theory which includes speculation not only with respect to the origin of mitochondria but to that of chloroplasts as well-considered to be “unrealistic” in this context. 7. The almost universal acceptance of the SET and its heuristic value.

The last item can be disposed of in short order. It can hardly be considered a scientific argument; the validity of scientific theories is not to be adjudicated on the basis of popularity contests, and the history of science is replete with examples in which unpopular, minority views-sometimes held originally with lethal consequences for their proponents-gained eventual acceptance as more representative models of physical reality. Conversely, examples are plentiful in which reliance on, or appeals to, a universally held doctrine have been used to cast into outer darkness apparently heretical views, and in consequence slowed or even halted scientific progress. It is precisely because of these considerations that we felt impelled to put an alternative view of the matter before the scientific community in the first place-not because we held it to be ‘‘more correct” than the endosymbiont theory, but because we had become convinced that the other side had not been adequately presented in the contemporary literature and that this absence of a dialogue and the resultant acceptance of one theory by default was unhealthy for scientific progress. Item 6 similarly cannot really withstand close logical scrutiny. We purposely focused our attention on the origin of the mitochondrion, based on our belief that currently the factual i n f o ~ a t i o available, n at least to us, permitted a clearer exposition of the evidence in this instance. This body of i n f o ~ a t i o nmight permit informed discussion of+nd perhaps might even result in a considered preference for-one of various opposing theories. In general, the more precise and restricted a hypothesis or model, the better the chance for the formulation of critical experiments designed to render its continued acceptance untenable-the only possible and defensible way of dealing with scientific hypotheses or theories (Platt, 1964). Conversely, the more comprehensive and all-inclusive a model, the more diffuse and difficult the marshaling of hard evidence for its elimination. As pointed out by Taylor himself, various theories are possible in which the origin of chloroplasts and mitochondria need not be parallel-in any event the steps leading to their evolution probably

100

HENRY R. MAHLER AND RUDOLF A. RAFF

did not take place concurrently. If some of the ideas presented here are directly applicable to chloroplasts-or, conversely, clearly are not-let those who are interested in the more general problem make whatever use appears appropriate. At this stage of development in the general problem area, and barring future evidence to the contrary, we believe that we are on reasonably firm ground in believing that evidence for or against a certain hypothesis for the origin of mitochondria need not necessarily apply with the same force to chloroplasts, and vice versa. As concerns item 5, we suspect it is largely a question of personal preference, predilection, and expertise. We agree that no theory should-or can-rest on a single line of evidence; in this spirit we have tried to adduce bits and pieces bearing on as many different aspects of the problem as we felt competent to provide. No doubt in the continuing dialogue and the resultant dialectic, which has been our aim to reinitiate, more will b e provided by many experts in as many areas. What we do wish to contend is that phylogenies based on biochemical interrelationships are at least equally as valid-and when diligently applied can provide quite detailed evidence and lead to results in accordance with-more classic approaches. Some examples have been provided in the body of this report, as have our attempts to utilize this approach to the problem of mitochondria1 evolution. Item 4 is a mixed bag. Some of it patently represents special pleading-such as the suggestion that one is obliged to make a detailed statement concerning the precise nature of the postulated bacterial protoeukaryotic ancestor to the extent of whether it is grampositive or not (extrapolating to the Precambrian) if one espouses one theory, but can remain silent with regard to the same question concerning the three different prokaryotic ancestors envisaged in the SET. If we are forced to make a choice, we would probably subscribe to the ancestor as some kind of Urulge (Klein and Cronquist, 1967)-see Table I1 and Fig. 1. Along somewhat the same line relatively little is gained b y the use of the pejorative conjunction of “ortuitous(1y) capture of a plasmid bearing precisely the right genes for the upproprinte protein synthesis.” None of the italicized words are ones employed by us. However, given the length of time, number of cells, frequency of reorganization of cytomembrane systems, and detachment of plasmids from the chromosome and their independent replication on the cell membrane, the particular combination of circumstances there envisaged is no more or less likely than thefortuitous endocytosis and capture of precisely the right precursor orga-

MITOCHONDRIA AND T H E ORIGIN OF EUKARYOTES

101

nisms to provide the appropriately useful potential endosymbiont for the right host in the right microenvironment. Incidentally, as concerns the latter, it is not incumbent on us to provide a solution for the logical dilemma facing adherents of the SET as to why a host already adapted to an aerobic microenvironment need necessarily rely on an endosymbiont, rather than on additional continuous evolutionary development, in order to utilize this environment at full efficiency. Furthermore, as concerns protein synthesis-and the systems catalyzing this essential step-one might inquire why in the SET it is considered to b e of selective advantage for just one of the three members of the postulated ancestral triad, namely the “host,” to have to undergo the transition from a “70s” (bacterial) to an “80s” (eukaryotic) type. After all, all three were of the former variety in the first instance, and since in the hypothesis its maintenance in the organelles was required to be accompanied by wholesale (all but the requisite RNAs) gene transfer to the host genophore, surely efficiency and control of this transfer and integration, as well as of its continued function, would have been much more facile if it involved homologous genes. To put the problem in another way: If, by the time of the establishment of the obligatory endosymbiosis, the host has already evolved an “eukaryotic” 80s system with all the requisite structural and regulatory genes, what is the justification for considering it still a prokaryote, and how in the absence of homologies can it integrate and regulate the incoming genes for the 70s system? If it has not reached this stage of evolution, what are the driving forces and constraints that lead to the evolution of parts of the nonintegrated genome into an 80s system, retaining the 70s pattern for another part? The question as to the selective advantages of a self-enclosed, and eventually partially self-replicating, respiratory organelle is of course an eminently valid one. It is Taylor in his commentary rather than ourselves who refers to it as a “metabolically specialized sac” and ascribes only “additional permeability control” to such an arrangement. In fact, the formation of cristae or thylakoidlike structures, sometimes combined with the budding off or blebbing of some of them into enclosed vesicles is-as we have mentioned-a device constantly employed by various species of contemporary prokaryotes in response to environmental stress. By this means the cell optimizes efficiency and regulation in its energy supply and transduction. All the potential pathways and advantages enumerated by Hall (1973) for mitochondria would already be present in the prokaryotic protoorganelle. It is only their further refinement and especially their

102

HENRY R. MAHLER AND RUDOLF A. RAFF

continued and efficient maintenance, synthesis, and integration-and the attendant advantages-that have led to their eventual fixation in the organelle. Items 3 and 1 require only brief comment, since they are only peripheral to our argument. The existence of cells containing a photosynthesizing inclusion apparently intermediate between chloroplasts and endosymbiotic blue-greens is indeed of utmost interest. As pointed out, if any of them should prove to be truly intermediate between pro- and eukaryotes in their organization, this might provide strong evidence against an SET for chloroplasts. However, they can tell us nothing about mitochondria. Nor can the very interesting-although generally unconfirmed-report of gene transfer from bacterial or plasmid to metazoan or metaphyte genomes, until more is known of the detailed structure of the genes responsible and their possible homologies with attachment sites between the two genomes. Finally, one might propose a single and straightforward, albeit biochemical, test to those who are interested in determining whether a given structure represents a degenerate blue-green symbiont or an emerging chloroplast (Sakano et al., 1974): Are both subunits of ribulose diphosphate carboxylase (“fraction I protein”) synthesized by, and thus susceptible to, inhibitors of 70s ribosomes or not? A similar test in the case of a presumptive mitochondria1 precursor could probably be provided by a comparison of two polypeptides in the Mg2+-requiring, DCCD-sensitive ATPase complex; one of the polypeptides interacting with the inhibitor (70s ribosomes), and one of the structural polypeptides of the ATPase itself (80s ribosomes). Finally, as concerns the question of elementary structural organization: although the origin of the dyads or triads, postulated in the theory (Taylor, 1974, Fig. l),is clearly different for alternate hypotheses, once formed and established, there is nothing that necessarily permits extrapolation backward to the nature of the ancestral monad(s) and the transition between them. These are and will remain the crux of the issue: One cell with one genome and its satellite plasmid, or a mosaic of a multiplicity of cells? We have left point 2 to the last, not only because it deserves most serious consideration, but also because some of the most damaging evidence against the SET emerges as a result of such a scrutiny. First, although the exposition includes the caveat that this view, which emerges as a logical consequence and extension of Robertson’s (1962, 1964) model of the cell, is to b e considered in dynamic rather than static terms, this structure is conveniently

MITOCHONDRIA AND T H E ORIGIN OF EUKARYOTES

103

forgotten when its consequences for the organelles are described (cf. Taylor, 1974, Fig. 2 , where there is nothing dynamic about the “outsidedness” of the mitochondrion and chloroplasts). But the milieu, which is itself in contact with the outside and within which these organelles are immersed, is composed of the endo(cyt0)membrane system of the cell, and w e know that structurally, functionally, and especially biosynthetically, this system is in continuous and dynamic equilibrium with soluble and small particulate elements of the cell sap. Second, the inner and the outer membranes of the mitochondria are not as completely independent of one another as they are made to appear in many, and particular in diagramatic, representations such as the above Fig. 2 (Taylor, 1974). Actually, in situ, there is good evidence for points of contact between them (Kellems et al., 1974a,b, 1975); and since all the proteins of the mitochondria1 matrix are provided b y cytoribosomes (probably attached to the outer membrane), such contacts may provide for the channels for their direct vectorial discharge from the site of their synthesis to that of their utilization. Third, there is no logical or mechanistic difference between the mode of biosynthesis of the bulk of the inner membrane-and in certain cases, such as anaerobic yeasts under glucose repression, and particularly in p- or po mutants under all growth conditions, that of the complete organelle-and that of uny other cell membrane; they are encoded in nuclear genes and synthesized on cytoribosomes and thus wholly beholden to the “host cell” and its “80s matrix” (Taylor, 1974). Fourth, as discussed earlier, prokaryotes themselves are, under appropriate circumstances, perfectly capable of “blebbing off” enclosed, functionally specialized vesicles (e.g., chromatophores), attaching them to these membranes, and then, under different physiological conditions, reabsorbing the whole cytomembrane system and reconverting it again to a simple plasmalemma with or without mesosomes. They are thus capable not only of bringing about the key steps postulated in the model, but of all the changes usually ascribed to the eukaryotic cell in its “Robertsonian” attributes. Fifth, there is no longer any obvious and fundamental difference either in topology or mode of biosynthesis of the mitochondrion on the one hand, and that of other organelles such as the nuclear and cytomembranes, as well as the mitotic apparatus, flagellae, and cilia on the other. The evolution of some or all of these organelles must have involved an event as discontinuous and sudden as has been postulated for the mitochondrion. Yet, at least Taylor (1974), although not Margulis (1970), now feels constrained to ascribe their origin to the

104

HENRY R. MAHLER AND RUDOLF A. RAFF

host, and even considers them representative characteristics of its ancestral functions. This reasoning, together with the one presented under point 4 above, might suggest to an unbiased observer that application of Occam’s razor renders unnecessary a separate set of postulates for one particular set of organelles-that it indeed constitutes, in Uzzell and Spolsky’s (1974) phrase, a resuscitation of “special creation.” The SET requires a long and continuous series of evolutionary events, subsequent to the initial engulfment of the future endosymbiont, during which there has taken place a stepwise abdication of biosynthetic capability of the latter, and a transfer of the genes responsible from its own to the nuclear genophore of the host. An examination of contemporary eukaryotes from the most primitive to the most complex discloses no evidence for such an evolutionary change. Instead there is observed a remarkable constancy of the qualitative nature of the major organellar gene products whether RNA or polypeptide. In the latter instance there is constancy also in their quantitative aspects of number, kind, and function; in the former with the rRNAs, there are indications, as we have shown, of considerable evolution-but only in size and composition, never in their localization on the mitochondria1 genophore.

B. VALIDITY

OF THE MOLECULARAPPROACH

Regardless of the mode of origin of the eukaryotic cell and its organelles, whether by the symbiotic association model or by the plasmid model, both the nuclear-cytoplasmic and nuclear-mitochondrial systems of the eukaryotic cell have undergone over a billion years of coevolution to their present forms. Because of the extreme antiquity of the events we are attempting to reconstruct, evidence relevant to the problem of the origin of eukaryotic organelles is difficult to obtain and even more difficult to interpret. The almost complete lack of a fossil record bearing on these events requires us to find our clues by unraveling the nature of the contemporary systems available to us. The sheer mass of morphological, ultrastructural, ecological, taxonomic, biochemical, and genetic data available are almost overwhelming, and must be culled and sifted through by anyone attempting to study the problem. Various workers who have done this have, not surprisingly, emphasized particular lines of evidence (depending on their predilections and disciplines) over others. Unfortunately, this has resulted in a somewhat disjointed debate, since most of the evidence presented in favor of the symbiotic hy-

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

105

pothesis is drawn from cytological studies of cells and from comparative studies of living intracellular symbionts, organelles, and freeliving prokaryotes (e.g., Margulis, 1970; Taylor, 1974), while we have concentrated on the molecular-biological aspects of the problem. While we agree that many lines of evidence have already proved valuable in arriving at an understanding of cellular evolution, we suggest that appropriate comparative molecular studies offer the greatest potential in comprehending the origin of eukaryotic cells and organelles. Several types of molecular data provide useful evolutionary evidence. These include protein sequences, metabolic pathways, nucleic acid hybridization, and physical parameters of macromolecules and supramolecular structures. The most developed of these for evolutionary studies is the use of protein sequences, and indeed, the potential power of the molecular approach may be illustrated by the construction of phylogenies based on amino acid sequence comparisons. A comparison of a classic and a molecular phylogeny of the kingdoms is diagramed in Fig. 21 (see also Fig. 1). The classic phylogeny follows the five-kingdom scheme of Whittaker (1969).The animal kingdom is further divided into the deuterostome superphylum and the protostome superphylum (Barnes, 1963). These are, respectively, labeled vertebrates and insects for their most familiar representatives. This diagram is based on the sum of the data conventionally used in the construction of phylogenies (morphology, biochemistry, embryology, fossil record, etc.). The molecular phylogeny (for cytochrome c) is based on a phylogeny drawn by Dayhoff (1972) from sequence data. The relationships of the kingdoms and of the animal superphyla based on protein sequence is essentially the same as that based on conventional data. Note that the cytochrome c phylogeny lacks the protozoa, since no protozoan cytochrome c had been sequenced at the time this phylogeny was drawn (Dayhoff, 1972). Since then sequences of cytochrome c from two protozoans, Crithidia and Euglena, have been published (Petigrew, 1972, 1973; Lin et al., 1973). These differ very strikingly from any other eukaryotic cytochrome c and, if they were positioned into the dendrogram, they would appear as an early offshoot of the eukaryotic stem. Three general classes of molecular consequences may be predicted to have arisen from the evolutionary acquisition of an organelle such as the mitochondrion. First are primitive features characteristic of the ancestral prokaryote, which have been retained by the organelle.

106

HENRY R. MAHLER AND RUDOLF A. RAFF

I I I

I

FIG. 21. Classic and molecular phylogenies of the kingdoms. (a) A classic phylogeny based on Whittaker’s (1969) five-kingdom scheme. (b) A phylogeny of cytochrome c. See text for details.

Second are features that may have been characterized by rapid rates of evolution early in the history of the organelle and low rates of evolution subsequently. Simpson (1953) has pointed out that evolution may occur at an accelerated pace during evolutionary transitions between major adaptive zones. The origin of eukaryotic cells clearly was such a major evolutionary step which may have required a relatively short span of time. Since the innovations were subcellular and biochemical, this transition may have had a high rate of molecular evolution followed by a slowing of rates. Evidence that such variations occur in molecular evolution is available. For instance, a-lactoglobulin apparently had an increased rate of evolution subsequent to its divergence from lysozyme (Dickerson and Geis, 1969; Dickerson, 1971), and the acquisition of families of redundant nucleotide sequences in eukaryotic genomes may have occurred by

MITOCHONDRIA AND T H E ORIGIN OF EUKARYOTES

107

saltatory events while subsequent sequence divergence has been gradual (Britten and Kohne, 1970). Third are features indicating organellar divergence. This type of evolutionary change has been discussed in detail by Lewontin (1970) who has pointed out that the various genomes of an eukaryotic cell represent different units of selection and thus may evolve in divergent fashions. Examples of all three categories appear to exist in the data we have gathered on mitochondria. Into the first category (retention of prokaryotic characteristics) fall such features as the amino acid sequence of mitochondrial superoxide dismutase and the inhibitor sensitivity spectrum of the mitochondrial ribosome. These features have been considered by some to constitute powerful arguments for the symbiotic theory but, as pointed out by Uzzell and Spolsky (1974), such conservative features only provide confirmation of the ultimate prokaryotic origin of eukaryotic systems and provide no proof of mode of origin. While up to now examples of such strongly conservative characteristics have been limited to comparisons of organellar and bacterial systems, recent evidence is emerging which suggests that equally conservative characteristics exist when comparisons are made between extramitochondrial systems and bacteria. Several examples have been presented above. A particularly striking case is provided ~ al. (1974) on cytochrome P450 (Section IV,E). by the studies of D u et Other mitochondrial characters such as the structure of mitochondrial DNA may be best explained by the plasmid hypothesis, since mitochondrial DNA strongly resembles bacterial plasmids. There are probably several mitochondrial features that meet the second category (initial high rate of evolution, followed by low rates). Perhaps the best example is a member of the electron transport chain, cytochrome c, which has evolved slowly in eukaryotes, but very likely had an initial high rate since eukaryotic cytochromes c differ greatly from the prokaryotic species (Dickerson, 1971;Ambler, 1973). The third category (divergent mitochondrial evolution) provides some bizarre examples which indicate that very specialized evolutionary trends (Fig. 22) have occurred in mitochondria-trends that we feel are not consistent with the symbiotic model. Two notable examples are mitochondrial RNA polymerase and the physical characteristics of mitochondrial ribosomes. Mitochondria1 polymerases resemble neither nuclear nor bacterial species (which in fact resemble each other in several significant respects). Mitoribosomes, which have been characterized as bacterial in type because of their similar

108

HENRY R. MAHLER AND RUDOLF A. RAFF

MODERN PROK ARYOTIC MITOCHONDRIAL

+

EUK ARYOTIC

CYTOPLASM

4NIMAL ASCOMYCETE

i

w

I

F

ANCESTRAL PROKARYOTIC

I

I

I

I

I

1

0

EVOLUTIONARY DISTANCE (arbitrary units)

FIG. 22. Evolution of ribosomes: a simplified phylogeny of mitochondrial, eukaryotic, and prokaryotic ribosomes. For simplicity, it is assumed that modern prokaryotic ribosomes are identical to those of Precambrian prokaryotes. Organellar (mitochondrial) and eukaryotic cytoplasmic ribosomes have diverged in various ways from the basic prokaryotic pattern. Mitochondria1 ribosomes have diverged less than cytoplasmic ribosomes, but show significant differences from prokaryotic ribosomes and from each other. (From Raff and Mahler, 1972. Copyright 1972 by the American Association for the Advancement of Science.)

inhibitor sensitivities, differ among themselves and from bacterial ribosomes in size, and differ greatly from bacterial ribosomes in composition of rRNA and in lacking a 5s RNA (which, incidentally, is found in cytoribosomes). The existence of examples of molecular species fitting these categories indicates that mitochondrial evolution has been perhaps more complex and interesting than anyone has suspected.

c.

MITOCHONDRIA AS

ASSEMBLIES is true for much of the contem-

SUPRAMOLECULAR

Implicit throughout this article-as porary research in the field-has been the view that the problem of mitochondrial biogenesis is to be regarded properly as a particularly complex and challenging instance of supramacromolecular assembly.

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

109

That is, it differs only in degree but not in kind from processes such as the assembly of ribosomes and viruses. The last comparison appears particularly apt, because it focuses attention on the most outstanding characteristics of these assembly processes, namely, their ordered and sequential nature involving the coordinated participation of two systems of gene expression. The construction of a fully functional mitochondrion, competent to perform its prime cellular role as the transducing device between oxidation and energy generation, probably requires the process discussed earlier: the well-regulated and simultaneous vectorial discharge of nascent polypeptide chains from two populations of polyribosomes into the membrane system of the organelle; one of the cytoplasmic variety attached to the outer membrane and translating mRNA molecules transcribed from nuclear DNA, and the other peculiar to the organelle and attached to its inner membrane, charged with the translation of mRNAs originally encoded in mtDNA. The two groups of polyribosomes appear restricted in their respective localization to specialized regions where inner and outer membrane are in close apposition or contact, and may even be subject to temporary fusion. Out of this integrated and binary system of assembly there then arise the products of this joint venture, which we have seen involve participation by both systems at the level of the individual protein (macromolecule), enzyme complex (macromolecular aggregate), membrane, and organelle (assembly of macromolecular aggregates). Two additional points require emphasis. Although full competence requires participation by both systems, this requirement is not absolute; they may be decoupled from one another, and it is the mitochondrial system that can be rendered dispensable either by the use of inhibitors or, most convincingly, in DNAo mutants of S . cerevisiae. In such organisms the nuclear-cytosolic system alone is capable of specifying and constructing mitochondria that are not only topologically identical and morphologically analogous to the entities of normal cells, but retain many of their functional attributes as well, such as the localization of the enzymes of the citric acid cycle, the key steps in heme synthesis, the ability for active transport of certain metabolites, and so on (see, e.g., Perlman and Mahler, 1970; Mahler, 1973a). Thus these organelles are fully competent for maintenance, growth, and duplication, and constitute the source of the appropriate scaffolding or framework for the insertion of the products of the intramitochondrial system which can then be regarded as a source for mitochondria1 differentiation (Perlman and Mahler, 1974). Finally, we include a word concerning the extension of this frame-

110

HENRY R. MAHLER AND RUDOLF A. RAFF

work, the processes involved in mitochondrial growth and divis i o n - o r more properly: increases and duplication of the mitochondrial mass. Recent studies have shown that, at least in some organisms such as S . cerevisiae, these events are asynchronous and occur continuously throughout the cell division cycle (Grimes et al., 1974). Furthermore, the total mitochondrial substance of a cell is either localized in very few organelles or, if present in multiple organelles, it is capable of rapid equilibration by a process of fusion and fission. Similarly, mitochondrial DNA is present in many copies (Grimes et al., 1974) which may replicate independently and asynchronously. Thus mitochondrial duplication can b e regarded as a continuing and continuous process of extension involving many separate regions within one cell. The relevance of these considerations to the problem of mitochondrial evolution is simply this. We believe that it will prove possible eventually to reconstruct the process required for niitochondrial biogenesis in vitro. This constitutes a reasonable expectation for a supermolecular assembly, no matter how complex, but hardly for an obligatory endosymbiont.

D. A SPECULATION:PROTOEUKARYOTES AS LIVING FOSSILS Contemporary eukaryotes are separated from prokaryotes b y a wide and apparently unbridgeable gulf. Yet, transitional forms, protoeukaryotes, must have existed during the mid- to late Precambrian. In most cases transitional forms (missing links) between major groups are supplanted by their descendants and become extinct. Occasionally, a fossil record of transitional forms persists, for example, the mammallike reptiles and the Archaeopteryx, which, respectively, illustrate the transition from reptile to mammal and from reptile to bird. But it is unlikely that any such record of the transition between eukaryotes and prokaryotes will ever b e recognized. However, we wish to suggest that it is possible that protoeukaryotes may not be completely extinct, but may in fact survive in specialized niches. Such organisms may have been easily overlooked by workers used to the characteristics of the more usual prokaryotes and eukaryotes. While this may, on the face of it, seem improbable, we can only point out that the discovery of living forms apparently identical to the 1.9billion-year-old Gunflint chert fossil Kakabekia umbellata is equally improbable. Kakabekin is one of the most bizarre of the Gunflint organisms: it resembles a tiny (5-30 Fm) umbrella (Fig. 23a). A very similar (Fig. 23b) apparently prokaryotic organism has been isolated from soil from Wales and Alaska. This organism is ammonophilic

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

111

FIG. 23. Living and fossil cells of Kakabekia. (a and b) Micrographs of fossil Kakabekia from the 2 x 1O8-year-oldGunflint chert of Canada. (c) Micrograph of a living cell of Kakahakia from Wales. Photographs (a and b) courtesy of Dr. E. S. Barghoom, and (c) courtesy of Dr. S. M. Siegel.

and aerotolerant (Siegel and Giumarri, 1966; S. M. Siegel and Siegel, 1968; B. Z. Siegel and Siegel, 1970). Of course this in itself does not guarantee the survival of protoeukaryotes to the present, but it does suggest the possibility. Such survivors, if they exist, would provide us with a unique view of the evolutionary transition fiom prokaryote to eukaryote. NOTE ADDED

IN

PROOF

Among the numerous recent reports dealing with topics covered in this review four sets are of particular relevance: i. The apparently rapid rate of evolution of mitochondrial genes has been further documented for rRNA cistrons in different yeasts [Groot, G. S. P., Flavell, R. A., and Sanders, J. P. M., Biochim. Biophys. Acta 378,186 (1975)], the leucyl-tRNA cistrons in a number of mammals, chick,and yeast [Jakovcic, S., Casey, J., and Rabinowitz, M., Biochemistry 14, 2037 (1975)], and bulk sequences for the same vertebrate species [Jakovcic, S., Casey, J., and Rabinowitz, M., BiochBmistry 14,2043 (1975)l. ii. Unlike all other systems studied so far, the mitochondrial rRNA cistrons of S. cerevisiue (and S. curlshergensis) are genetically unlinked [Faye et al. (1973); Faye, C., Kujawa, C., and Fukuhara, H., J. Mol. B i d . 88, 185 (1974)], and physically separated by more than 25 x lo3 base pairs [Sanders, J. P. M., Heyting, C., and Borst, P., Biochem. Biophys. Res. Commun. 65, 699 (1975)l. It is therefore highly unlikely that they form a single transcriptional unit and that the RNAs are derived from a common precursor-results now well documented for E . coli [Ginsburg, D.,and Steitz, J. A., J. B i d . Chem. 250, 5647 (1975)l. iii. Short (chain length 15-50 nucleotides), 3’-terminal poly(rA) sequences have now been identified on bacterial mRNA [Nakazato, H., Venkatesan, S., and Edmonds, M., Nature (London) 256, 144 (1975); Ohta, N., Sanders, M., and Newton, A., Proc. Nat.

112

HENRY R. MAHLER A N D RUDOLF A. RAFF

Acad. Sci. U . S. 72, 2343 (1975)l. It is therefore highly likely that there exists a continuously increasing, evolutionarily significant, gradient of sizes of such sequences between pro- and eukaryotes, and for different classes of messages. iv. The mtDNA of certain yeasts, such as Kluyoeromyces lactis, is considerably smaller (MW = 20 x lo6; length, 11.5 pm circles) than that of Saccharomyces, as is the mtDNA of Acanthanioeba castellanii [Bohnert, H. J. and Hermiann, R. G. Ezcr. J . Biochem. 50,83 (1974)l.Thus, there is reason to believe that the actual informational content of mtDNA has remained relatively invariant during evolution.

REFERENCES Adelman, M. R., Sabatini, D. D., and Blobel, G . (1973).J.Cell Biol. 56, 206. Adman, R., Schultz, L. D., and Hall, B. D. (1972). Proc. Nut. Acad. Sci. U.S. 69, 1702. Agsteribbe, E., Datema, R., and Kroon, A. M. (1974). I n “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 305-314. Academic Press, New York. Amaldi, F. (1969). Nature (London) 221, 95. Ambler, R. P. (1973). Syst. Zool. 22, 554. Andreason, A. A,, and Stier, T. J. B. (1953).J. Cell. Comp. Physiol. 41, 23. Andreason, A. A., and Stier, T. J. B. (1954).J.Cell. Comp. Physiol. 43, 271. Andrews, T. M . and Tata, J. R. (1971).Biochem. J. 121, 683. Attardi, B., and Attardi, G. (1971).J.Mol. Biol. 55, 231. Attardi, G., and Amaldi, F. (1970).Annu. Reo. Biochem. 39, 183. Attardi, G., Constantino, P., England, J., Lynch, D., Murphy, W., Ojala, D., Posakony, J., and Storrie, B. (1975).Colloy. Genet. Biogenesis Mitochondria Chloroplasts 1st Colunihus, 1974. Ohio State Univ. Press, Columbus. Avadhani, N. G., and Buetow, D. E. (1972). Biochem. J . 128,353. Avadhani, N. G., Battula, N., and Rutman, R. J. (1973).Biochemistry 12,4122. Avadhani, N. G., Lewis, F. S., and Rutman, R. J. (1974).Biochemistry 13, 4638. Avers, C. J. (1971). Sub-cell. Biochem. 1, 25. Awramik, S. M. (1971).Science 174, 825. Bachop, W., Boatman, E. S., Mackler, B., and Ladda, R. L. (1972). Trans. Anier. Micros. Sac. 91, 169. Bandlow, W. (1972).Biochim. Biophys. Acta 282, 105. Barath, Z., and Kiintzel, H. (1972). Proc. Nut. Acad. Sci. U.S. 69, 1371. Barghoorn, E. S., and Schopf, J. W. (1966). Science 152, 758. Barnes, R. D. (1963). “Invertebrate Zoology.” Saunders, Philadelphia, Pennsylvania. Bazaral, M., and Helinski, D. R. (1970a).Biochemistry 9, 399. Bazaral, M., and Helinski, D. R. (1970b).J.M o l . Biol. 36, 185. Beattie, I). W., Patton, G. M., and Stuchell, R. N. (1970). J . Biol. Chem. 245, 2177. Beaud, G., and Hayes, D. H. (1971). Eur. J . Biochem. 19,323. Bell, P. R., and Muhlethaler, K. (1962).J.Ultrastruct. Res. 7, 452. Belozersky, A. H., and Spirin, A. S. (1958). Nature (London) 182, 111. Bendich, A. J., and McCarthy, B. J. (1970). Proc. Nat. Acad. Sci. U.S. 65, 349. Benveniste, R., and Davies, J. (1973).Annu. Reu. Biochem. 42,471. Berk, A. J., and Clayton, D. A. (1974).J.Mol. Biol. 86, 801. Berthelot, F., Bogdanovsky, D., Schapira, G., and Gros, F. (1973).Mol. Cell Biochem. 1, 63. Bird, C. W., Lynch, J . M., Pirt, F. J., Reid, W. W., Brooks, C. 1. W., and Middleditch, B. S. (1971). Nature (London) 230, 473.

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

113

Blamire, J., Cryer, D. R., Finkelstein, D. B., and Marmur, J. (1972).J.Mol. Biol. 67, 11. Bleeg, H. S., Leth Bak, A,, Christiansen, C., Smith, K. E., and Stenderup, A. (1972). Biochem. Biophys. Res. Commun. 47,524. Bloch, K. (1962). Fed. Proc., Fed. Amer. Sue. E x p . B i d . 21, 1058. Bloch, K. (1969).Accounts Chem. Res. 2, 193. Bloch, K., Baronowsky, P., Goldfine, H., Lennarz, W. I., Light, R., Norris, A. T., and Schenerbrandt, G. (1961).Fed. Proc., Fed. Amer. Soc. E x p . Biol. 20,921. Bloomfield, D. K., and Bloch, K. (1960).]. B i d . Chem. 235, 337. Bodley, J. W., Zieve, F. J., and Lin, L. (1970).J.Biol. Chem. 245, 5662. Bolotin, M., Coen, D., Deutsch, J., Dujon, B., Netter, P., Petrochilo, E., and Slonimski, P. P. (1971). Bull. Inst. Pasteur, Paris 69, 215. Borst, P. (1972).Annu. Reo. Biochem. 41, 333. Borst, P. (1974).Biochem. Soc. Trans. 2, 182. Borst, P., and Grivell, L. A. (1971).F E B S (Fed. Eur. Biochern. Soc.), Lett. 13, 73. Bowman, C. M., Dahlberg, J . E., Ikemura, T., Konisky, J., and Nomura, M. (1971). Proc. Nut. Acad. Sci. U S . 68, 964. Bottomley, W., Smith, H. J., and Bogorad, L. (1971).Proc. Nut. Acad. Sci. U.S. 68,2412. Bourque, D. P., Boynton, J. E., and Gillam, N. W. (1971).J.Cell Sci. 8, 153. Bragg, P. D., and Hou, C. (1972).F E B S (Fed. Eur. Biochem. Soc.),Lett. 28, 309. Britten, R. J., and Kohne, D. E. (1970).Sci. Amer. 222,24. Brodie, A. F., and Gutnick, D. L. (1971).In “Electron and Coupled Energy Transfer in Biological Systems” (T. E. King and M. Klingenberg, eds.), Vol. 1, Part B, pp. 599-681. Dekker, New York. Brodie, A. F., Hirata, H., Asano, A., Cohen, N. S., Hinds, T. R., Aithal, H. N., and Kalra, V. K. (1972).In “Membrane Research” ( C . F. Fox, ed.), pp. 44-72, Academic Press, New York. Brown, D. D., and Blackler, A. W. (1972).J . Mol. B i d . 63, 75. Brown, D. D., and Dawid, I. B. (1968). Science 160, 272. Brown, D. D., and Weber, C. S. (1968).j.Mol. B i d . 34, 661, 681. Brown, D. D., Wensink, P. C., and Jordan, E. (1972).J.Mol. B i d . 63, 57. Brownlee, G. G., Sanger, F., and Barrell, B. G. (1968).J.Mol. Biol. 34, 379. Brownlee, G. G., Cartwright, E., McShane, T., and Williamson, R. (1972).FEBS (Fed. Eur. Biochem. Soc.), Lett. 25, 8. Bunn, C. L., Mitchell, C. H., Lukins, H. B., and Linnane, A. W. (1970). Proc. Nut. Acad. Sci. U S . 67, 1233. Burgess, R. R. (1971).Annu. Reo. Biochem. 40, 711. Burke, J. P., and Beattie, D. S. (1973). Biochem. Biophys. Res. Commun. 51, 349. Burke, J. P., and Beattie, D. S. (1974).Arch. Biochem. Biophys. 164, 1. Calvayrac, R., Butow, R. A,, and Lefort-Tran, M. (1971).Exp. Cell Res. 71, 422. Campbell, P. N. (1965). Progr. Biophys. 15, 3. Capaldi, R. A. (1973).Biochem. Biophys. Res. Commun. 53, 1331. Carnevali, F., Leoni, L., Morpurgo, G., and Conti, G. (1971). Mutut. Res. 12, 357. Catterall, W. A., and Pedersen, P. L. (1971).J.B i d . Chern. 246, 4987. Chamberlin, M. J. (1974).In “The Enzymes” (P. D. Boyer, ed.), Vol. 10, pp. 333-374. Chambon, P. (1974).In “The Enzymes” (P. D. Boyer, ed.), Vol. 10, pp. 261331. Academic Press, New York. Chapman, D., and Leslie, R. B. (1970).In “Membranes of Mitochondria and Chloroplasts” (E. Racker, ed.), pp. 91-126. Van Nostrand-Reinhold, New York. Chua, N.-H., Blobel, G., Siekevitz, P., and Palade, G. E. (1973).Proc. Nut. Acud. Sci. U . S . 70, 1554. Cihik, A., and Cerria, J. (1972). FEBS (Fed. Eur. Biochem. Soc.), Lett. 23, 271.

114

HENRY R. MAHLER AND RUDOLF A. RAFF

Clark-Walker, G. D., and Linnane, A. W. (1966).Biochem. Biophys. Res. Commtrn. 25, 8. Clewell, D. B., and Helinski, D. R. (1970). Biochem. Biophys. Res. Commun. 41, 150. Cloud, P. E., Jr. (1968a).In “Evolution and Environment” (E. T. Drake, ed.), pp. 1-72. Yale Univ. Press. New Haven, Connecticut. Cloud, P. E., Jr. (1968b). Science 160, 729. Cloud, P. E. (1974).Amer. Sci. 62, 54. Cloud, P. E., and Gibor, A. (1970).Sci. Amer. 223, 110. Cloud, P. E., Jr., Licari, G. R., Wright, L. A., and Troxel, B. W. (1969).Proc. Nat. Accid. Sci. U.S. 62, 623. Cobon, G. S., and Haslam, J. M. (1973).Biochem. Biophys. Res. Commun. 52,320. Cohen, S . E. (1970).Amer. Sci. 58, 281. Cohen, S . S . (1973).Amer. Sci. 61, 437. Cohen, S. N., and Miller, C. A. (1969).Nature (London)224, 1273. Cooper, C. S., and Avers, C. J. (1974). In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 28-03. Academic Press, New York. Coote, J. L., and Work, T. S. (1971). Eur. J. Biochem. 23, 564. Costantino, P., and Attardi, G. (1973). Proc. Nat. Acad. Sci. U S . 70, 1490. Cottrell, S., Rabinowitz, M., and Getz, G. S. (1973). Biochemistry 12,4374. Crane, F. L., and Sun, F. F. (1971). I n “Electron and Coupled Energy Transfer in Biological Systems” (T. S . King and M. Klingenberg, eds.), Val. 1, Part B, pp. 477-588. Marcel Dekker, New York. Croxford, N. J. W., Janecek, J., Muir, M. D., and Plumb, K. A. (1973).Nature (London) 245, 28. Curtiss, R. (1969).Annu. Reo. Microbiol. 23, 69. Dalgarno, L., and Shine, J. (1973).Nature (London), New B i d . 245, 261. Darnell, J. E., Jelinek, W. R., and Molley, G. R. (1973). Science 181, 1215. Dawid, I. B. (1972a).J.Mol. B i d . 63, 201. Dawid, I. B. (1972b). Deoelop. Biol. 29, 139. Dawid, I . B., and Blackler, A. W. (1972).Deuelop. B i d . 29, 152. Dawid, I. B., and Chase, J. W. (1972).J.Mol. Biol. 63, 217. Dawid, I . B., and Horak, I. (1972). Carnegie Znst. Wash., Yearb. 71, 22. Dawidowicz, K. (1972).Ph.D. Thesis, Indiana Univ. Dayhoff, M. 0. (1972). “Atlas of Protein Sequence and Structure,” Vol. 5. Nat. Biomed. Res. Found., Silver Spring, Maryland. DeDuve, C. (1969).Ann. N . Y . Acad. Sci. 168,369. DeDuve, C., and Baudhuin, P. (1966).Physiol. Reo. 46, 323. Denslow, N . D., and O’Brien, T. W. (1974). Biochem. Biophys. Res. Commnn. 57, 9. de Vries, H., Arendzen, A. J., and Kroon, A. M. (1973). Biochim. Biophys. Acta 331, 264. Dezelee, S., and Sentenac, A. (1973). Eur. J. Biochem. 34,41. Dickerson, R. E. (1971).J.Mol. Eool. 1, 26. Dickerson, R. E., and Geis, I. (1969). “The Sruchire and Action of Proteins.” Harper, New York. Diegelmann, R. F., Bernstein, L., and Peterkofsky, B. (1973).J.B i d . Chem. 248,6514. Dietrich, W. E., Jr., and Biggins, J. (1971).J.Bacteriol. 105, 1083. diMauro, E., Hollenberg, C. P., and Hall, B. D. (1972).Proc. Nat. Acad. Sci. U.S. 69, 2818. Diver, W. L. (1974).Nature (London) 247, 361. Diuon, H., Kellerman, G. H., Mitchell, C. H., Towers, N. H., and Linnane, A. W. (1971) Biochern. Biophys. Res. Commun. 43,780.

MITOCHONDRIA AND T H E ORIGIN OF EUKARYOTES

115

Dott, R. A., Jr., and Batten, R. L. (1971). “Evolution of the Earth.” McGraw-Hill, New York. Dubin, D. T. (1974).J . Mol. Biol. 84,257. Dubin, D. T., and Friend, D. A. (1972).j.Mol. Biol. 71, 163. Dubin, D. T., and Montenecourt, B. S. (1970).J.Mol. Biol. 48, 279. Dubin, D. T., Jones, T. H., and Cleaves, G. R. (1974). Biochem. Biophys. Res. Commztn. 56, 401. DuBuy, B., and Weissman, S. M. (1971).J.Biol. Chem. 246, 747. Diijon, B., Slonimski, P. P., and Weill, L. (1974).Genetics 78, 415. Dunn, J. J., and Studier, F. W. (1973).Proc. Nut. Acud. Sci. U S . 70, 3296. Dus, K., Litchfield, W. J., and Miguel, A. G. (1974). Biochem. Biophys. Res. Commun. 60, 15. Eccleshall, T. R., and Criddle, R. S. (1974).In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 31-46. Academic Press, New York. Echelin, P. (1970a).In “Phytocheniical Phylogeny” (J. B. Harborn, ed.), pp. 1-19. Academic Press, New York. Echelin, P. (1970b). I n “Organization and Control i n Prokaryotic and Eukaryotic Cells” (H. P. Charles and B. C. J. G . Knight, eds.), pp. 221-248. Cambridge Univ. Press, London antl New York. Edelnian, M., Verma, I. M., and Littauer, U. Z. (1970).J.Mol. B i d . 49, 67. Edelman, M., Vernia, I. M., Herzog, R., Galun, E., and Littauer, U. Z . (1971). Eur. J. Biocheni. 19, 372. Emerson, R., antl Held, A. A. (1969).Anier. J. Bot. 56, 1103. Erdmann, V. A., Fahnestock, S., Higo, K., and Nomura, M. (1971). Proc. Nut. Acad. Sci. U . S . 68, 2932. Fanica-Gaignier, M., and Clement-Metral, J. (19734.Enr. J. Biocheni. 40, 13. Fanica-Gaignier, M., and Clement-Metral, J. (1973b). Eur. J . Biochem. 40, 19. Farber, J. L., and Farmar, R. (1973). Biochem. Biophys. Res. Commun. 51, 626. Farron, F. (1970).Biochemistry 9, 3823. Faye, G., Fukuhara, H., Grandchamp, C., Lazowska, J., Michel, F., Casey, J., Getz, G. S., Locker, J., Rabinowitz, M., Bolotin-Fukuhara, M., Coen, D., Deutsch, J., Dujon, B., Netter, P., and Slonimski, P. P. (1973).Biochimie 55, 779. Fee, J. A,, and Teitelbaum, H. D. (1972). Biochem. Biophys. Res. Commun. 49, 150. Fellner, P., Ehreniann, C., Stiegler, P., and Ebel, J. P. (1972).Nature (London),N e w Biol. 239, 1. Firtel, R. A,, Jacobson, A,, and Lodish, H. F. (1972).Nature (London),New B i o l . 239, 225. Fischer, A. G. (1965). Proc. Nut. Acud. Sci. U.S. 53, 1205. Flavell, R. A,, Borst, P., and Ter Schegget, J. (1972).Biochim. Biophys. Actu 272, 341. Flavell, R. A., and Trampe, P. 0. (1973).Biochim. Biophys. Actu 308, 101. Forget, B. G., and Weissman, S . M. (1969).J.B i d . Chem. 244,3148. Forrester, I. T., Nagley, P., and Linnane, A. W. (1970). FEBS (Fed. Eur. Biochori. Soc.),L e t f . 11, 59. Freeman, K. B., Mitra, R. S., and Bartoov, B. (1973). Sub-Cell. Biochem. 2, 183. Freifelder, D. (1968).]. M o l . Biol. 35, 95. Fridovich, I. (1972).Accounts Chem. Res. 5, 321. Fridovich, I. (1974).Life Sci. 14, 819. Fukuhara, H. (1970). Mol. Gen. Genet. 107, 58. Fnkuhara, H., and Kujawa, C. (1970). Biocheni. Biophys. Res. Commun. 41, 1002. Ganoza, M. C., and Williams, C. A. (1969).Proc. N o t . Acud. Sci. U . S . 63, 1370. Garrett, P. (1970).Science 169, 171.

116

HENRY R. MAHLER AND RUDOLF A. RAFF

Gingold, E. G., Saunders, G. W., Lukins, H. B., and Linnane, A. W. (1969). Genetics 62, 735. Gissinger, F., and Chambon, P. (1972). Eur. J. Biochem. 28, 277. Glaessner, M. F. (1962). B i d . Reu. Cumbridge Phil. Soc. 37, 467. Glaessner, M. F. (1966). Earth Sci. Reo. 1, 29. Glaessner, M. F. (1968). Can. J. Earth Sci. 5, 585. Glaessner, M. F. (1971). Ceol. Soc. Amer. Bull. 82,509. Goebel, W. (1973). Angew. Chem. 12, 517. Goebel, W., and Sclirempf, H. (1973). Nature (London),New B i d . 245, 39. Goldberg, B., and Green, H. (1964).J. Cell Biol. 22, 227. Goldfine, H., and Bloch, K. (1963). In “Control Mechanisms in Respiration and Fermentation” (B. Wright, ed.), pp. 81-103. Ronald, New York. Golubic, S. (1973). In “The Biology of the Blue-Green Algae” (N. G. Carr and R . A. Whittaker, eds.), pp. 434-472. Blackwell, Oxford. Goscin, S. A., and Fridovich, I. (1972). Biochitn. Biophys. Acto 289, 276. Granboulan, N., and Schemer, K. (1969). Eur. J. Biochem. 9, 1. Grandi, M., Helms, A., and Kiintzel, H. (1971).Biocheni. Biophys. Res. Commtin. 44, 864. Gray, P. N., and Attardi, G. (1973). Anier. Soc. Cell B i d . , 13th Meet. 120a (Abstr.). Gray, P. N., and Monier, R. (1971). F E B S (Fed. Eur. Biochem. Soc.), Lett. 18, 145. Gray, P. N . , Bellemare, G., Monier, R., Garrett, R. A., and Stoffler, G. (1973). J. M o l . Biol. 77, 133. Gregory, E. M., and Fridovich, I. (1973).J. Bucteriol. 114, 1193. Gregory, E. M., Yost, F. J., Jr., and Fridovich, I. (1973).J. Bucteriol. 115, 987. Gregory, E. M., Goscin, S. A,, and Fridovich, I. (1974).J. Bacteriol. 117, 456. Griffiths, D. E., and Houghton, R. L. (1974). Eur. J. Biochem. 46, 157. Grimes, G. W., Mahler, H. R., and Perlman, P. S. (1974).J. Cell B i d . 61, 565. Grivell, L. A., and Metz, V. (1973). Biochem. Biophys. Res. Commun. 55, 125. Grivell, L. A., and Walg, H. L. (1972). Bioclzem. Biophys. Res. Commun. 49, 1452. Grivell, L. A., Netter, P., Borst, P., and Slonimski, P. P. (1973). Biochim. Biophys. Acta 312, 358. Groot, P. H. E., Aaij, C., and Borst, P. (1970). Biochem. Biophys. Res. Conmuti. 41, 1321. Groot, G. S. P., Flavell, R. A., Van Ommen, G . J. B., and Grivell, L. A. (1974). Nature (London) 252, 167. Grossman, L. I., and Watson, R., and Vinograd, J. (1973). Fed. Proc., Fed. Amer, Soc. E r p . B i d . Abstr. No. 1747, p. 529. Guiard, B., and Lederer, F. (1973). Int. Congr. Biochem., Stockholm, Proc. 9th, Abstr. No. 2n7, p. 92. Cuillory, R. J. (1964). Biochini. BiopAys. Acta 89, 197. Halbreich, A., and Rabinowitz, M. (1971). Proc. Nut. Acud. Sci. U.S. 68, 294. Hall, J. B. (1973).J. Theor. BioZ. 38, 413. Harmon, H. J., Hall, J. D., and Crane, F. L. (1974). Biochim. Biophys. Acta 344, 119. Harold, F. M. (1972). Bucteriol. Reo. 36, 172. Hartmann, G., Honikel, K. O., Kniisel, F., and Niiesch, J. (1967). Biochim. Biophys. Acta 145, 843. Haselkorn, R., and Rothman-Denes, L. B. (1973). Annu. Reo. Biochem. 42, 397. Hatefi, Y., Hanstein, W. G., Davis, K. A,, and You, K. S. (1973). Ann. N.Y. Acud. S c i . 227, 504. Hayaishi, O., and Nozaki, M. (1969). Science 164, 389. Held, A. A. (1970). Mycologin 62, 339.

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

117

Helser, T. L., Davies, J. E., and Dahlberg, J. E. (1971).Nature (London),New Biol. 233, 12. Henning, R., Kaulin, H. D., and Stoffel, W. (1970). Hoppe-Seyler’s Z. Physiol. Chem. 351, 1191. Hicks, S. J., Drysdale, J. W., and Munro, H. N. (1969).Science 164, 584. Highland, J. H., Lin, L., and Bodley, J. W. (1971). Biochemistry 10,4404. Hindley, J., and Page, S. M. (1972).F E B S (Fed. Eur. Biochem. SOC.), Lett. 26, 157. Hinkle, D. C., Mangel, W. F., and Chamberlin, M. J. (1972).J.Mol. Biol. 70, 209. Hirsch, M., and Penman, S. (1973).J.Mol. Biol. 80, 379. Hirsch, M., Spradling, A., and Penman, S. (1974).Cell 1, 31. Hochachka, P. W., and Mustafa, T. (1973).Amer. Zool. 13, 543. Hoffman, H. P., and Avers, C. S. (1973).Science 181, 749. Hollenberg, C. P., Borst, P., and van Bruggen, E. F. J. (1970).Biochim. Biophys. Actu 209, 1. Holloway, P. W., and Wakil, S. J. (1970).J.Biol. Chem. 245, 1862. Horak, I., and Dawid, I. B. (1972). Carnegie Inst. Wash., Yearb. 71,24. Horio, T., and Kamen, M. D. (1970).Annu. Reo. Microbiol. 24, 399. Horne, J. R., and Erdmann, V. A. (1973). Proc. Nat. Acad. Sci. U.S. 70, 2870. Howell, D. G. (1971).J.Poleontol. 45,48. Hrycay, E. G., and O’Brien, P. J. (1974).Arch.Biochem. Biophys. 160,230. Huang, M., Biggs, R. D., Clark-Walker, G. D., and Linnane, A. W. (1966). Biochim. Bioplzys. Acta 114, 434. Hughes, D. E., Lloyd, D., and Brightwell, R. (1970).In “Organization and Control i n Prokaryotic and Eukaryotic Cells” (H. P. Charles and B. C. J. G . Knight, eds.), pp. 29-22. Cambridge Univ. Press, London and New York. Hungate, R. E. (1966). “The Rumen and Its Microbes.” Academic Press, New York. Hurley, P. M., Pinson, W. H., Nagy, B., and Teska, T. M. (1972).Earth Planet. Sci. Lett. 14, 360. Ikeda, H., and Tomizawa, J.-I. (1968).Cold Spring Harbor Symp. Quant. Biol. 33, 791. Inselburg, J., and Fuke, M. (1971). Science 169, 590. Jacob, S. T., Sajdal, E. M., and Munra, H. N. (1970). Biochem. Biojhys. Res. Comnzun. 38, 765. Jacob, S. T., Schindler, D. G., and Morris, H. P. (1972).Science 178, 639. Jakovcic, S., Getz, G. S . , Rabinowitz, M., Jakob, H., and Swift, H. (1971).J.Cell Biol. 48, 490. John, P., and Whatley, F. R. (1975).Nuture 254,495. Jukes, T. H., and Holmquist, R. (1972a).J.Mol. Biol. 64, 163. Jukes, T. H., and Holmquist, R. (1972b). Science 177, 530. Kadenbach, B., and Hadvary, P. (1973). Eur. J . Biochem. 32, 343. Kagawa, Y. (1972).Bioclzim. Bioplzys. Acta 265, 297. Kedinger, C., and Chambon, P. (1972).Eur. I . Biochem. 28, 283. Kedinger, C., Gniazdowski, M., Mandel, J.-L., Gissinger, F., and Chambon, P. (1970). Biochem. Biophys. Res. Comniun. 38, 165. Kedinger, C., Gissinyer, F., Gniazdowski, M., Mandel, J.-L., and Chambon, P. (1972). Eur. J . Biochem. 28, 269. Keele, B. B., Jr., McCord, J. M., and Fridovich, I. (1970).J.B i d . Chem. 245, 6176. Keele, B. B., Jr., hlcCorc1, J. M., and Fridovich, I. (1971).J . B i d . Clzem. 246, 2875. Keenan, T. W., and Morre, D. J. (1970).Biocheniistry 9, 19. Keith, A. D., Waggoner, A. S . , and Griffith, 0. H. (1968).Proc. Nut. Acod. Sci. U.S. 61, 819. Keith, A. D., Wisnieski, B., Henry, S., and Williams, J. C. (1972). I n “Biological

118

HENRY R. MAHLER AND RUDOLF A. RAFF

Membranes of Eucaryotic Microbes” (J. Erwin, ed.), pp. 259-321. Academic Press, New York. Kellems, R. E., Allison, V. F., and Butow, R. A. (1974a). In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 511-551. Academic Press, New York. Kellems, R. E., Allison, V. F., and Butow, R. A. (197413).J. B i d . Chem. 249, 3297. Kellenis, H.E., Allison, V. F., and Butow, R. A. (1975)J.Cell Biol. 65, 1. Kenyon, D. H., and Steinman, G . (1969).“Biochemical Predestination.” McGraw-Hill, New York. Kimmel, C. B. (1969).Biochim. Biophys. Actu 182, 361. Kimura, M . , and Ohta, T. (1973).Nature (London),New Biol. 243, 199. Klagsbrun, M. (1973).J.B i d . Clzem. 248, 2612. Klein, R., and Cronquist, N. (1967). Quart. Reo. B i d . 43, 205. Kleinig, H. (1970).]. Cell Biol. 46, 396. Kline, B. C. (1974).Biochemistry 13, 139. Knowles, A. F., and Penefsky, H. S. (1972).J.B i d . Chem. 247, 6617. Koike, K., and Kobayashi, M. (1973). Biochim. Biophys. Actu 324,452. Kokileva, L., Mladenova, I., and Tsanev, R. (1971).F E B S (Fed. Ettr. Biochem. Soc.), Lett. 16, 17. Kozlov, I . A., and Mikelsaar, H. N. (1974).F E B S (Fed. Eitr. Biochem. Soc.),Lett. 43, 212. Kroon, A. M., Agsteribbe, E., and d e Vries, H. (1972). I n “The Mechanism of Protein Synthesis and Its Regulation” (L. Bosch, ed.), pp. 539-582. North-Holland Piil>l., Amsterdam. Kroon, A. M., Arendzen, A. J., and d e Vries, H. (1974). In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 395-402. Academic Press, New York. Knhuyaina, M., Yong, F. C., and King. T. E. (1972).]. B i d . Chem. 247, 6375. Kiintzel, H. (1971).Curr. Top. Microhiol. Immunol. 54, 92-120. Kiintzel, H., and Blossey, H. C. (1974).Eur. J . Biochem. 47, 165. Kiintzel, H., and Schifer, K. P. (1971).Nature (London),New B i d . 231, 265. Kiintzel. H . , Bmxtli, Z.. A l i , I., Kind, J., Althaiis, H.-H., and Blossey, H. C. (19731,).ZII U.S. 70, 1574. Kiintzel, € l . ,Baratlt, Z., Ali, I., Kind, J., Althaus, H.-H., and Blossey, H. C. (19731)). f i t “Regulation of Transcription and Translation in Enkaryotes” ( E . K. F. Bautz, P. Karlson, and H. Kersten, eds.), pp. 195-210. Springer-Verlag, Berlin and New York. Kiintzel, H., Ah, I., and Blossey, H. C. (1974).In “The Biogenesis of Mitochondria” (A. M. G o o n and C. Saccone, eds.), pp. 71-78. Academic Press, New York. Kupersztoch, Y. M., and Helinski, D. R. (1973).Biochem. B i o p h y s . Res. Cornniun. 54, 1451. Kuriyanxi, Y., and Luck, D. J. L. (1973a).J.MoZ. B i d . 73, 425. Knriyama, Y., and Luck, D. J. L. (1973b).J. Cell B i d . 59, 776. Kuriyania, Y., and Lnck, D. J. L. (1974a).J.Mol. Biol. 83, 253. Kuriyam;i, Y., and Lnck, D. J. L. (1974b). In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 117-133. Academic Press, New York. Kiizela, S . , Knlarov, J., and Krempasky, V. (1973). Biochem. B i o p h ! / s . Res. Cornmrrti. 54, 9. Lai, C. J., and Weisl)lum, B. (1971).Proc. Nut. Acad. Sci. U S . 68, 856. Lava-Sanchez, P. A., Amaldi, F., and LaPosta, A. (1972).J.Mol. Eool. 2, 44.

MITOCHONDRIA AND THE OFUCIN OF EUKARYOTES

119

Lavelle, F., Michelson, A. M., and Dimitrijeruc, L. (1973). Biochem. Biophys. Res. Commun. 55,350. Lederman, M., and Attardi, G. (1973).J. Mol. Biol. 78, 275. Lee, S. G., and Evans, W. R. (1971).Science 173, 241. Levin, W., Ryan, D., West, S., and Lu, A. Y. H. (1974).J. Biol. Cheni. 249, 1747. Lewontin, R. C. (1970).Annu. Reu. Ecol. Syst. 1, 1. Licari, G . R., and Cloud, P. (1972).Proc. Nat. Acud. Sci. U.S. 69, 2500. Lien, S., and Gest, H. (1973).Arch. Biochem. Biophys. 159, 730. Lin, D. K., Niece, R. L., and Fitch, W. M. (1973).Nature (London)241, 533. Lindell, T. J., Weinberg, F., Morris, P. W., and Roeder, R. G . (1970). Science 170,447. Linnane, A. W., Haslam, J. M., Lukins, H. B., and Nagley, P. (1972). Annu. Reo. Microhiol. 26, 163. Linnane, A. W., Gobon, G . , and Marzuki, S. (1973). Znt. Spec. S y m p Yeasts, 3rd, Part 11, pp. 34S367. Otaniemi, Helsinki. Lizardi, P. M., and Luck, D. J. L. (1971). Nature (London),New Biol. 229, 140. Loening, U. E. (1968).J. M o l . Biol. 38, 355. Loening, U. E. (1970). In “Organization and Control in Prokaryotic and Eukaryotic Cells” (H. P. Charles and B. C. J. G . Knight, eds.), Cambridge Univ. Press, London and New York. Loening, U. E., and Ingle, J. (1967).Nature (London)215, 363. Logan, B. W. (196U.J.Geol. 69, 517. Logan, B. W., Rezak, R., and Ginsburg, R. N. (1964).J.Geol. 72,68. Lowe, D., and Hallinan, T. (1973).Biochem. J. 136,825. Lowry, C. V., and Dahlberg, J. E. (1971). Nature (London),New Biol. 232, 52. Lu, A. Y, H., and Levin, W. (1974). Biochim. Biophys. Acta 344, 205. Lukins, H. B., Tate, J. R., Saunders, G . W., and Linnane, A. W. (1973). M o l . Gen. Genet. 120, 17. McConkey, E. H. (1974).Proc. Nat. Acud. Sci. U.S. 71, 1379. McConkey, E. H., and Hopkins, J. W. (1969).J.Mol. Biol. 39, 545. McCord, J. M., Keele, B. B., Jr., and Fridovich, I. (1971).Proc. Nut. Acad. Sci. U.S. 68, 1024. McLaughlin, P. J., and Dayhoff, M. 0. (1970). Science 168, 1469. McMurray, W. C., and Dawson, R. M. C. (1969). Biochem. J. 112,91. Mahler, H. R. (1973a).CRC Crit. Rev. Biochem. 1, 381. Mahler, H. R. (1973b).J.Suprumol. Struct. 1, 449. Mahler, H. R., and Bastos, R. N. (1974a).FEBS (Fed. Eur. Biochem. Soc.),Lett. 39, 27. Mahler, H. R., and Bastos, R. N. (1974b). Proc. Nat. Acad. Sci. U.S. 71, 2241. Mahler, H. R., and Dawidowicz, K. (1973). Proc. Nat. Acad. Sci. U.S. 70, 111. Mahler, H. R., and Perlman, P. S. (1973).Mol. Gen. Genet. 121, 285. Malloy, P. L., Howell, N., Plummer, D. T., Linnane, A. W., and Lukins, H. B. (1973). Biochem. Biophys. Res. Conimun. 52, 9. Margulis, L. (1970).“Origin of Eukaryotic Cells.” Yale Univ. Press, New Haven, Connecticut. Margulis, L. (1971).Sci. Amer. 225,48. Marrs, B., and Gest, H. (1973).J.Bacteriol. 114, 1052. Marrs, B., Stahl, C. L., Lien, S., and Gest, H. (1972). Proc. Nat. Acad. Sci. U.S. 69, 916. Matsunioto, L., Kasamatsu, H., Piko, L., and Vinograd, J. (1974).J. Cell Biol. 63, 146. Meilhac, M., Tysper, Z., and Chambon, P. (1972).Eccr. J. Biochem. 28, 291. Melandri, B. A., Baccarini-Melandri, A., San Pietro, A., and Gest, H. (1971). Science 174, 514.

120

HENRY R. MAHLER AND RUDOLF A. RAFF

Meyer, R. R. (1973).J.Theor. Biol. 38,647. Meyer, R. R., Probst, G . S., and Keller, S. J. (1972).Arch. Biochem. Biophys. 148, 425, Michel, R., and Neupert, W. (1973). Eur. J . Biochem. 36, 53. Michel, R., and Neupert, W. (1974). In “The Biogenesis of Mitochondria” (A. M , Kroon and C. Saccone, eds.), pp. 315-326. Academic Press, New York. Midgley, J. E. M. (1962). Biochim. Biophys. Acta 61, 513. Mitchell, P. (1973a). In “Mechanisms in Bioenergetics” (G. F. Azzone, L. Ernster, A. Papa, E. Quagliariello, and N. Siliprandi, eds.), pp. 177-201. Academic Press, New York. Mitchell, P. (1973b). FEBS (Fed. Eur. Biochem. Sac.),Lett. 33, 267. Mitchell, P. (1974).F E B S (Fed. Eur. Biochem. Sac.), Lett. 43, 189. Mitchell, P. J., and Rogers, P. R. (1974).J. Bacterial. 119, 653. Miura, K. (1962). Biochim. Biophys. Acta 55, 62. Mizumo, S., Yamazaki, A., Nitta, K., and Umezawa, H. (1967). Biochim. Biophys. Acta 157, 322. Modolell, J.. Vazquez, D., and Monro, R. E. (1971a). Nature (London),New Biol. 230, 109. Modolell, J., Cabrer, B., Parmeggiani, A., and Vazquez, D. (1971b). Proc. Nut. Acad. Sci. U.S. 68, 1796. Mondal, H., Gaguly, A., Das, A., Mandal, R. K., and Biswas, B. B. (1972). Eur. J. Biochem. 28, 143. Monier, R. (1972). In “The Mechanisms of Protein Synthesis and Its Regulation” (L. Bosch, ed.), pp. 353-394. North-Holland Publ., Amsterdam. Moorbath, S., O’Nions, R. K., and Pankhurst, R. J. (1973).Nature (London) 245, 138. Moustacchi, E. (1971).Mol. Gen. Genet. 114,50. Muir, M. D. (1974a).Origins Lije 5, 105. Muir, M. D. (197413).Nature (London) 248, 730. Mullinix, K. P., Strain, G. C., and Bogorad, L. (1973).Proc. Nut. Acad. Sci. U.S. 70, 2386. Murray, D. R.. and Linnane, A. W. (1972). Biochem. Biophys. Res. Commun. 49, 855. Nagy, L. A. (1974).Science 183,514. Nanninga, N., Meyer, M., Sloof, P., and Reijnders, L. (1972). J . Mol. Biol. 72, 807. Nass, S. (1969). I n t . Rev. Cytol. 25, 55. Nelson, N., Kanner, B. I., and Gutnick, D. L. (1974). h o c . Nut. Acad. Sci. U S . 71, 2720. Neupert, W., and Ludwig, G . D. (1971).Eur. J. Biochem. 19,523. Newton, J. W., and Kamen, M. D. (1961).In “The Bacteria” (I. C. Gunsalus and R. Y. Stanier. eds.), Vol. 2, pp. 397-423. Academic Press, New York. Niederman, R. A. (1974).J.Bacterial. 117, 19. Nielsen, M. H., Ludvik, J.. and Nielsen, R. (1966).J.Microsc. (Paris) 5, 229. Nieuwenhuis, F. J. R. M., Kanner, B. I., Gutnick, D. L., Postma, P. W., and Van Dam, K. (1973). Biochim. Biophys. Acta 325, 62. Nikolaev, N., Silengo, L., and Schlessinger, D. (1973). Proc. Nut. Acad. Sci. U.S. 70, 336 1. Nikolaev, N., Schlessinger, D., and Wellauer, P. K. (1974).J.Mo2. B i d . 86,741. Nisioka, T., Mitani, M., and Clowes, R. (1969).J . Bacterial. 97, 376. Nobrega, F. G., and Ozols, J. (1971),J.Biol. Chem. 246, 1706. Noll, H. (1970). In “Control of Organelle Development” (P. L. Miller, ed.), pp. 419447. Academic Press, New York. Novick, R. P., Smith, K., Shcehy, R. J., and Murphy, E. (1973). Biochem. Biophys. Res. Comrnurr. 54. 1460.

MITOCHONDRIA AND T H E ORIGIN OF EUKARYOTES

121

Oelze, J., and Drews, G. (1972). Biochim. Biophys. Acta 265,209. Ojala, D., and Attardi, G. (1972). J . Mol. B i d . 65, 273. . U.S. 71, 563. Ojala, D., and Attardi, G. (1974a). Proc. Nat. A c Q ~Sci. Ojala, D., and Attardi, G. (1974b).J. Mol. B i d . 82, 151. Ojala, D., and Attardi, G. (1974~). J . Mol. Biol. 88, 205. Olson, J. M. (1970). Science 168,438. Omura, T., Sanders, E., Estabrook, R. W., Cooper, D. Y., and Rosenthal, 0. (1966). Arch. Biochem. Biophys. 117, 660. Oshino, N., Imai, Y., and Sato, R. (1971).J. Biochem. (Tokyo) 69, 155. Otaka, T., and Kaji, A. (1973). Eur. J. Biochem. 38, 46. Ozols, J. (1974). Biochemistry 13,426. Ozols, J., and Strittmatter, P. (1967). Proc. Nut. Acad. Sci. U.S. 58,264. Pace, N. R. (1973). Bacteriol. Reti. 37, 562. Palade, G. E. (1966).J. Amer. Med. Ass. 198, 815. Paltauf, F., and Schatz, G. (1969). Biochemistry 8, 335. Parsons, D. F., and Yano, Y. (1967). Biochim. Biophys. Acta 135,362. Payne, P. I., and Dyer, T. A. (1971). Biochem. J . 124, 83. Penman, S. (1968).J. Mol. B i d . 34, 49. Perlman, P. S., and Birky, C. W., Jr. (1974). Proc. Nat. Acad. Sci. U.S. 71,46124616. Perlman, P. S., and Mahler, H. R. (1970).J. Bioenerg. 1, 113. Perlman, P. S., and Mahler, H. R. (1974). Arch. Biochem. Biophys. 162,248. Perlman, S., Abelson, H. T., and Penman, S. (1973). Proc. Not. Acad. Sci. U S . 70,350. Perry, R. P. (1973). In “Molecular Cytogenetics” (B. A. Hamkalo and J. Papaconstantinou, eds.), pp. 133-146. Plenum, New York. Pestka, S. (1971a).Annu. Reu. Biochem. 40,697. Pestka, S. (1971b).Annu. Reti. Microbiol. 25,487. Peters, T., Jr. (1962).J. B i d . Chem. 237, 1186. Pettigrew, G. W. (1972). F E B S (Fed. Eur. Biochem. Soc.),Lett. 22,64. Pettigrew, G. W. (1973). Nature (London) 241, 531. Pigott, G. H., and Carr, N. G. (1972). Science 175, 1259. Platt, J. R. (1964). Science 146, 347. Ponta, H., Ponta, U . , and Wintersberger, E. (1972). Eur. J . Biochem. 29, 110. Porra, R. J., Barnes, R., and Jones, 0. T. G. (1972). Hoppe-Seyler’s Z. Physiol. Chem. 353, 1365. Poyton, R. O., and Schatz, G. (1975).J. Biol. Chem. 250, 752. Preer, J. R., Jr. (1971). Annu. Reti. Genet. 5 , 361. Proudlock, J. W., Haslam, J. M., and Linnane, A. W. (1971). Bioenergetics 2, 327. Racker, E. (1972). In “Horizons of Bioenergetics” (A. San Pietro and H. Gest, eds.), pp. 53-72. Academic Press, New York. Raff, R. A. (1970). Curr. Mod. B i d . 3, 250. Raff, R. A., and Mahler, H. R. (1972). Science 177, 575. Raff, R. A., and Mahler, H. R. (1975). Soc. E x p . Biol. Symp. 29. Raff, R. A,, and Raff, E. C. (1970). Nature (London) 228, 1003. Ratner, M. I., and Walker, J. C. G. (1972).J. Atmos. Sci. 29, 803. Raven, P. H. (1970). Science 169, 641. Ray, T. K., Skipski, V. P., Barclay, M., Essner, E., and Archibald, F. M. (1969).J. Biol. Chem. 244,5528. Razin, S. (1972). Biochim. Biophys. Acta 265, 241. Redman, C. M. (1967).J. B i d . Chem. 242,761. Redman, C. M. (1969). J . B i d . Chem. 244, 4308. Redman, C. M., and Sabatini, D. D. (1966). Proc. Nut. A c Q ~Sci. . U . S . 56,608.

122

HENRY R. MAHLER AND RUDOLF A. RAFF

Redman, C. M., Siekevitz, P., and Palade, G . E. (1966).J. Biol. Cheni. 241, 1150. Reid, B. D., and Parsons, P. (1971). Proc. Nut. Acad. Sci. U.S. 68, 2830. Reijnders, L., Sloof, P., Sival, J., and Borst, P. (1973a). Biochim. Biophys. Acta 324, 320. Reijnders, L., Sloof, P., and Borst, P. (1973b).Eur. J. Biochem. 35, 266. Reilly, C., Fuhrmann, G.-F., and Rothstein, A. (1970). Biochirn. Biophys. Acta 203, 583. Remsen, C. C., Valois, F. W., and Watson, S. W. (1967).J.Bacteriol. 94, 422. Reusch, V. M., Jr., and Burger, M. M. (1973). Biochim. Biophys. Acta 300,79. Rhoads, D. C., and Morse, J. W. (1971).Lethaia 4,413. Richmond, M. H. (1970). In “Organization and Control in Prokaryotic and Eukaryotic Cells” (H. P. Charles and B. C. J. G . Knight, eds.), pp. 249-277. Cambridge Univ. Press, London and New York. Richter, D., and Lipmann, F. (1970). Biochemistry 9,5065. Richter, D., Lin, L., and Bodley, J. W. (1971).Arch. Biochem. Biophys. 147, 186. Richter, D., Herrlich, P., and Schweiger, M. (1972).Nature (London),New BioZ. 238, 74. Robberson, D., Aloni, Y.,Attardi, G., and Davidson, N. (1971).J. MoZ. BioZ. 60, 473. Robberson, D. L., Kasamatsu, H., and Vinograd, J. (1972).Proc. Nut. Acad. Sci. U.S. 69, 737. Roberts, J. W. (1969).Nature (London) 224, 1168. Robertson, J. D. (1962).Sci. Amer. 151, 3. Robertson, J. D. (1964). In “Cellular Membranes in Development” (M. Locke, ed.), pp. 1-81. Academic Press, New York. Roeder, R. G. (1974).J.Biol. Chem. 249, 241. Roeder, R. G., and Rutter, W. J. (1969).Nature (London)224,234. Roeder, R. G., and Rutter, W. J. (1970a).Proc. Nut. Acad. Sci. U.S. 65, 765. Roeder, R. G., and Rutter, W. J. (1970b). Biochemistry 9,2543. Rogall, G., and Wintersberger, E. (1974). FEBS (Fed. Eur. Biochem. Soc.), Lett. 46, 333. Rolleston, F. S. (1972). Biochem. J. 129, 721. Rolleston, F. S. (1974).Sub-CeZl. Biochem. 3, 91. Rosbash, M., and Penman, S . (1971).J.Mol. B i d . 59,227. Russell, R. L., Abelson, J. N., Laudy, A., Getter, M. L., Brenner, S., and Smith, J. D. (1970).J.Mol. B i d . 47, 1. Rutten, M. G. (1971). “The Origin of Life by Natural Causes.” Elsevier, Amsterdam, Sagan, L. (1967).J. Theor. B i d . 14, 225. Sakano, K., Kung, S. D., and Wildman, S. G . (1974). Mol. Gen. Genet. 130, 91. Salim, M., and Maden, B. E. H. (1973).Nature (London)224, 334. Sankoff, D., Morel, C., and Cedergren, R. J. (1973). Nature (London),New B i d . 245, 232. Sarathy, P. V., and Siddiqi, 0. (1973a).J.Mol. Biol. 78, 427. Sarathy, P. V., and Siddiqi, 0. (1973b).J.Mol. B i d . 78, 443. Scaller, T. J., Dean, W. J., and Schuster, M. W. (1968). J. B i d . Chern. 243, 5205. Schatz, G., and Mason, T. (1974).Annu. Rev. Biochem. 43,51. Schnebli, H. P., Vatter, A. E., and Abrams, A. (1970).J. Biol. Chem. 245, 1122. Schnepf, E., and Brown, R. M., Jr. (1971). In “Origin and Continuity of Cell Organelles” (J. Reinert and H. Ursprung, eds.), Springer-Verlag, New York. Schopf, J . W. (1970).Biol. Rev. Cmibridge Phil. Soc. 45, 319. Schopf, J. W., and Blacic, J . M. (1971).J.Paleontol. 45, 925. Schopf, J. W., and Fairchild, T. R. (1973).Nature (London) 242, 537.

MITOCHONDRIA AND THE ORIGIN OF EUKARYOTES

123

Schol)f, J. W., Oehler, D. Z., Horodyski, R. J., and Kvenvolden, K. A. (1971).J.Paleontol. 45, 477. Schopf, J. W., Haugh, B. N., Malnar, R. E., and Satterthwait, D. F. (1973). J. Paleontol. 47, 1. Scott, N. S., and Smillie, R. M. (1969).Curr. Mod. Biol. 2,339. Scragg, A. H. (1971). Biochem. Biophys. Res. Comrnun. 45,701. Scragg, A. H. (1974). In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 47-57. Academic Press, New York. Sebald, W., Machleidt, W., and Otto, J. (1973).Eur. J. Biochem. 38, 311. Senior, A. E. (1973).Biochim. Biophys. Acta 301, 249. Senior, B. W., and Holland, I. B. (1971).Proc. Nat. Acad. Sci. U.S. 68,959. Shannon, C., Enns, R., Whellis, L., Burchiel, K., and Criddle, R. S . (1973).J. Biol. Chem. 248,3004. Shively, J. M., Ball, F., Brown, D. H., and Saunders, R. E. (1973). Science 182, 584. Siegel, B. Z., and Siegel, S. M. (1970).Proc. Nat. Acad. Sci. U.S. 67, 1005. Siegel, S. M., and Giumarri, C. (1966).Proc. Nat. Acad. Sci. U.S. 55, 349. Siegel, S. M., and Siegel, B. Z. (1968).Arner. J. Bot. 55, 684. Sierra, M. F., and Tzagoloff, A. (1973). Proc. Nat. Acad. Sci. U.S. 70, 3155. Simpson, G. G. (1953).“The Major Feature of Evolution.” Columbia Univ. Press, New York. Sinclair, J. H., and Brown, D. D. (1971). Biochemistry 10, 2761. Sinha, A. K. (1972).Earth Planet. Sci. Lett. 14, 360. Sogin, S. J., Sogin, M. L., and Woese, C. R. (1972).J.Mol. Euol. 1, 173. South, D. J., and Mahler, H. R. (1968).Nature (London) 218, 1226. Spirin, A. S. (1964).“Macromolecular Structure of Ribonucleic Acids .” Van NostrandReinhold, Princeton, New Jersey. Stanier, R. Y. (1970). Symp. SOC. Gen. Microbiol. 20, 1. Stanier, R. Y., Doudoroff, M., and Adelberg, E. A. (1970). “The Microbial World,” 3rd Ed. Prentice-Hall, Englewood Cliffs, New Jersey. Stanley, S. M. (1973). Proc. Nat. Acad. Sci. U.S. 70, 1486. Stein, H., and Hausen, P. (1970).Eur. J . Biochem. 14,270. Steinman, H. M., and Hill, R. L. (1973). Proc. Nat. Acad. Sci. U.S. 70, 3725. Storti, R. V., and Sinclair, J. H. (1974). Biochemistry 13,4447. Swanson, R. F. (1971).Nature (London)231, 31. Sy, J., and McCarthy, K. S. (1968).Biochim. Biophys. Acta 166, 571. Tabak, H. F., and Borst, P. (1970). Biochim. Biophys. Acta 217, 356. Takagi, M., and Ogata, K. (1968). Biochem. Biophys. Res. Commun. 33,55. Tanaka, M., Haniu, M., Yasunobu, K. T., Dus, K., and Gunsalus, I. C. (1974).J.Biol. Chem. 249,3689. Tang, F. L. M., and Krogmann, D. W. (1972).Plant Physiol. 49, 264. Tata, J. R. (1971).Sub-cell. Biochem. 1, 83. Taylor, F. J. R. (1974). Tuxon 23,229. Tchen, T. T., and Bloch, K. (1957).J.B i d . Chem. 226,921. Timberlake, W. E., and Griffin, D. H. (1973). Biochern. Biophys. Res. Commun. 54, 216. Timberlake, W. E., Hagen, G., and Griffin, D. H. (1972). Biochem. Biophys. Res. Commun. 48,823. Towe, K. M. (1970). Proc. Nat. Acad. Sci. U.S. 65, 781. Towers, N. R., Kellerman, G. M., Raison, J. K., and Linnane, A. W. (1973). Biochini. Biophys. Acta 299, 153. Trembath, M. K., Bunn, C. L., Lukins, H. B., and Linnane, A. W. (1973).Mol. Gen. Genet. 121, 35.

124

HENRY R. MAHLER AND RUDOLF A. RAFF

Tsai, M. J., Michaelis, G., and Criddle, R. S. (1971).Proc. Nat. Acad. Sci. U.S. 68,473. Tzagoloff, A., and Akai, A. (1972).J . Biol. Chem. 247, 6517. Tzagoloff, A., Rubin, M. S., and Sierra, M. F. (1973).Biochim. Biophys. Acta 301, 71. Tzagoloff, A,, Akai, A., and Rubin, M. S. (1974). In “Biogenesis of Mitochondria” (A. M. b o o n and C. Saccone, eds.), pp. 405-421. Academic Press, New York. Ullrich, V. (1972).Angew. Chem., Int. Ed. Engl. 11, 701. Uzzell, T., and Spolsky, C. (1974).Amer. Sci. 62,334. Vail, J. R., and Dodson, M. H. (1970). Trans. Geol. SOC. S . Afr. 72,79. Vaughan, M. H., Soeiro, R., Warner, J. R.,and Darnell, J. E. (1967).Proc. Nut. Acad. Sci. U S . 58, 1527. Verma, I. M., Edelman, M., Henberg, M.,and Littauer, U. Z. (1970).J.Mol. Biol. 52, 137. Vesco, C., and Penman, S. (1969).Proc. Nut. Acad. Sci. US.62,218. Visconti, N., and Delbriick, M. (1953).Genetics 38, 5. Walsby, A. E. (1972).Bacteriol. Rev. 36, 1. Watson, S. W., and Remsen, C. C. (1970).J,Ultrastruct. Res. 33, 148. Watson, S. W., and Waterbury, J. B. (1971).Arch. Mikrohiol. 77, 203. Watson, S. W., Graham, L. B., Remsen, C. C., and Valois, F. W. (1971).Arch. Mikrobiol. 76, 183. Weaver, R. F., Blatti, S. P., and Rutter, W. J. (1971).Proc. Nat. Acad. Sci. U.S. 68,2994. Weisiger, R. A., and Fridovich, 1. (1973a).J.Biol. Chem. 248,3582. Weisiger, R. A,, and Fridovich, I. (197313).J . Biol. Chem. 248,4793. Weiss, H., and Ziganke, B. (1974a).Eur. J . Biochem. 41, 63. Weiss, H., and Ziganke, B. (197413).In “Biogenesis of Mitochondria” (A. M. Goon and C. Saccone, eds.), pp, 491-500. Academic Press, New York. Weltman, J. K., and Dowben, R. M. (1973).Proc. Nat. Acad. Sci. US.70,3230. Weser, U. (1973). In “Structure and Bonding” (J. D. Dunitz. P. Hemmerich, J. C. Ihers, C. K. Jergesen, J. G . Neilands, D. Reinen, and R. J. P. Williams, eds.), pp. 2-65. Springer-Verlag, Berlin and New York. White, D. A., Lennarz, W. J., and Schnaihnan, C. A. (1972).J . Bacteriol. 109, 686. Whittaker, P. A., Hammond, R. C., and Luha, A. A. (1972).Nature (London),New Biol. 238,266. Whittaker, R. H. (1969).Science 163, 150. Wilkie, D. (1963).J.Mol. Biol. 7, 527. Wilkie, D., and Thomas, D. Y. (1973).Genetics 73,367. Willett, J. D., Sharpless, K. B., Lord, K. E., Van Tamelen, E. E., and Clayton, R. B. (1967).]. Biol. Chem. 242,4182. Williams, P. H., Boyer, H. W., and Helinski, D. R. (1973).Proc. Nut. Acad. Sci. U.S. 70,3744. Williamson, D. H., Maroudas, N.G., and Wilkie, D. (1971).Mol. Gen. Genet. 111,209. Wintersberger, E. (1970).Biochem. Biophys. Res. Commun. 40, 1179. Woese, C. R. (1961).Nature (London) 189,920. Woese, C. R., and Bleyman, M. A. (1972).J . Mol. Euol. 1,223. Wolk, C . P. (1973).Bacteriol. Rev. 37,32. Wong-Staal, F., Mendelsohn, J., and Goulian, M. (1973). Biochem. Biophys. Res. Commun. 53, 140. Wu, G.-J., and Dawid, I. B. (1972).Biochemistry 11,3589. Wu, M., Davidson, N., Attardi, G., and Aloni, Y. (1972).J.Mol. Biol. 71,81. Yost, F. J., Jr., and Fridovich, I. (1973).J . B i d . Chem. 248,4905. Yuan, C., and Bloch, K. (196l).J.B i d . Chem. 236, 1277. Zylber, E., Vesco, C., and Penman, S. (1969).J . Mol, Biol. 44, 195.

Biochemical Studies of Mitochondrial Transcription and Translation

c. SACCONE AND

E. QUACLIARIELLO

Institute of Biological Chemistry, University of Bari, Bari, Italy

I. Introduction

.

.

.

.

.

XI. Mitochondrial Transcription .

.

.

.

.

.

.

.

.

.

A. Studies in Vitro . . . . . . . . B. Studies in Vivo . . . . . . . . 111. Mitochondrial Translation . . . . . . A. The Protein-Synthesizing Machinery of Mitochondria B. The Translation Products . . . . . . Note Added in Proof . . . . . . . References . . . . . . . . .

. . . .

.

,

. . .

125 127 127 142 152 152 157 160 160

The field of mitochondrial biogenesis has expanded greatly in the last 10 years and is at the moment one of the most active areas of cell biology. For this reason a complete review of the subject is difficult, and would not be of great use to those who wish to delve more deeply into this interesting field. Furthermore, every conceivable aspect of the subject has been discussed in recent symposia and in several reviews (Borst and Kroon, 1969; Ashwell and Work, 1970; Kuntzel, 1971; Borst, 1972; Boardman et al., 1971; Sager, 1972; Kroon and Saccone, 1974). Therefore we feel that it is more suitable to limit this article to two aspects of mitochondrial biogenesis, namely, the transcription process, which has not yet been extensively reviewed and which is treated here in detail with special emphasis on the different biochemical experimental approaches and techniques used, and the translation process, which has in contrast been widely discussed on many occasions. Only some of the most recent developments in this area are described. Furthermore, the important contributions of genetic experiments to these aspects of mitochondrial biogenesis will not be treated here.

I. Introduction It is well known that mitochondria, like chloroplasts, are semiautonomous organelles. This implies that two genetic systems cooperate in their biosynthesis: an extramitochondrial system and an in125

126

C. SACCONE AND E. QUAGLIARIELLO

tramitochondrial system. The extramitochondrial system is formed by the nuclear genome which codes for the majority of mitochondrial proteins through the formation of mRNA translated mostly or completely at the level of the cytoplasmic machinery for protein synthesis. The intramitochondrial system is formed by a mitochondrial genome whose genetic continuity and expression are ensured by the existence, within the organelle itself, of enzymes responsible for DNA and RNA synthesis, and a complete protein-synthesizing machinery with properties completely different from those of its cytoplasmic counterpart. What is the significance of the cooperation of two different genetic systems in the biosynthesis of cellular organelles? What is the advantage of eukaryotic cells having preserved a separate, different, intraorganelle genetic system during evolution? These and other questions can be raised in relation to this interesting and stimulating problem. As far as the evolutionary origin of mitochondria is concerned, we only mention here that two hypotheses have been put forward (Borst, 1972): the endosymbiotic hypothesis, according to which mitochondria originate from aerobic bacteria in symbiosis with primitive probably anaerobic eukaryotic cells, and that in the course of evolution hand over the major part of their autoreplication capacity to the nucleus; and the episome hypothesis, according to which mitochondrial DNA started out as an episome which covered itself with a membrane formed b y respiratory chain components. The numerous experimental data on mitochondrial biogenesis now available support either hypothesis and sometimes both. These data, however, owing to the multiplicity of interpretations offered, suggest that probably we never shall be able to establish definitely the origin of the mitochondria within cells. However, even if this aspect of the problem remains unresolved, the study of mitochondrial biogenesis is of great importance in elucidating some of the more basic and important problems of biology, such as the structural and functional organization of the cell and its replication and differentiation. The study of mitochondrial biogenesis started naturally with that of the major component of the genetic system of the organelle, that is, mitochondrial DNA. It has been very accurately characterized, and most of its properties have been extensively discussed (Borst and Kroon, 1969; Borst and Flavell, 1972; Borst and Grivell, 1973). The major problem is, however, to establish the genetic content of mitochondrial DNA. One way to determine this is to identify its primary gene products, that is, to study the transcription and translation of the mitochondrial genome.

MITOCHONDRIAL TRANSCRIPTION AND TRANSLATION

127

11. Mitochondrial Transcription

Two approaches are used in the study of mitochondrial transcription:

1. Studies in vitro. In this case the transcription of the mitochondrial genome can be followed (a) with isolated organelles, (b) with isolated mitochondrial DNA and a heterologous DNA-dependent RNA polymerase enzyme, and (c) with isolated mitochondrial DNA and its homologous DNA-dependent RNA polymerase enzyme. 2. Studies in vivo. These studies are essentially based on identification of the cellular RNA species homologous to mitochondrial DNA. Both the in vitro and in vivo approaches have advantages and disadvantages, and only an integrated analysis of the results obtained by both methods can give satisfactory answers. In this report we summarize the main results obtained in the study of mitochondrial transcription with different organisms, those most commonly used being yeast, Neurospora crassa, Tetrahymena pyriformis, HeLa cells, Xenopus, and rat liver.

A.

STUDIESin Vitro

1. Mitochondrial Transcription in lsolated Organelles The study of mitochondrial transcription with isolated organelles presents several difficulties and suffers from several limitations. Some difficulties are due to the fact that the rate of mitochondrial RNA synthesis in vitro, using isolated organelles, is rather low and rigorous criteria must be used to exclude the possibility of activity being due to other fractions (e.g., the nuclear fraction or bacteria) present as contaminants in mitochondrial preparations. Furthermore, isolated mitochondria possess enzyme activities, other than DNAdependent RNA polymerase, responsible for the incorporation of labeled nucleotides into acid-insoluble material (see also Section II,A,3). The presence of such activities could explain most of the discrepancies reported in the literature concerning the main characteristics of mitochondrial RNA synthesis. However, this experimental approach, although having many limitations and often producing results that are difficult to interpret, has been of great help in the initial characterization of the mitochondrial transcription process. Since 1964 it has been reported from several laboratories that

128

C. SACCONE AND E. QUAGLIARIELLO

isolated mitochondria, incubated in an appropriate medium containing a labeled precursor, are able to synthesize radioactive RNA by a mechanism which displays many properties of a transcription process (Wintersberger, 1964; Kalf, 1964; Luck and Reich, 1964; Neubert et al., 1966; Saccone et al., 1967). The synthesis is inhibited by well-known transcription inhibitors, but does not require addition of exogenous DNA since it is supported by endogenous DNA which acts as a template. Many properties differentiate the process of mitochondrial RNA synthesis from that occurring in nuclei. We recall that the mitochondrial system of RNA synthesis is almost completely RNase- and DNase-resistant, since the molecules of these enzymes cannot penetrate the intact mitochondrial membrane. The same situation applies for some inhibitors, such as actinomycin D. I n order to demonstrate the sensitivity of the reaction to these inhibitors in some systems, such as rat liver (Neubert et al., 1966; Saccone et al., 1967), it is necessary to use mitochondria pretreated to alter the inner membrane permeability. For such purposes phosphate-swollen mitochondria or sonicated preparations are often used in the study of mitochondrial RNA synthesis. Table I shows several characteristics and the sensitivity to inhibitors of RNA synthesis in isolated rat liver mitochondria. It can be seen that the reaction is completely insensitive to a-amanitin, whereas it is specifically inhibited by ethidium TABLE I PROPERTIES OF

RNA

SYNTHESIS IN ISOLATED

FROM

Optimum temperature Optimum pH (tris-HC1 buffer) Optimum divalent cation concentration (d) K m

Km

ATP ( 4 4 ) UTP ( 4 4 )

Dependence on DNA Dependence on nucleotides Inhibition by RNase Inhibition by DNase Inhibition by actinomycin Inhibition by acriflavin Inhibition by rifampicin Inhibition by a-anianitin Inhibition by ethidium bromide

" n.m., Not measured.

MITOCHONDRIA

RAT LIVER

37°C 7.8 n.m. 60 60

30°C 7.4 2MgZt+ 2MnZt 35 60 -

n.m. -

+

MITOCHONDRIAL TRANSCRIPTION AND TRANSLATION

129

bromide and by rifampicin, although there is still some argument about this characteristic. Most discrepancies could result from the different experimental conditions used by various investigators in isolating the organelles and/or differences in the incubation mixture. We come back to this point in Section II,A,3. It must be stressed that the size and nature of mitochondrial RNA synthesized in vitro b y isolated organelles have been determined only in a few cases. In yeast mitochondria, Wintersberger (1966) first reported the incorporation of ATPJH into a RNA which in sucrose gradient sedimented as rRNA-like RNA (23 and 16s)and as a tRNAlike RNA species. Suyama and Eyer (1968), using isolated mitochondria from T . p yriforrnis, demonstrated that RNA ranging from 14 to 18S, but not tRNA, was synthesized. This RNA was hybridizable with mitochondrial DNA and contained no more than 10% mitochondrial rRNA. RNA synthesized by isolated rat liver mitochondria (Saccone et al., 1969) appeared polydisperse when analyzed in a sucrose gradient, radioactivity being the most concentrated in the region 14-8s. The product of synthesis by rat liver mitochondria has been extensively characterized by hybridization studies (Aaij et al., 1970). It was demonstrated that more than 85% of the RNA synthesized in witro specifically hybridized with rat liver mitochondrial DNA. By using isolated complementary strands of mitochondrial DNA separated in an alkaline cesium chloride gradient, it was shown that the RNA synthesized in witro preferentially hybridizes with the heavy (H) strand, although it also hybridizes to a significant extent with the light (L) strand of mitochondrial DNA. This property, together with the observation that about 20% of the newly synthesized RNA could be converted into a double-stranded form by annealing in the presence of excess cold mitochondrial RNA, clearly suggests that the transcription process can be, at least partly, symmetric. We discuss this question again in Section II,B,2. With regard to the nature of the RNA synthesized by isolated rat liver organelles, hybridization-competition experiments have shown that the RNA extracted in vivo from mitochondria efficiently inhibits the hybridization reaction between RNA synthesized in vitro and mitochondrial DNA. Significant inhibition is also observed using RNA extracted from purified mitochondrial ribosomes, clearly demonstrating that the in witro transcripts mostly contain sequences identical to mitochondrial rRNA (Saccone, 1973). Synthesis of ribosomelike RNA species in vitro by isolated organelles from rat liver had been shown by Fukamaki et al. (1970) also. The product of transcription displayed an electrophoretic mobility of 21 and 12s on agarose-acryla-

130

C. SACCONE AND E. QUAGLIARIELLO

mide gels, and its synthesis was inhibited by ethidium bromide. In addition to stable RNA species, isolated mitochondria seem to be able to synthesize an unstable species which is probably mRNA. When mitochondria are incubated in the presence of precursors of RNA for a fixed time and RNA synthesis is then blocked by adding inhibitors of the transcription process, part of the radioactive material that was insoluble in acid rapidly becomes acid-soluble. The time dependence of this process can be used to measure the half-life of the unstable RNA synthesized in vitro. Half-life values obtained by this method are essentially in agreement with those found by measuring the protein-synthesizing capacity of isolated organelles in the presence of inhibitors of the transcription process. Half-lives of mitochondrial mRNA, obtained with isolated mitochondria from various organisms and with different techniques, are summarized in Table 11. They range from 1.4 minutes up to 1 hour or more in HeLa cells. Data obtained with experiments in vivo also give values ranging from less than 10 minutes in yeast (Weislogel and Butow, 1971) to 1 3 hours in HeLa cells (Zylber et al., 1971).The large differences in half-life values reported for mitochondria1 mRNA are clearly due to the different approaches and/or experimental conditions used by the TABLE I1 HALF-LIFEVALUES OF mRNA MEASUREDIN ISOLATEDMITOCHONDRIA

Source

Saccharom yces cerevisiae Physarum polycephalum P . polycephalum Paracentrotus lividus Rat heart Rat liver Rat liver Rat liver Rat brain HeLa cells

Half-life values (minutes)

Technique'

Reference

< 15

a

2-3

C

Wintersberger (1966) Grant and Poulter (1973) Grant and Poulter (1973) Gadaleta and Saccone (1973) Gamble and McCluer (1970) Gadaleta and Saccone (1974) Gadaleta and Saccone (1974) Gadaleta and Saccone (1973) Gadaleta and Saccone (1973) Lederman and Attardi (1970)

<5

<5 1.4 4.8 -3 <5 6.9 60-90

a C

b b a C

b

C

The techniques used were: (a) measure of acid-insoluble radioactivity degradation after blocking RNA synthesis with inhibitors of the transcription process, (b) measure of protein synthesis decay in the presence of inhibitors of the transcription process, and (c) measure of loosing ability to synthesize proteins by mitochondria pretreated with inhibitors of RNA synthesis.

MITOCHONDRZAL TRANSCRIPTION AND TRANSLATION

131

various investigators, and probably to differences in the source of mitochondria.

2. Transcription of Mitochondria1 DNA by Heterologous RNA Polymerase It is well known that purified RNA polymerase from Escherichia coli can transcribe several heterologous templates in vitro with considerable fidelity. A specific transcription can lead to a complete biochemical gene mapping. The transcription process in vitro usually requires intact DNA, recognition of promoter sites by RNA polymerase, and the use of a RNA polymerase preparation free of DNA endonuclease activity and with a functional (+ factor. For the study of mitochondrial DNA transcription in vitro these requirements can b e fulfilled only in some cases, largely because for some organisms, such as protists, it is difficult to isolate the template in its intact native form. Five-micrometer intact circles of mitochondrial DNA can be readily isolated from rat liver and other animal cells and are useful tools for experiments on in vitro transcription using E . coli RNA polymerase. Transcription of the mitochondrial genome by heterologous RNA polymerase has been extensively investigated with mitochondrial DNA from yeast, N . crassa, Artemia salina, and rat liver. Transcription of mitochondrial DNA from yeast with E . co2i or yeast nuclear DNA-dependent RNA polymerase has been accomplished by Michaelis et al. (1972). The DNA was isolated by chromatography on hydroxylapatite, but no information was given b y the investigators about the size and the physical state of the DNA molecules. In order to detect which genes and how much of the isolated yeast mitochondrial genome are transcribed in vitro, Michaelis et al. hybridized newly synthesized RNA with mitochondrial DNA both in the absence and in the presence of excess cold RNA extracted from purified mitochondrial ribosomes. Using this approach they demonstrated that the E . coli RNA polymerase transcribes the ribosomal genes of mitochondrial DNA. This type of experiment was also used to demonstrate that the altered mitochondrial DNA from one petite strain (D 243-2B-R1-6)maintains all or some part of the ribosomal genes. Furthermore, by observing the hybridization of labeled RNA synthesized in vitro with nuclear and mitochondrial DNA of yeast, these workers showed that very little homology exists between the nuclear and mitochondrial genomes. Schafer et al. (1971) studied the transcription of intact mito-

132

C. SACCONE AND E. QUACLIAFUELLO

chondrial DNA from N . crussa by DNA-dependent RNA polymerase from E . coli. For this experiment they used a homogeneous DNA preparation consisting of linear molecules with a contour length of 25-26 pm, probably derived from circular molecules existing in uivo (Agsteribbe et al., 1972). These workers demonstrated that bacterial RNA polymerase recognizes mitochondrial DNA sequences resembling bacteriophage initiation sites. The number of binding sites conferring rifampicin resistance per mitochondrial genome (molecular weight 50 to 60 x lo6)has been estimated to be eight to nine. Competition-hybridization experiments showed that about 50% of the sequences synthesized in vitro and hybridizing with mitochondrial DNA are homologous to RNA species isolated from intact mitochondria, probably representing stable RNA species like rRNA and tRNA. Furthermore the product synthesized in vitro contained self-complementary regions since 25%of the sequences formed double strands after annealing for 16 hours at 62°C.These regions did not contain sequences complementary to the stable RNA species present in d u o . These latter data again suggest that the mechanism of symmetric transcription probably operates in Neurosporu mitochondria also and give further support to the idea of Aloni and Attardi (1971a), which is discussed later, that both strands of mitochondria DNA are copied but that only the products of the H strand and of a small portion of the L strand remain undegraded and therefore are represented in the stable RNA species in uivo. Tabak and Borst (1970) demonstrated that RNA polymerase from E . coli can transcribe intact circular mitochondrial DNA from rat liver, even when the m factor is inactivated. The RNA transcribed in vitro was about onethird self-complementary, that is, it showed resistance to degradation by pancreatic RNase after self-annealing. The remaining singlestranded RNA, when incubated with purified complementary strands of mitochondrial DNA isolated in an alkaline cesium chloride gradient, exclusively hybridized with the L strand of mitochondrial DNA. The data clearly demonstrated that in such a system a symmetric transcription process takes place and that the L strand of mitochondrial DNA is preferentially transcribed. Since Borst and Aaij (1969) had previously demonstrated that rat liver mitochondrial RNA, pulselabeled in uivo, hybridized only with the H strand of mitochondrial DNA, these results were interpreted as indicating a loss of fidelity in the transcription of mitochondrial DNA by the bacterial polymerase. Now these data can b e reinterpreted in light of the data of Attardi (see Section II,B,2).A situation similar to that described for rat liver occurs

MITOCHONDFUAL TRANSCRIPTION AND TRANSLATION

133

for the transcription of Xenopus mitochondrial DNA by E . coli RNA polymerase. Also in this case a preferential transcription ofthe L strand in the region of ribosomal and 4s antisequences is observed (Dawid, 1972b). RNA polymerase I and I1 extracted from Xenopus nuclei transcribe almost symmetrically both native or denatured mtDNA (I. B. Dawid and R. H. Roeder, unpublished data). Using the 5-pm circular mitochondrial DNA from A. salina cysts, Schmitt et al. (1974) attempted to transcribe it with purified RNA polymerase from E , coli. They demonstrated that the addition of (T factor to core enzyme greatly stimulated the transcription process. In the presence of rifampicin, there was only limited transcription, dependent on the concentration of (T factor in the reaction mixture. Since it is known that antibiotic rifampicin blocks the initiation step of DNA transcription by E . coli RNA polymerase and permits only one round of transcription before the reaction is inhibited, the results with A. salina mitochondrial DNA thus suggest that in this case also u factor is involved in the initiation reaction and that it recognizes specific promoter sites. With regard to the product of transcription, it was shown that in the presence of u factor the RNA mass was about 1.5 x lo6daltons and that no synthesis of low-molecular-weight RNA occurred. From the results of hybridization, hybridization-competition experiments, and measurement of RNase resistance following self-annealing, these investigators showed that the in vitro transcription of mitochondrial DNA with E . coli RNA polymerase partially occurred on both DNA strands in opposite directions, and that about 15% of the transcript was homologous to mitochondrial rRNA. Studies on the transcription of mitochondrial DNA by heterologous polymerase enzyme have again allowed a demonstration that the mitochondrial genome of all organisms studied contains the cistrons for mitochondrial rRNAs. With regard to the mechanism of transcription, it has quite clearly emerged that symmetric or partially symmetric transcription takes place. Much experimental evidence suggests that this mechanism probably reflects that which occurs in vivo, although it is difficult to determine if the transcription in uitro mimics with great fidelity the situation in vivo, mostly because only stable RNAs, such as rRNA and tRNA, are usually extracted from isolated mitochondria, whereas other species, such as mRNA or possible precursor sequences removed during synthesis, are absent or present at a very low concentration. This of course makes it difficult to judge how specific is the transcription of mitochondrial DNA by heterologous RNA polymerase.

TABLE 111

PROPERTIESOF PURIFIED MITOCHONDW RNA POLYMERASES Optimal cation concentration Subunits Source Yeast Yeast Neurospora crassa Xenopus laevis

ovaries Rat liver Rat liver

Inhibition

(mM)

Mi?+

Number

MW x105

MnP+

1 2 3 2 1

0.59-0.63 1.45; 2.1 1.5; 2; 2.6 1.35; 1.9 0.64

1-3 2 4 1 1.6

None

1

0.46

None

10

1 1

0.60-0.64 0.66

1

3 4

1

20 10 3 30

High ionic strength"

Rifampicin

a-Amanitin"

Reference

+

-

a b

+ + + +

-

-

n.m.

+

-

+ +

-

n.m.

d

-

e

-

f

n.m.

+

+

+

C

a n.m., Not measured. (a) Scragg (1974); (b) Eccleshall and Criddle (1974); (c) Kiintzel and Schafer (1971); (d) Wu and Dawid (1972); (e) Gallerani and Saccone (1974); (f) Mukerjee and Goldfeder (1973). (Modified from Saccone et al., 1974a.)

MITOCHONDRIAL TRANSCRIPTION AND TRANSLATION

135

3. Transcription of Mitochondrial DNA by Homologous RNA Polymerase a. Mitochondrial DNA-Dependent RNA Polymerase. The enzyme responsible for mitochondrial transcription has only recently been obtained in purified form from yeast (Scragg, 1971; Tsai et al., 1971), Neurospora (Kuntzel and Schafer, 1971), Xenopus ovaries (Wu and Dawid, 1972), and rat liver (Gallerani et al., 1972; Mukerjee and Goldfeder, 1973). Partial purification of the enzyme from Ehrlich ascites tumor cell mitochondria has also been reported (Jackisch et al., 1972). Although the enzyme is rather unstable, it can be solubilized by sonication, digitonin, or treatment with detergent. With the exception of the procedure employed by Kuntzel and Schafer (1971), who used a series of glycerol gradients in order to purify RNA polymerase from N . crussa mitochondria, the purification of other mitochondrial polymerase enzymes generally includes high-speed centrifugation, ammonium sulfate precipitation, and the use of a DEAESephadex or a DEAE-cellulose column. The enzymes are then eluted from the ion-exchange column by stepwise increases in salt concentration (potassium chloride) or linear gradients of the same salt. The principal properties of the purified polymerase enzymes are shown and compared in Table 111. I t can be seen that in one case, yeast, controversial results have been obtained from two groups. Eccleshall and Criddle (1974) and Wintersberger (1972) claim that yeast mitochondria, like the nuclei of the same cells, contain several polymerase enzymes, some of which resemble nuclear polymerases. Scragg (1974), however, was unable to repeat these experiments and showed the presence in mitochondria of only one enzyme which very closely resembled mitochondrial polymerases purified from other organisms. From the data of Table 111, it can be deduced that all mitochondrial RNA polymerases so far purified, with the exception of Criddle’s enzymes, have several properties in common, such as: (1) relatively low molecular weight; the enzyme seems to b e composed of a single polypeptide chain of about 60,000 daltons (46,000 for Xenopus enzyme), which at a low salt concentration tends to form aggregates of higher molecular weight; (2) inhibition at high ionic strength; (3) requirement for low Mn2+concentrations (not exceeding 2-3 d); (4) insensitivity to a-amanitin; and ( 5 ) inhibition by rifampicin (with the exception of the Xenopus enzyme which is inhibited by SV rifamycin). Rifampicin sensitivity is still one of the major controversial points in the characterization of the mitochondrial transcription process. The controversy concerning this property (see Table IV) can be sum-

TABLE IV SENSITWITYTO RIFAMYCINOR RIFAMPICIN OF DIFFERENT MITOCHONDRIAL RNA POLYMERA~ES ~

Native enzyme"

Source Neurospora crassa Neurospora crassa Yeast Yeast Yeast Rat liver Rat liver Rat liver Rat liver Rabbit liver Xenopus ovaries Sea urchin eggs Ehrlich ascites Yoshida ascites a

Antibiotic concentration (dml) n.s.

20 40 20 76 10 SO 10 20

-

20 100

Purified enzyme

Inhibition

Antibiotic concentration

Inhibition

(%)

(aW

(%)"

References

n.s. 4 10 7 32

6 20 10 20

90

Kiintzel and Schafer (1971) Wintersberger (1972) Tsai et al. (1971); Eccleshdl and Criddle (1974) Wintersberger (1972) Scragg (1971, 1974) Shmerling (1969) Gadaleta et al. (1970);Gallerani and Saccone (1974) Reid and Parsons (1971) Mukejee and Goldfeder (1973) Wintersberger (1972) Wu and Dawid (1972) Cantatore et al. (1974) Jackisch et al. (1972) de Montalvo et al. (1974)

90 80 45 n.m. 2 n.m. 45 n.m. 60

10 10

50

-

100 -

n.m. 3-8 n.m. 95 n.m. 70 35 80 n.m. 0 n.m. 18 n.m.

n.s., Not specified; n.m., not measured. (Modified from Saccone et al., 1974a.)

MITOCHONDRIAL TRANSCRIPTION AND TRANSLATION

137

marized as follows. By using isolated organelles it was demonstrated (Gadaleta et al., 1970) that the rat liver enzyme is significantly inhibited by this drug which is known to inhibit specifically the bacterial polymerase enzyme. Under the same conditions, however, some investigators claim to be unable to detect any sensitivity (Wintersberger, 1972). In the case of N . crassa, inhibition by rifampicin can be shown only by using the purified enzyme and not isolated organelles (Kiintzel and Schafer, 1971). Mitochondria1 RNA polymerase purified from Xenopus ovaries also seems to be rifampicin-insensitive (Wu and Dawid, 1972). In yeast, Scragg (1971) found a sensitive enzyme, whereas Tsai et aE. (1971) report that neither of the three separate RNA polymerase enzymes isolated from the mitochondrial preparation is rifampicin-sensitive. These discrepancies can be explained in one of the following ways. In using isolated organelles as the source of the enzyme, the insensitivity to rifampicin may be due to the fact that the drug cannot freely reach the DNA-enzyme complex. Another possibility, in the system mentioned above, is that under the conditions used by some investigators the enzyme is only able to lengthen the RNA chains already present. Since it is known that rifampicin inhibits initiation of the chain (Lill et al., 1970), this could explain the lack of inhibition by rifampicin. A third possibility, according to Kiintzel and Schafer (1971), at least in the case of Neurospora, is that the organelle or the native enzyme contains a cofactor which protects it against rifampicin. As far as the sensitivity of the purified enzyme is concerned, rifampicin sensitivity can be lost during purification. In our first attempts to purify the enzyme, we often observed that DNA dependence and rifampicin sensitivity were lost simultaneously, probably because the active enzyme is often converted into a form which, although still able to polymerize the nucleotides, appears to be neither DNA-dependent nor inhibited by rifampicin. This possibility is also suggested by Scragg (1974) to explain the rifampicin insensitivity of yeast enzyme(s) found by other workers. It has recently been suggested by several investigators (Busiello et al., 1973) that the inhibition of polymerase reactions by rifampicin or rifampicin derivatives may be nonspecific, since rifampicin can simultaneously produce several other effects such as inhibition of nucleotide uptake and of RNA metabolism in mammalian cells. It must be stressed, however, that nonspecific effects are always observed when a high molar ratio of drug to enzyme molecules is used. In the case of mitochondrial RNA synthesis in rat liver, we observe inhibition by rifampicin both in isolated organelles synthesizing a DNA-like RNA and during all the purification steps.

138

C. SACCONE AND E. QUAGLIARIELLO

TABLE V SENSITIVITY OF PUFUFIEDMITOCHONDFUALRNA POLYMERASE FROM RAT LIVERTO SEVERAL INHIBITORS" Experimental conditions Complete system Plus cordycepin Plus cycloheximide Plus ethidium bromide

Inhibitor concentration (CLdml)

Specific activity

Inhibition

6 60 10

2380 2195 2350 1890

-

100

880

21 63

4

2270 1840

23

-

40

(W 8

5

" For technical details, see Gallerani and Saccone (1974). Furthermore, the drug concentrations used in our experiments were in the range employed in the studies concerned with prokaryote and eukaryote polymerase enzymes. Therefore, from our own and other studies, we are tempted to suggest that rifampicin sensitivity is an intrinsic property peculiar to all mitochondrial RNA polymerase enzymes. With regard to sensitivity to other inhibitors, not very much is known about the purified enzyme. Table V details the sensitivity of rat liver mitochondrial RNA polymerase to several inhibitors. It is interesting to note that high concentrations of cycloheximide strongly affect the mitochondrial polymerase enzyme, whereas sensitivity to ethidium bromide is very low. No information on inhibition by ethidium bromide is available for other purified mitochondrial polymerases. However, sensitivity to ethidium bromide may depend on the physical state of the template used. b. PoZy(A) Polymerase, Another enzyme probably connected with transcription and translation of mitochondrial DNA is the poly(A) polymerase activity recently described in rat liver mitochondria. In 1972, Jacob and Schindler (1972) reported the solubilization fiom rat liver mitochondria of an enzyme which, using ATP as precursor, incorporates AMP into a chain of poly(A). The solubilized enzyme does not need DNA as a template and, although the primer requirement is not very clear, it seems that the bulk of the mitochondrial RNA does not work as a primer. Moreover, the enzyme is insensitive to the action of the usual inhibitors of RNA polymerases. It was emphasized that within the mitochondria there is a specific nuclease which degrades the product synthesized by this enzyme, so that removal of

MITOCHONDRIAL TRANSCRIPTION AND TRANSLATION

139

the nuclease by sonication and centrifugation is necessary before enzyme activity can be detected. Successively, a purification of this enzyme from both rat liver and Morris hepatoma by phosphocellulose and DEAE Sephadex chromatography has been reported (Jacob et al., 1974). The purified enzymes require the presence of a primer that can be extracted from mitochondria or the addition of poly(A). The same authors have also shown that the activity of poly(A) polymerase is lower in a series of poorly differentiated hepatoma probably because of the low level of the enzyme in the tumors and/or to an inefficiency of the primer. Philips and Parsons (1973) reported that there is in rat liver an enzyme that incorporates ATP into a material which is acid-insoluble and pronase-sensitive. In the extract obtained by solubilizing mitochondria with Brij 58 detergent, more than one enzymic activity seems to be present. In fact, using 8 mM Mn2+ or 15 mM Mg2+ the incorporation of ATP is stimulated by dithiothreitol (DTT) above 4 mM, and by CTP, GTP, and UTP, whereas when 2 mM Mgz+ is used DTT and the other nucleoside triphosphates have no effect. But, whatever the concentration of divalent cation used, the acid-insoluble radioactivity is almost completely hydrolyzed by pronase, These investigators think that the products of the reaction are fragments of ATP attached to a pronasesensitive receptor, identified as adenosine when 8 mM Mn2+ or 2 mM Mg2+ concentrations were used and identified as a mixture of adenosine (20%)and AMP (80%)at 15 mM Mg2+.According to these workers, the existence in rat liver organelles of such activities whose nature and function are completely unknown must be taken into particular account when studying poly(A) synthetase activity, since this is often characterized by methods that do not preclude the formation of this type of product. In our laboratory we extracted from rat liver mitochondria an enzyme that incorporates ATP into acid-insoluble material (Saccone et al., 1974a).That the product of the enzymic reaction is a chain of poly(A) is based on the following observations: (1) The acid-insoluble material is hydrolyzed by 0.3 M potassium hydroxide. (2) It is insensitive to pancreatic RNase but sensitive to snake venom phosphodiesterase. (3)The product is retained on Millipore filters in 0.5 M potassium chloride. The enzyme is copurified with DNA-dependent RNA polymerase at least up to the ammonium sulfate step and resembles the enzyme described by Jacob and Schindler (1972) in several properties. It is inhibited by cordycepin, and it is essentially insensitive to most inhibitors of the DNA-dependent RNA polymerase. More recently, we also succeeded in a further

140

C. SACCONE AND E . QUAGLIAFUELLO

purification of this enzyme from rat liver (manuscript in preparation). The partially purified enzyme requires, as reported by Jacob et al. (1974), the presence of an endogenous primer of ribonucleotide nature. Although the role of a poly(A) polymerase within the mitochondrion remains to be clarified, such activity could be correlated with the presence of poly(A) segments linked to the mitochondrial RNA recently described by several investigators. Table VI shows the experimental evidence now available for the presence of poly(A)containing RNA species within mitochondria. It can be seen that in one case (HeLa cells) it has been demonstrated that poly(A)-containing RNA species are made on mitochondrial DNA as template. In the case of yeast controversial results have been reported. According to Cooper and Avers (1974) RNA molecules containing tracts of poly(A) and possessing messenger activity can be extracted from mitochondrial polysomes. On the other hand, Groot et al. (1974) were unable to find poly(A)-containing RNA species in yeast mitochondria and attribute the results of Cooper and Avers (1974) to contamination of the mitochondrial preparation with intact protoplasts (see also Section II,B and Section III,A,l). c. Produ,cts of in Vitro Transcription of Mitochondria1 DNA by Homologous DNA-Dependent RNA Polymerase. Characterization of the product of transcription of mitochondrial DNA by its homologous polymerase enzyme has been achieved only with yeast, Xenopus

TABLE VI POLY(A)-CONTAINING RNAs IN MITOCHONDRIA

Source

HeLa cells

Yeast

Ehrlich ascites

Length of poly(A) sequence (number of nucleotides)"

Hybridizability with mitochondrial DNA"

Messenger activity"

70 57

+ +

n.m. n.m.

n.m.

n.m.

+

150-180

n.m.

n.m.

n.m., Not measured. (Modified from Saccone et al., 1974a.)

Reference Attardi et al. (1974) Hirsch and Penman (1973) Cooper and Avers (1974) contrast Groot et al. (1974) Avadhani et (JZ. (1973)

MITOCHONDRIAL TRANSCRIPTION AND TRANSLATION

141

ovaries, and rat liver. The results obtained are summarized in Table VII. The transcription of yeast mitochondrial DNA has been accomplished by Michaelis et al. (1972) by using two enzymes with different properties from yeast mitochondria, polymerase I and I11 (Section II,A,3). In both cases newly synthesized RNA seemed to contain sequences of RNA extracted from purified mitochondrial ribosomes. Labeled RNA was 16-19% resistant to RNase, and this suggests a certain degree of symmetric transcription. Transcription of mitochondrial DNA with the enzyme isolated by Scragg (1974) produced 2 0 4 s RNA when analyzed by polyacrylamide gel electrophoresis, which was 70% sensitive to RNase. The ability of this RNA to direct protein synthesis was studied by adding labeled RNA to a cell-free system from E . coli capable of transcribing and translating small DNAs such as T7 and 6x174. The protein products, analyzed by precipitation with antisera prepared against mitochondrial membrane proteins, and by sodium dodecyl sulfate (SDS) gel electrophoresis, were compared with the membrane proteins labeled in vivo in the presence of cycloheximide. The protein synthesized in vitro yielded on analysis seven discrete species, three of which were similar to those membrane proteins labeled in vivo; the remainder were detected as minor in vivo components. The product of transcription of X. laevis mitochondrial DNA by mi-

TABLE VII In Vitro TRANSCRIPTION PRODUCTSOF MITOCHONDFUAL DNA IN DIFFERENT ORGANISMS

Source

Sedimentation coefficient of producp

n.m.

+

30 19

19, 14, and 4s

n.m.

n.m.

12-4s

+ ( H strand)

37

Xenopus ovaries

'l

RNase resistance (%)"

20-4s n.m.

Yeast Yeast Rat liver

Hybridization with mitochondrial DNA"

n.m., Not measured.

Reference Scragg (1974) Michaelis et al. (1972) Gallerani and Saccone (1974) Dawid and Wu (1974)

142

C. SACCONE AND E. QUAGLIAFUELLO

tochondrial RNA polymerase depends on the secondary structure of the template (Dawid and Wu, 1974). By using mitochondrial DNA I (closed, circular DNA), both strands were transcribed and the H strand was preferred. With mitochondrial DNA I1 (open or nicked DNA), the H strand was almost completely preferred. By denaturation of the template, however, strand prefereuce was completely altered, since only 20% of labeled mitochondrial RNA hybridized with the H strand of mitochondrial DNA. Competition-hybridization experiments demonstrated that 15% of labeled RNA transcribed in vitro was homologous to mitochondrial DNA. This value, which nicely corresponds to the fraction of the H strand of mitochondrial DNA homologous to mitochondrial rRNA, clearly shows that mitochondrial RNA polymerase transcribes all sequences of the H strand with about equal frequency. RNA synthesized in vitro by mitochondrial RNA polymerase contains a fraction that is RNase-insensitive and which may represent, according to Dawid and Wu (1974), a helical region formed during the transcription process between one strand of the template and the nascent RNA. Using the above-mentioned system, Dawid and Wu (1974) showed furthermore that ATP is the most frequent initiator of RNA chains, followed by GTP. Pyrimidine nucleotides initiate very rarely, if at all, and mitochondrial RNA polymerase initiates on mitochondrial DNA at about six and probably more sites. Transcription of mitochondrial DNA of rat liver by homologous polymerase was carried out by Gallerani and Saccone (1974).The product was 25% RNAase-resistant and produced three main peaks on analysis. Two peaks were of high molecular weight: 22 and 18s in a sucrose gradient, and 19 and 14s on gel electrophoresis analysis; the third peak was in the range 4-5s. From these experiments it can be concluded that the study of mitochondrial DNA transcription with its homologous polymerase enzyme is still in its infancy. This is probably because the enzyme responsible for the transcription is unstable, and nothing is known about the factors and the other enzymes involved in the mitochondrial process. Studies now in progress in many laboratories will probably soon throw light on this matter. B. STUDIESin Vivo

1. Transcription of Mitochondria1 DNA in Lower Eukaryotes a. Yeast. It is well known that yeast mitochondrial DNA differs in properties, organization, and size from animal mitochondrial DNA.

MITOCHONDFUAL TRANSCRIPTION AND TRANSLATION

143

The molecular weight of yeast mitochondrial DNA appears to be about 50-60 million daltons, more than five times that of animal mitochondrial DNA. Yeast mitochondrial DNA is characterized by a low GC content which varies from 23 to 17% according to different investigators. Although most of the mitochondrial DNA molecules appear to be linear after extraction from yeast cells, it has been suggested that normal mitochondrial DNA consists of closed, circular molecules of a mean length of about 25 pm, which give rise to linear molecules during extraction (Hollenberg et al., 1970). According to Locker et al. (1974), however, circular mitochondrial DNA molecules in monomeric or oligomeric form can be extracted from petite strains. The size of the monomer varied according to the different strains and ranged from 0.13 to 5.5 pm. Hybridization studies have clearly established that a yeast mitochondrial rRNA is coded for by mitochondrial DNA. Furthermore, as for animal mitochondrial DNA, only one cistron for each of the two rRNAs is present on each DNA molecule (Reijnders et al., 1972). These ribosomal mitochondrial genes furthermore seem to be responsible of the antibiotic resistance observed in several yeast mutants (Grivell et d . , 1973; Faye et d . ,1974). Yeast mitochondria contain tRNA species differing from the corresponding cell sap tRNAs, which hybridize with mitochondrial DNA. Reijnders and Borst (1972) demonstrated that hybridization of the mitochondrial 4s RNA-32P fraction from Saccharomyces cerevisiae with mitochondrial DNA gives a plateau of 0.9 p g RNA/100 p g DNA when carried out in the presence of excess unlabeled highmolecular-weight mitochondrial RNA. From these values, and assuming a molecular weight of 55 x lo6 for mitochondrial DNA, it can be concluded that the hybridization plateau corresponds to about 20 tRNA genes. The Rabinowitz's group has also reported that the sequence of at least fourteen tRNAs is encoded in mitochondrial DNA (Casey et al., 1969, 1972, 1974). These values could indicate that in yeast mitochondria, in contrast to animal mitochondria, a complete set of tRNAs for the 20 amino acids is present; however, since it is difficult to exclude the possibility of code degeneracy in the case of yeast mitochondria, the number of 20 tRNAs is always restrictive and the presence of additional tRNA coded by nuclear genes and imported into the mitochondria cannot be excluded. b. Neurospora crassa. Neurospora, like other primitive eukaryotes, contain a mitochondrial genome which is at least five times larger than that of animal cells. A molecular weight of about 60 x 10'

144

C. SACCONE AND E. QUAGLIARIELLO

daltons has been calculated from renaturation data and agrees very well with the molecular weight expected from a contour length of a circular molecule of about 20-25 pm (Agsteribbe et al., 1972; Clayton and Brambl, 1972). An established genetic function for this DNA is that of coding rRNA and tRNA species. This conclusion is supported by hybridization studies carried out by labeling in viuo RNA b y adding labeled precursors to a culture medium of N . crussu. Although Wood and Luck (1969) first suggested that mitochondrial DNA contains at least four genes for each rRNA species, it was subsequently clearly demonstrated that no more than one gene for each rRNA species is present on the mitochondrial genome (Schafer and Kuntzel, 1972). The study of rRNA synthesis in Neurosporu mitochondria has been carried out with the pulse-labeling experiments by Kuriyama and Luck (1973). The method used by these investigators was based on continuous labeling of cells with 32Pin order to detect the stable component and, after 14 hours of preculture, on pulse-labeling for varying times with ~ r a c i l - ~in H order to detect the precursor molecules. Using this technique these workers demonstrated that rRNA species are first transcribed from mitochondrial DNA as a high-molecular-weight precursor which is cleaved to give both a large and a small RNA subunit according to the scheme in Fig. 1. It is interesting to mention here that in the mitochondria of p o k y mutants (Kuriyama and Luck, 1974) there is an alteration in the processing of the ribosomal precursor, which results in a deficient amount of ribosomal small subunits. This deficiency seems to be correlated with an alteration in the methylation process. I n these mutants a small accumulation of 32s RNA and precursors of 19s (P 19s) is firstly observed but most of these 19s precursors are degraded before they can be assembled into small subunits. However, although 25 and 19s RNA of p o k y mitochondria are clearly less

-

-1.6 x 10'

<

(P 255)

Precursor 2.4 X lo8

(P 19s)

1.28 x 10' 255

-

-0.9 x lo6

19s 0.72 X 10'

FIG.1. Scheme of N . crassa mitochondrial rRNA synthesis. (Reprinted by permission from Kuriyama and Luck, 1974.)

MITOCHONDRIAL TRANSCRIPTION AND TRANSLATION

145

methylated than their counterparts in control cells, nevertheless, the mitochondrial ribosomal subunits containing these undermethylated species are active in protein synthesis, Although Barnett and Brown (1966, 1967) reported that the mitochondria of Neurospora contain a full complement of tRNAs different from those of their cytoplasmic counterparts, the number of genes for tRNA species present on mitochondrial DNA is not as yet exactly known. Furthermore, no conclusion about the coding capacity of the two strands is possible, because of the difficulty of separation. The presence of genes for mRNA on mitochondrial DNA of Neurospora has been shown elegantly in studies with the abn-1 stopper growth mutant of N . crassa by Kuntzel et al. (1973, 1974). These investigators found that this mutant has a particulate fraction, not detectable in the wild type, which appears under the electron microscope as polymorphic vesicles with a dense nucleoid surrounded by a membrane envelope. These particles, called viruslike particles (VLP), contain single-stranded RNA sedimenting at 33s and, after heat treatment, as 7-9s with a base composition different from mitochondrial and cytoplasmic rRNA. This RNA hybridizes efficiently with mitochondrial DNA from wild-type cells, reaching a saturation plateau at 4.5% of the genome, and saturates a considerably higher portion of mitochondrial DNA from the abn-1 mutant. Competition experiments clearly suggest that the cistron coding for VLP RNA is different from the ribosomal cistron, but that it is also transcribed in wild-type mitochondria, especially in older cells, although no significant amount of 33s RNA is detected in the wild type. Since this RNA is able to direct the synthesis of a polypeptide component of molecular weight 11,000, which is probably similar to one of the four components (MW 8000, 11,000, 41,000, and 47,000) formed in the same cell-free system in the presence of the product of the transcription in vitro of mitochondrial DNA, it is concluded that this RNA has messenger activity. From these and other studies of the mutant, these investigators conclude that mitochondrial DNA from Neurospora carries a cistron for 33s RNA which is transcribed in vivo both in the wild type and in abn-1, but only in the mutant is it incorporated into VLP together with phospholipids and protein. The existence of a 33s mRNA codifying a 11,000-dalton protein suggests that one has to consider an extensive processing of RNA in Neurospora mitochondria not only for rRNA but also for mRNA. A tentative scheme for mitochondrial cistrons and their products in N . crassa is shown in Fig. 2.

146

C. SACCONE AND E. QUACLIARIELLO

mitochondrial cistrons 1

...

..I

32s RNA

I

I

- import?

I

I I

abn- 1

I

t+ I

I I

I I

subunits of cyt. b, CO, ATPase

I I

I

I.

regulatory peptides?

I

I

export

FIG. 2. Tentative presentation of mitochondrial cistrons and their products. (Reprinted by permission from Kiintzel et al., 1974.)

c. Tetrahymena pyriformis. I n this organism also it has been shown that the constituent RNAs of mitochondrial ribosomes are products of the mitochondrial genome. The number of genes for rRNA on mitochondrial DNA approaches unity, showing that in this organism also there is no redundancy of the rRNA cistron (Suyama, 1967; Chi and Suyama, 1970; Schutgens et al., 1973). It has also been demonstrated in this organism that mitochondria contain tRNA species differing from the cytoplasmic tRNAs (Suyama, 1969). Although it is not yet clear how many genes for tRNA are present on mitochondrial DNA, Chiu et al. (1974) recently showed for the first time that there exists in Tetrahymena mitochondria rather extensive degeneracy in the coding.

2 . Transcription of Mitochondria1 DNA in Animal Cells a. HeLa Cells. The transcription of mitochondrial DNA in this cell line has been extensively studied in Attardi’s and Penman’s laboratories. It has been clearly shown that the two species of rRNA in HeLa cells (Vesco and Penman, 1969a,b; Attardi et al., 1970; Robberson et al., 1971) having sedimentation coefficients in a sucrose gradient of 16 and 12s and behaving on polyacrylamide gel electrophoresis as

MITOCHONDRIAL TRANSCRIPTION AND TRANSLATION

147

21 and 18s RNA, respectively, are gene products of mitochondrial DNA. The studies of Attardi’s group have also demonstrated that the two mitochondrial rRNA species are coded for by the H strand of mitochondrial DNA. Hybridization experiments, furthermore, indicate that one gene for each of the two mitochondria-specific rRNA species is located on the H strand (Aloni and Attardi, 1971b). Twelve species of rRNA have been identified so far in mitochondria from HeLa cells. Nine genes for 4 s RNA appear to be located on the H strand, and three on the L strand of mitochondrial DNA (Wu et al., 1972). The observation that the mitochondrial DNA of HeLa cells codes for only 12 tRNA molecules, together with the evidence that rat liver mitochondrial DNA contains information for at least 4 tRNA species and Xenopus mitochondrial DNA for 15 tRNA species, raises the question whether animal cell mitochondria utilize an incomplete set of endogenous tRNA species for protein synthesis or whether the missing tRNA molecules are imported from the cytoplasm. Costantino and Attardi (1973) have suggested that mitochondrial translation products are deficient in about eight of the amino acids normally present in polypeptides, notably alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, and lysine. Most of these, it should be noted, have charged polar residues. However, if we consider the possibility of an extensive degeneracy of the genetic code in mRNA of animal mitochondria as demonstrated for Tetrahymena mitochondria (Chiu et al., 1974), we realize that 12 tRNAs are still not sufficient for the incorporation of 12 amino acids. How animal mitochondria can read their amino acid-specifying codons with such a limited number of specific tRNA species therefore remains an open question. The possibility that mitochondrial DNA of HeLa cells contains cistrons for mRNA has been recently suggested by the studies of Perlman et al. (1973) and Ojala and Attardi (1974a,b), in which the occurrence of poly(A) sequences of a distinctive size in HeLa mitochondrial RNA was first demonstrated. I n 1974, Attardi et al. (1974) reported the isolation from the mitochondrial polysome region of two discrete poly(A)-containing RNA molecules with sedimentation coefficients of 7 and 9S, corresponding to molecular weights of 8.5 x lo4 and 1.5 x lo5 daltons, respectively. The isolation of two discrete species from a fairly uniform distribution of components covering the range from 6 to 16s was possible using denaturing conditions, since poly(A)-containing RNA molecules have a tendency to aggregate and cosediment with heavier molecules. Hybridization experiments demonstrate that these poly(A)-containing RNA species

148

C. SACCONE AND E. QUAGLIARIELLO

are synthesized on mitochondrial DNA as a template. It is interesting that the 9s component is coded for by the H strand of mitochondrial DNA, while the 7s component is coded for by the L strand. This again implies that the symmetric transcription mechanism may operate within the mitochondria and suggests a possible amplification of the informational content of animal mitochondrial DNA via transcription of both strands. Very recently, Ojala and Attardi (1974c), using polyacrylamide gel electrophoresis after formaldehyde treatment, resolved the poly(A)-containing RNA, which hybridizes with the H strand of mitochondrial DNA, into seven components with molecular weights ranging between 2.6 and 5.3 x lo5 daltons. Although it has not yet been demonstrated that these poly(A)-containing RNA species possess template activity for in vitro protein synthesis, the most plausible significance is that they represent mitochondrial DNA-coded mRNAs, possibly individual messenger species. It is interesting to note that both the number and the molecular-weight range of the poly(A)-containing RNA components are in accordance with the number and size of the products of mitochondrial protein synthesis in vivo in HeLa cells that Costantino and Attardi (in preparation) report: 10 discrete species with a molecular weight between 10,000 and 50,000 daltons. According to Hirsch and Penman (1973), the size of poly(A) found in mitochondrial RNA is 4s; the polyadenylic acid sequence is approximately 56 nucleotides in length and is located at the 3’ end of the RNA molecule. This RNA hybridizes with mitochondrial DNA and after isolation can be resolved into eight distinct species b y acrylamide gel electrophoresis. Furthermore, these investigators showed that the addition of 4 s poly(A) to mitochondrial RNA is a posttranscriptional event (Hirsch and Penman, 1974). It is interesting that the presence of a poly(A) sequence in a messengerlike mitochondrial RNA differentiates the mitochondrial and bacterial macromolecular-synthesizing systems, since no poly(A) has yet been found in prokaryote cells. This could argue against the hypothesis that mitochondria are derived from primordial prokaryotes. It must be recalled however that according to Groot et al. (1974) only animal mitochondria contain poly(A)-mRNA (see also p. 140) and this could mean according to authors an adaption of a prokaryotic type-genetic system to the cell organization of higher eukaryotes. It has been demonstrated by Aloni and Attardi (1971a) that in HeLa cells, and probably in all mitochondria, a particular transcription mechanism operates, that is, a complete or almost complete symmetric transcription of both strands of mitochondrial DNA followed

MITOCHONDRIAL TRANSCRIPTION AND TRANSLATION

149

by specific degradation which leaves undegraded only certain RNA species. The hybridization of mitochondrial RNA from HeLa cells exposed to short pulses of uridine-S3H showed that the labeled RNA hybridizes with both the L and H strands of mitochondrial DNA to an extent that depends on the labeling time. In particular, when the pulse is very short ( 1 5 minutes), the hybridization of RNA is about equal for the two strands, but becomes more and more predominant for the H strand on increasing the pulse length. Furthermore, the pulse-labeled, fast-sedimenting mitochondrial RNA forms a structure resistant to RNase when self-annealed or annealed with an excess of unlabeled mitochondrial RNA. These results have been interpreted to mean that mitochondrial DNA is transcribed in HeLa cells symmetrically, and that the major portions of the L-strand product are successively rapidly degraded or removed from the mitochondrial fraction. Products of the transcription of the L strand have been so far identified as three genes for mitochondrial tRNA and probably the gene for 7s mRNA (see above). Other evidence has suggested that the H strand of mitochondrial DNA is transcribed in the form of continuous long chains (Attardi et al., 1970). This has been more directly demonstrated by the isolation and characterization of the transcription complexes of mitochondrial DNA as long as expected for complete transcripts (Aloni and Attardi, 1972). By electron microscopy of RNA-DNA and DNA-DNA hybrids, it has been possible to construct a genetic map of HeLa cell mitochondrial DNA (Attardi et al., 1974). The two genes for 16 and 12s rRNA have lengths of 0.47 A 0.07 and 0.26 I ~0.04 I pm, respectively. They map on the H strand and are separated by a region of DNA corresponding only to 160 ~t60 base pairs. On the H strand there are nine genes for 4 s tRNA. One of them is located between the 12 and 16s genes, another in the region immediately adjacent to the 12S, and a third immediately after the 16s gene. The other genes are more-or-less uniformly distributed along the H strands, only two being grouped in the same region. The three genes for tRNAs localized on the L strand are separated from each other by 2280 and 3900 nucleotides. Figure 3 shows a circular genetic map which includes information derived from the biochemical studies discussed above. b. Xenopus laevis. From the study of mitochondrial DNA transcription in this organism, it appears that rRNA and 4s RNA are coded on mitochondrial DNA. No evidence for the presence of mitochondrial mRNA has been so far found in this organism. Hybridization experiments have clearly shown that each circular molecule of mitochondrial DNA having a molecular weight of

150

C. SACCONE AND E. QUAGLIARIELLO

H8 HI

H strand

FIG.3. Circular map of the positions of the complementary sequences for 4 s RNAs on the H and L strands of HeLa mitochondrial DNA, and of the 12 and 16s rRNA genes on the H strand. (Reprinted by permission from Attardi et al., 1974.)

11.7 x los contains one gene for each mitochondrial rRNA molecule (Dawid, 1972a), whose estimated molecular weight is 5.3 X lo5 for large rRNA and 3 x lo5 for small rRNA (Dawid and Chase 1972), and that these genes are localized on the H strand of mitochondrial DNA (Dawid, 1972a). These molecular-weight values closely resemble those determined by electron microscopy for the mitochondrial rRNA in HeLa cells (5.5 and 3.5 x 1@), suggesting that they are common to all mitochondrial rRNA of animal cells. A 4s RNA hybridizing with mitochondrial DNA can be isolated from mitochondria of X . Zaevis. It represents a tRNA species whose molecular weight has been estimated at about 28,000 daltons. Hybridization-saturation experiments performed to determine what fraction of DNA is transcribed into tRNA species suggest that each molecule of X . Zaevis mitochondrial DNA contains 15 sequences coding for 4s RNA. Most of these sequences for 4s RNA seem to be located on the H strand of mitochondrial DNA, although some hybridization of the L strand with 4s mitochondrial RNA has been detected (Dawid, 1972a). The number of tRNA genes found in mitochondrial DNA of Xenopus also resembles that found in HeLa cells (12 genes) and implies the same problem raised in relation to mitochondrial translation in HeLa cells. In this case also, Dawid suggests two possibilities: (1)mitochondria use only a limited set of 15 tRNAs for protein synthesis, and (2) mitochondria import additional tRNA molecules from the cytoplasm. The latter possibility is supported by the finding that mitochondrial preparations contain cytoplasmic tRNAs; however, whether or not these tRNAs are always contaminants of the extramitochondrial cytoplasmic fraction is not known.

MITOCHONDRIAL TRANSCRZPTION AND TRANSLATION

151

RNA and tRNA sequences together account for about 20% of the information content of mitochondrial DNA in Xenopus ovaries. According to Dawid, another 16%of DNA is represented in rare, poorly identified RNA molecules (probably precursors), whereas the transcripts of the remaining sequences of mitochondrial DNA in uiuo are absent or very rare. The work of Dawid on Xenopus suggests also either that mitochondrial DNA is not transcribed in its entirety in the ovary, or that a large fraction of the RNA turns over so fast that it is rarely found in a steady-state population. In this respect Dawid (1972a) suggests that the only function of mitochondrial DNA can be to code for the rRNA and tRNA species, the remaining sequences may be transcribed partly as precursor molecules and partly as species with no coding ability. This implies that mitochondrial protein synthesis takes place on mRNAs imported from the nucleus (Swanson, 1971; Grivell and Metz, 1973). Such a possibility does not take into account the fact that protein synthesis with isolated mitochondria of animal cells is inhibited b y actinomycin D or rifampicin inhibitors of the mitochondrial transcription, which suggests that the process requires the concomitant transcription of the mitochondrial genome. Furthermore, it remains to b e substantiated by other experimental evidence. c. Rat Liver. From in uiuo labeling studies, Aaij and Borst (1970) concluded in 1970 that rat liver mitochondria contain three components that hybridize with mitochondrial DNA. They suggested that these components represent the stable mitochondrial RNA species with apparent molecular weights of approximately 6.6 x lo5, 3.4 x lo5, and 27 x103 as measured by electrophoretic mobility on polyacrylamide gel. Further studies (see Section III,A, 1) confirmed that the two larger species derive from mitochondrial ribosomes which in rat liver sediment with a 50-60s sedimentation coefficient, as do probably all mitochondrial ribosomes from animal cells. The smaller component migrating as RNA, with a molecular weight of 27 X lo3, represents tRNA better characterized by Nass and Buck (1970). Although from previous studies Borst and Aaij (1969) suggested that the stable mitochondrial RNA species labeled in uivo hybridizes only with the H strand of mitochondrial DNA, on the basis of further experiments Nass and Buck (1970) demonstrated that the transcription sites of individual tRNAs are located on either one of the complementary strands of mitochondrial DNA. In particular it was found that leucyl and phenylalanyl tRNA hybridize exclusively with the H strand, whereas tyrosyl and seryl tRNA hybridize exclusively with the L strand of mitochondrial DNA. Here again the situa-

152

C. SACCONE AND E. QUAGLIARIELLO

tion recalls that described b y Attardi et al. for mitochondria of HeLa cells. In this respect it seems probable that in all mitochondrial DNA molecules from animal cells the L strand of the mitochondrial genome specifies some tRNA species, whereas its coding capacity for mRNA remains restricted so far only to HeLa cells.

111. Mitochondrial Translation A. THE PROTEIN-SYNTHESIZING MACHINERY OF MITOCHONDRIA

1. Mitochondrial Ribosomes Because of the sensitivity of mitochondrial ribosomes to many antibiotics that interfere with 70s prokaryotic ribosomes, whereas they are insensitive to inhibitors of cell sap protein synthesis, it was initially suggested that mitochondria contain “bacterialtype” ribosomes. However, the present availability from many organisms of purified fractions of mitochondrial ribosomes that synthesize protein very actively has allowed extensive and rigorous characterization and has also settled most of the controversies previously reported in the literature. Much of the older literature on this subject has been reviewed excellently by Borst and Grivell (1971),and recent results have been discussed at a conference (Kroon and Saccone, 1974). We summarize here the better-documented data and discuss some topics of current controversy. Mitochondrial ribosomes from all organisms studied have several properties in common, such as dissociation at rather high ratios of Mg2+ to monovalent cations, a rather loosely folded structure of rRNA, and a high protein/RNA ratio (see Borst, 1972, for review). As regards other properties, there appears to b e some difference between mitochondrial ribosomes from lower eukaryotes and from animal cells. The two groups are therefore treated separately. a. Ribosomes from Lower Eukaryotes. It seems generally accepted that in yeast cells, from the rate of sedimentation, mitochondrial ribosomes appear to be generally slightly larger than ribosomes from E . coli. Mitochondrial ribosomes synthesizing protein very actively, isolated by Grivell et al. (1971a) from Saccharomyces curlsbergensis, appeared as 74s particles which dissociated into 50 and 37s subunits containing RNA sedimenting in a sucrose gradient as 22 and 15S, respectively. Mitochondrial rRNA in yeast, as in other organisms, has an unusual secondary structure, and its molecular

MITOCHONDRIAL TRANSCRIPTION AND TRANSLATION

153

weight can be determined only by using methods not affected by variation in the secondary structure of RNA. Average molecular weights of 1.30 and 0.70 x lo6 for the two species of rRNA were obtained by Reijnders et al. (1973), using different methods. These Values are higher than those for the rRNAs from E . coli (1.10 and 0.56 X lo6). In the case of yeast, it is recalled that altered mitochondrial ribosomes have been isolated from mutants resistant to several antibiotics (Grivell et aZ., 1971b) as the result of a mutation in mitochondrial DNA. Identification of the components altered in these mutants could give information concerning the genetic function of mitochondrial DNA. Thus far, however, no alteration in ribosomal proteins from mutant cells has been discovered, and it has been suggested that mitochondrial rRNA is the component altered (see also Section II,B,l). Extensive characterization of mitochondrial ribosomes from Candida utilis has been reported by Vignais et al. (1972). According to these workers, mitochondrial ribosomes sediment as a 72s particle dissociating into 50 and 36s subunits, and an artifactual dimerization of these particles could account for the accumulation, under certain conditions, of 80s particles reported several times to be mitochondrial ribosomes. From mitochondria of N . crassa, two types of ribosomes with sedimentation values of 73 and 80s have been recently isolated b y Agsteribbe et al. (1974). Although their function and physical characterization indicate that both particles are of mitochondrial origin, according to these investigators only the 80s particle, the monomer form found in mitochondrial polysomes actively involved in protein synthesis, represents the native mitochondrial ribosome. An 80s sedimentation coefficient is also displayed by mitochondrial ribosomes from T . pyrijormis (Suyama, 1969; Stevens et al., 1974). They, however, can be easily distinguished from their cytoplasmic counterparts by analysis of their heavy and light rRNA components, by their buoyant density in cesium chloride, and by their fine structure. An unusual property of the Tetrahymena mitochondriaI ribosome is its dissociation into subunits with identical sedimentation coefficients (Chi and Suyama, 1970; Stevens et al., 1974). Extraction of functional polysomes from mitochondria has also been recently reported. Cooper and Avers (1974) isolated mitochondrial polysomes from yeast and demonstrated the presence in these particles of a poly(A)-containing mRNA (see Section II,A,3). From Neurospora, Agsteribbe et al, (1974) isolated mi-

154

C. SACCONE AND E. QUAGLIARIELLO

tochondrial polysomes whose mRNA directed protein synthesis when tested in a cell-free system from E . coli. Extraction of active polysomes from E . grucilis was also obtained by Avadhani and Butow (1972). Recent data of Moorman and Grivell (1974), however, raise serious doubts on these reports. The authors claim that it is possible to demonstrate the presence of a polysomal fraction only using mitochondria prepared in Mg-containing media as performed in the above mentioned papers (Avadhani and Butow, 1972; Cooper and Avers, 1974; Agsteribbe et al., 1974). In this condition, however, the mitochondria contain a 80:20% mixture of cytoplasmic and mitochondrial RNAs probably because magnesium promotes aggregation of microsomal membrane and association of cytoplasmic ribosomes with mitochondria. On the basis of these results the authors conclude that the polysomes present in Mg-washed mitochondria are of cell-sap origin and suggest, as already proposed by others (Bunn et a1., 1970) that mitochondrial ribosomes are sufficiently attached to the mitochondrial membrane to prevent the isolation of the mRNAribosome complex. It must be added, however, that typical polysomes structures in mitochondria have been shown by electron microscopy (Vignais et ul., 1969) and reported also from EDTAwashed mitochondria (Ojala and Attardi, 1972; Michel and Neupert,

1973). b. Ribosomes from Animal Cells. Mitochondria1 ribosomes from animal cells have been called “miniribosomes” because of their unusually low sedimentation coefficient (55-60s). Also, the availability of purified preparations of mitochondrial ribosomes has led to better physicochemical and functional characterization of these particles. From the studies of various investigators who used different approaches (Klienow et al., 1974; O’Brien et al., 1974; d e Vries and Kroon, 1974; Saccone et al., 1974), it has emerged quite clearly that “miniribosomes” are less “mini” than their rRNA (16-17s and 12-14s) suggests. The unusual composition of these particles, in particular their low RNA content and/or disproportionately high protein content, could account for their low sedimentation coefficient, low buoyant density, and electrophoretic behavior. In the electron microscope mitochondrial ribosomes from animal cells appear larger than E . coli ribosomes, and of a size comparable to that of cytoplasmic ribosomes (Greco et al., 1974). It must be stressed, however, that often the dimensions of animal mitochondrial ribosomes in tissue section appear smaller than in isolated preparations (Klienow et al., 1974), probably because of the difficulty in fixing particles of peculiar structure and chemical composition in situ. Greco et al. (1973)

MITOCHONDRIAL TRANSCRIPTION AND TRANSLATION

155

isolated from rat liver mitochondria a ribosomal fraction highly active in poly(U)-directed polyphenylalanine synthesis when assayed in the presence of supernatant factors from E . coli. By using this system the effect of several antibacterial antibiotics whose action at the level of animal mitochondrial ribosomes was controversial has been tested. Table VIII summarizes data on the sensitivity of mitochondrial protein synthesis to some antibiotics in yeast and rat liver. From these data it can clearly be seen that most antibiotics that affect mitochondrial ribosomes from protists also affect animal mitochondrial ribosomes, although to a different extent (Saccone et al., 1974). The suggestion of de Vries et al. (1973) that only a slight difference in this property exists between the two classes of mitochondrial ribosomes from lower and higher eukaryotes is more likely than the proposal of Linnane's group that there is a phylogenetic difference (Davey et al., 1970; Towers et al., 1972). The resistance of mammalian mitochondrial protein synthesis to the same antibiotics must therefore be due in some cases to the impermeability of the mitochondrial membrane (Kroon and de Vries, 1970). c. Origin of Mitochondrial Ribosomal Proteins. As regards the origin of proteins of mitochondrial ribosomes, several workers showed that the bulk of ribosomal protein is synthesized on cell sap ribosomes (Kuntzel, 1969; Schmitt, 1972; Lizardi and Luck, 1972; Neupert et al., 1969). However, the possibility that some ribosomal proteins can b e synthesized (Millis and Suyama, 1972) on mitochondrial

SENSITIVITY

OF

TABLE VIII MITOCHONDRIAL PROTEIN SYNTHESIS Mitochondria"

Antibiotic

Yeast

Rat liver

TO

ANTIBIOTICS Mitochondrial ribosomes, rat liveP

Lincomycin Erythromycin Oleandomycin Carbomycin Spiramycin Oxytetracycline Neomycin Kanamycin ~

a ++, 50-100% inhibition; +, 30-50% inhibition; 2, 15-30% inhibition; -, >15% inhibition; n.m., not measured. (a) = Firkin and Linnane (1969); (b) = Goon and de Vries (1971);(c) = S x c o n e e t a / . (197413);(d) = Ibrahim and Beattie (1973);( e )= Towers et al. (1972); (f) = Clark-Walker and Linnane (1966); (9) = Davey et al. (1970).

156

C. SACCONE AND E. QUAGLIARIELLO

ribosomes has also been put forward. Recently, Groot (1974) demonstrated in yeast that probably all the ribosomal proteins necessary for a functional mitochondrial ribosome are synthesized on cytoplasmic ribosomes. According to him, the component with a molecular weight of 35,000 that he demonstrated to be synthesized on mitochondrial ribosomes is not necessary for ribosomal function and may be an initiation factor or a link between ribosomes and the mitochondrial membrane.

2. Enzymes and Factors lnvolved in Mitochondria1 Protein Synthesis Very little is known about factors and enzymes involved in mitochondrial protein synthesis. Mitochondria-specific aminoacyl RNA synthetases have been reported to occur in N. crassa (Barnett et al., 1967) and in T . pyriformis (Suyama and Eyer, 1967). The enzymes are distinct from their cytoplasmic counterparts. It is well established that mitochondrial ribosomes from yeast, Neurospora, and rat liver exhibit high activity in a poly(U)-directed cell-free system for protein synthesis when combined with supernatant enzymes from bacteria (Grivell et al., 1971a; Kuntzel, 1969; Greco et al., 1973). This suggests that mitochondria that, like bacteria, use N-formyl methionine as an initiator in polypeptide synthesis (Smith and Marcker, 1968; Epler et al., 1970) contain factors similar to those of prokaryote cells. Mitochondrial factors G and T have been isolated from some organisms and separated using hydroxylapatite chromatography (Richter and Lipman, 1970; Scragg, 1971; Grandi and Kuntzel, 1970). According to Richter and Lipman (1970), mitochondrial elongation factors of yeast are of the prokaryote type and differ from cytoplasmic elongation factors in molecular weight and mobility on chromatographs. Furthermore, these workers showed that mitochondrial factors and ribosomes are interchangeable with bacterial factors and ribosomes, whereas cytoplasmic ribosomes respond only to the mitochondrial binding factor T as they respond to the bacterial T. The interchangeability of mitochondrial and cytoplasmic T factors was not, however, confirmed by the work of Scragg (1971), who demonstrated an absolute specificity of mitochondrial elongation factors for 70S-type ribosomes. Preliminary results seem to indicate that the mitochondrial T factor can be split into subfractions, Ts and Tu (Richter, unpublished data). The molecular weight of the mitochondrial T factor is estimated to be about 60,000 (Albrecht et d., 1970). Also, in N. crassa the mitochondrial system can be in-

MITOCHONDRIAL TRANSCRIPTION AND TRANSLATION

157

terchanged with the E . coli system, but not with the cytoplasmic system from the same cell or from rat liver. Similar results demonstrating a interchangeability between mitochondrial and E . coli systems has been recently obtained with systems from X . laevis ovary (Swanson, 1973) and E . gracilis (Avadhani and Butow, 1974). As regards the origin of the translation factors involved in mitochondrial protein synthesis, it has been shown that in yeast (Parisi and Cella, 1971; Scragg, 1971; Richter, 1971) and in Neurospora (Barath and Kuntzel, 1972) the genetic information for polypeptide chain elongation factors of mitochondria is encoded by nuclear DNA and such information is translated on cytoplasmic ribosomes. Similar data have been reported relating to the origin of a mitochondrial translation factor from Chlorella vulgaris (Ciferri et al., 1974). B. THE TRANSLATION PRODUCTS

The experimental approaches used to study mitochondrial translation products have been extensively illustrated and discussed in several reviews and papers (Ashwell and Work, 1970; Borst, 1972; Wheeldon, 1973; Schatz and Mason, 1974) and are not considered here, where only some discussed data are summarized. With regard to the role of the proteins synthesized using mitochondrial DNA as template, it has been suggested that they can have two kinds of function: a repressor-type function at the level of the nuclear genome, which should ensure coordination between nuclear and mitochondrial biosynthesis processes (Barath and Kuntzel, 1972), and a constitutive function as part of the protein component of the inner mitochondrial membrane. It is well known that the products of mitochondrial protein synthesis so far identified are subunit proteins of enzymes of the mitochondrial inner membrane. Studies from several laboratories performed with yeast and Neurospora indicate that three proteins of cytochrome c oxidase (Sebald et al., 1973; Mason et al., 1973) and four proteins of the rutamycin-sensitive ATPase complex (Tzagoloff et al., 1974) are translated on mitochondrial ribosomes. To these four candidates we now add, from the recent studies of Ross et al. (1974), a protein necessary for the assembly of cytochrome c and, from the studies of Weiss and Ziganke (1974) on Neurospora, one or two components of the cytochrome b complex. There is not yet any direct evidence that the genes for these proteins are located on mitochondrial DNA, however, there is also no clear support for translation at the level of mitochondrial ribosomes of imported cytoplasmic mRNA (Mahler and Dawidowicz, 1973). Therefore, if we assume that the genes for the

158

C. SACCONE AND E . QUAGLIARIELLO

various polypeptides mentioned before are localized on mitochondrial DNA, we can calculate on the basis of their assumed molecular weights about 4-5 x los daltons of organelle DNA is involved in the synthesis of these components. This represents a maximum value for the information content of animal mitochondrial DNA (MW 10 x los daltons), although in animal cells almost nothing is known about the product of mitochondrial protein synthesis. It has been well established that the products of mitochondrial protein synthesis are extremely hydrophobic (Tzagoloff and Akai, 1972). Attardi et al. (1974) have even suggested that mitochondrial translation products are deficient in about eight of the amino acids normally present in polypeptides (see also Section II,B,2), These facts, together with the finding that the proteins are subunit parts of mitochondrial enzymes, raised several interesting questions, such as:

1. What is the mechanism by which mitochondrial translation products are released from ribosomes and then transferred from their site of synthesis to their site of function? 2. What is the assembly process of mitochondrial and cytoplasmic products of protein synthesis that gives rise to the final functional enzymes? In Michel and Neupert’s (1973) opinion, the systems of mitochondrial transcription and translation have been maintained (according to the endosymbiont theory) or created (according to the episomic theory) just because the translation products are so hydrophobic they cannot be transported through the cytoplasm and have to be delivered directly to their site of function (the inner membrane). With reference to this, these investigators studied the properties of mitochondrial translation products before and after their integration into the membrane. They found that nascent polypeptide chains at the level of mitochondrial ribosomes have an apparently uniform molecular weight. This implies that completed chains are processed after their synthesis, so that the mitochondrial translation products found in the enzyme complex, such as cytochrome aa,, cytochrome b, and ATPase are not original translation products but are generated by the conversion of peptides with lower apparent molecular weights. Evidence for the synthesis from mitochondria of a single product of relatively low-molecular-weight also derives from the studies of Tzagoloff and Akai (1972). These investigators found that when yeast is incubated in a radioactive medium under conditions in which cytoplasmic protein synthesis is blocked, five mi-

MITOCHONDFUAL TRANSCRIPTION AND TRANSLATION

159

tochondrial proteins appear to be labeled, and the major product appears to be a protein with an apparent molecular weight of 45,000. This protein, however, undergoes a reduction in size when the membranes are treated with an organic solvent or depolymerized in SDS in an alkaline medium. Under these conditions almost all components appear in the region of a polypeptide with an apparent molecular weight of 7800. Based on these results, these workers suggested that in final products this polypeptide is either present in polymeric form or associated with other components having higher molecular weights. Recent papers of Kuntzel and Blossey (1974) and Wheeldon et al. (1974) seem in agreement with this proposal. In v i t r o studies of Wheeldon et al. (1974) show that the major product of mitochondrial protein synthesis in rat liver is not finished protein but nascent peptide bound to mitochondrial ribosomes. This product has an exceptional grade of hydrophobicity and is released by heat treatment of mitochondrial ribosomes as a fraction with an apparent molecular weight of 12,000-13,000 daltons in SDS-gel electrophoresis. Kuntzel and Blossey (1974) also report that the major translation products of a submitochondrial fraction from N . crussa are two polypeptides having molecular weight of 10,000 and 12,000 daltons. These products, that show a high tendency to aggregate, correspond in apparent molecular weight to the two products synthesized in v i t r o by an E . coli cell-free system in the presence of mitochondrial mRNA transcribed by mitochondrial DNA with E . coli polymerase enzyme. Synthesis of “proteolipids” from mitochondria has also been reported b y Hadvary and Kadenbach (1973), Murray and Linnane (1972), and Burke and Beattie (1973). According to Hadvary and Kadenbach (1973), rat liver mitochondria should synthesize a number of different proteins soluble in chloroform and having a molecular weight in the range from 8,000 to 30,000 daltons, while Burke and Beattie (1973) identified seven peaks: only one of these bands, having a molecular weight of 40,000 daltons, was extracted mainly with chloroform: methanol. On the other hand, Murray and Linnane (1972) demonstrated that proteolipids are not the sole products of protein synthesis in yeast mitochondria. T h e significance of these “proteolipids” synthesized and extracted from mitochondria is still unknown, and, as pointed out by Schatz and Mason (1974),they require better characterization. As regards the hypothesis that mitochondria are able to synthesize one single product of relatively low molecular weight, we would like to mention here that the finding of Attardi et al. (1974) and Hirsch and Penman (1974) that several different species of mRNA are present in the mitochondria of HeLa cells

160

C. SACCONE AND E. QUAGLIAFUELLO

does not support the proposal of Neupert and Tzagoloff. As far as the assembly process of mitochondrial and cytoplasmic products of protein synthesis is concerned, this has been particularly well studied in yeast and Neurospora (see Schatz and Mason, 1974, for review). It has clearly emerged that a coordination between the processes of mitochondrial and cytoplasmic protein synthesis must exist (Ross et al., 1974; Mahler et al., 1974; Beattie et al., 1974). Little is also known about the problem of the transfer of the proteins synthesized by cytoplasmic ribosomes to mitochondria. Among the hypotheses raised we would like to mention that of Butow (Kellems et al., 1974), according to which there is a selective synthesis of mitochondrial proteins by a special class of mitochondrial-associated cytoplasmic ribosomes which could allow a vectorial discharge of polypeptides into mitochondria. This hypothesis, however, although attractive, requires further experimental substantiation. NOTE ADDED IN PROOF Since this review was completed, new literature has appeared relevant to the subjects discussed. In particular, by concerted genetic and biochemical experiments it has been possible to start to construct, in several organisms, a map of the mitochondrial DNA as far as the rRNA and tRNA genes are concerned. Great progress has also been made in the identification of the proteins synthesized in the mitochondria by immunological methods. On the other hand, other problems have received less attention, for example, the identification of mitochondrial mRNA species, of the enzymes, factors, and mechanism of mitochondrial transcription, as well as the problem of the transport of the cytoplasmically synthesized mitochondrial proteins within the organelles, require concerted efforts by different experimental techniques. Nevertheless we hope that our goal in this review, the description of the biochemical approaches to the problem of mitochondrial transcription and translation, has been attained, and that it may serve to encourage further efforts with those problems that remain outstanding.

REFERENCES Aaij, C., and Borst, P. (1970).Biochim. Biophys. Acta 217, 560. Aaij, C., Saccone, C., Borst, P., and Gadaleta, M. N. (1970). Biochim. Biophys. Acta 199, 373. Agsteribbe, E., Kroon, A. M., and Van Bruggen, E. F. J. (1972). Biochim. Biophys. Actu 269, 299. Agsteribbe, E., Datema, R., and Kroon, A. M. (1974). In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 305314. Academic Press, New York. Albrecht, U., Prenzel, K., and Richter, D. (1970).Biochemistry 9, 316. Aloni, Y., and Attardi, G. (1971a).Proc. Nut. Acud. Sci. U.S. 68, 1757. Aloni, Y., and Attardi, G. (1971b).J. Mol. Biol. 55, 271. Aloni, Y., and Attardi, G. (1972).J. Mol. Biol. 70, 363. Ashwell, M., and Work, T. S . (1970).Annu. Rev. Biochem. 39, 251. Attardi, G., Aloni, Y., Attardi, B., Ojala, D., Pica-Mattoccia, L., Robberson, D., and Stonie, €3. (1970). Cold Spring Harbor Symp. Quant. Biol. 35, 599.

MITOCHONDRIAL TRANSCRIPTION AND TRANSLATION

161

Attardi, G., Costantino, P., and Ojala, D. (1974).In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 9-29. Academic Press, New York. Avadhani, N. G., and Buetow, D. E. (1971). Biochem. J. 140,13. Avadhani, N. G., and Buetow, D. E. (1972) Biochem. J., 128,353. Avadhani, N. G., Kaun, M., VanDer Lign, P., and Rutman, R. J. (1973). Biochem. Biophys. Res. Commun. 51, 1090. Barath, Z., and Kuntzel, H. (1972). Proc. Nut. Acud. Sci. U S . 69, 1371. Barnett, E., Brown, D. H., and Epler, J. L. (1967). Proc Nut. Acad. Sci. U.S.57, 1775. Barnett, W. E., and Brown, D. H. (1966). Science 154,417. Barnett, W. E., and Brown, D. H. (1967). Proc. Nat. Acud. Sci. U.S. 57,452. Beattie, D. S., Lin, L.-F. H., and Stuchell, R. N. (1974). In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 4 6 M 7 5 . Academic Press, New York. Boardman, N. K., Linnane, A. W., and Smillie, R. M. (1971). “Autonomy and Biogenesis of Mitochondria and Chloroplasts.” North-Holland Pub]., Amsterdam. Borst, P. (1972).Annu. Reu. Biochem. 41, 333. Borst, P., and Aaij, C. (1969).Biochem. Biophys. Res. Commun. 34, 358. Borst, P., and Flavell, R. A. (1972).In “Mitochondria: Biogenesis and Bioenergetics; Biomembranes: Molecular Arrangements and Transport Mechanisms” (S. G. Van den Bergh, P. Borst, L. L. Van Deenen, J. C. Riemersma, E. C. Slater, and J. M. Tager, eds.), Vol. 28, pp. 1-19. North-Holland/Amer. Elsevier, Amsterdam. Borst, P., and Grivell, L. A. (1971).F E B S (Fed. Eur. Biochem. Soc.), Lett. 13, 73. Borst, P., and Grivell, L. A. (1973).Biochimie 55, 801. Borst, P., and Kroon, A. M. (1969). Int. Rev. Cytol. 26, 107. Bunn, C. L., Mitchell, C. H., Lukins, H. B., and Linnane, A. W. (1970).Proc. Nat. Acad. Sci. U.S. 67, 1233. Burke, J . P., and Beattie, D. S. (1973). Biochem. Biophys. Res. Commun. 51, 349. Busiello, E., Di Girolamo, A., Di Girolamo, M., Fischer-Fantuzzi. L., and Vesco, C. (1973).Eur. J. Biochem. 35,251. Cantatore, P., Nicoba, A., Loria, P., and Saccone, C. (1974) Cell Different. 3, 45. Casey, J., Fukuhara, H., Getz, G. S., and Rabinowitz, M. (1969).J.Cell. Biol. 43, 18A. Casey, J., Cohen, M. Rahinowitz, M., Fukuhara, H., and Getz, G. S. (1972).J. Mol. Biol. 63, 431. Casey, J., Hsu, H. J., Getz, S. G., Rabinowitz, M., and Fukuhara, H. (1974).J. Mol. Biol. 88, 735. Chi, J . C. H., and Suyama, Y. (1970).J.Mol. Biol. 53, 531. Chiu, N., Chiu, A. 0. S., and Suyarna, Y. (1974).In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 383-394. Academic Press, New York. Ciferri, O., Tiboni, O., Lazar, G., and Van Etten, J. (1974). In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 107-115. Academic Press, New York. Clark-Walker, G. D., and Linnane, A. W. (1966). Biochem. Biophys. Res. Conmiin. 25, 8. Clayton, D. A,, and Brambl, R. M. (1972). Biochem. Biophys. Res. Commun. 46, 1477. Cooper, C. S., and Avers, C. J. (1974).In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 289-303. Academic Press, New York. Costantino, P., and Attardi, G. (1973).Proc. N u t . Acud. Sci. U.S. 70, 1490. Davey, P. J., Haslam, J. M., and Linnane, A. W. (1970).Arch.Biochem. Biophys. 136,54. Dawid, I. B. (19724.J. Mol. Biol. 63, 201. Dawid, I. B. (1972b).Deoelop. B i d . 23, 139.

162

C. SACCONE AND E. QUAGLIAFUELLO

Dawid, I. B., and Chase, J . W. (1972).J. MoZ. Biol. 63, 217. Dawid, I. B., and Wu, G . J. (1974).In “The Biogensis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 79-88. Academic Press, New York. de Montalvo, A., Guerritore, D., and Gadaleta, M. N. (1974). MoZ. Cell. Biochem. de Vries, H., and Kroon, A. M. (1974).In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 357365. Academic Press, New York. de Vries, H., Arendzen, A. J., and Kroon, A. M. (1973).Biochim. Biophys. Acta 331,264. Eccleshall, T. R., and Criddle, R. S. (1974). In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 31-46. Academic Press, New York. Epler, J., Shugart, L., and Barnett, W. (1970). Biochemistry 9, 3575. Faye, G. Kujawa, C., Fukuhara, H, (1974)./. MoZ. Biol. 88, 195. Firkin, F. C., and Linnane, A. (1969). F E B S (Fed. Eur. Biochem. S O C . ) , Lett. 2, 330. Fukamaki, S., Bartoov, B., Mitra, R. S., and Freeman, K. B. (1970).Biochem. Biophys. Res. Coinmun. 40, 852. Gadaleta, M. N., and Saccone, C. (1973).Boll. Soc. Ital. Biol. Sper. 49, 806. Gadaleta, M. N., and Saccone, C. (1974).I n “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 85-88. Academic Press, New York. Gadaleta, M. N., Greco, M., and Saccone, C. (1970).FEBS (Fed. Eur. Biochem. S O C . ) , Lett. 10, 54. Gallerani, R., and Saccone, C. (1974).In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 59-69. Academic Press, New York. Gallerani, R., Saccone, C., Cantatore, P., and Gadaleta, M. N. (1972).FEBS (Fed. Eur. Biocheni. Soc.), Lett. 22, 37. Gamble, G . J., and McCluer, H. R. (1970).]. MoZ. Biol. 53, 557. Grandi, M., and Kuntzel, H. (1970). FEBS (Fed. Eur. Biochem. Soc.), Lett. 10, 25. Grant, W. D., and Poulter, T. M. (1973).]. MoZ. Biol. 73, 439. Greco, M., Cantatore, P., Pepe, G., and Saccone, C. (1973).Eur. J. Biochem. 37, 171. Greco, M., Pepe, G., and Saccone, C. (1974). In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 367476. Academic Press, New York. Grivell, L. A., and Metz, V. (1973).Biochem. Biophys. Res. Commun. 55, 125. Grivell, L. A., Reijnders, L., and Borst, P. (1971a). Biochim. Biophys. Acta 247, 91. Grivell, L. A., Reijnders, L., and de Vries, H. (1971b).FEBS (Fed. Eur. Biochem. Soc.), Lett. 16, 159. Grivell, L. A., Netter, P., Borst, P., and Slonimski, P. P. (1973).Biochim. Biophys. Acta 312, 358. Groot, G . S . P. (1974).In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 443-452. Academic Press, New York. Groot, G. S. P., Flavell, R. A., Van Ommen, G. J. B., and Grivell, L. A. (1974).Nature (London),New Biol. 252, 167. Hadvary, P., and Kadenbach, B. (1973).Eur.1. Biochem. 39, 11. Hirsch, M., and Penman, S. (1973).J.Mol. Biol. 80, 379. Hirsch, M., and Penman, S. (1974).J.MoZ. Biol. 83, 131. Hollenberg, C. P., Borst, P., and Van Bruggen, E. F. J. (1970). Biochim. Biophys. Acta 209, 1. Ibrahim, N . G., and Beattie, D. S. (1973).FEBS (Fed. Eur. Biochem. Soc.), Lett. 36, 102. Jackisch, R., Jung, A., Schlegel, W., and Mayer, D. (1972).Hoppe-SeyZer’s 2. PhysioZ. Chem. 353, 1705. Jacob, S. T., and Schindler, D. G. (1972).Biochem. Biophys. Res. Commun. 48, 126. Jacob, S. T., Rose, K. M., and Morris, H. P. (1974).Biochim. Biophys. Acta 361, 312.

MITOCHONDFUAL TRANSCRIPTION AND TRANSLATION

163

Kalf, G. F. (1964).Biochemistry 3, 1702. Kellems, R. E., Allison, V. F., and Butow, R. W. (1974).J. Biol. Chem. 249,3297. Kleinow, W., Neubert, W., and Miller, F. (1974).I n “The Biogenesis of Mitochondria“ (A. M. Kroon and C. Saccone, eds.), pp. 337-346. Academic Press, New York. Kroon, A. M., and de Vries, H. (1970).In “Control of Organelle Development” (P. L. Miller, ed.), pp. 181-199. Cambridge Univ. Press, London and New York. Kroon, A. M.. and de Vries, H. (1971). In “Autonomy and Biogenesis of Mitochondria and Chloroplasts” (N. K. Boardman, A. W. Linnane, and R. M. Smillie eds.), pp. 318-327. North-Holland Publ., Amsterdam. Kroon, A. M. and Saccone, C., eds. (1974). “The Biogenesis of Mitochondria.” Academic Press, New York. Kuntzel, H. (1969). Nature (London) 222, 142. Kiintzel, H. (1971).Curr. Top. Microbiol. Immunol. 54, 94. Kiintzel, H., and Blossey, H. C. (1974).Eur. J. Biochem., 47, 165. Kiintzel, H., and Schafer, K. P. (1971). Nature (London),New Biol. 231,265. Kiintzel, H., Barath, Z., Ah, I., Kind, S., and Althons, H. H. (1973). Proc. Nut. Acad. Sci. US.70, 1574. Kiintzel, H., Ali, I., and Blossey, H. C. (1974). In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 71-78. Academic Press, New York. Kuriyama, Y., and Luck, D. J . L. (1973).J. Mol. Biol. 73, 425. Kuriyama, Y., and Luck, D. J. L. (1974).In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 117-133. Academic Press, New York. Lederman, M., and Attardi, G. (1970). Biochem. Biophys. Res. Commun. 40, 1492. Lill, H., Lill, U., Sippel, A., and Hartmann, G. (1970). I n “RNA Polymerase and Transcription” (L. Silvestry, ed.), pp. 55-64. North-Holland Publ., Amsterdam. Lizardi, P. M., and Luck, D. J. L. (1972).J. Cell Biol. 54,56. Locker, J., Rabinowitz, M., and Getz, G. S. (1974).J.Mol. Biol. 88, 489. Luck, D. J. L., and Reich, E. (1964). Proc. Nut. Acad. Sci. U S . 52, 931. Mahler, H. R., and Dawidowicz, K. (1973). Proc. Nat. Acad. Sci. U S . 70, 111. Mahler, H. R., Feldman, F., Phan, S. H., Hamill, P., and Dawidowicz, K. (1974).I n “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 423441. Academic Press, New York. Mason, T., Poyton, R. O., Wharton, D. C., and Schatz, G. (1973).J. Biol. Chem. 248, 1346. Michaelis, G., Douglass, S., Tsai, M. J., Burchiel, K., and Criddle, R. S. (1972).Biochemistry 11,2026. Michel, R., and Neupert, W. (1973). Eur. J. Biochem. 36,53. Millis, A. J. T., and Suyama, Y. (1972).J.Biol. Chem. 247, 4063. Moorman, A. F. M., and Grivell, L. A. (1974). In “Nucleocytoplasmic Relationships during Cell Morphogenesis in some Unicellular Organisms.” Elsevier, Amsterdam. Mukerjee, H., and Goldfeder, A. (1973).Biochemistry 12,5096. Murray, D. R., and Linnane, A. W. (1972).Biochem. Biophys. Res. Commun. 49,855. Nass, M. M. K., and Buck, C. A (1970).J. Mol. Biol. 54, 187. Neubert, D., Helge, H., and Tescke, S. (1966). Naunyn-Schmiedebergs Arch. E r p . Pathol. Phannakol. 252,452. Neupert, W., Sebald, W., Schwab, A. J., Massinger, P., and Bucher, T. (1969). Eur. J . Biochem. 10,589. O’Brien, T. W., Denslow, N. D., and Martin, G. R. (1974). I n “The Biogenesis of Mitochondria’’ (A. M. Kroon and C. Saccone, eds.), pp. 347-356. Academic Press, New York.

164

C. SACCONE AND E. QUACLIARIELLO

Ojala, D., and Attardi, G . (1972).J.Mol. Biol. 65, 273. Ojala, D., and Attardi, G. (1974a).J.Mol. Biol. 82, 151. Ojala, D., and Attardi, G. (1974b). Proc. N u t . Acad. Sci. U.S. 71, 563. Ojala, D., and Attardi, G . ( 1 9 7 4 ~ J. ) . Mol. Bio2. 88, 205. Parisi, B., and Cella, R. (1971). F E B S (Fed. Eur. Biochem. Soc.), Lett. 14, 209. Perlman, S., Abelson, H. T., Penman, S. (1973). Proc. Nat. Acad. Sci. U.S. 70, 350. Philips, D. P., and Parsons, P. (1973). Biochem. Biophys. Res. Commun. 55, 945. Reid, B. D., and Parsons, P. (1971). Proc. Nat. Acud. Sci. U.S. 68, 2830. Reijnders, L., and Borst, P. (1972).Biochem. Biophys. Res. Commun. 47, 126. Reijnders, L., Kleisen, C. M., Grivell, L. A., and Borst, P. (1972).Biochim. Biophys. Acta 272, 396. Reijnders, L., Sloff, P., and Borst, P. (1973). Eur. J. Biochem. 35, 266. Richter, I>. (1971).Biochemistry 10,4422. Richter, I)., and Lipnian, F. (1970). Biochemistry 9, 5065. Robberson, D., Aloni, Y., Attardi, G . , and Davidson, N. (1971).J. Mol Biol. 60, 473. Hoss, E., Ebner, E., Poyton, R. O., Mason, T. L., Ono, B., and Schatz, G . (1974). Z t i “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 477-489. Academic Press, New York. Saccone, C. (1973). In “Atti del Seminario di Studi Biologici” (E. Quagliariello, ed.), Vol. V, pp. 91-111. Adriatica Editrice, Bari. Saccone, C., Gadaleta, M. N., and Quagliariello, E. (1967). Biochim. Biophys. Acta 138, 474. Saccone, C., Gadaleta, M . N., and Gallerani, R. (1969). Eur. J . Biochem. 10, 61. Saccone, C., De Giorgi, C., Cantatore, P., and Gallerani, R. (1974a).In “BiomembranesArchitecture, Biogenesis, Bioenergetics, and Differentiation” (L. Packer, ed.), pp. 87-99. Academic Press, New York. Saccone, C., Greco, M., and Gadaleta, M. N. (1974b). F E B S Meeting, 9th, Buahpest. Abstracts, p. 458. Sager, R. (1972). “Cytoplasmic Genes and Organelles.” Academic Press, New York. Schafer, K. P., and Kiintzel, H. (1972). Biochem. Biophys. Res. Commun. 46, 1312. Schafer, K. P., Bugge, G., Grandi, M., and Kiintzel, H. (1971).Eur.1. Biochem. 21,478. Schatz, G., and Mason, T. L. (1974). Annu. Reo. Biochem. 43, 51. Schmitt, H . (1972). F E B S (Fed. Eur. Biochem. Soc.),Lett. 26,215. Schmitt, H., Grossfeld, H., Beckmann, J. S., and Littauer, U. Z. (1974). In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 135-146. Academic Press, New York. Schutgens, R. B. H., Reijnders, L., Hoekstra, S. P., and Borst, P. (1973). Biochim. Biophys. Acta 308, 372. Scragg, A. H. (1971).Biochem. Biophys. Res. Commun. 45, 701. Scragg, A. H. (1974).I n “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 47-57. Academic Press, New York. Sebald, W., Machleidt, W., and Otto, J. (1973). Eur. J. Biochem. 38, 311. Shmerling, 2. G. (1969).Biochem. Biophys. Res. Commun. 37, 965. Smith, A., and Marcker, K. (1968).J . Mol. Biol. 38, 241. Stevens, B. J., Curgy, J. J., Ledoigt, G., and Andri., J. (1974). In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 327335. Academic Press, New York. Suyama, I’. (1967). Biochemistry 6, 2829. Suyama, Y. (1969).I n “Atti del Seminario di Studi Biologici” (E. Quagliariello, ed.), Vol. IV, pp. 83-141. Adriatica Editrice, Bari.

MITOCHONDRIAL TRANSCRIPTION AND TRANSLATION

165

Suyama, Y., and Eyer, J. (1967). Biochem. Biophys. Res. Commun. 28, 746. Suyama, Y., and Eyer, J. (1968).J. Biol. Chem. 243,320. Swanson, R. F. (1971). Nature 231, 31. Swanson, R. F. (1973). Biochemistry 12,2142. Tabak, H. F., and Borst, P. (1970). Biochim. Biophys. Acta 217, 356. Towers, N. R., Dixon, H., Kellerman, G . M., and Linnane, A. W. (1972).Arch. Biochem. Biophys. 151,361. Tsai, M. J . , Michaelis, G . , and Criddle, R. S. (1971). Proc. Nut. Acad. Sci. U S . 68,473. Tzagoloff, A,, and Akai, A. (1972).J. B i d . Chem. 247, 6517. Tzagoloff, A., Akai, A., and Robin, M. S. (1974).In “The Biogenesis of Mitochondria” (A. M. Kroon and C. Saccone, eds.), pp. 405419. Academic Press, New York. Vesco, C., and Penman, S. (1969a).Proc. Nut. Acad. Sci. U.S. 62,218. Vesco, C., and Penman, S. (1969b).Nature (London) 224, 1021. Vignais, P. V., Huet, J., and Andr6, J. (1969).FEBS (Fed. Eur. Biochem. Soc) Lett. 3, 177. Vignais, P. V., Stevens, B. J., Huet, J., and Andr6, J. (1972)./. Cell Biol. 54, 468. Weislogel, P. O., and Butow, R. A. (1971).J. Biol. Chem. 246, 5113. Weiss, H., and Ziganke, B. (1974).In “The Biogenesis of Mitochondria” (A. M. Goon and C. Saccone, eds.), pp. 491-500. Academic Press, New York. Wheeldon, L. W. (1973).Biochimie 55, 805. Wheeldon, L. W., Dianoux, A. C. Bof, M., and Vignais, P. V. (1974). Eur. J . Biochem., 46, 189. Wintersberger, E. (1964).Hoppe-Seyler’s 2. Physiol. Chem. 336,285. Wintersberger, E. (1966). Regul. Metab. Processes Mitochondria, Proc. Symp., Bari, 1965 pp. 439453. Wintersberger, E. (1972). Biochem. Biophys. Res. Commun. 48, 1287. Wood, D. D., and Luck, D. J. L. (1969).J.Mol. Biol. 41, 211. Wu, G . J., and Dawid, I. B. (1972).Biochemistry 11, 3589. Wu, M., Davidson, N., Attardi, G . , and Aloni, Y. (1972).J. Mol. Biol. 71, 81. Zylber, E. A., Perlman, S., and Penman, S. (1971).Biochem. Biophys. Res. Commun. 40, 1492.

This Page Intentionally Left Blank

The Evolution of the Mitotic Spindle DONNAF. KUBAI’ Department of Zoology, University of Wisconsin, Madison, Wisconsin

I. Introduction

.

.

.

.

.

11. Some Evolutionary Considerations

111.

IV.

V.

VI.

VII.

. .

. .

. .

. .

.

. .

A. Evolution of Cellular and Nuclear Organization . B. Evolution of Microtubular Function . . . . The Possible Involvement of the Nuclear Envelope in . . . . . . . Chromosome Movement A. “Closed” Divisions . . . . . . . . B. Extranuclear Spindles . . . . . . . . . . C. A Tentative Outline of Spindle Evolution The Participation of Microtubules in Typical Mitosis. . A. Chromosome Behavior . . . . . . . B. Models for Microtubule Function in Chromosome . . . . . . . . . Movement . The Possibility of an Unconventional Role for Microtubules . . . . . . in Chromosome Movement . A. Nuclear Divisions without Microtubule-Chromosome . . . . . . . . . Connections B. Some Possible Sources of Misinterpretation . . . Nuclear Divisions with Microtubule-Mediated Chromo. . . . . . . . some Movements . A. Conventional Metaphase . . . . . . . B. Unconventional Metaphase Arrays . . . . . Final Remarks . . . . . . . . . References . . . . . . . . . .

167 168 168 169 171 171 171 193 194 194 195 197 197 203 205 205 206 220 22 1

I. Introduction Among lower organisms many variations in the details of nuclear division have been noted (Bglir, 1926; Grell, 1964); but the prevalent approach has been to emphasize the generality of the phenomenon of nuclear division, treating differences as superficial modifications of a common mechanism. This viewpoint is exemplified by Grell’s (1964) urging: “While proper attention should be paid to the differences, it would seem more important to find a common denominator for all types,” Such an approach is of course consistent with the unifying principles of the theory of evolution and serves as the framework for important generalizations. In this context, then, we recognize that there is a “standard plan” of mitosis whereby chromoPresent address: Department of Zoology, Duke University, Durham, North Carolina. 167

168

DONNA F. KUBAI

somes condense and become aligned at the equator of the mitotic spindle before they are moved to opposite poles under the influence of the mitotic apparatus. This sequence of events is a process that is seen as an essential aspect of cell division in the majority of organisms “from algae to orchids and amoeba to man” (Ris, 1955).However, evolutionary theory provides an equally strong basis for the expectation that we may find important differences among biological organisms; minimization of the significance of any differences noted in unusual modes of nuclear division could surely lead one to formulate procrustean analogies with the events of standard mitosis. As an example of this sort of error, we recall the attempts made to describe the distribution of the bacterial genophore in the terminology of typical mitosis (DeLamater, 1953).In the hope that we might gain some insight into the evolutionary past of the mitotic spindle, we review evidence that nuclear reproduction in some of the “lower forms”-algae, protozoa, and fungi-cannot be considered to conform to the standard plan of mitosis. Similar examinations have been undertaken by Pickett-Heaps (1969c, 1972b, 1974) and by Heath

(1973). 11. Some Evolutionary Considerations A. EVOLUTIONOF CELLULAR AND NUCLEAR ORGANIZATION Today, as a result of the ultrastructural investigations of the past two decades, we can appreciate that there is a fundamental dichotomy between the cellular organization of prokaryotes (bacteria and blue-green algae) and eukaryotes (all other cells) (Chatton, 1937; Stanier and van Niel, 1962; Stanier, 1970). Among the array of differences that distinguish these two classes of cells are profound differences in organization of their genetic material (Ris and Chandler, 1963) and parallel differences in the manner in which they distribute their genetic heritage to daughter cells. In prokaryotes a single DNA molecule encodes the essential hereditary information to be passed on to daughter cells, and it appears that the simple attachment of replicated genophores to independent sites on the cell membrane is sufficient to account for the separation of daughter genophores if growth of the membrane is restricted to the region between attachment sites (Cuzin and Jacob, 1967; Ryter, 1968). In eukaryotes, in which the genetic material is typically distributed among numerous independent genophores (the chromosomes) and in which, therefore, coordinate movement of a set of chromosomes is necessary to

EVOLUTION OF THE MITOTIC SPINDLE

169

achieve distribution of equal genetic complements to daughter cells, the mitotic spindle apparatus is responsible for chromosome movements (Luykx, 1970; Nicklas, 1971; Bajer and Molh-Bajer, 1972). This mitotic apparatus is in large part composed of microtubules, organelles never encountered in prokaryotes. In typical eukaryotes (i.e., higher plant and animal cells), at least some of the microtubules are found to b e intimately associated with a specific, structurally differentiated region of the chromosome, the kinetochore (Brinkley and Stubblefield, 1970; Luykx, 1970). Thus, at the level of the structures that appear to be most directly involved in genophore distribution, we may distinguish prokaryotes as organisms in which membrane phenomena play the predominant role, and eukaryotes as organisms with microtubule-associated chromosome movements. Differences in cellular organization of prokaryotes and eukaryotes might be taken to indicate that these cell types are the products of independent lines of evolution, were it not for the fact that we have equally striking demonstrations of a profound similarity in the cellular biochemistry of these cell types, extending even to the most basic level, that of the genetic code (Woese, 1970). Therefore it is reasonable to consider both descendants of a common progenitor. Regardless of the class of theory one favors to explain this evolutionary relationship of prokaryotes and eukaryotes, whether it be the endosymbiote theory (which suggests that eukaryotes arose as the result of symbiotic association between several lines of prokaryotes) (Mereschkowsky, 1905; Famintzin, 1907; Ris, 1961; Sagan, 1967; Cohen, 1970, 1973; Margulis, 1970; Raven, 1970; Stanier, 1970; Flavell, 1972), or one that proposes a more direct route (deriving eukaryotes from a single prokaryote ancestor) (Raff and Mahler, 1972; Uzzell and Spolsky, 1974), a corollary question concerns the manner in which eukaryotic mitosis developed and requires an explanation of the pathway by which responsibility for genophore distribution was transferred from the membrane to the microtubules of the mitotic apparatus. B.

EVOLUTIONO F MICROTUBULAR FUNCTION

Among eukaryotic organisms microtubules are ubiquitous organelles which appear to make at least two general kinds of functional contribution (Porter, 1966). (1) Microtubules may serve as cytoskeletal elements, providing a structural framework for the maintenance or change of cell shape; and insofar as this stems from the structural characteristics of the long, cylindrical microtubules, their shape-defining capabilities must be considered a static property. (2)

170

DONNA F. KUBAI

In addition, microtubules seem to be intimately involved in a wide spectrum of cellular and organellar motility phenomena, either in force production or force transmission, and may therefore be considered to exhibit dynamic properties. At the time of nuclear division in all eukaryotes, a new configuration of microtubules makes its appearance. In the mitosis of higher plants and animals, this takes the form of the mitotic spindle apparatus (Luykx, 1970; Nicklas, 1971; Bajer and Molk-Bajer, 1972), in which microtubules are found in more-or-less parallel array within the familiar bipolar shape of the mitotic apparatus. In terms of their disposition within the spindle, these microtubules are seen to occur as two classes: (1) chromosomal microtubules which impinge on a specific region of the chromosome, the kinetochore, and are directed toward the spindle poles, and (2) continuous microtubules which lie on the pole-to-pole axis of the spindle but are not attached to chromosomes. Whether this structural distinction reflects a corresponding functional difference in the two types of microtubules is not known. Nevertheless, the mitotic spindle, like the microtubules which are one of its major components, is a structure that displays both static and dynamic attributes. Simply by virtue of its overall form, the spindle defines the opposite poles toward which sister chromosomes must move from their metaphase orientation at the spindle equator. Its dynamic influence is evident in its role in chromosome movements, both those of metakinesis which bring chromosomes to their metaphase position, and those of anaphase separation which move sets of chromosomes toward opposite poles, Clearly, until the detailed mechanisms of the function of microtubules and of the spindle are understood, there is no way of knowing just how artificial a distinction between their static and dynamic properties is. From an evolutionary perspective, however, it seems reasonable to think that these may be separable characteristics which were acquired independently. For example, microtubules may have originally come to be associated with the dividing nucleus solely in their cytoskeletal capacity, only secondarily assuming the functions of producing and/or transmitting forces. In attempting to trace the evolution of the “typical” eukaryotic spindle, w e then ask if there are any examples in which microtubules concerned in nuclear reproduction seem to play only (or predominantly) a cytoskeletal role. If so, it is to be expected that the direct responsibility for chromosome movement resides in another structure, perhaps a membrane as in prokaryotes.

EVOLUTION OF THE MITOTIC SPINDLE

171

111. The Possible Involvement of the Nuclear Envelope in Chromosome Movement A. “CLOSED”DIVISIONS

We might expect that if there exist any organisms in which membranes play a role in chromosome movement it is the membranes of the nuclear envelope that would be implicated. In contrast to higher eukaryotes, in which the nuclear envelope disperses as the mitotic spindle appears, in most lower eukaryotes the nuclear envelope persists throughout the period of nuclear division (cf. Tables 1-111). In many organisms the nuclear envelope appears to remain totally intact (Table 11), while in others there is some localized disruption of the membrane system (Table 111), most commonly seen as polar gaps or fenestrae. Such a closed (Jenkins, 1967) configuration of the dividing nucleus obviously has important bearing on the question of whether division-related microtubules are cytoplasmic or nuclear in origin (Pickett-Heaps, 1969a,b) and, in some plants at least, it seems that persistence of the nuclear envelope has an effect in determining whether cleavage of the cytoplasm will involve a phragmoplast or a phycoplast (Pickett-Heaps, 1972b; Pickett-Heaps and Marchant, 1972). However, with respect to the mechanisms of chromosome movement, the persistence of the nuclear envelope seems to be of little absolute significance. In many closed divisions an apparently normal spindle is formed intranuclearly and chromosomes interact with this spindle in the standard manner, engaging spindle microtubules at a differentiated kinetochore and aligning at the spindle equator in the metaphase array (cf. Tables I-VI). The myxomycetes are exemplary in this respect. In these organisms either closed or open divisions may be found, depending on the stage in the life cycle, yet no essential modification of the mitotic mode of chromosome distribution is apparent in either case (Aldrich, 1969). PickettHeaps (1974) has suggested that the persistence of the nuclear envelope in the plasmodia1 mitosis of myxomycetes is simply a mechanism to guard against fusion of several spindles during this multinucleate phase of the organism’s life cycle (see also Ross, 1968). B. EXTRANUCLEAR SPINDLES It is not always easy to discern the characteristics of conventional mitosis in closed divisions, however, and several organisms exhibit striking departures from the sort of chromosome behavior we define

172

DONNA F. KUBAI

TABLE I LOWERORGANISMS IN WHICH NUCLEARENVELOPEDISPERSES DURING NUCLEARDIVISION

Organism" Fungi M yxomycota M yxomycetes Caoostelium apophysatum (amoeboflagellate) Physarum jlaoicomum (myxamoebal mitosis) Eumycota Z ygomycotina Zygomycetes (Entomophthorales) Basidiobolus ranarum

Metaphase plate

References

+

Flirtado and Olive (1970)

+

Aldrich (1969)

+

Tanaka (1970); Sun and Bowen

(1972) Basidiomycotina H ymenomycetes Coprinus radiatus (meiosis)

Lerbs and Thielke (1960); Lerbs

(1971) Coprinus lagopus (meiosis) Polystictus oersicolor Arniillaria niellea Boletus nibinellus (meiosis) Deuteromycotina Hyphomycetes Penicillium striatum (meiosis) Protozoa Mastigophora Phytomas tigina Chrysomonadina Prymnesiuin parvum Ochromonas danica Sarcodina Rhizopoda Arnoebina Pelomyxa carolinensis Pelomyxa illinoisensis Sporozoa Telosporidia Gregarinida Stylocephalus longicollus

t(?)

Lu (1967); Raju and Lu (1973) Girbardt (1968, 1971) Motta (1969) McLaiighlin (1971)

Laane (1970)

+ +

Manton (1964) Slankis and Gibbs (1972);Bouck and Brown (1973)

+ +

Roth and Daniels (1962) Daniels and Roth (1964)

+

Desportes (1970)

EVOLUTION OF T H E MITOTIC SPINDLE

173

TABLE I (Continued)

Organism" Algae Chlorophyceae Volvocales Pyramimonas sp. Ulotrichales Klebsormidium subtilissimum Klebsormidium flaccidum Ulothrix fimbriata Conjugales Desmidioideae Micrasterias americana Closterium littorale Cosmarium botrytis Charales Chara sp. Nitella missouriensis Chrysophyceae Chrysomonadales Prymnesium parvum Ochromonas danica Bacillarioph yceae Centrales Lithodesmium undulatumb (mitosis and meiosis) Cryptophyceae Chroomonas salina Of uncertain taxonomic position Cyanophora paradoxa

Metaphase plate

References

Norris and Pearson (1973)

+

Pickett-Heaps ( 1 9 7 2 ~ )

+ +

Floyd et al. (1972b) Floyd et al. (1972a)

+

+ +

+

+ +

Ueda (1973) Pickett-Heaps and Fowke (1970b) Pickett-Heaps (1972f) Pickett-Heaps (1967) Turner (1968)

Manton (1964) SIankis and Gibbs (1972); Bouck and Brown (1973)

Manton et al. (1969a,b, 1970); Manton (1970)

+ +

Oakley and Dodge (1973) Pickett-Heaps (1972d)

" Classification is: for fungi according to the scheme of Ainsworth (1971); for protozoa, according to Kudo (1954); and for algae, according to Fritsch (1935). * Spindle forms extranuclearly prior to nuclear envelope dispersal.

as mitotic. I n at least some of these, the nuclear membrane is strongly suspected to b e directly involved in chromosome movement. The most compelling evidence for such behavior is found in dinoflagellates and hypermastigote flagellates (Fig. 1) in which microtubules, although present, never invade the nuclear volume and are seen to be effectively excluded from any direct interaction with chromosomes during at least a portion of chromosome movement.

174

DONNA F. KUBAI TABLE I1 LOWERORGANISMS IN WHICH NUCLEARENVELOPEREMAINS INTACT DURING NUCLEARDIVISION

Organism" Fungi M yxomycota M yxomycetes

Clastoderina debaryanum (plasmodial mitosis)

Physarum flavicomum (plasmodia1 mitosis) Eumycota Mastigomycotina Ch ytridiomycetes

Catenaria anguillulae Blastocladiella emersonii Allomyces arbusculus H yphochytridiomycetes

Rhizidiomyces apophysatus

Metaphase plate

References

+

McManus and Roth (1968)

+

Aldrich (1969)

+

Ichida and Fuller (1968) Lessie and Lovett (1968) Turian and Oulevey (1971)

+

Fuller and Reichle (1965)

+

Heath and Greenwood (1968,1970) Howard and Moore (1970)

Oomycetes

Saprolegnia ferax Saprolegnia terrestris (mitosis and meiosis)

Aphanomyces euteiches Thraustotheca clauata

Hoch and Mitchell (1972) Heath (1974)

Z ygomycotina Zygomycetes

Ph ycom yces bla kesleea nus Mucor hiemalis

Franke and Reau (1973) McCully and Robinow (1973)

Ascomycotina Hemiascomycetes

Saccharom yces cerevisiae Saccharomyces cerevisiae

Robinow and Marak (1966) Moens (1971); Rapport (1971); Peterson et al. (1972) Moens and Rapport (1971)

(meiosis)

Saccharomyces cerevisiae (mitosis and meiosis)

Schizosaccharomyces pombe Wickerhamia fluorescens

McCully and Robinow (1971) Rooney and Moens (1973)

(meiosis) Plectomycetes

Erysiphe graminis hordei Pyrenom ycetes

Xylosphaera pol ymorpha Podospora anserina

McKeen (1972)

+ +

Beckett and Crawford (1970) Zickler (1970)

(mitosis and meiosis)

Podospora setosa Neurospora crassa

4-

Zickler (1970) Van Winkle et al. (1971)

175

EVOLUTION OF THE MITOTIC SPINDLE

TABLE I1 (Continued)

Organisma Discomycetes Ascobolus immersus (mitosis and meiosis) Ascobolus stercorarius Pustularia cupularis Pyronema domesticum Deuteromycotina Hyphomycetes Aspergillus nidulans Fusarium oxysporum Protozoa Mastigophora Phytomastigina Euglenoidina Astasia longa

Euglena gracilis Scytomonas pusilla lsonema nigricans Dinoflagellatab Woloszynskia micra Amphidinium carteri Blastodinium chattonii Crypthecodinium cohnii Anaoebophrya (several species) Glenodinium foliaceum Prorocentrum (several species) Exuuiella (several species) Amphidinium sp. Oodinium fritillariae Syndinium sp. Haplozoon axiothellae Zoomastigina Rhizomastiginab Histomonas maleagridis Protomonadina Leishmania tropica Trypanosoma gambiense Trypanosoma rhodesiense Trypanosoma raiae Trypanosoma cruzi

Metaphase plate

References

+

Zickler (1970)

+

Zickler (1970); Wells (1970) Schrantz (1967, 1970) Hung and Wells (1971) Robinow and Caten (1969) Aist and Williams (1972)

Blum et al. (1965); Sommer and Blum (1965) Blum et al. (1965); Leedale (1968) Mignot (1966) Schuster et al. (1968) Leadbeater and Dodge (1967) Dodge and Crawford (1968) Soyer (1969, 1971) Kubai and Ris (1969) Cachon and Cachon (1970) Dodge (1971) Dodge and Bibby (1973) Dodge and Bibby (1973) Oakley and Dodge (1974) Cachon and Cachon (1974) Ris and Kubai (1974) Siebert and West (1974) Schuster (1968) Bianchi et al. (1969) Inoki and Ozeki (1969) Vickerman and Preston (1970) Vickerman and Preston (1970) de Souza and Meyer (1974)

(Continued)

176

DONNA F. KUBAI TABLE I1 (Continued)

Organism"

Metaphase plate

Polyniastiginab Chilomastix aulastomi Dientamoeba fragilis Trichomonas termopsidis Hypermastiginab Joenia duboscqui Barbulanympha ufalula

Brugerolle (1973) Camp et al. (1974) HolIande and Valentin (196813) Hollande and Valentin (1968b) Hollande and Valentin (1968a); Hollande and Carruette-Valentin (1971) Hollande and Carruette-Valentin (1970,1971);Kubai (1973) Hollande and Carruette-Valentin (1971) Hollande and Carruette-Valentin (1971) Hollande and Carruette-Valentin (1971) Hollande and Carruette-Valentin (1972)

Trichonympha agilis Staurojoenina caulleryi Spirotrichonympha psammotermi tidis Holomastigotoides hemigymnum Lophornonas striata Sarcodina Rhizopoda Amoebina Naegleria gruberi Foraminifera Allogromia laticollaris Myxotheca arenilega (gametogenic mitosis) (meiosiq) lridia lucida Actinopoda Radiolaria Collozoom pelogfcum Sporozoa Telosporidia Coccidia Eimeria maxima

Eimeria falciformis Eimerda necatrix Eimeria magna Eimeria callospermophili Eimeria aubernensis Eimeria ninakohlyakimovae Toxoplasma gondii

References

+

Fulton and Dingle (1971) Schwab (1972)

+

Schwab (1968,1969) Schwab (1973) Cesana (1971) Hollande et al. (1969)

Mehlhorn (1972);Mehlhorn et al. ( 1972) Mehlhorn et al. (1972) Dubremetz (1971,1972, 1973) Danforth and Hammond (1972); Hammond et al. (1973) Roberts et al. (1970);Hammond et al. (1973) Hammond et al. (1969) Kelley and Hammond (1972,1973) Sheffield and Melton (1968)

177

EVOLUTION OF THE MITOTIC SPINDLE TABLE I1 (Continued) Organism" Haemosporidia Plasmodium berghei Plasmodium gallinaceum Plasmodium elongatum Plasmodium ftoridense Plasmodium brazilianum Plasmodium cynoinolgi Haemoproteus colombae Parahoemoproteus velum Cnidos poridia Microsporidia Thelohania bracteata Plistophora sp. Metchnikovella hovassei Glugea sp. Nosema vivieri Ciliata H olotricha Gymnostomata lchthyophtirius mrtltifiliis (micronucleus) Loxodes magnus (micronucleus) Nassula sp. (micronucleus) (macronucleus) Nassula ornata (micronucleus) (macronucIeus) Trichostomata Paramecium uurelia (micronuclear mitosis) (meiosis) (macronucleus) Alloizona trizona (micronucleus) Isotricha sp. (macronucleus) Hymenostomata Tetrahymena pyriformis (macronucleus)

Metaphase plate

References

Aikawa et al. (1972); Howells and Davies (1971); Canning and Sinden (1973) Terzakis et al. (1967); Aikawa and Beaudoin (1968); Aikawa et al. (1972) Aikawa et al. (1967) Aikawa and Jordan (1968) Sterling et al. (1972) Terzakis (1971) Bradbury and Trager (1968) Desser (1972) Vavra (1965) Vavra (1965) Vivier (1965) Sprague and Vernick (1968) Vinckier et al. (1971)

Hauser (1972, 1973) Raikov (1973) Tucker (1967) Tucker (1967) Raikov (1966) Raikov (1966) Jurand and Selman (1970);Stevenson and Lloyd (1971a) Stevenson (1972) Jurand and Selman (1970); Stevenson and Lloyd (1971b) Grain (1966) Grain (1966) Falk et al. (1968); Ito et al. (1968) (Continued)

178

DONNA F . KUBAI

TABLE I1 (Continued)

Organism" Spirotricha Heterotricha Blepharisma sp. (micronucleus) ( macronucleus)* Blepharisma wardsii (micronucleus) (macronucleus)b Oligotricha Diplodinium sp. (micronucleus and macronucleus) Peritricha Campanella umbelloria (macronucleus) Epistylis anastatico (micronucleus) Vorticella nebulifera (micronucleus) Suctoria Parucineta limbata (micronucleus) (macronucleus) Tokophrya infusionum (micronucleus and macronucleus) Acineto tuberosa (macronucleus) Algae Chloroph yceae Chaetophorales Stigeocloneum helueticum Siphonales Vaucherio litorea Rhodoph yceae Grifjthsia flosculosa

Metaphase plate

+

References

Jenkins (1967) Jenkins (1969) Inaba and Sotakawa (1968) Inaba and Sotakawa (1968) Roth and Shigenaka (1964)

Carasso and Favard (1965) Carasso and Favard (1965) Carasso and Favard (1965)

+

Hauser (1968, 1972) Hauser (1972) Millecchia and Rudzinska (1971)

Bardele (1969)

+

Floyd et al. (1972a)

+

Ott and Brown (1972)

+

Peyriere (1971)

Classification is: for fungi according to the scheme of Ainsworth (1971); for protozoa, according to Kudo (1954); and for algae, according to Fritsch (1935). Extranuclear spindle or microtubules.

1. Dinoflagellat a In coining the terms dinomitosis and dinokaryon, Chatton (1920) emphasized the distinctive nature of the dinoflagellate nucleus and its mode of reproduction; and Grasse (1952) was the first to recognize that in dinoflagellates (as in some other organisms) the persistent

TABLE 111 LOWERORGANISMS IN WHICH FENESTRAE OR CAPS APPEAR ON NUCLEARENVELOPEDURING NUCLEARDIVISION

Organism"

Metaphase plate

Fungi M yxom ycota Hydrom yxomycetes Lahyrinthula sp. (mitosis)

M yxomycetes Arcyria cineria (sporanginm cleavage) Physarum polycephalum (plasmodial mitosis) Physarum flaoicomum (meiosis) Plasmodiophoromycetes Sorosphaera oeronicae (plasmodia1 mitosis) Eumycota Mastigomycotina Ch ytridiomycetes Phlyctochytrium irregulare Ascomycotina Pyrenomycetes Xylaria polymorpha Basidiomycotina Teliomycetes Leucosporidium scotti Rhodosporidium sp. Aessosporon salmonicolor H ymenom ycetes Schizophyllum commune Protozoa Sporozoa Telosporidia Gregarinida Diplauxis hatti Algae Chlorophyceae Volvocales Chiamydomonas reinhard Tetraspora sp. Chlorococcales Kirchneriella liinaris Hydrodictyon reticulatum Tetraedron hitridens

References

Perkins and Amon (1969); Porter (1972)

+ +

Mims (1972)

+

Guttes et al. (1968); Goodman and Ritter (1969);Ryser (1970); Sakai and Shigenaga (1972) Aldrich (1967)

+

Braselton and Miller (1973)

+

McNitt (1973)

Schrantz (1970)

McCully and Robinow (19724 McCully and Robinow (1972b) McCully and Robinow (1972b) Raudaskoski (1970)

Prensier (1972)

+ + + + +

Johnson and Porter (1968) Pickett-Heaps (1973) Pickett-Heaps (1970) Marchant and Pickett-Heaps (1970) Pickett-Heaps (1972a) (Con tinried)

180

DONNA F. KUBAI

TABLE 111 (Continued)

Organism" Ulotrichales Ulva mutabilis Microspora sp. Cladophorales Cladophora fracta Oedogoniales Oedogonium sp. Oedogonium curdiacum Conjugales Euconjugatae Spirogyra sp.b Spirogyra inajusculab Mougeotia sp. Chloromonadincac Vacuolaria virescens Phaeophyceae Dictyota dichotoma Padina pavonica Zonaria farlowii Dictyopteris zonarioides Rhodoph yceae Men1branop tern plat y p h y l la

Metaphase plate

+(?)

+ + + +

References

L i ~ l i eand Brdten (1970) Pickett-Heaps (1972e) Mughal and Godward (1973) Pickett-Heaps and Fowke (1969) Pickett-Heaps and Fowke (1970a)

+ +

Fowke and Pickett-Heaps (1969) Mughal and Godward (1973) Bech-Hansen and Fowke (1972)

+

Heywood (1973) Neushul Neushul Neushul Neushul

+

and and and and

Dahl Dahl Dahl Dahl

(1972) (1972) (1972) (1972)

McDonald (1972)

Classification is: for fungi according to the scheme of Ainsworth (1971); for protozoa, according to Kudo (1954); and for algae, according to Fritsch (1935). Complete dispersal of the nuclear envelope is accomplished at anaphase; prior to this polar fenestrae are present in the otherwise intact nuclear envelope. I'

nuclear envelope might not only account for anomalies of nuclear division but indeed even play a role in chromosome movement. With the acquisition of ultrastructural information, these ideas have been reinforced. In several dinoflagellates the microtubules which make their appearance during the process of nuclear division are confined to the cytoplasm and never assume a spindlelike configuration. Rather, they are found as several separate bundles distributed in a number of cytoplasmic invaginations of the nuclear surface (Leadbeater and Dodge, 1967; Dodge and Crawford, 1968; Kubai and Ris, 1969; Soyer, 1969, 1971; Dodge, 1971; Dodge and Bibby, 1973; Cachon and Cachon, 1974; Oakley and Dodge, 1974; Siebert and West, 1974). In one species of free-living dinoflagellate, Crypthecodinium cohnii [formerly Gyrodiniurn cohnii (Javornick9, 1962; Keller et al., 1968)1, the process of formation of these microtubule-containing invaginations was followed in serial sections (Kubai and Ris, 1969). It

181

EVOLUTION OF T H E MITOTIC SPINDLE

TABLE IV LOWERORGANISMS POSSESSING SINGLE- OR MULTILAY ERED DISCLIKEKINETOCHORES Organism" Fungi M yxomycota M yxomycetes Physarum polycephalum Eumycota Mastigom ycotina Chytridiomycetes Phlyctochytrium irregulare Oomycetes Saprolegnia ferax Thruustotheca clauata Zygomycotina Zygomycetes Basidiobolus runarum Protozoa Mastigophora Phytomastigina Dinoflagellata Syndinium sp. Amphidinium sp. Oodinium fritilluriae Haplozoon axiothellae Zoomastigina Hypermas tigina Lophomonas striata Trichonympha agilis Barbulanympha ufalula

Staurojoenina caulleryi Spirotrichonympha psammotermitidis Sarcodina Actinopoda Radiolaria Collozoom pelagicum Sporozoa Telosporidia Gregarinida Stylocephalus longicollus Coccidia Eimeria necatrix

References

Ryser (1970)

McNitt (1973) Heath and Greenwood (1970) Heath (1974)

Tanaka (1970)

Ris and Kubai (1974) Oakley and Dodge (1974) Cachon and Cachon (1974) Siebert and West (1974) Hollande and Carruette-Valentin (1972) Hollande and Carruette-Valentin (1970, 1971); Kubai (1973) Hollande and Valentin (1968a); Hollande and Carruette-Valentin (1971) Hollande and Carruette-Valentin (1971) Hollande and Carruette-Valentin (1971)

Hollande et al. (1969)

Desportes (1970) Dubremetz (1973) (Continued)

182

DONNA F. KUBAI TABLE IV (Continued) Organism"

References

Haemosporidia

Plasmodium berghei

Howells and Davies (1971);Canning and Sinden (1973);Aikawa et al.

Plasmodium gallinaceurn

Aikawa and Beaudoin (1968);Sterling and Aikawa (1973) Aikawa et al. (1967) Sterling et al. (1972) Bradbury and Trager (1968) Desser (1972)

(1972)*

Plasmodium elongatum Plasmodium brazilianum Haemoproteus colombae Parahaemoproteus velans Ciliata Holotricha Gymnostomata

Loxodes magnus

Raikov (1973)

Algae Chloroph yceae Chlorococcales

Hydrodictyon reticulatum

Marchant and Pickett-Heaps (1970)

Ulotrichales

Microspora sp.

Pickett-Heaps (1972e)

Cladophorales

Cladophora fracta

Mughal and Godward (1973)

Oedogoniales

Oedogonium sp. Oedogonium cardiacum

Pickett-Heaps and Fowke (1969) Pickett-Heaps and Fowke (1970a)

Conjugales Euconjugatae

Spirogyra majuscula

Mughal and Godward (1973)

Charales

Nitella missouriensis

Turner (1968)

Dinoph yceae See above: Protozoa, Dinoflagellata Rhodoph yceae

Membranoptera platyphylla

McDonald (1972)

" Classification is: for fungi, according to the scheme of Ainsworth (1971); for protozoa, according to Kudo (1954); and for the algae, according to Fritsch (1935). * This report establishes the identity of multilaininar kinetochores; in earlier studies these structures in the Haemosporidia were postulated to be chromosomes.

appears that nuclear deformation arises as a single large sheaf of microtubules pushes into the nucleus in a direction perpendicular to the long axis of its component microtubules. Smaller bundles of microtubules then split off from the main bundle, and the nuclear membrane closes around these separate bundles so that the nucleus is ultimately traversed by several parallel, microtubule-containing,

EVOLUTION OF T H E MITOTIC SPINDLE

183

TABLE V LOWERORGANISMS POSSESSING GLOBULAROR CONE-SHAPEDKINETOCHORES Organism" Fungi Eumycota Ascom ycotina Plectomycetes Erysiphe graminis hordei Pyrenomycetes Xylosphaeru polymorpha Deu terom ycotina Hyphomycetes Fusarium oxysporum Algae Chlorophyceae Conjugales Euconjugatae Mougeotia sp. Desmidioideae Micrasterias americanu Chlorornonadineae Vaucherin cirescens

References

McKeen (1972) Beckett and Crawford (1970)

Aist and Williams (1972)

Bech-Hansen and Fowke (1972) Ueda (1973) Heywood and Godward (1972)

" Classification is: for fungi, according to the scheme of Ainsworth (1971); for protozoa, according to Kudo (1954); and for algae, according to Fritsch (1935).

cytoplasmic channels which persist until the separation of daughter nuclei is complete. There is no apparent change in the length of microtubules at any stage in division, and no discontinuities in the nuclear envelope are found at any time. Since the intact nuclear envelope serves as a barrier between microtubules and chromosomes throughout division, it is difficult to conclude that microtubules play any direct role in chromosome movement. While no microtubule-chromosome interaction was seen in C. cohnii, an association between chromosomes and the nuclear envelope was noted, as has also been described for other species (Bouligand et al., 1968; Soyer, 1969, 1971; Dodge, 1971).These chromosome-membrane associations were examined in both interphase and dividing cells of C. cohnii (Kubai and Ris, 1969). In the nondividing cell, short, sausage-shaped chromosomes are scattered throughout the nuclear volume, and only the relatively few at the nuclear periphery make contact with the nuclear envelope. During division a dramatically different situation prevails. Chromosomes, now longer and thinner, are V-shaped, and the V apexes are attached to the nuclear envelope. Moreover, it is only in a specific area of the nuclear envelope that these associations occur, namely, the area of the nu-

184

DONNA F. KUBAI

TABLE VI TERMINATING I N CHROMATIN: LOWER ORGANISMS DISPLAYINGMICROTUBULES

N o KINETOCHORE DIFFERENTIATION Organism' Fungi Myxomycota H ydromyxomycetes Labyrinthula sp. Myxomycetes Arcyria cineria Physarum polycephalum (plasmodial mitosis) Physarum jlaoicomum (myxameba1 and plasmodial mitoses) Clastoderma debaryanum Plasmodiophoromycetes Sorosphaera oeronicae (plasmodial mitosis) Eumycota Mastigomycotina Chytridiomycetes Catenaria anguillulae Oomycetes Saprolegnia terrestris Ascomycotina Hemiascomycetes Saccharomyces cerevisiae Pyrenomycetes Podospora anserina Podospora setosa Neurospora crassa Disconiycetes Ascobolus stercorarius Ascobolus immersus Basidiom ycotina Teliom ycetes Leucosporidium scotti H ymenomycetes Armillaria mellea Coprinus lagopus (meiosis) Protozoa Mastigophora Phytomastigina C hrysomonadina Ochromonas danica C yptomonadina Chroomonas salina

References

Porter (1972) Mims (1972) Sakai and Shigenaga (1972); Guttes et a / . (1968);Goodman and Ritter (1969) Aldrich (1969) McManus and Roth (1968) Braselton and Miller (1973)

Ichida and Fuller (1968) Howard and Moore (1970)

Peterson and Ris (personal communication) Zickler (1970) Zickler (1970) Van Winkle et al. (1971) Zickler (1970); Wells (1970) Zickler (1970)

McCully and Robinow (1972a) Motta (1969) Lu (1967)

Slankis and Gibbs (1972) Oakley and Dodge (1974)

185

EVOLUTION OF T H E MITOTIC SPINDLE TABLE VI (Continued) Organism" Sarcodina Rhizopoda Arnoebina Pelomyxa carolinensis Pelomyxa illinoisensis Foraminifera Allogromia laticollaris lridia lucida Sporozoa Telosporidia Coccidia Eimeria ninakohlyakimotiae Cnidosporidia Microsporidia Clugea sp. Nosema uiuieri Ciliata Holotricha Gymnos tomata Nassula sp. Trichostomata Paramecium aurelia (micronuclear mitosis) (meiosis) Spirotricha Heterotricha Blepharisma sp. (micronuclear mitosis) Algae Chlorophyceae Volvocales Tetraspora sp. Chlorococcales Kirchneriella lunaris Tetraedron bitridens Ulotrichales Ulva mutabilis Conjugales Euconjugatae Spirogyra sp. Desmidioideae Cosmarium botrytis Closterium littorale Siphonales Vaucheria litorea

References

Roth and Daniels (1962) Daniels and Roth (1964) Schwab (1972) Cesana (1971)

Kelley and Hammond (1972, 1973)

Sprague and Vernick (1968) Vinckier et al. (1971)

Tucker (1967)

Jurand and Selman (1970); Stevenson and Lloyd (1971a) Stevenson (1972)

Jenkins (1967)

Pickett-Heaps (1973) Pickett-Heaps (1970) Pickett-Heaps (1972a) L0vlie and Briten (1970)

Fowke and Pickett-Heaps (1969) Pickett-Heaps (1972f) Pickett-Heaps and Fowke (1970b)

Ott and Brown (1972) (Continued)

186

DONNA F. KUBAI

TABLE VI (Continued) ~

Organism“ Charales Chara sp. Chrysophyceae Ochroinonas danica Cryptophyceae Chroomonas salina Of uncertain taxoironric pobition Cyanolihora paradoxu

~~

References

Pickett-Heaps (1967) Slankis and Gibbs (1972) Oakley and Dodge (1973) Pickett-Heaps (19724

Classification is: for fungi, according to the scheme of Ainsworth (1971); for protozoa, according to Kudo (1954); and for algae, according to Fritsch (1935).

clear envelope at the boundary of channels. On these grounds, Kubai and Ris proposed that chromosome movement in dinoflagellates is probably membrane-mediated [see also Soyer (1969)], leaving the cytoplasmic microtubules to serve primarily as cytoskeletal elements, defining polarity. This is exactly the sort of microtubule-plusmembrane division mechanism one might expect to find in the early transition from the prokaryotic to the eukaryotic condition, and allows us to view dinoflagellate mitosis as a prokaryote-eukaryote intermediate. This interpretation is in accord with earlier demonstrations that the structure (Giesbrecht, 1962; Ris, 1962; de Haller et al., 1964; Grass6 et al., 1965; Kubai, 1965) and chemistry (Ris, 1962; Dodge, 1964; Kubai, 1965) of dinoflagellate chromosomes are prokaryotelike, while in terms of their overall cellular organization dinoflagellates are generally eukaryotic, an intermediate Dodge (1966) has called “mesocaryotic.” For additional discussion of nuclear division in dinoflagellates, please see Section 111,B,3. 2. Hypermastigina Another clear-cut demonstration of membrane involvement in the processes of chromosome movement is found in the closed nuclear divisions of hypermastigote flagellates. Here also, as in dinoflagellates, microtubules are found only extranuclearly. However, since these microtubules are massed in a fairly typical spindlelike structure, the predominant viewpoint has been that of Cleveland et al. (1934), who interpreted this form of nuclear reproduction in the terms of standard mitosis. Accordingly, the only departures from classic mitosis are perceived to be the trivial consequences of spatial separation of the main body of the spindle and intranuclear chromosomes. Thus, while this condition requires that interaction of spindle

EVOLUTION OF THE MITOTIC SPINDLE

187

b

C

FIG. 1. The various forms of extranuclear spindles. (a) In dinoflagellates extranuclear microtubules occur in several parallel cytoplasmic channels which traverse the nucleus. Chromosomes are attached to the nuclear envelope surrounding channels. In some species a dense knoblike differentiation is found in the region of chromosome-membrane contact, and a single microtubule terminates at this structure (as illustrated in lower channel; cf. Fig. 5 ) . In other species no such indications of microtubule-chromosome contact have been found (upper channel). (b) In hypermastigotes extranuclear microtubules are massed in a well-defined spindle. During a significant portion of chromosome movement, kinetochores are enclosed in pouchlike evaginations of the intact nuclear envelope and are not in contact with microtubules (cf. Figs. 2 and 3). (c) In Syndiniurn sp. the extranuclear spindle occurs within a single cytoplasmic channel which traverses the nucleus. Kinetochores protrude through porelike openings in the nuclear envelope and are connected to centrioles via chromosomal microtubule?. Chromosome movement is produced as intercentriolar microtubules elongate (cf. Fig. 4).

fibers and kinetochores occur at the nuclear surface and at the same time prevents metaphase assembly of chromosomes a t the spindle equator, chromosome movement is considered to remain under the control of spindle fibers. Grass6 (1952), however, maintained that the presence of an intact nuclear membrane prevents orthodox involve-

188

DONNA F. KUBAI

ment of spindle fibers in chromosome movement, so that chromosomes must move as their kinetochores glide beneath the nuclear envelope. Although Grassk’s suggestion was dismissed by Hollande and Carruette-Valentin (1970, 1971; also Hollande and Valentin, 1968a,b) on the basis of their demonstrations that kinetochores actually protrude through openings in the nuclear envelope and make direct contact with extranuclear microtubules, more recent evidence favors Grassk’s interpretation for at least some chromosome movement. Through examination of the entire sequence of events during mitosis in Trichonyrnpha agilis, Kubai (1973) showed that kinetochores remain in an intranuclear condition, associated with the inner layer of the completely intact nuclear envelope throughout significant stages of chromosome movement (Fig. 2). I n the earliest stages sister kinetochores are easily recognized as pairs distributed over the entire nuclear surface. Before kinetochores insert into openings of the nuclear envelope, kinetochore pairs disjoin and there is some indication that they become grouped in two areas of the nuclear surface

FIG.2. Trichonympha agilis. During early division, when sister kinetochore pairs are found to disjoin, kinetochores (arrow) are enclosed in pouchlike evaginations of the nuclear envelope and are incapable of direct interaction with the extranuclear microtubules. Such an observation is the basis of the suggestion that membrane phenomena are responsible for at least the early phases of chromosome movement in hypermastigotes. x 68,500. (Micrograph from Kubai, 1973, reproduced by permission of The Company of Biologists, Ltd.)

EVOLUTION OF THE MITOTIC SPINDLE

189

closest to the poles of the fully developed spindle. Only after these movements are accomplished does the nuclear envelope open at kinetochores to allow contact between kinetochores and microtubules (Fig. 3). Hypermastigote mitosis thus clearly provides another example of membrane-mediated chromosome movement. Cleveland et al.’s (1934) observational evidence that elongation of the central spindle is responsible for the forces of karyokinesis suggests that there are at least some dynamic properties associated with this collection of microtubules; but Kubai has suggested that the microtubules that actually interact with kinetochores may have little influence on chromosome movement, serving simply as an anchoring device so as to maintain an already accomplished chromosome distribution during the phase of active central spindle elongation leading to the actual division of the nucleus. By means of

FIG. 3. Trichonympha agilis. In later phases of division, kinetochores (arrows) become inserted in openings of the nuclear envelope and are the site of termination for chromosomal microtubules. At this time therefore microtubules may have a direct role in chromosome movement. x 68,500. (Micrograph from Kubai, 1973, reproduced by permission of The Company of Biologists, Ltd.)

190

DONNA F. KUBAI

polarized-light and differential interference microscopy, Ritter and Inoub (personal communication) followed the progress of nuclear division in another hypermastigote, living Barbulanympha, and found that grouping of kinetochores near the spindle poles occurs before the central spindle elongates (an elongation that leads to a fivefold increase in the interpolar dimension of the spindle).

3. Syndinium sp. On the basis of the foregoing evidence from dinoflagellates and hyperniastigotes, one might b e tempted to generalize that an exclusively extranuclear arrangement of division-related microtubules is indicative of at least some degree of microtubule-independent chromosome movement. That this is emphatically not the case is clear from the recent description of mitosis in Syndinium sp., an organism that parasitizes radiolaria (Ris and Kubai, 1974). In Syndinium, through all stages of the division cycle, including interphase, kinetochores are found to project through pores in the nuclear envelope and are the site of termination for microtubules which radiate from a pair of centrioles. During interphase the kinetochore insertion points are confined to a distinct area of the nuclear surface where an indentation of the nuclear envelope forms a cuplike depression containing the two centrioles. As nuclear division begins, additional microtubules grow between the centrioles, separating them and ultimately converting the centriole-containing cup into a single cytoplasmic channel running through the center of the nucleus. During this process the microtubular centriole-kinetochore connection is maintained (Fig. 4); and, most significantly, the length of the kinetochore microtubules remains constant. This latter finding prompted the conclusion of Ris and Kubai that the force for chromosome separation is generated entirely through elongation of intercentriole microtubules, while kinetochore microtubules passively anchor chromosomes to the separating poles. If this interpretation is correct, Syndinium may be the first instance in which we can easily distinguish two sets of microtubules that function quite differently, intercentriole microtubules playing an active role in chromosome movement, while kinetochore microtubules have only a static function. Syndinium sp. is an organism that has been classified as a dinoflagellate. Clearly, there is a striking contrast between mitosis in this parasitic species, in which elongation of intercentriole microtubules seems to account for both channel formation and kinetochore separation, and nuclear division in the free-living dinoflagellate C. cohnii (Section III,B,l), in which channels pinch off around fully formed

EVOLUTION OF THE MITOTIC SPINDLE

191

FIG.4. Syndiniuin sp. A single cytoplasmic channel passing through the nucleus is formed as microtubules elongate between the separating centrioles. Kinetochores (arrows) protrude through openings in the nuclear envelope and are connected to the centrioles via chromosomal microtubules. No shortening of chromosomal microtubules is noted at any time during division. x42,OOO. (Micrograph from Ris and Kubai, 1974, reproduced by permission of The Rockefeller University Press.)

microtubules and chromosome separation gives no evidence of being directly dependent on microtubule function. A further difference between Syndinium and typical dinoflagellates exists at the level of chromosome structure and chemistry (Xis and Kubai, 1974). Syndinium chromosomes are composed of D N A plus basic protein and consequently are not at all ultrastructurally similar to typical dinoflagellate chromosomes in which protein-free D N A is arranged in a characteristic pattern. These distinctions were the basis for a suggestion by Xis and Kubai that Syndinium may not be closely allied to the dinoflagellates. A recent report by Cachon and Cachon (1974; see also Cachon, 1964), however, suggests that such differences, at least with respect to chromosome structure and chemistry, may be related to the parasitic versus free-living life-styles of these two organisms. In Oodinium fritilluriue, an appendicularian parasite, these investigators claim, chromosome-associated basic proteins are present only during the vegetative and earliest sporogenetic stages, progressively disappearing as chromosomes assume an increasingly dino-

192

DONNA F. KUBAI

flagellatelike ultrastructure. Ultrastructural changes related to stage in the life cycle are also known in parasitic Blastodinium (Soyer, 1971) and in free-living Noctiluca miliaris (Soyer, 1970), but no evidence of such a transition has been found in Syndinium, either by Ris (personal communication) or by Manier et al. (1971), who examined another species of Syndinium. Final resolution of the question of whether or not chromosome differentiation is a general phenomenon among parasitic dinoflagellates awaits further investigation. In any case 0. fritillariae is a parasitic species having typical dinoflagellate chromosomes during at least part of its life cycle; and in this organism Cachon and Cachon (1974) demonstrated a Syndinium-like connection between cytoplasmic microtubules and a dense knoblike differentiation of the nuclear envelope at the site where chromosomes contact the membrane. Similar structures were found in another parasitic species Haplozoon axiothellae (Siebert and West, 1974), as well as in a free-living dinoflagellate, Amphidinium sp. (Fig. 5) (Oakley and Dodge, 1974). In both 0.fritillariae and Amphidinium sp., the conformation of the nuclear envelope in the region where microtubules impinge on kinetochores suggests that the microtubules transmit a tension to this site. Thus at least some species of dinoflagellates, parasitic and free-living, seem to undergo microtubule-mediated chromosome movements; and S yndinium may be more closely related to dinoflagellates than supposed b y Ris and Kubai (1974). Recent studies of dinoflagellate nuclear division raise several questions regarding whether or not a single mechanism of chromosome movement may be considered to characterize the dinoflagellates. In this context we must inquire whether or not C. cohnii, studied by Kubai and Ris (1969), or any of the other dinoflagellates in which chromosome-membrane connections have so far been considered to bear direct responsibility for all chromosome movement (Soyer, 1969, 1971; Dodge, 1971), exhibits cytoplasmic microtubule termination at the site where chromosomes attach to the nuclear envelope. This may be the case at least certain stages of the life cycle overlooked by these investigators. Conversely, the question whether or not the direct influence of microtubules is required for all stages of chromosome movement in the Amphidinium species studied by Oakley and Dodge (1974), 0.fritillariae (Cachon and Cachon, 1974), and H . axiothellae (Siebert and West, 1974) remains open. Further, even if it is found that all these species have in common a microtubule-mediated mechanism of chromosome movement, the extent of its similarities to nuclear division in Syndinium sp. must be deter-

EVOLUTION OF THE MITOTIC SPINDLE

193

FIG. 5 . Amphidinium sp. Some of the extranuclear microtubules found in cytoplasmic channels passing through the dividing nucleus terminate at dense differentiations at the site of chromosomenuclear envelope contact (arrows). Note that the conformation of nuclear envelope in this region is consistent with the possibility that these microtubules are exerting a tension on the chromosome-membrane connection. x 40,000. (Micrograph from Oakley and Dodge, 1974, reproduced by permission of The Rockefeller University Press.)

mined. Centrioles, structures that serve as polar attachment sites for chromosomal microtubules in Syndinium, definitely do not occur in C . cohnii, H. axiothellae, 0 .fritillarae, or Amphidinium sp., and so some variations in the details of nuclear division will probably continue to distinguish the various species of dinoflagellates.

C. A TENTATIVEOUTLINE OF SPINDLE EVOLUTION The diversity in nuclear division found among dinoflagellates and in hypermastigotes must certainly be accepted as compelling evidence that it is not possible to define a single standard mode of chromosome distribution as characteristic of eukaryotes. Even more significant, although perhaps surprising, is the fact that these orga-

194

DONNA F. KUBAI

nisms provide an almost schematic example of one possible sequence in the early evolution of mitosis:

1. In the early stages of eukaryote evolution, the attachment of chromosomes to the nuclear membrane was a necessary condition for their distribution to daughter cells, and microtubules were restricted to a static, cytoskeletal role (e.g., C . cohnii; see Section III,B,l). 2. Later, chromosome-membrane interaction continued to be important in at least early stages of chromosome movement, but specific microtubule-chromosome associations are found at the kinetochore and might have some bearing on later stages of chromosome distribution (e.g., T . ugilis; see Section III,B,2). 3. In the later stages of evolution, chromosome movements became totally microtubule-mediated, but only some of the microtubules function actively in force production (for spindle pole separation), while another group of microtubules makes only a static contribution in maintaining a chromosome-spindle pole connection (e.g., Syndinium sp.; see Section III,B,3). IV. The Participation of Microtubules in Typical Mitosis The conclusion that microtubules are not direct participants in the actual movement of chromosomes in some dinoflagellates and hypermastigotes is almost unavoidable in view of the finding that the nuclear envelope is a physical barrier between chromosomes and microtubules during nuclear division. Similar inferences have been drawn for some other eukaryotes that display unusual features of mitosis. However, in these cases the intrunuclear appearance of microtubules renders such an interpretation less obvious from the start, and we must question further whether or not the function of these microtubules differs significantly from microtubular filnction in typical mitosis.

A. CHROMOSOME BEHAVIOR In examining any problematic division, it is useful to recall some of the salient features of mitosis as it occurs in higher plants and animals (see Table VII, footnote b ) (Luykx, 1970; Nicklas, 1971; Bajer and Mok-Bajer, 1972). As already mentioned, microtubules included in the mitotic spindle occur in two classes, interpolar and chromosomal. Regardless of the formative history of the spindle, whether it is of the central spindle, nuclear spindle, or chromosomal

EVOLUTION OF THE MITOTIC SPINDLE

195

spindle type, ultimately both classes of microtubules intermingle and acquire a parallel disposition which delineates the division axis. Although each chromosome is capable of movement within the spindle as a semiindependent individual, all chromosomes are constrained to move in a direction generally parallel to the interpolar axis (i.e., parallel to the microtubule long axis), both during prometaphase when conjoined sister chromatids move toward the spindle equator to form the metaphase plate (metakinesis), and in anaphase when sister chromosomes separate and move to opposite spindle poles. In both phases of chromosome movement, metakinesis as well as anaphase, a restricted chromosomal region, the kinetochore, is recognized as the site of application of motion-producing force and, ultrastructurally, as the locus of attachment of chromosomal microtubules. Until metaphase, each chromosome is equipped with two kinetochores, one per sister chromatid. Because these premetaphase kinetochore pairs are positioned on opposite faces of the chromosome, the opposite-pole orientation of sister chromatids is a direct consequence of chromosome (i.e., kinetochore) structure; with the kinetochore of one chromatid oriented toward a given spindle pole under the agency of a kinetochore-pole connection by chromosomal microtubules, the oppositely directed sister kinetochore is obliged to face the other pole. This is the chromosome orientation that is fully achieved as a result of metakinesis, a series of movements which brings all chromosomes to the metaphase position equidistant between division poles, with sister kinetochores symmetrically arranged across the spindle equator. Subsequent chromosome movement, that of anaphase, accompanies the separation of sister kinetochores and has the effect of imposing an increasing distance between equivalent sets of sister chromosomes. In at least some mitoses, spindle elongation is a prominent event and gives the impression that one component of anaphase sister chromatid divergence may be the consequence of chromosomes maintaining a fixed connection with separating spindle poles. It is more often found, however, that the progress of anaphase involves the actual movement of chromosomes toward the spindle poles, evident as a shortening of the chromosome-to-pole distance.

MODELS FOR MICROTUBULE FUNCTIONIN CHROMOSOME MOVEMENT Even in purely descriptive terms, comparison of the details of certain nuclear divisions with the prominent features of mitosis outlined in the foregoing paragraphs suggests that the anomalies of these B.

196

DONNA F. KUBAI

unusual divisions reflect atypical modes of chromosome movement. For a real understanding of how unorthodox divisions may be evolutionarily related to the mitotic process, it is necessary to extend comparisons to the level of molecular behavior, especially with regard to the function of microtubules which are such an ubiquitous element of the division apparatus. Unfortunately, w e are not yet equipped to exploit this approach, as investigation of the molecular function of microtubules is only now in an early stage (Olmsted and Borisy,

1973). Suggestions of possible mechanisms of microtubular function within the mitotic spindle are, however, available. One of these is the dynamic equilibrium model proposed by Inouk and Sato (1967), which considers chromosome movement the direct effect of microtubule formation and disassembly, that is, their lengthening and shortening. Experiments involving in vitro assembly of the microtubule precursor protein tubulin demonstrate that microtubules have the capacity to elongate in this manner (Weisenberg, 1972; Borisy and Olmsted, 1972; Shelanski et al., 1973; Borisy et al., 1974) and, further, that the isolated mitotic spindle apparatus can grow in a medium containing tubulin (Inouk et al., 1974; Cande et al., 1974; Rebhun et al., 1973). A different conception of spindle function attributes movement to an interaction between microtubules in which chromosomal microtubules, pulling their attached chromosomes, move along on a scaffolding of interpolar microtubules, either by “sliding” (McIntosh et al., 1969; Nicklas, 1971) or “zipping” (Bajer, 1973) between interpolar and chromosomal microtubules. In contrast to the dynamic equilibrium model which presupposes no functional modification of microtubules beyond their capacity for forming specific associations with chromosomes and for length change, both sliding and zipping models require an additional component to account for the transmission of force laterally between parallel microtubules. The “bridges” or “arms” found to link microtubules in many systems (e.g., cilia and flagella, axostyles and axopods; for refs. see McIntosh, 1974), including the mitotic spindle (Wilson, 1969; Hepler et al., 1970; Brinkley and Cartwright, 1971; McIntosh, 1974), are seen as likely participants in any sliding or zipping mechanism. Experimental evidence for the sliding of microtubules in ciliary and flagellar movement is available in the works of Satir (1965, 1968) and Summers and Gibbons (1971). Cross-bridge models of spindle function offer a background for evolutionary speculation. As suggested earlier (Section II,B), func-

EVOLUTION OF THE MITOTIC SPINDLE

197

tional evolution of microtubules may have involved their acquisition of an entirely new dynamic property as an adjunct to preexisting static characteristics. The appendage of force-producing crossbridges to microtubules previously capable only of simple cytoskeletal functions conforms to such a scheme and suggests a simple basis for functional differentiation between classes of microtubules. Just such a distinction between coexisting chromosomal and interpolar microtubules is an important feature of Nicklas’ (1971) modification of the sliding-microtubule model of mitosis. I n the Nicklas model active cross-bridges occur only on interpoIar microtubules and transmit poleward forces to nearby chromosomal microtubules, thereby generating poleward chromosome motion. Spindle elongation is not provided for in Nicklas’ model of cross-bridging, but may be ascribed to growth of interpolar microtubules. The Nicklas model, then, presents the idea that there may b e two types of microtubular function involved in the activity of the mitotic spindle. Moreover, if we concede that a mechanism strictly related to microtubular growth should be considered more primitive, evolutionarily speaking, than one dependent on cross-bridging between microtubules, this model also alludes to the possibility that the spindle retains some relatively primitive features in its highly adapted mechanisms of chromosome distribution.

V. The Possibility of an Unconventional Role for Microtubules in Chromosome Movement A. NUCLEAR DIVISIONSWITHOUT MICROTUBULE-CHROMOSOME CONNECTIONS Whether or not any of the current models are found to give valid representation of the details of spindle function, they serve to underscore the importance of two classes of microtubules, both interpolar and chromosomal, in the production of chromosome movement during mitosis. By contrast, there exist claims of systems of nuclear division wherein the division-related microtubules seem to belong to only a single category and, most significantly, are never found to engage in the sort of specific chromosome-microtubule association found at the kinetochore in typical mitosis. The closed nuclear divisions of euglenoids, trypanosomes, the ciliate macronucleus, and some fungi are examples in which no signs of specific termination of microtubules at chromosomes have been found. The diversity of these divisions is striking.

198

DONNA F. KUBAI

1. Euglenoidina In Euglena gracilis (Leedale, 1968), for example, microtubules appear in bundles which span the nucleus parallel to the nuclear long axis. Concomitant with nuclear elongation, the persistent nucleolus elongates and, at a stage Leedale equates with metaphase, discrete chromosomes are massed about the midportion of the cylindrical nucleolus, lying with their long axes parallel to the common nuclear and nucleolar long axes. Some microtubule bundles are associated with the nucleolar surface, and the remainder pass hetween chromosomes, terminating near the nuclear envelope in undifferentiated polar nucleoplasm. It has not been determined whether microtubules are continuous from pole to pole; neither, however, have they ever been seen to terminate at chromosomes. Similar observations have been made for other euglenoids (Blum et al., 1965; Sommer and Blum, 1965; Mignot, 1966; Schuster et al., 1968). This apparent absence of any kinetochorelike microtubule-associating sites on the euglenoid chromosomes is indicative that the division is unconventional. Additionally, there are light microscope observations that the time of separation of sister chromatids is variable in different species of Euglena (Leedale, 1958, 1966, 1967, 1968), in E . grucilis occurring even before chromosomes congregate near the spindle equator. In the absence of any positive indications that microtubules influence chromosome distribution, Leedale (1968) has suggested that euglenoid chromosomes move “autonomously,” microtubules playing only an indirect role as directional guides. If microtubules indeed are important in euglenoid nuclear division simply as a structural framework either for nuclear elongation or for chromosome movement derived from a force-generating system unrelated to microtubules, this is another example of the postulated most primitive stage in the history of involvement of microtubules in nuclear division. However, no suggestion of the existence of an alternative forcegenerating system has been uncovered, so it seems more likely that these microtubules play a more dynamic, albeit unconventional, role in chromosome movement. If the close lateral associations between microtubule bundles and chromosomes that are obvious in some micrographs of euglenoid mitosis [e.g., Figs. 56 and 60 of Leedale (1968)l are of significance, a cross-bridge interaction between microtubules and chromatin may account for chromosome movement in the manner discussed at length by Heath (1973).

EVOLUTION OF THE MITOTIC SPINDLE

199

2. Protomonida I n reproducing trypanosomes intranuclear microtubules are massed in a single fairly discrete bundle which is centrally placed and spans the length of the elongating nucleus (Bianchi et al., 1969; Inoki and Ozeki, 1969; Vickerman and Preston, 1970; de Souza and Meyer, 1974). This bundle may ensheath the persistent nucleolus, as in Trypanosoma gambiense (Inoki and Ozeki, 1969) or T . rhodesiense (Vickerman and Preston, 1970), or not, as in T . cruzi (de Souza and Meyer, 1974) or T . raiae (Vickerman and Preston, 1970), in which the nucleolus appears to disperse during division. In culture forms of T. rhodesiense (Vickerman and Preston, 1970) and T. cruzi (de Souza and Meyer, 1974), chromatin, easily recognized in the nondividing nucleus as dense submembrane masses, usually becomes dispersed during division, with the result that neither chromatin nor discrete chromosomes are recognizable. Such chromatin dispersion is not so pronounced in the bloodstream forms of T . rhodesiense examined by Vickerman and Preston (1970); however, the chromatin appears even during division to remain distributed over the entire inner surface of the nuclear envelope, well-separated from the central microtubular mass. Trypanosomes thus represent another class of organisms in which no specific interaction of microtubules and chromatin is discernible, and so Vickerman and Preston tentatively suggest that the observed close apposition of chromatin and membrane may signal membrane involvement in genophore distribution.

3. Ciliophora In these protozoa genetic function is partitioned between two nuclei, the micronucleus and the macronucleus. The micronucleus distributes a diploid genetic complement to daughter cells at each asexual generation, but appears to have no further vegetative function. Its major contribution seems to be in the recombination of genetic material after meiosis. These micronuclei display fair1y typical closed mitotic division. The macronucleus, however, bears sole responsibility for the vegetative functions of ciliates and is reproduced via plainly atypical division. For a more detailed discussion of the differences in biological activity of these two types of nuclei, consult Nanney and Rudzinska (1960) and Raikov (1969). a. Micronucleus. The best developed ultrastructural descriptions of ciliate micronuclear divisions are concerned with the holotrichs Paramecium aurelia (Jurand and Selman, 1970; Stevenson and

200

DONNA F. KUBAI

Lloyd, 1971a), Loxodes magnus (Raikov, 1973), and Nassula sp. (Tucker, 1967), and with the spirotrich Blepharisma sp. (Jenkins, 1967). In these species chromosomes become aligned in metaphase configuration at the equator of a mitotic spindle comprising both interpolar and chromosomal microtubules. In P . aurelia and Blepharisma sp., chromosomal fibers are recognized as bundles of microtubules abutting on a localized region of the chromosome, and in Nassula sp. occasional association of chromosomes and microtubules is noted; but a differentiated kinetochore has not been demonstrated in these species. In another ciliate, L. magnus, the region of microtubular contact with chromosomes is distinctly layered and so resembles strongly the kinetochore of higher animals. Chromosomes in L. magnus being highly condensed and very small, the kinetochore “region” extends over the entire poleward face of the chromosome. During prometaphase, Raikov (1973) found chromosomal niicrotubules in contact with the kinetochore before chromosomes became aligned in the metaphase plate, and so bundles of chromosomal microtubules are at this time in varying orientations and not parallel to the interpolar microtubules. This finding is consistent with the role chromosomal fibers appear to play in chromosome orientation and metakinesis in higher organisms (Nicklas, 1967; Nicklas and Staehly, 1967). In general, then, micronuclear mitosis in ciliates is quite similar to typical mitosis. As for the actual mechanism by which microtubules implement chromosome movement in micronuclear division, the question remains open, as it does for the mitosis of all eukaryotes. In Blepharisma sp. (Jenkins, 1967), Nassula sp. (Tucker, 1967), P . aurelia (Jurand and Selman, 1970; Stevenson and Lloyd, 1971a), and Diplodinium sp. (Roth and Shigenaka, 1964), however, we have examples of enormous spindle elongation (e.g., the approximately 10-fold elongation of the spindle in P . aurelia). Since this elongation occurs concomitantly with chromosome separation in Blepharisma and at the same time there is little or no diminution of the chromosome-to-pole distance, Jenkins (1967) has‘ suggested that spindle elongation may be the primary factor in chromosome movement in these organisms. I n L. magnus (Raikov, 1973) and Nassula sp. (Tucker, 1967), in contrast, spindle elongation is not considerable, and chromosomal microtubules are reported to shorten during anaphase. b. Macronucleus. Among the several characters that distinguish the two types of nuclei in ciliates is the fact that macronuclear DNA content is usually very much greater than that of the micronucleus.

EVOLUTION OF THE MITOTIC SPINDLE

20 1

Since the macronucleus is derived by transformation from a micronucleus following meiosis, this increased DNA content has been interpreted as reflecting a high degree of ploidy (for references, see Raikov, 1969); but in at least some ciliates the relationship between the genetic complement of macro- and micronuclei may be less straightforward. I n the spirotrich StyZonychia sp. (Prescott and Murti, 1973), for example, not all DNA sequences of the micronuclear complement are found in the macronucleus. Thus it appears that the large quantity of macronuclear DNA represents only a fraction of the micronuclear genome which is replicated many times. Given that the ciliate macronucleus seems to be provided with a large excess of the DNA sequences important for the organism’s vegetative survival, regardless of the source of this redundancy, we might expect that a division mechanism less precise than mitosis might prove sufficient for species survival. The most general statement that may be made regarding the microtubular arrays that appear at the time of ciliate macronuclear division is that they are usually found to have no association with the chromatinic elements in the nucleus. Otherwise, among the various species examined, there appears to b e no consistent disposition of these microtubules. In several ciliates, relatively short microtubule bundles are distributed throughout the nucleus, scattered among the chromatinic elements, and have no preferred orientation relative to each other or to the direction of nuclear elongation [Diplodinium sp.: Roth and Shigenaka (1964); C . umbeZZaria: Carasso and Favard (1965); Zsotricha sp.: Grain (1966); and P . aurelia: Stevenson and Lloyd (1971b)l. As division progresses in P . aurelia (Stevenson and Lloyd, 1971b) and C . umbellaria (Carasso and Favard, 1965), the unoriented bundles lengthen and assume an orientation parallel to the long axis of the elongating nucleus. In P . aurelia (Jurand and Selman, 1970), microtubules apparently eventually become closely packed in a single bundle restricted to the central constriction of the dumbbell-shaped nucleus, and only a few scattered microtubules are found in the chromatinic knobs. A somewhat different disposition of microtubules is noted in Nassula sp. (Tucker, 1967) in which, from their first appearance, macronuclear microtubules are oriented parallel, singly or in small bundles, in the midregion of an already slightly elongate nucleus. Finally, these become grouped as a single bundle within the isthmus of the constricting nucleus, a configuration quite like that reported for the last stage of Paramecium division. Again, in the absence of any demonstration of specific association between microtubules and chromatin during these macronuclear

202

DONNA F . KUBAI

divisions (but see Falk et al., 1968), the possibility that microtubules make no effective contribution to genophore movement is contemplated (Stevenson and Lloyd, 1971b). This idea is strongly reinforced b y the findings for yet another ciliate, Blepharisma sp. (Inaba and Sotokawa, 1968; Jenkins, 1969), in which intranuclear microtubules are never found during division. Instead, cytoplasmic microtubules come to lie adjacent to the nuclear membrane, oriented parallel to the direction of nuclear elongation. Accordingly, microtubules, whether intra- or extranuclear, may function solely in macronuclear elongation, in order to position daughter nuclei at opposite ends of the dividing cell (Tucker, 1967). A “passive” means of chromatin distribution such as would result from simple elongation and constriction of the ciliate macronucleus could be effective in delivering at least the required minimum number of DNA sequences to daughter cells if highly repeated sequences are distributed at random within the dividing macronucleus. At each generation randomization of the replicated chromatin would be required, a process that certainly must involve chromatin movement. Evidence that such movement does take place during interphase is available in the autoradiographic studies of Kimball and Prescott (1962), which demonstrate that replicated DNA is redistributed before division in the hypotrich Euplotes eurystomus, and in the cinematographic studies of the suctorian Tokophrya lemnarum (Heckmann, 1966),which show a vortexlike movement of the nuclear contents. Ultrastructural investigations of the suctorians Tokophrya infusionum (Millecchia and Rudzinska, 1971) and Acineta tuberosa (Bardele, 1969) are suggestive that microtubules may have a role in this sort of movement. During interphase in these species, numerous microtubule bundles are found running between chromatin strands; and, as division commences, their number decreases. As in macronuclear division, no kinetochorelike regions of microtubular termination in chromatin are demonstrated, so it is again possible to question whether or not cross-bridging between microtubules and chromatin (Heath, 1973) plays a role in these interphase chromatin motions. Of course, similar conjectures may be made for the intranuclear microtubules scattered between chromatin bodies in the dividing macronuclei described above; early in division crossbridge-generated movement on haphazardly oriented microtubules would serve in randomization of the nuclear contents while at later stages, after microtubules are in parallel array, a similar mechanism of chromatin movement on microtubular pathways would tend to distribute chromatin to opposite lobes of the dumbbell-shaped nucleus.

EVOLUTION OF THE MITOTIC SPINDLE

203

4. Fungi I n this kingdom (Whittaker, 1969), we encounter several organisms in which ultrastructural studies indicate that only a single type of microtubule is involved in nuclear division. The most striking examples are reported for the mucorine zygomycetes Phycomyces blakesleeanus (Franke and Reau, 1973) and Mucor hiemalis (McCully and Robinow, 1973), in which chromosomal microtubules are not found. The only microtubules observed during nuclear division in these organisms form a compact bundle composed of relatively few microtubules, which comes to occupy the central axis of the elongating nucleus. In P . blakesleeanus in particular, the pole-topole continuity of these microtubules seems unquestionable. Since chromosomal microtubules have not been discovered in either species, some authors naturally consider the possible existence of mechanisms of chromatin distribution that do not require the direct interaction of chromatin and microtubules, that is, movement of chromatin by virtue of its attachment to the nuclear envelope (Franke and Reau, 1973) or to the spindle poles (McCully and Robinow, 1973).It is appropriate to defer an evaluation of such suggestions until we can consider them in the context of a more complete enumeration of the unusual features observed in fungal mitosis (Section VI,B,l). B.

SOME POSSIBLESOURCESOF MISINTERPRETATION

As outlined above, we are confronted with a collection of organisms in which the single type of microtubules concerned with nuclear division appears to form no specific associations with chromatin and for which, therefore, the possibility of extraordinary mechanisms of chromatin distribution has been entertained. I n considering any proposition based on such negative ultrastructural findings, we must take full account of the uncertainties inherent in the method. One of the most disquieting aspects of any ultrastructural investigation is the necessity for reliance on a given fixation procedure to provide satisfactory preservation of several chemically dissimilar cellular components. For the study of nuclear division, these include at least microtubules, chromatin, and membrane, as well as any specific associations formed between them. In the case of microtubules, the critical importance of fixation is well illustrated by the fact that little more than a decade ago the available preparative procedures were adequate for the preservation of only certain microtubules. Only after the introduction of glutaraldehyde fixatives (Sabatini et

204

DONNA F . KUBAI

al., 1963) did their widespread occurrence become apparent. Fixation properties are thus indicative that microtubules respond differently to differing chemical treatment; and that differences in susceptibility to a given chemical treatment distinguish interpolar and chromosomal spindle microtubules is suggested by experiments involving treatment of the mitotic spindle with agents such as chloral hydrate (Ris, 1949; Mok-Bajer, 1969) and Colcemid (Brinkley et al.,

1967). In addition to misinterpretations which might stem from difficulties in preservation for electron microscopy, the thin-section technique is a source of potential error, The most prevalent approach involves examination of a rather limited number of random sections taken in random orientation, and it is not unlikely that subtle details such as relatively miniscule and/or poorly differentiated kinetochores or low numbers of chromosomal microtubules may escape detection in such a sample. A comparison of the studies of closed nuclear division in plasmodia of Physarum polycephalum is illustrative of the disparate results that can be obtained in ultrastructural studies. In earlier investigations (Guttes et al., 1968; Goodman and Ritter, 1969), microtubules were not found in the interzone between separating anaphase chromosome sets; and it thus appeared that interpolar microtubules were absent in this mitosis. Microtubules seen between chromosomes and poles were considered chromosomal microtubules, although no kinetochorelike differentiations were found on the chromosomes. A painstaking reexamination of P . polycephalum was undertaken by Ryser (1970); and through study of serial sections of more favorably fixed material, this investigator demonstrated well-defined disclike kinetochores, each being the site of termination for one or two chromosomal microtubules. In serial cross sections 115 microtubules were traced. On the basis of the expectation that 75 to 80 of these must be associated with kinetochores, it is estimated that there occur 30 to 40 interpolar microtubules in the P . polycephalum spindle. Because there are examples such as P . polycephalum in which significant details can be revealed only with the most favorable preservation and after searching exploration, we must remain skeptical of all suggestions of unusual mechanisms of genophore distribution that have as their sole foundation the negative evidence that microtubules cannot be found to interact with chromatin in the conventional manner. All interpretations that chromosome movement in the above-described euglenoid, trypanosome, ciliate, and fungal nuclear

EVOLUTION OF THE MITOTIC SPINDLE

205

divisions is unconventional must therefore remain tentative in the absence of concrete demonstrations of alternative means for force production.

VI. Nuclear Divisions with Microtubule-Mediated Chromosome Movements

A. CONVENTIONAL METAPHASE As we are aware from the studies of T . agilis (Kubai, 1973) and Syndinium sp. (Ris and Kubai, 1974) (see Sections III,B,2 and B,3), the mere existence of distinct kinetochores as the chromosomal sites of microtubule termination is not certification that these microtubules participate actively or conventionally in chromosome movement. Thus, even for those nuclear divisions in which kinetochores and/or chromosomal microtubules are clearly demonstrable (Tables IV-VI), a key for their comparison with so-called typical mitosis remains the analysis of actual chromosome movements. A most striking aspect of orthodox mitotic chromosome behavior is the fact that chromosome movement occurs in two phases, separation of sister chromosomes taking place only after metakinesis which brings conjoined sister chromosomes to the metaphase position midway between the poles of a well-formed spindle. Since the metaphase configuration can be understood as a crucial condition in orienting chromosomes preparatory to their final separation, the formation of a metaphase plate seems to b e one valid indicator for conventional chromosome behavior. By using this criterion nuclear divisions in many lower organisms do not appear to deviate significantly from typical mitosis (Tables 1-111). Although not yet possible, it is only precise comparisons of their molecular behavior that will reveal whether there are fundamental differences in function among these “typical” spindles. Nonetheless, it bears noting that the lower organisms encompass several examples of mitosis in which spindle elongation is impressive, while little or no decrease in the chromosome-to-pole distance can be discerned, for example, Pelomyxa carolinensis and PeZom yxa illinoisensis (Daniels and Roth, 1964); Paramecium aurelia (Stevenson and Lloyd, 1971a); Blepharisma sp. (Jenkins, 1967); Klebsormidium sub; jlaccidum (Floyd tilissimum (Pickett-Heaps, 1 9 7 2 ~ )Klebsormidium et al., 197213);and Vaucheria litorea (Ott and Brown, 1972). Such observations are consistent with suggestions (e.g., Jenkins, 1967; Floyd et al., 1972b; Pickett-Heaps, 1972c, 1974) that chromosome niove-

206

DONNA F. KUBAI

ment in these cases is a function solely of spindle elongation and chromosomal microtubules have no active role beyond anchoring chromosomes to the separating poles. Without further information, however, it is impossible to distinguish this from spindle function in which rates of spindle elongation and rates of movement of chromosomes toward the poles (e.g., sliding) are balanced such that an apparently constant chromosome-pole relationship is maintained (Pickett-Heaps, 1 9 7 2 ~ ) . B.

UNCONVENTIONAL METAPHASEARRAYS

Just as the occurrence of typical metakinesis and its end result, the metaphase plate, can b e taken as a minimal indication of conventional mitosis, its absence or modification might arouse suspicion that atypical mechanisms of chromosome distribution operate. Again, dinoflagellates, Syndinium sp., and T . agilis (Section II1,B) can be offered as examples, since their unusual division modes are causally related to our failure to observe the metaphase chromosome configuration in these organisms. Because the fungi constitute a group of organisms in which unusual metaphase formations are common, it is worthwhile to examine this kingdom rather extensively. And, while the details of nuclear division in sporozoa have been less thoroughly studied, a certain correspondence between mitotic features in fungi and sporozoans encourages their comparison.

1. Fungi Among the fungi small size and the presence of a cell wall which interferes with fixation and staining have contributed to the difficulty in studying mitosis by means of light microscopy and are responsible for earlier characterizations of fungal nuclear divisions as amitotic. With improved techniques, it became apparent that the “taffy pull” appearance of the dividing nucleus, which had served as the basis for the amitotic interpretation, was the consequence of overstaining (Aist and Wilson, 1968). Nevertheless, even with the more delicate differentiations of spindle and chromatin achieved using methods developed in large part by Robinow and his collaborators (Robinow and Marak, 1966; Robinow and Caten, 1969), the impression that many filngal mitoses are atypical has persisted. A major generalization which has emerged regards the frequent absence of a typical metaphase plate in somatic mitoses of ascomycetes and basidiomycetes. Instead, a so-called two-track configuration of chromatin is seen. The prevalence of this condition is emphasized by Day’s (1972) estimate that this configuration is illustrated, even if not iden-

EVOLUTION OF THE MITOTIC SPINDLE

207

tified as such, in 70430% of all reports on basidiomycete nuclear division. Robinow and Caten’s (1969) investigation of Aspergillus nidulans demonstrates very clearly some of the characteristics of chromatin behavior in such mitoses. At the outset of division, several chromatin bodies appear in a cluster. These later become stretched along either side of a narrow spindle, and the existence of chromatin strands which connect the several chromatin bodies at each side of the spindle gives this stage its two-track or double-bar appearance. Finally, a transverse break appears at the middle of each track, and daughter nuclei are reconstituted from the thus separated blocks of chromatin. Since there is poor correspondence between the number of chromatin bodies observed and the number of linkage groups known from genetic studies, identification of individual chromatin bodies as chromosomes is impossible. I t is this sort of light microscope observation that has been the foundation for persisting ideas that unusual forms of nuclear division may be characteristic of fungi. Ultrastructural studies have revealed that in the fungal kingdom mitosis is, with only few exceptions (Table I), intranuclear, the persistent nuclear membrane remaining either totally intact throughout division (Table 11) or having some localized dispersion of the nuclear envelope occurring either at the poles or along the sides of the elongated nucleus (Table 111). As found in protists and lower algae, intranuclear mitosis can proceed in a manifestly orthodox manner. Examples are also found among the fungi, in which the ultrastructural demonstration of a reasonably typical intranuclear spindle composed of both interpolar and chromosomal microtubules, as well as the congregation of chromosomes in a well-defined metaphase plate configuration, suggests that there is nothing extraordinary about these divisions. Such clear-cut demonstrations are most consistently found among the so-called lower fungi and include representatives of myxomycetes [ P . pol ycephalum, Ryser (1970) and see above Section V,B; Clastoderma debaryanum, McManus and Roth (1968)], chytrids [Catenaria anguillulae, Ichida and Fuller (1968); Phlyctochytrium irregulare, NcNitt (1973)],hyphochytrids [Rhizidiomyces apophysatus, Fuller and Reichle (1965)1, and entomophthorale zygomycetes [Basidiobolus ranarum, Tanaka (1970);Sun and Bowen (1972)l (cf. Tables 11-VI). I n the remainder of the fungal species so far examined, similarly unambiguous ultrastructural indicators of typical mitosis are not always immediately apparent. As alluded to in Section V,A,4, these include species in which it has been impossible to detect chromo-

208

DONNA F. KUBAI

soma1 microtubules, that is, microtubules terminating in chromatin. This is the situation for the zygomycete M . hiemalis (McCully and Robinow, 1973) and several species of hemiascomycetes (for references, consult Table 11), organisms in which chromatin is not condensed and so does not form identifiable chromosomes. Even where condensed chromatin masses are obviously recognizable during division, it is often the case that microtubule-chromatin interactions are not found, as in another zygomycete, P . blakesleeanus (Franke and Reau, 1973), and a variety of ascomycetes and basidiomycetes (for references, consult Tables I-VI). In such absence of direct evidence that microtubules are associated with chromosomes during division, alternative explanations for chromosome movement have been sought; and in this regard attention has been focused on the peculiar differentiations found to be associated with the nuclear envelope in the polar regions of dividing fungal nuclei. These take various forms in the different classes of fungi (Table VII) and can appear, depending on the species, as (1) extranuclear electron-dense masses of variable shape, globular, dumbbell-shaped, plaquelike, or even L-shaped; (2) intranuclear electron-dense masses or layers found just subjacent to the nuclear envelope; (3) pluglike, layered, dense bodies protruding through a porelike opening in the nuclear envelope; or even (4)a simple increased electron density in one or both membranes of the nuclear envelope in the region of the spindle poles. Because these diverse structures are invariably positioned at opposite ends of the intranuclear spindle during division, the general designation spindle pole body (SPB)has been adopted [First International Mycological Congress (1971), cited in Aist and Williams (1972)l for all forms and has the merit of avoiding the structural and functional connotations of such earlier terms as centriolar plaque, microtubule organizing center, and kinetochore equivalent. It is of course the very specific functional implications of the last-mentioned term, kinetochore equivalent, that are of interest to us as an attempt to provide an explanation of chromosome distribution in fungi. Girbardt’s (1971) development of the idea that the SPB functions as a kinetochore equivalent is based on his studies of nuclear division in the basidiomycete Polystictus versicolor but was intended to be more generally applicable to all fungi. In P . versicolor, the dumbbell-shaped SBP sinks into the nuclear space as the nuclear envelope breaks down. Microtubules soon grow between the two globular ends of the SPB to form a spindle having a single globular element at each pole, the globular elements then being pushed

EVOLUTION OF THE MITOTIC SPINDLE

209

progressively farther apart as microtubules elongate. On the basis of his findings that throughout interphase the extranuclear SPB maintains a tight, contiguous relationship with the nuclear envelope and lies in a position opposite the site of association of a patch of chromatinlike electron-dense material and the inner leaflet of the nuclear envelope, Girbardt has suggested that the SPB is the single primary motile center for all the chromatin in the nucleus, that is, a single kinetochore equivalent. This would require that all chromatin of the fungal nucleus be connected to form a single genophore which would in turn be attached to the globular elements of the SPB. Lengthening of microtubules that separate the globular ends of the SPB would then accomplish distribution of all genetic material to two daughter cells. Similar schemes have been contemplated by other investigators (Robinow and Caten, 1969; Franke a n d . Reau, 1973; McCully and Robinow, 1973; see Section V,A,4), and the manner in which such a mechanism could account for the formation of the two-track configuration often seen in light microscopy of fungal division has been considered by Day (1972). Certainly, such a conception of the SPB as a kinetochore equivalent is untenable for the number of fungal species in which the orthodoxy of mitosis is evident from association of chromosomal microtubules with the kinetochore region of individual chromosomes which align conventionally on the metaphase plate (cf. Tables I-VI). Even the more limited view that the SPB might serve as the single motive center for all chromatin only in those species in which chromatin-microtubule associations are not demonstrable and/or in which the two-track configuration is a characteristic of mitosis is contradicted by the ultrastructural demonstrations of kinetochores and chromosomal microtubules in Fusarium oxysporum (Aist and Williams, 1972) and in Thraustotheca clavata (Heath, 1974). By use of serial sections, Aist and Williams showed for F . oxysporum that individually distinct chromatids bear a small differentiated kinetochore region and that each kinetochore is connected to a single chromosomal microtubule. Moreover, this is a species in which the twotrack configuration is easily observed in the light microscope. Aist and Williams established unequivocally that this condition arises because chromosomes at metaphase assume a staggered disposition along the length of the spindle, rather than congregating in the equatorial plane to form the metaphase plate, and that the kinetochores are displaced toward the spindle poles as division proceeds. In studying T. clavata, Heath (1974) took advantage of serial sectioning to reveal the attachment of solitary microtubules to kinetochores. By

2 10

DONNA F. KUBAI

TABLE VII DIFFERENTIATIONS FOUNDA T S P I N D L E POLES Polar structure

Org;tni*ni" Mastigoniycotinab Chytridioniycetes Catenaria angirillulae Blastocladiella eniersonii

Phlyriochytriuiii irreyirlare

Ooniycetes Suprolegnia ferax

Saprolegnio terrestria

Aphanosiyces euteiches Thruustotheru cluorrlri

Zygoinycotina Zygomycetes (Mucorales) Phyconiycr~sblakesleeonvs Mucnr hiemulir Zygoinycetes (Entoinophthoralesj Busidioholus ranuruin Ascomycotiria Heniinscomycetes S a c d i ~ m , t n ~ ~ cereoisiae crs

Wickerharnio fluoreseem Plectomycetes Erysiphv graininis hortli~i Pyrenom ycetes Xylario polyniorpha Xylosphoera polyinorpha Podospirru onserina Podospirro setosu Nrirros~iorocrn,wu

IN

EUMYCOTA Reference

Centrioles. Electron-dense layer, 50 nm lchida and Fuller (1968) thick, subjacent to nuclear envelope Centrioles. Increased electron density of Lessie and Lovett (1968) nuclear envelope. Layer of diffuse material subjacent to nuclear envelope Centrioles in pocketlike depression of McNitt (1973) nuclear envelope. Nuclear envelope ultimately disperses in this area to form fenestrae Heath and Greenwood (1968, 19701 Centrioles paired end to end (180") iii pocketlike depression of noclear envelone. Increased electron density of nuclear envelope. Layer of diffiise material. 30 nni thick, snbjacent to nuclear envelope Howard and Moore (1970) Centrioles paired end to end (180') in pocketlike depression of nuclear envelope. Membranes of nuclear envelope slightly thickened. Fihrnus layer snbjacent to nuclear envelope Centrioles Hoch aiid Mitchell (1972) Centrioles paired end to end (lea-) in Heath (1974) pocketlike depression of nnclear envelope

.Intranuclear electron-dense knob

Franke and Reau (1973)

suhjacent to nuclear envelope lntranuclear electron-dense body snbjacent to nnclear envelope

McCully and Hohinow (1973)

Centriole'

Sun and Bowen (1972)

Multilamniar plaque in porelike opening Rohinow and Marak (1966); Moeiis of nilclear envelope (1971): Rapport (1971): Moens and Rapport (1971): Peterson et a l . (1972) Extranuclear multilaminar plaqne. McCully and Hobinow (1971) Intrannclear electron-dense niaterial subjacent to nuclear envelope Thickening of both inner and outer Rooney and Moenr 71973) membranes of nuclear envelope Extranuclear plaque

McKeen (1972)

Extrannclear Extranuclear Extrannclear Extranoclear Extranuclear

Schrantz (1970) Beckett and Crawford (1970) Zickler (1970) Zickler (1970) Van Winkle et 01. (1971)

plaque plaque ("archontosonie") L-shaped body L-shaped body diglolnilnr structure

211

EVOLUTION OF THE MITOTIC SPINDLE

TABLE VII (Continued) Organism" Uiscomycetes Ascobolus ininiersirs Ascobolus siercororiirs

Pustulurilr cupuloris Pyronemu domesticum

Polar structure

Reference

Extranuclear plaque. Intranuclear fibrous Zickler (1970) layer subjacent to nuclear envelope Extranuclear plaque. Intranuclear fibrous Zickler (1970);Wells (1970) layer suhjacent to nuclear envelope Extranuckar plaque Schrantz (1967. 1970) Plaque (position uncertain because of in- Hung and Wells (1971) sufficient quality of micrographs)

Basidioinycotina Telioniycetes hucosporidiutn scottii

Rhodosporidiirm sp.

Aessosrmrori sulnionicolor

Basidioinycotina Hynienomycetes Coprinir~rudiatus Coprinus lirgopus

Polysiictirs oersicolotArrnilloriu melleo Boletus nrbinelltrs Schizophyllrrni conitiiirtie

Deuteroniycotina H yphoniycetes Aspergillus iiidulons Petririlliirin striotirni Firanriurn ox!isporunl

Extranuclear diglobular structure enters nucleoplasm as gaps appear in iiuclear envelope Extranuclear double-bar structure enters nucleoplasm as gaps appear in nuclear envelope Extranuclear diglobular structure enters nucleoplasm as gaps appear in nuclear envelope

McCully and Robinow (1972a)

Diglobular structure

Lerbs and Thielke (1969); Lerbs (1971) Lu (1967):Raju and Lu (1973)

Monoglobular structure becomes diglohu lar Monoglobular structure becomes diglobular (see text, Section V1,B.l) Globular structure Diglobular struchlre Globular finely filamentous body occurring at polar fenestrae of nucleiir envelope

Plaquelike region of nuclear envelope Extranuclear plaque found in interphase fate in mitosis uncertain Monoglohular body

McCully and Robinow (1972b)

McCully and Robinow (197%)

Girbardt (1968, 1971) Motta (1969) McLaughlin (1971) Raiidaskoski (1970)

Robinow and Caten (1969) Laane ( 1970) Aist and Willimm\ (1972)

'' Claasifivatioii according to Ainvworth (1971). Pickett-Heaps (1971) has developed the argument that centrioles present at spindle poles are not an integral component of the mitotic apparatus but rather are related to the occurrence of a motile stage in the life cycle of the organism. Centrioles ultimately function as basal bodies for flagella (cf. higher plants i n which IIO centriole5 are found and higher animals in which centrioles occur at spindle poles). Thus, in Mastigomycotina, the presence of centrioles is a reflection of the occurrence of a motile stage in the life cycle. T h e body found at the bpindle pole by Sun and Bowen (1972)was termed a centriole, but typical cross-sectional centriolar morphology was never denronstrated. Tanaka (1970) fouiid no centrioles in this organism, as would he expected since there is no motile stage in t h r life cycle (cf. Pickett-Heaps, 1971).

recording the positions of kinetochores relative to the spindle poles, he showed that although T . clavata kinetochores are not arranged in a flat plate at metaphase they are distributed throughout a rather restricted zone nearer the middle of the spindle. Later, chromosomal fibers shorten and the kinetochores move closer to the spindle poles. These studies on T . clavnta and F . oxysporum are outstanding ex-

212

DONNA F. KUBAI

amples of the importance of technique in uncovering subtle details of mitosis. In both, the use of serial sections was crucial for the analysis of the small, minimally differentiated kinetochores each of which is associated with but a single microtubule. In addition, Aist and Williams (1972) have underscored the significance of the proper choice of fixation conditions in their report that overlong postfixation with osmium tetroxide seems to decondense metaphase chromosome of F . orysporum so that they can no longer be recognized in electron microscope preparations. Thus further investigation with improved techniques will probably yield evidence of microtubule-chromatin associations for many of the species in which previous searches for such evidence have been unfruitful. In summary, the demonstrations by Aist and Williams (1972) and Heath (1974) that chromosomal microtubules are directly involved in anaphase chromosome movement in F. oxysporum and T . clavata, when taken together with the several observations of kinetochores and/or chromosomal microtubules in various other species of fungi (including several of the so-called higher fungi, Tables IV-VI), leave little justification for Girbardt’s conjecture that fungal SPBs may be generally analogous to kinetochores, taking an active role in anaphase chromosome movement. Several lines of evidence are suggestive that early phases of chromosome movement and the orientation of chromosomes relative to the spindle poles may be unusual in fungi. These relate to the numerous indications from light microscopy that the main mass of interphase chromatin remains connected to the SPB by means of a chromatinic strand and to parallel ultrastructural indications such as those presented by Aist and Williams (1972) and by Girbardt (1971), who found that a mass of chromatinlike material is closely associated with the nuclear membrane just subjacent to the SPB during interphase in P. versicolor and F . oxysporum. Aist and Williams speculate that this may represent an accumulation of kinetochore-associated heterochromatin, possibly marking a continuing interphase connection between SPB and kinetochores, and that “as the kinetochore microtubules grow during prophase, the specific connections between the chromatids and the SPB’s . . . would b e maintained via the kinetochore microtubules.” Unfortunately, virtually no ultrastructural information is available regarding the early phases of chromosome condensation, prometaphase movements, and the processes of kinetochore-spindle microtubule engagement in these species. Although Fig. 7 of Aist and Williams (1972) illustrates condensed chromosomes closely as-

EVOLUTION O F THE MITOTIC SPINDLE

213

sociated with the microtubules of the very short, just-forming spindle in F . oxysporum, these investigators gathered no information as to whether or not chromosomal microtubules join kinetochores and poles at this stage. In other fungi there are, however, observations indicating that such a connection is present very soon after the beginning of spindle formation. In Saprolegnia ferax (Heath and Greenwood, 1970), for example, the spindle poles are identifiable as centriole-containing, pocketlike indentations of the nuclear envelope, which move progressively farther apart on the nuclear surface as intranuclear spindle microtubules are laid down between them. Before this separation is very advanced, and even before a well-defined spindle has developed, very short microtubules are seen to extend from the polar pockets to nearby kinetochorelike structures (Fig. 6). Essentially the same findings have been made for another species of oomycete, T. clauata (Heath, 1974); and, in addition, Heath has

FIG. 6. Snprolegnia ferax. At the earliest stages of division, when centrioles have just begun to separate on the nuclear surface, kinetochorelike structures (arrows) are already found to be connected to the division poles via very short microtubules. x 74,800. (Micrograph from Heath and Greenwood, 1970, reproduced by permission of The Journal of General Microbiology.)

214

DONNA F. KUBAI

reported (but not illustrated) that at a time during interphase, even before centriole replication signals impending mitosis, kinetochorelike structures are connected to the centriolar pocket by very short microtubules. Although little more than suggestive, observations such as these on S. ferax and T. clavata present the possibility that an early connection between kinetochores and division poles, a connection that may even be maintained throughout interphase, is required for early phases of chromosome movement. Under such a system the chromosomal microtubules joining kinetochores and division poles need function simply as anchors, while actual chromosome movement at this time could derive solely from the forces causing separation of the SPBs. If such separation of SPBs were related to the growth of interpolar microtubules, as may be the case in T . clauata (Heath, 1974), S. ferux (Heath and Greenwood, 1970), and P . versicolor (Girbardt, 1968, 1971), similarity to chromosome movement in Syndinium sp. (Section 111,B,3) would be obvious. Thus early phases of chromosome movement in these fungal species might involve no active functioning of the chromosomal microtubules; rather, the direct role in force production for chromosome movement would derive from elongation of interpolar microtubules. In yet other fungi it has been shown that SPBs move apart on the nuclear envelope before interpolar microtubules span the distance between them; and the possibility that SPB separation is caused by nuclear envelope growth or flow must be considered (McCully and Robinow, 1971). This sort of evidence is available for the hemiascomycetes Schizosaccharomyces pombe (McCully and Robinow, 1971) and Sacchuromyces cerevisiae (Moens and Rapport, 1971; Byers and Goetsch, 1973). In S. cerevisiae, for example, the two justseparated SPBs lying side by side on the nuclear surface are not joined by interpolar microtubules and therefore have probably moved apart without the influence of microtubules. A t this stage each SPB is, however, already the focus of a fanlike radiation of microtubules into the nucleoplasm. Because S. cerevisiae is a species in which individual chromosomes are not readily distinguished in the conventionally fixed, thin-sectioned nucleus, these investigators could not determine if chromatin is already attached to the microtubules at this early stage in spindle formation. However, after examining the fully formed spindle of S. cerevisiue by means of high-voltage electron microscopy, Peterson and Ris (personal communication) found that only a few microtubules are continuous from pole to pole (interpolar microtubules), and have concluded therefore

EVOLUTION OF T H E MITOTIC SPINDLE

215

that the majority of microtubules in the S. cerevisiae spindle are probably chromosomal microtubules. Additional observations indicate that most of the microtubules emanating from each division pole in the fully formed spindle terminate in chromatinlike material (Fig. 7) and are present in sufficient number to allow one chromosomal microtubule per linkage group (as determined from genetic studies). From these findings of Peterson and Ris, we may infer that most if not all of the microtubules found to be connected to the justseparated SPBs of S . cerevisiae are destined to become, if they are not already, attached to chromatin. It is to be hoped that the techniques of Peterson and Ris will prove equally useful in revealing the relationship between microtubules and chromatin at the earliest stages of SPB separation. If it were found that here also, as in T . clavata and S. ferax, microtubular connection between chromosomes and poles is already established at the earliest stages of SPB separa-

FIG. 7. Snccknroinyces cereuisiae. Most of the microtnhriles that emanate from the SPB are found to terminate at chromatinlike masses (arrows). Cell wall removed by glusulase digestion; fixation in paraformaldehyde-glutaraldehydefollowed by osmium tetroxide. x 53,500. (Unpublished micrograph, courtesy of Joan Peterson, Zoology Deparhnent, University of Wisconsin.)

216

DONNA F. KUBAI

tion, it would be difficult to reject an interpretation that early stages of chromosome movement may occur with no dynamic component of microtubule function; membrane phenomena causing polar separation could serve alone for the fundamental force production, while the contribution of microtubules need be only static in that they anchor chromosomes to the moving poles. Aside from its implications for the possible mechanisms of early chromosome movement, the relative time at which an association between kinetochores and poles is irrevocably established has important consequences for other aspects of chromosome behavior. For example, we may suppose that, once the connection of sister kinetochores to opposite poles is secured, appropriate chromosome orientation is achieved and the continued connection of sister kinetochores is no longer required. Thus the very early attachment of kinetochores to spindle poles as in T. clavata may explain Heath’s (1974) finding that at “metaphase” in this species there is no close association of sister kinetochores, that is, kinetochores d o not occur as pairs at this stage as they do at metaphase in higher cells (Nicklas, 1971). The finding of paired kinetochores at a corresponding stage of division in F. oxysporum (Aist and Williams, 1972) is of course not contradictory to a possible early chromosome-spindle pole attachment, since there would be no requirement that sister kinetochores disjoin immediately following establishment of their appropriate orientation. Further unconventional aspects of chromosome behavior would be expected if the connection between kinetochores and poles were not simply early but were persistent (cf. Syndinium sp., Section 11I,B,3), remaining unbroken throughout the cell cycle and merely changing its outward manifestations as chromosome microtubules grow between poles and kinetochores, as suggested by Aist and Williams (1972). If this were the case, it is not unreasonable to think that all kinetochores of one parental chromosome set remain attached to the “parental” SPB, while all kinetochores of the newly replicated chromosomes are obliged to associate with a newly formed SPB. The resulting strict coordination of the orientation of all chromosomes of a given generation to a given SPB would be strikingly different from the independent orientations of individual chromosomes, which we consider a characteristic of the prometaphase behavior of chromosomes in orthodox mitosis; but in species such as the hemiascomycetes S. cerevisiae (Moens, 1971; Moens and Rapport, 1971) and Wickerhamia fluorescens (Rooney and Moens, 1973), or the oomycete Saprolegnia terrestris (Howard and Moore, 1970), in

EVOLUTION OF THE MITOTIC SPINDLE

217

which both meiosis I1 spindles are formed within a single nuclear envelope, there would be advantages for a system that maintains the integrity of complete chromosomal sets and thereby prevents the intermingling of two chromosomal generations. Autoradiographic observations of the distribution patterns of radioactively labeled DNA in A. nidulans indicate that an extremely strict coordination of chromosome orientation can occur in fungal divisions (Rosenberger and Kessel, 1968). When conidiospores fully labeled with adenine-3H were germinated and the resulting hyphal growth followed through three generations in nonradioactive medium, only two nuclei of each hypha were found to retain the label, the remainder of the nuclei being nonradioactive. These results indicate that at each division the originally labeled chromosomes segregate as a set from the newly replicated nonradioactive chromosomes, exactly the sort of coordinated chromosome movement that might result if all chromosomes of a set remain attached to a single structure such as the SPB. From the foregoing we can see that there are several fungi whose nuclear divisions give no sign of differing significantly from typical mitosis. Nevertheless, tantalizing fragments of evidence are available for several species of fungi, which suggest there may b e peculiarities in early chromosome behavior deriving from the manner in which chromosomes become associated with the division poles. These indications have profound bearing on the question whether or not proper chromosome distribution in these fungi requires a period of prometaphase orienting activity similar to the metakinesis of typical mitosis, and on the question whether or not there may be a contribution of membrane-mediated force production in early stages of chromosome movement. So, for judgment of the extent to which fungal nuclear divisions resemble typical mitosis, we must await investigations that direct major attention to discovering the true nature of the relationship between chromatin and spindle poles through interphase and at the earliest stages of nuclear division.

2. Sporozoa Numerous ultrastructural studies have dealt with nuclear division at various stages in the complex life cycle of organisms belonging to the parasitic protozoan class Sporozoa. With the exception of Desportes’ (1970) report of mitosis in Stylocephalus Zongicollus, there is general agreement that these divisions are intranuclear and that spindle microtubules terminate at characteristic polar structures. (In analogy with similar structures in fungi, we refer to these polar differentiations as SPBs.)

218

DONNA F. KUBAI

SPBs of the sporozoa take a different characteristic form in each of the major orders of these organisms. In Gregarinida (Premier, 1972), they are seen as ill-defined, electron-dense masses of cytoplasmic origin. From this mass microtubules radiate into the nucleoplasm through polar openings of the nuclear envelope. In Coccidia the electron-dense SPBs have a distinctly conical form, hence the term centrocone for this formation which is peculiar to the coccids. Details of the obviously intimate relationship between the centrocone (Fig. 8) and nuclear envelope have not been thoroughly elucidated, but the most satisfactory illustrations (Dubremetz, 1973; Hammond et al., 1973) indicate that the centrocone is an extranuclear structure, albeit outlined by foldings of the nuclear envelope [the claim of Hammond e t al. (1973) that the centrocone is enclosed in the perinuclear cisterna is contradicted by their own diagrammatic illustration]. Spindle microtubules pass through pores in the nuclear envelope and continue into the body of the centrocone. In Haemosporidia, the

FIG.8. Eitneriu ninakohlyakimooae. Continuous interpolar microtubules are found to span the distance between centrocones (arrows). x 21,500. (Micrograph from Kelley and Haminond, 1972, reproduced by permission of Springer-Verlag.)

EVOLUTION OF THE MITOTIC SPINDLE

219

SPB is a plaquelike structure of considerable electron density; again, the relationship with nuclear envelope is ill-defined, these SPBs being defined variously as situated “in” or “on” the nuclear envelope, possibly within a porelike opening of this membrane system (for references, consult Table 11). Information relating to chromosome behavior in sporozoa is fragmentary at best. Nevertheless, for each of the major groups there is evidence that both interpolar and chromosomal microtubules contribute to spindle structure (for references, consult Tables IV-VI). Small kinetochores are also found in each group (Tables IV and V); and, with the exception of the gregarine S. longicollus (Desportes, 1970) in which a bundle of many microtubules abuts at the stratified kinetochore, only one or at most very few microtubules connect to these kinetochores. Such indications, together with reports that the distribution of these kinetochores relative to the spindle poles changes as division progresses, moving from a more-or-less equatorial to a juxtapolar position (Aikawa et al., 1972; Canning and Sinden, 1973; Dubremetz, 1973), leave little reason to question that a microtubule-mediated movement of chromosomes occurs in the sporozoa and that therefore these divisions must b e considered to share a significant aspect of typical mitosis. Although little attention has been devoted to the question of chromosome behavior in early sporozoan division, there is some indication that kinetochores may maintain a special relationship to SPBs, a condition that would b e expected to have some effect on early chromosome movements. The most direct evidence pertains to Eimeria necatrix (Dubremetz, 1973), in which it is claimed that spindle formation is initiated extranuclearly, microtubules being laid down between two slightly separated pairs of centrioles. At this stage centrocones are not yet differentiated, but centriolar position indicates the sites on the nuclear surface where they will soon appear. Closely associated with the nuclear envelope in this region are several kinetochore-like layered structures. Somewhat later, spindle microtubules become internalized in the nucleoplasm, and kinetochores are found to be dissociated from the nuclear envelope, lying deep in the nucleoplasm and connected to centrocones via chromosomal microtubules. Finally, kinetochores, which have achieved their polar position and are no longer associated with chromosomal microtubules, are once again found in close association with the nuclear envelope in the region underlying the centrocone. Here they form a submembrane layer of intermediate density, and Dubremetz indicates that the centrocone-kinetochore relationship is maintained as

220

DONNA F. KUBAI

centrocones become yet further separated on the nuclear surface. These observations thus suggest that a specific relationship of kinetochores and centrocone-nuclear membrane complex is established after anaphase chromosome separation; and since a similar nuclear envelope-kinetochore relationship is also found at the earliest stages of division, the possibility that this relationship is maintained through interphase must be considered. For another group of sporozoans, Haemosporidia, similar although less direct inferences may be drawn from the observations that numerous spindles occur at the same time in the very large microgametocyte (Bradbury and Trager, 1968) or oocyst (Howells and Davies, 1971; Canning and Sinden, 1973) nuclei. As suggested for certain fungal meiotic divisions in which more than one spindle per nucleus is also observed (see Section VI,B,l), the necessity that individual chromosomes of a set be restricted to interaction with only the appropriate spindle favors a system in which entire sets of chromosomes are physically constrained to an appropriate site. Such would of course be the case if all kinetochores of a given generation had a persistent connection to the SPB. Like fungi, then, sporozoans constitute a group of organisms having distinctive division-related differentiations associated with the nuclear membrane (SPBs), and there are some indications that a specific relationship between kinetochores and SPBs might be maintained throughout the cell cycle. In the case of the fungi, we have pointed out findings consistent with the idea that an intimate connection of kinetochores and SPBs may govern unusual aspects of chromosome behavior, that is, coordinated movement of entire sets of chromosomes and the lack of association between sister kinetochores at “metaphase.” Information relating to such questions is unfortunately not available for sporozoans, but the parallels already demonstrated for nuclear division in these two groups are suggestive that in sporozoa, as in fungi, significant variations may be found if attention is directed to discovering the mode of early chromosome movement. Again, there is the possibility that early phases of chromosome movement are tied to separation of the SPBs (perhaps membrane-mediated), and that therefore a sequence of prometaphase orienting movements of individual chromosomes is not required.

VII. Final Remarks Earlier in this review we presented the idea that a recounting of the evolutionary past of the mitotic processes of chromosome movement would entail tracing a gradual shift from membranes to micro-

EVOLUTION OF THE MITOTIC SPINDLE

22 1

tubules as the primary producer of forces for chromosome movement. In addition, it was suggested, microtubules themselves might in the course of evolution have acquired the capacity for force production as an adjunct to preexisting static properties. That at least remnants of some of the intermediate stages in such transitions probably survive in unusual forms of nuclear division should be apparent from the foregoing account. Thus we find among these not only organisms such as C. cohnii (Section III,B,I) in which membrane phenomena may yet exert the sole direct force for chromosome movement, but also forms such as hypermastigote flagellates (Section 111,B,2) in which membranes and microtubules may both take a direct role in chromosome movement. As for the notion that functional evolution of microtubules accompanied their increasingly more direct influence on chromosome movement, indications are less compelling. Nevertheless, there are certainly indications that microtubules can assume roles in nuclear division quite unrelated to force production, as in C . cohnii in which a cytoskeletal role for the exclusively extranuclear microtubules seems most likely, and in S yndinium sp. (Section III,B,3) in which chromosomal microtubules effect a permanent connection of kinetochores and poles but seem not to function actively in chromosome movement. Such signs that possible intermediates in the evolution of the mitotic apparatus still exist among living organisms, rather than having been casualties of the selective process, should give us confidence that at least some of the unusual patterns of nuclear division of other lower organisms such as euglenoids, trypanosomes, ciliates, fungi, and sporozoa are of evolutionary significance. ACKNOWLEDGMENTS

I thank Dr. Hans Ris for his critical review of the manuscript. I am also grateful to John D. Dodge, I. B. Heath, Gary L. Kelley, and Joan B. Peterson who kindly provided micrographs, to Cheryl Hughes and Donald Chandler who were responsible for the preparation of Fig. 1, and to Ann Chambers and Terry Rybold who typed the manuscript. During the preparation of this chapter I was supported by a United States Public Health Service grant (CM04738) awarded to Dr. Hans Xis. REFERENCES Aikawa, M., and Beaudoin, R. L. (1968).J.Cell Biol. 39, 749. Aikawa, M., and Jordan, H. B. (1968).J.Purusitol. 54, 1023. Aikawa, M., Huff, C. G., and Sprinz, H. (1967).J. Cell B i d . 34, 229. Aikawa, M., Sterling, C. R., and Rabbege, J. (1972). Proc. Helrninthol. Soc. Wush. 39, Spec. Issue, 174. Ainsworth, C. G. (1971). “Dictionary of the Fungi,” 6th Ed. Commonw. Mycol. Inst., Kew, Surrey, England.

222

DONNA F. KUBAI

Aist, J . R., and Williams, P. H. (1972).J. Cell Biol. 55, 368. Aist, J. R., and Wilson, C. L. (1968). Phytopathology 58, 876. Aldrich, H. C. (1967). Mycologia 59, 127. Aldrich, H. C. (1969). Amer. J. Botany 56, 290. Bajer, A. S. (1973). Cytobios 8, 139. Bajer, A. S., and Molb-Bajer, J . (1972). “Spindle Dynamics and Chromosome Movement,” International Review of Cytology, Suppl. 3, 271 pp. Academic Press, New York. Bardele. C. F. (1969). Z . Zellforsch. Mikrosk. Anat. 93, 93. Bech-Hansen, C. W., and Fowke, L. C. (1972). C a n . ] . Botany 50, 1811. Beckett, A., and Crawford, R. M. (1970).J. Gen. Microbiol. 63, 269. B&u, K. (1926). Ergebn. Fortschr. Zool. 6, 1. Bianchi, L., Rondanelli, E. G., Carosi, G . , and Gerna, G . (1969).J. Parasitol. 55, 1091. Blum, J. J., Sommer, J. R., and Kahn, V. (1965).]. Protozool. 12,202. Borisy, G . G., and Olmsted, J. B. (1972). Science 177, 1196. Borisy, G . G . , Olmsted, J . B., Marcum, J . M., and Allen, C. (1974).Fed. Proc., Fed. Arner. SOC. E x p . Biol. 33, 167. Bouck, G . B., and Brown, D. L. (1973).J. Cell Biol. 56, 340. Bouligand, Y., Puiseux-Dao, S., and Soyer, M. 0. (1968). C . A. Acud. Sci., Ser. D 266, 1287. Bradbury, P. C., and Trager, W. (1968).J. Protozool. 15, 700. Braselton, J. P., and Miller, C. E. (1973). Mycologia 65, 220. Brinkley, B. R., and Cartwright, J. (1971). J. Cell Biol. 50,416. Brinkley, B. R, and Stubblefield, E. (1970). Adoan. Cell Biol. 1, 119-185. Brinkley, B. R., Stubblefield, E., and Hsu, T. C. (1967). J . Ultrastruct. Res. 19, 1. Brugerolle, G . (1973).J. Protozool. 20, 574. Byers, B., and Goetsch, L. (1973). Cold Spring Harbor Symp. Quant. Biol. 38, 123. Cachon, J. (1964). Ann. Sci. Natur. Zool. 6, 1. Cachon, J., and Cachon, M. (1970). Protistologica 6, 57. Cachon, J., and Cachon, M. (1974). C . R. Acad. Sci., Ser. D 278, 1735. Camp, R. R., Mattern, C. F. T., and Honigberg, B. M. (1974). J. Protozool. 21, 69. Cande, W. Z., Snyder, J., Smith, D., Summers, K., and McIntosh, J. R. (1974). Proc. Nat. Acad. Sci. U S . 71, 1559. Canning, E. U., and Sinden, R. E. (1973). Parasitology 67,29. Carasso, N., and Favard, P. (1965).J . Microsc. (Paris)4, 395. Cesana, D. (1971). C . R. Acad. Sci., Ser. D 272, 3057. Chatton, E. (1920). Arch. Zool. E x p . Gen. 59, 1. Chatton, E. (1937). “Titres et Travaux Scientifique.” S&e, Sottano. Cleveland, L. R., Hall, S. R., Sanders, E. P., and Collier, J. (1934).Mem. Amer. Acad. Arts Sci. 17, 185. Cohen, S. S . (1970). Amer. Sci. 58, 281. Cohen, S. S. (1973). Amer. Sci. 61, 437. Cuzin, F., and Jacob, F. (1967). In “Regulation of Nucleic Acid and Protein Biosynthesis” (V. V. Koningsberger and L. Bosch, eds.), pp. 39-50. Elsevier, Amsterdam. Danforth, H. D., and Hammond, D. M. (1972).J. Protozool. 19,454. Daniels, E. W., and Roth, L. E. (1964).J. Cell Biol. 20, 75. Day, A. W. (1972). Can. J . Botany 50, 1337. de Haller, G . , Kellenberger, E., and Rouiller, C. (1964). J. Microsc. (Paris) 3, 627. DeLamater, E. D. (1953). Znt. Reo. Cytol. 2, 158.

EVOLUTION OF THE MITOTIC SPINDLE

223

de Souza, W., and Meyer, H. (1974).J. Protozool. 21,48. Desportes, I. (1970). Ann. Sci. Natrcr. Zool. 12, 73. Desser, S . S. (1972). Can. J. Zool. 50, 477. Dodge, J. D. (1964). Arch. Mikrobiol. 48, 66. Dodge, J . D. (1966). In “The Chromosomes of the Algae” (M. B. E. Godward, ed.), pp. 96-1 15. Arnold, London. Dodge, J. D. (1971). Protoplasma 73, 145. Dodge, J. D., and Bibby, B. T. (19731.J. Linn. Soc. London, Bot. 67, 175. Dodge, J. D., and Crawford, R. M. (1968). Protistologica 4, 231. Dubremetz, J.-F. (1971).J. Microsc. (Paris) 12, 453. Dubremetz, J.-F. (1972).J. Microsc. (Paris) 14, 42a. Duhremetz, J.-F. (1973).J. Ultrastruct. Res. 42, 354. Falk, H., Wunderlich, F., and Franke, W. W. (1968).J. Protozool. 15, 776. Famintzin, A. (1907). B i d . Centralhl. 27, 353. Flavell, R. (1972). Biochem. Genet. 6, 275. Floyd, G. L., Stewart, K. D., and Mattox, K. R. (1972a).J. Phycol. 8, 68. Floyd, G. L., Stewart, K. D., and Mattox, K. R. (1972b).J. Phycol. 8, 176. Fowke, L. C., and Pickett-Heaps, J. D. (1969).J. Phycol. 5,240. Franke, W. W., and Reau, P. (1973). Arch. Mikrohiol. 90, 121. Fritsch, F. E. (1935). “The Structure and Reproduction of the Algae.” Cambridge Univ. Press, London and New York. Fuller, M. S., and Reichle, R. (1965). Mycologia 57, 946. Fulton, C., and Dingle, A. D. (1971).J. Cell B i d . 51,826. Furtado, J. S., and Olive, L. S. (1970). Cytobiologie 2, 200. Giesbrecht, P. (1962). Zentrulbl. Bakteriol., Parasitenk., Infektionskr. Hyg., A h . I: Orig. 187, 452. Girbardt, M. (1968). Symp. Soc. E x p . Biol. 22, 249-259. Girbardt, M. (1971).J. Cell Sci. 9, 453. Goodman, E. M., and Ritter, H. (1969). Arch. Protistenk. 111, 161. Grain, J. (1966). Protistologica 2(2), 5. Grass&, P. P. (1952). “Trait6 de Zoologic" Vol. I, Part 1. Masson, Paris, Grass&,P. P., Hollande, A., Cachon, J., and Cachon-Enjumet, M. (1965). C . R. Acad. Sci. 260, 1743. Grell, K. G. (1964). In “The Cell” (J. Brachet and A. E. Mirsky, eds.), Vol. 4, pp. 1-79. Academic Press, New York. Guttes, S., Guttes, E., and Ellis, R. A. (1968).J. Ultrastruct. Res. 22, 508. Hammond, D. M., Scholtyseck, E., and Chobotar, B. (1969). Z. Parasitenk. 33, 65. Hammond, D. M., Roberts, W. L., Youssef, N. N., and Danforth, H. D. (1973). J. Parasitol. 59, 581. Hauser, M. (1968). Z. Naturforsch. B 23, 887. Hauser, M. (1972). Chromosoma 36, 148. Hauser, M. (1973). Chromosomu 44, 49. Heath, I. B. (1973).I n “The Nucleus” (H. Busch, ed.), pp. 487-515. Academic Press, New York. Heath, I. B. (1974).J. Cell Biol. 60,204. Heath, I. B., and Greenwood, A. D. (1968).J. Gen. Microbiol. 53, 287. Heath, I. B., and Greenwood, A. D. (1970).J. Gen. Microbiol. 62, 139. Heckmann, K. (1966). Puhl. Wiss. Filmen Inst. Wiss. Film, Goettingen l A , 475. Hepler, P. K., McIntosh, J. R., and Cleland, S. B. (1970).J. Cell B i d . 45, 438. Heywood, P. (1973).J. Phycol. 9, Suppl., 15.

224

DONNA F. KUBAI

Heywood, P., and Godward, M. B. E. (1972). Chrornosoma 39, 333. Hoch, H. D., and Mitchell, J. E. (1972). Protoplasma 75, 113. Hollande, A,, and Carruette-Valentin, J. (1970). C. R. Acad. Sci., Ser. D 270, 1476. Hollande, A,, and Carruette-Valentin, J. (1971). Protistologica 7, 5. Hollande, A., and Carruette-Valentin, J. (1972). Protistologica 8, 267. Hollande, A., and Valentin, J . (1968a). C. R. Acad. Sci., Ser. D 266, 367. Hollande, A., and Valentin, J. (196813). C. R. Acad. Sci., Ser. D 267, 1383. Hollande, A., Cachon, J., and Cachon, M. (1969). C. R . Acad. Sci., Ser. D 269, 179. Howard, K. L., and Moore, R. T. (1970). Botan. Gaz. 131,311. Howells, R. E., and Davies, E. E. (1971). Ann. Trop. Med. Parasitol. 65, 451. Hung, C.-Y., and Wells, K. (1971).J. Gen. Microbiol. 66, 15. Ichida, A. A., and Fuller, M. S. (1968). Mycologia 60, 141. Inaba, F., and Sotokawa, Y. (1968).J. Protozool. 15, Suppl., 28. Inoki, S., and Ozeki, Y. (1969). Biken J. 12, 31. Inoui., S., and Sato, H. (1967).J. Gen. Physiol. 50, Suppl., 259. Inoui., S., Borisy, G . G, and Kiehart, D. P. (1974).J. Cell Biol. 62, 175. Ito, J., Lee, Y.C., and Scherbaum, 0. H. (1968). E x p . Cell Res. 53,85. Javornickf, P. (1962). Preslia 34, 98. Jenkins, R. A. (1967).J . Cell Biol. 34,463. Jenkins, R. A. (1969).J. Protozool. 16, Suppl., 10. Johnson, U. G . , and Porter, K. R. (1968).J. Cell B i d . 38,403. Jurand, A., and Selman, G. G. (1970).J . Gen. Microbiol. 60, 357. Keller, S. E., Hutner, S. H . , and Keller, D. E. (1968).J. Protozool. 15, 792. Kelley, G. L., and Hammond, D. M. (1972). Z. Parasitenk. 38, 271. Kelley, G . L., and Hammond, D. M. (1973).J. Parasitol. 59, 1971. Kimball, R. F., and Prescott, D. M. (1962).J. Protozool. 9,88. Kubai, D. F. (1965). M.S. Thesis, Univ. of Wisconsin, Madison. Kubai, D. F. (1973).J. Cell Sci. 13, 511. Kubai, D. F., and Ris, H. (1969).J. Cell Biol. 40, 508. Kudo, R. R. (1954). “Protozoology,” 4th Ed. Thomas, Springfield, Illinois. L a n e , M. M. (1970). Hereditas 65, 133. Leadbeater, B., and Dodge, J. D. (1967). Arch. Mikrobiol. 57, 239. Leedale, G. F. (1958). Arch. Mikrobiol. 32, 32. Leedale, G. F. (1966). In “The Chromosomes of the Algae” (M. B. E. Godward, ed.), pp. 78-95. Arnold, London. Leedale, G. F. (1967). “Euglenoid Flagellates.” Prentice-Hall, Englewood Cliffs, New Jersey. Leedale, G. F. (1968). In “The Biology of Euglena” (D. E. Buetow, ed.), Vol. 1, pp. 185-242. Academic Press, New York. Lerbs, V. (1971).Arch. Mikrobiol. 77, 308. Lerbs, V., and Thielke, C. (1969).Arch. Mikrobiol. 68, 95. Lessie, P. E., and Lovett, J. S. (1968). Amer. J. Botany 55, 220. L@vlie,A., and Briten, T. (1970).J. Cell Sci. 6, 109. Lu, B. C. (1967).J. Cell Sci. 2, 529. Luykx, P. (1970).“Cellular Mechanisms of Chromosome Distribution,” International Review of Cytology, Suppl. 2, 173 pp. Academic Press, New York. McCully, E. L., and Robinow, C. F. (1971).J. Cell Sci. 9,475. McC~illy,E. K., and Robinow, C. F. (1972a).J. Cell Sci. 10, 857. McCully, E. K. and Robinow, C. F. (1972b).J. Cell Sci. 11, 1. McCully, E. K., and Robinow, C. F. (1973). Arch. Microbiol. 94, 133.

EVOLUTION OF THE MITOTIC SPINDLE

22s

McDonald, K. (1972).J. Phycol. 8, 156. McIntosh, J. R. (1974).J. Cell Biol. 61, 166. McIntosh, J. R., Hepler, P. K., and Van Wie, D. G. (1969).Nature (London) 224, 659. McKeen, W. E. (1972).Can. J. Microbiol. 18, 1915. McLaughlin, D. J. (1971).J. Cell Biol. 50, 737. McManus, M. A., and Roth, L. E. (1968).Mycologia 60, 426. McNitt, R. (1973). Can. J. Botany 51, 2065. Manier, J. F., Fize, A., and Grizel, H. (1971). Protistologica 7, 213. Manton, I. (1964).J. Roy. Microsc. Soc. 83, 317. Manton, I. (1970).J.Cell Sci. 7, 407. Manton, I., Kowallik, K., and von Stosch, H. A. (1969a).J. Microsc. (Oxford) 89, 295. Manton, I., Kowallik, K., and von Stosch, H. A. (1969b).J. Cell Sci. 5, 271. Manton, I., Kowallik, K., and von Stosch, H. A. (1970).J. Cell Sci. 6, 131. Marchant, H. J., and Pickett-Heaps, J. D. (1970).Aust. J . B i d . Sci. 23, 1173. Margulis, L. (1970). “Origin of Eukaryotic Cells.” Yale Univ. Press, New Haven, Connecticut. Mehlhorn, H. (1972). 2. Parusitenk, 40, 243. Mehlhorn, H., Senaud, J., and Scholtyseck, E. (1972).C . R. Acad. Sci., Ser. D 275,835. Mereschkowsky, C. (1905). Biol. Centralbl. 25, 593. Mignot, J. P. (1966). Protisologica 2(3), 51. Millecchia, L. L., and Ruszinska, M. A. (1971). Z. Zellforsch. Mikrosk. Anat. 115, 149. Mims, C. W. (1972).J. Gen. Microbiol. 71, 53. Moens, P. B. (1971). Can. J. Microbiol. 17, 507. Moens, P. B., and Rapport, E. (1971).J. Cell Biol. 50,344. Molil-Bajer, J. (1969). Chromosoma 26, 427. Motta, J. J. (1969). Mycologia 61, 873. Mughal, S., and Godward, M. B. E. (1973). Chromosoma 44, 213. Nanney, D. L., and Rudzinska, M. A. (1960). In “The Cell” (J. Brachet and A. E. Mirsky, eds.), Vol. 4, pp. 109-150. Academic Press, New York. Neushul, M., and Dahl, A. L. (1972).Amer. J. Botany 59,401. Nicklas, R. B. (1967). Chromosoma 21, 17. Nicklas, R. B. (1971).Aduan. Cell B i d . 2, 225-297. Nicklas, R. B., and Staehly, C. A. (1967).Chromosorna 21, 1. Norris, R., and Pearson, B. R. (1973).J. Phycol. 9, Suppl., 16. Oakley, B. R., and Dodge, J. D. (1973).Nature (London) 244,521. Oakley, B. R., and Dodge, J. D. (1974).]. Cell Biol. 63,322. Olmsted, J. B., and Borisy, G. G . (1973).Anntr. Rev. Biochem. 42, 507. Ott, D. W., and Brown, R. M. (1972). Brit. Phycol. J. 7, 361. Perkins, F. O., and Amon, J . P. (1969).J. Protozool. 16, 235. Peterson, J. B., Gray, R. H., and Ris, H. (1972).J. Cell Biol. 53,837. Peyriilre, M. (1971). C . R . Acad. S c i . , Ser. D 273,2071. Pickett-Heaps, J. D. (1967).Aust. J , Biol. Sci. 20,883. Pickett-Heaps, J . D. (1969a).Aust. J. Biol. Sci. 22,375. Pickett-Heaps, J . D. (1969b).J. Ultrastruct.Res. 27, 24. Cytobios 1,257. Pickett-Heaps, J. D. (1969~). Pickett-Heaps, J. D. (1970). Protoplasma 70,325. Pickett-Heaps, J. D. (1971). Cytobios 3, 205. Pickett-Heaps, J. D. (1972a).Ann. Bot. (London) 36,693. Pickett-Heaps, J . D. (1972b). Cytobios 5, 59. Pickett-Heaps, J. D. (1972~). Cytohios 6, 167.

226

DONNA F. KUBAI

Pickett-Heaps, J. D. (1972d). New Phytol. 71, 561. Pickett-Heaps, J. D. (1972e). New Phytol. 72,347. Pickett-Heaps, J. D. (19720.J . Phycol. 8,343. Pickett-Heaps, J. D. (1973). Ann. Bot. (London)37, 1017. Pickett-Heaps, J . D. (1974). Biosystems 6, 37. Pickett-Heaps, J . D., and Fowke, L. C. (1969). Aust. J . Biol. Sci. 22,857. Pickett-Heaps, J. D., and Fowke, L. C. (1970a). Aust. J. B i d . Sci. 23, 71. Pickett-Heaps, J. D., and Fowke, L. C. (1970b).J. Phycol. 6, 189. Pickett-Heaps, J. D., and Marchant, H. J. (1972). Cytobios 6, 255. Porter, D. (1972). Protoplasma 74,427. Porter, K. R. (1966). Principles Biomol. Organ., Ciba Found. Symp., 1965 pp. 308-345. Prensier, G. (1972).J. Microsc. (Paris) 14, A82. Prescott, D. M., and Murti, K. G. (1973). c o l d Spring Harbor S!ymp. Quant. B i d . 38, 609. Raff, R. A., and Mahler, H. R. (1972). Science 177, 575. Raikov, I. B. (1966). Arch. Protistenk. 109, 71. Raikov, I. B. (1969). In “Research in Protozoology” (T.-T. Chen, ed.), Vol. 3, pp. 1-128. Pergarnon, Oxford. Raikov, I. (1973). C. R. Acad. Sci., Ser. D 276, 2385. Raju, N. B., and Lu, B. C. (1973).J. Cell Sci. 12, 131. Rapport, E. (1971). Can. J. Genet. Cytol. 13, 55. Raudaskoski, M . (19 Raven, P. H. (1970). Rehhun, L., Lefehvre, P., and Rosenbaum, J . (1973). Biol. Bull. 145, 451. Ris, H. (1949). Biol. Bull. 96, 90. Ris, H. (1955). In “Analysis of Development” (B. H. Willier, P. A. Weiss, and V. Hamburger, eds.), pp. 91-125. Academic Press, New York. Ris, H. (1961). Con. J. Genet. Cytol. 3, 95. Ris, H. (1962). In “The Interpretation of Ultrastructure‘” (R. J. C. Harris, ed.), Symposia of the International Society for Cell Biology, pp. 69-87. Academic Press, New York. Ris, H., and Chandler, B. L. (1963). Cold Spring Harbor S y m p Quant. Biol. 28, 1. Ris, H., and Kubai, D. F. (1974).J . Cell Biol. 60, 702. Roberts. W. L., Hammoncl, D. M., Anderson, L. C., Speer, C. A. (1970). J. Protozool. 17, 584. Robinow, C. F., and Caten, C. E. (1969).J . Cell Sci. 5, 403. Rohinow, C. F., and Marak, J. (1966).J. Cell Biol. 29, 129. Rooney, L., and Moens, P. B. (1973). Can. J. Microbiol. 19, 1383. Rosenberger, R. F., and Kessel, M. (1968).J.Bocteriol. 96, 1208. Ross, I. K. (1968). Protoplusma 66, 173. Roth, L. E., and Daniels, E. W. (1962).J. Cell Biol. 12, 57. Roth, L. E., and Shigenaka, Y. (1964).J. Cell Biol. 20, 249. Hyser, U . (1970). Z . Zellforsch. Mikrosk. Anut. 110, 108. Ryter, A . (1968). Bacteriol. Reo. 32, 39. Sabatini, D. D., Bensch, K., and Barriiett, R. J. (1963).J. Cell Biol. 17, 19. Sagan, I,. (1967).J. Theor. Biol. 14,225. Sakai, A., and Shigenaga, M. (1972). Chromosomu 37, 101. Satir, P. (1965).J. Cell Biol. 26,805. Satir, P. (1968).J. Cell Biol. 39, 77. Schrantz, J.-P. (1967). C . R . Acud. Sci., Ser. D 264, 1274.

EVOLUTION OF T H E MITOTIC SPINDLE

227

Schrantz, J.-P. (1970). Rev. Cytol. Biol. Veg. 33, 1. Schuster, F. L. (1968).J. Parasitol. 54, 725. Schuster, F. L., Goldstein, S., and Hershenov, 9. (1968).Protistologica 4, 141. Schwab, D. (1968).Naturwissenschaften 55,88. Schwab, D. (1969). Z . Zellforsch. Mikrosk. Anat. 96,295. Schwab, D. (1972). Protoplasma 75, 79. Schwab, D. (1973). Protoplasma 78,339. Sheffield, H. G., and Melton, M. L. (1968).J. Parasitol. 54, 209. Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973). Proc. Nat. Acad. Sci. U.S. 70, 765. Siebert, A. E., Jr., and West, J. A. (1974).Protoplasma 81, 17. Slankis, T., and Gibbs, S. P. (1972).J.Phycol. 8, 243. Sommer, J. R., and Blum, J. J. (1965). E x p . Cell Res. 39, 504. Soyer, M. 0. (1969). C. R. Acad. Sci., Ser. D 268,2082. Soyer, M. 0. (1970). C. R. Acad. Sci., Ser. D 271,1003. Soyer, M. 0. (1971). Chromosoma 33, 70. Sprague, V., and Vernick, S. H. (1968).J. Protozool. 15, 547. Stanier, R. Y. (1970). Symp. Soc. Gen. Microbiol. 20, 1-38. Stanier, R. Y., and van Niel, C. 9. (1962).Arch. Mikrohiol. 42, 17. Sterling, C. R., and Aikawa, M. (1973).J. Protozool. 20, 81. Sterling, C. R., Aikawa, M., and Nussenzweig, R. S. (1972). Proc. Helminthol. Soc. Wash. 39, Spec. Issue, 109. Stevenson, I. (1972).Aust. J. Biol. Sci. 25, 775. Stevenson, I., and Lloyd, F. P. (1971a).Aust. J . Biol. Sci. 24, 963. Stevenson, I., and Lloyd, F. P. (1971b).Aust. J. BioZ. 24,977. Summers, K. E., and Gibbons, I. R. (1971).Proc. Nat. Acad. Sci. U.S. 68, 3092. Sun, N. C., and Bowen, C. C. (1972). Caryologia 25,471. Tanaka, K. (1970). Protoplasma 70,423. Terzakis, J. A. (1971).J. Protozool. 18, 62. Terzakis, J. A., Sprinz, H., and Ward, R. A. (1967).J. Cell Biol. 34, 311. Tucker, J. 9. (1967).J. Cell Sci. 2, 481. Turian, G., and Oulevey, N. (1971). Cytobiologie 4, 250. Turner, F. R. (1968).J. Cell Biol. 37, 370. Ueda, K. (1973). Shokuhutsugakii Zasshi 85,263. Uzzell, T., and Spolsky, C. (1974).Amer. Sci. 62,334. Van Winkle, W. B., Biesele, J. J., and Wagner, R. P. (1971). Can. J. Genet. Cytol. 13, 873. Vavra, J. (1965). C. R. Acad. Sci. 261, 3467. Vickerman, K., and Preston, T. M. (1970).J. Cell Sci. 6, 365. Vinckier, D., Devauchelle, G . , and Premier, G. (1971). Protistologica 7, 273. Vivier, E. (1965).J.Microsc. (Paris)4, 559. Weisenberg, R. C. (1972). Science 177, 1104. Wells, K. (1970). Mycologia 62, 761. Whittaker, R. H. (1969). Science 163, 150. Wilson, H. J. (1969).J. Cell Biol. 40, 854. Woese, C. R. (1970). Symp. Soc. Gen. Microbiol. 20, 39-54. Zickler, D. (1970). Chromosoma 30, 287.

This Page Intentionally Left Blank

Germ Plasm and the Differentiation of the Germ Cell Line E. M. EDDY Department of Biological Structure, Unioersity of Washington, Seattle, Washington

I. Historical Background

.

.

.

.

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

11. AnimalsReportedto HaveGermPlasm 111. Polar Granules and Insect Development A. Descriptive Studies

.

IV. V.

VI.

VII.

. . . . . . .

. . . . . . .

B. Experimental Studies C. Fine-Structural Studies D. Cytochemical Studies Germinal Determinants and Chaetognath Development Germinal Plasm and Amphibian Development , A. Descriptive Studies . . . . . . . B. Experimental Studies . . . . . . C. Fine-Structural Studies . . . . . . D. Cytochemical Studies . . . . . . The Nuage and Germ Cells . . . . . . A. Animals with Germ Cells Containing a Nuage , B. Composition of the Nuage and of Germ Plasm . C. Origin of the Nuage . . . . . . . Conclusions . . . . . . . . . References . . . . . . . . .

.

. . . . . . . . , . . . . . . . . . .

229 230 231 231 234 237 239 240 24 1 24 1 244 247 250 250 250 263 269 271 275

I. Historical Background According to Wilson (1896),it was first suggested that the germ cell line was continuous from one generation to the next by Owen in 1849, who thought that not all the progeny of the primary impregnated germ cell were used for forming the body, but that some remained unchanged and contributed to the development of another individual. Weismann (1892) reports that Jager proposed a similar idea in 1877, that part of the Keim Protoplasma of the animal formed the individual and the rest formed the reproductive material, However, Nussbaum (1880), studying germ cells in the trout and in the frog, was the first to state definitively that there was a continuity between germ cells of succeeding generations. He thought that the fertilized egg was divided into the cells of the individual and the cells that preserve the species, either at the beginning of embryonic development or before any histological differentiation takes place. The germ cells were thus not derived from the individual in which they were located, according to this theory, but rather had a common origin with it. 229

230

E. M. EDDY

Subsequently, Weismann (1892), because of his studies on the formation of germ cells in hydroids, came to the conclusion that rather than continuity of germ cells there was continuity of a substance handed down from parent germ cells to the germ cells of the new individual. He termed this substance Keimplasma (germ plasm) and believed that it was responsible for the transfer of hereditary tendencies from generation to generation, being uninfluenced by what happened to the individual bearing it. He also suggested that the transmission of germ plasm from the ovum to the place of origin of the reproductive cells took place through a definite series of cells he called the Keintbahn or germ track. Although when Weismann wrote of the continuity of germ plasm he was referring to genetic material of the nucleoplasm, this account forms the basis of the present germ plasm hypothesis. Several early investigators noted that oocytes and primordial germ cells contained visible substances with similar staining characteristics in their cytoplasm. Hegner (1911)traced granules from the pole plasm of insect oocytes of one generation to the germ cells in the next generation, which he referred to as Keimbahn determinants (Hegner, 1914a). He suggested that Keimbahn determinants marked the position of the part of the egg substance that controlled the production of primordial germ cells and that they later identified the cells of the germ track. He believed that Keimbuhn determinants were the visible evidence of specialized differentiation in the cytoplasm, and that the localization of these bodies was determined by the organization of the cytoplasm (Hegner, lQl4a,c). A similar material was reported in amphibian eggs by Bounoure (1931). He observed a substance in the cortical cytoplasm of the vegetal pole of fertilized eggs of Rana pipiens, which he initially called determinante germinal. He traced it from the fertilized egg to the floor of the blastocoel and later into the primordial germ cells. From his descriptive and experimental studies, he concluded that this region of the cytoplasm was related to germ cell differentiation and thus referred to the material as cytoplasme germinal (Bounoure, 1939). 11. Animals Reported to Have Germ Plasm

For the purposes of this article, germ plasm is defined as a substance present in the cytoplasm of gametes, which is segregated into specific cells during blastulation and determines that those cells shall become the progenitors of the germ cell line during subsequent development. Table 1 lists animals in which germ plasm has been

GERM PLASM

23 1

experimentally detected or has been reported to be visible at the light microscope level. It typically stains with basic dyes, is best observed in fertilized or cleaving eggs, and can be traced through embryogenesis to the primordial germ cells of the next generation. Most of the animals listed in Table I were reported in earlier reviews (Hegner, 1914c; Bounoure, 1939) and are not discussed individually in detail here. However, in recent years the germ plasms of insects, chaetognaths, and amphibians have been subjected to experimental studies and to fine-structural analyses which have resulted in some interesting findings and new observations about this material. Thus this article concentrates on the work that has been done on these animals in order to provide a better understanding of the role of germ plasm in germ cell formation.

111. Polar Granules and Insect Development A.

DESCRIPTIVE STUDIES

Studying the embryos of Chironomus sp., Ritter (1890) was the first to relate the polar plasm of insect eggs to the subsequent origin of primordial germ cells. He referred to the darkly staining masses in the pole cells as Keimwulst and suggested that they might have an important role in the segregation of germ cells. Subsequently, Kahle (1908)followed germ cells of Miastor metraloas from one generation to the next by the presence of polares Plasma in the egg, in the pole cells, and then in the primordial germ cells. Because of this, he suggested that the polar plasm represented the Keimplasma of insect eggs. About this time, Hegner was beginning his studies on the formation of germ cells and the role of polar plasm in this process. In a series of experimental studies (discussed in Section III,B), he determined that eggs of chrysonielid beetles, from which the polar plasm had been removed, developed into embryos without germ cells (Hegner, 1908, 1909a,b, 1911). I n other studies he also traced the polar plasm from the egg to the germ cells in histological sections of embryos of several species of Diptera, Coleoptera, and Hymenoptera (Hegner, 1914a,b, 1915). H e summarized these studies and the observations of others on suspected germinal plasms in The Germ Cell ) ~ indicated that the history of Cycle of Animals (Hegner, 1 9 1 4 ~and the Kiembahn determinants in insects involved localization of a visible substance in the oocyte or mature egg, association of cleavage nuclei with the substance to form primordial germ cells, distribution of the substance between daughter germ cells by mitotic division,

E. M. EDDY

232

TABLE I ANIMALS WITH GERMPLASM _

_

_

~

Phylum Rotifera Asplanchna ebbesbornii Asplanchna priodonta Nematoda Ascaris megalocephala Annelida Polychaeta Salmacina dysteri Oligochaeta Lemnidrites sp. Psamiosmonas sp. Tubifex sp. Mollusca Paludina sp. Lymnaea stagnalis Sphaerium striatinum Arthropoda Branchiopoda Polyphemus pediculus Copopoda Cyclops (1 1 species) Diaptomus (3 species) Canthocamptus staphylinus Heterocope saliens Cyclops brevicornis Insecta Coleoptera Acanthoscelides obtectus Calligrapha multipunctata, C. lunata, C. bigsbyana Leptinotarsa decemlineata Lema trilineata Hymenoptera Copidosoma buyssoni Leitomastix truncatellus Ageniaspis fusciocollis Copidosoma sp. Copidosoma gelechiae Apanteles glomeratus

Diastrophus nebulosus Tricogramma evanescans Lepidoptera Smerinthus populi Homoptera Pseudococcus medanieli

~

___

References

Tannreuther (1920) Nachtwey (1925) Boveri (1909)

Malaquin (1924a,b, 1925) Iwanoff (1928) Iwanoff (1928) Iwanoff (1928) Gatenby (1919a) Gatenby (1919b) Woods (1932)

Kuhn (1912) Amma (1911) Amma (1911) Amma (1911) Amma (1911) Haecker (1897)

Mulnard (1947, 1950) Hegner (1908, 1909a,b) Hegner (1909a,b, 1911) Hegner (1909b) Silvestri (1914) Silvestri (1906) Martin (1914) Hegner (1914b) Hegner (1915) Hegner (1915); Gatenby (1920); Muckerji (1930) Hegner (1915) Gatenby (1918) Gatenby (1917) Shinji (1919)

233

GERM PLASM

TABLE I (Continued)

H eteren c e s

Phylum

Lecanodiaspis pruinosa lcerya purchai Diptera Drosophila melanogaster

Muscu uiciniu Wachtliella persicariae Dacus tryoni Sciara sp. Chironomus confinus, C . riparius Miastor sp. Mayetiola destructor Coelopa frigida Lucilia cuprina Simulium pinctipes Chaetognatha Sagitta bipunctata Sagitta sp. Spadella cephaloptera Spadella inflata Chordata Amphibia Rana temporaria Rana esculenta Rana pipiens Rana dalmatia Xenopus laeuis

Bufo bufo Discoglossus pictus Phrynobotruchus natalensis Ambystoma mexicanum

Shinji (1919) Shinji (1919) Huettner (1923); Howland (1941);Rabinowitz (1941); Aboim (1945); Poulson (1947); Counce and Selman (1955); Imaizumi (1958); Poulson and Waterhouse (1960); Hathaway and Selman (1961); Mahowald (1962); Counce (1963);Gill (1964); Ullmann (1965); Jazdowska-Zagrodzinska (1966); Mahowald (1968, 1970, 1971a,b); Mahowald and Strassheim (1970) Bruiyan and Shafiq (1959) Geyer-Duszynska (1959); Wolf (1967) Anderson (1962) DuBois (1932); Metz (1938) Hasper (1911) Nicklas (1959) Bantock (1961) Schwalm et al. (1971) Poulson and Waterhouse (1960) Gambrel1 (1933) Elpatievsky (1909) Stevens (1910) Vasiljev (1925); Ghirardelli (1968) Ghirardelli (1968)

Bounoure (1931, 1934, 1939); Aubry (1953a,b); Bounoure et al. (1954); Blackler (1958) Padoa (1963); Hammer, cited in Blackler (1966) Di Berardino (1961); Smith (1966); Mahowald and Hennen (1971); Williams and Smith (1971) Gipouloux (1971) Nieuwkoop and Faber (1956); Blackler (1958, 1970); Nieuwkoop and Suminski (1959); Czoloska (1969, 1972); Buehr and Blackler (1970); Kalt (1973) Blackler (1958) Gipouloux (1962); Librera (1964) Balinsky (1966) Williams and Smith (1971)

234

E. M. EDDY

disappearance of the substance in the oogonia and spermatogonia, and reappearance of the substance in the oocyte or mature egg to begin the cycle again. Because of this sequence, he proposed that the granules of the polar plasm either were the germ cell determinants for insects or were at least the visible sign of the germ cell determinant region. During and after the period of Hegner’s studies, other investigators observed the same sequence of events in other species. Hasper (1911) followed germ cell determinants in the posterior region of eggs of Chironomus confinus and C . riparius through the early period of embryonic development until they reached the gonads. Similar observations were made by Gatenby for Trichogramma evanescens (Gatenby, 1918) and for Apanteles glomeratus (Gatenby, 1920), and by Anderson (1962) for Dacus tryoni. However, Huettner (1923), studying Drosophila melanogaster, doubted that the polar granules exerted an influence on the differentiation of the pole cells and believed that their nature remained to be determined. Some workers have since favored the more noncommittal term polar granules to the term germ cell determinants for these cytoplasmic inclusions (Rabinowitz, 1941; Sonnenblick, 1950). Indeed, there have been studies indicating that some of the pole cells probably give rise to a specific region of the midgut (Poulson, 1947; Poulson and Waterhouse, 1960; Counce, 1963).

B. EXPERIMENTALSTUDIES Experimental studies on the polar plasm of insects (Diptera and Coleoptera) have repeatedly indicated that it serves an essential role in germ cell determination. Hegner (1908) found that, if the freshly laid eggs of Calligrapha multipunctata and C . lunata were pricked in the center of the posterior end (the region of the egg containing the polar plasm), the turgidity of the egg caused a small droplet of the contents to be forced out. The embryos and larvae produced from these eggs appeared normal, but sections of them seemed to show that less than the characteristic number of germ cells was present. In a second set of experiments, Hegner (1911) touched the posterior part of the egg with a hot needle to cauterize the cytoplasm containing the polar granules. H e reported that the blastoderm developed normally, except at the posterior end, and that the germ cells failed to form in that region. He concluded from this study that the polar granules or the substances in which they are embedded are responsible for the formation of germ cells. Similar results have also been obtained by cauterizing the region of the pole plasm in eggs of Wachtliella persicariae L. (Geyer-Duszynska, 1959).

GERM PLASM

235

Another approach used to study germ cell formation in insects with some success is ultraviolet irradiation of the eggs. Geigy (1931) subjected the posterior pole of D. melanogaster eggs to ultraviolet light and obtained adult flies that lacked germ cells. These experiments were repeated by Aboim (1945) with the same results. Using ultraviolet microbeam irradiation, Geyer-Duszynska (1959) also prevented germ cell formation, but at the same time produced substantial nuclear and cytoplasmic damage in the eggs. In most of these experiments, the nuclei were intentionally destroyed, either before or after they entered the pole plasm. However, Bantock (1961) was careful to irradiate only the extreme posterior ends of Mayetiola destructor eggs, while shielding the remainder, before the two nuclei migrated in. H e found that the two nuclei subsequently took up their normal position near the polar granules but underwent chromosome elimination during the fifth mitosis. The adult female flies that resulted were sterile and lacked recognizable germ cells in their gonads. However, there is some difficulty in determining the specificity of the ultraviolet effect on the polar granules. Gunther (1971) reported that Pimpula turionellae failed to form pole cells but developed germ cells after ultraviolet treatment. Also, in one experiment by Geyer-Duszynska (1959), the cytoplasmic zone just above the polar plasm was irradiated and it was noted that the cleavage nuclei were incapable of crossing the irradiated zone for some time. This too allowed all the nuclei to undergo chromosome diminution and prevented normal germ cell formation. In another study the number of germ cells was reduced i n D . melanogaster embryos, whether the posterior pole was irradiated before or after the arrival of the nuclei (Hathaway and Selman, 1961). Other experiments on the pole plasm of insect eggs have used physical methods to displace the polar granules or to prevent nuclei from migrating into the polar cytoplasm. Hegner (1909b) was the first to attempt this approach in insects by centrifuging eggs of C. multipiinctata and several other beetles with their posterior end toward the center of rotation. The polar granules moved together into the anterior part of the egg, but the subsequent development was too abnormal to determine whether or not germ cells were formed. In subsequent studies by Howland (1941) on D. melanogaster and by Nicklas (1959) on Miastor sp., the polar granules were not displaced by centrifugation. However, when these studies were repeated by Imaizumi (1958) and Jazdowska-Zagrodzinska (1966) on D. nzelunogaster, by Geyer-Duszynska (1959) and Wolf (1969) on eggs of W. persicariae, and b y Bantock (1961) on eggs of M . destructor, they found that chromosome elimination occurred in all nuclei and that

236

E. M. EDDY

germ cells failed to form. Furthermore, when eggs were constricted with a fine hair or a nylon thread to prevent nuclei from migrating into the polar cytoplasm until after chromosome elimination, germ cell formation did not proceed (Geyer-Duszynska, 1959; Bantock, 1961). These studies may be interpreted as showing that chromosome retention and formation of germ cells in the pole plasm are influenced by the polar plasm and are not properties of the nuclei that migrate into it (Gurdon and Woodland, 1968). Whether the factor responsible for preventing chromosome elimination is the same as that which appears to promote germ cell formation remains to be determined (Achtelig and Krause, 1971; Counce, 1973). Another approach used to study polar granules in insects is to transplant cellular components from one embryo to another. Zalokar (1973) transplanted nuclei from strain yw D. melanogaster preblastodermic embryos to the posterior ends of fertilized eggs of strain vibw flies. This yielded mosaics with donor-type characters and, in one case, donor-type gonads, as determined b y the production of progeny with the donor genotype. This indicated that the implanted nuclei can migrate into the polar plasm and give rise to germ cells. Also relevant is an interesting series of experiments reported recently by Illmensee and Mahowald (1973, 1974). They transplanted polar plasm from wild-type Drosophila embryos to the anterior tip of e mhw-strain embryos, producing cells containing polar granules in the micropyle region. When these cells were in turn introduced into the polar region of y w sn3 embryos and the resultant flies mated to y w sns partners, heterozygous wild-type flies were found in a few cases. Thus their results demonstrated that the posterior pole plasm has a determinative quality for germ cells, which retains its capacity with transfer, and further showed that germ line initiation is not an exclusive property of certain blastoderm nuclei. In a similar study, Okada, Kleinman, and Schneiderman (1974) transplanted polar plasm from donor eggs of Drosophila to recipient eggs that had been ultraviolet-irradiated. When the polar plasm was transplanted to the posterior pole of irradiated eggs, 21 of 50 adults from eggs that survived the treatment showed well-developed gonads, while only 1 of 83 flies from eggs that had been irradiated but did not receive germ plasm had normal gonads. Transplantation of cytoplasm from the anterior pole of donor eggs to the posterior pole of irradiated eggs did not restore fertility. They also observed that ultraviolet irradiation delays the time of migration of cleavage nuclei into the posterior part of the egg, and that the cytoplasmic protrusions do not become isolated to form pole cells. In addition, they

GERM PLASM

237

reported that germ cell determinants are not species-specific, as evidenced by the successful restoration of fertility in eggs of one species of Drosophila with polar plasm from another species. C. FINE-STRUCTURAL STUDIES Electron microscope studies of polar granules of insects have added considerably to the knowledge of these structures. Much of this work has been done by Mahowald, who was also the first to describe the fine structure of polar granules (Mahowald, 1962). Examining polar granules during oogenesis, in preblastoderm stages of development, and in pole cells of the blastoderm stage of D. melanogaster embryos, he characterized them as dense organelles, 0.2-0.5 pm in diameter, lacking a limiting membrane, and often having a light core. During oogenesis and preblastoderm stages, they were composed of clusters of granules the size of ribosomes and a smaller granular or fibrillar component, but at the blastoderm stage of development the large granular component was not visible. Mahowald noted that this structural change might account for the decreased staining intensity of blastoderm-stage polar granules reported b y Rabinowitz (1941). Many small, dense bodies were found attached to the nuclear envelope of oogonia, which resembled polar granules, but definite polar granules were not seen until the early stage of vitellogenesis in oocytes. During the ovarian growth phase, the polar granules became attached to mitochondria, but this contact was lost after fertilization (Mahowald, 1962). Ullmann ( 1965) confirmed the description of the fine-structural appearance of polar granules in fertilized eggs and pole cells of D. melanogaster and extended these observations to D . willistoni and D. virilis as well. In addition, in W. persicariae (Wolf, 1967) polar granules were present as loosely woven strands of fibrous material distributed throughout the posterior region of the egg. I n another electron microscope study of the changes in the structure of the polar granules during embryogenesis of several species of Drosophila, Mahowald (1968) confirmed the earlier observation that polar granule distribution varies between species (Counce, 1963). He also found that the size of the granules differs in several species and at different stages of development of the same species. During the latter part of oogenesis, polar granules were attached to mitochondria in every species examined except D . hydei. However, just prior to pole cell formation, when the polar granules had lost their association with mitochondria, the granules became clumped together in groups in every species except D . willistoni. Still, in all

238

E. M. EDDY

species examined there was fragmentation of the polar granules into small, dense clumps at the time of pole cell formation. These became recondensed into large polar granules in the blastoderm stage, but fragmented again in primordial germ cells and were seen there as small clumps in association with the nuclear envelope. Because of evidence for the presence of RNA in polar granules (Poulson and Waterhouse, 1960; Mahowald, 1962; Counce, 1963), Mahowald suggested that it might be mRNA stored during oogenesis, which at the proper developmental stage is used to direct specific protein synthesis needed for pole cell formation. Polar granule fragmentation could then b e explained as the mechanism by which RNA becomes available for the synthetic activity necessary for pole cell determination and germ cell formation (Mahowald, 1968). In a later study, Mahowald (1971a) confirmed light microscope observations (Counce, 1963)that polar granules become attached to the nuclear envelope at the time the pole cells move to the presumptive gonadal region. H e also extended these observations by showing that fibrous bodies associated with the nuclear envelope and apparently derived from polar granules are present in primordial germ cells throughout the larval and pupal stages of development in Drosophila. Another variation in organization of the polar granules was noted as well; during the formation of the blastoderm, the fibrous polar granules changed to regularly arranged, crystalline structures (Mahowald, 1968). However, later during gastrulation the reverse occurred and the crystalline polar granules fragmented into fibrous bodies which became associated with the nuclear envelope. Since similar organelles were found in germ cells during oogenesis, until typical polar granules formed during vitellogenesis (Mahowald, 1962), the substance of the polar granules appears to exist continuously in one form or another in the germ cell line throughout the life cycle of Drosophila (Mahowald, 1971a). Changes in the fine structure of polar granules were also noted during the early stages of embryonic development in another dipteran, Coelopa frigida (Schwalm et al., 1971). Polar granules were composed of globular subunits until shortly before blastema formation, when they were transformed into groups of parallel, dense rods. With pole cell formation these in turn associated to form one large complex. It was suggested that these alterations might b e conformational changes of the molecules in the polar granules to expose chemically active sites or to bind formerly active sites, enabling them to carry out their presumed morphogenetic role. Evidently, polar granule material continues to be formed b y the

GERM PLASM

239

germ cells during the larval and pupal stages of the Drosophila life cycle, because the amount of fibrous material on the nuclear envelope and free in the cytoplasm does not decrease, even though the oogonia increase in number (Mahowald, 1971a). Furthermore, all 16 cells in the germarium derived from a stem oogonium have the same kind of fibrous material attached to the nuclear envelope (Mahowald and Strassheim, 1970). At the time the pro-oocytes differentiate into nurse cells, the true oocyte loses the fibrous material associated with its nuclear membrane into the cytoplasm. During the remainder of early oogenesis, the fibrous material is associated only with the nuclear membrane of nurse cells and is not found on the oocyte nuclear membrane (Mahowald and Tiefert, 1970). Because it was observed in Miastor and in Drosophila (Mahowald, 1971a) that the fibrous material on the nurse cell nuclear envelope passed into the cytoplasm of the oocyte at the time the nurse cells break down, it was proposed that polar granules are derived from nurse cells and possibly other precursors already present in the oocyte cytoplasm (Mahowald, 1971a). Furthermore, it has been postulated that the proteinaceous portion of polar granules is continuous in the germ cell line, but that it acquires RNA and then accumulates in the posterior tip of the egg during the final stages of oogenesis (Mahowald, 1970). In addition to these studies, mutants and genetic markers have been used to demonstrate the localization of germ cell-determining factors. Gehring (1973) studied the grandchildless mutant of Drosophila subobscura, in which the females lay eggs that do not form pole cells and give rise to sterile adults. Fine-structural analysis of the wild-type and mutant eggs (Mahowald and Gehring, 1974, quoted in Gehring, 1973) determined that polar granules are present in the mutant as well as in the wild-type eggs and appear to have the same structure. However, as was reported earlier by Fielding (1967), cleavage nuclei from the central region do not enter the polar cytoplasm in the mutants as they do in the wild type, but rather nuclei from the lateral periplasm migrate into the polar region secondarily. Although these nuclei associate with polar granules, pole cells do not form. It appears that the granules in the mutant do not function normally, however, and that biochemical or transplantation studies will be needed to determine the specific defect (Gehring, 1973).

D. CYTOCHEMICAL STUDIES Early workers were aware that the polar granules of insects had an affinity for basic dyes, and later Mulnard (1947), observing that they were preferentially stained by Pyronine in germ cells of

240

E. M. EDDY

Acanthoscelides obtectus, suggested that they were rich in RNA. Using the same staining reaction, Bruiyan and Shafiq (1959) came to the same conclusion for polar granules in fully grown oocytes of Musca oinca. Other workers used azure B at pH 4 to stain the polar granules and showed that the staining could b e prevented by subjecting sections of Drosophila eggs and embryos to RNase digestion or trichloracetic acid extraction prior to staining (Nicklas, 1959; Counce, 1963; Mahowald, 1971b). In addition, Mahowald (1971b) used the indium trichloride reaction for nucleic acids of Watson and Aldridge (1961) at the electron microscope level and observed that the polar granules of mature eggs were of the same electron density as organelles known to contain nucleic acids. Furthermore, this reactivity was eliminated by perchloric acid extraction. In another study, Poulson and Waterhouse (1960) irradiated the polar plasm of preblastoderm embryos of D. melanogaster and Lucilia cuprina, before nuclei entered, with an ultraviolet beam at wavelength 2536 A to produce sterile flies. They suggested that the principal absorbing material was probably RNA. Thus the evidence available favors the idea that polar granules of insect eggs and embryos contain RNA. However, it is interesting that Mahowald (1971b) observed that, following fertilization the ability of polar granules to bind indium or to stain with basophilic dyes decreased until the blastoderm stage, when they ceased to react, suggesting that RNA was no longer present.

IV. Germinal Determinants and Chaetognath Development Butschli (1873) reported that the germinal cells of chaetognaths were present in early stages of embryonic development and identified a group of cells at the bottom of the archenteron cavity in gastrulas of Sagitta as the primordium of the germ line. Hertwig (1880) further determined that this anlage was first formed b y only two cells. However, it remained for Elpatievsky (1909) to observe that a body in the cytoplasm of the primordial germ cells was also present in the eggs of Sagitta bipunctata. H e called it the besondere Korper and indicated that it appeared in fertilized but unsegmented eggs before pronuclear fusion. This characteristic body was also observed by Stevens (1910) in Sagitta at nearly the same time as Elpatievsky reported it, and was later observed b y Vasiljev (1925) in eggs of Spadella cephaloptera. Elpatievsky (1909)described the besondere Korper as a small, regular mass at the vegetal pole of the egg. During the first five divisions, the body remains intact and is segregated into one cell, but at

GERM PLASM

24 1

the 64-cell stage it divides to enter the two primordial germ cells. Although the somatic cells continue to divide, there are only two primordial germ cells present until the latter part of gastrulation, when they divide once more. The chaetognaths are hermaphroditic and, after hatching, two of the primordial germ cells give rise to oogonia, while the other two produce spermatogonia (Elpatievsky,

1909)* Because of its similarity in appearance and distribution to the polar plasms of insects and germinal plasms of amphibians, Ghirardelli (1953) referred to the besondere Korper as the germinal determinant in S . cephaloptera. Subsequently, he showed that when the blastomere containing the germinal determinant was destroyed with a fine glass needle the other blastomere gave rise to a gastrula lacking germ cells (Ghirardelli, 1954). Although the germinal determinant is not visible in living cells and the destruction was done randomly, histological studies showed that half of the embryos so treated lacked germinal determinant. Ghirardelli (1966) also studied the germinal determinant with the electron microscope and observed that it usually lies near the periphery of the egg and appears to be a mass of irregularly shaped dense threads, granules, and aggregates. It lacks a surrounding membrane, but has a well-defined boundary. When the germinal determinant is divided between primordial germ cells, it becomes disaggregated and many small plaques lie first throughout the cytoplasm and later around the nucleus. It has also been noted that the accumulation of plaques of germinal determinants around the nuclei of primordial germ cells is similar to what is seen in young oocytes, and it has been suggested that this may be indicative of the origin of the germ cell determinant in chaetognaths (Ghirardelli, 1968). I n S . cephaloptera, Ghirardelli (1953) observed that the germinal determinant in ripe eggs stained intensely with Pyronine. H e also showed that, after digesting sections with RNase or after treating them with warm 1 N hydrochloric acid, staining did not occur. He concluded from this that RNA was a major constituent of the germinal determinant of chaetognaths (Ghirardelli, 1968).

V. Germinal Plasm and Amphibian Development A.

DESCRIPTIVE STUDIES

I n a series of articles on the early development of R . temporaria embryos, Bounoure (1927, 1931, 1934) reported the presence of a cytoplasmic substance with distinctive staining characteristics, which

242

E. M. EDDY

was apparently associated with cells initiating the germ line. He first observed it in fertilized but still unsegmented eggs as small islets of dense material aligned along the membrane of the vegetal pole. During cleavage the substance was segregated into the vegetal blastomeres nearest the vegetal pole, and then during gastrulation the cells containing the substance moved into the presumptive endoderm on the floor of the blastocoel. These cells later acquired the appearance and behavior of primordial germ cells, migrating out of the endoderm and into the genital ridges. Bounoure was well aware of the studies on the polar plasm in insects, which indicated that it served as the Keimbahn determinant (Hegner, 1914c) and was sensitive to ultraviolet irradiation (Giegy, 1931). H e applied this technique to frog eggs and showed that their cytoplasmic substance probably had a similar role (Bounoure, 1937). Because of these findings, he termed the cytoplasmic substance in frog embryos the cytoplasme germinal and, in L’Origin des Cellules Reproductrices et la Probleme de la Lignbe Germinale, suggested that it was responsible for determination of the germ cell line (Bounoure, 1939). These observations were the only evidence of a possible germinal plasm in vertebrates until 1956, when a report mentioned a similar plasm in the early embryos of Xenopus laevis (Nieuwkoop and Faber, 1956). This was soon followed by another study in which eggs, embryos, and larvae of R . temporaria, R . esculenta, Bufo bufo, and X . laevis were examined specifically for the presence of germinal plasm (Blackler, 1958). For R . temporaria, germinal plasm was not seen in unfertilized eggs, but was present in fertilized and uncleaved eggs among the yolk platelets in the vegetal part of the cytoplasm. Eggs of this species fixed immediately prior to the first cleavage contained islets of germinal plasm concentrated at the vegetal pole and surrounded by mitochondria. In the early to midblastula stages, germinal plasm was present within five to seven cells lying between the floor of the blastocoel and the vegetal pole, each cell usually containing a single islet up to three times the size of the nucleus. In late blastulas germinal plasm often enveloped the nucleus as a cap and, by the time gastrulation was under way, the material lay next to the nuclei in the cells. During gastrulation 11 to 23 cells contained germinal plasm, and they moved from the floor of the endoderm to the midendoderm of the young neurula. During subsequent development the cells moved around the archenteron to the dorsal crest of the endoderm and were identifiable there at the time of hatching by the corona of germinal plasm surrounding their large, clear nucleus. By the time the primordial germ cells reached the

GERM PLASM

243

dorsal mesentery, they numbered between 30 and 50. It was reported that the germinal plasm progressively lost its affinity for the stains employed, but not before the cells containing it were identified as primordial germ cells on the basis of location in the gonadal rudiments, size, abundance of juxtanuclear mitochondria, and persistence of yolk platelets. Similar observations were made on embryos of B. bufo, germinal plasm first being observed in the vegetal pole region of fertilized eggs fixed prior to and during the first cleavage (Blackler, 1958). Cells containing germinal plasm were seen in blastulas, gastrulas, and neurulas, and in the dorsal mesenteries and germinal ridges of tadpoles. Particularly noticeable in the later stages was the density of the mitochondria1 population around the nuclei of the cells containing germinal plasm. Studies involving X. Zaevis embryos resulted in much the same findings, except that the germinal plasm was more widely distributed in blastulas and that by the tadpole stage primordial germ cells were not so clearly demarcated as in the other species. In Rana esczrlenta, germinal plasm was not detected in gastrulas (the earliest stages examined), neurulas, or tadpoles, even though primordial germ cells could be readily identified in the latter stage by other criteria. However, germinal plasm has since been reported in this species by Hammer (cited in Blackler, 1966). Blackler believed that these observations confirmed Bounoure’s findings and showed that certain cells in the blastula stage embryo containing germinal plasma were direct ancestors of primordial germ cells (Blackler, 1958). Germinal plasm has also been observed in embryos of other species, that in blastulas and gastrulas of R. pipiens (Di Berardino, 1961) occupying a more peripheral position in the earlier stage and surrounding the nucleus in the latter stage. In Discoglossus pictus, Gipouloux (1962) saw germinal plasm first in mature eggs as three or four bodies 20-30 pni in diameter. In blastulas germinal plasm was observed in seven or eight cells between the vegetal pole and the floor of the blastocoel, and with gastrulation the cells moved into the endoderm an equal distance from the ventral surface of the embryo and the floor of the archenteron. During this time the germinal plasm became closely applied to the nucleus, but with neurulation decreased in staining intensity. However, the density of the mitochondria and the appearance of the nucleus allowed the primordial germ cells to be followed during later stages of development until they lodged in the genital crests. Also, germ plasm has been traced from the vegetal pole of unfertilized eggs, through blastulation and

244

E. M. EDDY

gastrulation, to the primordial germ cells of tadpoles of Bufo reguluris (Amer, 1966). More recently, Czoloska (1969) examined fertilized eggs, unfertilized eggs, and oocytes ovulated in uiuo and in uitro, as well as ovarian oocytes, from X . laevis to determine the origin of the germinal cytoplasm. Cytoplasmic islands with the staining characteristics of germinal plasm were seen in all these groups, but they were smaller, more dispersed, and less numerous in ovarian oocytes than in ovulated oocytes. This investigator suggested that the formation of germinal plasm takes place during oogenesis, and that a possible “informative” component is added after the breakdown of the germinal vesicle (Czoloska, 1969). Thus light microscope observations, relying on the staining characteristics of germinal plasm for its identification, have demonstrated that germinal plasm is present in ovarian oocytes (Czoloska, 1969), unfertilized eggs (Gipouloux, 1962; Czoloska, 1969), cleaving eggs (Bounoure, 1934; Nieuwkoop and Faber, 1956; Blackler, 1958), and certain cells in blastulas (Bounoure, 1934; Blackler, 1958; Di Berardino, 1961; Gipouloux, 1962), gastrulas (Bounoure, 1934; Blackler, 1958; Di Berardino, 1961; Gipouloux, 1962), and neurulas (Bounoure, 1934; Blackler, 1958; Gipouloux, 1962) in several amphibian species. Furthermore, these cells acquire other characteristics which allow them to be observed after the germinal plasm loses its affinity for the stains employed, and they can be identified in the endoderm, dorsal mesentery, and germinal ridges in later stages of development as primordial germ cells (Bounoure, 1934; Blackler, 1958; Gipouloux, 1962). B. EXPERIMENTAL STUDIES Several investigators have attempted to determine the role of germinal plasm in anuran eggs b y experimentally altering the cytoplasm in which it is contained. One method employed has been to remove surgically some of the vegetal pole cytoplasm from early embryos and then to examine the animals that develop from these embryos for the presence of germ cells. Nieuwkoop and Suminski (1959) removed vegetal pole cytoplasm from early four-cell-stage embryos of X . lueuis by making an X-shaped incision at the junction of the blastomeres with a glass needle and allowing some of the underlying cytoplasm to leak out. Of the 122 embryos so treated, 27 survived and 8 were allowed to grow to stage 40-41 before they were fixed and their primordial germ cells counted. They contained an average of 19 germ cells, while a control group had an average of 22 germ cells,

GERM PLASM

245

and it was concluded that the removal of the vegetal pole plasm at the early four-cell stage did not significantly influence the formation of primordial germ cells. However, rather different results have since been obtained by other investigators. Librera (1964) punctured the vegetal pole of fertilized but uncleaved eggs and of four-cell-stage embryos of D. pictus with a micropipet and withdrew as much as one-fourth of the egg volume. She found that the animals reared from these eggs and embryos often had only a few germ cells or were completely sterile. Also, Gipouloux (1971) made several micropunctures in the vegetal pole of eggs and of two- and four-cell-stage embryos of Rana dalmatia and B . bufo and found that most of the animals that survived had few or no germ cells. In another series of experiments, Buehr and Blackler (1970) repeated this approach using two- and four-cellstage embryos of X laevis. They made small X-shaped incisions at the vegetal pole when the first or second cleavage furrow was seen to circumscribe the egg completely. This study differed from the others, however, in that some of the experimental embryos were fixed after 20 minutes and others at the 8- and 16-cell stages of development and they and their associated exudates examined histologically for the presence of germinal plasm. Of these embryos about one-third had lost essentially all their germinal plasm into the exudate, while others had lost only part or none of their germinal plasm. When tadpoles raised from experimentally treated embryos were examined histologically for the presence of germ cells, about one-third lacked germ cells, while others had diminished numbers. Thus this study indicated that there was a good correlation between the amount of germinal plasm lost and the number of germ cells that subsequently formed. A more recent study (Tanabe and Kotani, 1974) also concluded that the amount of germinal plasm present was instrumental in determining the number of primordial germ cells that formed in X . Zaevis tadpoles. Another approach used for the study of germ plasm in anurans has been to expose the vegetal pole to ultraviolet irradiation. The sterilizing effect of ultraviolet light on frog eggs was first observed by Bounoure (1937), and has since been used by other investigators interested in the role of germinal plasm. H e irradiated the vegetal pole of early frog embryos with ultraviolet light and reported that some failed to develop germ cells during subsequent embryogenesis. A problem with the early use of ultraviolet light was that only about one-third or less of the eggs so treated yielded sterile frogs. Aubry (1953a,b) improved this to about 60% sterility in R . temporaria by

246

E. M. EDDY

flattening the vegetal pole against a quartz microscope slide before irradiation. Other studies by Bounoure, Aubry, and Huck (1954) showed that the success rate could be increased to slightly greater than 70% by compressing the lower pole of the eggs so that they formed a flat cap against the quartz support and b y irradiating just before the first division, about 3 hours after fertilization. These investigators indicated that this was because the germinal plasm is spread as a rather extensive cap at the lower pole of each egg until just before the first division, when it becomes concentrated at the pole itself in a more restricted zone, allowing the ultraviolet irradiation to b e more effective then than at other times. Following confirmation of the sterilizing effect of ultraviolet irradiation by Padoa (1963) using R . esculenta eggs, Smith (1966) extended these studies with R . pipiens eggs. He observed that irradiation of the animal pole had no detectable effect on the formation of primordial germ cells, but that the ultraviolet effect on the vegetal pole was dose-dependent. Eggs irradiated at the vegetal pole with 7700 ergs/mm2 developed into larvae completely lacking primordial germ cells, while irradiation with 2600 ergs/mm2 resulted in reduced numbers of germ cells in more than half the larvae. Smith (1966) also observed that the effect of ultraviolet irradiation decreased after the first division and that, by the eight-cell stage, irradiation did not decrease the number of primordial germ cells seen in most larvae. Differences in sensitivity among different batches of X . Zaevis eggs were noted b y Blackler (1970), who suggested that they might be due to differences in the number and distribution of pigment granules in the cortical region rather than variations in germinal plasm. This investigator also noted that irradiated germinal plasm in X . Zaevis did not appear cytologically different from unirradiated germinal plasm, although the later development of germ cells was plainly abnormal. The most convincing experimental evidence for a possible role of germinal plasm in determination of the germ cell line in frogs comes from studies b y Smith (1966). He reasoned that, since the germinal plasm was apparently lost from eggs following surgical incision, it was free in the subcortical cytoplasm and thus could be transferred between eggs. Accordingly, subcortical cytoplasm from the vegetal pole of unirradiated eggs was injected with a micropipet into the same region of R . pipiens eggs which had been ultraviolet-irradiated at a dose of 15,000 ergs/mm2. Forty-seven percent of the ultravioletirradiated four-cell-stage embryos receiving injections of vegetal pole cytoplasm from unirradiated embryos developed into larvae that

GERM PLASM

247

possessed germ cells. Although there were fewer germ cells in these animals than in unirradiated controls, there were no germ cells in larvae reared from ultraviolet-irradiated eggs that received no injections or in larvae from ultraviolet-irradiated eggs receiving injections of animal pole cytoplasm. Thus it is possible to accomplish restitution of the germ cell line in irradiated eggs by transferring only about 3% of the total volume of the unirradiated egg cytoplasm.

C. FINE-STRUCTURAL STUDIES A better understanding of the form and distribution of germinal plasm in amphibia came when it was examined by electron microscopy. While studying eggs of Phrynohatrachus natalensis fixed 10 minutes after fertilization, Balinsky (1966) noted that mitochondria occurred in large groups near the vegetal pole and were acconipanied by peculiar rounded bodies. The bodies were electron-dense and appeared to consist of aggregations of particles smaller than ribosomes. He suggested that the groups of mitochondria and accompanying dense bodies might represent the basophilic areas of cytoplasm Bounoure (1934) had observed. This has since been confirmed in several careful studies specifically concerned with the fine structure of the germinal plasm in amphibian eggs and in progenitors of primordial germ cells in amphibian embryos. Mahowald and Hennen (1971) studied germinal plasm by electron microscopy in four different stages of the life cycle of R . pipiens: unfertilized eggs, two-cell- and blastula-stage embryos, and tadpoles. In unfertilized eggs the germinal plasm was characterized as accumulations of distinctive, electron-dense bodies 0.2-0.3 pm in diameter, lying in the vegetal cytoplasm. They were usually round, appeared to be formed of a fine meshwork of fibrils, and lacked a surrounding membrane. The dense bodies were frequently in contact with mitochondria and grouped in islands of the subcortical cytoplasm which were free of cortical granules, pigment granules, and all but the mitochondrial yolk granules. The germinal plasm appeared much the same at the two-cell (Fig. 1) and blastula stages of development, except that the contact with mitochondria seemed less frequent and larger bodies of germinal plasm were present in blastulas. By the time the tadpole stage was reached, germinal plasm was present in cells along the median ridge of the dorsal portion of the mesentery as large accumulations of irregularly shaped fibrous material situated adjacent to the nucleus in regions filled with mitochondria. Some of the germinal plasm was applied to outer mitochondrial membranes while other portions were located along the nuclear envelope, sepa-

FIG. 1. The cortical cytoplasm at the vegetal pole of two-cell-stage embryos of the anrphibi;in R . pipiews contains regions that are relatively free of yolk. A higher niagnification \. iew of part of o i i c of' these areas (within the square) is shown i n the inset.

GERM PLASM

249

rated 50-100 p m from it. The dense bodies identified as germinal plasm in this study were found in the same location as the germinal plasm reported in light microscope studies, were unique structures not seen elsewhere in eggs or embryos, and were distinctive in their appearance by electron microscopy (Mahowald and Hennen, 1971). This study was shortly followed by another in which germinal plasm was examined in eggs and early-cleavage-stage embryos of R . pipiens and in fertilized eggs of Ambystoma mexicanum (Williams and Smith, 1971). Germinal plasm regions were identified at the light microscope level in R . pipiens as finely granular areas free of inclusions in the vegetal cytoplasm, rather than by staining characteristics. At the electron microscope level, these areas were seen to contain distinctive electron-dense, spherical bodies associated with large clusters of mitochondria. These bodies appeared to b e composed of fibrillar and particulate components and were twice as large in 16-cell-stage embryos as they were in eggs. Although germinal plasm regions were not seen at the light microscope level prior to fertilization, electron microscope examination revealed the presence of typical dense bodies at that stage. Similar bodies were observed in the fertilized eggs of A. mexicanum. They were larger than the bodies present at the same stage in R . pipiens and were associated with mitochondria. These investigators concluded that these unique bodies, which they called germinal granules, are a general feature of the germinal plasm in amphibian eggs and probably represent germ cell determinants (Williams and Smith, 1971). More recently, two studies have been reported on the fine structure of the germinal plasm of X . Zaevis. In the first, fertilized eggs with the first cleavage furrow completed or nearly completed were examined by Czoloska (1972) for the presence of germinal plasm. It appeared much the same as that in other species, except for more variation in size and shape. The number of dense bodies varied from island to island and from egg to egg, with a maximum of about 30 bodies per 100 pm2, the size of the bodies ranging from 0.2 to 0.9 p m in diameter. Kalt (1973) examined unfertilized eggs and twocell- and blastula-stage embryos and observed discrete cytoplasmic bodies consisting of irregularly shaped, dense aggregates of particles.

These areas contain numerous small, dense, fibrogranular masses which usually lie in association with mitochondria. Although these dense masses vary in appearance, apparently depending on factors such as fixation, the stage examined, and the species of amphibian examined, they have usually been referred to as germinal plasm (Mahowald and Hennen, 1971; Williams and Smith, 1971; Czoloska, 1972). ~ 3 6 0 0 . Inset: X 16,000.

250

E. M. EDDY

They were often associated with membranous elements and frequently surrounded by ribosomes. He suggested that the initial manifestation of germ cell development occurs in the postvitellogenic oocyte, in which these areas of germinal plasm are first discerned. Kalt also examined primordial germ cells in sexually undifferentiated gonads of tadpoles, oogonia and spermatogonia in recently sexually differentiated tadpoles, and germ cells present in subsequent stages of differentiation in male and female animals. In all cases he demonstrated dense bodies with the same particulate structure and general association with mitochondria as the germinal plasm of mature eggs and early embryos. Because these structures are common to germ cells and are not present in somatic cells, h e suggested that they may b e related to the maintenance of the germ line (Kalt, 1973).

D. CYTOCHEMICAL STUDIES Studies on the composition of germinal plasm in amphibians have indicated that RNA is a major component. Blackler (1958), working with R . temporaria, R . esculentu, B . bufo and X . lueois, found that germinal plasm in fertilized eggs and blastulas stained with Pyronine and that RNase digestion prevented staining. However, gastrulas did not show positively staining material. Czoloska (1969) repeated these studies on X . laevis eggs and observed staining in the vegetal pole region of ovarian eggs as well. Furthermore, the dense fibrous bodies in unfertilized eggs and two-cell-stage embryos of R . pipiens that Mahowald and Hennen (1971) identified at the electron microscope level as germinal plasm were also stained by the indium reaction. In this study eggs and embryos were extracted with cold perchloric acid as a control and, when this was done, the germinal plasm was unstained. Using a different approach, Padoa (1963) reported that ultraviolet irradiation apparently produced sterility in R . esculenta b y damaging the germinal plasm and speculated that ultraviolet light affected the RNA. Further evidence for this was obtained by Smith (1966), who observed that the most effective wavelength for producing sterility in R . pipiens was 2540& a wavelength known to affect RNA.

VI. The Nuage and Germ Cells A. ANIMALS WITH GERMCELLS CONTAINING A Nuage Many investigators have reported the presence of densely staining bodies in the cytoplasm of germ cells of numerous animals. Although

GERM PLASM

25 1

these were sometimes noted in studies by light microscopists, they have been observed more frequently since the advent of electron microscopy. Table I1 is a compilation of studies in which dense bodies have been observed by electron microscopy. As may be seen, they have been referred to by a variety of different names. However, several workers have used the term nuage, the French word for cloud, originally employed by Andrd and Rouiller (1957), and since this is a neutral term as far as composition or function is concerned, and yet is quite descriptive, it is used here. Although Table I1 lists over 80 animals in eight phyla that have a nziuge in their germ cells, it is undoubtedly incomplete. The nuage is often mentioned only in passing, if at all, as a curiosity in studies on other features of germ cells. Furthermore, in some animals listed, the nuage was illustrated but not discussed by the investigator. However, on examining the literature on the fine structure of germ cells, one is impressed by the frequency with which the nuage has been observed. It is almost always reported in studies concerned with the earlier stages of development of germ cells, and often in studies on later stages of gametogenesis as well. It has been observed most commonly in germ cells of female animals, often in young oocytes, and sometimes in oogonia or in nurse cells originally derived from the germ cell line. Although reports of the nuage in male germ cells are less common in this list, it has been seen most often in animals in which the premeiotic and meiotic phases of spermatogenesis were closely examined. Indeed, the chromatoid body was well known to light microscopists as a feature of male germ cells; when these earlier reports are considered, the nuage seems to be a reasonably common feature of male germ cells as well (see Sud, 1961b, for a review). Thus what has already been proposed for mammalian germ cells, that the nuage is a characteristic morphological feature (Eddy, 1974), may be true for other germ cells as well. The nuage is of special interest because of its striking similarity to the polar granules of insects, the germinal determinant of chaetognaths, and the germinal plasm of frogs in fine structure and distribution. In nearly every animal listed in Table 11, the nuage appears as a discrete, dense fibrous cytoplasmic organelle which lacks a surrounding membrane (Figs. 2-8). Furthermore, it is frequently seen in association with mitochondria1 clusters or immediately adjacent to the nuclear envelope of germ cells. It has been observed in primordial germ cells, oogonia, oocytes, spermatogonia, spermatocytes, and spermatids. As discussed below, it also appears to b e similar to germ plasms in composition. However, it must be kept in mind that the nuage has not been followed throughout the entire life history of the

ANIMALS WITH

Phylum Platyhelminthes Gorgoderina attenunta (trematode) Polycelis tenius, P. nigna (planaria) Rotifera Asplanchna brightwelli (rotifer) Asplanchna sieboldi (rotifer) Annelida Nereis pelagica (polychaeta) Enchytraeus albidus (oligochaeta) Eisenia foetida (oligochaeta) Nematoda Rhabditis pellio (nematode)

TABLE I1 GERM CELLS CONTAINLYC Nuage

Name

Stage

References

Nucleoluslike body Nuclear emissions

Maturing oocytes Spermatogonia

Koulish (1965) Franquinet and Lender (1973)

Nuclear extrusions Nuclear extrusions

Young oocytes Young oocytes

Bentfeld (1971a,b) Bentfeld (1971a,b)

Cytoplasmic aggregates Dense granular material Unnamed

Young oocytes Stage I1 oocytes Young oocytes

Dhainaut (1970) Dumont (1969) Lechenault (1968)

Dense body

Spermatogonia, spermatids

Beams and Sekhon (1972)

Nuclear extrusions, intermitochondria1 cement Granular material, basophilic body Basophilic regions

Previtellogenic oocytes Young and maturing oocytes Young oocytes

Dhainaut and Richard (1972)

Anderson (1969)

Perinuclear particles

Young oocytes

Gerin (1971)

Perinuclear electron opacities Nuage

Early oogonia

Reger (1970)

Oocytes

AndrC and Rouiller (1957)

Mollusca Sepia officinalis (cephalopod)

Mopalia mucosa (amphineuran) Chaetopleura apiculata (amphineuran) llyanassa obsoleta (gastropod) Arthropoda Arachnida Leiobunum sp. (harvestman) Tegeneria domestica (spider)

Anderson (1969)

Crustacea Libinia emarginata (spider crab) Orconectes oirilis (crayfish)

Cambarus sp. (crayfish) Homarus sp. (lobster) Panulirus sp. (lobster) Insecta Libellula pulchella (dragonfly) Periplaneta americana (roach) Rhodnius prolixus (reduvid bug) Tipula oleracea (fly) Drosophila melanogaster (fruit fly) E3

01 0

Dytiscus marginalis (diving beetle)

Hinsch and Cone (1969) Hinsch (1970) Beams and Kessel (1963); Kessel and Beams (1968) Beams and Kessel (1963) Kessel (196%) Kessel (196%)

Granular material Dense material Masses of granules, cytoplasmic nuage Granular masses Granular masses Granular aggregates

Previtellogenesis Early vitellogenesis Young oocytes

Cytoplasmic mass, dense mass Dense clusters Granular aggregates Dense masses

Oogonia, young oocytes Small oocytes Nurse cells Nurse cells, early oocytes Spermatids, ovarian nurse cells

Kessel and Beams (1969)

Ovarian nurse cells

Bertolini and Urbani (1964);Ficq and Urbani (1969) Favard-Skrkno (1968)

Amorphous body, dense plasmosomal masses, clouds of ribosomal particles Nuclear material

Oocytes Small oocytes Small oocytes

Gryllus bimaculatus, G . mitratus, G . scapsipedus (crickets) Sciara coprophilia (fungus gnat) Acheta domestica (cricket) Gerris remigis (water strider)

Pseudonucleoli

Oocytes

Unnamed Fascicles Chromatoid body

Acrida lata (grasshopper) Oncopeltus fasciatus (milkweed bug) Drosophila cirilis (fruit fly) Philaenus spumarius (spittle bug) Water boatman

Chromatoid body Nuclear material Chromatoid body Nuclear extrusion Dense juxtanuclear material

Ovarian nurse cells Oocytes Spermatocytes, spermatids Spermatids Early spermatocytes Spermatocytes Young spermatids Spermatocytes

Anderson (1964) Anderson and Beams (1956) Lima-de-Faria and Moses (1966) Rasmussen (1973);King (1960);Dapples and King (1970)

Phillips (1967) Allen and Cave (1968) Tandler and Moriber (1965) Yasuzumi et al. (1970) Barker and Riess (1966) Ito (1960) Maillet and Gouranton (1965) Tandler and Hoppel (1972)

(Continued)

TABLE I1 (Continued) Phylum Myripoda Polydesmus angustus (millipede)

Lithobius forfieatus (centipede) Echinodermata Ophiuroidea Ophioderma paramensis (brittle star) Echinacea Arbacia punctulata (sea urchin)

Name

References

Cytoplasmic aggregates, dense cement Fibrogranular material

Young oocytes

Petit (1973)

Young spermatocyte

Descamps (1971)

Aggregates of granules

Growing oocytes

Kessel (1968a)

Unnamed, heavy bodies

Early primary oocytes, mature eggs Oogonia, perivitellogenic oocytes Mature oocytes Mature eggs Fertilized eggs

Verhey and Moyer (1967); Bal et a / . (1968); Conway (1971)

Arbacia lixula (sea urchin)

Dense aggregates

Lytechinus pictus (sea urchin) Lytechinus uerigntus (sea urchin) Strongylocentrotus purpuratus (sea urchin) Paracentrotus lioidus (sea urchin)

Heavy bodies Heavy bodies Heavy bodies

Echinus esculentus (sea urchin) Holothuroidea Thyone briureus (sea cucumber) Chordata Ascidiacea Boltenia uillosu (tunicate) Cionu intestinulis (tunicate) Pisces Sygnatlzus fiisctis (pipefish)

Stage

Dense aggregates

Millonig et al. (1968) Verhey and Moyer (1967) Verhey and Moyer (1967); Conway (1971) Harris (1967) Millonig et al. (1968)

Heavy bodies

Oogonia, perivitellogenic oocytes Oocytes

Nucleolar material

Young oocytes

Kessel and Beams (1963); Kessel (1966b)

Unnamed Dense granular material

Young oocytes Young oocytes

Hsu (1962) Kessel (1966a)

Dense particles

Small oocytes

Anderson (1967, 1968)

Afzelius (1957)

Fundulus heteroclitus (killifish) Hippocampus erectus (sea horse) Xiphophorus helleri (swordtail) Lebistes reticularis (guppy)

Salmo gairdneri (trout) Carassius auratus L. (goldfish) Mollienisia sphenops (molly) Equidens latifrons Brachydanio rerio (zebra fish) Gardonus rutilus (roach fish)

N2

01

cn

Pimephales notatus Gasterosteus aculeatus (stickleback) Oryzias latipes (medaka) Protopterus aethiopicus (lungfish) Aniphibia Rana temporaria Rana esculenta Rana catesbeiana Rana clamitans Rana nigromaculata Rana dalmatia

Dense particles Dense granules Nucleolar material Nuclear extrusions Mitochondrial clusters Dense granules Mitochondrial rosettes Nuclear material Nuclear material Nuclear material, nuclear extrusions, intermitochondrial cement Nuclear emissions Chromatoid bodies Nuclear extrusions Electron-opaque substance Bodies Dense material, intermitochondrial cement Dense aggregates Intermitochondrial cement Perinuclear nuage, dense perinuclear material Dense perinuclear material Nuclear emissions Dense granular material, intermitochondrial cement

Oogonia Young oocytes Young oocytes Early oocytes Spermatocytes Young oocytes Small oocytes Oocytes Oocytes Oocytes, young oocytes

Anderson (1968) Anderson (1967) Wegman and Gotting (1971) Follenius (1965) Droller and Roth (1966) Beams and Kessel (1973) Yamamoto and Onozato (1965) Zahnd and Porte (1966) Zahnd and Porte (1966) Zahnd and Porte (1966); Ulrich (1969)

Primary spermatocytes Spermatogonia Spermatocytes Young oocytes Young oocytes

Clerot (1971)

Oogonia, oocytes, spermatocytes Oocytes Spennatocytes Oogonia, oocytes Small primary oocytes Young oocytes Oocytes

Schjeide et al. (1972) Follenius (1965) Yamamoto (1964) Scharrer and Wurzelmann (1969) Clbrot (1968) Wartenberg (1962) Cldrot (1968) Cl6rot (1968);Massover (1968); Eddy and Ito (1971) Eddy and Ito (1971) Takamoto (1966) Delbos et al. (1971)

(Continued)

TABLE I1 (Continued) Phylum Rana pipielis Xenopus laecis

5

Name Nucleoluslike body, dense perinuclear material Nucleoluslike body, chromatoid body, nuage, nuclear emissions, granular material

Stage Young oocytes, oogonia Primordial germ cells, spermatogonia, spermatocytes, spermatids, oogonia, early oocytes Medium-sized oocytes Medium-sized oocytes Immature oocytes

References Kessel (1969); Eddy and Ito (1971) Balinsky and Devis (1963);Al-Mukhtar and Webb (1971); Reed and Stanley (1972); Van Gansen and Weber (1972); Coggins (1973); Kalt (1973)

Amblystoma mexicanurn

Nucleolar granules

Tritrrrus ciridescens

Nucleolar granules

Tritrrrus alpestris

Nucleolus-derived dense material Expelled material Dense mass

Young oocytes Young spermatids

Takamoto (1966) Picheral (1972)

Nuage, fibrogranular material

Pyriform cells (nurse cells)

Neaves (1971)

Strands of granules

Small oocytes

Schjeide et al. (1965); Greenfield (1966)

Dense bodies, mitochondrial clusters, chromatoid body

Primordial germ cells, oocytes, spermatocytes, spermatids

Odor and Blandau (1969); Fawcett et al. (1970); Zamboni (1970); Comings and Okada (1972); Spiegelman and Bennett (1973)

m

Triturus pyrrliogaster Pleurodeles waltlii Reptilia Anolis carolinensis

Aves Domestic chicken Manimalia Mouse

Lane (1967) Lane (1967) Franke and Scheer (1970)

Rat

Mitochondrial clusters, chromatoid body

Primordial germ cells, oogonia, oocytes, gonocytes, spermatogonia, spermatocytes, spermatids

Guinea pig

Mitochondrial clusters, chromatoid body Mitochondrial clusters

Oocytes, spermatocytes, spermatids Oocytes, spermatocytes Spermatocytes, spermatids Oocytes, spermatogonia, spermatocytes Spermatid Spermatid Oocytes, spermatogonia, spermatocytes, spermatids Oogonia, spermatogonia, spermatocytes, spermatids

Hamster Chinchilla Rabbit

Mitochondrial clusters, chromatoid body Mitochondrial clusters, dense body

E3

Ei:

Cat Ram Monkey

Chromatoid body Chromatoid body Mitochondrial clusters, chromatoid body

Human

Dense bodies, mitochondrial clusters

Watson (1952);Swift (1956); Odor (1960); Watson and Aldridge (1961); Andre (1962); Brokelmann (1963); Franchi and Mandl (1964,1966); Roosen-Runge and Leik (1968);Novi and Saba (1968);Eddy (1970); Susi and Clermont (1970); Fawcett et al. (1970); Gondos and Conner (1973); Eddy (1974); Kang (1974) Fawcett and Ito (1958);Adams and Hertig (1964); Fawcett et al. (1970) Odor (1965); Weakley (1967, 1969); Fa\\ cett et al. (1970); Weakley (1971) Fawcett et al. (1970) Blanchette (1961); Nicander and Ploen (1969); Gondos et al. (1973) Burgos and Fawcett (1955) Courot and Loir (1968) Hope (1965); Szollosi (1969); Fawcett et al. (1970); Gondos and Zemjanis (1970) Burgos et al. (1970);Gondos and Hobel (1971);Wartenberg et al. (1971);Szollosi et al. (1972)

FIG.2. The nrccigc appears to be issuing from nuclear pores in a spermatid of'the insect Lepyroniu quundrungularis (Hemiptera, Cercopidae) in the vicinity of the developing axoneme. It has been reported that this material contains RNA (Maillet and Gouranton, 1965). X 18,700.

GERM PLASM

259

animals listed or subjected to experimental test to demonstrate that it has a role in determining germ cell formation. The nuage has been subjected to the closest scrutiny in germ cells of amphibians and mammals, and thus they are discussed here as examples of animals possessing this material. Since germinal plasm has also been identified and examined b y electron microscopy in amphibians, the form and distribution of the nuage in amphibians is considered first. As noted previously, the germinal plasm of amphibians typically consists of accumulations of dense bodies, frequently in contact with mitochondria or the nuclear envelope (Mahowald and Hennen, 1971; Williams and Smith, 1971; Czoloska, 1972; Kalt, 1973).The nuage in primordial germ cells of amphibians also consists of small patches of dense, granular material associated with nuclear pores (Al-Mukhtar and Webb, 1971), or larger accumulations of dense material either lying free in the cytoplasm or associated with clusters of mitochondria (Al-Mukhtar and Webb, 1971; Reed and Stanley, 1972; Kalt, 1973). Kalt (1973)previously pointed out the similarity between the nuage seen at this stage and the germinal plasm seen in eggs and early embryos. I n oogonia the nuage is present in small accumulations lying in the interstices of mitochondria1 clusters (Eddy and Ito, 1971; Al-Mukhtar and Webb, 1971; Coggins, 1973), and as larger accumulations in the cytoplasm (Al-Mukhtar and Webb, 1971). Similarly, the numerous observations of the nuage in amphibian oocytes recorded in Table I1 usually indicate that it may be found either associated with mitochondria or nuclear pores or present in larger accumulations lying free in the cytoplasm (reviewed by Eddy and Ito, 1971). Also, in spermatogonia (Reed and Stanley, 1972; Kalt, 1973) and spermatids (Picheral, 1972; Kalt, 1973), the nuage may be seen associated with mitochondria or nuclear pores, as well as in larger, discrete aggregates in the cytoplasm in much the same fashion as in oocytes. These studies indicate that in amphibians the nuage is a characteristic feature of germ cells of all stages of development and closely resembles germinal plasm in its morphology and associations with other cytoplasmic components (Figs. 9 and 10). Recently the germ cells of the rat were studied by electron microscopy from the time they were present in the epithelium of the embryonic gut until near the time of production of mature gametes in the adult (Eddy, 1974; Eddy and Clark, 1975). Particular attention was paid to the form and location of the nuage during this period of

GERM PLASM

26 1

the life cycle. In primordial germ cells the nuage was present as a discrete accumulation of fibrous material, usually situated in the perinuclear region (Figs. 11-13). A similar arrangement was noted in oogonia and gonocytes in fetal gonads, but the nuage was more commonly associated with mitochondrial clusters in primary oocytes, spermatogonia, and spermatocytes. The nuage ccntinued to be found in mitochondrial clusters well into the later stages of oogenesis, but in the male the nuage previously present in mitochondrial clusters aggregated to form the chromatoid body characteristic of spermatocytes and spermatids (Fawcett et al., 1970). On occasion the nuage assumed a transient association with pores in the nuclear envelope, and nuclear granules seemed to be entering the cytoplasm in the vicinity of small masses of nuage material (Eddy, 1974). In addition, the nuage has been observed in several different stages of germ cell formation in other mammals (Table 11). It may be seen in published micrographs of primordial germ cells of mouse, oogonia of human, and oocytes of rat, mouse, rabbit, guinea pig, hamster, and monkey. It has been observed in gonocytes in fetal male rats and in spermatogonia of juvenile and adult rabbit, monkey, and human. The nuage is also present in spermatocytes of rat, mouse, rabbit, guinea pig, chinchilla, Chinese hamster, and monkey. In addition, it has been demonstrated at the electron microscope level in spermatids of rat, cat, guinea pig, mouse, ram, chinchilla, Chinese hamster, monkey, and human (Table 11). In all these animals the nuage is quite like that present throughout much of the life cycle of the rat in form and distribution. It is typically a small, dense accumulation of fibrous material lying in the perinuclear cytoplasm. In oogonia, spermatogonia, and early spermatocytes, it is usually associated with mitochondria, while in primordial germ cells, late spermatocytes, and spermatids it is present as a discrete mass. Based on these observations, it has been suggested that the nuage is a characteristic morphological feature of mammalian germ cells (Eddy, 1974).

FIG.3. The nuuge in a young oocyte of the tunicate Ascidiu callosu (Urochordata, Ascidiacea) is of two different densities after processing for electron microscopy, a more dense and tightly packed form, here closely associated with nuclear pores, and a less dense and more loosely organized form, l y i w nearby. x 12,600. FIG.4. The nuuge in a young oocyte of the tunicate Pyura huustor (Urochordata, Ascidiacea) is located adjacent to the nuclear envelope around much of its profile. Although of uniform density, the nuage may be either loosely organized or more compactly arranged. x 13,900.

GERM PLASM

263

B. COMPOSITION OF THE Nuage AND OF GERM PLASM Morphology alone is not a totally satisfactory way of identifying a cytoplasmic component such as the nuage. Although it is considerably similar in form and distribution to the polar granules of insects, the germinal determinants of chaetognaths, and the germinal plasm of amphibians, these are not sufficient reasons to equate the nuage to germ plasm. There are other similarities, however, which are also consistent with such a possibility. An important factor to be considered is the composition of the nuage and of the germ plasm. The germ plasm hypothesis, indicating that the germ cell line is determined by an agent present in mature germ cells, suggests that the agent contains stored information capable of influencing the formation of primordial germ cells. It is useful therefore to review what is known about the composition of the nuage to see if it contains substances that play such a role. RNA has been reported to be present in the nuage of germ cells in a variety of animals. Dhainaut (1970) stained thin sections of young oocytes of the polychaete Nereis pelagica by the EDTA-uranyl acetate reaction of Bernhard (1969) and concluded that RNA was contained in the nuage. The investigator also injected animals with uridine-H3 and observed that in 5 days it was incorporated into the nuage, five times more silver grains being present in radioautographs viewed by electron microscopy in the vicinity of the nuage than in other areas of the cytoplasm. In another study, Petit (1973) digested thin sections of young oocytes of the millipede Polydesmus angustus with RNase and reported that particles were removed from the nuage, leaving a less dense matrix visible in the electron microscope. It had earlier been reported that the heavy bodies in oocytes of several species of sea urchins stained with Pyronine but did not stain after RNase digestion (Afzelius, 1957).These observations were supported by a more recent study showing that the heavy bodies of sea urchin oocytes stained with azure B and toluidine blue at an acid pH and that this basophilia was eliminated by RNase (Conway, 1971). Furthermore, it was found in the latter study that the nuage was FIG. 5. In a yolk-containing oocyte of the scallop Pecten caurinus (Mollusca, Bivalvia) the nziage is ropy and loosely arranged. Other organelles interdigitate into the interstices of the rather large body of nuage material. x7800. FIG. 6. An oocyte of the chiton Katharina tunicata (Mollusca, Amphinenra) contains two other forms of the nuage, one having a rather dense center surrounded by a tight zone, and another being of uniform texture and less density. Mitochondria are often sihiated adjacent to the first form. x26,600.

GERM PLASM

265

reduced in electron opacity in thin sections digested with RNase and that the usual positive reaction to the indium stain was eliminated by prior RNase digestion. However, not all studies have detected RNA in the nuage. Clbrot (1968) used RNase to digest thin sections of frog oocytes and reported that the nuage was unaffected. Also, Eddy and Ito (1971) were unable to demonstrate incorporation of uridine-H3 into the nuage of frog oocytes, and they observed that actinomycin D had no effect on nuage fine structure. Furthermore, the indium reaction was used to stain structures known to contain RNA, including ribosomes and nucleoli. However, after this treatment the nuage was found to be of no greater density than structures composed of protein, and its staining was unaffected b y RNase digestion (Eddy and Ito, 1971). Similarly, Kalt (1973) was unable to detect RNA in the nuage of primordial germ cells, oogonia, spermatogonia, or oocytes in X. laevis using either the indium reaction, the Bernhard reaction, or RNase digestion at the electron microscope level. In mammals it was earlier suggested that the nuage in hamster oocytes (Weakley, 1971) and in rat spermatocytes and spermatids (Daoust and Clermont, 1955; Sud, 1961a) contained RNA because of its stainability with Pyronine and RNase sensitivity. However, in studies at the electron microscope level using the indium reaction, the Bernhard reaction, and RNase digestion, this was not confirmed in germ cells of male rats, mice, or guinea pigs (Eddy, 1970), and it has since been suggested that clusters of ribosomes often associated with the nuage may be sufficient to give the staining reaction visible at the light microscope level (Susi and Clermont, 1970). There is evidence of a protein component in the nuage present in germ cells of several animals. Sud (1961a) reported that the nuage in spermatocytes and spermatids of the rat stained with acid dyes and probably contained basic proteins. Using pronase and pepsin digestion on thin sections, Clbrot (1968) extracted the nuage present in frog oocytes while leaving most other structures intact. The same technique was used to remove the nuage from sections of oocytes of the millipede (Petit, 1973), and of the newt Pleurodeles waltlii FIG. 7. In a young oocyte of the sea urchin Strongylocentrotus droebakhiensis (Echinodermata, Echinacea), nuuge is usually associated with clusters of mitochondria, apparently “cementing” them together. x30,000. FIG. 8. In a stage of oocyte development later than that shown in Fig. 7 for the sea urchin S. droebachiensis (Echinodermata, Echinacea), the nuage is organized into small, round bodies which lie in the cytoplasm either singly or in groups. ~21,200.

GERM PLASM

267

(Picheral, 1971). Other evidence for protein comes from the study of Eddy and Ito (1971) on frog oocytes, in which it was shown by electron microscope radioautography that tritium-labeled amino acids were incorporated into the nuage. It was observed that labeling of the nuage increased during the first 6 hours the precursor was available, but apparently leveled off after that time. This indicated that at least some of the protein of the nuage was being manufactured during that stage of oogenesis, and also that there was probably a turnover of that protein component of the nuage. It was interesting that, of the amino acids used, phenylalanine appeared to be the one most heavily incorporated into the nuage (Eddy and Ito, 1971). This correlated well with the observation that the nuage could be extracted with pepsin (Clkrot, 1968), an endopeptidase that preferentially cleaves the peptide bonds of aromatic amino acids. As has been noted, available evidence indicates that RNA is probably also present in the polar granules of insects, the germinal determinant of chaetognaths, and the germinal plasm of amphibians. However, this appears to be true only of eggs and early embryos, since RNA is no longer present b y the blastoderm stage of development in Drosophila (Mahowald, 1971b) or by the gastrula stage of development in several amphibians (Blackler, 1958).Since it also has been reported that polar granule material is visible at the electron microscope level throughout the life cycle of Drosophila (Mahowald, 1971a), and that the germinal plasm of R . pipiens embryos is also visible well after RNA disappears (Mahowald and Hennen, 1971), it is apparent that other substances are present in these bodies as well. Nicklas (1959) reported that polar granules contained tyrosine, detectable with the Millon reaction, and Mahowald (1971b) noted that the polar granules stained densely at pH 8 with azure B and toluidine blue, presumably indicating the presence of basic proteins. Because FIG.9. In a small primary oocyte of the amphibian Rana clamitans, the nuage is seen to be present as many small, dense bodies present in the perinuclear zone of the cytoplasm. Radioautographic studies indicate that this is an active time of synthesis of the protein component of the nuage. x6000. (Reprinted by permission, Eddy and Ito, 1971, The Journal of Cell B i o l o g y . ) FIG. 10. A portion of a small oocyte of the amphibian R . pipiens is shown. The nuage is present near the nuclear envelope in close association with mitochondria. The perinuclear cytoplasm contains small tufts, and streamers of dense material are seen near nuclear pores. These features and the presence of small, dense granules in the nucleus caused earlier workers to suggest that cytoplasmic nuage had a nuclear origin. x31,800. (Reprinted by permission, Eddy and Ito, 1971, The Journal of Cell Biology.)

GERM PLASM

269

of these various observations, it has been proposed that polar granules are a site of localization of maternal mRNA used during early embryogenesis (Mahowald, 1968, 1971b), the proteinaceous fibrous material present throughout the life cycle serving as the matrix on which the RNA becomes localized (Mahowald, 1971a). Although what is known about the composition of germ plasm and of the nuage is relatively superficial, it appears that they may both contain RNA and a basic protein. However, as has been noted, the RNA is probably present in polar granules of insects and germinal plasm of amphibians during only a small part of the germ cell cycle. Perhaps a similar situation is true for the nuage, with the time of appearance of RNA varying somewhat between species. This would explain why RNA has been detected in oocytes of polychaetes, millipedes, and sea urchins, but not in young oocytes of frogs or spermatocytes of mammals. In the frog, in which the evidence that the nuage and the germinal plasm may be one and the same appears the strongest (Kalt, 1973), this seems likely to be the case. However, other explanations may b e possible, and it remains to be seen if the nuage always has an RNA component at some point in its development.

c.

ORIGIN OF THE

Nuage

Since the nuage is quite similar in form, distribution, and composition to the polar granules of insects, the germinal determinant of chaetognaths, and the germinal plasm of amphibians, other features of the image may be similar as well. If this is the case, what is known about the nuuge may be useful in understanding something more about polar granules, germinal determinant, and germinal plasm. One situation in which this should be considered is the origin of the nuage in the cytoplasm of germ cells.

FIGS.11-13. Primordial germ cells are present in the epithelium o f t h e hindgut of the rat on the tenth day of embryonic development. Figure 11 is a low-magnification electron micrograph showing this. Figure 13 is a higher magnification view of a portion o f t h e same area seen in Fig. 11 in which two primordial germ cells are present. Th e primordial germ cells are included by the basal lamina o f t h e gut epithelium. Figure 12 is a yet higher magnification view of a section through the primordial germ cells (the area of the Imx in Fig. 13). The nricige is present a s a sinall, discrete aggregation of dense fibrous material, as it is in g e m cells in more advanced stages of gametogenesis. Fig. 11: x1000. Fig. 12: x28,OOO. Fig. 13: x6200. (Reprinted by permission, Eddy, 1974, The At~otonticcrlRecord.)

270

E. M. EDDY

Several investigators have observed the nuage in the perinuclear zone of the cytoplasm of amphibian oocytes. They have usually interpreted it as being a product of nucleocytoplasniic transfer (Pollister et aZ., 1954; Ornstein, 1956; Wischnitzer, 1958; Lanzavecchia, 1962; Miller, 1962; Wartenberg, 1962; Merriam, 1962; Balinsky and Devis, 1963; Swift, 1965; Takamoto, 1966; Lane, 1967; Clkrot, 1968; Hay, 1968a; Massover, 1968; Kessel, 1969; Franke and Scheer, 1970; Eddy and Ito, 1971). Linear arrays of 30- to 35-nm-diameter granules seen within the nucleus of amphibian oocytes have been suggested to move through the nuclear pores and subsequently to aggregate in larger masses of dense material in the cytoplasm (Swift, 1965). The fine-structural similarity of the nuclear granules to ribosomes and components of nucleoli led some investigators to propose that the granules may be ribonucleoprotein particles enroute to the cytoplasm (Swift, 1965; Lane, 1967; Franke and Scheer, 1970). It was suggested by Swift (1965)that the granules formed on the lampbrush loops of the oocyte chromosomes and then nioved to the periphery of the nucleoplasm for their eventual release into the cytoplasm. However, Lane (1967) observed streams of nuclear granules lying between the nucleoli and the nuclear pores, both in intact cells and in isolated nuclei, and suggested that they had a nucleolar origin. Also, Miller (1962) noted that the dense material was similar to the fibrous components of the nucleolus, and other investigators have since implicated the nucleolus in the formation of the nuage (Hay, 1968a; Kessel, 1969). More recent studies indicate that RNA probably is not present in the nuage of primary oocytes in amphibians (Eddy and Ito, 1971; Kalt, 1973), but tend to support the idea of a nuclear origin for the material. Association of the nuage with nuclear pores has been observed in other animals a s well, and has usually been believed to be evidence for nucleocytoplasmic exchange (Wischnitzer, 1973). The term nuage was originally used by Andri. and Rouiller (1957) to describe inaterial apparently issuing from the nuclear pores of spider oocytes. Electron micrographs in many of the studies listed in Table I1 show the same types of images in germ cells of other animals. As may be seen, they have been referred to as nucleoluslike bodies, nuclear extrusions, nuclear material, pseudonucleoli, nucleolar material, nuclear emissions, nucleolar granules, or expelled material by various investigators convinced of their nuclear origin. In yet other studies listed in Table 11, the nuage was given a more generalized name, but the investigators also suggested a possible nuclear origin. From

GERM PLASM

27 1

these observations it appears that in most animals the nuage in the cytoplasm probably forms from granules originally present in the nucleus of germ cells. If the nuage forms from nuclear granules, it becomes of interest to know where and when these precursors of the cytoplasmic nziage form. This question was addressed in a general way in the study of Eddy and Ito (1971) in which pieces of frog tadpole ovaries were incubated in a culture medium containing radioactive compounds to be incorporated into the nuage. Electron microscope radioautography after extended incubatio ti with tritiated uridine, thymidine, and actinomycin D did not reveal incorporation of these compounds into the nuage, as would be expected if they contained nucleic acid. However, there was significant incorporation of the amino acids leucine, arginine, lysine, tryptophan, and phenylalanine, indicating that the nuage contained protein. Although there was incorporation of all the amino acids into nuclear and cytoplasmic structures, as well as into the nuage, phenylalanine had a greater uptake into the nuage than the other amino acids. It was found that the greatest amount of labeling was apparent after 6 hours of incubation with isotope followed by 6 hours of chase, and that there was no evident increase of labeling when the incubation was extended to 24 hours. This suggested that the material was synthesized elsewhere in the cell and required about 6 hours to concentrate in the nuage. In addition, it indicated that the nuage not only was being formed during this stage but also contained a protein component which apparently had a rapid turnover. However, further studies will b e necessary to determine where the precursors of the nuage form and whether there is a phase during which turnover no longer occurs.

VII. Conclusions The history of the study of germ plasm has been an erratic one. Although the books by Hegner ( 1 9 1 4 ~and ) Bounoure (1939) on the subject denote periods of interest, it has not been until the last decade or so that widespread and sustained research efforts have been mounted to test the germ plasm hypothesis. While the first two eras involved primarily morphological studies, more recent work has been experimental in nature. It seems likely that this type of activity will continue and that such techniques as irradiation, microsurgical manipulation, and the use of mutants and genetic markers will con-

272

E. M. EDDY

tinue to be useful in determining the role of germ plasm in the life history of the germ cell line. Another tool that has provided valuable information is electron microscopy, by which it has been shown that germ plasm is a discrete and easily recognizable structure, is quite similar in form and distribution in both invertebrates and vertebrates, and is apparently unique to germ cells. Indeed, it appears that this is a topic ripe for detailed investigation; not only are the tools and approaches available, but the characteristics of germ plasm make it particularly amenable to study in a variety of ways. Although there has been encouraging progress in recent years, there are several important questions remaining about germ plasm that deserve thorough investigation. One is: What is the specific time of origin of germ plasm? If germ plasm has a role in the determination of the germ cell line, this effect is probably programmed at the time the germ plasm is synthesized. As noted in this article, germ plasm has been reported to be identifiable in oocytes in some species and to appear during early cleavage stages of development in other species. In the latter case it may be that germ plasm is present but not organized into a recognizable form prior to cleavage. However, it is important to know for sure whether germ plasm is a product of the previous generation or arises during embryogenesis in the new generation. Another important question is: What is the composition of germ plasm? Since the germ plasm hypothesis implies some type of information storage in germ plasm, most investigators have been rather willing to accept the histochemical evidence for the presence of RNA in polar granules of insects and germinal plasm of amphibians. This evidence also indicates that RNA is present only in eggs and early embryos, while a protein component is present throughout the germ cell cycle. If this is the case, it suggests that germ plasm carries a message for only a brief part of the life cycle of germ cells (Mahowald, 1971a). However, this question should be examined with techniques that are more specific and sensitive than histochemistry. It may be that there are several RNA components and that small amounts of some persist beyond the early embryonic stage. Furthermore, it would be useful to know more about the composition of the protein component, particularly whether or not it has some unique characteristics. This information would also be valuable in understanding how and when germ plasm might influence germ cell differenti at'ion. Good morphological and experimental evidence for the presence of germ plasm is available for only a few animal species. Most of the

GERM PLASM

273

animals in Table I reported to have germ plasm were identified as the result of purely descriptive studies. Thus the question should be posed: Is germ plasm characteristic of all germ cells? Since it is present in such widely separated classes as insects and amphibians, there is some indication that it might be. However, other animal species suggested to have germ plasm on morphological grounds should be studied experimentally to see if this indication holds. Also, evidence is needed that germ plasm is present in germ cells of animals other than those few listed in Table I. Should the nuage be shown to b e a stage in the life cycle of germ plasm, the list of animals possessing germ plasm would be considerably increased by the addition of those in Table 11. Furthermore, the information acquired about the form, distribution, composition, and origin of the nuage could be applied to the study of germ plasm if this were the case. The cardinal question is, of course: What is the role of germ plasm? The morphological observations and the experimental findings reported here are consistent with the hypothesis that germ plasm determines the germ cell line, but it would be premature to accept this as a proven phenomenon at the present time. The evidence is still circumstantial, and it will be necessary to answer questions raised here, and probably others before the role of germ plasm can b e fully explained. However, if it is established that germ plasm is a specific determinant serving to separate the germ cell line from the somatic cell line during development, it will then be quite interesting to learn how this is accomplished. Presumably, germ plasm would act by altering the cell potential in some way. One can envisage two alternate mechanisms which might be involved. The first would be a positive influence, with germ plasm acting on an uncommitted precursor cell nucleus to cause initiation of the germ cell line, perhaps by derepressing a specific segment of the genome. The other alternative would be a negative influence, with germ cells being formed because germ plasm prevented them from becoming somatic cells, perhaps by repression of a specific segment of the genome. While considering the role of germ plasm, it should be kept in mind that there may be other or additional functions that it serves. For example, there is some evidence that germ plasm is involved in preventing the elimination of chromosomes from the germ cells of certain invertebrates, including insects, at the time they are eliminated from the somatic cells (Nicklas, 1959; Geyer-Duszynska, 1959; Bantock, 1961, 1970). Also, since at least some germ plasm components persist and apparently continue to be formed well after the germ cell line is formed, perhaps other events unique to game-

274

E. M. EDDY

togenesis, such as meiosis, are influenced by germ plasm. In addition, there are two cases we are aware of in which material identical to germ plasm in form and distribution is apparently found in somatic cells. The first is Drosophila, in which some pole cells containing polar granules do not migrate to the gonads but are incorporated into the posterior midgut to form part of the epithelium (Poulson, 1947). The other is planaria, in which dense, cytoplasmic bodies have been observed in association with the nuclear pores of neoblast cells (Sauzin, 1966, 1968; Le Moigne, 1966) and of hlastema cells participating in regeneration (Hay, 1968b; Morita et al., 1969). Although the first case may be only an example of vestigial development, the latter could b e of more general significance. I t has been reported that, during regeneration in planaria, the gonads are derived from accumulations of neoblasts (Ghirardelli, 1965a). Also, it has been noted that the ultrastructure of spermatogonia is identical to that of neoblasts, and nuclear emissions and dense material have been seen in association with mitochondria in both (Franquinet and Lender, 1973). This may be of importance, because it has been suggested that there might be a rather close connection between regenerative power and the stage at which segregation of the germ line occurs, animals with precocious determination of primordial germ cells having limited regenerative ability (Ghirardelli, 1965b). Thus it will be of interest to learn whether materials similar in morphology to germ plasm bear other likenesses as well. The final question is: What is the general significance of the germ plasm hypothesis? There has long been an interest in the cytoplasmic control of nuclear activity in animal development (Gurdon and Woodland, 1968; Davidson, 1968) and, more recently, in the cytoplasmic control of general cellular events (Harris, 1974). If the germ plasm hypothesis is confirmed, it may well be found that this method of cell line determination during development is unique to germ cells. However, there is the possibility that a more general regulatory process is involved and that germ plasm represents a conspicuous example of the influence of cytoplasmic structures on nuclear events. If this is the case, germ plasm, because of its structural integrity and its distinctive appearance, offers considerable promise for the study of cell regulatory phenomena by means of the various approaches of experimental morphology and biochemical analysis. ACKNOWLEDGMENTS

I gratefully acknowledge the contributions of Judy M. Clark i n the studies that led to this article. I n addition, I thank the editors and publishers of the following joiirnals for permission to reproduce some of' the figures: Journal of Cell Biology, Figs. 9 and

GERM PLASM

275

10; Anatomical Record, Figs. 11-13. Appreciation is also expressed to A. 0. D. Willows for the opportunity to work at the Friday Harbor Laboratories of the University of Washington. This work was supported in part by USPHS Grants HD-06161 and GM-16598 from the National Institutes of Health.

REFERENCES Aboim, A. N. (1945). Reu. Suisse Zool. 52, 53. Achtelig, M., and Krause G. (1971). Wilhelm Roux’ Arch, Entwicklungsmech. Organismen 167, 164. Adams, E. C., and Hetig, A. T. (1964).J. Cell Biol. 21,397. Afzelius, B. A. (1957).Z. Zellforsch. Mikrosk. Anat. 45, 660. Allen, E. R., and Cave, M. C. (1968). 2.Zellforsch. Mikrosk. Anat. 92, 477. Al-Mukhtar, K. A. K., and Webb, A. C. (1971). J. Embryol. Exp. Morphol. 26, 195. Amer, F. I. (1966).Zool. Anz. 176, 420. Amma, K. (1911).Arch. Zellforsch. 6,498. Anderson, D. T. (1962).j. Embryol. Exp. Morphol. 10, 248. Anderson; E. (1964).J. Cell Biol. 20, 131. Anderson, E. (1967).J. Cell Biol. 35, 193. Anderson, E. (1968).J. Morphol. 125, 23. Anderson, E. (1969).J. Morphol. 129, 89. Anderson, E., and Beams, H. W. (1956).J. Biophys. Biochem. Cytol., Suppl. 2, 439. Andri., J. (1962).J. Ultrastruct. Res., Suppl. 3, 31. Andre, J., and Rouiller, C. (1957). Electron Microsc., Proc. Stockholm Conf., 1956 pp. 162-164. Aubry, R. (1953a). C. R. Acad. Sci. 236, 1101. Aubry, R. (195313).C. R. SOC. Biol. 147,893. Bal, A. K., Jubinville, F., Cousineau, G. H., and Inoue, S. (1968).J. Ultrastruct. Res. 25, 15. Balinsky, B. I. (1966).Acta Ernbryol. Morphol. Exp. 9, 132. Balinsky, B. I., and Devis, R. J. (1963).Acta Embryol. Morphol. Exp. 6, 55. Bantock, C. R. (1961).Nature (London) 190,466. Bantock, C. R. (1970).J. Embryol. Exp. Morphol. 24,257. Barker, K. R., and Riess, R. W. (1966). Cellule 66,41. Beams, H . W., and Kessel, R. G. (1963).J. Cell Biol. 18, 621. Beams, H. W., and Kessel, R. G. (1973).Amer. J. Anat. 136, 105. Beams, H. W., and Sekhon, S. S. (1972).J. Ultrastruct.Res. 38, 511. Bentfeld, M. E. (1971a).Z. Zellforsch. Mikrosk. Anat. 115, 165. Bentfeld, M. E. (1971b). Z. Zellforsch. Mikrosk. Anat. 115, 184. Bernhard, M. W. (1969). C . R. Acad. Sci., Ser. D 267,2170. Bertolini, B., and Urbani, E. (1964).Atti Accad. Naz. Lincei, C1. Sci. Fis., M a t . Natur., Rend. 36, 240. Blackler, A. W. (1958).J. Embryol. Exp. Morphol. 6,491. Blackler, A. W. (1966). I n “Advances in Reproductive Physiology” (A. McLaren, ed.), pp. 9-28. Logos Press, London. Blackler, A. W. (1970).In “Current Topics in Developmental Biology” (A. A. Moscona and A. Monroy, eds.), Vol. 5, pp. 71-87. Academic Press, New York. BlancheRe, E. J. (1961).J. Ultrastruct. Res. 5, 349. Bounoure, L. (1927). C . R. Acad. Sci. 185, 1304. Bounoure, L. (1931). C. R. Acad. Sci. 193, 402. Bounoure, L. (1934).Ann. Sci. Nut. Zool. 17, 67.

276

E. M. EDDY

Bounoure, L. (1937). C. R. Acad. Sci. 204, 1837. Bounoure, L. (1939). “L’Origin des Cellules Reproductrices et la Probleme de la Lignee Germinale.” Gauthier-Villars, Paris. Bounoure, L., Aubry, R., and Huck, M. L. (1954).J. Embryol. E x p . Morphol. 2, 245. Boveri, T. (1909).Arch. Zellforsch. 3, 181. Brokelmann, J. (1963).2. Zellforsch. Mikrosk. Anat. 59,820. Bruiyan, N. I., and Shafiq, S. A. (1959). Erp. Cell Res. 16, 427. Buehr, M. C., and Blackler, A. W. (1970).]. Embryol. E x p . Morphol. 23, 375. Burgos, M. H., and Fawcett, D. W. (1955).J . Biophys. Biochem. Ctyol. 1,287. Bnrgos, M. H., Vitale-Calpe, R., and Aoki, A. (1970). In “The Testis” (A. D. Johnson, W. R. Comes, and N. L. Vandemark, eds.), Vol. 1, pp. 339-432. Academic Press, New York. Biitschli, 0. (1873).Z . Wiss. Zool., Abt. A 23,409. Clerot, J. C. (1968).J. Microsc. (Paris) 7, 973. Clbrot, J. C. (1971).J. Ultrastruct. Res. 37, 690. Coggins, L. W. (1973).J.Cell Sci. 12, 71. Comings, D. E., and Okada, T. A. (1972).J. Ultrastruct. Res. 39, 15. Conway, C. A. (1971).J. Cell Biol. 51, 889. Counce, S. J. (1963).J. Morphol. 112, 129. Counce, S. J. (1973). In “Developmental Systems: Insects” (S. J. Counce and C. H. Waddington, eds.), Vol. 2, pp, 2-156. Academic Press, New York. Counce, S. J., and Selman, G. G. (1955).J.Embryol. E x p . Morphol. 3, 121. Courot, M., and Loir, M. (1968). Congr. Int. Reprod. Anim. Insem. Artif., 6th, Paris 1, 125-127. Czoloska, R. (1969).J. Embryol. Erp. Morphol. 22, 229. Czoloska, R. (1972). Wilhelm Rozix’ Arch. Entwicklungsmech. Organismen 169, 335. Daoust, R., and Clermont, Y. (1955).Amer. J. Anat. 96,255. Dapples, C. C., and King, R. C. (1970). 2. Zellforsch. Mikrosk. Anat. 103,34. Davidson, E. H. (1968). “Gene Activity in Early Development.” Academic Press, New York. Delbos, M., Gipouloux, J. D., and Camber, R. (1971). C . R. Acad. Sci., Ser. D 272, 2372. Descamps, M. (1971).Z . Zellforsch. Mikrosk. Anat. 121, 14. Dhainaut, A. (1970).J. Microsc. (Paris) 9, 99. Dhainaut, A., and Richard, A. (1972). C . R . Acad. Sci., Ser. D 275,417. Di Berardino, M. A. (1961).J. Embryol. Erp. Morphol. 9, 507. Droller, M. J., and Roth, T. F. (1966).]. Cell Biol. 28, 209. DuBois, A. M. (1932).J. Morphol. 54, 161. Dumont, J. N. (1969).J. Morphol. 129, 317. Eddy, E. M. (1970).Biol. Reprod. 2, 114. Eddy, E. M. (1974).Anat.Rec. 178,731. Eddy, E. M., and Clark, J. M. (1975).I n “Electron Microscopic Concepts of Secretion. The Ultrastructure of Endocrine and Reproductive Organs” (M. Hess, ed.), Chapter 9, pp. 151-167. Wiley (Interscience),New York. Eddy, E. M., and Ito, S. (1971).]. Cell Biol. 49, 90. Elpatievsky, W. (1909).Anat. Anz. 35, 226. Favard-SPrbno, C. (1968).J. Microsc. (Paris) 7, 205. Fawcett, D. W., and Ito, S. (1958).J. Biophys. Biochem. Cytol. 4, 135. Fawcett, D. W., Eddy, E. M., and Phillips, D. M. (1970). Biol. Reprod. 2, 129. Ficq, A., and Urbani, E. (1969). Exp. Cell Res. 55, 243. Fielding, C. (1967).J. Embryol. Erp. Morphol. 17, 375.

GERM PLASM

277

Follenius, E. (1965). C. R. Acad. Sci., Ser. D 261, 4849. Franchi, L. L., and Mandl, A. M. (1964).J. Embryo/. E x p . Morphol. 12, 289. Franchi, L. L., and Mandl, A. M. (1966). Proc. Roy. Soc., Ser. B 165, 136. Franke, W. W., and Scheer, U. (1970).J. Ultrastruct. Res. 30,317. Franquinet, R., and Lender, T. (1973). Z. Mikrosk. Anat. Forsch. 87,4. Gambrell. F. L. (1933).Ann. Entomol. Soc. Amer. 26, 641. Gatenby, J. B. (1917). Quart. J . Microsc. Sci. 62,407. Gatenby, J. B. (1918). Quart. J . Microsc. Sci. 63, 161. Gatenby, J. B. (1919a). Quart. J . Microsc. Sci. 63, 401. Gatenby, J. B. (1919b). Quart. J . Microsc. Sci. 63,445. Gatenby, J. B. (1920). Quart. J . Microsc. Sci. 64, 133. Gehring, W. J. (1973). In “Genetic Mechanisms of Development” (F. H. Ruddle, ed.), pp. 103-128. Academic Press, New York. Geigy, R. (1931). Rev. Suisse Zoo/. 38, 187. Gerin, Y. (1971).J. Embryol. Exp. Morphol. 25, 423. Geyer-Duszynska, I. (1959).J. E x p . Zool. 141, 391. Ghirardelli, E. (1953). Ptibbl. Stuz. Zool. Napoli 24, 332. Ghirardelli, E. (1954). Pubbl. Staz. Zool. Napoli 25, 444. Ghirardelli, E. (1965a). Z n “Regeneration in Animals and Related Problems” (V. Kiortsis and H. A. L. Trampusch, eds.), pp. 177-184. North-Holland Publ., Anisterdam. Ghirardelli, E. (1965b). I n “Regeneration in Animals and Related Problems” (V. Kiortsis and H. A. L. Trampusch, ecls.), pp. 177-184. North-Holland Publ., Amsterdam. Ghirardelli, E. (1966).Acta Med. Romano 4, 243. Ghirardelli, E. (1968).Aduan. Mar. B i d . 6, 271375. Gill, K. S. (1964).J. Ex),. 2001. 155, 91. Gipouloux, J. D. (1962). C. R. Acatl. Sci. 254, 2433. Gipouloux, J. D. (1971). C. R. Acad. Sci., Ser. D 273, 2627. Gondos, B., and Conner, L. A. (1973). Amer. J . Anat. 136,23. Condos, B., and Hobel, C. J. (1971).2. Zellforsch. Mikrosk. Anat. 119, 1. Condos, B., and Zemjanis, R. (1970).J. Morphol. 131,431. Condos, B., Renston, R. H., and Conner, L. A. (1973).Amer. J . Anat. 136,427. Greenfield, M. L. (1966).J. Etnbryol. E x p . Morplio/. 15, 297. Gunther, J. (1971).Zool. Jahrb., Abt. Anat. Ontog. Tiere 88, 1. Gurdon, J . B., and Woodland, H. H. (1968). Biol. Rev. Cambridge Phil. Soc. 43, 233. Haecker, V. (1897). Arch. Mikrosk. Anat. 49, 35. Harris, H. (1974). “Nucleus and Cytoplasm,” 2nd Ed. Oxford Univ. Press (Clarendon), London and New York. Harris, P. (1967). E x p . Cell Res. 21, 569. Hasper, M. (1911). Zool. Jnhrb.,Anut. Abt. Ontog., Tiere 31, 543. Hathaway, D. S., and Selman, C. C . (1961). J . E t i h - ! / d . E x p . Morphol. 9, 310. Hay, E. D. (l968a). In “The Nucleus” (A. J . Dalton and T. Haguenau, eds.), pp. 1-79. Academic Press, New York. Hay, E. D. (1968b).I n “The Stability o f t h e Differentiated State” (H. Ursprung, ed.), Springer-Verlag, Berlin and New York. Hegner, R. W. (1908). B i o l . Brill. 16, 19. Hegner, R. W. (1909a).J. Morphol. 20, 231. Hegner, R. W. (1909b).J. E x p Z o o / . 6, 507. Hegner, R. W. (1911). Biol. Bull. 20, 237. Hegner, H.W. (1914a).J. Morphol. 25, 375.

278

E. M. EDDY

Hegner, R. W. (1914b).A n d . Anz. 46, 51. Hegner, R. W. ( 1 9 1 4 ~ )“The . Germ Cell Cycle in Animals.” Macmillan, New York. Hegner, R. W. (1915).J. Morphol. 26, 495. Hertwig, 0. (1880).Jenu Ges. Med. Nutnrtoiss. 14, 196. Hinsch, C.. W. (1970).J. Cell Biol. 47, 531. Hinsch, G. W., and Cone, M. V. (1969).J. Cell B i d . 40, 336. Hope, J. (1965).J . Ultrustrzrct. Res. 12, 592. Howland, R. B. (1941). Proc. Amer. Phil. Soc. 84, 605. HSLI,W. S. (1962). Z . Zellforsch. Mikrosk. Anut. 58, 660. Huettner, A. F. (1923).J. Morphol. 37, 385. Illmensee, K., and Mahowald, A. P. (1973).J. Cell B i d . 59, 154a. Illmensee, K., and Mahowald, A. P. (1974). Proc. Nut. Acud. Sci. U.S. 71, 1016. Imaiznrni, T. (1958). Cytologiu 23, 286. Ito, S. (1960).J. B i o p h y s . Biochenr. Cytol. 7, 433. Iwanoff, P. P. (1928). Z . Moryihol. Oekol. Tiere 10, 62. Jazdowska-Zagrodzinska, B. (1966).J . Emhryol. Exp. Morphol. 16,391. Kahle, W. (1908). Zookigica (Stuttgurt) 21, 1. Kalt, M. R. (1973).Z. Zellforsch. Mikrosk. Anut. 138, 41. Kang, V.-H. (1974). Amer. J. Anut. 139, 535. Kessel, R. G. (1966a).J . Ultrustnict. Res. 15, 181. Kessel, R. G. (1966b).J. Ultrustruct. Res. 16, 305. Kessel, H. G. (1968a).J. Ultrustruct. Res. 22, 63. Kessel, H. G. (196813).Z . Zellforsch. Mikrosk. Anat. 89, 17. Kessel, R. G. (1969).J. Ultrustruct. Res. 28, 61. Kessel, R. G., and Beams, H. W. (1963).E x p . Cell Res. 32, 612. Kessel, R. G., and Beams, H. W. (1968).J. Cell Biol. 39, 735. Kessel, R. G., and Beams, H. W. (1969).J. Cell B i d . 42, 185. King, R. C. (1960). Growth 24,265. Koulish, S. (1965). Develop. Biol. 12, 248. Kuhn, A. (1912). Zoo/. Juhrb., A b t . Anut. Ontog. Tiere 35, 243. Lane, N . J. (1967).J. Cell Biol. 35, 421. Lanzavecchia, G. (1962). Electron Microsc., Proc. Int. Congr., 5th, Philudelphiu 2, WW-143. Lechenault, H. (1968). Z . Zdlforscli. Mikrosk. Anat. 90, 96. Le Moigne, A. (1966). C . R. Acud. Sci. 263, 550. Librera, E. (1964). Actu Embryo/. Exit. Morphol. 7, 217. Lima-de-Faria, A., and Moses, M. J. (1966).J. Cell Biol. 30, 177. Mahowald, A. P. (1962).J. E x p . Zool. 151, 201. Mahowald, A. P. (1968).J. E x p . Zool. 167, 237. Mahowald, A. P. (1970). I n “Results and Problems in Cell Differentiation. Vol. 2: Origin and Continuity of Cell Organelles” (J. Reinert and H. Ursprung, eds.), pp. 158-169. Springer-Verlag, Berlin and New York. Mahowald, A. P. (1971a).J. E q i . Zool. 176, 329. Mahowald, A. P. (1971b).J. E x p . Zool. 176, 345. Mahowald, A. P., and Hennen, S. (1971). Develop. Biol. 24, 37. Mahowald, A. P., and Strassheim, J . M. (1970).J. Cell Biol. 45, 306. Mahowald, A. P., and Tiefert, M. (1970). Wilhelm Roux’ Arch. Entruicklrrr~jisttlee~~. Orgunisnien 165, 8. Maillet, P. L., and Gouranton, J. (1965). C . R . Acud. Sci., Ser. D 261, 1417. Malaquin, A. (19244. C . R. Acud. Sci. 179, 1348.

GERM PLASM

279

Malaquin, A. (1924b).C. R. Acud. Sci. 179, 1636. Malaquin, A. (1925).C . R. Acud. Sci. 180, 324. Martin, F. (1914).Z. Wiss. Zool., A h . A 110, 419. Massover, W. H. (1968).J. Ultrustruct. Res. 22, 159. Merriam, R. W. (1962).J. Cell Biol. 12, 79. Metz, C. W. (1938).Amer. Nutur. 72, 485. Miller, 0. L. (1962).Electron Microsc., Proc. Znt. Congr., 5th, Philadelphia 2, “-8. Millonig, G., Bosco, M., and Giambertone, L. (1968).J. Exp. Zool. 169,293. Morita, M., Best, J. B., and Noel, J. (1969).J. Ultrustruct. Res. 27, 7. Muckerji, R. N. (1930).Proc. Roy. Soc., Ser. B 106, 131. Mulnard, J. (1947).C. R. Ass. Anat. 34e Reunion p. 393. Mulnard, J. (1950).Bull. C1. Sci., Acad. Roy. Belg. 36, 767. Nachtwey, R. (1925).Z. Wiss. Zool., A h . A 126, 239. Neaves, W. B. (1971).Anat. Rec. 170,285. Nicander, L., and Ploen, L. (1969).Z. Zellforsch. Mikrosk. Anat. 99, 221. Nicklas, R. B. (1959).Chromosoma 10, 301. Nieuwkoop, P. D., and Faber, J. (1956).“Normal Table of Xenopris laeois (Daudin).” North Holland-Publ., Amsterdam. Nieuwkoop, P. D., and Suminski, E. H. (1959).Arch. Anut. Microsc. Morphol. Exp. 48, Suppl., 189. Novi, A. M., and Saba, P. (1968).Z. Zellforsch. Mikrosk. Anut. 86, 313. Nussbaum, M. (1880).Arch. Mikrosk. Anut. 18, 1. Odor, D. L. (1960).J. Biophys. Biochem. Cytol. 7, 567. Odor, D. L. (1965).Amer. J . Anat. 116,493. Odor, D. L., and Blandail, R. J. (1969).Amer. J , Anat. 125, 177. Okada, M., Kleinman, F. A,, and Schneiderman, H. A. (1974).Deoelop. Biol. 37, 43. Ornstein, L. (1956).J. Biophys. Biochem. Cytol., s t c p p l . 2,351. Padoa, E. (1963).Motiit. Zool. Itul. 70-71, 238. Petit, J. (1973).J . Microsc. (Paris) 17, 41. Phillips, D. M. (1967).J. Cell Biol. 33, 73. Picheral, B. (1971).J. Microscop. (Paris) 12, 107. Picheral, B. (1972).Z. Zellforsch. Mikrosk. Anut. 131, 371. Pollister, A. W., Gettner, M., and Ward, R. (1954).Science 120, 789. Poulson, D. F. (1947).Proc. Nut. Acad. Sci. U.S.33, 182. Poulson, D. F., and Waterhouse, D. F. (1960).Aust. J . Biol. Sci. 13,541. Rabinowitz, M. (1941).J. Morphol. 69, 1. Rasmussen, S. W. (1973).Z. Zellforsch. Mikrosk. Anut. 140, 125. Reed, S. C., and Stanley, H. P. (1972).J. Ultrastruct. Res. 41, 277. Reger, J. R. (1970).J . Suhmicrosc. Cytol. 2, 1. Ritter, R. (1890).Z. Wiss. Zool., Abt. A 50, 408. Roosen-Runge, E. C., and Leik, J. (1968).Amer. J . Anat. 122,275. Sauzin, M. J. (1966).C. R. Acad. Sci., Ser. D 263,605. Sauzin, M. F. (1968).C. R. Acud. Sci., Ser. D 267, 1146. Scharrer, B., and Wurzelmann, S. (1969).Z. Zellforsch. Mikrosk. Anut. 96, 325. Schjeide, 0. A., McCandless, R. G . , and Munn, R. (1965).Nature (London) 205, 156. Schjeide, 0. A., Nicholls, T., and Graham, G. (1972). Z. Zellforsch. Mikrosk. Anut 129, 1. Schwalm, F. E., Simpson, R. S., and Bender, H. A. (1971). Wilhelm Roux’ Arch. Entwicklungsmech. Organisinen 166,205. Shinji, G. 0. (1919).J. Morphol. 33, 73.

280

E. M. EDDY

Silvestri, F. (1906).Ann. Roy. Scu. Super. Agr. Portici 1, 17. Silvestri, F. (1914). Anat. Anz. 47,45. Smith, L. D. (1966). Deuelop. Biol. 14, 330. Sonnenblick, B. P. (1950).In “Biology of Drosophila” (M. Demerec, ed.), pp. 6 2 1 6 7 . Wiley, New York. Spiegelman, M., and Bennett, D. (1973).]. Embryol. Exp. Morphol. 30, 97. Stevens, N. M. (191O).J. Morphol. 21, 279. Sud, B. N. (1961a). Quart. J . Microsc. Sci. 102, 495. Sud, B. N. (1961b). Quart. J . Microsc. Sci. 102, 51. Susi, F. R., and Clerniont, Y. (1970). Amer. J . Anat. 129, 177. Swift, H. (1956).J. Biophys. Biochem. Cytol., S t r p p l . 2, 415. Swift, H. (1965). Chromosome; Stnrct. Funct. Aspects, Symp., Miami, Flu. pp. 26-49. Szollosi, D. (1969).J . Cell Biol. 43, 143a. Szollosi, D., Calarco, P. G., and Donahue, R. P. (1972). Anat Rec. 174, 325. Takamoto, K. (1966).Nature (London) 211,772. Tanabe, K., and Kotani, M. (1974).]. Embryol. E x p Morphol. 31, 89. Tandler, B., and Hoppel, C. L. (1972). “Mitochondria,” pp. 1-59. Academic Press, New York. Tandler, B., and Moriber, L. G. (1965).2. Zellforsch. Mikrosk. Anat. 68, 301. Tannreuther, G. (1920). J . Morphol. 33, 389. Ullmann, S. L. (1965).J. Embryol. Exp. Morphol. 13, 73. Ulrich, E. (1969).J. Microsc. (Paris) 8, 447. Van Gansen, P., and Weber, A. (1972). Arch. Biol. 83, 215. Vasiljev, A. (1925). B i d . Gen. 1, 249. Verhey, C. A., and Moyer, F. H. (1967).J. Exp. 2001. 164, 195. Wartenberg, H. (1962). 2. Zellforsch. Mikrosk. Anat. 58, 427. Wartenberg, H., Holstein, A. F., and Vossmeyer, J. (1971). 2. Anat. Entwicklnngsgesch. 134, 165. Watson, M. L. (1952).J. Biophys. Biochem. Cytol. 4, 475. Watson, M. L., and Aldridge, W. G. (1961). J. Biophys. Biochem. Cytol. 11, 257. Weakley, B. S. (1967). 2. Zellforsch. Mikrosk. Anat. 83, 582. Weakley, B. S. (1969). 2. Zellforsch. Mikrosk. Anat. 101, 394. Weakley, B. S. (1971). 2. Zellforsch. Mikrosk. Anat. 112, 69. Wegnxin, I., and Gotting, K. J . (1971). 2. Zellforsch. Mikrosk. Anat. 119, 405. Weismann, A. (1892). “Das Keimplasm. Eline Theorie der Vererhurg.” Fischer, Jena. Williams, M. A,, and Smith, L. D. (1971). Deoelop. B i d . 25, 568. Wilson, E. B. (1896). “The Cell in Development and Inheritance.” Macmillan, New York. Wischnitzer, S. (1958).J.Ultrastruct. Res. 1,201. Wischnitzer, S. (1973). lnt. Reo. Cytol. 34, 1-48. Wolf, R. (1967). Wilhelin Rour’ Arch. Entwicklungsmech. Organismen 158,459. Wolf, R. (1969). Wilhelni Rorrx’ Arch. Entwicklungsmech. Organismen 163,40. Woods, F. H. (1932).J. Morphol. 53, 345. Yamamoto, K., and Onozato, H. (1965).Mem. Fac. Fish. Hokkaido Uniu. 13, 79. Yamamoto, M. (1964).]. Foe. Sci., Unio. Tokyo, Sect. 4 10, 335. Yasuzumi, G., Sugiska, T., Tsubo, I., Yasuzumi, F., and Matano, Y. (1970). 2. Zellforsch. Mikrosk. Anut. 110, 231. Zahnd, J. P., and Porte, A. (1966). C. R. Acad. Sci., Ser. D 262, 1977. Zalokar, M. (1973). Deoelop. Biol. 32, 189. Zamboni, L. (1970). Biol. Reprod., Suppl. 2, 44.

Gene Expression in Cultured Mammalian Cells RODY P. COX' AND JAMES

c. KING2

. . . . . . . I. General Introduction . . . 11. The Discovery of Gene Regulation in Bacteria . 111. Difficulties in Applying the Bacterial Model to Higher Forms . . . . . . . . . . . . IV. Differences between Prokaryotes and Eukaryotes . A. DNA and Chromosomes . . . . . . B. RNA . . . . . . . . . . C. Protein Synthesis . . . . . . . . D. Mitochondria . . . . . . . . . V. Regulation of Specific Protein Synthesis in Mammalian Cells . . . . . . . . . . . A. Determination and Epigenotype . . . . . B. Cell Functions in Vitro . . . . . . . C. Gene Expression in Synchronous Cell Cultures . . . VI. Differentiated Functions in Mammalian Cell Culture A. General Aspects . . . . . . . . B. Myogenesis in Culture . . . . . . . C. Established Cell Lines with Differentiated Functions . . . . D. Long-Term Lymphocyte Cultures. VII. Hormonal Effects on Gene Expression in Cultured Cells . A. General Aspects . . . . . . . B. Induction of TAT in Hepatoma Cell Cultures . C. Glutamine Synthetase Induction in Embryonic Retina D. Alkaline Phosphatase Induction in HeLa Cells . . E. Regulation of the Activity of Metalloenzymes by Hor. . . . . . . . . . mones VIII. Regulation of the Activity of Metalloenzymes by Agents Other than Hormones . . . . . . . A. Aryl Hydrocarbon Hydroxylase . . . . . B. Alcohol Dehydrogenase in Maize . . . . . . . IX . Regulation of the Rate of Protein Degradation . A. General Aspects . . . . . . . . B. Measurement of Protein Degradation in Cell Culture . . . X. Protein Modification Altering Gene Expression XI. Control of Enzyme Activity of Intact Cells-Role of Sub. . . . . . . strates and Cofactors . XII. Genetic Control of Intracellular Localization of Enzymes XIII. Gene Expression in Heterokaryons of Mammalian Cells . A. Cell Fusion . . . . . . . . . B. Complementation in Heterokaryons . . . . C. Gene Activity and Differentiation in Heterokaryons .

. .

.

282 283 284 285 286 290 292 293 293 293 294 307 308 308 310 311 312 313 313 314 317 318 328 329 329 330 331 331 332 333 334 335 336 336 337 339

Division of Human Genetics, Departments of Medicine and Pharmacology, New York University Medical Center, New York, New York. * Division of Human Genetics, Department of Microbiology, New York University Medical Center, New York, New York. 28 1

282

RODY P. COX AND JAMES C. KING XIV. Gene Expression in Synkaryons of Mammalian Cells A. Nuclear Fusion . . . . . . . B. Gene Expression in Synkaryons , , , . XV. Reconstruction of Mammalian Cells . . . . XVI. Conclusions . . . . . . . . . References . . . . . . . . .

.

.

. . . .

339 339 340 343 343 344

I. General Introduction Cell and tissue culture provides a simple system for investigating a variety of fundamental problems involved in gene expression. Cells can b e grown under relatively controlled environmental conditions that permit analysis of the physiology, biochemistry, and genetics of cells in culture. New methods of cell fusion and hybridization allow some aspects of recombination analysis in mammalian cell culture, and the recent development of methods for enucleating mammalian cells with the recovery of cytoplasm and nuclei separately permits reconstruction of cells by fusing nuclear and cytoplasmic components derived from different cell types. Mammalian cells grown in culture can be classified in several ways. Established cell lines can be continuously cultivated in vitro and are usually heteroploid. Cell strains are euploid and have a finite life-span in culture, which can b e related to the age of the donor (Hayflick, 1965; Martin et al., 1970). Cells in culture also can be classified into those that exhibit differentiated cell functions, for example, nerve or muscle cells, and those that are less specialized, for example, skin fibroblasts. The tissue of origin is also of importance; fibroblastic cell cultures can be derived from nearly all tissues but, until recently, most cells with special differentiated functions were derived from tumors (i.e., hepatomas, endocrine tumors, or melanomas). It is difficult to establish that any in vitro observation of cell function is an accurate reflection of the in vivo state, and some of the most compelling evidence is obtained from comparative studies on cell cultures derived from “normal” subjects and those with genetic diseases. This article emphasizes studies on mutant cell cultures, as well as examples of enzyme regulation and metabolic control that reflect gene expression in cultured mammalian cells. The literature on gene regulation in mammalian cell culture is too large and diffuse to permit an adequate general review, therefore we intend to present an overview of the current status of gene expression by analyzing several examples of well-studied systems using

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

283

mammalian cell culture. This approach necessitates selecting specific examples from among a large number of important observations. Before beginning our discussion of gene expression in mammalian cell culture, it seems appropriate to compare briefly regulation in those prokaryotic systems that are well understood to the emerging concepts of control in eukaryotic cells.

11. The Discovery of Gene Regulation in Bacteria Although the control of gene action or inaction is a process still imperfectly understood, almost all that we do know was discovered in the last 2 decades. The concept of mRNA was not developed until the late 1950s, and our knowledge of the mechanisms of protein synthesis came later. One of the most valuable contributions of Beadle’s (1945) one gene-one enzyme hypothesis was the clear distinction between the autocatalytic activity of the gene (replication) and its heterocatalysis (formation of a gene product). In earlier discussions these two processes were often regarded as merely different aspects of a single action, and this old way of thinking died slowly. It was not until the elucidation of the mechanism of protein synthesis in bacteria in 1961 (Brenner et al., 1961; Jacob and Monod, 1961) that gene action began to be understood at the molecular level. Rather suddenly at this time a clear picture of gene action in bacteria came into focus. The gene was composed of DNA existing in a Watson-Crick double helix. At one point in the cell cycle, this gene was replicated, semiconservatively, so that each new gene contained one strand from the original. In a process distinct from replication, the gene could be transcribed-its information transferred, nucleotide by nucleotide, to a molecule of mRNA which was then translated on a ribosome into a correspondingly specified polypeptide chain. At one end of the gene was an operator which could be blocked, thus repressing or turning off transcription, or opened or derepressed, thus allowing transcription to take place. When transcription occurred, translation followed. In Escherichia coli two types of gene control were recognized. A set of genes coding for enzymes involved in lactose metabolism was repressed, except when lactose or a compound of very similar structure was present in the medium. The presence of such a compound caused derepression of the gene and induction of the synthesis of the enzymes. When the inducing substance disappeared from the medium, the gene became repressed again. Another set coding for

284

RODY P. COX AND JAMES C. KING

enzymes necessary for the synthesis of tryptophan was transcribed in the absence of this amino acid. The resulting mRNA was translated into enzymes that synthesized the tryptophan necessary for growth. If, however, the medium contained tryptophan, the genes were repressed and neither mRNA nor tryptophan-synthesizing enzymes were produced until the exogenous tryptophan was exhausted. Then, in the absence of tryptophan the genes became derepressed and resumed production of mRNA and of the enzymes for synthesizing tryptophan. The picture was simple, elegant, and aesthetically satisfying-almost Panglossian. Enzymes of an anabolic pathway were produced constitutively and were turned off only when an exogenous supply of the metabolite made producing them unnecessary and wasteful. Enzymes of a catabolic pathway were naturally repressed, but were automatically turned on when the substance to be catabolized turned up in the environment. When the windfall was exhausted, the gene was rerepressed. It was inevitable that this beautiful system of gene regulation and control in bacteria should b e invoked as a model for explaining gene action and differentiation in metazoa. Exactly this was done by Monod and Jacob (1961) at the Cold Spring Harbor Symposium in 1961. In the same year Parker and Bearn (1961a,b), in two papers, discussed the application of the Jacob-Monod model to human inherited biochemical variants. In 1965, Tschudy et al. (1965), in describing elevated levels of 8-aminolevulinic acid synthetase as the biochemical abnormality responsible for acute intermittent porphyria, considered a constitutive operator mutation a possible mechanism for explaining the disease. 111. Difficulties in Applying the Bacterial Model to Higher Forms

Although the bacterial system provided an admirable base for constructing speculative models for explaining gene action in metazoa, experimental data supporting the speculations did not appear. It gradually became apparent that there were two very potent reasons why the understanding of gene action in higher forms was so slow in coming. One was the profound difference in the type of genetic analysis possible in bacteria and in higher plants and animals. The vast numbers of bacteria that can be grown and the effective methods for screening them as to phenotype provide an inexhaustible supply of

GENE EXPRESSION I N CULTURED MAMMALIAN CELLS

285

genetic variants. In the case of higher plants and animals, an entirely different technical situation obtains. Even though methods of cell culture make it possible to grow individual cells in numbers orders of magnitude greater than is possible for whole organisms, nevertheless, the methods available simply do not produce the mutants in the numbers and variety with which they appear in bacteria. Even in yeasts, the higher organisms whose behavior in culture most nearly resembles that of bacteria, no operator mutants, so well known in E . coli, have been identified. As a result, nothing like the fruitful interplay between genetic and biochemical analysis, so characteristic of bacterial and viral studies, is available to the investigators of higher forms. The second big block in understanding gene action in higher forms involves the fact that, while genetic information is coded in essentially the same way in all organisms, higher forms possess not only vastly greater numbers of bits than bacteria and their methods of storage and retrieval are so much more complex that their total functioning cannot be understood by a mere transfer of concepts based on knowledge of the operation of simpler organisms. This difference in the organization of genetic information is one of the fundamental taxonomic characters dividing simpler organisms or prokaryotes from higher organisms or eukaryotes. These terms derive from the presence of a nucleus bounded by a membrane within the cells of the latter and its absence in those of the former, but this distinction is diagnostic of more fundamental differences in the organization of the genetic material. Eukaryotes possess an elaborate mechanism-mitosis-which they employ for separating the two copies of the genetic material following its replication. In addition, the DNA of eukaryotes is found in complex structures, the chromosomes-which do not exist in prokaryotes. Furthermore, the DNA of eukaryotes is differentiated into sequences, some of which are repeated, and others of which are unique. This type of differentiation is not found in prokaryotes.

IV. Differences between Prokaryotes and Eukaryotes

The terms prokaryote and eukaryote date back to 1937 (Chatton, 1937), but the profound taxonomic and evolutionary significance of the differences between the two groups became evident only with the development of molecular biology. An article b y Stanier and Van Niel (1962) clarified the problem elegantly. It is worthwhile to re-

286

RODY P, COX AND JAMES C. KING

view the differences in some detail here, for only with a clear picture of these differences in mind can one begin to assess the possibilities of parallelism and homology of gene action in the two systems.

A. DNA AND CHROMOSOMES In bacteria genetic information exists in the linear extension of one double helix of DNA in a circular configuration. This very long loop is compacted by folding into a very small space. The degree to which the folding is patterned or random is not clear, but the resulting mass lies naked in intimate contact with the other constituents of the cell. Certain protein molecules-RNA polymerase, repressors, activating factors-associate noncovalently and dissociate from time to time at certain specific points on the DNA, and polymerases travel along it to effect transcription. But these associated proteins in no sense envelope the DNA. At one point there is a definite association with the cell membrane. Replication of the DNA seems to start here and to proceed in two directions around the circle until the two replicating forks join on the other side (Bird et al., 1972). Separation of the two genomes resulting from replication appears to be produced b y growth of the membrane between the two resulting attachment points (Korn and Thomas, 1971). As the bacterium goes through its cycle, some enzymes are synthesized continuously and others appear and disappear at certain times (Mitchison, 1969),but transcription and translation can occur during DNA replication, for in rapidly growing cells replication may be practically continuous. In the bacterium there appears to be no point in the cycle at which transcription is completely shut down. In eukaryotic cells the DNA, instead of consisting of a single circle, is partitioned into two or more portions, each portion corresponding to a chromosome. These may be roughly equal or very unequal in DNA content, but in the cells of a given species their number is the same, and every one has an individuality as to size, morphology at metaphase, and the genetic information it contains. All the chromosomes together carry the full informational complement. Although some base sequences in the DNA are repeated-some of them many thousands of times-no organism is normally viable without one complete set of chromosomes. Organisms with one set are termed haploid, but all mammals and the vast majority of higher animals and plants have two complete sets of chromosomes and are said to be diploid. The chromosomes of diploids are consequently present in pairs, the members of which are homologous as to size and informational content. Thus in a diploid genetic

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

287

information is present in two complete copies or versions, one having been present in each of the two gametes that fused to produce the individual. These versions may differ at certain loci; such differences constitute heterozygosity. To form the chromosome a substantial quantity of protein is intimately associated with the DNA. From eukaryotic cells the DNA and associated protein can be isolated without destroying the association. The isolate, known as chromatin, can be dissociated into DNA and two distinct types of protein. One of these, the histones, are characterized by the fact that they contain a large proportion of basic amino acids, arginine, and lysine. Histones from all eukaryotes can be separated into five classes of small monomers with molecular weights ranging from about 10,000 to 20,000, and the amino acid sequences of the monomers of corresponding classes show astonishingly little divergence even when extracted from distantly related organisms. The DNA and histone fractions of chromatin are approximately equal in weight and, although their precise structural relationships are unknown, there appears to be a repeating unit in which eight histone monomers, some of which are combined as dimers, are associated with every stretch of 200 nucleotides (Kornberg, 1974; Kornberg and Thomas, 1974). The histones were suggested as suppressors of gene action as early as 1950 (Stedman and Stedman), and some experiments in the early 1960s showed that chromatin deprived of histones fomented transcription and translation in a cell-free system of a specific protein which did not appear when undismantled chromatin was used (Huang and Bonner, 1962). But during the 1960s evidence rapidly accumulated that specific control of gene repression or expression was effected by another group of proteins found in chromatin, the nonhistones. These are rich in neither arginine nor lysine and are much more variable in size and composition than histones. Furthermore, nonhistones vary greatly in amount and composition from species to species, from tissue to tissue in the same organisms, and from one stage to another in the cell cycle. Thus the nonhistone portion of the chromosome is highly heterogeneous and is in constant flux, different components constantly being synthesized, added, subtracted, and degraded in continuous turnover (Stein et al., 1974). Chromatin that has been separated into its three components, DNA, histones, and nonhistones, can be reconstituted by bringing the three back together under the proper conditions. Stein and Farber extracted chromatin from mitotic HeLa cells, which show a very low rate of RNA synthesis, and from S-phase HeLa cells in

288

RODY P. COX A N D JAMES C. KING

which the rate of RNA synthesis is much higher. Histones and nonhistones were separated from both types of chromatin. Chromatin was then reconstituted using DNA from exponentially growing HeLa cells, pooled histones from both mitotic and S-phase cells, and nonhistones from either mitotic or S-phase cells. When these two reconstituted chromatins were subjected to a cell-free RNA-synthesizing system containing E . coli RNA polymerase, that containing the nonhistones from the S-phase cells showed the higher rate of template activity. When the nonhistones were pooled and chromatin was reconstituted using histones from either mitotic or S-phase cells, the two reconstituted chromatins showed no difference in template efficiency (Stein and Farber, 1972). This seems to indicate strongly that the template characteristics of chromatin are specified by nonhistone proteins, although the mechanism of action probably involves interaction among all three components. So the eukaryote chromosome, instead of being a snarl of naked DNA with some associated protein molecules here and there, is a highly organized structure composed of repeated units organized by the histones and further structured by coiling and folding at various levels of complexity by the attractive forces of the many nonhistones. The latter must include not merely repressors but also structural elements, enzymes of various sorts, not merely transcriptases but others involved in modifying, repairing, and degrading the various elements of the whole chromosome structure, and finally, recognition sites for regulatory macromolecules. The activity in the whole chromosome seems to be bound up with the phosphorylation and dephosphorylation of nonhistone proteins, and many component enzymes must be engaged in this function (Stein et a ] . , 1974). In addition to being more complex structures than bacterial DNA, eukaryotic chromosomes are provided with a separate compartment, the nucleus, set off from the rest of the cell by a membrane. Their structure and their segregation in the nucleus makes the process of chromosome replication a vastly more complicated one than replication of the bacterial genome. What triggers the start of replication in bacteria is obscure. Cells in an environment nutritionally unfavorable suspend replication, and when provided with fresh medium dally for some time before resuming. But during growth under optimal conditions, replication is a rapid process-it may be completed in less than ?hhour-with little inhibiting effect on general metabolism. Chromosome replication is different. Except during the early rapid cleavages of the embryo, after division, a eukaryotic cell in growing

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

289

tissue or in culture enters a period known as G, during which no DNA is synthesized. This may last for hours. It is terminated when some event which has not yet been identified triggers DNA synthesis and ushers in the S phase. During this second phase of the cycle, the DNA is replicated in each chromosome individually. Within each chromosome there are many starting points at which replication begins and proceeds in both directions until it reaches the point to which replication from the adjacent starting point extends (Callan, 1974). Whether or not replication starts at all points simultaneously, it does not finish everywhere at the same time. The segments associated with the different starting points are not equal in length, and often extended regions or whole chromosomes finish replicating later than others. When the DNA has been replicated-completely, accurately, and exactly once for every segment of every chromosome-the S phase is ended and the cell enters the third phase of the cycle, G z . In preparation for the division to follow, the chromosomes gradually contract to their densest and most individual configurations. During this time, to the extent that it was not done during replication, each half-new strand of DNA must be provided with a complete complement of proteins. The contracted chromosomes take their place in a plane known as the metaphase plate, the membrane defining the nucleus dissolves, and the members of the pairs of replicated chromosomes are pulled apart by the mitotic apparatus. At the approach of mitosis, the contracting chromosomes become inactive and transcription falls to a minimum. It begins again only after a new nucleus, complete with a reconstituted membrane, has formed around each of the two groups of chromosomes separated by mitosis. As the chromosomes begin to unfold, the daughter cells enter the G, of a new cycle. That this complicated cycle of eukaryotic cells has a powerful influence on gene expression is shown by the fact that inducing agents are often effective only during precise periods in the cycle. In synchronized rat hepatoma cell cultures, dexamethasone phosphate induces tyrosine aminotransaminase (TAT) (EC 2.6.1.5) but only when present during the latter part of GI or at any time during S. It is ineffective during M, G2, or early G, (Martin et al., 1969). In HeLa S3 cell cultures, hydrocortisone induces a higher level of alkaline phosphatase activity if present during the second half of GI or the first half of the S phase. It is without effect in late S, M, GP,or early G, (Cox, 1971). Even when divested of all its associated proteins, which determine the functionally dynamic structure of the chromosome, eukaryotic

290

RODY P. COX A N D JAMES C. KING

DNA can be distinguished from that of prokaryotes by the presence in the former of sequences which are repeated many many times. Such sequences are entirely lacking in prokaryotes. DNA from E . coli sheared to fragments of known mean length, dissociated by heat treatment, and then allowed to reassociate gives a smooth reassociation curve when percent reassociated is plotted against the log of the product of time and concentration (Cot). Eukaryotic DNA so treated, however, never gives a smooth curve, but one that breaks one or more times, indicating that some nucleotide sequences are repeated and others are unique (Britten and Kohne, 1968). Different organisms give different curves, depending on the number of copies of different repeated sequences and the proportion of total DNA involved in repetition. By using other methods in addition to the Cot curves, it has been possible to obtain estimates of the proportion, length, and distribution of the repeated and unique sequences. Such analysis has been carried out in greatest detail for an echinoderm (Strongylocentrotus purpuratus) and an amphibian (Xenopus laeuis), but the work done on other organisms, including mammals, fits together in one fairly consistent picture. About a quarter of the DNA reassociates very rapidly. This includes identical sequences present in great numbers, some of which occur in clusters. About half the DNA is composed of repetitive sequences averaging 300 nucleotides in length, present in sets of somewhat smaller numbers and not so perfectly matched within the sets, interspersed with unique sequences of from 700 to 1100 nucleotides. The remaining quarter differs from the middle half primarily in having much longer unique sequences, of at least 4000 nucleotides (Davidsori and Britten, 1973). RNA extracted from polysomes, which must be in the process of translation, hybridizes only with unique sequences, showing that these must correspond to structural genes and that a structural gene as a rule is present in a single copy. The information in repetitive sequences appears not to be destined for translation into protein; so some kind of regulatory function seems most likely. But in spite of elaborate and ingenious proposed modeIs (Davidson and Britten, 1973; Crick, 1971; Tsanev and Sendov, 1971; Wallace and Kass, 1974), a conclusive demonstration of how these sequences regulate has yet to be presented. B. RNA In many ways the RNAs of prokaryotes and eukaryotes are remarkably similar. Both originate through transciption of DNA. Both exist in three functionally different fonns: mRNA which contains in-

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

291

formation for synthesizing specific proteins; rRNA, one of the principal components of the ribosomes which travel along the mRNA in the process of protein synthesis; and tRNAs, smaller molecules which bring amino acids to the points called for by the mRNA as the peptide chain is formed. So similar are these molecules functionally, that in a cell-free system eukaryotic mRNA can be translated b y ribosomes and tRNAs derived from a prokaryote-E. coli, for example. But there are also differences between the rRNAs of the two types of organisms. One clear-cut difference is in the morphology of the ribosomes. Those of eukaryotes are larger and heavier and can be clearly distinguished. But there are other differences which are probably more important with respect to gene action, and these are found in the way the RNAs are synthesized and processed. In prokaryotes, in which the DNA is not separated from other cellular components, newly synthesized RNA in immediately available to ribosomes and translation begins at one end of the molecule as transcription is still proceeding toward the other. In eukaryotes, however, transcription occurs in the intact nucleus, and the resulting mRNA is unavailable to ribosomes until it has been transported through the nuclear membrane into the cytoplasm (Miller et al., 1971). Nonribosomal and non-tRNA extracted from the eukaryote nucleus is heterogeneous in length and consists of few identical sequences. This is known as heterogeneous nuclear RNA (HnRNA) and differs from mRNA isolated from polysomes in having much greater average length. Within the nucleus HnRNA turns over very rapidly. Much of it therefore is never exported from the nucleus and never becomes mRNA. This strongly suggests regulatory processing of the nuclear products of transcription before they are ready for translation. Molecules of mRNA from the polysomes consist of three parts: beginning at the 5’ end, (1) a short section-fewer than 100 nucleotides-transcribed from a repeated sequence, (2) a segment variable in length but of at least several hundred nucleotides transcribed from a unique sequence, and ( 3 ) at the 3’ end a sequence of about 200 nucleotides all of which are adenylic acid (Dina et al., 1974). This poly A is added to the 3‘ e n d of the molecule after transcription, and it decreases in length with the time that the mRNA molecule remains in the cytoplasm. Whether the HnRNA becomes mRNA merely by selective degradation-a process that must occur to account for the high nuclear turnover-or whether some very long molecules of HnRNA contain two tandem transcripts of unique sequences which are separated by cleavage is not clear, but there is no doubt that the metamorphosis of HnRNA to mRNA provides an

292

RODY P. COX A N D JAMES C. KING

opportunity for informational processing which prokaryotes do not enjoy (Darnel1 et al., 1973).

C . PROTEINSYNTHESIS Although the process of linking amino acids into polypeptides is so similar in prokaryotes and eukaryotes that cell-free systems from either may synthesize protein on mRNA derived from the other, the intact eukaryotic cell is so constructed that it has greater possibilities for programming when and where translation will occur and what will be the fate of the products. Ribosomes do not exist within the nucleus, so in order to be translated mRNA must cross the nuclear membrane. What is known of the synthesis of histones clearly shows the potentiality of eukaryotes for controls of greater complexity. During the S phase of the cell cycle, histones are built into the chromatids as the D N A is replicated. They are synthesized on ribosomes in the cytoplasm. The mRNA is transcribed in the nucleus, crosses the nuclear membrane, and is translated; the resulting polypeptide recrosses the membrane into the nucleus. No poly-A segment has ever been found on a histone mRNA. All other mRNA in polysomes is so provided. The precise significance of these facts is not understood, but it is hard to believe that they are not related to the regulation of the synthesis and transport of these highly specialized polypeptides (Darnell et al., 1973). Another mechanism involved in the control of translation derives from the deployment of eukaryotic ribosomes. Some lie free in the cytoplasm; others are attached to the membrane of the endoplasmic reticulum. These two classes of ribosomes translate polypeptides having different functions. The free ribosomes translate proteins necessary for intracellular growth and metabolism. Those bound to the membrane synthesize proteins destined for secretion, for segregation in vesicles, or for incorporation into the membrane (Ganoza and Williams, 1969; Redman and Sabatini, 1966; Chua et al., 1973). Again, the detailed functioning of this system is still a mystery but, clearly, ways exist to ensure that an mRNA molecule is transported to the proper ribosome for translation, and very likely the ribosomes are more precisely classified than as merely free or bound. Even before the discovery of mRNA in bacteria, it has been shown that in an alga protein synthesis under nuclear control could continue for days after excision of the nucleus (Brachet et al., 1955). Later experiments with erythroid cells (Marks and Kovach, 1966) and with calf lens cells (Papaconstaninou, 1967) strongly suggested that mammalian mRNA could persist in the cell in functional form much

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

293

longer than mRNA in bacteria. Work with developing embryos (Gross and Cousineau, 1964) indicated that eggs contained mRNA transcribed in the maternal ovary, which remained quiescent until fertilization and then directed translation. Spirin (1966) suggested the name informasome for such "masked" mRNA. Recently, Gross et al. (1973) isolated such messenger from unfertilized sea urchin eggs and showed that in a cell-free system it synthesizes histones indistinguishable from those produced in vivo. All this indicated that eukaryotic mRNA is not short-lived, like that of prokaryotes, and that it may sometimes remain inactive in a cell for an indefinite period and later become active in protein synthesis, although the methods employed for its inactivation and reactivation have not yet been elucidated. D.

MITOCHONDRIA

Finally, another way in which eukaryotes differ from prokaryotes is in having included in their cytoplasm two types of organelles which are to a certain degree genetically autonomous. These are the plastids and the mitochondria. The former occur only in plants, but the latter are indispensable to all eukaryote cells as the seat of oxidative phosphorylation, hence the source of available energy. They defy the generalization that genetic information is confined to the nucleus, for they contain DNA and their own ribosomes which differ from those in the cytoplasm. It is therefore possible for an error of metabolism to arise from an informational defect in the mitochondrial DNA (see Section XV). Nevertheless, many of the enzymes found to b e integrated into membranes within the mitochondria are proteins defined in the nuclear DNA and translated in the cytoplasm on mRNA of nuclear origin. I n fact, about 90% of the protein mass of the mitochondria is of nuclear definition. The intimate coordination that must exist between mitochondrial and cytoplasmic protein synthesis and thus be part of the control of gene expression is simply not understood (see Schatz and Mason, 1974, for a recent review).

V. Regulation of Specific Protein Synthesis in Mammalian Cells A. DETERMINATION AND EPIGENOTYPE

The morphological characteristics, functional capacities, and metabolic potentials of cells are determined by the kind and quantities of proteins they synthesize. The regulation of gene expression is the fundamental process that controls the array of proteins made by a

294

RODY P. COX AND JAMES C. KING

particular cell. The nuclei of all cells of higher animals contain the complete genetic information of the species, and the commitment of a cell to a specific differentiated function is called determination. Whether a cell exhibits the differentiated phenotype frequently depends on environmental conditions or on the milieu. The term epigenotype has recently been proposed by Abercrombie (1967) to designate that part of the genome that under appropriate conditions can be expressed by a particular differentiated cell. Thus cells with different determinations have different epigenotypes. Studies of nuclear transplantation (Gurdon, 1970) clearly show that the epigenotype is sometimes reversible in that amphibian somatic nuclei support the normal development of enucleated eggs. Therefore the epigenotype is probably a result of different regulatory states rather than a genetic alteration or mutation. The mechanism leading to selective expression of different parts of the genome is not known, although several interesting hypotheses have been proposed recently (Britten and Davidson, 1969; Tsanev and Sendov, 1971). B. CELL FUNCTIONS in Vitro Ephrussi (1972) has emphasized that cell functions can be divided into ubiquitous functions, that is, metabolic functions essential for maintenance and growth of any cell, and differentiated functions which are necessary for survival of multicellular organisms but not for that cell. H e has called the gene products performing these two sorts of functions “household items” and “luxury items,” respectively. In cultured cells the stability of differentiated or luxury functions has been the exception, however, recent improvements of cell culture techniques and the use of neoplastic cells derived from tumors with differentiated function provide cultures that can be propagated without losing the differentiated functions characteristic of the tissue of origin.

1. Adaptations to Cell Culture a. Nutrition. Most cells in culture have similar nutritional requirements (Eagle, 1960, 1965; Rothblat and Cristofalo, 1972a). Only 28 growth factors appear to be required for the sustained propagation of mammalian cells in culture (Eagle, 1965). One poorly defined factor is required b y nearly all animal cells in culture, and that is serum protein or a factor derived from serum protein. The active component is probably a small molecule, either a polypeptide or a substance bound to protein. The nutritional requirement of cells de-

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

295

pends on cell density and is probably related to Ieakage of Iimited metabolites from cells at low population densities. For example, serine is required for the efficient cloning of cells at a density of less than 10 to 100 per millimeter (Lockart and Eagle, 1959). At higher population densities the cells can maintain the concentrations of serine necessary to achieve growth. There are several other examples of population density-dependent growth requirements (Eagle, 1965). b. Enzymic Alterations in Adaptation to Cell Culture. The adaptation of cells to culture has been associated with alterations in the enzymic profile, which presumably reflect alterations in gene expression. Lieberman and Ove (1958)determined the relative activities of a series of enzymes in four putatively different established cell lines. They found the specific activity of each of the 13 different enzymes appeared to be nearly identical among these cell lines, and that they differed strikingly from the enzyme activities of the tissues from which each line was derived. Since contamination of primary cell cultures with established cell lines, for example, L cells or HeLa cells, was commonly not recognized at that time, and the enzyme compositions reported resemble those of L cells, this report must be viewed with some skepticism. Burlington (1959) also reported alterations in the enzyme pattern of primary cultures of cat kidney cells compared to trypsinized kidney cell suspensions. In unpublished experiments, R, P. Cox carried out a similar study using human kidney and as a enzymic marker the constitutive alkaline phosphatase (AP) (EC 3.1.3.1) content of trypsinized cell suspension and primary cultures. The purpose was to determine if alterations in the enzyme pattern of primary cultures signify a population shift, with the selection of a minority cell type or a repatterning of enzyme activities within individual cells. In human kidney high constitutive AP activity is limited to the proximal tubule cell. In cell suspensions derived from trypsinized kidney, the AP specific activity is high. However, when these cells are inoculated into culture, the enzyme activity is lower in cells that adhere to the substratum. The cells that remain floating in the medium have high AP activity. It seems probable that proximal tubule cells do not adhere to glass or grow in culture. This finding suggests selection as the basis for low AP activity in primary human renal cell cultures rather than altered gene expression in culture. However, in other instances the selection hypothesis may not be adequate to explain the experimental findings. Ebner et al. (1961) studied primary monolayer cultures of bovine mammary gland and observed a series of biosynthetic and enzymic alterations

296

RODY P. COX A N D JAMES C. KING

related to the length of time in culture. Lactose production declined precipitously, while the capacity to synthesize p-lactoglobulin fell more slowly. This differential loss of individual functions suggests a modulation of gene expression rather than a cell population selection. It seems clear that a definitive choice between gene modulation and cell selection cannot be made at this time, and it is probable that under different circumstances each can occur (Harris, 1964).

2. Enzymic and Metabolic Characteristics of Cells in Culture Mammalian cells in culture exhibit polymorphism for a variety of different enzymes. The isozyme patterns of some cultured cells have shown no alteration from the intact tissue from which they were derived with respect to esterase, lactate dehydrogenase (LDH), and phosphoglucomutase (Paul and Fottrell, 1961; Davidson et al., 1965; Spencer et al., 1964). However, skin fibroblasts in culture showed a leucine aminopeptidase component of intermediate mobility not present in human skin (Beckman et al., 1967). The process of in vitro cultivation apparently changes the isozyme pattern for this marker. The new isozyme of leucine aminopeptidase is different from the isozymes found in a variety of adult organs, and raises the possibility that this may be a fetal form of the enzyme. Walker et al. (1972) showed that in cultures of adult liver cells the isozyme pattern for hexokinase, aldolase, and pyruvate kinase shifts to the fetal forms of the enzymes. Thus cells from adult liver in culture resemble fetal liver. Elson and Cox (1969) showed that the AP produced by HeLa cells differed in its chemical and physical properties from the enzymes found in adult organs and tissues, but closely resembled the fetal form of the enzyme found in human placenta. Human placental AP exhibits genetic polymorphism which is determined by the genotype of the fetus, and this enzyme is the product of a single genetic locus (Robson and Harris, 1965). There are several common alleles at the placental AP locus, and conveniently HeLa cells are heterozygous for the F and S alleles. Thus HeLa cell AP has a distinctive triplebanded pattern when electrophoresis is carried out by the methods of Robson and Harris (1965). Immunochemical studies also show the near identity of HeLa and placental enzyme (Elson and Cox, 1969; Ghosh and Cox, 1975). It is noteworthy that, after many years of culture, HeLa cells retain a heterozygous expression for AP. This suggests that during the process of heteroploidy, at least for this pair of alleles, there has been no loss of genetic information. It is not known whether the production of a fetal form of AP by HeLa cells represents an activation by the tissue culture environment of normally repressed genes, or whether the neoplastic cervical cells that

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

297

gave rise to HeLa cells express the placental AP phenotype. Normal human cervix does not contain a fetal form of AP (Elson and Cox, 1969). It should be noted that there are distinctive differences among various cell lines and strains in the physical and chemical properties of their APs (Cox and Griffin, 1967). Fibroblast cultures derived from fetuses retain the expression of their original developmental state. Condon et al. (1971) established diploid fibroblast cell strains from the skin of four fetuses of 3 months’ gestation and from children and adults. Fetal cell cultures exhibited enhanced activity of the hexose monophosphate shunt when compared to cultures prepared from children or adults. The differences in glucose metabolism in fetal cells in culture may be due to developmental characteristics of certain enzymes. Thus, with respect to carbohydrate metabolism, cell culture systems may be useful in extending our understanding of the biochemistry of fetal development.

3. Gene Expression in Cultured Cells Derived from Human Biochemical Mutants a. Fidelity of Gene Expression in Culture. Diploid fibroblastic cell strains can be derived from tissue (usually skin) of patients with inborn errors of metabolism, and these cells frequently exhibit the enzyme deficiency in culture (Krooth and Weinberg, 1960; Krooth and Sell, 1970; Mellman, 1973). It should be appreciated that only a fraction of inherited biochemical disorders express themselves in fibroblast cultures. Many that do not, involve enzymes with restricted or tissue-specific localization. For example, the enzyme defect in phenylketonuria (PKU) is not expressed in fibroblasts, since phenylalanine hydroxylase-the deficient enzyme-is normally found only in liver and kidney. A valid in vitro model requires the culture of liver cells, unless a method is found to “turn on” the gene responsible for PKU in fibroblast cultures. Such a possibility is under investigation (see Section XIV,B). The consistency of the quantitative levels of gene expression in cultured mammalian cells is well illustrated by studies on enzyme levels in fibroblast cultures derived from patients with classic forms of inborn errors and variant forms of these diseases. Maple syrup urine disease (MSUD) is the result of a deficiency in the activity of branched-chain a-keto acid (BCKA) decarboxylase, an enzyme required for the degradation of three branched-chain amino acids, leucine, isoleucine, and valine. I n the classic form of this disorder, symptoms, neurological signs, and a maple syrup odor of the urine

298

RODY P. COX AND JAMES C . KING

appear within the first week of life. Without treatment, death commonly ensues within a few weeks. Variant forms of this disease show a delayed onset of symptoms, and in some patients they appear only during times of stress, for example, infection. Neurological damage is not so severe or is absent in the variants. Both classic and variant forms of the disease involve a deficiency in BCKA decarboxylase activity. Differences in the severity of the disease are related to differences in tolerance for branched-chain amino acids in the diet and probably reflect differences in residual BCKA decarboxylase activity. Dancis et al. (1972) measured BCKA decarboxylase activity in skin fibroblast cultures derived from six classic and six variant forms of MSUD (Fig. 1). The level of residual decarboxylase activity exhibited in fibroblast cultures correlated precisely with the clinical phenotype and was predictive of the dietary tolerance for branchedchain amino acids. The enzyme levels presented in Fig. 1 are given as a percentage of the means of control subjects. Each point represents the average of at least two separate determinations and, with the exception of subject U.F., the results were quite consistent. The constancy of gene expression in cultured cells is exemplified by the

j

CaCu 9 WaBi

9

MeCad

G,d

I

lsoleucine

0 Valine

I

&1 &/

EmTa 9

I

ThAd d

MiAd d NiRo

9

ElEd

p

UnFa 9 l 0

~

~ 5

~

l ~ t l 10 15 Enzyme activlly 1% of normall

t

~

l

~

~

~

~

FIG. 1. Decarboxylase activity in MSUD as percent of normal. The mean ratios in the control subjects are considered 100%.Brackets indicate sibling pairs. [Reproduced with permission from Dancis et al. (1972).]

l

l

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

299

finding that the maximum deviation from the average of replicate determinations at different times was less than 1% for 9 of the 12 subjects. The genetic control for expression of this enzyme in cell culture is further emphasized by the similarity of results in cells derived from siblings with MSUD, who can be assumed to have the same alleles at the locus. Subjects M.C. and K.C. have barely detectable decarboxylase activity, whereas the brothers T.A. and M.A. have levels about 4% of normal. Genetic heterogeneity within this interesting inborn error of metabolism is further discussed in Section XII1,B. The fidelity of gene expression is less constant in differentiated cell cultures. For example, Peterson (1974) found that various clonal lines of rat hepatoma cells have widely varying production of albumin. This variability is not random but is clustered around certain distinct values which fit a geometric progression. Moreover a survey of 23 of 28 enzymes in hepatoma cells shows a similar distribution. Clonal lines of myeloma cells also show phenotypic variability (Coffino and Scharff, 1971). This variability is unlikely to be the result of mutation, since it occurs with a frequency of per cell generation. Moreover, studies on the effect of ploidy on rate of mutation in hamsters (Harris, 1971) and frogs (Mezger-Freed, 1972) are also incompatible with a mutational mechanism. Taken together, these results suggest a regulatory mechanism operative in certain mammalian cells, which modulates certain luxury functions. These regulatory mechanisms are discussed in Section XIV,B. b. Enzyme Induction in Inborn Errors of Metabolism. Table I lists the human biochemical defects expressed in skin fibroblast cultures. These enzymic deficiencies provide important markers for studying gene expression in culture. For example, hereditary orotic aciduria is a rare recessive disorder characterized by megaloblastic anemia and developmental retardation. This disorder is associated with deficient activity of two enzymes that catalyze sequential steps in the de nouo biosynthesis of uridine 5’-monophosphate (UMP) (see Table I). The enzymes affected are orotidine 5’-monophosphate (OMP) pyrophosphorylase and OMP decarboxylase (Pinsky and Krooth, 1967a). Diploid fibroblasts derived from homozygous mutant subjects have about 1% of normal activity for both enzymes. When mutant cells are grown in a medium containing either 5-azaorotic acid (a competitive inhibitor of OMP pyrophosphorylase) or 5azauridine (an inhibitor of OMP decarboxylase), the mutant cells develop near-normal activity for both enzymes (Pinsky and Krooth, 1967a,b). Barbituric acid, which is not an inhibitor of either

TABLE I HUMANBIOCHEMICAL DISORDERSEXPRESSEDIN SKIN FIBROBLAST CULTURES ~~

Disorder Amino acid metabolism Maple syrup urine disease

Enzyme defect

References

Dancis et al. (1972)

Argininosuccinic aciduria Citrullinemia

Branched-chain amino acid decarboxylase Lysine-ketoglutarate reductase Argininosuccinase Arginosuccinate synthetase

Homocystinuria

Cystathionine synthetase

Methylmalonic acidemia Ketotic hyperglycinemia Cystinosis

Methylmalonyl CoA reductase Propionyl CoA carboxylase Cystine transport or metabolism

Morrow et al. (1969) Hsia et al. (1971) Schneider et ul. (1967)

Galactose-1-PO, uridyl trans feras e Galactokinase

Tedesco and Mellman (1969) Monteleone et al. (1971)

u-1,4-Glucosidase (lysosomal) a-1,6-Glucosidase (debrancher) a-1,6-Glucan, a-1,4-glucan, 6-glycosyl transferase (brancher) Glucose-6-P04 dehydrogenase (GB-PD)

Cox et al. (1970) Justice et al. (1970)

Phosphohexose isomerase

Krone et al. (1970)

a-L-Iduronidase Sulfoiduronate sulfatase Heparin sulfate sulfatase N-Acetyl-a-glucosaminidase a-L-Iduronidase

Neufeld (1974)

Hyper1ysinemia

Dancis et al. (1969) Shih et al. (1969) Tedesco and Mellman (1967) Uhlendorf and Mudd

(1968)

Carbohydrate metabolism Galactosemia, classic Galactosemia, kinase type Glycogen storage Type I1 Type I11 Type IV

Nonspherocytic hemolytic anemia (Mediterranean form) Nonspherocytic hemolytic anemia Mucopol ysaccharidoses Hurler Hunter Sanfilippo A Sanfilippo B Scheie Maroteaux Lamy Atypical I-cell disease Generalizcd gangliosidosis Krabbe's disease Tay-Sachs disease Sandhoff's disease

Howell et a / . (1971) Nitowsky et al. (1965)

? P-Glucuronidase Enzyme that adds carbohydrate moiety to proteins P-Galactosidase Galactocerebroside P-galactos i das e Hexosaminidase A Hexosaminidase A and B

Neufeld (1974) Pinsky et al. (1970) Suzuki and Suzuki (1971) Okada et al. (1971) Okada et al. (1971)

GENE EXPRESSION IN CULTURElD MAMMALIAN CELLS

301

TABLE I (Continued) Disorder Gaucher’s disease Fabry’s disease Lactos ylceramidosis Fucosidosis Lipid Metabolism Niemann-Pick disease Refsum’s disease Metachromatic leukodystrophy Wolman’s disease Familial hypercholesterolemia Nucleic acid metabolism Lesch-Nyhan syndrome Orotic aciduria

Enzyme defect

References

Glucocerebrosidase Ceramidetrihexosidase Lactosylceramide galactosylhydrolase a-I-Fucosidase

Beutler et al. (1971) Brady et al. (1971) Dawson and Stein (1970) Patel et al. (1972)

Sphingomyelinase Phytanic acid oxidase Arylsulfatase A

Sloan et al. (1969) Herndon et al. (1969) Porter et al. (1969)

Acid lipase

Patrick and Lake (1969) Brown and Goldstein (1974)

Deficiency of cell surface receptor for LDL

Hypoxanthine phosphoribosyl Seegmiller et al. transferase (1967) Orotidylic pyrophosphorylase Pinsky and Krooth and decarboxylase (19674 Excision of thymine dimers Cleaver (1970)

Xeroderma pigmentosum Miscellaneous Acatalasia Catalase Acid phosphatase Acid phosphatase deficiency Congenital errythropoietic Uroporphyrinogen 111 porph yria synthetase

Krooth et al. (1962) Nadler and Egan (1970) Romeo et al. (1970)

enzyme, also causes mutant cells to develop near-normal activity for the deficient enzymes. A third enzyme dihydroorotase, which catalyzes the reaction preceding the enzymic deficiency, is not deficient in mutant cells, nor is its activity affected b y the agents described above (Wuu and Krooth, 1968). It is not yet clear how these agents alter enzyme activity. There is some evidence that the increased activity is due to increased enzyme within the cells (Pinsky and Krooth, 1967a).It has been suggested that the accumulation of precursors of UMP, for example, dihydroorotic acid, may in some ways activate mutant genes or a second set of normally dormant loci needed for UMP synthesis. In any case this system exemplifies the challenges and opportunities for studying gene expression in biochemically deficient cells. c. Expression of Dominant Traits in Cell Culture. Recent studies on fibroblast cultures from patients with familial hypercholes-

302

RODY P. COX AND JAMES C. KING

terolemia have elucidated one possible mechanism for the effect of a dominant disorder in man. Nearly 500 different dominantly inherited mutations are known to cause disease in man, but most of them have morphological or behavioral expression and very little is known about the biochemical mechanisms b y which these mutant genes act. Brown and Goldstein (1974), in a series of experiments, developed a cell culture system for the study of familial hypercholesterolemia, a dominantly inherited disorder which causes an increase in blood cholesterol and the development of arteriosclerotic disease. This system has provided evidence that the mutation involves a genetic abnormality in a regulatory protein which in the heterozygote is reduced to a 50% deficiency. Fibroblasts cultures of normal subjects show the rate of cholesterol synthesis is regulated by the presence of low-density lipoproteins (LDL) in the culture medium. I n order to suppress cholesterol synthesis, LDL must first bind to a specific highaffinity receptor on the cell surface, and this binding inhibits the synthesis of 3-hydroxyl-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) the rate-controlling enzyme in cholesterol biosynthesis. The surface receptor for LDL can be measured by the extent of radioactive LDL binding and by the suppression of HMG CoA reductase activity. In addition, the receptor also regulates LDL degradation. In cell cultures from heterozygotes for the mutant gene, there is a 60% reduction in the number of LDL receptors. The deficiency of cell receptors in heterozygous cells results in a defective concentration-dependent regulation of cholesterol synthesis. Attainment of equal rates of cholesterol synthesis and LDL degradation in normal and heterozygous cultures requires a two to threefold higher concentration of LDL in the heterozygote. It is noteworthy that these studies in cell culture elucidated the mechanism of gene expression in familial hypercholesterolemia and also provided insight into the pathogenetic mechanism of this form of dominant inheritance, which may well be applicable to other diseases (Brown and Goldstein, 1974). d. Cell Communication in Culture. The importance of mammalian cell culture in understanding gene expression is further documented by studies on the Lesch-Nyhan syndrome. The critical biochemical lesion is overproduction of uric acid, and this is associated with severe retardation, spasticity, and compulsive self-mutilation. Seegmiller et al. (1967) cultured skin from normals and from Lesch-Nyhan subjects and then studied the inhibition of purine synthesis by 6-mercaptopurine (6-MP). They found that the drug blocked purine biosynthesis in normal cells, but not in those from

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

303

Lesch-Nyhan patients. This was a key observation, since it was known the 6-MP must form the ribonucleotide prior to developing the inhibiting activity. The enzyme responsible for forming the ribonucleotide of 6-MP was hypoxanthine phosphoribosyl transferase (HPRT), an enzyme that functions to “salvage” hypoxanthine and guanine by converting them to the ribonucleotides inosinic acid and guananylic acid. Direct measurement of HPRT activity in Lesch-Nyhan cells clearly demonstrated that it was deficient. Thus cell culture studies elucidated the biochemical defect in this disease. The mutant phenotype of Lesch-Nyhan cells can be detected at the cellular level by radioautographic studies. Normal cells (HPRT+) incorporate large amounts of radioactivity intracellularly (Fig. 2b) when they are grown in a medium containing tritiated hypoxanthine or guanine, while Lesch-Nyhan cells (HPRT-) incorporate little or no label under similar conditions (Fig. 2a). However, when Lesch-Nyhan cells are mixed with equal numbers of normal human cells, grown to confluency and labeled with hyp~xanthine-~H, nearly all cells incorporate label (Fig. 3c). Cell contact is apparently required, since HPRT- cells grown close to, but not in contact with, normal cells do not incorporate hyp~xanthine-~H. This correction of the mutant (HPRT-) phenotype by contact with normal cells was thought possibly to be due to an alteration in the expression of the mutant HPRT gene in Lesch-Nyhan cells. It was hypothesized that a regulatory molecule might be passed from normal to mutant cell by

FIG. 2. Metabolic cooperation between normal hum;in fibroblasts and cells from patients with the Lesch-Nyhan syndrome. Radioautographs of human skin fibroblast monolayer cultures incubated for 3 hours with 100 pCi/ml of hyp~xanthine-~H at 37°C. (a) Lesch-Nyhan fibroblasts; (b) normal fibroblasts; (c) 1 : 1 mixture of cocultured Lesch-Nyhan and normal fibroblasts, showing correction of the mutant phenotype. [Reproduced with permission of Wiley Interscience Press from “Cell Communication,” edited by R. P. Cox (1974).]

304

RODY P. COX AND JAMES C. KING

Inducer removed 60

0 c

0

5

I

I

I

10

15

20

Ttme (hours)

FIG.3. Induction of TAT in HTC cells by dexaniethasone phosphate. A culture of HTC cells, grown to a density of about 8 x 105 cells/ml, was resuspended in fresh medium and divided into two portions. To the first portion dexamethasone phosphate, M . The a synthetic adrenal steroid, was added to a final concentration of 5 x other portion was used as a control. Enzyme activity was assayed and is expressed as milliunits of enzyme per milligram of cell protein. [Reproduced with permission from Tomkins et u1. (1969). Copyright 1969 by the American Association for the Advancement of Science.]

contact and that this regulatory molecule might activate the HPRT locus. Other possibilities included endowment of the mutant cell with the ability to synthesize functional HPRT by the transfer of episomal DNA or informational RNA from normal to mutant cell. It was also considered possible that normal HPRT+ cells provide HPRT- mutant cells with preformed enzyme. A final possibility was that normal cells, incubated with tritiated hypoxanthine, synthesize radioactive nucleotides or their derivatives which are then transferred to mutant cells. If the last-mentioned mechanisms were operative, one would predict that the incorporation of radioactivity into mutant cells would cease promptly after separation of' HPRT- cells from normal cells. If one of the first three mechanisms were operative, incorporation of radioactive label into separated mutant cells should continue for as long as functional enzyme persists. Experiments carried out by Cox et al. (1971b) and Pitts (1972) clearly showed that HPRT- cells promptly revert to the mutant phenotype when separated from normal cells. The unlikely possibility that HPRT is extremely unstable was excluded in cell culture by inhibiting protein synthesis and determining the rate of fall of enzyme activity (Cox et al., 1971b). Since transfer of nucleotide, or nucleo-

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

305

tide derivative, appears to be the most reasonable basis for this form of cell communication, the structure that allows this exchange must have properties different from the normal cytoplasmic membrane which is not permeable to the compounds. The specialized membrane structure necessary for contact transfer appears to be the gap or low-resistance junction, which also mediates the ionic coupling of cells (Gilula, 1974). The production of a gap junction apparently requires close apposition of membranes of two cells (Cox et al., 1972). This cell-to-cell communication may be extremely important for regulating gene expression, since it provides channels for exchanging regulatory substances, for example, cyclic nucleotides known to mediate gene activity (Cox et al., 1974a,b). From an evolutionary view cell-to-cell interactions provide multicellular organisms with an important mechanism for homeostasis and control of metabolic activity. The interconnected cell system, rather than individual cells, may sometimes constitute the functional unit for gene expression. e. Control of the Degradation of Intracellular Macromolecules in Culture. The value of mammalian cell cultures in understanding the basis of gene expression is elegantly exemplified by investigations on mucopolysaccharidoses. These comprise a group of rare disorders classified on the basis of certain consistent clinical findings, of the mode inheritance, and of the nature of urinary mucopolysaccharides (McKusick et al., 1965). Lysosomes are filled with mucopolysaccharides (Van Hoof and Hers, 1964) and fibroblasts cultured from the skin of patients store sulfated mucopolysaccharides which can be demonstrated by metachromatic staining (Danes and Bearn, 1966) or by chemical analysis (Matalon and Dorfman, 1966), hence the name lysosomal storage disease. In a series of experiments, Neufeld and her associates showed that the defect is in the degradation of mucopolysaccharides (Neufeld, 1974). Moreover, if fibroblasts from a patient with mucopolysaccharidosis are cocultured with normal cells, or even with cells from a patient with another type of mucopolysaccharidosis, there is a “corrrection” of the defect in degradation. Unlike the previously described studies on the Lesch-Nyhan syndrome (see Section V,B,d), cell contact is not required, since the interaction could be observed by using a medium preincubated with cells of one genotype placed on cells of another genotype or in mixed cultures in which the cells are separated from one another. Clearly, fibroblasts elaborated some substance into the medium which reduced the accumulation of mucopolysaccharides in cells of another genotype. These “corrective factors” were invaluable for

306

RODY P. COX AND JAMES C. KING

classifying and diagnosing this group of diseases. Purification of these factors indicated they were proteins, and their characterization showed that they were lysosomal enzymes required for the degradation of specific linkages of the mucopolysaccharides (see Table I). The uptake of a lysosomal enzyme from the medium by a mutant cell is responsible for correction of the phenotype. Interestingly, the uptake mechanism proved to be specifically selective. For example, Hurler's fibroblasts took up nearly half the activity of a-Liduronidase supplied in the medium, but only traces of a l b ~ m i n - ' ~ ~ I (Bach et al., 1972). The uptake of lysosomal enzymes apparently involves specific recognition. This important observation was confirmed by studies on skin fibroblast cultures from patients with inclusion cell (I-cell) disease. I cells are fibroblasts with large lysosomes filled with granular debris readily observed on phase microscopy. These cells have a multiplicity of enzyme defects including ones involving P-galactosidase, a-L-fucosidase, and aryl sulfatase; certain other hydrolases have reduced activity (1030%) (Neufeld, 1974). The cells therefore accumulate both mucopolysaccharides and glycolipids. Some of the enzymes that are deficient in the fibroblasts are present in considerable excess over normal in cell culture media, and also in the serum of I-cell disease patients (Wiesmann et al., 1971).Although I cells accumulate mucopolysaccharide, they do not cross-correct when treated with corrective factors from other mucopolysaccharidoses. This is to be expected, since I cells exhibit multiple enzyme deficiencies. Suprisingly, however, corrective factors from I cells are unable to correct the degradative abnormality in other mucopolysaccharidoses, for example, Hurler cells, despite the presence of large amounts of a-L-iduronidase in the medium. This suggests that the I-cell enzyme may be abnormal. Neufeld (1974) and associates showed that, although I-cell enzymes have normal catalytic functions, they apparently lack the recognition site necessary for uptake by cells. This was shown directly by the finding that N-acetyl-pglucosaminidase derived from I cells was unable to be taken u p by normal or mutant cells (Sandhoff and Jatzkewitz, 1972). However, I cells were able to take up and retain lysosomal enzymes derived from normal cells. The efficient uptake of lysosomal enzymes apparently requires a specific marker on the enzyme. I-cell hydrolases lack this marker and therefore accumulate outside the cell in the body fluid of patients and in the media of cell cultures. These observations suggest that fibroblasts have no intracellular mechanism for channeling hydro-

GENE EXPRESSION I N CULTURED MAMMALIAN CELLS

307

lases from the site of synthesis to their eventual lysosomal localization. This implies that lysosomal enzymes of fibroblasts are recaptured secretory protein whose uptake requires a specific recognition marker. The enzyme(s) required for the synthesis or coupling of the recognition marker is apparently the specific defect in I-cell disease. Present evidence suggests that the recognition site is a carbohydrate moiety (Neufeld, 1974) and may be analogous to the terminal glycosides present on plasma glycoproteins required for their recognition by liver cell membranes and eventual sequestration into lysosomes (Morel1 et al., 1971).

c.

GENE EXPRESSIONI N SYNCHRONOUS CELL CULTURES In mammalian cells gene expression may be regulated by events occurring as cells enter different stages of the cell cycle. The patterns of enzyme synthesis can be classified into two broad groups, depending on whether synthesis is continuous or periodic during the cell cycle (Mitchison, 1971). The synthesis of certain enzymes appears to b e restricted to periods of the cell cycle characteristic for each enzyme, and does not follow the common pattern of continuous increase in total cellular protein. Enzymes synthesized periodically during one or more stages of the cell cycle show a “step” pattern if the enzyme is stable, or a “peak” if the enzyme is rapidly degraded. Enzymes synthesized continuously during the various stages of the cell cycle show either an exponential or linear increase. In general, enzymes concerned with DNA synthesis are synthesized periodically at a stage of the cycle that often occurs during, or just preceding, the S phase. Mammalian cells appear to differ from lower forms of eukaryotes (yeasts) in that a greater percentage of the ubiquitous (household) enzymes thus far studied in mammalian cells show a continuous pattern of synthesis (about half), while in yeast only onefourth show this pattern (Mitchison, 1971). There also appear to be differences among different strains of mammalian cells in patterns of enzyme synthesis. For example, LDH shows three peaks of periodic synthesis in synchronized diploid Chinese hamster cells, while in heteroploid hamster cells the enzyme appears to show continuous synthesis (Klevecz, 1969a). Klevecz (1969b) clearly demonstrated by immunochemical methods that the oscillations in LDH activity in diploid hamster cells were the result of intermittent synthesis. Alkaline phosphatase in HeLa cells exhibits a continuous pattern of synthesis (Regan, 1966; Griffin and Ber, 1969; Cox, 1971). To date only a few enzymes have been examined in synchronous mammalian cell cultures, but one can anticipate many more studies in the near future

308

RODY P. COX AND JAMES C. KING

(Mitchison, 1971).These studies on the pattern of enzyme synthesis in synchronous cells are important in understanding gene regulation in mammalian cells. The cell cycle is a series of ordered biochemical events and not merely a steady state of uniform activity. Thus the composition of a cell constantly changes as it traverses the cycle. The cell cycle also plays a major role in determining the response to inducers of enzymes in mammalian cells. Certain of these studies are described in Section VII. Cell cycle events also play a major role in differentiated cell functions, and these are described in Section VI.

VI. Differentiated Functions in Mammalian Cell Culture

A. GENERALASPECTS Differentiated functions are potentially of great importance in investigating certain aspects of gene regulation. Cell populations that carry out differentiated functions are represented by both established cell lines capable of continuous growth in vitro, and cell strains that are usually euploid and have a finite lifetime in culture. Several excellent reviews on differentiated cell physiology have been published recently (Green and Todaro, 1967; Rothblatt and Cristofalo, 1972b), and these should be consulted for amplification and further details. Table I1 shows some differentiated functions which have been described in cell culture. Holtzer and his associates (1972) defined the core problem in cell differentiation as understanding the mechanism whereby cells acquire both determination and specialized functions in response to inducers that are lacking in the progenitor cell. The cell cycle has two functions. One is proliferation, in which both determined and progenitor cells have similar biosynthetic activities. The second is quanta2 functions in which the determined cell exhibits specialized activities lacking in its progenitors. Quanta1 functions reflect a cascade of events in which the first establishes a lineage (determination), and further steps are required to produce the requisite biosynthetic machinery necessary to produce all the molecules characteristic of fully differentiated cells. Little is known about the mechanisms by which the emerging synthetic programs are coupled to the replication of DNA. However, the problems are complex in that in some cells full differentiation precludes further cell division, for example, muscle (Holtzer et al., 1972), while other cell types, such as melanocytes, manifest the fully

TABLE I1 DIFFERENTIATED FUNCTIONS EXPRESSEDIN CELL CULTURE Cell Fibroblast

Differentiated products Collagen Hyaluronic acid Chondroitin sulfate

Muscle, striated Muscle, cardiac Muscle, smooth Chondrocyte Osteoblast Mast Melanocyte, retinal

Melanoma Erythroblast Granulocyte Macrophage Lymphoblast

Hepatic (adult) Hepatoma Teratocarcinoma

Actomyosin, creatine kinase Myosin, creatine kinase Elastin Chondroitin sulfate, a,-collagen, hyaluronic acid Collagen, bone-type AP Serotonin, histamine Melanin, dopaoxidase, tyrosinase S-100 protein Melanin, dopaoxidase, tyrosinase Hemoglobin Inhibitor of macrophage and granulocyte development Inhibitor of granulocyte and macrophage development Immunoglobulins (B cells), mediators of cell immunity (T cells) Tyrosine transaminase induction by steroids Tyrosine transaminase induction by steroids Multipotential differentiation

T-locus antigen Human chorionic gonadotrophin, multipotential differentiation S-100 protein, axone formation Neuroblas toma Neuromuscular junction Neurotransmitters

Choriocarcinoma

Lens

Cry stallin

Leydig (tumor) Pinealoma

Steroid synthesis H ydroxyindole-0-methyl transferase ACTH, prolactin, growth hormone Steroids Parathormone Synthesis ofvon Willebrand factor Spermatids

Pituitary tumor Adrenal cortical tumor Parathyroid tumor Endothelial Sertoli

References Green and Goldberg (1965) Green and Hamerman (1964) Matalon and Dorfman (1966) Holtzer et al. (1972) Goldstein et al. (1974) Ross (1971) Holtzer et al. (1972) Singh (1974) Day and Green (1962) Cahn and Cahn (1966) Wenger and Friedman (1970) Silagi (1969) Holtzer et al. (1972) Paron et al. (1969) Ichikawa et al. (1967) Bloom et al. (1973) Gerschenson et al. (1970) Tomkins et al. (1969) Finch and Ephrussi (1967) Artzt e t al. (1974) Pattillo et al. (1968) Seeds et al. (1970) Robbins and Yonezawa (1971) Braverman and Katoh (1971) Yasumuraet al. (1966a) Wells et al. (1966) Yasumura et al. (1966a) Yasumura et (11. (1966b) Deftos et al. (1968) Jaffe et al. (1974) Kodani and Kodani (1966)

310

RODY P. COX AND JAMES C. KING

differentiated character during proliferation (Cahn and Cahn, 1966). In the model in which differentiation is coupled to DNA replication, Holtzer et al. (1972) speculate that the coupling of cell generations with the developmental program permits control of the readout of information. This functions to preclude the development of cells with mutually exclusive synthetic activities, for example, hemoglobin, myosin, and albumin synthesis. This view supports the notion that on a molecular level there are no biochemically “undifferentiated” gentically unprogrammed cells. This is consistent with the findings that HeLa cells after 25 years in culture produce an AP that is normally confined to the placenta (Elson and Cox, 1969), and when iron is added to HeLa cultures they synthesize ferritin, a special iron-containing protein (Richter, 1964). The interrelationship of DNA synthesis and differentiated function enforces a stepwise and slow emergence of differentiated function. This provides a stringent control on the inappropriate synthesis of differentiated molecules, a luxury function previously referred to in the discussion of epigenotype (Section V).

B. MYOCENESISIN CULTURE Myogenesis represents a model system for describing several aspects of gene expression in differentiated cell culture. Our understanding of the process is the result of studies from many laboratories. In our summary we emphasize the work of Holtzer and his associates, who have made many distinguished contributions in this area. Presumptive myoblasts proliferating in tissue culture do not synthesize contractile protein. Cessation of DNA synthesis is a prerequisite for expression of the differentiated phenotype (Holtzer, 1970). Cell fusion that produces multinucleated myotubes is not an obligatory process for translation of differentiated or contractile proteins. There appears to be coordinate synthesis of the specialized contractile proteins characteristic of differentiated function (Bischoff and Holtzer, 1969). The programming of myogenesis is intrinsic to the cells and apparently does not require exogenous factors, for example, nerve innervation, hormones, or other agents (Holtzer et al., 1972). Only a small number of cells from stage-17 to -18 chick somite have the option in culture to differentiate into myoblasts, chondroblasts, and fibroblasts (Dienstman et al., 1974). Most of the cells that comprise the early somite and limb bud cells are defined as progenitor cells. Proof of the phenotypic options open to differentiated cells in the early stages of determination has been given by studies on the

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

311

progeny of single cells cloned from 8-day-old leg muscle (Abbott et

al., 1974). A myogenic clone derived from such single cells contains myotubes and mononuclear cells. When this clone is subcultured, large numbers of mononucleated cells are recovered. The mononucleated cells have a fibroblast phenotype morphologically, and they synthesize (a1)2 a2 chains of collagen, hyaluronic acid, and chondroitin sulfate, which are the specialized products characteristic of fibroblasts. These findings are of importance, since they directly demonstrate the presence of progenitor cells in 8-day-old muscle. Interestingly, attempts to induce chondral formation from these progenitors was not successful, indicating that this cell lineage was beyond the stage of yielding chondrogenic cells (Abbott et al., 1974). The potential of cultured differentiated cells in analyzing gene expression is clearly documented b y studies on myogenesis.

c.

ESTABLISHEDCELL LINES WITH DIFFERENTIATED FUNCTIONS Diploid cell strains derived from embryonic tissues, or from differentiated tissues, often have limited growth potential, complicating long-term studies on cell differentiation. It is possible, using techniques developed by Sat0 and his colleagues, to culture tumor cells exhibiting differentiated function by starting with transplantable tumors (Buonassisi et al., 1962). These lines have retained particular differentiated functions for many years in culture and provide important models for studying gene expression. Hormone-secreting cells derived from tumors include pituitary cells which secrete ACTH, prolactin, and growth hormone (Yasumura et al., 1966a), and adrenal cortical cells which secrete steroids and respond to ACTH (Yasumura et al., 1966b). Human parathyroid cells have been transformed by SV40 virus, and a cell line producing parathormone has been established (Deftos et al., 1968). The mouse testicular teratocarcinoma originally isolated by Stevens (1958) and adapted to cell culture provides a multipotential cell which can express several differentiated phenotypes (Finch and Ephrussi, 1967). This cell line has been of particular importance i n approaching the process of determination (see Section V), since it maintains a state of multipotentiality in cell culture and under appropriate conditions these cells oscillate between overt differentiation and a state of covert maintenance of the potentiality to express the same functions when stimulated (Finch and Ephrussi, 1967).Artzt et al. (1974) recently showed that a surface antigen found on mouse teratocarcinoma cells is also expressed on the surface of mouse

312

RODY P. COX AND JAMES C. KING

sperm and embryonic morula. The genetic locus controlling this antigen is the T locus in the mouse. The T locus has been implicated in a series of moi-phogenetic abnormalities in the mouse (Bennett, 1964).The importance of this association is that embryonic cell surface components probably play a key role in mediating morphogenetic events by means of differential surface conformation. These surface components provide recognition devices by which embryonic cell associations and migratory patterns may be regulated. The primitive teratocarcinoma cell provides a tissue culture system for investigating factors controlling differential gene expression. Other tissue culture systems are also available for studying similar problems. A human gestational choriocarcinoma (BeWO) has been established in culture, and these cells synthesize human chorionic gonadotropin. The cultures also exhibit multipotential differentiation and retain many of the metabolic and growth characteristics of human placenta (Pattillo et al., 1968). Differentiated cell cultures have been used to investigate the role of effector molecules in the expression of differentiated function. Cahn and Cahn (1966) fractionated chick embryo extract on Sephadex columns and studied the effects of various factors on the growth and expression of differentiation in cartilage and retina cells from chick embryos. Medium supplemented with the low-molecularweight fraction (<5000) supported differentiation, while the highmolecular-weight fraction (> 10,000) suppressed expression of differentiation but stimulated growth and plating efficiency. Nevo and Dorfman (1972) showed that the addition of chondromucoprotein to chondrocyte cultures stimulated the synthesis of chondromucoprotein. D.

LONG-TERMLYMPHOCYTECULTURES

The recently discovered techniques of lymphocyte transformation by infection with Epstein-Barr virus, and the establishment of permanent cultures from small samples of peripheral blood from individual donors, provide an important source for studying gene activation and expression in cell culture (Gerber, 1973). Once established, lymphocyte cultures can b e cloned and shown to have characteristics of either thymus-dependent T cells which produce mediators of cellular immunity, or bone marrow-derived B cells which produce immunoglobulin. Many of these lymphocytoid lines maintain the normal chromosomal complement (Hamerton, 1973) and exhibit the same phenotype for polymorphic enzymes as their donor (Harris, 1973). Lymphocytoid cell lines of the B type synthesize gamma

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

313

globulin (Bloom et al., 1973) and are therefore excellent subjects for studying the molecular biology of gamma globulin synthesis. The gamma globulin produced by a single clonal strain may contain heavy chains of all three major classes-gamma, mu, and alpha. These cell populations provide an important tool in the study of cell differentiation. Lymphocytoid cell lines established from patients with certain inborn errors of metabolism exhibit the enzymic deficiency, and therefore can be used in the preparation and isolation of mutant proteins from hereditary diseases (Singer et al., 1973). The ease of establishing lymphocytoid cell lines from human donors, and the face that they can be grown in suspension culture to high population densities, make the cells particularly useful for a variety of studies on gene expression.

VII.

Hormonal Effects on Gene Expression in Cultured Cells A. GENERALASPECTS

The regulation of gene expression implies alteration of either the amount or kind of proteins synthesized by cells. The spectrum and amount of protein made determine the cell’s character and metabolism. Proteins may have structural and functional (i.e., enzymic) characteristics, and the combination of subcellular localization, time of appearance during development, and microheterogeneity characterizes the cell’s epigenotype. Hormones are effector molecules produced by specialized cells which alter the physiological responses of other cells, and in the process they may also alter gene expression. During embryonic development and in adaptation to physiological changes, the protein composition of animal cells undergoes a series of ordered alterations which may be attributed to sequential gene action. Hormones are frequently the initiators of modulation of proteins, isozymes, and metazymes. These effects are specific and in some cases are the results of an increase in the synthesis of a particular protein without an appreciable change in total protein synthesis. However, the complexity of regulation in animal cells affords many potential sites at which hormonal interaction can alter the biosynthetic capabilities of cells or their protein content. For example, alterations in an enzyme’s activity may occur either through increasing the amount of enzyme protein or through altering the catalytic efficiency of the enzyme. An increase in the concentration of an enzyme may occur

3 14

RODY P. COX AND JAMES C. KING

through accelerated synthesis or through delayed enzyme degradation or both. Cell culture provides a relatively simple model for investigating mechanisms of hormonal induction of enzyme activity. In the following discussion several different mechanisms of hormone regulations of gene expression in mammalian cell culture have been selected for analysis. In hepatoma cells grown in culture, adrenal glucocorticoid hormones have been shown to increase the synthesis and decrease the degradation of TAT and tryptophan pyrolase, leading to the accumulation of enzyme protein within the cell (Granner et al., 1968; Johnson and Kenny, 1973). This accounts for the enhanced enzyme activity noted in these cultures. However, increased synthesis of enzyme is not the only mechanism whereby steroid hormones can increase the specific activity of an enzyme. Cortisol induction of AP in HeLa cells has been shown to be due to enhanced catalytic efficiency of the enzyme rather than increases in the amount of enzyme protein (Griffin and Cox, 1966a; Cox, 1971; Cox et al., 1971a; 1975).The mechanism of this induction appears to have a general relevance to control of the activity of several metalloenzymes.

B.

INDUCTIONOF TAT IN HEPATOMACELL CULTURES

1. Physiology of the Induction of TAT When adrenal steroids are administered to intact animals, the liver enzyme TAT, which catalyzes the rate-limiting reaction in tyrosine degradation is induced (Lin and Knox, 1958).A line of rat hepatoma cells (HTC) has been adapted to cell culture and can b e propagated indefinitely. This cell line exhibits induction of TAT when grown in a medium with hydrocortisone or its analogs (Thompson et al., 1966). The induction occurs when steroids are added to either growing or stationary HTC cultures, and the level of TAT rises rapidly so that after 6-12 hours a new steady state is achieved which is 5 to 15 times higher than the basal activity (Fig. 3 ) (Thompson et al., 1966). This induction was clearly shown to be due to an increase in the rate of TAT synthesis (Granner et al., 1968). Antiserum was prepared against rat liver TAT, and this antiserum was used to show that hepatoma TAT and the rat liver enzyme are immunologically identical. Quantitative precipitation was carried out with this antiserum using enzyme preparations from control and steroid-treated HTC cells, and these studies showed that the steroid-induced increase in TAT activity is accompanied by a corresponding increase in the enzyme protein of induced cells. Measurements of the rate of TAT synthesis by

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

315

labeling the enzyme with radioactive amino acids and specifically precipitating it with antiserum at various times after adding inducer showed that the steroid stimulated the rate of amino acid incorporation into the enzyme about 15-fold without measurably affecting total protein synthesis (Granner et al., 1968). Under the conditions of induction used by Tomkins and his associates, there was no detectable change in the rate of TAT degradation (Auricchio et al., 1969). Therefore the kinetics of induction seen in Fig. 3 can b e attributed to an increase in the rate of enzyme synthesis. However, using different conditions of culture or a different hepatoma cell line, Kenney (1967) and Grossman and Mavrides (1967) showed that the steroid also delays TAT degradation in their systems.

2. Interaction of Hormone with Receptors Studies relating steroid structures to their effectiveness as inducers of TAT strongly suggest that the hormone reacts with a steroid receptor, and that the conformation of the complex mediates the extent of induction (Samuels and Tomkins, 1970). The receptor is probably cytoplasmic, and an allosteric protein which has a different conformation depending on the type of steroid with which it is complexed. In intact cells inducer-receptor complexes accumulate in the nucleus. However, steroids that inhibit induction (antiinducers, for example, progesterone) when complexed with receptor remain in the cytoplasm (Baxter et al., 1972). The nuclear inducer-receptor complex binds to a limited number of sites on the nucleus, and DNA is a component of these binding sites. A temperature-dependent step is apparently necessary for modification of the cytosol receptor-inducer complex prior to nuclear uptake and binding (Baxter et al., 1972; Rousseau et al., 1972). Dexamethasone interaction with HTC cells is very similar to that described for other steroid hormones and their target tissues (O’Malley et al., 1972). In general, steroid hormones appear to initiate their effects on steroid-responsive cells by combining with a specific hormone receptor system as demonstrated in HTC. These receptor proteins bind steroid, and this specific binding is diminished by steroid antagonists. The affinity of the steroid for the receptor correlates directly with the biological effectiveness of the hormone. Studies on hormone-receptor interactions emphasize the role of these complexes in mediating hormone action, and this interaction is probably the first step in determining hormone effects. Cytosol receptors are heat-labile proteins with sedimentation coefficients of 3-5s at high ionic strength (Jensen and Jacobson, 1962;

316

RODY P. COX AND JAMES C. KING

Rubin and O’Malley, 1969; Bruchovsky and Wilson, 1968; Baulieu and Jung, 1970). Nuclear steroid receptors are different from cytoplasmic receptors in their physiochemical properties, although most evidence suggests that nuclear receptors are formed b y transfer of the cytosol receptor-steroid complex into the nucleus. Probably the best understood example of the interaction of a hormone and its target cell receptors is the binding of estrogen to the mammalian uterus. Studies by Jensen and his associates (1968) showed that estrogen combines with a specific cytoplasmic receptor to form a 4s estrogen-cytosol receptor complex. The 4s hormone-receptor complex is then transferred to the nucleus, and the nuclear form has a sedimentation constant of 5s. Apparently, the 4s form must be converted to the 5s form prior to nuclear uptake. O’Malley et al. (1972), in a series of elegant experiments, showed that progesterone interacts with chick oviduct in a similar manner. Moreover, the nuclear form of the steroid-receptor complex in the oviduct system is associated with specific acidic proteins in the target chromatin. Following this binding to chromatin, quantitative and qualitative alterations in nuclear RNA synthesis occur, leading to the induction of synthesis of a specific oviduct protein, avidin. The binding of the hormone-receptor complex to chromatin is confined to target tissues, and this in conjunction with the distribution of hormone receptors provides the specificity of response and determines the “target character” of individual cells or tissues.

3. Role of the Cell Cycle in Znduction of TAT As described in Section V,C, cell cycle events play a major role in regulating gene expression. Martin et al. (1969) studied the effects of the cell cycle on the synthesis and hormone-mediated inducibility of TAT in hepatoma cells. The generation time of the cultures was not affected by the steroid. TAT was found to be continuously synthesized in HTC cells throughout the cell cycle. However, when dexamethasone was added to synchronous cultures at various times during the cell cycle, it was found that only cells in late GI or in S were inducible. During Gz, M, and early GI, the hormone did not induce TAT. These results could not be explained by differences in steroid uptake or binding. These results and other evidence to b e described have been interpreted to indicate that, in the interval between mitosis and DNA synthesis when the enzyme becomes inducible, a repressor of enzyme synthesis is produced which acts after transcription.

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

317

4. Theories on the Mechanism of TAT Induction in HTC Cells Tomkins and his associates (1969) have proposed an ingenious model to explain their experimental results. They suggest that steroids in their system antagonize a posttranscriptional repressor which inhibits messenger translation and promotes messenger degradation. The noninducible phases of the cell cycle are the result of repression of the transcription of TAT by a process insensitive to the steroids. Just before DNA synthesis this repression is lifted. During the inducible period the regulatory gene appears to synthesize the repressor at a constant rate. I t is assumed that the repressor is a protein, and that it and its mRNA are more labile than the product of the TAT structural gene. The repressor is believed to inhibit reversibly the translation of TAT mRNA. This reversibility is supported by the phenomenon of “superinduction” produced by actinomycin D (Tomkins et al., 1966). That is, addition of actinomycin D to fully induced HTC cells produces a further increase in enzyme synthesis, putatively b y inhibiting the transcription of the labile repressor. The already synthesized repressor is inactivated, and the repressed TAT messenger can dissociate to liberate active mRNA which is translated. It is postulated that the action of the repressor is antagonized by steroid inducers. Thus, in the presence of the inducer, the repressor is inactivated, TAT messenger is translated, and degradation of messenger is prevented. The concentration of “active” TAT messenger is increased, and its translation leads to a proportionate increase in enzyme. Further experimental support for this model is described in an excellent review by Tomkins et al. (1969). Gene expression for TAT inducibility in somatic hybrids is discussed in Section XV. C. GLUTAMINESYNTHETASEINDUCTION IN EMBRYONIC RETINA Moscona and his collaborators developed a system for the induction of glutamine synthetase by hydrocortisone in embryonic neural retina as a model for the regulation of specific gene expression in embryonic cells (reviewed by Moscona, 1973). This induction has been shown by immunochemical studies to be due to an increase in the rate of enzyme synthesis and not to a delay in enzyme degradation or activation of enzyme precursors (Moscona et al., 1972). The data are consistent with the view that the induced increase in glutamine synthetase synthesis is due to the accumulation of active RNA templates for the enzyme. This suggestion was confirmed by precipitat-

318

RODY P. COX AND JAMES C. KING

ing retina polysomes involved in the translation of glutamine synthetase with antiserum against the enzyme and demonstrating that there was an approximately threefold increase in the treated retina (Sarkar and Moscona, 1973). The molecular mechanisms of interaction of hydrocortisone with embryonic retinas have been characterized, and they resemble those described for steroid interaction with target tissues and dexamethasone effects on HTC cells. Space limitation prohibits a more detailed description of this interesting embryonic cell model which has been recently reviewed (Moscona, 1973).

D. ALKALINE PHOSPHATASEINDUCTION

IN

HELA CELLS

1. Comparison of Control Mechanisms f o r Metalloenzymes and Nonmetallonenzymes Diversity in gene expression and in cell regulatory mechanisms in eukaryotic cells is illustrated by control of the activity of certain metalloenzymes. In Sections VII,B and VII,C, hormonal induction of nonmetalloenzymes, TAT in hepatoma cells, and glutamine synthetase in embryonic retina were conclusively demonstrated to be the result of an increase in the synthesis of the enzymes with a proportionate increase in enzyme activity. However, this increased synthesis of enzymes is not the only mechanism whereby steroids increase the specific activity of an enzyme. Griffin and Cox (1966a) proposed that, in HeLa cells, the induction by cortisol of AP, a zinc-containing metalloenzyme, is due to enhanced catalytic efficiency of the enzyme rather than an increase in the amount of enzyme protein in induced cells. This induction requires both RNA and protein synthesis; however, immunological and other methods clearly show that the increased catalytic activity is not accompanied b y an increase in the enzyme protein content of the induced cells (Cox et al., 1971a). Moreover, it appears as though granulocyte AP (Bottomley et al., 1969) and fetal intestinal A P (Etzler and Moog, 1966) may be similarly regulated. Control of the activity of several metalloenzymes may involve similar mechanisms. The following discussion describes the induction of AP b y cortisol in HeLa cells as a model for investigating the regulation of metalloenzymes in mammalian cell culture, and then briefly considers other model systems which appear to b e similarly controlled.

2. Nature of the HeLa Cell AP AP synthesized by H e L e J cells is a membrane-associated fetal protein and is apparently the product of a single genetic locus which

GENE EXPRESSION I N CULTURED MAMMALIAN CELLS

319

in HeLa cells is heterozygous for the F and S alleles of placental AP (Elson and Cox, 1969). T h e highest specific activity of HeLa AP is found in the membranes of the smooth endoplasmic reticulum and plasma membranes, although all membrane fractions appear to have enzyme activity (Tu et al., 1972).

3. Relation of Hormone Structure to Induction of AP On the basis of the level of AP activity they induce in HeLa6, cells, steroid hormones can be separated into optimal inducers suboptimal inducers, and noninducers. (Melnykovych, 1962; Cox, 1971). Certain steroids (i.e., progesterone) that are unable to induce HeLa cell AP competitively block the induction mediated by cortisol and therefore are antiinducers. It seems probable that optimal inducers and antiinducers have the same site of interaction in HeLa cells, and this is supported by competition studies in which the concentrations of inducer and antiinducer are varied (Cox, 1971). Antiinducers do not affect the uptake of cortisol by HeLa cells, nor do they affect RNA or protein synthesis at the concentrations studied. This work suggests that steroids initiate increased AP activity in HeL%,, cells by interacting with a putative hormone receptor, and that the structure of the steroid determines the extent of induction. It is noteworthy that the relative effectiveness of the various steroid hormones in inducing AP activity in HeLa cells is the same as that previously described for TAT induction in HTC cells (Samuels and Tomkins, 1970) and glutamine synthetase induction in embryonic retina (Moscona, 1973). Thus the initial interaction of cortisol with the various cell types is similar, despite the differences in the mechanism of induction in the AP system and the other two enzymes (see Section VII,B,2).

4 . AP Induction in Asynchronous Cell Cultures The kinetics of induction of AP by cortisol or its analogs in asynchronous HeLa cell cultures are complex, as shown in Fig. 4. There is a 12- to 20-hour lag phase after adding the steroid before the rapid increase in AP activity. The lag in induction cannot be explained b y a delay in the uptake of cortisol, since maximum accumulation of hormone is achieved within 30 minutes (Griffin and Ber, 1969; Cox, 1971). The duration of the lag phase varies directly with the doubling time of the culture and is followed by a linear increase in AP activity reaching a level 5- to 20-fold greater than controls after 50-90 hours of growth in a medium containing the hormone. This level of enzyme activity is maintained in the presence of

320

RODY P. COX AND JAMES C. KING

//--

0.1 o.l[1

o.olv-;-;--;

0.08

~

"

0.02

10

30

50

70

90

HOURS

FIG.4. Kinetics of AP induction in HeLasJ cell suspension cultures grown in media with and without cortisol. AP activity is in micromoles of p-nitrophenol released per 30 minutes per milligram of cell protein. Control (open circles) cultures grown in Eagle's spinner medium with 7% fetal calf serum. Induced (solid circles) replicate cultures were grown in a medium with 3 &ml hydrocortisone hemisuccinate. Cycloheximide (1.0 pg/ml) was added to control (open triangles) and induced (solid triangles) cultures. [Reproduced with permission from Cox et al. (1971a).]

cortisol. When the hormone is removed, AP activity decreases over the following 3-4 days as a result of both enzyme degradation and dilution by cell multiplication (Griffin and Cox, 1966b). The kinetics of AP induction (Fig. 4) should be compared with the kinetics of TAT induction (Fig. 3). The prolonged growth in the presence of steroid before maximal induction of AP is achieved may result from differences in the turnover of the enzyme, intracellular location (being membrane-bound, for example), and fundamental differences in the mechanism of the induction as described in Section VII,B,l.

5. Requirement f o r RNA and Protein Synthesis No gross changes in RNA or protein synthesis are observed in suspension cultures of HeL+, cells grown in a medium with cortisol (Griffin and Cox, 1966a,b). However, the synthesis of both of these macromolecules is required for hormone-mediated enhancement of AP activity (Griffin and Cox, 1966a,b; Cox et al., 1971a). Actinomycin D (0.1 pg/ml) blocks the increase in AP activity when added as late as 8 hours after the hormone (Griffin and Cox, 1966b). However, actinomycin D does not block induction during the most rapid phase of increase in AP activity, suggesting translation of preformed mRNA. The role of RNA as a necessary intermediate for the induction of increased AP activity was further substantiated by blocking protein synthesis in the presence of the hormone and studying the accumulation of mRNA which expressed itself promptly after cycloheximide was removed by washing (Cox et al., 197la). The effect of inhibiting protein synthesis at various times during

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

321

induction of AP by cortisol is shown in Fig. 4. Cycloheximide added as late as 7 hours after the addition of hormone completely inhibits the induction. If protein synthesis is inhibited during the phase of rapid increase in enzyme activity, further increase is blocked and there is a gradual decrease in AP activity.

6. AP Induction in Synchronous Cultures The lag or initial slow increase in AP activity may be the result of cells having to pass through a critical event in the cell cycle before AP can be induced. HeLas5 cells were collected in mitosis by selective detachment of the rounded dividing cells from the monolayer (Griffin and Ber, 1969). When cortisol was added to synthronized cell cultures in mid-GI, AP induction occurred during the midportion of the S phase of the cell cycle, about 6-7 hours after the hormone was added. When, however, the hormone was added to synchronous cultures in late S phase, or in G2, induction was delayed until the cells reentered the S phase, about 18-20 hours after the addition of cortisol. The time of onset of induction, the rate of increase in AP activity, and the magnitude of induction were similar under both conditions. Apparently, the hormone interacts with the cell to promote induction only when the cell traverses the S phase of the cell cycle (Griffin and Ber, 1969; Cox, 1971). Although the period of inducibility of AP in HeLa cells and TAT in HTC cells is apparently somewhat different, there are again many similarities despite the basic differences in mechanism of induction.

7. Measurement of the AP Content in Cells by Immunochemical Methods As described in Sections VII,B and VII,C, the induction of increased activity of TAT in hepatoma cells or glutamine synthetase in embryonic retina by steroid hormones is associated with a proportional increase in the amount of enzyme protein as measured by specific immunological methods. In both the above systems, the increased enzyme content is the result of increased enzyme synthesis. In contrast, measurement of the AP content in HeLa cells by immunochemical methods showed that the enzyme protein content was similar to that of control cultures despite a fivefold difference in specific enzyme activity. The antiserum used in these studies was thoroughly characterized and was shown to b e highly specific (Cox et al., 1971a). Figure 5 shows the titration of AP prepared from control and prednisolone-treated cultures. The amount of enzyme activity remaining in the supernatant is plotted, and the bar graph represents the AP

322

RODY P. COX AND JAMES C. KING

>. 0.30

a

0.20

r

:0.10 0

1128164 132 116 18 14 12 ANTISERUM DILUTION

FIG.5. Precipitation of base-level and induced AP preparations by increasing concentrations of antiserum. The enzyme preparations were n-butanol extracts of HeLass cells grown in the presence or absence of prednisolone (1.5 pg/ml). The enzyme was further purified by gel filtration on Sephadex G-200. The final purification was approximately 120-fold. Antigen (0.2 ml containing about 22 p g of protein) was incubated with 0.2 in1 of diluted antiserum for 2 hours at 37°C. The reaction mixture was then incubated at 4°C for 24 hours. The precipitate was removed by centrifugation, and the supernate decanted. The precipitate was washed twice with ice-cold 0.9% NaCl and then resuspended in 0.4 ml of 0.05 M tris-HC1 (pH 7.4). AP activity of supernate-control (open circles) and induced (solid circles) are shown with AP activity of precipitate-control (open bars) and induced (solid bars). [Reproduced with permission from Coxet a!. (1971a).]

activity of the antigen-antibody precipitate when constant amounts of control and induced AP are reacted with increasing concentrations of antiserum. With antibody dilutions of one to eight and less, enzyme activity was nearly quantitatively recovered in the antigen-antibody precipitate. Both the base-level and induced preparations are completely precipitated at the same antibody concentration (one to four), despite a fivefold difference in phosphatase activity. This provides strong evidence that the enzyme protein content of cells grown in the presence and absence of prednisolone is the same, despite the marked differences in AP activity. These findings further suggest that increased catalytic efficiency of induced enzyme molecules is responsible for the increase in activity.

8. Comparison of Kinetic Properties of Base-Level and lnduced HeLa Cell AP An explanation for the increased catalytic activity of the induced form of HeLa AP was sought by comparing the kinetic properties of base-level and induced enzymes both purified 250-fold (Ghosh et al., 1972). Michaelis constants (K,) of both base-level and induced APs were 18 mM for the substrate phenylphosphate in 0.05 M carbonate-bicarbonate buffer at p H 10.7. However, the maximal velocities (V) for base-level and induced APs were markedly different.

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

323

For the base-level enzyme V was 1.3 nmoles of substrate hydrolyzed per minute, whereas for the induced form V was 27.0 nmoles per minute. Since changes in the affinity of induced enzyme for substrate cannot explain the increase in catalytic efficiency, the firstorder rate constant k3 was measured to determine the rate of decomposition of enzyme-substrate complex into products. In using the purified enzyme preparations and phenylphosphate as substrate, the mean k, of base-level enzyme was 150.0 X 10+ and that of induced enzyme was 963.2 x determined by the method of Veibel and Lillelund as applied to P-glucosidase by Nath and Rydon (1954). The increased k3 value for induced AP suggests that the 0-P bond in an induced enzyme-substrate complex is more easily cleaved by hydroxyl ions in alkaline medium.

9. Comparison of Physical and Chemical Properties of Base-Level and Induced AP Immunological evidence clearly shows that the hormone-mediated induction of increased AP activity in HeLa,, cells is not accompanied by an increase in the enzyme content of induced cells but is due to enhanced catalytic efficiency. This is supported by studies on purification to apparent homogeneity of AP from control and induced cells. Enzyme preparations from control and steroid-treated cultures purified by preparative disc gel electrophoresis or by isoelectric focusing gave a single protein band when analyzed by gel electrophoresis, and both preparations contained similar amounts of protein despite a five- to tenfold difference in AP activity. Characterization of the molecular-weight variants of base-level and induced forms of HeLa AP by gel filtration on Sephadex G-200 and by sedimentation velocity in a sucrose gradient showed that both preparations could be resolved into two variants with approximate molecular weights of 240,000 rt 1000 daltons and 120,000 t 5000 daltons {Cox et al., 1971a). The first variant is apparently a tetramer and the second a dimer. The base-level and induced forms of the enzyme are identical with respect to the above determinations and resemble placental AP. The electrophoretic mobilities of control and induced AP are also identical (Cox and Griffin, 1967; Griffin and Bottomley, 1969; Ghosh et al., 1972). The identical electrophoretic mobilities and isozyme patterns of base-level and induced enzyme support the concept that they are the products of the same gene locus. Moreover, when equal amounts of base-level and induced enzyme protein are applied to the gel, the induced form is much more enzymically active when stained b y his-

324

RODY P. COX AND JAMES C. KING

tochemical methods, showing that no electrophoretically separable modulatory molecule is present in either enzyme preparation.

10. Zinc Content of Base-Level and Znduced AP AP is a metalloenzyme which requires zinc at the catalytic site for enzyme activity. In microorganisms it has been shown that differences in catalytic efficiency of AP in various mutant strains of E . coli can be attributed to differences in the zinc content of the enzyme (Schlesinger, 1967). The possibility that differences between catalytic efficiency in base-level AP and that in induced AP of HeLa cells might b e the result of differences in the zinc content of the enzymes was studied b y further purifying the dimeric peaks from a Sephadex G-200 column by preparative gel electrophoresis. Zinc content was assayed by atomic adsorption spectroscopy. The baselevel enzyme contained an average of 4.8p g of zinc per milligram of protein, and the induced had 3.7 p g of zinc. The agreement between two different purifications was well within experimental error and showed that the control and induced forms of AP contain similar amounts of zinc. These results suggest that differences in catalytic activity between these metalloproteins are not due to differences in the number of zinc atoms at the active site (Cox et al., 1971a). The binding of zinc to control and induced apoenzyme was studied by comparing the kinetics of enzyme inactivation by various concentrations of EDTA (Cox et al., 1971a). The initial inactivation of control enzyme by EDTA is consistently much greater than that observed with induced AP preparations. One minute after adding 1 mM EDTA to control enzyme, approximately 40% of its activity was lost, while induced enzyme lost only 1 5 2 0 % under the same conditions. After a l-hour incubation with EDTA, the extent of inactivation of both enzyme preparations was similar. It should be noted that the enzyme preparations were purified by column chromatography and contained similar amounts of zinc and protein. As time of incubation with EDTA increases, the differences between inactivation of control and induced enzyme become less. These findings suggest that the zinc binding forces at the active site of the control enzyme may be weaker than those in the induced. The formation of alternate bonds between apoenzyme zinc in the induced cells might lower the energy requirements of the enzyme-substrate transition state and thereby increase the catalytic efficiency (Cox et al., 1971a). Vallee and Williams (1968) have described alterations in the binding of metals to metalloenzymes that change the catalytic activity of the enzyme b y altering the conformation of the catalytic site (“entatic” effects).

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

325

11. Comparison of the Kinetics of AP Znduction with the Kinetics of AP Synthesis Several hypotheses can be advanced to explain the requirement for RNA and protein synthesis during induction of increased catalytic efficiency of AP without an increase in enzyme protein. For example, the hormone could directly alter the conformation of AP during its synthesis, or the hormone might induce the synthesis of a modifier which alters AP during its synthesis. Figure 6 presents the results of an experiment designed to test these possibilities (Cox et al., 1971a). Immunological precipitation of radioactively labeled AP was used to compare the rates of increase of enzyme (Fig. 6a) with the kinetics of enzyme synthesis (Fig. 6b). As expected from previous results from more indirect methods, the rates of enzyme synthesis and degradation in control and steroidtreated cultures are the same when measured by incorporation of

0

50

100

140

HOURS

FIG. 6. Precipitation of radioactive AP by antiserum during induction of increased enzyme activity by prednisolone. (a) AP specific activity (in micromoles of p-nitrophenol released per minute per milligram of protein) in enzyme-antibody precipitate. Open circles, control; solid circles, induced. (b) Specific radioactivity (in counts per minute times per milligram of protein) in enzyme-antibody precipitate. Open triangles, control; solid triangles, induced. HeLa cells grown in monolayer culture in Waymouth’s medium containing 10% calf serum. At zero time, 100 nCi of leucine-l‘*C per milliliter was added to all cultures and 1.5 p g of prednisolone per milliliter was added to the steroid-treated cultures. Thereafter, cultures were harvested at the times indicated. After 48 hours the medium was decanted and replaced with fresh medium containing radioactive leucine. At 96 hours the radioactive medium was decanted from the remaining cultures, and the cell monolayers were subcultured into a nonradioactive medium. Prednisolone was readded to the hormone-treated cultures. AP preparations were extracted from cells and reacted with antiserum as described in the legend to Fig. 5. [Reproduced with permission from Cox et al. (1971a).]

326

RODY P. COX AND JAMES C. KING

radioactive label into enzyme-antibody precipitate (Fig. 6b). The half-life of the base-level and that of the induced enzyme also appear to be the same, approximately 16-20 hours. An unexpected observation of unusual interest is that a near plateau in radioactivity in the enzyme-antibody complex is achieved at about 30 hours, long before maximal induction of AP activity is reached. This finding suggests that the rate of increase in enzyme activity is not directly proportional to the rate of synthesis of AP. The discrepancy between the kinetics of AP synthesis and the induction of increased AP activity suggests that the modification of the enzyme responsible for its enhanced catalytic efficiency is complex.

12. Modification of HeLa AP as the Mechanism of Induction The observations described above suggest that the induction of increased AP activity in HeLa cells by steroid hormones may be the result of a modification of the enzyme, which requires both RNA and protein synthesis. In Section X examples of enzyme modifications are discussed that do not require RNA or protein synthesis, but these alterations also change the activity or substrate specificity of the enzyme. The hypothetical modification induced by cortisol, as suggested by our experimental results, might enhance the catalytic activity of HeLa cell AP by a chemical modification of the enzyme. This possibility was studied by measuring the phosphate content of highly purified base-level and induced forms of AP, both of which were found to be phosphoprotein. Nine different AP preparations, two prepared by isoelectric focusing and seven purified by preparative acrylamide gel electrophoresis, were analyzed for tightly bound inorganic phosphate. Base-level enzyme had between 5 and 6.5 inorganic phosphate residues per enzyme molecule, while induced enzyme had only between 2.7 and 3.3 (Cox et al., 1975). The enzyme preparations used for phosphate determination were apparently homogeneous, as shown by a symmetric peak on isoelectric focusing and a single protein band on analytical acrylamide disc gel electrophoresis. The difference in amount of tightly bound inorganic phosphates is the most striking difference between the physical properties of base-level and induced forms of AP. For example, these enzyme preparations gave similar amounts of hexosamine (8 ? l), sialic acid ( 1 0.5),and free-sulfhydryl(4 f 1)molecules per enzyme of 120,000 daltons. The simplest assumption is that the degree of phosphorylation of the enzyme regulates its AP activity, presumably by altering the binding of zinc at the catalytic site and thereby

*

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

327

enhancing its catalytic efficiency by an entatic effect. It is noteworthy that in a recently completed study the level of CAMP was found not to be increased in HeLa cells during the ninety-six hours of growth in a medium with cortisol, nor can this cyclic nucleotide induce an increase in AP activity (Griffin et al., 1974).

13. Speculations on the Mechanisms of Induction of AP in HeLa Cells Several models for the induction of increased AP activity in HeLa cells can be proposed. The models must be compatable with the following observations.

1. Three agents have been reported to induce an increase in AP activity in HeLa cells. In addition to cortisol they are sodium butyrate (Griffin et al., 1974) and increased medium osmolarity (Nitowsky et aZ., 1963). One possible common site of interaction of these diverse inducers is the cell membrane. 2. HeLa AP is a membrane-bound enzyme, and can be induced only in growing cells in which new membrane synthesis occurs. 3. Several lines of evidence indicate that HeLa AP is a stable enzyme, and it takes several days of growth in the presence of inducers to achieve a maximum increase in enzyme activity. Therefore, once the enzyme is integrated into membranes, its intrinsic AP activity apparently cannot b e changed. 4. Inhibition of protein synthesis during induction results in prompt cessation of the increase in AP activity, suggesting that only newly synthesized AP can b e modulated by phosphorylation or dephosphorylation. 5. The requirement of RNA synthesis and of cell cycle events as necessary prerequisites for induction suggests that a mRNA required for mediating the increase in AP synthesis may be synthesized only at a specific point in the cell cycle, for example mid-S. This might account for the observed requirement that cells pass through DNA synthesis in the presence of hormone before manifesting the induced phenotype. 6. Completion of the induction of increased AP activity appears to b e temporally independent of the synthesis of the enzyme. 7. The catalytic activity of HeLa AP is apparently regulated by the degree of phosphorylation of enzyme protein, and this may affect the bond between the protein ligand and zinc at the active site. The induced form of HeLa AP is therefore a metazyme as defined by Horecker (1975).

328

RODY P. COX AND JAMES C. KING

One possibility is that cortisol, and perhaps other inducers, alter

the synthesis of membrane sites or membrane subunits necessary for integration of AP into the membrane mosaic. Altered membranes could favor incorporation of AP with reduced phosphorylation. Alternatively, cortisol or a cortisol-induced mediator may inhibit or repress a protein kinase which phosphorylates AP, or may stimulate a phosphoprotein phosphatase which hydrolyzes phosphate groups on the enzyme after they are added. In either case there is restricted time for phosphorylation or dephosphorylation, suggesting that modifications of AP preferentially occur during enzyme synthesis or before its insertion into the membrane mosaic. Obviously, the above models are highly speculative, and much more work is required to differentiate between them or to develop alternate models to account for the induction.

E. REGULATION OF THE ACTIVITY OF METALLOENZYMESBY HORMONES AP Other than HeLa The production of increased enzyme activity by a mechanism that increases the catalytic activity of the enzyme is also found with AP from human leukocytes, fetal mouse intestine, and rat liver. Human leukocyte AP is increased when steroid hormones, for example, hydrocortisone, are administered to patients (McCoy et al., 1966). Bottomley and his associates (1969) purified AP preparations from base-level and induced leukocytes and shown similar amounts of enzyme protein in the two preparations with markedly different specific AP activities. The biophysical, biochemical, and immunological properties of each of these APs are also similar. Using specific antiserum and quantitative precipitation techniques, these investigators showed that the induced and base-level enzyme preparations had a similar amount of enzyme protein despite marked differences in the specific activity of the antigen-antibody precipitates. These results indicate that changes in the AP activity of leukocytes with steroid administration are produced by marked changes in the catalytic activity of the enzyme rather than by changes in the amount of enzyme protein, a finding analogous to that observed in HeLa cells. Etzler and Moog (1966) have reported that during differentiation of embryonic mouse intestine an enzymically inactive protein, which reacts immunologically with an antibody against intestinal AP, is converted to the active enzyme. The activation normally occurs under the influence of endogenous corticoid hormones secreted by the developing adrenal, and results in a 19fold increase in this en-

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

329

zyme’s specific activity (Moog, 1953). Moog (1971), in an excellent review of corticoid induction of intestinal AP, suggests that these hormones influence the structure of membrane components, resulting in consequent effects on the catalytic properties of membrane-bound enzymes (e.g., AP), on permeability, and on cell metabolism in general. This view is similar to one recently proposed for the induction of AP in HeLa cells. Ferwerda and Stepan (1973) studied the marked induction of rat liver AP associated with extrahepatic bile duct obstruction. The AP from normal and bile duct-obstructed rat livers was purified over lOOO-fold, and the incorporation during synthesis of radioactive amino acids into the proteins of these enzymes was also measured. The enzyme protein contents of control and induced AP preparations were the same, and the amount of C14-labeled amino acid incorporated into base-level and induced liver AP was also similar despite a three- to fourfold increase in enzyme specific activity. Moreover, preparative and analytic disc gel electrophoresis showed similar amounts of protein for control and inducible enzymes, despite a marked difference in their catalytic activity. These investigators concluded that the induction of liver AP following bile duct obstruction is due to the synthesis of an enzyme with increased catalytic efficiency, rather than an increase in the enzyme protein content of induced livers. The similarities to the induction of HeLa AP are obvious. The alteration in membrane lipids following bile duct obstruction and the effect of regurgitated bile salts on the membrane mosaic (Poltorak and Vorobeva, 1966) may mediate a modification of liver AP, which enhances its catalytic activity. It appears that several different forms of AP in different mammalian tissues exhibit similarities in their mode of regulation and induction. Moreover, as described in the following sections, regulation of the activities of several metalloenzymes may occur through alterations in the catalytic efficiency of the enzyme secondary to changes in the binding of the metal at the active site, rather than through an increase in the number of enzyme molecules.

VIII. Regulation of the Activity of Metalloenzymes by Agents Other than Hormones

A. ARYL HYDROCARBON HYDROXYLASE

Aryl hydrocarbon hydroxylase (AHH) is one of several cytochromes P-450-mediated enzymes inducible by either aromatic hydrocarbons or phenobarbital. Nebert and his associates (1973)

330

RODY P. COX AND JAMES C. KING

studied AHH induction by polycyclic hydrocarbons in certain inbred strains of mice and showed that inducibility is controlled by a single autosomal dominant gene designated a h (Gielen et al., 1972). The genetic expression of inducibility is specific for hydrocarbons and does not affect inducibility b y phenobarbital. Induction of AHH also can be studied in second-passage fetal cell cultures derived from hamsters or various inbred mouse strains, or in fetal rat liver cells in culture (Nebert and Bausserman, 1970; Gielen and Nebert, 1972). The maximum induction of hydroxylase by benz ( a ) anthracene is approximately 10-fold greater than base level and requires about 20 hours of growth in the presence of inducer. The entrance of inducer into cells of all genotypes is rapid and independent of temperature. Benz ( a ) anthracene binds to nucleus and to cytoplasmic macromolecules. Induction can be divided into several phases. In the initial state induction-specific RNA is formed for which protein synthesis is not required. Subsequently, translation involving this induction-specific RNA occurs. Induction is associated with a shift in the spectral properties of microsomal CO-binding cytochrome P-450 to a distinct form, P1-450 or P-448. The alteration in spectral activity is caused by a change in the iron of the cytochrome from a low-spin to a high-spin state. Nebert and his associates (1973) have presented evidence that this change in the state of the metal is related to membrane differences in or near the catalytic site, which are regulated at the genetic level by the a h locus. Nebert interprets his data b y suggesting that, in the presence of inducer, cells synthesize a modifier molecule which interacts with the cytochrome P450-enzyme complex and alters the conformation of the iron at the active site. This changes the state of ferrous electron orbitals to favor a high-spin state and is associated with increased catalytic efficiency. It is of great interest that gene expression that controls the induction of AHH by polycyclic hydrocarbons is regulated by a single locus.

B. ALCOHOL DEHYDROGENASE IN MAIZE Although studies on the genetic control of alcohol dehydrogenase (ADH) (EC 1.1.1.1) in corn are not strictly concerned with gene expression in mammalian cell culture, the findings in this system are of unusual interest in interpreting the changes in the activity of metalloenzymes described above. ADH is a zinc-containing enzyme. Maize is polymorphic for ADH as determined by a locus designated Adh. Four alleles have been described at the structural gene locus (Schwartz and Endo, 1966), which specify enzymes with different electrophoretic mobilities in the scutellum of corn kernels. ADH is a

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

331

dimer, and in heterozygotes three isozymes are formed. Efrom (1970) carried out genetic studies on two inbred strains of maize that differed in the electrophoretic mobility of the ADH isozymes and in the catalytic efficiency of the enzymes. These studies clearly showed that the gene that specifies the activity of the enzyme is not allelic to the Adh structural gene that specifies the electrophoretic mobility of the enzyme. The regulatory locus alters the catalytic activity of ADH when the Adh locus is occupied by either one of two of the four structural gene alleles and is located 17 crossover units from the structural gene locus. These findings are of interest in interpreting the previously described findings on AP and AHH induction in cell culture. Apparently, modifiers can interact with metalloenzymes to alter their catalytic activity, and in maize the gene determining this modification can be clearly separated from the structural gene for the enzyme.

IX. Regulation of the Rate of Protein Degradation A.

GENERALASPECTS

Schimke and his associates (1965; Schimke, 1964) have emphasized that the level of an enzyme in animal tissues represents a steady state, and that alterations in an enzyme level can be achieved by altering the rate of enzyme degradation as well as by altering the rate of synthesis. The mechanism by which enzymes are degraded is complex and, in some cases, is controlled by a specific locus. For example, protein degradation can be easily demonstrated in intact cells (Schimke et al., 1965), but may not occur in tissue homogenates. Moreover, proteolytic inactivation of enzyme activity in vitro has not provided valid information on protein degradation in vivo (Schimke et al., 1965). Rechcigl and Heston (1963, 1967) and Ganschow and Schimke (1969) studied inbred strains of mice with differences in catalase levels in liver and kidney. They showed that the variations in catalase activity are due to differences in enzyme content and result from differences in the rate of catalase degradation. The level of catalase activity can be controlled by alleles at the structural gene locus for this enzyme, which affect the catalytic activity of individual enzyme molecules, presumably by an entatic effect. For example, specific activities of purified catalase from C57BL/6 and C57BL/Ha strains of mice are only 60% that of the DBN2 strain. A second locus controls the rate of catalase degradation in the liver. Liver catalase of C57BL/Ha mice is degraded in vivo at a rate one-

332

RODY P. COX AND JAMES C. KING

half that of liver catalase of mice of the DBAI2 strain. Therefore the liver of C57BL/Ha mice contains twice as many catalase molecules as those of the other two strains (Ganschow and Schimke, 1969). The locus controlling catalase degradation shows a specificity of expression in that it does not affect the turnover of kidney enzyme nor does it alter the metabolism of peroxisomes or of liver protein in general. It should be emphasized that this second gene locus controls catalase degradation rather than synthesis and thereby regulates the level of the enzyme in tissues. Thus gene expression can be modulated by specific control of the rate of enzyme degradation, as well as by altering rates of enzyme synthesis. Several studies including those described above show that the degradation of specific proteins is carefully regulated. It may be that the characteristic half-life of some proteins, for example, catalase, is determined by loci coding for specific proteins that render labile or degrade other proteins. In other instances it is the tertiary structure of the protein as a substrate that determines its rate of degradation. Thus the nature of the peptide bonds exposed, as determined by the tertiary structure of the protein and as affected by conformational changes induced by small molecules or macromolecular structures, may determine the protein’s susceptibility to degradation. Physiological states, the presence of substrates and cofactors, and genetic differences are known to affect rates of protein degradation. Therefore changes in the level of protein in a cell can be affected by changing rates of synthesis or rates of degradation.

B. MEASUREMENTOF PROTEINDEGRADATION IN CELL CULTURE As previously described, the rates of degradation of TAT (Tomkins et al., 1969; Kenney, 1967) and AP (Cox et al., 1971a) have been measured in cell cultures during induction by cortisol and in control cultures (Sections VII,B and C). The turnover of specific proteins in diploid cell strains has not been so extensively investigated. Mellman et al. (1972) measured the rates of synthesis and degradation of catalase in cultures of human diploid fibroblasts. These cells have the advantage that at confluency there is little cell division and the protein content of cultures is essentially static, so that studies of protein synthesis and degradation can be carried out with a constant cell mass. Studies on catalase synthesis and turnover were facilitated by measuring the recovery of catalase activity after inactivation of the enzyme with the irreversible inhibitor aminotriazole. The return of

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

333

activity to its original steady state is the result of synthesis of the enzyme, and the rate of synthesis (k,)is constant during this period. The rate of approach to this steady state is a function of the rate constant of degradation &), and the return of activity following aminotriazole has been described as an exponential function (Price et al., 1962). By using these methods, catalase was found to have a turnover half-time of 27 hours. The turnover was affected by the presence of serum, the nutritional state of the cultures, and other factors. The level of catalase in these experiments also was measured by immunoprecipitation with specific antiserum, and differences in enzyme activity were directly correlated with the enzyme content of cells. The experiments of Mellman et al. (1972) indicate the applicability of methods for measuring protein turnover to cell culture systems. It is of interest that the rate of catalase degradation in fibroblasts is similar to that in liver and different from that found in the enucleate metabolically inactive red cell.

X. Protein Modification Altering Gene Expression The activity or substrate specificity of certain enzymes can be changed by chemical modification of the enzyme protein (Anderson et al., 1970; Kingdon et al., 1967; and Shapiro et al., 1967).I n order to survive, living organisms must accommodate to changes constantly occurring in the environment. These modulations must occur rapidly in eukaryotes without the delay imposed by gene activation, transcription, or translation. Special effectors have evolved which permit rapid adjustments in metabolism. These include hormones and “second messengers” which have been identified as the cyclic nucleotides cyclic 3’,5’-adenosine monophosphate (CAMP) and 3’-5’guanosine monophosphate (cGMP). Sutherland and his associates (Robison et al., 1971) demonstrated that cAMP mediates the action of many hormones by initiating a series of protein modifications. Krebs and his associates (Walsh et al., 1968) showed that a family of similar enzymes-protein kinases-are activated by CAMP. Protein kinases consist of at least two subunits, one catalytic and the other regulatory or inhibitory; when the two are complexed together, the enzyme is inactive. Activation occurs when cAMP binds to the inhibitory subunit, permitting the catalytic subunit to phosphorylate certain proteins. Phosphorylation involves the transfer of phosphate from ATP to serine residues of the protein. When phosphorylated, certain enzymes, for example, phosphorylase kinase, are activated. The activated phosphorylase kinase in turn catalyzes phosphorylation by

334

RODY P. COX AND JAMES C. KING

ATP of the final enzyme in this cascade, glycogen phosphorylase, which catalyzes phosphorylation by ATP of glycosyl units of glycogen into glucose l-phosphate, which in the liver becomes transformed into glucose. Thus the modification of a series of enzymes by phosphorylation activates the catalytic functions of each of these proteins, producing a shift to a different metabolic state. Inactivation of each enzyme in the sequence involves the removal of the phosphate groups from the enzyme, a process catalyzed by phosphoprotein phosphatases. The protein kinase is inactivated when its inhibitor subunit recombines with its catalytic subunit. Inactivation of protein kinases is regulated by the level of CAMP in the cell. The above modulations occur quickly in the cell and permit rapid metabolic adjustment to changes in the level of substrates, cofactors, and products. Modifications of this kind should be contrasted to those described for HeLa cell AP (Section VII,C,12) in which RNA and protein synthesis are required and in which only newly synthesized enzyme or nascent peptides on polysomes can be modified. The alteration in catalytic activity of AP mediated by cortisol requires several days of growth in the presence of the hormone.

XI. Control of Enzyme Activity in Intact Cells-Role of Substrates and Cofactors Measurement of enzyme activity is usually carried out with optimal concentrations of substrates, cofactors, and metal activators, so that maximum catalytic activity is achieved. Such conditions are essential for determining the enzyme content of cells and for studying the kinetics of enzyme reactions. However, in intact cells conditions may be quite different and the amount of enzyme may not be the controlling factor in regulating the amount of product formed. Differences between the in vivo and in vitro conditions may confuse interpretation of gene expression viewed at the level of the phenotype. An important example of this has recently been described by Dancis and his associates (1973). A family was reported in which each of two sisters had a son with no detectable hypoxanthine phosphoribosyl transferase (HPRT) in erythrocytes, a finding considered pathognomonic of the Lesch-Nyhan syndrome. However, neither boy had the phenotype for this disease; one was neurologically normal and the other had an unrelated seizure disorder which caused mild retardation. Both subjects had increased excretion of oxypurines and slight increases in serum uric acid. In contrast to the absent HPRT activity of red cells, white cell lysates had 10-15% of normal activity,

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

335

suggesting continued synthesis of an unstable enzyme. The discrepancy between the biochemical findings and the near-normal phenotype was explained by studying the activity of HPRT in intact cells of the mutant subjects and normals. When intact cells of mutant, heterozygous carriers and normals are incubated with labeled hypoxanthine at optimal concentrations but without added cosubstrate phosphoribosyl phosphate (PRPP), all genotypes produce similar amounts of product, inosinic acid. These findings strongly suggest that the enzyme concentration is not rate-limiting, whereas the concentration of cosubstrate PRPP is. Appropriate tests completely support this conclusion. It is noteworthy that studies on intact cells explained the discrepancy between the biochemical findings and the clinical phenotype. A similar explanation may apply to other genetic diseases in which incomplete but severe enzyme deficiencies are found in clinically normal individuals. Holland et al. (1974), in a recent study, developed a method of studying purine metabolism in culture using skin fibroblasts from patients with different degrees of HPRT deficiency, subjects with overproduction gout, and normals. Using intact cell assays, she extended the previous findings of Dancis et al. (1973) and demonstrated that cells from HPRTdeficient individuals leak purines into media while cells from patients with overproduction gout who have normal HPRT activity do not. Chan et al. (1973) have also reported that HPRT-deficient cells excrete xanthine and hypoxanthine into media. This system is therefore a model for investigating abnormalities of purine metabolism in culture and for characterizing various mutations that alter purine biosynthesis. It is likely that other models can be developed using mutant fibroblasts from patients with inborn errors to study other metabolic pathways and correlate biochemical aberrations with clinical findings.

XII. Genetic Control of Intracellular Localization of Enzymes

The localization of an enzyme within a cell is a major factor influencing expression of the activity of that enzyme in the metabolism of the cell. The predilection of enzymes for certain intracellular sites derives in part from the chemical structure of the enzyme and its mode of assembly. However, in some cases specific loci determine the localization of an enzyme. Ganschow and Paigen (1967) showed that P-glucuronidase localization in mouse liver is determined by a locus distinct from that of the structural gene for the enzyme. In

336

RODY P. COX A N D JAMES C. KING

normal mouse liver about 60% of P-glucuronidase activity is lysosomal, and 40% is tightly bound to endoplasmic reticulum. In liver cells derived from the YBR strain of inbred mice, only the lysosomal enzyme was present. The distribution of other microsomal enzymes, for example, glucose-6-phosphatase, was normal in the mutant strains, as were the electrophoretic patterns of microsomal enzyme preparations. The physical and chemical properties of the pglucuronidase from mutant strains appeared normal, suggesting that the structure of the enzyme was not responsible for its failure to be incorporated into microsomal membranes. This conclusion was confirmed by breeding experiments with another inbred strain having a form of glucuronidase distinguishable from that of the YBR strain. These studies showed that in the F, generation the YBR enzyme was incorporated into the endoplasmic reticulum. Backcrosses clearly established that a second locus distinct from that of the glucuronide structural gene was responsible for the failure of glucuronidase to attach to the microsomal membrane of the YBR strain. This finding suggests that the intracellular compartmentalization of enzymes may be controlled by factors other than the primary structure of the enzyme. Thus gene expression dictates not only the primary structure of enzymes but, in some cases, also their subcellular localization. The effects of these genes appear to b e tissue-specific, since the locus affecting the subcellular location of liver P-glucuronidase does not affect the location of the enzyme in spleen or in cell cultures.

XIII. Gene Expression in Heterokaryons of Mammalian Cells A. CELL FUSION Cell fusion provides an important method for investigating gene regulation and for studying complementation in mutant mammalian cells. The most commonly employed agent to induce cell fusion under controlled laboratory conditions is inactivated Sendai virus (Okada, 1962; Okada and Murayama, 1965; Harris, 1970). Although the technique is effective, virus preparations give variable results. In our experience the fusion rate with virus varied from 7 to 30%,with an average of 15-2070.Recently, a new method of cell fusion using a crystalline natural product extracted from the beetle Paederus fuscipes was shown to induce consistently a rate of cell fusion of from 40 to 60% in human diploid fibroblasts grown in a monolayer culture (Levine et al., 1974). Heterokaryons are produced between human

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

337

diploid fibroblasts and HeLa or mouse cells. The pederine fusion technique is simple and, with levels of the compound (1to 4 ng/ml) that produce 4 0 4 0 % fusion, there is relatively little toxicity. Lysolecithin has also been used for cell fusion, as it is a well-defined chemical which acts directly on the cell membrane without metabolic intervention (Poole et al., 1970; Croce et al., 1971). However, it damages cells, and its effectiveness in causing fusion of diploid fibroblasts is variable.

B. COMPLEMENTATION IN HETEROKARYONS Following the fusion of cells derived from different lines of descent, two or more separate nuclei share a common cytoplasm, and interactions between the genomes can be analyzed. These multinucleate cells or heterokaryons, if they contain genetically different nuclei, may live for a few weeks in culture, but their continued reproduction depends on the formation of a cell containing a single nucleus (Harris, 1970). Synkaryon formation and hybridization are discussed in Section XIV. Heterokaryons provide an important system for studying gene activation and expression. A potentially useful application of heterokaryon formation is in the study of genetic heterogeneity exhibited by many inborn errors of metabolism. As previously described (Section V,B,3), MSUD is a disorder characterized by an inability to degrade by decarboxylation the three BCKAs, and the enzymic defect is expressed in fibroblast cultures (Dancis et al., 1972). Genetic heterogeneity in MSUD was investigated by using Sendai virus-induced fusion of skin fibroblasts derived from unrelated patients (Lyons et al., 1973). If fusing two different mutant cells corrects their common BCKA decarboxylase deficiency, it is assumed that they have different genetic lesions. In MSUD it was found that with fusion of some cell strains the efficiency of complementation is high and can be detected without the selective system method required for synkaryons. The ability of MSUD fibrobIasts to complement one another bears no apparent relationship to the clinical phenotype of the donor (classic or variant), or to the level of residual BCKA decarboxylase activity (Lyons et al., 1973). Some cell strains complemented with several other MSUD fibroblasts derived from both classic and variant forms of the disease, while others did not complement. Complementation occurs with the fusion of some cell strains but not with others, as would be expected if the genetic lesion exists in different forms. Complementation must have restored BCKA decarboxylase activity to near-normal levels in the 18% of virus-treated

338

RODY P. COX AND JAMES C. KING

cells that were heterokaryons. In MSUD the enzyme resulting from complementation had catalytic activities that differed from the wildtype in that the keto acid derived from leucine was more readily decarboxylated than that derived from valine. It is not possible to distinguish between intergenic or interallelic complementation in these studies. The differences in kinetic properties of the complemented enzyme suggest interallelic complementation (Lyons et al., 1973). De Weerd-Kastelein and his collaborators (1972) studied complementation in heterokaryons produced by fusing into pairs cultured fibroblasts from patients with the classic form of xeroderma pigmentosum and fibroblasts from patients with another form of this disease which involves neurological and mental deficiency (the De Sanctis-Cacchione syndrome). Xeroderma pigmentosum is an autosomal recessive disease characterized by extreme hypersensitivity of the skin to ultraviolet radiation due to impaired DNA repair. The difference in symptoms between the classic and the De Sanctis-Cacchione form suggests that the genetic defect resulting in xeroderma pigmentosum might involve more than one gene. Cell fusion studies confirm this conclusion. Mixed heterokaryons obtained by fusing xeroderma pigmentosum cells with De Sanctis-Cacchione cells marked by sex chromosome differences are better able to repair DNA damaged by ultraviolet radiation than are heterokaryons obtained by fusing pairs of xeroderma or De Sanctis cells. Furthermore, both nuclei in the mixed heterokaryons repair DNA damage, as evidenced by incorporation of t h ~ r n i d i n e - ~ after H irradiation. The simplest interpretation of these findings is that two different loci, each specifying different proteins, are required for repair of radiation-damaged DNA, and that in classic xeroderma pigmentosum one gene is inactivated and in De Sanctis the other gene is inactive. Recent more extensive studies have extended these findings and have shown that DNA repair involves four distinct complementation groups, indicating that at least four mutations can cause defective DNA repair (Robbins et al., 1974). Complementation analysis of heterokaryons provides a potentially important method for studying the genetic heterogeneity observed in many human biochemical disorders. For example, complementation can reveal functional differences between mutations of independent origin. By using these techniques it may be possible to construct complementation maps of various inborn errors and eventually to understand the molecular basis of the disease at the level of the deficient enzyme.

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

339

c.

GENE ACTIVITYAND DIFFERENTIATION IN HETEROKARYONS Harris (1970) has summarized his important contributions on gene activity and expression in heterokaryons in a recent monograph which should be consulted for details. Harris fused several differentiated cells (rabbit macrophage, rat lymphocyte, and chicken erythrocyte) with actively dividing human and mouse cells and with each other. Using autoradiographic studies of RNA and DNA synthesis by nuclei of the three differentiated cell types in heterokaryons, he demonstrated that, if either original cell normally synthesizes RNA, then RNA synthesis will occur in both nuclei even if one of the original cells normally does not synthesize RNA. The same general rule applies to DNA synthesis; however, if neither original cell synthesized DNA, then DNA synthesis will not take place in the heterokaryon. When a cell that synthesizes a particular nucleic acid is fused with one that does not, the active cell initiates the synthesis of this nucleic acid in the inactive partner. In no case does the inactive partner suppress synthesis in the active cell. Synthesis of DNA and RNA in inactive nuclei begins soon after fusion with a dividing or active cell. However, expression of genes in the inactive nucleus has to await the development of a full-sized nucleolus (Sidebottom and Harris, 1969). The expression of genetic information from the erythrocyte nuclei requires 10-14 days. Survival of heterokaryons for this length of time requires that the nuclei of the active host cell have its mitotic cycle suppressed by irradiation prior to fusion. Once the nucleolus had developed in a reactivated chick nucleus, Harris demonstrated the expression of chick protein in the heterokaryons. The nucleolus is apparently required for transport to the cytoplasm of the RNA synthesized b y the reactivated nucleus. Both chicken surface antigens and enzyme markers have been demonstrated in heterokaryons.

XIV. Gene Expression in Synkaryons of Mammalian Cells

A. NUCLEARFUSION Multinucleate cells produced by cell fusion are apparently not able to multiply, or do so only rarely in cell culture. Survival of a hybrid cell therefore depends on the formation of a cell with a single nucleus. Binucleate cells are more likely to give rise to viable synkaryons than are cells with more than two nuclei. The two nuclei of a

340

RODY P. COX AND JAMES C. KING

binucleate cell enter mitosis together, a single spindle is formed, all the chromosomes become aligned along one metaphase plate, and cell division gives rise to mononucleate daughters which contain within a single nucleus the chromosomes of both original cells. Other forms of mitosis are also seen, especially bipolar and tetrapolar mitosis, which give rise to variable numbers of daughter cells, some of which may be binucleate. I n other cases the nuclei in the heterokaryon may enter mitosis, but cell division may not occur. Postmitotic reconstitution may collect all the chromosomes of the cell into a single large nucleus containing both original sets of chromosomes (Harris, 1970). Thus mononucleate cells containing genetic components from two different sources may be formed from multinucleate cells in several ways. Barski et al. (1960) first reported synkaryon formation using two heteroploid lines of mouse cells grown in culture. Cocultivation of these lines gave rise to a new cell type which contained nearly all the chromosomes of both lines, including all the distinctive marker chromosomes that characterized each line. There was initially some skepticism concerning these results, since the superposition of two parental metaphases would be expected to produce similar karyological findings. However, Barski’s findings were soon confirmed by Ephrussi and his associates (see review, Ephrussi, 1972).Allosynkaryons have been extensively used for studying gene expression and in gene localization. Studies on gene localization in synkaryons have been recently reviewed and are not considered in this presentation (Bergsma, 1974; Ruddle, 1973; Ephrussi, 1972). B. GENE EXPRESSIONIN SYNKARYONS Ephrussi (1972) has emphasized that in synkaryons between genetically dissimilar cells the household (see Section V,B) functions (ubiquitous enzymes) of both original lines are expressed as would be expected if there were no interaction or regulatory mechanism. Therefore complementation of nonallelic deficiencies is observed in crosses between different mutant cells. For example, Weiss and Green (1967) used the complementing activity of household enzymes to localize the human thymidine kinase gene, by fusing diploid human cells with thymidine kinase-deficient mouse cells and selecting for allosynkaryons with thymidine kinase activity (Littlefield, 1964). Human chromosome number 17 has been shown to correlate with the presence of human thymidine kinase in these cells (Miller et al., 1971). Ruddle and his collaborators and other investigators utilized similar techniques to localize many of the human

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

341

gene loci to particular human chromosomes (see monograph, Bergsma, 1974). Puck and his collaborators utilized synkaryon formation in Chinese hamster cells deficient in nutritional biosynthetic capabilities to study complementation between independent mutations (Kao et al., 1969). Siniscalco et al. (1969) demonstrated intergenic complementation for X-linked mutations in allosynkaryons derived from human male diploid fibroblasts. The synkaryons synthesized both normal gene products, indicating that both X chromosomes may be functionally active in fused human cells. Nadler et al. (1970) has described intraallelic complementation in synkaryons derived from fibroblasts of different patients with galactosemia. In contrast to the findings with ubiquitous enzymes, fusion affects differentiated functions in a more variable manner. In many cases in which a differentiated cell is fused to an “undifferentiated” one, the allosynkaryon does not exhibit the differentiated phenotype. This has been described as extinction of the luxury function (Ephrussi, 1972). Loss of differentiated function has been observed in synkaryons between melanoma cells and mouse fibroblasts (Davidson et al., 1968), rat pituitary cells and mouse fibroblasts (Sonnenschein et al., 1968), glial cells and mouse fibroblasts (Benda and Davidson, 1971), hepatoma and mouse fibroblasts (Schneider and Weiss, 1971; Peterson and Weiss, 1971), and immunoglobulin-producing cells and mouse fibroblasts (Coffin0 et al., 1971). However, in some allosynkaryons the differentiated function is retained in cells or is regained in some after further chromosome loss. The last-mentioned examples provide important insights into gene regulation. Peterson (1974) has described some exceptional synkaryons between rat hepatoma cells and mouse fibroblasts which continue to produce albumin. These lines each show characteristic rates of albumin production, but they do not occur randomly. Rather there is a discontinuous distribution of albumin-producing synkaryons which clusters around distinct values which can be fitted to a geometric progression. Synkaryons also exhibit a decrease in albumin production with increasing cell generations, which contrasts with the stable phenotype of rat hepatoma lines in continuous culture. The clustering of amounts of albumin produced by individual clones of synkaryons suggests a regulation of gene expression. Such regulation could occur by duplication of structural genes, however, selective reiteration of other structural genes has not been found, for example, for hemoglobin (Bishop et al., 1972) or ovalbumin (Sullivan et al., 1973). Alternatively, the different levels of albumin synthesis could reflect different degrees of duplication of regulatory elements rather than structural genes. Weiss and

342

RODY P. COX AND JAMES C. KING

Chaplain (1971) have described reexpression of TAT inducibility in a clone of synkaryon mouse fibroblasts and rat hepatoma cells following prolonged growth in culture. These cells have apparently “segregated” certain mouse chromosomes and thereby have reduced the total number of chromosomes of the undifferentiated cell so that reexpression of differentiated function (TAT inducibility) occurs. It appears that certain loci on the undifferentiated mouse cell suppress expression of differentiated function by the genome of the rat hepatoma cell, In rat hepatoma-human hybrids suppression of TAT inducibility was correlated with the presence of the human X chromosome (Croce et al., 1973). In synkaryons that lost the human X chromosome, restoration of TAT induction b y corticosteroid hormones occurred. Somewhat similar results have been reported for esterase production by a renal adenocarcinoma cell when fused to a diploid human fibroblast (Klebe et al., 1970). In many synkaryons the renal esterase isozyme was not produced. However, following loss of human chromosomes, certain clones reexpressed the renalspecific esterase. Karyological analysis of the various synkaryon clones suggested that a human chromosome of the C group, probably number 10, appears to correlate with suppression or extinction of renal esterase. In clones that segregated this chromosome esterase production resumed. It appears that in synkaryons certain loci are not expressed for many cell generations, because a specific chromosome derived from the other line contains loci that prevent its expression. When the suppressing locus is lost, specialized function can reappear in the segregated progeny. Peterson and Weiss (1971) have described a remarkable synkaryon derived from rat hepatoma and mouse 3T3 fibroblasts. Certain cells produce both rat and mouse albumin as shown by immunochemical methods. Apparently, in the synkaryon the differentiated rat hepatoma cell genome has activated the mouse locus for albumin synthesis, one of the most specific of all liver finctions. In this interspecific synkaryon the regulatory signals for albumin synthesis in the rat cells appear to be recognized by the mouse genome. The relative genetic contribution of the rat hepatoma cells seems to determine activation of the mouse albumin locus. Fusion of mouse 3T3 cells with hypertetraploid hepatoma cells frequently leads to the production of mouse albumin, while fusion of mouse cells with hyperdiploid hepatoma cells does not usually activate mouse albumin synthesis. It should be noted that other liver functions, for example, TAT inducibility and synthesis of aldolase type B were not expressed in these synkaryons, indicating separate regulation of differentiated liver functions.

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

343

XV. Reconstruction of Mammalian Cells Cytochalasin B causes the nucleus of animal cells in monolayer culture to migrate to the margin of the cell and form an outpocketing which frequently remains attached to the body of the cell only by a thin cytoplasmic stalk (Carter, 1967). The nucleus can be pulled away from the cell by centrifugal force (Prescott et al., 1972). The enucleated cells (cytoplasts) recover from the effects of cytochalasin B and are viable for several days. The nuclei (karyoplasts) surrounded by a thin shell of cytoplasm and plasma membrane can be recovered, and by using Sendai virus can be fused with heterologous cytoplasts (Veomett et aZ., 1974). Reconstructed cells with heterologous nuclei and cytoplasm provide a potentially important tool for studying gene expression. Recently, partial reconstruction of mouse cells in culture has been used to study the mode of inheritance of chloramphenicol resistance (Bunn et aZ., 1974). A chloramphenicol-resistant mouse cell line was isolated, and the mutant cells were enucleated with cytochalasin B. Enucleated chloramphenicol-resistant cytoplasm was fused with nucleated chloramphenicol-sensitive mouse cells, and the resulting fusion products were found to be chloramphenicol-resistant. They were capable of continuous growth in culture and maintained the chloramphenicol-resistant phenotype. Thus the genetic information for drug resistance resides in the cytoplasm of mouse cells and is probably encoded in the mitochondria1 DNA. It has been shown in other studies that chloramphenicol resistance is expressed at the level of mitochondria1 protein synthesis in HeLa cells (Kislev et al., 1973). The potential for studying cytoplasmic inheritance in cell culture using partial reconstruction is obvious. The breadth of the topic gene expression in mammalian cells precludes further discussion of related topics. For example, the expression of foreign DNA in mammalian cell culture recently has been authoritatively reviewed (Ottolenghi-Nightingale, 1974).

XVI. Conclusions Cell culture systems provide the opportunity to investigate molecular mechanisms that control gene expression, as detected by the cellular phenotype. A major challenge is to understand the basis of determination and the control of the expression of differentiated functions. Studies on mutant mammalian cells, particularly those that show a temperature sensitivity toward expressing differentiated

344

RODY P. COX AND JAMES C. KING

activities, may be useful in separating and analyzing the required biochemical events. Difficulty in selecting for such mutant cells is a serious limitation, but recent studies on cell lines selected for temperature-sensitive differences in contact inhibition of cell growth confirm the resolving power of such methods. Cell lines derived from mutant subjects and from multipotential tumors are particularly informative for investigating basic mechanisms of gene expression and determination. Cell fusion and the subsequent analysis of complementation in heterokaryons and synkaryons may provide insight into the nature of mutations at the levels of the defective protein and interactions of the regulatory process. As an example of the refinements that tissue culture systems permit in analyzing basic molecular events, the effects of hormones on metallo- and nonmetalloenzymes have been emphasized in this article. This seems appropriate, since our understanding of these controls has recently been greatly increased. A hormone may induce an increase in the activity of an enzyme. In one cell type this effect may be due to increased synthesis and accumulation of an enzyme protein; in another situation the hormone may increase the catalytic efficiency of an enzyme without changing the enzyme protein content. It appears that the former mechanism is more characteristic of nonmetalloenzymes, while the latter is observed with metalloenzymes. The diversity and complexity of control mechanisms in eukaryotic cells is exemplified by these differences in regulation of metal and nonmetal enzymes. We have attempted a survey of some of the mechanisms of gene expression in cultured cells rather than an in-depth analysis of a few narrow areas. This has necessitated selecting only a few examples of the many well-studied systems. It is unavoidable and perhaps desirable that out personal biases affected our choices for emphasis. We have also attempted to document the complexity and variety of different mechanisms whereby mammalian cells modulate their activities. Tissue culture systems provide important and invaluable techniques for exploring the molecular basis of cell control.

REFERENCES Abbott, J., Schiltz, J., Dienstman, S., and Holtzer, H. (1974). Proc. Nut. Acud. Sci. U.S. 71, 1506. Abercrombie, M . (1967). In “Cell Differentiation” (A. V. S. D e Reuck and J. Knight, eds.), pp. 3-12. Churchill, London. Anderson, W. B., Hennig, S. B., Ginsburg, A., and Stadtman, E. R. (1970). Proc. N u t . Acad. Sci. U . S . 67, 1417.

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

345

Artzt, K., Bennett, D., and Jacob, F. (1974).Proc. Acad. Sci. U.S. 71,811. Auricchio, F., Martin, D., and Tomkins, G. M. (1969). Nature (London)221, 806. Bach, G., Freidman, R., Weissman, B., and Neufeld, N. F. (1972).Proc. Nut. Acad. Sci. U.S. 69, 2048. Barski, G., Sorieul, S., and Comefert, F. (1960). C . R. Acad. Sci. 251, 1825. Baulieu, E. E., and Jung, I. (1970). Biochem. Biophys. Res. Commun. 38,599. Baxter, J. D., Rousseau, G. G., Benson, M. C., Garcea, R. L., Ito, J., and Tomkins, G. M. (1972). Proc. Nut. Acad. Sci. U.S. 69, 1892. Beadle, G . W. (1945). Chem. Rev. 37, 15. Beckman, L., Bergman, S., and Lundgren, E. (1967).Acta Genet. 17, 304. Benda, P., and Davidson, R. L. (1971).J.Cell. Physiol. 78, 209. Bennett, D. (1964). Science 144,263. Bergsma, D. (ed.). (1974). “Human Gene Mapping.” Intercontinental Med. Book Corp., New York. Beutler, E., Kuhl, W., Trinidad, F., Teplitz, R., and Nadler, H. (1971). Amer. J . Hum. Genet. 23, 62. Bird, R. W., Louarn, J., Martuscelli, J., and Caro, L. (1972). J . Mol. Biol. 70, 549. Bischoff, R., and Holtzer, H. (1969).J.Cell Biol. 41, 188. Bishop, J. O . , Pemberton, R., and Baglioni, C. (1972).Nature (London),New Biol. 235, 231. Bloom, A. D., Wong, A., and Tsuchimoto, T. (1973). “Long Term Lymphocyte Cultures in Human Genetics,” pp. 62-72. Nat. Found.-March of Dimes Press, New York. Bottomley, R. H., Lovig, C. A., Holt, R., and Griffin, M. J. (1969). Cancer Res. 29, 1866. Brachet, J., Chantrenne, H., and Vanderhaeger, F. (1955). Biochim. Biophys. Acta 18, 544. Brady, R. O . , Uhlendorf, B., and Jacobson, C. B. (1971). Science 172, 174. Braverman, M., and Katoh, A. (1971). Nature (London)230,392. Brenner, S., Jacob, F., and Mudson, M. (1961). Nature (London) 190, 576. Britten, J., and Davidson, E. H. (1969).Science 165,349. Britten, R. J., and Kohne, D. E. (1968).Science 161,529. Brown, M. S., and Goldstein, J. L. (1974). Science 185, 61. Brown, M. S., Segal, A,, and Stadtman, E. R. (1971). Proc. Nut. Acad. Sci. U.S. 68, 2949. Bruchovsky, N., and Wilson, J. D. (1968).J.Biol. Chem. 213, 5953. Bunn, C. L., Wallace, D. C., and Eisenstadt, J. M. (1974). Proc. Nat. Acad. Sci. U.S. 71, 1681. Buonassisi, V., Sato, G., and Cohen, A. L. (1962). Proc. Nat. Acad. Sci. U S . 48, 1184. Burlington, H. (1959).Amer. J. Physiol. 197, 68. Cahn, R. D., and Cahn, M. B. (1966).Proc. Nut. Acad. Sci. U.S. 55, 106. Callan, H. G. (1974).Cold Spring Harbor Symp. Quant. Biol. 38, 195. Carter, S. B. (1967).Nature (London) 213,261. Chan, T. S., Ishii, K., Long, C., and Green, H. (1973).J.Cell. Physiol. 81, 315. Chatton, E. (1937).“Titres et Travaux Scientifiques.” Sotano, SBte. Chua, N.-H., Blobel, G., Siekevitz, P., and Palade, G. E. (1973). Proc. Nut. Acad. Sci. U.S. 70, 1554. Cleaver, J. E. (1970).Int. J. Radiat. Biol. 18, 557. Coffino, P., and Scharff, M. D. (1971). Proc. Nut. Acad. Sci. U.S. 68,219. Coffino, P., Knowles, B., Nathenson, S. G., and Scharff, M. D. (1971). Nature (London),New Biol. 231, 87.

346

RODY P. COX AND JAMES C. KING

Condon, M. A. A., Oski, F. A., Di Mauro, S., and Mellman, W. J. (1971). Nature (Londoii),New Biol. 229, 214. Cox, R. P. (1971).Ann. N.Y. Acad. Sci. 179, 596. Cox, R. P., and Griffin, M. J. (1967). Arch. Biochem. Biophys. 122,552. Cox, R. P., Douglas, G., Hutzler, J., Lynfield, J., and Dancis, J. (1970). Lancet 1, 893. Cox, R. P., Elson, N. A., Tu, S. H., and Griffin, M. J. (1971a).J . Mol. Biol. 58, 197. Cox, R. P., Krauss, M. R., Balis, M. E., and Dancis, J. (1971b). Proc. Nat. Acad. Sci. U . S . 67, 1573. Cox, R. P., Krauss, M. R., Balis, M. E., and Dancis, J. (1972).E x p . Cell Res. 74, 251. Cox, R. P., Krauss, M. R., Balis, M. E., and Dancis, J. (1974a).J.Cell. Physiol. 84, 237. Cox, R. P., Krauss, M. R., Balis, M. E., and Dancis, J. (1974b). In “Cell Communication” (R. P. Cox, ed.), pp. 67-95. Wiley (Interscience), New York. Cox, R . P., Ghosh, N. K., Bazzel, K., and Griffin, M. J. (1975). In “Isozymes I Molecular Structure” (C. L. Markert, ed.), pp. 343-365. Academic Press, New York. Crick, F. (1971).Nature (London)234,25. Croce, C . M., Sawick, W., Kitchevsky, D., and Koprowski, H. (1971).E x p . Cell Res. 67, 427. Croce, C. M., Litwack, G., and Koprowski, H. (1973). Proc. Nat. Acad. Sci. U . S . 70, 1268. Dancis, J., Hutzler, J., Cox, R. P., and Woody, N. C. (1969).J . Clin. Znuest. 48, 1447. Dancis, J., Hutzler, J., Synderman, S. E., and Cox, R. P. (1972).J . Pediat. 81, 312. Dancis, J., Yip, L. C., Cox, R. P., Piomelli, S., and Balis, M. E. (1973).J.Clin. Znuest. 52,2068. Danes, B. S., and Bearn, A. G. (1966).J.Erp. Med. 123, 1. Darnell, J. E., Jalinck, W. R., and Milloy, G. R. (1973).Science 181, 1215. Davidson, E. H., and Britten, R. J. (1973).Quart. Reu. B i d . 48, 565. Davidson, R. L., Ephrussi, B., and Yamamoto, K. (1968).J . Cell. Physiol. 72, 115. Davidson, R. G., Fieldes, R. A., Glenn-Bott, A. M., Harris, H., and Robson, E . B. (1965).Ann. Hum. Genet. 29, 5. Dawson, G., and Stein, A. 0. (1970).Science 170, 556. Day, M., and Green, J. P. (1962).J.Physiol. (London) 164, 210. Deftos, L. J,, Robson, A. S., Buckle, R. M., Aurbach, G. D., and Potts, J. T. (1968). Science 159,435. De Weerd-Kastelein, E. A., Keijzer, W., and Bootsma, D. (1972). Nature (London) 238, 80. Dienstman, S., Biehl, J., Holtzer, S., and Holtzer, H. (1974). Develop. Biol. 39, 83. Dina, D., Meza, I., and Crippa, M. (1974). Nature (London)248,486. Eagle, H. (1960). Proc. Nut. Acad. Sci. U.S. 46, 427. Eagle, H. (1965). Science 148, 42. Ebner, K. E., Hageman, E. C., and Larson, B. L. (1961). E x p . Cell Res. 25, 555. Efrom, Y. (1970). Science 170, 751. Elson, N. A., and Cox, R. P. (1969). Biochem. Genet. 3, 549. Ephrussi, B. (1972). “Hybridization of Somatic Cells,” pp. 52-70. Princeton Univ. Press, Princeton, New Jersey. Etzler, M . E., and Moog, F. (1966).Science 154, 1037. Ferwerda, W., and Stepan, J. (1973). Hoppe-Seyler’s Z. Physiol. Chem. 354, 1462. Finch, B. W., and Ephrussi, B. (1967).Proc. Nut. Acad. Sci. U S . 57, 615. Ganoza, M. C., and Williams, C. A. (1969). Proc. Nat. Acad. Sci. U S . 63, 1370. Ganschow, R. E., and Paigen, K. (1967).Proc. Nut. Acad. Sci. U.S. 58, 938. Ganschow, R. E., and Schimke, R. T. (1969).J.Biol. Chem. 244,4649.

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

347

Gerber, P. (1973). In “Long-Term Lymphocyte Cultures in Human Genetics” (D. Bergsma, ed.), pp. 20-30. Nat. Found.-March of Dimes Press, New York. Gerschenson, L. E., Anderson, M., Molson, J., and Okigaki, T. (1970). Science 170, 859. Ghosh, N. K., and Cox, R. P. (1975).Enzyme 20,35. Ghosh, N. K., Ruckenstein, A., Baltimore, R., and Cox, R. P. (1972). Biochim. Biophys. Acta 286, 175. Gielen, J. E., and Nebert, D. W. (1972).J. B i d . Chem. 247, 7591. Gielen, J. E., Goujon, F. M., and Nebert, D. W. (1972). J. B i d . Chem. 247, 1125. Gilula, N. B. (1974). In “Cell Communication” (R. P. Cox, ed.), pp. 1-38. Wiley (Interscience), New York. Goldstein, M. A., Claycomb, W. C . , and Schwartz, A. (1974). Science 183, 212. Granner, D. K., Hayashi, S., Thompson, E. B., and Tomkins, G . M. (1968).J.Mol. Biol. 35, 291. Green, H., and Goldberg, B. (1965).Proc. Nut. Acad. Sci. U.S. 53, 1360. Green, H., and Hamerman, D. (1964).Nature (London)201, 710. Green, H., and Todaro, G . J. (1967). Annu. Rev. Microbiol. 21, 573. Griffin, M. J., and Ber, R. (1969).J.Cell Biol. 40, 297. Griffin, M. J.. and Bottomley, R. H . (1969). Ann. N.Y. Acad. Sci. 166, 417. Griffin, M. J., and Cox, R. P. (1966a).Proc. Nut. Acad. Sci. U.S. 56, 946. Griffin, M. J., and Cox, €3. P. (1966b).J.Cell Biol. 29, 1. Griffin, M. J., Price, G. H., Bazzell, K. L., Cox, R. P., and Ghosh, N. K. (1974).Arch. Biochem. Biophys. 164, 619. Gross, K. W., Jacobs-Lorena, M., Baglioni, C., and Gross, P. R. (1973).Proc. Nat. Acad. Sci. U.S. 70, 2614. Gross, P. R., and Cousineau, G. H. (1964). E x p . Cell Res. 33, 368. Grossman, A., and Mavrides, G. (1967).J.Biol. Chem. 242, 1398. Gurdon, J. B. (1970).Proc. Roy. Soc., Ser. B 176,303. Hamerton, J. L. (1973). In “Long-Term Lymphocyte Cultures in Human Genetics” (D. Bergsma, ed.), pp. 183-187. Nat. Found.-March of Dimes Press, New York. Harris, H. (1970).“Cell Fusion.” Harvard Univ. Press, Cambridge, Massachusetts. Harris, H. (1973). In “Long-Term Lymphocyte Cultures in Human Genetics” (D. Bergsma, ed.), pp. 132-137. Nat. Found.-March of Dimes Press, New York. Harris, M. (1964).“Cell Culture and Somatic Variation,” pp. 159-166. Holt, New York. Harris, M. (1971).J.Cell. Physiol. 78, 177. Hayflick, L. (1965).E x p . Cell Res. 37, 614. Herndon, J. H., Sternberg, D., and Uhlendorf, B. W. (1969).J. Clin. Znoest. 48, 1017. Holland, J., DiLorenzo, A. M., Cox. R. P., Dancis, J., and Balis, M. E. (1974).Clin. Res. 22,697a. Holtzer, H. (1970). In “Cell Differentiation” (0. Schjeide and J. D e Villis, eds.), pp. 476-493. Van Nostrand-Reinhold, Princeton, New Jersey. Holtzer, H., Weintraub, H., Mayne, R., and Mochan, B. (1972).In “Current Topics in Developmental Biology” (A. A. Moscona and A. Monroy, eds.), pp. 229-256. Academic Press, New York. Horecker, B. L. (1975). In “Isozymes I Molecular Structure” (C. L. Marked, ed.), pp. 11-38. Academic Press, New York. Howell, R. R., Kaback, M. M., and Brown, B. I. (1971).J.Pediat. 78, 638. Hsia, Y. E., Scully, K. J., and Rosenberg, L. E. (1971).J.Clin. Inoest. 50, 127. Huang, R. C., and Bonner, J. (1962).Proc. Nut. Acad. Sci. U.S. 48, 1216. Ichikawa, Y., Pluznik, D. H., and Sachs, L. (1967).Proc. Nut. Acad. Sci. U.S. 58, 1480.

348

RODY P. COX A N D JAMES C. KING

Jacob, F., and Monod, J. (1961).J.Mol. Biol. 3,318. Jaffe, E. A., Hoyer, L. W., and Nachman, R. L. (1974).Proc. N a t . Acad. Sci. U . S . 71, 1906. Jensen, E. V., and Jacobson, H. I. (1962). Recent Progr. Horm. Res. 18,387. Jensen, E. V., Suzuki, T.,Kawashima, T., Stumpf, W. E., Jungblut, P. W., and De Sombre, E. R. (1968). Proc. Nut. Acad. Sci. U . S . 59,632. Johnson, R. W., and Kenney, F. T. (1973).J . Biol. Chem. 248,4528. Justice, P., Ryan, C., Hsia, D. Y., and Krmpatik, E. (1970). Biochem. Biophys. Res. Commun. 39, 301. Kao, F. T., Chasin, L., and Puck, T. T. (1969). Proc. Nut. Acad. Sci. U S . 64, 1284. Kenney, F. T. (1967). Science 156, 525. Kingdon, H. S., Shapiro, B. M., and Stadtman, E. R. (1967). Proc. Nat. Acad. Sci. U S . 58, 1703. Kislev, N., Spolsky, C. M., and Eisenstadt, J. M. (1973).J . Cell Biol. 57, 571. Klebe, R. J., Chen, T., and Ruddle, F. H. (1970). Proc. Nut. Acad. Sci. U.S. 66, 1220. Klevecz, R. R. (1969a).Science 166, 1536. Klevecz, R. R. (1969b).J.Cell Biol. 43,207. Kodani, M., and Kodani, K. (1966). Proc. Nat. Acad. Sci. U.S. 56, 1200. Korn, D., and Thomas, M. (1971). Proc. Nat. Acad. Sci. U.S. 68, 2047. Kornberg, R. D. (1974). Science 184, 868. Kornberg, R. D., and Thomas, J. 0. (1974). Science 184, 865. Krone, W., Schneider, G., Schutz, D., Arnold, H., and Blume, K. G. (1970).Humangentick 10, 224. Krooth, R. S., and Sell, E. K. (1970).J.Cell. Physiol. 76, 311. Krooth, R. S., and Weinberg, A. N. (1960). Biochem. Biophys. Res. Commun. 3, 518. Krooth, R. S., Howell, R. R., and Hamilton, H. H. (1962).J . E x p . Med. 115, 313. Levine, M. R., Dancis, J., Pavan, M., and Cox, R. P. (1974). Pediat. Res. 8, 606. Lieberman, I., and Ove, P. (1958).J.Biol. Chem. 233,634. Lin, E. C. C., and Knox, W. E. (1958).J.B i d . Chem. 233, 1186. Littlefield, J. (1964).Science 145, 709. Lockart, R. Z., and Eagle, H. (1959). Science 129, 252. Lyons, L. B., Cox, R. P., and Dancis, J. (1973).Nature (London),New Biol. 243, 533. McCoy, E. E., Ebodi, M., and England, J. (1966). Pediatrics 38, 996. McKusick, V. A,, Kaplan, D., Wise, D., Hanley, W. B., Suddarth, S. B., Sevick, M. E., and Maumenee, A. E. (1965). Medicine (Baltimore)44,445. Marks, P. A., and Kovach, J. S. (1966).In “Current Topics in Developmental Biology” (A. A. Moscona and A. Monroy, eds.), Vol. 1, pp. 213-252. Academic Press, New York. Martin, D., Tomkins, C.M., and Granner, D. K. (1969).Proc. Nat. Acad. Sci. U . S . 62, 248. Martin, G . M., Sprague, C. A., and Epstein, C. J. (1970). Lab. Invest. 23, 86. Matalon, R., and Dorfman, A. (1966). Proc. Nut. Acad. Sci. U . S . 56, 1310. Mellman, W. J. (1973). In “Human Genetics” (H. Harris and K. Hirshhorn, eds.), pp. 259-306. Plenum, New York. Mellman, W. J., Schimke, R. T., and Hayflick, L. (1972). E x p . Cell Res. 73, 399. Melnykovych, G. (1962).Biochem. Biophys. Res. Commun. 8, 81. Mezger-Freed, L. (1972). Nature (London),New Biol. 235, 245. Miller, 0. J., Allderdice, P. W., Miller, D. A., Breg, W. R., and Migeon, B. R. (1971). Science 173, 244. Miller, 0. L., Jr., Beatty, B. R., Hamkalo, B. A., and Thomas, C. A., Jr. (1970). Cold

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

349

Spring Harbor Symp. Quant. Biol. 35, 505. Mitchison, J. M. (1969). Science 165, 657. Mitchison, J. M. (1971). “Biology of the Cell Cycle,” pp. 159-180. Cambridge Univ. Press, London and New York. Monod, J., and Jacob, F. (1961). Cold Spring Harbor Symp. Quant. Biol. 26, 389. Monteleone, J. A., Beutler, E., Monteleone, P. L., Utz, C. L., and Cosey, E. C. (1971). I . Pediat. 78, 1067. Moog, F. (1953).]. E x p . Zool. 124, 329. Moog, F. (1971).In “Hormones in Development” (M. Hamburgh and E. J. W. Barrington, eds.), pp. 143-160. Appleton, New York. Morell, A. G., Gregoriadis, G., Scheinberg, I. H., Hickman, J., and Ashwell, G. (1971). J. Biol. Chem. 246, 1461. Morrow, G. J., Mellman, W. J., and Barness, L. A. (1969).Pediat. Res. 3,217. Moscona, A. A. (1973). In “Biochemistry of Cell Differentiation” (A. Monroy and R. Tsanev, eds.), Federation of European Biochemical Societies, Vol. 24, pp. 1-23. Academic Press, New York. Moscona, M., Frenkel, N., and Moscona, A. A. (1972).Deuelop. Biol. 28,229. Nadler, H. L., and Egan, T. J. (1970).New Engl.]. Med. 282,302. Nadler, H. L., Chacko, C. M., and Rachmeler, M. (1970).Proc. Nut. Acad. Sci. U.S. 67, 976. Nath, R. L., and Rydon, H. N. (1954).Biochem. /. 57, 1. Nebert, D. W., and Bausserman, L. L. (1970).]. Biol. Chem. 245,6373. Nebert, D. W., Considine, N., and Kon, H. (1973). Drug Metab. Disposition 1, 231. Neufeld, E. (1974).In “Cell Communication” (R. P. Cox, ed.), pp. 217-231. Wiley (Interscience), New York. Nevo, Z., and Dorfman, A. (1972).Proc. Nat. Acad. Sci. U.S. 69,2069. Nitowsky, H. M., Herz, F., and Geller, S.(1963).Biochem. Biophys. Res. Commun. 12, 293. Nitowsky, H. M., Davidson, R. G., Soderman, D. D., and Childs, B. (1965).Johns Hopkins M e d . ] . 117, 363. Okada, S., Beath, M. L., LeRoy, J., and O’Brien, J. S. (1971).Amer. J. H u m . Genet. 23, 55. Okada, Y. (1962).E x p . Cell Res. 26, 98. Okada, Y., and Murayama, F. (1965). E x p . Cell Res. 40, 154. O’Malley, B. W., Spelsberg, T. C., Schroder, W. T., Chytil, F., and Steggles, A. W. (1972).Nature (London)235, 141. Ottolenghi-Nightingale, E. (1974). In “Cell Communication” (R. P. Cox, ed.), pp. 233-254. Wiley (Interscience), New York. Papaconstaniou, J. (1967).Science 156, 338. Parker, W. C., and Bearn, A. G . (1961a).Amer.1.Hum. Genet. 15, 159. Parker, W. C., and Bearn, A. G. (1961b).Amer. I . Med. 34, 180. Paron, M., Ichikawa, Y., and Sachs. L. (1969).Proc. Nat. Acad. Sci. U.S. 62, 81. Patel, V., Itaru, W., Zeman, W. (1972).Science 176, 426. Patrick, A. D., and Lake, B. D. (1969).Nuture (London)222, 1067. Pattillo, R. A., Gey, G. O., Delfs, I?,., and Mattingly, R. F. (1968). Science 159, 1467. Paul, J., and Fottrell, P. G. (1961).Ann. N.Y. Acad. Sci. 94, 668. Peterson, J. A. (1974). Proc. Nat. Acad. Sci. U.S. 71, 2062. Peterson, J. A., and Weiss, M. C. (1971). Proc. Not. Acad. Sci. U.S. 69, 571. Pinsky, L., and Krooth, R. S. (1967:1).Proc. Nat. Acad. Sci. U.S. 57, 925. Pinsky, L., and Krooth, R. S. (196711).Proc. Nut. Acad. Sci. U.S. 57, 1267.

350

RODY P. COX AND JAMES C. KING

Pinsky, L., Powell, E., and Callahan, J. (1970).Nature (London) 228, 1093. Pitts, J. D. (1972). I n “Third Lepetit Colloquium on Cell Interaction” (L. G. Silvestri, ed.), pp. 277-285. North-Holland Publ., Amsterdam. Poltorak, 0. M., and Vorobeva, E. S. (1966).Zh. Fis. Khim. 40, 1665. Poole, A. R., Howell, J . I., and Lucy, J. A. (1970).Nature (London) 227, 810. Porter, M. T., Fluharty, A. L., and Kihara, H. (1969).Proc. Nat. Acad. Sci. U.S. 62,887. Prescott, D. M., Myerson, D., and Wallace, J. (1972). Exp. Cell Res. 71, 480. Price, V. E., Sterling, W. R., Tarantola, V. A., Hartley, R. W., and Rechcigl, M. (1962). J. Biol. Chem. 237, 3468. Regan, J. D. (1966). Experientia 22,708. Rechcigl, M., and Heston, W. E. (1963).J. Nut. Cancer Inst. 30, 855. Rechcigl, M., and Heston, W. E. (1967). Biochem. Biophys. Res. Commun. 27, 119. Redman, C. M., and Sabatini, D. D. (1966).Proc. N a t . Acad. Sci. U.S. 56, 608. Reynolds, J. A., and Schlesinger, M. J. (1969). Biochemistry 8, 588. Richter, G. W. (1964). Brit. J . E x p . Pathol. 45, 88. Robbins, N., and Yonezawa, T. (1971). Science 172,395. Robbins, J. H., Kraemer, K. H., Lutzner, M. A., Festoff, B. W., and Coon, H. G. (1974). Ann. Intern. Med. 80,221. Robison, G. A., Butcher, R. W., and Sutherland, E. W. (1971).I n “Cyclic AMP” (G. A. Robison, R. W. Butcher, and E. W. Sutherland, eds.), pp. 17-47. Academic Press, New York. Robson, E. B., and Harris, H. (1965).Nature (London) 207, 1257. Romeo, G., Kaback, M. M., and Levin, E. Y. (1970).Biochem. Genet. 4,659. Ross, R. (1971).J.Cell Biol. 50, 172. Rothblat, G. H., and Cristofalo, V. J., eds. (1972a). “Growth, Nutrition and Metabolism of Cells in Culture,” Vol. 1, pp. 17-426. Academic Press, New York. Rothblat, G. H., and Cristofalo, V. J., eds. (197213).“Growth, Nutrition and Metabolism of Cells in Culture,” Vol. 2, pp. 57-286. Academic Press, New York. Rousseau, G. G., Baxter, J. D., and Tomkins, G. M. (1972).J.Mol. Biol. 67,99. Rubin, M . M., and O’Malley, B. W. (1969).Int. Biophys. Congr. Int. Union Pure Appl. Biophys., 3rd, pp. 27-38. MIT Press, Cambridge. Ruddle, F. H. (1973).Nature (London) 242, 165. Samuels, H. H., and Tomkins, G. M. (1970).J. Mol. Biol. 52, 57. Sandhoff, K., and Jatzkewitz, H. (1972).“Sphingolipidoses and Allied Diseases” (B. W. Volk and S. M. Aronson, eds.), p. 305. Plenum, New York. Sarkar, P. K., and Moscona, A. A. (1973).Proc. Nut. Acad. Sci. U . S . 70, 1667. Schatz, G., and Mason, T. L. (1974). Annu. Reu. Biochem. 43, 51. Schimke, R. T. (1964).J.Biol. Chem. 239, 3808. Schimke, R. T., Sweeney, E. W., and Berlin, C. M. (1965).J. Biol. Chem. 240, 322. Schlesinger, M. J. (1967).J.Biol. Chem. 242, 1604. Schneider, J. A., and Weiss, M. C. (1971).Proc. Nat. Acad. Sci. U S . 68, 127. Schneider, J. A., Rosenbloom, F. M., and Bradley, K. H. (1967). Biochem. Biophys. Res. Commun. 29, 527. Schwartz, D., and Endo, T. (1966).Genetics 53, 709. Seeds, N . G., Gilman, A. G., Amano, T., and Nirenberg, M. W. (1970).Proc. N u t . Acad. Sci. U .S. 66, 160. Seegmiller, J. E., Rosenbloom, F. M., and Kelley, W. N. (1967). Science 155, 1682. Shapiro, B. M., Kingdon, H. S., and Stadtman, E. R. (1967).Proc. Nut. Acad. Sci. U S . 58, 642. Shih, V., Littlefield, J. W., and Moser, H. W. (1969). Biochem. Genet. 3,81.

GENE EXPRESSION IN CULTURED MAMMALIAN CELLS

351

Sidebottom, E., and Harris, H. (1969).J.Cell Sci. 5, 351. Silagi, S. (1969)./. Cell Biol. 43, 263. Singer, J. D., Sachdeva, S., Dowben, R., Smith, G. F., and Hsia, D. Y. Y. (1973). I n “Long-Term Lymphocyte Cultures in Human Genetics” (D. Bergsma, ed.), pp. 55-60. Nat. Found.-March of Dimes Press, New York. Singh, I. (1974). Personal communication. Siniscalco, M., Klinger, H. P., Eagle, H., Koprowski, H., Fujimoto, W. Y., and Seegmiller, J . E. (1969). Proc. Nut. Acad. Sci. U.S. 62, 793. Sloan, H. R., Uhlendorf, B. W., Jacobson, C. B., and Fredrickson, D. S. (1969). Pediat. Res. 3, 532. Sonnenschein, C., Tashjian, A., and Richardson, U. (1968). Genetics 60, 227. Spencer, N., Hopkinsson, D. N., arid Harris, H. (1964). Nature (London)204, 742. Spirin, A. S. (1966). In “Current Topics in Developmental Biology” (A. A. Moscona and A. Monroy, eds.), Vol. 1, pp. 1 3 8 . Academic Press, New York. Stanier, R. Y., and Van Niel, C. B. (1962).Arch. Mikrohiol. 42, 17. Stedman, E., and Stedman, E. (1950).Nature (London) 166, 780. Stein, G., and Farber, J. (1972). Proc. Nut. Acad. Sci. U.S. 69,2918. Stein, G. S., Spelsberg, J. C., and Heinsmith, L. J. (1974). Science 183, 817. Stevens, L. C. (1958)./. Nut. Cancer Ztist. 20, 1257. Sullivan, D., Palacios, R., Stavnezer, J., Taylor, J. M., Faros, A. J., Kiely, M. L., Summers, N. M., Bishop, J. M., and Schimke, R. T. (1973).J.B i d . Chem. 248, 7530. Suzuki, Y., and Suzuki, K. (1971).Science 171, 73. Tedesco, T. A., and Mellman, W. J. (1967). Proc. Nut. Acud. Sci. U.S. 57, 829. Tedesco, T. A., and Mellman, W. J. (1969)./. Clin. Inoest. 48, 2390. Thompson, E. B., Tomkins, G. M., and Curran, J. F. (1966). Proc. Nut. Acud. Sci. U.S. 56,296. Tomkins, G. M., Thompson, E. B., Hayashi, S., Gelehrter, T. D., Granner, D. K., and Peterkofsky, B. (1966). Cold Spring Harbor Symp. Qunnt. Biol. 31, 349. Tomkins, G. M., Gelehrter, T. D., Gronner, D., Martin, D., Samuels, H. H., and Thompson, E. B. (1969). Science 166, 1474. Tsanev, R., and Sendov, B. (1971).J.Theor. Biol. 30,337. Tschudy, D. P., Perbroth, M. G., Marver, H . S., Collins, A., Hunter, G., Jr., and Rechcigl, M., Jr. (1965).Proc. Nut. Acud. Sci. U.S. 53, 841. TLI,S. H., Nordqnist, R. E., and Griffin, M. J. (1972). Biochim. Bioph!/S. Acta 290, 92. Uhlendorf, B. W., and Mudd, S. H. (1968). Science 160, 1007. Vallee, B. L., and Williams, R. J. P. (1968).Proc. Nut. Acud. Sci. U.S. 59, 498. Van Hoof, F., and Hers, H. G. (1964).C. R . Acud. Sci. 259, 1281. Veomett, G., Prescott, D. M., Shay, J., and Porter, K. R. (1974).Proc. Nut. Acud. Sci. U.S. 71, 1999. Walker, P. R., Bonney, R. J., Becher, J. E., and Potter, V. R. (1972). In Vitro 8, 107. Wallace, B., and Kass, T. L. (1974). Genetics 77, 541. Walsh, D. A,, Perkins, J. P., and Krebs, E. G. (1968).J.Biol. Chem. 243, 3763. Weiss, M. C., and Chaplain, M. (1971).Proc. Nut. Acad. Sci. U.S. 68, 3026. Weiss, M. C., and Green, H. (1967).Proc. Nut. Acud. Sci. U.S. 58, 1104. Wells, S. A,, Wurtman, R. J., and Rabson, A. S. (1966). Science 154, 278. Wenger, B., and Friedman, H. (1970).Nuture (London) 228, 1214. Wiesmann, U., Vassella, F., and Herschkowitz, N. (1971).New Engl. J . Med. 285, 1090. Wuu, K., and Krooth, R. S. (1968).Science 160, 539. Yasumura, Y., Tashjian, A. H., and Sato, G. (1966a).Science 154, 1186. Yasumura, Y., Buonassisi, V., and Sato, G. (1966b). Cancer Res. 26, 529.

This Page Intentionally Left Blank

Morphology and Cytology of the Accessory Sex Glands in Invertebrates K. G . ADIYODI AND R. G . ADIYODI Department of Zoology, Calicut University, Kerala, India

I. Introduction . . . . . . . . . 11. General Survey . . . . . . . . A. Gonadal Glands. . . . . . . . B. Ductal Glands . . . . . . . . C. Seminal Glands . . . . . . . . D. Spermathecal Glands , , , E. Genitoatrial Glands . . . . . . . F. Penial Glands . . . . . . . . G. Male Collateral Glands . . . . . . H. Female Collateral Glands . . . . . . 111. Evolutionary Aspects . . . . . . . IV. Maturation . . . . . . . . . V. Differentiation and Secretion: The Role of Hormones VI. Concluding Remarks . . . . , , References . . . . . . . . .

.

.

.

.

.

.

.

.

.

. . . . . . . . .

353 354 355 357 364 365 367 367 368 380 388 390 392 393 396

I. Introduction In multicellular animals gametes escape to the exterior either by rupture of the body wall, as in the more primitive forms, or through conduits which may or may not be associated with the gonads. Accessory reproductive glands are either absent or reduced in animals that shed their gametes into the ambient water (e.g., echinoderms, coelenterates). Many invertebrates secrete semen to ensure the survival and safe transport of sperm. Rhynchobdellid and pharyngobdellid Hirudinea, Onychophora, copepod, euphasid, and decapod Crustacea, many Arachnida, M yriapoda, and Insecta, and some Mollusca deliver the sperm in protected envelopes, spermatophores. The latter may serve also as copulatory plugs to hold the semen in place in forms that have evolved internal fertilization. In Bombyx mod, secretions from the glandula prostatica are required for the activation of sperm (Wigglesworth, 1950). It is becoming increasingly recognized that male accessory sex secretions influence aspects of female reproductive physiology and behavior in Diptera (Fuchs et al., 1968). In many female species there is a need for protective membranes or shells for the eggs, and for jellies or mucus to hold the eggs together and also for cementing the eggs to the sub353

354

K. G. ADIYODI AND R. G. ADIYODI

stratum. Accessory glandular elements, particularly abundant in Mollusca among invertebrates, evolved as ectodermal (ectadenes) or mesodermal (mesadenes) contributions in association with the male and female genital systems to meet these crucial demands in reproduction. Accessory sex glands have sometimes come to acquire other functions: for example, storage excretion as in the uricose glands of Dictyoptera (Roth, 1967), venom secretion as in Hymenoptera (see King and Copland, 1969, for details), and milk secretion as in tsetse flies (Section II,H,4). Many glands producing secretions auxiliary to reproductive function can be cited in invertebrates: epidermal glands of nemertines which produce the gelatinous strings in which the eggs are enclosed, silk glands of spiders secreting silk to suspend egg cases, salivary glands of Periplaneta americana whose secretion is used to glue the ootheca to the substratum, mucous cells o f many nemertines and some polychaetes secreting a mucous sheath which envelopes the copulating members, and the femoral cement glands in male pycnogonids serving to fasten the eggs. These auxiliary sex glands, and also the exocrine glands that produce sex attractants and aphrodisiacs are beyond the scope of this article. Glandular elements that provide “alimentary” nourishment to the gametes, zygotes, or embryos come under the purview of accessory reproductive glands. But the nurse cells and follicular cells of invertebrate ovaries, vitelline glands of Platyhelminthes and Rotifera, the “gland cells” adjacent to the ovary of Entoprocta (cf. Mathews, 1962), ovisacs of Polychaeta, yolk cells of Lingula, nutritive tissue (yolk glands) of Gastrotricha, and the food ova of Nemertina, Antarctic Crinoidea, many Polychaeta, and prosobranch Gastropoda have been excluded inasmuch as they are directly involved in the supply of yolk which forms a structural component of the oocytes themselves. 11. General Survey

Accessory glandular elements vary from primitive secretory epithelial cells along the wall of the genital tract to complicated glandular masses consisting of many different types of tubules specialized to elaborate a spectrum of specific secretions. The classification of invertebrate accessory sex glands is beset with difficulties because their origin, structure, biochemistry, and functions, which could serve as criteria, are only incompletely understood; also, tubules differing in structure, biochemistry, and physiology may be grouped together to form composite glands (e.g., the mushroom gland in

ACCESSORY SEX GLANDS IN INVERTEBRATES

355

cockroaches). Some glands probably secreting the same types of substances (jelly or mucus, for example) have been given confusingly different names, (see Section II,H) and, conversely, glands whose homologies are not certain have been grouped under catch-all terms such as prostate. Our classification of accessory reproductive glands into gonadal, ductal, seminal, spermathecal, genitoatrial, penial, male collateral, and female collateral is tentative and only one of convenience, based mostly on locational morphology of the glandular elements in relation to the genital (or rarely urogenital) system. A. GONADALGLANDS The gonads, although not glandular in organization, may be endowed with secretory cells whose secretion may aid in the production of viable gametes. In Daphnia magna, the giant cells developed directly by polyploidy from spermatogonia apparently provide nourishment for the testicular cells (Zaffagnini, 1965). I n the crayfish Pontastacus leptodactylus, the cells of the testicular acini are secretory; the alcianophilic secretory substances appear to move down the vasa efferentia (Adiyodi and Adiyodi, 1974 unpublished data). In Lepidoptera, Diptera, Orthoptera, and Heteroptera, the apical cells or Verson’s cells, located usually apically in the testicular follicles, have a trophic function. In Chironomus plumosus, large trophocytes in the testis may be derived from whole germ cells; smaller trophocytes may result from division of germ cells. Nutritive cytoplasm is colored orange-red with Heidenhain’s azan, whereas germ cell cytoplasm stains bluish-red (Wensler and Rempel, 1962). Lusis et al. (1970) observed during spermatogenesis in the testis of the cockroaches B yrsothria fumigata and Gromphadorhina portentosa a mucoproteinaceous or mucopolysaccharide (Table I) secretion product in spaces among the developing gametes. Tubular epithelium is formed of thin cells with flat, elongated nuclei. The intratubular secretory product appears to be derived from epithelial cells, as some of the latter show inclusions with tinctorial characteristics similar to those of intratubular material. The neck region of each testicular tube contains groups of modified epithelial cells (neck cells) which secrete a lipoproteinaceous substance (Table I). During the presecretory phase the cells 25-30 pm in diameter have large, round, rather chromophobic nuclei approximately 15 p m in diameter. In cells in their secretory phase, the nuclei tend to become irregular or kidney-shaped, and the chromatin to be peripherally dispersed. Interestingly, intratubular material and neck cell secretion in cockroach testis show no acid mucopolysaccharides (acidic glycosamino-

356

K. G . ADIYODI AND R. G . ADIYODI

TABLE I NATUREOF SECRETORYMATERIALS IN THE TESTIS OF Byrsothria furnigata AND Cromphadorhina portentosao” Histochemical test

Neck cells

Intratubular material

Sudan black B (frozen sections) Sudan black B (paraffin sections) Nile blue sulfate Schultz test Digitonin Acid hematein Acid hematein after pyridine extraction Lux01 fast blue PAS PAS after saliva extraction Alcian blue (acid) Hale’s test MBE at pH 2.6-4.1 Toluidine blue metachromasia, below pH 4 Millon’s reaction Bromphenol blue Tetrasotized benzidine coupled with p-naphthol Coupled tetrazonium Dinitrofluorobenzene (DNFB) Dihydroxydinaphthyl disulfide Performic acid/alcian blue Performic acimictoria blue Aldehyde fuchsin, oxidation with KMNO, Aldehyde fuchsin, without prior oxidation Aldehyde thionine, oxidation with KMNO, Azo dye for acid phosphatase Calcium cobalt for alkaline phosphatase Naphthol AS acetate for esterases Steroid SP-dehydrogenase Ferric hydroxyaniline for P-glucuronidase Methyl green-Pyronine for RNA and DNA Methyl green-Pyronine control with RNase

” From Lusis et al. (1970). Reproduced by permission of the National Research Council of Canada. +, Positive; w+, weakly positive; -, negative. glucuronoglycans). It could be presumed from the nature of distribution of the mucoproteinaceous or mucopolysaccharide material that it serves during the formation and maturation of spermatozoa; the lipoproteinaceous material from the neck cells probably helps in the maintenance of mature spermatozoa, as this product accompanies the spermatozoa into the sperm duct. I n the mollusks Littorina and Montacuta, there is a close association between the cytophores (nurse

ACCESSORY SEX GLANDS IN INVERTEBRATES

357

cells) and developing spermatozoa. The cytophores and spermlike cells contribute to the nutrition of spermatozoa; in Littorina and Montacuta, cytophore-sperm association persists until after transference to the female (Fretter and Graham, 1964). Gonadal secretory cells may furnish nourishment to the female germ cells for their growth and maintenance. I n Coelenterata (Hydrozoa) and Turbellaria, some young oocytes are pressed into service, whereas in Cladocera some nurse cell-like entities, and in Stomatopoda the ovarian epithelial cells, serve this function. I n some nemertines the ovarian wall is provided with secretory cells; the secretions form capsules in which the eggs are accumulated. In Pseudoscorpionidea the ovarian secretory cells arranged in between the oocytes supply nourishment to the embryos. Accordingly, these cells become active after fertilization of the eggs within the ovarian cavity; the incubation chamber developed on the ventral surface of the female from secretions of special glands is in uninterrupted contact with the ovary, facilitating the flow of nourishment from the secretory cells to the developing embryos. In Areneida the internal epithelial cells of the ovary develop into oogonia, follicle, or secretory cells; the last-mentioned supply nourishment to the derived oocytes. Toward oviposition, the epithelial cells become particularly large and active and secrete into the ovarian lumen a cement which wraps and binds the eggs in the cocoon. In Nepidae (Hemiptera) a substance cementing the eggs originates in the ovarioles (Hinton,

1961). It may b e recalled here that in higher vertebrates the Sertoli cells of the testis secrete the testicular fluid which has a remarkable composition and a nutritive function (Setchell, 1970). Subsurface epithelial structures in the outer cortex of the canine ovary, which are in the form of solid and hollow groups and cords of epithelial cells, secrete mucin. Acid mucins containing sialic acid were observed by O’Shea (1966) as intracytoplasmic secretion droplets in subsurface epithelial structures.

B. DUCTALGLANDS 1. Spermiductal Glands Spermiductal secretions may serve, depending on the species, as semen, as spermatophore material, and for transportation of sperm or spermatophores. The vas deferens of hermit crabs is indeed very complex, having as many as nine functional regions (Mouchet, 1931); seven intergrading functional zones have been recognized in Pagurus

358

K. G . ADIYODI AND €3. G . ADIYODI

novae-zealandiae (Greenwood, 1972). The glandular epithelium, which is of varying thickness in different regions of the vas deferens, is enveloped by a thin muscular coat with overlying connective tissue. The sperm mass, as it passes down the vas deferens, is ensheathed by different secretions, producing a spermatophore. Studies by Kessel et al. (1969) indicate that inosine-5’-diphosphatase is apparently synthesized in the endoplasmic reticulum (ER) and transported to the Golgi saccules via Golgi vesicles in the crayfish vas deferens. The apparent presence also of glucose-6-phosphatase activity in the Golgi saccules suggests that Golgi complexes of ductal epithelial cells may be the loci where carbohydrates in the seminal plasma are synthesized. The spermiductal glandular epithelium of the crayfish P . leptodactylus shows clear seasonal cyclic activity, being much reduced in winter when the amount of semen stored in the sperm duct is low and the number of spermatozoa therein are few, whereas in autumn the sperm ducts are full of secretion (Adiyodi and Adiyodi, 1974 unpublished data). According to Melis (1966), the sperm duct of P . americana has a “monostratified” epithelium composed of secretory and cuticulogenous cells. Secretion in the sperm duct is said to consist of neutral polysaccharides. In houseflies and culicine mosquitoes, male accessory secretion passed out during copulation induces loss of female sexual receptivity and also enhances oviposition (see Riemann, 1973, for references). In houseflies (Musca), which have no accessory glands, unlike culicine mosquitoes, the accessory secretion composed of several electrophoretically resolvable protein fractions (Terranova et al., 1972) is produced and stored extracellularly in roughly the anterior third of the coiled ejaculatory duct. The cytology and cytochemistry of the glandular ejaculatory duct of Musca domest i c ~have been studied by Leopold (1970), and the ultrastructure in relation to maturation by Riemann (1973). The cells are mostly cuboidal in shape, 15-20 pm in depth, and have spherical nuclei and prominent nucleoli in pharate and newly emerged adults. Some synthesis of secretion as indicated by intense nucleolar and cytoplasmic pyroninophilia of the epithelial cells begins at about emergence, although storage is not evident before 4 hours after eclosion (Fig. 1). Toward the intima the plasma membranes of adjacent cells are often branched and bound by septate desmosomes. The cytoplasm of the duct cell has vesiculate rough endoplasmic reticulum (RER) enclosing fibrous material, numerous free ribosomes, many Golgi units having vesicles enclosing electron-dense material, sparsely distributed microtubules, and also some large vacuoles and lysosomelike

ACCESSORY SEX GLANDS I N INVERTEBRATES

359

FIG. 1. Transverse sections of the ejaculatory duct of Mnsco. (a) Pharate adult; (11) 12-hour postemergence adult; ( c ) 7-day postemergence virgin. Note the gradual increase in the storage space for secretion between the cells and the intima (arrows). (From Riemann, 1973.)

multivesiculate bodies. In pharate adults the apical surface of the duct cell, generally apposed to the intima, separates in some places, leaving spaces between the plasma membrane and intima containing electron-dense material. The duct cells become villate and actively secretory b y about 4-8 hours after emergence; storage space for a secretory product develops by a splitting off of the apical cytoplasm and its lysis below the intima. Leopold et al. (1971) showed by autoradiography that the ejaculatory ducts of M . domesticu incorporate into their stored secretion tritiated arginine and lysine. The degree

360

K. G . ADIYODI AND R. G . ADIYODI

of uptake of tritiated arginine into the ductal epithelium depends on the number of previous matings. Results of administration of actinomycin D and cycloheximide indicate that the different fractions of the accessory secretion are apparently synthesized on stable mRNA templates, and also that the production of stable mRNA begins shortly after emergence. From the account given by Riemann (1973), the different duct cells of M . domestica appear to make use of merocrine, apocrine, and holocrine methods of secretion. Unlike those of Musca, the paired ejaculatory ducts in the midge C. plumoms are glandular over their entire length, each being divided into four sections separated by plugs of tissue (Wensler and Rempel, 1962). Sections 1 and 3 are dark, 2 is light, and 4 is opalescent in unstained preparations. Tinctorial behavior and structure of secretory vacuoles in the epithelial cells, and luminal secretions, differ in each section, suggesting different types of secretions, although their specific functions are not understood. In the meloid coleopteran Lyttanuttali, the bulk of the spermatophore (the portion composed of jellylike material) is produced from the dilated glandular regions of the vas deferens lined by cuboidal-to-short columnar secretory cells (Gerber et al. 1971b).The vas deferens functions in semen production in Trichoptera also (Khalifa, 1949). The ability of the vas deferens to produce semen and spermatophoral components is perhaps widespread among insects. In the striped slug, Philomycus carolinianus, the epithelial lining of the vas deferens emerging from the spermoviduct is composed of high, columnar, apocrine cells with eosinophilic globules (Kugler, 1965). The secretions serve as a coating for the thread of semen. That some of the secretory cells in the spermiductal epithelium may be holocrine is indicated by the presence of cell nuclei similar to those of the epithelial cells in the semen coat. PAS-positive, amylase-fast, large, flask-shaped mucous cells, containing both acid and neutral mucopolysaccharides and voiding their contents into the lumen of the spermatic groove by thin necks interpolated in the lining epithelium, have also been reported in this species (Kugler, 1965). Identical cells are distributed from the loop of the hermaphroditic duct to the fertilization sac. Another type of subepithelial mucous cell, smaller in size and more numerous, but similar to the larger cells in staining reactions, has also been described in P. carolinianus. These cells, endowed with an efferent tubule having an inverted, funnelshaped opening, discharge their secretions into the spermatic groove, and also into the basilar ends of the prostatic tubules. In the snails Helix pomatia (Breucker, 1964) and Lymnaea stagnalis

ACCESSORY SEX GLANDS IN INVERTEBRATES

36 1

(Joosse et al., 1968), the gland cells in the wall of the spermoviduct are capable of resorbing the spermatozoa.

2. Oviductal Glands An increase in the complexity of female genital systems in multicellular animals resulted in a need for the safe transport of sex cells, zygotes, and embryos from the interior of the body to the exterior via complicated duct systems. One of the methods by which the safe transport of these products was ensured was to suspend them in a fluid medium or give them a fluid or semifluid coating. The substance generally used for this purpose is mucus or mucus mixed with other substances. The oviducts in general are profusely supplied with secretory cells which may also be aggregated in specific regions of the duct. In addition to mucus-secreting cells or glands, the oviductal epithelium may harbor secretory cells which provide protective membranes and nutritive substances for the ovum, zygote, or embryo. The presence of unicellular glands along the gonoducts is known even in Entoproctit. In Balanus balanoides, oviductal glands provide the egg sacs. The glands, which are located toward the terminal part of the oviduct, arise as epidermal invaginations soon after metamorphosis. Toward the breeding season the cells, which are columnar in shape, secrete a transparent, elastic material enclosed in a saclike structure. The secreted material later separates, and the sac attached to the mouth of the oviduct receives the eggs at oviposition (Walley, 1965). In insects the presence of glandular regions is reported in acridids in which the anterior part of the oviduct develops into a tubular gland. In Delphacidae the oviductal gland cells are known to secrete a foamy mucus which is white, blue, or red in color (Striibing, 1956). In L. nuttali, the ovarian calyces and oviducts are lined by epithelial cells which secrete a carbohydrate-protein complex, coating the eggs (Gerber et al., 1971a). In the wasp Dahlbominus fuscipennis, oviductal epithelial cells produce a lubricant for the passage of eggs (Wilkes, 1965), as do probably the median columnar cells in the common oviduct of mymarid Hymenoptera (King and Copland, 1969). P. E. King and J. G. Richards (unpublished observations, cited in King and Copland, 1969) maintain that the oviductal epithelial cells in Nasonia vitripennis may function in oosorption, based on their claim of the occurrence of acid phosphatase, leucine aminopeptidase, and esterase activity; it is also stated that the degree of reaction depended on how recently the eggs had been laid. Investigations along this line will be of much interest, inasmuch as in insects it is the follicle cells that are known

362

K. G . ADIYODI AND R. G . ADIYODI

to divide amitotically and invade the oocytes in their new capacity as vitellophages and absorb the yolk, possibly by enzymic lysis. In terrestrial slugs the hermaphroditic duct consists of two highly glandular tubes; the female duct is provided with oviductal glands which reach their peak development prior to oviposition (see Runhani and Hunter, 1970, for review). In pulmonate mollusks the section of the oviduct modified as the uterus is provided with glandular ciliated epithelium containing mucocytes. In aquatic gastropods the oviduct appears to originate as an open pallial groove richly provided with gland cells embedded in its thick walls (Fretter and Graham, 1964). In Arion ater, the oviduct secretes the outer calcium carbonate coating of the egg (Smith, 1965). Oviductal glands supply a membrane or capsule for the egg in cephalopods. Cuneiform ciliated cells may be interpolated at the luminal end of the oviductal glandular epithelial cells, as in Physa (Fig. 2A), in mollusks. Interestingly, in vertebrates the oviductal fluids play a vital role in sperm viability (Blandau, 1969). In the newt Notophthalmus uiridescens, the egg is enclosed in a “jelly” coat having five structurally and cytochemically different layers successively deposited by five rather distinct secretory zones in the oviduct (Humphries, 1966). In the frog Rana pipiens, the oviduct is richly supplied with jellysecreting glands and is divisible into six histochemically different zones. The jelly contains large amounts of acid, sulfated, and neutral mucopolysaccharides (Shivers and James, 1970). The goblet cells lining part of the avian oviduct secrete mucin containing both acid and neutral mucopolysaccharides (Guzsal, 1968). In the rabbit the oviductal fluid, which has a composition different from blood serum, is apparently secreted by the oviductal cells (Feigelson and Kay,

1972). Ultrastructural studies on invertebrate oviductal glands are few. Investigations on the fine structure of the chicken oviduct show that protein and carbohydrate fractions of ovomucin and ovalbumin are synthesized in the RER and Golgi complex, respectively (Trainis,

1968). Speciul Jell y-Secreting Glands. In the polychaete Scoloplos armiger, terminal glandular regions in the approximately 100 pairs of the nephridia of the female secrete a jelly which wraps the worms and their eggs (Chapman, 1965). The gland cells distributed around the nephridiopores are large and become engorged during the breeding period with aggregations (20-30 pm in diameter) of rod-shaped bodies, each 5-10 pm along the long axis. The cocoon formed from the jelly is pear-shaped or globular (1.0-1.5 cm in diameter) and may

ACCESSORY SEX GLANDS IN INVERTEBRATES

363

.cc

.gc'

A

^^

dtgc

E

-cc

"9C

gc

I

F

FIG.2. Accessory sex glands c r f Mollusca. (A) A portion of the oviductal epitheliuni of Phystr (Pulnionata); (B) a poItion of the epithelium of the prostate gland of an opisthobranch; ( C ) penial gland of Littorina (Prosobranchia); (D) strrichire of the preputial gland of P. fontinalis (Pulmonata); (E) albumen gland cells of Tritonia (Opisthobranchia); (F) a gland cluster of the capsule gland of Nucella (Prosobranchia). cc, Cuneiform ciliated cell; ctc, connective tissue cell; dgc, discharged gland cell; dtgc, discharging tip of gland cell; ep, epidermis; gc, gland cell; lp, lumen of prepuce; mg, main gland; ml, muscle layer, ngc, neck of gland cell; sg, subsidiary glands; wp, wall of prepuse. (After various sources from Hyman, 1967.)

have about 500 to 1200 eggs. Organic substances, which constitute only 1% of the jelly, are in the form of a mucoprotein having 15 amino acid residues in the protein moiety, the carbohydrate part consisting of glucosamine, galactosamine, glucose, and fucose. I n many

364

K. G. ADIYODI AND R. G . ADIYODI

lower prosobranch mollusks, special subepithelial gland clusters called jelly glands, which resemble structurally the capsule gland of higher Prosobranchia, are located at the terminal part of the right ureter in the female (Fig. 12C). These apparently provide a gelatinous coat for the eggs as they leave the right ureter and nephridiopore.

C. SEMINALGLANDS In the male, although the sperm duct itself can serve for storage of sperm, portions of the sperm duct may be specially modified for this purpose into one or more vesicles or pouches; rarely, seminal vesicles may also arise from the ejaculatory duct. Epithelium of the seminal vesicle may be thin and one-layered as in P . americana, or multilayered as in Schistocerca gregaria. The swollen seminal vesicles of balanomorph barnacles may reach about 4% of the size of the adult barnacle. In Balanus balanus the seminal plasma, rich in cystine, is a yellowish fluid with an ionic composition similar to that of sea water, with the exception of potassium. Forty percent of the total solids in the seminal plasma consists of inorganic ions, and of the rest 35% is protein (Barnes, 1962). The seminal plasma from the vesiculae seminales of P . americana has glycogen, glucose, phospholipids, PAS-positive substances not destroyed by acetylation, and neutral and bound lipids, but is deficient in tyrosine-containing protein and acidic nonsulfated and sulfated mucopolysaccharides (Vijayalekshmi and Adiyodi, 1 9 7 3 ~ ) The . seminal vesicle of the guinea pig secretes sizable amounts of free glucose and fructose (Fouquet, 1971), whereas that of the giant fruit bat, Pteropus giganteus, secretes only fructose (Rajalakshmi and Prasad, 1970). The seminal plasma of the drone honeybee, derived from the seminal vesicles, mucous glands, and penis bulb, is reported to contain at least three free sugars, namely, fructose, glucose, and trehalose (Blum et al., 1962). Our studies show that the epithelium of the seminal vesicle of P . americana is low in protein and deficient in neutral lipids and acid mucopolysaccharides, but rich in glycogen and phospholipids. The near histochemical identity between the epithelium of the vesiculae seminales on the one hand, and the seminal plasma on the other (Table 111), suggests that the seminal plasma of P . americana may be, if not fully at least in part, intrinsic in origin. Seminal vesicles of some species may function more as sperm reservoirs and show little recognizable secretion, for example, gland 16 (a functional seminal vesicle) described by Odhiambo (1969a) in the desert locust, S . gregaria. RER and Golgi units are sparse and small. These features, together with the presence of free ribosomes and

ACCESSORY SEX GLANDS IN INVERTEBRATES

365

their aggregates in the cytoplasm, suggest that the cells are mainly concerned with their own growth and maintenance and not in the production of any secretion for export. However, Odhiambo’s (1969a) observation that the somewhat abundant smooth-surfaced E R cisternae of gland 16 contains a secretory product of unknown composition is of some interest. Membrane-bound vacuities with varied contents and presumably representing remnants of a necrotic process are present in the cytoplasm, particularly apically adjoining the gland lumen. Slightly acidophilic vesicular bodies released into the gland lumen may represent these cytoplasmic vacuities, or fragmented and lysed apical cytoplasm.

D. SPERMATHECALGLANDS Secretions of the spermatheca provide nutrients for the sperm stored therein (Bhatnagar and Musgrave, 1971) and maintain sperm viability (Tulsyan, 1966). Spermathecal cells also have the capacity to resorb excess germ cells. In the black widow spider, Latrodectus curacaviensis, spermathecal secretion may increase the hydrostatic pressure and help eject the sperm through the fertilization tubes (Bhatnagar and Rempel, 1962). The cells in the secretory portion of the spermatheca of Rhodnius prolixus are columnar, having a smooth intima and possible interdigitations between adjacent cells. The ovoid nuclei located basally are frequently lobulated and have prominent nucleoli (Davey and Webster, 1967). The cells are actively secretory; osmiophilic droplets first appear in the midregion of the cell above the nucleus and coalesce to form a distinctly PASpositive conical region surrounded by a less dense PAS-negative zone. Apically, the dense material fragments into secondary strands which merge into a zone of medium density. The secretion in the lumen is a mucoprotein or glycoprotein. Studies by Bhatnagar and Musgrave (1971) on the spermathecal gland of the granary weevil, Sitophilus granarius, indicated in the gland, as well as in the lumina, glycogenous polysaccharides, glycoproteins, and phospholipids, but no acid mucopolysaccharides (Table 11). In the midge C. plumosus, the spermathecae are in the form of spherical capsules lined with a thick chitinous intima; the wall is formed of columnar epithelial cells having vacuolated cytoplasm and large spherical nuclei (Wensler and Rempel, 1962). The wall of the duct, which is surrounded by longitudinal muscles, is composed of (1)small epithelial cells which probably secrete the chitinous intima of the duct, and ( 2 ) large special secretory cells having one or two prominent nuclei and one or two flame-shaped ductules to drain the secretions. Ultrastructural studies on the spermatheca of the mosquito Aedes

K. G. ADIYODI AND R. G. ADIYODI

366

TABLE I1 HISTOCHEMISTRY OF THE SPERMATHECAL GLANDOF THE GRANARY WEEVIL, Sitophilus granarius" ~

~

Staining method

Indicated activity

Result

Polysaccharides, adjacent Strongly positive (only weakly positive after glycol or aminohydroxy salivary amylase groupings digestion Positive Best's carmine, Benseley Glycogen in tissue modification sections Negative Alcian blue, Mowry and Acid mucopolysaccharides Johnson modification Negative Acid mucopolysacMayer's test charides Positive Millon's reaction, Benseley and Proteins containing Gersch modification tyrosine Weakly positive Mercury-bromphenol blue, Proteins after Bonhag Positive Proteins containing Diazotization coupling, after Glenner and Lillie tyrosine Nuclei strongly positive Feulgen reaction, after Feulgen DNA; cytoplasmic and (extranuclear Feulgenthe basic proteins and Rossenbeck positive material) Strongly positive Congo red, after Bennhold Glycoprotein (amyloid) material Positive Crystal violet, after Lieb Glycoprotein (amyloid) material Positive Sudan black B, after McManus Lipids and lipoproteins Weakly positive Fettrot 7 (B), after Pearse Neutral fats Positive (negative after Acid hematein Phospholipids pyridine extraction)

PAS, after McManus

a

Adapted from Bhatnagar and Musgrave (1971).

aegypti show the presence of a group of gland cells located at the origin and scattered along the proximal part of the spermathecal duct. The secretory cells release their products through cuticle-lined ductules; at one end the gland cells possess microvilli. Protein synthesis occurs in the ribosome-rich gland cells (Clements and Potter, 1967). In the honeybee, Apis mellifica, the spermathecal gland cells have basophilic cytoplasm rich in protein. Intracellular canaliculi positive to paraaminosalicylic acid, collect and drain the secretion into the lumen. The intima is in the form of an amorphous cytoplasmic mass (De Camargo and Mello, 1970; Poole, 1970). Immunologically identical protein fractions were detected in the spermathecal fluid and blood (Lensky and Allimot, 1969),suggesting the possible absorption

ACCESSORY SEX GLANDS IN INVERTEBRATES

367

of proteins by the sperniathecal cells from the hemolymph. In Opisthobranchia and Pulmonata the spermathecal secretion can dissolve the wall of the spermatophore (Quick, 1960). I n the slug P. carolinianus, the spermathecal epithelium shows apocrine secretion. The secretory products contain glycogen and an alcianophilic substance (Kugler, 1965).

E. GENITOATRIALGLANDS In the wall of the genital atrium of P. caroliniunus occurs, beneath the muscularized connective tissue layer, a thick layer enclosing the venters of large flask-shaped mucous cells with large multinucleolated nuclei; proximal to the genital aperture, the large cells are replaced by smaller flask-shaped ones with compact, deeply stained nuclei. The mucous cells, both large and small, in the genital atrium show some alkaline phosphatase activity. They are presumed to contain neutral but not acidic mucopolysaccharides, inasmuch as these cells are PAS-positive and amylase-fast, but exhibit little thionine metachromasia and react negatively with alcian blue. Secretions of genitoatrial glands may facilitate oviposition, and also eversion of the penis during copulation, both by lubrication, and also serve as a fluid transit medium for sperm received during copulation (Kugler, 1965). The spermatophoral receptacle, which is formed as an evagination of the vagina and is attached to it by a duct, serves for the storage of spermatophores and the digestion and absorption of spermatophoral materials in the meloid beetle L. nuttalli. A cuticular intima limits the cuboidal and columnar epithelial cells (Gerber et al., 1971a). It is not known whether or not secretion of digestive enzymes is the function of only certain cells. Nuclei of the epithelial cells are large, and the cytoplasm shows irregular masses of basophilic material. Some cells show near the apex many vacuoles which are not positive to Millon’s test, hematoxylin, or the PAS reaction. The latter may represent not a distinctive cell type but only a different phase of secretion of the same cell type in the gland, according to Gerber et al. (1971b). These investigators observed cells indicating secretory and absorptive activity in close proximity to one another in the spermatophoral receptacle epithelium, but furnish no cytological criteria for distinguishing them.

F. PENIALGLANDS I n some prosobranch mollusks (e.g., Littorinidae), the penis may bear a row of glands. The penial glands range from simple invagina-

368

K. G . ADIYODI AND R. G . ADIYODI

tions to complicated formations consisting of a pyriform main gland into the lumen of which may open a ring of subsidiary glands (Fig. 2C) (see Hyman, 1967, for review). Glands may also be attached to accessory copulatory organs. Preputial glands formed of groups of long-necked subepithelial cells with their necks filled with secretion granules have been described by Duncan (1958) in the penial apparatus of the snail Physa fontinalis (Fig. 2D). The term sarcobelum has been applied to the glandular hillock, which is apparently stimulatory in function and projects into the prepuce in pulmonates such as Ancylidae, Lymnaeidae, and Planorbidae. A preputial organ in the form of a cup-shaped glandular body with a coiled duct is present in Planorbidae. It opens at the junction of the prepuce and the penis sheath and probably serves as a holdfast in copulation. Resembling the penial glands, although not homologous, are the gland cells in the (pedi)palpal (copulatory) organs of spiders (Osterloh, 1922; Harm, 1951; Bhatnagar and Rempel, 1962). Rosetteshaped glands surrounding the seminal receptacle in Segestria bavarica are believed b y Harm (1951) to pour their secretions into the bulb and force the exit of sperm. Many globules and granules are present in the columnar gland cells distributed along the inner margins of the fundus and the reservoir in the black widow spider, L. curacaviensis (Bhatnagar and Rempel, 1962). GLANDS G. MALE COLLATERAL Many invertebrates show a variety of specialized accessory glands, paired or unpaired and generally ectodermal or mesodermal in origin, as adjuncts to the genital tract. The gross morphology, histology, histochemistry, and ultrastructure of male collateral glands have been explored in greater detail in insects than in other groups. Studies dealing with the cytomorphology of male collateral glands in different orders of insects include those of Khalifa (1950), Callahan and Cascio (1963), Suseela (1965), Roth (1967), Odhiambo (1969a), Gerber et al. (1971b), Louis and Kumar (1971), Hartmann (1971), Jaiswal and Naidu (1972), and Vijayalekshmi and Adiyodi (1973b,c). 1. Prostate The term prostate has been applied to male accessory sex glands of Platyhelminthes, Rotifera, Oligochaeta, Hirudinea, Gastropoda Mollusca, and some Insecta (e.g., the glandula prostatica in B . mori), although the homologies and analogies of these glands are uncertain, The prostatic apparatus of Turbellaria (called prostatoids, pyriform organs, musculoglandular organs, or apioid organs, when multiple)

ACCESSORY SEX GLANDS IN INVERTEBRATES

369

consists of gland cells (prostatic or granule glands) which void their contents into the ejaculatory duct, penis base, or a muscular prostatic vesicle. The chemistry of the generally granular secretion of these gland cells is not known. In some species prostatic secretion is of two types: finely and coarsely granular. Information available on aspects of the morphology and location of the prostate in Platyhelminthes has been summarized b y Hyman (1951) and in Oligochaeta by Avel (1959), but we know little about the cytology and ultrastructure of these glands. In the male rotifer Asplunchnu brightwelli, the secretions of the prostate gland serve apparently to form a bond between the copulatory organ of the male and the integument of the female during copulation (Aloia and Moretti, 1973). The prostate, composed of unicellular exocrine glands, reminiscent of individual epithelial goblet cells, extends in a spiral fashion over the whole length of the copulatory organ and shows some regional differences in ultrastructural details. Sections through the posterior zone of the prostate gland reveal several autophagic vacuoles, 3-5 p m in diameter and containing degenerating mitochondria, concentric membranous whorls, and vesicles bound by single membrafles (Fig. 3). The limiting membrane of the autophagic vacuoles is in spatial intimacy with small single membrane-bound vesicles and Golgi cisternae. Secretion granules differ in electron density, indicating that they either may represent different stages of maturation or are different in chemical composition. The mechanism of secretion appears to be a form of exocytosis. In actively secreting prostate gland cells, a large apical vacuole filled with some amorphous material is found at the apex of the gland cell; the amorphous material in the apical vacuole is continuous with a similar substance covering the microvillar formations basally in the urethra (Fig. 4) (Aloia and Moretti, 1973). The prostate of mollusks is an enlarged glandular region, or in the form of a few to many diverticula in the male duct, or in the form of a tubular gland appended to the common duct with a cavity often called the male groove. The gland enlarges before copulation, and the sperm mix with the prostatic secretion. I n opisthobranch mollusks (Fig. 2B) the prostatic portion of the sperm duct is formed of tall secretory cells which alternate with cuneiform ciliated cells. Secretory granules in the prostate vary in size and are often eosinophilic; in some species such as Elysia, the cells may present a “vacuolated’ appearance (Hyman, 1967, for review). In acetonid opisthobranchs prostatic cells become purple-blue and nuclei bright red with Mallory and Heidenhain stains. In Weigert iron-hema-

370

K. G . ADIYODI AND R. G. ADIYODI

FIG.3. Electron micrographs of the posterior zone of the prostate gland cell of the rotifer A. brightwelli. The autophagic vacuoles [three are shown in (b)] contain many mitochondria and also membranous bodies. The cytoplasm of the cell shows numerous Golgi apparatuses sectioned transversely (G), and also secretory granules (SG) differing in electron density. A lone strand of RER can b e seen running parallel to the gland cell periphery. N, Nucleus. (a) x 3520. (b) x 7600. (From Aloia and Moretti, 1973.)

toxylin and van Gieson preparations, the cytoplasm shows yellowbrown spherules (Rudman, 1972). In P. carolinianus, the tubules of the prostate gland, which are unbranched and compact, have a cytology similar to those of the Opisthobranchia (cf. Kugler, 1965). Cell membranes, as well as the cytoplasmic reticulum of the prostate cells of this species, are rich in alkaline phosphatase. Two types of epithelial cells, one secretory and the other nonsecretory, evenly interspersed and differing in their ultrastructure, have been distinguished in the prostate of some Gastropoda (Quattrini, 1967; Quattrini and Fondi, 1969). In Aplysia depilans, the prostate is not well differentiated; the cells distributed along folds in the wall of the oviductal groove of its common duct reportedly resemble the prostatic cells of Milax gagates, Vaginulus borellianus, and Laevicaulis alte. Joosse et

ACCESSORY SEX GLANDS IN INVERTEBRATES

37 1

FIG. 4. Electron micrograph of the prostate gland cells from a copulating rotifer, A . brightwelli. Note the apical vacuole (AV) situated apically in the gland cell. The vacuole is filled with an amorphous secretory product, which is continuous with (arrow) a similar substance in the urethra (UR). X and Y are empty secretory granules, but Y has apparently the same density as the apical vacuole and also contains a similar amorphous product. x6720. (From Aloia and Moretti, 1973.)

al. (1968) found in L. stagnulis that starvation results in a regular decrease in the weight of the prostate, the decrease being rapid during the fifth week of inanition. It has not been explained whether the loss in weight, which is regained on refeeding, is due to stoppage or slowing down of the synthesis and/or release of secretions by prostatic cells, or the dissolution of the cells themselves to maintain the deteriorating metabolic pool.

2. Mushroom Gland Besides the functional seminal vesicle, we have distinguished four types of tubules (Vijayalekshmi and Adiyodi, 1973b,c) in the mushroom gland of P . umericana based on morphological, cytological, and cytochemical parameters: utriculi majores (UM), utriculi breviores (UB), utriculi intermedii (UI), and utriculi translucentes (UT). The histochemistry of the mushroom gland as described by Vijayalekshmi and Adiyodi (1973~) is summarized in Table 111. Mucopolysaccharides appear to be one of the important constituents of

TABLE I11 HISTOCHEMISTRYOF THE MUSHROOM-SHAPED (MSG) AND CONGLOBATE GLANDS (CG) OF P. americanaa*b MSG

vs

UB Stain or histochemical technique employed Feulgen reaction Mercury-bromphenol blue Millon's reaction Oil red 0 Sudan 111 and IV Acetone Sudan black Nile blue Best's carmine Best's carmine after digestion in saliva at 37°C for 3 hours Alcian blue, after Steedman Alcian blue, 1.0 procedure PAS Alcian blue followed by PAS Red Bluish-red Blue

UI

Secretion

Epithelia

++ ++

++ + +

+ -

_-

ND ND

*

ND ND

+?

2

+? +?

Seminal plasma

Epithelia

++

"

++

+* ++ ++

Epithelia

++

* *

ND ND

+++

++ ++ +*

++ ++*

++*

+++

+*

-

ND ND

++

++

++

++

+ + ++

++

ND ND

ND

ND

ND

ND

--

ND

ND

ND

++

+++ ++

++

---

+

++

++? ++

++f

++ -

+

++

++

+

________

a

Secretion

UM

From Vijayalekshmi and Adiyodi ( 1 9 7 3 ~ ) . --, Trace; -, low; +, medium; +, fairly strong;

+

++

-

Secretion

++ ++f

+++ +++ ++

++*

Epithelia

Secretion

f f

ND ND

f

ND ND

-

+ ++* +"

?

+* -

*

++ ++ ++

+++ +

++ + + +++

++

+ + + ++*

ND

ND

ND

ND

ND

''

ND

ND

ND

+

+

~ _ _ _

++ strong; +++, very strong; ?,

doubtful; N D , not detected.

-

++ ++ ++

+

+ +

++

+*

-

+

++ ++

Epithelia

+++ ++*

++ +

+

Epithelia

++ * +

?

+

CG -

UT

++

-

++

+?

ND

ND

ND

ND

++

+

ACCESSORY SEX GLANDS IN INVERTEBRATES

373

the accessory secretions in male insects. In Leptinotarsa decemlineata (De Loof and Lagasse, 1972) and S . gregaria (accessory reproductive gland 4),secretion is in the form of a neutral mucopolysaccharide; in Schistocerca, some gland types possibly also produce acid mucopolysaccharides. Some tubules of the mushroom gland of P . americana contain acidic sulfated and nonsulfated mucopolysaccharides (Table 111). The wall of the spermatophore is kneaded mainly out of secretions of the UM, although secretions of the seminal vesicles and apparently also of the UT and the UB enter the spermatophore. Secretion appears to be merocrine in the mushroom gland, and the storage of secretion mostly extracellular. This is a point in which the mushroom gland differs from the conglobate gland. The latter stores most of its secretion intracellularly in the thick glandular epithelium and releases the secretion probably under

FIG.5. Electron micrograph of the epithelium of the UM in the mushroom gland of male P. americana. Note the involutions of basal plasmalemma (PI) and intranuclear granules (ING) in the nucleus (NU). M, mitochondrion; T, T system. (From R. G. Adiyodi and K. G. Adiyodi, 1974.)

374

K. G. ADIYODI AND R. G . ADIYODI

ACCESSORY SEX GLANDS IN INVERTEBRATES

375

copulatory stimuli. In the mushroom gland the ulpastructure of only the UM has been studied (Adiyodi and Adiyodi, 1974). The tubules are lined by a single layer of gland cells having basal plasmalemmal infoldings and apical microvilli (Fig. 5). The enveloping layer of thin, slow-acting muscle, which probably plays some role in the extrusion of the luminal contents, is predominantly monomyofibrillar and has a poorly developed T system, sarcoplasmic reticulum, and striations. Monomyofibrillar muscles have also been reported in the male accessory sex gland complex of S . gregaria (Odhiambo, 1970) and L. decemlineata (De Loof and Lagasse, 1972). The proteinsynthesizing and -exporting machinery of the U M is extensively developed as judged by the abundance of RER and active Golgi elements (Fig. 6). Secretory granules pinched off from layered cisternae of the Golgi complex are more abundant apically in the glandular epithelium, that is, in the vicinity of the luminal surface. Inasmuch as the cytoplasm adjacent to the utricular lumen contains mostly free ribosomes and little RER, much of the synthesis of secretory proteins presumably takes place in the interior of the cell. The sulfonation of acidic mucopolysaccharides possibly occurs in the Golgi complex (cf. Peterson and Leblond, 1964). The mode of release of secretory granules is not known, but it is likely that the microvilli present on the U M and also in the male accessory reproductive glands of Schistocerca (Odhiambo, 1969a) and Leptinotarsa (De Loof and Lagasse, 1972) provide an adequate surface area for secretory transfer in ways not yet understood. Differences in size, shape, and electron density of luminal secretory bodies in the U M (Fig. 7) of P . americana may be related to the chemical diversity of these products (Table 111); possibly, the secretory granules in the epithelial cells undergo some structural and/or chemical change on release into the lumen.

3. Collateral Gland Complex of Locusts In the males of S . gregaria, the accessory sex gland apparatus consists of two gland masses, each composed of 16 glands which are of 9 distinct types based on histological, histochemical, and phase-con-

FIG. 6. Electron micrograph of the secretory epithelium of the UM in the mushroom gland of male P . arnericana. Note the budding off of dense secretory granules (SG) from parallel cisternae of the Golgi complex (G), RER, Rough endoplasmic reticulum. (From R. G. Adiyodi and K. G. Adiyodi, 1974.) FIG. 7. Electron micrograph showing secretory bodies (SS) embedded in a “coagulum” (GS) in the lumen of the UM of P . americana. (From R. G. Adiyodi and K. G . Adiyodi, 1974.)

376

K. G. ADIYODI AND R. G . ADIYODI

trast features, and on ultrastructural details of the glandular epithelia and the secretions produced by them (Odhiambo, 1969a). Gland 1 produces a proteinaceous crystalline secretion; glands 2 (Fig. 8), 4, and the “homogeneous” glands (7-10 and 13-15) a minutely fibrous secretory product; gland 3 an acidic lipoprotein secretion; and glands 6, 11, and 12 a globular secretion; gland 16 constitutes the functional seminal vesicle as described in Section I1,C. Specificity in the subcellular organization of the secretory machinery can only be expected, in view of the specificity of the secretion of different types of glands. Thus Golgi elements are obscure in gland 3 which secretes an acidic lipoprotein complex; in gland 4 (Fig. 9), the secretion of which is in the form of a neutral mucopolysaccharide, Golgi elements are well developed and widespread. Similarly, in homogeneous glands, which produce a glycoproteinaceous secretion, the RER is largely in the form of flattened parallel arrays of cisternae, whereas in gland 1, which produces a highly crystalline glycoproteinaceous secretion, the RER is massive and is composed of enormously swollen cisternae. Golgi elements of gland 1 consist of stacks of saccules, centrally located condensing vacuoles, and a constellation of small, smooth-surfaced peripheral vesicles. The RER and Golgi complex appear to b e jointly involved in the production of glycoproteinaceous secretion in ways not yet fully understood. It is not clear how exportable proteins synthesized by membrane-associated ribosomes find their way into the Golgi apparatus; possibly, the constellation of peripheral smooth vesicles act as “shuttle carriers” between the RER, the smooth-surfaced ER adjoining the Golgi apparatus, and the condensing vacuoles (Odhiambo, 1969a). An extrusion process of the macroapocrine type has been observed in gland 2 (Fig. 8). Multivesicular bodies containing several large secretory vesicles and many small, smooth vesicles develop at the luminal plasmalemma in between the microvilli. Finely fibrous secretion contained in the vesicles is released into the lumen by the rupture of the multivesicular bodies and the apparent lysis of the vesicles. Microtubules are of common occurrence in mature accessory reproductive glands of s. gregaria (Odhiambo, 1969b); some have also been encountered in the UM of P . americana (Adiyodi and Adiyodi, 1974). The microtubules may serve a mechanical function, forming a scaffolding for the cytoplasm of the gland cells, as it were. It is possible that the microtubules also function as channels of transport for small molecules within the cell, as suggested by the spatial intimacy between microtubules and some cytoplasmic organelles (Odhiambo,

1969b).

ACCESSORY SEX GLANDS IN INVERTEBRATES

377

FIG.8. Diagram of the fine structure of a gland-2 cell in the accessory reproductive gland complex of the locust S. gregariu. bv, Coated vesicle; cb, cytoplasmic body; cv, condensing vacuole of the Golgi complex; er, endoplasmic reticulum; G , Golgi apparatus; lum, lumen; ly, lysosonielike body; m, mitochondrion; mv, microvilliis; mvb, multivesicular body; mtb, bundle of mucrotubules; np, nuclear pore; rb, ribosome; tb, terminal bar, v, vesicle. (From Odhiambo, 1969a.)

FIG.9. Diagram of the fine structure of the gland-4 cell in t h e accessory reproductive gland complex of S. gregnria. cb, Cytoplasmic body; er, endoplasmic reticulum; G , Golgi apparatus; m, mitochondrion; mtb, bundle of microtubules; n, nucleus; pm, plasma membrane; SG, secretory vesicle; tw, terminal web region; v, vesicle. (From Odhiambo, 1969a.)

ACCESSORY SEX GLANDS IN INVERTEBRATES

379

4 . Conglobate Gland The conglobate (phallic) gland of cockroaches is a club-shaped ectodermal structure which opens separately to the outside by a duct. The lumen of the gland is lined with cuticle containing epi-, exo-, and endocuticles and a layer of epidermal cells. Ductules penetrate the cuticular layer and establish continuity with intracellular ductules in gland cells. The epicuticular layer of the ductule becomes highly reticulated on entering the gland cell, possibly for the passage of secretion. The microvilli abut on this reticulate layer. I n the luminar border of the gland cells, rows of small vesicles occur rather frequently in association with the microvilli; these small vesicles apparently constitute units of a transport system (Beams et al., 1962). The microvilli-ductule system of the conglobate gland is comparable to the “end apparatus” described by Mercer and Brunet (1959) in type I1 and type I11 cells in the colleterial gland of the cockroach. Ergastoplasm of the conglobate gland consists of vesicular elements and numerous rosettes or aggregations of small ribosomes. Golgi elements are not particularly abundant in the gland cell. Mitochondria1 accumulations occur below and in close proximity to the base of the microvilli. Secretion bodies with regions of parallel lamellae may achieve large proportions (Beams et al., 1962). The epithelium of the conglobate gland is moderately positive to Millon’s test and mercury-bromphenol blue. Oil red 0 and Sudan I11 and IV staining procedures indicate low to moderate amounts of neutral lipids, whereas with the acetone-Sudan black procedure moderately high amounts of bound lipids are demonstrated. Free aldehydes are scarce or absent; the abundant PAS-positive reaction of the gland epithelium appears to be related to phospholipids and possibly also unsaturated lipids (Vijayalekshmi and Adiyodi, 1973~). Plasmalogens abound in the conglobate gland of P . americana (Adiyodi and Adiyodi, 1972). The epithelium is also fairly rich in periodate-reactive alcianophilic components (Table 111). The function of conglobate gland secretions is not known, although Gupta (1946) maintains that the outer layer of the spermatophore is contributed by the conglobate gland. We have histochemically detected peroxidase activity in this gland in mature adults (Adiyodi and Adiyodi, 1970); homogenates of gland tissue yielded two peroxidase isozymes under acid (pH 4.5) and two under alkaline (pH 8.9) conditions of electrophoresis on polyacrylamide gels.

380

K. G . ADIYODI AND R. G . ADIYODI

H. FEMALE COLLATERALGLANDS 1. Mehli’s Glands Mehli’s gland, although often referred to as the shell gland, apparently secretes no shell. The gland, which may serve in the lubrication of the female sex duct, possibly occurs in Platyhelminthes, encircling the part of the sex duct generally known as the ootype. In the trematode Corpop yrum, two types of secretory cells were light microscopically detected. One type located near the vitellogenic duct appears basophilic with azan, and the other, which is more numerous, is amphiphilic (Del Conte, 1970).All the cells contain alcian blue-positive and testicular hyaluronidase-resistant substances which are metachromatic with toluidine blue. The basophilic cells have in addition abundant glycogen and possibly also mucosubstances. The secretory cells possess elongated processes which terminate in between the epithelial cells of the ootype. In the final stages of release, the secretion appears to be in the form of acid epithelial mucins and neutral mucosubstances (Del Conte, 1970). In the liver fluke, Fasciola hepatica, the gland shows peripherally large cells which appear to be secretory, and also some smaller cells in the interior of the gland. The peripheral cells possess large nucleoli, cytoplasmic basophilia, ER, secretory granules, and sizable numbers of ribosomes. Mitochondria are common, and small vesicles and structures resembling Golgi complexes are randomly observed in close association with secretory granules and mitochondria. The cells appear to be holocrine in nature. The centrally located cells show few signs of secretory activity, but are basophilic and contain ribosomes, mitochondria, and a few dense bodies (Walborg and Bjorkman, 1965). In Huematolochus medioplexus, Mehli’s gland shows acid phosphatase and glucosaminidase activity (Bogitsch, 1970). Ultrastructural studies show that the acid phosphatase activity is associated with multivesicular bodies in secretory cells which produce membranous bodies, and also in the plasma membranes of the gland cell ducts. The multivesicular bodies, which apparently originate from the Golgi area, supposedly have a lytic function and control, as it were, the overproduction of secretory products. The second type of secretory cell associated with Mehli’s gland produces dense bodies which are positive for macromolecular diglycols. Possibly, these bodies are responsible for the mucus secreted into the ootype (Bogitsch, 1970).

ACCESSORY SEX GLANDS IN INVERTEBRATES

38 1

2. Clitellar Glands The clitellar glands of Oligochaeta resemble the molluskan albumen glands (Section I,5) in that one of their major secretory products is albumen which is likewise used as a nutritive substance during embryonic development. The secretion is also used as mucus in copulation and in cocoon wall formation. The position of the clitellar region varies. It is located either in the same region as the genital pores, as in aquatic forms, or posterior to the gonopores, as in lumbricids. The clitellar region is characterized by the presence of swollen epidermis containing unicellular glands. This modified epidermal region encircles the body almost completely, or only partially (dorsolaterally). Three types of secretory cells can be distinguished in the clitellum: mucocytes, cocoon-secreting cells, and the more numerous albumen-secreting cells. The mucus elaborated by the mucocytes wraps the mating pair. The cocoon-secreting cells are in the form of unicellular glands situated below the epidermal layer; their secretion is globular, rich in amino sugars. The wall of the cocoon has the physical properties of chitin, although this is not borne out by x-ray diffraction studies. The albumen-secreting glands, which are located beneath the other two types of cells, are in the form of large cells arranged in several layers. The thickness of the clitellum is mainly due to the albumen-secreting gland layer. The secretion, rich in albumen, also includes mucus and amino sugars or their derivatives (see Avel, 1959, for review). The clitellar glands are not directly connected to the genital system and are therefore not strictly accessory sex glands, but we include them here in view of their often close association with genital segments and the key role of their secretions in reproduction. Aspects of cocoon formation in leeches have been summarized by Mann (1962). In the leech Erpobdella, sucker glands are known to secrete materials which plug the holes at either end of the egg capsule (Knight and Hunt, 1974).

3. Colleterial Glands Colleterial glands, which may vary in location, structure, and function, occur in association with the female reproductive system of many insects. Their products coat the surface of eggs and help in their attachment to the substratum, as in Hemiptera, Neuroptera, Lepidoptera, and Hymenoptera, or furnish a gelatinous coat which swells on contact with water, as in Plecoptera, Ephemeroptera, Trichoptera, Chironomidae, and some Odonata, or produce an elaborate silky egg cocoon, as in the Hydrophilidae (King and Copland, 1969).

382

K. G . ADIYODI AND R. G . ADIYODI

In Dictyoptera the eggs are enclosed in an ootheca, the raw materials used in its formation being the products of a pair of accessory sexglands, the right and left colleterials. In P . americana these glands are located in the dorsal vestibular wall and formed as invaginations of the glandular epithelium. The gland generally consists of dichotomously branched tubules. The left gland has a milky white appearance, whereas the right is comparatively translucent or yellowish. The right and left glands also differ in size, and in the number of branches present. Depending on the requirements of the individual, the colleterial glands are either enlarged or atrophied (Rosay, 1968). In Leucophaea muderae, which produces thin ootheca, the colleterial glands are small in size, while P . americana, which produces dark, thick ootheca, has proportionately large glands. In P . americana, each tubule of the left colleterial gland contains four types of cells: basal epidermal cells which line the main duct of the gland, structural protein-secreting cells, oxidase-secreting cells, and structural protein with calcium oxalate-secreting cells. The structural protein-secreting cells resemble closely the active proteinsecreting cells of mammals. A well-developed RER and tiny, rounded, smooth vesicles are observed. The apex of the secretory cell has an end apparatus. The invaginated surface of the cell forms elongated, narrow, and radially arranged processes directed into a funnellike structure formed from the intima lining the glandular lumen. Small globular secretory substances are visible in the cavity of the end apparatus. The chitinogenic cells, in keeping with the polysaccharide nature of their products, have a slightly different structure, RER being not so well developed. The cells supposedly responsible for the production of oxidase possess cytoplasm rich in mitochondria. It appears that the mitochondria have a role in the formation of the enzyme and pass it on to the tubules of the end apparatus (Brunet, 1952; Mercer and Brunet, 1959). The major oothecal proteins of P . umericana secreted by the left colleterial gland are of two types: water-soluble and water-insoluble. The latter have a globular form under the electron microscope (Pau et al., 1971). Unlike P . americana, the colleterial gland of mymarid Hymenoptera has only one type of cell (King and Copland, 1969). Also an end apparatus has not been observed in these cells. Chitinogenic cells are also apparently absent, the cuticular lining of the lumen probably originating from the gland cells themselves. The major duct of the right colleterial gland of P . americana is lined with cuboidal epithelial cells endowed with a chitinous intima. The secretory cells are confined to the smaller tubes. The right gland

ACCESSORY SEX GLANDS IN INVERTEBRATES

383

is known to contain P-glucosidase, and the left gland a glucoside of protocatechuic acid, along with one or two unidentified glucosides (Stay and Roth, 1962).

4. M i l k Gland In the tsetse fly, Glossina, which exhibits adenotrophic viviparity, the larva in the uterus is nourished by branched tubular milk glands whose secretions reach the uterus through a common collecting duct. The proximal and distal regions of the milk gland show cyclic cytophysiological changes correlated with the pregnancy cycle, the changes being more conspicuous in distal regions, according to Tobe et al. (1973). These investigators consider the proximal and distal regions distinct glands, but this seems to be hardly justified as the differences in cytological details between the two regions are not particularly significant, and the transition from proximal to distal “cell types” is gradual. A spinose intima, presumably produced by epidermal cells, limits the lumen of the proximal region; the cuticular intiina is bereft of spines in the distal region. The gland cells show extensive vacuolation during the secretory phase and have secretory vesicles which communicate with the lumen through cuticle-lined ductules (Figs. 10 and ll).Cells in their secretory phase

FIG. 10. Transverse section through the distal region of the milk gland of Glossina austeni 5 days before the first laiviposition. Note the vacuolated cytoplasm and the large apical vacuoles (AV) which connect to the lumen (L) by a ductule (D). E, Epidermal cell. ~ 7 5 0(From . Tobe et al., 1973.)

384

K. G . ADIYODI AND R. G. ADIYODI

FIG.11. Longitudinal section through the tip of the distal region of the milk gland of G. austeni 3 days before the first laviposition. Note the distention of the apical vesicles (AV) with secretion and the presence of refractile bodies in the lumen (L). ~ 7 5 0(From . Tobe et al., 1973.)

are much enlarged and have large nuclei and prominent nucleoli, highly vacuolated cytoplasm, swollen secretory vesicles, and prominent ductules. Injection of horseradish peroxidase has shown that the secretion of the milk gland contains no exogenous protein, the protein component of the milk being synthesized by the secretory cells themselves (Tobe et al., 1973).

5 . Albumen Gland In female Gastropoda the albumen gland (Fig. 2E) is a delimited or somewhat diffuse region of the pallial section of the oviduct and is composed usually of tall epithelial cells of two or three types. In terrestrial slugs this gland produces embryonic nutrients in the form of albumen which is supplied as a coating for the zygote. Secretory activity in the glandular epithelium is closely associated with periods of egg laying. In P . carolinianus, the secretory tubules of the

ACCESSORY SEX GLANDS IN INVERTEBRATES

385

albumen gland are PAS- and Best’s carmine-positive, but not salivalabile. The epithelium of the albumen canal is positive for glycogen. The cell membrane and the cytoplasmic reticulum possess high alkaline phosphatase activity. The secretory granules of the albumen gland and the albuminous coat of eggs show an orthochromatic blue color with thionine (Kugler, 1965). In H . pomatia, the albumen gland is a tubular organ composed of centrotubular cells and secretory cells which produce large PASpositive globules. In the environs of the Golgi zone, small vesicles with a homogeneous matrix coalesce to form secretory droplets which enlarge and accumulate galactogen in the form of granules 200 A in size (Nieland and Goudsmit, 1969). In Helix aspersa and H . pomatia, an acid P-galactosidase which can hydrolyze o-nitrophenylP-D-glucopyranoside, lactose, and galactogen has been detected in the albumen gland (Barnett, 1971). Histoenzymic investigation of the albumen glands of H . pomatia and Lymnaea stagnalis has shown the presence of uridyl transferase and epimerase, although other metabolic pathways of galactogen synthesis are not ruled out (Fantin and Gervaso, 1971). The galactan detected in the albumen gland of Bromphalaria glabrata has a highly branched structure. In this gland D- (64%)and L-galactopyranose (36%)residues are linked P-( 1-3) and /3-(1-6)in more or less the same proportion. Mild hydrolysis resulted in the release of 3-O-galactopyranosylgalactopyranoseand 6-O-galactopyranosylgalactopyranose (Correa et al., 1967). The albumen gland cells appear to be holocrine in nature. I n addition to albumen and galactogen, the secretion also contains calcium, possibly along with other minerals. Galactogen appears to be the major energy source for the embryo. In Lymnaea, there is a notable increase in the weight of the albumen gland, nidamental gland, and oviduct following prolonged periods of starvation, possibly as a result of accumulation of secretory substances due to nonutilization (Joosse et al., 1968). Information on the albumen glands of Hirudinea is meager.

6. Nidamental Gland Nidamental glands are reported only in certain mollusks. The capsule gland of Prosobranchia (Fig. 2F), the mucous gland of Opisthobranchia (Fig. 12B), and the oothecal gland of some Pulmonata (Fig. 12A) are comparable in histology to the nidamental glands of Cephalopoda and other mollusks. The albumen gland, jelly gland, and capsule gland may coexist, as in Littorina. The secretory substances released from the nidamental gland produce the lamellated capsules and other tertiary envelopes of the egg. I n P. carolinianus, the gland

386

K. G. ADIYODI AND R. G . ADIYODI

A

B

C

D

FIG. 12. Accessory sex glands of Mollusca. (A) A portion of the epithelium of the oothecal gland of Plnnorbnrius cornelm (Pulmonata); (B) a portion of the epithelium of the mucous gland of Tritonin (Opisthobranchia); (C) a gland cluster of the ureter jelly gland of a trochid; (D) R portion of the shell gland of an aplacophoran mollusk. cc, Cuneiform ciliated cell; gc, gland cell. (From different soiirces after Hyman, 1967.)

ACCESSORY SEX GLANDS IN INVERTEBRATES

387

surrounds the epithelial lining of the oviductal groove. The secretory cell, which is pear-shaped and provided with an apical nucleus, liberates mucus into the oviductal groove through a thin neck interpolated between the epithelial cells. Secretory cells of the nidamental gland are PAS-positive and amylase-fast, and have a remarkable capacity to absorb water. They acquire a metachromatic purple color with thionine and stain intensely blue with alcian blue, indicating the possible presence of neutral and acid mucopolysaccharides (Kugler, 1965). I n Cephalopoda, albumen and tertiary membranes are secreted by nidamental glands associated with the mantle cavity, while the oviductal gland furnishes a membrane or capsule. The capsule gland occurs in Prosobranchia that secrete a capsule around the albumen-coated eggs. This is wholly or partly replaced by the jelly gland in prosobranchs like many Littorinidae that deposit eggs in gelatinous masses. The capsule gland has very swollen lateral walls in which occur many invaginated clusters (Fig. 2F) of pear-shaped gland cells endowed with long necks which reach out of the lumen. Wedge-shaped ciliated cells are interpolated near the luminal surface among the tips of the gland cells. The composition of the secretions of the capsule gland is likely to be complex in view of the reported occurrence of different staining zones in the gland in different species (see Hyman, 1967, for references). The mucous gland of Opisthobranchia is lined with tall, granular, columnar cells which are positive for mucin; these cells alternate with cuneiform ciliated cells, as in the albumen gland (Fig. 12B). In Limapontiidae the gland shows four staining zones, each apparently exuding a chemically different type of mucus. In Pulmonata the oothecal gland (Fig. 12A) is attached to the uterus or incorporated into its wall. The gland consists of mucocytes and also of cells that produce the egg capsules. In L. stagnalis, the oothecal gland has two zones. One zone has cells containing only acid mucopolysaccharides; there are two cell types in the second zone; one type contains mucopolysaccharides and the other sulfated mucopolysaccharides, proteins, and glycogen (Fantin and Vigo, 1968). Columnar mucocytes constitute the muciparous gland in Pulmonata; these cells become dense purplish-red when stained with thionine.

7. Other Tertiary Membrane Secreting Glands I n solenogasters the distal portions of the gonoducts are modified to form shell (precloacal) glands which open into the cloaca separately, or may be fused and open by a single aperture, or even dis-

388

K. G . ADIYODI AND R. G . ADIYODI

charge to the exterior separately or after fusion (Fig. 12D). The cells in the shell gland epithelium are much elongated and highly secretory. At their distal extremity the gland cells alternate with wedgeshaped ciliated cells. In Pachymenia abyssorurn, the proximal part of the gonoduct is provided with several multicellular glands which replace the glandular epithelium of the shell glands. It is not known whether these glands have the ability to secrete shells. I n chitons a thick hull or shell which envelopes the egg is secreted by a layer of follicle cells which remain in the shed egg. The diverse patterns encountered in the hull result from varied patterns of enlargement and secretion of these torn-out cells. In Artemia salina the shell gland contains mucopolysaccharides, basic proteins, and lipids; the secretion has a role in the formation and differentiation of the tertiary membranes (Fautrez and FautrezFirlefyn, 1971). The gland is composed of bunches of cells, two to four in number, connected to a secretion-transporting canalicula. Secretion originates toward the central region of the intermediate cell walls of two cells. During the latter part of secretion formation, acid phosphatase-positive secretory vesicles, apparently originating from the Golgi apparatus, accumulate adjacent to the intermediate cellular membranes. The acid phosphatase produced is believed to play a role in loosening the intercellular junctions (De Maeyer-Criel, 1971). Secretory activity of the gland shows variation in relation to reproductive behavior (De Maeyer-Criel, 1970).

111. Evolutionary Aspects

The foregoing brief survey of the accessory reproductive glands of invertebrates shows that the primitive ability of the ductal epithelial cells to secrete mucus has been retained by many forms, including higher vertebrates. This can be explained by the fact that mucus serves one or more of the very same functions in all animals: provision of nutrients and protection of germ cells, transportation of gametes, capacitation of sperm, consolidation of the fertilization membrane, promotion of sperm adhesion, sperm penetration through jelly envelopes of the egg, and lubrication during copulation. Cytological adjustments in keeping with the type of mucous secretion related probably to the fiinctional needs of the species have, however, been made in the course of evolution as evidenced from variation in the composition of the mucus in different species. Also, in some cases mucus production has been at least partly relegated from genital ducts to specialized gland formations. Specific functional needs of

ACCESSORY SEX GLANDS IN INVERTEBRATES

389

the animal have led to the acquisition, by epithelial cells, of the capacity to secrete some rather unconventional products which may fit admirably well into a fiinctional biochemical plan, for example, protocatechuic acid glucoside, laccase, and calcium oxalate in the left colleterial, and P-glucosidase in the right colleterial of cockroaches for sclerotization of the egg case. When the secretions from the two glands mix, the P-glucosidase hydrolyzes the glucoside and liberates the phenol which in turn is oxidized to its 0-quinone. The 0-quinone is believed to cross-link the protein molecules, leading to sclerotization of the structural protein (Willis and Brunet, 1966). The degree of sclerotization of the ootheca varies with oviposition behavior and ranges from the hard, chestnut-brown egg cases of oviparous forms to the thin, soft, lightly colored ones characteristic of ovoviviparous and viviparous species. This is partly attributable to differences in the quantity of structural proteins secreted, and possibly also to the relative proportion of phenol produced, although the type or types of phenol involved are by themselves perhaps not so important. We have no knowledge of the cytology of the colleterial glands of any species other than P. americana (Brunet, 1952), making it impossible to reconstruct the modifications in cytological architccture related to the functional needs of ovoviviparity and viviparity. Stabilization of protein by phenolic cross-linking probably also occurs in the egg cocoons of the leech Erpobdella octoculata (Knight and Hunt, 1974). Many collateral glands appear to be specializations of the secretory epithelium of the male and female genital tracts, as borne out by intergeneric comparisons. The housefly, Musca, and the midge Chironomus have no discrete accessory glands associated with the male duct; the ejaculatory duct epithelium itself is secretory, and in the midge (Wensler and Rempel, 1962) is modified into four regions differing in structure and staining properties. However, separate accessory glands are present in Drosophila melanoguster (Bairati, 1968) and have two types of secretory cells, main and secondary cells. The secondary cells, which produce filamentous tubular bodies, have no homologs in the housefly. The main cells are comparable in ultrastructure and secretory physiology to the secretory cells in the housefly ejaculatory duct. The only major point of difference is the absence in the accessory sex gland cells of Drosophila of lysosomelike bodies which are a major constituent of the actively secreting ejaculatory duct cells of the housefly. Specializations to form discrete glands from primitive ductal glands and glandular outpouchings can be traced in the slime sac of the oviduct and prostate of mollusks, and in the spermathecal gland of insects.

390

K. G . ADIYODI AND R. G. ADIYODI

The presence of ciliated cells wedged among gland cells or aggregated in close association with glandular areas is a common feature of many invertebrate accessory sex glands (e.g., prostate, albumen gland, mucous gland, shell gland); the spatial intimacy between gland cells and ciliated cells suggests that translocation of secretion may be assisted at least in part by ciliary action. In many tubular accessory sex glands, the more distally located cells tend to be involved in most of the synthesis and secretion, the proximal region of the gland adjoining the duct serving mainly for the storage and transport of secretions. Monomyofibrillar musculature found enveloping accessory sex gland tubules of some insects (Odhiambo, 1970; De Loof and Lagasse, 1972; Adiyodi and Adiyodi, 1974) may be under nervous control and affect the extrusion of their luminal contents. Muscular coats consisting of circular and longitudinal fibers are often present in genital ducts having ductal glands; muscular action may assist in the mechanical transport of gametes and accessory sex secretions, and in the formation (cf. Greenwood, 1972) and transport of spermatophores.

IV. Maturation The cytological aspects of maturation have been studied in glands such as the mushroom gland (Vijayalekshmi and Adiyodi, 197313)and the conglobate gland (Suseela, 1965) of P . americana; ultrastructural aspects of the maturation of the accessory reproductive gland complex of the desert locust ( S . gregaria) have been investigated by Odhiambo (1971). The primordium of the mushroom gland of P . arnericana is small, of the size of a pinhead in early larval stages; in the last larva cell proliferation and utricular differentiation are already well underway by the premolt stage. All glandular utricles of the mushroom gland are thick-walled and have basophilic cytoplasm in cockroaches at the larval-adult molt stage, with the result that it is difficult to distinguish the utricles from one another, the UT with their particularly thick walls and increased epithelial basophilia being the only exception. Cell borders are not distinct at the light microscope level in the mushroom gland through much of early adulthood. In day-0 individuals the nuclei are closely packed and contain reticulate or loosely granular chromatin; the nucleoli are not distinct. Although secretion of some sort can be demonstrated in sections of the mushroom gland, even in the last larval stage differentiation of the appropriate synthetic and secretory machinery becomes manifest at the earliest only by day 2, when the utricles are iden-

ACCESSORY SEX GLANDS IN INVERTEBRATES

39 1

tified based on tinctorial relations of the secretions (Vijayalekshmi and Adiyodi, 1973b). The maturation of the mushroom gland involves a trimming of the wall of the utricles b y a rearrangement of the constituent cells, and also the acquisition of adult tinctorial characteristics b y their secretions, both of which proceed through early adulthood and become complete by about day 14. The seminal plasma becomes tinctorially similar to that of adults by about day 3, but the dark-blue granules encountered in the lumina of the seminal vesicles in adult cockroaches become distinct only at a later stage, suggesting that the seminal plasma also requires a period of maturation. Ultrastructural evidence for comparable maturation of secretion with aging has been presented by Odhiambo (1971) for gland 1 of S. gregaria. Aspects of maturation of the secretory machinery of gland 1 of S. gregaria which produces a protein-neutral mucopolysaccharide secretion complex, have been described by Odhiambo (1971). At emergence the gland is immature and diminutive and shows secretion neither in the cytoplasm nor in the occluded lumen; ribosomes are mostly free, and RER cisternae very rare. Vesicular smooth-surfaced ER elements, however, are rather abundant in the cytoplasm. Golgi complexes are few, small, and inactive, with no condensing vacuoles and few peripheral vesicles. Many small mitochondria are present; they have few cristae and intramitochondrial granules. Also, large clumps of chromatin occur in the nucleus, suggesting that the cells are not active in secretion. Fairly extensive development of the ER is discernible by 3 days after emergence; most of the free ribosomes are grouped into ribosomal aggregates or polyribosomal configurations, also, ribosomes attach themselves to vesicles of the ER. Golgi complexes and mitochondria acquire condensing vacuoles and intramitochondrial granules, respectively. Apically, in the cytoplasm there are many small vesicles, some of which are within the microvilli themselves. By day 5 conspicuous developments have taken place in the ER, Golgi complex, and nucleus. The ER, which comes to occupy most of the gland cell cytoplasm, is composed of swollen vesicles, some containing a fibrous material resembling that appearing in the Golgi condensing vacuoles. Golgi units, with their large condensing vacuoles, occupy a sizable area in the cell. In the region of the luminal plasmalemma, pinocytotic vesicles are produced; secretory vesicles release their contents from in between the microvilli. In the nucleus the chromatin is in a more dispersed state, indicating that some organizational changes have also taken place at the level of the nucleus (Odhiambo, 1971).

392

K. G. ADIYODI AND R, G . ADIYODI

V. Differentiation and Secretion: The Role of Hormones Control of accessory sex gland function by gonadotropins and/or sex hormones can only be expected, inasmuch as secretory products from accessory sex glandular elements are often indispensable for reproduction. Such controls, however, are not absolute, some glands, particularly in the males [e.g., the accessory sex gland in male L. maderue (Scharrer, 1946)l being independent of at least the conventionally implicated reproductive hormones. The analage of the accessory sex gland, which is diminutive in early developmental stages, differentiates into one or more gland cell types and acquires the competence to produce characteristic secretions only if the gland matures in a milieu of the developmental hormones in insects. Acquisition of competence need hardly involve much subcellular differentiation; for example, the late fifth instar accessory reproductive gland of the desert locust has many or all of the features characteristic of undifferentiated or embryonic cells. According to Odhiambo (1971), glands at this stage show nuclear chromatin in large clumps; the many ribosomes are free in the cytoplasm, there being no RER; also, the Golgi elements have a simple structure. Differentiation of the secretory machinery, which proceeds through early adulthood (see Section IV), takes place with an increase in the content of the corpus allatum hormone (juvenile hormone, JH). Maturation of all the accessory reproductive glands in the desert locust, except gland 16, appears to be under the control of JH, The secretory apparatus of the accessory reproductive glands does not develop completely, and the polyribosomes fail to appear if the locusts are allatectomized on emergence as adults (see Odhiambo, 1971, for references). In invertebrates undergoing sex reversal, a search for the mode of differentiation of the accessory reproductive glands and the underlying control mechanisms would be rewarding, but this has hardly been attempted. Among invertebrates some evidence for gonadal control of accessory reproductive glands has been presented in mollusks (see Blum, 1970, for review and references). In Cryptochiton stelleri (Polyplacophora), cyclic changes in the size of the glandular oviduct accompany variation in the size of the gonad (Lawrence et al., 1965). In Lirnax maximus, complete castration leads to regression of the albumen gland and genital ducts. Androgens are believed to control the extent of the ribosomal population of ergastoplasmic membranes in target epithelial cells such as those of the seminal vesicle, prostate, and vas deferens in the rat (Szirmai and van der Linde, 1965).

ACCESSORY SEX GLANDS IN INVERTEBRATES

393

Maturation of the seminal vesicle is apparently independent of JH in Schistocerca (Odhiambo, 1971), but it is not known whether the testis plays a role in the maturation of seminal vesicles. The problem has to be examined in view of the reports of the occurrence of steroid 17P-dehydrogenase which converts testosterone to androst-4-ene-3, 17-dione in the testis of the cricket Gyrllus domesticus (Lehoux et al., 1968). Degeneration is complete in the inner layer and partial in the outer layer of gland cells following allatectomy in the accessory reproductive gland of L. decemlineata ( D e Loof and Lagasse, 1972). There is a swelling of the RER, and autophagic vacuoles appear in the gland cells. Microvillous formations develop between the inner and outer layers of gland cells. The plasmalemma and the nuclei of the outer gland cells remain intact, although there is some dispersion of the chromatin masses; the ER of these cells, however, breaks down into swollen vesicles. Plasma membranes of the inner cells degenerate completely. The chromatin masses of the nuclei form large clumps, and the nuclei together with the cytoplasmic organelles flow into the lumen of the gland. In cockroaches secretion of protocatechuic acid glucoside (Willis and Brunet, 1966), and the structural proteins of the ootheca (Adiyodi, 1968) by the epithelium of the left colleterial gland, are controlled by JH, a control not mediated b y the ovaries. The influence of J H on the nuclei of the left colleterial gland is likely to b e that of increasing activity at the transcriptional level (Nair and Menon, 1972). Adult males of P . americana lack some factor required for glucoside formation; left colleterials transplanted into adult males can synthesize protein, but not protocatechuic glucoside. Parabiosis experiments show that adult males produce enough J H to support the synthesis of both protein and glucoside in the female (Shaaya and Bodenstein, 1969). J H probably acts on some specific female gene or genes and influences transcription of specific mRNA concerned with the synthesis of some enzyme or enzymes crucial to the biosynthesis of glucoside (Adiyodi and Adiyodi, 1974a).

VI. Concluding Remarks Gland cells secreting chemically different molecules have been grouped either vertically, each type of gland spatially sufficiently separated from the others and specialized to produce a specific secretion (e.g., accessory reproductive glands of male locusts), or horizontally, with different secretions being produced by different

394

K. G . ADlYODI AND R. G . ADIYODI

cells in the same glandular epithelium (e.g., left colleterial of female cockroaches) or by the same cells with or without temporal succession. The complexity of the secretory behavior is well illustrated by L. nuttalli in which the first pair of accessory sex glands of the male produces six different secretions, all of which are used in spermatophore formation. Zone I of the glands secretes substances 1 to 4, zone I1 substance 5 besides substances 1 to 4, and zone I11 only substance 6 (Gerber et al., 1971b). Substance 1 is believed to be a carbohydrate-neutral lipid complex, 2 and 3 phospholipoproteins, 4 a lipoprotein having neutral lipid as its lipid moiety, 5 one or more complexes containing carbohydrate, protein, and phospholipid, and 6 a carbohydrate-protein complex. Cytoplasm of cells of zones I and 11, producing the tyrosine-rich basophilic substances 3 and 5, stains darkly throughout the cell; substances 1 and 4,which are nonacidic, are secreted by cells that stain fairly darkly at the base but a light gray apically. The cells in zone 111, producing substance 6, have highly basophilic cytoplasm and stain extremely densely. Amphibian oviducts, however, have cytochemically distinct glandular zones, each zone secreting a chemically specific layer of the jelly coat of the egg (Humphries, 1966; Shivers and James, 1970). We have already seen how the secretory apparatus of accessory sex gland cells is modified to produce their characteristic secretions (Section II,G,3). Gland cells that look alike morphologically may produce different secretions, for example, the ejaculatory duct epithelial cells of Musca secrete at least 12 proteins (Terranova et al., 1972). Similarly, there are several chemical components in the secretion of the epithelium of the gland cells of the UM in the mushroom gland of P . americana (Vijayalekshmi and Adiyodi, 1973c; Adiyodi and Adiyodi, 1974). A degree of plasticity in the activity of the secretory apparatus may be expected only in these multipotent cells. It is not known how the activity of the different components of the secretory apparatus is precisely regulated in these cells according to the type or types of secretion required to be produced at any one time. The various types of secretions encountered in some adult glands may be the result of accumulation by a successional and or differential type of secretory activity. The number of types of cells in the left colleterial gland of the cockroach Nauphoeta cinerea is not known, but the gland shows a progressive accumulation of the electrophoretically resolvable protein fractions, which can be correlated with the ovarian cycle (Adiyodi, 1968).There are only three secretory proteins, all present in traces (fractions 5 to 7), at the start of the new ovarian cycle after parturition in this species (basal oocytes 672-820 pm in

ACCESSORY SEX GLANDS IN INVERTEBRATES

395

length); fraction 3 has made its appearance by the time the basal oocyte has grown to about 1.50 mm; the remaining proteins, namely, fractions 1,2, and 4 can be recognized in females with oocytes in the range of 2.88 mm. The fact that there is a progressive increase in the density of many fractions in the left colleterial gland with time, associated with the ovarian cycle, suggests that there is “temporal spacing” in the initiation of the secretory machinery for different species of proteins and that, once started, the processes could probably continue simultaneously. Conversely, the similarity in the chemistry of glandular cells and their products in accessory sex glands with diverse origins, as between the mushroom gland (possibly mesodermal) and the conglobate gland (ectodermal) in P . arnericana (Table 111; see Vijayalekshmi and Adiyodi, 1973a,c, for details), is also of great interest. Conceivably, some of the same macromolecules could be synthesized and secreted by gland cells in different locales under the influence of analogous driving forces. Many studies have appeared on the subcellular enzymology of accessory sex glands in vertebrates (see Mann, 1964, for review; Sirakov and Kochakian, 1970; and others), but unfortunately few in invertebrates (Srivastava and Das, 1965; Kessel et al., 1969; Bogitsch, 1970). In some arthropod groups, growing oocytes in the course of vitellogenesis are known to sequester preferentially blood proteins-sex-specific vitellogenins-synthesized elsewhere (Adiyodi and Adiyodi, 1974b, for review). It will be interesting to learn whether or not some of the accessory sex gland cells that rapidly build up large protein reserves can similarly pinocytotically ingest presynthesized protein molecules, although Tobe et al. (1973) have shown this to b e unlikely in the milk gland of the tsetse fly. In the UM of the mushroom gland of P . arnericana, basal plasmalemma of the gland cells is thrown into folds which permeate the cell body rather deeply (Adiyodi and Adiyodi, 1974). The basal lamina located in between the secretory epithelium and the muscle layer, however, does not follow the plasmalemma. In contrast to the situation in the distal convoluted tubule of the guinea pig kidney described b y Fawcett (1967), the mitochondria1 population is rather small in these infoldings, and the ones present are not intimately associated with the plasmalemmal infoldings. These infoldings resemble formations in the cell base of many epithelial cells engaged in ion and water transport (Gorbman and Bern, 1962; Fawcett, 1967; D e Robertis et al., 1970).It could therefore be speculated that some of the raw materials needed for the production of secretory substances enter the secretory epithelium of the UM by this route.

396

K. G. ADIYODI AND R. G. ADIYODI

Accessory sex gland function being indispensable for successful reproduction in nearly all advanced animal groups, it is indeed surprising that correlative studies on the structure, biochemistry, and physiology of the accessory glandular elements are comparatively few. There are also many challenging problems in the secretory dynamics of accessory sex glands to be investigated, such as the effect of possible feedback from secretory products on secretory activity factors influencing rates of cyclic synthesis and output of secretions, and the mechanisms that maintain synchrony or differential asynchrony, as the case may be, of the glandular epithelium related to functional needs. REFERENCES lnsect Physiol. 14,309. Adiyodi, K. G. (1968).J. Adiyodi, K.G., and Adiyodi, R. G. (1970).Indian J . Exp. B i d . 8, 55. Adiyodi, K. G., and Adiyodi, R. G. (1972).Proc. Int. Congr. Nistochem. Cytochem., 4th, Kyoto p. 255. Adiyodi, K. G., and Adiyodi, R. G. (19744.J . Sci. Itid. Res., India 33,343. Adiyodi, K, G.,and Adiyodi, R. G. (197415).Aduan. C o ~ n pPhysiol. . Biochem. 5, 37. Adiyodi, R, G.,and Adiyodi, K. G. (1974).Z . Zelljorsch. Mikrosk. An&. 147, 433. Aloia, R. C.,and Moretti, R. L. (19731.J. Morphol. 140,285. Avel, M. (1959).In ‘‘Trait6 d e Zoologie” (P. Grasse, ed.), Vol. 5, 224.Masson, Paris. Bairati, A. (1968).Montt. Zool. Ital. 2, 105. Barnes, H.(1962).J.Exp. Biol. 39,345. Barnett, J. E.G. (1971).Cowap. Biochem. Phystol. 40, 585. Beams, H.W.,Anderson, E., and Kessel, R. G. (1962).J . Roy. Microsc. Soc. 81, 85. Bhatnagar, R. D. S., and Musgrave, A. J. (1971).Can. J. Zool. 49,275. Bhatnagar, R. D. S., and Rempel, J. G. (1962).Can. J. 2001.40,465. Blandau, R.J. (1969).In “The Mammalian Oviduct” (E. S. E. Hafez and R. J. Blandau, eds.), p. 129.Univ. of Chicago Press, Chicago, Illinois. Blum, M. S. (1970).In “The Testis” (A. D. Johnson, W. R. Comes, and N. L. Van Demark, eds.), Vol. 2,p. 393.Academic Press, New York. Blum, M. S., Glowsh, Z., and Taber, S. (1962).Ann. Entomol. SOC. Amer. 55, 135. Bogitsch, B. J. (1970).J. Purosttol. 56, 1084. Breucker, H.(1964).Protoplusma 58, 1. Brunet, P. C. J. (1952).Quart. J. Microsc. Sci. 93,47. Callahan, P. S., and Cascio, T. (1963).Ann. Entomol. SOC. Amer. 56,535. Chapman, G . (1965).B i d . Bull. 128, 189. Clements, A. N.,and Potter, S. A. (1967).J. Insect Physdol. 13, 1825. Cornea, J. B. C., Dmytraczenko, A., and Duarte, J. H. (1967).Carbohyd. Res. 3,445. Davey, K. G.,and Webster, G. F. (1967).Can. J . 2001.45,653. De Carnargo, J. M. F., and Mello, M. L. S. (1970).Apidologie 1,351. Del Conte, E. (1970).Arch. Anut. Microsc. Morphol. E x p . 59,Q. De Loof, A., and Lagasse, A. (1972).Z. Zelljorsch. Mikrosk. Anat. 130,545. D e Maeyer-CrieI, G.(1970).Arch. Biol. 81,491. D e Maeyer-Criel, G.(1971).Arch. Biol. 82, 163. De Robertis, E. D. P., Nowinski, W. W.,and Seez, F. A. (1970).“Cell Biology.” Saunders, Philadelphia, Pennsylvania.

ACCESSORY SEX GLANDS IN INVERTEBRATES

397

Duncan, C. J. (1958). Proc. 2001.Soc. London 131, 55. Fantin, A. M. B., and Cervaso, M. V. (1971). Histochemie 28,88. Fantin, A. M. B., and Vigo, E. (1968).Histochemie 15,300. Fautrez, J., and Fautrez-Firlefyn, N . (1971). Arch. Biol. 82,41. Fawcett, D. W. (1967). “The Cell.” Saunders, Philadelphia, Pennsylvania. Feigelson, M., and Kay, E. (1972).Biol. Reprod. 6,244. Fouquet, J. P. (1971).C o m p . Biochem. Physiol. 40A, 305. Fretter, V., and Graham, A. (1964). In “Physiology of Mollusca” (K. M. Wilbur and C. M. Yonge, eds.), Vol. 1, p. 127. Academic Press, New York. Fuchs, M. S., Craig, G. B., Jr., and Hiss, E. A. (1968). Life Sci. 7, 835. Gerber, G. H., Church, N. S., antl Rempel, J. G. (1971a). Can. J. Zool. 49, 523. Gerber, G. H., Church, N. S., and Rempel, J. G . (1971b). Can. J. Zool. 49, 1595. Gorbman, A., and Bern, H. A. (1962). “A Text Book of Comparative Endocrinology.” Wiley, New York. Greenwood, J. G. (1972).J.Natur. Hist. 6, 561. Gupta, P. D. (1946). ZndianJ. Entomol. 8, 79. Guzsal, E. (1968).Acta V e t . (Budapest) 18, 251. Harm, M. (1951).2. Morphol. Oukol. Tiere 22,629. Hartmann, R. (1971). Z. Vergl. Physiol. 74, 190. Hinton, H. E. (1961).J.Znsect Physiol. 7, 224. Humphries, A. A., Jr. (1966). Detelop. B i d . 13, 214. Hyman, L. H. (1951). “The Invertebrate$. Vol. 11: Platyhelminthes and Rhynchocoela.” McGraw-Hill, New York. Hyman, L. H. (1967). “The Inbertebrates. Mollusca I.” Vol. 6. McGraw-Hill, New York. Jaiswal, A. K., and Naidu, M. B. (1972).J.Anim. Morphol. Pyhsiol. 19, 1. Joosse, J., Boer, M. H., and Cornelisse, C. J. (1968).Symp. Zool. Soc. London 22,213. Kessel, R. G., Panje, W. R., antl Decker, M. L. (1969). J. Ultrastrtcct. Res. 27, 319. Khalifa, A. (1949).Trans. Roy. Entomol. Soc., London 100, 449. Khalifa, A. (1950).Proc. Roy. Entomol. Soc., London, Ser. A 25, 53. King, P. E., and Copland, M. J. W. (1969).J.Natur. Hist. 3, 349. Knight, D. P., and Hunt, S. (1947). C o m p . Biochem. Physiol. 47A, 871. Kugler, 0. E. (1965).J.Morphol. 116, 117. Lawrence, A. L., Lawrence, J. M., and Giese, A. C. (1965). Science 147, 508. Lehoux, J. G., Sandor, T., and Lanthier, A. (1968). Proc. Znt. Congr. Endocrinol., 3 r d Mexico C i t y p. 115. Lensky, Y., and Allimot, E. (1969). Comp. Biochem. Physiol. 30,519. Leopold, R. A. (1970).J . Znsect Physiol. 16, 1859. Leopold, R. A., Terranova, A. C., Thorson, B. J., and Degrugillier, M. E. (1971).J.Znsect Physiol. 17, 987. Louis, D., and Kumar, R. (1971). Ann. Entomol. Soc. Amer. 64, 977. Lusis, O., Sandor, T., and Lehoux, J.-G. (1970).Can. J . Zool. 48, 25. Mann, K. H. (1962). “Leeches (Hirudinea): Their Slructure, Physiology, Ecology and Embryology.” Pergamon, Oxford. Mann, T. (1964). “The Biochemistry of Semen and of the Male Reproductive Tract.” Methuen, London. Mathews, L. H. (1962).In “Ovary” (S. Zuckerman, ed.), Vol. 1, p. 89. Academic Press, New York. Melis, G. (1966).Atti Accad. Fisiocrit Siena-Sez. Med.-Fis. 15, 107. Mercer, E. H., and Brunet, P. C. J. (1959).J. Biophys. Biochem. Cytol. 5,257. Mouchet, S. (1931).Ann. Sta. Oceanogr. Salummho 6, 1.

398

K. G . ADIYODI AND R. G . ADIYODI

Nair, K. K., and Menon, M. (1972). Experientiu 28, 577. Nieland, M. L. L., and Goudsmit, E. M. (1969).J. Ultrustruct. Res. 29, 119. Odhiambo, T. R. (1969a). Phil. Trans. Roy. Soc London, Ser. B 256,85. Odhiambo, T. R. (1969b). Tissue Cell 1, 325. Odhiambo, T. R. (1970). Tissue Cell 2, 233. Odhiambo, T. R. (1971). Tissue Cell 3, 309. O’Shea, J. D. (1966).J. Morphol. 120, 347. Osterloh, A. (1922). Z . Wiss. Zool. 119, 326. Pau, R. N., Brunet, P. C. J., and Williams, M. J. (1971). Proc. Roy. Soc., Ser. B 177,565. Peterson, M., and Leblond, C. P. (1964).J. Cell B i d . 21, 143. Poole, H. K. (1970). Ann. Entomol. Soc. Amer. 63, 1625. Quattrini, D. (1967). Monit. Zool. Ztal. 1,235. Quattrini, D., and Fondi, R. (1969). Monit. Zool. Ztal. 3, 117. Quick, H. E. (1960). Brit. Mus. Natur. Hist., Bull. Dep. Zool. 6, 105. Rajalakshmi, M., and Prasad, M. R. N. (1970).J. Endocrinol. 46, 413. Riemann, J. G. (1973).J. Znsect Physiol. 19, 213. Rosay, B. (1968).J. Appl. Entomol. 5 , 478. Roth, L. M. (1967). Ann. Entomol. Soc. Amer. 60, 1203. Rudman, W. B. (1972).J. Notur. Hist. 6, 603. Runham, N. W., and Hunter, P. J. (1970). “Terrestrial Slugs.” Hutchinson, London. Scharrer, B. (1946). Endocrinology 38,46. Setchell, B. P. (1970). In “The Testis” (A. D. Johnson, W. R. Gomes, and N. L. Van Demark, eds.), Vol. 1, p. 101. Academic Press, New York. Shaaya, E., and Bodenstein, D. (1969).J. Exp. Zool. 170,281. Shivers, C . A., and James, J. M. (1970). Anat. Rec. 166, 541. Sirakov, L. M., and Kochakian, C. D. (1970). Biochim. Biophys. Acta 204,359. Smith, B. J. (1965). Ann. N.Y. Acad. Sci. 118, 997. Srivastava, M. D. L., and Das, C. C. (1965). Proc. N u t . Acad. Sci., India, Sect. B 35, 441. Stay, B., and Roth, L. M. (1962). Ann. Entomol. Soc. Amer. 55, 124. Striibing, H. (1956). Zool. Beitr. 2, 331. Suseela, A. P. (1965).J. Annamalai Unio. 26, 169. Szirmai, J. A., and van der Linde, P. C. (1965).J. Ultrustruct. Res. 12, 380. Terranova, A. C., Leopold, R. A., Degrugillier, M. E., and Johnson, J. R. (1972).J. Znsect Phusiol. 18, 1573. Tobe, S. S., Davey, K. G., and Huebner, E. (1973).Tissue Cell 5, 633. Trainis, K. V. A. (1968). Arkh. Anat., Gistol. Embriol. 55, 17. Tulsyan, G. P. (1966). Ann. Mag. Natur. Hist. 9, 681. Vijayalekshmi, V. R., and Adiyodi, K. G. (1973a). I n d i a n ] . Exp. Biol. 11, 512. Vijayalekshmi, V. R., and Adiyodi, K. G. (1973b).IndiunJ. Exp. Biol. 11,515. Vijayalekshini, V. R., and Adiyodi, K. G. ( 1 9 7 3 ~ )ZndianJ. . E x p . Biol. 11, 521. Walborg, T., and Bjorkman, N. (1965). Z . Parasitenk. 26,63. Walley, L. J. (1965).J. Mar. Biol. Ass. U . K . 45, 115. Wensler, R. J. D., and Rempel, J. G. (1962). C a n . J. Zool. 40, 199. Wigglesworth, V. B. (1950). “The Principles of Insect Physiology.” Methuen, London. Wilkes, A. (1965). Can. Entomol. 97, 647. Willis, J. H., and Brunet, P. C. J. (1966).J. Exp. Biol. 44, 363. Zaffagnini, F. (1965).Curyologia 18, 1.

Subject Index A

between prokaryotes and eukaryotes,

Accessory sex glands, invertebrate, differentiation and secretion, 392-

286290 Ductal glands, morphology and cytology,

357364

393 evolutionary aspects, 388-390 general survey, 354-388 maturation, 390-391 Adenosine triphosphatase(s), energy coupling and, 30-33 Alcohol dehydrogenase, maize, regulation of, 330-331 Alkaline phosphatase, induction in HeLa cells, 318-328 Amphibian development, germinal plasm and, cytochemistry,.250 descriptive studies, 241-244 experimental studies, 244-247 fine-structure, 247-250 Aryl hydrocarbon hydroxylase, regulation of, 329-330

E Electron transport, sequences, 2 2 3 0 Embryonic retina cells, glutamine synthetase induction in, 317-318 Enzymes, control of activity, substrates and cofactors and, 334-335 intracellular localization, genetic control, 335-336 Epigenotype, mammalian cell cultures,

293-294 Eukaryotes, aerobic adaptation and survival of primitive functions, 11-12 anaerobic metabolic requirements,

17-18 electron transport sequences, 22-30 energy coupling and ATPases, 30-33 heme biosynthesis, 20-22 oxygen and biosynthetic patterns,

B Bacteria, gene regulation in, 283-284 application to higher forms, 284-285

C Chaetognaths, development, germinal determinants and, 240-241 Chromosome(s), differences between prokaryotes and eukaryotes, 286-290 mitotic behavior, microtubules and,

194-197 movement,

nuclear

envelope

18-20 protection

oxygen

toxicity,

differences from prokaryotes, 285-286 DNA and chromosomes, 286-290 mitochondria, 293 protein synthesis, 292-293 RNA, 290-292 origin, Precambrian earth conditions, 3 4 Precambrian fossil record, 4-7 rates of molecular evolution, 7-9 Evolution, accessory sex glands, 388-390

and,

G

171-194 Collateral glands, morphology and cytology, female, 380388 male, 368-379

Gene expression, alteration, protein modification and,

333-334 hormonal effects, alkaline phosphatase

D Deoxyribonucleic

from

12-17

acid,

318328 differences

general aspects, 313-314

399

induction,

400

SUBJECT INDEX

glutaniine synthetase induction, 317318 induction of TAT, 314317 regulation of metalloenzynies 328329 mammalian cell heterokaryons, cell fusion, 336337 complementation, 337338 differentiation, 339 mammalian cell synkaryons, 340-342 nuclear fusion, 339-340 synchronous cell cultures, 307308 Gene regulation, bacterial, 283-284 application to higher forms, 284-285 Genitoatrial glands, morphology and cytology, 367 Germ cells, nuoge-containing, animals with, 250-262 composition, 263-269 origin, 269-271 Germinal determinants, chaetognath development and, 240-241 Germ plasm, amphibian development and, cytochemistry, 250 descriptive studies, 241-244 experimental studies, 244-247 fine-structure, 247-250 animals containing, 230-231 differentiation, background, 229-230 conclusions, 271-274 Glntamine synthetase, induction in embryonic retina, 317318 Gonadal glands, morphology and cytology, 355-357

H HeLa cells, alkaline phosphatase induction in, 318328 Heme, biosynthesis, 20-22 Hepatoma cells, tyrosine amino transferase induction in, 314-317 Heterokaryons, gene expression in, cell fusion, 336-337 complementation, 337338 differentiation, 339

Hormones, accessory sex glands and, 392-393

I Insect development, polar granules and, cytochemistry, 239-240 descriptive studies, 231-234 experimental studies, 234-237 fine-structure, 237-239 Invertebrates, accessory sex glands, differentiation and secretion, 392393 evolutionary aspects, 388390 general survey, 354388 maturation, 390391

1 Lipids, mitochondria1 function and, 75-77 Lymphocytes, long-term cultures, differential functions 312-313

M Maize, alcohol dehydrogenase, regulation of, 3 3 0 3 31 Mammalian cell(s), reconstruction of, 343 Mammalian cell cultures, differentiated functions, established cell lines, 311312 general aspects, 308-310 long-term lymphocyte cultures, 312-313 myogenesis and, 310-311 gene expression i n heterokaryons, cell fusion, 336337 complementation in, 337-338 gene expression and differentiation, 339 gene expression in synkaryons, 340342 nuclear fusion, 339-340 hormonal effects on gene expression, alkaline phosphatase induction, 318328 general aspects, 313314

401

SUBJECT INDEX glutainine synthetase induction, 317-318 induction of TAT, 314-317 regulation of metalloenzymes, 328-329 regulation of specific protein synthesis, cell function i n uitro, 294-307 determination and epigenotype, 293-294 gene expression in synchroiious cultures, 307308 types of, 282-283 Metalloenzymes, regulation, hormones, 328329 other agents, 329-331 Microtubules, chromosome movement and, 194-197 function, evolution of, 169-170 possible unconventional role, 197-205 Mitochondria, differences between prokaryotes and eukaryotes, 293 functions, autonomy in biogenesis, 83 inner membrane, 78-83 lipids, 75-77 matrix, 78 topography and, 74-75 gene expression, messenger RNA, 60-65 protein synthesis, 65-74 ribosomal RNA, 46-60 genes and their transcription, 3 3 3 4 DNA-dependent RNA polymerases, 4046 genome and, 34-39 genetic control of, 126126 nonsymbiotic origin, elements of model, 8 6 9 7 model of, 83-85 as supramolecular assemblies, 108-110 transcription, in uitro, 127-142 in O ~ U O , 142-152 translation, products, 157-160 protein-s ynthesizing machinery, 152157 Mitochondria1 origin,

background, 2-3 molecular approach, validity, 104-108 Mitotic spindle, evolution, background, 167-168 cellular and nuclear organization, 168-169 microtubular function, 169-170 Mitosis, microtubule participation, chromosome behavior, 194-195 models for, 195-197 Molecular evolution, rates, prokaryote-eukaryote divergence and, 7-9 symbiotic theory and its consequences, 9-11 two theories: contrast and outlook, 98-104 Myogenesis, cell cultures and, 310-311

N Nuuge, genn cells and, animals containing, 25C262 composition, 263-269 origin, 269-271 Nuclear envelope, chromosome movement and, “closed” divisions, 171 extranuclear spindles, 171-193 outline of spindle evolution, 193-194 Nuclei, division with microtubules, conventional metaphase, 205-206 unconventional metaphase arrays, 206-220 division without microtubules, 197-203

0 Oxygen, biosynthetic patterns and, 18-20 toxicity, enzymic protection from, 12-17

P Penial glands, morphology and cytology, 367-368 Polar granules,

402

SUBJECT INDEX

insect development and, cytochemistry, 239-240 descriptive studies, 231-234 experimental studies, 234-237 fine-structure, 237-239 Precambrian, earth conditions, 3-1 fossil record, 4-7 Prokaryotes, differences from eukaryotes, 285-286 DNA and chromosomes, 286-290 mitochondria, 293 protein synthesis, 292-293 RNA, 290-292 Protein(s), degradation rate regulation, general aspects, 331-332 measurement in cell cultures, 332333 modification, gene expression and, 333-334 synthesis, differences between prokaryotes and eukaryotes, 292-293 mitochondrial, 65-74, 152-160 regulation in mammalian cells, 293-308 Protoeukaryotes, as living fossils: a speculation, 110-111

R Ribonucleic acid, differences between prokaryotes and eukaryotes, 290-292 mitochondrial, messenger, 60-65 ribosomal, 46-60 Rihonucleic acid polymerases, mitochondrial, 40-46

S

Seminal glands, morphology and ctyolO ~ Y364-365 , Sex glands, accessory, types and function, 353-354 Spermathecal glands, morphology and cytology, 365-367 Synkaryons, gene expression in, 340-342 nuclear fusion, 339-340

T Tyrosine amino transferase, induction in hepatoma cells, 314-317

Contents of Previous Volumes Ascorbic Acid and Its Intracellular Localization, with Special Reference Some Historical Features in Cell Biology to Plants-J. CHAYEN -ARTHUR HUGHES Aspects of Bacteria as Cells and as OrNuclear Reproduction-C. LEONARD ganisnis-STuAm MUDD AND EDWARD HUSKINS D. UELAMATER Enzymic Capacities and Their Relation Ion Secretion in Plants-J. F. SUTCLIFFE to Cell Nutrition in himals-GEoRGE Multienzynie Sequences in Soluble W. KIDDER Extracts-HENRY R. MAHLER The Application of Freezing and Drying The Nature and Specificity of the FeulTechniques in Cytology-L. G. E. gen Nucleal Reaction-M. A. LESSLER BELL Quantitative Histochemistry of PhosphaEnzymatic Processes in Cell Membrane taSeS-wILLL4M L. DOYLE ROSENBERG AND w. Alkaline Phosphatase of the NucleusPenetration-TH. WILBRANDT AND H. FIRKET M. CHBVREMONT Bacterial Cytology-K. A. BISSET Gustatory and Olfactory Epithelia-A. F. Protoplast Surface Enzymes and AbsorpBARADIAND G. H. BOURNE tion of Sugar-R. BROWN Growth and Differentiation of Explanted Reproduction of Bacteriophage-A. D. Tissues-P. J. GAILLARD HERSHEY Electron Microscopy of Tissue SectionsThe Folding and Unfolding of Protein A. J. DALTON Molecules as a Basis of Osmotic Work A Redox Pump for the Biological Per-R. J. GOLDACRE formance of Osmotic Work, and Its Nucleo-Cytoplasmic Relations in AmphibRelation to the Kinetics of Free Ion ian Developmeent-G. FRANK-HAUSER Diffusion across Membranes-E. J. Structural Agents in Mitosis-M. M. CONWAY SWA" A Critical Survey of Current Approaches Factors Which Control the Staining of in Quantitative Histo- and CytochemTissue Sections with Acid and Basic istry-DAvm GLICK Dyes-MARCUS SINGER Nucleo-cytoplasmic Relationships in the The Behavior of Spermatozoa in the Development of AcetabuZoria-J. HAMNeighborhood of Eggs-Lorn ROTHSMERLINC

Volume 1

CHILD

The Cytology of Mammalian Epidermis and Sebaceous Glands-WILLIAM MONTAGNA The Electron-Microscopic Investigation of Tissue Sections-L. H. BRETSCH-

AUTHOR INDEX-SUB

JECT INDEX

Volume 3

NEIDER

The Histochemistry GOMORI AUTHOR INDEX-SUB

Report of Conference of Tissue Culture Workers Held at Cooperstown, New York-D. J. HETHERINGTON

of

Esterases-G.

JECT INDEX

Volume 2

Quantitative Aspects of Nuclear Nucleoproteins-HEwsoN SWIFT

403

The Nutrition of Animal Celk-cHARITY WAYMOUTH Caryometric Studies of Tissue CulturesOTTO BUCHER The Properties of Urethan Considered in Relation to Its Action on MitosisIVORCORNMAN

404

CONTENTS OF PREVIOUS VOLUMES

Composition and Structure of Giant Evidence for a Redox Pump in the Active Transport of Cations-E. J. CONWAY Chromosomes-MAx ALFERT How Many Chromosomes in Mammalian AUTHOR INDEX-SUB JECT INDEX Somatic Cells?-R. A. BEATTY The Significance of Enzyme Studies on Volume 5 L. Isolated Cell Nuclei-ALEXANDER Histochemistry with Labeled Antibody DOUNCE -ALBERT H. COONS The Use of Differential Centrifugation The Chemical Composition of the Bacin the Study of Tissue Enzymesterial Cell W a l C C . S. CUMMINS CHR. DE DUVEAND J. BERTHET Theories of Enzyme Adaptation in MicroEnzymatic Aspects of Embryonic Differorganisms-J. MANDELSTAM entiation-TRYGGvE GUSTAFSON The Cytochondria of Cardiac and Azo Dye Methods in Enzyme HistochemW. HARMON Skeletal MUSCI~--JOHN istry-A. G. EVERSONPEARSE The Mitochondria of the NeuronMicroscopic Studies in Living MamWARRENANDREW mals with Transparent Chamber The Results of Cytophotometry in the Methods-ROY G. WILLIAMS Study of the Deoxyribonucleic Acid The Mast Cell-G. ASBOE-HANSEN (DNA) Content of the NucleusElastic Tissue-EDWARDS w. DEMPSEY R. VENDRELY AND C. VENDRELY A N D ALBERT I. LANSING Protoplasmic Contractility in Relation to The Composition of the Nerve Cell Gel Structure: Temperature-Pressure Studied with New Methods-SvENExperiments on Cytokinesis and OLOEB R A T T G ~AND D HOLCERHYDEN Amoeboid Movement - DOUGLAS MARSLAND AUTHOR INDEX-SUBJECT INDEX Intracellular pH-PETER C. CALDWELL The Activity of Enzymes in Metabolism Volume 4 and Transport in the Red Cell-T. A. J. PRANKERD Cytochemical Micrurgy-M. J. KOPAC Uptake and Transfer of Macromolecules Anioebocytes-L. E. WACCE by Cells with Special Reference to Problems of Fixation in Cytology, HisGrowth and Development-A. M. tology, and Histochemistry-M. WOLSCHECHTMAN MAN Bacterial Cytology-ALFRED MARSHAK Cell Secretion: A Study of Pancreas and c. 1. JUNQUEIRA Histochemistry of Bacteria-R. VENDRELY Salivary Glands-L. AND G. C. HIRSCH Recent Studies on Plant MitochondriaThe Acrosome Reaction-JEAN c. DAN DAVIDP. HACKETT Cytology of Spermatogenesis-VIsHwA The Structure of Chloroplasts-K. NATH MUHUTHALER The Ultrastructure of Cells, as Revealed Histochemistry of Nucleic Acids-N. B. by the Electron Microscope-hfTIoF KURNICK S. SJOSTRAND Structure and Chemistry of NucleoliAUTHOR INDEX-SUBJECT INDEX W. S. VINCENT On Goblet Cells, Especially of the InVolume 6 testine of Some Mammalian SpeciesHARALDMOE The Antigen System of Paramecium Localization of Cholinesterases at H. BEALE aurelia-G. Neuromuscular Junctions-R. Cou- The Chromosome Cytology of the Ascites TEAUX Tumors of Rats, with Special Ref-

CONTENTS OF PREVIOUS VOLUMES

405

erence to the Concept of the Stemline The Structure and Innervation of Lamellibranch M u s c l e J . BOWDEN Cell-SAJIRo M m O Hypothalamo-neurohypophysial NeuroThe Structure of the Golgi ApparatusC. SLOPEFI ARTHUR W. POLLISTERAND PRISCHIA secretion-J. Cell Contact-P.wL WEISS F. POLLISTER An Analysis of the Process of Fertiliza- The Ergastoplasm: Its History, Ultrastructure, and Biochemistry--tion and Activation of the EggCOISE HAGUENAU A. MONROY Anatomy of Kidney Tubules-JomNNEs The Role of the Electron Microscope in RHODIN Virus Research-ROBLEY c. WILLIAMS Structure and Innervation of the Inner The Histochemistry of PolysaccharidesEar Sensory Epithelia-Ham ENGARTHUR J. HALE STROM AND JAN WERSHLL The Dynamic Cytology of the Thyroid The Isolation of Living Cells from Gland-J. GROSS Animal Tissues-L. M. RINALDINI Recent Histochemical Results of Studies on Embryos of Some Birds and Mam- AUTHOR INDEX-SUB JECT INDEX mals-ELI0 BORGHESE Carbohydrate Metabolism and Embryonic Volume 8 Determination-R. J. O'CONNOR Enzymatic and Metabolic Studies on Isolated N u c l e i 4 . SIEBERTAND R. M. S. The Structure of C y t o p l a s m - C w s OBERLING SMELLIE Wall Organization in Plant Cells-R. D. Recent Approaches of the Cytochemical PRESTON Study of Mammalian Tissues-GEORGE H. HOCEBOOM,EDWARDL. KUFF, AND Submicroscopic Morphology of the Synapse-EDuARDo DE ROBERTIS WALTERC. SCHNEIDER The Cell Surface of Paramecium-C. F. The Kinetics of the Penetration of NonEHRET AND E. L. POWERS electrolytes into the Mammalian ErythThe Mammalian Reticulocyte-LEAH rocyte-Fmm BOWYER MIRIAMLOWENSTEIN AUTHOR INDEX-SUB JECT INDEX The Physiology of ChromatophoresCUMULATIVE SUBJECT INDEX MILTONFINGERMAN (VOLUMES 1-5) The Fibrous Components of Connective Tissue with Special Reference to the Elastic Fiber-Davm A. HALL Volume 7 Experimental Heterotopic OssificationJ. B. BRIDGES Some Biological Aspects of Experimental A Survey of Metabolic Studies on IsoRadiology: A Historical Review-F. G. lated Mammalian Nuclei-D. B. SPEAR ROODYN The Effect of Carcinogens, Hormones, Trace Elements in Cellular Functionand Vitamins on Organ Cultures-ILsE BERT L. VALLEE A N D FREDERIC L. LASNITZKI HOCH Recent Advances in the Study of the Osmotic Properties of Living CellsKinetochore-A. LIMA-DE-FARIA D. A. T. DICK Autoradiographic Studies with S"-Sulfate Sodium and Potassium Movements in -D. D. DZIFWIATKOWSKI Nerve, Muscle, and Red Cells-I. M. The Structure of the Mammalian SperGLYNN matozoon-DoN W. FAWCETT Pinocytosis-H. HOLTER The Lymphocyte--O. A. TROWELL AUTHOR INDEX--SUB JECT INDEX

406

CONTENTS OF PREVIOUS VOLUMES

Volume 0

Volume 11

The Influence of Cultural Conditions on F. WILJCINSON Bacterial Cytology-J. AND J. P. DUGUID Organizational Patterns within Chromosomes-BERWIND p. KAUFMANN, HELEN GAY, AND MARGARW R. MCDONALD Enzymic Processes in Celh-JAY BOYD BEST The Adhesion of CellS-LEONARD WEISS Physiological and Pathological Changes in Mitochondria1 Morphology-CH. ROUILLER The Study of Drug Effects at the Cytological Level-G. B. WILSON Histochemistry of Lipids in OogenesisV I S H ~ ANATH Cyto-Embryology of Echinoderms and Amphibia-Kwrsum DAN The Cytochemistry of Nonenzyme Proteins-RONALD R. COWDEN

Electron Microscopic Analysis of the Secretion Mechanism-K. KUROSUMI The Fine Structure of Insect Sense Organs-ELEANOR H. SLIFER Cytology of the Developing E y e ALFRED J. COULOMBRE The Photoreceptor Structures-J. J. WOLKEN Use of Inhibiting Agents in Studies on Fertilization Mechanisms-cmmLm B. METZ The Growth-Duplication Cycle of the Cell-D. M. PRESCOTT Histochemistry of Ossification-ROMuLo L. CABRINI Cinematography, Indispensable Tool for Cytology-C. M. POMERAT AUTHOR INDEX-SUBJECT

INDEX

Volume 12

Sex Chromatin and Human ChromoL. HAMERTON somes-JOHN Chromosomal Evolution in Cell Populations-T. C. Hsu Volume 10 Chromosome Structure with Special Reference to the Role of Metal IonsThe Chemistry of Shiffs ReagentDALE M. STEFFENSEN FREDERICK H. ~ S T E N Electron Microscopy of Human White Spontaneous and Chemically Induced Blood Cells and Their Stem CellsChromosome Breaks-AnuN KUMAR MARCELBESSISAND JEAN-PAUL THIERY SHARMAAND ARCHANASHARMA In Vioo Implantation as a Technique in The Ultrastructure of the Nucleus and J. L. Skeletal Biology-WILLIAM Nucleocytoplasmic RelationsPAuL FELTS WISCHNITZER The Nature and Stability of Nerve The Mechanics and Mechanism of CleavMyelin-J. B. FINEAN age-LEwxs WOLPERT Fertilization of Mammalian Eggs in The Growth of the Liver with Special Vitru-C. R. AUSTIN Reference to Mammals-F. DOLJANSKI Physiology of Fertilization in Fish Eggs Cytology Studies on the Affinity of the -Tom-o YAMAMOTO Carcinogenic Azo- Dyes for Cyto- AUTHOR INDEX-SUBJECT INDEX NAGAplasmic Components-YosAUTHOR INDEX-SUB

JECT INDEX

TAN1

Epidermal Cells in Culture-A. GEDEON Volume 13 MATOLTSY The Coding AUTHOR INDEX-SUB

JECT INDEX

CUMULATIVE SUBJECT INDEX

(VOLUMES 1-9)

Hypothesis-MARTYNAS

YCAS

Chromosome Reproduction-J. TAYLOR

HERBERT

407

CONTENTS OF PREVIOUS VOLUMES

Sequential Gene Action, Protein Synthesis, and Cellular DifferentiationREED A. FLICKINCER The Composition of the Mitochondria1 Membrane in Relation to Its Structure and Function-ERIC G. BALL AND CLIFFE D. JOEL Pathways of Metabolism in Nucleate A. and Anucleate Erythrocytes-H. SCHWEICER Some Recent Developments in the Field of Alkali Cation Transport-W. WILBRANDT

Chromosome Aberrations Induced by Ionizing Radiations-H. J. EVANS Cytochemistry of Protozoa, with Particular Reference to the Golgi Apparatus and the MitochondriaVISHWANATH AND G. P. DUTTA Cell Renewal-FELIX BERTALANFFY AND CHOSENLAU AUTHOR INDEX-SUB

Volume 14

JECT INDEX

The Tissue Mast Wall-Doucus SMITH AUTHOR INDEX-SUB

E.

JECT INDEX

Volume 15

The Nature of Lampbrush Chromosomes -H. G. CALIAN The Intracellular Transfer of Genetic Information-J. L. SIRLIN Mechanisms of Gametic Approach in PlantS-LEONARD MACHLISAND ERIKA RAWITSCHER-KUNKEL The Cellular Basis of Morphogenesis and Sea Urchin Development-T. GUSTAFSON AND L. WOLPERT Plant Tissue Culture in Relation to DeR. PARvelopment Cytology-CARL TANEN

Regeneration of Mammalian LiverNANCYL. R. BUCHER Collagen Formation and Fibrogenesis with Special Reference to the Role of Ascorbic Acid-BEmAm S. GOULD The Behavior of Mast Cells in Anaphylaxis-Ivm MOTA Lipid Absorption-ROBERT M. WOTTON

Inhibition of Cell Division: A Critical AUTHOR INDEX-SUBJECT INDEX and Experimental Analysis-SEYMOUR GELFANT Electron Microscopy of Plant Protoplasm Volume 16 -R. BUVAT Cytophysiology and Cytochemistry of the Ribosomal Functions Related to Protein Synthesis-Tom HULTIN Organ of Corti: A Cytochemical A. VINNIKOV Physiology and Cytology of Chloroplast Theory of Hearing-J. Formation and "Loss" in EugknaAND L. K. TITOVA M. GRENSON Connective Tissue and Serum ProteinsCell Structures and Their Significance R. E. MANCINI for Ameboid Movement-K. E. WOHLThe Biology and Chemistry of the Cell FARTH-BOTTERMAN Walls of Higher Plants, Algae, and Microbeam and Partial Cell Irradiation H. NORTHCOTE Fungi-D. -C. L. SMITH Development of Drug Resistance by Nuclear-Cytoplasmic Interaction with Staphylococci in Vitro and in VivoIonizing Radiation-M. A. LESSLER MARYBARBER Cytological and Cytochemical Effects of In Vioo Studies of Myelinated Nerve Fibers-Cam CASKEYSPEIDEL Agents Implicated in Various Pathological Conditions: The Effect of Respiratory Tissue: Structure, Histophysiology, Cytodynamics. Part I: Viruses and of Cigarette Smoke on Review and Basic Cytomorphologythe Cell and Its Nucleic Acid-CEcnm AND RUDOLFLEUCHFELIXD. BERTALANFFY LEUCHTENBERGER TENBERGER

AUTHOR INDEX-SUB

JECT INDEX

408

CONTENTS OF PREVIOUS VOLUMES

Volume 17

Volume 19

The Growth of Plant Cell Walls-K. WILSON Reproduction and Heredity in Trypanosomes: A Critical Review Dealing Mainly with the African Species in J. WALKER the Mammalian Host-P. The Blood Platelet: Electron Microscopic Studies-J. F. DAVID-FERREIRA The Histochemistry of Mucopolysaccharides-ROBERT c. CURRAN Respiratory Tissue Structure, Histophysiology, Cytodynamics. Part 11. New Approaches and Interpretations -FELIX D. BERTALANFFY The Cells of the Adenohypophysis and Their Functional Significance-MARC HERLANT

“Metabolic” DNA: A Cytochemical Study-H. ROELS The Significance of the Sex ChromatinL. BARR MURRAY Some Functions of the Nucleus-J. M. MITCHISON Synaptic Morphology on the Normal and Degenerating Nervous System-E. G. GRAYAND R. W. GUILLERY Neurosecretion-W. BARGMANN Some Aspects of Muscle RegenerationE. H. BETZ, H. FIRKET,AND M. REZNIK The Gibberellins as Hormones-P. W. BRIAN Phototaxis in PlantS-wOLFGANG HAUPT Phosphorus Metabolism in Plants-K. S.

AUTHOR INDEX-SUB

JECT INDEX

ROWAN AUTHOR INDEX-SUBJECT

INDEX

Volume 18 The Cell of Langerhans-A.

S. BREATH-

NACH

The Structure of the Mammalian EggROBERTHADEK Cytoplasmic Inclusions in OogenesisM. D. L. SRIVASTAVA The Classification and Partial Tabulation of Enzyme Studies on Subcellular Fractions Isolated by Differential Centrifuging-D. B. ROODYN Histochemical Localization of Enzyme Activities by Substrate Film Methods: Ribonucleases, Deoxyribonucleases, Proteases, Amylase, and Hyaluronidase -R. DAOUST Cytoplasmic Deoxyribonucleic Acid-P. B. GAHANAND J. CHAYEN Malignant Transformation of Cells in VitfO-KATHERINE K. SANFORD Deuterium Isotope Effects in CytologyE. FLAWMENHAFT, S. BOSE, H. I. CRESPI, AND J. J. KATZ The Use of Heavy Metal Salts as Electron Stains-C. RICHARDZOBEL AND MICHAELBEER AUTHOR INDEX-SUB

JECT INDEX

Volume 20 The Chemical Organization of the Plasma H. Membrane of Animal Cells-A. MADDY Subunits of Chloroplast Structure and Quantum Conversion in Photosynthesis-RoDERIc B. PARK Control of Chloroplast Structure by Light -LESTER PACKER AND PAUL-AND& SIEGENTHALER The Role of Potassium and Sodium Ions as Studied in Mammalian BrainH. HILLMAN Triggering of Ovulation by Coitus in the Rat-cLAUDE ARON,GITTA ASCH, AND JAQUELINE Roos Cytology and Cytophysiology of NonMelanophore Pigment CeIIs-JOSEPH T. BAGNARA The Fine Structure and Histochemistry of Prostatic Glands in Relation to Sex Hormones-DavIo BRANDES Cerebellar Enzymology-LucE ARVY AUTHOR INDEX-SUB

JECT INDEX

CONTENTS OF PREVIOUS VOLUMES

409

Volume 23

Volume 21

Histochemistry of Lysosomes-P. B. Transformationlike Phenomena in Somatic Cells-J. M. OLENOV GAHAN Physiological Clocks-R. L. BRAHM- Recent Developments in the Theory of ACHARY Control and Regulation of Cellular ~rocesses-~oBERT ROSEN Ciliary Movement and Coordination in Cikates-Bma PARDUCA Contractile Properties of Protein Threads from Sea Urchin Eggs in Relation to Electromyography: Its Structural and Cell Division-HiKoIcHr SAKAI Neural Basis-Jom V. BASMA JIAN Cytochemical Studies with Acridine Electron Microscopic Morphology of oogenesis-ARNE NI~RREvANc Orange and the Influence of Dye Contaminants in the Staining of Dynamic Aspects of Phospholipids during Nucleic Acids-FmERICK H. KASTEN Protein Secretion--LowELs. E. HOKXN Experimental Cytology of the Shoot The Golgi Apparatus: Structure and Apical Cells during Vegetative Function-H. W. BEAMSAND R. G. NouGrowth and Flowering-A. KESSEL CARBDE The Chroinosomal Basis of Sex DeterNature and Origin of Perisynaptic Cells minatiOn-~ENNETH R. LEWIS AND of the Motor End Plate-T. R. SHANBEI~NARD JOHN THAVEERAPPA AND G. H. BOURNE AUTHOR INDEX-SUB JECT INDEX AUTHOR INDEX-SUB

JECT INDEX

Volume 24 Volume 22 Synchronous Cell DifferentiationCurrent Techniques in Biomedical ElecGEORGEN. PADILLAAND IVANL. tron Microscopy-SnuL 'UJISCHNITZER CAMERON The Cellular Morphology of Tissue Re- Mast Cells in the Nervous Systempair-R. M. H. MCMINN YNEVE OLSSON Structural Organization and Embryonic Development Phases in Intermitosis and the Preparation for Mitosis of Mam~ f f e ~ n ~ i a t i o n ~ A V, ~ ~SHERBET NAN AND M. S , LAKSHMI ~ malian Cells in V i t m - B u ~ o ~ A. NE&KOVIC The Dynamism of Cell Division dun'ng Early Cleavage Stages of the EggAntimito€ic Suhstancw-Gw DEYSSON N. FAUTREZ-FIUFYN AND 1. FAUTREZ The form and F u n c t ~ ~o€ n the Sieve Lymphopoiesis in the Thymus and Other Tube: A Problem in ReconciliationP. E. WMTEimLEY AND R. P. c. Tissues: Functional Implications-N. 3. EVERETTAND RUTH W. TYLER J o x i N s m ( CAFFREY ) Analysis of Antibody Staining Patterns Obtained with Striated Myofibrils in Structure and Organization of the MyoFluorescence Microscopy and Electron neural Junction-42 COERS Microscopy-FRANK A. PEPE The Ecdysial Glands of ArthropodsWILLIAMS. HERMAN Cytology of Intestinal Epithelial. CellsP ~ E G, R TONER Cytokinins in Plants-B, I, SAF~AI &UVASe TAVA Liquid Junction Potentials and Their Effects on Potential Measurements in AUTHOR INDEX-SUB J E T INDEX Biology Systems-P. C. C A L ~ W E ~ L CUMULATKVE SUBJECT INDEX AUTHOR WDEX-SUBjECr INDEX (WLUWS 1-22)

410

CONTENTS OF PREVIOUS VOLUMES

Volume 25

Volume 27

Cytoplasmic Control over the Nuclear Events of Cell Reproduction-NOEL DE TERRA Coordination of the Rhythm of Beat in Some Ciliary Systems-M. A. SLEIGH The Significance of the Structural and Functional Similarities of Bacteria and Mitochondria-SYLVAN NASS The Effects of Steroid Hormones on Macrophage Activity-B. VERNONROBERTS The Fine Structure of Malaria Parasites -MARIA A. RUDZINSKA The Growth of Liver Parenchymal Nuclei and Its Endocrine Regulation -RITA CARRIERE Strandedness of Chromosomes-SHELDON WOLFF Isozymes : Classification, Frequency, and Significance-CmRms R. SHAW The Enzymes of the Embryonic Nephron -LUCIE ARVY Protein Metabolism in Nerve Cells-B. DROZ Freeze-Etching-HANS MOOR

Wound-Healing in Higher PlantsJACQUESLIPETZ Chloroplasts as Symbiotic OrganellesDENNISL. TAYLOR The Annulate Lamella-SAUL WISCHNITZER

Gametogenesis and Egg Fertilization in Planarians-G. BENAZZI LENTATI Ultrastructure of the Mammalian Adrenal Cortex-SIMON IDELMAN The Fine Structure of the Mammalian Lymphoreticular SyStem-IAN CARR Immunoenzyme Technique: Enzymes as Markers for the Localization of Antigens and Antibodies-STRATIS AVRAMEAS AUTHOR INDEX-SUB

JECT INDEX

Volume 28

The Cortical and Subcortical Cytoplasm Of LymnUf?U Egg-CHRISTIAAN P. RAVEN The Environment and Function of Invertebrate Nerve Cells-J. E. AND R. B. MORETON TREHERNE AUTHOR INDEX-SUB JECT INDEX Virus Uptake, Cell Wall Regeneration, and Virus Multiplication in Isolated C. COCKING Plant Protoplasts-E. Volume 20 The Meiotic Behavior of the Drosophih OOCyk-ROBERT c. KING A New Model for the Living Cell: A The Nucleus: Action of Chemical and Summary of the Theory and Recent SIMARD Physical Agents-RENk Experimental Evidence in Its Support The Origin of Bone Cek-MAUREEN -GILBERT N. LING OWEN The Cell Periphery-LEONARD WEISS Regeneration and Differentiation of Mitochondria1 DNA: Physicocheniical Sieve Tube Elements-WILLIAM P. Properties, Replication, and Genetic JACOBS Function-P. BORSTAND A. M. KROON Cells, Solutes, and Growth: Salt AcMetabolism and Enucleated Cells-KoNcumulation in Plants ReexaminedRAD KECK F. C. STEWARDAND R. L. M o m Stereological Principles for Morphometry AUTHOR INDEX-SUB JECT INDEX in Electron Microscopic CytologyEWALDR. WEIBEL Some Possible Roles for Isozyniic Substi- Volume 20 tutions during Cold Hardening in Gram Staining and Its Molecular MechPlants-I). W. A. ROBERTS anism-B. B. BISWAS,P. S. BASU,AND AUTHOR INDEX-SUB JECT INDEX M. K. PAL

411

CONTENTS OF PREVIOUS VOLUMES

The Surface Coats of Animal Cells-A. MART~EZ-PAUIMO Carbohydrates in Cell Surfaces-Rrcum J. WINZLER Differential Gene Activation in Isolated Chromosomes-MAms LEZZI Intraribosomal Environment of the Nascent Peptide Chain-HmEKO KAJI Location and Measurement of Enzymes in Single Cells by Isotopic Methods Part I-E, A. BARNARD Location and Measurement of Enzymes in Single Cells by lsotopic Methods Part I I - G . C. BUDD Neuronal and Glial Perikarya Preparations: An Appraisal of Present Methods AND BETTY -PATRICIA V. JOHNSTON I. ROOTS Functional Electron Microscopy of the Hypothalamic Median EminenceHIDESHIKOBAYASHI, TOKUZOMATSUI, AND S u s u ~ rISHII Early Development in Callus CulturesMICHAELM. YEOMAN

Morphological and Histochemical Aspects of Glycoproteins at the Surface of Animal Cells-A. RAMBOURG DNA Biosynthesis-H. S. JANSZ,D. VAN DER MEI, AND G. M. ZANDVLUX Cytokinesis in Animal Cells-R. RAPPAPORT

The Control of Cell Division in Ocular Lens-C. V. HARDING, J. R. REDDAN, N. J, UNAICAR, AND M. BACCHI The Cytokinins-HANS KENDE Cytophysiology of the Teleost Pituitary -MARTIN SAGE AND HOWARDA. BERN AUTHOR INDEX-SUB

JECT INDEX

Volume 32

Highly Repetitive Sequences of DNA in Chromosomes-W. G. FLAMM The Origin of the Wide Species Variation in Nuclear DNA Content-H. REES AND R. N. JONES Polarized Intracellular Particle Transport: AUTHOR INDEX-SUB JECT INDEX Saltatory Movements and Cytoplasmic Streaming-LroNEL I. REBHUN Volume 30 The Kinetoplast of the HemoflagellatesLARRYSIMPSON High-pressure Studies in Cell BiologyTransport across the Intestinal Mucosal ARTHUR M. ZIMMERMAN S. Cell: Hierarchies of Function-D. Micrurgical Studies with Large FreePARSONS AND C. A. R. BOYD Living Amebas-K. W. JEON AND Wound Healing and Regeneration in the J. F. DANIEUX Crab Pamtelphusa hgdrodromousThe Practice and Application o f Electron RITA G. ADIYODI Microscope Autoradiography-J. JACOB The Use of Ferritin-Conjugated AntiApplications of Scanning Electron bodies in Electron MicroscopyMicroscopy in Biology-K. E. CARR COUNCILMAN MORGAN Acid Mucopolysaccharides in Calcified Metabolic DNA in Ciliated Protozoa, Tissues--SHIN JIRO KOBAYASHI Salivary Gland Chromosomes, and AUTHOR INDEX-SUB JECT INDEX Mammalian Cells-S. R. PELC CUMULATIVE SUBJECT (VOLUMES

INDEX

1-29)

AUTHOR INDEX-SUBJECT

INDEX

Volume 31

Volume 33

Studies on Freeze-Etching of Cell Membranes-KmT M~~HLETHALER Recent Developments in Light and Electron Microscope Radioautography -G. C. BUDD

Visualization of RNA Synthesis on Chromosomes-0. L. MILLER,JR. AND BARBARA A. HAMKALO Cell Disjunction ( “Mitosis”) in Somatic Cell Reproduction-ELAINE G. DIA-

412

CONTENTS OF PREVIOUS VOLUMES

SCOTT HOLLAND, AND PAULINEPECORA Neuronal Microtubles, Neurofilaments, and Microfilaments-RAYMOND B. WUERKERA N D JOEL B. KIRKPATRICK Lymphocyte Interactions in Antibody Responses-J. F. A. P. MILLER Laser Microbeams for Partial Cell Irw. BERNS AND radiation-MlcmEL CHRISTIAN SALET Mechanisms of Virus-Induced Cell Fusion-GEORGE POSTE Freeze-Etching of Bacteria-CHARLES c. REMSENAND STANLEYW. WATSON The Cytophysiology of Mammalian Adipose Cells-BERNARD G. SLAVIN

Synthetic Activity of Polytene Chromosomes-HANS D. BERENDES Mechanisms of Chromosome Synapsis at Meiotic Prophase-PETER B. MOENS Structural Aspects of Ribosomes-N. NANNINCA Comparative Ultrastructure of the Cerebrospinal Fluid-Contacting NeuronsB. VICH AND I. VIGH-TEICHMA" Maturation-Inducing Substance in Starfishes-Hmuo KANATANI The Lirnoniurn Salt Gland: A Biophysical and Structural Study-A. E. HILL AND B. S. HILL Toxic Oxygen Effects-HAROLD M.

AUTHOR INDEX-SUBJECT

AUTHOR INDEX-SUBJECT

CUMAKOS,

INDEX

Volume 34

SWART2 INDEX

Volume 36

Molecular Hybridization of DNA and HENNIC RNA in SitU-WOLFGANG The Relationship between the PlasmaNITZER lemma and Plant Cell Wall-JEANThe Energy State and Structure of IsoCLAUDEROLAND lated Chloroplasts: The Oxidative Recent Advances in the Cytochemistry Reactions Involving the Water-Splitand Ultrastructure of Cytoplasmic ting Step of Photosynthesis-ROBERT Inclusions in Mastigophora and L. HEATH Opalinata (Protozoa)-G. P. DUTTA A. Chloroplasts and Algae as Symbionts in Transport in Neurospora-GENE SCARBOIIOUGH MOIIUSCS-LEONARDMUSCATINEAND Mechanisms of Ion Transport through RICHARDW. GREENE Plant Cell Membranes-EMANuEL The Macrophage-SAIMoN GORDONAND ERSTEIN ZANVIL A. COHN Cell Motility: Mechanisms in Proto- Degeneration and Regeneration of Neuroplasmic Streaming and Ameboid secretory Systems-HORsr-DIETER Movement-H. KOMNICK,W. STOCDELLMANN KEM, AND K. E. WOHLEFARTH- AUTHOR INDEX-SUB JECT INDEX BOTKERMANN The Gliointerstitial System of MolluscsGHISLAINNICAISE Volume 37 Colchicine-Sensitive Microtuhles-Lm MARGULIS Units of DNA Replication in ChromoAUTHOR INDEX-SUB JECT INDEX somes of Eukaroytes-J. HERBERT TAYLOR Viruses and Evolution-D. C. REANNEY Electron Microscope Studies on SpermioVolume 35 genesis in Various Animal SpeciesGONPACHIRO YASUZUMI The Structure of Mammalian ChromoMorphology, Histochemistry, and BioSomeS--ELTON STUBBLEFIELD

The Subniicroscopic Morphology of the Interphase Nucleus-SAuL WISCH-

CONTENTS OF PREVIOUS VOLUMES

413

chemistry of Human Oogenesis and Nucleocytoplasmic Interactions in Development of Amphibian HybndsOvulation-SmvL S. GURAYA STEPHENSUBTELNY Functional Morphology of the Distal The Interactions of Lectins with Animal LUng-bYE H. -URN Cell Surfaces-GARTH L. NICOLSON Comparative Studies of the Juxtaglomerular Apparatus-Hmomm SOKAFIE Structure and Function of Intercellular Junctions-L. ANDREW STAEHELIN AND MXZUHO OGAWA The Ultrastructure of the Local Cellular Recent Advances in Cytochemistry and Ultrastructure of Cytoplasmic IncluCARR Reaction to Neoplasia-Im sions in Ciliophora (Protozoa)-G. P. AND J. C. E. UNDERWOOD DUTTA Scanning Electron Microscopy in the Ultrastructural Analysis of the Mam- Structure and Development of the Renal Glomerulus as Revealed by Scanning malian Cerebral Ventricular SystemElectron Microscopy-FRANC0 SPID. E. SCOTT, G. P. KOZLOWSYCI,AND NELLI M. N. SHERIDAN Recent Progress with Laser Microbeams AUTHOR INDEX-SUB JECT INDEX -MICHAEL W. BERNS The Problem of Germ Cell Determinants Volume 38 -H. W. BEAMSAND R. G . KESSEL SUBJECT INDEX

Genetic Engineering and Life Synthesis: An Introduction to the Review by R. Widdus and C. AU~-JAMES F. DANIELLI Progress in Research Related to Genetic Engineering and Life Synthesis-Roy WIDDUS AND CHARLES R. AULT The Genetics of C-Type RNA Tumor Viruses-J. A. WYKE Three-Dimensional Reconstruction from Projections: A Review of AlgorithmsRICHARD GORDON AND GABOR T. HERMAN The Cytophysiology of Thyroid CellsVLADXMXR R. PANTIC The Mechanisms of Neural Tube Formation--PERRY KARFUNXEL The Behavior of the XY Pair in Mam~ ~ ~ S - A L B E R T OJ. SOLARI Fine-Structural Aspects of Morphogenesis in Acetabu1uria-G. WERZ Cell Separation by Gradient CentrifugaHARWOOD tion-R. SUBJECT INDEX

Volume 39

Volume 40 B-Chromosome Systems in Flowering Plants and Animal Species-R. N.JONES The Intracellular Neutral SH-Dependent Protease Associated with Inflammatory Reactions- HIDEO HAYASHI The Specificity of Pituitary Cells and Regulation of Their Activities - VLAI)IMIH R. PANTIC Fine Structure of the Thyroid GlandH i s m FUJITA Postnatal Gliogenesis in the Mammalian Brain -A. PRIVAT Three-Dimensional Reconstruction from Serial Sections - RANDLE W. WARE AND VINCENT LOPHESTI SUBJECT INDEX

Volume 41 The Attachment of the Bacterial Chromosome to the Cell Membrane-PAUL J. LEIBOWITZAND MOSELIO SCHAECHTEH

Regulation of the Lactose Operon in Androgen Receptors in the Nonhistone Escherichia coli by CAMP-G. CAnProtein Fractions of Prostatic ChroPENTEH AND B. H. SELLS matin-TUNG YUE WANG ANI) LEROY Regulation of Microtubules in TetmM. NYBERG hgrnena - NORMAN E. WILLIAMS

414

CONTENTS OF PREVIOUS VOLUMES

Ultrastructure of' Mammalian CliromoCellular Receptors and Mechanisms of ANI) some Aberrations - B. H.BRINKLEY Action of Steroid Hormones - SHUTWALTER N. HITTELMAN SUNG LIAO Computer Processing of Electron MicroA Cell Culture Approach to the Study of graphs: A Nonmathematical AccountAnterior Pituitary Cells- A. TIXIEHVIDAL, D. COUHUJI, A N D c. TOUGAHD P. W. HAWKES Cyclic Changes in the Fi n e Structure of Immunohistochemical Demonstration of the Epithelial Cells of Human EndoNeurophysin in the Hypothalamoneumetrium- MILDHEU GOHIION rohypophysial System- W. B. WATKINS T h e Ultrastructure of the Organ of The Visual System of the Horseshoe Corti - ROBERT S. KIMURA Crab Limulus polyphernus - WOLF H. Endocrine Cells o f t h e Gastric MucosaFAHHENBACH ENRICO SOLCIA, CAHLO CAPELLA, SUBJECT INDEX GARHIELE VASSALLO, AND ROBERTO Volume 42 BUFFA Regulators of Cell Division: Endogenous Membrane Transport of' Purine and Mitotic Inhibitors of Mammalian Pyrimidine Bases and Nucleosides in Cells- BISMARCK B. LOZZIO,CARMEN Animal Cells- RICHARI) D. BERLIN B. LOZZIO, ELENA G. BAMBERGER, AND AND J A N E T M. OLIVER STEPHENV. LArn SUBJECT INDEX

A 5 6 6

c 7 D B E 9

F O

G 1 H Z 1 3 J

4