International Review of Cytology, Volume 83

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME83

ADVISORY EDITORS DONALD G. MURPHY H. W. BEAMS ROBERT G. E. MURRAY HOWARD A. BERN RICHARD NOVICK GARY G. BORISY ANDREAS OKSCHE PIET BORST MURIEL J. ORD BHARAT B. CHATTOO VLADIMIR R. PANTIC STANLEY COHEN W. J. PEACOCK RENE COUTEAUX DARRYL C. REANNEY MARIE A. DIBERARDINO LIONEL I. REBHUN CHARLES J. FLICKINGER JEAN-PAUL REVEL OLUF GAMBORG M. NELLY GOLARZ DE BOURNE JOAN SMITH-SONNEBORN WILFRED STEIN YUKIO HIRAMOTO HEWSON SWIFT YUKINORI HIROTA K. TANAKA K. KUROSUMI DENNIS L. TAYLOR GIUSEPPE MILLONIG TADASHI UTAKOJI ARNOLD MITTELMAN AUDREY MUGGLETON-HARRIS ROY WIDDUS ALEXANDER YUDIN

INTERNATIONAL

Review of Cytology EDITED BY

G. H. BOURNE

J. F. DANIELLI

St. George’s University School of Medicine

Danielli Associates Worcester, Massachusetts

St. George’s, Grenada

West lndies

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

VOLUME83

1983

ACADEMIC PRESS

A Subsidiary of Harcourt Brace Jovanovich. Publishers

New York London Paris San Diego San Francisco SBo Paulo Sydney Tokyo Toronto

0

COPYRIGHT 1983, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY F O R M OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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NUMBER: 52- 5203

I S B N 0-12-364483-6 PRINTED IN THE UNITED STATES OF AMERICA 83 84 85 86

9 8 1 6 5 4 3 2 1

Contents CONTRIBUTORS .............................................................

vii

Transposable Elements in Yeast VALERIE MOROZWILLIAMSON

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yeast Transposable Element Ty ................................. Other Transposable Elemen .................. ...... IV. Discussion ...... ................................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. 111.

1

2 20 21 23

Techniques to Study Metabolic Changes at the Cellular and Organ Level ROBERTR. DEFURIAA N D MARYK. DYGERT

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nuclear Magnetic Resonance Spectroscopy as a Method to Study Adenosine Triphosphate Metabolism at the Organ Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Relationship of the Energetic State of Cells and Their Biology.. . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

27

11.

28 46 60

Mitochondrial Form and Function Relationships in Vivo: Their Potential in Toxicology and Pathology ROBERTA. SMITHA N D MURIELJ. ORD

............... 1. Introduction ........................ 11. The Chondriome of the Eukaryotic Cell.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................ 111. Mitochondrial Cristae c Activity ....................... IV . The Form of the Chon V. Mitochondrial Inclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . The Potential of Modem Staining Methods in Monitoring Mitochondrial Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Mitochondriagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................................ VIII. Concluding Remarks References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Note Added in Proof . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .

V

63 64 76 85 101

109 118

122 125 134

vi

C0NT EN T S

Heterogeneity and Territorial Organization of the Nuclear Matrix and Related Structures M. BOUTEILLE, D. BOUVIER, AND A. P. SEVE

............................................... I. Introduction 11. Definitions . ..... 111. Toward an Anatomy of atin St ........................... IV. Role of Nonchromatin Structures in Nuclear Organization. . . V. Involvement of Nonchromatin Structures in Gene E VI. Three-Dimensional Organization of Nonchromatin Structures . . . . . . . . . . . . . . . . . VII. Prospects on Nonchromatin Structure Characterization Based on ......... Fractionation Experiments. ............................... VIII. Conclusion . . . . . . . . . . . . . ... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

135 137

160 166 177

Changes in Membrane Properties Associated with Cellular Aging A. MACIEIRA-COELHO

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

183

11. Cell Volume.. . . . . . . . . . . . . . . . . 111. Adhesion.

IV. V. VI. Vil.

Mechanis Relationship between Cell-Substra Putative Mechanisms for Membrane-Dependent Manifestations of Aging., . . . . . . .......... Conclusions . . . . . . . . . . . . . . . . . . References .....................................................

212 215 217

Retinal Pigment Epithelium: Proliferation and Differentiation during Development and Regeneration OLGAG . STROEVAA N D VICTOR I. MITASHOV Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Proliferation in Growth and Differentiation of the Retinal Pigment Epithelium in High Vertebrates and Humans.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Proliferation of Pigment Epithelium Cells in Process of Retinal and Lens Regeneration. . . ........... IV. General Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 11.

INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTSOF RECENTVOLUMESAND SUPPLEMENTS. .............................

22 I 223 263 285 287 295 299

Contributors

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

M. BOUTEILLE ( 1 35), Laboratoire de Pathologie Cellulaire, Institut Biome'dical des Cordeliers, 75270 Paris Cedex 06, France D. BOUVIER ( 1 3 9 , Laboratoire de Pathologie Cellulaire, Institut Biome'dical des Cordeliers, 75270 Paris Cedex 06, France ROBERTR. DEFURIA(27), Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, Massachusetts 01609 MARY K . DYGERT(27), Department of Chemistry, Smith College, Northampton, Massachusetts 01060 A. MACIEIRA-COELHO ( 183), Dipartement de Pathologie Cellulaire, Institut de Cancirologie et dlmmunogine'tique (INSERM U 50), 94804 Villejuif, Cidex, France VICTORI. MITASHOV (22 1 ), N. K. Koltzov Institute of Developmental Biology, Academy of Sciences of the USSR, Moscow 11 7808, USSR MURIEL J . ORD(63), Department of Biology, University of Southampton, Southampton SO9 3TU, England, and MRC Toxicology Research Unit, Carshalton, Surrey, England A. P. SEVE( 1 3 3 , Laboratoire de Pathologie Cellulaire, Institut Biome'dical des Cordeliers, 75270 Paris Cedex 06, France

ROBERTA. SMITH(63),Department of Anatomy, University of Glasgow, Glasgow GI2 8QQ, Scotland OLGA G. STROEVA(221), N. K . Koltzov Institute of Developmental Biology, Academy of Sciences of the USSR, Moscow 117808, USSR VALERIE MOROZWILLIAMSON ( l ) , ARC0 Plant Cell Research Institute, Dublin, California 94568

vii

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 83

Transposable Elements in Yeast VALERIEMOROZWILLIAMSON ARC0 Plant Cell Research Institute, Dublin, California Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yeast Transposable Element Ty . . . . . . . . . . . . . . . . . . . . . . A. Physical Structure . . . . . . . . . . . . . . . . . . . . ......... B. Transcription . . . . . . . . . C. Transposition . . . . . . . . . D. Effects of Tyl Insertion ............... E. Associated Gene Conversion. .................... F. Transposable Elements a 111. Other Transposable Elements in Yeast.. ....................... IV. Discussion . . . . . . . . . . . . . . . ................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

1

11.

10

20 21 23

I. Introduction Transposable elements are DNA sequences that move to new genomic locations at a much higher rate than that of the bulk of the cellular DNA. Such mobile elements were first defined genetically as controlling elements in maize (McClintock, 1952, 1957) and have been studied on the molecular level in diverse organisms, such as bacteria, yeast, and fruit flies (Kleckner, 1977: Carlos and Miller, 1980; Green, 1980; Shapiro and Cordell, 1982). Certain general properties characterize these elements. Physically, these transposable DNAs have a direct and/or inverted repeat of DNA sequence at each end. The ability to cause deletions or chromosomal rearrangements is characteristic of these elements, and many have been shown to affect the expression of chromosomal genes by inserting adjacent to or into these genes. The first transposable element found in the yeast Saccharomyces cerevisiae was named Tyl by Cameron et al. (1979). It was observed as a moderately repetitive DNA sequence, one copy of which was present adjacent to a tyrosine tRNA gene in one yeast strain but not in others. Analysis of the hybridization spectrum of this element to DNA from various laboratory yeast strains indicated that its locations in the yeast genome varied from strain to strain, a finding suggesting that it was transposable. Evidence for transposition was also obtained after prolonged growth at 37°C. Soon after, analysis of mutations in expression of the HIS4 gene, which codes for activities required for histidine biosynthesis, revealed that two unstable His- mutations were caused by insertion of DNA

Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-364483-6

2

VALERIE MOROZ WILLIAMSON

sequences homologous to Tyl upstream from the structural gene (Roeder et al., 1980). Other mutations, such as one that resulted in 20-fold overproduction of iso-2-cytochrome c (Errede et al., 1980a,b) and seven different mutations that altered the expression of alcohol dehydrogenase (Ciriacy, 1976, 1979, Williamson et al., 1981), were also found to be due to insertion of Tyl-like sequences into their regulatory regions. Other genetic phenomena such as deletions, translocations, and inversions were observed in connection with Tyl-like DNA sequences in yeast (Chaleff and Fink, 1980). Similarities are apparent in the features of Tyl , Drosophila transposable elements such as copia, and integrated proviral forms of retroviruses in birds and mammals (see Cold Spring Harbor Symp. Quant. Biol. 45, 1980). These elements are each composed of an internal DNA segment of several thousand base pairs flanked by a direct repeat of a DNA sequence that is several hundred base pairs long. They are bounded by the terminal dinucleotides 5’TG . . . CA3’ and are flanked by direct repeats 4-6 base pairs long, which appear to have been generated by duplication of a target DNA sequence upon integration of the element. All are transcribed into nearly full length polyadenylated RNA. There is evidence that copia, Ty, and proviruses can each alter the expression and regulation of adjacent genes (Bingham etal., 1981: Hayward etal., 1981; Payne etal., 1982). Similarities between Ty, copia, and proviruses are a recurring theme in this article and point to the likelihood that these fascinating elements have a common origin. Saccharomyces cerevisiae is very useful as a eukaryotic model system for molecular biologists. Features of the yeast system that make this organism particularly advantageous include ease of growth, small genome size, availability of selectable markers, ease of genetic manipulation, and availability of techniques for transformation with exogenous DNA (Beggs, 1978: Hinnen et al., 1978). A number of useful shuttle vectors are available, and it is relatively easy to integrate DNA sequences into the yeast genome by homologous recombination. Using these techniques, investigators can, for example, replace a chromosomal DNA sequence with one that has been specifically altered in vitro (Scherer and Davis, 1979). Because of the advantages mentioned earlier, much has been and can be learned from the study of the properties of transposable elements in this organism. 11. Yeast Transposable Element Ty

A. PHYSICAL STRUCTURE Ty is defined as a family of disperse, repetitive DNA sequences, each of which is homologous to the original Ty 1 sequence discovered by Cameron et al. (1979). Ty family members consist of an internal 5.3-kilobase (kb) fragment of DNA [epsilon (E) DNA] bounded by copies of the direct repeat sequence delta

3

TRANSPOSABLE ELEMENTS IN YEAST

RNA

Y M

5.0

15'

FIG. 1. General structure of Ty element DNA. Black bars represent the delta ( 8 ) sequences, which are present in directly repeated orientation; white bar represents the epsilon (e) region, and dashed lines represent chromosomal DNA sequence. A 5-base pair sequence of target DNA is duplicated upon transposition and one copy is present at each end after Ty integration. Arrows represent length and direction of the major RNA transcripts that have been characterized (Elder et a / ., 1982). Numbers represent the lengths of the nucleic acid sequences shown in kilobases (kb).

( 6 ) , which is about 0.3 kb long (Fig. 1). Members of this family have been observed as single units on several different chromosomes in yeast. Cameron et al. (1979) isolated from a yeast genomic library DNA clones that contain parts of two adjacent elements, a finding indicating that Ty copies also occur either tandemly or as free circles. Approximately 30 copies of the complete element per haploid genome are present in most laboratory strains of S. cerevisiae, although the number and,distribution of the copies varies from strain to strain (Cameron er al., 1979; Eibel et al., 1980; Fink et al., 1980). The number of Ty copies in wild-type isolates of S . cerevisiae is in general lower than in laboratory strains. Eibel et al. (1980) examined 21 natural isolates and found that the number of copies of Ty ranged from 4 to 20. Saccharomyces norbensis, which is closely related to standard laboratory strains of S. cerevisiae and produces viable spores when crossed with S. cerevisiae strains, appears to have no sequences that strongly hybridize to the epsilon region of Ty (Fink et al., 1980). Its genome does, however, contain sequences that hybridize with a probe for the delta sequence (Roeder and Fink, 1982b). Numerous Ty elements have been cloned, and many others have been studied indirectly by analyzing Southern hybridization profiles using adjacent DNA sequences as probes. The length of most of the Ty DNA sequences is about 5.9 kb. However, others may differ in length; for example, the cloned element Tyl-17 (Kingsman et a l . , 1981) is somewhat longer because of an insertion near its right end. A comparison of the restriction enzyme cleavage maps of several cloned Ty elements is given in Fig. 2. Significant sequence heterogeneity, as reflected by restriction site polymorphism, exists among Ty elements. The restriction maps shown, as well as Southern analyses and heteroduplex analyses, allow division of these elements into two broad classes. The first class is exemplified by element Tyl(S13) (Fig. 2). Elements of this class generally contain one or two EcoRI sites. Those that have been tested cross-hybridize strongly to the original Tyl, which is adjacent to the Tyr tRNA-coding gene. Elements of the second

4

VALERIE MOROZ WILLIAMSON

Class 1 (Tyl) X Bg S

EE

b

b

Bg S

H

E

Bg

S

Bg

1

I

I

I

1

1

I

I

I

I

I

1

1

Bg

EE

I

1

1

I

I

1 1

I

I

I

H

EE

Bg

I

1

I

1 1

I

I

I

I

&l

I

EE

Bg

S

Bg

I 1

I

I

1

I

I

I

E I I

Bg I

I

I

I

1

1

X Bg S

b

H

B

I

I

H

B

X

& Ty ADH2-2';

I

I

I I

Class 2 (Ty2) X Bg S

S Bg

I

I

TyCYC7-H2

I

I I

I

Ty912

S Bg

I

1

XBgS

x

h T y l (S13)

I

X Bg S

x

X

Bg

Bg

Bg

I

I

I

I

Ty AD1

X Ty ADH2-6';

Ty AD

X

v//////n

Ty 1-161

X

r///////////

rx//7v/A

b

H

Ty 1-17; Ty 917

X

d Ty ADH2-3'

FIG.2. Comparison of cloned Ty elements. Solid bars represent delta sequence; white bars represent epsilon regions. Crosshatched areas indicate the approximate locations of regions that are nonhomologous to Tyl because of insertions (in the case of Tyl-161) or substitutions (for the Class 2 elements). Restriction enzyme cleavage sites are designated as follows: B, BarnHI: Bg, BgllI; E, EcoR1; H, HindIII, S, SalI; and X, XhoI. Only restriction enzyme cleavage sites that have been determined for all the elements shown are presented here. References for these restriction maps are Tyl(S13). Cameron et a/.. 1979, and Eibel et al.. 1980; Ty912 and Ty917, Fink eta/.. 1980, and Roeder and Fink, 1982b; TyCYC7-H2, Errede et al., 1980a,b; TyADH2-2', -3c, -fF, -7c, and -8c, Williamson et al., 1983; and Tyl-161 and Tyl-17, Kingsman e t a / . , 1981.

class contain no EcoRI sites but contain a BarnHI restriction site in the corresponding region. Elements belonging to Class 1 or Class 2 are often referred to as Tyl or Ty2, respectively. In cases where heteroduplex analysis has been done between Tyl and Ty2 elements, two substitution loops are seen (Kingsman et al., 1981 ; Williamson et al., 1983). One substitution of about 1 kb begins about 2 kb from the left end of the Ty elements and encompasses the EcoRI sites of the Class 1 elements and the HindIII and BarnHI sites of the Class 2 elements. Closer examination indicates that there may be some homology within this substitution (V. M. Williamson, unpublished). The second substitution loop is about 2 kb

TRANSPOSABLE ELEMENTS IN YEAST

5

long and begins about 3 kb from the left end of the Ty element. Fink et al. (1980) have shown that a Clul fragment about 1.6 kb long from this region of Ty9 17 does not cross-hybridize to Class 1 Ty elements. Heteroduplex and Southern hybridization studies have shown that this 2-kb region is conserved among Class 1 Ty elements: however, it has not been determined whether the corresponding region is conserved among Class 2 elements. The BglII restriction site shown near the left delta is conserved in all published examples. In fact, the left-most kilobase of DNA sequence appears to be conserved among all Ty elements. The five-base pair (bp) nucleotide sequence TACCA is present in direct repeat orientation at the ends of the epsilon region (i.e., at the delta-epsilon junctions) in both classes of Ty elements (Farabaugh and Fink, 1980; Gafner and Philippsen, 1980; Williamson et a l . , 1983). The proportion of elements in each of the two classes and of restriction site variants within each class varies considerably from strain to strain. For example, genomic blotting analysis by Eibel et al. (1980) indicates that in some yeast strains none of the DNA fragments that cross-hybridize with Tyl appear to contain restriction sites for EcoRI. Two Australian yeast strains that they examined lack at least two of the three BgllI restriction sites shown in Fig. 2 within their Ty elements. Similar experiments by Cameron et al. (1979) suggest that none of the Ty family members in strain S288C contain Hind111 restriction sites and that the fraction of Ty elements containing one versus two EcoRI sites varies from strain to strain. The 330-bp DNA sequence delta, which is found at the ends of all Ty elements, also occurs at other loci in the genome and is present in about 100 copies per genome (Cameron et al., 1979). It is difficult to obtain a good estimate of the number of deltas in a genome because these repetitive DNA sequences occur both as single and clustered units. Cameron et al. (1979) found several delta sequences in a 12.5-kb DNA fragment containing a tyrosine tRNA gene ( S U M ) : sequence analysis has shown that five delta sequences are present as two pairs of inverted sequences and one single delta (Gafner and Philippsen, 1983; Rothstein and Helms, 1982). Another problem in estimating the number of delta sequences in a genome is that some solo delta sequences have diverged in sequence to such an extent that hybridization to a single prototype delta would not be observed (P. Philippsen, personal communication). Recombination between delta sequences at the ends of Ty elements is one potential mechanism for generation of solo delta sequences. In fact, several cases have been observed where a Ty element is lost from a particular place in the yeast genome, leaving behind a single (solo) delta sequence (Roeder et ul., 1980; Ciriacy and Williamson, 1981). It is possible that deltas themselves are transposable, but no evidence for this has been obtained. The DNA sequences of several Ty-associated delta elements have been determined; these are compared in Fig. 3. The deltas at opposite ends of each Ty are

6

VALERIE MOROZ WILLIAMSON 100

TGAGAAAlGGGTGAATGTTGAGATAAlTGTTGGGATlCCAlTGTlGAlAAAGGCTAlAAlATTAGGTATACAGAAl~TAClAGAAGlTClCCTCGAGGAl

AG

G T TG G T TGGT GG AA 1

RAT AAT

AG

1 AT A

1

1

T

A

T T

A A

-

GT

A A

Gl

-

pi1

GG A A 1

T

1

A G AG T

A

.

GT

-

Gl

G

-

2w llAGGAAlCCAlAAAAGGGAAlClGCAATTClACACAAllClATAAAlAlTATl-AlCAl--CGllTlAlATGllAAlAllCAllGAlCClAlTACAllA TC 1 A1 T 1 C AA 1 CG CC TC 1

A

917

1

A 1C TC T A

917 L i R

1

A

AOHZ-ZC,

8C

T 1

TC TC

A A

AT T

AT T

T C AA T C AA

T CG

1 CG

CC CC

T...

.

1 1

lCAAlCCTlGCGlTlCAGCllCCAClAAlllAGAlGACTAlTTCTCAlCATTlGCGlCAlCTTCl-AACACCGlATAlGAlAAlAlAClAGlAACGTAAA 1

Annz-Ic 1 GA

T GA

8C

TA TA T

C C

lACTAGTT~GlAGAlGAlAGT*GAlllTlATlCCAACA

c

A

T T

G G

A A GA

C C

G

G C

FIG. 3. Comparison of the DNA sequence of deltas at the ends of Ty elements. The complete sequence of the TyADH2-2[ delta is shown, and nucleotides that differ from this in the other delta sequences that have been determined are indicated. Ty elements are identified on the left margin, and the sequence is presented such that the 5' to 3' strand (left to right) of Fig. 2 is shown. Where the left (L) and right (R) deltas of a particular Ty element differ, both are presented. A dash (-) indicates that a nucleotide is not present in the delta sequence shown. References for these sequences are as follows: ADH2-2r, -3c, -6l, -70 7 0 .

dorsal

I

,

,

,

,

,

6 8 1 0 1 2 days

IIb

T

ventral

FIG. 36. Change of [3H]TdR labeling index of iris inner (la,Ib) and outer (IIaJIb) layer cells in tests. In the pulse-labeling test [3H]TdR was injected on the pulse-labeling (0)and reutilization (0) second to the twelfth day after lens removal and retinectomy. (a) Single injection of [3H]TdR dorsal part of iris; (b) three injections of ['HITdR (every 3 hours], ventral part of iris. Eyes were fixed 3 hours after a single injection of 13H]TdR or 3 hours after the last injection of [3H]TdR in the test with three injections. In the reutilization test, ['HITdR was injected once (a) or three (b) times before lens removal and retinectomy, then lens and retina were removed 3 hours after [3H]TdR injection. Eyes were fixed on the fourth to the twelfth day after injection of [3H]TdR. Each point represents four to six eyes. Bars give means for each day; vertical lines are standard errors of these means. From Parshina and Mitashov (1978).

27 8

OLGA G . STROEVA AND VICTOR 1. MITASHOV 100

-

T = 23.5 hrs

f -

-F K

"

I

10

Y

,

,

,

,

,

,

,

,

I

,

I

I

,

FIG.37. Labeled mitoses curve for lens rudiment in adult newt 7'. crisfatus for the fourteenth to the sixteenth day after lens removal and retinectomy. For each point all cells (six eyes) in metaphase through telophase were scored for the presence of silver grains. From Mitashov (1969~).

fourth to seventh day after the operation. Eguchi and Shingai (1971) revealed two elevations in the [3H]TdR index in the dorsal iris on the seventh and twelfth day after removal of the lens. Following formation of the lens rudiment and its subsequent development, the cells of the iris end the proliferation cycle. Numerous proliferating cells exist in the various portions of the iris not directly involved in the formation of the regenerating lens, as was established when determining the proliferating cell index by using a method that takes into account the phenomenon of reutilization of the labeled precursors of DNA synthesis (Fig. 36) (Parshina and Mitashov, 1978), in contrast to tests involving a single [3H]TdR injection. Determined for the period of active proliferation by the cells of the dorsal iris and the lens rudiment were the duration of mitotic cycles and their parameters on the sixth to tenth and the fourteenth to sixteenth day after the operation (Fig. 37) (Eisenberg-Zalik and Yamada, 1967; Mitashov, 1969c; Yamada et al., 1975). The most substantial results of these few studies done in different laboratories are given in Table VII. The data in that table demonstrate that in this system of regeneration, just as with the regeneration of the retina, the total duration of mitotic cycles and other related parameters of the proliferating cells become greatly reduced, the reduction accompanying an increase of the relative duration of the S phase in the mitotic cycle as the cell population of the dorsal iris follows a new pathway of differentiation. As in the case of regeneration of the neural retina from RPE cells, these data show that the changes in the parameters of the

PARAMETERS OF CELL CYCLES IN

Iris or lens region

Time after operation (days) 6- 10 6-10 6-10 6-10 14-16

TABLE VII DIFFERENTREGIONSOF THE IRIS AND LENS RUDIMENTDURING LENSREGENERATION (HOURS)

Cells not involved in lens formation Ventral Dorsal Dorsal zone forming lens cells Lens rudiment cells

aFor Norophrhalmus viridescens. bFor Trirurus cristatus.

+

b % ~

29.94 18.35 9.40 11.14 5.50

tS

40.50 32.95 27.70 27.09 16.00

tG,

+ 1/2tM 8.11 8.35 7.60 7.60 2.00

T

?SIT

References

78.55 59.65 44.70 45.85 23.50

0.51 0.55 0.63 0.59 0.68

Yamada er al. (1975p Yamada et al. (1975) Yamada e r a!. (1975) Yamada ef a!. (1975) Mitashov ( 1 9 6 9 ~ ) ~

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OLGA G. STROEVA AND VICTOR I . MITASHOV

proliferating cells of the iris and lens rudiment may serve as an indicator of changes in some of the iris cells’ properties during the process of their gradual transdifferentiation. One of the most crucial moments in the lens regeneration-the dedifferentiation of the dorsal region of the iris, as manifested by the depigmentation of the cells and cytoplasmic shedding-begins at a late phase of the first mitotic cycle and ends in the fourth mitotic cycle (Yamada et al., 1975). Upon completion of dedifferentiation, the cells that are to become the primary lens fibers must pass through another one to two mitotic cycles before they go over to the terminal stage of fiber differentiation in which accumulation of lens crystallins takes place without DNA replication. Also different is the duration of the mitotic cycles of the cells located in different areas of the iris. Only those cells of the dorsal iris that have the shortest duration of the mitotic cycle become lens fibers (EisenbergZalik and Yamada, 1967; Mitashov, 1969c; Yamada et al., 1975). It is probable that there are alternative paths of iris cell development during lens regeneration, paths that are controlled by mitotic cycles differing in duration (Yamada, 1977). This means that the prolongation of the cell cycle and the corresponding completion of a smaller number of cell divisions may cause the absence of lens differentiation. Indeed, in vivo the lens is not formed from the cells of the ventral and proximal zones of the dorsal iris, which during regeneration complete fewer mitotic cycles. Another feature in the proliferative activity of the iris cells can be detected if the results of iris cell cultivation in vitro are compared with regeneration in vivo. 2. Transdifferentiation of the Iris Cells in Vitro Not all reports on iris cell cultivation contain information about cell proliferation. The first experiments on iris cultivation failed to produce lens (Stone and Galagher, 1958; Eguchi, 1967; Eisenberg-Zalik and Meza, 1968; EisenbergZalik and Scott, 1969; Yamada et al., 1973; Yamada and McDevitt, 1974). The first positive results were obtained when advanced stages of lens regeneration were used for cell cultivation (Eisenberg-Zalik and Meza, 1968; Eisenberg-Zalik and Scott, 1969) or when the iris was cultivated in the presence of a frog hypophysis (Connelly et al., 1973; Reese, 1973) or frog NR (Yamada et al., 1973). When transplanted for cultivation, the cells of the newt NR are in the lag phase for the first 20 days, after which they shift into the logarithmic growth phase, with a cell doubling time of 150 hours (Horstman and Zalik, 1974). The cell doubling time, when cultivating iris extracted from the newt eye 10 days after the preliminary removal of the lens, is shorter: 85 hours. Similar values of cell doubling time (84 hours) were obtained for the cell line drawn from the dorsal iris in a culture maintained for several years (Reese et al., 1976). Horstman and Zalik (1974) determined the duration of the mitotic cycle phase when cultivating iris cells and a 10-day iris (the iris cells were placed under cultivation

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TABLE VIII PARAMETERS OF CELLCYCLES(HOURS) I N CELLSOF DIFFERENT REGIONS OF IRIS DURING CULTIVATION in Vitro (Noiophrhalmus viridescens)

Whole iris Dorsal iris Ventral iris Dorsal + ventral iris

26.0 46.6 42.0 44.0

36.6 34.6 34.7 35.1

7.0 7.4 6.9 7.3

69.6 88.6 83.6 86.4

Horstman and Zalik (1974) Yamada and Beauchamp (1978) Yamada and Beauchamp (1978) Yamada and Beauchamp (1978)

conditions 10 days after the preliminary removal of the lens from the eye). Similar duration values for iris cell mitotic cycles under cultivation were obtained by Yamada and Beauchamp (1978); see Table VIII. It is noteworthy that no differences were detected in the duration of the mitotic cycles of the cultivated iris cells of the dorsal and ventral zones. Following the dissociation of the iris cells of the dorsal and ventral zones and their separate cultivation, it is possible to obtain lentoid bodies from any zone of the iris (Eguchi et al., 1974), although under in vivo conditions the cells of the dorsal iris alone have this capacity, as was noted earlier (Fig. 35). A comparison of mitotic cycle duration values for in vivo lens regeneration (Table VII) and cell culture regeneration (Table VIII) revealed that with mitotic cycles of different duration under these conditions, one of the most important factors for detecting the signs of lens differentiation is the number of mitotic cycles the proliferating cells have to complete. A rough assessment indicates that for the lens to undergo regeneration under cell culture conditions, just as under in vivo conditions, the cells of the iris must complete six mitotic cycles (Yamada et al., 1975). Because the reduction of the parameters of the mitotic cycles takes place in the presence of the retina, it was suggested that some signal issuing from the retina may speed up proliferation and shorten the mitotic cycles rather than initiate cell transformation (Yamada et al., 1975; Yamada and Beauchamp, 1978).

C. COMPARISON OF THE CHARACTERISTICS OF EYECELLPROLIFERATION DURING RETINAAND LENS REGENERATION IN NEWTS A comparison of the proliferative activity of RPE cells and cells of the retinal rudiment, of the iris, and of the lens rudiment in the process of NR and lens regeneration in newts reveals much that is common and similar. First of all, during the transdifferentiation of the pigmented cells of the eye, a large reduction both of the total duration of mitotic cycles and of their individual parameters during proliferation takes place. Another feature in proliferation is that the cells

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must complete a certain number of mitotic cycles-from 6 to 10-before characteristics of another type of differentiation are revealed in their descendants. During cultivation, the pigmented cells of the eye pass through approximately the same number of mitotic cycles as they do in situ before the characters of another type of differentiation are revealed in their descendants. Here two types of proliferation in cell cultivation must be distinguished. The first proliferative wave is the proliferation of cells in primary cultures, where cell transdifferentiation is not yet manifested. This period is characterized above all by the accumulation of a certain number of differentiated cells. It is only the next proliferative cycle of a new generation of cells, initially still intensively pigmented, that leads to the differentiation of lentoid bodies. In this final proliferative cycle of clones arising from a single pigmented cell, a breed of cells that form lentoid bodies after the passage of perhaps 10 mitotic cycles within 12 days of cultivation is obtained (Eguchi and Okada, 1973; Eguchi, 1976, 1979). Whether a shortening of both the total mitotic cycle and of its individual parameters takes place in this final cycle of proliferation is unknown. Even when it becomes possible to shorten the lag phase during the cultivation of the RPE cells of quails in the presence of 0.5 mM phenylthiourea, the earlier manifestation of lensdifferentiated cells again takes place only after previous proliferation (Eguchi, 1976, 1979). Though this preliminary work by Eguchi does not define the number of mitotic cycles required, one may conclude from the indicated time of lentoid body differentiation that their presumed number is not more than 10. However, the most important thing for an understanding of the mechanisms of cellular transdifferentiation is to reveal the causal relationship between cell proliferation and the formation of NR and lens in the process of eye restoration. This relationship is probably manifested in the dependence of the dedifferentiation of RPE and iris cells on proliferation, because cytoplasmic detachment and melanin granule expulsion occur only during the cells' passage through the mitotic cycles. There is probably also a causal relationship between the shortening time of mitotic cycles in the cells of the retinal and lens rudiments and their formation. The causal relation is manifested in that the final loss of specific features by the pigmented cells takes place only against the background of their intensive proliferation. Is it possible to single out from the aforementioned specific features in the proliferation of the eye's pigmented cells and the cells of the rudiment of the retina and lens forming from them those features that are most essential for the transdifferentiation process? The most important, perhaps, is the process of cell dedifferentiation against the background of their intensive proliferation given the certain completion of a definite number of mitotic cycles. Are these processes influenced by some extraneous factors? Such factors are most probable during the transdifferentiation of the cells of the iris into the lens. It is presumed that some substances secreted by the retina of the adult newt speed up the prolifera-

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tion of iris cells (Yamada et al., 1975; Yamada and Beauchamp, 1978) but do not initiate the transdifferentiation process itself. Under in vivo conditions, the vitreous body may be the conductor of such substances (Gulati, 1980, 1981; Gulati and Reyer, 1980; Gulati et al., 1981). Similar results were obtained by Mitashov et al. (1983) in the experimental investigation of [3H]tryptophanincorporation in the process of lens and NR regeneration in adult newts. During transdifferentiation of pigmented cells under in vitro cultivation conditions, the external signals may come from some kind of factors in the culture medium. However, it is not known whether there exist some specific phases in the structure of the mitotic cycle to which these suggested factors may respond. Still less elaborated are the conceptions about the action of extraneous factors during in situ transdifferentiation of the pigmented epithelium in adult newts. So far such factors have not been detected in the process of NR regeneration. IN SPECIFIC SYNTHESES DURING EYEREGENERATION D. CHANCES

Another essential aspect for understanding cell transdifferentiation mechanisms in the process of eye regeneration concerns changes in the differentiation of the initial cells during the formation of the retinal and lens rudiment. We shall examine in this section the macromolecular syntheses that help to assess the changes in the differentiation of transdifferentiating cells. Let us examine these events in eye regeneration that are in the background of changing cell proliferation. Melanin synthesis is a specific characteristic of the pigmented cells of newt eyes. As already noted (Section III,A), the precursor of melanin synthesis is known-it is the amino acid DOPA, which under the effect of the enzyme tyrosinase forms melanin in the pigmented cells of newts, just as in those of mammals (Model, 1973; Moran et al., 1973). This is deposited on special protein structures, the protein matrix-premelanosomes. The simultaneous use of [3H]TdR and [3H]DOPA revealed in the RPE and iris the subpopulation of cells incorporating both precursors during different stages of eye regeneration (Grigoryan and Mitashov, 1979; Mitashov and Grigoryan, 1980a,b). Figure 28 gives the index values of cells containing [3H]TdR and [3H]DOPA for different zones of the pigmented epithelium as the restoration of the eye proceeds. The following paragraph provides a brief comparison of the basic data for the pigmented epithelium and the iris. We have already considered the details of the spatial distribution of cells in the pigmented epithelium once melanin synthesis has been initiated in them. This enabled a determination of the number of RPE cells participating in the restoration of NR (Section 111,A). We shall merely note here that against a background of melanin biosynthesis the peripheral zones of the pigmented epithelium contain only 4-6% of cells with [3H]TdR (Fig. 28). The RPE cells in the area of the fundus oculi contain no [3H]DOPA throughout the entire period of transdifferen-

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tiation into the retinal rudiment, whereas the index of [3H]TdR-labeled cells reaches nearly 50% (Fig. 28). Thus, in this area with a background of a high RPE cell proliferation level, the synthesis of specific melanin granules comes to an end. The autoradiographic data about the distribution and location in the iris of cells in the phase of melanin synthesis indicate that melanin synthesis occurs in the cells of the outer lamina of the ciliary zone throughout the process of lens regeneration (Figs. 28 and 35). Just as in the peripheral regions of the pigmented epithelium, the metabolism of melanin being synthesized is most active in this zone of the iris (Mitashov, 1978). Along with the formation, growth, and pinching off of the regenerating lens, the number of melanin-synthesizing cells sharply increases in the outer lamina of the ciliary zone. There are no cells that incorporate [3H]DOPA in the inner lamina of the ciliary zone because all the cells are nonpigmented. During lens rudiment formation, the number of cells in the melanin-synthesizing phase drops sharply in the outer lamina of the pupillary zone, whereas the cells in the inner lamina of this zone do not synthesize melanin at all. Regarding the drop in the number of cells in the melanin-synthesizing phase in the outer and inner laminae of the pupillary zone of the dorsal iris, it is important to note the location of the cells synthesizing and not synthesizing melanin. The most noteworthy result was that no labeled cells were detected at all in the region of transition of the outer into the inner lamina of the dorsal iris (i.e., in the zone of lens rudiment formation) (Figs. 28 and 35). Thus, the transformation of iris cells into cells of another type of differentiation, just as in the case of NR regeneration from the pigmented epithelium (Mitashov et al., 1978), takes place upon termination of synthesis of specific products characteristic of the initial cell type. The termination of melanin synthesis in the iris occurs long before the detection of protein (a-, p-, and y-crystallins) specific for the lens differentiation cells in the descendants of the iris cells (Takata er al., 1964, 1965, 1966). What merits special attention is the fact that the most significant changes in the [3H]DOPA-labeledcell index occur in the dorsal iris. This is quite natural because the cells in this zone are the most depigmented in the process of lens formation. What proved most amazing and unexpected was that in the absence of [3H]DOPA incorporation in the cells of the pupillary zone of the iris forming the lens, and in the cells of the lens, there is increased activity of tyrosinase (the key enzyme of melanin biosynthesis) in these portions of the regenerating system (Achazi and Yamada, 1972). No other functions of this enzyme are known apart from the hydroxylation of tyrosine and dehydroxyphenylalanine by way of melanin biosynthesis. The absence of [3H]DOPA incorporation in lens cells, combined with high tyrosinase activity, is probably determined by the absence of the structual component premelanosomes on which melanin is synthesized and deposited. The results concerning the changes of [3H]DOPA incorporation in the cells of the dorsal area of the iris in comparison

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with data about tyrosinase activity mean that at the molecular level pigmented cells do not lose all their characteristics during their transformation into cells of another differentiation type. What is the correlation between proliferation processes during transdifferentiation and the new proteins appearing in the lens rudiment? The specific marker proteins of the lens are the crystallins, which have been discovered only in the lens and have not been detected in any other tissues of the eye. The method of immunofluorescence has been used to determine the time of synthesis of the lens antigens during its regeneration (Takata et al., 1964, 1965, 1966). The specific proteins of lens differentiation appear for the first time in a small number of would-be lens fibers during the fourth stage of regeneration. By the simultaneous use of [3H]TdR autoradiography and immunofluorescence, Yamada (1966) demonstrated that the appearance of a-and P-crystallins takes place not earlier than 3 hours after the proliferating cells of the regenerating lens complete the final S phase; y-crystallins in the same cells of the regenerating lens appear still later: 1.5 days after completion of the last S phase. Yamada also demonstrated that along with the growth of the regenerating lens the y-crystallins are synthesized in the lens fibers only, whereas a- and P-crystallins are synthesized both in the fibers and in the lens epithelium. Thus, in this system, mutually exclusive events between the synthesis of DNA and y-crystallin in the lens fibers were discovered. At the same time, there exists a combination of proliferation and specific synthesis in the lens epithelium where DNA synthesis as well as that of a-and P-crystallins can proceed in the same cells. Thus, the transdifferentiation of pigmented cells of the eye into the retinal and lens rudiments proceeds against a background of canceled synthesis of melanin, which supplements very well the previously discovered phenomenon of cytoplasmic shedding (Dumont and Yamada, 1972) in the process of lens regeneration, The experimental results demonstrate that the changes in the indicators of differentiation of the initial cells during their transdifferentiation are probably regulated at the levels of transcription and translation.

IV. General Conclusions Recent investigations revealed that cell proliferation is of great importance in the development and transdifferentiation of the pigmented epithelia of the eye. The melanotic differentiation of the RPE melanocytes as compared to that of other pigment cells has been reviewed by Whittaker (1974) and Garcia et al. (1979b). The data discussed in this article revealed some new aspects of the regulatory mechanisms of melanotic differentiation of pigment cells. The studies in vivo have shown that ontogenic melanization of the RPE is not a process of self-differentiation. In RPE development of both chick embryos and postnatal rats, two phases of melanin synthesis were found.

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Because BUdR does not prevent the onset of the first phase of the RPE pigmentation in chick embryos, the mechanism proposed by Benson and Triplett (1974) seems to operate in the initial events of melanin synthesis. They showed that in Rana pipiens embryos, tyrosinase is synthesized as early as during neurulation and is stored in the inactive state by being bound to protein-inhibitor. The subsequent involvement of tyrosinase in melanogenesis is regulated at the posttranslational level by the specific proteases (Barisas, 1974; Slaughter and Triplett, 1975a,b). It seems that the same holds true for mammals. The data on the timing of X chromosome inactivation in mosaic mice support the idea that determination of RPE melanotic differentiation occurs no later than at the neural plate stage (Deol and Whitten, 1972). The onset of the second phase of RPE melanization in chick embryos is controlled by new transcriptional events, and in postnatal rats it seems to be melanotropin dependent. However, more studies are needed to discover the molecular mechanisms of the processes under investigation. In both species, differentiative signals for the onset of the second phase of melanin synthesis act during intensive cell proliferation, which is eye-growth independent. Conversely, the expression of the final phase of RPE melanotic differentiation proceeds against a background of low-level proliferation, which is eye-growth dependent. Causal relationships between RPE melanotic differentiation and proliferation during intrauterine life in rats still remain unknown. Finally, the finding that RPE cells of adult chick are incapable of repigmentation in long-term cell cultures suggests that gene activity necessary for melanin synthesis becomes irreversibly repressed as soon as the program of definitive melanization of the RPE has been accomplished. A peculiar type of melanin synthesis regulation was found during eye regeneration in adult newts. In the course of NR regeneration, two subpopulations of RPE cells were observed. At the RPE periphery, melanin synthesis goes on during NR restoration, and only a few cells with [3H]TdR can be distinguished. The cells of this zone are not involved in the formation of the retinal rudiment. On the contrary, RPE cells of the fundus oculi area do not synthesize melanin throughout the entire period of their transdifferentiation, which coincides in time with a high level of RPE cell proliferation. It is very important to note that RPE cells that incorporate no labeled melanin precursor are those containing some pigment. Only after the retinal rudiment has been formed do RPE cells of this zone begin to resynthesize melanin to restore the original level of melanization. Similar events were discovered during lens regeneration. The molecular mechanism of these phenomena remains obscure. Thus, a change in kinetics of cell populations seems to be one of the regulatory mechanisms of the expression of RPE melanotic differentiation. In adult newts during NR and lens regeneration, both dedifferentiation and transdifferentiation of pigment cell progeny are related not only to an increase in

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the proportion of cyclic cells as well as to a shortening of the total cell cycle, but also to a change in specific duration of the cell cycle phases. The capacity of chick RPE for transdifferentiation into neural retina correlates with intensive proliferation and short cell cycle in situ, whereas the stabilization of RPE cells coincides in time with a decrease in a proportion of cyclic cells that replicate with prolonged generation time. Further experiments are needed for detailed analysis of such a phenomenon. Assuming that both protyrosinase and retina cognins are synthesized in all eye rudiment cells, it may .be possible to account for retinal differentiation of the RPE as well as for the appearance of pigmented cells in clonal cultures of the neural retina cells (for references, see Okada, 1980). The expression of either eye cell phenotype would depend, therefore, on cell interactions within a particular pattern of morphogenesis and environmental conditions (see, for example, Clayton et al., 1977; Prichard, 1981; de Pomerai and Gali, 1981) and ought to be dependent on the cell cycle events. The state of unstable determination (pigmented cells versus lentoid ones in long-term cell culture) is preserved as long as RPE cells of donor chick embryos have not completely withdrawn from reproduction. Thus, the state of a nondividing RPE cell population in situ is a prerequisite of manifestation of the stable RPE cell determination in culture. It seems possible to conclude, therefore, that cell proliferation not only is closely related to cell multiplication and reduction of cell volume, but is essential for RPE melanotic differentiation and for transdifferentiation into other cell types. We believe the preceding conclusions open the way for a detailed analysis of these phenomena.

ACKNOWLEDGMENTS We have the pleasure of expressing our gratitude to Professor Bruce Carlson and to our colleagues and friends Dr. A. Kostomarova and Mrs. G. Losovskaya for reading and helpful revising of the manuscript. We are also grateful to our colleagues Mrs. I. Panova and Mrs. E. Grigoryan for their valuable technical assistance during preparation of the manuscript for publication.

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Index

A

C

Adenosine triphosphatase activity, NMR and, 40-4 1 Adenoslne triphosphate measurement, potential for restoration at cellular level and, 58-59 some specific costs, 55-58 study at organ level by NMR applicability to reactions occurring in vivo, 33-34 ATPase activity, 40-41 computer modeling of Bloch equations, 42-44 measurement of chemical exchange, 29-33 overview, 28-29 studies of creatine phosphokinase, 34-40 summary and conclusions, 44-46 Adenosine triphosphate/adenosine diphosphate, compartmentation, cell cycle and, 50-52 Aging cell volume and, 186-190 putative mechanisms for membrane-dependent manifestations of, 212-215 Amphibia, proliferation of pigment cells in, 263 changes in specific synthesis during regeneration, 283-285 comparison of eye cell regenerations in newts, 281-283 regeneration of lens, 275-281 regeneration of neural retina, 264-275

Calcium, localization, staining methods for, 1 I6 Cell(s) changes in energy demands within, mitochondria and, 87 in culture, mitochondria of, 90-94 relationship of energetic state to biology ATP/ADP compartmentation and cell cycle, 50-52 difficulties in interpretation of experimental data, 48-50 measurement of ATP and its potential for restoration at cellular level, 58-59 normal values for energetic state, 52-55 overview, 46-48 some specific ATP costs, 55-58 respiration, chemical manipulation of, 94-98 Cell adhesion aging and cell-cell, 194-195 cell-ligand, 195-203 cell-substratum, 190- 194 cytoskeleton and, 203-204 mechanisms of, 204-207 Cell cycle, ATP/ADP compartmentation and, 50-52 Cell division, cell-substratum adhesion and, 208-2 I 1 Cell-substratum adhesion, function and, 207-208 differentiation, 21 1 division, 208-21 1 malignancy, 21 1-212 movement, 208

B Bloch equations, computer modeling of, 42-44

295

2 96

INDEX

Cell types, different, differences in energy demands, 85-87 Cell volume, aging and, 186-190 Chemical exchange, measurement, NMR and, 29-33 Chick, retinal pigment epithelium, differentiation and proliferation, 247-259 Chondriome, see also Mitochondria of eukaryotic cells enlargement, 73-76 numbers, 64-73 form and metabolic activity cells in culture, 90-94 changes in energy demand within cells, 87 chemical manipulation of cell respiration, 94-98 differences in energy demand of different cell types, 85-87 form changes in virro, 87-89 form changes in vivo, 89-90 form and metabolic activity, manipulation by microinjection into living cells, 98-101 Chromatin, see also Nonchromatin structures higher order structures, nonchromatin structures and, 155- 157 Chromosomes, arrangement at interphase, nonchromatin structures and, 155-157 Creatine phosphokinase, exchange reaction, NMR and, 34-40 Cristae, mitochondrial, 76-85 Cytoskeleton, cell adhesion and, 203-204

D Deoxyribonucleic acid rearrangement, transposable elements and, 16-20 superstructures, nonchromatin structures and, 152-153 Differentiation, cell-substratum adhesion and. 211

E Energetic state, of cells, normal values for, 52-55 Enzymes, oxidative and reductive of mitochondria, staining methods for, 109-1 15

Eukaryotic cells, chondriome enlargement, 73-76 numbers, 64-73 Eye cell proliferation, comparison of characteristics during regeneration, 28 1-283 Eye regeneration, changes in specific synthesis during, 283-285

F Fluorescent stains, for mitochondria of living cells, 116-1 18

G Gene conversion, transposable element Ty and, 14-16 Gene expression effects of insertion of Ty 1 on, 10-14 involvement of nonchromatin structures in posttranscriptional events, 158- 160 transcription, 157- 158 Glycoproteins, in nonchromatin structures, 174-176

H HeLa cells, histone-depleted nuclei organization of nonchromatin structures in, 160-165 Human, retinal pigment epithelium, differentiation and proliferation, 259-262

I Interchromatin matrix, anatomy of, 146- 150 Intraocular pressure, in human RPE development, 261-262 L

Lens, regeneration in amphibia, 275-281

M Malignancy, cell-substratum adhesion and, 211-212 Melanization, proliferation and capacity of RPE cells for neural retina differentiation during uterine life, 224-227

297

INDEX Melanogenesis, in RPE, during postnatal development, 240-246 Melanniic differentiation, of RPE, proliferation and, 252-253 Mitochondria, see also Chondriome cristae of, 76-85 inclusions, 101-102 crystalline, 106- 109 granular, 102-106 microinjection into living cell, 98-101 Mitochondriagenesis, mechanism of, 118-122 Mitochondria1 function, potential of modem staining methods in monitoring calcium localization and Ca2+ -binding proteins, I16 fluorescent stains for living cells, 116-118 oxidative and reductive enzymes, 109- 115 phosphatases, 115-1 16 Movement, cell-substratum adhesion and. 208

N Neural retina, regeneration in amphibia, 264-275 Nonchromatin structures anatomy and extensive interchromatin matrix, 146- 150 lamella/lamina concept, 142-145 nuclear membranes and pore complexes, 137-142 nuclear shell or cortex, 145-146 nucleolar skeleton, 150- 151 characterization based on fractionation experiments, 166-168 achromatic and histone-depleted nuclei, 168 extraction from isolated nuclear territories, 168-174 glycoproteins in, 174-176 involvement in gene expression posttranscriptional events, 158- 160 transcription, 157- 158 role in nuclear organization, 151- 152 chromosome arrangement at interphase, 155-157 DNA superstructures, 152- 153 higher order chromatin structures, 153- 155 three-dimensional organization of in HeLa cell histone-depleted nuclei, 160- 165

operational media necessary to maintain organization, 165- 166 Nuclear membrane, pore complexes and, anatomy of, 137-142 Nuclear magnetic resonance spectroscopy, in study of ATP metabolism at organ level applicability to reactions occurring in vivo, 33-34 ATPase activity, 40-44 computer modeling of Bloch equations, 42-44 measurement of chemical exchange, 29-33 overview, 28-29 studies of creatine phosphokinase, 34-40 summary and conclusions, 44-46 Nuclear shell or cortex, anatomy of, 145-146 Nucleolar skeleton, anatomy of, 150-151 Nucleus(i) achromatic and histone-depleted, nonchromatin structure organization and, 168 nonchromatin structures, definitions, 136-137

P Phosphatases, mitochondrial, staining methods for, 115-116 Pigment epithelium cells, in amphibian retina and iris, 263 changes in specific syntheses during regeneration, 283-285 comparison of eye cell regenerations in newts, 281-283 regeneration of lens, 275-281 regeneration of neural retina, 264-275 Posttranscriptional events, nonchromatin structures and, 158-160 Proteins, Ca2+ -binding, staining methods for, I I6

R Rat, retinal pigment epithelium, differentiation and proliferation, 223-247 Retinal pigment epithelium differentiation and proliferation in chick, 247-259 in human, 259-262 in rat, 223-247

298

INDEX

growth and development in human, 259-261 proliferation in normal development, 247-250 dependence on intraocular pressure, 251-252 proliferation in postnatal development, formation of binucleated cells, 227-234 transdifferentiation into neural retina and lens, 253-256 transdifferentiation of human cells in culture, 262 S Scleral part, growth, RPE and, 234-240

T Transcription, nonchromatin structures and, 157- 158 Transposable elements, other, in yeast, 20-21

Transposable element Ty, in yeast associated gene conversion, 14-16 DNA rearrangements and, 16-20 effects of Ty 1 insertion on gene expression, 10-14 physical structure, 2-7 transcription, 7-9 transposition, 9- 10 ~

Y Yeast other transposable elements in, 20-21 transposable element Ty associated gene conversion, 14- 16 DNA rearrangements and, 16-20 effects of Ty I insertion on gene expression, 10-14 physical structure, 2-7 transcription, 7-9 transposition, 9-10

Contents of Recent Volumes and Supplements Volume 70

*

Volume 72

Cycling Noncycling Cell Transitions in Tissue Aging, Immunological Surveillance, Transformation, and Tumor GrowthSEYMOUR GELFANT The Differentiated State of Normal and Malignant Cells or How to Define a “Normal” Cell in CUltUre-MlNA J. BISSELL On the Nature of Oncogenic Transformation of CellS4ERALD L. CHAN Morphological and Biochemical Aspects of Adhesiveness and Dissociation of Cancer C e l l s HIDEOHAYASHI AND YASUJI ISHIMARU The Cells of the Gastric MUCOSB-HERBERT F. HELANDER Ultrastructure and Biology of Female Gametophyte in Flowering Plants-R. N. KAPILA N D A. K. BHATNAGAR

Microtubule-Membrane Interactions in Cilia and Fktgek3-wILLIAM L. DENTLER The Chloroplast Endoplasmic Reticulum: Structure, Function, and Evolutionary Significance-SARAH P. GIBES DNA Repair-A. R . LEHMANNA N D P. KARRAN Insulin Binding and Glucose Transport-RusSELL HILF.LAURIE K. SORGE.A N D ROGER J . GAY Cell Interactions and the Control of Development in Myxobacteria Populations-DAVID WHITE Ultrastructure, Chemistry, and Function of the Bacterial Wall-T. J. BEVERIDGE INDEX

INDEX

Volume 73 Volume 71 Protoplasts of Eukaryotic Algae-MARTHA D. BERLINER Integration of Oncogenic Viruses in Mammalian Polytene Chromosomes of Plants-WALTER CeIk