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Studies on the cell cycle in Paramecium tetraurelia Rasmussen, Colin Dale 1984

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STUDIES ON THE CELL CYCLE IN PARAMECIUM TETRAURELIA by COLIN DALE RASMUSSEN B . S c , U n i v e r s i t y o-f B r i t i s h C o l u m b i a , 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE THE FACULTY OF GRADUATE STUDIES in the Department o-f Z o o l o g y < We a c c e p t t h i s t h e s i s a s con-forming to the r e q u i r e d s t a n d a r d . THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1?84 © Co l i n Dale Rasmussen, 1984 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements fo r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of ZOOLOGY The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date April 24, 1984 DE-6 (3/81) Abstract Several aspects o-f the ce l l cycle in Parameciurn were studied using the temperature-sensitive mutations ccl and cc2. and the recessive gene mutation tamG. Cell cycle progression can be blocked by the ccl de-fect up to 0.72 o-f the cel l cycle. There was no progression through the cel l cycle at the restr ic t ive temperature, nor excess delay o-f cel l division once ce l l s were returned to permissive conditions. The ccl de-fect also reversibly inhibits macronuclear DNA synthesis throughout the cel l cycle. Macronuclear DNA synthesis is not required -for cel l division past 0.72 of the ce l l cycle. Downward regulation of ce l l mass was examined. Cell mass is regulated by the concerted action of two mechanisms. F i r s t , the rate of growth becomes limited by gene dosage when cel l mass increases. Second, the cel l cycle is shortened when in i t i a l cel l mass is increased, at the expense of Gl. Cel ls with an in i t i a l cel l mass greater than 120% of normal i n i t i a l mass have a cel l cycle duration equal to the normal length of S + nuclear d iv is ion. Both cel l mass and parental DNA content influence the timing of DNA synthesis in i t i a t ion . As i n i t i a l cel l mass increases, the length of Gl decreases. When in i t i a l cel l mass exceeds the normal value at the start of S, DNA synthesis begins immediately after f i s s ion, without a Gl period. Gl DNA content has no effect on the timing of DNA synthesis in i t i a t ion . However, ce l l s with low parental DNA content appear to enter S phase ear l ier than ce l l s with high parental DNA content. ii A s i m p l e computer s i m u l a t i o n model has been d e v e l o p e d w h i c h c o n s i s t e n t w i t h the e x p e r i m e n t a l o b s e r v a t i o n s . The model i s ba s e d the u n s t a b l e i n h i b i t o r model of Y e a s e t a l . <1945>. ni TABLE OF CONTENTS ABSTRACT .ii -LIST OF TABLES .' vi ii LIST OF FIGURES < ix ACKNOWLEDGEMENTS x"i CHAPTER I. REGULATION OF THE CELL CYCLE IN EUKARYOTES 1 Introduct i on 2 Coordination o-f cel l growth and DNA repl i cat i on . . . .3 General view o-f the ce l l cycle ...3 Experimental systems -for cel l cycle study 4 The cel l cycle in Saccharomyces cerevis iae.. 5 Start 6 Coordination o-f growth and DNA synthesis 7 Nature o-f Gl in Saccharomyces.... .....8 The cel l cycle in Schizosaccharomyces pombe 9 Regulation o-f ce l l mass and mitosis ? Start 11 Nature o-f Gl in Schizosaccharomyces. 11 The cel l cycle in Mammalian ce l l cultures 12 Coordination o-f growth and DNA synthesis.... 13 E-ffect of mass on cel l cycle progression 14 Cell cycle regulation models 15 Concentration models • • 17 Positive vs. negative control mechanisms 18 Structural model s 1? iv The cel l cycle in Paramecium tetraurel ia: The subject o-f this study. 20 Aims o-f this study 21 CHAPTER II. A FAST-ACTING TEMPERATURE SENSITIVE CELL CYCLE MUTATION OF PARAMECIUM TETRAURELIA AFFECTING DNA SYNTHESIS AND CELL CYCLE PROGRESSION, DURING BOTH Gl AND S: IMPLICATIONS FOR CELL CYCLE REGULATION... 27 Introduct i on 28 Experimental design... . . 30 Results 31 E-f-fect o-f temperature shi-ft on macronuclear DNA synthesis 31 Delay o-f in i t iat ion by heat treatment in Gl. .31 Completion o-f DNA synthesis is not a precondition -for ce l l division ...33 Di scussi on 35 E-f-fect on DNA synthesis.. 35 E-f-fect on cel l cycle progression 36 E-f-fect o-f DNA synthesis inhibition on cel l division 38 CHAPTER III. REGULATION OF CELL SIZE IN PARAMECIUM TETRAURELIA: EFFECTS OF INCREASED CELL SIZE, WITH OR WITHOUT INCREASED DNA CONTENT, ON THE CELL CYCLE 47 Introduct i on .48 Experimental design 52 Resul ts 53 v Analysis of cel.; Increase in eel 1 size alone .53 Growth and DNA synthesis during heat treatment.... 53 Cell cycle dura t ions . . . . . . . . . . . . . . . . . . .53 DNA content and DNA synthesis. 54 Cell mass and growth rate 54 Analysis of cc2: Increase in cel l mass and DNA content 55 Growth and DNA synthesis during heat treatment . . . . . . .55 Cell cycle durations 55 DNA content and DNA synthesis ...56 Cell mass and growth ra te . . . . ....56 Long-term downward regulation of cel l s ize...57 Discussion 58 Control of growth and DNA synthesis 58 Control of ce l l cycle length... 60 CHAPTER IV. INITIATION OF DNA SYNTHESIS IN PARAMECIUM TETRAURELIA: THE EFFECT OF CELL MASS AND PARENTAL DNA CONTENT ON THE TIMING OF DNA SYNTHESIS INITIATION 83 Introduct i on .84 Ex per imental desi gn .86 Effect of ce l l mass on in i t iat ion 86 Effect of parental DNA content on i n i t i at i on 86 Results 88 E f f e c t of c e l l mass on i n i t i a t i o n of DNA synthes i s 88 E f f ec t of parental DNA content on i n i t i a t i o n of DNA synthes i s . . . .8? Di scussi on .91 E f f ec t of c e l l mass on the t iming of i n i t i a t i o n of DNA synthes i s 91 E f f ec t of parental DNA content on the t iming of DNA synthes i s i n i t i a t i o n . . . 92 Nature of the Gl per iod in Paramecium 93 CHAPTER V. SUMMARY: A CELL CYCLE MODEL FOR PARAMECIUM 104 REFERENCES I l l APPENDIX 1. MATERIALS AND METHODS 124 Stocks and cu l tu re 125 Synchronizat ion of c e l l s 125 Cyto log ica l procedures 126 Microf luor i me t ry 126 L a b e l l i n g and autoradiography . . . .127 S t a t i s t i c a l procedures . . 127 APPENDIX II. PARAMECIUM CELL CYCLE MODEL 128 Ce l l cyc le s imulat ion BASIC program 131 vVt LIST OF TABLES 2-1. Effect o-f heat treatment on ce l l cycle progression during Gl 40 4- 1. Comparison of pre-f iss ion DNA content in tamG ce l l s with labelled or non-labelled nuclei , at 0.25 of the cel l cycle 95 5- 1. Summary of observations on ce l l cycle regulation in Paramecium and models consistent with each observat i on 109 5-2. Comparison of observed consequences of changes in DNA content and cel l mass with results predicted by the ce l l cycle model 110 LIST OF FIGURES 1-1. C e l l c y c l e o r g a n i z a t i o n i n s e v e r a l e u k a r y o t e s . . . . . . . . 2 3 1- 2. C e l l c y c l e o r g a n i z a t i o n i n Paramecium t e t r a u r e l i a . . . . 2 5 2- 1. Rate o-f m a c r o n u c l e a r DNA s y n t h e s i s a f t e r t e m p e r a t u r e s h i f t s . . . . . ...41 2-2. C e l l c y c l e l e n g t h v s . l e n g t h o f heat t r e a t m e n t 43 2- 3. P o r t i o n o f the c e l l c y c l e r e m a i n i n g a f t e r the l e n g t h o f heat t r e a t m e n t i s a c c o u n t e d f o r v s . the l e n g t h o f heat t r e a t m e n t 45 3- 1. E f f e c t o f heat t r e a t m e n t on the l e n g t h o f the 1 s t and 2nd c e l l c y c l e s i n c c l / c c l c e l l s ....65 3-2. 61 m a c r o n u c l e a r DNA c o n t e n t i n h e a t - t r e a t e d c c l / c c l c e l l s 67 3-3. P o s t - f i s s i o n c e l l mass i n h e a t - t r e a t e d c c l / c c l c e l l s 69 3-4. P r o t e i n a c c u m u l a t i o n d u r i n g the 2nd c e l l c y c l e . i n h e a t - t r e a t e d c c l / c c l c e l l s 71 3-5. E f f e c t o f heat t r e a t m e n t on the l e n g t h o f the 1 s t and 2nd c e l l c y c l e s i n c c 2 / c c 2 c e l l s 73 3-6. Time r e q u i r e d f o r c c 2 / c c 2 c e l l s t o complete the c e l l c y c l e a f t e r the end of h e a t t r e a t m e n t 75 3-7. Gl m a c r o n u c l e a r DNA c o n t e n t i n h e a t - t r e a t e d c c 2 / c c 2 c e l l s 77 3-8. P o s t - f i s s i o n p r o t e i n c o n t e n t i n h e a t - t r e a t e d c c 2 / c c 2 c e l l s 79 3-9. Downward r e g u l a t i o n o f c e l l mass i n e x p e r i m e n t a l l y e n l a r g e d c c l / c c l c e l l s 81 ix 4-1. The effect of initial cell mass on cell cycle durat i on 96 4-2. Effect of initial cell mass on the length of the Gl period 98 4-3. Cumulative percentage plot of parental macronuclear DNA content vs. DNA synthesis state at 0.25 of the cell cycle 100 4-4. Cumulative percentage plot of parental macronuclear DNA content vs. DNA synthesis state at 0.35 of the cell cycle 102 x Acknowledgement I would like to sincerely thank Dr. J.D. Berger -for his advice, support and enthusiasm during the entire course of this study. I would also thank Dr. T. Grigliatti and Dr. D. Holm for their help in preparing this thesis, and Ada Ching for the use of her unpublished results. I would also like to thank my family, for everything they have done over the years. Finally, but most importantly, I would very much like to thank my wife Graciela for her constant love, support and encouragment. Research funds for this study were provided by a Natural Sciences and Engineering Research Grant #67-6300 to Dr. J.D. Berger. CHAPTER I. Regulation o-f the C e l l Cycle in Eukaryotes 1 Introduct i on 0 omnis cellula e cellula 8 - RudoH Virchow, 1858 The cell cycle is a -fundamental unit o-f development in all organisms and can itsel-f be considered as a developmental pathway, the completion o-f which depends on many interrelated events. Development in higher organisms depends on two things, growth and organization. Without growth , organisms cannot increase in size or number. However, growth in itsel-f is insu-f f i c i ent. Growth must be organized so that the relationships between cells and tissues allow normal development and maintenance o-f the organism. Study o-f the cell cycle has provided in-formation on how cells grow, and on how growth is organized to create a functional eel 1. The most important aspect o-f the cell cycle is the ability o-f cells to coordinate their rates o-f growth and cell division. The control o-f prol i-f erat i on is especially important in mul t i-eel 1 ul ar organisms, where tissues depend on precise organization o-f cells to per-form speci-fic -functions. In these cases the strict regulation o-f cellular proliferation is a vital component o-f developmental organization. For example, the primary feature of oncogenesis is the apparent loss of control over the rate of cell proliferation. Thus, the most important decision made by a cell is between cycling or quiescence. Once this decision has been made, the cell is irreversibly commited to completing the cell cycle. The cell cycle itself consists 2 of a number of coordinated processes with discrete control points. This chapter reviews cell cycle control in the yeasts Saccharomyces (budding-yeast) and Schizosaccharomyces <fission-yeast), and in mammalian tissue culture cells. The major emphasis has been placed on the points of cell cycle control identified so far, and the models which have been devised to explain the experimental observations. This serves as a background for the subsequent experimental work with Paramec i urn. Coordination of cell orowth and DNA replication A proliferating cell must duplicate both its cytoplasmic and nuclear components. Not only must the cell ensure that daughter cells are qualitatively and quantitatively equal, but that each is a functional representative of the mother. Under most circumstances, growth occurs continuously while DNA synthesis is periodic, and doubling of cell mass requires more time than DNA replication. Thus, for cycling cells the major problem is the coordination of cell growth with DNA synthesis. Without coordination, succesive rounds of DNA replication would quickly outstrip the cell's capacity to grow, thereby leading to a rapid decrease in cell mass in subsequent generations. This does not occur. Consequently it may be concluded that all cells coordinate these important cell cycle events. General view of the cell cycle The eukaryotic cell cycle consists of two interdependent cycles; the DNA-division cycle and the cell-growth cycle (Mitchison, 1971). The DNA-division cycle is separable into three parts. S phase, the 3 period of DNA replication, 62 a period where the cell awaits suitable conditions for initiation of nuclear division, and nuclear division, the partitioning of the nucleus to each daughter cell, ln contrast, the cell-growth cycle is not considered divisible since growth continues throughout the cell cycle. Experimental systems for cell cycle study Several organisms have proven useful for studying cell cycle events. Yeasts, slime molds, ciliates and mammalian tissue culture cells have been the systems favoured. Several features of the biology of the lower eukaryotes make them particularly advantageous for studying cell cycle problems. First, the culture conditions for the cells are very similar to the natural situation. Thus, disruption due to growth conditions is minimal. Second, the rate of cell division is high, and cells typically have cell cycles of less than 6 hours. Yeasts have the advantage of being useful for biochemical and genetic studies, while ciliates, because of their large size, can be used to examine the effects of perturbations on individual cell lineages. Another important consideration is the ease with which cells can be synchronized. To examine stage-specific processes adequate synchronization is crucial. Synchronization methods are generally of three types: natural, by selection or by induction. Natural synchrony is the ideal situation because there is no experimental perturbation. There are some cell types that possess natural mitotic synchrony (e.g. Physarum. and other myxomycetes), but organisms of this type are rare and usually have atypical cell cycles. There are several methods of selection synchrony, usually based on size (e.g. filtration, membrane 4 elution, sedimentation) or some aspect o-f morphology <e.g. hand-selection o-f dividing cells, mitotic selection). These methods usually give excellent synchrony, with a minimum o-f perturbation to cells. The yields, however, are characteristically low. Synchrony by induction produces the largest yields, but also the greatest disruption to the cell. There are several methods for inducing synchrony in cell cultures, e.g. thymidine block, heat shock, or specific inhibitors (reviews; Mitchison, 1971; Lloyd et al ., 1982). The cell cycle in Saccharomyces cerevisiae Saccharomyces cerevisiae reproduces by formation of a bud from the side of the mother cell. The cell has a mass-doubling time of 75 minutes under optimal conditions (Carter, 1981), and a typical cell cycle organization (Figure 1-1A). There are several, well-defined landmarks throughout the cell cycle (e.g. bud emergence, DNA synthesis initiation, nuclear division and cell division), and cell cycle stage can be estimated by the ratio of bud to parent cell volume (Carter, 1981). Genetically, the cell cycle of Saccharomyces is the best studied to date. This is due to the availabilty of eel 1-division-cycle (ede) mutations, originally described by Hartwell et al. (1970). These temperature-sensitive (ts) mutants can divide at the permissive temperature (23 C) but are unable to complete the cell cycle at the restrictive temperature (36 C) and arrest at a characteristic stage. Originally, 148 mutations of 32 different genes were isolated and characterized (Hartwell et al ., 1973). Each gene has been shown to have a 5 characteristic execution point (transition point). The execution point is the stage after which the mutation no longer blocks cell division in the current eel! cycle (Hartwell et al., 1970). There are now a total of 50 cdc genes that have been characterized (review; Pringle & Hartwell, 1982). From studies of the cdc mutants, a functional sequence map of gene events neccesary for completion of the cell cycle has been developed. Start In terms of cell cycle regulation, the most important regulatory point in the Saccharomyces cell cycle is the start function. Start was originally defined as the step mediated by the cdc28 gene product. The execution point of cdc28 is in Gl (Hartwell, 1974). The Gl period in Saccharomyces is quite variable, with a minimum duration of ten minutes (Tyson et al., 1979). In contrast, S + G2 + M is of fairly constant length for cell cycle durations from 75 minutes to 9 hours (Jagadish & Carter, 1977). Cells in Gl have a variety of alternate developmental fates. Haploid cells can traverse the cell cycle, remain in stationary phase or conjugate. Diploid cells, in addition to going through a cell cycle can undergo meiosis. Each of these fates are determined before the completion of start (Hereford & Hartwell, 1974; Shilo et al., 1978). Execution of the cdc28 step (start) commits the cell to completion of the cell cycle (Hartwell, 1974). Since the execution of start is prerequisite to cell cycle initiation, it is considered an important regulatory event in the yeast cell cycle. Several factors are required for the completion of start. 6 1) completion of the previous- nuclear division; 2) presence o-f nutrients; 3> presence or absence o-f mating factor and 4) attainment of a critical cell mass. Under optimal conditions, start is executed when cell volume exceeds 27 l»m (Hartwell, 1974; Johnston et al., 1977). Both bud emergence and DNA synthesis initiation are dependent on start (Hartwell, 1978). Coordination of Growth and DNA synthesis Under stable growth conditions, cells tend to maintain a constant size from one cell division to the next. Thus, on average, mass exactly doubles during the course of each cell cycle and cell cycle duration remains constant. However, when growth conditions decline (e.g. nitrogen starvation in yeast) cell cycle length increases. In Saccharomyces. most of this lengthening occurs in Gl before start (Jagadish & Carter, 1977). Since execution of start appears to depend on reaching a critical cell mass (Johnston et al., 1977), cells below this mass remain in 61 until sufficient growth allows completion of start. Thus, the length of Gl is a function of initial cell mass. Some observations can not be accounted for by the simple assumption that the rate of cell division is controlled entirely at start. At slow growth rates, the duration of the budded interval increases at a rate of 1.7 minutes for every 10 minute increase in generation time, suggesting that some dependent event in the cell cycle other than start may become rate-limiting at low growth rates (Pringle & Hartwell, 1982). The volume at which cells complete start also varies with growth rate and age. At slow growth rates the size at bud emergence is less than in cells grown under optimal conditions 7 (Johnston et al ., 1979; Lorincz & Carter, 1979). Thus, the criteria •for start can be altered according to nutritional state. In mother cells, volume at bud emergence increases about 17Xwith each successive cell cycle (Johnston et al., 1979). Therefore, mother cells have a growth requirement for start, even though their volume at division is the same as the cell volume at the previous start event (Pringle & Hartwell, 1982). All of these observations indicate that cells are not strictly monitoring cell size, but some parameter correlated with size. The mechanism which executes start is unknown. Earlier evidence suggested that completion of start depends on attaining a certain cell volume. However, recent studies indicate that a stronger correlation exists between start and accumulation of stable mRNA (Bedard et al., 1980). It has also been proposed that the doubling of the spindle pole body (SPB) commits cells to the cell cycle and demarcates the completion of start by the accumulation of a set number of SPB (Pringle 6c Hartwell, 1982). This suggests that the rate of SPB subunit synthesis might control entry through the cell cycle, and that SPB production should be correlated with cell mass. It has been shown that in diploids, which-have 2 times as many SPB's, the mean cell mass is also approximately twice the haploid amount (Byers, 1982). Nature of Gl in Saccharomyces As in most eukaryotes, Gl is the most variable portion of the cell cycle in Saccharomyces. In addition to being a period of general growth for the cell, Gl contains specific functions required for completion of the cell cycle. Start is one such function. This step always occurs after the end of the previous DNA-division cycle (nuclear 8 division) and before the next (onset of S phase) and is therefore not part of the chromosome cycle. There are two other genes that function specifically in Gl. The cdc4 and cdc7 genes act after start but before DNA synthesis and are required for completion of the cell cycle (Hartwell, 1978). Therefore, unlike bacteria, which have no Gl-specific functions (Cooper, 1979), there are at least three such events in Saccharomyces. The cell cycle in Schizosaccharomyces pombe S. pprobe is the best understood organism in terms of cell cycle regulation. This organism divides by transverse fission rather than by budding. It grows in length during the entire cell cycle, while the width does not change appreciably. Thus, cell length can be used as an indicator of both cell mass and cell cycle stage (Mi tenison, 1970). Under optimal conditions, the cell cycle is about 90 minutes. S. pombe has a typical cell cycle organization (Figure 1-1B). One unusal feature is that DNA synthesis initiation occurs coincident with cytokinesis (Nurse, 1975). Several ts ede mutations have also been isolated in S. pombe (Nurse et al., 1976). A total of 90 mutations defining 26 genes have been isolated to date. In addition, there is a class of mutants called wee. which divide at a smaller size than wild-type cells at the restrictive temperature (Nurse, 1975). Regulation of cell mass and mitosis Of the 52 wee mutant al1 eles recovered, all but one map to a 9 single locus, weel. At the restrictive temperature, wee causes mitosis to be initiated at a reduced cell mass <Nurse, 1975). It has been suggested that the wee de-fect alters the size threshold for initiation of mitosis. Cells normally initiate mitosis when cell mass exceeds 20 pg/cell of protein. In the wee mutant, however, mitosis begins when cell mass exceeds 10 pg/cell of protein (Nurse, 1975). ln addition to the effect on mitosis, wee also delays initiation of DNA synthesis in the subsequent cell cycle. DNA synthesis normally occurs concurrent with cell division, but is delayed by 0.29 of a cell cycle by the action of the wee mutant (Nurse, 1975). This observation can be explained by the existence of a cryptic size threshold for initiation of DNA synthesis. Under normal conditions cells complete the size requirement before the end of mitosis, and enter S after a minimum 61. However, wee cells, because they initiate mitosis and divide at a smaller than normal mass, must grow an extra 0.29 of a cell cycle in order to reach the size threshold (7 pg/cell protein)(Nurse, 1975). Cells from stationary phase, upon refeeding, do not initiate DNA synthesis until they pass a similar size threshold (Nurse & Thuriaux, 1977). Thus, there are two regulatory points in the S. pombe cell cycle. Cells with an initial mass greater than 15 pg are regulated by the size threshold at mitosis, followed by a minimum 61 of 0.1 cell cycles. In cells with an initial mass less than 15 pg, the cell cycle is regulated by the size threshold for initiation of DNA synthesis (Nasmyth et al., 1979). Also, in 62 there is evidence for a RNA synthesis rate doubling step that is prerequisite for completion of the cell cycle (Barnes et al., 1979). 10 Start In S. pombe there is a -function that operates the same as the start -function described in Saccharomyces <Nurse & Bissett, 1981). The cdc2 and cdclO genes are required in 61 -for commitment to cell division. S.  pombe cells deprived of nitrogen behave the same as Saccharomyces cells treated the same way. Proliferation stops, and cells accumulate in 61. If conditions allow, the cells will undergo conjugation and sporulation. Most cdc mutations which block in 61 are unable to conjugate at the restrictive temperature. However, cdc2 and cdclO cells are able to conjugate at the restrictive temperature and therefore must block before commitment to cell division <Nurse & Bissett, 1981). Both cdc2 and cdc10 are required for cells to pass start. Recently, the cdc28 gene from Saccharomyces. and the cdc2 gene from Sch i zosaccharomyces have been cloned (Beach et al., 1982). These two genes appear to be both functionally and structurally homologous. The cdc28 gene is able to complement cdc2 but the reverse is not true (Beach et al., 1982). Nature of 61 in Schizosaccharomyces In rapidly growing cultures, cells have a minimum Gl of 0.10 cell cycles. As growth rate declines, the length of Gl increases. However, the length of S + 62 remains constant over a 6-fold range of generation times (Nasmyth, 1979). Thus, the length of the 61 period is largely determined by cell mass. The start function observed in S. pombe demonstrates the existence of 61-specific functions as previuosly demonstrated for Saccharomyces. At present, yeasts are the only organisms in which 61-specific functions have been clearly demonstrated. 11 The cell cycle in Mammalian Cell Cultures Unlike lower eukaryotes, tissue culture cells are studied in an unnatural environment. Generation times o-f these cells are also much longer than those of yeast and ciliates. In spite of these limitations, tissue cultures fulfill a pressing need, namely the understanding of cell cycle regulation in animals. As in other systems, ts-mutations have been used to approach the underlying problems of the cell cycle. Mutants with a variety of defects in S phase entry, DNA synthesis, mitosis and cell division have been isolated (reviews; Basilico, 1977; Lloyd et al., 1982). The general method for isolation has been to grow mutagenized cultures at the restrictive temperature in the presence of agents that kill proliferating cells. Cells are then shifted to permissive conditions to allow the putative ts mutants to grow (Lloyd et al., 1982). The largest fraction of mutations recovered are blocked in Gl, although mutations representative of other cell cycle phases have also been obtained (Hirschberg et al., 1980). This suggests that there are many genes which act during Gl and make it amenable to mutational di ssecti on. Several general conclusions can be drawn from available studies. DNA synthesis and cell division are dependent on completion of mitosis. Mutants which block in M cease DNA synthesis and cytokinesis. However, 12 all the steps- o-f mitosis are not required since some cells continue to synthesize DNA even though mitosis is incomplete. In addition, DNA synthesis and cell division must become unlinked -for endoredup i cat i on to occur. Coordination o-f orowth and DNA synthesis Like all cells, mammalian cells tend to maintain a constant size -from one cell cycle to the next, when growth conditions are stable. Therefore, higher eukaryotes must also possess a means of coordinating growth and DNA synthesis. Studies of a wide variety of cell types have shown that the Gl period is the most variable while S + G2 + M tend to remain constant <Prescott, 1982). This same pattern has been observed in lower eukaryotes. Several lines of evidence suggest that cells begin DNA synthesis after attaining a critical cell mass. Smaller, post-fission cells have a longer Gl than do larger cells (Killander & Zetterberg, 1965a; Yen et al., 1975; Shields et al., 1978). When growth is restricted by nutritional limitation, cell mass is reduced, and the length of Gl increases <Kimball et al., 1971; Yen et al., 1975; Shields et al., 1978). Starvation characteristically causes cells to arrest in Gl (Kimball et al., 1971). Microphotometric measurements of cell mass demonstrate that variation in cell mass is lowest just after the start of DNA synthesis (Killander & Zetterberg, 1965b; Kimball et al., 1971). This is consitent with the predicted effect of a mass-related control over entry into S phase. 13 Effect o-f cell mass on cell cycle progression While the observations described above suggest that increase in mass is responsible for initiation of DNA synthesis, increase in mass is not an absolute requirement for progress through the cell cycle. Once DNA synthesis has been initiated, cell cycle progression is moderately insensitive to the action of growth inhibitors (Prescott, 1982). It has also been demonstrated that cells can be induced to enter S phase and will complete mitosis and cytokinesis, in the absence of any increase in mass (Zetterberg & Engstrom, 1983; Ronning & Lindmo, 1983; Mercer et al., 1984). Cell growth cannot be entirely excluded as a contributing factor in cell cycle regulation. The rate of cellular proliferation has been correlated with the rate of protein accumulation (Baxter 6c Stanner, 1978), and it has been suggested that some aspect of cell growth is required for initiation of DNA synthesis (Liskay et al., 1980). Cells which complete this requirement by the end of mitosis start DNA synthesis almost immediately, while cells which have not, have an intervening Gl period. V79-8 Chinese Hamster cells have no measurable Gl under optimal growth conditions (Liskay, 1977). However, when the rate of protein synthesis is decreased by treatment with cycloheximide, a Gl period is observed and the length of S + G2 + M is unchanged (Liskay et al., 1980). This is consistent with the suggestion that Gl is not a part of the DNA-division cycle, and exists only to satisfy a growth-related requirement for initiation of DNA synthesis (Stancel et al., 1981). The rate of DNA synthesis can also be reduced without affecting cell growth, by treating cells with hydroxyurea. Thus, S phase can be 14 extended without an increase in mass doubling time. The length o-f the cell cycle is unchanged, but Gl is decreased by an amount equal to the extra time required -for replication <Stancel et al ., 1981). Thus, it appears that the Gl phase of the mammalian cell cycle exists only when the cell growth cycle is longer than the time required to complete the DNA-division cycle. These observations are consistent with a mass-related control over entry into S phase. Cell cycle regulation models The evidence so far suggests that cell mass, initiation of DNA synthesis and the timing of cell division are under the control of homeostatic mechanisms. Three classes of theoretical models for the regulation of cell division have been proposed recently (Fantes & Nurse, 1981). 1) Cell division mass. Division could be triggered by the attainment of a critical cell mass. Cells would all divide at a predetermined size. Thus, large cells would have short cell cycles, while small cells would have long cell cycles. 2) Increment in mass between divisions. Regulation could be by incremental growth during the cell cycle. All cells would grow the same amount, thus reducing variation by one-half by partitioning to daughter cells. 15 3) Cell cycle duration. Cells might grow -for a -fixed length of time. However, since cells grow exponentially, cell mass will double and there will be no reduction in the amount of variation. Two more specific classes of models, concerned with the mechanisms of regulation have also been proposed (Fantes et al., 1975). These models, and most models devised, are based on three assumptions. 1) Cells tend to maintain a constant ratio of mass to genome, alternately referred to as the nucleocytoplasmic <n/c) ratio, or gene dosage. The timing of cell cycle events is assumed to be a response to the constraints imposed by this ratio becoming either too high (excess mass) or too low (deficient mass). All models are an attempt to describe the mechanisms that cells utilize to maintain a constant gene dosage. 2) Evidence indicates that the factor(s) regulating cell cycle events are cytoplasmic. Thus, cell mass should be strongly correlated with the control exerted by the regulatory substances. 3) Nuclei in a common cytoplasm initiate DNA synthesis and divide synchronously. Sister cells show less variation in the time of DNA synthesis onset and cell division than do unrelated cells. Regulatory models can be roughly divided into two classes, concentration and structural. Concentration models describe the effect of regulating substances as a function of their cellular concentration. Structural models postulate that accumulation of subunits to form a completed structure mediates the occurrence of cell cycle events. 16 Concentration models Concentration models postulate that cell cycle events are triggered when the cytoplasmic concentration o-f an effector molecule reaches a critical threshold concentration. There are two variations of this model, simple concentration, or active decay of an inhibitor. The simple concentration model predicts that events occur when 1) the concentration of an activator rises above a critical threshold (activator accumulation) or 2) the concentration of an inhibitor falls below a critical threshold (inhibitor dilution). These models are mathematically equivalent (Fantes et al., 1975). In the activator accumulation model, the rate of activator production will be correlated with cell mass, since the rate of activator synthesis should be a function of cell growth. This prediction is consistent with the observation of growth requirements for initiation of DNA synthesis (Liskay, 1977). This model is also consistent with the size-related thresholds observed at the start of S phase in several eukaryotes (Nurse, 1975; Johnston et al., 1977; Nasmyth, 1979). In the inhibitor dilution model, the rate of synthesis is nil for most of the cell cycle, and decrease in inhibitor concentration occurs by passive dilution as cytoplasmic volume increases. The observations regarding size thresholds are also consistent with this model. There is evidence suggesting an inhibitor regulation of DNA synthesis onset at least in quiescent cells (Yanishevsky & Stein, 1981; Burmer et al., 1983; Polunovsky et al., 1983). However, both concentration models are contradicted by studies which show that DNA synthesis can be initiated in the absence of net growth (Zetterberg & Engstrom, 1983; Ronning & 17 Lindmo, 1983). The unstable inhibitor model was first proposed by Yeas et al. (1965). The model suggests that cell cycle regulation is under the control o-f an inhibitor substance whose absolute amount is proportional to gene dosage. Rather than the amount being solely a -function o-f dilution, the inhibitor is proposed to decay at a constant rate, independent o-f its concentration. Cell cycle events are triggered when the amount o-f inhibitor -falls below a critical threshold. Thus, the regulatory e-f-fects exerted by the inhibitor will be dependent on both growth and gene dosage. This model is consistent with the growth-related requirement -for initiation o-f DNA synthesis <Liskay, 1977), and the existence o-f size-thresholds -for cell cycle events (Nurse, 1975; Johnston et al., 1977; Nurse &Thuriaux, 1977; Nasmyth, 1979). Positive vs. Neoative control mechanisms Most o-f the controversy about cell cycle regulation concerns the nature o-f the control mechanism. Are cell cycle events triggered by activating substances or modulated by inhibitors ? (i.e. positive or negative control). There is evidence supporting both views. Most o-f the evidence supporting positive control mechanisms is derived -from cell -fusion studies. Mammalian culture cells, when treated with polyethylene glycol and Sendai virus, -fuse to form a heterodi karyon. When cells in S phase were -fused with Gl cells, the Gl nucleus was induced to begin DNA synthesis (Rao & Johnson, 1970). G2 cells -fused with Gl cells do not have this e-f-fect. This suggests that 18 a substance, present only in S phase nuclei, is responsible for initiating DNA synthesis. It is important to note, however, that this does not exclude the possibilty that initiation of DNA synthesis may still be under negative control even though the maintenance of DNA synthesis under positive control. Other cell fusion experiments have given different results. Quiescent human diploid cells <HDC) in Gl fused to cells in S phase are not induced to synthesize DNA (Yanishevsky ic Stein, 1980). In addition, several other cell types when fused to HDC, not only fail to induce DNA synthesis in the HDC, but are themselves inhibited from entering S phase <Yansihevsky & Stein, 1980). Finally, inhibition of protein synthesis in quiescent NIH-3T3 and human diploid fibroblast-1ike cells, prevents these cells from inhibiting DNA synthesis in proliferating cells (Burmer et al., 1982; Polunovsky et al., 1983). Thus, it appears that, at least in quiescent cells, initiation of DNA synthesis is under the negative control of a protein or a group of proteins. Structural Models Structural models propose that cell cycle events are triggered by the completion of a particular structure. The structure is composed of subunits, so that the rate of assembly depends on the rate of subunit synthesis. In turn this will be proportional to cell growth. At least two cases have suggested a structural model for regulation of the cell cycle. In Tetrahymena. observations are consistent with cytoplasmic subunits control 1 ing eel 1 division <Zeuthen & Ui11iams, 1969). In Saccharomyces. the spindle pole body has been suggested as the structure 19 regulating completion o-f the start -function, since the SPB is completed just prior to start and its number is proportional to cell mass <Pringle & Hartwell, 1982). The cell cycle in Paramecium tetraurelia; The subject o-f this study The cell cycle in Paramec i urn is similar to that o-f most eukaryotes. There is a substantial Gl phase which extends from -fission to 0.25 o-f the cell cycle. Macronuclear DNA synthesis starts abruptly at this point, and continues until nuclear division at 0.87 <Berger, 1971)<Figure 1-2). There is no macronuclear G2. In Paramec i urn the coefficient of variation in cell mass is typically 15-20 V. <Kimball, 1959; Berger, 1982). Until recently, very little was known about cell cycle regulation in Paramec i urn. Early work by Kimball <1959, 1967) demonstrated a strong correlation between macronuclear DNA content and cell mass over a wide range of values. The only other study of cell mass regulation showed that small clonal differences in cell mass were maintained over several cell cycles (Kimball, 1967). However, this slow regulation of mass does not explain the maintenance of a constant variance in cell mass from one cell cycle to the next. Some other mechanism must be functioning to keep the variation which is introduced at each fission within limit. In contrast to the lack of understanding of cell mass regulation, the regulation of DNA content is much better understood. Several 20 studies indicate that cells make a standard amount o-f DNA during the cell cycle regardless of initial DNA content <Berger & Schmidt, 1978; Berger, 1978, 1979). The amount made is equal to the mean DNA content of the population (Berger, 1978, 1979). Thus, regulation of DNA content operates by an incremental mechanism (Fantes & Nurse, 1981), so that half the variation in DNA content is removed in each cell cycle. The cclccl and cc2 mutations of Paramec iurn have proven to be very useful in examining the coordination between DNA synthesis and cell growth in this organism. Both mutants are temperature-sensitive and block cell division. In addition, cc2 partially inhibits DNA synthesis while cclccl totally inhibits DNA synthesis (Peterson & Berger, 1982). Aims of this Study This study has five objectives. 1) To further characterize the cell-cycle mutant cclccl. 2) To examine the consequences of cell cycle blockage on cell mass, DNA content and cell division. 3) To examine the regulation of cell mass, after cells have been experimentally enlarged, and the effects of excess mass on cell cycle duration, macronuclear DNA content and cell mass in subsequent cell cycles. 21 4) To determine the effect of initial cell mass and parental DNA content on initiation of DNA synthesis. 5) To develop a simulation model of the Paramec i urn cell cycle, that can account for the experimental observations. The availabilty of the ts cell cycle mutants cclccl and cc2, which interfere with cell cycle progression and DNA synthesis, and the recessive mutation tamG. which alters the distribution of the macronucleus to daughter cells, allow these problems to be studied. 22 Figure 1-1. Cell cycle organization in several eukaryotes. 0 = start o-f cell cycle; 1 = end o-f cell cycle. ND = nuclear division, S = period of DNA synthesis. 1-1A. Cell cycle organization in Saccharomyces cerevisiae. 1-1B. Cell cycle organization in Schizosaccharomyces pombe. 1-1C. Cell cycle organization in a typical mammalian tissue culture line (Balb-c/3T3 cells). 23 24 Figure 1-2. Cell cycle organization in Paramecium tetraurelia. 0 = start o-f cell cycle; 1 = end o-f cell cycle; 0.25 = normal time o-f DNA synthesis initiation; 0.87 = end o-f S and start of macronuclear division (MND). 25 G1 Macronuclear S MND 0.0 .25 .87 1.0 26 CHAPTER II. A Fast-acting Temperature-sensitive Cell Cycle Mutation Paramecium tetraurel i a Af f ec t i no DNA Synthesis and Cell Cycle Progression, During both Gl and S: Implicati for Cell Cycle Regulation. 27 Introduct i on The cell cycle in eukaryotes is composed o-f two interdependent pathways; the cell growth and DNA division cycles (Mitchison, 1971). Normally these two pathways are coordinated such that one cycle does not outstrip the other. Many temperature-sensitive cell cycle mutations, which inter-fere with a wide variety o-f -functions have been isolated in eukaryotes (review; Simchen, 1978). The use o-f these mutations has revealed much about the relationships between the cell growth and DNA division cycles (Hartwell, 1978; Fantes, 1982). In particular, mutations which block DNA synthesis while allowing nearly norma) patterns of cell growth to continue are useful in examining the coordination of DNA synthesis with cell growth and cell cycle progression. One such mutation, ccl. has been isolated in Paramecium (Peterson & Berger, 1976). This mutation blocks macronuclear DNA synthesis and cell cycle progression at 34.4 C. This study examines three additional aspects of the ccl defect: namely a) kinetics of inactivation and reactivation of macronuclear DNA synthesis; b) blockage of cell cycle progression in 61 and; c) separation of the cell division and DNA synthesis pathways late in the cell cycle. The results indicate that ccl causes rapid inhibition of both macronuclear DNA synthesis and cell cycle progression at the restrictive temperature. This inhibition is reversible, and cells resume normal 28 I rates o-f DNA synthesis a-fter being returned to permissive conditions. Cell cycle progression is blocked even when the heat treatment lies totally within the Gl period. There is neither progression through the cell cycle at the restrictive temperature, nor excess delay o-f cell division when cells are returned to permissive conditions. Macronuclear DNA synthesis is not required -for the completion o-f cell cycle past 0.72 of the cell cycle. This point of commitment to cell division coincides with the micronuclear mitosis and the onset of oral morphogenesis in Paramecium. 29 Experimental Desicm Protocol -for Temperature Shift Experiments Shift from 27 C to 34.4 C Synchronous samples of ccl cells were incubated at 27 C for three hours after fission to allow the cells to reach mid S phase. Cells were then placed in a water bath at 34.4 C. Ten minutes prior to fixation, medium with I3H1 -thymidine labelled bacteria, pre-warmed to 34.4 C was added to the sample. At the scheduled time, samples were removed and cells killed in a solution of ethanol/Cerophyl (.3/. v/v) to prevent further incorporation of label into the macronuclei. The rate of DNA synthesis was determined by direct grain counts as desrcibed earlier (Berger, 1971), and compared to an untreated (27 C) control group. Shift from 34.4 C to 27 C DNA synthesis was first completely blocked by incubating ccl cells in mid S phase at 34.4 C for one hour. Cells were then returned to 27 C, the permissive temperature, and transfered to medium with [3Hl-thymidine labelled E. coli. Samples were fixed at 10 minute intervals. The rate of DNA synthesis was determined and compared to an untreated control group. 30 Results Effect of temperature shift on macronuclear DNA synthesis Following a rapid shift from 27 C to 34.4 C the rate of macronuclear DNA synthesis declined to 50% of initial activity within 6 minutes and was completely inhibited within 30 minutes (Figure 2-1; open circles). The rapid inhibition of DNA synthesis at 34.4 C was found to be reversed with equal rapidity when cells were transferred from restrictive to permissive conditions. The rate of macronuclear DNA synthesis in heat-treated ccl cells rose to 50% of the control level within 5 minutes, and was normal within 20 minutes after return to permissive conditions (Figure 2-1; filled circles). Delay of initiation by heat treatment in Gl When ccl cells were exposed to heat treatment at 34.4 C and subsequently returned to permissive conditions the cell cycle was extended (Figure 2-2, solid line). The duration of this extended cell cycle, less the length of heat treatment was always nearly equal to the length of the control cell cycle (96.8±2.5%; n=13)(Figure 2-3). In these experiments, heat treatment always began 30 minutes after fission (early in Gl), suggesting that the ccl defect, in addition to blocking DNA synthesis, also blocked progression through the cell cycle in 61. If ccl 31 blocked only DNA synthesis- then a cell cycle delay would be expected only when heat treatment extended past the normal point o-f DNA synthesis initiation (Figure 2-2, dashed line). The observation that cell cycle duration is increased when heat treatment is wholly contained wi thin the 61 period (Figure 2-2) supports the -first alternat ive. I-f ccl blocked progression through 61, then heat treatment would delay initiation o-f DNA synthesis. On the other hand, if heat treatment in 61 causes cell cycle elongation only through interference with DNA synthesis, then cells heat treated in 61 should initiate DNA synthesis at the normal time when heat treatment is contained completely within the 61 period. Such cells would also be expected to initiate DNA synthesis very soon after return to permissive conditions if the period of heat treatment extended past the normal time of initiation, since during heat treatment cells would have accumulated at the 61/S interface. To distinguish between these two alternatives, the timing of macronuclear DNA synthesis onset was determined in synchronous samples of ccl cells under different conditions. A control sample was grown continuously at 27 C, while, starting 30 minutes after fission, other samples were subjected to either a 1.0 hour, a 3.0 hour or a 5.0 hour heat treatment. Comparison of the time elapsed between the end of heat treatment and initiation of DNA synthesis in the experimental cells, with the duration of the portion of the Gl period remaining at the start of the heat treatment shows that there was no progression through Gl during heat treatment (Table 2-1). This conclusion is supported by the observation that the difference between the duration of the remaining portion of the 61 period and the time from the end of heat treatment to 32 initiation o-f DNA synthesis did not increase when the duration o-f the heat treatment was increased. Completion o-f DNA synthesis is not a precondition -for cell division Studies on P. tetraurelia suggest that cells synthesize a standard amount of DNA during the cell cycle <Berger, 1979). This is true even in cells which begin the cell cycle with abnormally low or high macronuclear DNA contents. Consequently it is important to know whether completion o-f the -full schedule of DNA synthesis is a precondition for cell division in this organism. The complete and rapid blockage of macronuclear DNA synthesis at restrictive conditions by the ccl defect makes it possible to directly test whether blockage of DNA synthesis during the latter part of the macronuclear S period is associated with blockage of cell division. The transition point (execution point) for cell division in ccl was estimated to occur at 0.73 of the normal 27 C cell cycle by Peterson & Berger (1976). This study, using the residual cell division method of Howell et al. (1975), estimates the transition point at 0.72+ 0.03 of the normal cell cycle. After the transition point, incubation of ccl cells at 34.4 C does not prevent cell division in the current cell cycle. To test whether DNA synthesis was inhibited after the transition point, synchronous samples of ccl cells were shifted to the restrictive temperature at four points (0.64, 0.72, 0.80, 0.88) in the cell cycle, and incorporation of tritiated thymidine into macronuclear DNA was examined after transfer of cells to the restrictive temperature. Earlier samples contained a significant number of cells which had not yet 33 reached the transition point, and were blocked, while in the later samples most o-f the ce l l s had passed the transition point and completed the ce l l cycle on schedule. There was no incorporation o-f label into macronuclei o-f either group o-f ce l l s , while controls at 27 C showed normal label l ing. Thus, macronuclear DMA synthesis is not precondition for cel l division after 0.72 of the ce l l cycle. 34 Discussion The ccl phenotype appears to be the result o-f a mutation in a single gene mutation that is pleiotropic (Peterson & Berger, 1976). At the restrictive temperature, DNA synthesis is rapidly inhibited, and progression through the cell cycle is arrested provided that the cells are shi-fted to restrictive conditions prior to 0.72 o-f the cell cycle. This e-f-fect occurs i-f cells are in either the Gl or the S phase o-f the cell cycle. Cell growth is not a-f-fected and cell mass increases normally while cells are at the restrictive temperature (Rasmussen & Berger, 1982). Effect on DNA Synthesis Several mutat i ons wi th temperature-sensitive de-fects in DNA synthesis have been described in Saccharomyces (review; Pringle ic Hartwell, 1982) and Schizosaccharomyces (review; Mi tenison, 1971), ciliates (Peterson & Berger, 1976; review: Berger, 1984) and mammalian cell cultures (e.g. Basilico, 1977; Sheinin & Lewis, 1980; Hyodo & Suzuki, 1982; McCracken, 1982). Many ts-mutants have defects in metabolite uptake or production. These characteristically produce a slow inactivation of DNA synthesis (McCracken, 1982). The rapid inactivation and reactivation of DNA synthesis in the ccl mutant is 35 unusual, and suggests that the mutation does not inter-fere with metabolite uptake or production but ma/ represent a defect in the rep 1icat ion mechanism itself. i A similar defect is observed in the cdc8 mutant of Saccharomyces  cerevisiae (Arendes et al., 1983). The cdc8 gene is required for DNA synthesis and in vitro replication of yeast 2 ion plasmid. The cdc8 gene product has recently been isolated and has been shown to be a single-stranded DNA binding protein. After 20 min. incubation at the restrictive temperature, more than 80% of the DNA binding and 90% of the complementation activity were lost (Arendes et al., 1983). However, unlike ccl. the effect of heat treatment is not reversible. Also, ccl blocks progression through Gl while cdc8 blocks only after the initiation of DNA synthesis. Effect on Cell Cycle Progression Cell cycle progression can be blocked by incubating cells in the presence of metabolic inhibitors such as cycloheximide (Polunovsky et al., 1983) or hydroxyurea (Worthington et al., 1976; Stancel et al., 1981; Singer & Johnston, 1983), or through the action of ts-mutations (Hartwell, 1970; Nurse et al., 1976; Peterson & Berger, 1976; Hyodo & Suzuki, 1982). However, cell cycle blockage is commonly either incomplete, and allows some progression through the cell cycle under restrictive conditions, or imposes excess delay (Prescott, 1982). The ccl mutation is unusual in that it blocks progression through the cell cycle completely, with neither residual progression nor excess delay. This study indicates that an indispensible cell function, altered by the 36 action o-f ccl at the restrictive temperature, is required -for progression through the 61 portion o-f the cell cycle, as well as for maintenance o-f DNA synthesis. Cell growth has been suggested as a major determinant o-f cell cycle progression <Liskay et al., 1980; Stancel et al., 1981). Attainment o-f a critical minimum cell mass is correlated with initiation of DNA synthesis in many eukaryotes (Prescott, 1956; Killander & Zetterberg, 1965; Nurse, 1975; Johnston et al., 1977; Nasmyth, 1979; Nasmyth et al., 1979; Rasmussen & Berger, 1982). However, the mechanisms coordinating cell growth with cell cycle events are not fully understood and the available evidence is conflicting. The effect of critical cell mass on control of initiation of DNA synthesis has been rationalized as a consequence of either accumulation of an activator <Rao & Johnson, 1970; Prescott, 1982) or dilution of an inhibitor (Stein & Yanishevsky, 1981; Burmer et al., 1982; Polunovsky et al., 1983). In each case, i n i t iat ion of DNA synthesi s i s a funct i on of cell growth. Cell growth also plays a role in determining the timing of other cell cycle events, e.g. . nuclear division (Hartwell, 1974; Nurse, 1975; Nurse & Thuriaux, 1977; Craigie & Cavalier-Smith, 1982). The present experiments indicate that cell growth by itself is not sufficient to bring about DNA synthesis in Paramecium. Since ccl cells continue to grow normally at the restrictive temperature, there should be no delay in the onset of DNA synthesis if initiation of DNA synthesis is dependent solely on attainment of a critical cell mass. Other observations have demonstrated that initiation of DNA synthesis and progression through the entire mitotic cell cycle can occur in the 37 absence of a significant increase in cell mass (Zetterberg & Engstrom, 1983; Ronning & Lindmo, 1983). Effect of DNA Synthesis Inihibition on Cell Division Earlier studies suggested that completion of the full schedule of DNA synthesis may be required to initiate cell division in Paramec ium (Berger & Schmidt, 1978; Berger, 1978; 1979). All cells, regardless of initial DNA content, synthesize a standard amount of DNA. Cells with very low initial DNA contents have extremely long cell cycles with greatly prolonged S periods. Our results show that the full schedule of DNA synthesis is not required for the initiation and completion of cell division or nuclear division. When ccl cells were shifted to restrictive conditions after the cell division transition point, they completed the cell cycle in the absence of any measureable macronuclear DNA synthesis, and post-fission DNA content was reduced by the expected amount (Ching, unpublished results). Similar divergence of the DNA division cycle from the cell division cycle (Mitchison, 1971) has been observed in other organisms (Hartwell, 1978; Mitchison, 1974). In most of these cases the point at which cell division becomes independent of the DNA division cycle occurs after the start of nuclear division. In Paramec i urn, however, the point at which blockage of DNA synthesis no longer blocks cell division (0.72 of the cell cycle) occurs at the start of stage 1 of oral morphogenesis, as defined by Kaneda & Hanson (1974). This point also coincides with the beginning of micronuclear mitosis, but occurs substantially prior to the start of macronuclear amitosis (0.87). Similar, though less precise observations have been made with 38 Tetrahymena (Anderson, 1972; Jeffery, 1972; worthington et al ., 1976). Thus the coordinated initiation of oral morphogenesis and micronuclear mitosis appears to define a commitment point for cell division in both Paramec i urn and Tetrahymena (review; Berger, 1984). 39 Table 2-1. E-f-fect o-f heat treatment on cell cycle progression dur i ng 61. Length oi Control Remainder o-f Time -from HT 61 61 at SHT EHT to S DIFF 1.0 hr 1.66 hr 1.16 hr 1.06 hr 0.10 hr 3.0 hr 1.38 hr 0.88 hr 0.85 hr 0.03 hr 5.0 hr 1.47 hr 0.97 hr 0.90 hr 0.07 hr HT = Heat Treatment SHT = Start o-f Heat Treatment EHT = End o-f Heat Treatment DIFF = (Remainder o-f 61 at SHT)-(EHT to S phase) 40 Figure 2-1. Rate of macronuclear DNA synthesis a-fter temperature shifts. Open circles = cells shifted -from 27 C to 34.4 C; Filled circles = cells shifted from 34.4 C to 27 C. 41 0 10 20 30 40 Min. After Temp. Shift 41 Figure 2-2. Cell cycle length vs. length o-f heat treatment. Dashed line = expected result if heat treatment extends cell cycle by interfering with cell cycle progression in S phase; Solid line = expected result if heat treatment blocks cell cycle progression in Gl; Open circles = existing data. Length of heat treatment is expressed as a percentage of the control cell cycle duration. 43 175 Figue 2-3. Portion o-f the cell cycle remaining a-fter the length o-f heat treatment is accounted -for vs. the length o-f heat treatment. Both variables are expressed as a percentage o-f the control cell cycle duration. 45 CD 3 CO Extended cycle - Length of HT o o _L ro . 01 f i o 6 to o o X CD 0) CD A) 3 CD 3 01. o cn O o o o o o CHAPTER III. Downward regulation o-f Cell Size in Paramecium tetraurelia: Effects of Increased Cell Size, With or Without Increased DNA Content, on the Cell Cycle. 47 Introduction In a typical eukaryotic cell the nuclear division mechanism ensures that daughter cells receive quantitatively and qualitatively identical chromosomal complements. The partitioning o-f cell mass (cytokinesis) is not as precise, and sister cells typically differ in size (e.g. Killander & Zetterberg, 1965a). Rates of growth and generation times also vary slightly. As a result, variation in cell mass is introduced during each cell cycle. If no regulatory mechanisms existed, the variance in cell mass within a cell population would increase without limit. Obviously this does not happen and cell mass shows only a limited variation (Killander & Zetterberg, 1965a; Killander, 1965; Kimball, 1967; Kimball et al., 1971; Yen et al. 1975; Fantes, 1977). Thus, one may conclude that regulation of cell mass occurs in all organisms. Regulation of cell mass has been demonstrated in several organisms including mammalian cell cultures (Prescott, 1956; Kimball et al., 1971), and the yeasts Schizosaccharomyces pombe (Fantes, 1977; Fantes & Nurse, 1977) and Saccharomyces cerevisiae. (Johnston et al. 1977). These studies show that cells tend to maintain a constant cell mass under stable growth conditions. However, when growth conditions are altered, cell mass is modulated. Studies in S. pombe (Fantes & Nurse, 1977), Saccharomyces (Johnston et al., 1980), mammalian cell cultures (Kimball et al., 1971) and Tetrahymena (Zalkinder 1979a, 1979b) show that 48 cell mass decreases as growth conditions decline and increases when conditions improve. Cell mass, or some cell mass related factor, appears to play an important role in regulating the cell cycle. Several studies have shown a correlation between the attainment of a critical cell size and the onset of DNA synthesis (Killander & Zetterberg, 1965b; Nurse, 1975; Yen et al. 1975; Fantes, 1977; Johnston et al., 1979; Nasmyth, 1979; Nasmyth et al., 1979). A similar relationship exists between cell size and the triggering of cell division (Frazier, 1973; Fantes & Nurse, 1977, 1978; Johnston et al., 1977; Zalkinder, 1979a, 1979b; Craigie & Cavalier-Smith, 1982; Soil et al., 1982). The mechanism that translates cell mass into a regulatory signal for the cell is, as yet, unknown. Variability in the generation times of sister cells is universal. Much of the work in cell cycle kinetics has been undertaken to explain how genetically identical cells can vary in their rates of growth, and generation times. The result of such studies suggests that in proliferating cells, the length of S + G2 + M tends to remain constant over a wide range of growth rates. (Kimball et al., 1971; Johnston et al., 1979; Nasmyth, 1979; Nasmyth et al., 1979). Most, if not all, of the variation in cell-cycle length is a consequence of variation in the Gl phase of the cell cycle (Prescott, 1976). There are contrasting explanations for the observed variability in the length of Gl. Paramecium is no exception to the rule. The generation time of exponentially growing cells in Cerophyl medium' (Kimball et al., 1959) ranges from 5.5 - 6.5 hours at 27 C, and sister cells typically differ in their generation times, cell mass and DNA contents. Paramec i um is 49 particularly useful for examining the cell cycle and cell mass regulation as it is possible to isolate and study individual cell lines. Further, the use of fluorochrome staining procedures in conjunction with a computerized system for quantifying staining intensity, makes it possible to do rapid and accurate measurements of both cell mass (protein) and macronuclear DNA content (Berger, 1979). In addition, the use of tritiated thymidine label led E. coli (Berger, 1964) is a sensitive assay for DNA synthesis and allows direct determination of the length of Gl (Berger, 1971; Rasmussen & Berger, 1982). Parameciurn tetraurelia is a relatively large protist with a volume 50 - 100 times that of a typical diploid eukaryotic cell. It has a large polygenomic macronucleus, which can tolerate extreme variation in DNA content without the occurence of genie imbalance. Cell mass also varies considerably, with a coefficient of variation on the order of 15 - 20% (Kimball et al., 1959; Rasmussen, unpublished data). In spite of the normal variation in both macronuclear DNA content and cell mass, there is usually a high correlation between mean cell mass and mean DNA content in Paramec iurn populations (Kimball, 1967; Berger, 1982a). These observations suggest that Paramec i urn can regulate cell mass, and that regulation of cell mass and DNA content are interrelated processes (Berger, 1982a). This chapter is concerned with the effect of heat treatment on two temperature-sensitive (ts) cell-cycle mutants, and examines the kinetics of cell mass regulation in Parameciurn and its relation to the cell cycle. The ts mutation ccl blocks macronuclear DNA synthesis and cell division at the restrictive temperature (34.4 C). However, protein synthesis and accumulation continue, so that cells with excess cell mass 50 and normal DNA content can be produced. A second ts mutation, cc2, also blocks cell division, but allows macronuclear DNA synthesis to continue at the restrictive temperature (34.25 C), although at a reduced rate. These cells show an increase in both cell mass and DNA content at the restrictive temperature, although DNA content increases at a lower rate than cell mass under these conditions. These mutations make it possible to experimentally generate cells o-f increased size, with or without a concommitant increase in macronuclear DNA content. The kinetics o-f downward regulation o-f cell mass were examined, and the e-f-fects o-f increased DNA content and cell mass on the rates o-f growth and DNA synthesis, cell-cycle duration and the changes in DNA content and cell mass over the course o-f a recovery cycle a-fter the return o-f experimental cells to permissive conditions were studied. In addition experimentally enlarged ccl cells were -followed over several recovery cell cycles to examine long term downward regulation o-f cell mass. 51 Experimental Pesion Two groups o-f approximately 20 dividing cells were hand-selected prior to fission. One group, the control sample, was incubated at the permissive temperature <27 C). This group was used to determine the length of the normal cell cycle, and the protein and DNA contents of untreated cells. The other sample, the experimental group, was incubated at 27 C for 0.5 h before the start of heat treatment to ensure that all cells had completed division prior to incubation at the restrictive temperature. Heat treatments were carried out in a water-bath with an electronic temperature controller, which maintained water temperature to within 0.02 C of the desired temperature. At the end of the heat treatment cells were returned to the permissive temperature and allowed to reach division. After the first division, one of the daughter cells was fixed, and the other was allowed to progress through the next cell cycle. After the second cell cycle, both daughter cells were fixed. The cell cycle that included the heat treatment was designated the first, or experimental, cell cycle and the recovery cell cycle was designated the second. The permissive temperature for both ts mutants was defined as 27 C. The restrictive temperature for ccl was 34.4 C, and for cc2. 34.25 C. 52 Results Analysis o-f ccl: Increase in cell size alone Growth and DNA synthesis durino heat treatment At 34.4 C, ccl cells were unable to synthesize macronuclear DNA. However, the rate o-f growth was only slightly less than that of wild-type cells at 34.4 C (Peterson & Berger, 197d). Cell mass, as estimated by protein content, increased linearly during heat treatment while DNA content remained at the Gl level. The rate of growth (protein accumulation) was nearly normal as compared to cells grown at the permissive temperature (27 C). Cell cycle durations Heat treatment caused an extension of the cell cycle proportional to the length of the heat treatment (open circles, Figure 3-1). The duration of the experimental cell cycle less the duration of the heat treatment was consistently 90-100% of the control cycle length (n = 13; mean = 96.8± 2.53%). This suggests that ccl cells did not progress through the cell cycle while at the restrictive temperature when heat treatment began in early Gl. It is not known if the same effect occurs when heat treatment is started at other points in the cell cycle. Presumably the same effect on cell cycle progression should occur up to the transition point at 0.72. 53 The second cycle was quite different from the f i r s t ( f i l l e d c i rc le s , Figure 3-1). There was a linear decrease in the length of the second ce l l cycle in ce l l s heat treated for up to 30'/. of the control cel l cycle length. For ce l l s treated longer than this, a consistent second ce l l cycle duration of 75% was obtained. DNA content and DNA synthesis Heat treatment of ccl ce l l s during the f i r s t cel l cycle caused no change in macronuclear DNA content (open c i r c le s , Figure 3-2). The post-f ission DNA content of heat treated ce l l s was never s ign i f icant ly different than the control sample. Therefore, ccl ce l l s synthesized the normal amount of macronuclear DNA once returned to the permissive temperature even though their cel l mass was up to twice the normal amount. The DNA content was s t i l l at the normal level following the second ce l l cycle ( f i l l e d c i r c l e s , Figure 3-2). Thus, the normal amount of DNA was made, even though this cel l cycle was up to 25% shorter than normal, and despite increased ce l l mass. Cell mass and growth rate. Since growth continued during heat treatment, ccl ce l l s were larger than normal, the increase in ce l l mass was d irect ly proportional to the length of the heat treatment (open c i r c le s , Figure 3-3). Cell mass was reduced during the second ce l l cycle, ( f i l l e d c i r c l e s , Figure 3-3). Cel ls heat treated for less than 0.3 of a cel l cycle returned to normal mass, while those heat treated longer than 0.3 of a ce l l cycle retained greater than normal mass. The amount of cel l mass reduction increased with increasing i n i t i a l ce l l mass. The amount of protein 54 accumulation during the second cell cycle closely parallels the pattern of decreased cell cycle length as initial cell mass increased (Figure 3-4) (compare filled circles in Figure 3-2 and Figure 3-4). Thus although the amount of protein synthesized in cells with different initial masses varied, this was a consequence of differences in the length of the second cell cycle and not due to the rate of protein accumulation which was constant and independent of cell mass. Analysis of cc2: Increase in cell mass and DNA content Growth and DNA synthesis durino heat treatment At 34.25 C, cc2 cells did not undergo cell division. However, unlike ccl. the cc2 cells were able to synthesize macronuclear DNA at the restrictive temperature. Cell mass increased linearly throughout heat treatment at 77% of the normal rate. Macronuclear DNA content also increased linearly, but only when heat treatment extended past 0.25 of the cell cycle. This point coincides with initiation of DNA synthesis in normal cells (Berger, 1971). Cell cycle durations The first cell cycle was extended by heat treatment (open circles, Figure 3-5). However, unlike ccl cells, the increase in cell cycle duration was less than the length of the heat treatment. The time between the end of heat treatment and cell division decreased as the duration of the heat treatment increased (Figure 3-6). This suggests that cc2 cells progress through the cell cycle while at the restrictive 55 temperature. The rate of progression was estimated to be about 40% of the normal rate. The duration of heat treatment also affected the length of the second cell cycle (Figure 3-5). The pattern was nearly identical to that of ccl cells. There was a linear decrease in the duration of the second cell cycle with heat treatments up to 30% of normal cell cycle length. Longer heat treatments produced a second cell cycle 75% of normal length. DNA content and DNA synthesis When heat treatment was restricted to the Gl portion of the cell cycle, there was no significant change in DNA content. However, when heat treatment extended into macronuclear S phase, DNA content increased (Figure 3-7). The rate of accumulation was about 40% of normal. Thus, although DNA synthesis occured in cc2 cells at the restrictive temperature, the rate was reduced. During the second cell cycle there was a strong correlation between the initial DNA content and the rate of macronuclear DNA synthesis, so that the increased DNA content was maintained (Figure 3-7)(r = 0.97). Cell mass and orowth rate Like ccl. cc2 cells had an extended cell cycle at the restrictive temperature. The cells continued to grow normally, and so were larger than normal at the end of the first cell cycle (open circles, Figure 3-8). The increase in cell mass was directly proportional to the duration of the heat treatment. Reduction in cell mass occured during the second cell cycle 56 (filled circles, Figure 3-8). Cells heat treated entirely within the Gl period of the first cell cycle returned to normal size, while those given heat treatments longer than 0.3 of a cell cycle were still larger than normal. The amount of cell mass reduction during the second cell cycle increased with increasing inital cell mass. Unlike ccl cells, in which the rate of growth during the second cell cycle was constant, the rate of growth in cc2 cells showed a strong positive correlation between Gl DNA content and growth rate (r = 0.92). This observation, and the fact that the macronuclear DNA content was always relatively less than the cell mass, suggests that macronuclear gene dosage is the rate-limiting variable for cell growth and DNA synthesis when cell mass is increased relative to macronuclear DNA content. Long-term downward regulation of cell size Synchronous samples of ccl cells were heat treated for several hours in order to produce cells with increased cell mass. Cell mass at the start of the second cell cycle was about 150% of normal. Cell mass returned to the control level within two cell cycles while DNA content remained at the control level throughout (Figure 3-9). 57 Di scussi on Downward regulation of cell mass in Paramecium involves the combined action of two processes which control both the rates of cell growth and DNA synthesis, and the length of the cell cycle. Each process is linked to different aspects of the macronuclear replication cycle. The rates of cell growth and DNA synthesis appear to be controlled by macronuclear gene dosage and cell mass, while the length of the cell cycle appears to be regulated by a eel 1-mass-dependent control over the intitiation of macronuclear DNA synthesis as shown in the subsequent chapter. This two-part homeostatic mechanism allows cells with a mass of up to 1.18 times normal to regulate back to normal mass within one cell cycle. Since normal variation in cell mass is less than 20%, most, if not all variation in cell mass can be removed by the next f i ssi on. Control of growth and DNA synthesis The results of this study are consistent with the hypothesis that the rates of cell growth and DNA synthesis are jointly determined by cell mass and macronuclear DNA content. When one variable is increased relative to the other, the smaller of the two becomes rate-limiting (Berger, 1978, 1982a). In the present study, cell mass was increased 58 relative to DNA content in both heat treated ccl and cc2 cells. In ccl. where DNA content remains at normal levels, the rates o-f cell growth and DNA synthesis were also normal. By comparison, in cc2, where DNA content was also increased but to a lesser extent than cell mass, the rates o-f cell growth and DNA synthesis were increased at a rate proportional to initial macronuclear DNA content. The inverse situation has been previously studied. Cells with experimentally reduced DNA content showed reduced cell growth and DNA synthesis (Berger, 1979). It appears that regulation o-f growth and DNA synthesis involves a coordinated interaction between cell mass and DNA content. The coordination is probably -fairly stringent, in order to account -for the strong correlation between mean DNA content and mean cell mass (Kimball, 1967; Morton & Berger, 1978; Berger, 1982a), and the rapid downward regulation o-f cell mass in ccl. reported in this study. The relationship between protein synthesis rate and gene dosage in Paramec iurn has been more thoroughly examined by Berger (1982a). At low DNA content, the rate o-f protein synthesis is proportional to DNA content. The rate o-f protein synthesis increases with increasing gene dosage up to a critical level. The critical gene dosage is on average 8% higher than the mean DNA content (Berger, 1982a). Further increase in gene dosage does not increase the rate of protein synthesis and the rate of protein synthesis reaches a plateau. There is a strong correlation between the plateau level of protein synthesis and cell mass in Paramecium (Berger, 1982a). The means by which Paramec iurn regulates growth and DNA synthesis can thus be explained in terms of a saturation hypothesis. When gene dosage is below the critical level, as in cells with normal or reduced 59 DNA content, the rate o-f cell growth will be limited by the availabiliy o-f DNA template. Above the critical level, the rate o-f cell growth might be limited by the size o-f the protein-synthesizing system o-f the cell, or by a concentration-dependent -feedback system as has been suggested as the basis -for cell mass regulation in the -fission yeast, Schizosaccharomyces (Fraser & Nurse, 1978, 1979; Barnes et al., 1979). The present data are insu-f-f ic ient to exclude one hypothesis in -favour o-f the other, but the implications -from this study are clear. In experimentally enlarged ccl and cc2 cells, cell mass is always relatively greater than DNA content. Thus, the rate o-f cell growth is expected to be limited by the gene dosage o-f the cell. The -finding that growth rate is correlated with initial DNA content is consistent with this expectat i on. The involvement o-f cell mass in the regulatory system is not -fully understood. It may be that a -feedback mechanism exists which prevents cell mass -from going above or below a certain level without influencing the DNA content of the cell. Paramecium must have some means of coordinating DNA content and cell mass in order to explain the simultaneous reduction of both variables in chemostat cultures at low growth rates (Ching & Berger, unpublished results). It is likely that the level of protein synthesis activity is a major component in the regulation of DNA content and cell mass. Control of cell cycle lenoth The second aspect of cell mass regulation in Paramec i um involves the length of the cell cycle. As initial cell mass increases there is a 60 c o r r e s p o n d i n g d e c r e a s e in c e l l c y c l e d u r a t i o n , t o a minimum o-f 0 .75 o-f i t s normal l e n g t h . F u r t h e r i n c r e a s e in c e l l mass has no e f f e c t on c e l l c y c l e d u r a t i o n . M a c r o n u c l e a r S phase i s known t o s t a r t a t 0 .25 o f the c e l l c y c l e in normal c e l l s ( B e r g e r , 1971 ) . T h e r e f o r e , S + M ( t h e r e i s no m a c r o n u c l e a r G2 in Paramec ium) e q u a l s 0 .75 o f the normal c e l l c y c l e . The minimum c e l l c y c l e l e n g t h o b s e r v e d in c e l l s w i t h i n c r e a s e d mass s u g g e s t s t h a t Gl i s r e d u c e d as i n i t i a l c e l l mass i n c r e a s e s . I f the i n i t i a t i o n o f DNA s y n t h e s i s r e q u i r e s the a t t a i n m e n t o f a c r i t i c a l c e l l mass , then the mass j u s t r e q u i r e d to p roduce a c e l l c y c l e o f minimum d u r a t i o n s h o u l d be equa l t o the c e l l mass a t the t ime o f i n i t i a t i o n in normal c e l l s . C e l l mass in normal c e l l s at the on se t o f S phase i s about 120% o f i n i t i a l c e l l mass ( B e r g e r , 1982a ) . T h i s i s c o n s i s t e n t w i t h the o b s e r v a t i o n t h a t p r o t e i n c o n t e n t i n c r e a s e s e x p o n e n t i a l l y ( K i m b a l l e t a l . , 1959 ) . These o b s e r v a t i o n s sugge s t t h a t DNA s y n t h e s i s in Paramecium b e g i n s when c e l l mass e x c e e d s 120% o f the mean i n i t i a l v a l u e . When the i n i t i a l c e l l mass i s equa l t o or g r e a t e r than t h i s t h r e s h o l d , the Gl phase o f the c e l l c y c l e i s e l i m i n a t e d . L a b e l l i n g e x p e r i m e n t s r e p o r t e d l a t e r in t h i s s t u d y ( C h a p t e r IV) show t h i s t o be the c a s e . E l i m i n a t i o n o f Gl f o l l o w i n g the p r o d u c t i o n o f a b n o r m a l l y l a r g e c e l l s th rough b l o c k a g e o f DNA s y n t h e s i s has been o b s e r v e d in T e t r a h y m e n a ( W o r t h i n g t o n e t a l . , 1976) and in some s t u d i e s on mammalian c e l l s (Ge rne r e t a l . , 1976; C r e s s & G e r n e r , 1977 ) . In a d d i t i o n , two r e c e n t s t u d i e s have shown t h a t t r e a t m e n t o f C h i n e s e hamster c e l l s ( S t a n c e l e t a l . , 1981) or S a c c h a r o m y c e s ( S i n g e r & J o h n s t o n , 1981) w i t h h y d r o x y u r e a can cau se a l e n g t h e n i n g o f S phase at the expense of G l . The l e n g t h of the c e l l c y c l e r e m a i n e d unchanged at low i n h i b i t o r l e v e l s , so 61 that cells are o-f normal size. Prescott (1982) suggests that 61 is not a part of the chromosome cycle, but rather a component of the growth cycle. Cells will only have a 61 phase when the cell mass doubling time is greater than the time required to complete S phase. Early mouse embryos in cleavage stage lack a 61 phase <6amow & Prescott, 1970). Only later, when the rate of cell division decreases, is a 61 period introduced. There are also several cultured cell lines which lack a 61 (these are termed 61-) (Robbins it Scharf, 1967; Liskay & Prescott, 1978; Prescott, 1982). One of these, Chinese hamster line V79-8, has been studied extensively. The results indicate that 61 is dispensible and the 61- condition is phenotypically dominant (Prescott, 1981). The fact that Paramec iurn appears to delete 61 when initial cell mass is increased agrees with the interpretation that 61 is only required to prevent the chromsome cycle from out-stripping the time required to double cell mass. It is not known whether initiation of DNA synthesis in eukaryotes is strictly determined by cell mass, or is a consequence of some other aspect of cell growth (Liskay et al., 1980). There is, however strong evidence that some cell types have rigid cell mass requirements for initiation of DNA synthesis, the length of 61 being negatively correlated with cell mass (Johnston et al., 1977; Nurse & Thuriaux, 1977; Killander & Zetterberg, 1965a, 1965b; Kimball et al. 1971; Yen et el. 1975; Schafer & Cleffman, 1982). This section has only addressed the question of downward regulation of cell mass in experimentally enlarged cells. It is not known whether the observed correlation between cell mass and initiation of DNA synthesis is absolute. Evidence suggests that cells which are smaller than normal 62 are able to traverse the cell cycle. The ts mutation sm2 interferes with cell surface proliferation and causes a reduction in cell mass and DNA content (Jones & Berger, 1?82; Berger, 197?). At the restrictive temperature cells continue to traverse the cell cycle, but become progressively smal1er at each succesive fission. It appears that reduction of cell surface by the sm2 defect, causes a shortening of subsequent cell cycles, thus limiting the amount of protein and DNA accumulation (Rasmussen, unpublished results). The two regulative processes revealed by this study - regulation of growth rate and shortening of the cell cycle in enlarged cells - act to quickly attenuate experimental increases in cell mass. The limitation of growth rate to the level of the initial DNA content when cell mass is increased results in the loss of 50% of the excess mass per cell cycle. This occurs by partitioning of the excess mass between the two daughter cells produced at fission. The shortening of the cell cycle accounts for a further reduction in cell mass by restricting the length of time available for growth. A computer-simulation model of the combined regulatory processes produces a good fit to the observed consequences of increasing cell mass (Berger, 1982a). The model predicts that increases in cell mass of up to 20% are accomodated within one cell cycle. Since the coefficient of variation in cell mass is normally about 20% (Kimball et al. 1959; Berger, 1978), most,if not all of the normally introduced variation in cell mass is removed within one cell cycle of its occurence. This study suggests that eel 1-mass-dependent initiation of DNA synthesis is an important regulatory component of the cell cycle in Parameciurn. Recent data indicate that there may also be a regulatory 63 point for cell division at or near 0.72 of the cell cycle (Chapter II). It is apparent that the effect of increased cell mass on the timing of DNA synthesis is not just a function of absolute cell mass. Doublet cells have twice the normal cell mass yet begin DNA synthesis at 0.25 of the cell cycle (Morton & Berger, 1978). Incomplete doublets are smaller, but still maintain cell and nuclear sizes that are greater than those in normal singlet cells (Sonneborn, 1963). This suggests that the cell cortex may play an important role in the regulation of cell mass and DNA content. The observation that reduction of the cell surface in sm2 cells causes a shortening of the subsequent cell cycle at the expense of S phase suggests that the cell cortex may mediate its effect quite late in the cell cycle (Rasmussen, unpublished data). A control mechanism is also required for termination of the cell cycle. In Paramecium this system is not time-dependent. Cells with highly decreased DNA content require three to five times the normal cell cycle duration to reach fission (Berger, 1979), even though the timing of DNA synthesis is normal (Berger, 1982b). The observations that these cells synthesize the normal amount of DNA (Berger, 1979) and reach at least normal cell mass at division suggest that cell division depends on growth and DNA synthesis and may also be dependent on cell mass as in fission yeast (Fantes & Nurse, 1977). The uncoupling of DNA synthesis and cell division after the transition point in ccl may be a consequence of the completion of a DNA synthesis-related event required to initiate the cell division pathway. 64 Figure 3-1. Effect of heat treatment on the length of the 1st and 2nd cell cycles in ccl/ccl cells. Open circles = 1st cell cycle; Filled circles = 2nd cell cycle. Cell mass is expressed as a percentage of the initial cell mass of control cells. 65 100 125 150 175 200 Initial Cell Mass 66 Figure 3-2. Gl macronuclear DNA content in heat-treated ccl/ccl cells. Open circles = start o-f 2nd cell cycle; Filled circles = start o-f 3rd cell cycle. Cell mass is expressed as a percentage o-f the initial mass o-f control cells. DNA content is expressed as a percentage o-f the Gl DNA content o-f control cells. 67 150-i o o — n — 50-100 125 150 175 200 Initial Cell Mass 68 Figure 3-3. Post—fission cell mass in heat-treated ccl/ccl cells. Open circles = start o-f 2nd cell cycle; Filled circles = start o-f 3rd cell cycle. Mass is expressed as a percentage o-f initial cell mass o-f control cells. Length o-f heat treatment is expressed as a percentage o-f the control cell cycle duration. 69 7o Figure 3-4. Protein accumulation during the 2nd cell cycle in heat-treated ccl/ccl cells. Cell mass is expressed as a percentage o-f the initial mass o-f control cells. 71 125 o w 100-u o < w OT co 2 = 7 5 O ( ) V r 50-100 125 150 175 200 Initial Cell Mass 7Z Figure 3-5. E-f-fect o-f heat treatment on the length o-f the 1st and 2nd cell cycles in cc2/cc2 cells. Open circles = 1st cell cycle; Filled circles = 2nd cell cycle. Cell mass is expressed as a percentage of the initial mass of control cells. 73 200 150 Length of Heat Treatment 100 125 150 Initial Ce l l Mass 175 200 74-Figure 3-6. Time required -for cc2/cc2 cells to complete the cell cycle after the end o-f heat treatment. 75 Figure 3-7. Gl macronuclear DNA content in heat-treated cc2/cc2 cells. Open circles = start o-f 2nd cell cycle; Filled circles = start o-f 3rd cell cycle. Cell mass is expressed as a percentage o-f the initial mass o-f control cells. 77 100 125 150 175 200 Initial Cell Mass 78 Figure 3-8. Post—fission protein content in heat-treated cc2/cc2 cells. Open circles = start o-f 2nd cell cycle; Filled circles = start o-f 3rd eel 1 cycle. 79 Figure 3-9. Downward regulation o-f cell mass in experimentally-enlarged ccl/ccl cells. Cell mass is expressed as a percentage of initial cell mass of control cells. 81 82 CHAPTER IV. Initiation o-f DNA synthesis in Paramec i um tetraureli a; The e-f-fect o-f Cell Mass and Parental DNA content on the Timing o-f DNA synthesis Initiation. 83 Introduct i on For most cells the time needed to double cell mass is greater than the time required -for DNA replication. Mechanisms must exist to ensure that succesive rounds o-f DNA synthesis are not initiated be-fore the cell has completed sufficient growth, otherwise cell mass would quickly decrease in subsequent generations. Thus, the central problem for cycling cells is coordination of cell growth with DNA synthesis. A large body of evidence suggests that attainment of a critical cell mass prior to initiation of DNA synthesis is a major mechanism through which this coordination is achieved <Ki11ander & Zetterberg, 1965; Nurse, 1975; Yen et al., 1975; Johnston et al., 1977; Nasmyth, 1979; Rasmussen & Berger, 1982). If initiation of DNA synthesis is solely dependent on attainment of a critical cell mass, then the length of the Gl period should represent the time required to reach the size threshold, and should vary with cell size. The Gl phase is the most variable portion of the cell cycle while the remainder <S + G2 * M) is of relatively constant duration (Precott, 1976), suggesting that the Gl/S boundary is the major coordination point for the cell cycle. However, the mechanisms responsible for this coordination are not understood. There is evidence that 61 can be eliminated in Parameciurn when cells begin the cell cycle with increased cell mass (Rasmussen & Berger, 84 1982). Further, in cleavage stage embryos Gl is absent but appears as cell size is reduced, and the rate of cellular proliferation decreases (Gamow & Prescott, 1970). In mammalian tissue culture cells, mutants have been obtained which lack a Gl period. However, a Gl period can be introduced by reduction of growth rate <Liskay et al., 1980). These studies raise the question as to whether there are any Gl-specific events, or whether the presence of a Gl period merely reflects a requirement for cell growth before DNA synthesis initiation. Paramecium is a useful organism for studying coordination of growth and DNA synthesis since both cell mass and gene dosage can be varied over a wide range. The availability of gene mutations affecting 1) cell division and DNA synthesis, and 2) distribution of macronuclear DNA at fission, make it possible to independently vary either cell mass or gene dosage, in order to study the effects of change in either variable on initiation of DNA synthesis. Cell mass has been correlated with initiation of DNA synthesis in many eukaryotes, including Paramecium (e.g. Nurse, 1975; Rasmussen & Berger, 1982). While gene dosage in Gl has no effect on the timing of DNA synthesis onset, increase in the variance of gene dosage is accompanied by increased variabilty in the timing of DNA synthesis initiation (Berger, 1982b). This suggests that parental gene dosage may affect the timing of DNA synthesis in the subsequent cell cycle. This study examines the effects of initial cell mass and parental gene dosage on initat ion of DNA synthesis in Paramec i um. 85 Experimental Pes ion Effect o-f cell mass on initiation Experimentally enlarged cells were produced by heat treating synchronous samples of ccl cells for 5% to 35% of a normal cell cycle. This produced groups of cells with mean initial cell masses from 5% to 33% above normal. From these groups, several sub-samples of dividing cells were hand selected at fission and these synchronous cell samples were then placed into medium containing C3H]-thymidine labelled bacteria. At 15 minute intervals, samples of 20-30 cells were removed, washed in unlabel led Cerophyl medium, and cells fixed individually onto a slide. One group was fixed immediately after fission as a cell mass control. The length of Gl was determined as described previously (Rasmussen & Berger, 1982), and compared to the mean initial cell mass. Effect of parental DNA content on initiation Individual tamG cells were selected at division and both daughters isolated in medium containing C3HI -thymidine labelled bacteria for times ranging from 30 minutes to 3 hours before fixation. The time between collection and fixation for each pair of daughter cells was recorded and the stage in the cell cycle determined. DNA content was determined as described previously (Rasmussen ic Berger, 1982), and the parental DNA content derived by summing the Gl DNA contents of the two daughter cells. The results were sorted according to cell cycle stage, parental DNA content and whether or not the cell had initiated DNA 86 synthesis, as determined by autoradiography (Berger, 1971). 87 Results Effect of cell mass on initiation o-f DNA synthesis Synchronous samples of ccl cells were heat treated for varying lengths of time to produce abnormally large cells. At the end of the heat-treated cell cycle two groups of dividing cells were collected. One group was fixed immediately after fission as a measure of initial cell mass, while the second group was allowed to complete the subsequent recovery cell cycle to determine the cell cycle duration after experimentally increasing cell mass. Both the initial cell mass and duration of the recovery cell cycle were scaled relative to control values from a parallel experiment involving non-heat-treated cells. It was found that the duration of the subsequent cell cycle decreased as initial cell mass increased (Figure 4-1). There was a strong negative correlation between the two variables <r=-0.87). Cell cycle duration approached a minimum of 75% of the control value when initial cell mass exceeded 120% of the normal post-fission value. Gl normally encompasses 25% of the cell cycle, while S phase and nuclear division (there is no G2) require 75% of the cell cycle for completion. Thus, the data indicate that increasing initial cell mass shortens the cell cycle by reducing the Gl period. This idea was tentatively examined in an earlier paper (Rasmussen & Berger, 1982). This hypothesis was tested in detail by directly comparing the 88 l e n g t h of 61 ( a s opposed to c e l l c y c l e d u r a t i o n ) , w i t h i n i t i a l c e l l mass in hea t t r e a t e d g r o u p s o f c c l c e l l s . The p o i n t o f DNA s y n t h e s i s i n i t i a t i o n was taken as the t ime a t wh i ch 50% o f the n u c l e i in a group showed s i g n i f i c a n t i n c o r p o r a t i o n of l a b e l . T y p i c a l l y , the median 50% o f the p o p u l a t i o n i n i t a t e DNA s y n t h e s i s w i t h i n a 7 m inu te i n t e r v a l . The r e s u l t s a r e c l e a r . I n c r e a s e in i n i t i a l c e l l mass l e a d s to a s h o r t e n i n g in the l e n g t h o f the 61 phase o f the c e l l c y c l e ( F i g u r e 4 - 2 ) . T h e r e was a h i g h n e g a t i v e c o r r e l a t i o n between i n i t i a l c e l l mass and l e n g t h o f 61 up t o c e l l masses o f 118% o f normal <r = - 0 . 9 2 ) . The 61 phase o f the c e l l c y c l e was c o m p l e t e l y e l i m i n a t e d when i n i t i a l c e l l mass e x c e e d e d 118.5% o f n o r m a l . C e l l s wh i ch s t a r t the c e l l c y c l e w i t h an i n i t i a l mass g r e a t e r than t h i s amount i n i t i a t e m a c r o n u c l e a r DNA s y n t h e s i s w i t h i n 5 m i n u t e s a f t e r f i s s i o n . E f f e c t o f p a r e n t a l DNA c o n t e n t on i n i t i a t i o n o f DNA s y n t h e s i s T o t e s t the e f f e c t o f p a r e n t a l DNA c o n t e n t on i n i t i a t i o n o f DNA s y n t h e s i s , tamG c e l l s were c o l l e c t e d a t f i s s i o n and each p a i r o f daugh te r e e l Is w a s i n d i v i d u a ] l y i s o l a t e d in medium c o n t a i n i n g [ 3 H ] - t h y m i d i n e l a b e l l e d Ej_ c o l i . A t v a r i o u s t i m e s between 0.20 and 0.40 o f the c e l l c y c l e , each p a i r o f c e l l s was f i x e d o n t o a s l i d e . The p a r e n t a l DNA c o n t e n t was d e t e r m i n e d by summing the i n d i v i d u a l DNA c o n t e n t s o f each d a u g h t e r c e l l . A u t o r a d i o g r a p h s were made, and the s t a t e o f DNA s y n t h e s i s ( l a b e l l e d v s . n o n - l a b e l l e d ) was d e t e r m i n e d f o r each p a i r o f d a u g h t e r s . The d a t a f rom s e v e r a l e x p e r i m e n t s were p o o l e d and s o r t e d a c c o r d i n g t o c e l l c y c l e s t a g e and the s t a t e o f DNA s y n t h e s i s . The c u m u l a t i v e d i s t r i b u t i o n s o f DNA c o n t e n t in l a b e l l e d and 89 non-labelled nuclei were compared at two different cell cycle stages; 0.20-0.30 and 0.30-0.40 (Figure 4-3). The results indicate that, at the mean time of initiation (0.25 of the cell cycle), the average macronuclear DNA content of cells which have initiated DNA synthesis is 0.79 times that of cells which have not yet started macronuclear S. Thus, it appears that cells with smaller nuclei initiate DNA synthesis slightly earlier than do cells with larger nuclei. Later in the cell cycle (at 0.35), there is no significant difference between the distributions of labelled and unlabelled macronuclei. 90 Di scussi on Effect of ce l l mass on the timino o-f DNA synthesis The results demonstrate a strong correlation between cel l mass and DNA synthesis in i t iat ion in Paramecium. Cel ls in i t iate DNA synthesis when cel l mass exceeds 118.5% of the normal post-fission mass. This is the cel l mass that would be expected in at ce l l s at 0.25 of the cel l cycle if growth occurs exponentially as has been observed (Kimball et a l . , 1959). Correlations between cel l mass and timing of DNA synthesis have been observed in several other eukaryotes (Prescott, 1956; Killander & Zetterberg, 1965; Kimball et a l . , 1971; Nurse, 1975; Yen et a l . , 1975; Nurse ficThuriaux, 1977; Johnston et a l . , 1977; Nasmyth, 1979). The popular interpretation suggests that ce l l s must attain a c r i t i c a l cel l mass or volume, before DNA synthesis can begin. Other studies show that cel l mass alone is insufficient to trigger DNA synthesis. In cases where growth is inhibited by serum deprivation, ce l l s can enter S phase and traverse the ce l l cycle in the absence of ce l l growth (Zetterberg & Engstrom, 1983; Ronning & Lindmo, 1983). Also, ccl ce l l s blocked early in Gl do not begin DNA synthesis immediately upon return to permissive conditions, but complete the remainder of Gl , even though ce l l mass at the end of heat treatment is greater than the size at which ce l l s normally begin DNA synthesis (Chapter II). Thus, while initation of DNA synthesis appears to be related to some aspect of ce l l growth, as has been previously suggested, (Liskay et a l . , 1980; Singer & Johnston, 1981; Stancel et a l . , 1981), cel l mass alone is not suff ic ient for entry into S phase. 91 Effect o-f parental DNA content on the timino of DNA synthesis  in i t iat ion Paramec i un> is unusual compared with most eukaryotes in that it has a large polygenomic macronucleus which carries out the somatic functions of the c e l l . Since the macronucleus divides ami totical1y, DNA content, and consequently, gene dosage varies. The action of the tamG gene mutation causes this variation to increase considerably (Berger & Schmidt, 1978). Variation in Gl gene dosage has no effect on the timing of DNA synthesis in i t iat ion (Berger, 1982b; this study). However, this study shows that DNA synthesis in i t iat ion is influenced by parental gene dosage. This effect is not due to cel l mass since cel l mass was not s ign i f iant ly different in labelled vs. non-labelled ce l l s . This observation is consistent with the interpretation that in i t iat ion of DNA synthesis is under the control of an inhibitory factor, produced in the previous ce l l cycle, and whose amount is proportional to the rate of growth. The effect of gene dosage on protein synthesis has been previously discussed (Berger, 1982a). Protein synthesis rate in ce l l s with normal cel l mass, but greater than normal gene dosage is limited by ce l l mass, so that the amount of inhibitor produced wi l l be normal. Protein synthesis in ce l l s with with normal mass, but low parental gene dosage is limited by gene dosage and thus, the amount of inhibitor produced wi l l be proportional to gene dosage. Thus, the effect of gene dosage on in i t iat ion of DNA syntheis should only be observed in ce l l s where gene dosage is rate- l imit ing for growth, i .e. in ce l l s with low 92 parental DNA content. Recent observations in human tissue culture cells also suggests inhibitor control over DNA synthesis initiation (Yanishevsky & Stein, 1980 j Burmer et al ., 1982; Polunovsky et al ., 1983). Blockage o-f protein synthesis in quiescent -fibroblasts using cycloheximide makes these cells unable to inhibit DNA synthesis in proliferating -fibroblasts (Polunovsky et al ., 1983). Thus, it appears that entry into S in quiesent cells is prevented by a protein inhibitor o-f DNA synthesis initiation. Howver, functions controlling entry of cells from quiescent to cycling state are not necessarily the same functions which control initiation of DNA synthesis in cycling cells. The recent isolation of a ts-mutant, specific for entry into the cell cycle from 60, suggests very strongly that these are indeed separate mechanisms (Ide et al., 1984). Nature of the 61 period in Paramecium Normally, the cell mass doubling time is longer than the time required to complete DNA replication. Therefore, a mechanism must exist to coordinate growth and DNA synthesis. Otherwise succeeding rounds of DNA synthesis would outpace the cell's ability to increase in size, and cell mass would decrease. In most, but not all, cycling cells each round of DNA synthesis is preceded by a 61 period. It has been suggested that 61 reflects a requirement by the cell to attain a critical cell size before DNA synthesis can begin (Liskay et al., 1979). Cells which have completed this requirement by the end of mitosis initiate DNA synthesis with minimum or no 61 phase (Prescott, 93 1981). Cells which have not completed the size requirement -for initiation by the end o-f mitosis, nescesarily have an intervening Gl period. DNA synthesis begins once the critical cell mass has been reached. Studies in which the rate o-f DNA synthesis was reduced, so that the time required -for replication more nearly equaled the cell mass doubling time, showed that as the length o-f S phase increased, there was a corresponding decrease in the length o-f Gl. Cell cycle duration was unaffected (Singer & Johnston, 1981; Stancel et al., 1981). In Paramecium. the Gl period can be reduced or eliminated by increasing initial cell mass. This is consistent with the model that Gl is a general period of growth required for cell mass to keep pace with succeeding rounds of DNA synthesis, and is not an intrinsic part of the DNA division cycle (Prescott, 1982). 94 Table 4-1. Comparison o-f pre—fission DNA content in tamG cells with labelled or non-labelled nuclei, at 0.25 o-f the cell cycle. DNA content is expressed as percentages o-f the Gl DNA content of control cells. DNA Synthesis state Mean Cell Mass Mean DNA Content Labelled 114.5% 169.3% Non-labelled 117.9% 214.3% 95 Figure 4-1. The effect o-f inital cell mass on cell cycle duration. Initial cell mass is expressed as a percentage of the initial mass of control cells. Cell cycle duration is expressed as a percentage of control cell cycle duration. 96 100-# 7 ( H 100 120 140 160 180 Initial Cell Mass 97 Figure 4-2. Effect of i n i t i a l cel l mass on the length of the Gl period. In i t ia l ce l l mass is expressed as a percentage of the i n i t i a l mass of control ce l l s . Gl duration is expressed as a percentage of control cel l cycle duration. F i l l e d c i r c le s = length of Gl determined direct ly by autoradiography} Open c i r c l e = Onset of DNA synthesis estimated cytof1uorimetrical 1 y (see Chapter 111). 98 3 0 H Initial Cell Mass 99 Figure 4-3. Cumulative percentage plot o-f parental macronuclear DNA content vs. DNA synthesis state at 0.25 o-f the cell cycle. Open circles = cells not synthesizing DNA; Closed circles = cells which are synthesizing DNA. Parental DNA content is expressed as a percentage o-f the initial DNA content o-f a control sample. 100 0 100 200 300 400 500 Parental DNA Content lot Figure 4-4. Cumulative percentage plot o-f parental macronuclear DNA content vs. DNA synthesis state at 0.35 o-f the cell cycle. Open circles = cells not synthesizing DNA; Closed circles = cells which are synthesizing DNA. Parental DNA content is expressed as a percentage o-f the initial DNA content o-f a control sample. 102 0 100 200 300 400 500 P a r e n t a l DNA Content I03 CHAPTER V. Summary: A Ce l l Cycle Model -for Paramecium 104 This study has revealed three major points about cell cycle regulation in Paramec i urn. First, the present data show that there is a mass-related control over DNA synthesis initiation (Chapter IV). Second, DNA synthesis is not required -for completion of cell division past 0.72 of the cell cycle (Chapter II). Finally, there is evidence which suggests that the timing of DNA synthesis initiation is / determined in the previous cell cycle (Berger, 1982b; Chapter IV). Recent observations indicate that the onset of DNA synthesis does not depend strictly on the attainment of a critical cell mass through cell growth (Ronning & Lindmo, 1983; Zetterberg & Engstrom, 1983; Chapter II). We have shown in this study that varying gene dosage between cells influences the kinetics of entry into S phase, such that cells with lower than average gene dosage initiate DNA synthesis earlier than cells with higher than average gene dosage. However, sister cells initiate DNA synthesis at the same time despite larger differences in gene dosage (Berger, 1982a). These observations suggests that the timing of DNA synthesis initiation is determined in the preceding cell cycle. The point of determination must be prior to the ccl transition point (0.72) since inhibition of DNA synthesis in ccl cells after this point reduces gene dosage, but does not affect the timing of DNA synthesis initiation in the subsequent cell cycle (Ching, unpublished results). There are four major observations concerning the cell cycle in Parameciurn. 1) The ccl defect blocks cell cycle progression completely 105 during the Gl period. Thus some -function uncovered by this de-fect is required -for cell cycle progression. 2) Gl gene dosage has no e-f-fect on the timing o-f DNA synthesis initiation. Sister cells with extreme differences in DNA content, caused by the action of the tamG mutation, initiate DNA synthesis at the same time. 3) There is a cell mass related threshold for initiation of DNA synthesis in Paramecium. 4) The timing of DNA synthesis initiation appears to be determined in the previous cell cycle, and is affected by parental gene dosage. Table 5-1 summarizes these observations, and lists the cell cycle regulation models consistent with each observation. Only one model, the unstable inhibitor model, is consistent with all observations. A simple computer model describing the cell cycle in Paramecium has been discussed previously <Berger, 1982a). This model makes four assumptions; (1) DNA synthesis begins when a critical cell mass is reached; (2) Cell division is triggered once sufficient macronuclear DNA has been synthesized; <3) Gene dosage and cell interact to control the rate of protein synthesis; <4) The rate of protein synthesis is determined by the relatively smaller of gene dosage or cell mass. This model has been further refined in order to account for the present observations. The revised model closely parallels the unstable inhibitor model of Fantes et al. <1975). The BASIC code for the model is shown in Appendix II. The criteria for initiation of DNA synthesis and start of the cell division pathway have been altered. DNA synthesis begins when the level of inhibitor falls below 0.85. Inhibitor concentration at the start of the cell cycle is set at 1.00. The inhibitor is pulse synthesized at the point of commitment to cell division. In normal 106 cells this occurs at 0.72 of the cell cycle. The amount of inhibitor produced is proportional to a rate-limiting-variable, whose value is equal to either gene dosage or cell mass, whichever is relatively smaller. The inhibitor decays at a constant rate such that the amount will be reduced by 1/2 during the course of a normal duration cell cycle. One assumption remains. The cell division pathway is triggered when DNA content passes 168% of the initial amount. This is an a priori assumption based on the estimated DNA content in cells at 0.72 of the cell cycle. For cells with increased cell mass, or increased DNA content, the values predicted by the simulation model closely parallel the experimental observations (Table 5-2). In conclusion, this study has demonstrated four components of cell cycle regulation in Paramecium tetraurelia. (1) There is a cell mass-related control over initiation of DNA synthesis. Cells enter S phase when cell mass exceeds 118.5% of normal mass. (2) There is a commitment point for cell division at 0.72 of the cell cycle. After this point cell division can be completed in the absence of macronuclear DNA synthesis. (3) There is an indispensible function in Gl required for progression through the cell cycle. (4) There is evidence that the timing of DNA synthesis initiation is determined in the previous cell cycle. There are several questions which remain unanswered. What is the mechanism which controls initiation of DNA synthesis ? What is the event which causes the cell to become commited to cell division, before replication is complete in the macronucleus ? How does upward regulation of cell mass occur ? What are the effects of nutritional limitation on the organization of the cell cycle ? These are all 107 questions which need to be examined in order to develop a -fuller understanding o-f how cell cycle events are determined in Parameciurn and other organisms. 108 Table 5-1. Summary o-f Observations on Cell Cycle Regulation in Paramec i urn and Models Consistent with each Observation. <1) Blockage o-f cell cycle progression by ccl. <a) Activator accumulation - assumes that ccl prevents activator production. <b) Unstable inhibitor - assumes that ccl prevents inhibitor decay. <c) Structural model - assumes that ccl prevents subunit production. <2) Gl gene dosage has no ef-fect on initiation o-f DNA ... synthesi s. (a) Inhibitor dilution - assumes near normal growth in cells with small nuclei. <b) Unstable inhibitor - decay constant is not a-f-fected by rate of growth in current cell cycle. <3) Cell mass threshold at start of S phase. (a) Activator accumulation - activator synthesis proportional to growth. (b) Inhibitor dilution - inhibitor is diluted as cell volume increases. (c) Unstable inhibitor - production of inhibitor proprtional to mass in previous cell cycle. <d) Structural model - subunit production is a function of growth. <4) Parental effect on initiation of DNA synthesis. (a) Inhibitor dilution - inhibitor produced late in previous cell cycle, amount depends on growth rate before fission. <b) Unstable inhibitor - same as inhibitor dilution. 10? Table 5-2. Comparison of observed consequences of changes in DNA content and cell mass with results predicted by the cell cycle model (Appendix II). D = macronuclear DNA content; P = size of protein synthesis system; M = cell mass. Means are shown with standard errors in parentheses. Both DNA content and cell mass are expressed as percentages of initial control values. a) Increased DNA Content b) Increased Cell Mass M Initial Observation 193 <8.6) - 109 <3) Values Simulation 200 100 100 Final Observation 165 <15.5) - 98.2 <2) Values Simulation 150 100 100 Cycle Observation 91.4'/. 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In: The Molecular Biolooy of the Yeast Saccharomyces: Life Cycle & Inheritance. J.N. Strathern, E.W. Jones & J.R. Broach eds. Harbor Lab. Cold Spring Harbor, New York. Rao, P.N. & R.T. Johnson (1970). Mammalian cell fusion: Studies on the regulation of DNA synthesis and mitosis. Nature (Lond.) 225:159-170. 120 Rasmussen, CD. it J.D. Berger (1982). Downward regulation o-f cell size in Paramecium tetraurelia: Ef-fects o-f increased cell size, with or without increased DNA content, on the cell cycle. J. Cel 1 Sci. 57:315-329. Robbins, E.R. & M.D. Schar-f (1967). The absence o-f a detectable 61 phase in a cultured strain o-f Chinese hamster lung cell. J. Cel 1  Biol. 34:684-688. Ronning, O.W. ic T. Lindmo (1983). Progress through 61 and S in relation to net protein accumulation in human NHIK 3025 cells. Exp. Cell Res. 144:171-179. Scha-fer, E. ic 6. Cleffman (1982). Division and growth kinetics of the division mutant conical of Tetrahymena. Exp. Cell Res. 137:277-286. Seyfert, H. -M. (1977). A short 61 period is correlated with low macronuclear DNA content in Tetrahymena. Exp. Cel1 Res. 108:456-459. Shields, R., R.F. Brooks, P.N. Riddle, D.E. Cape11aro& D. Delia (1978). Cell size, cell cycle and transition probability in mouse fibroblasts. Cel1 15:469-474. Sheinin, R. ic P.N. Lewis (1980). DNA and histone synthesis in mouse cells which exhibit temperature-sensitive DNA synthesis. Som. Cel1  Genet. 6:225-239. Shilo, B., 6. Simchen & A.B. Pardee (1978). Regulation of cell cycle initiation in yeast by nutrients and protein synthesis. J. Cel 1  Physiol• 97:177-188. Simchen, 6. (1978). Cell cycle mutants. Ann. Rev. Genet. 12:161-191. Singer, R.A. & 6.C. Johnston <1983). Growth and the cell cycle of the yeast Saccharomyces cerevisiae. Exp. Cell Res. 149:15-26. Sokal , R.R. 6e F.J. Rohl-f (1969). Biometry. San Francisco, Freeman. Soil, D.R., G. Bedell, J. Thiel & M. Brummel <1981). The dependency oi nuclear division on volume in the dimorphic yeast Candida  albicans. 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Bacter i ol. 138:92-98. 122 Worth ington, D.H., M. Salamone ic D.S. Nachtwey (1976). Nucleocytoplasmic ratio requirments -for the initiation of DNA replication and fission in Tetrahymena. Cell Tiss. Kinet. 9:119-130. Yanishevsky, R.M. ic G.H. Stein (1981). Regulation of the cell cycle in eukaryotic cells. Int. Rev. Cytol . <S9:223-259. Yeas, M., M. Sugita ic A. Clarkson (1975). A model of cell size regulation. J. Theor. Biol. 9:444-476. Zalkinder, V. (1979a). Correlation between cell nutrition, cell size and division control I. Biosystems 11:295-308. Zalkinder, V. (1979b). Correlation between cell nutrition, cell size and division control II. Biosystems 11:309-322. Zetterberg, A. & W. Engstrom (1983). Induction of DNA synthesis and mitosis in the absence of cellular enlargement. Exp. Cell Res. 144:199-207. Zeuthen, E. (1974). A Molecular Model for Repetitive and Free-running Synchrony in Tetrahymena and Schizosaccharomyces. In: Cel 1 Cycle Controls. G.M. Padilla, I.L. Cameron fit A. Zimmerman, eds. pp.1-30. Academic Press, New York. Zeuthen, E. ic H.E. Williams (1969). Division-1imiting Morphogenetic Processes in Tetrahymena. In: Nucleic Acid Metabolism. Cel1  Different i at ion and Cancer Growth. E.V. Cowdry and S. Seno eds. Academic Press, New York. 123 APPENDIX I. Materials and Methods 124 Stocks & Culture Paramecium tetraurelia (Sonneborn, 1975), was grown in phosphate-buffered Cerophyl medium (Sonneborn, 1970) with Enterobacter  aerooenes as the -food organism. Culture medium was grown up overnight and the pH adjusted to 6.8-6.9 prior to use. Heat treatments were carried out in a water bath equipped with an electronic thermostat which maintained the water bath within 0.02 C o-f the desired temperature. Three mutant stocks, all derived -from the wild-type stock 51-S were used. Stock d4-1001 carries the ts mutation ccl. which completely blocks macronuclear DNA synthesis and cell division at 34.4 C (Peterson & Berger, 1976). Stock d4-1003 carries the ts mutation cc2, which completely blocks cell division but only partially blocks macronuclear DNA synthesis at 34.25 C (Peterson & Berger, 1976; Rasmussen & Berger, 1982). Stock d4-1031 carries the recessive mutation tamG. which causes missegregation and unequal division of the macronucleus at amitosis. Synchronization of cells Synchronous samples of cells were obtained by hand-selection of dividing cells from exponential growth-phase cultures. For each sample, cells were collected for a period of 5 minutes or less and 40-50 dividing cells collected per sample. 125 Cytolooical Procedures Cells were placed by mi cropipette on albumin-coated slides, and most o-f the culture medium removed to ensure maximal -flattening o-f cells as they dried. Cells were -fixed in ethanol/gl ac i al acetic acid (3:1) -for 30 minutes, and stained by the two-color method described by Cornel isse & Ploem (1976) using the -following procedure: 1) RNA was removed by hydrolysis in 5N HC1 at room temperature -for 45 minutes. 2) Cells were stained in an Acri-flavine solution -for 30 minutes. The solution is o-f the -following composition: 0.05 gm Acr i-f 1 av ine-HCl 2.0 gm Sodium metabi sul-f i te 30 ml IN HC1 170 ml disti11ed water 3) Unbound stain was removed by rinsing cells -for 1 minute in an acid-ethanol rinse o-f the -following composition. 70 ml 95% Ethanol 62 ml Disti11ed water 68 ml IN HC1 4) Cells were then stained in a 0.10 mM solution o-f Primulin -for 20 minutes. Mi cro-f 1 uor imetry Micro-f luor imetry was used to estimate DNA and protein content in single cells. The microscope was equipped with an incident light 126 illuminator. For measurement of fluoresence of Acrif1avine-stained DMA, a 490 nm low-pass excitation filter, 500 nm dichroic reflector and 520 nm barrier filter were used. For measurement of Primulin-stained protein, a UG3 excitation filter, 400 nm dichroic reflector and 430 nm barrier filter were used. Each fluorescence measurment was obtained by taking the difference between the intensity of the field with the specimen and one over an adjacent area without the specimen. The output from the photomultiplier was monitored, by a computer (Berger, 1979). The measurement sequence was initiated at a fixed interval (0.1 sec) after the excitating radiation was switched on, and consisted of a series of 32 digitizations of the output from the photomultiplier, averaged to give an intensity reading. Label lino & Autoradiography Label 1ing with tritiated thymidine-1abel1ed bacteria, and autoradiography have been described previously (Berger, 1971). Statistical Procedures Statistical procedures were performed as described in Sokal & Rohlf (1969). 127 APPENDIX I I . Paramec i urn Ce11 Cyc1e Mode 1 128 A simple BASIC computer simulation o-f the Paramec i urn cell cycle was developed to incorporate the experimental observations to date. In this model, both the rate o-f growth and DNA synthesis are a -function o-f the protein synthesis rate. Protein synthesis is in turn limited by either cell mass or gene dosage, depending on the relative amount o-f theses two variables. DNA synthesis initiation is controlled by an unstable inhibitor <DECF), which decays at a rate independent o-f its concentration. The initial level o-f inhibitor is set at 1.00. DNA synthesis begins when the inhibitor decays to 0.85 o-f the starting amount. The rate o-f decay <DECR), is set so that the inhibitor decays by one-hal-f during the course o-f a normal cell cycle. Nuclear division is triggered when the scheduled amount o-f DNA is made. This is an assumption, and does not account -for the observation that DNA synthesis is not required after 0.72 of the cell cycle. In the absence of a demonstrable correlation between mass or DNA content and the triggering of the cell division sequence, it is difficult to use either variable as a component in causing cell division to begin. The value for the decay rate of protein synthesis activity (PDEC) was determined by Berger (1982a). Each pass through the program loop equals \V. of a normal cell cycle. The rate of increase in DNA content (DRATE) is set to produce a doubling in amount during the course of a normal S phase (from 0.25 to 0.87). The rates of increase in protein synthesis activity (PRATE) and cell mass (MRATE) were set so that each exactly doubles during a normal duration cell cycle. The input values 129 are expressed as a percentage o-f normal initial values. The output values are expressed as a percentage o-f initial values at the start o-f the subsequent cell cycle. The e-f-fect o-f changes in initial DNA content or cell mass were tested by altering initial values. There were no other changes made during the simulations. 130 ProQrarn Cell Cycle Simulation BASIC Program 1000 REM *** Constants & control variables *** X=0:ZZ=0:Z=0 1001 PDEC=.89o63:PRATE=.1125:DRATE=.01145:MRATE=.007345 DECF=1.:DECR=.9930925 REM *** Starting conditions *** 2100 PRINT CHR$<12) PRINT "Paramecium Cell Cycle Model" PRINT " ":PRINT 2000 INPUT "Initial DNA content ";D INPUT "Initial synthesis activity ";P INPUT "Initial cell mass " ;M PRINT INPUT "* oi cell cycles ";Z2 DECF=DECF/<M/100) REM *** The cell cycle begins *** INPUT "For continual output ENTER 1 ";X:G0T0 2001 X=0 2001 T=l:N=0 PRINT CHR*<12> DI=D PI=P DST0P=D+P 131 REM *** Final DNA content is set *** 3000 N=N+1 IF M>D THEN RLM=D ELSE RLV=M REM *** Rate limiting variable determined *** P=P*PDEC+ (RLv"*PRATE) REM *** Protein synthesizing system grows *** IF DECF<.85 THEN D=D+<P*DRATE) REM *** DNA synthesis if critical size is reached *** DECF=DECF*DECR REM *** Unstable Inhibitor Decays 1500 M=M+(P*MRATE) REM *** Cell Growth *** IF XO0 THEN PRINT N,D,M,DECF IF D<DST0P THEN GOTO 3000 ND FOR 1=1 TO 13 M=M+<P*MRATE> P=P*PDEC+ < RLV*PRATE) N=N+1 DECF=DECF*DECR IF M>D THEN RLV=D IF D>M THEN RLV=M IF XO0 THEN PRINT N,D,M,DECF NEXT I PRINT 2=2+1 REM *** The cell divides *** DECF=DECF*RLvY100 M=M/2:D=D/2:P=P/2 132 PRINT CHR$<12) PRINT :PRINT PRINT "Cell cycle # ";Z PRINT " " :PRINT PRINT "Cell Cycle length ";N PRINT "DNA Content ";D PRINT "Cell Mass " ;M PRINT "Synthesis System ";P PRINT :PRINT IF Z<ZZ THEN INPUT "Press <RETURN) to continue...0;A IF Z<ZZ THEN GOTO 2001 PRINT "DONE" 9??? END 133 

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