Genetic control of the cell division cycle in yeast

J. Mol. Biol. (1971) 59, 183-194

Genetic Control of the Cell Division Cycle in Yeast II.? Genes Controlling

DNA Replication

LELAND

and its Initiation

H. HARTWELL

Department of Genetics Ulaiversity of Washington, Seattle, Wash. 98105, U.S.A. (Received 30 September 1970, and in revised foma 10 March 1971) Temperature-sensitive mutations occurring in two unlinked complementation groups, c&4 and cdc8, are recessive and result in a defect in DNA replication at the restrictive temperature. Results obtained with synchronous cultures suggest that c&4 functions in the initiation of DNA replication and cdcd functions in the propagation of DNA replication. From the behavior of mutant strains carrying lesions in cdc4, or in cdc8, or in both genes it is concluded that : (1) nuclear division and cell separation in yeast are dependent upon prior DNA replication; (2) a cellular clock controls bud initiation and the running of this clock is independent of the other events in the cycle, DNA replication, nuclear division and cell separation: (3) premature bud initiation is normally prevented as a consequence of the successful initiation of DNA

replication.

1. Introduction Saccharomyces cerevisiae is a yeast that reproduces by budding. A cell of S. cerevisiae begins a new cell cycle by initiating a bud on its surface. The bud grows in size as the cell division cycle progresses; DNA replication occurs while the bud is small, nuclear division when it is about three-fourths the size of the mother cell, and cell separation when the bud is approximately equal in size to that of the mother cell (Fig. 3). Since the size of the bud is correlated with the position of the cell in the cell division cycle, the developmental stage of individual cells is revealed by their morphology. This fact has been exploited in screening temperature-sensitive mutants of yeast by timelapse photomicroscopy, to detect those mutants that are defective in genes that function at specific times in the cell division cycle (Hartwell, Culotti & Reid, 1970). This technique provides two types of information about a mutant. First, it allows an approximate determination of the time in the cell division cycle when the temperature-sensitive gene product completes its function at the permissive temperature, defined as the execution point. Second, it reveals the morphological stage at which cells terminate their development when the temperature-sensitive gene product does not perform its function at the restrictive temperature. Among a number of cell division cycle (cdc) mutants detected in this manner, seven, belonging to two different complementation groups, have proved in subsequent studies to be defective in DNA replication. Genetic studies (in preparation) demonstrate that the two complementation groups represent two unlinked nuclear genes and that all seven of the mutations t Paper I in this series is Hartwell, Culotti & Reid, 1970 183

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L. H. HARTWELL

are recessive. Although these two genes, cdc4 and cdc8, both control the same cellular process, their products exhibit different execution points, and cells defective in one or the other of these products terminate at different abnormal stages of development. It is the purpose of this report to describe these differences and to examine their implications.

2. Materials and Methods (a) Yeast strains and mef&z The mutant strains 198D1 and 314D5 are diploid strains that are homozygous for the temperature-sensitive lesions, c&4-1 and c&b-l, respectively. The diploid strains and the haploid double mutant H104-7-1 were constructed by standard procedures (Mortimer & Hawthorne, 1966). The composition of synthetic media (Hartwell, 1970) and YEPDTAU plates have been described previously (Hartwell, 1967). (b) Monitoring events in synchronowr cu1ture.a Synchronous cultures were obtained by centrifuging cells from an asynchronous culture to equilibrium in RenograGn density-gradients and collecting the cells of greatest or least density (Hartwell, 1970). Nuclear division, bud initiation, cell number, execution and the incorporation of [2J4C]uracil into RNA and DNA were determined by standard procedures (Hartwell, 1967).

3. Results (a) Morp7udogical

development at the restrictive

temperature

The morphological development of cells of strain 198Dl and 314D5 following a shift from the permissive to the restrictive temperature is recorded in Table 1 and summarized in Figure 3. Cells of strain 198Dl with buds smaller than 0.4 that of t,he parent ceil at the time of the temperature shift cease development as a single cell TABLE

1.

Moq~h~logkal development of individual cells of mutant strains 19801 and 31405 following a shift to the restrictive temperature

strain

Bud sizet at time of 36°C shift 0

198Dl

O-o.2 0.2-0.4 04-0~6 0.6-0.8 0.8-1.0

0 314D5

O-6.2 0.2-0.4 0.4-0.6 0.6-0.8 0.8-1.0

Cells that terminato$ TOM lU3: One cell Two ~011s 16 30 60 26 0 1

0 0 1 33 45 43

16 30 51 59 45 44

17 11 21 22 14 4

25 11 21 22 14 4

t Length of bud divided by length of parent cell. 3 Cells of strain 198Dl terminate as one or two cells eaoh with a large bud attached. strain 314D5 terminate as one or two cells each with 1 to 6 elongated buds attaohed.

Cells of

I II. Morphological development of cells of a double mut,ant strain, HI 04.7. ;e temperature. Cells of strain H104.7-l growing asynchronously at the permis WWC? transferred to an ager plate warmwl to t,he restrict~ive twnperat.rwc~ tphv taken at the time of the shift (0) and 9 hr later are shown.

t,h(> em,to-

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with a large bud. Cells with buds larger than 06 that of the parent cell cease development only after forming two cells, each with a large bud. The thermolabile gene product in strain 198Dl apparently completes whatever function is necessary for the cell cycle in progress when the bud is approximately O-5 as large as the parent cell. This time is called the execution poi& for that gene product. All cells of strain 198Dl terminate at the restrictive temperature with a nucleus that remains undivided but is located at the isthmus between parent cell and bud (Plate I(a)). Cells of strain 314D5 that have buds at the time of the temperature shift form two cells at the restrictive temperature, whereas some cells without buds terminate as a single cell (Table 1 and Pig. 3). The execution point for the thermolabile gene product in this mutant strain is therefore early in the cell cycle, before bud initiation. Cells of strain 314D5 continue to undergo bud initiation at the restrictive temperature so that most cells terminate with three to five elongated buds attached to the parent cell. The nucleus, however, remains undivided in these cells (Plate I(b)). (b) Monitoring

cell-cycle events in syachronou.s cultures

Synchronous cultures of mutants 198Dl and 314D5 were examined for cell separation, bud initiation, nuclear division, DNA synthesis, and RNA synthesis, at the permissive and restrictive temperatures ; loss of viability was monitored in t,he culture at the restrictive temperature and the execution point was monitored in the culture at the permissive temperature. The results from a synchronous culture of mutant 198Dl are presented in Figure 1. Due to the timing of the execution point in this mutant, the synchronous culture was begun from the cells of lightest density. At the permissive temperature, cellseparation occurs at 30 minutes (50% of the cells have undergone cell separation by this time), bud initiation at 50 minutes, nuclear division at 155 minutes and the second cell separation by 195 minutes. In previous synchrony experiments with the parent strain, a marked decrease in the rate of DNA synthesis between the synthetic periods of the first and second cycles was observed and allowed a relatively clear delineation of the beginning and end of the first DNA synthetic period (Hartwell, 1970). In these experiments the gap between the two DNA synthetic periods at the permissive temperature is less obvious, although the degree of cell synchrony was quite good. Perhaps this mutant has a prolonged DNA synthetic period at the permissive temperature. We can, however, estimate the approximate limits of the first synthetic period to be from 43 to 110 minutes at 23°C. RNA synthesis is continuous throughout the cell cycle. The execution point was determined by shifting samples from the permissive to the restrictive temperature and following their subsequent development by photomicroscopy as in Table 1. By 108 minutes 50% of the cells have developed past the execution point at the permissive temperature. In the culture of 198Dl shifted to the restrictive temperature at the beginning of the experiment, the cells undergo cell separation at 45 minutes and bud initiation at 70 minutes. The first wave of DNA synthesis does not occur at the restrictive temperature nor does the subsequent nuclear division. Ninety per cent of the cells terminate with nuclei elongated between bud and parent cell as in Plate I(a) and the completion of nuclear division, cell separation, and further bud initiation does not occur. Viability begins to be lost at the restrictive temperature at about 70 minutes. The rate and extent of RNA synthesis are approximately the same at the restrictive and permissive temperatures. DNA synthesis continues at the restrictive temperature at a rate about

186

L. CS BI

EX

ND

CS

H.

HARTWELL 3

23

I

-

DNA’.

f r /

I

,

i loo

200

100 Time

200

(mid

FIG. 1. Monitoring cell cycle events in a synchronous culture of strain 198D1 rtt the permissive (23’C) and restrictive (36°C) temperatures. Approximately 5 x IO9 cells growing in synthetio medium were collected by centrifugation and banded isopycnically in a Renografin density-gradient. Approximately 1 x 10s of the lightest cells were resuspended in 100ml. of synthetic medium containing 1 $Zi [2-**C]urecil/ml. (final specific activity 39 mCi/m-mole) and the culture was divided into two portions, one of which remained at 23°C (left panel) while the other was placed at 36% (right panel). Samples were removed at various times and monitored for the increase in cell number to determine the time of cell separation (CS), for the percentage of the cells that had completed bud initiation (BI), and nuclear division (ND), and for the percentage of the cells with dividing nuclei (DN). DNA synthesis ws,s monitored by the incorporation of radioactivity into DNA, and RNA synthesis by the incorporation of radioactivity into RNA. Samples from the 23°C culture were transferred to agar plates et 36”C, photographed immediately and again several hours later to determine the time of execution (EX). Samples from the 36°C culture were removed, diluted and plated onto agar plates at 23°C to determine the number of viable cells; percentage viable cells is calculated relative to a value of 100% at the beginning of the experiment. Arrows designate the time at which 60% of the cells complete a particular event.

10% of that occurring at the permissive temperature. This slow synthesis of DNA might be due to leakiness of the mutation or to the synthesis of mitochondrial DNA which is known to account for between 5 and 20% of the total DNA of the cell. Although nuclear division did not occur at the restrictive temperature in the culture shifted to 36”C, it is clear from the fact that the execution point considerably precedes nuclear division that cells of mutant 198Dl will undergo one nuclear division at the restrictive

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t

0

100

200

0 Time (min)

100

200

Fro. 2. Monitoring cell cycle events in a synchronous culture of strain 314D5 at the permissive (23°C) and restrictive (3tYC) temperatures. Legend is the same as for Fig. 1 except that the densest cells were selected for the synchrony experiment.

temperature if they have been allowed to complete DNA synthesis at the permissive temperature. These results suggest that the primary defect in mutant 198Dl involves DNA replication and that if DNA replication is permitted at the permissive temperature the other events of the cell cycle, nuclear division, cell separation, and bud initiation, can occur once at the restrictive temperature, The results from a synchronous culture of mutant 314D5 are presented in Figure 2. In this case it was necessary to begin the synchronous culture by selecting cells of greatest density, since the execution point in this mutant is close to the time of minimum density. At the permissive temperature the first event, nuclear division, occurs at 62 minutes, cell separation at 82 minutes, bud initiation at 128 minutes and the second cell separation at 250 minutes. The second nuclear division was not monitored. The first DNA synthetic period occurs from approximately 90 to 150 minutes during the first cyole. The execution point in this mutant is at about 97 minutes. RNA synthesis is continuous throughout the cell cycle. At the restrictive temperature cells of mutant 314D5 undergo nuclear division at

188

L. H.

HARTWELL

37 minutes, cell separation at 60 minutes and bud initiation at 100 minutes. The first wave of DNA synthesis doea not occur at the restrictive temperature. RNA synthesis is aa rapid at 36°C as at 23°C. Cells slowly accumulate in a stage of nuclear division although the maximum percentage with an apparently dividing nucleus reached only 60°% in this mutant. Nuclear division is not completed, nor is further cell separation. However, as pointed out above, in this mutant, bud initiation continues at the restrictive temperature. Some loss of viability occurs at 36°C but less rapidly than in mutant 198Dl. In mutant 314D5 a slow rate of DNA synthesis is apparent

TABLE

Timing

of events in the cell division cycle of rnutant and uon-mutant Strain

A364AD6 314D5 198Dl t The timing separations.

2.

Ever&F DS

CS

BI

0 0 0 0

0.19 0.22 0.27 0.12

of events is recorded

Abbreviations used: CS, cellseparation; DS, DNA synthesis.

0~11-0.41 0.07-0.44 0.05-0.40 0.08-0.48

as the fraotion

ND

CS

EX

0.72 0.81 0.88 0.76

1.0 1.0 1.0 1.0

0.09 0.47

of a cell cycle between

BI, bud initiation;

strains

two successive cell

ND, nuclear division;

EX, execution;

FIG. 3. Properties of mutant strains defeotive in cdc4 or cdcd determined by shifting cells from the permissive to the restrictive temperature (Figs 2 and 3 and Table 2). The morphologioal development of mutant oells defective in genes cdc4 or cdcd following a shift from the permissive to the restrictive temperature is indicated by the arrowa designated 4 and 8 that exit from the normal cycle. Abbreviations: DS, DNA synthesis; BI, bud initiation; ND, nuclear division; CK, oytokinesia; CS, cell separation; max and min p are the times of maximum and minimum cell density.

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at the restrictive temperature, and again this might be accounted for by leakiness of the mutation or by mitochondrial DNA replication. These results demonstrate that mutant 314D5 can undergo nuclear division, cell separation, and bud initiation at the restrictive temperature after prior DNA replication at the permissive temperature but that the mutant is apparently defective in DNA replication. It is noteworthy, however, that the execution point in mutant 314D5 coincides approximately with the beginning of DNA replication rather than with its completion as was the case with mutant 198Dl. This is more easily seen in Table 2 where the events occurring during the cell cycle at the permissive temperature have been recorded as fractions of a cell cycle; data from two previous experiments with a non-temperature-sensitive (ts +) strain have been included for comparison. While there is some question as to the exact beginning and completion of the DNA synthetic period in these mutants, it is quite clear that execution occurs between cell separation and bud initiation for mutant 314D.5, whereas execution occurs between bud initiation and nuclear division for mutant 198Dl. These results suggest that the gene product defective in mutant 198Dl is needed throughout the period of DNA replication whereas the gene product defective in mutant 314D5 is needed only at the beginning of DNA replication. The execution points for the gene products of cdc4 and cdc8 and the morphological states that are attained by mutant cells defective for these gene products following their exit from the normal sequence of cell cycle events are summarized in Figure 3. (c) Temperature shifts during the DNA

synthetic period

A defect in the initiation of DNA replication should be distinguishable from a defect in the propagation of DNA replication by shifting synchronous cultures to the restrictive temperature after DNA replication has been initiated at the permissive temperature but before replication has been completed. A mutant defective in initiation should be capable of completing a round of DNA replication at the restrictive temperature whereas a mutant defective in propagation should not, Even under my conditions of less than perfect synchrony, one would predict that a culture of an initiation mutant should synthesize considerably more DNA at the restrictive temperature than would a culture of a propagation mutant. Synchronous cultures of 198Dl and 314D5 were shifted to the restrictive temperature approximately 30% of the way through the DNA synthetic period and the effect on DNA synthesis was measured. The results of this experiment are presented in Figure 4. Following the temperature shift very little additional DNA synthesis occurs in mutant 198D1, whereas a considerable increase occurs in mutant 314D5. (d) Periodicity of bud initiation restrictive

in mutant 31405 at the temperature

Although both mutants cease DNA replication, nuclear division, and cell separation following a shift to the restrictive temperature, they differ in their subsequent behavior with respect to bud initiation. Mutant 314D5 continues to initiate buds, forming several elongated buds that remain attached to the parent cell, but mutant 198Dl does not. As a first step in characterizing the regulation of bud initiation, it was of interest to determine whether or not the initiation of successive buds in mutant 314D5 at the restrictive temperature was periodic or random in time. An asynchronous culture of 314D5 growing at the permissive temperature was spotted onto an agar

L. H.

190

0

100

HARTWELL

0 Time (min)

100

FIQ. 4. Temperature shift during the DNA synthetic period in synchronized cultures of strain 198Dl and 314DS. Synchronous cultures were prepared by selecting approximately 1 x lo8 of the lightest cells from a Renografin density-gradient. The cells were resuspended in synthetic medium containing 1 pCi [2-14C]uracil/ml. and incubated at 23’C @al specific activity 39 mCi/m-mole). Samples were removed at various times and analyzed for the incorporation of radioactivity into DNA. The arrow marks the time at which a portion of the culture was removed and shifted to 36°C.

plate prewarmed to the restrictive temperature, and individual cells were followed by time-lapse photomicroscopy. Pictures were taken of ten different fields of cells every ten minutes for 520 minutes. Individual cells were then scored for the time of appearance of buds, and the difference in time between two successive bud initiation events was calculated. Since execution in this mutant is before bud initiation, some cells form one morphologically normal bud at the restrictive temperature before they begin forming abnormal elongated buds that do not separate from the parent cell. Consequently the intervals of time from the f?rst normal to the fist abnormal bud, from the birth of a normal bud to the formation of its first abnormal bud, from the first abnormal bud to the second, from the second abnormal bud to the third, and from the third abnormal bud to the fourth were monitored. The number of cells displaying a given interval of time is plotted against the time interval in Figure 5 for each of these classes. It is clear that a periodicity of bud initiation is maintained at, the restrictive temperature. It should be mentioned that the number of cells scored for a particular class is not an accurate indication of the number of cells that undergo that many cycles of bud initiation, since the complexity of the interweaving elongated buds prohibits scoring some cells after the first few buds are formed. This experiment shows that the timing of bud initiation remains periodic at the restrictive temperature for at least four cycles of budding and that the interval of periodicity is roughly constant with most of the cells initiating another bud between 100 and 150 minutes after the time of the previous bud initiation. This period of time is about the

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200

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I

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Time (min)

Fm. 5. Intervals of bud initiation in cells of 314DS incubated at the restrictive temperature. An asynchronously growing culture of strain 314D6 was transferred to an agtar plate prewarmed to 36°C. The plate was maintained at 36°C and photographs were taken of the same field of cells every 10 min. The number of cells displaying a particular interval is plotted against the interval of time between two bud initiation events. (a) Time from first normal bud initiation to Grst abnormal bud initiation; (b) time from birth of a normal bud until it initiates its first abnormal bud; (c) time from first abnormal bud initiation until second abnormal bud initiation; (d) time from seoond abnormal bud initiation until third abnormal bud initiation; (e) time from third abnormal bud initiation until fourth abnormal bud initiation.

same as the doubling time of this mutant at the permissive temperature (150 minutes). Thus it is apparent that a cellular clock controls bud initiation and that this clock continues to run in the absence of DNA replication, nuclear division or cell separation. Cells eventually stop budding at the restrictive temperature and many lyse, for reasons that are not understood. (e) Bud initiation

in a cdc4-cdc8 double mutant

A haploid strain (HlOP7-1) carrying both the cdc4-I and &8-l mutations was obtained as a segregant from a diploid heterozygous for both mutations, and it(s genetic constitution was confirmed by its inability to complement with haploids carrying either lesion alone. A clone of the double mutant was isolated, grown at the permissive temperature, spotted onto an agar plate at the restrictive temperature, and the development of individual cells was followed by time-lapse photomicroscopy. It is clear that some cells in the population form elongated buds characteristic of a mutant containing only the cdc4 lesion ; other cells end up with a single large bud attached to the parent cell characteristic of a mutant containing only the cdc8 lesion (Plate II). Individual cells were measured for the length of the parent cell and the length of the bud along their longest axis at the time of the temperature shift; the ratio, length of bud divided by length of parent cell, has been tabulated for the cells

192

L. H. HARTWELL TABLE 3. Bud initiation

at 36°C in the double mutant strain (H104-7-l) Bud size at time of 36°C shift

Bud initiation

at 36’Ct +

0

0

o-o.2 0.2-0.4 04-0.6 0~6-04

4 14

3 0 0 5 27 28

M-1.0

19 2

5

t A minus indicates that cells do not continue bud initiation (analogous to strain 196Dl), a plus indicetes the cell does continue bud initiation (ancblogous to strain 314D5).

while

that do and those that a0 not continue ha initiation at the restrictive temperature (Table 3). There is a strong correlation showing that most of the cells with small buds do not continue bud initiation, whereas most of the cells with larger buds do continue bud initiation. This result suggests that cells of the double mutant strain that have passed the execution point for cdc4 but not for cdc8 at the time of the temperature shift do not continue bud initiation at the restrictive temperature whereas the rest of the population of cells does. This finding indicates that at the restrictive temperature, the difference in bud initiation between strains 314D5 and 198Dl is a consequence of the different positions in the cell cycle at which they are arrested and is not due to a direct effect of either thermolabile gene product upon bud initiation. 4. Discussion The product of gene cdc4 is apparently required for the initiation of DNA replication, while the product of c&8 is necessary for DNA synthesis throughout the period of replication. In fractions of a cell cycle, the execution points for these gene products are 0.09 and 0.47, respectively, suggesting that 0.38 of the cell division cycle is taken up by the DNA synthetic period. This estimate is slightly longer but in reasonably good agreement with earlier studies by Williamson (1965) employing autoradiography. Temperature-sensitive DNA synthesis mutants analogous to the two types reported here have been described in bacteria (Kohiyama, Lamfrom, Brenner t Jacob, 1963; Menclelson & Gross, 1967; Fangman & Novick, 1968; Hirota, Ryter & Jacob, 1968; Kuempel, 1969 ; Hirota, Mordoh & Jacob, 1970; Karamata & Gross, 1970). A temperature-sensitive mutant of Ustilago naaydis has been described (Unrau & Holliday, 1970) that undergoes an inhibition of DNA synthesis at the restrictive temperature, but the properties of this mutant appear to be analogous to yeast mutants defective in nuclear division (Culotti & Hartwell, manuscript in preparation). The effects of these blocks in DNA replication upon subsequent cellular processes are quite striking and provide some insight into the coordination of the events of the cell division cycle. In the absence of DNA replication, resulting from a block either in the initiation of DNA replication or in its propagation, subsequent nuolear division

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is prevented. Since both of these mutants are capable of nuclear division at the restrictive temperature following the completion of DNA replication at the permissive temperature, this result strongly suggests that DNA replication is a prerequisite for the completion of nuclear division. It is interesting, however, that both mutants terminate with the nucleus elongated between parent cell and bud, a morphology characteristic of a dividing nucleus; this result suggests that processes leading to the initiation of nuclear division are not dependent upon prior DNA replication but that at some later stage the apparatus responsible for nuclear division requires duplicated chromosomes. Neither mutant is capable of cell separation at the restrictive temperature after a block in DNA replication, but both can undergo cell separation at 36°C following the completion of DNA replication at the permissive temperature. Cell separation appears, therefore, to be dependent either upon DNA replication itself, or, more likely, upon the successful completion of nuclear division. Perhaps the most striking observation of these studies was the continued initiation of buds by mutant 314D5 at the restrictive temperature, in contrast with the complete cessation of bud intiation by mutant 198Dl. In the former mutant, bud initiation remained periodic at the restrictive temperature with cells attaining as many as five new buds after the inhibition of DNA replication. It is clear from this result that bud initiation is not dependent upon prior DNA replication, nuclear division, or cell separation. Furthermore, the periodicity of bud initiation displayed by this mutant suggests the presence of a cellular clock that controls bud initiation and continues keeping time independently of DNA replication, nuclear division, or cell separation. The control of bud initiation by a clock that runs independently of the completion of other events in the cell cycle might require additional regulatory processes to ensure coordination of the cycle. Somehow the cell must ensure that this clock does not run ahead or behind the completion of the other cell cycle events, so that the cell initiates a new bud once and only once in each cycle. The finding that cells of a double mutant, defective in both cdc4 and cd&, do or do not continue bud initiation at the restrictive temperature, depending upon their position in the cell division cycle at the time of the temperature shift, provides some insight into the mechanism of this regulation. This observation can be interpreted to indicate that bud initiation by the clock is permitted during the period of the cycle prior to the initiation of DNA replication, but that the successful completion of the initiation of DNA replication prevents further bud initiation by the clock. The nature of the clock is at present a mystery although one possibility presents itself. It has been suggested that the stepwise appearance of various enzymic activities during the yeast cell cycle can be explained by an ordered transcription of the yeast genome (Tauro, Halvorson $ Epstein, 1968). Such ordered transcription could provide the elements of a clock with the beginning and end of the cycle determined by the beginning and end of transcription rounds. Evidence for a cellular clock controlling division in Euglena that can be phased by a light pulse (Jarrett & Edmunds, 1970) and results consistent with the periodic synthesis of an initiator protein of DNA synthesis in E. coli (Rosenberg, Cavalieri & Ungers, 1969) suggest that the presence of a clock controlling the cell cycle may be a general phenomenon. The author thanks Dr Walt Fangman for helpful discussions during the course of this work and for his comments on the manuscript, Dr Herschel Roman for his help with the 18

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manueoript, and Mrs Susan He&y and Miss Mary Hubbard for technical assistance. This work was supported by grant number GB8028 from the National Scienoe Foundation and by U.S. Publio Health Service research grant number GM17709-01 (National Institute of General Medical Sciences). REFERENCES Fangman, W. & Novick, A. (1968). Genetics, 60, 1. Hartwell, L. H. (1967). J. Bact. 93, 1662. Hartwell, L. H. (1970). J. Bad. 104, 1280. Hartwell, L. H., Culotti, J. & Reid, B. (1970). Proc. Nat. Acad. Sci., Wash. 66, 352. Hirota, Y., Mordoh, J. & Jacob, F. (1970). J. MOE. Biol. 53, 369. Hirota, Y., Ryter, A. & Jacob, F. (1968). Cold Spr. Harb. Syrup. Quant. Biol. 33, 677. Jarrett, R. M. & Edmunds, L. N., Jr. (1970). Science, 167, 1730. Keramata, D. & Gross, J. D. (1970). Mol. Gem. Genetics, 108, 277. Kohiyama, M., Lamfrom, H., Brenner, S. & Jacob, F. (1963). C. R. Acad. Sci., Pubis, 265, 1820. Kuempel, P. (1969). J. Bact. 100, 1302. Mendelson, N. t Gross, J. (1967). J. Bact. 94, 1603. Mortimer, R. K. & Hawthorne, D. C. (1966). Genetics, 53, 165. Rosenberg, B. H., Cavalieri, L. F. & Ungers, G. (1969). PYOC. Nat. Acad. Sci., Wash. 63, 1410. Tauro, P., Halvorson, H. 0. & Epstein, R. L. (1968). Proc. Nut. dcud. Sci., Wash. 59, 277.

Unrau, P. & Holliday, R. (1970). Cenet. Res., Camb. 15, 157. Williamson, D. (1965). J. Cell Biol. 25, 517.