Planta
Planta (Berl.) 130, 131 - 136 (1976)
9 by Springer-Verlag 1976
Induction of Meiosis in Saccharomyces cerevisiae at Different Points in the Mitotic Cycle A. F. Croes, H.J. D o d e m o n t , a n d C. S t u m m Departments of Botany and Microbiology, University, Toernooiveld, Nijmegen, Netherlands
S u m m a r y . Saccharomyces cells i n d u c e d to u n d e r g o meiosis when in late G1 or early S-phase, proceed mitotically until a p o i n t between c o m p l e t i o n of the S-phase a n d n u c l e a r division. F r o m that point, the cells start meiotic d e v e l o p m e n t w i t h o u t i n t e r v e n t i o n of a r o u n d of premeiotic D N A replication. Cells i n d u c e d at any other p o i n t in the cell cycle, enter meiosis from G1.
Introduction W h e n yeast cells at the end of the e x p o n e n t i a l growth phase are transferred into a s p o r u l a t i o n m e d i u m , the cells enter meiosis from the Gt-interval of mitosis. This is inferred from the fact that there is one r o u n d of D N A replication in that p r o p o r t i o n of the cells which eventually completes meiosis a n d ascus forma t i o n (Croes, 1966). It is u n c e r t a i n if meiosis can be initiated from outside the Gl-phase. Several a u t h o r s have investigated the b e h a v i o r of cells transferred into s p o r u l a t i o n m e d i u m at different points in the mitotic cycle (Haber a n d H a l v o r s o n , 1972; Sando et al., 1973; T s u b o i a n d Y a n a g i s h i m a , 1974; M i l n e [cited by Hartwell, 1974]). Some evidence has been o b t a i n e d that all b u d d e d cells a n d even cells which are a b o u t to form a b u d at the time of the m e d i u m change, complete mitosis in s p o r u l a t i o n m e d i u m . As the event of b u d emergence coincides with the i n i t i a t i o n of D N A replication (Witliamson, 1965), this would m e a n that meiosis can only be initiated in G t (Hartwell, 1974). We wish to present evidence that yeast cells i n d u c e d to u n d e r g o meiosis in late G 1 or early S are arrested with respect to mitotic d e v e l o p m e n t after c o m p l e t i o n of D N A synthesis. O u r data indicate that these cells enter meiosis from the p o i n t of arrest w i t h o u t interv e n t i o n of a r o u n d of premeiotic D N A replication.
*
Abbreviations: see "Material and Methods"
Material and Methods Saccharomyces cerevisiae Hansen, strain CBS 5525, clone nr. 10 (Croes, 1967) was maintained at 30 ~ on slants of PSP~ medium (see below) solidified with agar. In alI experiments, the yeast was grown in aerated culture at 30 ~ The first presporulation medium (PSP0 was the VHG medium of Tsuboi and Yanagishima (1973) with 2% glucose instead of 5 %. This medium allows cells in the exponential growth phase to sporulate upon transfer into the sporulation medium (SPM). The composition of the SPM has been described earlier (Croes, 1967). The second presporulation medium (PSPII) differed from the PSPI in having a glucose concentration of 0.5 % and in containing 10 gg/ml of chloramphenicol added immediately before use. Yeast cells were inoculated in PSP~ at a density of 5 x 104 cells per ml. The yeast was harvested 14h later when the density was between 3.7 and 4.2 x 107 cells per ml. The cells were spun down for 1 min at 4,000 x g and separated on a 27-34 % Renografin gradient essentially according to Hartwell (1970). The uppermost fraction containing approximately 1.5 % of the cells was removed from the gradient and seeded in PSPn at a density of 2 x 10v cells per ml. The yeast was transferred to SPM after different periods of synchronous growth in PSPII.Cells transferred after x rain in PSPn are referred to as "x-min cells". The time of inoculation in SPM is To, T, is n hours later. As a routine, the frequency of asci was determined at T24.The sporulation percentage somewhat varied with the time of transfer into SPM but it was always in the range of 60-80%. All cell counting was done with an electronic particle counter (Coulter Electronics). The cell nucleus was stained according to Pontefract and Miller (1962)with minor modifications (Croes, 1967). The frequency of cells in different stages of the mitotic cycle was determined in at least 1,000 cells. Incorporation of [2-14C]uracil (New England Nuclear, 58 mCi/ mmol) into the nucleic acids was measured for RNA according to Hartwell (1970) and for DNA according to Simchen et al. (1972). In some experiments, the specific activity of the precursor was reduced by addition of cold uracil.
Results Cell Cycle D a t a o n the mitotic cycle of strain CBS 5525 in P S P n are presented in F i g u r e 1. The S-phase starts after 50 m i n of i n c u b a t i o n a n d is t e r m i n a t e d 35 m i n later.
132
A.F. Croes et al.: Induction of Meiosis in S a c c h a r o m y c e s
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120 150 time(rain) Fig. l. Characteristics of the mitotic cycle of strain CBS 5525. After density centrifugation, cells from the uppermost portion of the gradient were seeded in PSPn supplemented with [2-14C]uracil (spee. act. 3.6 mCi/mmol) at an activity of 0.25 ~tCi/ml. Samples of 0.5 ml were withdrawn at intervals for determination of cell count and of radioactivity in RNA and DNA. ND: nuclear division 0
30
50,000
/// i.f~.
0
/7 ff NO ,g // / // !/ 9 ~ 9 / /
90
Table 1. Frequency distribution of cells in different stages of mitosis in asynchronous culture in PSPH Stage
Frequency (~o)
I II III IV
54 11 8 27
Other features of the mitotic cycle can be traced microscopically after Giemsa staining (Fig. 2). The 4 stages we distinguish are: I. budded or unbudded cell with a single nucleus not located at the isthmus; II. budded cell with a single nucleus located at the isthmus; III. nucleus in division; IV. budded cell with 2 separate nuclei. Stage III is observed in 40 ~o of the cells at 90 min after inoculation in PSPII. This frequency which is high in comparison with the percentage of stage III cells in asynchronous culture (Table 1), is indicative of the degree of synchrony obtained. There is a period of about 5 min between the end of the Sphase and the time of nuclear division. The G2-period may actually last somewhat longer as synchrony is not perfect. Cytologically, the Gz-phase falls within stage II which occupies approximately 10 ~o of the cell cycle as judged from its frequency in asynchronous culture (Table 1). Cytological Observations The behavior of cells transferred at intervals from PSPII into SPM is presented in Figure 3 and Table 2.
In all cell populations the percentages of stage II and stage III cells gradually decrease and fall to virtually zero at T4 and Ts. This indicates that mitotic activity in SPM is restricted to the first few hours and that during this period all cells in stage II and stage III pass to stage IV. The observation that there is little or no increase in cell number except in the 0-min cells (Table 2), shows that apart from the 0-min cells no stage IV cells loose their buds and thus proceed to stage I. The constant percentage of stage I cells in most populations considered in combination with the absence of cell separation indicates that no cells in stage I enter nuclear division in SPM. The behavior of the 80-min cells is a notable exception in this respect. The histograms show that in this population not only the stage II and stage III cells present at To, but also part of the stage I cells eventually accumulate in stage IV. These stage I cells which are in late S-phase at the time of transfer (Fig. 1) are, apparently, committed to complete mitosis in SPM. As no stage I cells in the 60-min population pass to stage IV, these cells are obviously not committed to complete mitosis although a large proportion of them is in early S-phase at the time of transfer to SPM (Fig. 1). This means that mitotic development in these cells is arrested somewhere between the initiation of D N A replication and nuctear division and that they are bound to enter meiosis from the point of arrest. It would appear that all other cells which are in Gx, G z or late S-phase at the time of the medium shift, enter meiosis from G1. However, a category of cells in late G1 follow a different pattern as will be pointed out below.
D N A Synthesis The question arises whether early S-phase cells complete the S-phase after change of the medium and, if so, whether there is an additional round of premeiotic D N A replication in these cells. To obtain an answer to this question and to compare the behavior of early S-phase cells with those in other parts of the cycle we followed the pattern of D N A synthesis in cells transferred to SPM after prelabeling with [2-a~C] uracil added to PSP n. The results are shown in Figure 4. The number of counts in D N A at To is subtracted from all values in the same experiment. The resulting increase in count number is divided by the number of counts in R N A at T3. This is done to correct for differences in cell number and in specific activity in the R N A which is the source of nucleotide precursors needed for premeiotic D N A synthesis (Simchen et al., 1972). The reference point for the determination of cpm in R N A is T3 which is well ahead of the start of premeiotic D N A replication
A. F. Croes et al.: Induction of Meiosis in Saccharomyces
133
Fig. 2. Stages in the mitotic cycle: I. cell with or without bud having a single nucleus not located at the isthmus; II. budded cell with a single nucleus located at the isthmus; III. nuclear division; IV. budded cell with 2 separate nuclei. Arrows mark cells which are typical for the indicated stage. Giemsa staining. Magnification: x 2,150
(Croes, 1966; E s p o s i t o et al., 1969; K a d o w a k i a n d H a l v o r s o n , 1971; Simchen et al., 1972). F i g u r e 4 shows t h a t there is a rise in the rate of D N A synthesis after 5 or 6 h in S P M which is especially p r o m i n e n t in the 0, 20 a n d 40-min cells. The rise m a r k s the onset of
p r e m e i o t i c synthesis. D N A r e p l i c a t i o n d u r i n g the earlier p e r i o d is c o n s i d e r e d to be m i t o t i c (Simchen et al., 1972). The p a t t e r n s of the D N A counts in the 20, 40 a n d 60-min cells show early synthesis to increase a n d
134 80- CeLLCount(%) I ]IIII]K
A. F. Croes et al.: Induction of Meiosis in Saccharomyces
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Fig. 3. Frequency distribution of cells in different stages of the mitotic cycle during the first 5 h in sporulation medium_ Abscissa: Time of preincubation in PSP H. Ordinate: Cell count ( ~ )
synthesis after Ts to decrease correspondingly with time of pre-incubation in PSP]~. This inverse relationship suggests that in the single cell mitotic D N A synthesis in SPM and premeiotic D N A synthesis are mutually exclusive. Mitotic D N A synthesis is at a maximum in the 60-min cells which indicates that
cells transferred in early S-phase complete mitotic DNA synthesis in SPM. The 40-min cells are not in S-phase at the time of the medium shift (Fig. 1) and nevertheless start to synthesize D N A immediately after transfer. This reveals that at least part of these cells are committed to enter the mitotic S-phase
A. F. Croes et al.: Induction of Meiosis in Saccharomyces
135
Discussion
after 4 0 m i n in PSPII. The 40-min cells that are responsible for the early rise in D N A counts are presumably in late Gt, as the 20-min cells do not synthesize D N A mitotically and go through a round of premeiotic D N A replication after T6. A considerable proportion of the cells pre-incubated in PSP n for 80 min or longer are beyond the S-phase when transferred and complete mitosis before entering meiosis (Fig. 3). The ratio of mitotic vs. meiotic D N A replication would be expected to be lower than in the 60-min cells and this has actually been found (Fig. 4). The increase in D N A counts in the 0-min cells during the first hours in SPM is surprising. One possibility is that synthesis is related to cell multiplication which occurs only in these cells (Table 2). A similar unexpected synthesis of D N A in cells seeded in SPM directly after synchronization was observed by Tsuboi and Yanagishima (1974).
An accurate estimate of the initiation point of the mitotic S-phase is of critical importance for the interpretation of our experiments. D N A synthesis in PSP H starts after 50 min of incubation and proceeds during an interval of 35 min. The generation time calculated from the midpoints of the slopes in the growth curve resulting from the 1st and 2 "d synchronous cell separation is 115 min. F r o m this it follows that the S-phase occupies a fraction of 0.30 of the cell cycle. This figure is only 0.05 higher than the value obtained from examinations on single cells in asynchronous culture (Williamson, 1965), the difference amounting to less than 6 min. This means that the cells in PSPzl are well synchronized with respect to the S-phase. Hence, there is little doubt that most of the ceils have started D N A replication and thus are in early S after 60 min in PSPII. It is equally clear that not only these cells but also some of the 40-rain cells, presumably those which are in late G z at the time of transfer, synthesize D N A mitotically in SPM. The low rate of D N A synthesis after T5 in the 60-min population compared with the high rate in the 20-min cells suggests that a specifically premeiotic D N A synthesis is initiated only in cells which are in G t at T5 . In the other cells meiosis ensues after a round of D N A replication which has been initiated as part of a mitotic cycle. The cytological observation that early S-phase cells may enter meiosis without completing the mitotic cycle fits well into this pattern. Earlier reports provide evidence that the switch from mitosis to meiosis can only be made in G~ (Sando et al., 1973; Milne,
Table 2. Increase in cell number in populations transferred into sporulation medium after various periods of growth in PSPn Time of incubation in PS Pii(min)
Increase in cell number (%) during the interval
0 20 40 60 80 100 120
3 5 3 0 5 3 0
18 2 0 0 0 0 0
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Fig. 4. DNA synthesis in cells transferred to SPM after differentperiods of pre-incubation in PSP. in the presence of [2-1*C]uracil. The activity of label in PSPn varied with time of incubation: 20 min, 1.9 gCi/ml; 40 rain, 0.50 gCi/ml; 60, 80, 100, 120 min, 0.25 gCi/ml. When 0-min cells were to be labeled, radioactive uracil was added to PSP~ 1.5 h before harvest of the cells at an activity of 0.07 gCi/ml. Samples of 0.5 ml were taken each hour and processed for counting. Abscissa: Time of incubation in SPM. Ordinate: Increase in DNA; A cpm in DNA: cpm in DNA at Tx minus cpm in DNA at To; cpm in RNA; cpm in RNA at T3
136
1974). In the fission yeast Schizosaccharomyces pombe where there is no measurable Gl-phase in the normal cell cycle, the diploid cells go through a Gl-phase before entering an azygotic meiosis (Egel and EgelMitani, 1974). There is no easy way to reconcile these conflicting results. It seems unlikely that a different behavior with respect to the co-ordination of events in the division cycles can be attributed to differences in strain or other experimental conditions. The similarity of mitotic and meiotic DNA replication has been a point of discussion for some time. A number of genetic factors that control mitotic DNA synthesis, have been found to function also in meiosis (Simchen, 1974). There are strong indications that a round of DNA replication initiated as a premeiotic process may be followed immediately by mitosis (Simchen et al., 1972). The reverse situation has been described in the present study. From these data the picture emerges that meiotic and mitotic DNA are synthesized in essentially the same way. Only the mechanisms of initiation seem to be different (Roth and Lusnak, 1970).
References Croes, A. F.: Duplication of DNA during meiosis in baker's yeast. Exp. Cell Res. 41, 452-454 (1966) Croes, A. F.: Induction of meiosis in yeast. I. Timing of cytological and biochemical events. Ptanta (Berl.) 76, 209-226 (1967) Egel, R., Egel-Mitani, M.: Premeiotic DNA synthesis in fission yeast. Exp. Cell Res. 88, 127-134 (1974)
A.F. Croes et al.: Induction of Meiosis in Saccharomyces Esposito, M. S., Esposito, R. E., Arnaud, M., Halvorson, H. O.: Acetate utilization and macromolecular synthesis during sporulation of yeast. J. Bact. 100, 180-186 (1969) Haber, J. E., Halvorson, H. O.: Cell cycle dependency of sporulation in Saccharomyces cerevisiae. J. Bact. 109, 1027-1033 (1972) Hartwell, L. H.: Periodic density fluctuation during the yeast cell cycle and the selection of synchronous cultures. J. Bact. 104, 1280-1285 (1970) Hartwell, L. H.: Saccharomyces cerevisiae cell cycle. Bact. Rev. 38, 164-198 (1974) Kadowaki, K., Halvorson, H. O.: Appearance of a new species of ribonucleic acid during sporulation in Saccharomyces cerevisiae. J. Bact. 105, 826-830 (1971) Pontefract, R. D., Miller, J. J.: The metabolism of yeast sporulation. IV. Cytological and physiological changes in sporulating cells. Canad. J. Microbiol. 8, 573-584 (1962) Roth, R., Lusnak, K.: DNA synthesis during yeast sporulation: Genetic control of an early developmental event. Science 168, 493-494 (1970) Sando, N., Maeda, M., Endo, T., Oka, R., Hayashibe, M.: Induction of meiosis and sporulation in differently aged cells of Saccharomyces cerevisiae. J. gen. Appl. Microbiol. 19, 359-373 (1973) Simchen, G.: Are mitotic functions required in meiosis? Genetics 76, 745-753 (1974) Simchen, G., Pifion, R., Salts, Y.: Sporulation in Saccharomyces cerevisiae: Premeiotic DNA synthesis, readiness and commitment. Exp. Cell Res. 75, 207-218 (1972) Tsuboi, M., Yanagishima, N.: Effect of cyclic AMP, theophylline and caffeine on the glucose repression of sporulation in Saccharomyces cerevisiae. Arch. Mikrobiol. 93, 1-12 (1973) Tsuboi, M., Yanagishima, N.: Changes in sporulation ability during the vegetative cell cycle in Saccharomyces cerevisiae. Bot. Mag. (Tokyo) 87, 183-193 (1974) Williamson, D. H.: The timing of deoxyribonucleic acid synthesis in the cell cycle of Saccharomyces cerevisiae. J. Cell Biol. 25, 517-528 (1965)
Received 8 January; accepted 20 January 1976
Planta (Berl.) 130, 131 - 136 (1976)
9 by Springer-Verlag 1976
Induction of Meiosis in Saccharomyces cerevisiae at Different Points in the Mitotic Cycle A. F. Croes, H.J. D o d e m o n t , a n d C. S t u m m Departments of Botany and Microbiology, University, Toernooiveld, Nijmegen, Netherlands
S u m m a r y . Saccharomyces cells i n d u c e d to u n d e r g o meiosis when in late G1 or early S-phase, proceed mitotically until a p o i n t between c o m p l e t i o n of the S-phase a n d n u c l e a r division. F r o m that point, the cells start meiotic d e v e l o p m e n t w i t h o u t i n t e r v e n t i o n of a r o u n d of premeiotic D N A replication. Cells i n d u c e d at any other p o i n t in the cell cycle, enter meiosis from G1.
Introduction W h e n yeast cells at the end of the e x p o n e n t i a l growth phase are transferred into a s p o r u l a t i o n m e d i u m , the cells enter meiosis from the Gt-interval of mitosis. This is inferred from the fact that there is one r o u n d of D N A replication in that p r o p o r t i o n of the cells which eventually completes meiosis a n d ascus forma t i o n (Croes, 1966). It is u n c e r t a i n if meiosis can be initiated from outside the Gl-phase. Several a u t h o r s have investigated the b e h a v i o r of cells transferred into s p o r u l a t i o n m e d i u m at different points in the mitotic cycle (Haber a n d H a l v o r s o n , 1972; Sando et al., 1973; T s u b o i a n d Y a n a g i s h i m a , 1974; M i l n e [cited by Hartwell, 1974]). Some evidence has been o b t a i n e d that all b u d d e d cells a n d even cells which are a b o u t to form a b u d at the time of the m e d i u m change, complete mitosis in s p o r u l a t i o n m e d i u m . As the event of b u d emergence coincides with the i n i t i a t i o n of D N A replication (Witliamson, 1965), this would m e a n that meiosis can only be initiated in G t (Hartwell, 1974). We wish to present evidence that yeast cells i n d u c e d to u n d e r g o meiosis in late G 1 or early S are arrested with respect to mitotic d e v e l o p m e n t after c o m p l e t i o n of D N A synthesis. O u r data indicate that these cells enter meiosis from the p o i n t of arrest w i t h o u t interv e n t i o n of a r o u n d of premeiotic D N A replication.
*
Abbreviations: see "Material and Methods"
Material and Methods Saccharomyces cerevisiae Hansen, strain CBS 5525, clone nr. 10 (Croes, 1967) was maintained at 30 ~ on slants of PSP~ medium (see below) solidified with agar. In alI experiments, the yeast was grown in aerated culture at 30 ~ The first presporulation medium (PSP0 was the VHG medium of Tsuboi and Yanagishima (1973) with 2% glucose instead of 5 %. This medium allows cells in the exponential growth phase to sporulate upon transfer into the sporulation medium (SPM). The composition of the SPM has been described earlier (Croes, 1967). The second presporulation medium (PSPII) differed from the PSPI in having a glucose concentration of 0.5 % and in containing 10 gg/ml of chloramphenicol added immediately before use. Yeast cells were inoculated in PSP~ at a density of 5 x 104 cells per ml. The yeast was harvested 14h later when the density was between 3.7 and 4.2 x 107 cells per ml. The cells were spun down for 1 min at 4,000 x g and separated on a 27-34 % Renografin gradient essentially according to Hartwell (1970). The uppermost fraction containing approximately 1.5 % of the cells was removed from the gradient and seeded in PSPn at a density of 2 x 10v cells per ml. The yeast was transferred to SPM after different periods of synchronous growth in PSPII.Cells transferred after x rain in PSPn are referred to as "x-min cells". The time of inoculation in SPM is To, T, is n hours later. As a routine, the frequency of asci was determined at T24.The sporulation percentage somewhat varied with the time of transfer into SPM but it was always in the range of 60-80%. All cell counting was done with an electronic particle counter (Coulter Electronics). The cell nucleus was stained according to Pontefract and Miller (1962)with minor modifications (Croes, 1967). The frequency of cells in different stages of the mitotic cycle was determined in at least 1,000 cells. Incorporation of [2-14C]uracil (New England Nuclear, 58 mCi/ mmol) into the nucleic acids was measured for RNA according to Hartwell (1970) and for DNA according to Simchen et al. (1972). In some experiments, the specific activity of the precursor was reduced by addition of cold uracil.
Results Cell Cycle D a t a o n the mitotic cycle of strain CBS 5525 in P S P n are presented in F i g u r e 1. The S-phase starts after 50 m i n of i n c u b a t i o n a n d is t e r m i n a t e d 35 m i n later.
132
A.F. Croes et al.: Induction of Meiosis in S a c c h a r o m y c e s
CeUs/m[
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cpmin DNA
cpminRNA
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150,000
9 ,/-//d,)' 2000
1000
.- 9
100,000
// ._%7'z ......... l
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9
60
~
120 150 time(rain) Fig. l. Characteristics of the mitotic cycle of strain CBS 5525. After density centrifugation, cells from the uppermost portion of the gradient were seeded in PSPn supplemented with [2-14C]uracil (spee. act. 3.6 mCi/mmol) at an activity of 0.25 ~tCi/ml. Samples of 0.5 ml were withdrawn at intervals for determination of cell count and of radioactivity in RNA and DNA. ND: nuclear division 0
30
50,000
/// i.f~.
0
/7 ff NO ,g // / // !/ 9 ~ 9 / /
90
Table 1. Frequency distribution of cells in different stages of mitosis in asynchronous culture in PSPH Stage
Frequency (~o)
I II III IV
54 11 8 27
Other features of the mitotic cycle can be traced microscopically after Giemsa staining (Fig. 2). The 4 stages we distinguish are: I. budded or unbudded cell with a single nucleus not located at the isthmus; II. budded cell with a single nucleus located at the isthmus; III. nucleus in division; IV. budded cell with 2 separate nuclei. Stage III is observed in 40 ~o of the cells at 90 min after inoculation in PSPII. This frequency which is high in comparison with the percentage of stage III cells in asynchronous culture (Table 1), is indicative of the degree of synchrony obtained. There is a period of about 5 min between the end of the Sphase and the time of nuclear division. The G2-period may actually last somewhat longer as synchrony is not perfect. Cytologically, the Gz-phase falls within stage II which occupies approximately 10 ~o of the cell cycle as judged from its frequency in asynchronous culture (Table 1). Cytological Observations The behavior of cells transferred at intervals from PSPII into SPM is presented in Figure 3 and Table 2.
In all cell populations the percentages of stage II and stage III cells gradually decrease and fall to virtually zero at T4 and Ts. This indicates that mitotic activity in SPM is restricted to the first few hours and that during this period all cells in stage II and stage III pass to stage IV. The observation that there is little or no increase in cell number except in the 0-min cells (Table 2), shows that apart from the 0-min cells no stage IV cells loose their buds and thus proceed to stage I. The constant percentage of stage I cells in most populations considered in combination with the absence of cell separation indicates that no cells in stage I enter nuclear division in SPM. The behavior of the 80-min cells is a notable exception in this respect. The histograms show that in this population not only the stage II and stage III cells present at To, but also part of the stage I cells eventually accumulate in stage IV. These stage I cells which are in late S-phase at the time of transfer (Fig. 1) are, apparently, committed to complete mitosis in SPM. As no stage I cells in the 60-min population pass to stage IV, these cells are obviously not committed to complete mitosis although a large proportion of them is in early S-phase at the time of transfer to SPM (Fig. 1). This means that mitotic development in these cells is arrested somewhere between the initiation of D N A replication and nuctear division and that they are bound to enter meiosis from the point of arrest. It would appear that all other cells which are in Gx, G z or late S-phase at the time of the medium shift, enter meiosis from G1. However, a category of cells in late G1 follow a different pattern as will be pointed out below.
D N A Synthesis The question arises whether early S-phase cells complete the S-phase after change of the medium and, if so, whether there is an additional round of premeiotic D N A replication in these cells. To obtain an answer to this question and to compare the behavior of early S-phase cells with those in other parts of the cycle we followed the pattern of D N A synthesis in cells transferred to SPM after prelabeling with [2-a~C] uracil added to PSP n. The results are shown in Figure 4. The number of counts in D N A at To is subtracted from all values in the same experiment. The resulting increase in count number is divided by the number of counts in R N A at T3. This is done to correct for differences in cell number and in specific activity in the R N A which is the source of nucleotide precursors needed for premeiotic D N A synthesis (Simchen et al., 1972). The reference point for the determination of cpm in R N A is T3 which is well ahead of the start of premeiotic D N A replication
A. F. Croes et al.: Induction of Meiosis in Saccharomyces
133
Fig. 2. Stages in the mitotic cycle: I. cell with or without bud having a single nucleus not located at the isthmus; II. budded cell with a single nucleus located at the isthmus; III. nuclear division; IV. budded cell with 2 separate nuclei. Arrows mark cells which are typical for the indicated stage. Giemsa staining. Magnification: x 2,150
(Croes, 1966; E s p o s i t o et al., 1969; K a d o w a k i a n d H a l v o r s o n , 1971; Simchen et al., 1972). F i g u r e 4 shows t h a t there is a rise in the rate of D N A synthesis after 5 or 6 h in S P M which is especially p r o m i n e n t in the 0, 20 a n d 40-min cells. The rise m a r k s the onset of
p r e m e i o t i c synthesis. D N A r e p l i c a t i o n d u r i n g the earlier p e r i o d is c o n s i d e r e d to be m i t o t i c (Simchen et al., 1972). The p a t t e r n s of the D N A counts in the 20, 40 a n d 60-min cells show early synthesis to increase a n d
134 80- CeLLCount(%) I ]IIII]K
A. F. Croes et al.: Induction of Meiosis in Saccharomyces
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Fig. 3. Frequency distribution of cells in different stages of the mitotic cycle during the first 5 h in sporulation medium_ Abscissa: Time of preincubation in PSP H. Ordinate: Cell count ( ~ )
synthesis after Ts to decrease correspondingly with time of pre-incubation in PSP]~. This inverse relationship suggests that in the single cell mitotic D N A synthesis in SPM and premeiotic D N A synthesis are mutually exclusive. Mitotic D N A synthesis is at a maximum in the 60-min cells which indicates that
cells transferred in early S-phase complete mitotic DNA synthesis in SPM. The 40-min cells are not in S-phase at the time of the medium shift (Fig. 1) and nevertheless start to synthesize D N A immediately after transfer. This reveals that at least part of these cells are committed to enter the mitotic S-phase
A. F. Croes et al.: Induction of Meiosis in Saccharomyces
135
Discussion
after 4 0 m i n in PSPII. The 40-min cells that are responsible for the early rise in D N A counts are presumably in late Gt, as the 20-min cells do not synthesize D N A mitotically and go through a round of premeiotic D N A replication after T6. A considerable proportion of the cells pre-incubated in PSP n for 80 min or longer are beyond the S-phase when transferred and complete mitosis before entering meiosis (Fig. 3). The ratio of mitotic vs. meiotic D N A replication would be expected to be lower than in the 60-min cells and this has actually been found (Fig. 4). The increase in D N A counts in the 0-min cells during the first hours in SPM is surprising. One possibility is that synthesis is related to cell multiplication which occurs only in these cells (Table 2). A similar unexpected synthesis of D N A in cells seeded in SPM directly after synchronization was observed by Tsuboi and Yanagishima (1974).
An accurate estimate of the initiation point of the mitotic S-phase is of critical importance for the interpretation of our experiments. D N A synthesis in PSP H starts after 50 min of incubation and proceeds during an interval of 35 min. The generation time calculated from the midpoints of the slopes in the growth curve resulting from the 1st and 2 "d synchronous cell separation is 115 min. F r o m this it follows that the S-phase occupies a fraction of 0.30 of the cell cycle. This figure is only 0.05 higher than the value obtained from examinations on single cells in asynchronous culture (Williamson, 1965), the difference amounting to less than 6 min. This means that the cells in PSPzl are well synchronized with respect to the S-phase. Hence, there is little doubt that most of the ceils have started D N A replication and thus are in early S after 60 min in PSPII. It is equally clear that not only these cells but also some of the 40-rain cells, presumably those which are in late G z at the time of transfer, synthesize D N A mitotically in SPM. The low rate of D N A synthesis after T5 in the 60-min population compared with the high rate in the 20-min cells suggests that a specifically premeiotic D N A synthesis is initiated only in cells which are in G t at T5 . In the other cells meiosis ensues after a round of D N A replication which has been initiated as part of a mitotic cycle. The cytological observation that early S-phase cells may enter meiosis without completing the mitotic cycle fits well into this pattern. Earlier reports provide evidence that the switch from mitosis to meiosis can only be made in G~ (Sando et al., 1973; Milne,
Table 2. Increase in cell number in populations transferred into sporulation medium after various periods of growth in PSPn Time of incubation in PS Pii(min)
Increase in cell number (%) during the interval
0 20 40 60 80 100 120
3 5 3 0 5 3 0
18 2 0 0 0 0 0
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Fig. 4. DNA synthesis in cells transferred to SPM after differentperiods of pre-incubation in PSP. in the presence of [2-1*C]uracil. The activity of label in PSPn varied with time of incubation: 20 min, 1.9 gCi/ml; 40 rain, 0.50 gCi/ml; 60, 80, 100, 120 min, 0.25 gCi/ml. When 0-min cells were to be labeled, radioactive uracil was added to PSP~ 1.5 h before harvest of the cells at an activity of 0.07 gCi/ml. Samples of 0.5 ml were taken each hour and processed for counting. Abscissa: Time of incubation in SPM. Ordinate: Increase in DNA; A cpm in DNA: cpm in DNA at Tx minus cpm in DNA at To; cpm in RNA; cpm in RNA at T3
136
1974). In the fission yeast Schizosaccharomyces pombe where there is no measurable Gl-phase in the normal cell cycle, the diploid cells go through a Gl-phase before entering an azygotic meiosis (Egel and EgelMitani, 1974). There is no easy way to reconcile these conflicting results. It seems unlikely that a different behavior with respect to the co-ordination of events in the division cycles can be attributed to differences in strain or other experimental conditions. The similarity of mitotic and meiotic DNA replication has been a point of discussion for some time. A number of genetic factors that control mitotic DNA synthesis, have been found to function also in meiosis (Simchen, 1974). There are strong indications that a round of DNA replication initiated as a premeiotic process may be followed immediately by mitosis (Simchen et al., 1972). The reverse situation has been described in the present study. From these data the picture emerges that meiotic and mitotic DNA are synthesized in essentially the same way. Only the mechanisms of initiation seem to be different (Roth and Lusnak, 1970).
References Croes, A. F.: Duplication of DNA during meiosis in baker's yeast. Exp. Cell Res. 41, 452-454 (1966) Croes, A. F.: Induction of meiosis in yeast. I. Timing of cytological and biochemical events. Ptanta (Berl.) 76, 209-226 (1967) Egel, R., Egel-Mitani, M.: Premeiotic DNA synthesis in fission yeast. Exp. Cell Res. 88, 127-134 (1974)
A.F. Croes et al.: Induction of Meiosis in Saccharomyces Esposito, M. S., Esposito, R. E., Arnaud, M., Halvorson, H. O.: Acetate utilization and macromolecular synthesis during sporulation of yeast. J. Bact. 100, 180-186 (1969) Haber, J. E., Halvorson, H. O.: Cell cycle dependency of sporulation in Saccharomyces cerevisiae. J. Bact. 109, 1027-1033 (1972) Hartwell, L. H.: Periodic density fluctuation during the yeast cell cycle and the selection of synchronous cultures. J. Bact. 104, 1280-1285 (1970) Hartwell, L. H.: Saccharomyces cerevisiae cell cycle. Bact. Rev. 38, 164-198 (1974) Kadowaki, K., Halvorson, H. O.: Appearance of a new species of ribonucleic acid during sporulation in Saccharomyces cerevisiae. J. Bact. 105, 826-830 (1971) Pontefract, R. D., Miller, J. J.: The metabolism of yeast sporulation. IV. Cytological and physiological changes in sporulating cells. Canad. J. Microbiol. 8, 573-584 (1962) Roth, R., Lusnak, K.: DNA synthesis during yeast sporulation: Genetic control of an early developmental event. Science 168, 493-494 (1970) Sando, N., Maeda, M., Endo, T., Oka, R., Hayashibe, M.: Induction of meiosis and sporulation in differently aged cells of Saccharomyces cerevisiae. J. gen. Appl. Microbiol. 19, 359-373 (1973) Simchen, G.: Are mitotic functions required in meiosis? Genetics 76, 745-753 (1974) Simchen, G., Pifion, R., Salts, Y.: Sporulation in Saccharomyces cerevisiae: Premeiotic DNA synthesis, readiness and commitment. Exp. Cell Res. 75, 207-218 (1972) Tsuboi, M., Yanagishima, N.: Effect of cyclic AMP, theophylline and caffeine on the glucose repression of sporulation in Saccharomyces cerevisiae. Arch. Mikrobiol. 93, 1-12 (1973) Tsuboi, M., Yanagishima, N.: Changes in sporulation ability during the vegetative cell cycle in Saccharomyces cerevisiae. Bot. Mag. (Tokyo) 87, 183-193 (1974) Williamson, D. H.: The timing of deoxyribonucleic acid synthesis in the cell cycle of Saccharomyces cerevisiae. J. Cell Biol. 25, 517-528 (1965)
Received 8 January; accepted 20 January 1976
