Synchronized meiosis and recombination in fission yeast - Springer Link

Current Genetics

Curr Genet (1991) 19:445-451

9 Springer-Verlag 1991

Synchronized meiosis and recombination in fission yeast: observations with patl-ll4 diploid cells Jiirg Biihler

1,

Peter Schuehert

2,

Christian Grimm 3, and Jiirg Kohli 1

1 Institute of General Microbiology,University of Berne, Baltzer-Strasse4, CH-3012 Bern, Switzerland 2 Institut f/Jr Zoologic, Rheinsprung9, CH-4051 Basel, Switzerland 3 ZoologischesInstitut, Abteilung Entwicklungsbiologie,Baltzer-Strasse 4, CH-3012 Bern, Switzerland Received February 11, 1991

Summary. The mutation patl-II4 has been used to synchronize meiosis in the fission yeast Schizosaccharomyces pombe. We have investigated several aspects of such synchronized meiotic cultures. In both patl-ll4 and patl + diploids, meiotic landmark events are initiated at the same time after meiosis induction, but synchrony is much more pronounced in the patl-ll4-driven meiosis. Commitment to recombination and to meiosis have been timed at 2 h after meiotic induction. Due to a seven-fold reduction of intragenic recombination frequency in the ade6 region ofpatl-ll4 diploids, physical analysis of recombination has not been possible. We have distinguished three factors that inflhence intragenic recombination frequencies: temperature, azygotic versus zygotic meiosis, and the nature of the patl allele. Differences and similarities in the timing of meiotic landmarks in S. cerevisiae and S. pombe are discussed. Key words: Synchronized meiosis = Schizosaccharomyces pombe - Meiotic recombination - p a t l - l l 4

Introduction Sexual reproduction in eukaryotes is dependent on meiosis. Recombination is highly increased and specifically regulated during this process. In the haploid unicellular ascomycete Schizosaccharomyces pombe, zygotes result from fusion of two cells of different mating-type. The zygotes undergo meiosis immediately to form four haploid spores. This sexual differentiation is induced by nutritional starvation (particularly of nitrogen, for a review see Egel 1989). Diploid cells can be obtained by transfer of zygotes from nitrogen-free medium to growth medium before commitment to meiosis (Egel 1973). In order to define the time and duration of various meiotic events (S-phase, prophase, meiosis I and II), to

Offprint requests to: J. B/ihler

search for recombination intermediates, and for biochemical studies on meiotic processes, a synchronized meiosis is helpful. Meiosis can be induced in S. pombe by shifting a diploid strain, heterozygous for mating-type, from growth medium to sporulation medium (Egel and Egel-Mitani 1974). However, synchrony of meiosis in this system is poor. Highly synchronized meiosis can be obtained in haploid strains containing the temperature-sensitive mutation patI-i14 (Iino and Yamamoto 1985a; Nurse 1985; P. Szankasi, personal communication). The pati gene encodes a protein kinase that plays a central regulatory role in a cascade leading to the initiation of meiosis. Inactivation of the pati protein leads to activation of the mei2 gene which is necessary for meiosis (for a review see Egel 1989). Inpati-114 mutants a rapid and well-synchronized meiosis can be induced by shifting the cells from the permissive (25 ~ to the restrictive temperature (32 34 ~ Sexual differentiation then becomes independent of mating-type and nutritional starvation. In the present study several features of the patl-114-induced meiosis have been analyzed in diploid cells and compared with a starvation-induced meiosis. Recent developments in molecular biology have rendered it possible to create well-defined genetic intervals in which recombination can be followed directly, by isolation of DNA from cells undergoing recombination (see Haber et al. 1988 for a review). A system with polymorphic restriction sites has allowed the detection of physically recombined DNA I to 2 h after commitment to intragenic recombination in S. cerevisiae (Borts et al. 1984). Recombination intermediates have also been characterized. Specific double-strand breaks were detected at the time of recombination in budding yeast (Sun et al. 1989; Game et al. 1989; Cao et a1.~1990). We have attempted the physical detection of a recombination product at the ade6 locus during meiosis and the identification of recombination intermediates, especially DNA breaks. However, this turned out to be impossible due to decreased recombination frequencies in the patl-ll4-induced meiosis. We then analyzed three factors contributing to the decrease of recombination frequency. In addition, an ex-

446

Table 1. Strains used in this study Strain

Genotype

Source

PS5 GP179 JB1 JB2 JB3 JB4 JB5 JB6

h +/h- ade6-M26/+ +/ura4-294 h-/h- argl-2/ + ade6-M26/ade6-M210 ura4-294/ + patl-ll4/pat-l l4 h + ade6-M26 leul-32 h + ade6-M26 leul-32 patl-ll4 h- ade6-469(2RIpm) ura4-D18 h- ade6-469(2RIpm) ura4-D18 patl-ll4 h- /h- ade6-469(2RIpm)/ade6-M26 ura4-DlS/ + + /leul-32 patl-ll4/patl-ll4 h +/h- ade6-469(2RIpm)/ade6-M26 ura4-D18/+ +/leul-32

This study A. S. Ponticelli This study This study This study This study This study This study

p e r i m e n t was c a r r i e d o u t to d e t e r m i n e the time o f c o m m i t m e n t to r e c o m b i n a t i o n in S. p o m b e meiosis.

ed on YEA (determination of viable spores) as well as on YEA supplemented with 200 mg/l guanine (selection for adenine prototrophs: Cummins and Mitchison 1967; Ponticelli and Smith 1989).

Materials and methods

Southern analysis. Genomic DNA was prepared according to Wright et al. (1986). Zymolyase 100T (1 mg/ml) was used for DNA extraction from meiotic cells to reduce the time of incubation (10 rain, 37~ Denaturing gels and Southern analysis were performed as described by Maniatis et aL (1982). For nick translations 100 gCi[e-32p]dCTP and 50-100 ng plasmid-DNA (probe indicated in Fig. 2, integrated into pUC8) were used to obtain specific activities of 108 cpm/p.g DNA. Some films were strongly overexposed for detection of weakly hybridizing fragments.

Strains and media. The genotypes of the strains used in this study are indicated in Table 1. The different alleles of ade6 are described by Szankasi et al. (1988). PS5 was constructed according to Flores da Cunha (1970) freshly for every experiment. JB3 was derived from GC4 (Grimm 1990) which contained an artificially inserted EcoRI site near the 5'-end of ade6. A further EcoRI site, lying 2.3 kb upstream of the inserted site, was destroyed on appropriate plasraids by filling the cleaved ends with the Klenow fragment of DNA polymerase I and subsequent religation of the blunt ends. Using the method of gene disruption and replacement described by Grimm et al. (1988), the destroyed restriction site was introduced into the genome of GC4, yielding JB3 (2RIpm: polymorphism of two EcoRI sites, see Fig. 2). JB5 and JB6 were constructed from the parental strains JB2/JB4 and JBI/JB3, respectively, according to Gutz et al. (1974). JB5 was then obtained by screening for mitotic segregants that have become homozygous for mating-type (h-/h-). Standard genetic methods and media were described by Gutz et al. (1974). The following abbreviations were used: MML and MMA for liquid and solid minimal medium, YEL and YEA for liquid and solid yeast extract medium and MEA for solid malt extract medium, respectively. Media for ade-strains were supplemented with 80rag/1 adenine. Meios& induction. The patl + diploids were induced to sporulate as described by Egel and Egel-Mitani (1974). The patl-ll4 dipIoids were kept on MMA at 22 ~ and transferred onto fresh plates every 2 weeks. Fresh single colonies were grown in 10 ml MML at 25 ~ for 1 day. 2 ml of this preculture was used to inoculate 200 ml MML (or YEL). When a cell titer of 2 - 4 x 10 6 cells/ml (in MML) or 4 - 7 x 106 cells/ml (in YEL) was reached, the temperature was shifted to 34 ~ (or 32 ~ for some experiments). In order to shift all cells as rapidly as possible, the flask was shaken and rinsed with 40-50 ~ water for 1 min before transferring it to the shaking water bath at the restrictive temperature. If not stated otherwise, patl-ll4-induced sporulation was carried out in MML at 34 ~ Since even small changes of parameters influence sporulation, care was taken to maintain identical conditions for all experiments. Nuclear staining with DAPI. Cell suspensions were centrifuged briefly, fixed with an equal volume of 70% ethanol and stored at 4 ~ 100 gl of cells were then added to 1 ml of water, centrifuged, and resuspended in a solution of DAPI (4',6-diamidino-2-phenylindole, Boehringer Mannheim, FRG) at 2 gg/ml (40 x lens) or 1020 gg/ml (100 x lens). Fluorescence was observed in an epi-fluorescence microscope. Determination of meiotic recombination frequencies. Mature asci were treated with snail enzyme (Industrie Biologique Frangaise) to obtain pure spore suspensions. Spore titers were determined in a counting chamber. After appropriate dilutions the spores were plat-

Commitment to recombination. A synchronized meiosis was induced as described above. Half of the culture in 200 ml MML was kept growing mitotically (25 ~ Samples of 1 ml from mitotic and meiotic cells were taken at different times, centrifuged, and used to inoculate 10 ml MML. A first enrichment for adenine prototrophs lasted 5 days at 25 ~ After that time there remained many adeninedependent cells which had been able to divide for a few mitotic cycles. Therefore, 0.5 ml of these precultures were used to inoculate again 10 ml of fresh MML. After a further 2 days these cultures had reached stationary phase and genomic DNA was extracted from them, followed by Southern analysis.

Results Timing o f meiotic events and observations with p a t l - l l 4 diploids T h e kinetics o f f o u r m e i o t i c events ( c o m m i t m e n t to m e i o sis, meiosis I, meiosis II, a n d f o r m a t i o n o f f o u r - s p o r e d asci) were d e t e r m i n e d in d i p l o i d s h o m o z y g o u s for pat1114 (Fig. 1). Cells g r o w i n g at 25 ~ were shifted to 34 ~ a n d a l i q u o t s were t h e n t a k e n at r e g u l a r intervals for o b s e r v a t i o n o f the events. T h e results are c o m p a r e d w i t h t h o s e o b t a i n e d w i t h a d i p l o i d h o m o z y g o u s for pat1 § (shift to s p o r u l a t i o n m e d i u m at 30 ~ O l s o n et al. 1978 a n d o w n results). C o m m i t m e n t to meiosis was i n v e s t i g a t e d b y shifting a l i q u o t s o f s t r a i n JB 5 ( h o m o z y g o u s for patt-114) b a c k to the permissive t e m p e r a t u r e (25 ~ at different times after the m e i o s i s - i n d u c i n g shift to 34 ~ Twelve h o u r s later the relative a m o u n t s o f asci were d e t e r m i n e d in a c o u n t i n g c h a m b e r . T h e first cells b e c a m e c o m m i t t e d to meiosis at 2 h after the t e m p e r a t u r e rise. F o u r h o u r s after the initial shift to 34 ~ 9 5 % o f all cells were c o m m i t t e d to meiosis (Fig. 1). I n a n o t h e r e x p e r i m e n t ( d a t a n o t shown), d i l u t e d cell s u s p e n s i o n s o f JB5 t a k e n at h o u r l y intervals after the

447

100%

Bo .

y

60% 40%

20%

0~1 o

T'~

3

4

#~-

s 7 8 hours offer temperature shift

9

10

Fig. 1. Timecourse of meiosis and sporulation ofpatl-lI4 diploid strains. (=) Commitmentto meiosisof JB5 shiftedto 34~ (A) Two ore more nucleivisible(terminationof meiosis I). (.) Three or more nuclei visible (termination of meiosis II). (e) Spore formation as visualized by light microscopy. Meiosis I + lI and spore formation were determined with strain GP179 shifted to 32~ upshift were plated on YEA + phloxinB to determine the ploidy of the growing colonies (Kohli et al. 1977). The first haploid colonies (identified as lightly staining) were observed at 2 h indicating again that this is the time at which the first cells become committed to and will complete meiosis, even at 25 ~ This experiment also showed that the viability of JB5 cells at 34~ decreases after commitment to meiosis. The titer of living cells approximately doubled within the first 2 h after the upshift (from 2.5 x 106 to 4.8 x 106 cells/ml), but decreased afterwards to about 1 x 106 cells/ml (from 3-8 h after shift). Finally the titer increased again to 2 x 106 cells/ml at 26 h after the shift. Thus, many cells (about 80%) die upon entry into a patl-lI4 driven meiosis at 34 ~ Cell viability remained higher when the temperature was shifted back to 25 ~ after commitment to meiosis (4 h after upshift), or when meiosis was carried out at 32 ~ instead of 34~ Yet synchrony was more pronounced at 34 ~ Meiosis at temperatures higher than 35 ~ did not yield any viable spores (data not shown). Cell titers also influenced the efficiency of meiosis. If titers at the time of the upshift were higher than 7 x 106 cells/ml (in MML) or 1.2 x 10 v cetls/ml (in YEL) there was hardly any sporulation. The late exponential phase of JB5 cells is optimal for the shift to 34~ (see Materials and methods). The influence of cell titer on meiosis shows that inactivation of the patl114 gene product by high temperature is not always sufficient to start immediate sporulation. Nuclear staining with DAPI was performed in a patli14-induced meiosis with the diploid strain GP179 (shift to 32 ~ During the first 3 h after the temperature shift the nuclei showed the special morphology of mitotic cells (Toda et al. 1981). Sectors of the nuclei stained with different intensities, thus giving a half-moon appearance. This mitotic morphology disappeared after 3 h and the nuclei became elongated or even showed the so called ,,horse-tail" appearance (Robinow 1977); the staining became more intense and showed filamentous structures. These changes in nuclear morphology indicate meiotic

prophase I (Robinow 1977; Olson et al. 1978). About 10% of the cells had two nuclei at this time point. This fraction increased rapidly after 5 h (start of meiosis I), reaching 90% within a further 2 h (Fig. 1). Meiosis II started at 5.5 h after the shift and was almost completed in the population after 8 h. Mature asci became visible after 6 h and reached saturation at 9 h after shift. Some asci contained more than four spores (up to eight), probably resulting from cells that had completed mitosis but not cell division ("twin meiosis"). Spores were generally smaller, more heterogenous, and less bright than wildtype spores. Commitment to meiosis of strain PS5 (homozygous forpatl--) was measured by plating appropriate dilutions of the cells onto YEA at different times after the initial shift to sporulation medium. The induction of meiosis was performed as described by Egel and Egel-Mitani (1974). As the diploid strain PS5 contains one ade6-M26 allele, haploidization could be monitored by the appearance of sectored and red colonies on YEA. The first cells being committed to meiosis were detected at 2 h after the shift to liquid sporulation medium. But maximal haploidization was not observed before 10 h. After 6 h the first asci became visible by light microscopy, but the maximal yield was reached only after 14 h (data not shown). Synchronization of meiosis is, therefore, poor with this strain. This difference in synchrony between patl + and pati-114 cells might reflect the simplified and more direct initiation of the sexual pathway in patl-Ii4 strains.

Molecular analysis of recombination during synchronized meiosis The diploid strain JB5 (with two EcoRI restriction site heterologies in the 5'flanking region of the ade6 gene) was constructed for the timing of recombination events during a synchronous meiosis. It is possible with this system to detect conversion at ade6 by the appearance of a novel restriction fragment of 2.3 kb length (Fig. 2). In order to raise the level of meiotic recombination in this region, one of the ade6 alleles carried the M26 mutation. The chromatid carrying M26 almost always acts as a recipient for wild-type information during conversion. As a result more than 90% of aberrant tetrads are of the 3 +: 1 M26 type and they make up about 5% of total tetrads (Gutz 1971). Since the EcoRI site near the Y-end of the gene is co-converted in about 70% of the conversion events at M26 (Grimm 1990), the recombinant 2.3 kb fragment was expected to be generated at a frequency of about 0.7% during meiosis. After the shift of strain JB5 to 34~ genomic DNA was extracted at 0, 1, 2, 2V2, 3, 3 '/2, 4, 4 V2, 5, 6, 8 and 10 h, digested with EcoRI, and then analyzed by Southern blot and hybridization with the probe shown in Fig. 2. The recombinant 2.3 kb band was never detected. The detection limit was below 0.7% of total hybridizing DNA in these experiments as revealed by reconstruction experiments involving serial dilutions of the genomic DNA (data not shown). The recombinant 2.3 kb band was well detectable, however, if the haploid strains JB1 and JB3 (patl +) were crossed at 25~ on

448 < RI /

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RI 1.4kb

ode6

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RI

M26

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5.6 kb

RI

Meiotic intragenic recombination frequencies at ade6 are reduced in p a t l - l l 4 diploids

RI

}

469 1.4 kb

RI

4

probe

Fig. 2. System for detection of meiotic recombination by Southern

analysis in the diploid strain JB5. One chromosome contains the wild-type EcoRI pattern in the ade6 region (top). The other chromosome has one EcoRI site artificially inserted and another EcoRI site destroyed (below). The system is symmetrical, resulting in only two parental fragments of 3.6 and 1.4 kb. Arrow, conversion of this EcoRI site leads to the recombinant 2.3 kb fragment. Its frequency is strongly increased by M26 (see text). The probe used for hybridization is shown below

Meiotic intragenic recombination frequencies (expressed by the frequency of prototrophs per 106 spores) were determined in crosses between ade6-M26 and ade6-469 (Table 2). The haploid strains JB1 and JB3 (patl+), as well as JB2 and JB4 (patl-ll4), were crossed at 25 ~ and 34~ Prototroph frequencies were also determined in azygotic meiosis of the diploid strains JB5 (patl-ll4) and JB6 (patl +) at 25~ and 34~ The frequency of 7 270 prototrophs/106 spores resulting from the cross between the haploidpatl + strains at 25 ~ was used as the control frequency. All other values were lower. The increase of temperature reduces recombination frequency. Azygotic meiosis (JB6) also lowers the recombination frequency in comparison with the mating of haploids (JB1 x JB3) while the pati-114 mutation seems to reduce recombination at 25~ (JB2 x JB4 versus JBI x JB3). At the restrictive temperature patI-114 might reduce recombination still further, as can be inferred from the strong reduction of recombination frequency in JB5 at 34~ (ten-fold on MEA, seven-fold in MML). Alternatively, homozygosity at the mating-type locus ( h - / h - ) , which is the only difference between JB5 and all the other configurations, might further reduce recombination. We found similar reductions of intragenic recombination frequencies between ade6-706 and ade6-469 in pati-114 diploids sporulated at 34 ~ (200 prototrophs/106 spores) versus patl § diploids sporulated at 25 ~ (605 prototrophs/106 spores). Thus, the reduction of intragenic recombination is not restricted to crosses involving ade6-M26.

Timing of commitment to recombination during meios& Fig. 3A, B. Correlation between recombination and formation of the 2.3 kb fragment. A Strong correlation between formation of ade + cells and the 2.3 kb fragment during meiotic recombination. The haploid strains JB1 and JB3 were crossed at 25~ on MEA. The resulting spores were treated with snail enzyme and aliquots of them were grown in YEL + adenine (left) of in M M L (right), respectively. Genomic D N A was extracted and digested with EcoRI. The probe used for hybridization is shown in Fig. 2. B Weak correlation between formation of ade + cells and the 2.3 kb fragment during mitotic recombination. Six independent colonies of JB5 were grown at 25 ~ for 2 days in MML+adenine, inoculated into MML, and after 5 days at 25 ~ inoculated again into fresh M M L and grown to stationary phase at 25 ~ for 7 days. Genomic D N A was digested with EcoRI and probed as shown in Fig. 2. The kind of the events, and their relative time of occurrence, strongly influenced the intensity of the 2.3 kb band. The bands at 1.6 and 2.8 kb are cross hybridizations appearing on some filters depending on hybridization and washing conditions

MEA, and genomic D N A was analyzed from the germinated spores (Fig. 3A). This indicates that recombination frequencies at the ade6 locus are reduced in the patl-ll4driven meiosis. This result may also explain the failure of experiments aimed at the detection of D N A breaks in the ade6 region ofpatl-ll4-driven meiosis in strains GPI79 and JB5 (native and denaturing gels were used). We also failed to find single- or double-strand breaks in the asynchronous meiosis of strain PS5 (data not shown).

The following experiment shows that commitment to meiotic recombination does not occur before commitment to meiosis. Fig. 3A, and additional experiments (data not shown), confirms that the recombinant 2.3 kb fragment is strongly enriched in ade § spores. As discussed above this is due to the marker effect of M26 (Gutz 1971; Grimm 1990). But this marker effect is restricted to meiotic recombination (Ponticelli et al. 1988). Therefore, mitotic recombination might result in a less pronounced correlation of adenine prototrophy with the recombinant 2.3 kb fragment. That this is the case is shown in Fig. 3B. Six independent mitotic cultures of JB5 were subjected to selection for adenine prototrophy in MML. D N A from the resulting populations of ade + cells was checked for the presence of the 2.3 kb fragment which was prominent only in two of the six clones. The great majority of ade + recombinants in the other clones are derived from events that (unlike meiotic recombination) do not produce the 2.3 kb fragment. The mitotic recombination frequencies are low. We measured about ten prototrophs/106 diploid patl § cells of constitution M26/469. After the shift of JB5 to 34~ a few percent of cells committed to meiotic recombination will, therefore, be enough to result in an increase of the 2.3 kb band over the mitotic background (except for the few JB5 clones

449 Table 2. Meiotic intragenic recombination frequencies between ade6-M26 and ade6-469 Temperature

25 ~ 34~

Strain JBI x JB3 (n patl +)

JB2 x JB4 (n patl-ll4)

JB6 (2n patI +)

JB5 (2n patl-ll4)

JB5 (MML) (2n patl-ll4)

7 270 (3) 4 200 (2)

5 630 (2) -

5 415 (4) 3 870 (2)

620 (2)

1 010 (7)

The values indicate prototrophs x 10 .6 spores and are the mean of independent measurements given in parentheses, n, crosses; 2n, azygotic meiosis. All experiments were performed on MEA except for JB5 which (as in the meiotic experiments) was also sporulated in MML. Recombination frequency for JB2 x JB4 at 34 ~ could not

be measured because these cells enter a haploid meiosis at this temperature rather than mating. The frequencies of JB5 at 25 ~ were not determined because this strain (h-/h-) sporulates only at the resctrictive temperature

The same kind of experiment was performed twice with JB6 (homozygous for patl +) sporulated in N-free medium at 300C. In both cases there was an evident increase in the intensity of the 2.3 kb band at 2 h after the medium shift (data not shown). Thus, c o m m i t m e n t to meiotic recombination does not occur before commitment to meiosis in patl + diploids as well.

Discussion

Fig. 4. Commitment to recombination in pati-114-driven meiosis. Until 1.5 h after the temperature shift only the mitotic background of the 2.3 kb band is visible. Due to meiotic recombination there is an increase of the 2.3 kb band (and of the 1.4 kb band, see Fig. 2) at 2 h after the shift. The 3.6 kb band does not become weaker after commitment to recombination since the relative decrease of that band is much smaller compared with the relative increase of the 2.3 kb band

that experienced an early mitotic event yielding the 2.3 kb fragment). Therefore, aliquots of cells were taken at different times after the shift of JB5 to 34 ~ and used to inoculate fresh M M L lacking adenine followed by incubation at 25 ~ Genomic D N A was then prepared, digested with EcoRI, and analyzed by Southern hybridization. This experiment was repeated ten times. In five experiments c o m m i t m e n t to recombination occurred 2 h after the shift. Figure 4 shows one of these experiments with an increase of the 2.3 kb band over the mitotic background at 2 h. In two cases c o m m i t m e n t occurred 3 to 4 h after the shift. In these latter experiments meiosis was delayed, as judged microscopically by the appearance of visible spores. C o m m i t m e n t to recombination could not be timed in the remaining three experiments because the 2.3 kb band of mitotic origin was too strong. As a control, aliquots from parallel cultures continually grown at 25 ~ were investigated in the same way. N o enrichment of the 2.3 kb band was observed in these mitotic cells (data not shown).

This project has involved the study of meiosis and recombination induced by a temperature shift in diploid cells homozygous for patl-ll4. The m a j o r result is the high synchrony of meiotic events in comparison with the azygotic meiosis ofpatl + and matings of haploid strains (Egel and Egel-Mitani 1974; Olson et al. 1978; Beach et al. 1985; this work). U n d e r optimal conditions it takes only 2 h from the appearance of the first asci to completion of sporulation versus 8 h for the patl + diploid strain PS5. Thus, patl-ll4-driven meiosis m a y be helpful for all studies that are facilitated by a good synchrony of meiosis (e.g., cytology of meiotic prophase and isolation of stage-specific enzymes). Even though there is a clear difference in the degree of synchrony between patl-ll4 and patl + diploids, the earliest occurrence of all meiotic events is identical in populations of the two cell types. This indicates that the basic processes of meiosis are not altered in patl-ll4 diploids. The cell titer nearly doubles during the first 2 h after the shift. Since the majority of exponentially growing S. pombe cells are in the G2 phase (Hayles and Nurse 1989) most cells have to finish the mitotic cycle to reach G1, which for patl-ll4 cells is probably also the only phase from where entry into meiosis is possible (Iino and Y a m a m o t o 1985b). The spread of an event over 2 h in patl-ll4 diploids coincides with the length of the mitotic cycle in late exponential phase after a temperature shift. Better synchrony of meiosis might only be achieved by starting with synchronous mitotic cells (P. Szankasi, personal communication). A shift to 34 ~ is optimal for synchrony of meiosis. The cost of high synchrony is 80% cell death during meiosis. This damage can be prevented to some extent by shifting the cells back to 25 ~ after c o m m i t m e n t to meiosis. N o viable spores are produced after shifts to temperatures above 35 ~

450 R

S. oombe

i

~M

M

s ~ 0

[]

~

9 hours after shift"

i

u

I

,

,

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o

Fig. 5. Comparison of the start points of meiotic events in S. cerevisiae and S. pombe. S. cerevisiae: S-phase, commitment to meiotic

recombination, and to meiosis and spore formation, have been determined by Resnick et al. (1984) in the rapidly and synchronously sporulating strain SK-1. Earlier attempts to determine commitment to recombination and to meiosis are reviewed by Esposito and Klapholz (1981). Commitment to meiotic recombination is not a distinct point, its timing depends on the chromosomal interval investigated. Duration of S-phase, beginning of meiotic prophase I, and first and second meiotic division, have been determined in SK-1 by Williamson et al. (1983). S. pombe." The correlation between commitment to meiosis and premeiotic S-phase has been described by Beach et al. (1985). Egel and Egel-Mitani (1974) timed the beginning of S-phase at 2.5 h after the shift to sporulation medium. Prophase I, both meiotic divisions, as well as spore formation, have been determined by Olson et al. (1978) for a meiosis induced by N-starvation. The same start points (but not the same kinetics) have been observed in this study for a patl-i14-induced meiosis (Fig. 1). The correlation between commitment to meiosis and to recombinatin has been established in this study. R: commitment to recombination; S: premeiotic S-phase; M: commitment to meiosis; o: begin of meiotic prophase I; n: completed first meiotic division; :completed second meiotic division; o: visible spore bodies A summary of the timing of meiotic events in S. cerevisiae and S. pombe is given in Fig. 5. For this comparison, the time points are shown at which the first cells of the population complete a given event. These time points are independent of the degree of synchrony. The relative timing of the events is more constant than the absolute time points after the induction of meiosis, which depend on the specific experimental conditions. The data for S. cerevisiae are obtained from SK-1 and strains derived from it (about 4 h from the start of sporulation to completion, Resnick et al. 1984). With the exception of commitment to meiosis, the timing of events is very similar in the two yeasts. This difference in the timing of commitment to meiosis might reflect the different life cycles of budding and fission yeast. S. cerevisiae prefers diploidy and will, therefore, delay the decision to enter meiosis (haploidization) as long as possible. In S. pombe on the other hand, conjugation and meiosis are tightly co-regulated and zygotes will normally enter meiosis without intervening mitotic cycles. The main decision to differentiate sexually seems to be made by S. pombe before conjugation and not before meiosis. Commitment to recombination and meiosis in budding yeast have been determined by the "return-to-growth" protocol first applied by Sherman and Roman (1963). Cells removed at an appropriate time from meiotic conditions will remain diploid, revert to mitosis, and perform recombination with meiotic frequencies. Commitment to recombination and to meiosis are, therefore, separable processes in S. cerevisiae. Commitment to intragenic recombination occurs coincidently with, or shortly after, initiation of

premeiotic D N A replication (Resnick et al. 1984), whereas commitment to meiosis does not occur before spindle pole body separation after pachytene (for a review see Esposito and Klapholz 1981). In S. pombe commitment to meiosis coincides closely with the onset of premeiotic D N A replication (Beach et al. 1985), much earlier than in S. cerevisiae. Our experiments show that commitment to M26-stimulated recombination at the ade6 gene coincides with a commitment of cells to meiosis. Commitment to recombination might occur early during premeiotic S-phase in fission yeast (as in budding yeast). Alternatively commitment to recombination might just reflect commitment to meiosis in S. pombe and has no physiological significance per se. With our experiment we cannot exclude that commitment to recombination occurs very shortly before commitment to meiosis. The delay of 2 h until the first cells irreversibly enter meiosis in S. pombe might be a consequence of the time it takes for the activation, or inactivation, of gene products necessary to differentiate sexually. The detection and timing of a specific product of recombination at the ade6 locus, and of intermediates of recombination in the synchronous patl-114-driven meiosis, has not been possible. This is explained by the sevenfold reduction of recombination frequency at ade6 in the synchronous meiotic system. We have demonstrated that the expected recombination product does occur at low frequency (Fig. 3A). But it is too low to be detected by the chosen experimental approach in aliquots of cells retrieved from the synchronous meiosis. The same holds forpatl + diploids that recombine at higher frequency but show poor synchrony. Other approaches are, therefore, necessary for the physical study of recombination during meiosis. Three factors have been shown to reduce intragenic recombination inpatl-ll4-driven meiosis: increased temperature, azygotic meiosis (versus mating of haploids), and the patl-ll4 mutation at 25 ~ all individually reduce recombination frequency to 70% of the control (mating of haploid patl + strains at 25 ~ Table 2). It is surprising that zygotic meiosis with haploid patl-ll4 strains shows reduced recombination frequencies compared with patl+ strains. This observation indicates that, even at 25 ~ (permissive temperature), patl-ll4 strains are not completely comparable with wild-type. To explain the strong reduction of recombination in JB5 diploids at 34~ one has to assume that patl-ll4 either has a stronger reducing effect at 34 ~ than at 25 ~ or that the additional homozygosity of mating-type in JB5 contributes to the final reduction, or else that the three factors act synergistically. Intergenic recombination also seems to be reduced. The genetic linkage between lys3 and ural is reduced about three-fold in a patI-ll4-induced meiosis compared to a starvation-induced meiosis (Iino and Yamamoto 1985b). The reduction of recombination frequencies may reflect physiological differences during prophase I caused by high temperature, azygotic meiosis, the patl-ii4 mutation, and homozygosity of mating-type. Entry into sexual development is normally a continuous process depending on the gradual decrease of nutrients and stepwise

451 inactivation o f the patI protein (Egel 1989; Nielsen and Egel 1990). It m a y be that starvation and mating-type heterozygosity are required n o t only to turn off the pati gene p r o d u c t , but have additional tasks in sexual differentiation. Full induction o f the mei2 gene is achieved only by a patI-independent p a t h w a y involving nitrogen starvation (Watanabe et al. 1988). Therefore, inactivation o f the pati protein by temperature shift p r o b a b l y does n o t fully imitate the natural situation.

Acknowledgements. We thank Dr. A. S. Ponticelli for providing the strain GP179 and Dr. P. Szankasi for helpful discussions. This work was supported by the Swiss National Science Foundation.

References Beach D, Rodgers L, Gould J (1985) Curr Genet 10:297-311 Borts RH, Lichten M; Hearn M, Davidow LS, Haber JE (1984) Cold Spring Harbor Syrup Quant Biol 49:67-76 Cao L, Alani E, Kleckner N (1990) Cell 61:1089-1101 Cummins JE, Mitchison JM (1967) Biochim Biophys Acta 136:108-120 Eget R (1973) Mol Gen Genet 121:277-284 Egel R (1989) In: Nasim A, Young P, Johnson BF (eds) Molecular biology of the fission yeast. Academic Press, San Diego, California, pp 31-71 Egel R, Egel-Mitani M (1974) Exp Cell Res 88:127 134 Esposito RE, Klapholz S (1981) In: Strathern JN, Jones EW, Broach JR (eds) The molecular biology of the yeast Saceharomyces cerevisiae(life cycle and inheritance). Cold Spring Harhour Laboratory, Cold Spring Harbor, New York, pp 211-287 Flores da Cunha M (1970) Genet Res 34:306-310 Game JC, Sitney KC, Cook VE, Mortimer RK (1989) Genetics 123:695-713 Grimm C (1990) Molecular analysis of homologous recombination in the ade6 gene of S. pombe. PhD thesis, Berne

Grimm C, Kohli J, Murray J, Maundrell K (1988) Mol Gen Genet 215:81-86 Gutz H (1971) Genetics 69:317-337 Gutz H, Heslot U, Leupold U, Loprieno N (1974) In: King RC (ed) Handbook of genetics, vol 1 Plenum Press, New York, pp 395446 Haber JE, Borts RH, Connolly B, Lichten M, Rudin N, White CI (1988) Progr Nucleic Acid Res Molec Biol 35:209-259 Hayles J, Nurse P (1989) Exp Cell Res 184:273-286 Iino Y, Yamamoto M (1985a) Mol Gen Genet 198:416-421 Iino Y, Yamamoto M (1985b) Proc Natl Acad Sci USA 82:24472451 Kohli J, Hottiger H, Munz P, Strauss A, Thuriaux P (1977) Genetics 87:471-489 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Nielsen O, Egel R (1990) EMBO J 9:1401-1406 Nurse P (1985) Mol Gen Genet 198:497 502 Olson LW, Ed6n U, Egel-Mitani M, Egel R (1978) Hereditas 89:189-199 Ponticelli AS, Smith GR (1989) Genetics 123:45-54 Ponticelli AS, Sena EP, Smith GR (1988) Genetics 119:491-497 Resnick MA, Sugino A, Nitiss J, Chow T (1984) Mol Cell Biol 4:2811-2817 Robinow CF (1977) Genetics 87:491-497 Sherman F, Roman H (1963) Genetics 48:255-261 Sun H, Treco D, Schultes NP, Szostak JW (1989) Nature 338: 8790 Szankasi P, Heycr WD, Schuchert P, Kohli J (1988) J Mol Biol 204:917-925 Toda T, Yamamoto M, Yanagida M (1981) J Cell Sci 52:271 287 Watanabe Y, Iino Y, Furuhata K, Shimoda C, Yamamoto M (1988) EMBO J 7:761-767 Williamson DH, Johnston LH, Fennell DJ, Simchen G (1983) Exp Cell Res 145:209 217 Wright APH, Maundrell K, Shall S (1986) Curr Genet 10:503-508 C o m m u n i c a t e d by K. W o l f