Estrogen delays entry of the yeast Saccharomyces cerevisiae into meiosis.

Estrogen has been suggested to influence the cell cycle of haploid yeast cells in the early G1 phase of mitosis, its effect possibly being mediated by control of the level of cAMP (TANAKA, S. et al. (1989). Cell, 57: 675-681). Therefore, we were interested in whether estrogen also affects the meiotic phase of diploid yeast cells. Accordingly, we measured the amounts of adenylate cyclase mRNA and intracellular cAMP, the proportions of dividing cells and 4n cells and the doubling times of diploid yeast cells during the presporulation stage in the presence and absence of estrogen. The amount of adenylate cyclase mRNA was found to decrease rapidly within 24 hours after inoculation of cells onto sporulation-promoting plates (YPA plates). The cAMP level of these cells also decreased rapidly. Mitotic cell division continued for 18 hours after cell inoculation, but about 24 hours after inoculation, the amount of cAMP per cell had decreased to a minimum and the cells began to enter meiosis. By contrast, when the cells were inoculated onto YPA plates in the presence of estrogen, their intracellular cAMP and adenylate cyclase mRNA levels became higher than those in control cultures without estrogen and cell division continued for 24 hours. But after 30 hours their intracellular cAMP level decreased to a minimum and they began to enter meiosis. These results show that estrogen delayed the entry of diploid yeast cells into meiosis on sporulation-promoting plates and suggest that its effect may be mediated by control of the level of cAMP.     

The functional domain of adenylate cyclase associated with entry into meiosis in Saccharomyces cerevisiae.

Diploid yeast cells that carry a part of the CYR1 gene deficient in a region coding for the N-terminal domain of adenylate cyclase were growth arrested and accumulated unbudded cells after inoculation into complete medium or nitrogen-free medium, but produced many cells which had one or more buds after incubation in sporulation medium. The cells incubated in sporulation medium had abnormal spindles which were free from the spindle pole bodies, larger in size, or frequently distributed in cytoplasm. The levels of cyclic AMP in these cells did not decrease to the wild-type level after transfer to the sporulation medium and remained at a constant level. The results suggest that the N-terminal domain of adenylate cyclase is associated with the regulatory function for sporulation. The environmental signals for sporulation may be transferred to the adenylate cyclase system through a factor that negatively interacts with the N-terminal domain of this enzyme.     

Recombinationless meiosis in Saccharomyces cerevisiae.

We have utilized the single equational meiotic division conferred by the spo13-1 mutation of Saccharomyces cerevisiae (S. Klapholtz and R. E. Esposito, Genetics 96:589-611, 1980) as a technique to study the genetic control of meiotic recombination and to analyze the meiotic effects of several radiation-sensitive mutations (rad6-1, rad50-1, and rad52-1) which have been reported to reduce meiotic recombination (Game et al., Genetics 94:51-68, 1980); Prakash et al., Genetics 94:31-50, 1980). The spo13-1 mutation eliminates the meiosis I reductional segregation, but does not significantly affect other meiotic events (including recombination). Because of the unique meiosis it confers, the spo13-1 mutation provides an opportunity to recover viable meiotic products in a Rec- background. In contrast to the single rad50-1 mutant, we found that the double rad50-1 spo13-1 mutant produced viable ascospores after meiosis and sporulation. These spores were nonrecombinant: meiotic crossing-over was reduced at least 150-fold, and no increase in meiotic gene conversion was observed over mitotic background levels. The rad50-1 mutation did not, however, confer a Rec- phenotype in mitosis; rather, it increased both spontaneous crossing-over and gene conversion. The spore inviability conferred by the single rad6-1 and rad52-1 mutations was not eliminated by the presence of the spo13-1 mutation. Thus, only the rad50 gene has been unambiguously identified by analysis of viable meiotic ascospores as a component of the meiotic recombination system.     

The role of Saccharomyces cerevisiae type 2A phosphatase in the actin cytoskeleton and in entry into mitosis.

We have prepared a temperature-sensitive Saccharomyces cerevisiae type 2A phosphatase (PP2A) mutant, pph21-102. At the restrictive temperature, the pph21-102 cells arrested predominantly with small or aberrant buds, and their actin cytoskeleton and chitin deposition were abnormal. The involvement of PP2A in bud growth may be due to the role of PP2A in actin distribution during the cell cycle. Moreover, after a shift to the non-permissive temperature, the pph21-102 cells were blocked in G2 and had low activity of Clb2-Cdc28 kinase. Expression of Clb2 from the S.cerevisiae ADH promoter in pph21-102 cells was able to partially bypass the G2 arrest in the first cell cycle, but was not able to stimulate passage through a second mitosis. These cells had higher total amounts of Clb2-Cdc28 kinase activity, but the Clb2-normalized specific activity was lower in the pph21-102 cells compared with wild-type cells. Unlike wild-type strains, a PP2A-deficient strain was sensitive to the loss of MIH1, which is a homolog of the Schizosaccharomyces pombe mitotic inducer cdc25+. Furthermore, the cdc28F19 mutation cured the synthetic defects of a PP2A-deficient strain containing a deletion of MIH1. These results suggest that PP2A is required during G2 for the activation of Clb-Cdc28 kinase complexes for progression into mitosis.     

The Ubp15 deubiquitinase promotes timely entry into S phase in Saccharomyces cerevisiae

The anaphase-promoting complex in partnership with its activator, Cdh1, is an E3 ubiquitin ligase responsible for targeting cell cycle proteins during G1 phase. In the budding yeast Saccharomyces cerevisiae, Cdh1 associates with the deubiquitinating enzyme Ubp15, but the significance of this interaction is unclear. To better understand the physiological role(s) of Ubp15, we examined cell cycle phenotypes of cells lacking Ubp15. We found that ubp15∆ cells exhibited delayed progression from G1 into S phase and increased sensitivity to the DNA synthesis inhibitor hydroxyurea. Both phenotypes of ubp15∆ cells were rescued by additional copies of the S-phase cyclin gene CLB5. Clb5 is an unstable protein targeted for proteasome-mediated degradation by several pathways. We found that during G1 phase, the APC^Cdh1-mediated degradation of Clb5 was accelerated in ubp15∆ cells. Ubp15 interacted with Clb5 independent of Cdh1 and deubiquitinated Clb5 in a reconstituted system. Thus deubiquitination by Ubp15 counteracts APC activity toward cyclin Clb5 to allow Clb5 accumulation and a timely entry into S phase.     

Competing crossover pathways act during meiosis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae the MSH4-MSH5, MLH1-MLH3, and MUS81-MMS4 complexes act to promote crossing over during meiosis. MSH4-MSH5, but not MUS81-MMS4, promotes crossovers that display interference. A role for MLH1-MLH3 in crossover control is less clear partly because mlh1Delta mutants retain crossover interference yet display a decrease in crossing over that is only slightly less severe than that seen in msh4Delta and msh5Delta mutants. We analyzed the effects of msh5Delta, mlh1Delta, and mms4Delta single, double, and triple mutants on meiotic crossing over at four consecutive genetic intervals on chromosome XV using newly developed computer software. mlh1Delta mms4Delta double mutants displayed the largest decrease in crossing over (13- to 15-fold) of all mutant combinations, yet these strains displayed relatively high spore viability (42%). In contrast, msh5Delta mms4Delta and msh5Delta mms4Delta mlh1Delta mutants displayed smaller decreases in crossing over (4- to 6-fold); however, spore viability (18-19%) was lower in these strains than in mlh1Delta mms4Delta strains. These data suggest that meiotic crossing over can occur in yeast through three distinct crossover pathways. In one pathway, MUS81-MMS4 promotes interference-independent crossing over; in a second pathway, both MSH4-MSH5 and MLH1-MLH3 promote interference-dependent crossovers. A third pathway, which appears to be repressed by MSH4-MSH5, yields deleterious crossovers.     

Stable denaturation of chromosomal DNA from Saccharomyces cerevisiae during meiosis.

Partial denaturation of Saccharomyces cerevisiae chromosomal DNA was found to occur spontaneously during meiosis. Short regions of strand separation (300 base pairs long) were seen in DNA molecules prepared for electron microscopy by the aqueous spreading technique. These regions were clustered along the DNA. The time course of their appearance indicated that the denatured regions were present during the periods of premeiotic DNA replication and recombination. A similar pattern of denaturation was also detected in the DNA from vegetatively grown cells of a conditional cdc8 mutant, which is defective in DNA replication.     

DNA polymerases, deoxyribonucleases, and recombination during meiosis in Saccharomyces cerevisiae.

We utilized strains of Saccharomyces cerevisiae that exhibit high efficiency of synchrony of meiosis to examine several aspects of meiosis including sporulation, recombination, DNA synthesis, DNA polymerase I and II, and Mg2+-dependent alkaline DNases. The kinetics of commitment to intragenic recombination and sporulation are similar. The synthesis of DNA, as measured directly with diphenylamine, appears to precede the commitment to recombination. Both DNA polymerase I and II activities and total DNA-synthesizing activity in crude extracts increase two- to threefold before the beginning of meiotic DNA synthesis. Increases of 10- to 20-fold over mitotic levels are found for Mg2+-dependent alkaline DNase activity in crude extracts before and during the commitment to meiotic intragenic recombination. Of particular interest is the comparable increase in a nuclease under the control of the RAD52 gene; this enzyme has been identified by the use of antibody raised against a similar enzyme from Neurospora crassa. Since the RAD52 gene is essential for meiotic recombination, the nuclease is implicated in the high levels of recombination observed during meiosis. The effects observed in this report are meiosis specific since they are not observed in an alpha alpha strain.     

Events associated with restoration by zinc of meiosis in apomictic Saccharomyces cerevisiae.

The effects of nutritional alterations (carbon source and zinc) on nuclear division and protein synthesis during apomictic and meiotic development in Saccharomyces cerevisiae 19e1 were investigated. Unlike cells cultivated under meiosis-promoting conditions, cells cultured under apomixis-promoting conditions exhibited extensive protein synthesis during the first 3 h of incubation in sporulation medium, and nuclear divisions were evident during this time. Cycloheximide treatment of the latter cells induced meiosis, and maximum yields of meiotic asci resulted when this treatment was given for the first 3 h in sporulation medium. The results indicate that the decision concerning which developmental route cells will follow is made shortly after transfer to sporulation medium. Electrophoretic analysis of labeled proteins synthesized during sporulation revealed bands unique to both developmental routes.     

Conservative duplication of spindle poles during meiosis in Saccharomyces cerevisiae

During sporulation in diploid Saccharomyces cerevisiae, spindle pole bodies acquire the so-called meiotic plaque, a prerequisite for spore formation. Mpc70p is a component of the meiotic plaque and is thus essential for spore formation. We show here that MPC70/mpc70 heterozygous strains most often produce two spores instead of four and that these spores are always nonsisters. In wild-type strains, Mpc70p localizes to all four spindle pole bodies, whereas in MPC70/mpc70 strains Mpc70p localizes to only two of the four spindle pole bodies, and these are always nonsisters. Our data can be explained by conservative spindle pole body distribution in which the two newly synthesized meiosis II spindle pole bodies of MPC70/mpc70 strains lack Mpc70p.     

Cyclic variations in sensitivity to X-irradiation during meiosis in Saccharomyces cerevisiae

Cyclic variation in mutation induction and lethality was found following X-irradiation during meiosis in Saccharomyces cerevisiae. An enhanced mutagenic response was found in meiotic G1 phase cells in comparison to cells later in meiosis, similar to the response shown during mitosis, but meiotic G1 phase cells appeared more resistant to the lethal effects of X-irradiation than mitotic G1 phase cells. Resistance to the lethal effects of X-rays was found during meiotic DNA synthesis in the strain SK1, which may indicate the operation of a sister-chromatid exchange repair mechanism. A difference was found between gene conversion which appeared to be at a maximum by the end of meiotic DNA synthesis and reciprocal recombination, which could be induced up to prophase I.     

A role for MMS4 in the processing of recombination intermediates during meiosis in Saccharomyces cerevisiae.

The MMS4 gene of Saccharomyces cerevisiae was originally identified due to its sensitivity to MMS in vegetative cells. Subsequent studies have confirmed a role for MMS4 in DNA metabolism of vegetative cells. In addition, mms4 diploids were observed to sporulate poorly. This work demonstrates that the mms4 sporulation defect is due to triggering of the meiotic recombination checkpoint. Genetic, physical, and cytological analyses suggest that MMS4 functions after the single end invasion step of meiotic recombination. In spo13 diploids, red1, but not mek1, is epistatic to mms4 for sporulation and spore viability, suggesting that MMS4 may be required only when homologs are capable of undergoing synapsis. MMS4 and MUS81 are in the same epistasis group for spore viability, consistent with biochemical data that show that the two proteins function in a complex. In contrast, MMS4 functions independently of MSH5 in the production of viable spores. We propose that MMS4 is required for the processing of specific recombination intermediates during meiosis.     

Differential function and expression of Saccharomyces cerevisiae B-type cyclins in mitosis and meiosis.

We have studied the patterns of expression of four B-type cyclins (Clbs), Clb1, Clb2, Clb3, and Clb4, and their ability to activate p34cdc28 during the mitotic and meiotic cell cycles of Saccharomyces cerevisiae. During the mitotic cell cycle, Clb3 and Clb4 were expressed and induced a kinase activity in association with p34cdc28 from early S phase up to mitosis. On the other hand, Clb1 and Clb2 were expressed and activated p34cdc28 later in the mitotic cell cycle, starting in late S phase and continuing up to mitosis. The pattern of expression of Clb3 and Clb4 suggests a possible role in the regulation of DNA replication as well as mitosis. Clb1 and Clb2, whose pattern of expression is similar to that of other known Clbs, are likely to have a role predominantly in the regulation of M phase. During the meiotic cell cycle, Clb1, Clb3, and Clb4 were expressed and induced a p34cdc28-associated kinase activity just before the first meiotic division. The fact that Clb3 and Clb4 were not synthesized earlier, in S phase, suggests that these cyclins, which probably have a role in S phase during the mitotic cell cycle, are not implicated in premeiotic S phase. Clb2, the primary mitotic cyclin in S. cerevisiae, was not detectable during meiosis. Sporulation experiments on strains deleted for one, two, or three Clbs indicate, in agreement with the biochemical data, that Clb1 is the primary cyclin for the regulation of meiosis, while Clb2 is not involved at all.     

Partial deprivation of GTP initiates meiosis and sporulation in Saccharomyces cerevisiae

Summary We have investigated the physiological conditions under which meiosis and the ensuing sporulation of Saccharomyces cerevisiae are initiated. Initiation of sporulation occurs in response to carbon, nitrogen, phosphorus, or sulfur deprivation, and also, when met auxotrophs are partially starved for methionine, but not after starvation of other amino acid auxotrophs. It also occurs after partial starvation of pur or gua auxotrophs for guanine but not after starvation of ura auxotrophs for uracil. Under all these sporulation conditions the concentrations of both guanine nucleotides (GTP) and S-adenosylmethionine (SAM) decrease whereas those of other nucleotides show no trend. We show that the decrease of guanine nucleotides is essential for the initiation of meiosis and sporulation: when a gua auxotroph, also lacking one of the two SAM synthetases, is starved for guanine but supplemented with 0.1 mM methionine, GTP decreases while SAM slightly increases and yet the cells sporulate.