Cyclin B-cdk activity stimulates meiotic rereplication in budding yeast

Haploidization of gametes during meiosis requires a single round of premeiotic DNA replication (meiS) followed by two successive nuclear divisions. This study demonstrates that ectopic activation of cyclin B/cyclin-dependent kinase in budding yeast recruits up to 30% of meiotic cells to execute one to three additional rounds of meiS. Rereplication occurs prior to the meiotic nuclear divisions, indicating that this process is different from the postmeiotic mitoses observed in other fungi. The cells with overreplicated DNA produced asci containing up to 20 spores that were viable and haploid and demonstrated Mendelian marker segregation. Genetic tests indicated that these cells executed the meiosis I reductional division and possessed a spindle checkpoint. Finally, interfering with normal synaptonemal complex formation or recombination increased the efficiency of rereplication. These studies indicate that the block to rereplication is very different in meiotic and mitotic cells and suggest a negative role for the recombination machinery in allowing rereplication. Moreover, the production of haploids, regardless of the genome content, suggests that the cell counts replication cycles, not chromosomes, in determining the number of nuclear divisions to execute.     

Yeast meiotic mutants proficient for the induction of ectopic recombination.

A screen was designed to identify Saccharomyces cerevisiae mutants that were defective in meiosis yet proficient for meiotic ectopic recombination in the return-to-growth protocol. Seven mutants alleles were isolated; two are important for chromosome synapsis (RED1, MEK1) and five function independently of recombination (SPO14, GSG1, SPOT8/MUM2, 3, 4). Similar to the spoT8-1 mutant, mum2 deletion strains do not undergo premeiotic DNA synthesis, arrest prior to the first meiotic division and fail to sporulate. Surprisingly, although DNA replication does not occur, mum2 mutants are induced for high levels of ectopic recombination. gsg1 diploids are reduced in their ability to complete premeiotic DNA synthesis and the meiotic divisions, and a small percentage of cells produce spores. mum3 mutants sporulate poorly and the spores produced are inviable. Finally, mum4-1 mutants produce inviable spores. The meiotic/sporulation defects of gsg1, mum2, and mum3 are not relieved by spo11 or spo13 mutations, indicating that the mutant defects are not dependent on the initiation of recombination or completion of both meiotic divisions. In contrast, the spore inviability of the mum4-1 mutant is rescued by the spo13 mutation. The mum4-1 spo13 mutant undergoes a single, predominantly equational division, suggesting that MUM4 functions at or prior to the first meiotic division. Although recombination is variably affected in the gsg1 and mum mutants, we hypothesize that these mutants define genes important for aspects of meiosis not directly related to recombination.     

Meiotic effects of DNA-defective cell division cycle mutations of Saccharomyces cerevisiae

The meiotic effects of several cell division cycle (cdc) mutations of Saccharomyces cerevisiae have been investigated by electron microscopy and by genetic and biochemical methods. Diploid strains homozygous for cdc mutations known to confer defects on vegetative DNA synthesis were subjected to restrictive conditions during meiosis. Electron microscopy revealed that all four mutants were conditionally arrested in meiosis after duplication of the spindle pole bodies but before spindle formation for the first meiotic division. None of these mutants became committed to recombination or contained synaptonemal complex at the meiotic arrest. — The mutants differed in their ability to undergo premeiotic DNA synthesis under restrictive conditions. Both cdc8 and cdc21, which are defective in the propagation of vegetative DNA synthesis, also failed to undergo premeiotic DNA synthesis. The arrest of these mutants at the stage before meiosis I spindle formation could be attributed to the failure of DNA synthesis because inhibition of synthesis by hydroxyurea also caused arrest at this stage. — Premeiotic DNA synthesis occurred before the arrest of cdc7, which is defective in the initiation of vegetative DNA synthesis, and of cdc2, which synthesizes vegetative DNA but does so defectively. The meiotic arrest of cdc7 homozygotes was partially reversible. Even if further semiconservative DNA replication was inhibited by the addition of hydroxyurea, released cells rapidly underwent commitment to recombination and formation of synaptonemal complexes. The cdc7 homozygote is therefore reversibly arrested in meiosis after DNA replication, whereas vegetative cultures have previously been shown to be defective only in the initiation of DNA synthesis.     

A chromosome rearrangement in Neuvospora that produces viable progeny containing two nucleolus organizers

In rearrangement T(VLIVL)AR33 the segment of chromosome 2 bearing the nucleolus organizer is translocated to the end of chromosome 4. When AR33 is crossed by Normal sequence (N), one third of the viable progeny contain a stable nontandem duplication with two organizers per nucleus. The organizer-deficient complementary products are inviable. Chromosomes and nucleoli have been examined during meiosis and postmeiotic nuclear divisions in the ascus, comparing heterozygous AR33 × N crosses with N × N and with crosses heterozygous for other interchanges. When AR33 is heterozygous, asci are of three types having the nucleolus organizer duplicated in 0, 1 or 2 of the meiotic products. Frequencies of the ascus types are as expected from the known positions of rearrangement break points. Nucleoli formed by two organizers frequently fuse. Deficiency nuclei that contain no nucleolus organizer may form one or more small nucleolus-like bodies.     

Involvement of 14-nm Filament-Forming Protein and Tubulin in Gametic Pronuclear Behavior during Conjugation in Tetrahymena

We have studied in detail the immunofluorescence localizations of Tetrahymena 14-nm filament-forming protein (49-kDa protein) in relation to tubulin in conjugating wild-type Tetrahymena thermophila (B strain) pairs and in pairs between B strain and star strains with defective micronuclei. The results suggest that germ nuclear behavior during conjugation may involve the following cytoskeletal structures: (1) during meiosis, microtubule structures are involved in micronuclear elongation and meiotic division; (2) at the postmeiotic stage, 49-kDa protein network structures that are formed independently of the existence of pronuclei are involved in the selection and the survival of one of four meiotic products; (3) during the third prezygotic division, gametic pronuclear transfer, and zygote formation, a cytoskeletal structure in which the 49-kDa protein colocalizes with microtubules and which is dependent on the existence of a normal gametic pronucleus is involved in gametic pronuclear behavior, and (4) during the postzygotic divisions, the microtubules are involved in nuclear behavior.     

Meiotic S-phase damage activates recombination without checkpoint arrest

Checkpoints operate during meiosis to ensure the completion of DNA synthesis and programmed recombination before the initiation of meiotic divisions. Studies in the fission yeast Schizosaccharomyces pombe suggest that the meiotic response to DNA damage due to a failed replication checkpoint response differs substantially from the vegetative response, and may be influenced by the presence of homologous chromosomes. The checkpoint responses to DNA damage during fission yeast meiosis are not well characterized. Here we report that DNA damage induced during meiotic S-phase does not activate checkpoint arrest. We also find that in wild-type cells, markers for DNA breaks can persist at least to the first meiotic division. We also observe increased spontaneous S-phase damage in checkpoint mutants, which is repaired by recombination without activating checkpoint arrest. Our results suggest that fission yeast meiosis is exceptionally tolerant of DNA damage, and that some forms of spontaneous S-phase damage can be repaired by recombination without activating checkpoint arrest.     

OSD1 Promotes Meiotic Progression via APC/C Inhibition and Forms a Regulatory Network with TDM and CYCA1;2/TAM

Cell cycle control is modified at meiosis compared to mitosis, because two divisions follow a single DNA replication event. Cyclin-dependent kinases (CDKs) promote progression through both meiosis and mitosis, and a central regulator of their activity is the APC/C (Anaphase Promoting Complex/Cyclosome) that is especially required for exit from mitosis. We have shown previously that OSD1 is involved in entry into both meiosis I and meiosis II in Arabidopsis thaliana; however, the molecular mechanism by which OSD1 controls these transitions has remained unclear. Here we show that OSD1 promotes meiotic progression through APC/C inhibition. Next, we explored the functional relationships between OSD1 and the genes known to control meiotic cell cycle transitions in Arabidopsis. Like osd1, cyca1;2/tam mutation leads to a premature exit from meiosis after the first division, while tdm mutants perform an aberrant third meiotic division after normal meiosis I and II. Remarkably, while tdm is epistatic to tam, osd1 is epistatic to tdm. We further show that the expression of a non-destructible CYCA1;2/TAM provokes, like tdm, the entry into a third meiotic division. Finally, we show that CYCA1;2/TAM forms an active complex with CDKA;1 that can phosphorylate OSD1 in vitro. We thus propose that a functional network composed of OSD1, CYCA1;2/TAM, and TDM controls three key steps of meiotic progression, in which OSD1 is a meiotic APC/C inhibitor.     

Genetics of CHLAMYDOMONAS REINHARDTII Diploids I. Isolation and Characterization and Meiotic Segregation Pattern of a Homozygous Diploid

A strain of Chlamydomonas reinhardtii has been investigated which, when mated with known wild-types, produces very few viable germination products and transmits its Mendelian markers to more than half of those products. Cytogenetic observations, fluorometric measurements of DNA and genetic data all suggest that the strain, d mt^-ery-M3a sr-u-1 is a stable homozygous diploid. This strain has twice as many nuclear chromatin bodies at metaphase and twice as much DNA as its haploid progenitor, and the phenotypes of its meiotic progeny are consistent with predictions based on triploid meiosis. Data from crosses involving d mt^-ery-M3a sr-u-1 and from crosses involving hybrid diploids indicate that the frequency of second division segregation increases in triploid zygotes and that mitotic segregation following triploid meiosis is a frequent event which may more often result from mitotic recombination than from chromosome loss.     

Dual role of the Cdc7-regulatory protein Dbf4 during yeast meiosis

The Dbf4-dependent Cdc7 kinase (DDK) is essential for chromosome duplication in all eukaryotes, but was proposed to be dispensable for yeast pre-meiotic DNA replication. This discrepancy led us to investigate the role of the unstable Cdc7-regulatory protein Dbf4 in meiosis. We show that, when Dbf4 is depleted at the time of meiotic induction, cells enter the meiotic program but do not replicate their chromosomes. Surprisingly when Dbf4 is depleted after the initiation of DNA synthesis, S phase goes to completion, but most cells arrest before anaphase I. Deletion of the cohesin Rec8 suppresses this phenotype, suggesting a distinct role of DDK for meiotic chromosome segregation. As after Cdc5 depletion, a fraction of cells undergo a single equational division suggesting a failure to mono-orient sister kinetochores. Our results demonstrate that Dbf4 is essential for DNA replication during meiosis like in vegetative cells and provide evidence for an additional role in setting up the reductional division of meiosis I.     

Distinct temporal requirements for autophagy and the proteasome in yeast meiosis

Meiosis is a special type of cellular renovation that involves 2 successive cell divisions and a single round of DNA replication. Two major degradation systems, the autophagy-lysosome and the ubiquitin-proteasome, are involved in meiosis, but their roles have yet to be elucidated. Here we show that autophagy mainly affects the initiation of meiosis but not the nuclear division. Autophagy works not only by serving as a dynamic recycling system but also by eliminating some negative meiotic regulators such as Ego4 (Ynr034w-a). In a quantitative proteomics study, the proteasome was found to be significantly upregulated during meiotic divisions. We found that proteasomal activity is essential to the 2 successive meiotic nuclear divisions but not for the initiation of meiosis. Our study defines the roles of autophagy and the proteasome in meiosis: Autophagy mainly affects the initiation of meiosis, whereas the proteasome mainly affects the 2 successive meiotic divisions.     

Variation of thymidine incorporation patterns in the alternating vegetative and sexual life cycles of Chlamydomonas reinhardtii

The cellular distribution of thymidine kinase activities in Chlamydomonas reinhardtii, as manifested by the in vivo incorporation of exogenous thymidine and 5-bromodeoxyuridine into different DNA species, appeared to be organelle specific and varied with different developmental stages in the life cycle of this organism. During vegetative growth and gametogenic differentiation, thymidine and 5-bromodeoxyuridine were shown to be selectively incorporated into chloroplast but not nuclear DNA. On the other hand, during zygotic germination in which meiosis occurs and the ensuing vegetative divisions of meiotic products, thymidine as well as 5-bromodeoxyuridine were incorporated into both nuclear and chloroplast DNA. These results suggest that, in addition to the thymidine kinase activity that is constantly present in the chloroplast, a cytoplasmic thymidine kinase is derepressed only during the sexual reproductive cycle of C. reinhardtii.     

The Role of the SPO11 Gene in Meiotic Recombination in Yeast

Several complementary experimental approaches were used to demonstrate that the SPO11 gene is specifically required for meiotic recombination. First, sporulating cultures of spo11-1 mutant diploids were examined for landmark biochemical, cytological and genetic events of meiosis and ascosporogenesis. Cells entered sporulation with high efficiency and showed a near-doubling of DNA content. Synaptonemal complexes, hallmarks of intimate homologous pairing, and polycomplex structures appeared during meiotic prophase. Although spontaneous mitotic intra- and intergenic recombination occurred at normal levels, no meiotic recombination was observed. Whereas greater than 50% of cells completed both meiotic divisions, packaging of the four meiotic products into mature ascospores took place in only a small subset of asci. Haploidization occurred in less than 1% of viable colony-forming units. Second, the Rec- meiotic defect conferred by spo11-1 was confirmed by dyad analysis of spores derived from spo13-1 single-division meiosis in which recombination is not a requirement for viable ascospore production. Diploids homozygous for the spo13-1 mutation undergo meiotic levels of exchange followed by a single predominantly equational division and form asci containing two near-diploid spores. With the introduction of the spo11-1 mutation, high spore viability was retained, whereas intergenic recombination was reduced by more than 100-fold.     

Induction of Blocks in Nuclear Divisions and Overcondensation of Meiotic Chromosomes with Cycloheximide During Conjugation of Tetrahymena thermophila

During conjugation, the micronucleus of Tetrahymena thermophila undergoes five consecutive nuclear divisions: meiosis, third prezygotic division (pregamic mitosis) and two postzygotic mitoses of the synkaryon. The four products of the synkaryon differentiate into macronuclear anlagen and new micronuclei and the old macronucleus is resorbed. The protein synthesis inhibitor cycloheximide, applied during conjugation, induced several developmental blocks. Pairs shifted to the drug during early meiotic prophase (stages I–III) were arrested at prophase. Cycloheximide applied to cells at pachytene (stages IV-VI) to metaphase arrested the conjugants at the stage of modified prometaphase/metaphase with overcondensed, swollen bivalents. In contrast to other systems, in the presence of cycloheximide, separation of chromatids, decondensation of chromosomes and exit from metaphase I were inhibited in both diploid and haploid cells. Pairs shifted to the drug after metaphase I were arrested at postmeiotic interphase after completing one nuclear cycle. The same rule applied to the subsequent cycle; then cells were arrested at the stage of pronuclei, and those pairs with functional pronuclei and synkarya were arrested at the stage of two products of the first postzygotic division (pronuclei were not arrested in nuclear transfer and karyogamy). Only pairs with two products of the first postzygotic division were arrested at the same stage after the cycloheximide treatment. Pairs shifted to cycloheximide during the second postzygotic division were arrested in development of macronuclear anlagen and resorption of old macronuclei. The postmeiotic conjugants pulse-treated with cycloheximide (2 h) yielded heterokaryons retaining parental macronuclei (i.e. they exhibited macronuclear retention).     

Cdc28 and Ime2 possess redundant functions in promoting entry into premeiotic DNA replication in Saccharomyces cerevisiae.

In the budding yeast Saccharomyces cerevisiae initiation and progression through the mitotic cell cycle are determined by the sequential activity of the cyclin-dependent kinase Cdc28. The role of this kinase in entry and progression through the meiotic cycle is unclear, since all cdc28 temperature-sensitive alleles are leaky for meiosis. We used a "heat-inducible Degron system" to construct a diploid strain homozygous for a temperature-degradable cdc28-deg allele. We show that this allele is nonleaky, giving no asci at the nonpermissive temperature. We also show, using this allele, that Cdc28 is not required for premeiotic DNA replication and commitment to meiotic recombination. IME2 encodes a meiosis-specific hCDK2 homolog that is required for the correct timing of premeiotic DNA replication, nuclear divisions, and asci formation. Moreover, in ime2Delta diploids additional rounds of DNA replication and nuclear divisions are observed. We show that the delayed premeiotic DNA replication observed in ime2Delta diploids depends on a functional Cdc28. Ime2Delta cdc28-4 diploids arrest prior to initiation of premeiotic DNA replication and meiotic recombination. Ectopic overexpression of Clb1 at early meiotic times advances premeiotic DNA replication, meiotic recombination, and nuclear division, but the coupling between these events is lost. The role of Ime2 and Cdc28 in initiating the meiotic pathway is discussed.     

Identification of yeast meiosis-specific genes by differential display

Meiosis, a specialized cell division process, occurs in all sexually reproducing organisms. During this process a diploid cell undergoes a single round of DNA replication followed by two rounds of nuclear division to produce four haploid gametes. In yeast, the meiotic products are packaged into four spores that are enclosed in a sac known as an ascus. To enhance our understanding of the meiotic developmental pathway and spore formation, we followed differential expression of genes in meiotic versus vegetatively growing cells in the yeast Saccharomyces cerevisiae. Such comparative analyses have identified five different classes of genes that are expressed at different stages of the sporulation program. We identified several meiosis-specific genes including some already known to be induced during meiosis. Here we describe one of these previously uncharacterized genes, SSP1, which plays an essential role in meiosis and spore formation. SSP1 is induced midway through meiosis, and the homozygous mutant-diploid cells fail to sporulate. In ssp1 cells, meiosis is delayed, nuclei fragment after meiosis II, and viability declines rapidly. The ssp1 defect is not related to a microtubule-cytoskeletal-dependent event and is independent of two rounds of meiotic divisions. Our results suggest that Ssp1 is likely to function in a pathway that controls meiotic nuclear divisions and coordinates meiosis and spore formation. Functional analysis of other uncharacterized genes is underway.     

Un ménage à quatre: the molecular biology of chromosome segregation in meiosis

Sexually reproducing organisms rely on the precise reduction of chromosome number during a specialized cell division called meiosis. Whereas mitosis produces diploid daughter cells from diploid cells, meiosis generates haploid gametes from diploid precursors. The molecular mechanisms controlling chromosome transmission during both divisions have started to be delineated. This review focuses on the four fundamental differences between mitotic and meiotic chromosome segregation that allow the ordered reduction of chromosome number in meiosis: (1) reciprocal recombination and formation of chiasmata between homologous chromosomes, (2) suppression of sister kinetochore biorientation, (3) protection of centromeric cohesion, and (4) inhibition of DNA replication between the two meiotic divisions.     

Suppression of DNA replication via Mos function during meiotic divisions in Xenopus oocytes.

Meiosis is characterized by the absence of DNA replication between the two successive divisions. In Xenopus eggs, the ability to replicate DNA develops during meiotic maturation, but is normally suppressed until fertilization. Here we show that development of the DNA-replicating ability depends on new protein synthesis during meiosis I, and that mere ablation of the endogenous c-mos product Mos allows maturing oocytes to enter interphase and replicate DNA just after meiosis I. Moreover, we demonstrate that during normal maturation cdc2 kinase undergoes precocious inactivation in meiosis I and then premature reactivation before meiosis II; importantly, this premature cdc2 reactivation absolutely requires Mos function and its direct inhibition by a dominant-negative cdc2 mutant also results in nuclear reformation and DNA replication immediately after meiosis I. These findings indicate that suppression of DNA replication during meiotic divisions in Xenopus oocytes is accomplished by the Mos-mediated premature reactivation of cdc2 kinase. We suggest that these mechanisms for suppressing DNA replication may be specific for meiosis in animal oocytes, and that the ultimate biological function, including the well known cytostatic factor activity, of Mos during meiotic maturation may be to prevent undesirable DNA replication or parthenogenetic activation before fertilization.     

Developmental regulation of a sporulation-specific enzyme activity in Saccharomyces cerevisiae.

An alpha-glucosidase activity (SAG) occurs in a/alpha Saccharomyces cerevisiae cells beginning at about 8 to 10 h after the initiation of sporulation. This enzyme is responsible for the rapid degradation of intracellular glycogen which follows the completion of meiosis in these cells. SAG differs from similar activities present in vegetative cells and appears to be a sporulation-specific enzyme. Cells arrested at various stages in sporulation (DNA replication, recombination, meiosis I, and meiosis II) were examined for SAG activity; the results show that SAG appearance depends on DNA synthesis and some recombination events but not on the meiotic divisions.     

The Role of Radiation (rad) Genes in Meiotic Recombination in Yeast

In yeast, the functions controlled by radiation-repair genes RAD6, RAD50, RAD52 and RAD57 are essential for normal meiosis; diploids with lesions in these genes either fail to sporulate (rad6) or sporulate but produce inviable spores (rad50, 52, 57). Since RAD genes may control aspects of DNA metabolism, we attempted to define more precisely the role of each gene in meiosis, especially with regard to possible roles in premeiotic DNA replication and recombination. We constructed diploids singly homozygous for each of the four rad mutations, heteroallelic at his1 and heterozygous for a recessive canavanine-resistance marker. Each strain was exposed to sporulation-inducing conditions and monitored for (1) completion of mitotic cell cycles, (2) cell viability, (3) utilization of acetate for mass increases, (4) premeiotic DNA synthesis, (5) intragenic recombination at his1, and (6) formation of viable haploid spores. Control strains heterozygous for the rad mutations completed mitosis, metabolized acetate, replicated their DNA, and showed typically high levels of gene conversion and viable-spore formation. The mutant diploids also completed mitosis, utilized acetate, and carried out premeiotic DNA replication. The mutants, however, showed little or no meiotic gene conversion. The rad50, 52 and 57 strains sporulated, but the spores were inviable. The rad6 strain did not sporulate. The rad50, 52 and 57 strains exhibited viability losses that coincided with the period of DNA synthesis, but not with later meiotic events; the rad6 strain did not lose viability. We propose that the normal functions specified by RAD50, 52 and 57 are not essential for either the initial or terminal steps in meiosis, but are required for successful recombination. The rad6 strain may be recombination-defective, or it may fail to progress past DNA replication in the overall sequence leading to formation and recovery of meiotic recombinants.