The PSO4 gene of S. cerevisiae is important for sporulation and the meiotic DNA repair of photoactivated psoralen lesions

We have evaluated the effect of the Saccharomyces cerevisiae pso4-1 mutation in sporulation and DNA repair during meiosis. We have found that pso4-1 cells were arrested in an early step of meiosis, before premeiotic DNA synthesis, and hence did not produce spores. These results suggest that the PSO4 gene may act at the start point of the cell cycle, as do some SPO and CDC genes. The pso4-1 mutant cells are specifically sensitive to 8-MOP- and 3-CPs-photoinduced lesions, and are found to be severely affected in meiotic recombination as well as impaired in the mutagenic response, as previously described for mitosis. This means that the PSO4 gene is important for the repair 8-MOP-photoinduced lesions, mainly double-strand breaks, and the processing of these lesions into recombinogenic intermediates.     

Meiotic recombination in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

The faithful segregation of homologous chromosomes during meiosis is dependent on the formation of physical connections (chiasma) that form following reciprocal exchange of DNA molecules during meiotic recombination. Here we review the current knowledge in the Caenorhabditis elegans meiotic recombination field. We discuss recent developments that have improved our understanding of the crucial steps that must precede the initiation and propagation of meiotic recombination. We summarize the pathways that impact on meiotic prophase entry and the current understanding of how chromosomes reorganize and interact to promote homologous chromosome pairing and subsequent synapsis. We pay particular attention to the mechanisms that contribute to meiotic DNA double-strand break (DSB) formation and strand exchange processes, and how the C. elegans system compares with other model organisms. Finally, we highlight current and future areas of research that are likely to further our understanding of the meiotic recombination process.     

Synchronized meiosis and recombination in fission yeast: observations with pat1-114 diploid cells

Summary The mutation pat1-114 has been used to synchronize meiosis in the fission yeast Schizosaccharomyces pombe. We have investigated several aspects of such synchronized meiotic cultures. In both pat1-114 and pat1 ⁺ diploids, meiotic landmark events are initiated at the same time after meiosis induction, but synchrony is much more pronounced in the pat1-114-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 of pat1-114 diploids, physical analysis of recombination has not been possible. We have distinguished three factors that influence intragenic recombination frequencies: temperature, azygotic versus zygotic meiosis, and the nature of the pat1 allele. Differences and similarities in the timing of meiotic landmarks in S. cerevisiae and S. pombe are discussed.     

Behavior of chloroplast genes during the early zygotic divisions of Chlamydomonas reinhardtii

Summary Chloroplast mutations in the green alga Chlamydomonas reinhardtii exhibit a predominantly maternal pattern of inheritance and this pattern can be perturbed by UV irradiation of the maternal gametes prior to mating. In a series of crosses over a range of UV doses, the transmission, segregation, and recombination of mutations at three closely linked chloroplast loci have been examined by pedigree analysis of products arising from the first three post-zygotic divisions. Stocks used in these crosses were constructed to permit identification of the nuclear products of each of the two meiotic divisions and the first post-meiotic mitotic division.A bias toward maternal alleles at all three chloroplast loci was observed in all pedigrees and in zygote clones analyzed from the same crosses many generations after meiosis. This bias decreased with increasing UV dose and with each subsequent division. Segregation of chloroplast genes was rapid during the first three post-zygotic divisions. The type of segregation event from which a given heteroplasmic cell arose had a significant effect on its most likely segregation. pattern in the subsequent division. The results presented here have been discussed in terms of published models of chloroplast gene segregation.     

The multiple roles of the Mre11 complex for meiotic recombination

During the first meiotic prophase, numerous DNA double-strand breaks (DSB) are formed in the genome in order to initiate recombination between homologous chromosomes. The conserved Mre11 complex, formed of Mre11, Rad50 and Nbs1 (Xrs2 in Saccharomyces cerevisiae) proteins, plays a crucial role in mitotic cells for sensing and repairing DSB. In meiosis the Mre11 complex is also required for meiotic recombination. Depending on the organisms, the Mre11 complex is required for the formation of the DSB catalysed by the transesterase Spo11 protein. It then plays a unique function in removing covalently attached Spo11 from the 5′ extremity of the breaks through its nuclease activity, to allow further break resection. Finally, the Mre11 complex also plays a role during meiosis in bridging DNA molecules together and in sensing Spo11 DSB and activating the DNA damage checkpoint. In this article the different biochemical functions of the Mre11 complex required during meiosis are reviewed, as well as the consequences of Mre11 complex inactivation for meiosis in several organisms. Finally, I describe the meiotic phenotypes of several animal models that have been developed to model hypomorphic mutations of the Mre11 complex, involved in humans in some genetic instability disorders.     

The effect of rec-2 on repeat-induced point-mutation (RIP) and recombination events that excise DNA sequence duplications at the his-3 locus in Neurospora crassa

In Neurospora crassa, duplicated DNA suffers both extensive repeat-induced point-mutation (RIP) and also excision by recombination events during the dikaryotic phase of the life cycle that precedes karyogamy and meiosis (reviewed by Selker 1990). This paper describes experiments designed to test the effect of rec-2, a gene known to modulate the local level of meiotic recombination at his-3, on RIP and the excision of tandem duplications. Duplications carrying his-3 sequences and a marker, hug ^r, that confers hygromycin resistance were constructed by targeted transformation. RIP and excisive recombination were assessed from the progeny of crosses heterozygous for a duplication and having different combinations of rec-2 alleles. In the presence of rec-2 ⁺, excision of hyg ^r was reduced to about half of that in crosses homozygous for rec-2. In contrast, rec-2 ⁺ had little influence on the frequency of duplications that escaped RIP. Thus, in addition to reducing recombination between his-3 alleles during meiosis, rec-2 ⁺ also influences recombination events that lead to the excision of duplications carrying his-3. However, RIP may be independent.     

Multiple branches of the meiotic recombination pathway contribute independently to homolog pairing and stable juxtaposition during meiosis in budding yeast

A unique aspect of meiosis is the segregation of homologous chromosomes at the meiosis I division. Homologs are physically connected prior to segregation by crossing over between nonsister chromatids. Crossovers arise from the repair of induced double-strand breaks (DSBs). In many organisms, more DSBs are formed than crossovers in a given nucleus. It has been previously suggested that repair of DSBs to noncrossover recombination products aids homolog alignment. Here we explore how two modes of the meiotic recombination pathway (crossover and noncrossover) and meiotic telomere reorganization contribute to the pairing and close juxtaposition of homologous chromosomes in budding yeast. We found that intermediates in the DSB repair pathway leading to both crossover and noncrossover recombination products contribute independently to close, stable homolog juxtaposition (CSHJ), a measurable state of homolog pairing. Analysis of the ndj1delta mutant indicates that the effect of meiotic telomere reorganization on CSHJ is exerted through recombination intermediates at interstitial chromosomal loci, perhaps through the noncrossover branch of the DSB repair pathway. We suggest that transient, early DSB-initiated interactions, including those that give rise to noncrossovers, are important for homolog recognition and juxtaposition.     

A developmentally regulated translational control pathway establishes the meiotic chromosome segregation pattern

Production of haploid gametes from diploid progenitor cells is mediated by a specialized cell division, meiosis, where two divisions, meiosis I and II, follow a single S phase. Errors in progression from meiosis I to meiosis II lead to aneuploid and polyploid gametes, but the regulatory mechanisms controlling this transition are poorly understood. Here, we demonstrate that the conserved kinase Ime2 regulates the timing and order of the meiotic divisions by controlling translation. Ime2 coordinates translational activation of a cluster of genes at the meiosis I–meiosis II transition, including the critical determinant of the meiotic chromosome segregation pattern CLB3. We further show that Ime2 mediates translational control through the meiosis-specific RNA-binding protein Rim4. Rim4 inhibits translation of CLB3 during meiosis I by interacting with the 5′ untranslated region (UTR) of CLB3. At the onset of meiosis II, Ime2 kinase activity rises and triggers a decrease in Rim4 protein levels, thereby alleviating translational repression. Our results elucidate a novel developmentally regulated translational control pathway that establishes the meiotic chromosome segregation pattern.     

The axial element protein HTP-3 promotes cohesin loading and meiotic axis assembly in C. elegans to implement the meiotic program of chromosome segregation

Faithful transmission of the genome through sexual reproduction requires reduction of genome copy number during meiosis to produce haploid sperm and eggs. Meiosis entails steps absent from mitosis to achieve this goal. When meiosis begins, sisters are held together by sister chromatid cohesion (SCC), mediated by the cohesin complex. Homologs then become linked through crossover recombination. SCC subsequently holds both sisters and homologs together. Separation of homologs and then sisters requires two successive rounds of chromosome segregation and the stepwise removal of Rec8, a meiosis-specific cohesin subunit. We show that HTP-3, a known component of the C. elegans axial element (AE), molecularly links these meiotic innovations. We identified HTP-3 in a genetic screen for factors necessary to maintain SCC until meiosis II. Our data show that interdependent loading of HTP-3 and cohesin is a principal step in assembling the meiotic chromosomal axis and in establishing SCC. HTP-3 recruits all known AE components to meiotic chromosomes and promotes cohesin loading, the first known involvement of an AE protein in this process. Furthermore, REC-8 and two paralogs, called COH-3 and COH-4, together mediate meiotic SCC, but they perform specialized functions. REC-8 alone is necessary and sufficient for the persistence of SCC after meiosis I. In htp-3 and rec-8 mutants, sister chromatids segregate away from one another in meiosis I (equational division), rather than segregating randomly, as expected if SCC were completely eliminated. AE assembly fails only when REC-8, COH-3, and COH-4 are simultaneously disrupted. Premature equational sister separation in rec8 mutants of other organisms suggests the involvement of multiple REC-8 paralogs, which may have masked a conserved requirement for cohesin in AE assembly.     

Caenorhabditis elegans Ce-rdh-1/rad-51 functions after double-strand break formation of meiotic recombination

During meiotic prophase 1, homologous recombination is accompanied by dynamic chromosomal changes. The Ce-rdh-1/rad-51 gene is the only bacterial recA-like gene in the nematode C. elegans genome. Upon depletion of Ce-rdh-1/rad-51 using the RNA interference method, abnormal kinked chromosomes can be observed in mature oocytes at diakinesis, whereas synapsis between homologous chromosomes during the pachytene stage is normal. Following fertilization, Ce-rdh-1/rad-51-depleted embryos die early in embryogenesis, and their nuclei exhibit abnormal chromosome fragments and bridges. From epistasis analyses with Ce-spo-11 defective mutant and ionizing radiation, it is indicated that Ce-rdh-1/rad-51 functions after double-strand break (DSB) formation of meiotic recombination. Under the Ce-chk-2 defective condition, whose meiotic synapsis and meiotic recombination between homologous chromosomes are completely inhibited, the Ce-rdh-1/rad51 is normally expressed in the gonadal cells. Moreover, it seems that exogenous DSBs in the Ce-chk-2 defective nuclei at the pachytene stage can be repaired between sister chromatids in a Ce-rdh-1/rad-51-dependent manner. These results indicate that Ce-rdh-1/rad51 functions after both endogenous and exogenous DSB formation during meiosis, but not as pairing centers for meiotic synapsis.     

Achiasmate segregation of X and B univalents in males of the grasshopperEyprepocnemis plorans is independent of previous association

B chromosomes proved to be more frequent in males than females of the grasshopperEyprepocnemis plorans collected from the population in Jete (Granada, Spain) in 1992. The meiotic behaviour of the X and B univalents was analysed in a high number of 1B males collected from this population in 1991 and 1992, and in males from another population (Salobreña, Granada, Spain) for comparison. These two chromosomes showed a significant tendency to migrate to opposite poles in the Jete population, during the 2 years analysed, but separated randomly in the Salobreña population. Thus, sex differences in the B frequency in Jete seemed to be due to the non-random X-B segregation during male meiosis. The analysis of association patterns between the two univalents over several stages of the first meiotic division indicated a heterochromatic affinity rather than association by chiasmata because most X-B associations had resolved by metaphase I. The X and B chromosomes share two different DNA sequences, so that some associations during prophase I undoubtedly involve homologous DNA sequences. The frequency with which X and B migrated to opposite poles at anaphase I in Jete, however, did not show any significant dependence on previous association at zygotene, diplotene or metaphase I.accepted for publication by J. S. (Pat) Heslop-Harrison     

Common genes and pathways in the regulation of the mitotic and meiotic cell cycles of Schizosaccharomyces pombe

Summary Cell division cycle mutants defective in G₁, DNA replication or nuclear division were tested for sporulation at semi-restrictive temperatures. In cdc1-7, cdc5-120, cdc17-L16 and cdc18-46 no abnormalities were observed; cdc10-129, cdc20-M10, cdc21-M6B, cdc23-M36 and cdc24-M38 formed four-spored asci but with a low efficiency; cdc22-M45 was completely defective in meiosis, but could conjugate and formed zygotes with a single nucleus. Mutants defective in the mitotic initiation genes cdc2, cdc25 and cdc13 were blocked in meiosis II. None of the wee1-50, adh.nim1 ⁺ and win1 ⁺ alleles had any affect on sporulation, suggesting that their interactions with cdc25 and cdc2 are specific to mitosis. The meiotic function of cdc13 is TBZ-sensitive and probably exerted downstream of cdc2. Single mutants in cut1 or cut2 did not effect sporulation, whereas the double mutant cut1 cut2 formed two-spored asci. The results demonstrate that the cell division cycle and the meiotic developmental pathway share common genes and regulatory cascades.     

Genetic evidence for different RAD52-dependent intrachromosomal recombination pathways in Saccharomyces cerevisiae

Mutations in the RecA-like genes RAD51 and RAD57 reduce the frequency of gene conversion/reciprocal exchange between inverted repeats 7-fold. However, they enhance the frequency of deletions between direct repeats 5–12-fold. These induced deletions are RAD1- and RAD52-dependent. On the basis of these results it is proposed that there are several RAD52-dependent pathways of recombination: the recombinational repair pathway of gene conversion/reciprocal exchange dependent on RAD51 and RAD57; a RAD1-and RAD52-dependent pathway exclusively responsible for deletions that are induced in rad51 and rad57 mutants; and finally, it is possible that the gene conversion/reciprocal exchange events observed in rad51 and rad57 strains represent another RAD52-dependent recombination pathway of gene conversion/reciprocal exchange that does not require Rad51 and Rad57 functions. It is also shown that the RAD10 excision-repair gene is involved in long gene conversion tracts in homologous recombination between inverted repeats, as previously observed for RAD1. Finally, an analysis of meiotic recombination reveals that deletions are induced in meiosis 100-fold above mitotic levels, similar to intrachromosomal gene conversion/reciprocal exchange, and that, in contrast to intrachromosomal meiotic gene conversion (50% association), intrachromosomal meiotic gene conversion is not preferentially associated with reciprocal exchange (12–30% of association).     

Functional interactions among members of the meiotic initiation complex in fission yeast

DNA double-strand breaks (DSBs) initiate meiotic recombination in Schizosaccharomyces pombe and in other organisms. The Rec12 protein catalyzes the formation of these DSBs in concert with a multitude of accessory proteins the role of which in this process remains to be discovered. In an all-to-all yeast two-hybrid matrix analysis, we discovered new interactions among putative members of the meiotic recombination initiation complex. We found that Rec7, an axial-element associated protein with homologies to Saccharomyces cerevisiae Rec114, is interacting with Rec24. Rec7 and Rec24 also co-immunoprecipitate in S. pombe during meiosis. An amino acid change in a conserved, C-terminal phenylalanine in Rec7, F325A interrupts the interaction with Rec24. Moreover, rec7F325A shows a recombination deficiency comparable to rec7Δ. Another interaction was detected between Rec12 and Rec14, the orthologs of which in S. cerevisiae Spo11 and Ski8 interact accordingly. Amino acid changes Rec12Q308A and Rec12R309A disrupt the interaction with Rec14, like the according amino acid changes Spo11Q376A and Spo11RE377AA loose the interaction with Ski8. Both amino acid changes in Rec12 reveal a recombination deficient rec12 ⁻ phenotype. We propose that both Rec7–Rec24 and Rec12–Rec14 form subcomplexes of the meiotic recombination initiation complex.     

<Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> Elongin BC complex is essential for cell proliferation and chromosome condensation and segregation during mitosis and meiotic division II

The ubiquitin-mediated protein degradation system is involved in a wide variety of cellular functions. The RING-H2 finger protein RBX1 is a common subunit of Cullin-based ubiquitin ligases. Caenorhabditis elegans RBX1 and CUL2 are essential for regulating chromosome condensation and segregation during mitosis and meiosis and are also critical for cell proliferation. Here, we demonstrate that Elongin B (ELB1) and C (ELC1) form a stable complex, and that depletion of either gene product by RNA-mediated interference (RNAi) causes pronounced defects in the second meiotic division. Embryos and adults that escape meiotic arrest have several irregular phenotypes. These include defects in mitotic chromosomal condensation and segregation, pronuclear rotation, and germ cell proliferation, abnormal cortical protrusion, and accumulation of the cyclin-dependent kinase inhibitor CKI1. All these defects are consistent with those found after depletion of CUL2. In addition, direct interaction between ELC1 and CUL2 is revealed by bacterial two-hybrid analysis. Thus, the RBX1/CUL2/ELC1/ELB1 complex acts as an E3 ubiquitin ligase in C. elegans and is essential for diverse functions relevant to chromosomal dynamics and cell cycle control.     

Neurospora mutants sensitive both to mutagens and to histidine

Summary Previous work in this and other laboratories showed that histidine strongly inhibits growth of mutants at ten out of 20 known mutagen-sensitivity loci in Neurospora, and that nine of the histidine-sensitive mutants disturb meiosis when homozygous. These and other results suggested that histidine affects recombination or DNA repair. Current work with the histidine-sensitive mutant uvs-6 shows that it is also inhibited by several other metabolites but none of them is as effective as histidine. On minimal medium without histidine or other inhibitors, uvs-6 first grows normally, then slows drastically and begins stop-start growth. Conidia from stop-start uvs-6 mycelia produce rejuvenated cultures. The stop-start growth, UV-sensitivity, histidine-sensitivity, and recessive meiotic characters of uvs-6 segregated together in crosses, and reverted together. In tests on other mutagen-sensitive mutants, sensitivity to histidine was strongly correlated with stop-start growth and with sensitivity to other metabolites. Histidine induces premature stop-start growth in at least two mutants. Several possible explanations for the histidine-sensitivity have been excluded.     

Autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast

Autophagy is a conserved intracellular degradation system, which contributes to development and differentiation of various organisms. Yeast cells undergo meiosis under nitrogen‐starved conditions and require autophagy for meiosis initiation. However, the precise roles of autophagy in meiosis remain unclear. Here, we show that autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast. Autophagy‐defective strains bearing a mutation in the autophagy core factor gene atg1, atg7, or atg14 exhibit deformed nuclear structures during meiosis. These mutant cells require an extracellular nitrogen supply for meiosis progression following their entry into meiosis and show delayed meiosis progression even with a nitrogen supply. In addition, they show frequent chromosome dissociation from the spindle together with spindle overextension, forming extra nuclei. Furthermore, Aurora kinase, which regulates chromosome segregation and spindle elongation, is significantly increased at the centromere and spindle in the mutant cells. Aurora kinase down‐regulation eliminated delayed initiation of meiosis I and II, chromosome dissociation, and spindle overextension, indicating that increased Aurora kinase activity may cause these aberrances in the mutant cells. Our findings show a hitherto unrecognized relationship of autophagy with the nuclear structure, regulation of cell cycle progression, and chromosome segregation in meiosis.     

Meiotic double-strand breaks in Schizosaccharomyces pombe

Meiotic DNA double-strand breaks (DSBs) are associated with recombination hot spots in the yeast Saccharomyces cerevisiae and are believed to initiate the process of recombination. Until now, meiosis-induced breaks have not been shown to occur regularly in other organisms. Here we show, by pulsed-field gel electrophoresis of DNA, that meiotic DSBs occur transiently in all three chromosomes of the fission yeast Schizosaccharomyces pombe. In a repair defective mutant, carrying a mutation in the RecA homolog gene rhp51, meiotic DSBs accumulate. In contrast to expectation from the genetic map of S. pombe, however, many chromosomal DNA molecules remain unbroken during meiosis. Received: 27 February 2000 / 12 March 2000