Sensitivity of meiotic yeast cells to ultraviolet light

Sporulating cells of Saccharomyces cerevisiae show an increasing sensitivity to ultraviolet irradiation. Maximum sensitivity is reached at a time comparable to meiotic prophase. Sensitivity is expressed as reduced sporulation after the irradiation. The uv effect can be efficiently reversed by photoreactivating light. Viability is also more severely affected during premeiotic DNA synthesis and during meiosis than in earlier stages in sporulation. Cells left in sporulation medium after the irradiation show a reduced viability compared with the cells plated immediately after the irradiation. Non-sporulating diploids do not acquire sensitivity when exposed to sporulation medium, hence the sensitivity is related to the sporulation process. That meiosis itself is affected, rather than spore formation alone, is evident from experiments in which the uv irradiation interferes with the uncovering of a recessive marker and with commitment to meiosis. It is proposed that during meiotic prophase, the DNA repair system is different from that found in vegetative cells.     

Differential effect of UV irradiation on induction of intragenic and intergenic recombination during commitment to meiosis in Saccharomyces cerevisiae

A comparison was made between the induction of intragenic and intergenic recombinations during meiosis in a wild-type diploid of Saccharomyces cerevisiae. Under non-irradiated normal conditions, production of both intragenic and intergenic recombinants greatly increased in the cells with commitment to meiosis. The susceptibility of cells to the induction of both the spontaneous intra- and intergenic recombinations in meiotic cells was similar. However, under condition of UV irradiation, there were striking differences between intra- and intergenic recombinations. Susceptibility to induction of intragenic recombination by UV irradiation was not enhanced at meiosis compared with mitosis, and was not altered through commitment to meiotic processes. In contrast, however, susceptibility to the induction of intergenic recombination by UV irradiation was enhanced markedly during commitment to meiosis compared with mitosis. Genetic analysis suggested that the enhanced susceptibility to recombination during meiosis is specifically concerned with reciprocal-type recombination (crossing-over) but not non-reciprocal-type recombination (gene conversion). Hence it is concluded that the meiotic process appears to be intimately concerned with the mechanism(s) of induction of recombination, especially reciprocal-type recombination.     

Commitment to meiosis in fission yeast

Summary Mutants of Schizosaccharomyces pombe blocked during meiosis were analysed with respect to the induction of diploid mitotic division. Wild type zygotes of this yeast can form diploid colonies with a low probability (ca. 1%) when they are transferred to fresh growth medium. Mutants of three genes affecting meiosis responded to the shift by forming diploid colonies with high yield (ca. 80% of the zygotes). Hence, commitment to meiosis could not be reached by such zygotes. Zygotes of a fourth meiosis-I-deficient mutant were unable to yield diploid colonies more effectively than wild type zygotes. Morphological changes were prominent in the mutant zygotes not reaching commitment (as well as in mutant cells that could not complete conjugation), indicating that activities needed during early stages of conjugation were not turned off in these zygotes.This work was supported by NIH grant GM 13234 initially, and by DFG (SFB 46).     

Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis

Mouse oocytes are reversibly inhibited from resuming meiotic maturation in vitro by cAMP phosphodiesterase inhibitors such as 3-isobutyl-1-methyl xanthine (IBMX) and cAMP analogs such as dibutyryl cAMP (dbcAMP). Oocytes cultured in IBMX-containing medium were transferred to and cultured in IBMX-free medium for various periods of time prior to their return to either IBMX- or dbcAMP-containing medium. Results from these experiments defined a period of time in which oocytes became committed to resuming meiosis. Forskolin, which elevated the intracellular oocyte cAMP concentration, transiently inhibited oocytes from resuming meiosis. Levels of cAMP were determined in oocytes incubated in medium that allows resumption of meiosis. The level of oocyte cAMP decreased significantly during the time in which oocytes become committed to resuming meiosis. This decrease in oocyte cAMP was not observed in oocytes inhibited from resuming meiosis by IBMX. In addition, cAMP levels were determined in preovulatory antral follicles, cumulus cell-oocyte complexes, and oocytes during gonadotropin-induced resumption of meiosis in vivo. A decrease in oocyte cAMP preceded resumption of meiosis as manifested by germinal vesicle breakdown (GVBD). This decrease apparently occurred before or during a period of time in which follicle and cumulus cell cAMP were increasing. Associated with commitment to resume meiosis was a characteristic set of changes in oocyte phosphoprotein metabolism that preceded GVBD. These changes are, to date, some of the first reported biochemical changes that precede GVBD. Results from these experiments are discussed in terms of a possible role cAMP may play in regulation of resumption of meiosis in mammals.     

Meiosis Can Induce Recombination in rad52 Mutants of SACCHAROMYCES CEREVISIAE

The RAD52 and RAD50 genes have previously been shown to be required for normal meiotic recombination and for various types of recombination occurring in mitotic cells. Recent evidence suggests that rad52 mutants might be defective in an intermediate recombination step; we therefore examined recombination during meiosis in several rad52 mutants at several different loci and in genetic backgrounds that yield efficient sporulation and synchronous meiosis. Similar to previous reports, spores from rad52 diploids are inviable and meiotic recombination is greatly reduced by rad52 mutations. However, intragenic recombinants were detected when cells were plated on selective media during meiosis; rad52 mutants experience induction of recombination between homologues under these special conditions. The frequencies of recombination at four loci were considerably greater than the mitotic controls; however, they were still at least 20 times lower than corresponding Rad+ strains. The prototrophs induced by meiosis in rad52 mutants were not typical meiotic recombinants because incubation in nutrient-rich medium before plating to selective medium resulted in the complete loss of recombinants. We propose that previously observed single-strand breaks that accumulate in rad52 mutants may be associated with recombinational intermediates that are resolved when cells are returned to selective mitotic media and that the meiosis-induced recombination in rad52 cells does not involve double-strand breaks.     

Initiation of meiosis in cell cycle initiation mutants of Saccharomyces cerevisiae

Control of the initiation of meiosis in yeast was examined in diploids homozygous for one of four different temperature-sensitive mutations that affect “start” of the mitotic cell cycle. Two of the mutations, cdc28 and tra3, bring about deficiencies in the initiation of meiosis, while cdc25 and cdc35 do not prevent initiation of normal meiosis at both permissive and restrictive temperatures. Moreover, diploids homozygous for the latter two mutations are capable of initiating meiosis in rich growth media upon transfer to the high, non-permissive temperature. This unique feature contrasts with the behavior of other yeast strains which require a starvation sporulation medium for initiation of meiosis. It is suggested that the initiation of meiosis includes functions that are shared with “start” of the mitotic cell cycle, as well as functions related to the choice between the two processes. Meiosis in vegetative media at the restrictive temperature (in cdc25 or cdc35 homozygotes) may be important for the study of chemical and physiological phenomena resulting from the meiotic process and not from adaptation to the sporulation medium.     

Nutritional regulation of late meiotic events in Saccharomyces cerevisiae through a pathway distinct from initiation.

The IME1 gene is essential for initiation of meiosis in the yeast Saccharomyces cerevisiae, although it is not required for growth. Here we report that in stationary-phase cultures containing low concentration of glucose, cells overexpressing IME1 undergo the early meiotic events, including DNA replication, commitment to recombination, and synaptonemal complex formation and dissolution. In contrast, later meiotic events, such as chromosome segregation, commitment to meiosis, and spore formation, do not occur. Thus, nutrients can repress the late stages of meiosis independently of their block of initiation. Cells arrested at this midpoint in meiosis are relatively stable and can resume meiotic differentiation if transferred to sporulation conditions. Resumption of meiosis does not require repression of IME1 expression, since IME1 RNA levels stay high after transfer of the arrested cells to sporulation medium. These results suggest that meiosis in S. cerevisiae is a paradigm of a differentiation pathway regulated by signal transduction at both early and late stages.     

Meiosis as a source of spontaneous mutations in Schizophyllum commune

Spontaneous mutation frequencies were determined for two loci in the fungus Schizophyllum commune, at meiosis and at mitosis. For both loci the meiotic frequency is significantly higher than the mitotic frequency. No correlation was found between meiotic mutagenesis and recombination of markers bracketing the mutant site. The meiotic temperature affected the spontaneous mutation frequency but not the recombination frequency in the cross examined.A number of suppressor mutations were detected for both loci examined. Almost all the suppressors are closely linked to the site they suppress. The distribution of mutations among the suppressor sites was different at meiosis and at mitosis.     

Commitment to meiosis: what determines the mode of division in budding yeast?

In budding yeast, commitment to meiosis is attained when meiotic cells cannot return to the mitotic cell cycle even if the triggering cue (nutrients deprivation) is withdrawn. Commitment is arrived at gradually, and different aspects of meiosis may be committed at different times. Cells become fully committed to meiosis at the end of Prophase I, long after DNA replication and just before the first meiotic division (M_I). Whole‐genome gene expression analysis has shown that committed cells have a distinct and rapid response to nutrients, and are not simply insulated from environmental signals. Thus becoming committed to meiosis is an active process. The cellular event most likely to be associated with commitment to meiosis is the separation of the duplicated spindle‐pole bodies (SPBs) and the formation of the spindle. Commitment to the mitotic cell cycle is also associated with the separation of SPBs, although it occurs in G1, before DNA replication.     

The Role of the Ameiotic1 Gene in the Initiation of Meiosis and in Subsequent Meiotic Events in Maize

Understanding the initiation of meiosis and the relationship of this event with other key cytogenetic processes are major goals in studying the genetic control of meiosis in higher plants. Our genetic and structural analysis of two mutant alleles of the ameiotic1 gene (am1 and am1-praI) suggest that this locus plays an essential role in the initiation of meiosis in maize. The product of the ameiotic1 gene affects an earlier stage in the meiotic sequence than any other known gene in maize and is important for the irreversible commitment of cells to meiosis and for crucial events marking the passage from premeiotic interphase into prophase I including chromosome synapsis. It appears that the period of ameiotic1 gene function in meiosis at a minimum covers the interval from some point during premeiotic interphase until the early zygotene stage of meiosis. To study the interaction of genes in the progression of meiosis, several double meiotic mutants were constructed. In these double mutants (i) the ameiotic1 mutant allele was brought together with the meiotic mutation (afd1) responsible for the fixation of centromeres in meiosis; and with the mutant alleles of the three meiotic genes that control homologous chromosome segregation (dv1, ms43 and ms28), which impair microtubule organizing center organization, the orientation of the spindle fiber apparatus, and the depolymerization of spindle filaments after the first meiotic division, respectively; (ii) the afd1 mutation was combined with two mutations (dsy1 and as1) affecting homologous pairing; (iii) the ms43 mutation was combined with the as1, the ms28 and the dv1 mutations; and (iv) the ms28 mutation was combined with the dv1 mutation and the ms4 (polymitotic1) mutations. An analysis of gene interaction in the double mutants led us to conclude that the ameiotic1 gene is epistatic over the afd1, the dv1, the ms43 and the ms28 genes but the significance of this relationship requires further analysis. The afd gene appears to function from premeiotic interphase throughout the first meiotic division, but it is likely that its function begins after the start of the ameiotic1 gene expression. The afd1 gene is epistatic over the two synaptic mutations dsy1 and as1 and also over the dv1 mutation. The new ameiotic*-485 and leptotene arrest*-487 mutations isolated from an active ROBERTSON's Mutator stocks take part in the control of the initiation of meiosis.     

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.     

Sisters Unbound Is Required for Meiotic Centromeric Cohesion in Drosophila melanogaster

Regular meiotic chromosome segregation requires sister centromeres to mono-orient (orient to the same pole) during the first meiotic division (meiosis I) when homologous chromosomes segregate, and to bi-orient (orient to opposite poles) during the second meiotic division (meiosis II) when sister chromatids segregate. Both orientation patterns require cohesion between sister centromeres, which is established during meiotic DNA replication and persists until anaphase of meiosis II. Meiotic cohesion is mediated by a conserved four-protein complex called cohesin that includes two structural maintenance of chromosomes (SMC) subunits (SMC1 and SMC3) and two non-SMC subunits. In Drosophila melanogaster, however, the meiotic cohesion apparatus has not been fully characterized and the non-SMC subunits have not been identified. We have identified a novel Drosophila gene called sisters unbound (sunn), which is required for stable sister chromatid cohesion throughout meiosis. sunn mutations disrupt centromere cohesion during prophase I and cause high frequencies of non-disjunction (NDJ) at both meiotic divisions in both sexes. SUNN co-localizes at centromeres with the cohesion proteins SMC1 and SOLO in both sexes and is necessary for the recruitment of both proteins to centromeres. Although SUNN lacks sequence homology to cohesins, bioinformatic analysis indicates that SUNN may be a structural homolog of the non-SMC cohesin subunit stromalin (SA), suggesting that SUNN may serve as a meiosis-specific cohesin subunit. In conclusion, our data show that SUNN is an essential meiosis-specific Drosophila cohesion protein.     

Concerted Evolution in the Ribosomal RNA Cistron

Background

Gene conversion is the mechanism proposed to be responsible for the homogenization of multigene families such as the nuclear ribosomal gene clusters. This concerted evolutionary process prevents individual genes in gene clusters from accumulating mutations. The mechanism responsible for concerted evolution is not well understood but recombination during meiosis has been hypothesized to play a significant role in this homogenization. In this study we tested the hypothesis of unequal crossing over playing a significant role in gene conversion events within the ribosomal RNA cistron during meiosis, mitosis or both life stages in the fungal tree pathogen Ceratocystis manginecans.

Methods

Ceratocystis manginecans, a haploid ascomycete, reproduces homothallically and was found to have two distinct sequences within the internally transcribed spacer (ITS) region of the ribosomal RNA cistron. The different ITS types were scored using PCR-RFLP assays and chi-square analyses to determine the level of significance of the changes in the ratios of the ITS types.

Results

The relative ratios of the two ITS sequence types changed when the fungal isolates were cultured vegetatively or allowed to produced sexual structures and spores. These active changes were shown to occur more frequently during meiosis than mitosis.

Conclusion

The evidence presented provides concrete support for homogenization in the rRNA gene clusters found in this fungus and that the most reasonable explanation for this process is unequal crossing over.     

The CYCLIN-A CYCA1;2/TAM Is Required for the Meiosis I to Meiosis II Transition and Cooperates with OSD1 for the Prophase to First Meiotic Division Transition

Meiosis halves the chromosome number because its two divisions follow a single round of DNA replication. This process involves two cell transitions, the transition from prophase to the first meiotic division (meiosis I) and the unique meiosis I to meiosis II transition. We show here that the A-type cyclin CYCA1;2/TAM plays a major role in both transitions in Arabidopsis. A series of tam mutants failed to enter meiosis II and thus produced diploid spores and functional diploid gametes. These diploid gametes had a recombined genotype produced through the single meiosis I division. In addition, by combining the tam-2 mutation with AtSpo11-1 and Atrec8, we obtained plants producing diploid gametes through a mitotic-like division that were genetically identical to their parents. Thus tam alleles displayed phenotypes very similar to that of the previously described osd1 mutant. Combining tam and osd1 mutations leads to a failure in the prophase to meiosis I transition during male meiosis and to the production of tetraploid spores and gametes. This suggests that TAM and OSD1 are involved in the control of both meiotic transitions.     

Frameshift mutagenesis by ultraviolet light effects of broth and caffeine in the post-irradiation plating medium

Ultraviolet-induced back mutation yields were studied in the frameshift strain of Salmonella typhimurium, LT2 hisC3076. The numbers and frequencies per 10⁸ survivors of small and large revertant colonies were found to be affected significantly by plating density, but it was possible to detect a considerable enhancement of mutation frequency when broth (2.5%, v/v) was present in the post-irradiation plating medium. Caffeine also significantly enhanced the yields of UV-induced frameshift mutations, but not of γ-induced frameshifts, indicating that the UV-induced pre-mutational lesions which lead to frameshift mutations may be treated in a similar way by the excision-repair system to those which lead to base-pair substitutions.     

Role for rodent Smc6 in pericentromeric heterochromatin domains during spermatogonial differentiation and meiosis

Chromatin structure and function are for a large part determined by the six members of the structural maintenance of chromosomes (SMC) protein family, which form three heterodimeric complexes: Smc1/3 (cohesin), Smc2/4 (condensin) and Smc5/6. Each complex has distinct and important roles in chromatin dynamics, gene expression and differentiation. In yeast and Drosophila, Smc6 is involved in recombinational repair, restarting collapsed replication forks and prevention of recombination in repetitive sequences such as rDNA and pericentromeric heterochromatin. Although such DNA damage control mechanisms, as well as highly dynamic changes in chromatin composition and function, are essential for gametogenesis, knowledge on Smc6 function in mammalian systems is limited. We therefore have investigated the role of Smc6 during mammalian spermatogonial differentiation, meiosis and subsequent spermiogenesis. We found that, during mouse spermatogenesis, Smc6 functions as part of meiotic pericentromeric heterochromatin domains that are initiated when differentiating spermatogonia become irreversibly committed toward meiosis. To our knowledge, we are the first to provide insight into how commitment toward meiosis alters chromatin structure and dynamics, thereby setting apart differentiating spermatogonia from the undifferentiated spermatogonia, including the spermatogonial stem cells. Interestingly, Smc6 is not essential for spermatogonial mitosis, whereas Smc6-negative meiotic cells appear unable to finish their first meiotic division. Importantly, during meiosis, we find that DNA repair or recombination sites, marked by γH2AX or Rad51 respectively, do not co-localize with the pericentromeric heterochromatin domains where Smc6 is located. Considering the repetitive nature of these domains and that Smc6 has been previously shown to prevent recombination in repetitive sequences, we hypothesize that Smc6 has a role in the prevention of aberrant recombination events between pericentromeric regions during the first meiotic prophase that would otherwise cause chromosomal aberrations leading to apoptosis, meiotic arrest or aneuploidies.     

Acetate as Sole Carbon and Energy Source for Growth of Methanosarcina Strain 227

Methanosarcina strain 227 grew rapidly and produced methane on a mineral medium containing acetate as the sole added organic substrate. Cell yields but not doubling times were affected by the presence or absence of yeast extract. Greater cell yields occurred in yeast extract medium than in mineral medium. Radioactive labeling studies showed that acetate was decarboxylated in mineral medium, as was shown previously in complex medium. The specific radioactivity of methane produced per specific acitvity of acetate added was not significantly different in yeast extract medium compared with mineral medium. Unequivocal evidence indicates that the cleavage of acetate to methane and carbon dioxide provided the energy for growth in the presence or absence of other organic compounds; these latter compounds do not serve as energy sources, electron donors, or significant sources of methane during this aceticlastic reaction.