Novel requirements for HAP2-mediated gamete fusion in Tetrahymena.

The ancestral gamete fusion protein, HAP2, catalyzes sperm-egg fusion in a broad range of taxa dating to the last eukaryotic common ancestor. Remarkably, HAP2 orthologs are structurally related to the class II fusogens of modern-day viruses, and recent studies make clear that these proteins utilize similar mechanisms to achieve membrane merger. To identify factors that may regulate HAP2 activity, we screened mutants of the ciliate Tetrahymena thermophila for behaviors that mimic Δhap2 knockout phenotypes in this species. Using this approach, we identified two new genes, GFU1 and GFU2, whose products are necessary for the formation of membrane pores during fertilization and show that the product of a third gene, namely ZFR1, may be involved in pore maintenance and/or expansion. Finally, we propose a model that explains cooperativity between the fusion machinery on apposed membranes of mating cells and accounts for successful fertilization in T. thermophila's multiple mating type system.

Ionic liquids as advantageous solvents for headspace gas chromatography of compounds with low vapor pressure.

The potential of ionic liquids as solvents for headspace gas chromatography was investigated. Three compounds with boiling points above 200 degrees C were selected to demonstrate the feasibility of the concept described. 2-Ethylhexanoic acid, formamide, and tri-n-butylamine as examples of acidic, neutral, and basic analytes were dissolved in acidic 1-n-butyl-3-methylimidazolium hydrogen sulfate (1), neutral 1-n-butyl-2,3-dimethylimidazolium dicyanamide (2), and 2 containing 1,8-diazabicyclo[5.4.0]undec-7-ene to adjust basic conditions. All analytes could be determined with limits of detection and limits of quantification in the low-ppm concentration range.

A Caenorhabditis elegans cohesion protein with functions in meiotic chromosome pairing and disjunction.

We have studied four Caenorhabditis elegans homologs of the Rad21/Scc1/Rec8 sister-chromatid cohesion protein family. Based on the RNAi phenotype and protein localization, it is concluded that one of them, W02A2.6p, is the likely worm ortholog of yeast Rec8p. The depletion of C. elegans W02A2.6p (called REC-8) by RNAi, induced univalent formation and splitting of chromosomes into sister chromatids at diakinesis. Chromosome synapsis at pachytene was defective, but primary homology recognition seemed unaffected, as a closer-than-random association of homologous fluorescence in situ hybridization (FISH) signals at leptotene/zygotene was observed. Depletion of REC-8 also induced chromosome fragmentation at diakinesis. We interpret these fragments as products of unrepaired meiotic double-stranded DNA breaks (DSBs), because fragmentation was suppressed in a spo-11 background. Thus, REC-8 seems to be required for successful repair of DSBs. The occurrence of DSBs in REC-8-depleted meiocytes suggests that DSB formation does not depend on homologous synapsis. Anti-REC-8 immunostaining decorated synaptonemal complexes (SCs) at pachytene and chromosomal axes in bivalents and univalents at diakinesis. Between metaphase I and metaphase II, REC-8 is partially lost from the chromosomes. The partial loss of REC-8 from chromosomes between metaphase I and metaphase II suggests that worm REC-8 might function similarly to yeast Rec8p. The loss of yeast Rec8p from chromosome arms at meiosis I and centromeres at meiosis II coordinates the disjunction of homologs and sister chromatids at the two meiotic divisions.

The Saccharomyces cerevisiae MUM2 gene interacts with the DNA replication machinery and is required for meiotic levels of double strand breaks.

The Saccharomyces cerevisiae MUM2 gene is essential for meiotic, but not mitotic, DNA replication and thus sporulation. Genetic interactions between MUM2 and a component of the origin recognition complex and polymerase alpha-primase suggest that MUM2 influences the function of the DNA replication machinery. Early meiotic gene expression is induced to a much greater extent in mum2 cells than in meiotic cells treated with the DNA synthesis inhibitor hydroxyurea. This result indicates that the mum2 meiotic arrest is downstream of the arrest induced by hydroxyurea and suggests that DNA synthesis is initiated in the mutant. Genetic analyses indicate that the recombination that occurs in mum2 mutants is dependent on the normal recombination machinery and on synaptonemal complex components and therefore is not a consequence of lesions created by incompletely replicated DNA. Both meiotic ectopic and allelic recombination are similarly reduced in the mum2 mutant, and the levels are consistent with the levels of meiosis-specific DSBs that are generated. Cytological analyses of mum2 mutants show that chromosome pairing and synapsis occur, although at reduced levels compared to wild type. Given the near-wild-type levels of meiotic gene expression, pairing, and synapsis, we suggest that the reduction in DNA replication is directly responsible for the reduced level of DSBs and meiotic recombination.

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.

Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin.

It has been proposed but never proven that cohesion between sister chromatids distal to chiasmata is responsible for holding homologous chromosomes together while spindles attempt to pull them toward opposite poles during metaphase of meiosis I. Meanwhile, the mechanism by which disjunction of homologs is triggered at the onset of anaphase I has remained a complete mystery. In yeast, cohesion between sister chromatid arms during meiosis depends on a meiosis-specific cohesin subunit called Rec8, whose mitotic equivalent, Sccl, is cleaved at the metaphase to anaphase transition by an endopeptidase called separin. We show here that cleavage of Rec8 by separin at one of two different sites is necessary for the resolution of chiasmata and the disjunction of homologous chromosomes during meiosis.

Meiotic segregation, synapsis, and recombination checkpoint functions require physical interaction between the chromosomal proteins Red1p and Hop1p.

In yeast, HOP1 and RED1 are required during meiosis for proper chromosome segregation and the consequent formation of viable spores. Mutations in either HOP1 or RED1 create unique as well as overlapping phenotypes, indicating that the two proteins act alone as well as in concert with each other. To understand which meiotic processes specifically require Red1p-Hop1p hetero-oligomers, a novel genetic screen was used to identify a single-point mutation of RED1, red1-K348E, that separates Hop1p binding from Red1p homo-oligomerization. The Red1-K348E protein is stable, phosphorylated in a manner equivalent to Red1p, and undergoes efficient homo-oligomerization; however, its ability to interact with Hop1p both by two-hybrid and coimmunoprecipitation assays is greatly reduced. Overexpression of HOP1 specifically suppresses red1-K348E, supporting the idea that the only defect in the protein is a reduced affinity for Hop1p. red1-K348E mutants exhibit reduced levels of crossing over and spore viability and fail to undergo chromosome synapsis, thereby implicating a role for Red1p-Hop1p hetero-oligomers in these processes. Furthermore, red1-K348E suppresses the sae2/com1 defects in meiotic progression and sporulation, indicating a previously unknown role for HOP1 in the meiotic recombination checkpoint.

Cohesin ensures bipolar attachment of microtubules to sister centromeres and resists their precocious separation.

The multisubunit protein complex cohesin is required to establish cohesion between sister chromatids during S phase and to maintain it during G2 and M phases. Cohesin is essential for mitosis, and even partial defects cause very high rates of chromosome loss. In budding yeast, cohesin associates with specific sites which are distributed along the entire length of a chromosome but are more dense in the vicinity of the centromere. Real-time imaging of individual centromeres tagged with green fluorescent protein suggests that cohesin bound to centromeres is important for bipolar attachment to microtubules. This cohesin is, however, incapable of resisting the consequent force, which leads to sister centromere splitting and chromosome stretching. Meanwhile, cohesin bound to sequences flanking the centromeres prevents sister chromatids from completely unzipping and is required to pull back together sister centromeres that have already split. Cohesin therefore has a central role in generating a dynamic tension between microtubules and sister chromatid cohesion at centromeres, which lasts until chromosome segregation is finally promoted by separin-dependent cleavage of the cohesin subunit Scc1p.

Centromere clustering is a major determinant of yeast interphase nuclear organization.

During interphase in the budding yeast, Saccharomyces cerevisiae, centromeres are clustered near one pole of the nucleus as a rosette with the spindle pole body at its hub. Opposite to the centromeric pole is the nucleolus. Chromosome arms extend outwards from the centromeric pole and are preferentially directed towards the opposite pole. Centromere clustering is reduced by the ndc10 mutation, which affects a kinetochore protein, and by the microtubule poison nocodazole. This suggests that clustering is actively maintained or enforced by the association of centromeres with microtubules throughout interphase. Unlike the Rabl-orientation known from many higher eukaryotes, centromere clustering in yeast is not only a relic of anaphase chromosome polarization, because it can be reconstituted without the passage of cells through anaphase. Within the rosette, homologous centromeres are not arranged in a particular order that would suggest somatic pairing or genome separation.

Bouquet formation in budding yeast: initiation of recombination is not required for meiotic telomere clustering.

Fluorescence in situ hybridization in combination with synaptonemal complex and spindle pole body immunostaining to both spread and structurally preserved nuclei from time course experiments disclosed prominent telomere clustering during meiotic prophase of the yeast Saccharomyces cerevisiae. It was found that centromere clustering, which dominates vegetative nuclear structure, is rapidly lost after induction of meiosis. Telomeres tightly clustered during leptotene/zygotene-equivalent stages in the vicinity of the spindle pole body, giving rise to a classical chromosomal bouquet arrangement. This arrangement dissolved later during prophase. Painting of chromosomes XI revealed that initially compacted chromosome territories adopt an outstretched morphology in bouquet nuclei. This conformational state was associated with alignment and pairing. Chromosome condensation during pachytene rendered condensed and compact bivalents, and dispersed telomeres. Both the spo11 and rad50S recombination mutants formed bouquets, demonstrating that bouquet formation is recombination and synapsis independent.

Meiotic pairing and segregation of translocation quadrivalents in yeast.

Meiotic pairing and segregation were studied in three different heterozygous reciprocal translocation strains of the baker's yeast, Saccharomyces cerevisiae. Pachytene translocation quadrivalents were identified by a combination of immunofluorescence and fluorescence in situ hybridization and the karyotypes of meiotic products were determined by pulsed-field gel electrophoresis. The translocations differed with respect to the relative sizes of the chromosomes involved and the positions of translocation breakpoints, and produced translocation quadrivalents of widely different shapes. This allowed us to study the influence of the morphology of quadrivalents on their segregation behaviour. In all cases alternate predominated over adjacent segregation. 3:1 disjunction of chromosomes was more frequent when translocation breakpoints were close to the centromeres. If a translocation breakpoint was distant from the centromere, the occurrence of an intervening chiasma influenced the pattern of segregation. In general, quadrivalent formation and segregation resembled the behaviour of translocation heterozygotes in most higher eukaryotes. We therefore conclude that, although chromosome condensation does not occur in yeast metaphase, centromere orientation and chromosome disjunction are governed in a way similar to that of higher eukaryotes.

Interlaboratory study of the analysis of benzylpenicillin by liquid chromatography.

A liquid chromatography method for analysis of benzylpenicillin was examined in a collaborative study involving seven laboratories. The method comprised an isocratic part, which is used in the assay. The isocratic part corresponds to the assay method for benzylpenicillin used by a manufacturer. When the isocratic part is combined with gradient elution, the method is suitable for purity control. Five samples of benzylpenicillin (sodium and potassium salts) were analysed. The main component and the impurities were determined. An analysis of variance proved the absence of consistent laboratory bias. The laboratory-sample interaction was not significant. Estimates for the repeatability and reproducibility of the method, expressed as standard deviations (S.D.) of the result of the determination of benzylpenicillin, were calculated to be 0.71 and 0.80, respectively.

Yeast nuclei display prominent centromere clustering that is reduced in nondividing cells and in meiotic prophase.

Chromosome arrangement in spread nuclei of the budding yeast, Saccharomyces cerevisiae was studied by fluorescence in situ hybridization with probes to centromeres and telomeric chromosome regions. We found that during interphase centromeres are tightly clustered in a peripheral region of the nucleus, whereas telomeres tend to occupy the area outside the centromeric domain. In vigorously growing cultures, centromere clustering occurred in approximately 90% of cells and it appeared to be maintained throughout interphase. It was reduced when cells were kept under stationary conditions for an extended period. In meiosis, centromere clusters disintegrated before the emergence of the earliest precursors of the synaptonemal complex. Evidence for the contribution of centromere clustering to other aspects of suprachromosomal nuclear order, in particular the vegetative association of homologous chromosomes, is provided, and a possible supporting role in meiotic homology searching is discussed.

Switching yeast from meiosis to mitosis: double-strand break repair, recombination and synaptonemal complex.

BACKGROUND: When Saccharomyces cerevisiae cells that have begun meiosis are transferred to mitotic growth conditions ('return-to-growth', RTG), they can complete recombination at high meiotic frequencies, but undergo mitotic cell division and remain diploid. It was not known how meiotic recombination intermediates are repaired following RTG. Using molecular and cytological methods, we investigated whether the usual meiotic apparatus could repair meiotically induced DSBs during RTG, or whether other mechanisms are invoked when the developmental context changes. RESULTS: Upon RTG, the rapid disappearance of meiotic features--double-strand breaks in DNA (DSBs), synaptonemal complex (SC), and SC related structures-was striking. In wild-type diploids, the repair of meiotic DSBs during RTG was quick and efficient, resulting in homologous recombination. Kinetic analysis of double-strand breakage and recombination indicated that meiotic DSB formation precedes the commitment to meiotic levels of recombination. DSBs were repaired in RTG in dmc1, but not rad51 mutants, hence repair did not occur by the usual meiotic mechanism which requires the Dmc1 gene product. In haploids, DSBs were also repaired quickly and efficiently upon RTG, showing that DSB repair did not require the presence of a homologous chromosome. In all strains examined, SC and related structures were not required for DSB repair or recombination following RTG. CONCLUSIONS: At least two pathways of DSB repair, which differ from the primary meiotic pathway(s), can occur during RTG: One involving interhomologue recombination, and another involving sister-chromatid exchange. DSB formation precedes commitment to recombination. SC elements appear to prevent sister chromatid exchange in meiosis.

Karyotype variability in yeast caused by nonallelic recombination in haploid meiosis.

Chromosomes of altered size were found in the meiotic products of a haploid Saccharomyces cerevisiae strain by pulsed field gel electrophoretic separation of whole chromosomes. About 7% of haploid meioses produced chromosomes that differed by > or = 10 kb from their wild-type counterparts. Chromosomes most often became enlarged or shortened due to recombination events between sister chromatids at nonallelic sequences. By this mechanism chromosome III acquired tandem arrays of up to eight extra copies of the approximately kb MAT-HMR segment during repeated rounds of haploid meioses. Enlarged chromosomes III were unstable and changed their size during meiosis more often than remaining unchanged. Altered chromosomes appeared also as the products of intrachromatid recombination and of reciprocal translocations caused by ectopic recombination between nonhomologous chromosomes. In diploid meiosis, chromosomes of altered size occurred at least 10 times less frequently, whereas in mitotic cultures cells with altered karyotypes were virtually absent. The results show that various forms of ectopic recombination are promoted by the absence of allelic homologies.

Morphology of a human-derived YAC in yeast meiosis.

In meiosis of human males DNA is packaged along pachytene chromosomes about 20 times more compactly than in meiosis of yeast. Nevertheless, a human-derived yeast artificial chromosome (YAC) shows the same degree of compaction of DNA as endogenous chromosomes in meiotic prophase nuclei of yeast. This suggests that in yeast meiosis, human and yeast DNA adopt a similar organization of chromatin along the pachytene chromosome cores. Therefore meiotic chromatin organization does not seem to be an inherent chromosomal property but is governed by the host-specific cellular environment. We suggest that there is a correlation between the less dense DNA packaging and the increased rate of recombination that has been reported for human-derived YACs as compared with human DNA in its natural environment.

The yeast MER2 gene is required for chromosome synapsis and the initiation of meiotic recombination.

Mutation of the MER2 gene of Saccharomyces cerevisiae confers meiotic lethality. To gain insight into the function of the Mer2 protein, we have carried out a detailed characterization of the mer2 null mutant. Genetic analysis indicates that mer2 completely eliminates meiotic interchromosomal gene conversion and crossing over. In addition, mer2 abolishes intrachromosomal meiotic recombination, both in the ribosomal DNA array and in an artificial duplication. The results of a physical assay demonstrate that the mer2 mutation prevents the formation of meiosis-specific, double-strand breaks, indicating that the Mer2 protein acts at or before the initiation of meiotic recombination. Electron microscopic analysis reveals that the mer2 mutant makes axial elements, which are precursors to the synaptonemal complex, but homologous chromosomes fail to synapse. Fluorescence in situ hybridization of chromosome-specific DNA probes to spread meiotic chromosomes demonstrates that homolog alignment is also significantly reduced in the mer2 mutant. Although the MER2 gene is transcribed during vegetative growth, deletion or overexpression of the MER2 gene has no apparent effect on mitotic recombination or DNA damage repair. We suggest that the primary defect in the mer2 mutant is in the initiation of meiotic genetic exchange.

Simulation of chromosomal homology searching in meiotic pairing.

A cellular automaton model has been developed to simulate some aspects of chromosome behaviour during meiotic prophase when homologous chromosomes search for each other so that they can pair. Simulations allow one to compare the relative efficiencies of random searching by chromosome shuffling along the inner nuclear membrane or within the lumen of the nucleus, the effects of biased movements and of chromosome clustering (bouquet or synizetic knot), different numbers of homology recognition sites per chromosome and different chromosome numbers on the efficiency of the pairing process. Parameters of the simulated models can be easily adjusted to fit experimentally obtained figures.

Meiotic chromosome pairing in triploid and tetraploid Saccharomyces cerevisiae.

Meiotic chromosome pairing in isogenic triploid and tetraploid strains of yeast and the consequences of polyploidy on meiotic chromosome segregation are studied. Synaptonemal complex formation at pachytene was found to be different in the triploid and in the tetraploid. In the triploid, triple-synapsis, that is, the connection of three homologues at a given site, is common. It can even extend all the way along the chromosomes. In the tetraploid, homologous chromosomes mostly come in pairs of synapsed bivalents. Multiple synapsis, that is, synapsis of more than two homologues in one and the same region, was virtually absent in the tetraploid. About five quadrivalents per cell occurred due to the switching of pairing partners. From the frequency of pairing partner switches it can be deduced that in most chromosomes synapsis is initiated primarily at one end, occasionally at both ends and rarely at an additional intercalary position. In contrast to a considerably reduced spore viability (approximately 40%) in the triploid, spore viability is only mildly affected in the tetraploid. The good spore viability is presumably due to the low frequency of quadrivalents and to the highly regular 2:2 segregation of the few quadrivalents that do occur. Occasionally, however, quadrivalents appear to be subject to 3:1 nondisjunction that leads to spore death in the second generation.