Morphology of mitochondrial nucleoids, mitochondria, and nuclei during meiosis and sporulation of the yeast Saccharomycodes ludwigii

The morphology of mitochondrial nucleoids (mt-nucleoids), mitochondria, and nuclei was investigated during meiosis and sporulation of the diploid cells of the ascosporogenic yeast Saccharomycodes ludwigii. The mt-nucleoids appeared as discrete dots uniformly distributed in stationary-phase cells as revealed by 4',6-diamidino-2-phenylindole (DAPI) staining. Throughout first and second meiotic divisions, the mt-nucleoids moved to be located close to the dividing nuclei with the appearance of dots. On the other hand, mitochondria, which had tubular or fragmented forms in stationary-phase cells, increasingly fused with each other to form elongated mitochondria during meiotic prophase as revealed by 3,3' -dihexyloxacarbocyanine iodide [DiOC(6)(3)] staining. Mitochondria assembled to be located close to dividing nuclei during first and second meiotic divisions, and were finally incorporated into spores. During the first meiotic division, nuclear division occurred in any direction parallel, diagonally, or perpendicular to the longitudinal axis of the cell. In contrast, the second meiotic division was exclusively parallel to the longitudinal axis of the cell. The behavior of dividing nuclei explains the formation of a pair of spores with opposite mating types at both ends of cells. In the course of this study, it was also found that ledges between two spores were specifically stained with DiOC(6)(3).     

Stage-Specific Effects of X-Irradiation on Yeast Meiosis

Previous work has shown that cdc13 causes meiotic arrest of Saccharomyces cerevisiae following DNA replication by a RAD9-dependent mechanism. In the present work, we have further investigated the implicit effects of chromosomal lesions on progression through meiosis by exposing yeast cells to X-irradiation at various times during sporulation. We find that exposure of RAD9 cells to X-irradiation early in meiosis prevents sporulation, arresting the cells at a stage prior to premeiotic DNA replication. rad9 meiotic cells are much less responsive to X-irradiation damage, completing sporulation after treatment with doses sufficient to cause arrest of RAD9 strains. These findings thereby reveal a RAD9-dependent checkpoint function in meiosis that is distinct from the G(2) arrest previously shown to result from cdc13 dysfunction. Analysis of the spores that continued to be produced by either RAD9 or rad9 cultures that were X-irradiated in later stages of sporulation revealed most spores to be viable, even after exposure to radiation doses sufficient to kill most vegetative cells. This finding demonstrates that the lesions induced by X-irradiation at later times fail to trigger the checkpoint function revealed by cdc13 arrest and suggests that the lesions may be subject to repair by serving as intermediates in the recombination process. Strains mutant for chromosomal synapsis and recombination, and therefore defective in meiotic disjunction, were tested for evidence that X-ray-induced lesions might alleviate inviability by promoting recombination. Enhancement of spore viability when spo11 (but not hop1) diploids were X-irradiated during meiosis indicates that induced lesions may partially substitute for SPO11-dependent functions that are required for the initiation of recombination.     

Differential Mitotic Stability of Yeast Disomes Derived from Triploid Meiosis

The frequencies of recovered disomy among the meiotic segregants of yeast (Saccharomyces cerevisiae) triploids were assessed under conditions in which all 17 yeast chromosomes were monitored simultaneously. The studies employed inbred triploids, in which all homologous centromeres were identical by descent, and single haploid testers carrying genetic markers for all 17 linkage groups. The principal results include: (1) Ascospores from triploid meiosis germinate at frequencies comparable to those from normal diploids, but most fail to produce visible colonies due to the growth-retarding effects of high multiple disomy. (2) The probability of disome formation during triploid meiosis is the same for all chromosomes; disomy for any given chromosome does not exclude simultaneous disomy for any other chromosome. (3) The 17 yeast chromosomes fall into three frequency classes in terms of disome recovery. The results support the idea that multiply disomic meiotic segregants of the triploid experience repeated, nonrandom, post-germination mitotic chromosome losses (N + 1 leads to N) and that the observed variations in individual disome recovery are wholly attributable to inherent differences in disome mitotic stability.     

Detection of the loss of 1 chromosome from pair III in Saccharomyces cerevisiae yeasts

A diploid strain of Saccharomyces cerevisiae, T6 is described which monitors both mitotic crossing over and induction of aneuploidy. The chromosome III carries recessive markers: rgh12 of "rough colony" phenotype closely linked to centromere, the left arm is marked with his4, the right arm is marked both with thr4 and the locus of mating type alpha. Expression of all the markers on chromosome III leads to formation of colonies which are rough, require histidine and threonine, and are of alpha mating type. These colonies arise as a result of the loss of a chromosome during mitosis, which makes the strain allow detection of monosomic cells formation. Chromosome XV carries two phenotypically distinguishable and recessive alleles of the gene ade2: ade2-192 (causes red coloration of colonies) and ade2-G45 (causes pink coloration of colonies). Mitotic crossing over generates two reciprocal products which can be revealed together in colonies as pink and red sectors in double-spotted colonies. Both double-spotted and monosomic colonies have been obtained after treatment with gamma-rays. The frequency of mitotic crossing over after irradiation by 1000-3000 Gray increased up to 2-3.2% (the spontaneous level was 0.006%), the frequency of aneuploidy was 0.12 to 0.57% at plating immediately after irradiation (the spontaneous monosomics were not observed among 1.5 X 10(5) cells scored). Induction of mitotic crossing over and aneuploidy by benomyl was rather slight (up to 0.05 and 0.006%, respectively).     

Alphoid variant-specific FISH probes can distinguish autosomal meiosis I from meiosis II non-disjunction in human sperm

Over the past few years, several groups have used fluorescence in situ hybridization (FISH) to study aneuploidy in human sperm. Several important observations have derived from these studies, including the demonstration of chromosome-specific variation in non-disjunction frequencies, and the possible association of aneuploidy with environmental agents and with increasing paternal age. However, an important technical limitation of these studies has been the inability to distinguish between autosomal non-disjunction occurring at meiosis I and meiosis II. In the present report, we describe a simple FISH-based approach designed to overcome this limitation. Using oligonucleotide probes capable of distinguishing subtle differences in the alpha satellite sequences of chromosome 17, we demonstrate that (in appropriate heterozygotes) it is possible to simultaneously identify disomic sperm and to determine the meiotic stage of origin of the additional chromosome. This novel approach has important implications for future FISH sperm studies, since the ability to distinguish between meiosis I and meiosis II non-disjunction will make it possible to determine whether putative etiological agents affect chromosome segregation at both, or only one, of the two meiotic stages. Received: 19 March 1997 / Accepted: 12 June 1997     

Distributive Disjunction of Authentic Chromosomes in Saccharomyces Cerevisiae

Distributive disjunction is defined as the first division meiotic segregation of either nonhomologous chromosomes that lack homologs or homologous chromosomes that have not recombined. To determine if chromosomes from the yeast Saccharomyces cerevisiae were capable of distributive disjunction, we constructed a strain that was monosomic for both chromosome I and chromosome III and analyzed the meiotic segregation of the two monosomic chromosomes. In addition, we bisected chromosome I into two functional chromosome fragments, constructed strains that were monosomic for both chromosome fragments and examined meiotic segregation of the chromosome fragments in the monosomic strains. The two nonhomologous chromosomes or chromosome fragments appeared to segregate from each other in approximately 90% of the asci analyzed, indicating that yeast chromosomes were capable of distributive disjunction. We also examined the ability of a small nonhomologous centromere containing plasmid to participate in distributive disjunction with the two nonhomologous monosomic chromosomes. The plasmid appeared to efficiently participate with the two full length chromosomes suggesting that distributive disjunction in yeast is not dependent on chromosome size. Thus, distributive disjunction in S. cerevisiae appears to be different from Drosophila melanogaster where a different sized chromosome is excluded from distributive disjunction when two similar size nonhomologous chromosomes are present.     

Disomic and diploid meiotic products in Saccharomyces cerevisiae. Effect of vincristine, vinblastine, adriamycin, bleomycin, mitomycin C and cyclophosphamide

The effect of some antineoplastic drugs on the induction of disomic and diploid meiotic products in Saccharomyces cerevisiae was evaluated. Vincristine and vinblastine turned out not to be effective on any of the four genetic phenomena studied (sporulation, recombination, disomic induction and diploid induction). Adriamycin showed only slight activity in inducing diploids, particularly during the first meiotic division. Cyclophosphamide was active on both phenomena leading to the formation of disomic and diploid spores. Mitomycin C and bleomycin were effective on the induction of diploids. In all of these inductions, the origin of diploids was due to failure of the second meiotic division. No significant effects were found on recombination frequency. As a general conclusion, one may assume that formation of aneuploid and diploid gametes are two distinct phenomena, not necessarily correlated from the point of view of the mechanism and of the specificity of induction.     

Trichlorfon effects on mouse oocytes following in vivo exposure

Trichlorfon (TCF) is a widely used pesticide, which according to some epidemiological and experimental data, is suspected of being aneugenic in human and mouse cells. In particular, in vitro studies in mouse oocytes showed the induction of aneuploidy and polyploidy at the first meiotic division and of severe morphological alterations of the second meiotic spindle. We have tested the hypothesis that an acute treatment of mice with TCF might similarly affect chromosome segregation in maturing oocytes. Superovulated MF-1 mice were intraperitoneally injected with 400mg/kg TCF or orally administered with 600mg/kg TCF either at the time of or 4h after human chorionic gonadotrophin (HCG) injection. Oocytes were harvested 17h after HCG and metaphase II chromosomes were cytogenetically analyzed. No significant increase of aneuploid or polyploid cells was detected at any treatment condition. A significant (p<0.001) decrease of metaphases showing premature chromatid separation or premature anaphase II in all TCF-treated groups with respect to controls suggested that TCF treatment may have delayed the first meiotic division. To evaluate possible effects of the pesticide upon the second meiotic division, a group of females orally treated with 600mg/kg TCF at resumption of meiosis was mated with untreated males and zygotes were collected for cytogenetic analysis. No evidence of aneuploidy induction was obtained, but the frequency of polyploid zygotes was increased fivefold over the control level (p<0.01). Such polyploid embryos might have arisen from fertilization of oocytes that were either meiotically delayed and still in metaphase I at fertilization or progressed through anaphase II without cytokinesis. These findings show that in vivo studies on aneuploidy induction in oocytes may yield results different from those obtained by in vitro experiments and that both kinds of data may be necessary for risk assessment of environmentally relevant exposures.     

Substructural studies on sporulation of Saccharomycopsis lipolytica

During sporulation of diploids from crosses between different strains of the yeast Saccharomycopsis (Candida) lipolytica irregular numbers of ascospores per ascus have been observed. Using the serial section method it could be shown now by means of electron microscopy that in one-, two-, and three-spored asci unenclosed "naked" nuclei occur additionally to nuclei incorporated in mature spores. It was demonstrated that the production of less than four spores per ascus in this yeast is not the result of a lack of meiotic products but of the nonutilization of nuclei from meiosis. In 2--4 spored asci usually four products of meiosis in form of enclosed and free nuclei could be demonstrated which indicate a normal meiotic division. All ascospores derived from asci with different spore numbers are uninuclear. It is assumed that a defect in spore formation caused by structural changes of chromosomes or aneuploidy should give rise to the occurrence of non incorporated nuclei and spore irregularity. It was concluded that meiosis and spore formation in Saccharomycopsis lipolytica seem to represent parallel and coordinated processes which generally resemble those recorded for Saccharomyces cerevisiae and Hansenula species.     

Allelic and Ectopic Interactions in Recombination-Defective Yeast Strains

Ectopic recombination in the yeast Saccharomyces cerevisiae has been investigated by examining the effects of mutations known to alter allelic recombination frequencies. A haploid yeast strain disomic for chromosome III was constructed in which allelic recombination can be monitored using leu2 heteroalleles on chromosome III and ectopic recombination can be monitored using ura3 heteroalleles on chromosomes V and II. This strain contains the spo13-1 mutation which permits haploid strains to successfully complete meiosis and which rescues many recombination-defective mutants from the associated meiotic lethality. Mutations in the genes RAD50, SPO11 and HOP1 were introduced individually into this disomic strain using transformation procedures. Mitotic and meiotic comparisons of each mutant strain with the wild-type parental strain has shown that the mutation in question has comparable effects on ectopic and allelic recombination. Similar results have been obtained using diploid strains constructed by mating MATa and MAT alpha haploid derivatives of each of the disomic strains. These data demonstrate that ectopic and allelic recombination are affected by the same gene products and suggest that the two types of recombination are mechanistically similar. In addition, the comparison of disomic and diploid strains indicates that the presence of a chromosome pairing partner during meiosis does not affect the frequency of ectopic recombination events involving nonhomologous chromosomes.     

Recombination and Chromosome Segregation during the Single Division Meiosis in SPO12–1 and SPO13–1 Diploids

This paper reports a study of chromosome segregation and recombination during sporulation of spo12–1 and spo13–1 diploid strains of S. cerevisiae. These strains undergo a single division to form asci containing two diploid or near-diploid spores. The segregation of centromere-linked markers in the two-spored (dyad) products indicates that the division is generally equational. However, in a small percentage of the spo12–1 and spo13–1 cells, it appears that a meiosis I-like division occurs. Aberrant segregation of the MAT locus on chromosome III, yielding a monosomic and a trisomic spore pair, occurs in 12% of all dyads. The segregation patterns of markers at various distances from their centromeres and several pairs of markers on the same chromosome indicate that recombination takes place in both strains at nearly standard meiotic levels.     

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.     

Me14, a Yeast Gene Required for Meiotic Recombination

Mutants at the MEI4 locus were detected in a search for mutants defective in meiotic gene conversion. mei4 mutants exhibit decreased sporulation and produce inviable spores. The spore inviability phenotype is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division. The MEI4 gene has been cloned from a yeast genomic library by complementation of the recombination defect and has been mapped to chromosome V near gln3. Strains carrying a deletion/insertion mutation of the MEI4 gene display no meiotically induced gene conversion but normal mitotic conversion frequencies. Both meiotic interchromosomal and intrachromosomal crossing over are completely abolished in mei4 strains. The mei4 mutation is able to rescue the spore-inviability phenotype of spo13 and 52 strains (i.e., mei4 spo13 rad52 mutants produce viable spores), indicating that MEI4 acts before RAD52 in the meiotic recombination pathway.     

The Rec8 Gene of Schizosaccharomyces Pombe Is Involved in Linear Element Formation, Chromosome Pairing and Sister-Chromatid Cohesion during Meiosis

The fission yeast Schizosaccharomyces pombe does not form tripartite synaptonemal complexes during meiotic prophase, but axial core-like structures (linear elements). To probe the relationship between meiotic recombination and the structure, pairing, and segregation of meiotic chromosomes, we genetically and cytologically characterized the rec8-110 mutant, which is partially deficient in meiotic recombination. The pattern of spore viability indicates that chromosome segregation is affected in the mutant. A detailed segregational analysis in the rec8-110 mutant revealed more spores disomic for chromosome III than in a wild-type strain. Aberrant segregations are caused by precocious segregation of sister chromatids at meiosis I, rather than by nondisjunction as a consequence of lack of crossovers. In situ hybridization further showed that the sister chromatids are separated prematurely during meiotic prophase. Moreover, the mutant forms aberrant linear elements and shows a shortened meiotic prophase. Meiotic chromosome pairing in interstitial and centromeric regions is strongly impaired in rec8-110, whereas the chromosome ends are less deficient in pairing. We propose that the rec8 gene encodes a protein required for linear element formation and that the different phenotypes of rec8-110 reflect direct and indirect consequences of the absence of regular linear elements.     

The spindle checkpoint protein Mad2 regulates APC/C activity during prometaphase and metaphase of meiosis I in Saccharomyces cerevisiae

In many eukaryotes, disruption of the spindle checkpoint protein Mad2 results in an increase in meiosis I nondisjunction, suggesting that Mad2 has a conserved role in ensuring faithful chromosome segregation in meiosis. To characterize the meiotic function of Mad2, we analyzed individual budding yeast cells undergoing meiosis. We find that Mad2 sets the duration of meiosis I by regulating the activity of APC(Cdc20). In the absence of Mad2, most cells undergo both meiotic divisions, but securin, a substrate of the APC/C, is degraded prematurely, and prometaphase I/metaphase I is accelerated. Some mad2Δ cells have a misregulation of meiotic cell cycle events and undergo a single aberrant division in which sister chromatids separate. In these cells, both APC(Cdc20) and APC(Ama1) are prematurely active, and meiosis I and meiosis II events occur in a single meiotic division. We show that Mad2 indirectly regulates APC(Ama1) activity by decreasing APC(Cdc20) activity. We propose that Mad2 is an important meiotic cell cycle regulator that ensures the timely degradation of APC/C substrates and the proper orchestration of the meiotic divisions.     

Incidence of mosaic cell lines in vivo and malsegregation of chromosome 21 in lymphocytes in vitro of trisomy 21 patients: detection by fluorescence in situ hybridization on binucleated lymphocytes

In order to detect aneuploidy in interphase human lymphocytes, both in vivo and in vitro, fluorescence in situ hybridization (FISH) was carried out on binucleated cells cytokinesis-blocked by cytochalasin B at the first mitosis after phytohemagglutinin stimulation. A pericentric chromosome-21-specific DNA probe prepared from yeast artificial chromosome clone 881D2 by the polymerase chain reaction was employed. One thousand binucleated cells per individual were scored from cultures from twelve trisomy 21 patients aged 0.01-8.9 years (mean 4.3 years) and 20 normal children of similar age. Of trisomy 21 patients, increased frequencies of disomic cells in vivo (1.690+/-1.070%) and cells containing six signals with nondisjunction (0.822+/-0.554%) were found, compared with those of monosomic 21 cells in vivo (0.265+/-0.130%) and cells containing four signals with nondisjunction in normal children (0.369+/-0.250%; P=0.000 and P=0.000, respectively). These results show that malsegregation of chromosome 21 occurs more often in trisomic 21 cells than in disomic cells from normal children. The frequency of nondisjunction was significantly higher than the loss of chromosome 21 in both cultured trisomic (0.822+/-0.554% vs 0.043+/-0.049%, P=0.000) and disomic (0.369+/-0.250% vs 0.010+/-0.30%, P=0.000) cells. Comparisons of in vivo and in vitro data on aneuploidy indicate that a cell selection mechanism may exist in vivo. All these results show that FISH, with a chromosome-specific probe, on binucleated lymphocytes is a powerful tool for simultaneously detecting mosaic cell lines in vivo and malsegregation (loss and nondisjunction) of a corresponding chromosome in vitro in the same cell population.     

The Rec102 Mutant of Yeast Is Defective in Meiotic Recombination and Chromosome Synapsis

A mutation at the REC102 locus was identified in a screen for yeast mutants that produce inviable spores. rec102 spore lethality is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division. The rec102 mutation completely eliminates meiotically induced gene conversion and crossing over but has no effect on mitotic recombination frequencies. Cytological studies indicate that the rec102 mutant makes axial elements (precursors to the synaptonemal complex), but homologous chromosomes fail to synapse. In addition, meiotic chromosome segregation is significantly delayed in rec102 strains. Studies of double and triple mutants indicate that the REC102 protein acts before the RAD52 gene product in the meiotic recombination pathway. The REC102 gene was cloned based on complementation of the mutant defect and the gene was mapped to chromosome XII between CDC25 and STE11.     

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.     

The essential role of yeast topoisomerase III in meiosis depends on recombination

Yeast cells mutant for TOP3, the gene encoding the evolutionary conserved type I-5' topoisomerase, display a wide range of phenotypes including altered cell cycle, hyper-recombination, abnormal gene expression, poor mating, chromosome instability and absence of sporulation. In this report, an analysis of the role of TOP3 in the meiotic process indicates that top3Delta mutants enter meiosis and complete the initial steps of recombination. However, reductional division does not occur. Deletion of the SPO11 gene, which prevents recombination between homologous chromosomes in meiosis I division, allows top3Delta mutants to form viable spores, indicating that Top3 is required to complete recombination successfully. A topoisomerase activity is involved in this process, since expression of bacterial TopA in yeast top3Delta mutants permits sporulation. The meiotic block is also partially suppressed by a deletion of SGS1, a gene encoding a helicase that interacts with Top3. We propose an essential role for Top3 in the processing of molecules generated during meiotic recombination.     

Dis1: A Yeast Gene Required for Proper Meiotic Chromosome Disjunction

Mutants at a newly identified locus, DIS1 (disjunction), were detected by screening for mutants that generate aneuploid spores (chromosome VIII disomes) at an increased frequency. Strains carrying the partially dominant alleles, DIS1-1 or DIS1-2, generate disomes at rates up to 100 times the background level. Mitotic nondisjunction is also increased 10- to 50-fold over background. Half-tetrad analysis of disomes for a marked interval on chromosome VIII yields wild-type map distances, indicating that a general recombination deficiency is not the cause of nondisjunction. Meiotic nondisjunction in DIS1 mutants is not chromosome specific; 5% of the spores disomic for chromosome VIII are also disomic for chromosome III. Although only one disomic spore is found per exceptional ascus most of the disomes appear to be generated in the first meiotic division because recovered chromosome VIII disomes contain mostly nonsister chromosomes. We propose that disome generation in the DIS1 mutants results from precocious separation of sister centromeres.