Mutations in Saccharomyces Cerevisiae That Block Meiotic Prophase Chromosome Metabolism and Confer Cell Cycle Arrest at Pachytene Identify Two New Meiosis-Specific Genes Sae1 and Sae3

Two new meiosis-specific genes, SAE1 and SAE3, have been identified in a screen for mutations that confer an intermediate block in meiotic prophase. Such mutations confer a block to spore formation that is circumvented by addition of a mutation that eliminates meiotic recombination initiation and other aspects of chromosome metabolism, i.e., spo11. We show that sae1-1 and sae3-1 mutations each confer a distinct defect in meiotic recombination. sae1-1 produces recombinants but very slowly and ultimately to less than half the wild-type level; sae3-1 makes persistent hyper-resected meiotic double-strand breaks and has a severe defect in formation of recombinants. Both mutants arrest at the pachytene stage of meiotic prophase, sae1-1 temporarily and sae3-1 permanently. The phenotypes conferred by sae3-1 are similar to those conferred by mutation of the yeast RecA homologue DMC1, suggesting that SAE3 and DMC1 act at the same step(s) of chromosome metabolism. These results provide further evidence that intermediate blocks to prophase chromosome metabolism cause cell-cycle arrest. SAE1 encodes a 208-residue protein homologous to vertebrate mRNA cap-binding protein 20. SAE3 corresponds to a meiosis-specific RNA encoding an unusually short open reading frame of 50 codons.     

The Sep1 Mutant of Saccharomyces Cerevisiae Arrests in Pachytene and Is Deficient in Meiotic Recombination

Strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae promotes homologous pairing of DNA in vitro and sep1 mutants display pleiotropic phenotypes in both vegetative and meiotic cells. In this study, we examined in detail the ability of the sep1 mutant to progress through meiosis I prophase and to undergo meiotic recombination. In meiotic return-to-growth experiments, commitment to meiotic recombination began at the same time in wild type and mutant; however, recombinants accumulated at decreased rates in the mutant. Gene conversion eventually reached nearly wild-type levels, whereas crossing over reached 15-50% of wild type. In an assay of intrachromosomal pop-out recombination, the sep1, dmc1 and rad51 single mutations had only small effects; however, pop-out recombination was virtually eliminated in the sep1 dmc1 and sep1 rad51 double mutants, providing evidence for multiple recombination pathways. Analysis of meiotic recombination intermediates indicates that the sep1 mutant is deficient in meiotic double-strand break repair. In a physical assay, the formation of mature reciprocal recombinants in the sep1 mutant was delayed relative to wild type and ultimately reached only 50% of the wild-type level. Electron microscopic analysis of meiotic nuclear spreads indicates that the sep1δ mutant arrests in pachytene, with apparently normal synaptonemal complex. This arrest is RAD9-independent. We hypothesize that the Sep1 protein participates directly in meiotic recombination and that other strand exchange enzymes, acting in parallel recombination pathways, are able to substitute partially for the absence of the Sep1 protein.     

Mitotic and Meiotic Gene Conversion of Ty Elements and Other Insertions in Saccharomyces Cerevisiae

We examined meiotic and mitotic gene conversion events involved in deletion of Ty elements and other insertions from the genome of the yeast Saccharomyces cerevisiae. We found that Ty elements and one other insertion were deleted by mitotic gene conversion less frequently than point mutations at the same loci. One non-Ty insertion similar in size to Ty, however, did not show this bias. Mitotic conversion events deleting insertions were more frequently associated with crossing over than those deleting point mutations. In meiosis, conversion events duplicating the element were more common than those that deleted the element for one of the loci (HIS4) examined.     

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 (~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.     

Analysis of Meiotic Recombination Pathways in the Yeast Saccharomyces Cerevisiae

In the yeast, Saccharomyces cerevisiae, several genes appear to act early in meiotic recombination. HOP1 and RED1 have been classified as such early genes. The data in this paper demonstrate that neither a red1 nor a hop1 mutation can rescue the inviable spores produced by a rad52 spo13 strain; this phenotype helps to distinguish these two genes from other early meiotic recombination genes such as SPO11, REC104, or MEI4. In contrast, either a red1 or a hop1 mutation can rescue a rad50S spo13 strain; this phenotype is similar to that conferred by mutations in the other early recombination genes (e.g., REC104). These two different results can be explained because the data presented here indicate that a rad50S mutation does not diminish meiotic intrachromosomal recombination, similar to the mutant phenotypes conferred by red1 or hop1. Of course, RED1 and HOP1 do act in the normal meiotic interchromosomal recombination pathway; they reduce interchromosomal recombination to ~10% of normal levels. We demonstrate that a mutation in a gene (REC104) required for initiation of exchange is completely epistatic to a mutation in RED1. Finally, mutations in either HOP1 or RED1 reduce the number of double-strand breaks observed at the HIS2 meiotic recombination hotspot.     

Effects of Homology, Size and Exchange on the Meiotic Segregation of Model Chromosomes in Saccharomyces Cerevisiae

In most eukaryotic organisms, chiasmata, the connections formed between homologous chromosomes as a consequence of crossing over, are important for ensuring that the homologues move away from each other at meiosis I. Some organisms have the capacity to partition the rare homologues that have failed to experience reciprocal recombination. The yeast Saccharomyces cerevisiae is able to correctly partition achiasmate homologues with low fidelity by a mechanism that is largely unknown. It is possible to test which parameters affect the ability of achiasmate chromosomes to segregate by constructing strains that will have three achiasmate chromosomes at the time of meiosis. The meiotic partitioning of these chromosomes can be monitored to determine which ones segregate away from each other at meiosis I. This approach was used to test the influence of homologous yeast DNA sequences, recombination intiation sites, chromosome size and crossing over on the meiotic segregation of the model chromosomes. Chromosome size had no effect on achiasmate segregation. The influence of homologous yeast sequences on the segregation of noncrossover model chromosomes was negligible. In meioses in which two of the three model chromosomes experienced a crossover, they nearly always disjoined at meiosis I.     

Competition between Adjacent Meiotic Recombination Hotspots in the Yeast Saccharomyces Cerevisiae

In a wild-type strain of Saccharomyces cerevisiae, a hotspot for meiotic recombination is located upstream of the HIS4 gene. An insertion of a 49-bp telomeric sequence into the coding region of HIS4 strongly stimulates meiotic recombination and the local formation of meiosis-specific double-strand DNA breaks (DSBs). When strains are constructed in which both hotspots are heterozygous, hotspot activity is substantially less when the hotspots are on the same chromosome than when they are on opposite chromosomes.     

The ATM Signaling Cascade Promotes Recombination-Dependent Pachytene Arrest in Mouse Spermatocytes

Most mutations that compromise meiotic recombination or synapsis in mouse spermatocytes result in arrest and apoptosis at the pachytene stage of the first meiotic prophase. Two main mechanisms are thought to trigger arrest: one independent of the double-strand breaks (DSBs) that initiate meiotic recombination, and another activated by persistent recombination intermediates. Mechanisms underlying the recombination-dependent arrest response are not well understood, so we sought to identify factors involved by examining mutants deficient for TRIP13, a conserved AAA+ ATPase required for the completion of meiotic DSB repair. We find that spermatocytes with a hypomorphic Trip13 mutation (Trip13^mod/mod) arrest with features characteristic of early pachynema in wild type, namely, fully synapsed chromosomes without incorporation of the histone variant H1t into chromatin. These cells then undergo apoptosis, possibly in response to the arrest or in response to a defect in sex body formation. However, TRIP13-deficient cells that additionally lack the DSB-responsive kinase ATM progress further, reaching an H1t-positive stage (i.e., similar to mid/late pachynema in wild type) despite the presence of unrepaired DSBs. TRIP13-deficient spermatocytes also progress to an H1t-positive stage if ATM activity is attenuated by hypomorphic mutations in Mre11 or Nbs1 or by elimination of the ATM-effector kinase CHK2. These mutant backgrounds nonetheless experience an apoptotic block to further spermatogenic progression, most likely caused by failure to form a sex body. DSB numbers are elevated in Mre11 and Nbs1 hypomorphs but not Chk2 mutants, thus delineating genetic requirements for the ATM-dependent negative feedback loop that regulates DSB numbers. The findings demonstrate for the first time that ATM-dependent signaling enforces the normal pachytene response to persistent recombination intermediates. Our work supports the conclusion that recombination defects trigger spermatocyte arrest via pathways than are genetically distinct from sex body failure-promoted apoptosis and confirm that the latter can function even when recombination-dependent arrest is inoperative. Implications of these findings for understanding the complex relationships between spermatocyte arrest and apoptosis are discussed.     

Analysis of the kar3 Meiotic Arrest in Saccharomyces cerevisiae

The motor protein Kar3p and its associated protein Cik1p are essential for passage through meiosis I. In the absence of either protein, meiotic cells arrest in prophase I. Experiments were performed to determine whether the arrest was caused by a structural inability to proceed through meiosis, or by a regulatory mechanism. The data demonstrate that the meiotic arrest is not structural; kar3 and cik1 mutants are able to form normal looking bipolar spindles and divide their DNA into two masses in spo11 mutant backgrounds. To identify the regulatory system necessary for the kar3/cik1 meiotic arrest, we tested whether the arrest could be bypassed by eliminating the pachytene checkpoint or the spindle checkpoint. The arrest is not solely dependent upon the pachytene checkpoint that monitors recombination and aspects of chromosome synapsis. Elimination of the spindle checkpoint failed to allow kar3 mutants to undergo meiosis I nuclear division, but phenotypes of the kar3/spindle checkpoint double mutants suggest that the kar3 meiotic arrest may be mediated by the spindle checkpoint.     

Physical Lengths of Meiotic and Mitotic Gene Conversion Tracts in Saccharomyces Cerevisiae

Physical lengths of gene conversion tracts for meiotic and mitotic conversions were examined, using the same diploid yeast strain in all experiments. This strain is heterozygous for a mutation in the URA3 gene as well as closely linked restriction site markers. In cells that had a gene conversion event at the URA3 locus, it was determined by Southern analysis which of the flanking heterozygous restriction sites had co-converted. It was found that mitotic conversion tracts were longer on the average than meiotic tracts. About half of the tracts generated by spontaneous mitotic gene conversion included heterozygous markers 4.2 kb apart; none of the meiotic conversions included these markers. Stimulation of mitotic gene conversion by ultraviolet light or methylmethanesulfonate had no obvious effect on the size or distribution of the tracts. Almost all conversion tracts were continuous.     

Nonsense Mutations in Essential Genes of Saccharomyces Cerevisiae

A new method for isolating nonsense mutations in essential yeast genes has been used to develop a collection of 115 ochre mutations that define 94 complementation groups. The mutants are isolated in a genetic background that includes an ochre suppressor on a metastable plasmid and a suppressible colony-color marker on a chromosome. When the parental strain is plated on a rich medium, the colonies display a pattern of red, plasmid-free sectors on a white background. Mutants containing an ochre mutation in any essential yeast gene give rise to nonsectoring, white colonies, since cell growth is dependent on the presence of the plasmid-borne suppressor. Analysis of the data suggests that mutations are being recovered from a pool of approximately 250 genes.     

Identification of New Genes Required for Meiotic Recombination in Saccharomyces Cerevisiae

Mutants defective in meiotic recombination were isolated from a disomic haploid strain of Saccharomyces cerevisiae by examining recombination within the leu2 and his4 heteroalleles located on chromosome III. The mutants were classified into two new complementation groups (MRE2 and MRE11) and eight previously identified groups, which include SPO11, HOP1, REC114, MRE4/MEK1 and genes in the RAD52 epistasis group. All of the mutants, in which the mutations in the new complementation groups are homozygous and diploid, can undergo premeiotic DNA synthesis and produce spores. The spores are, however, not viable. The mre2 and mre11 mutants produce viable spores in a spo13 background, in which meiosis I is bypassed, suggesting that these mutants are blocked at an early step in meiotic recombination. The mre2 mutant does not exhibit any unusual phenotype during mitosis and it is, thus, considered to have a mutation in a meiosis-specific gene. By contrast, the mre11 mutant is sensitive to damage to DNA by methyl methanesulfonate and exhibits a hyperrecombination phenotype in mitosis. Among six alleles of HOP1 that were isolated, an unusual pattern of intragenic complementation was observed.     

Meiotic Nondisjunction and Recombination of Chromosome III and Homologous Fragments in Saccharomyces Cerevisiae

A yeast strain, in which nondisjunction of chromosome III at the first-meiotic division could be assayed, was constructed. Using chromosome fragmentation plasmids, chromosomal fragments (CFs) were derived in isogenic strains from six sites along chromosome III and one site on chromosome VII. Whereas the presence of the CFs derived from chromosome III increased considerably the meiosis I nondisjunction of that chromosome, the CF derived from chromosome VII had no effect on chromosome III segregation. The effects of the chromosome III-derived fragments were not linearly related to fragment length. Two regions, one of 12 kb in size located at the left end of the chromosome, and the other of 5 kb, located at the center of the right arm, were found to have profound effects on chromosome III nondisjunction. Most disomics arising from meioses in strains containing chromosome III CFs did not contain the CF; thus it appears that the two chromosome III homologs had segregated away from the CF. Among the disomics, recombination between the homologous chromosomes III was lower than expected from the genetic distance, while recombination between one of the chromosomes III and the fragment was frequent. We suggest that there are sites along the chromosome that are more involved than others in the pairing of homologous chromosomes and that the pairing between fragment and homologs involves recombination among these latter elements.     

Cycloheximide-Resistant Temperature-Sensitive Lethal Mutations of Saccharomyces Cerevisiae

We describe the isolation and preliminary characterization of a set of pleiotropic mutations resistant to the minimum inhibitory concentration of cycloheximide and screened for ts (temperature-sensitive) growth. These mutations fall into 22 complementation groups of cycloheximide resistant ts lethal mutations (crl). None of the crl mutations appears to be allelic with previously isolated mutations. Fifteen of the CRL loci have been mapped. At the nonpermissive temperature (37°), these mutants arrest late in the cell cycle after several cell divisions. Half of these mutants are also unable to grow at very low temperatures (5°). Although mutants from all of the 22 complementation groups exhibit similar temperature-sensitive phenotypes, an extragenic suppressor of the ts lethality of crl3 does not relieve the ts lethality of most other crl mutants. A second suppressor mutation allows crl10, crl12, and crl14 to grow at 37° but does not suppress the ts lethality of the remaining crl mutants. We also describe two new methods for the enrichment of auxotrophic mutations from a wild-type yeast strain.     

Ty Element-Induced Temperature-Sensitive Mutations of Saccharomyces Cerevisiae

Temperature-sensitive mutants of Saccharomyces cerevisiae were isolated by insertional mutagenesis using the HIS3 marked retrotransposon TyH3HIS3. In such mutants, the TyHIS3 insertions are expected to identify loci which encode genes essential for cell growth at high temperatures but dispensable at low temperatures. Five mutations were isolated and named hit for high temperature growth. The hit1-1 mutation was located on chromosome X and conferred the pet phenotype. Two hit2 mutations, hit2-1 and hit2-2, were located on chromosome III and caused the deletion of the PET18 locus which has been shown to encode a gene required for growth at high temperatures. The hit3-1 mutation was located on chromosome VI and affected the CDC26 gene. The hit4-1 mutation was located on chromosome XIII. These hit mutations were analyzed in an attempt to identify novel genes involved in the heat shock response. The hit1-1 mutation caused a defect in synthesis of a 74-kD heat shock protein. Western blot analysis revealed that the heat shock protein corresponded to the SSC1 protein, a member of the yeast hsp70 family. In the hit1-1 mutant, the TyHIS3 insertion caused a deletion of a 3-kb DNA segment between the delta 1 and delta 4 sequences near the SUP4 locus. The 1031-bp wild-type HIT1 DNA which contained an open reading frame encoding a protein of 164 amino acids and the AGG arginine tRNA gene complemented all hit1-1 mutant phenotypes, indicating that the mutant phenotypes were caused by the deletion of these genes. The pleiotropy of the HIT1 locus was analyzed by constructing a disruption mutation of each gene in vitro and transplacing it to the chromosome. This analysis revealed that the HIT1 gene essential for growth at high temperatures encodes the 164-amino acid protein. The arginine tRNA gene, named HSX1, is essential for growth on a nonfermentable carbon source at high temperatures and for synthesis of the SSC1 heat shock protein.     

Length and Distribution of Meiotic Gene Conversion Tracts and Crossovers in Saccharomyces Cerevisiae

We have measured gene conversion tract length in strains of the yeast Saccharomyces cerevisiae containing three to six restriction site heterozygosities in a 9-kb interval. Tetrads containing a conversion were identified genetically by nonmendelian segregation of a marker in the middle of the interval. Gene conversions accompanied by a crossover have a tract length of 1.4 kb +/- 0.7 kb, which is indistinguishable from a tract length of 1.6 +/- 0.8 for conversions without an associated exchange. Among tetrads identified first as having a crossover in the interval, the average gene conversion tracts were apparently significantly shorter (0.71 +/- 1). We provide evidence that this apparent difference is due to the method of measuring conversion tracts and does not reflect a real difference in tract length. We also provide evidence that the number and position of restriction site markers alters the apparent distribution of the conversion tracts. More than ninety percent of the conversion tracts spanning three or more sites were continuous.     

Mutations Affecting the Trna-Splicing Endonuclease Activity of Saccharomyces Cerevisiae

Two unlinked mutations that alter the enzyme activity of tRNA-splicing endonuclease have been identified in yeast. The sen1-1 mutation, which maps on chromosome 12, causes temperature-sensitive growth, reduced in vitro endonuclease activity, and in vivo accumulation of unspliced pre-tRNAs. The sen2-1 mutation does not confer a detectable growth defect, but causes a temperature-dependent reduction of in vitro endonuclease activity. Pre-tRNAs do not accumulate in sen2-1 strains. The in vitro enzyme activities of sen1-1 and sen2-1 complement in extracts from a heterozygous diploid, but fail to complement in mixed extracts from separate sen1-1 and sen2-1 haploid strains. These results suggest a direct role for SEN gene products in the enzymatic removal of introns from tRNA that is distinct from the role of other products known to affect tRNA splicing.     

Identification and Characterization of Mutations Affecting Sporulation in Saccharomyces Cerevisiae

Mutations affecting the synthesis of the sporulation amyloglucosidase were isolated in a homothallic strain of Saccharomyces cerevisiae, SCMS7-1. Two were found, both of which were deficient in sporulation at 34 degrees. One, SL484, sporulated to 50% normal levels at 30 degrees but less than 5% at 34 degrees or 22 degrees. The other, SL641, failed to sporulate at any temperature. Both mutants were blocked before premeiotic DNA synthesis, and both complemented spo1, spo3, and spo7. Genetic analysis of the mutation in SL484 indicated linkage to TRP5 and placed the gene 10 map units from TRP5 on chromosome VII. A plasmid containing an insert which complements the mutation in SL484 fails to complement SL641. We therefore conclude that these two mutations are in separate genes and we propose to call these genes SPO17 and SPO18. These two genes are (with SPO7, SPO8, and SPO9) among the earliest identified in the sporulation pathway and may interact directly with the positive and negative regulators RME and IME.