Meiotically Induced Rec7 and Rec8 Genes of Schizosaccharomyces Pombe

The Schizosaccharomyces pombe rec7 and rec8 genes, which are required for meiotic intragenic recombination but not for mitotic recombination, have been cloned and their DNA sequences determined. Genetic and physical analyses demonstrated that the cloned fragments contained the rec genes rather than rec mutation suppressors. A 1.6-kb DNA fragment contained a functional rec7 gene, and a 2.1-kb fragment contained a functional rec8 gene. The nucleotide sequences of these fragments revealed open reading frames predicting 249 amino acids for the rec7 gene product and 393 amino acids for the rec8 gene product. Northern hybridization analysis showed that both rec gene mRNAs were detectable only at 2-3 hr after induction of meiosis. The absence of these mRNAs in mitosis and their disappearance at 4 hr and later in meiosis suggest that the rec7 and rec8 gene products may be involved primarily in the early steps of meiotic recombination in S. pombe.     

Mutational Analysis of Cdc19p, a Schizosaccharomyces Pombe Mcm Protein

The cdc19(+) gene encodes an essential member of the MCM family of replication proteins in Schizosaccharomyces pombe. We have examined the structure and function of the Cdc19p protein using molecular and genetic approaches. We find that overproduction of wild-type Cdc19p in wild-type cells has no effect, but cdc19-P1 mutant cells do not tolerate elevated levels of other MCM proteins or overexpression of mutant forms of Cdc19p. We have found genetic interactions between cdc19(+) and genes encoding subunits of DNA polymerase {delta small} and the replication initiator cdc18(+). We have constructed a series of point mutations and sequence deletions throughout Cdc19p, which allow us to distinguish essential from nonessential regions of the protein. Not surprisingly, conserved residues in the MCM homology domain are required for protein function, but some residues outside the core homology domain are dispensable.     

Stf1: Non-Wee Mutations Epistatic to Cdc25 in the Fission Yeast Schizosaccharomyces Pombe

In Schizosaccharomyces pombe, cdc25 is a cell cycle regulated inducer of mitosis. wee1 and phenotypically wee alleles of cdc2 are epistatic to cdc25. Mutant alleles of a new locus, stf1 (suppressor of twenty-five), identified in a reversion analysis of conditionally lethal cdr1-76 cdc25-22 and cdr2-96 cdc25-22 double mutant strains, also suppress both temperature-sensitive and gene disruption alleles of cdc25. These mutants, by themselves, are phenotypically indistinguishable from wild type strains; hence they represent the first known mutations that are epistatic to cdc25 and do not display a wee phenotype. stf1 genetically interacts with other elements of mitotic control in S. pombe. stf1-1 is additive with wee1-50, cdc2-1w and cdc2-3w for suppression of cdc25-22. Also, like wee1- and cdc2-w, stf1- suppression of cdc25 is reversed by overexpression of the putative type 1 protein phosphatase bws1+/dis2+. Interaction with various mutants and plasmid overexpression experiments suggest that stf1 does not operate either upstream or downstream of wee1. Similarly, it does not operate through cdc25 since it rescues the disruption. stf1 appears to encode an important new element of mitotic control.     

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.     

Meiotic Recombination-Deficient Mutants of Schizosaccharomyces Pombe

A mutant screen employing the ade6-M26 recombination hotspot was developed and used to isolate Schizosaccharomyces pombe mutants deficient in meiotic recombination. Nine rec mutations were recessive, defining six complementation groups, and reduced ade6 meiotic recombination 3-fold to greater than or equal to 300-fold when homozygous. Three recessive rec mutations analyzed further also reduced meiotic intragenic recombination at ura4 on chromosome III and intergenic recombination between pro2 and arg3 on chromosome I. The observed non-co-ordinate reductions of the recombinant frequencies in the three test intervals suggest a degree of locus (or intragenic vs. intergenic) specificity of the corresponding rec+ gene products. None of the mutations specifically inactivated the ade6-M26 hotspot. Additional rec genes may be identified with these methods.     

Respiration deficient mutants of Schizosaccharomyces Pombe I

Summary By use of N-methyl-N-nitro-N-nitrosoguanidin (NG) respiration deficient (RD) mutants were induced. They could be selected by replica-plating on glycerol medium. RD mutants were also induced by UV irradiation, enriched by use of 2,3,5-triphenyltetrazolium chloride (TTC) and tested for their inability to grow on glycerol medium. The RD mutants were characterized enzymatically for their decrease or loss in cytochrome c oxidase activity and in succinate- cytochrome c reductase activity. These assays allowed the localization of the mutational blocks in complexes II, III and IV of the respiratory chain. Tetrad analysis and random spore analysis demonstrated that all mutants contained chromosomal defects.     

Isolation and Characterization of Schizosaccharomyces Pombe Mutants Affected in Mitotic Recombination

A haploid Schizosaccharomyces pombe strain carrying a heteroallelic duplication of the ade6 gene was used to isolate mitotic recombination-deficient mutants. Recombination between the different copies of the ade6 gene can lead to Ade+ segregants. These are observed as growing papillae when colonies of a suitable size are replicated onto selective medium. We isolated mutants which show an altered papillation phenotype. With two exceptions, they exhibit a decrease in the frequency of mitotic recombination between the heteroalleles of the duplication. The two other mutants display a hyper-recombination phenotype. The 12 mutations were allocated to at least nine distinct loci by recombination tests. Of the eight rec mutants analyzed further, six were also affected in mitotic intergenic recombination in the intervals cen2-mat or cen3-arg 1. No effect on mitotic intragenic recombination was observed. These data suggest that mitotic gene conversion and crossing over can be separated mutationally. Meiotic recombination occurs at the wild-type frequency in all mutants investigated.     

An Analysis of Interference in the Fission Yeast Schizosaccharomyces Pombe

The evaluation of three-point crosses at the tetrad and random spore level leads to the conclusion that both chiasma and chromatid interference are absent in the fission yeast Schizosaccharomyces pombe.     

Double-Strand Break-Induced Mitotic Intrachromosomal Recombination in the Fission Yeast Schizosaccharomyces Pombe

The Saccharomyces cerevisiae HO gene and MATa cutting site were used to introduce site-specific double-strand breaks (DSBs) within intrachromosomal recombination substrates in Schizosaccharomyces pombe. The recombination substrates consisted of nontandem direct repeats of ade6 heteroalleles. DSB induction stimulated the frequency of recombinants 2000-fold. The spectrum of DSB-induced recombinants depended on whether the DSB was introduced within one of the ade6 repeats or in intervening unique DNA. When the DSB was introduced within unique DNA, over 99.8% of the recombinants lacked the intervening DNA but retained one copy of ade6 that was wild type or either one of the heteroalleles. When the DSB was located in duplicated DNA, 77% of the recombinants were similar to the deletion types described above, but the single ade6 copy was either wild type or exclusively that of the uncut repeat. The remaining 23% of the induced recombinants were gene convertants with two copies of ade6 and the intervening sequences; the ade6 heteroallele in which the DSB was induced was the recipient of genetic information. Half-sectored colonies were isolated, analyzed and interpreted as evidence of heteroduplex DNA formation. The results are discussed in terms of current models for recombination.     

Seventeen Complementation Groups of Mutations Decreasing Meiotic Recombination in Schizosaccharomyces Pombe

We have analyzed 43 recessive mutations reducing meiotic intragenic recombination in Schizosaccharomyces pombe. These mutations were isolated by a screen for reduced plasmid-by-chromosome recombination at the ade6 locus. Sixteen of the mutations define 10 new complementation groups, bringing to 17 the number of genes identified to be involved in meiotic recombination. The mutations were grouped into three discrete classes depending on the severity of the recombination deficiency in crosses involving the ade6-M26 recombination hotspot. Class I mutations caused at least a 1000-fold reduction in M26-stimulated intragenic recombination at the ade6 locus. Class II mutations reduced M26-stimulated recombination approximately 100-fold. Class III mutations caused a 3-10-fold reduction in either M26-stimulated or non-hotspot recombination. We obtained multiple alleles of class I and class II mutations, suggesting that we may be nearing saturation for mutations of this type. As a first step toward mapping, we used mitotic segregation to assign fourteen of the rec genes to chromosomes. Mutations in the six rec genes tested also caused a decrease in intragenic recombination at the ura4 locus; five of these mutations also reduced intergenic recombination between the pro2 and arg3 genes. These results indicate that these multiple rec gene products are required for high level meiotic recombination throughout the S. pombe genome.     

Active and Inactive Transplacement of the M26 Recombination Hotspot in Schizosaccharomyces Pombe

The ade6-M26 mutation of the fission yeast Schizosaccharomyces pombe creates a meiotic recombination hotspot that elevates ade6 intragenic recombination ~10-15-fold. A heptanucleotide sequence including the M26 point mutation is required but not sufficient for hotspot activity. We studied the effects of plasmid and chromosomal context on M26 hotspot activity. The M26 hotspot was inactive on a multicopy plasmid containing M26 embedded within 3.0 or 5.9 kb of ade6 DNA. Random S. pombe genomic fragments totaling ~7 Mb did not activate the M26 hotspot on a plasmid. M26 hotspot activity was maintained when 3.0-, 4.4-, and 5.9-kb ade6-M26 DNA fragments, with various amounts of non-S. pombe plasmid DNA, were integrated at the ura4 chromosomal locus, but only in certain configurations relative to the ura4 gene and the cointegrated plasmid DNA. Several integrations created new M26-independent recombination hotspots. In all cases the non-ade6 DNA was located >1 kb from the M26 site, and in some cases >2 kb. Because the chromosomal context effect was transmitted over large distances, and did not appear to be mediated by a single discrete DNA sequence element, we infer that the local chromatin structure has a pronounced effect on M26 hotspot activity.     

Genetic and Molecular Analysis of Cdr1/Nim1 in Schizosaccharomyces Pombe

The cdr1 gene in Schizosaccharomyces pombe was identified as a mutation affecting the nutritional responsiveness of the mitotic size control. cdr1 alleles have been further analyzed for genetic interactions with elements of the mitotic control pathway and cloned by plasmid rescue of a conditional lethal cdr1-76 cdc25-22 double mutant. These analyses show that the cdr1 gene is allelic to nim1, a gene identified as a high copy number plasmid suppressor of the mitotic control gene, cdc25. The gene structure for cdr1 differs from the described nim1 gene in the carboxyl-terminal portion of the gene. The published nim1 sequence encoded a product of predicted Mr 45,000, and included 356 amino acids from the amino-terminal region of the gene and 14 amino acids from a noncontiguous carboxyl-terminal fragment. The cdr1 sequence includes an additional 237 amino acids of the contiguous fragment and encodes a product of predicted Mr 67,000. The sequence shows a high level of identity with protein kinases over the amino-terminal catalytic domain, and limited identity with yeast protein kinases SNF1, KIN2 and KIN1 over part of the carboxyl-terminal domain. The effect of overexpression of the full length gene has been examined in various genetic backgrounds. These data show that the full length gene product is required to give a normal cell cycle response to nitrogen starvation. A detailed examination of the genetic interaction of cdr1 mutants with various mutants of mitotic control genes (cdc2, cdc25, wee1, cdc13) demonstrated strong interactions with cdc25, some cdc2 alleles, and with cdc13-117. Overall, the results are interpretable within the framework of the existing model of cdr1/nim1 action in mitotic control, i.e., cdr1 functions upstream of wee1 to relieve mitotic inhibition.     

The Mutator Gene Swi8 Effects Specific Mutations in the Mating-Type Region of Schizosaccharomyces Pombe

The swi8(+) gene of Schizosaccharomyces pombe appears to be involved in the termination step of copy synthesis during mating-type (MT) switching. Mutations in swi8 confer a general mutator phenotype and, in particular, generate specific mutations in the MT region. Sequencing of the MT cassettes of the h(90) swi8-137 mutant revealed three altered sites. One is situated at the switching (smt) signal adjacent to the H1 homology box of the expression locus mat1:1. It reduces the rate of MT switching. The alteration at the smt signal arose frequently in other h(90) swi8 strains and is probably caused by gene conversion in which the sequence adjacent to the H1 box of mat2:2 is used as template. This change might be generated during the process of MT switching when hybrid DNA formation is anomalously extended into the more heterologous region flanking the H1 homology box. In addition to the gene conversion at mat1:1, two mutations were found in the H3 homology boxes of the silent cassettes mat2:2 and mat3:3.     

Genetic and Physical Analysis of the M26 Recombination Hotspot of Schizosaccharomyces Pombe

The ade6-M26 mutation of Schizosaccharomyces pombe has previously been reported to stimulate ade6 intragenic meiotic recombination. We report here that the ade6-M26 mutation is a single G----T nucleotide change, that M26 stimulated recombination within ade6 but not at other distinct loci, and that M26 stimulated meiotic but not mitotic recombination. In addition, M26 stimulated recombination within ade6 when M26 is homozygous; this result demonstrates that a base-pair mismatch at the M26 site was not required for the stimulation. These results are consistent with the ade6-M26 mutation creating a meiotic recombination initiation site.     

The Ade6-M26 Mutation of Schizosaccharomyces Pombe Increases the Frequency of Crossing over

The ade6-M26 mutation of Schizosaccharomyces pombe increases conversion frequency in comparison with the nearby mutation ade6-M375. In order to investigate the effect of ade6-M26 on crossover frequency, heteroallelic ade6 duplications were constructed by integration of plasmids carrying the marker gene ura4. One ade6 gene carries either of the mutations M26 or M375 while the other ade6 copy carries the L469 mutation in both duplications. The duplication with ade6-M26 yields Ade(+) recombinants at significantly higher frequencies in meiosis, but not in mitosis. Tetrad analysis and physical characterization of spore clones from recombination tetrads demonstrate that conversions, unequal crossovers and intrachromatid exchanges occur at higher frequencies but with unaltered proportions among them. The conversion events show a pronounced bias when M26 is involved: they take place preferentially at the M26 allele. Thus the ade6-M26 mutation not only enhances conversion frequency as demonstrated before, but also crossover frequency. It displays the properties expected for a preferred site of initiation of general meiotic recombination. The duplications also yielded new information on ectopic recombination in S. pombe: ectopic crossovers occur in the duplications at much higher frequency than among naturally dispersed homologous sequences.