Initiation of sporulation in Saccharomyces cerevisiae. Mutations causing derepressed sporulation and G1 arrest in the cell division cycle

Mutants of Saccharomyces cerevisiae that are derepressed for meiosis and spore formation have been isolated and characterized genetically. All are the result of single, recessive nuclear mutations that fall into four linkage groups. Three of these groups are represented by spd1, spd3 and spd4 mutations, which in homozygous diploids confer poor growth and extensive sporulation on a range of non-fermentable media. Haploids carrying any of these mutations are arrested under these conditions in the G1 phase of the cell division cycle as large unbudded cells. The alleles of the spd2 mutation complemented all other mutations but were very closely linked to the spd1 locus. The fourth linkage group was represented by a mutation conferring temperature-sensitive growth and derepressed sporulation on homozygous diploids grown between 25 degrees C and 30 degrees C on media containing galactose or glycerol, but not glucose, as energy source. Above 30 degrees C this mutant lysed on all media. The mutation it carried failed to complement available cdc25 mutations. These data bring to five the number of loci at which mutation can lead to derepressed sporulation (spd1, spd3, spd4, cdc25 and cdc35). The spd1 locus has been mapped 13.9 cM to the left of the centromere on chromosome XV, adjacent to the SUP3 gene. Diploid strains homozygous for spd mutations are genetically unstable, giving rise to asporogenous mutants at high frequency, usually as the result of a second mutation unlinked to the spd mutation. Diploids homozygous for these mutations, and for spd mutations, show an altered regulation of the formulation of at least three polypeptides normally subject to carbon source repression.     

Abnormal amino acid metabolism in mutants of Saccharomyces cerevisiae affected in the initiation of sporulation

Amino acid metabolism was examined during sporulation in a wild-type strain of Saccharomyces cerevisiae and in a mutant homozygous for the spd1 mutation (derepression of sporulation). In the wild-type initiation of sporulation involves a rise in intracellular glutamate. In the derepressed mutant there was no further increase of intracellular glutamate: glutamate was already present at a greater concentration than in the wild-type. These elevated glutamate levels are apparently due to reduction in the activity of 2-oxoglutarate dehydrogenase. One consequence of the elevated glutamate levels in diploids homozygous for the spd1 mutation is the production of considerable amounts of glycyl-L-proline, glutathione and saccharopine.     

13C NMR analysis of a developmental pathway mutation in Saccharomyces cerevisiae reveals a cell derepressed for succinate dehydrogenase.

13C nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of [2-13C]acetate in a diploid strain of Saccharomyces cerevisiae homozygous for the spo50 mutation. This mutation results in failure to initiate sporulation and suppresses spd mutations (which cause derepressed sporulation). By analysing the pattern of 13C-labelling in glutamate it was deduced that the glyoxylate cycle is responsible for most of the acetate utilization and that there is very little tricarboxylic acid cycle activity. The labelling of alpha,alpha'-trehalose indicated that gluconeogenesis and the hexose monophosphate pathway operate in a similar way to the wild-type. The mutant strain has higher levels of succinate dehydrogenase than the wild-type. All of the physiological alterations caused by the spo50 mutation can be explained by this difference.     

The Role of the SPO11 Gene in Meiotic Recombination in Yeast

Several complementary experimental approaches were used to demonstrate that the SPO11 gene is specifically required for meiotic recombination. First, sporulating cultures of spo11-1 mutant diploids were examined for landmark biochemical, cytological and genetic events of meiosis and ascosporogenesis. Cells entered sporulation with high efficiency and showed a near-doubling of DNA content. Synaptonemal complexes, hallmarks of intimate homologous pairing, and polycomplex structures appeared during meiotic prophase. Although spontaneous mitotic intra- and intergenic recombination occurred at normal levels, no meiotic recombination was observed. Whereas greater than 50% of cells completed both meiotic divisions, packaging of the four meiotic products into mature ascospores took place in only a small subset of asci. Haploidization occurred in less than 1% of viable colony-forming units. Second, the Rec- meiotic defect conferred by spo11-1 was confirmed by dyad analysis of spores derived from spo13-1 single-division meiosis in which recombination is not a requirement for viable ascospore production. Diploids homozygous for the spo13-1 mutation undergo meiotic levels of exchange followed by a single predominantly equational division and form asci containing two near-diploid spores. With the introduction of the spo11-1 mutation, high spore viability was retained, whereas intergenic recombination was reduced by more than 100-fold.     

A study of the role of the hexose monophosphate pathway with respect to fatty acid biosynthesis in sporulation of Saccharomyces cerevisiae

13C NMR was used to study the pattern of label incorporation from [2-13C]acetate into trehalose during sporulation in Saccharomyces cerevisiae. A wild-type strain and a strain homozygous for the zwf1 mutation (which affects glucose-6-phosphate dehydrogenase) were used. In the wild-type it was possible to deduce the cycling of glucose 6-phosphate around the hexose monophosphate pathway whilst in the mutant strain this did not occur. The requirement of the hexose monophosphate pathway for providing NADPH for fatty acid biosynthesis was examined using 13C NMR and GC/MS. The wild-type strain produced a typical profile of fatty acids with palmitoleic acid being the most abundant whereas the mutant contained only one-quarter the amount of total fatty acid. As zwf1 homozygous diploids are able to sporulate this indicates that the large amount of fatty acid biosynthesis observed in sporulation of wild-type strains is not essential to the process.     

Allelism of PSO4 and PRP19 links pre-mRNA processing with recombination and error-prone DNA repair in Saccharomyces cerevisiae.

The radiation-sensitive mutant pso4-1 of Saccharomyces cerevisiae shows a pleiotropic phenotype, including sensitivity to DNA cross-linking agents, nearly blocked sporulation and reduced mutability. We have cloned the putative yeast DNA repair gene PSO4 from a genomic library by complementation of the blocked UV-induced mutagenesis and of sporulation in diploids homozygous for pso4-1. Sequence analysis revealed that gene PSO4 consists of 1512 bp located upstream of UBI4 on chromosome XII and encodes a putative protein of 56.7 kDa. PSO4 is allelic to PRP19, a gene encoding a spliceosome-associated protein, but shares no significant homology with other yeast genes. Gene disruption with a destroyed reading frame of our PSO4 clone resulted in death of haploid cells, confirming the finding that PSO4/PRP19 is an essential gene. Thus, PSO4 is the third essential DNA repair gene found in the yeast S.cerevisiae.     

A mutation affecting lipoamide dehydrogenase, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities in Saccharomyces cerevisiae

Summary In Saccharomyces cerevisiae a nuclear recessive mutation, lpd1, which simultaneously abolishes the activities of lipoamide dehydrogenase, 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase has been identified. Strains carrying this mutation can grow on glucose or poorly on ethanol, but are unable to grow on media with glycerol or acetate as carbon source. The mutation does not prevent the formation of other tricarboxylic acid cycle enzymes such as fumarase, NAD⁺-linked isocitrate dehydrogenase or succinate-cytochrome c oxidoreductase, but these are produced at about 50%–70% of the wild-type levels. The mutation probably affects the structural gene for lipoamide dehydrogenase since the amount of this enzyme in the cell is subject to a gene dosage effect; heterozygous lpd1 diploids produce half the amount of a homozygous wild-type strain. Moreover, a yeast sequence complementing this mutation when present in the cell on a multicopy plasmid leads to marked overproduction of lipoamide dehydrogenase. Homozygous lpd1 diploids were unable to sporulate indicating that some lipoamide dehydrogenase activity is essential for sporulation to occur on acetate.     

Protein Degradation, Meiosis and Sporulation in Proteinase-Deficient Mutants of SACCHAROMYCES CEREVISIAE

During the process of sporulation, a/α diploids degrade about 50% of their vegetative proteins. This degradation is not sporulation specific, for asporogenous diploids of a/a mating type degrade their vegetative proteins in a fashion similar to that of their a/α counterparts. Diploids lacking carboxypeptidase Y activity, prc1/prc1, show about 80% of wild-type levels of protein degradation, but are unimpaired in the production of normal asci. Diploids lacking proteinase B activity, prb1/prb1, show about 50% of wild-type levels of protein degradation. The effect on degradation of the proteinase B deficiency is epistatic to the degradation deficit attributable to the carboxypeptidase Y deficiency. The prb1 homozygotes undergo meiosis and produce spores, but the asci and, possibly, the spores are abnormal. Diploids homozygous for the pleiotropic pep4–3 mutation show only 30% of the wild-type levels of degradation when exposed to a sporulation regimen, and do not undergo meiosis or sporulation. Neither proteinase B nor carboxypeptidase Y is necessary for germination of spores.——Approximately half of the colonies arising from a/a or α/α diploids exposed to the sporulation regiment that express an initially heterozygous drug-resistance marker (can1) appear to arise from mating-type switches followed by meiosis and sporulation.     

Mutations Leading to Expression of the Cryptic HMR a Locus in the Yeast SACCHAROMYCES CEREVISIAE

Mutations leading to expression of the silent HMRa information in Saccharomyces cerevisiae result in sporulation proficiency in mata1/MAT alpha diploids. An example of such a mutation is sir5-2, a recessive mutation in the gene SIR5. As expected, haploids carrying the sir5-2 mutation are nonmaters due to the simultaneous expression of HMRa and HML alpha, resulting in the nonmating phenotype of an a/alpha diploid. However, sir5-2/sir5-2 mata1/MAT alpha diploids mate as alpha yet are capable of sporulation. The sir5-2 mutation is unlinked to sir1-1, yet the two mutations do not complement each other: mata1/MAT alpha sir5-2/SIR5 SIR1/sir1-1 diploids are capable of sporulation. In this case, recessive mutations in two unlinked genes form a mutant phenotype, in spite of the presence of the normal wild-type alleles. The PAS1-1 mutation, Provider of a Sporulation function, is a dominant mutation tightly linked to HMRa. PAS1-1 does not affect the mating ability of a strain, yet it allows diploids lacking a functional MATa locus to sporulate. It is proposed that PAS1-1 leads to partial expression of the otherwise cryptic a1 information at HMRa.     

Hop1: A Yeast Meiotic Pairing Gene

The recessive mutation, hop1-1, was isolated by use of a screen designed to detect mutations defective in homologous chromosomal pairing during meiosis in Saccharomyces cerevisiae. Mutants in HOP1 displayed decreased levels of meiotic crossing over and intragenic recombination between markers on homologous chromosomes. In contrast, assays of the hop1-1 mutation in a spo13-1 haploid disomic for chromosome III demonstrated that intrachromosomal recombination between directly duplicated sequences was unaffected. The spores produced by SPO13 diploids homozygous for hop1 were largely inviable, as expected for a defect in interhomolog recombination that results in high levels of nondisjunction. HOP1 was cloned by complementation of the spore lethality phenotype and the cloned gene was used to map HOP1 to the LYS11-HIS6 interval on the left arm of chromosome IX. Electron microscopy revealed that diploids homozygous for hop1 fail to form synaptonemal complex, which normally provides the structural basis for homolog pairing. We propose that HOP1 acts in meiosis primarily to promote chromosomal pairing, perhaps by encoding a component of the synaptonemal complex.     

A New Mapping Method Employing a Meiotic Rec- Mutant of Yeast

A rapid new mapping method has been developed for localizing a dominant or recessive mutation to a particular chromosome of yeast. The procedure utilizes the ability of strains homozygous for the spo11-1 mutation to undergo chromosome segregation without appreciable recombination during sporulation. The level of sporulation in spo11-1/spo11-1 diploids is reduced and asci are often immature or abnormal in appearance; spore viability is less than 1%. The first step of the mapping procedure is the construction of a haploid spo11-1 strain carrying a recessive drug-resistance marker and the unmapped mutation(s). This strain is crossed to a set of three spo11-1 mapping tester strains containing, among them, a recessive marker on each chromosome. The resulting spo11-1/spo11-1 diploids are sporulated and plated on drug-containing medium. Viable meiotic products that express the drug-resistance marker due to chromosome haploidization are selectively recovered. These meiotic products are haploid for most, but generally not all, chromosomes. The level of disomy for individual chromosomes averages 19%. Each of the recessive chromosomal markers is expressed in approximately a third of the drug-resistant segregants. Ninety-eight percent of these segregants show no evidence of intergenic recombination. Thus, two markers located on the same chromosome, but on different homologs, are virtually never expressed in the same drug-resistant clone. The utility of this mapping procedure is demonstrated by confirming the chromosomal location of seven known markers, as well as by the assignment of a previously unmapped mutation, spo12-1, to chromosome VIII. In addition, the analysis of the products of spo11-1 meiosis indicates that several markers previously assigned to either chromosome XIV or chromosome XVII are actually on the same chromosome.     

Initiation of meiosis in cell cycle initiation mutants of Saccharomyces cerevisiae

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

bgs2+, a sporulation-specific glucan synthase homologue is required for proper ascospore wall maturation in fission yeast

The formation of the ascospore cell wall of Schizosaccharomyces pombe requires the co-ordinated activity of enzymes involved in the biosynthesis of its components, such as glucans. We have cloned the bgs2+ gene. bgs2+ belongs to the glucan synthase family of S. pombe and is homologous to the Saccharomyces cerevisiae FKS1 and FKS2 genes. Deletion or overexpression of this gene does not lead to any apparent defect during vegetative growth, but homozygous bgs2Delta diploids do show a sporulation defect. Although meiosis takes place normally, ascospores are unable to mature, and their wall differs from that of wild-type ascospores. Moreover, bgs2Delta zygotes were not able to release ascospores spontaneously, and the ascospores were unable to germinate. We show that expression of bgs2+ is restricted to sporulation and that a bgs2-green fluorescent protein (GFP) fusion protein localizes to the ascospore envelope. The glucan synthase activity in sporulating diploids bearing a bgs2 deletion was diminished in comparison with that of the wild-type diploids, a fact that underscores the importance of the bgs2+ gene and glucan synthesis for the proper formation and maturation of the ascospore wall.     

Phage-mediated cloning of bldA, a region involved in Streptomyces coelicolor morphological development, and its analysis by genetic complementation.

Streptomyces coelicolor bald (bld) mutants form colonies of vegetative substrate mycelium, but do not develop aerial hyphae or spore chains. The bldA strains form none of the four antibiotics known to be produced by the parent strain. With a vector derived from the temperate bacteriophage phi C31, a 5.6-kilobase fragment of wildtype DNA was cloned which restored sporulation to five independent bldA mutants when lysogenized with the recombinant phage. The cloned gene(s) was dominant over the mutant alleles. Phage integration by recombination of the cloned bldA+ DNA with the bldA region of each mutant produced mainly sporulating colonies, presumably heterozygous bldA+/bldA partial diploids for the insert DNA. However, a minority of these primary transductants were bald and were apparently homozygous bldA/bldA mutant partial diploids, formed by some homogenetization process. The phages released from the bald lysogens carried bldA mutations and were used to show that bldA+ sequences had been cloned and that fine mapping of the region could be performed.     

Functional differences between RSC1 and RSC2, components of a for growth essential chromatin-remodeling complex of Saccharomyces cerevisiae,during the sporulation process

RSC, a for growth essential chromatin-remodeling complex of Saccharomyces cerevisiae, is composed of 15 subunits. Rsc1p and Rsc2p are highly homologous proteins and are contained in distinct RSC complexes. We found that both rsc1Delta and rsc2Delta homozygous diploids showed reduced sporulation with decreased expression of IME2 and that rsc1Delta, but not rsc2Delta, produced aberrant asci containing one to three spores. Overexpression of RSC2 in rsc1Delta recovered the sporulation efficiency but not the production of aberrant asci. In contrast, overexpression of RSC1 in rsc2Delta did not alleviate its sporulation defect. These results suggest that both Rsc1p and Rsc2p share overlapping functions on IME2 expression, with a prominent role for Rsc2p, whereas Rsc1p has an additional function in the late steps of the sporulation process.     

Yeast meiotic mutants proficient for the induction of ectopic recombination.

A screen was designed to identify Saccharomyces cerevisiae mutants that were defective in meiosis yet proficient for meiotic ectopic recombination in the return-to-growth protocol. Seven mutants alleles were isolated; two are important for chromosome synapsis (RED1, MEK1) and five function independently of recombination (SPO14, GSG1, SPOT8/MUM2, 3, 4). Similar to the spoT8-1 mutant, mum2 deletion strains do not undergo premeiotic DNA synthesis, arrest prior to the first meiotic division and fail to sporulate. Surprisingly, although DNA replication does not occur, mum2 mutants are induced for high levels of ectopic recombination. gsg1 diploids are reduced in their ability to complete premeiotic DNA synthesis and the meiotic divisions, and a small percentage of cells produce spores. mum3 mutants sporulate poorly and the spores produced are inviable. Finally, mum4-1 mutants produce inviable spores. The meiotic/sporulation defects of gsg1, mum2, and mum3 are not relieved by spo11 or spo13 mutations, indicating that the mutant defects are not dependent on the initiation of recombination or completion of both meiotic divisions. In contrast, the spore inviability of the mum4-1 mutant is rescued by the spo13 mutation. The mum4-1 spo13 mutant undergoes a single, predominantly equational division, suggesting that MUM4 functions at or prior to the first meiotic division. Although recombination is variably affected in the gsg1 and mum mutants, we hypothesize that these mutants define genes important for aspects of meiosis not directly related to recombination.     

Effects of Mutagen-Sensitive Mus Mutations on Spontaneous Mitotic Recombination in Aspergillus

Methyl methane-sulfonate (MMS)-sensitive, radiation-induced mutants of Aspergillus were shown to define nine new DNA repair genes, musK to musS. To test mus mutations for effects on mitotic recombination, intergenic crossing over was assayed between color markers and their centromeres, and intragenic recombination between two distinguishable adE alleles. Of eight mutants analyzed, four showed significant deviations from mus+ controls in both tests. Two mutations, musK and musL, reduced recombination, while musN and musQ caused increases. In contrast, musO diploids produced significantly higher levels only for intragenic recombination. Effects were relatively small, but averages between hypo- and hyperrec mus differed 15-20-fold. In musL diploids, most of the rare color segregants resulted from mitotic malsegregation rather than intergenic crossing over. This indicates that the musL gene product is required for recombination and that DNA lesions lead to chromosome loss when it is deficient. In addition, analysis of the genotypes of intragenic (ad+) recombinants showed that the musL mutation specifically reduced single allele conversion but increased complex conversion types (especially recombinants homozygous for ad+). Similar analysis revealed differences between the effects of two hyperrec mutations; musN apparently caused high levels solely of mitotic crossing over, while musQ increased various conversion types but not reciprocal crossovers. These results suggest that mitotic gene conversion and crossing over, while generally associated, are affected differentially in some of the mus strains of Aspergillus nidulans.