Meiosis Can Induce Recombination in rad52 Mutants of SACCHAROMYCES CEREVISIAE

The RAD52 and RAD50 genes have previously been shown to be required for normal meiotic recombination and for various types of recombination occurring in mitotic cells. Recent evidence suggests that rad52 mutants might be defective in an intermediate recombination step; we therefore examined recombination during meiosis in several rad52 mutants at several different loci and in genetic backgrounds that yield efficient sporulation and synchronous meiosis. Similar to previous reports, spores from rad52 diploids are inviable and meiotic recombination is greatly reduced by rad52 mutations. However, intragenic recombinants were detected when cells were plated on selective media during meiosis; rad52 mutants experience induction of recombination between homologues under these special conditions. The frequencies of recombination at four loci were considerably greater than the mitotic controls; however, they were still at least 20 times lower than corresponding Rad+ strains. The prototrophs induced by meiosis in rad52 mutants were not typical meiotic recombinants because incubation in nutrient-rich medium before plating to selective medium resulted in the complete loss of recombinants. We propose that previously observed single-strand breaks that accumulate in rad52 mutants may be associated with recombinational intermediates that are resolved when cells are returned to selective mitotic media and that the meiosis-induced recombination in rad52 cells does not involve double-strand breaks.     

Proteinase Mutants of SACCHAROMYCES CEREVISIAE

Fifty-nine mutants with reduced ability to cleave the chymotrypsin substrate N-acetyl-DL-phenylalanine beta-naphthyl ester have been isolated in S. cerevisiae. All have reduced levels of one or more of the three well-characterized proteinases in yeast. All have reduced levels of proteinase C (carboxy-peptidase Y). These mutations define 16 complementation groups.     

Small-Sized Mutants of SACCHAROMYCES CEREVISIAE

The isolation of mutants of Saccharomyces cerevisiae that divide at approximately half the size of the wild type is described. Three mutants have been isolated in which the small size at bud initiation is due to a mutation in a single nuclear gene.     

Inositol-Requiring Mutants of SACCHAROMYCES CEREVISIAE

Fifty-two inositol-requiring mutants of Saccharomyces cerevisiae were isolated following mutagenesis with ethyl methanesulfonate. Complementation and tetrad analysis revealed ten major complementation classes, representing ten independently segregating loci (designated ino1 through ino10) which recombined freely with their respective centromeres. Members of any given complementation class segregated as alleles of a single locus. Thirteen complementation subclasses were identified among thirty-six mutants which behaved as alleles of the ino1 locus. The complementation map for these mutants was circular.—Dramatic cell viability losses indicative of unbalanced growth were observed in liquid cultures of representative mutants under conditions of inositol starvation. Investigation of the timing, kinetics, and extent of cell death revealed that losses in cell viability in the range of 2-4 log orders could be prevented by the addition of inositol to the medium or by disruption of protein synthesis with cycloheximide. Mutants defective in nine of the ten loci identified in this study displayed these unusual characteristics. The results suggest an important physiological role for inositol that may be related to its cellular localization and function in membrane phospholipids. The possibility is discussed that inositol deficiency initiates the process of unbalanced growth leading to cell death through the loss of normal assembly, function, or integrity of biomembranes.—Part of this work has been reported in preliminary form (Culbertson and Henry 1974).     

Isolation and Characterization of Mms-Sensitive Mutants of SACCHAROMYCES CEREVISIAE

We have isolated mutants sensitive to methyl methanesulfonate (MMS) in Saccharomyces cerevisiae. Alleles of rad1, rad4, rad52, rad55 and rad57 were found amoung these mms mutants. Twenty-nine of the mms mutants which complement the existing radiation-sensitive (rad and rev) mutants belong to 22 new complementation groups. Mutants from five complementation groups are sensitive only to MMS. Mutants of 11 complementation groups are sensitive to UV or X rays in addition to MMS, mutants of six complementation groups are sensitive to all three agents. The cross-sensitivities of these mms mutants to UV and X rays are discussed in terms of their possible involvement in DNA repair. Sporulation is reduced or absent in homozygous diploids of mms mutants from nine complementation groups.     

A Method for Obtaining Antibiotic-Sensitive Mutants in SACCHAROMYCES CEREVISIAE

We have isolated a temperature-sensitive mutant of S. cerevisiae which has proven useful for the isolation of antibiotic-sensitive strains.     

Mutants of the Formyltetrahydrofolate Interconversion Pathway of SACCHAROMYCES CEREVISIAE

Thirteen mutants of Saccharomyces cerevisiae that lack one or more of the three enzyme activities of the pathway for interconversion of tetrahydrofolate coenzymes at the formate level of oxidation have been isolated. They do not require adenine. All fail to complement mutations in the ade3 locus. Mutations that greatly reduce activity for one enzyme also reduce activity for the other two interconversion enzymes. The three enzyme activities cochromatograph on TEAE-cellulose columns. A mutation that eliminates synthetase activity also alters the chromatographic behavior of the remaining cyclohydrolase and dehydrogenase activities. It is suggested that the three activities reside in an enzyme complex encoded by the ade3 locus.     

Increased Spontaneous Mitotic Segregation in Mms-Sensitive Mutants of SACCHAROMYCES CEREVISIAE

Methyl methanesulfonate (MMS)-sensitive mutants of Saccharomyces cerevisiae belonging to four different complementation groups, when homozygous, increase the rate of spontaneous mitotic segregation to canavanine resistance from heterozygous sensitive (canr/+) diploids by 13-to 170-fold. The mms8-1 mutant is MMS and X-ray sensitive and increases the rate of spontaneous mitotic segregation 170-fold. The mms9-1 and mms13-1 mutants are sensitive to X rays and UV, respectively, in addition of MMS, and increase the rate of spontaneous mitotic segregation by 13-fold and 85-fold, respectively. The mutant mms21-1 is sensitive to MMS, X rays and UV and increases the rate of spontaneous mitotic segregation 23-fold.     

Mutants Showing Heterothallism from a Homothallic Strain of SACCHAROMYCES CEREVISIAE

Reverse and forward mutation, induced by photoaddition of 8-methoxypsoralen (8-MOP) and 3-carbethoxypsoralen (3-CPs) or ultraviolet light (UV), are reduced in three pso mutants of Saccharomyces cerevisiae. The pso1–1 strain exhibits a lower frequency of spontaneous reversion (antimutator) and is almost entirely unaffected by the three agents in both the haploid and diploid states. The pso2–1 strain demonstrates very reduced frequencies of 8-MOP and 3-CPs plus 365 nm radiation-induced mutations in happloid and diploid cells. UV-induced mutations are slightly reduced, whereas survival is almost normal. The pso3–1 strain is mutable by 8-MOP and 3-CPs photoaddition only in the low-dose range. After UV treatment, survival of pso3–1 is nearly normal, whereas the frequencies of induced mutants are diminished as compared to the normal PSO⁺. An analogue of adenine, 6-N-hydroxyaminopurine, is capable of inducing reversions in wild type, as well as in pso and rad6–1 mutant strains, indicating that this drug may act as a direct mutagen in yeast. The comparison of photoaddition of the bifunctional agent (8-MOP) to that of the monofunctional one (3-CPs) confirms that cross-links, as well as monoadditions, are mutagenic in S. cerevisiae. Repair, of the recombinational type, taking place in diploid cells or in haploid cells in G2 phase leads to higher survival, but appears to be error-free.     

Genetic Analysis of Petite Mutants of SACCHAROMYCES CEREVISIAE : Transmissional Types

We have studied a number of petite [rho^- ] mutants of Saccharomyces cerevisiae induced in a wild-type strain of mitochondrial genotype [ome^- CHL ^R ERY^S OLI^S_1,2,3 PAR^S] by Berenil and ethidium bromide, all of which have retained two mitochondrial genetic markers, [CHL^R] and [ERY^S], but have lost all other known markers. Though stable in their ability to retain these markers in their genome, these mutants vary widely among themselves in suppressiveness and in the extent to which the markers are transmitted on crossing to a common wild-type tested strain. In appropriate crosses all of the strains examined in this study demonstrate mitochondrial polarity, and thus have also retained the [ome^-] locus in a functional form; however, five different transmissional types were obtained, several of them quite unusual, particularly among the strains originally induced by Berenil. One of the most interesting types is the one that appears to reverse the parental genotypes with [CHL^R ERY^S] predominating over [CHL^S ERY^R] in the diploid [rho+] progeny, rather than the reverse, which is characteristic of analogous crosses with [rho+] or other petites. Mutants in this class also exhibited low or no suppressiveness. Since all of the petites reported here are derived from the same wild-type parent, and so have the same nuclear background, we have interpreted the transmissional differences as being due to different intramolecular arrangements of largely common retained sequences.     

Sensitivity of Tryptophan, Tyrosine and Phenylalanine Mutants of SACCHAROMYCES CEREVISIAE to Phenethyl Alcohol

Phenethyl alcohol inhibits the growth of many microorganisms. It is believed that the growth inhibition is mediated by its effect on the cell membrane. Differences between sensitive and resistant strains are suggested to be due to alterations in membrane structure. We report that, in some strains, an unexpected relationship exists between auxotrophy for tryptophan, tyrosine and phenylalanine and sensitivity to phenethyl alcohol.     

Isolation of Suppressive Sensitive Mutants from Killer and Neutral Strains of SACCHAROMYCES CEREVISIAE

Dominant sensitive mutants were isolated from a killer and a neutral strain of Saccharomyces cerevisiae by treatment with nitrosomethylurethane. Genetic studies suggest that these sensitives arose by mutation of the wild-type cytoplasmic genetic determinants (k) or (n) to a mutant form (s). (s) determinants lack wild-type (k) and (n) activity but are retained in the cell and suppress the replication or activity of the wild-type determinants, converting killer and neutral cells to the sensitive phenotype. These mutants show an obvious similarity in behavior to suppressive petite mutants of yeast.     

Protein Synthesis in Relation to Sporulation and Meiosis in Yeast

The dependence upon protein synthesis of physiological and biochemical events occurring during yeast sporulation was investigated. Protein synthesis was inhibited by cycloheximide. There was an early, irreversible sensitivity to inhibition with respect to cell viability and ascus formation; inhibition was reversible only if the cells were inhibited after, but not prior to, 2 to 3 h in sporulation medium. Interruption of protein synthesis of any time during sporulation inhibited all measurable metabolic and sporulation-specific processes except protein breakdown and, to some extent, ribonucleic acid synthesis. The time interval between the occurrence of an event and the protein synthesis necessary for that event was determined to be 2 to 3 h for ascus formation, 相似文献   

A Cluster of Three Genes Responsible for Allantoin Degradation in SACCHAROMYCES CEREVISIAE

Mutant strains of Saccharomyces cerevisiae unable to utilize allantoin as sole nitrogen source were isolated and divided into three groups on the basis of their biochemical and genetic characteristics. The three loci associated with these mutant classes were designated dal1 (allantoinase minus), dal2 (allantoicase minus) and dal4 (allantoin transport minus). All three loci are located in a cluster that is proximal to the lys1 locus on the right arm of chromosome IX. The gene order and intergenic distances were estimated to be: dal1--2.5 cM--dal4--1.9cM--dal2--4.6cM-lys1.     

Isolation and Characterization of pso Mutants Sensitive to Photo-Addition of Psoralen Derivatives in SACCHAROMYCES CEREVISIAE

We have isolated mutants sensitive to photo-addition of bi-functional and mono-functional derivatives of psoralen in Saccharomyces cerevisiae. Three of these pso mutants were analyzed in detail. They segregate in meiosis like Mendelian genes and complement each other, as well as existing radiation-sensitive (rad and rev) mutants. The study of heterozygous diploid strains (PSO⁺/pso) indicates that the three pso genes are recessive. The mutant pso1–1 demonstrates a cross-sensitivity to UV and γ-rays, whereas mutants pso2–1 and pso3–1 are specifically sensitive to photo-addition of psoralen derivatives. The comparison of exponentially growing cells to stationary-phase cells demonstrates that for the three mutants the defect in repair capacity of DNA cross-links and monoadducts concerns G1 and early S-phase cells. The pso2–1 mutant is, however, also defective in G2 repair and loses diploid resistance when it is in the homozygous state.—The block in repair capacity in these novel mutants is discussed in relation to the three other repair pathways known to be involved in the repair of furocoumarins photo-induced lesions in yeast DNA.     

Gene Duplication in SACCHAROMYCES CEREVISIAE

Five indepdendent duplications of the acid-phosphatase (aphtase) structural gene (acp1) were recovered from chemostat populations of S. cerevisiae that were subject to selection for in vivo hyper-aphtase activity. Two of the duplications arose spontaneously. Three of them were induced by UV. All five of the duplication events involved the transpositioning of the aphtase structural gene, acp1, and all known genes distal to acp1 on the right arm of chromosome II, to the terminus of an arm of other unknown chromosomes. One of the five duplicated regions of the right arm of chromosome II was found to be transmitted mitotically and meiotically with very high fidelity. The other four duplicated regions of the right arm of chromosome II were found to be unstable, being lost at a rate of about 2% per mitosis. However, selection for increased fidelity of mitotic transmission was effective in one of these strains. No tandem duplications of the aphtase structural gene were found.     

Genetic Analysis of Mutants of SACCHAROMYCES CEREVISIAE Resistant to the Herbicide Sulfometuron Methyl

Sulfometuron methyl (SM), a potent new sulfonylurea herbicide, inhibits growth of the yeast Saccharomyces cerevisiae on minimal media. Sixty-six spontaneous mutants resistant to SM were isolated. All of the resistance mutations segregate 2:2 in tetrads; 51 of the mutations are dominant, five are semidominant and ten are recessive. The mutations occur in three linkage groups, designated SMR1, smr2 and smr3. Several lines of evidence demonstrate that the SMR1 mutations (47 dominant and four semidominant) are alleles of ILV2 which encodes acetolactate synthase (ALS), the target of SM. First, SMR1 mutations result in the production of ALS enzyme activity with increased resistance to SM. Second, molecular cloning of the ILV2 gene permitted the isolation of mutations in the cloned gene which result in the production of SM-resistant ALS. Finally, SMR1 mutations map at the ILV2 locus. The smr2 mutations (four recessive, two dominant and one semidominant) map at the pdr 1 (pleiotropic drug resistance) locus and show cross-resistance to other inhibitors, typical of mutations at this locus. The smr3 mutations (six recessive and two dominant) define a new gene which maps approximately midway between ADE2 and HIS3 on the right arm of chromosome XV.