Twenty-Six Chromosomal Genes Needed to Maintain the Killer Double-Stranded RNA Plasmid of SACCHAROMYCES CEREVISIAE

The double-stranded RNA killer plasmid gives yeast strains carrying it both the ability to secret a protein toxin and immunity to that toxin. This report describes a new series of mutants in chromsomal genes needed for killer plasmid maintenance (mak genes). These mutants comprise 12 complementation groups. There are a total of at least 26 mak genes. Each mak gene product is needed for plasmid maintenance in diploids as well as in haploids. None of these mak mutations prevent the killer plasmid from entering the mak- spores in the process of meiotic sporulation. Complementation between mak mutants can be performed by mating meitoic spores from a makx/+ plasmid-carrying diploid with a maky haploid. If x = y, about half the diploid clones formed lose the killer plasmid. If x not equal to y, complementation occurs, and all of the diploid clones are killers.     

Two Chromosomal Genes Required for Killing Expression in Killer Strains of SACCHAROMYCES CEREVISIAE

The killer character of yeast is determined by a 1.4 x 10⁶ molecular weight double-stranded RNA plasmid and at least 12 chromosomal genes. Wild-type strains of yeast that carry this plasmid (killers) secrete a toxin which is lethal only to strains not carrying this plasmid (sensitives). ——— We have isolated 28 independent recessive chromosomal mutants of a killer strain that have lost the ability to secrete an active toxin but remain resistant to the effects of the toxin and continue to carry the complete cytoplasmic killer genome. These mutants define two complementation groups, kex1 and kex2. Kex1 is located on chromosome VII between ade5 and lys5. Kex2 is located on chromosome XIV, but it does not show meiotic linkage to any gene previously located on this chromosome. ——— When the killer plasmid of kex1 or kex2 strains is eliminated by curing with heat or cycloheximide, the strains become sensitive to killing. The mutant phenotype reappears among the meiotic segregants in a cross with a normal killer. Thus, the kex phenotype does not require an alteration of the killer plasmid. ——— Kex1 and kex2 strains each contain near-normal levels of the 1.4 x 10⁶ molecular weight double-stranded RNA, whose presence is correlated with the presence of the killer genome.     

Chromosomal and Nonchromosomal Mutations Affecting the "Killer Character" of SACCHAROMYCES CEREVISIAE

The "killer character" of Saccharomyces cerevisiae is a nonchromosomal genetic element which imparts to cells carrying it (a) the ability to kill cells which lack it, by secreting the soluble macromolecular killer substance and (b) the ability to resist the killing effects of the killer substance.-Mutants have been isolated from S. cerevisiae carrying the "killer character". Mutants were found in two nuclear genes (mak1 and mak2) involved in maintenance of the genetic element of the killer character. Mutants in three other nuclear genes could maintain the cytoplasmic genetic element normally; two of these were able to express resistance to killing, but not the ability to kill (kex1 and kex2), while the third could express the killing function, but was sensitive to killing and is thus a "suicide" strain (rex1). These five nuclear genes were all distinct.-Several mutants were detected which showed non-mendelian segregation indicating mutation of the "cytoplasmic" genetic element. Some such mutants had normal resistance to killing but were either unable to kill at all or showed marked reduction in the ability to kill. Others had lost both killing ability and resistance. None were suppressive.-Two nonkilling mutants, both of which showed cytoplasmic inheritance, were mated. A large, but variable, proportion of diploid colonies, plated after several generations of growth, showed normal killing which has then stably inherited on further subcloning.     

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.     

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.     

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 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.     

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.     

Population Studies in Microorganisms I. Evolution of Diploidy in SACCHAROMYCES CEREVISIAE

The relative adaptation of isogenic haploid and diploid strains of yeast was investigated in different sets of physiological conditions. When all nutrients were present in excess, no difference in the reproductive rates of isogenic haploid and diploid strains of yeast was detected in both optimal and non-optimal growth conditions. Competition between haploid and diploid strains of yeast was observed when growth was limited by the concentration of a single nutrilite. Under certain conditions when fitness (reproductive rate) is determined by transport of an essential nutrilite that exists in very low concentrations, diploid cells were selected against. These environmental conditions are similar to those found in offshore marine environments where nutrients are often present in extremely low concentrations. The fitness of diploid cells was estimated to be.93 +/-.02 (haploid fitness = 1). The reduced fitness of diploid cells in this environment can be explained by the reduced surface area/volume ratio possessed by diploid cells in comparison to haploid cells. The fitnesses of haploid and diploid cells in these environments are closely correlated with geometric variations in these strains. These results are consistent with the hypothesis that diploid cells are simply double haploids, and diploidy per se does not confer any direct adaptive advantage. The mechanism of the evolution of diploidy as a dominant phase in the life cycle of higher plants and animals remains obscure.     

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.     

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.     

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.     

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.     

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.     

Mapping Chromosomal Genes of SACCHAROMYCES CEREVISIAE Using an Improved Genetic Mapping Method

A triploid (3n) strain of Saccharomyces cerevisiae was constructed carrying a standard marker on each of chromosomes 1 through XVII in the -/+/+ configuration. This is called a "supertriploid." Meiotic spores from this strain (n + approximately n/2) were mated with a haploid (n) carrying an unmapped mutation. Meiotic analysis of each zygote clone (2n + approximately n/2) produced in this way resulted in elimination of an average of 4.2 chromosomes as the possible location of the unmapped marker. The distribution of extra chromosomes in the 2n + approximately n/2) strains was nearly random. Meiotic segregrants of these crosses carrying the unmapped mutation in the -/+ configuration were then crossed with multiply marked haploid strains to further narrow the possible location of the unmapped mutation to a single chromosome. Scoring of markers by complemention tests was simplified by mating spore clones with mixtures of a and alpha strains, each pair carrying the same set of markers. Using this new, more rapid method ("supertriploid mapping"), eight genes required for the maintenance of the killer plasmid were located on the genetic map of S. cerevisiae.     

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.     

Selection of lys2 Mutants of the Yeast SACCHAROMYCES CEREVISIAE by the Utilization of alpha-AMINOADIPATE

Normal strains of Saccharomyces cerevisiae do not use alpha-aminoadipate as a principal nitrogen source. However, alpha-aminoadipate is utilized as a nitrogen source by lys2 and lys5 strains having complete or partial deficiencies of alpha-aminoadipate reductase and, to a limited extent, by heterozygous lys2/+ strains. Lys2 mutants were conveniently selected on media containing alpha-aminoadipate as a nitrogen source, lysine, and other supplements to furnish other possible auxotrophic requirements. The lys2 mutations were obtained in a variety of laboratory strains containing other markers, including other lysine mutations. In addition to the predominant class of lys2 mutants, low frequencies of lys5 mutants and mutants not having any obvious lysine requirement were recovered on alpha-aminoadipate medium. The mutants not requiring lysine appeared to have mutations at the lys2 locus that caused partial deficiencies of alpha-aminoadipate reductase. Such partial deficiencies are believed to be sufficiently permissive to allow lysine biosynthesis, but sufficiently restrictive to allow for the utilization of alpha-aminoadipate. Although it is unknown why partial or complete deficiencies of alpha-aminoadipate reductase cause utilization of alpha-aminoadipate as a principal nitrogen source, the use of alpha-aminoadipate medium has considerable utility as a selective medium for lys2 and lys5 mutants.