Saccharomyces cerevisiae aldolase mutants.

Six mutants lacking the glycolytic enzyme fructose 1,6-bisphosphate aldolase have been isolated in the yeast Saccharomyces cerevisiae by inositol starvation. The mutants grown on gluconeogenic substrates, such as glycerol or alcohol, and show growth inhibition by glucose and related sugars. The mutations are recessive, segregate as one gene in crosses, and fall in a single complementation group. All of the mutants synthesize an antigen cross-reacting to the antibody raised against yeast aldolase. The aldolase activity in various mutant alleles measured as fructose 1,6-bisphosphate cleavage is between 1 to 2% and as condensation of triose phosphates to fructose 1,6-bisphosphate is 2 to 5% that of the wild-type. The mutants accumulate fructose 1,6-bisphosphate from glucose during glycolysis and dihydroxyacetone phosphate during gluconeogenesis. This suggests that the aldolase activity is absent in vivo.     

Cell-cycle mutations among the collection of Saccharomyces cerevisiae dna mutants

Abstract The temperature-sensitive dna mutants of the budding yeast Saccharomyces cerevisiae (Dumas et al. (1982) Mol. Gen. Genet. 187, 42–46) are more inhibited in DNA synthesis than in protein synthesis. These properties are also characteristics of many yeast mutations that inhibit progress through the cell cycle. Therefore we surveyed the collection of dna mutants for cell-cycle mutations. By genetic complementation we found that dna 1 = cdc 22, dna 6 = cdc 34, dna 19 = cdc 36, and dna 39 = dbf 3. Furthermore, by direct gene cloning we found that the dna26 mutation is allelic to prt1 mutations, which are known to exert primary inhibition on protein synthesis. This protein-synthesis mutation exerts a dna phenotype due to cell-cycle inhibition: prt1 mutations can block the regulatory step of the cell cycle while allowing significant amounts of protein synthesis to continue. Our non-exhausive screening suggests that the dna mutants may house other mutations that affect the yeast cell cycle.     

Chromosomal superkiller mutants of Saccharomyces cerevisiae.

Yeast strains carrying a 1.5 X 10(6)-dalton double-stranded RNA in virus-like particles secrete a protein toxin which is lethal to strains not carrying this species of double-stranded RNA. We find that recessive mutations in any of four chromosomal genes result in the superkiller phenotype, i.e., increased secretion of killer toxin activity by strains carrying the killer genome. These genes are designated ski1 through ski4 (for superkiller), ski3 and ski4 are located on chromosome XIV, and ski1 is on chromosome VII. A ski1 mutation results in a decreased rate of cell growth. The kex1 and kex2 mutations are epistatic to each ski mutation.     

Characterization of Saccharomyces cerevisiae ubiquinone-deficient mutants.

Ubiquinol (QH2) is a lipid-soluble molecule that participates in cellular redox reactions. Previous studies have shown that yeast mutants lacking QH2 are hypersensitive to treatment with polyunsaturated fatty acids (PUFAs) indicating that QH2 can function as an antioxidant in vivo. In this study the effect of 1 mM linolenic acid on levels of Q6 and Q6H2 is assessed in both wild-type and respiration-deficient (atp2 delta) strains. The response of Q-deficient mutants to other forms of oxidative stress is further characterized to define those conditions where QH2 acts as an antioxidant. Endogenous antioxidant defense systems were also assessed in wild-type, Q-deficient, and atp2 delta strains. Superoxide dismutase (SOD) activity decreased and catalase activity increased in both Q-deficient and atp2 delta mutants compared to wild-type cells, suggesting that such changes result from the loss of respiration rather than the lack of Q.     

Diphtheria toxin-resistant mutants of Saccharomyces cerevisiae.

We developed a selection procedure based on the observation that diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae (Murakami et al., Mol. Cell. Biol. 2:588-592, 1982); this procedure yielded mutants resistant to the in vitro action of the toxin. Spheroplasts of mutagenized S. cerevisiae were transformed in the presence of diphtheria toxin, and the transformed survivors were screened in vitro for toxin-resistant elongation factor 2. Thirty-one haploid ADP ribosylation-negative mutants comprising five complementation groups were obtained by this procedure. The mutants grew normally and were stable to prolonged storage. Heterozygous diploids produced by mating wild-type sensitive cells with the mutants revealed that in each case the resistant phenotype was recessive to the sensitive phenotype. Sporulation of these diploids yielded tetrads in which the resistant phenotype segregated as a single Mendelian character. From these observations, we concluded that these mutants are defective in the enzymatic steps responsible for the posttranslational modification of elongation factor 2 which is necessary for recognition by diphtheria toxin.     

Ethanol-sensitive mutants of Saccharomyces cerevisiae

Saccharomyces cerevisiae mutants unable to grow at ethanol concentrations at which the wild type strain S288C does grow, have been isolated. Some of them show additional phenotypic alterations in colony size, temperature sensitivity and viability in ethanol, which cosegregate with the growth sensitivity in ethanol. 21 selected monogenic ethanol-sensitive mutants define 20 complementation groups, denominated ETA1 to ETA20, which indicates that there is a high number of genes involved in the ethanol tolerance/sensitivity mechanism.Out of 21 selected monogenic mutants, 20 are not altered in the glycolytic pathway since, when maintained in glucosesupplemented medium, they can produce as much ethanol as the wild type and at about the same velocity. Nor do any of the mutants seem to be altered in the lipid biosynthetic pathway since, whether grown in the absence or in the presence of ethanol, their concentration of fatty acids and ergosterol is similar to that of the wild type under the same conditions. Therefore growth sensitivity to ethanol does not seem necessarily to be related to carbohydrate or lipid metabolism.Non-common abbreviations YP yeast extract peptone medium - YPD yeast extract peptone dextrose agar or medium - YPG yeast extract peptone glycerol agar - YPDE yeast extract peptone dextrose ethanol agar or medium - SD yeast nitrogen base dextrose agar - SPO yeast extract potassium acetate glucose agar - PD parental ditype - NPD non-parental ditype - TT tetratype     

Isolation of mutations synthetic-lethal to prohibitin 2 null mutants of Saccharomyces cerevisiae

Prohibitins are ubiquitous, abundant proteins found in a wide range of organisms and that have a high degree of sequence conservation. In yeast it has previously been demonstrated that prohibitin proteins form a complex and are involved in maintaining the morphological and functional integrity of mitochondria. We have used a colony-sectoring assay as a screen for mutants that are dependent upon the presence of functional Phb2p in the cell. Two classes of prohibitin dependent mutation (pbd1 and pbd2) were isolated and characterised. The effect of these mutations on replicative lifespan was determined, demonstrating that the pbd1 mutant slightly extended lifespan, whereas in contrast, the pbd2 mutation resulted in a shortening in both the mean- and the maximum-lifespan. The pbd1 mutation was also found to reduce chronological lifespan. Reducing the expression of the PHB2 gene in the pbd mutants was found to retard the rate of growth and to affect replicative lifespan. As the two mutants behave in a different manner they probably affect different aspects of prohibitin function.     

Aluminum-sensitive mutants of Saccharomyces cerevisiae

We are developing budding yeast, Saccharomyces cerevisiae, as a genetic system for the study of tolerance to the trivalent aluminum cation (Al³⁺). We have isolated eight mutants that are more sensitive to Al³⁺ than the wild type. Each mutant represented a different complementation group. A number of the mutants were pleiotropic, and showed defects in other stress responses, changes in tolerance to other metal cations, or abnormal morphology. Two mutants also showed increased dependence on supplemental Mg²⁺ and Ca²⁺. One mutant with a relatively specific sensitivity to Al³⁺ was chosen for molecular complementation. Normal Al³⁺ tolerance was restored by expression of the MAP kinase gene SLT2. Strains carrying deletions of the SLT2 gene, or of the gene for the corresponding MAP kinase–kinase SLK1, showed sensitivity to Al³⁺. These results indicate that the SLT2 MAP kinase signal transduction pathway is required for yeast to sense and respond to Al³⁺ stress.     

Adenosine utilization in cordycepin-sensitive mutants of Saccharomyces cerevisiae.

A purine-requiring, wild-type yeast strain was cordycepin resistant and failed to grow in medium containing adenosine; in contrast, a cordycepin-sensitive mutant (also purine requiring) grew well in medium containing adenosine. The cordycepin-sensitive mutant incorporated [8-14C]adenosine at nine times the wild-type rate, and adenosine completely fulfilled the purine requirement of the cells. Exogenous adenosine rapidly entered the mutant cells, apparently as free nucleoside, and was phosphorylated; uptake displayed concentration-dependent saturation kinetics (Km, 6 mM). Within 10 min 14C radioactivity was being incorporated into nucleic acids.     

Flocculation of Saccharomyces cerevisiae tup1 mutants.

Strains of Saccharomyces cerevisiae carrying a mutation in the TUP1 locus exhibited calcium-dependent flocculation. The flocculation had none of the characteristics of sexual agglutination. The flocculation differed from that exhibited by a FLO1 strain in the effect of pH on cation dependence and sensitivity to chemical inactivation.     

Adenine phosphoribosyltransferase mutants in Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae deficient in adenine phosphoribosyltransferase (A-PRT, EC 2,4,2,7) have been isolated following selection for resistance to 8-azaadenine in a prototrophic strain carrying the ade4-su allele of the gene coding for amidophosphoribosyltransferase (EC 2,4,2,14). The mutants were recessive and defined a single gene, apt1. They did not excrete purine when combined with ade4+. The mutants appeared to retain some A-PRT activity in crude extracts, and strains of the genotype ade2 apt1 responded to both adenine and hypoxanthine. Mutants deficient in adenine aminohydrolase (EC 3,5,4,2) activity, aah1, and hypoxanthine:guanine phosphoribosyltransferase (EC 2,4,2,8) activity, hpt1, were used to synthesize the genotypes apt1 hpt1 aah+ and apt1 hpt+ aah1. The absence of A-PRT activity in strains with these genotypes confirmed the hypothesis that the residual A-PRT activity of apt1 mutants was due to adenine aminohydrolase and hypoxanthine:guanine phosphoribosyltransferase acting in concert.