Genes Affecting the Regulation of SUC2 Gene Expression by Glucose Repression in SACCHAROMYCES CEREVISIAE

Mutants of Saccharomyces cerevisiae with defects in sucrose or raffinose fermentation were isolated. In addition to mutations in the SUC2 structural gene for invertase, we recovered 18 recessive mutations that affected the regulation of invertase synthesis by glucose repression. These mutations included five new snf1 (sucrose nonfermenting) alleles and also defined five new complementation groups, designated snf2, snf3, snf4, snf5, and snf6. The snf2, snf4, and snf5 mutants produced little or no secreted invertase under derepressing conditions and were pleiotropically defective in galactose and glycerol utilization, which are both regulated by glucose repression. The snf6 mutant produced low levels of secreted invertase under derepressing conditions, and no pleiotropy was detected. The snf3 mutants derepressed secreted invertase to 10-35% the wild-type level but grew less well on sucrose than expected from their invertase activity; in addition, snf3 mutants synthesized some invertase under glucose-repressing conditions.--We examined the interactions between the different snf mutations and ssn6, a mutation causing constitutive (glucose-insensitive) high-level invertase synthesis that was previously isolated as a suppressor of snf1. The ssn6 mutation completely suppressed the defects in derepression of invertase conferred by snf1, snf3, snf4 and snf6, and each double mutant showed the constitutivity for invertase typical of ssn6 single mutants. In contrast, snf2 ssn6 and snf5 ssn6 strains produced only moderate levels of invertase under derepressing conditions and very low levels under repressing conditions. These findings suggest roles for the SNF1 through SNF6 and SSN6 genes in the regulation of SUC2 gene expression by glucose repression.     

Genetic Analysis of Mutations Affecting Growth of SACCHAROMYCES CEREVISIAE at Low Temperature

A large number of genes control growth of the yeast Saccharomyces cerevisiae at low temperatures (< 10 degrees ). Approximately 47 percent of the mutants selected for inability to grow at 4-5 degrees C show increased sensitivity to cycloheximide. In 3 of 4 cases tested, supersensitivity to cycloheximide and inability to grow at the low temperature segregate together and thus appear to be effects of the same mutation. Since many cold-sensitive mutants of bacteria have been found to have altered ribosomes and since cycloheximide resistance in yeast can be caused by ribosomal changes, this suggests that the mutants having low-temperature-sensitive growth may be defective in ribosome-assembly processes at the low temperatures. Two of the lts loci, lts1 and lts3 have been located on chromosome VII and another two, lts4 and lts10 on chromosome IV. A mutation, cyh10, conferring cycloheximide resistance, but not cold sensitivity, has been located close to the centromere on chromosome II.     

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 Properties of Mutations at the PEP4 Locus in SACCHAROMYCES CEREVISIAE

Yeast cells that inherit mutations at the PEP4 locus exhibit a pronounced phenotypic lag in the expression of the mutant phenotype imparted by these mutations. This lag appears to extend to all of the enzymes that are affected by the pep4-3 mutation. For at least two of the enzymatic activities, phenotypic lag shows mitotic cosegregation. Phenotypic lag is found for meiotic progeny and for mitotic segregants from heterokaryons. The phenotypic lag in the expression of the carboxypeptidase Y deficiency is abolished by nonsense mutations in either PRC1, the structural gene for carboxypeptidase Y, or PRB1, the structural gene for proteinase B. Models to explain these observations are proposed.     

Mutations in the PHO80 Gene Confer Permeability to 5''-Mononucleotides in SACCHAROMYCES CEREVISIAE

Yeast mutants permeable to dTMP (tup) were selected and two new complementation groups (tup5 and tup7) were identified. Assay of the levels of both acid and alkaline phosphatase in cells grown under either repressing (5 mM PO4(-3) or derepressing (0.03 mM PO4(-3) conditions indicated that, in general, tup mutations cause cells to be defective in their regulation of phosphatase synthesis. In addition, three of the tup mutations (tup1, tup4 and tup7) displayed markedly elevated rates of inorganic phosphate transport. The tup7 locus was found to be tightly centromere-linked on the right arm of chromosome XV, and was shown to be allelic with the pho80 regulatory locus on the basis of both genetic and biochemical criteria. Analysis of other mutations known to affect phosphatase levels (pho) indicated that some also conferred permeability to dTMP. Possible allelic relationships between tup genes and certain of these pho mutations are discussed. Regardless of the culture conditions, wild-type strains were not permeable to dTMP; in contrast, it was found in the course of this work that normal yeast cells were permeable to dUMP and that dUMP permeability was regulated by the concentration of inorganic phosphate present in the medium used to grow the cells. Thus, permeability to 5'-mononucleotides appears to be under coordinate control with phosphatase synthesis.     

PEP4 Gene Function Is Required for Expression of Several Vacuolar Hydrolases in SACCHAROMYCES CEREVISIAE

The pep4-3 mutation results in a 90–95% reduction in the levels of five vacuolar hydrolases in yeast, including proteinases A and B, carboxypeptidase Y, RNase(s) and the repressible alkaline phosphatase. The mutation is without effect on two secreted glycoproteins, on an enzyme of the vacuolar membrane, and on a proteinase located outside of the vacuole. Mutations at the PEP4 locus exhibit a dosage effect on the levels of some, but not all, of the enzymes whose expression requires the function of the gene.     

Nonsense Mutations in the ADE3 Locus of SACCHAROMYCES CEREVISIAE

Fifty seven mutations at the ade3 locus have been crossed to ochre, amber and ochre-amber suppressors. 70% (39/56) of the mutations at this locus are nonsense mutations; 61% (34/56) are ochre mutations and 9% (5/56) are amber mutations. The frequency of nonsense mutations among ade3 alleles recovered is very high and raises the interesting possibility that only polar mutations at this locus are recovered. An hypothesis to explain these genetical findings as well as physiological properties of these mutations is proposed.     

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.     

Gene Conversion of the Mating-Type Locus in SACCHAROMYCES CEREVISIAE

Tetrad analysis of MATa/MAT alpha diploids of Saccharomyces cerevisiae generally yields 2 MATa:2MAT alpha meiotic products. About 1 to 1.8% of the tetrads yield aberrant segregations for this marker. Described here are experiments that determine whether the aberrant meiotic segregations at the mating-type locus are ascribable to gene conversions or to MAT switches, that is, to mating-type interconversions. Diploid strains incapable of switching MATa to MAT alpha, or the converse, nevertheless display changes of MATa to MAT alpha, or the reverse. These events must be attributed to gene conversion. Further, we suggest that MATa and MAT alpha alleles may represent nonhomologous sequences of DNA since they fail to display postmeiotic segregations.     

Expression of Radiation-Induced Mutations at the Arginine Permease (CAN1) Locus in SACCHAROMYCES CEREVISIAE

In the yeast Saccharomyces cerevisiae, the expression of resistance to the L-arginine analog, L-canavanine, after mutagenesis, is strongly dependent on the metabolic state of the cell. The frequency of mutations recovered after exposure to ultraviolet light or X rays was measured under a variety of culture conditions. The results indicate that the frequency of mutants recovered is determined by the following three factors: (1) The potential mutants still possess enough permease activity to take up some of the cell poison, and some are therefore killed before they can express the mutant genotype. The sensitivity is strongly influenced by the endogenous free arginine, which is in turn influenced by the growth medium. (2) The rapid decay of the permease molecules and the inability of the potential mutants to resynthesize this protein results in a rapidly increasing change of expression when selection is delayed. (3) During the time when the permease activity is decaying, repair of the mutagen-induced damage appears to occur.     

Mutations Affecting Sexual Conjugation and Related Processes in SACCHAROMYCES CEREVISIAE. II. Genetic Analysis of Nonmating Mutants

Rare diploids formed by sterile mutants have been studied by tetrad analysis. Sixteen classes of mutants representing at least five distinct genetic loci have been defined. One group of mutations, isolated only in alpha, maps at the mating-type locus, while none of the others shows any linkage to mating type. Some of the mutations are nonspecific for mating type, while others act only on a or alpha. In addition, mutations were found that prevent sporulation when heterozygous in diploids. These appear to be mutations of the mating-type alleles.     

Effects of the RAD52 Gene on Recombination in SACCHAROMYCES CEREVISIAE

Effects of the rad52 mutation in Saccharomyces cerevisiae on meiotic, γ-ray-induced, UV-induced and spontaneous mitotic recombination were studied. The rad52/rad52 diploids undergo premeiotic DNA synthesis; sporulation occurs but inviable spores are produced. Both intra and intergenic recombination during meiosis were examined in cells transferred from sporulation medium to vegetative medium at different time intervals. No intragenic recombination was observed at the his1–1/his1–315 and trp5–2/trp5–48 heteroalleles. Gene-centromere recombination also was not observed in rad52/rad52 diploids. No γ-ray- or UV-induced intragenic mitotic recombination is seen in rad52/rad52 diploids. The rate of spontaneous mitotic recombination is lowered five-fold at the his1–1/his1–315 and leu1–c/leu1–12 heteroalleles. Spontaneous reversion rates of both his1–1 and his1–315 were elevated 10 to 20 fold in rad52/rad52 diploids.—The RAD52 gene function is required for spontaneous mitotic recombination, UV- and γ-ray-induced mitotic recombination and meiotic recombination.     

Enhanced Gene Conversion and Postmeiotic Segregation in Pachytene-Arrested SACCHAROMYCES CEREVISIAE

Previous study has demonstrated that incubation of yeast cells of strain AP-1 in sporulation medium at 36° permits them to begin meiosis but that they become arrested at pachytene and undergo enhanced intragenic recombination between ade2 heteroalleles. Tetrad analysis was undertaken to characterize the altered program of meiotic recombination more widely. In one set of experiments, pachytene-arrested cells were permitted to resume sporulation upon transfer to the permissive temperature. In the resulting asci, both postmeiotic segregation and gene conversion were increased several-fold at a number of loci relative to unarrested controls, whereas reciprocal recombination increased two- to threefold. Another set of experiments analyzed the genetic consequences of inducing the pachytene-arrested cells to revert directly to mitotic growth without completion of meiosis. The appearance of homozygous sectors from heterozygous markers revealed that these cells had become committed to appreciable recombination but that reciprocal exchange was less frequent than in normal asci. Taken together, the data indicate that pachytene arrest rendered the cells committed to enhanced recombination upon resumption of sporulation but that most of the crossing over did not occur until release from the arrest. —The genetic basis of pachytene arrest by AP-1 was investigated by mating each of its parents with progeny of strain Y55, which is able to sporulate at 36°. Both of these diploids sporulated at 36°, and asci from the one studied further exhibited 2:2 segregation of the sporulation defect, indicating that pachytene arrest is dependent on a recessive, temperature-sensitive allele at a chromosomal locus.     

Mutations Affecting Sexual Conjugation and Related Processes in SACCHAROMYCES CEREVISIAE. I. Isolation and Phenotypic Characterization of Nonmating Mutants

Nonmating mutants were also isolated from haploid strains of yeast of both mating types. The mutants were characterized with respect to their ability to produce and respond to specific yeast sex factors, their ability to mate at low frequencies, and the ability of the low-frequency diploids to sporulate. Loss of the ability to mate by either mating type was invariably accompanied by the loss of one or more, and in some cases, all, of the above capabilities. The results strongly indicate that the sex factors are functionally involved in the conjugation process.     

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.     

Effect of Genes Controlling Radiation Sensitivity on Chemically Induced Mutations in SACCHAROMYCES CEREVISIAE

The effect of 16 different genes (rad) conferring radiation sensitivity on chemically induced reversion in the yeast Saccharomyces cerevisiae was determined. The site of reversion used was a well-defined chain initiation mutant mapping in the structural gene coding for iso-1-cytochrome c. High doses of EMS and HNO₂ resulted in decreased reversion of cyc1–131 in rad6, rad9 and rad15 strains compared to the normal RAD+ strains. In addition, rad52 greatly decreased EMS reversion of cyc1–131 but had not effect on HNO ₂-induced reversion; rad18, on the other hand, increased HNO ₂-induced reversion but did not alter EMS-induced reversion. When NQO was used as the mutagen, every rad gene tested, except for rad14 , had an effect on reversion; rad6, rad9, rad15, rad17, rad18, rad22, rev1, rev2 and rev3 lowered NQO reversion while rad1, rad2, rad3, rad4, rad10, rad12 and rad16 increased it compared to the RAD+ strain. The effect of rad genes on chemical mutagenesis is discussed in terms of their effect on UV mutagenesis. It is concluded that although the nature of the repair pathways may differ for UV- and chemically-induced mutations in yeast, a functional repair system is required for the induction of mutation by the chemical agents NQO, EMS and HNO₂.     

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.     

Patterns of Genetic and Phenotypic Suppression of lys2 Mutations in the Yeast SACCHAROMYCES CEREVISIAE

A total of 358 lys2 mutants of Saccharomyces cerevisiae have been characterized for suppressibility by the following suppressors: UAA and UAG suppressors that insert tyrosine, serine or leucine; a putative UGA suppressor; an omnipotent suppressor SUP46; and a frameshift suppressor SUF1–1. In addition, the lys2 mutants were examined for phenotypic suppression by the aminoglycoside antibiotic paromomycin, for osmotic remediability and for temperature sensitivity. The mutants exhibited over 50 different patterns of suppression and most of the nonsense mutants appeared similar to nonsense mutants previously described. A total of 24% were suppressible by one or more of the UAA suppressors, 4% were suppressible by one or more of the UAG suppressors, while only one was suppressible by the UGA suppressor and only one was weakly suppressible by the frameshift suppressor. One mutant responded to both UAA and UAG suppressors, indicating that UAA or UAG mutations at certain rare sites can be exceptions to the specific action of UAA and UAG suppressors. Some of the mutants appeared to require certain types of amino acid replacements at the mutant sites in order to produce a functional gene product, while others appeared to require suppressors that were expressed at high levels. Many of the mutants suppressible by SUP46 and paromomycin were not suppressible by any of the UAA, UAG or UGA suppressors, indicating that omnipotent suppression and phenotypic suppression need not be restricted to nonsense mutations. All of the mutants suppressible by SUP46 were also suppressible by paromomycin, suggesting a common mode of action of omnipotent suppression and phenotypic misreading.     

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).