Characterization of a Saccharomyces cerevisiae mutant with oversecretion phenotype

An oversecreting mutant of Saccharomyces cerevisiae was obtained from about 400 meiotic segregants derived from thediploid cells made by crossing the HBsAg-induced mutant NI-C with the wild-type strain Sey6211. When transformed with a plasmid containing mouse alpha-amylase cDNA, the mutant (NI-C-D4) exhibited an increased capacity (up to 13-fold) for the secretion of mouse alpha-amylase, higher than the parental strains and other standard wild-type strains. It was also shown that alpha-amylase secreted by the oversecreting mutant had a higher activity and contained more of the non-glycosylated form than the glycosylated form. This isolated oversecreting, low-glycosylation mutant may prove to be a potential S. cerevisiae host for the production of foreign proteins. Further genetic analysis suggested that the mutation responsible for the mutant's oversecretion was partially dominant and that both the oversecretion and low-glycosylation phenotypes were governed by a single chromosome mutation. These pleiotrophic phenotypes may be attributed to a defect in the synthesis of an ER-resident chaperone.     

Mutagenic and recombinagenic responses to defective DNA polymerase delta are facilitated by the Rev1 protein in pol3-t mutants of Saccharomyces cerevisiae

Defective DNA replication can result in substantial increases in the level of genome instability. In the yeast Saccharomyces cerevisiae, the pol3-t allele confers a defect in the catalytic subunit of replicative DNA polymerase delta that results in increased rates of mutagenesis, recombination, and chromosome loss, perhaps by increasing the rate of replicative polymerase failure. The translesion polymerases Pol eta, Pol zeta, and Rev1 are part of a suite of factors in yeast that can act at sites of replicative polymerase failure. While mutants defective in the translesion polymerases alone displayed few defects, loss of Rev1 was found to suppress the increased rates of spontaneous mutation, recombination, and chromosome loss observed in pol3-t mutants. These results suggest that Rev1 may be involved in facilitating mutagenic and recombinagenic responses to the failure of Pol delta. Genome stability, therefore, may reflect a dynamic relationship between primary and auxiliary DNA polymerases.     

Checkpoint-dependent activation of mutagenic repair in Saccharomyces cerevisiae pol3-01 mutants

The Saccharomyces cerevisiae DNA polymerase delta proofreading exonuclease-defective mutation pol3-01 is known to cause high rates of accumulating mutations. The pol3-01 mutant was found to have abnormal cell cycle progression due to activation of the S phase checkpoint. Inactivation of the S phase checkpoint suppressed both the pol3-01 cell cycle progression defect and mutator phenotype, indicating that the pol3-01 mutator phenotype was dependent on the S phase damage checkpoint pathway. Epistasis analysis suggested that a portion of the pol3-01 mutator phenotype involves members of the RAD6 epistasis group that function in both error-free and error-prone repair. These results indicate that activation of a checkpoint in response to certain types of replicative defects can result in the accumulation of mutations.     

Adaptive mutation in Saccharomyces cerevisiae

Adaptive mutation is a generic term for processes that allow individual cells of nonproliferating cell populations to acquire advantageous mutations and thereby to overcome the strong selective pressure of proliferation-limiting environmental conditions. Prerequisites for an occurrence of adaptive mutation are that the selective conditions are nonlethal and that a restart of proliferation may be accomplished by some genetic change in principle. The importance of adaptive mutation is derived from the assumption that it may, on the one hand, result in an accelerated evolution of microorganisms and, on the other, in multicellular organisms may contribute to a breakout of somatic cells from negative growth regulation, i.e., to cancerogenesis. Most information on adaptive mutation in eukaryotes has been gained with the budding yeast Saccharomyces cerevisiae. This review focuses comprehensively on adaptive mutation in this organism and summarizes our current understanding of this issue.     

Mitochondria-mediated nuclear mutator phenotype in Saccharomyces cerevisiae

Using Saccharomyces cerevisiae as a model organism, we analyzed the consequences of disrupting mitochondrial function on mutagenesis of the nuclear genome. We measured the frequency of canavanine-resistant colonies as a measure of nuclear mutator phenotype. Our data suggest that mitochondrial dysfunction leads to a nuclear mutator phenotype (i) when oxidative phosphorylation is blocked in wild-type yeast at mitochondrial complex III by antimycin A and (ii) in mutant strains lacking the entire mitochondrial genome (rho⁰) or those with deleted mitochondrial DNA (rho^–). The nuclear mutation frequencies obtained for antimycin A-treated cells as well as for rho^– and rho⁰ cells were ~2- to 3-fold higher compared to untreated control and wild-type cells, respectively. Blockage of oxidative phosphorylation by antimycin A treatment led to increased intracellular levels of reactive oxygen species (ROS). In contrast, inactivation of mitochondrial activity (rho^– and rho⁰) led to decreased intracellular levels of ROS. We also demonstrate that in rho⁰ cells the REV1, REV3 and REV7 gene products, all implicated in error-prone translesion DNA synthesis (TLS), mediate mutagenesis in the nuclear genome. However, TLS was not involved in nuclear DNA mutagenesis caused by inhibition of mitochondrial function by antimycin A. Together, our data suggest that mitochondrial dysfunction is mutagenic and multiple pathways are involved in this nuclear mutator phenotype.     

Asparaginase II of Saccharomyces cerevisiae. Characterization of the ASP3 gene

Purified preparations of asparaginase II of Saccharomyces cerevisiae exhibit two protein bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cloning and sequencing of the ASP3 gene, and partial amino acid sequencing as asparaginase II, imply that both bands are encoded by ASP3 but have different N termini. Northern blot analysis using the cloned ASP3 gene as a probe indicates that nitrogen catabolite repression of asparaginase II is achieved by alteration in mRNA levels. Deletion of sequences greater than 600 base pairs upstream from the initiation AUG codon results in an altered response to certain nitrogen sources in strains containing the truncated gene.     

Bleomycin-induced mutation and recombination in Saccharomyces cerevisiae

Clinical preparations of bleomycins (BM) were tested for their recombinogenicity and mutagenicity at relatively high survival levels in the simple eucaryote, Saccharomyces cerevisiae. More than a dozen test loci or genetic intervals were assayed for bleomycin-induced mutation or recombination. Treatments of stationary phase diploid yeast routinely resulted in 25–75% inactivation. The antibiotic was mildly to very highly recombinogenic and mutagenic, with one exception. The amount of bleomycin-induced mutation, gene conversion or crossing-over depended upon the particular genetic markers assayed. The drug was also potently recombinogenic in yeast cells growing in the presence of BM. These results contrast with the finding that this antitumor agent was not mutagenic in the Salmonella/mammalian microsome mutagenicity test; possible explanations of this difference are given.     

Characterization of a Saccharomyces cerevisiae mutant with pseudohyphae and cloning of a gene complementing the mutation

Screening for morphological mutants of a haploid strain of Saccharomyces cerevisiae was done on the basis of their cell-shape on a solid medium containing isoamyl alcohol, which causes cell elongation, to obtain information on the morphogenesis. Mutant J19, which had pseudohyphae in liquid medium even in the absence of isoamyl alcohol, had many elongated cells. Few reports exist of haploid cells growing as pseudohyphae in liquid culture. Cell-wall analysis showed that J19 had ordinary amounts of alkali-insoluble glucan and chitin, but that isoamyl alcohol in the medium caused structural changes in the cell wall. Addition of a DNA fragment that included the wild-type SCL1 gene to J19 complemented its morphological phenotype. Sequencing of J19 SCL1 showed that the glycine at position 226 in the Scl1 protein had been replaced by asparatic acid, suggesting that this mutation in the protein, a subunit of proteasomes, may be involved in the morphological change.     

Subunit interactions within the Saccharomyces cerevisiae DNA polymerase epsilon (pol epsilon ) complex. Demonstration of a dimeric pol epsilon

Saccharomyces cerevisiae DNA polymerase epsilon (pol epsilon) is essential for chromosomal replication. A major form of pol epsilon purified from yeast consists of at least four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. We have investigated the protein/protein interactions between these polypeptides by using expression of individual subunits in baculovirus-infected Sf9 insect cells and by using the yeast two-hybrid assay. The essential subunits, Pol2p and Dpb2p, interact directly in the absence of the other two subunits, and the C-terminal half of POL2, the only essential portion of Pol2p, is sufficient for interaction with Dpb2p. Dpb3p and Dpb4p, non-essential subunits, also interact directly with each other in the absence of the other two subunits. We propose that Pol2p.Dpb2p and Dpb3p.Dpb4p complexes interact with each other and document several interactions between individual members of the two respective complexes. We present biochemical evidence to support the proposal that pol epsilon may be dimeric in vivo. Gel filtration of the Pol2p.Dpb2p complexes reveals a novel heterotetrameric form, consisting of two heterodimers of Pol2p.Dpb2p. Dpb2p, but not Pol2p, exists as a homodimer, and thus the Pol2p dimerization may be mediated by Dpb2p. The pol2-E and pol2-F mutations that cause replication defects in vivo weaken the interaction between Pol2p and Dpb2p and also reduce dimerization of Pol2p. This suggests, but does not prove, that dimerization may also occur in vivo and be essential for DNA replication.     

Genetic basis of flocculation phenotype conversion in Saccharomyces cerevisiae

Two NewFlo-type flocculent transformants Saccharomyces cerevisiae YTS-S and YTS-L were obtained from a partial yeast genomic library. Even though both of the transformants displayed the same flocculation phenotype, they represented different physiological characteristics during detailed investigation. Analysis of the two transformants YTS-L and YTS-S confirmed the presence of FLONL and FLONS genes, respectively. The 3396-bp ORF of FLONS encoded a protein of 1132 amino acids. Meanwhile, the presence of a 1686-bp ORF encoding a 562-amino acid protein was revealed in FLONL. Both FLONL and FLONS showed high identity to FLO1 gene. Aligned with the intact FLO1 gene, FLONS lost two internal repeated regions, whereas one repeated sequence was inserted into the middle of the FLONL gene. All of the altered regions could be found in the middle repetitive sequence of the FLO1 gene. The results indicate that FLONL and FLONS are both derived forms of the FLO1 gene. Genetic variability triggered by tandem repeats in FLO1 gene is believed to be responsible for the differential phenotypic properties of the yeast strains YTS-S and YTS-L.     

Spontaneous mutation of leu2 in Saccharomyces cerevisiae

The data obtained indicate that spontaneous mutations in Saccharomyces cerevisiae are formed during DNA replication. With no DNA replication in the lag-period, in the stationary growth phase, spontaneous mutations are not formed in cell culture during the G1 phase of cell cycle. Experimental data show the absence of primary spontaneously occurring DNA lesion accumulation in the cell G1 phase. Spontaneous mutations of yeasts are formed in the S phase of cell cycle, apparently as DNA replication errors. It is established that the frequency of spontaneous reversions of the leu2 gene in Saccharomyces cerevisiae strain NA3-24 increases when the cells are cultivated on the culture medium with different concentrations of leucine.     

Identification of a glutaminyl-tRNA synthetase mutation Saccharomyces cerevisiae

Saccharomyces cerevisiae glutaminyl-tRNA synthetase mutants were isolated through systematic screening of tight Gln- derivatives of a leaky glutamine auxotroph. These mutations define a single nuclear gene, GLN4. The gln4-1 mutation is specific for Gln-tRNA synthetase and shows a dosage effect in heterozygous diploids. The wild-type Gln-tRNA synthetase exhibits a Km for glutamine of 25 microM; the gln4-1 mutation increases this value 20-fold. These observations strongly suggest that GLN4 encodes the Gln-tRNA synthetase.     

Characterization of methylglyoxal synthase in Saccharomyces cerevisiae

Methylglyoxal synthase in Saccharomyces cerevisiae was purified approximately 300 folds from cell extracts with 20% of activity yield. During purification procedures, polymorphic behaviours of the enzyme were observed. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis and consisted of a single polypeptide chain of Mr = 26,000. The enzyme was most active at pH 9.5-10.5 and strictly specific to dihydroxyacetone phosphate with Km = 3 mM. Phosphoenolpyruvate, glyceraldehyde-3-phosphate, orthophosphate and thiol compounds were potent inhibitors of the enzyme.     

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.     

Characterization of NADH kinase from Saccharomyces cerevisiae

At least two enzymes that phosphorylate diphosphopyridine nucleotides were detected in Saccharomyces cerevisiae: NADH-specific kinase was localized exclusively in the mitochondria, and NAD+-specific kinase was distributed in the microsomal and cytosol fractions but not in the mitochondria. The identity of NAD+ kinase detected in the two fractions remains equivocal. NADH kinase was highly purified 1,041-fold from the mitochondrial fraction. The Km values for NADH and ATP were 105 microM and 2.1 mM, respectively. The relative molecular mass was estimated to be 160,000 by means of molecular sieve chromatography. From inactivation studies with SH inhibitors and protection by NADH, it was demonstrated that a cysteine residue is involved in the binding site of NADH.     

Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae

In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.