Isolation and characterization of sulfite mutants of Saccharomyces cerevisiae

Sulfite-resistant and sulfite-sensitive mutants of Saccharomyces cerevisiae were isolated and characterized. Genetic analysis indicated that one and four genes were responsible for the resistant and sensitive responses, respectively, and suggested that defects in methionine and cysteine metabolism were not involved. Some resistant alleles, all of which were dominant, conferred greater resistance than others. Mutations conferring sensitivity were recessive and one co-segregated with impaired respiration. Two of the sensitive mutants exhibited cross-sensitivity to other metabolic inhibitors: sulfometuron methyl, cycloheximide, oligomycin, and antimycin A. A 50% glutathione deficiency in one sensitive mutant was not sufficient in itself to account for its sensitivity. Screening of other relevant mutants revealed that relative to wild-type, met8 and a thioredoxin null mutant are sensitive, and met3 and met14 mutants are not. Reduced production of extracellular acetaldehyde, a compound that detoxifies sulfite, was observed in three of the four sensitive mutants. However, acetaldehyde was also underproduced in the resistant mutant. Because sulfite is a reducing agent, cells were tested for coincident sensitivity or resistance to ascorbate, selenite, dithiothreitol, nitrite, thiosulfate, reduced glutathione, and cysteine. No consistent pattern of responses to these agents emerged, suggesting that the response to sulfite is not a simple function of redox potential.Technical Paper Number 9984 of the Oregon Agricultural Experiment Station     

Isolation and genetic study of Triethyltin-resistant mutants of Saccharomyces cerevisiae

Summary Three mutants of Saccharomyces cerevisiae resistant to triethyltin (an inhibitor of mitochondrial ATPase) on non-fermentative media, and non-resistant to this drug on fermentative media, were isolated and named TTR1, TTR2 and TTR3. Apart from triethyltin resistance, these mutants show the following common characteristics: (1) Increased intracellular cytochrome c concentration. (2) Increased respiration rate. (3) Decreased growth yield. (4) Increased growth sensitivity to several drugs inhibiting oxidative phosphorylation: namely, CCCP (permeabilizing inner mitochondrial membrane to protons), valinomycin (permeabilizing inner mitochondrial membrane to potassium) and oligomycin (inhibitor of mitochondrial ATPase). (5) Increased sensitivity to carbon source starvation. For each mutant, these characteristics appeared to be due to a single pleiotropic nuclear mutation. Mutation TTR1 causes additional phenotypic characteristics which do not appear in mutants TTR2 and TTR3: (1) Pinkish coloration of colonies which is more pronounced after a long growth period. (2) Inability of the cells to store glycogen. (3) Growth defect of the cells on a galactose-containing medium. (4) Inability of a diploid homozygote mutant strain to sporulate. All these phenotypic characteristics have already been described in yeast mutants deregulated in cAMP-dependant protein phosphorylation. Crossing of a strain bearing the TTR1 mutation with a strain mutated in the adenylate cyclase structural gene suggested that the TTR1 phenotype is due to a modification in regulation of cAPK by cAMP, making cell multiplication possible without intracellular cAMP.Abbreviations cAMP Cyclic Adenosine-3-5-monophosphate - cAPK cAMP-dependent protein kinase - CCCP carbonylcyanide m-chlorophenylhydrazone - OD Optical density - TET triethyltin chloride     

Isolation and characterization of an uncoupler-resistant mutant of Saccharomyces cerevisiae

Summary One mutant resistant to carbonylcyanide m-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, was isolated in Saccharomyces cerevisiae.Genetic analysis showed that a single nuclear gene is responsible for increased resistance; this gene was dominant.The mutant showed cross-resistance or collateral sensitivity to several chemically-unrelated inhibitors (cycloheximide, dinitrophenol, tributhyltin chloride, chloramphenicol).The resistance of the mutant is related to a decreased uptake of CCCP which is not expressed in glucose-starved cells. It was shown that glucose induced a CCCP efflux which was more efficient in the mutant than in the wild-type cells. This effect was correlated to a greater acidification of the internal pH by glucose addition in the mutant cells.It was proposed that resistance was not due to a change of permeability of the plasmic membrane itself but to the change of internal pH which determines the extent of accumulation of weak acids or bases.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - FDA fluoresceine diacetate - EGTA ethyleneglycol-bis((-aminoethyl ether) N,N,N,N-tetraacetic acid - DES diethylstilbestrol     

Uptake of DNA by fragile mutants of Saccharomyces cerevisiae

Summary When Saccharomyces cerevisiae SY15 rho° mutant cells grown in media stabilized with 10% sorbitol were suspended in 2% sorbitol solutions, 60–70% of the population did not lyse and became permeable to native high molecular weight DNA. Maximal incorporation of DNA to DNase resistant state was measured after 60 min of incubation in presence of 5 g/ml DNA and 10 mM CaCl₂. These results suggest that the fragile mutants might be tested as hosts for transformation of whole yeast cells.     

Isolation and characterization of Saccharomyces cerevisiae mutants resistant to T-2 toxin

Summary T-2 toxin, a trichothecene mycotoxin, inhibits the growth of Saccharomyces cerevisiae. We have isolated nine spontaneous S. cerevisiae mutants resistant to this toxin. The mutants were distinguished from the wild type according to their degree of resistance to T-2 toxin on media with dextrose or glycerol as the carbon source. Generation time, mutation stability and level of cross-resistance to roridin A, another trichothecene, were determined for each mutant. The T-2 toxin resistant mutants were further characterized by subsequent tests involving cross-resistance and collateral sensitivtiy to chlorampenicol, neomycin, paromomycin, ethidium bromide and thiolutin. Mutants have been placed into three subgroups and the mechanism of T-2 toxin resistance in each group has been postulated. Mutant HK1 is the first S. cerevisiae isolate resistant to roridin A. One particular isolate, mutant HK1 l , carries a single recessive nuclear mutation. This mutation was termed ttt (for T-2 toxin resistant).     

Isolation and characterization of pre-mRNA splicing mutants of Saccharomyces cerevisiae

In this study we report the isolation of temperature-sensitive mutants that affect pre-mRNA splicing. A bank of approximately 1000 temperature-sensitive Saccharomyces cerevisiae strains was generated and screened on RNA gel blots by hybridization with an actin intron probe. We isolated 16 mutants defining 11 new complementation groups prp(rna)17-prp(rna)27 with four phenotypic classes of mutants and 21 mutants in the prp2-prp11 complementation groups (formerly rna2-rna11). The majority of the complementation groups share a phenotype of pre-mRNA accumulation, seen in all of the prp(rna)2-prp(rna)11 mutants. Three novel classes of mutants were isolated in this study. One class, consisting of two complementation groups, exhibits an accumulation of the lariat intermediate of splicing, with no change in the levels of pre-mRNA. The second class, also represented by two complementation groups, shows an accumulation of the intron released after splicing. The third novel class, comprising one complementation group, accumulates both pre-mRNA and the released intron. All mutants isolated were recessive for the splicing phenotype. Only 2 of the 11 complementation groups, although recessive, were not temperature sensitive. This study, together with previous isolation of the prp(rna)2-prp(rna)11 groups and the spliceosomal snRNAs, puts at least 26 gene products involved directly or indirectly in pre-mRNA splicing.     

Sporulation in mitochondrial OXI3 mutants of Saccharomyces cerevisiae

Summary By use of a set of mitochondrial oxi3 mutants (mit ^–, defective in cytochrome oxidase) we have shown that sporulation is possible at a very low level of respiration (below l% of the wild type respiration). A specific role for oxygen in biosynthesis during sporulation is suggested. Correlation of these results with the genetic map of the OXI3 region reveals that one group of mutants, mapping in the central part of the OXI3 region, is capable of sporulation.     

Isolation and characterization of S. cerevisiae mutants deficient in amino acid-inducible peptide transport

Summary The transport of small peptides into the yeast Saccharomyces cerevisiae is subject to complex regulatory control. In an effort to determine the number, and to address the function, of the components involved in peptide transport and its regulation, spontaneous mutants resistant to toxic di- and tripeptides were isolated under inducing conditions. Twenty-four mutant strains were characterized in detail and fell into two phenotypic groups; one group deficient in amino acid-inducible peptide uptake, the other with a pleiotropic phenotype including a loss of peptide transport. Complementation analysis of recessive mutations in 12 of these strains defned three groups; ptr1 (nine strains), ptr2 (two strains), and ptr3 (one strain). Isolation and screening of 31 additional N-methyl-N-nitro-N-Nitrosoguanidine (MNNG)-induced, peptide transport-deficient mutants produced one ptr3 and 30 ptr2 strains: no additional complementation groups were detected. Uptake of radiolabeled dileucine was negligible in ptr1 and ptr2 strains and was reduced by 65% and 90% in the two ptr3 mutants, indicating that all strains were defective at the transport step. We conclude that the S. cerevisiae amino acid-inducible peptide transport system recognizes a broad spectrum of peptide substrates and involves at least three components. One gene, PTR3, may play an indirect or regulatory role since mutations in this gene cause a pleiotropic phenotype.     

The unusual inheritance of multidrug-resistance factors in Saccharomyces

The yeast PDR5 locus encodes a 160-kDa member of the ABC family of transport proteins. Strains bearing a deletion of this locus are drug hypersensitive. Resistant revertants arise when cells are plated on cycloheximide medium. About one-third of these are cross resistant to other agents, including oligomycin, fluconazole and sulfometuron methyl. Most of the revertants exhibit linkage to the PDR5 locus and map in three locations. Curiously, the multi-drug resistance is not due to a single mutation. Most of the revertants behave as though they contained several tightly linked resistance factors. Received: 11 March 1996 / 23 April 1996     

Isolation and characterization of three mutants with increased sensitivity to photoactivated 3-carbethoxypsoralen in Saccharomyces cerevisiae

The complementation and genetical analysis of yeast mutants sensitive to photoactivated 3-carbethoxy-psoralen define three novel recessive mutant alleles pso-5-1, pso6-1, and pso7-1. Their cross-sensitivity to UV_254nm, radiomimetic mutagens, and to chemicals enhancing oxidative stress suggest that these mutants are either impaired in metabolic steps protecting from oxidative stress or in mechanisms of the repair of oxygen-dependent DNA lesions. None of the three novel mutant alleles block the induction of reverse mutation by photoactivated mono- and bi-functional psoralens, nitrogen mustards, or UV_254nm.     

RPD3 (REC3) mutations affect mitotic recombination in Saccharomyces cerevisiae

Prior research identified the recessive rec3-1ts mutation in Saccharomyces cerevisiae which, in homozygous diploid cells, confers a conditional phenotype resulting in reduced levels of spontaneous mitotic recombination and loss of sporulation at the restrictive temperature of 36 °C. We found that a 3.4-kb genomic fragment that complements the rec3-1ts/rec3-1ts mutation and which maps to chromosome XIV, is identical to RPD3, a gene encoding a histone de-acetylase. Sporulation is reduced in homozygous diploid strains containing the rec3-1ts allele at 24 °C, suggesting that this allele of RPD3 encodes a gene product with a reduced function. Sporulation is abolished in diploid strains homozygous for the rpd3Δ or rec3-1ts alleles, as well as in rpd3Δ/rec3-1ts heteroallelic diploids, at the non-permissive temperature. Acid-phosphatase expression has been shown to be RPD3 dependent. We found that acid-phosphatase activity is greater in diploid strains homozygous for the temperature-sensitive rec3-1ts allele than in RPD3/RPD3 strains and increased further when mutant strains are grown at 36 °C. We also tested the rpd3Δ/rpd3Δ strains for their effects on spontaneous mitotic recombination. By assaying a variety of intra- and inter-genic recombination events distributed over three chromosomes, we found that in the majority of cases spontaneous mitotic recombination was reduced in diploid rpd3Δ/rpd3Δ cells (relative to a RPD3/RPD3 control). Finally, although 90% of mitotic recombinant events are initiated in the G₁ phase of the growth cycle (i.e., before DNA synthesis) we show that RPD3 is not regulated in a cell-cycle-dependent manner. These data suggest that mitotic recombination, in addition to gene expression, is affected by changes in chromatin architecture mediated by RPD3. Received: 17 July / 30 November 1998     

Recombinational analysis of OXI1 mutants and preliminary analysis of their translation products in S. cerevisiae

Summary Recombinational analysis of oxil mutants was performed using a and a mutant strains with the same mitochondrial and nuclear backgrounds, derived from strain 777-3A.In spite of minor inconsistencies the overall map of oxi1 mutations can be constructed on the basis of wild-type recombinant frequencies in the two-point oxi1 ^{– –} x oxi1 ^– crosses. The frequencies of wild-type recombinants varied in a wide range from 0.003% to 16%, reaching the maximal values expected for unlinked mitochondria) markers. No distinct clusters of mutants were observed.The analysis of translation products of oxil mutants showed that all but one of the oxil mutants studied are connected with the conspicuous changes of the polypeptide band corresponding to subunit 11 of cytochrome c oxidase in electrophoresis on polyacrylamide gels. The exceptional G565 mutant showed no conspicuous change in subunit II, but lacked subunit I of cytochrome c oxidase.Various oxi1 mutants seemed to carry premature chain termination mutations. Most of them show a correlation between the length of the putative fragment of subunit II synthesized and the position on the genetic map. The direction of translation is from the V2 to the V60 mutation. The V2 mutation is proximal to cap and V60 proximal to the par locus.     

Recombinational analysis of oxi2 mutants and preliminary analysis of their translation products in S. cerevisiae

Summary Genetic and biochemical studies were performed with mutants allocated to the mitochondrial oxi2 gene.Recombinational analysis of 19 oxi2 mutants was performed using and a mutant strains derived from the same genetic background. The frequencies of wild-type recombinants in oxi2 ^– × oxi2 ^– crosses varied from 0.002 to 17%. The map of oxi2 mutations constructed on the basis of these frequencies shows many internal inconsistencies. In the course of rho ^– deletion mapping five classes of oxi2 mutations were distinguished. The results of deletion analysis are in agreement with those of recombinational mapping.The analysis of mitochondrial translation products by SDS-polyacrylamide electrophoresis of 20 oxi2 mutants shows that 17 of them are connected with conspicuous changes of 22 kd polypeptide band corresponding to subunit III of cytochrome oxidase. At least four of them carried instead of subunit III clearly visible significantly shorter polypeptides (12.8 to 20.1 kd). These were, most likely, shorter fragments of subunit III resulting from chain termination mutations. Colinearity was observed between the lenght of new polypeptides and the positions of the respective mutations on the recombinational map. These data confirm hat oxi2 encodes subunit III of cytochrome oxidase and suggest that translation of the oxi2 gene is in the direction from V303 to V273.     

Characterization of blasticidin S — resistant mutants of Saccharomyces cerevisiae

Summary Blasticidin S-resistant mutants of S. cerevisiae were isolated and characterized. Resistant mutations were found to fall into two complementation groups. A single recessive nuclear gene was responsible for each group, donated as bls1 and bls2, respectively. A gene bls1 was linked to an ilv3 gene located on the right arm of chromosome X. The resistant phenotypes from both genes were not associated with ribosomes known to be target sites of Blasticidin S, when analyzed by poly(U)-directed polyphenylalanine synthesis. The resistant mechanisms of the mutations are discussed in this paper.     

Newly synthesised DNA of high molecular weight in the yeast Saccharomyces cerevisiae

Summary Two species of newly synthesised DNA larger than average replicons have been found in yeast. Their molecular weights are 60 million and 90 million daltons respectively. The exact nature of these molecules is not certain. They may represent entirely novel species of cellular DNA or they could be concatameric replication intermediates of some particular fraction of DNA, such as mitochondrial DNA or rDNA. Alternatively they could result from the fusion of adjacent completed replicons in a small cluster.     

Identification and functional analysis of a Kluyveromyces lactis homologue of the SPT4 gene of Saccharomyces cerevisiae

Sequence analysis of a DNA fragment containing the KlCOX18 gene originating from chromosome II of the yeast Kluyveromyces lactis revealed the presence of an adjacent open reading frame (ORF) for a protein exhibiting 78.4% identity with the Saccharomyces cerevisiae Spt4p. Based on the identical length (102 aa) and the conservation of the zinc-finger motif found in Spt4p we named this ORF KlSPT4. When expressed in S. cerevisiae the KlSPT4 gene complemented all spt4 mutant phenotypes. It is proposed that KlSpt4p, like its S. cerevisiae counterpart is a protein involved in the establishment or maintenance of the chromatin structure that influences the expression of many yeast genes. Received: 15 June / 31 August 1998     

In-vitro recombination in rad and rnc mutants of Saccharomyces cerevisiae

Summary Extracts of S. cerevisiae cells can catalyze homologous recombination between plasmids in vitro. Extracts prepared from rad50, rad52 or rad54 disruption mutants all have reduced recombinational activity compared to wild-type. The rad52 and rad54 extracts are more impaired in the recombination of plasmids containing double-strand breaks than of intact plasmids, whereas rad50 extracts are deficient equally for both types of substrate. The nuclease RhoNuc (previously designated yNucR), encoded by the RNC1 (previously designated NUC2) gene and regulated by the RAD52 gene, is not required for recombination when one substrate is single-stranded but is essential for the majority of recombination events when both substrates are double-stranded. Furthermore, elimination of this nuclease restores recombination in rad52 extracts to levels comparable to those in wild-type extracts.