<Emphasis Type="Italic">IXR1</Emphasis> and <Emphasis Type="Italic">HMO1</Emphasis> genes jointly control the level of spontaneous mutagenesis in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The yeast genes IXR1 and HMO1 encode proteins belonging to the family of chromatin nonhistone proteins, which are able to recognize and bind to irregular DNA structures. The full deletion of gene IXR1 leads to an increase in cell resistance to the lethal action of UV light, γ-rays, and MMS, increases spontaneous mutagenesis and significantlly decreases the level of UV-induced mutations. It was earlier demonstrated in our works that the hmo1 mutation renders cells sensitive to the lethal action of cisplatin and virtually does not affect the sensitivity to UV light. Characteristically, the rates of spontaneous and UV-induced mutagenesis in the mutant are increased. Epistatic analysis of the double mutation hmo1 ixr1 demonstrated that the interaction of these genes in relation to the lethal effect of cisplatin and UV light, as well as UV-induced mutagenesis, is additive. This suggests that the products of genes HMO1 and IXR1 participate in different repair pathways. The ixr1 mutation significantly increases the rate of spontaneous mutagenesis mediated by replication errors, whereas mutation hmo1 increases the rate of repair mutagenesis. In wild-type cells, the level of spontaneous mutagenesis was nearly one order of magnitude lower than that obtained in cells of the double mutant. Consequently, the combined activity of the Hmo1 and the Ixr1 proteins provides efficient correction of both repair and replication errors.     

Phenotype character of the methylglyoxal resistance gene in Saccharomyces cerevisiae: expression in Escherichia coli and application to breeding wild-type yeast strains

The gene responsible for the methylglyoxal resistance of Saccharomyces cerevisiae was cloned, and its phenotypic characteristics were investigated. S. cerevisiae cells with the gene could accumulate large amounts of glutathione in the medium and should remarkably high resistance to various toxic compounds such as methylglyoxal, tetramethylthiuram disulfide, iodoacetamide, and heavy-metal ions. The gene was also expressed in Escherichia coli cells, and the resistance of E. coli cells to toxic compounds also increased as observed for S. cerevisiae cells. The phenotypic characteristics of the gene were applicable to the selection of the transformants of wild-type yeast strains having no genetic markers.     

Phenotype character of the methylglyoxal resistance gene in Saccharomyces cerevisiae: expression in Escherichia coli and application to breeding wild-type yeast strains.

The gene responsible for the methylglyoxal resistance of Saccharomyces cerevisiae was cloned, and its phenotypic characteristics were investigated. S. cerevisiae cells with the gene could accumulate large amounts of glutathione in the medium and should remarkably high resistance to various toxic compounds such as methylglyoxal, tetramethylthiuram disulfide, iodoacetamide, and heavy-metal ions. The gene was also expressed in Escherichia coli cells, and the resistance of E. coli cells to toxic compounds also increased as observed for S. cerevisiae cells. The phenotypic characteristics of the gene were applicable to the selection of the transformants of wild-type yeast strains having no genetic markers.     

A Deletion Map of cyc1 Mutants and Its Correspondence to Mutationally Altered Iso-1-Cytochromes c of Yeast

Mutants arising spontaneously from sporulated cultures of certain strains of yeast, Saccharomyces cerevisiae, contained deletions of the CYC1 gene which controls the primary structure of iso-1-cytochrome c. At least 60 different kinds of deletions were uncovered among the 104 deletions examined and these ranged in length from those encompassing only two adjacent point mutants to those encompassing at least the entire CYC1 gene. X-ray-induced recombination rates of crosses involving these deletions and cyc1 point mutants resulted in the assignment of 211 point mutants to 47 mutational sites and made it possible to unambiguously order 40 of these 47 sites. Except for one mutant, cyc1-15, there was a strict colinear relationship between the deletion map and the positions of 13 sites that were previously determined by amino acid alterations in iso-1-cytochromes c from intragenic revertants.     

The Saccharomyces cerevisiae orthologue of the human protein phosphatase 4 core regulatory subunit R2 confers resistance to the anticancer drug cisplatin

The anticancer agents cisplatin and oxaliplatin are widely used in the treatment of human neoplasias. A genome-wide screen in Saccharomyces cerevisiae previously identified PPH3 and PSY2 among the top 20 genes conferring resistance to these anticancer agents. The mammalian orthologue of Pph3p is the protein serine/threonine phosphatase Ppp4c, which is found in high molecular mass complexes bound to a regulatory subunit R2. We show here that the putative S. cerevisiae orthologue of R2, which is encoded by ORF YBL046w, binds to Pph3p and exhibits the same unusually high asymmetry as mammalian R2. Despite the essential function of Ppp4c-R2 in microtubule-related processes at centrosomes in higher eukaryotes, S. cerevisiae diploid strains with homozygous deletion of YBL046w and two or one functional copies of the TUB2 gene were viable and no more sensitive to microtubule-depolymerizing drugs than the control strain. The protein encoded by YBL046w exhibited a predominantly nuclear localization. These studies suggest that the centrosomal function of Ppp4c-R2 is not required or may be performed by a different phosphatase in yeast. Homozygous diploid deletion strains of S. cerevisiae, pph3Delta, ybl046wDelta and psy2Delta, were all more sensitive to cisplatin than the control strain. The YBL046w gene therefore confers resistance to cisplatin and was termed PSY4 (platinum sensitivity 4). Ppp4c, R2 and the putative orthologue of Psy2p (termed R3) are shown here to form a complex in Drosophila melanogaster and mammalian cells. By comparison with the yeast system, this complex may confer resistance to cisplatin in higher eukaryotes.     

A molecular target for viral killer toxin: TOK1 potassium channels.

Killer strains of S. cerevisiae harbor double-stranded RNA viruses and secrete protein toxins that kill virus-free cells. The K1 killer toxin acts on sensitive yeast cells to perturb potassium homeostasis and cause cell death. Here, the toxin is shown to activate the plasma membrane potassium channel of S. cerevisiae, TOK1. Genetic deletion of TOK1 confers toxin resistance; overexpression increases susceptibility. Cells expressing TOK1 exhibit toxin-induced potassium flux; those without the gene do not. K1 toxin acts in the absence of other viral or yeast products: toxin synthesized from a cDNA increases open probability of single TOK1 channels (via reversible destabilization of closed states) whether channels are studied in yeast cells or X. laevis oocytes.     

Cellular morphogenesis under stress is influenced by the sphingolipid pathway gene ISC1 and DNA integrity checkpoint genes in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, replication stress induced by hydroxyurea (HU) and methyl methanesulfonate (MMS) activates DNA integrity checkpoints; in checkpoint-defective yeast strains, HU treatment also induces morphological aberrations. We find that the sphingolipid pathway gene ISC1, the product of which catalyzes the generation of bioactive ceramides from complex sphingolipids, plays a novel role in determining cellular morphology following HU/MMS treatment. HU-treated isc1Δ cells display morphological aberrations, cell-wall defects, and defects in actin depolymerization. Swe1, a morphogenesis checkpoint regulator, and the cell cycle regulator Cdk1 play key roles in these morphological defects of isc1Δ cells. A genetic approach reveals that ISC1 interacts with other checkpoint proteins to control cell morphology. That is, yeast carrying deletions of both ISC1 and a replication checkpoint mediator gene including MRC1, TOF1, or CSM3 display basal morphological defects, which increase following HU treatment. Interestingly, strains with deletions of both ISC1 and the DNA damage checkpoint mediator gene RAD9 display reduced morphological aberrations irrespective of HU treatment, suggesting a role for RAD9 in determining the morphology of isc1Δ cells. Mechanistically, the checkpoint regulator Rad53 partially influences isc1Δ cell morphology in a dosage-dependent manner.     

Whole-genome comparison reveals novel genetic elements that characterize the genome of industrial strains of Saccharomyces cerevisiae

Human intervention has subjected the yeast Saccharomyces cerevisiae to multiple rounds of independent domestication and thousands of generations of artificial selection. As a result, this species comprises a genetically diverse collection of natural isolates as well as domesticated strains that are used in specific industrial applications. However the scope of genetic diversity that was captured during the domesticated evolution of the industrial representatives of this important organism remains to be determined. To begin to address this, we have produced whole-genome assemblies of six commercial strains of S. cerevisiae (four wine and two brewing strains). These represent the first genome assemblies produced from S. cerevisiae strains in their industrially-used forms and the first high-quality assemblies for S. cerevisiae strains used in brewing. By comparing these sequences to six existing high-coverage S. cerevisiae genome assemblies, clear signatures were found that defined each industrial class of yeast. This genetic variation was comprised of both single nucleotide polymorphisms and large-scale insertions and deletions, with the latter often being associated with ORF heterogeneity between strains. This included the discovery of more than twenty probable genes that had not been identified previously in the S. cerevisiae genome. Comparison of this large number of S. cerevisiae strains also enabled the characterization of a cluster of five ORFs that have integrated into the genomes of the wine and bioethanol strains on multiple occasions and at diverse genomic locations via what appears to involve the resolution of a circular DNA intermediate. This work suggests that, despite the scrutiny that has been directed at the yeast genome, there remains a significant reservoir of ORFs and novel modes of genetic transmission that may have significant phenotypic impact in this important model and industrial species.     

Whole-genome sequencing of the efficient industrial fuel-ethanol fermentative Saccharomyces cerevisiae strain CAT-1

The Saccharomyces cerevisiae strains widely used for industrial fuel-ethanol production have been developed by selection, but their underlying beneficial genetic polymorphisms remain unknown. Here, we report the draft whole-genome sequence of the S. cerevisiae strain CAT-1, which is a dominant fuel-ethanol fermentative strain from the sugarcane industry in Brazil. Our results indicate that strain CAT-1 is a highly heterozygous diploid yeast strain, and the ~12-Mb genome of CAT-1, when compared with the reference S228c genome, contains ~36,000 homozygous and ~30,000 heterozygous single nucleotide polymorphisms, exhibiting an uneven distribution among chromosomes due to large genomic regions of loss of heterozygosity (LOH). In total, 58 % of the 6,652 predicted protein-coding genes of the CAT-1 genome constitute different alleles when compared with the genes present in the reference S288c genome. The CAT-1 genome contains a reduced number of transposable elements, as well as several gene deletions and duplications, especially at telomeric regions, some correlated with several of the physiological characteristics of this industrial fuel-ethanol strain. Phylogenetic analyses revealed that some genes were likely associated with traits important for bioethanol production. Identifying and characterizing the allelic variations controlling traits relevant to industrial fermentation should provide the basis for a forward genetics approach for developing better fermenting yeast strains.     

Direction of chromosome rearrangements in Saccharomyces cerevisiae by use of his3 recombinational substrates.

We used the his3 recombinational substrates (his3 fragments) to direct large interchromosomal (translocations) and intrachromosomal (deletions and tandem duplications) rearrangements in the yeast Saccharomyces cerevisiae. In strains completely deleted for the wild-type HIS3 gene, his3 fragments, one containing a deletion of 5' amino acid coding sequences and the other containing a deletion of 3' amino acid coding sequences, were first placed at preselected sites by homologous recombination. His+ revertants that arose via spontaneous mitotic recombination between the two his3 fragments were selected. This strategy was used to direct rearrangements in both RAD52+ and rad52 mutant strains. Translocations occurred in the RAD52+ genetic background and were characterized by orthogonal field alternating gel electrophoresis of yeast chromosomal DNA and by standard genetic techniques. An unexpected translocation was also identified in which HIS3 sequences were amplified. Two types of tandem duplications of the GAL(7, 10, 1) locus were also directed, and one type was not observed in rad52 mutants. Recombination mechanisms are discussed to account for these differences.     

Isolation and characterization of the Saccharomyces cerevisiae MIS1 gene encoding mitochondrial C1-tetrahydrofolate synthase

C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. In Saccharomyces cerevisiae, C1-tetrahydrofolate synthase is found in both the cytoplasm and the mitochondria. The gene encoding yeast mitochondrial C1-tetrahydrofolate synthase was isolated using synthetic oligonucleotide probes based on the amino-terminal sequence of the purified protein. Hybridization analysis shows that the gene (designated MIS1) has a single copy in the yeast genome. The predicted amino acid sequence of mitochondrial C1-tetrahydrofolate synthase shares 71% identity with yeast C1-tetrahydrofolate synthase and shares 39% identity with clostridial 10-formyltetrahydrofolate synthetase. Chromosomal deletions of the mitochondrial C1-tetrahydrofolate synthase gene were generated using the cloned MIS1 gene. Mutant strains which lack a functional MIS1 gene are viable and can grow in medium containing a nonfermentable carbon source. In fact, deletion of the MIS1 locus has no detectable effect on cell growth.     

The yeast multidrug transporter Qdr3 (Ybr043c): localization and role as a determinant of resistance to quinidine, barban, cisplatin, and bleomycin

Saccharomyces cerevisiae ORF YBR043c, predicted to code for a transporter of the major facilitator superfamily required for multiple drug resistance, encodes a plasma membrane protein that confers resistance to quinidine and barban, as observed before for its close homologues QDR1 and QDR2. This ORF was, thus, named the QDR3 gene. The increased expression of QDR3, or QDR2, also leads to increased resistance to the anticancer agents cisplatin and bleomycin. However, no evidence for increased QDR3 expression in yeast cells exposed to all these inhibitory compounds was found. Transport assays support the concept that Qdr3 is involved, even if opportunistically, in the active export of quinidine out of yeast cell. A correlation was established between the efficiency of quinidine active export mediated by Qdr3p, Qdr2p or Qdr1p, and the efficacy of the expression of the encoding genes in alleviating the deleterious action of quinidine, as well as of the other compounds (QDR2>QDR3>QDR1).     

A yeast model system for functional analysis of the Niemann-Pick type C protein 1 homolog, Ncr1p

Niemann-Pick disease type C (NP-C) is a progressive, ultimately fatal, autosomal recessive neurodegenerative disorder. The major biochemical hallmark of the disease is the endocytic accumulation of low-density lipoprotein-derived cholesterol. The majority of NP-C patients have mutations in the Niemann-Pick type C1 gene, NPC1. This study focuses on the Saccharomyces cerevisiae homolog of the human NPC1 protein encoded by the NCR1 gene. Ncr1p localizes to the vacuole, the yeast equivalent to the mammalian endosome-lysosome system. Here, we identify the first phenotype caused by deletion of NCR1 from the yeast genome, resistance to the ether lipid drug, edelfosine. Our results indicate that edelfosine has a cytotoxic, rather than cytostatic, effect on wildtype yeast cells. We exploit the edelfosine resistance phenotype to assess the function of yeast Ncr1 proteins carrying amino acid changes corresponding to human NPC1 patient mutations. We find that one of these amino acid changes severely compromises Ncr1p function as assessed using the edelfosine resistance assay. These findings establish S. cerevisiae as a model system that can be exploited to analyze the molecular consequences of patient mutations in NPC1 and provide the basis for future genetic studies using yeast.