SKY1 and IXR1 interactions, their effects on cisplatin and spermine resistance in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has been previously used as a model eukaryotic system to identify genes related to drug resistance. Deletion of the IXR1 gene increases resistance to cisplatin, and deletion of the SKY1 gene increases resistance to cisplatin and spermine. Three S. cerevisiae strains and their derivatives, carrying single Δixr1 and Δsky1 and double Δixr1Δsky1 deletions, were compared in terms of resistance against these compounds. We found that the effects of these deletions are highly dependent on the genetic background of the selected strains. These results are valuable in the selection of yeast strains to be used in genetic screenings of compounds with putative pharmacological interest.     

Cloning of two β-xylanase-encoding genes from Aspergillus niger and their expression in Saccharomyces cerevisiae

Two endo-β-1,4-xylanase-encoding genes were amplified from Aspergillus niger ATCC 90196 mRNA, inserted between the yeast ADH2 promoter and terminator sequences (genes designated XYN4 and XYN5) and expressed in Saccharomyces cerevisiae. The nucleotide sequences of the XYN4 and XYN5 genes revealed that both genes encode 211-amino acid proteins that are 92% identical to each other. Both the Xyn4 and Xyn5 enzymes have pH and temperature optima of pH 4 and 60°C, respectively. Autoselective S. cerevisiae strains were developed that allowed β-xylanase production and secretion in complex medium. This revised version was published online in November 2006 with corrections to the Cover Date.     

Cloning and characterization of Saccharomyces cerevisiae genes that confer L-methionine sulfoximine and tabtoxin resistance.

Pseudomonas tabaci produces a toxin, tabtoxin, that causes wildfire disease in tobacco. The primary target of tabtoxin is presumed to be glutamine synthetase. Some effects of tabtoxin in tobacco can be mimicked by methionine sulfoximine (MSO), a compound that is known to inactivate glutamine synthetase. To understand how organisms can be made resistant to tabtoxin and MSO, we used Saccharomyces cerevisiae. We demonstrate that yeast strains carrying the glutamine synthetase gene, GLN1, on a multicopy plasmid overproduced glutamine synthetase and showed increased drug resistance. These and other data indicate that glutamine synthetase is the primary target of tabtoxin and MSO in S. cerevisiae. We also isolated three S. cerevisiae DNA inserts of 2.1, 2.3, and 2.8 kilobases that conferred tabtoxin and MSO resistance when the inserts were present on a multicopy plasmid. These plasmids conferred resistance to MSO by blocking intracellular transport of the drug. Transport appeared to occur by one or more methionine permeases. Resistance to tabtoxin could also occur by blockage of intracellular transport, but the drug was transported by some permease other than a methionine permease. These drug resistance plasmids did not block transport of citrulline, indicating that they did not affect the general amino acid permease.     

New mutation in Saccharomyces cerevisiae SRM genes and some features of their phenotypic effects

The effects of the previously identified mutations in nuclear genes SRM8, SRM12, SRM15, and SRM17 on the maintenance of chromosomes and recombinant plasmids in Saccharomyces cerevisiae cells and on cell sensitivity to ionizing radiation were studied. The srm8 mutation caused instability of chromosome maintenance in diploid cells. In yeast cells with the intact mitochondrial genome, all examined srm mutations decreased the mitotic stability of a centromeric recombinant plasmid with the chromosomal ARS element. Mutations srm12, srm15, and srm17 also decreased the mitotic stability of a centromereless plasmid containing the same ARS element, whereas the srm8 mutation did not markedly affect the maintenance of this plasmid. Mutations srm8, srm12, and srm17 were shown to increase cell sensitivity to gamma-ray irradiation. The SRM8 gene was mapped, cloned, and found to correspond to the open reading frame YJLO76w in chromosome X.     

Efficient production of S-adenosyl-l-methionine from dl-methionine in metabolic engineered Saccharomyces cerevisiae

S-Adenosyl-l -methionine (SAM) is an important small molecule compound widely used in treating various diseases. Although l -methionine is generally used, the low-cost dl -methionine is more suitable as the substrate for industrial production of SAM. However, d -methionine is inefficient for SAM formation due to the substrate-specificity of SAM synthetase. In order to increase the utilization efficiency of dl -methionine, intracellular conversion of d -methionine to l -methionine was investigated in the type strain Saccharomyces cerevisiae BY4741 and an industrial strain S. cerevisiae HDL. Firstly, via disruption of HPA3 encoding d -amino acid-N-acetyltransferase, d -methionine was accumulated in vivo and no N-acetyl-d -methionine production was observed. Further, codon-optimized d -amino acid oxidase (DAAO) gene from Trigonopsis variabilis (Genbank MK280686) and l -phenylalanine dehydrogenase gene (l -PheDH) from Rhodococcus jostii (Genbank MK280687) were introduced to convert d -methionine to l -methionine, SAM concentration and content was increased by 110% and 72.1% in BY4741 (plasmid borne) and increased by 38.2% and 34.1% in HDL (genome integrated), by feeding 0.5 g/L d -methionine. Using the recently developed CRISPR tools, the DAAO and l -PheDH expression cassettes were integrated into the HPA3 and SAH1 loci while SAM2 expression was integrated into the SPE2 and GLC3 loci of HDL, and the resultant strain HDL-R2 accumulated 289% and 192% more SAM concentration and content, respectively, by feeding 0.5 g/L dl -methionine. Further, in a 10 L fed-batch fermentation process, 10.3 g/L SAM were accumulated with the SAM content of 242 mg/g dry cell weight by feeding 16 g/L dl -methionine. The strategies used here provided a promising approach to enhance SAM production using low-cost dl -methionine.     

Identification of genes affecting selenite toxicity and resistance in Saccharomyces cerevisiae

Recent studies associating dietary selenium with reduced cancer susceptibility have aroused interest in this substance. In the millimolar range, selenite is toxic and slightly mutagenic for yeast. We show that selenite-treated yeast cells tend to arrest as large budded cells and that this arrest is abolished in a rad9 mutant that is significantly sensitive to selenite. Interestingly, a rev3 mutant affected in the error-prone repair pathway is also sensitive to selenite, whereas mutations in the other DNA repair pathways do not strongly affect resistance to selenite. We propose that selenite treatment leads to DNA damage inducing the RAD9-dependent cell cycle arrest. Selenite-induced DNA damage could be converted to mutations by the Rev3p-dependent lesion bypass system, thus allowing the cell cycle to progress. We have also investigated the selenite detoxification mechanisms and identified three genes involved in this process. In the present study, we show that lack of the cadmium glutathione-conjugate vacuolar pump Ycf1p or overexpression of the sulphite resistance membrane protein Ssu1p enhance the capacity of yeast cells to resist selenite treatment. Finally, we show that overexpression of the glutathione reductase Glr1p increases resistance to selenite, suggesting that selenite toxicity in yeast is closely linked to its oxidative capacity.     

Cloning and screening of the putative hexokinase genes from Rhizopus oryzae and their heterologous expression in Saccharomyces cerevisiae

Molecular Biology Reports - A filamentous fungus, Rhizopus oryzae (R. oryzae) is one of the ideal candidates for ethanol and lactic acid production due to its ability to grow on renewable carbon...     

Cloning and characterization of nuclear genes for two mitochondrial ribosomal proteins in Saccharomyces cerevisiae.

The genes for two large subunit proteins, YmL8 and YmL20, of the mitochondrial ribosome of Saccharomyces cerevisiae were cloned by hybridization with synthetic oligonucleotide mixtures corresponding to their N-terminal amino acid sequences. They were termed MRP-L8 and MRP-L20, respectively, and their nucleotide sequences were determined using a DNA sequencer. The MRP-L8 gene was found to encode a 26.8-kDa protein whose deduced amino acid sequence has a high degree of similarity to ribosomal protein L17 of Escherichia coli. The gene MRP-L20 was found to encode a 22.3-kDa protein with a presequence consisting of 18 amino acid residues. By Southern blot hybridization to the yeast chromosomes separated by field-inversion gel electrophoresis, the MRP-L8 and MRP-L20 genes were located on chromosomes X and XI, respectively. Gene disruption experiments indicate that their products, YmL8 and YmL20 proteins, are essential for the mitochondrial function and the absence of these proteins causes instability of the mitochondrial DNA.     

Complex interplay among regulators of drug resistance genes in Saccharomyces cerevisiae

The Gal4p family of yeast zinc cluster proteins comprises regulators of multidrug resistance genes. For example, Pdr1p and Pdr3p bind as homo- or heterodimers to pleiotropic drug response elements (PDREs) found in promoters of target genes. Other zinc cluster activators of multidrug resistance genes include Stb5p and Yrr1p. To better understand the interplay among these activators, we have performed native co-immunoprecipitation experiments using strains expressing tagged zinc cluster proteins from their natural chromosomal locations. Interestingly, Stb5p is found predominantly as a Pdr1p heterodimer and shows little homodimerization. No interactions of Stb5p with Pdr3p or Yrr1p could be detected in our assays. In contrast to Stb5p, Yrr1p is only detected as a homodimer. Similar results were obtained using glutathione S-transferase pull-down assays. Importantly, the purified DNA binding domains of Stb5p and Pdr1p bound to a PDRE as heterodimers in vitro. These results suggest that the DNA binding domains of Pdr1p and Stb5p are sufficient for heterodimerization. Our data demonstrate a complex interplay among these activators and suggest that Pdr1p is a master drug regulator involved in recruiting other zinc cluster proteins to fine tune the regulation of multidrug resistance genes.     

Antiapoptotic role of S-adenosyl-l-methionine against hydrochloric acid induced cell death in Saccharomyces cerevisiae

Exposure of stationary phase cells of Saccharomyces cerevisiae to 10 mM HCl (pH approximately 2) resulted in cell death as a function of time (up to 6 h) with most (about 40%-65%) of the cells showing apoptotic features including chromatin condensation along the nuclear envelope, exposure of phosphatidylserine on the outer leaflet of cytoplasmic membrane, and DNA fragmentation. During the first 2 h of acid exposure there was an increase in reactive oxygen species (ROS) level inside cells, with subsequent elevation in the level of lipid peroxidation and decrease in reducing equivalents culminating in loss of mitochondrial membrane potential (DeltaPsi(m)). An initial (1 h) event of mitochondrial hyper-polarization with subsequent elevation of ROS level of the acid treated cells was also observed. S-adenosyl-l-methionine (AdoMet; 1 mM) treatment increased the cell survival of the acid stressed cells. It partially scavenged the increased intracellular ROS level by supplementing glutathione through the transsulfuration pathway. It also inhibited acid mediated lipid peroxidation, partially recovered acid evoked loss of DeltaPsi(m) and protected the cells from apoptotic cell death. S-adenosyl di-aldehyde, an indirect inhibitor of the AdoMet metabolic pathway, increased mortality of the acid treated cells. Incubation of acid stressed cells with the antioxidant, N-acetyl-cysteine (1 mM), decreased the cellular mortality, but the same concentration of AdoMet offered more protection by scavenging the free radicals. The ability of AdoMet to scavenge ROS mediated apoptosis may be an important function of this molecule in responding to cellular stress. The study could open a new avenue for detailed investigation on the curative potential of AdoMet against gastric ulcer.     

Cystathionine accumulation in Saccharomyces cerevisiae.

A cysteine-dependent strain of Saccharomyces cerevisiae and its prototrophic revertants accumulated cystathionine in cells. The cystathionine accumulation was caused by a single mutation having a high incidence of gene conversion. The mutation was designated cys3 and was shown to cause loss of gamma-cystathionase activity. Cysteine dependence of the initial strain was determined by two linked and interacting mutations, cys3 and cys1 . Since cys1 mutations cause a loss of serine acetyltransferase activity, our observation led to the conclusion that S. cerevisiae synthesizes cysteine by sulfhydrylation of serine with hydrogen sulfide and by cleavage of cystathionine which is synthesized from serine and homocysteine.     

Cloning and characterization of seven cDNAs for hyperosmolarity-responsive (HOR) genes of Saccharomyces cerevisiae

Yeast cells can respond and adapt to osmotic stress. In our attempt to clarify the molecular mechanisms of cellular responses to osmotic stress, we cloned seven cDNAs for hyperosmolarity-responsive (HOR) genes from Saccharomyces cerevisiae by a differential screening method. Structural analysis of the clones revealed that those designated HOR1, HORS, HOR4, HOR5 and HOR6 encoded glycerol-3-phosphate dehydrogenase (Gpd1p), glucokinase (Glklp), hexose transporter (Hxtlp), heat-shock protein 12 (Hsp12p) and Na⁺, K⁺, Li⁺-ATPase (Enalp), respectively. HOR2 and HOR7 corresponded to novel genes. Gpdlp is a key enzyme in the synthesis of glycerol, which is a major osmoprotectant in S. cerevisiae. Cloning of HOR1/GPD1 as a HOR gene indicates that the accumulation of glycerol in yeast cells under hyperosmotic stress is, at least in part, caused by an increase in the level of GPDH protein. We performed a series of Northern blot analyses using HOR cDNAs as probes and RNAs prepared from cells grown under various conditions and from various mutant cells. The results suggested that all the HOR genes are regulated by common signal transduction pathways. However, the fact that they exhibited certain distinct responses indicated that they might also be regulated by specific pathways in addition to the common pathways. Ca²⁺ seemed to be involved in the signaling systems. In addition, Hog1p, one of the MAP kinases in yeast, appeared to be involved in the regulation of expression of HOR genes, although its function seemed to be insufficient for the overall regulation of expression of these genes.     

Genome-wide screen for genes with effects on distinct iron uptake activities in Saccharomyces cerevisiae

We screened a collection of 4847 haploid knockout strains (EUROSCARF collection) of Saccharomyces cerevisiae for iron uptake from the siderophore ferrioxamine B (FOB). A large number of mutants showed altered uptake activities, and a few turned yellow when grown on agar plates with added FOB, indicating increased intracellular accumulation of undissociated siderophores. A subset consisting of 197 knockouts with altered uptake was examined further for regulated activities that mediate cellular uptake of iron from other siderophores or from iron salts. Hierarchical clustering analysis grouped the data according to iron sources and according to mutant categories. In the first analysis, siderophores grouped together with the exception of enterobactin, which grouped with iron salts, suggesting a reductive pathway of iron uptake for this siderophore. Mutant groupings included three categories: (i) high-FOB uptake, high reductase, low-ferrous transport; (ii) isolated high- or low-FOB transport; and (iii) induction of all activities. Mutants with statistically altered uptake activities included genes encoding proteins with predominant localization in the secretory pathway, nucleus, and mitochondria. Measurements of different iron-uptake activities in the yeast knockout collection make possible distinctions between genes with general effects on iron metabolism and those with pathway-specific effects.     

Cloning of Saccharomyces cerevisiae promoters using a probe vector based on phleomycin resistance.

Vectors that confer high levels of phleomycin (Ph) resistance to Saccharomyces cerevisiae have been constructed with the TEF1 and ENO1 promoters, the Tn5 ble gene and the CYC1 terminator. They are able to transform yeast cells grown on rich glucose medium containing a moderate level of Ph (10 micrograms/ml, corresponding to 100-fold the minimal inhibitory concentration). Frequencies of transformation are identical to those obtained with the URA3 marker on a defined medium. A promoter probe vector, based on the same ble marker, enabled us to isolate sequences from chromosomal yeast DNA that had promoter activities. These DNA fragments have been sequenced and those which promote the highest levels of Ph resistance have been found to be either A + T-rich or have a potentially new and more efficient translation start site.     

Caffeine resistance of Saccharomyces cerevisiae

Four caffeine-resistant haploid isolates, two resistant to 50 mM caffeine and two resistant to 100 mM caffeine, were genetically analyzed. Complementation and tetrad analysis indicated that all four mutations are alleles of the same locus. All four isolates demonstrated incomplete dominance when hybridized to the wild-type strain and dominance of high to low resistance when hybridized to one another. Differences in caffeine resistance were found between wild-type grande cells and its petite derivative.     

Enhanced S-adenosyl-l-methionine production in Saccharomyces cerevisiae by spaceflight culture, overexpressing methionine adenosyltransferase and optimizing cultivation

Aims: S‐adenosyl‐l ‐methionine (SAM) is an important biochemical molecule with great potential in the pharmacological and chemotherapeutic fields. In this study, our aims were to enhance SAM production in Saccharomyces cerevisiae. Methods and Results: Through spaceflight culture, a SAM‐accumulating strain, S. cerevisiae H5M147, was isolated and found to produce 86·89% more SAM than its ground control strain H5. Amplified fragment length polymorphism (AFLP) analysis demonstrated that there were genetic variations between strain H5M147 and its ground control. Through recombinant DNA technology, the heterologous gene encoding methionine adenosyltransferase was integrated into the genome of strain H5M147. The recombinant strain H5MR83 was selected because its SAM production was increased by 42·98% when compared to strain H5M147. Furthermore, cultivation conditions were optimized using the one‐factor‐at‐a‐time and Taguchi methods. Under optimal conditions, strain H5MR83 yielded 7·76 g l^?1 of SAM in shake flask, an increase of 536·07% when compared to the strain H5. Furthermore, 9·64 g l^?1 of SAM was produced in fermenter cultivation. Conclusions: A new SAM‐accumulating strain, S. cerevisiae H5MR83, was obtained through spaceflight culture and genetic modification. Under optimal conditions, SAM production was increased to a relative high level in our study. Significance and Impact of the Study: Through comprehensive application of multiple methods including spaceflight culture, genetic modification and optimizing cultivation, the yield of SAM could be increased by 6·4 times compared to that in the control strain H5. The obtained S. cerevisiae H5MR83 produced 7·76 g l^?1 of SAM in the flask cultures, a significant improvement on previously reported results. The SAM production period with S. cerevisiae H5MR83 was 84 h, which is shorter than previously reported results. Saccharomyces cerevisiae H5MR83 has considerable potential for use in industrial applications.     

Two alleles of the sulfite resistance genes are differentially regulated in Saccharomyces cerevisiae

The sulfite resistance gene, SSU1-R, is widely distributed in wine yeasts. This gene has an upstream region distinct from that of the allelic gene, SSU1 and SSU1-R is expressed at a much higher level than SSU1. We characterized the promoters of both of these genes by analysis of their activity using the LacZ gene as a reporter. FZF1, the activator gene of SSU1, was shown to regulate SSU1-R expression indirectly. SSU1-R expression was activated under microaerobic conditions, and four 76-bp repeats, present within the SSU1-R promoter region, was essential for high expression. These results indicate that SSU1-R expression is regulated in different manner from that of SSU1. By deletion analysis of the SSU1-R promoter region, we found that at least two of the 76-bp repeats are necessary for promoter activity, and that the number of 76-bp repeats influences the activity. Hence, it was suggested that the number of 76-bp repeats increases in wine yeasts that require strong sulfite resistance.     

Process of Pb2 accumulation in Saccharomyces cerevisiae

Most of the Pb2+ taken up by Saccharomyces cerevisae was deposited in the inner part of the cells after 2 h. In the Pb2 accumulation experiments, the time to reach an equilibrium state was significantly shortened from 96 h to 24 h as the cell dry weight increased from 0.56 g/l to 5.18 g/l. The penetration time of Pb2+ to reach on the interacellular region (2 h) was quite different from that on the extracellular region (3 min). In the case of S. cerevisiae, the first step which a Pb2+ binds to cell wall within 3[f]5 min is metabolism-independent and the second step within 24 h is metabolism-dependent followed by the third step which is metabolism-dependent or -independent after 24 h.