Genetic evidence for a functional interaction between Saccharomyces cerevisiae CDC24 and CDC42

Cdc24p and Cdc42p are involved in the control of cell polarity during the Saccharomyces cerevisiae cell cycle. Cdc42p is a member of the Ras superfamily of GTPases and Cdc24p displays limited amino-acid sequence similarity with the Dbl proto-oncoprotein, which acts to stimulate guanine-nucleotide exchange on human Cdc42p. We have performed several genetic experiments to test whether Cdc24p and Cdc42p interact within the cell. First, overexpression of Cdc24p suppressed the dominant-negative cdc42^D118A allele. Second, overexpression of wild-type CDC24 and CDC42 genes together was a lethal event resulting in a morphological phenotype of large, round, unbudded cells, indicating a loss of cell polarity. Third, a cdc24^ts cdc42^ts double mutant exhibited a synthetic-lethal phenotype at the semi-permissive temperature of 30°C. These data suggest that Cdc24p and Cdc42p interact within the cell and that Cdc24p may be involved in the regulation of Cdc42p activity.     

酿酒酵母与粟酒裂殖酵母融合子检出方法的研究

为了快速检出酿酒酵母与粟酒裂殖酵母的融合子,在双亲原生质体融合处理后,利用两亲本再生营养条件的差异及氧化邻苯二胺能力的不同,通过设计选择性底层培养基和鉴定性上层培养基,进行了融合子检出方法的研究,并确定了3株融合子。该方法简单易行,可用于酿酒酵母与粟酒裂殖酵母原生质体融合育种的研究。     

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae is glycosylated,sorted and matured in the fission yeast Schizosaccharomyces pombe

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae (CPY^sc) has been expressed in a Schizosaccharomyces pombe strain devoid of the endogenous equivalent peptidase, employing a 2 μ derived plasmid. Immunoblot analysis revealed that CPY^sc produced in the fission yeast has a higher molecular mass than mature CPY^sc produced by the budding yeast. CPY^sc is glycosylated when expressed in S. pombe and uses four N-linked glycosylation sites as shown by endoglycosidase H digestion. Carbohydrate removal leads to a protein moiety which is indistinguishable in size from deglycosylated CPY^sc produced by S. cerevisiae. CPY^sc isolated from S. pombe soluble extracts is enzymatically active and thus is presumed to undergo correct proteolytic maturation. Subcellular fractionation experiments showed a cofractionation of CPY^sc with the S. pombe endoproteinases PrA and PrB, suggesting that the protein is correctly sorted to the vacuole and that these peptidases might be responsible for zymogen activation.     

Malo-ethanolic fermentation in grape must by recombinant strains of Saccharomyces cerevisiae.

Recombinant strains of Saccharomyces cerevisiae with the ability to reduce wine acidity could have a significant influence on the future production of quality wines, especially in cool climate regions. L-Malic acid and L-tartaric acid contribute largely to the acid content of grapes and wine. The wine yeast S. cerevisiae is unable to effectively degrade L-malic acid, whereas the fission yeast Schizosaccharomyces pombe efficiently degrades high concentrations of L-malic acid by means of a malo-ethanolic fermentation. However, strains of Sz. pombe are not suitable for vinification due to the production of undesirable off-flavours. Heterologous expression of the Sz. pombe malate permease (mae1) and malic enzyme (mae2) genes on plasmids in S. cerevisiae resulted in a recombinant strain of S. cerevisiae that efficiently degraded up to 8 g/l L-malic acid in synthetic grape must and 6.75 g/l L-malic acid in Chardonnay grape must. Furthermore, a strain of S. cerevisiae containing the mae1 and mae2 genes integrated in the genome efficiently degraded 5 g/l of L-malic acid in synthetic and Chenin Blanc grape musts. Furthermore, the malo-alcoholic strains produced higher levels of ethanol during fermentation, which is important for the production of distilled beverages.     

Review: The Cct eukaryotic chaperonin subunits of Saccharomyces cerevisiae and other yeasts

All eight of the CCT1-CCT8 genes encoding the subunits of the Cct chaperonin complex in Saccharomyces cerevisiae have been identified, including three that were uncovered by the systematic sequencing of the yeast genome. Although most of the properties of the eukaryotic Cct chaperonin have been elucidated with mammalian systems in vitro, studies with S. cerevisiae conditional mutants revealed that Cct is required for assembly of microtubules and actin in vivo. Cct subunits from the other yeasts, Candida albicans and Schizosaccharomyces pombe, also have been identified from partial and complete DNA sequencing of genes. Cct8p from C. albicans, the only other completely sequenced Cct protein from a fungal species other than S. cerevisiae, is 72% and 61% similar to the S. cerevisiae and mouse Cct8 proteins, respectively. The C. albicans CCT8 sequence has been assigned the Accession Number U37371 in the GenBank/EMBL database.     

Characterization of a branched-chain amino-acid aminotransferase from Schizosaccharomyces pombe

The Saccharomyces cerevisiae genes for the cytosolic and mitochondrial branched-chain amino-acid aminotransferases (BCAT) were isolated recently. These genes show significant homology to mammalian ECA39, originally isolated as a gene regulated by the c-myc oncogene. We now report the isolation of the Schizosaccharomyces pombe eca39/BCAT gene. The S. pombe protein shows 47–52% identity to other eukaryotic BCAT proteins isolated from S. cerevisiae, nematode, mouse and man. A genetic growth assay for BCAT activity was established using an S. cerevisiae strain disrupted in both BCAT isoenzymes. Consequently, the activity of the S. pombe BCAT was demonstrated by genetic and biochemical means. Possible applications of BCAT-encoding genes as selection markers in yeast transformation are proposed. The sequence has been deposited in the GenBank data library under Accession Number U88029. © 1998 John Wiley & Sons, Ltd.     

Malolactic fermentation by engineered Saccharomyces cerevisiae as compared with engineered Schizosaccharomyces pombe

The ability of yeast strains to perform both alcoholic and malolactic fermentation in winemaking was studied with a view to achieving a better control of malolactic fermentation in enology. The malolactic gene of Lactococcus lactis (mleS) was expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. The heterologous protein is expressed at a high level in cell extracts of a S. cerevisiae strain expressing the gene mleS under the control of the alcohol dehydrogenase (ADH1) promoter on a multicopy plasmid. Malolactic enzyme specific activity is three times higher than in L. lactis extracts. Saccharomyces cerevisiae expressing the malolactic enzyme produces significant amounts of l-lactate during fermentation on glucose-rich medium in the presence of malic acid. Isotopic filiation was used to demonstrate that 75% of the l-lactate produced originates from endogenous l-malate and 25% from exogenous l-malate. Moreover, although a small amount of exogenous l-malate was degraded by S. cerevisiae transformed or not by mleS, all the exogenous degraded l-malate was converted into l-lactate via a malolactic reaction in the recombinant strain, providing evidence for very efficient competition of malolactic enzyme with the endogenous malic acid pathways. These results indicate that the sole limiting step for S. cerevisiae in achieving malolactic fermentation is in malate transport. This was confirmed using a different model, S. pombe, which efficiently degrades l-malate. Total malolactic fermentation was obtained in this strain, with most of the l-malate converted into l-lactate and CO₂. Moreover, l-malate was used preferentially by the malolactic enzyme in this strain also.     

Hut1 proteins identified in Saccharomyces cerevisiae and Schizosaccharomyces pombe are functional homologues involved in the protein-folding process at the endoplasmic reticulum

The Saccharomyces cerevisiae HUT1 gene (scHUT1) and the Schizosaccharomyces pombe hut1(+) gene (sphut1(+)) encode hydrophobic proteins with approximately 30% identity to a human UDP-galactose transporter-related gene (UGTrel1) product. These proteins show a significant similarity to the nucleotide sugar transporter and are conserved in many eukaryotic species, but their physiological functions are not known. Both scHUT1 and sphut1(+) genes are non-essential for cell growth under normal conditions, and their disruptants show no defects in the modification of O- and N-linked oligosaccharides, but are sensitive to a membrane-permeable reducing agent, dithiothreitol (DTT). Consistent with this phenotype, scHUT1 has genetic interaction with ERO1, which plays an essential role in the oxidation of secretory proteins at the endoplasmic reticulum (ER). Overexpression of the MPD1 or MPD2 genes, which were isolated as multicopy suppressors of protein disulphide isomerase (PDI) depletion, could not replace the essential function of PDI in Delta hut1 S. cerevisiae cells. Our results indicate that scHut1p and spHut1p are functional homologues, and their physiological function is to maintain the optimal environment for the folding of secretory pathway proteins in the ER.     

Mapping of gene controlling thiamine transport in Saccharomyces cerevisiae

A recessive mutation leading to complete loss of thiamine uptake in Saccharomyces cerevisiae was mapped on the left arm of chromosome VII, approximately 56cM centromere-distal to trp5. As the analysed locus is relatively distant from its centromere and from the markers used, its attachment to chromosome VII was confirmed by chromosome loss methods.     

酿酒酵母低渗敏感性突变株的选育

为促进酿酒酵母胞内产物的有效释放,以其野生型二倍体(2n)菌株Y_1为出发菌株,经紫外诱变处理,通过测定其胞外FDP浓度及核酸、蛋白质渗透率,对比各突变株在低渗条件下的自溶程度。结果表明:突变株Hs_5~*(n)低渗培养时,条件性自溶程度最高,其胞外FDP浓度达25.82μg/mL,低渗培养6h时,其核酸、蛋白质渗透率高达1.86,2.07。此外突变株Hs_2~*(n)、Hs_1(2n)也具有较好的条件性自溶能力。试验共筛选获得3株低渗敏感性突变株,与野生型菌株相比,条件性自溶的低渗敏感性突变株能有效促进胞内大分子物质的外泌。     

Combining mutations in the incoming and outgoing pheromone signal pathways causes a synergistic mating defect in Saccharomyces cerevisiae

Mating pheromones stimulate Saccharomyces cerevisiae yeast cells to form a pointed projection that becomes the site of cell fusion during conjugation. To investigate the role of mating projections, we screened for mutations that enhanced the weak mating defect of MAT a ste2‐T326 cells that are defective in forming pointed projections. These cells are also 10‐fold more sensitive to α‐factor pheromone because ste2‐T326 encodes truncated α‐factor receptors that are not regulated properly. Mutations in AXL1, STE6 and FUS3 were identified in the screen. AXL1 was studied further because it is required for efficient a ‐factor pheromone production and for selecting the site for bud morphogenesis. Mutation of AXL1 did not enhance the morphogenesis or pheromone sensitivity defects of ste2‐T326. Instead, the synergistic mating defect was apparently due to decreased a ‐factor production because the axl1Δ ste2‐T326 cells mated well with a sst2 α mating partner that is supersensitive to a ‐factor. When combined with a wild‐type mating partner, the ste2‐T326 axl1Δ cells failed to mate because they did not lock cell walls, one of the earliest steps in conjugation. Analysis of axl1Δ in combination with other mutations that cause defects in morphogenesis or pheromone sensitivity (e.g. bar1, sst2, afr1) indicated that both phenotypes of ste2‐T326 cells, supersensitivity to α‐factor and the defect in forming pointed projections, contributed to the synergistic mating defect. We suggest a model that the synergistic mating defect is caused by the combined effects of ste2‐T326 and axl1Δ on the presentation of a ‐factor to partner cells. Altogether, these results demonstrate an important linkage between the incoming and outgoing pheromone signals during the intercellular communication that promotes yeast mating. Copyright © 1999 John Wiley & Sons, Ltd.     

XV. Yeast sequencing reports. Sequence of a 10·27 kb segment on the left arm of chromosome XV from Saccharomyces cerevisiae includes part of the IRA2 gene and a putative new gene

A 10 270 bp fragment from the left arm of chromosome XV of Saccharomyces cerevisiae was sequenced and analysed. The sequence reveals the presence of two open reading frames (ORFs), one of them is the larger part of the previously sequenced gene IRA2 (YOL0951). The other ORF, YOL0950, has a length of 1245 nucleotides and exhibits no significant homology with any known gene, although there is some similarity of its upstream region to the corresponding region of the Schizosaccharomyces pombe cdr1/nim1 gene which is involved in the control of mitotic cell size. The sequence has been deposited in the EMBL data library under Accession Number X75449.     

A conditional suicide system for Saccharomyces cerevisiae relying on the intracellular production of the Serratia marcescens nuclease

A conditional lethal system for biological containment of genetically modified strains of Saccharomyces cerevisiae is described. This suicide system is based on the intracellular production of the Serratia marcescens nuclease in the yeast cell, aiming at the destruction of the host genetic material. The S. marcescens nuclease, encoded by the nucA gene, is normally secreted by the bacterium into the medium. In the present work, the nucA gene, devoid of its signal peptide coding sequence, was cloned in a yeast expression vector, under control of the glucose-repressed S. cerevisiae alcohol dehydrogenase 2 gene (ADH2) promoter. When transformed into S. cerevisiae, the recombinant plasmid proved to be effective in killing the host cells upon glucose depletion from the medium, and the nuclease activity was found in lysates prepared from the transformants. In addition, the nuclease degrading effect was shown to reach chromosomal DNA in the yeast host. The killing effect of the nucA plasmid was also demonstrated in soil microcosm assays, indicating that whenever the GMM escapes into the environment where glucose is scarce, the nucA gene will be expressed and the resulting nuclease will destroy the genetic material and kill the cells. In contrast to other suicide systems that target the cell envelope, the advantage of the one described here is that it disfavours horizontal gene transfer from recombinant yeast cells to other microorganisms found in the environment.     

Increased endocytosis in the Saccharomyces cerevisiae fragile mutant VY1160

The VY1160 mutant is characterized by cell lysis in hypotonic solutions and generally increased permeability to substances for which Saccharomyces cerevisiae cells are not permeable. Two mutations, srb1 and ts1, have been identified in VY1160 mutant, and previous studies (Kozhina et al., 1979) have shown srb1 to be responsible for cell lysis. We now present evidence that the ts1 mutation leads to increased endocytosis in VY1160 cells. The internalization of lucifer yellow carbohydrazide in VY1160 cells is time-, temperature- and energy-dependent and consistent with a fluid-phase mechanism of endocytosis. The rate of steady-state accumulation of the dye at 37 degrees C is 145 ng/micrograms DNA per h for VY1160 mutant and 23 ng/micrograms DNA per h for S288C parental strain. Studies with isogenic strains having either the srb1 or the ts1 mutation, or SRB1 TS1 wild-type alleles have shown that only ts1 strains possess increased endocytosis. Quantitation of endocytosis in cells grown at 24 degrees C and shifted at 38 degrees C shows that ts1 strains, but not srb1 and wild-type strains, increase ten-fold the internalization of lucifer yellow 2 h after the shift at 38 degrees C. The analysis of ts1 x wild-type crosses provides evidence that the temperature-sensitive phenotype segregates together with the enhanced endocytosis. It is concluded that the increased endocytosis might explain the generally increased permeability of VY1160 mutant cells.     

葡萄酒酿造过程中酿酒酵母乙醛代谢特征的研究

选用4株本土酵母(G3-3、NX8423、D3-4、NX349)与2株商业酵母(X16、F15)分别在干白与干红葡萄汁中进行发酵,研究各菌株在酒精发酵过程中的乙醛动态变化,并对各菌株产生乙醛的特征进行分析。结果显示：本土酿酒酵母与商业酿酒酵母具有相似的乙醛动态变化,但各菌株的乙醛特征参数差异显著(P＜0.05)。在干白葡萄酒的发酵过程中,D3-4表现出最低的乙醛峰值及末值含量(发酵终止时发酵液中的乙醛含量),分别为58.60mg/L和39.96mg/L,较X16显著降低了25.62%及25.92%。乙醛产生速率最快的为X16菌株。乙醛降解速率最快的则为NX8423,达到5.90mg/(L·d),较X16提高了2.09倍。在干红葡萄酒发酵中,乙醛峰值含量最低的为F15,乙醛产生速率最快的为G3-3,达到65.04mg/L,较F15显著提高了45.63%。NX349的乙醛降解速率最高且末值含量最低,分别为25.62mg/(L·d)及29.65mg/L。本土酿酒酵母菌株具有较商业菌株优良的乙醛代谢特征,通过开发本土优良酵母菌株能够优化葡萄酒酿造过程中的乙醛含量。     

Saccharomyces cerevisiae SCY1发酵kefir的工艺研究

本文采用Saccharomyces cerevisiaeSCY1和乳酸菌混合发酵牛乳制备kefir,分别研究了接种量、灭菌条件、发酵温度和加糖量对kefir风味的影响,最终确定最佳工艺条件为:XPL-1接种量为0.0400g/L,SCY1接种量为103个/mL;灭菌条件为80~85℃下灭菌10min;发酵温度为32℃;加糖量为4%。通过此工艺条件制备的kefir,具有独特的风味和较高的营养价值。     

RCS1, a gene involved in controlling cell size in Saccharomyces cerevisiae

Cloning and sequencing of RCS1, Saccharomyces cerevisiae gene whose product seems to be involved in timing the budding event of the cell cycle, is described. A haploid strain in which the 3'-terminal region of the chromosomal copy of the gene has been disrupted produces cells that are, on average, twice the size of cells of the parental strain. The critical size for budding in the mutant is similarly increased, and the disruption mutation is dominant in a diploid heterozygous for the RCS1 gene. Spores from this diploid have a reduced ability to germinate, the effect being more pronounced in the spores carrying the disrupted copy of RCS1. However, disrupted cells recover from alpha-factor treatment equally as well as wild-type cells.