Protein-tyrosine kinase activity in Saccharomyces cerevisiae

Saccharomyces cerevisiae was examined for tyrosine kinase activity in vitro because this organism offers molecular and genetic approaches for analyzing the role of tyrosine phosphorylation in cellular growth control that are unavailable in higher eukaryotes. Yeast extracts phosphorylated a random copolymer (glutamic acid:tyrosine, 80:20) at tyrosine in a reaction that was linear with respect to time and protein concentration. In the absence of added copolymer, phosphotyrosine was 0.1 percent of the total phosphoamino acids labeled with [gamma-32P]adenosine triphosphate in endogenous yeast proteins. However, specific activities of these reactions were low (approximately 1 percent of those in extracts of chick embryo fibroblasts). Lack of significant incorporation of label from [alpha-32P]adenosine triphosphate into the copolymer or endogenous yeast proteins demonstrated that nucleotide interconversion, adenylylation, and subsequent hydrolysis could not account for the generation of phosphotyrosine observed.     

Mutations of the adenylyl cyclase gene that block RAS function in Saccharomyces cerevisiae

The interaction between RAS proteins and adenylyl cyclase was studied by using dominant interfering mutations of adenylyl cyclase from the yeast Saccharomyces cerevisiae. RAS proteins activate adenylyl cyclase in this organism. A plasmid expressing a catalytically inactive adenylyl cyclase was found to interfere dominantly with this activation. The interfering region mapped to the leucine-rich repeat region of adenylyl cyclase, which is homologous to domains present in several other proteins and is thought to participate in protein-protein interactions.     

Saccharomyces cerevisiae: a diffusible sex factor

A hormone-like substance is secreted by alpha mating-type cells of heterothallic yeast strains. It induces in cells of the opposite mating type, a, a morphological change characteristic of the mating process. Secretion of this substance and mating ability have some common genetic determinants. In partially purified preparations, the substance has properties of an oligopeptide.     

糖化酵母糖化酶基因在酿酒酵母中的克隆与表达

[目的]使酿酒酵母高效表达糖化酵母糖化酶基因.[方法]利用PCR技术从糖化酵母中扩增出大小为2700 bp左右的带有启动子的糖化酶基因STA1.通过限制性内切酶BspDI与Acc65I双酶切,将目的基因STA1连接到穿梭质粒pRS416中构建重组质粒pRS416-sta1,转化酿酒酵母通过筛选.[结果]糖化酶活性最高为120 U/ml,酶的最适温度为60℃,最适pH为5.0,在酶催化反应2h后,酶剩余活力能达到约90％.[结论]通过基因工程手段得到高效表达糖化酶基因的酿酒酵母工程菌株.     

Maintenance of genome stability in Saccharomyces cerevisiae

Most human cancer cells show signs of genome instability, ranging from elevated mutation rates to gross chromosomal rearrangements and alterations in chromosome number. Little is known about the molecular mechanisms that generate this instability or how it is suppressed in normal cells. Recent studies of the yeast Saccharomyces cerevisiae have begun to uncover the extensive and redundant pathways that keep the rate of genome rearrangements at very low levels. These studies, which we review here, have implicated more than 50 genes in the suppression of genome instability, including genes that function in S-phase checkpoints, recombination pathways, and telomere maintenance. Human homologs of several of these genes have well-established roles as tumor suppressors, consistent with the hypothesis that the mechanisms preserving genome stability in yeast are the same mechanisms that go awry in cancer.     

mleA基因在酿酒酵母中的整合型表达

 【目的】进行中国自行筛选的专利菌种酒酒球菌 SD-2a（Oenococcus oeni SD-2a）的苹果酸－乳酸酶基因在酿酒酵母中的整合型表达,使葡萄酒生产过程中的酒精发酵和苹果酸－乳酸发酵（malolactic fermentation,MLF）同时进行。【方法】克隆Oenococcus oeni SD-2a的苹果酸－乳酸酶基因mleA,以PGK1强启动子和ADH1终止子为调控元件,以酵母整合型质粒YIp5为载体,构建重组表达质粒pYILmleA,并转化酿酒酵母（Saccharomyces cerevisiae）YS59,经筛选鉴定获得酵母转化子YS59/pYILmleA。进行转化子的营养缺陷型和交配型鉴定、菌落PCR鉴定、SDS-PAGE检测及斑点杂交检测,并对酵母转化子培养上清液进行L-苹果酸及L-乳酸含量的HPLC分析,检测目的基因的功能性表达。【结果】转化子的营养缺陷型和交配型鉴定、菌落PCR鉴定表明获得了阳性转化子;SDS-PAGE检测表明获得的转化子表达了目标蛋白,斑点杂交检测表明目的基因整合到受体菌中。模拟发酵表明mleA基因整合到受体菌中后进行了功能性表达,将培养基中的L-苹果酸转化成L-乳酸,使得培养液中的L-苹果酸含量极显著降低。转化子YS59/pYILmleA在添加5 648 mg?L-1 L-苹果酸和10%葡萄糖的SD/-Ura培养基中培养4 d,培养液上清中L-苹果酸的剩余含量与空载体转化子YS59/YIp5（对照）差异极显著,苹果酸的相对降低率为20.18%～20.85%。供试的转化子L-乳酸生成量为1 278～1 312 mg?L-1,对照未检出乳酸的生成。【结论】中国自主筛选的专利菌种O.oeni SD-2a的mleA基因的整合型表达质粒构建成功并在酿酒酵母中进行了功能性表达。     

复合载体对啤酒酵母细胞的固定化效果

以海藻酸钠-琼脂为复合载体,用包埋法对啤酒酵母细胞固定化.将固定化的啤酒酵母发酵液体培养基,以酒精度为指标,对细胞固定化条件进行了单因素试验.通过正交试验确定了细胞固定化的最佳制备条件.结果表明,固定化的最佳条件为载体浓度1.4g·40mL-1,复合载体之间比例1:1,温度46℃.     

The frequency dependence of osmo-adaptation in Saccharomyces cerevisiae

The propagation of information through signaling cascades spans a wide range of time scales, including the rapid ligand-receptor interaction and the much slower response of downstream gene expression. To determine which dynamic range dominates a response, we used periodic stimuli to measure the frequency dependence of signal transduction in the osmo-adaptation pathway of Saccharomyces cerevisiae. We applied system identification methods to infer a concise predictive model. We found that the dynamics of the osmo-adaptation response are dominated by a fast-acting negative feedback through the kinase Hog1 that does not require protein synthesis. After large osmotic shocks, an additional, much slower, negative feedback through gene expression allows cells to respond faster to future stimuli.     

后基因组时代的酿酒酵母研究策略

近年,随着基因工程技术的日益发展,酿酒酵母在分子生物学研究方面的应用越来越广泛。本文介绍了后基因组学用于酿酒酵母的方法,并概述了利用该手段对试验菌株与商业酿酒酵母进行生理功能分析、研究和改造的最新进展,展示了后基因组时代对酿酒酵母的影响及综合基因组信息、生物信息学和分子生物学技术的研究策略。最后,展望了现代生物技术手段在葡萄酒工业中的应用前景。     

Amino acid coding in Sarcina lutea and Saccharomyces cerevisiae

Aminoacyl-tRNA's from Sarcina lutea were tested for incorporation into protein in a heterologous system from Escherichia coli or for biniding in a homologous system from Sarcina lutea. Aminoacyl-tRNA's from Saccharomyces cerevisiae were tested for biniding in a homologous Saccharomyces cerevisiae system. Synthetic polyribonucleotides were used as messengers. The code which exists in Sarcina lutea and Saccharomyces cerevisiae is the same as in Escherichia coli.     

膜生物反应器封闭循环系统中酿酒酵母连续发酵动力学研究

研究了酿酒酵母在封闭循环膜生物反应器中的连续发酵动力学特征。结果表明,当发酵液中乙醇浓度升高时,细胞比生长率,基质比消耗速率和细胞得率系数都呈下降趋势,当细胞得率减小时产物得率倾向于增加。基于细胞生长和基质消耗的不同的线性抑制动力学模型能较好的描述实验结果。拟合数据表明当乙醇浓度超过66 g/L时,细胞生长受到完全抑制,如果超过82 g/L,细胞不再进行代谢活动消耗基质,这与硅橡胶膜生物反应器内观察到的实验现象相似。在长时间封闭循环发酵系统内,细胞最大比生长速率仅为0.022 h^-1,这可能与长期发酵过程副产物累积对细胞的抑制有关,也说明一定时间后系统达到动态平衡状态。     

CDC25: a component of the RAS-adenylate cyclase pathway in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae contains two functional homologues of the ras oncogene family, RAS1 and RAS2. These genes are required for growth, and all evidence indicates that this essential function is the activation of adenylate cyclase. In contrast, ras in mammalian cells does not appear to influence adenylate cyclase activity. To clarify the relation between ras function in yeast and in higher eukaryotes, and the role played by yeast RAS in growth control, it is necessary to identify functions acting upstream of RAS in the adenylate cyclase pathway. The evidence presented here indicates that CDC25, identified by conditional cell cycle arrest mutations, encodes such an upstream function.     

Effect of yeast Saccharomyces cerevisiae supplementation on serum antioxidant capacity,mucosal sIgA secretions and gut microbial populations in weaned piglets

This study was conducted to determine the effect of different forms of yeasts Saccharomyces cerevisiae supplementation on serum antioxidant capacity, mucosal secretory immunoglobulin A(s Ig A) secretions and gut microbial populations in weaned piglets. A total of 96 piglets weaned at 14 d of age were randomly allotted to 4 dietary treatments:(1) basal diet without yeast(Control);(2) basal diet supplemented with 3.00 g kg–1 live yeast(LY);(3) basal diet supplemented with 2.66 g kg–1 heat-killed whole yeast(HKY); and(4) basal diet supplemented with 3.00 g kg–1 superfine yeast powders(SFY). Each treatment had 4 replicates(pens), with 6 piglets per replicate. The experiment lasted for 3 wk. At d 7 and 21 of the experiment, the samples of serum, mucosa and mesenteric lymph node(MLN) from jejunum, and digesta from the ileum and cecum were collected for determinations. Compared with the Control, dietary SFY supplementation increased serum superoxide dismutase(SOD) activity and lysozyme levels at d 7, and jejunum mucosal s Ig A secretions at d 21 of the experiment(P0.05). Dietary LY supplementation increased serum SOD activity and jejunum mucosal s Ig A secretions, but decreased serum malondialdehyde(MDA) concentration at d 7 and 21(P0.05). Piglets fed diets supplemented with LY and SFY had lower p H values and decreased numbers of Escherichia coli in the ileum and cecum contents at d 21 compared with the Control(P0.05). Moreover, the ratio of Lactobacilli to E. coli in the ileum and cecum contents was increased by dietary LY and SFY supplementations(P0.05). Collectively, different forms of yeasts, especially LY and SFY, may modulate body antioxidant capacity and enhance the intestinal immunity by regulation of secretions of mucosal s Ig A and reduction of pathogenic bacteria colonization, thus improving intestinal health of weaned piglets.     

Mitochondrial DNA in yeast and some mammalian species

Yeast DNA, in a cesium chloride density gradient, shows a minor or satellite band with a density lower than that of the main nuclear component. The DNA isolated from purified mitochondria of yeasts corresponds in density to this satellite band. In solution, this DNA more easily undergoes renaturation as compared to DNA from cell nuclei. The ease of this renaturation is presumably due to a homogeneity greater than that of nuclear DNA. Mitochondrial DNA isolated from several mammalian species has the same or higher density than nuclear DNA, but differs in its ready renaturability.     

Correction of a defect in mammalian GPI anchor biosynthesis by a transfected yeast gene

Glycosylphosphatidylinositol (GPI) serves as a membrane anchor for a large number of eukaryotic proteins. A genetic approach was used to investigate the biosynthesis of GPI anchor precursors in mammalian cells. T cell hybridoma mutants that cannot synthesize dolichol-phosphate-mannose (Dol-P-Man) also do not express on their surface GPI-anchored proteins such as Thy-1 and Ly-6A. These mutants cannot form mannose-containing GPI precursors. Transfection with the yeast Dol-P-Man synthase gene rescues the synthesis of both Dol-P-Man and mannose-containing GPI precursors, as well as the surface expression of Thy-1 and Ly-6A, suggesting that Dol-P-Man is the donor of at least one mannose residue in the GPI core.     

拟南芥木糖异构酶在酿酒酵母的成功表达

酿酒酵母是工业上乙醇发酵的首选目标微生物,但是不能代谢木糖等五碳糖.通过导入外源木糖异构酶基因,可以赋予酵母利用木糖的能力.但是长期的研究发现很难将细菌木糖异构酶转入酿酒酵母得到活性表达.研究成功的在酿酒酵母里表达了拟南芥木糖异构酶,其活性可达到0.51 U/(mg·protein).拟南芥木糖异构酶与成功在酵母里表达的真菌木糖异构酶相比更加耐受木糖醇抑制(Ki=13.21 mM).本研究为改善酵母利用木糖提供了新的可用的木糖异构酶.     

酿酒酵母β-D-葡聚糖测定方法的研究

    针对酵母细胞破壁困难和酸碱不可溶性β-D-葡聚糖水解不彻底的问题,首先利用涡流微珠破壁法对酵母细胞进行破壁,使酵母细胞破壁率达到95.28%;其次采用高浓度酸预处理酸水解方法使酵母β-D-葡聚糖的酸解回收率高达98.76%;最后,由GOPOD法测定出酵母β-D-葡聚糖的含量.在此基础上建立了一个新的酵母β-D-葡聚糖的测定方法,新方法的相对标准差(RSD)和平均加样回收率分别为0.46%和99.96%.     

Control of gene expression by artificial introns in Saccharomyces cerevisiae

Artificial yeast introns that show cold-sensitive splicing have been constructed. These conditional introns can be inserted into a target gene as an "intron cassette" without disrupting the coding information, allowing expression of the gene to be cold sensitive. Insertion of these intron cassettes rendered the yeast URA3 gene cold sensitive in its expression. The advantage of this intron-mediated control system is that any gene can be converted to a controllable gene by simple insertion of an intron.