共查询到20条相似文献，搜索用时 15 毫秒
1.
McMahon KW Manukyan A Dungrawala H Montgomery M Nordstrom B Wright J Abraham L Schneider BL 《Yeast (Chichester, England)》2011,28(9):661-671
A consortium of yeast geneticists have created -6000 individual ORF deletions, representing > 96% of the currently verified or predicted ORFs in S. cerevisiae. Importantly, molecular barcodes (each a unique 20 bp sequence termed either Uptag or Downtag) were used as identifiers for every ORF deletion. Microarray analyses of pooled yeast deletions has been used to identify thousands of genes involved in general fitness, haploinsufficiency, drug resistance and DNA damage repair. However, application of this powerful technology requires considerable expense, expertise and specialized equipment. While standard PCR techniques and specifically designed PCR primers can be used to confirm that a given ORF is in fact deleted, this procedure cannot be used to identify unknown deletions. In theory, every ORF deletion could be determined by barcode sequencing. However, neither a consolidated barcode database nor a reliable search engine is currently available for this purpose. To address this need, we have adapted a FASTA sequence program that utilizes the unique barcode database to allow users to identify individual ORF deletions, based upon simple sequencing reactions of PCR amplifications of either Uptag or Downtag barcodes. In silico and practical testing of this application reveals that it is an inexpensive, reliable and reproducible method for rapidly identifying unknown deletions. This approach allows laboratories to conduct small- or large-scale genetic screens with pooled yeast deletion strains and identify or verify any ORF deletion without the need for microarray technology. 相似文献
2.
Yeasts used in bread making are exposed to air-drying stress during dried yeast production processes. To clarify the genes required for air-drying tolerance, we performed genome-wide screening using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 278 gene deletions responsible for air-drying sensitivity. These genes were classified based on their cellular function and on the localization of their gene products. The results showed that the genes required for air-drying tolerance were frequently involved in mitochondrial functions and in connection with vacuolar H(+)-ATPase, which plays a role in vacuolar acidification. To determine the role of vacuolar acidification in air-drying stress tolerance, we monitored intracellular pH. The results showed that intracellular acidification was induced during air-drying and that this acidification was amplified in a deletion mutant of the VMA2 gene encoding a component of vacuolar H(+)-ATPase, suggesting that vacuolar H(+)-ATPase helps maintain intracellular pH homeostasis, which is affected by air-drying stress. To determine the effects of air-drying stress on mitochondria, we analysed the mitochondrial membrane potential under air-drying stress conditions using MitoTracker. The results showed that mitochondria were extremely sensitive to air-drying stress, suggesting that a mitochondrial function is required for tolerance to air-drying stress. We also analysed the correlation between oxidative-stress sensitivity and air-drying-stress sensitivity. The results suggested that oxidative stress is a critical determinant of sensitivity to air-drying stress, although ROS-scavenging systems are not necessary for air-drying stress tolerance. 相似文献
3.
Masayoshi Iwahara Toshiatsu Tanaka Yoshiyuki Nomura 《Journal of the Institute of Brewing》2001,107(6):345-348
The effects of edible fruiting of Basidiomycetes on growth of Saccharomyces cerevisiae and other yeast strains were examined. The growth rates were significantly increased in the presence of fruiting bodies but there was no significant difference growth yield between cultures with and without fruiting bodies. Growth rates of yeast cells were promoted in both synthetic and natural media. 相似文献
4.
Tallada VA Daga RR Palomeque C Garzón A Jimenez J 《Yeast (Chichester, England)》2002,19(13):1139-1151
Genetic studies in yeasts enable an in vivo analysis of gene functions required for the cell division cycle (cdc genes) in eukaryotes. In order to characterize new functions involved in cell cycle regulation, we searched for genes causing cell division defects by overexpression in the fission yeast Schizosaccharomyces pombe. By using this dominant genetic strategy, 26 independent clones were isolated from a Sz. pombe cDNA library. The cloned cDNAs were partially sequenced and identified by computer analysis. The 26 clones isolated corresponded to 21 different genes. Among them, six were genes previously characterized in Sz. pombe, 11 were homologues to genes identified and characterized in other organisms, and four represented genes with unknown functions. In addition to known cell cycle regulators encoding inhibitory protein kinases (wee1, pka1) and DNA checkpoint proteins (Pcna, rad24), we have identified genes that are involved in a number of cellular processes. This includes protein synthesis (ribosomal proteins L7, L10, L29, L41, S6, S11, S17 and the PolyA-Binding Protein PABP), protein degradation (UBI3), nucleolar rRNA expression (fib, imp1, dbp2), cell cytoskeleton (act1) and glycolysis (pfk1). The interference caused in the cell cycle by overexpression of these genes may elucidate novel mechanisms coupling different cellular processes with the control of the cell division. The effect caused by some of them is described in more detail. 相似文献
5.
Van Driessche B Tafforeau L Hentges P Carr AM Vandenhaute J 《Yeast (Chichester, England)》2005,22(13):1061-1068
The one-step PCR-mediated technique used for modification of chromosomal loci is a powerful tool for functional analysis in yeast. Both Saccharomyces cerevisiae and Schizosaccharomyces pombe are amenable to this technique. However, the scarce availability of selectable markers for Sz. pombe hampers the easy use of this technique in this species. Here, we describe the construction of new vectors deriving from the pFA6a family, which are suitable for tagging in both yeasts owing to the presence of a nourseothricin-resistance cassette. These plasmids allow various gene manipulations at chromosomal loci, viz. N- and C-terminal tagging with 3HA (haemagglutinin) or 13Myc epitopes, GST (glutathione S-transferase), 4TAP (tandem affinity purification) and several GFP (green fluorescent protein) isoforms. For N-terminal modifications, the use of different promoters allows constitutive (PADH1) or regulatable (PGAL1) promoters for S. cerevisiae and derivatives of Pnmt1 for Sz. pombe expression. 相似文献
6.
通过缺失乙酰辅酶A水解酶（ACH）基因和过表达乙酰辅酶A合成酶（ACS）基因技术提高酿酒酵母合成乙酰辅酶A（acetyl- CoA）能力的同时,过表达醇酰基转移酶（ATF）基因,提高乙酸乙酯合成能力,并考察acetyl-CoA含量对酿酒酵母合成乙酸乙酯能力的影响。结果表明,敲除ACH1基因、且在敲除ACH1基因基础上过表达ACS1、ACS2基因均能提高酿酒酵母Acetyl-CoA含量,进而提高乙酸乙酯含量。较亲本菌株α5,缺失突变株α5ΔACH1、重组菌株α5-A1、α5-A2的Acetyl-CoA的含量均分别提高了52.5%、80.33%、52.79%,乙酸乙酯含量分别提高10.59%、26.12%、23.70%。在敲除ACH1基因、过表达ACS1和ACS2基因的基础上同时过表达ATF1基因,得到工程菌株A1-ATF1和A2-ATF1,较亲本菌株α5,乙酸乙酯含量分别提高226.09%、530.43%、289.57%,工程菌株A1-ATF1乙酸乙酯产量最高,为72.52 mg/L。研究表明,提高乙酰辅酶A含量能够促进乙酸乙酯的合成,为提高乙酸乙酯生成量提供了新思路。 相似文献
7.
The yeast Saccharomyces cerevisiae has proved to be an excellent model organism to study the function of proteins. One of the many advantages of yeast is the many genetic tools available to manipulate gene expression, but there are still limitations. To complement the many methods used to control gene expression in yeast, we have established a conditional gene deletion system by using the FLP/FRT system on yeast vectors to conditionally delete specific yeast genes. Expression of Flp recombinase, which is under the control of the GAL1 promoter, was induced by galactose, which in turn excised FRT sites flanked genes. The efficacy of this system was examined using the FRT site-flanked genes HSP104, URA3 and GFP. The pre-excision frequency of this system, which might be caused by the basal activity of the GAL1 promoter or by spontaneous recombination between FRT sites, was detected ca. 2% under the non-selecting condition. After inducing expression of Flp recombinase, the deletion efficiency achieved ca. 96% of cells in a population within 9 h. After conditional deletion of the specific gene, protein degradation and cell division then diluted out protein that was expressed from this gene prior to its excision. Most importantly, the specific protein to be deleted could be expressed under its own promoter, so that endogenous levels of protein expression were maintained prior to excision by the Flp recombinase. Therefore, this system provides a useful tool for the conditional deletion of genes in yeast. 相似文献
8.
Reid RJ Sunjevaric I Keddache M Rothstein R Kedacche M 《Yeast (Chichester, England)》2002,19(4):319-328
Gene disruptions are a vital tool for understanding Saccharomyces cerevisiae gene function. An arrayed library of gene disruption strains has been produced by a consortium of yeast laboratories; however their use is limited to a single genetic background. Since the yeast research community works with several different strain backgrounds, disruption libraries in other common laboratory strains are desirable. We have developed simple PCR-based methods that allow transfer of gene disruptions from the S288C-derived strain library into any Saccharomyces strain. One method transfers the unique sequence tags that flank each of the disrupted genes and replaces the kanamycin resistance marker with a recyclable URA3 gene from Kluyveromyces lactis. All gene-specific PCR amplifications for this method are performed using a pre-existing set of primers that are commercially available. We have also extended this PCR technique to develop a second general gene disruption method suitable for any transformable strain of Saccharomyces. 相似文献
9.
腐生酵母菌在鲜榨苹果汁中的生长速率预测模型 总被引：1，自引：0，他引：1
通过研究温度、pH和水分活度对鲜榨苹果汁中腐生酵母菌生长情况的影响 ,建立了最大生长速率和迟滞时间与生长限制因子之间关系的数学预测模型 ,并对Ratkowsky扩展模型和响应面模型进行了对比。结果表明 ,Ratkowsky扩展模型的相关系数优于响应面模型 ,响应面模型在较高温度 ( 2 0℃以上 )最大生长速率呈现下降趋势的预测结果与实际情况不符。应用Ratkowsky扩展模型对鲜榨苹果汁的货架期进行预测时发现 ,温度、pH和水分活度对货架期的影响有协同作用。通过模型验证证实Ratkowsky扩展模型预测结果与实测值有良好的拟合性 ,相对误差在±1 0 %左右 ,可以用于鲜榨苹果汁货架期的预测 相似文献
10.
目的研究不同硒浓度对啤酒酵母细胞生长的影响，确定适宜的硒添加量。方法用分光光度计测定不同硒浓度时酵母细胞培养液在不同时间的吸收度，绘制生长曲线，比较硒浓度不同时酵母细胞的生长情况。结果亚硒酸钠的添加浓度小于20μg／mL时，有促进酵母菌生长的作用；亚硒酸钠浓度高于30μg／mL时，对酵母菌生长有明显的抑制作用；亚硒酸钠浓度在20μg／mL时酵母菌可以正常生长。结论亚硒酸钠的添加浓度应在20μg／mL左右。 相似文献
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Chlorophenols are a class of chemicals commonly used in preservatives, disinfectants, algaecides, herbicides and pesticides. However, there is a growing evidence that these compounds are a threat to human health. This is alarming as many chlorophenols are common pollutants found in the global environment at potentially biohazardous levels. Despite chlorophenols being abundant, widely used and poisonous, we know relatively little about their mechanism of toxicity in eukaryotes. Thus, we performed genome-wide growth screens using Saccharomyces cerevisiae to understand the molecular basis of chlorophenol toxicity. Of ～4850 single gene knockout strains tested, 393 mutants showed growth sensitivity to treatment with 4-chlorophenol (4-CP), 2,4-dichlorophenol (2,4-DCP) or pentachlorophenol (PCP). Only eight mutants showed growth hypersensitivity to all the three treatments and harboured deletions in genes important for aromatic amino acid biosynthesis (ARO1, ARO7) or mitochondrial protein synthesis and respiration (ATP5, ISA1, RML2, GET2, SLS1, MRPL38). 相似文献
13.
A temperature gradient incubator has been used to determine the effect of temperature on the growth of strains of Saccharomyces cerevisiae and Saccharomyces uvarum (including lager brewing yeasts formerly classified as Saccharomyces carlsbergensis). The maximum temperatures for growth (Tmax) for all strains of S. cerevisiae examined were in the range 37.5°C-39.8°C and the optimum temperatures for the most rapid initial growth (Topt) were in the range 30.0°C-35.0°C. Strains of S. uvarum, however, formed two distinct groups: Group A (including all brewing strains of S. uvarum tested) had Tmax values 31.6°C-34.0°C and Topt values 26.8°C-30.4°C; Group B had Tmax values 38.2°C-40.0°C and Topt values 30.0°C-34.6°C. It is proposed, therefore, that the species name S. carlsbergensis should be re-introduced and applied to those strains of S. uvarum (Group A) which have the lower Tmax values. Minimum temperatures for growth (Tmin) of the yeasts were not investigated as initial studies had shown that they could not be measured satisfactorily. Measurements of the generation times for one brewing strain of S. cerevisiae and one brewing strain of S. uvarum (Group A) over the temperature range 6.0°C-22.0°C have shown that there are significant differences between the yeasts at the lower end of the temperature range and that the relationship between generation time (GT) and temperature (T) for both yeasts closely follows the mathematical expression: 相似文献
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通过PCR扩增了肺炎克雷伯氏菌（Klebsiella pneumoniae）和大肠杆菌（Escherichia coli）各自的葡萄糖脱氢酶基因KPgdh和ECgdh,构建了表达载体p ET-28a-KPgdh和p ET-28a-ECgdh,转化大肠杆菌E.coli BL21,获得了重组菌BL21（p ET-28a-KPgdh）和BL21（p ET-28a-ECgdh）。经IPTG诱导,聚丙烯酰胺凝胶电泳显示重组菌实现了葡萄糖脱氢酶的高效表达,且葡萄糖脱氢酶活性分别是空质粒对照菌E.coli BL21（p ET-28a）的8.5倍和12倍。重组菌的生长速度快于空质粒对照菌E.coli BL21（p ET-28a）,并与原代菌E.coli BL21持平。如果扣除质粒复制造成的代谢负荷,过表达葡萄糖脱氢酶促进了大肠杆菌的生长。 相似文献
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从土壤中分离纯化获得9株酵母菌株,并从中得到一株海藻糖产量较高酵母菌株,经初步菌种鉴定属瓶形酵母属。以其作为出发株,得出了以硫酸二乙酯(DES)诱变该株的致死率曲线,诱变剂用量为1%、诱变时间为50min时,该株的致死率达到75%。在该诱变条件下,采用三氯乙酸浸提酵母菌中的海藻糖,用硫酸蒽酮法在波长为620nm测定其海藻糖的含量。突变株经初筛和复筛,得到海藻糖高产菌株,其海藻糖含量较出发株高1.6倍,海藻样含量达到525μg/mL发酵液,干重比为19%,是目前报道海藻糖在酵母中干重比的1.2倍。 相似文献
18.
Mark S. Longtine Amos Mckenzie III Douglas J. Demarini Nirav G. Shah Achim Wach Arndt Brachat Peter Philippsen John R. Pringle 《Yeast (Chichester, England)》1998,14(10):953-961
An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kanr gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae. © 1998 John Wiley & Sons, Ltd. 相似文献
20.
The sterol contents of brewing yeasts harvested from an anaerobic fermentation and inoculated into air-saturated wort rise rapidly from ca. 1 mg/g dry yeast to ca. 10 mg/g and remain at this level until the oxygen is absorbed. After all oxygen is removed from the medium, sterol synthesis ceases and the concentration in the cells declines as growth occurs until a limiting value is reached, when reproductive growth ceases. In the presence of oxygen, sterols are present primarily as sterol esters; free sterols accumulate after oxygen uptake is complete. The total quantity of sterol which is synthesized in the presence of oxygen determines the extent of subsequent yeast growth. It is shown that the magnitude of sterol synthesis in the yeast population as a whole is influenced by the pitching rate. 相似文献