Identification of genes required for growth under ethanol stress using transposon mutagenesis in Saccharomyces cerevisiae

被引：75

作者：

Takahashi T. ^{[1
]}

Shimoi H. ^{[2
]}

Ito K. ^{[2
]}

机构：

[1] General Research Laboratories, Kiku-Masamune Sake Brewing Co. Ltd., Higashinada-ku, Kobe 658-0026

[2] National Research Institute of Brewing

来源：

Molecular Genetics and Genomics | 2001年 / 265卷 / 6期

关键词：

Ethanol sensitivity; Saccharomyces cerevisiae; Transposon mutagenesis;

D O I：

10.1007/s004380100510

中图分类号：

学科分类号：

摘要：

The yeast Saccharomyces cerevisiae exhibits high ethanol tolerance compared with other microorganisms. The mechanism of ethanol tolerance in yeast is thought to be regulated by many genes. To identify some of these genes, we screened for ethanol-sensitive S. cerevisiae strains among a collection of mutants obtained using transposon mutagenesis. Five ethanol-sensitive (ets) mutants were isolated from approximately 7000 mutants created by transforming yeast cells with a transposon (mTn-lacZ/LEU2)-mutagenized genomic library. Although these mutants grew normally in a rich medium, they could not grow in the same medium containing 6% ethanol. Sequence analysis of the ets mutants revealed that the transposon was inserted in the coding regions of BEM2, PAT1, ROM2, VPS34 and ADA2. We constructed deletion mutants for these genes by a PCR-directed disruption method and confirmed that the disruptants, like the ets mutants, were ethanol sensitive. Thus, these five genes are indeed required for growth under ethanol stress. These mutants were also more sensitive than normal cells to Calcofluor white, a drug that affects cell wall architecture, and Zymolyase, a yeast lytic enzyme containing mainly β-1,3- glucanase, indicating that the integrity of the cell wall plays an important role in ethanol tolerance in S. cerevistae.

引用

页码：1112 / 1119

页数：7

共 41 条

[1] Aguilera A., Benitez T., Ethanol-sensitive mutants of Saccharomyces cerevisiae, Arch Microbiol, 143, pp. 337-344, (1986)
[2] Alexandre H., Rousseaux I., Charpentier C., Ethanol adaptation mechanisms in Saccharomyces cerevisiae, Biotechnol Appl Biochem, 20, pp. 173-183, (1993)
[3] Baudin A., Ozier-Kalogeropoulos O., Denouel A., Lacroute F., Cullin C., A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae, Nucleic Acids Res, 21, pp. 3329-3330, (1993)
[4] Beaven M.J., Charpentier C., Rose A.H., Production and tolerance of ethanol in relation to phospholipid fatty-acyl composition in Saccharomyces cerevisiae NCYC431, J Gen Microbiol, 128, pp. 1447-1455, (1982)
[5] Berger S.L., Pina B., Silverman N., Marcus G.A., Agapite J., Regier J.L., Triezenberg S.J., Guarente L., Genetic isolation of ADA2: A potential transcriptional adaptor required for function of certain acidic activation domains, Cell, 70, pp. 251-265, (1992)
[6] Burns N., Grimwade B., Ross-Macdonald P.B., Choi E.Y., Finberg K., Roeder G.S., Snyder M., Large-scale analysis of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae, Genes Dev, 8, pp. 1087-1105, (1994)
[7] Chen G.C., Zheng L., Chart C.S.M., The LIM domain-containing Dbm1 GTPase-activating protein is required for normal cellular morphogenesis in Saccharomyces cerevisiae, Mol Cell Biol, 16, pp. 1376-1390, (1996)
[8] Chun K.T., Goebl M.G., The identification of transposon-tagged mutations in essential genes that affect cell morphology in Saccharomyces cerevisiae, Genetics, 142, pp. 39-50, (1996)
[9] Cid V.J., Duran A., Del Rey F., Snyder M., Nombela C., Sanchez M., Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae, Microbiol Rev, 59, pp. 345-386, (1995)
[10] Cid V.J., Cenamor R., Sanchez M., Nombela C., A mutation in the Rho1-GAP-encoding gene BEM2 of Saccharomyces cerevisiae affects morphogenesis and cell wall functionality, Microbiology, 144, pp. 25-36, (1998)

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