An improved integration replacement/disruption method for mutagenesis of yeast [Saccharomyces cerevisiae] essential genes

• Published in: Genes & Genetic Systems Vol. 75; no. 1; pp. 33 - 39
• Main Authors: Tohe, A. (Tokyo Univ. (Japan)), Oguchi, T
• Format: Journal Article
• Language: English
• Published: Japan The Genetics Society of Japan 01.02.2000; Japan Science and Technology Agency
• Subjects: Cell Division - genetics; CONTROL GENETICO; Deoxyribonuclease BamHI - genetics; Deoxyribonuclease BamHI - metabolism; Fungal Proteins - genetics; GAIN GENETIQUE; GENETIC CONTROL; GENETIC GAIN; Genetic Techniques; INTEGRATED CONTROL; LUCHA INTEGRADA; LUTTE GENETIQUE; LUTTE INTEGREE; MEJORA GENETICA; Membrane Proteins - genetics; MPC1 gene; MUTACION; Mutagenesis; MUTATION; Phosphotransferases (Alcohol Group Acceptor); SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae Proteins; Temperature; Yeasts - genetics; Yeasts - physiology
• ISSN: 1341-7568; 1880-5779
• DOI: 10.1266/ggs.75.33

We improved the integration replacement/disruption method (Shortle, D., Novic, P., and Botstein, D. Proc. Natl. Acad. Sci. USA 81: 4889-4893, 1984) for isolating mutants in any of essential genes of the yeast Saccharomyces cerevisiae by integrating mutagenized DNA into the wild type gene of interest. We adopted this method to isolate temperature-sensitive mutants of the MPC1 gene encoding the YLL031C ORF. To facilitate integration of the mutagenic plasmid at a site near the 5' end of the ORF, a BamHI site was created at 300 bp downstream of the 5' end of the truncated ORF to be mutagenized. The MPC1 gene was disrupted in the wild type haploid strain by integrating a 5'-truncated derivative of the gene with mutations induced by in vitro mutagenesis. Transformants thus obtained were subjected for diagnosis of conditional lethality by replica-plating onto an appropriate selection medium to detect mutants. A primary mutant isolated by this method reverted in a high frequency due to a tandem repeat created by mutagenic integration. We deviced a method to obtain a stable temperature-sensitive strain by disrupting the tandem duplication. Two stable temperature-sensitive mutants thus obtained were found to be remedial either with 1 M sorbitol or with 0.1 M Mg2+ and to be sensitive to local anestheticum, tetracaine, at 25°C.