Disruption of six novel genes from the left arm of chromosome XV ofSaccharomyces cerevisiae and basic phenotypic analysis of the generated mutants

✍ Scribed by Séron, Karin; Blondel, Marie-Odile; Haguenauer-Tsapis, Rosine

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 242 KB
• Volume: 15
• Category: Article
• ISSN: 0749-503X
• DOI: 10.1002/(sici)1097-0061(19990115)15:1<73::aid-yea341>3.0.co;2-q

✦ Synopsis

Six open reading frames (ORFs) of unknown function from the left arm of Saccharomyces cerevisiae chromosome XV were deleted in two genetic backgrounds by disruption cassettes with long flanking homology (LFH) , within the frame of the research project EUROFAN. The LFH disruption cassettes, obtained by PCR, were made by introducing the kanMX4 marker module between two fragments homologous to the promoter and terminator regions of a given ORF. Transformants resistant to geneticin (G418) were selected. The LFH disruption cassettes were cloned in a bacterial vector. Each cognate gene was also cloned in a centromeric plasmid. Correct deletion of each gene was verified by four different PCR reactions. Sporulation and tetrad analysis of heterozygous deletants revealed that ORF YOL102c is essential. The non-growing haploid spores gave rise to microcolonies. Basic phenotypic analyses were performed on haploid deletants of both mating types of the five non-essential ORFs, YOL018c, YOL098c, YOL101c, YOL104c and YOL105c. Plate growth tests on different media at 15 C, 30 C or 37 C did not reveal any significant differences between parental and mutant cells. Mating and sporulation efficiencies were not affected in any of the viable disruptants as compared to wild-type cells.