Inactivation of six genes from chromosomes VII and XIV ofSaccharomyces cerevisiae and basic phenotypic analysis of the mutant strains

✍ Scribed by Sartori, Geppo; Mazzotta, Gabriella; Stocchetto, Silvia; Pavanello, Anna; Carignani, Giovanna

• Publisher: John Wiley and Sons
• Year: 2000
• Tongue: English
• Weight: 176 KB
• Volume: 16
• Category: Article
• ISSN: 0749-503X
• DOI: 10.1002/(sici)1097-0061(200002)16:3<255::aid-yea520>3.0.co;2-#

✦ Synopsis

Within the frame of the EUROFAN project, aimed at the functional analysis of the novel ORFs revealed by the systematic sequencing of the Saccharomyces cerevisiae genome, we have inactivated six ORFs encoding putative proteins with unknown function in the two S. cerevisiae strains FY1679 and W303-1B. Five ORFs are located on chromosome VII (YGR250c, YGR251w, YGR260w, YGR262c, YGR263c) and one on chromosome XIV (YNL234w). The genes have been inactivated in the FY1679 strain by a strategy that makes use of deletion cassettes containing the kanMX4 module, which confers resistance to geneticin to yeast cells, and short ¯anking regions homologous to the target locus (SFH). Tetrad dissection of heterozygous mutants and basic phenotypic analysis of the spores revealed that ORF YGR251w is an essential gene, while the disruption of YGR262c causes a severe slowgrowth phenotype. Deletion of the remaining ORFs did not give rise to a detectable phenotype in the mutant strains.

For each ORF we have cloned, in the pUG7 plasmid, a replacement cassette that possesses long ¯anking regions homologous to the target locus (LFH) and, in the pRS416 plasmid, the cognate wild-type gene. The LFH replacement cassettes were used to inactivate the respective genes in the W303-1B strain.