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

Within the frame of the EUROFAN project, aimed at the functional analysis o f 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, YGR262 c, YGR263c) and one on chromosome XIV (YNL234w). The genes have been inacti vated in the FY1679 strain by a strategy that makes use of deletion cassett es containing the kanMX4 module, which confers resistance to geneticin to y east cells, and short flanking regions homologous to the target locus (SFH) . Tetrad dissection of heterozygous mutants and basic phenotypic analysis o f the spores revealed that ORF YGR251w is an essential gene, while the disr uption of YGR262c causes a severe slow-growth phenotype. Deletion of the re maining ORFs did not give rise to a detectable phenotype in the mutant stra ins. For each ORF we have cloned, in the pUG7 plasmid, a replacement casset te that possesses long flanking regions homologous to the target locus (LFH ) and, in the pRS416 plasmid. the cognate wild-type gene. The LFH replaceme nt cassettes were used to inactivate the respective genes in the W303-1B st rain. This work has been performed in the framework of the B0 Consortium of the EUROFAN I project. Copyright (C) 2000 John Wiley & Sons, Ltd.