Disruption and phenotypic analysis of six novel genes from chromosome IV of Saccharomyces cerevisiae reveal YDL060w as an essential gene for vegetative growth

The disruption of six novel genes (YDL059c, YDL060w, YDL063c, YDL065c, YDL0 70w and YDL110c), localized on the left arm of chromosome IV in Saccharomyc es cerevisiae, is reported. A PCR-based strategy was used to construct disr uption cassettes in which the kanMX4 dominant marker was introduced between two long flanking homology regions, homologous to the promoter and termina tor sequences of the target gene (Wach er nl., 1994). The disruption casset tes were used to generate homologous recombinants in two diploid strains wi th different genetic backgrounds (FY1679 and CEN.PK2), selecting for geneti cin (G418) resistance conferred by the presence of the dominant marker kanM X4. The correctness of the cassette integration was tested by PCR. After sp orulation and tetrad analysis of the heterozygous deletant diploids, geneti cin-resistant haploids carrying the disrupted allele were isolated. YDL060w was shown to be an essential gene for vegetative growth. A more detailed p henotypic analysis of the non-lethal haploid deletant strains was performed , looking at cell and colony morphology, growth capability on different med ia at different temperatures, and ability to conjugate. Homozygous deletant diploids were also constructed and tested for sporulation. Only minor diff erences between parental and mutant strains were found for some deletant ha ploids. Copyright (C) 1999 John Wiley & Sons, Ltd.