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, YDL070w and YDL110c), localized on the left arm of chromosome IV in Saccharomyces cerevisiae, is reported. A PCR-based strategy was used to construct disruption cassettes in which the kanMX4 dominant marker was introduced between two long flanking homology regions, homologous to the promoter and terminator sequences of the target gene (Wach et al., 1994). The disruption cassettes were used to generate homologous recombinants in two diploid strains with different genetic backgrounds (FY1679 and CEN. PK2), selecting for geneticin (G418) resistance conferred by the presence of the dominant marker kanMX4. The correctness of the cassette integration was tested by PCR. After sporulation and tetrad analysis of the heterozygous deletant diploids, geneticin-resistant haploids carrying the disrupted allele were isolated. YDL060w was shown to be an essential gene for vegetative growth. A more detailed phenotypic analysis of the non-lethal haploid deletant strains was performed, looking at cell and colony morphology, growth capability on different media at different temperatures, and ability to conjugate. Homozygous deletant diploids were also constructed and tested for sporulation. Only minor differences between parental and mutant strains were found for some deletant haploids.
Casalone
E
E
Department of Biomedical Science, University 'G. D'Annunzio' of Chieti, via dei Vestini 31, I-66100 Chieti, Italy. casalone@dbag.unifi.it
Barberio
C
C
Cavalieri
D
D
Ceccarelli
I
I
Riparbelli
M
M
Ugolini
S
S
Polsinelli
M
M
D017931:DNA Primers / Q000737:chemistry; D004271:DNA, Fungal / Q000737:chemistry; D017353:Gene Deletion; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic
D017931:DNA Primers; D004271:DNA, Fungal