Disruption of six open reading frames on chromosome X of Saccharomyces cerevisiae reveals a cluster of four essential genes

In this study we report the construction and basic phenotypic analysis of s ix Saccharomyces cerevisiae deletion mutants. The open reading frames (ORFs ) YJL008C (gene symbol CCT8), YJL010C, YJL011C, YJL012C, YJL017W, and YJL02 0C from chromosome X have been disrupted by integration of deletion cassett es, comprising the bacterial KanMX4 marker gene and terminal long (LFH) or short (SFH) flanking sequences that are homologous to the 5' and 3' untrans lated regions of the respective ORFs. For correct disruption of ORF YJL008C , it was necessary to construct a deletion cassette flanked by 300-350 bp l ong target guide sequences by LFH-PCR. Transformations using ORF YJL008C ge ne disruption cassettes synthesized by standard SFH-PCR exclusively resulte d in false-positive or multiple integration events, probably because seven additional genes homologous to CCT8 exist in the yeast genome. The other fi ve ORFs have been disrupted using cassettes generated by SFH-PCR, comprisin g terminal homologous regions of approximately 50 bp to each target site. C orrect genomic integration of the reporter modules was verified by analytic al PCR and Southern hybridization. Deletion of YJL008C, YJL010C, YJL011C, a nd YJL012C was found to be lethal, as shown by sporulation and tetrad analy sis. This result is in contrast to the finding that only 16-20% of the gene s in S. cerevisiae are estimated to be essential. The four essential genes described in this work are clustered, while the two other non-essential ORF s are separated by further ORFs. Although the two viable deletion mutants w ere tested against 60 different inhibitors, heavy metal ions and salts, no phenotype could be detected that co-segregated with the deletion during mei osis. Copyright (C) 1999 John Wiley & Sons, Ltd.