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

Abstract

In this study we report the construction and basic phenotypic analysis of six Saccharomyces cerevisiae deletion mutants. The open reading frames (ORFs) YJL008C (gene symbol CCT8), YJL010C, YJL011C, YJL012C, YJL017W, and YJL020C from chromosome X have been disrupted by integration of deletion cassettes, comprising the bacterial KanMX4 marker gene and terminal long (LFH) or short (SFH) flanking sequences that are homologous to the 5' and 3' untranslated regions of the respective ORFs. For correct disruption of ORF YJL008C, it was necessary to construct a deletion cassette flanked by 300-350 bp long target guide sequences by LFH-PCR. Transformations using ORF YJL008C gene disruption cassettes synthesized by standard SFH-PCR exclusively resulted in false-positive or multiple integration events, probably because seven additional genes homologous to CCT8 exist in the yeast genome. The other five ORFs have been disrupted using cassettes generated by SFH-PCR, comprising terminal homologous regions of approximately 50 bp to each target site. Correct genomic integration of the reporter modules was verified by analytical PCR and Southern hybridization. Deletion of YJL008C, YJL010C, YJL011C, and YJL012C was found to be lethal, as shown by sporulation and tetrad analysis. This result is in contrast to the finding that only 16-20% of the genes in S. cerevisiae are estimated to be essential. The four essential genes described in this work are clustered, while the two other non-essential ORFs are separated by further ORFs. Although the two viable deletion mutants were tested against 60 different inhibitors, heavy metal ions and salts, no phenotype could be detected that co-segregated with the deletion during meiosis.