Disruption of six novel ORFs on the left arm of chromosome XII reveals one gene essential for vegetative growth of Saccharomyces cerevisiae

Stephen Oliver, Nianshu Zhang, Thamir Ismail, Jian Wu, K. Cara Woodwark, David C J Gardner, Richard M. Walmsley, Stephen G. Oliver

    Research output: Contribution to journalArticlepeer-review

    Abstract

    Deletion via PCR-mediated gene replacement, together with basic functional and bioinformatic analyses, have been performed on six novel open reading-frames (ORFs) on the left arm of chromosome XII of Saccharomyces cerevisiae (YLL033w, YLL032c, YLL031c, YLL030c, YLL029w and YLL028w). ORF deletion was realized using either a short-flanking homology (SFH) or a long-flanking homology (LFH) replacement cassette in the diploid strain FY1679. Sporulation and tetrad analysis showed that YLL031c is the only essential gene of the six. Microscopic examination of the non-growing spores carrying a disrupted copy of the essential gene showed that most of them were blocked after one or two cell divisions with heterogeneous bud size. The standard EUROFAN growth tests failed to reveal any obvious phenotype resulting from the deletion of each the five non-essential ORFs. Bioinformatic analysis revealed that YLL029w is probably an aminopeptidase for mitochondrial or nuclear protein processing and YLL028w may be involved in drug resistance in S. cerevisiae. Replacement cassettes, comprising the promoter and terminator regions of each of the six ORFs, were cloned into pUG7 and demonstrated to efficiently mediate gene replacement in an alternative diploid strain, W303. All the cognate gene clones were constructed, using either PCR products amplified from genomic DNA, or gap-repair. All clones and strains generated have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt).
    Original languageEnglish
    Pages (from-to)1287-1296
    Number of pages9
    JournalYeast
    Volume15
    Issue number12
    DOIs
    Publication statusPublished - 15 Sept 1999

    Keywords

    • Chromosome XII
    • Essential facilitator gene
    • Functional genomics
    • Saccharomyces cerevisiae

    Research Beacons, Institutes and Platforms

    • Manchester Institute of Biotechnology

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