Molecular analysis of Saccharomyces cerevisiae chromosome I. On the number of genes and the identification of essential genes using temperature-sensitive-lethal mutations

Steven D. Harris, Judy Cheng, Tom A. Pugh, John R. Pringle

Research output: Contribution to journalArticle

30 Scopus citations

Abstract

Previous analyses of Saccharomyces cerevisiae chromosome I have suggested that the majority (>75%) of single-copy essential genes on this chromosome are difficult or impossible to identify using temperature-sensitive (Ts-) lethal mutations. To investigate whether this situation reflects intrinsic difficulties in generating temperature-sensitive proteins or constraints on mutagenesis in yeast, we subjected three cloned essential genes from chromosome I to mutagenesis in an Escherichia coli mutator strain and screened for Ts- lethal mutations in yeast using the "plasmid-shuffle" technique. We failed to obtain Ts- lethal mutations in two of the genes (FUN12 and FUN20), while the third gene yielded such mutations, but only at a low frequency. DNA sequence analysis of these mutant alleles and of the corresponding wild-type region revealed that each mutation was a single substitution not in the previously identified gene FUN19, but in the adjacent, newly identified essential gene FUN53. FUN19 itself proved to be non-essential. These results suggest that many essential proteins encoded by genes on chromosome I cannot be rendered thermolabile by single mutations. However, the results obtained with FUN53 suggest that there may also be significant constraints on mutagenesis in yeast. The 5046 base-pair interval sequenced contains the complete FUN19, FUN53 and FUN20 coding regions, as well as a portion of the adjacent non-essential FUN21 coding region. In all, 68 to 75% of this interval is open reading frame. None of the four predicted products shows significant homologies to known proteins in the available databases.

Original languageEnglish (US)
Pages (from-to)53-65
Number of pages13
JournalJournal of Molecular Biology
Volume225
Issue number1
DOIs
StatePublished - May 5 1992

Keywords

  • Saccharomyces cerevisiae
  • chromosome
  • gene-number paradox
  • mutagenesis
  • temperature-sensitive mutants

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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