Molecular analysis of Saccharomyces cerevisiae chromosome I. On the number of genes and the identification of essential genes using temperature-sensitive-lethal mutations.
Previous analyses of Saccharomyces cerevisiae chromosome I have suggested that the majority (greater than 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.
Harris
S D
SD
Department of Biology, University of Michigan, Ann Arbor 48109-1048.
Cheng
J
J
Pugh
T A
TA
Pringle
J R
JR
D000595:Amino Acid Sequence; D001483:Base Sequence; D015825:Chromosomes, Fungal; D004271:DNA, Fungal; D005656:Fungal Proteins / Q000235:genetics*; D005800:Genes, Fungal; D005804:Genes, Lethal; D008969:Molecular Sequence Data; D009154:Mutation; D009693:Nucleic Acid Hybridization; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D013696:Temperature
D004271:DNA, Fungal; D005656:Fungal Proteins