MOLECULAR ANALYSIS OF SACCHAROMYCES-CEREVISIAE CHROMOSOME-I - ON THE NUMBER OF GENES AND THE IDENTIFICATION OF ESSENTIAL GENES USING TEMPERATURE-SENSITIVE-LETHAL MUTATIONS

被引:31
作者
HARRIS, SD [1 ]
CHENG, J [1 ]
PUGH, TA [1 ]
PRINGLE, JR [1 ]
机构
[1] UNIV MICHIGAN,DEPT BIOL,ANN ARBOR,MI 48109
关键词
SACCHAROMYCES-CEREVISIAE; CHROMOSOME; MUTAGENESIS; TEMPERATURE-SENSITIVE MUTANTS; GENE-NUMBER PARADOX;
D O I
10.1016/0022-2836(92)91025-K
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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. © 1992.
引用
收藏
页码:53 / 65
页数:13
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