Molecular cloning of SNM1, a yeast gene responsible for a specific step in the repair of cross-linked DNA.
We have isolated yeast gene SNM1 via complementation of sensitivity towards bi- and tri-functional alkylating agents in haploid and diploid yeast DNA repair-deficient snm1-1 mutants. Four independent clones of plasmid DNA containing the SNM1 locus were isolated after transformation with a YEp24-based yeast gene bank. Subcloned SNM1-containing DNA showed (i) complementation of the repair-deficiency phenotype caused by either one of the two different mutant alleles snm1-1 and snm1-2ts; (ii) complementation in haploid and diploid yeast snm1-1 mutants by either single or multiple copies of the SNM1 locus; and (iii) that the SNM1 gene is at most 2.4 kb in size. Expression of SNM1 on the smallest subclone, however, was under the control of the GAL1 promotor. Gene size and direction of transcription was further verified by mutagenesis of SNM1 by Tn10-LUK transposon insertion. Five plasmids containing Tn10-LUK insertions at different sites of the SNM1-containing DNA were able to disrupt the function of genomic SNM1 after gene transplacement. Correct integration of the disrupted SNM1::Tn10-LUK at the genomic site of SNM1 was verified via tetrad analysis of the sporulated diploid obtained after mating of the SNM1::Tn10-LUK transformant to a haploid strain containing the URA3 SNM1 wild-type alleles. The size of the poly(A)+ RNA transcript of the SNM1 gene is 1.1 kb as determined by Northern analysis.
Haase
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Institut für Mikrobiologie Johann Wolfgang Goethe-Universität, Frankfurt/Main, Federal Republic of Germany.
Riehl
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Mack
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Brendel
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D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004260:DNA Repair; D004251:DNA Transposable Elements; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D009154:Mutation; D010641:Phenotype; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D004251:DNA Transposable Elements; D004271:DNA, Fungal