Isolation and characterization of the ydr1 conditional mutants in the yeast Saccharomyces cerevisiae

The human Dr1 (hDr1) gene represses transcription by interacting with TATA-binding protein and by preventing formation of transcription preinitiation complexes. The YDR1 gene of the yeast Saccharomyces cerevisiae, which is the counterpart of hDr1, is an essential gene for viability and its overexpression is deleterious to the cells. Nine ydr1 mutants were isolated either by error-prone PCR or by hydroxylamine mutagenesis followed by plasmid shuffle. Four ydr1 mutants were FOA-sensitive. suggesting that the ydr1 alleles are not functional. The other five ydr1 mutants were conditional mutants at 16 degrees C or 37 degrees C. DNA sequence analysis revealed the nature of the mutations. Three of the four FOA-sensitive ydr1 strains resulted from nonsense mutations, which are R34 (ydr1-4), S50 (ydr1-5), and R118 (ydr1-2). Among conditional mutants the ydr1-7 mutant (L48P) showed cs at 16 degrees C and the other four ydr1 alleles showed ts at 37 degrees C (E35K, ydr1-3; S40F, ydr1-1; E67L, ydr1-6; Q96L, ydr1-8). The five base substitutions were positioned within the histone-fold motif in the YDR1 gene, suggesting that the histone-fold motif is important for the Ydr1 function. The ydr1 alleles obtained in this project will be useful to generate revertants to uncover suppressor genes involved in transcription.