Characterization of transposable element-associated mutations that alter yeast alcohol dehydrogenase II expression

Seven cis-dominant, constitutively expressed mutations of the normally glucose-repressible isozyme of alcohol dehydrogenase (ADHII) from the yeast Saccharomyces cerevisiae are caused by insertion of transposable elements from the Ty1 family in front of the ADHII structural gene (ADR2) (V. M. Williamson, E. T. Young, and M. Ciriacy, Cell 23:605-614, 1981). We cloned ADR2 with its associated Ty1 element from five S. cerevisiae strains carrying these mutations. Comparison of the Ty1 elements by heteroduplex studies and restriction enzyme analyses indicated that four were very similar; the fifth, although the same size as the others (about 5.6 kilobases), differed by the presence of two large substitutions of approximately 1 and 2 kilobases. The DNA sequences of the terminal direct repeats (deltas) were very homologous but not identical and were similar to previously reported Ty1 element direct repeats. We determined the 5'-flanking sequences of the ADR2 gene isolated from a wild-type strain and from five Ty1-associated mutations. The 5-base pair target sequence at the site of Ty1 insertion was present at both ends of each Ty1 element. The sites of insertion of the elements were all different and occurred from 125 to 210 base pairs in front of the coding region of ADR2. The 5' end of the major transcript as determined by S1 mapping was the same in wild-type cells and in Ty1-associated constitutive mutants and was approximately 54 base pairs upstream from the coding region. ADR2 transcripts were not detected when a solo delta sequence was present in the 5'-flanking region of this gene.