Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene.

Abstract

The alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) gene (Adh) of D. melanogaster was isolated by utilizing a mutant strain in which the Adh locus is deleted. Adult RNA from wild-type flies was enriched in ADH sequences by gel electrophoresis and then used to prepare labeled c[complementary]DNA for screening a bacteriophage .lambda. library of genomic Drosophila DNA. Of the clones that hybridized in the initial screen, one clone was identified that hybridized with labeled cDNA prepared from a wild-type Drosophila strain but did not hybridize with cDNA prepared from an Adh deletion strain. This clone was shown to contain ADH structural gene sequences by 3 criteria: in situ hybridization, in vitro translation of mRNA selected by hybridization to the cloned DNA, and comparison of the ADH protein sequence with a nucleotide sequence derived from the cloned DNA. Comparison of the restriction site maps from clones of 3 different wild-type Drosophila strains revealed the presence of a 200-nucleotide sequence in 1 strain that was absent from the other 2 strains. The ADH mRNA sequences were located within the cloned DNA by hybridization mapping experiments. Two intervening sequences were identified within Adh by S1 nuclease mapping experiments.