The gene that codes for Drosophila alcohol dehydrogenase (ADH; alcohol:NAD' oxidoreductase, EC 1.1.1.1) was identified in a bacteriophage A library of genomic Drosophila DNA by using ADH cDNA cloned DNA as a probe. The DNA sequence of the protein encoding region was shown to be in agreement with the amino acid sequence of the ADH. Two intervening DNA sequences (introns) were identified within the protein encoding region: one was 65 nucleotides and located between the codons for amino acid residues 32 and 33, and one was 70 nucleotides and located between the codons for amino acid residues 167 and 168. Both contained the 5' G-T and 3' A-G dinucleotides characteristic of intron boundaries of eukaryotic genes. On the basis of secondary structure predictions, the first 140 amino acid residues of Drosophila ADH are in an alternating a8-sheet/a-helix arrangement which is characteristic of the coenzyme binding domain of dehydrogenases. The smaller ofthe two introns interrupts the domain predicted to bind the adenine portion ofthe coenzyme.
Alcohol dehydrogenase (ADH) activity inDrosophila larvae and adults is localized primarily in fat body, intestine, and Malpighian tubules. In adult males, it occurs furthermore in derivatives of the genital disk.When expressed as ADH activity/whole organism, increasing values are found throughout the larval stages. Around the time of puparium formation, the activity decreases, to increase again around hatching of the adult.As the genital disk undergoes metamorphosis, it synthesizes (or activates) ADH rather than acquiring the enzyme from elsewhere in the organism.
Two formaldehyde-induced, homozygous viable ADH-negative mutants, Adhfn4 and Adhfn6, possess no material that cross-reacts with antibody directed against ADH, no mature mRNA of wild-type size, and greatly reduced amounts of RNA that hybridizes with an Adh probe. We have cloned the genomic DNA sequences from these mutants in bacteriophage lambda Charon 4 and subcloned the Adh region into plasmid vector pBR327. Restriction analyses revealed one small deletion in each of these mutants and DNA sequencing showed that the splice junctions of the 65-base pair (bp) intervening sequence (IVS) were altered. Both cloned mutant Adh genes, as well as the wild-type gene, are capable of promoting correct specific transcription initiation in HeLa cell nuclear extracts in vitro. We conclude that Adhfn4 and Adhfn6 are defective in RNA processing. Our results provide evidence for the importance of the splice junction sequences in normal ADH RNA processing and stabilization in Drosophila. We also speculate that splicing of ADH RNA proceeds in a nonrandom manner: mutations in one of the intervening sequences appear to cause accumulation of a large ADH RNA containing at least one other IVS.
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