Deletions at intervening sequence splice sites in the alcohol dehydrogenase gene of Drosophila

Abstract

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.