Abstract
Expression of the choline acetyltransferase (ChAT) gene in Drosophila melanogaster is responsible for production of the neurotransmitter acetylcholine and is necessary for viability. In previous studies, we have shown that the regulatory region for normal ChAT expression is large and composed of multiple regulatory elements (Kitamoto et al., 1992; Kitamoto and Salvaterra, 1993). In this study, using various lengths of 5' flanking DNA fused to wild type ChAT cDNA, we have defined a 0.3 kilobase (kb) region of the cis-regulatory DNA, which is essential for restoring viability of Cha lethal mutants. DNase I footprinting analysis of this 0.3 kb DNA revealed a protected 22 bp sequence that contains an octamer-like motif (ATTCAAAT) with one base difference from the consensus octamer motif (ATGCAAAT). Electrophoretic mobility shift assays and Southwestern blot analysis confirmed the presence of specific binding factor(s) for the 22 bp sequence in embryo nuclear extracts, and competition studies established the importance of the octamer-like motif for high-affinity binding. Using the 22-mer as a probe, we have isolated a cDNA clone encoding the Drosophila POU homeo domain protein, dPOU-19/pdm-1, whose target genes and specific binding sequences have not been identified. We propose that vital expression of the Drosophila ChAT gene is regulated by a member of the dPOU-19/pdm-1 putative transcription factor family.
