For a number of years, we have been interested in
cha-1, the structural gene encoding choline acetyltransferase (ChAT), the enzyme which synthesizes acetylcholine. We have been able to clone the gene by transposon tagging, taking advantage of the fact that
cha-1 mutants are resistant to cholinesterase inhibitors such as the pesticide aldicarb. In a previous Gazette (WBG 10:3, p.48), we described the isolation, from Tc1-active strains, of more than 400 spontaneous aldicarb-resistant mutations, 4 of which were in
cha-1. All 4 have turned out to be associated with Tc1 insertions, and we have now cloned and analyzed an 8.5 kb genomic region which apparently contains all of
cha-1 as well as a small part of adjacent
unc-17 sequences. The region identifies DNA polymorphisms associated with 7 independent
cha-1 mutations: the 4 Tc1 insertions, 1 non-Tc1 insertion, and 2 deletions. It also identifies 3
unc-17-associated small insertions. Several of these mutations have been mapped genetically, which permits us to align the genetic and physical maps of the region. We now have genomic sequence data for approximately half of the cha- 1 region. We have also obtained complete sequence information for a nearly full-length cDNA isolated from Stuart Kim's library. This 2.2 kb cDNA is derived entirely from the
cha-1 region, and contains a complete open reading frame encoding a deduced protein of 627 amino acids with a molecular weight of 71.5 kd, in excellent agreement with our measured 71 kd for the ChAT protein. The amino acid sequence shows 37% identity (58% similarity) with the porcine ChAT sequence, and 28% identity (50% similarity) with Drosophila ChAT. (The Drosophila and porcine sequences are 32% identical and 51% similar.) There are some regions which are highly conserved, e.g. a 25-amino acid stretch of the C. elegans deduced protein is 80% identical (88% similar) to the porcine sequence. Using this cDNA as a probe, we have detected and are now characterizing a low abundance mRNA approximately 2 kb in size. We are now characterizing the
unc-17 region. We have cloned 18 kb of genomic DNA adjacent to
cha-1 which should include all of
unc-17, and we have recently isolated and are now analyzing 26 additional cDNAs (from the Barstead-Waterston library) hybridizing to
cha-1 and/or
unc-17 genomic sequences. We hope that we will soon understand the transcription patterns from the entire region.