Worm Breeder's Gazette 11(4): 26

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Cloning and Sequencing of cha-1

Aixa Alfonso, Kiely Grundahl and Jim Rand

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.