Worm Breeder's Gazette 9(3): 115
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
The neurotransmitter acetylcholine is synthesized by choline acetyltransferase (ChAT), and is hydrolyzed by acetylcholinesterase ( AChE); cha-1 is the structural gene for ChAT, and ace-1, presumed structural genes for AChE classes A, B, and C, respectively. Although neither ace-1, s lead to any major developmental aberrations singly or in pairwise combination, the ace-1 ace-3 triple mutant is lethal. Such Ace-lethal animals apparently undergo near-normal embryonic development, and many of them eventually hatch, but they are nearly paralyzed and they do not develop any further. In order to determine if mutations in cha-1 could suppress the Ace-lethality, we constructed a strain homozygous for ace-1, heterozygous for ace- 3 over the balancer mnC1. This strain was stable, and segregated one- quarter quadruple mutants, i.e., ace-1 ace-3 tes. Some (5-50%) of these quadruple mutants grew to maturity, and it was possible to establish a culture. The quadruple mutants are extremely small and hypercontracted, and can barely move. (In other words, we have constructed a four-gene synthetic dumpy.) They resemble wild-type animals treated with the AChE inhibitors aldicarb or lannate. They grow very slowly, sometimes requiring three weeks before they lay any eggs, and, in each generation, only a fraction of the worms which hatch reach maturity. Nevertheless, they are a dramatic improvement over the ace-1 ace-3 triple mutant. Because of our evidence that cha-1 and unc-17 are part of the same complex gene (Rand and Russell, Genetics 106:227, 1984), we tried to suppress the Ace-lethal phenotype with several different unc-17 as well as cha-1 alleles. We were able to establish viable quadruple mutant cultures using the cha-1 alleles p1152 and p1186, and the unc- 17 alleles e245, p279, and p300. However, we were not able to establish quadruple mutant strains using the lethal cha-1 allele m324 ( isolated by Teresa Rogalski) or the extreme unc-17 allele p1160. It thus appears that suppression of the Ace-lethal phenotype requires some residual level of functional ChAT activity. In one sense, it seems reasonable that a defect in acetylcholine synthesis can partially compensate for lethality due to a deficiency in acetylcholine hydrolysis. The viability of quadruple mutant strains argues that the Ace-lethal phenotype is due to the toxic accumulation of acetylcholine in these animals, rather than loss of the AChE proteins themselves. The result that unc-17 and cha-1 mutations both suppress the Ace-lethal phenotype supports our model that they are part of the same gene, although we have not yet investigated whether other unc mutations might also act as suppressors. In fact, it is possible that other mutations which interfere with acetylcholine synthesis or release might also suppress Ace-lethality.Since the quadruple mutants lack AChE classes A, B, and C, they should prove useful in the study of minor AChE forms, such as class D enzyme reported by Stern and Russell (Neurosci. Abstr. 11:370, 1985). Mutagenesis of quadruple mutants has led to the isolation of significantly better-growing variants; the nature of the additional mutation(s) in these stocks responsible for the improved growth has not yet been analyzed.