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.