Worm Breeder's Gazette 10(3): 48
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.
For quite some time, we have been attempting to clone the cha-1 n Linkage Group IV (the structural gene for choline acetyltransferase - ChAT). We planned to use Tc1 tagging, taking advantage of the fact that cha-1 and unc-17 animals are resistant to cholinesterase inhibitors such as the pesticide aldicarb. This seemed to be a straightforward approach, but it has proved to be more difficult than we originally expected. One of the difficulties is that more than a dozen genes can mutate to give drug resistance ( see below), so that only a (small) fraction of the resistance mutations we obtained were in the cha-1 complex. Another problem is that cha-1 is an essential gene; i.e., null mutants would be lethal, and only relatively severe hypomorphs would be drug-resistant. We were therefore selecting for a relatively narrow window of residual activity, and presumably only a small fraction of transposon insertions into cha-1 would spare the requisite amount of ChAT. And when we finally did obtain viable cha-1 mutants, a significant fraction of them (including the first three we examined) were not associated with an extra copy of Tc1 (or any of the other known transposons, Tc2-Tc6). It was therefore necessary to screen a very large number of lines and animals to obtain the right mutant(s). We started with 3 strains: Bergerac (EM1002), TR638 (a mut-3 strain constructed by John Collins and Phil Anderson), and a derivative of TR403 (a wild strain isolated in Phil Anderson's lab). We set up 50-80 independent lines at a time, let them grow for approximately 3 generations, and then transferred the starved worms to plates containing aldicarb (0.3 or 0.5 mM for Bergerac, 0.75 or 1.0 mM for TR403, and 0.5 or 1.0 mM for TR638). These aldicarb plates were then scored after 7, 14, and 21 days for better-growing, better-moving animals. By the time we were done, we had examined a total of 583 independent lines representing approximately 2.8x10+E6 haploid genomes, and we had obtained 427 independent aldicarb-resistant mutants. Complementation tests indicated that 10 of the new mutations in the cha-1 complex: 4 in the cha-1 sublocus and 6 in the unc-17 sublocus. The numerical summary of these results is as follows. Bergerac: 1. 1x10+E6 total haploid genomes in 220 lines, 15 aldicarb-resistance mutations, 1 unc-17 allele. TR403: 1.1x10+E6 total haploid genomes in 217 lines, 132 aldicarb-resistance mutations, 2 unc-17 alleles. TR638: 7.3x10+E5 total haploid genomes in 146 lines, 280 aldicarb- resistance mutations, 4 cha-1 and 3 unc-17 alleles. Clearly, TR638 had the highest mutation rate of these three strains, although the number of cha-1 mutants is too low to determine if the fraction of aldicarb-resistance mutations which were in the cha-1 complex is significantly different among the three strains (i.e., 1 in 15, 2 in 132, 7 in 280). Mutations in the cha-1 complex: All of the new cha-1 and unc-17 mutants were outcrossed to Bristol 8-10 times. We also examined two Bergerac-derived cha-1 alleles isolated in Dick Russell's lab and an unc-17 allele isolated from RW7090 by Ryuji Hosono. When genomic Southern blots of these outcrossed strains were then compared to Bristol, most of the new mutants had 2-5 additional copies of Tc1. For each of these mutations, we constructed + cha-1 + / unc-33 + vab-2 heterozygotes and from them we isolated 30-35 independent Vab nonUnc- 33 recombinants. Recombination events to the left of cha-1 (i.e., between unc-33 and cha-1) generated + cha-1 mes; events to the right of cha-1 (i.e., between cha-1 and vab-2) generated + + vab-2 chromosomes. This allowed us to map each of the additional Tc1 elements with respect to cha-1. The bad news was that 8 of the mutations (5 of the ones we had isolated, plus the 2 from Pittsburgh and the one from Japan) were clearly not correlated with a novel Tc1 band. The good news is that two of our mutations, md1067 and md1143, were perfectly correlated with specific novel Tc1-containing EcoRI fragments, of approximately 3. 5 and 4.0 kb, respectively. Both of these mutations have cha-1 genetic complementation properties. In addition, a spontaneous revertant of md1067 is missing the md1067-specific Tc1 fragment. More good news is that in preliminary experiments, the remaining 3 new mutations (2 cha-1 alleles and an unc-17 allele) appear to be associated with specific Tc1 elements, but these results have not yet been confirmed. We have been taking all the obvious steps with md1067 and md1143, and we hope/expect to have the region cloned quite soon. Other aldicarb-resistant loci: We are interested in mutants resistant to cholinesterase inhibitors because we expect some of them to be defective in cholinergic transmission or its regulation. Although mutations in unc-1, unc-11, unc-18, unc-36, unc-64, f course, cha-1-unc-17) have been previously shown to confer resistance to aldicarb (Brenner, Hodgkin, Carr, Hirsh, Culotti, Petersen, Russell, Johnson, and others), no systematic analysis of the genetics of aldicarb resistance in C. elegans has yet been conducted. We have therefore been examining the 417 non-cha-1 mutations in our collection to determine which genes are represented. Approximately one half of our new mutants are severely uncoordinated and/or paralyzed, one quarter are mild-to-moderately uncoordinated, and the rest have no behavioral abnormality (or are extremely subtle in their effect). Among the strains in our collection, there does not seem to be a major correlation between the degree of resistance and the degree of behavioral impairment. All of the genetic analysis thus far has involved the uncoordinated strains, but it will be of particular interest to learn whether the resistant mutants with normal coordination map to the same genes as the uncoordinated strains, or whether they define a new set of loci. More than one third of the new mutants have been mapped to specific linkage groups, and we are now assigning them to specific genes. So far, all linkage groups except III are represented. In addition, the collection of mutants on Linkage Group I includes several distinct phenotypes, suggesting that many (or most) of the known genes on I are represented. We have also identified 2 previously undescribed genes ( with several independent isolates of each): a locus very close to dpy- 10 on II, and a locus near unc-8 on IV. In addition, we have evidence for at least 2 more new loci: one on V and one on X. It therefore appears that we will ultimately have about 20 genes which can mutate to an aldicarb-resistant phenotype.