Worm Breeder's Gazette 14(3): 53 (June 1, 1996)
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.
University of Washington, Seattle, WA 98195
Two semi-dominant gain-of-function mutations in egl-2, n693 and n2656, cause Egl and Exp phenotypes due to the inability of the animals to contract their egg-laying and enteric muscles. In addition, these phenotypes can be rescued by tricyclic antidepressants such as imipramine. We are interested in learning how egl-2 regulates muscle excitation and the nature of it's interaction with tricyclics. In order to isolate additional alleles of egl-2, we mutagenized n693 and n2656 hermaphrodites and screened in the F2 generation for non-Exp animals. 12 EMS and 3 mutator revertants of n693, and 17 EMS revertants of n2656 were recovered. All alleles were inseparable from egl-2, suggesting that these alleles are intragenic revertants. In addition, the Egl phenotype co-reverted with the Exp phenotype in all cases, indicating that both phenotypes were due to the same mutation. The phenotype of all revertants was grossly wild type. In order to characterize the new alleles further, we took advantage of the semi-dominance of the egl-2 gain-of-function mutations. We measured percent enteric muscle contraction (EMC) during the defecation motor programs of various allele combinations. Wild type has 100 percent EMC, while egl-2(d) homozygotes are completely defective (0 percent EMC). egl-2(d)/Df is slightly less severe, and a wild-type copy of egl-2 in trans to a dominant allele further relieves the defect. These last two observations reveal the semi-dominance of n693 and n2656 and suggests that wild-type copies of egl-2 can interfere with the expression of the dominant phenotype. To characterize the new alleles, we put them in trans to the egl-2(d) allele from which they were generated and measured percent EMC. The new alleles of egl-2 fell into three general classes. Class I alleles (18 alleles) behaved like a deficiency of the locus, suggesting that they probably represent loss-of-function or null mutations. Class II alleles (6 alleles) behaved like a wild-type copy of egl-2. It is possible that these alleles are true revertants back to the wild-type sequence, but we think that this is unlikely given the frequency with which they were recovered. Alternatively, the Class II mutations are acting in cis to compensate for the dominant mutations or the gene product is suppressing to wild-type levels in trans. The Class III alleles (8 alleles) relieved the EMC defect of the dominant alleles to an even greater extent than a wild-type copy of egl-2. The fact that the Class III alleles can dominantly suppress the dominant mutations in trans suggests that the two gene products physically interact and that the egl-2 protein functions as a homomeric complex. If this were the case, the dominant suppression might be expected to be allele-specific. In fact, all Class III alleles can suppress n693 and n2656 equally well. One explanation for this is that n693 and n2656 have identical mutations in the egl-2 gene or that the mutations are functionally identical. Alternatively, the Class III alleles may suppress the dominant mutations via a more general mechanism. We have cloned and are sequencing the egl-2 gene. We plan to sequence the two gain-of-function alleles and members of each revertant class. This should provide valuble information about the structure and function of the egl-2 gene product and how multiple subunits might interact. genotype percent EMC +/+ 100 egl-2(d)/egl-2(d) 0 egl-2(d)/Df 7 egl-2(d)/+ 22 egl-2(d)/Class I 0-13 egl-2(d)/Class II 17-28 egl-2(d)/Class III 37-57