Worm Breeder's Gazette 11(2): 47
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 receptor for the potent anthelmithic drug levamisole is the cholinergic receptor on the body wall muscle (Lewis et al., Mol. Pharmacol. 31:185-193 (1987)). This may be the only cholinergic receptor found there (Lewis et al., Neuroscience 5:967-989 (1980)). Only five genes can mutate to Lev(r) at forward mutation rates: unc-29 I, unc-38 I, unc-74 I, unc-63 I, and lev-1 IV (Lewis et al., Genetics 95:905-928 (1980)). Nulls of the first four are extremely resistant and strongly Unc, and all alleles are recessive. Nulls of lev-1 are only slightly resistant and very slightly Unc, and recessive. But lev- 1 is unique among these five for also having a very rare class of semidominant mutant (semidominant for resistance, recessive for Unc) that are as resistant and Unc as the others when homozygous (Lewis et al., Genetics 95:905-928 (1980)). unc-29 and unc-38 encode subunits of the nematode neuromuscular acetylcholine receptor (Lewis, WBG 10(1) :130-131). From its genetic and pharmacological behavior, lev-1 was not expected to encode a subunit (Lewis et al., J. Neurosci. 7:3059- 3071 (1987)); something like a receptor-specific kinase seemed more likely. We have now shown that it does indeed encode a subunit, which makes its genetics and kinetics much more intriguing. We were interested in cloning the lev-1 gene in its own right and also to start a walk towards tra-3, which lies to the right (Bames, Meeting Abstracts 1989). Several useful alleles of lev-1 had been created for this purpose (Lewis, WBG 9(1):35-36). These were 5 TR679 alleles, 2 spontaneous RW7000 alleles and 4 gamma-ray alleles. Of several strains examined in detail, one of the TR679 alleles, x548, had a novel Tc1 that was inseparable from lev-1 after backcrossing against N2 and recombining separately with unc-30 on the left and dpy-4 on the right. At this time, a better map location derived for lev-1 placed it much further to the left than previously thought, near unc-26, and a re-examination of the data showed that lev-1 had not actually ever been ordered with respect to unc-26. Consistent with this, a strain carrying Bergerac sequences around tra-3 but no further to the left than lev-1, retained Bergerac DNA as far left as eP88 (see the physical map). Lurking in the physical interval where lev-1 had to lie (and to the left of unc-26) was acr-1, an acetylcholine receptor homologue isolated by John Fleming and sequenced by Mike Squire (MRC Molecular Neurobiology Unit, Cambridge). Probing a Southern of the TR679, gamma-ray and RW7000 alleles, plus 4 EMS alleles (2 gf, 2 lf), with the phage containing the gene revealed rearrangements in 3 out of 4 gamma-ray alleles and 4 out of 5 TR679 alleles, and nothing in the others. All rearrangements affected a 4.5kb HindIII fragment, which contains 97% of the gene (M.Squire and J. Fleming, pers. comm.). Two of the 4 affected TR679 alleles had a band larger by about 1.6kb than the 4.5kb WT fragment, and one of these was x548. The other had not been looked at. Hence lev-1 encodes an essentially dispensable subunit of the AChR, which subunit is uniquely mutable to a semidominant form. Apart from wondering which subunit substitutes for lev-1 in the nulls, one may speculate about why x21 and x61 exhibit dominance. Recall that x211+ is non-Unc, but partially Lev(r), and x211x21 is Unc and fully resistant. There are three possibilities: 1) An odd kind of haplo-insufficiency, i.e. half the complement of wild-type receptors is insufficient to fully depolarize the muscle in the presence of an excess of levamisole, but under normal physiological conditions they are sufficient. This seems unlikely; you'd expect it to be the other way around. Rejecting this idea leaves only ideas where lev-1(d) gets incorporated into more than its fair share of receptors (i.e. >50%) : 2)There are two or more lev-1 subunits per receptor, and having only one gf molecule in it is enough to jam it up. This also seems unlikely, as the subunit is essentially dispensable. 3)A thermodynamic/assembly thing, with one subunit per receptor. The binding studies of the gf homozygote suggest the gf receptor is locked into a high affinity and inactive (i.e. desensitized) state. By definition, this would be the conformation with the lowest free energy. In vertebrates, assembly is a lugubrious process where two- thirds of newly synthesized subunits are turned over before they can make it into receptors. Mutant subunits may therefore associate sooner with nascent receptors and thus avoid destruction; alternatively, they may simply be resistant to degradation. Knowing the lesion in the dominants will clearly be informative. NOTE:Any lev-1(x21) 05) chromosomes out there are highly suspect. They are probably unc(e2477) lev-1(x21) 33) chromosomes, unless you made them yourselves. e2477 is near lin-1 on IV, and complements unc-33 and unc-24. We haven't yet checked unc-17, but it doesn't look like e245.