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.