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