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.

Three classes of new egl-2 alleles may reveal structural and functional aspects of the gene product.

David Weinshenker , James H. Thomas

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

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