Worm Breeder's Gazette 13(4): 81 (October 1, 1994)
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.
New York University Medical Center, Department of Biochemistry, 550 First
Avenue, New York, New York 10016
We have recently identified unc-8 as a member of the deg-1 gene
family by genetic and phenotypic criteria, including the ability to
dominantly mutate to cause motorneuron swelling (WBG 13(3):77). deg-1,
mec-4, and mec-10 have been previously shown to be members of a gene
family with sequence and functional homology to the mammalian
amiloride-sensitive sodium channel. The strong visible phenotype of unc-8
makes it practical to screen for novel interacting genes and to find rare
missense mutations within unc-8 itself to better understand protein
interactions and regulation of these multimeric ion channels. Null mutations
at unc-8 do not cause any obvious mutant phenotype, while dominant
mutations fall into two classes. The alleles e15 and n491 confer a fully unc
phenotype, i.e. homozygous animals coil and cannot back, while animals
homozygous for the weaker alleles, e49 and lb109, are semi-Unc. Semi-Unc
animals also tend to coil, though less severely, and can back when tapped
on the head. All four alleles are stronger in trans to null than wild
type. During reversion screening of the strong unc-8 allele, n491, we
recovered a unique intragenic mutation, n491 lb82. n491 lb82 confers the
strong Unc, non-backing phenotype in trans to unc-8(+). While n491 lb82
homozygotes are non-Unc, n491 lb82/+ is fully Unc, unlike n491/+ which
is semi-Unc. Several interallelic trans-acting suppressor mutations have been
isolated, but n491 lb82 is the only mutation found to date that requires the
presence of normal unc-8 product to produce the Unc phenotype. This
unusual "enhancer" mutation led us to think that perhaps other mutations
affecting the normal function of these channels could similarly enhance the
weaker semi-unc phenotype of e49 and lb109.
To screen for enhancers, EMS mutagenized unc-8(e49) or unc-8(lb109)
animals were screened for F1 and F2 progeny more Unc than the parental
strain, i.e. unable to back. We expect to obtain several kinds of mutants:
second site missense mutations that exacerbate the channel defect or affect
its regulation, extragenic mutations that upregulate the expression of the
parental mutant genes, mutations in other putative channel components that
further compromise its normal function, as well as other unc mutations that
cause defective backing or coiling. In addition to the visible Unc phenotype,
a true enhancer might be expected to cause vacuole formation that could be
seen by Nomarski microscopy. From approximately 60,000 genomes
screened so far, 11 candidate enhancers were picked. All candidates were
tested for their dependence on the unc-8 genotype to eliminate unc
mutations in other genes. Two of the eleven proved to be mutations in
other unc genes. Of the remainder, the majority are closely linked to unc-8
and are likely to be intragenic second site missense mutations. One
enhancer mutation appears to be extragenic and is currently being
characterized.
More than 60 unc-8 suppressor mutations have thus far been isolated.
The vast majority are intragenic putative null mutations and at least seven
are additional alleles of mec-6. However, single alleles at three new
suppressor loci have also been isolated, including a recessive X-linked
mutation and dominant suppressor mutations on LGI and LGIV. These rare
extragenic mutations are recoverable by virtue of the very high efficiency of
visual revertant screening, which is not yet saturated.