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.