Mutations in the smg genes (smg-l through
smg-6) act as allele specific suppressors of mutations in a number of genes (Hodgkin et. al, 1990). The smg gene products are involved in the degradation of aberrant mRNA species (Pulak & Anderson, these abstracts). In the hopes of understanding this process in molecular detail, we are attempting to clone
smg-3. Transposon tagging of
smg-3. We (R. Pulak and B. Cali)have isolated three spontaneous
smg-3 alleles in a strain that contains
mut2(r459) and is, therefore, highly active for transposable elements. One of these alleles,
smg-3(
r929), is unstable, and we have isolated 3 independent
smg-3(+) revertants. Southern blot analysis identifies a 2.3 kb Tc1-containing EcoRI fragment that is present in the unstable mutant (in several independently backcrossed lines), but is missing in the single revertant analyzed thus far. This element has not been separated from
smg-3 by recombination with flanking markers, placing it less than 0.3 m.u. Ieft or right of
smg-3. We are in the process of cloning this element, because it either causes the
smg-3 mutation or is tightly linked to
smg-3. Probable null phenotype for
smg-3. The genetic properties of smg mutants suggest that the existing alleles are loss-of-function mutations (Hodgkin et. al, 1989). However, the genetic arguments are more persuasive for some smg genes than others. Of the 54 EMS-induced smg alleles originally isolated, only four were
smg-3 mutants. This relatively low number of alleles suggested that the loss-of-function phenotype may be more extreme than the phenotypes originally described. We undertook a non- complementation screen to address this question more directly. Using suppression of
unc-54(
r293) as a test for new
smg-3 alleles, we isolated three new EMS-induced alleles of
smg-3 among 9400 haploid genomes screened, indicating a forward mutation frequency of about 3 X 10-4. This frequency is similar to that of average-sized C. elegans genes. Of the three non-complementing alleles, two are strong alleles [based on their phenotypic suppression of
unc-54 (
r293)] and one is a weak allele. Both strong alleles are viable as homozygotes and phenotypically indistinguishable from previously isolated
smg-3 alleles (i.e.- hermaphrodites have protruding vulvas and males are Mab) . These data support the conclusion that the loss-of-function phenotype for
smg3 is as previously described.