Extragenic suppressors: I am seeking extragenic suppressors of
deg-1(
u38) by looking for tail touch sensitive revertants of a high-copy number, stable transformant strain. This strain (TU1191, Nature 345:410) arose after microinjection of a mixture of
deg-1 dominant mutant
u38 DNA and
unc-22 antisense DNA into wild type oocytes. Genetically, the Deg and Twi phenotypes of this strain show X-linkage. By Southern blot analysis, at least ten intact copies of the
u38 allele are present. The null phenotype of
deg-1 is wild type, and after EMS mutagenesis of
u38 animals, touch sensitive, intragenic revertants are readily isolated at the expected frequency for gene knock-out events. It is therefore tedious to look for rare extragenic reversion events against the background of higher frequency intragenic events. After mutagenesis of TU1191, however, touch sensitive progeny occur much less frequently. I chose to work with this strain because intragenic reversion in only one of the multiple copies of the
u38 transforming DNA would be unlikely to restore tail touch sensitivity. The Deg phenotype of TU1191 is in fact partially suppressed by
mec-6 mutations, thus I expect to be able to identify mutations in other genes which act as suppressors of
u38. Unfortunately, EMS mutagenesis seems to stimulate excision of the transforming DNA, perhaps as a result of activation of DNA repair systems. Several touch sensitive revertants have been obtained which no longer express the Twi phenotype, and which fail to segregate the Deg phenotype when outcrossed. One candidate so far meets the criteria of tail touch sensitivity, and co-segregation of both the Twi and Deg phenotypes after outcrossing. Intragenic revertants: Most pseudo-wild type revertants of
u38 behave like null alleles. I isolated one exceptional revertant,
u38 u424, which confers a wild type phenotype to homozygotes, but which also acts as a semi-dominant suppressor of the original
u38 mutation in trans. As shown in the graph, suppression is cold-sensitive ( animals were scored as gravid adults). Because the
u38 and
u38 u424 alleles both show dominant effects, I compared their DNA sequences around the region encoding the amino acid changes that Monica Driscoll and Martin Chalfie reported (E. Coast Worm Mtg.) constitute the dominant
mec-4 mutations. They also reported that
mec-4 and
deg-1 predicted protein sequences are highly homologous.
mec-4 dominant alleles and
deg-1(
u38) affect different neurons, but in each case affected cells undergo vacuolar degenerations which appear to be similar events. A comparison of the wild-type,
u38, and
u38 u424 sequences is shown below. In fact,
u38 contains the same ala->val change described for
mec-4(d). The double mutant
u38 u424 retains this change, and an additional mutation, gly->arg is present nearby. The ala->val mutation has little effect on computer predictions of protein structure. The gly->arg change, however, reduces the hydrophobicity of the possible membrane spanning domain and removes a predicted turn in the peptide backbone. The dominance of the suppressor mutation suggests that the double mutant protein may interact with
u38 molecules in a way that inactivates their cell killing activity, for example by disrupting complexes of
deg-1 proteins. The gly->arg mutation is likely to change the shape of the protein at the junction between the extracellular space and the membrane, or even prevent its proper insertion into the membrane. Either effect might prevent association of
deg-1 molecules. [See Figure 1]