Worm Breeder's Gazette 11(4): 104

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deg-1 Suppressors

Eve Wolinsky

Figure 1

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]

Figure 1