Worm Breeder's Gazette 11(3): 41

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Sequence Analysis of mec-7 Mutations

Cathy Savage and Marty Chalfie

Figure 1

mec-7 is a alpha-tubulin gene required for the production of 15-
protofilament microtubules, a unique feature of the touch cells.  54 
alleles of mec-7 have been isolated, and were originally characterized 
as strong or weak depending upon whether the touch insensitive 
phenotype in homozygotes is complete or partial.  In addition, about 
60% of the alleles are expressed dominantly or semi-dominantly.  
Genetic experiments have suggested that the strong recessive alleles 
are the result of a complete loss of gene activity, while the semi-
dominant and dominant alleles are the result of the synthesis of an 
abnormal alpha-tubulin gene product.
To analyze the relationship between the structure and function of 
mec-7, we have begun to sequence the mutations in mec-7.  We would 
like to correlate mec-7 mutant phenotypes and amino acid changes with 
available data on tubulin structure.  We hypothesize that dominant and 
semi-dominant effects are caused by mutant proteins that dimerize with 
alpha-tubulin, then disrupt polymerization.  Recessive alleles may be 
caused by defects in protein stability or alpha-tubulin binding.  We 
will test the stability and alpha-tubulin binding capacity of mec-7 
mutant proteins by Western blotting of non-denaturing protein gels.  
So far, we have identified mutations in 19 
alleles:
[See Figure 1]
We draw the following conclusions from these 
results:
1.  The localization of mutations in the coding region confirms that 
mec-7 does encode this alpha-tubulin.
2.  Loss of function alleles are recessive, because four recessive 
alleles are caused by stop mutations at amino acids 101, 279 and 280.
3.  Four dominant mutations cause changes within a seven amino acid 
region: pro (243) to leu, gly (244) to ser, asn (247) to ile, and asp (
249) to asn.  Since these amino acids are conserved in all sequenced 
alpha-tubulins, these mutants are probably defective in a conserved 
tubulin function.  The region of amino acids 243-249 may be very 
important for some interaction required for microtubule assembly or 
stability.
4.  Two alleles, which we had thought were deletions on the basis of 
altered EcoRI fragments, we now find are point mutations generating 
new EcoRI sites.  PCR-amplified mec-7 DNA from these strains (e1505 
and u156) was digested with EcoRI to reveal two new fragments whose 
total size was the same as that of the missing wild-type fragment.  
Sequence analysis of e1505 DNA confirms the existence of a novel EcoRI 
site.  Thus, we have found no deletions caused by EMS mutagenesis.
5.  There may be some variability in phenotypic effects of the same 
mutation.  Two mutations [pro (171) to leu and gln (280) to stop] were 
each identified in two different strains that were categorized as 
strong and weak.  This variability may be explained by difficulty in 
categorizing these strains, which were not extremely weak but rather 
on the borderline.  Another mutation [trp(101) to stop] was identified 
in a recessive and in a semi-dominant allele.  Although we are re-
examining these alleles, we do not believe that they were mis-
categorized.  These results suggest that other mutations in the 
background may affect the phenotypic expression of a given mutation.
6.  Alterations in two putative GTP-binding domains cause mec-7 
mutations.  One strong semi-dominant allele is the result of a gly (
109) to glu change which disrupts a putative GTP-binding domain.  
Consistent with this result, Sullivan has mutated this putative GTP-
binding domain in yeast and found the resulting tubulin gene to be 
dominant lethal.  One strong recessive mec-7 allele is caused by a gly 
to arg change at amino acid 148, in or near a putative GTP-binding 
region.  How this change causes a loss of function is as yet unclear.

Figure 1