Worm Breeder's Gazette 11(3): 41
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.
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.