Worm Breeder's Gazette 10(3): 37
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.
Mutations in the gene mec-7 result in touch-insensitive animals whose touch cell processes lack the 15-protofilament microtubules normally present in and specific to the touch cells. The mec-7 gene has been cloned and sequenced, and appears to encode a -tubulin which is necessary for the formation of these microtubules. One of the cloned C. elegans -tubulin genes (originally called tub- 2) is contained in a contig which maps by in situ hybridization to the region of the X chromosome near mec-7. We have used the cosmid R02A8 containing this -tubulin gene and a subfragment of this cosmid to probe genomic DNA from wild type and from mec-7 mutants. DNAs from seven mec-7 mutants exhibit restriction fragment length differences when probed with R02A8. Five mutants, two from EMS mutagenesis (e1505 and u156) and three from gamma-ray mutagenesis (u443, u448, and u453), contain deletions. The EMS mutations delete approximately 600bp and 300bp, respectively, of DNA from a fragment containing -tubulin homology. Two of the gamma-ray mutations (u443 and u448) delete approximately 28kb and 18kb, respectively, of DNA that includes the region of -tubulin homology. Animals homozygous for these mutations are completely touch insensitive but show no other behavioral defects. The third gamma-ray-induced mutation (u453) deletes more than 40kb of DNA and results in additional defects (partial lethality at 25 C and poor male mating). These additional phenotypes are probably associated with defects in one or more adjacent genes. DNAs from two other mutants (u382 and u388, both derived from TR679) contain insertions of approximately 3.5kb into the -tubulin sequences. The insertions are not seen in wild-type revertants derived from these animals. Taken together these data provide strong evidence that .mec-7 is the tub-2 -tubulin gene. The predicted tubulin sequence has 441 amino acids. Our finding that mec-7, a gene required for the production of 15-protofilament microtubules, codes for a -tubulin suggests that the constituent tubulins of the microtubule may affect the number of protofilaments. We have compared the deduced amino acid sequence of the mec-7 -tubulin with those of other -tubulins to identify regions of the mec-7 product that could potentially be required for polymerization into 15-protofilament microtubules. The mec-7 tubulin is 90-93% identical to vertebrate -tubulins. The carboxyl-terminal region beyond amino acid 430 is highly divergent in mec-7, as it is in all other -tubulins, and is the shortest of any reported -tubulin ( Sullivan and Cleveland, PNAS 83: 4327; Rudolph et al., Mol. Cell. Biol. 7: 2231; Burland et al., Mol. Cell. Biol. 8: 1275). In addition to the difference at the C-terminus, only seven residues in the mec-7 peptide sequence are not shared by other reported -tubulins, including two other worm -tubulins (tub-1 and ben-1) these are residues 35 (gln), 127 (thr), 198 (ser), 278 (asn), 293 (cys), 343 ( asp), and 429 (ala). Two of these changes, those at positions 127 and 343, are in amino acids that have hitherto been absolutely conserved. We do not know which of these differences are important for the unique structural role of the mec-7 -tubulin, but we suspect that two significant changes are the substitutions of a threonine for an absolutely conserved cysteine at residue 127 and of a mec-7-specific cysteine at residue 293. Free sulfhydryls have been implicated in the regulation of tubulin assembly (Nishida and Kobayashi, J. Biochem. ( Tokyo) 81: 343; Burchill et al., J. Cell Biol. 76: 439). In addition, a mutation in the testis-specific -tubulin of Drosophila at position 288 (i.e. 5 amino acids from the new cysteine in mec-7) prevents polymerization of the tubulin into functional microtubules without preventing heterodimer or protofilament formation (Rudolph et al., Mol. Cell. Biol. 7: 2231). This region may thus be important for interactions between protofilaments. It should be possible to test the importance of these residues in determining the protofilament number of microtubules by in vitro mutagenesis of the mec-7 gene.