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.

mec-7 Encodes a þ-tubulin

Cathy Savage, Michel Hamelin, Joe Culotti, Alan Coulson, Donna Albertson and Marty Chalfie

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 
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.