Worm Breeder's Gazette 11(2): 35
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.
The deb-1 gene has been cloned, sequenced, mapped to chromosome IV close to unc-44, and shown to encode the protein vinculin (1). The postulated function of vinculin in vertebrate muscle and non-muscle cells is to control the position and stability of the attachment of actin filaments to the membrane (2). In nematodes vinculin is found at the base of the muscle dense-bodies linking the thin filaments to the membrane, an analogous position to that in vertebrate tissues. In order to asses its function in the nematode we sought to recover mutations in the deb-1 gene. Since there were no known muscle Uncs in the region of deb-1, we suspected that mutations in the gene would be lethal, with morphology and behavior like that of lethal myo-3 mutants (3). We have now recovered two mutants with such a phenotype which map close to unc-44. One, deb-1(st555), does not express any vinculin as assayed by immunofluorescence of arrested embryos. The second, deb- 1(st385), expresses vinculin at a reduced level compared to wild type worms. Two recent methodologies made it feasible to recover DNA from arrested deb-1 mutants, locate the position of the mutations in the gene, and sequence the mutated site. To recover DNA from the mutant gene, arrested animals were picked off plates, digested with Proteinase K, and PCR fragments were made which covered all of the coding sequence. To map the position of the mutated site, the PCR fragments were mixed with corresponding end-labeled fragments from wild type DNA, denatured, and allowed to hybridize to generate heteroduplexes. The heteroduplexes were treated with hydroxylamine which results in the modification of any mismatched cytosine. Cleavage of the modified cytosines with piperidine followed by fractionation of the products on denaturing acrylamide gels allowed us to position and subsequently to sequence the mutations. The mutation deb-1(st555) was found to represent a change in a conserved G in a splice acceptor site adjacent to exon five, and deb-1(st385) was found to represent the introduction of an amber stop codon in exon eight. We have subsequently shown that sup-7 will rescue the early arrest phenotype of st385, yielding adults with disorganized muscle. We are currently attempting to rescue the mutant by transformation with a wild type copy of the deb-1 gene.