Worm Breeder's Gazette 10(3): 109
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.
In the body wall muscle of the nematode actin filaments are anchored to the sarcolemma through the dense-bodies. Two of the protein components of the dense-bodies have been characterized using monoclonal antibodies (Francis and Waterston, 1985). One of these components, p107b/110, is found at the base of the dense-bodies adjacent to the sarcolemma and, as we have previously reported, the deb-1 gene which encodes this antigen has been cloned from an expression library. We have completed the sequencing of this gene and an associated cDNA, and have now shown that it encodes a peptide that is highly similar to chicken vinculin. We have further shown that the size difference between chicken vinculin, Mr 130,000, and its nematode analog, Mr 107,000, is due in part to the absence from the nematode sequence of one of the three internal 110 amino acid repeats found in the chicken sequence. While the function of vinculin is not known, its location in structures which mediate the attachment of actin filaments to membranes as well as its biochemical properties has led to the speculation that it is important for the initiation of actin filament assembly. We are currently attempting to recover mutations in the deb-1 gene which should allow us to assess the developmental role of nematode vinculin. A second component of the dense-body, p107a, has been shown to be immunologically similar to the actin binding protein alpha-actinin. After failing to recover the gene for p107a using antibodies to screen an expression library, we decided to make use of two known alpha- actinin cDNA sequences from dictyostelium and chicken. Degenerate oligonucleotide probes to the conserved actin binding region of the known sequences were synthesized and used to screen a genomic library. A set of overlapping clones was recovered and sent to Cambridge where the clones were mapped to a single locus on chromosome V on the same contig as myo-3, the gene encoding the minor heavy chain myosin of the body wall muscle. Other than myo-3, there are no known muscle affecting genes in this region. We are currently sequencing a 3.8 kb cDNA for the gene. The sequence obtained thus far shows extensive similarity with other known alpha-actinin sequences. Moreover, when portions of the cDNA clone are fused to the E. coli -gal gene, a fusion protein is made which reacts with antibodies that recognize p107a. We are currently focusing on completing the sequence of the cDNA. Now that both the alpha-actinin and the vinculin genes are cloned, we are in a position to genetically dissect the interactions between these two proteins and their role in the initiation of thin filament assembly.