Worm Breeder's Gazette 11(1): 32
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 actin binding protein alpha-actinin is a major protein component of the Z disc in vertebrate muscle and the dense-body in nematode body wall muscle. It is also found associated with cytoskeletal actin filaments in vertebrate and invertebrate non-muscle tissue. Two previously reported alpha-actinin cDNA sequences from chick fibroblasts and Dictyostelium discoideum were used to design an oligonucleotide probe which was then used to isolate a putative C. elegans alpha-actinin gene. Sulston and Coulson mapped this gene to chromosome V, close to the sma-1 locus. A partial sequence of this gene revealed a region of DNA encoding 49 amino acids which are 86% homologous to a chick alpha-actinin region. A second oligonucleotide homologous to the nematode gene was used to screen a cDNA library. A 3.5 kb cDNA was recovered, which was 100 base pairs shorter than expected for a full length cDNA as calculated from the size of the mRNA (3.6 kb) on Northern blots. The complete sequence of the cDNA showed that it does not include the initiator ATG codon, but a comparison with the chick sequence indicates that greater than 95% of the coding sequence is present, representing a polypeptide of 908 amino acids (MW=105,535 daltons). Computer alignment of the nematode alpha-actinin sequence with chick alpha-actinin using the Staden program Diagon and the UWGCG program Bestfit, show an overall amino acid homology between these proteins of 68%, with a 50% amino acid identity. The first 250 amino acids, which contain the putative actin-binding region of the molecule, is the most conserved region, showing an 80% amino acid homology, with a 68% amino acid identity. The central region of the nematode sequence contains three 113 amino acid repeats which show weak and variable homology to each other and to similar internal repeats found in human erythroid spectrin and human dystrophin. The number of similar repeats found in the alpha-actinins from chick fibroblast and Dictyostelium are 4 and 2 respectively, and we have compiled a consensus repeat sequence based upon the 9 repeat units found in the alpha-actinins of these three species. The carboxy terminal region of the nematode protein has two putative EF hand motifs which are indicative of calcium binding sites. However, it is unlikely that the nematode protein binds calcium since each site is missing some of the oxygen-containing amino acid side chains necessary for chelating calcium. This lack of functional calcium binding sites in the nematode protein suggests that it may be of muscle origin since, unlike non-muscle alpha-actinin, actin binding to muscle alpha-actinin is not calcium regulated. The mapping of the putative alpha-actinin gene close to the sma-1 locus will help us design screens for isolating alpha-actinin mutants. In addition, the multiple isoforms of alpha-actinin found in vertebrates may also exist in C. elegans, and we are currently investigating whether other nematode cDNA clones we have isolated exhibit such diversity.