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.

Isolation of a Putative alpha-Actinin Gene in C. elegans alpha-Actinin cDNA

Lawrence Kleiman, Robert Barstead and Robert Waterston

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.