SIMONSON, M.E.; MYKLES, D.L.: Characterization of a cDNA encoding lobster muscle troponin-I

Skeletal muscle contraction is controlled by the regulatory proteins troponin and tropomyosin. Troponin is composed of three subunits: troponin-I, troponin-C, and troponin-T. At least five isoforms of troponin-I (TnI) have been identified in muscles from American lobster (Homarus americanus) using SDS-polyacrylamide gel electrophoresis. TnI isoform distribution varies between fast and slow muscles, which suggests that TnI isoforms are produced by differential fiber type-specific expression and/or mRNA processing of TnI gene(s). We hypothesize that the different isoforms are generated by alternative mRNA splicing of a single gene, as is the case with other arthropods. We screened cDNA libraries from fast and slow muscles with PCR using primers to highly-conserved sequences in TnI genes from diverse species. A partial sequence of 341 base pairs was obtained. The deduced amino acid sequence of the segment was 92% identical to a homologous region (amino acid residues #43-155) in the Pontastacus leptodactylus (narrow-clawed crayfish) TnI gene (accession #p05547). Northern blot analysis indicated that the gene was only expressed in skeletal muscle; mRNA was not detected in any other tissue, including cardiac muscle. Hybridization of the TnI probe produced a broad band at about 1.3-1.6 kb, indicating some heterogeneity in message sizes. Our eventual goal is to obtain full-length sequences of all five TnI isoforms using 3� and 5� RACE. Supported by NSF (IBN-0077422).