MENDOZA, P.; MYKLES, D.L.: Characterization of a cDNA encoding lobster muscle troponin-T
Three fiber types in decapod crustacean skeletal muscles differ in the expression of myofibrillar protein isoforms. Two slow types (slow-twitch or S1 and slow-tonic or S2) and one fast type are distinguished by differences in expression of three isoforms of troponin-T (TnT), the tropomyosin-binding subunit of the heterotrimeric troponin complex that regulates muscle contraction. During development of the American lobster (Homarus americanus), fiber-type switching occurs while the claws differentiate into cutter and crusher claws. This switching requires coordinated changes in gene expression and/or mRNA processing in conjunction with complete remodeling of the contractile apparatus. We hypothesize that the different TnT isoforms are produced by fiber-type-specific alternative mRNA splicing of a single gene. A cDNA library from fast muscle was screened by PCR using primers to highly-conserved sequences in TnT genes from other species. A partial sequence of 567 base pairs was initially obtained that was 70% identical to a homologous region (amino acid residues #195-242) in the Drosophila TnT gene (accession #S13251). Further PCR resulted in additional sequences encompassing about a 1.2-kb region of the gene. Using Northern blot analysis, two TnT mRNAs (1.1 and 1.6 kb) were expressed in skeletal muscle, but not other tissues. Our goal is to obtain full-length sequences of all three TnT isoforms to analyze their expression and mRNA processing during fiber switching. Supported by NSF (IBN-0077422).
