CLONING OF TROPOMYOSINS FROM LOBSTER (HOMARUS-AMERICANUS) STRIATED MUSCLES - FAST AND SLOW ISOFORMS MAY BE GENERATED FROM THE SAME TRANSCRIPT

Complementary DNAs encoding fibre-type-specific isoforms of tropomyosi n (Tm) have been isolated from lobster (Homarus americanus) striated m uscle expression libraries made from poly(A)(+) RNA purified from deep abdominal (fast-type) and crusher-claw closer (slow-type) muscles. A cDNA of slow-muscle Tm (sTm(1)), containing a complete open reading fr ame (ORF) and portions of the 5' and 3' untranslated regions (UTRs), e ncodes a protein of 284 amino acid residues with a predicted mass of 3 2 950, assuming acetylation of the amino terminus. The nucleotide sequ ence of a fast-muscle tropomyosin (fTm cDNA), which includes the entir e ORF and part of the 3' UTR, is identical to that of sTm(1) cDNA, exc ept in the region encoding amino acid residues 39-80 (equivalent to ex on 2 of mammalian and Drosophila muscle tropomyosin genes). The deduce d amino acid sequences, which display the heptameric repeats of nonpol ar and charged amino acids characteristic of alpha-helical coiled-coil s, are highly homologous to tropomyosins from rabbit, Drosophila, and shrimp (57% to 99% identities, depending on species). Northern blot an alysis showed that two transcripts (1.1 and 2.1 kb) are present in bot h fibre types. Mass spectrometry indicated that fast muscle contains o ne major isoform (fTm: 32 903), while slow muscle contains two major i soforms (sTm(1) and sTm(2): 32 950 and 32 884 respectively). Both Tm p reparations contained minor species with a mass of about 32 830. Seque nces of peptides derived from purified slow and fast Tms were identica l to the deduced amino acid sequences of the sTm(1) and fTm cDNAs, res pectively, except in the C-terminal region of fTm. The difference in m ass between that predicted by the deduced sequence (32 880) and that m easured by mass spectrometry (32 903) suggests that fTm is posttransla tionally modified, in addition to acetylation of the N-terminal methio nine. These data are consistent with the hypothesis that the fTm and s Tm(1) are generated by alternative splicing of two mutually-exclusive exons near the 5' end of the same gene. (C) Chapman & Hall Ltd.