Isolation and characterization of related cDNA clones encoding skeletal muscle beta-tropomyosin and a low-molecular-weight nonmuscle tropomyosin isoform.

We have isolated and characterized cDNA clones from chicken cDNA libraries derived from skeletal muscle, body wall, and cultured fibroblasts. A clone isolated from a skeletal muscle cDNA library contains the complete protein-coding sequence of the 284-amino-acid skeletal muscle beta-tropomyosin together with 72 bases of 5' untranslated sequence and nearly the entire 3' untranslated region (about 660 bases), lacking only the last 4 bases and the poly(A) tail. A second clone, isolated from the fibroblast cDNA library, contains the complete protein-coding sequence of a 248-amino-acid fibroblast tropomyosin together with 77 bases of 5' untranslated sequence and 235 bases of 3' untranslated sequence through the poly(A) tract. The derived amino acid sequence from this clone exhibits only 82% homology with rat fibroblast tropomyosin 4 and 80% homology with human fibroblast tropomyosin TM30nm, indicating that this clone encodes a third 248-amino-acid tropomyosin isoform class. The protein product of this mRNA is fibroblast tropomyosin 3b, one of two low-molecular-weight isoforms expressed in chicken fibroblast cultures. Comparing the sequences of the skeletal muscle and fibroblast cDNAs with a previously characterized clone which encodes the smooth muscle alpha-tropomyosin reveals two regions of absolute homology, suggesting that these three clones were derived from the same gene by alternative RNA splicing.     

Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation.

The molecular basis for the expression of rat embryonic fibroblast tropomyosin 1 and skeletal muscle beta-tropomyosin was determined. cDNA clones encoding these tropomyosin isoforms exhibit complete identity except for two carboxy-proximal regions (amino acids 189 to 213 and 258 to 284) and different 3'-untranslated sequences. The isoform-specific regions delineate the troponin T-binding domains of skeletal muscle tropomyosin. Analysis of genomic clones indicates that there are two separate loci in the rat genome that contain sequences complementary to these mRNAs. One locus is a pseudogene. The other locus contains a single gene made up of 11 exons and spans approximately 10 kilobases. Sequences common to all mRNAs were found in exons 1 through 5 (amino acids 1 to 188) and exons 8 and 9 (amino acids 214 to 257). Exons 6 and 11 are specific for fibroblast mRNA (amino acids 189 to 213 and 258 to 284, respectively), while exons 7 and 10 are specific for skeletal muscle mRNA (amino acids 189 to 213 and 258 to 284, respectively). In addition, exons 10 and 11 each contain the entire 3'-untranslated sequences of the respective mRNAs including the polyadenylation site. Although the gene is also expressed in smooth muscle (stomach, uterus, and vas deferens), only the fibroblast-type splice products can be detected in these tissues. S1 and primer extension analyses indicate that all mRNAs expressed from this gene are transcribed from a single promoter. The promoter was found to contain G-C-rich sequences, a TATA-like sequence TTTTA, no identifiable CCAAT box, and two putative Sp1-binding sites.     

Chicken cardiac tropomyosin and a low-molecular-weight nonmuscle tropomyosin are related by alternative splicing

We have isolated and characterized complementary DNAs (cDNAs) encoding chicken cardiac muscle tropomyosin and a low-molecular-weight nonmuscle tropomyosin. The cardiac muscle cDNA (pCHT-4) encodes a 284-amino acid protein that differs from chicken skeletal muscle alpha- and beta-tropomyosins throughout its length. The nonmuscle cDNA (pFT-C) encodes a 248-amino acid protein that is most similar (93-94%) to the tropomyosin class including rat fibroblast TM-4, equine platelet tropomyosin, and human fibroblast TM30pl. The nucleotide sequences of the cardiac and nonmuscle cDNAs are identical from the position encoding cardiac amino acid 81 (nonmuscle amino acid 45) through cardiac amino acid 257 (nonmuscle amino acid 221). The sequences differ both 5' and 3' of this region of identity. These comparisons suggest that the chicken cardiac tropomyosin and low-molecular-weight "platelet-like" tropomyosin are derived from the same genomic locus by alternative splicing. S1 analysis suggests that this locus encodes at least one other tropomyosin isoform.     

Amino acid sequence of chicken gizzard beta-tropomyosin. Comparison of the chicken gizzard, rabbit skeletal, and equine platelet tropomyosins

Chicken gizzard beta-tropomyosin has the same chain length (284 residues) as other muscle tropomyosins, and is most closely related to the beta component of rabbit skeletal muscle. The majority of the amino acid substitutions are restricted to two regions of the structure, residues 185-216 and 258-284. The altered sequences at the COOH-terminal ends (residue 258-284) of the two gizzard components are very similar to each other and to those in platelet tropomyosin and can be correlated with the reduced affinity of interaction of all three tropomyosins with skeletal troponin T and its T1 fragment. The virtually identical NH2-terminal sequences of all four muscle tropomyosin chains indicates that the gizzard proteins' greater ability to polymerize head-to-tail is due to the sequence changes at its COOH terminus. On the other hand, the weaker head-to-tail aggregation of the platelet protein must be due to its NH2-terminal sequence alterations. Examination of the distribution of amino acids and the frequency of their substitution in the a to g positions of the repeating pseudoheptapeptide for all five tropomyosin sequences (four muscle and one platelet) emphasizes the importance of Glu residues at position e. Examination of those features of the muscle sequences implicated in the stabilization of their coiled-coil structures and in their interactions with F-actin suggest only marginal differences among them, with the possible exception of the chicken gizzard gamma component.     

Rat embryonic fibroblast tropomyosin 1. cDNA and complete primary amino acid sequence

A cDNA expression library of approximately 80,000 members was prepared from rat embryonic fibroblast mRNA using the plasmid expression vectors pUC8 and pUC9. Using an immunological screening procedure and 32P-labeled cDNA probes, clones encoding rat embryonic fibroblast tropomyosin 1 (TM-1) were identified and isolated. DNA sequence analysis was carried out to determine the amino acid sequence of the protein. Rat embryonic fibroblast TM-1 was found to contain 284 amino acids and is most homologous to smooth muscle alpha-tropomyosin compared with skeletal muscle alpha- and beta-tropomyosins and platelet beta-tropomyosin. Among the various tropomyosins, two regions where the greatest sequence divergence is evident are between amino acids 185 and 216 and amino acids 258 and 284. Rat embryonic fibroblast TM-1 and chicken smooth muscle alpha-tropomyosin are most closely related from amino acids 185 and 216 compared with skeletal muscle and platelet tropomyosins. In contrast, rat embryonic fibroblast TM-1, smooth muscle alpha-tropomyosin, and platelet tropomyosin are most homologous from amino acids 258 and 284 compared with skeletal muscle tropomyosins. These differences in sequences at the carboxyl-terminal region of the various tropomyosins are discussed in relation to differences in their binding to skeletal muscle troponin and its T1 fragment.     

Cloning and characterization of a cDNA encoding transformation-sensitive tropomyosin isoform 3 from tumorigenic human fibroblasts.

We isolated a cDNA clone from the tumorigenic human fibroblast cell line HuT-14 that contains the entire protein coding region of tropomyosin isoform 3 (Tm3) and 781 base pairs of 5'- and 3'-untranslated sequences. Tm3, despite its apparent smaller molecular weight than Tm1 in two-dimensional gels, has the same peptide length as Tm1 (284 amino acids) and shares 83% homology with Tm1. Tm3 cDNA hybridized to an abundant mRNA of 1.3 kilobases in fetal muscle and cardiac muscle, suggesting that Tm3 is related to an alpha fast-tropomyosin. The first 188 amino acids of Tm3 are identical to those of rat or rabbit skeletal muscle alpha-tropomyosin, and the last 71 amino acids differ from those of rat smooth muscle alpha-tropomyosin by only 1 residue. Tm3 therefore appears to be encoded by the same gene that encodes the fast skeletal muscle alpha-tropomyosin and the smooth muscle alpha-tropomyosin via an alternative RNA-splicing mechanism. In contrast to Tm4 and Tm5, Tm3 has a small gene family, with, at best, only one pseudogene.     

Construction, expression and unexpected regulatory properties of a tropomyosin mutant with a 31-residue deletion at the C-terminus (exon 9)

The cDNA coding for human skeletal muscle beta-tropomyosin was expressed in Escherichia coli to produce an unacetylated beta-tropomyosin. This cDNA was deleted from the sequence corresponding to the exon 9 and expressed in E. coli to produce an unacetylated beta-tropomyosin mutant lacking the C-terminal residues 254-284. The main structural and functional properties of the two isolated proteins, designated tropomyosin-1 and des-(254-284)-tropomyosin, respectively, were characterized in comparison with those of the genuine rabbit skeletal muscle alpha beta-tropomyosin. The folding and thermal stability of the three tropomyosins were indistinguishable. Tropomyosin-1, but not des-(254-284)-tropomyosin, was polymerized in the presence of troponin and did bind to actin in the presence of the troponin complex. Despite its weak binding to actin, des-(254-284)-tropomyosin displayed a regulatory function in the presence of troponin with a marked activation of the actomyosin subfragment-1 ATPase in the presence of Ca2+ and low concentrations of subfragment-1. The data were interpreted in the light of the allosteric models of regulation and suggest the involvement of the sequence coded by exon 9 in the stabilization by tropomyosin of the off state of the thin filament.     

Factors determining the subunit composition of tropomyosin in mammalian skeletal muscle.

Adult rat fast-twitch skeletal muscle such as extensor digitorum longus contains alpha- and beta-tropomyosin subunits, as is the case in the corresponding muscles of rabbit. Adult rat soleus muscle contains beta-, gamma- and delta-tropomyosins, but no significant amounts of alpha-tropomyosin. Evidence for the presence of phosphorylated forms of at least three of the four tropomyosin subunit isoforms was obtained, particularly in developing muscle. Immediately after birth alpha- and beta-tropomyosins were the major components of skeletal muscle, in both fast-twitch and slow-twitch muscles. Differentiation into slow-twitch skeletal muscles was accompanied by a fall in the amount of alpha-tropomyosin subunit and its replacement with gamma- and delta-subunits. After denervation and during regeneration after injury, the tropomyosin composition of slow-twitch skeletal muscle changed to that associated with fast-twitch muscle. Thyroidectomy slowed down the changes in tropomyosin composition resulting from the denervation of soleus muscle. The results suggest that the 'ground state' of tropomyosin-gene expression in the skeletal muscle gives rise to alpha- and beta-tropomyosin subunits. Innervation by a 'slow-twitch' nerve is essential for the expression of the genes controlling gamma- and delta-subunits. There appears to be reciprocal relationship between expression of the gene controlling the synthesis of alpha-tropomyosin and those controlling the synthesis of gamma- and delta-tropomyosin subunits.     

In vitro phosphorylation of tropomyosin by a kinase from chicken embryo

A tropomyosin kinase has been partially purified from the leg muscle of 11-day-old chick embryos by ammonium sulfate precipitation and DEAE and phosphocellulose chromatography. The tropomyosin kinase requires Mg2+ for its activity, but Ca2+ and cyclic AMP are not needed. Increase in KC1 concentration decreased the tropomyosin kinase activity with over 90% inhibition at 0.2 M KC1. The alpha-tropomyosin subunit from rabbit and chicken skeletal muscle was phosphorylated about five times faster than the beta-tropomyosin subunit. Smooth muscle tropomyosin from chicken gizzard was not phosphorylated. The in vitro phosphorylation site in rabbit and chicken skeletal tropomyosins is a single serine residue close to the COOH terminus, a region intimately engaged in the head to tail polymerization of tropomyosin. Since the amino acid sequences of rabbit alpha- and beta-tropomyosin and chicken alpha-tropomyosin in this region are known, their phosphorylation sites can be unambiguously assigned as the penultimate residue, serine 283.