Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1989 Jan;9(1):185–192. doi: 10.1128/mcb.9.1.185

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

J A Bradac 1, C E Gruber 1, S Forry-Schaudies 1, S H Hughes 1
PMCID: PMC362160  PMID: 2927392

Abstract

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.

Full text

PDF
185

Images in this article

189Image
on p.189
190Image
on p.190

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adelstein R. S. Calmodulin and the regulation of the actin-myosin interaction in smooth muscle and nonmuscle cells. Cell. 1982 Sep;30(2):349–350. doi: 10.1016/0092-8674(82)90232-x. [DOI] [PubMed] [Google Scholar]
  2. Bretscher A. Smooth muscle caldesmon. Rapid purification and F-actin cross-linking properties. J Biol Chem. 1984 Oct 25;259(20):12873–12880. [PubMed] [Google Scholar]
  3. Bretscher A. Thin filament regulatory proteins of smooth- and non-muscle cells. Nature. 1986 Jun 19;321(6072):726–727. doi: 10.1038/321726b0. [DOI] [PubMed] [Google Scholar]
  4. Broschat K. O., Burgess D. R. Low Mr tropomyosin isoforms from chicken brain and intestinal epithelium have distinct actin-binding properties. J Biol Chem. 1986 Oct 5;261(28):13350–13359. [PubMed] [Google Scholar]
  5. Clayton L., Reinach F. C., Chumbley G. M., MacLeod A. R. Organization of the hTMnm gene. Implications for the evolution of muscle and non-muscle tropomyosins. J Mol Biol. 1988 Jun 5;201(3):507–515. doi: 10.1016/0022-2836(88)90633-x. [DOI] [PubMed] [Google Scholar]
  6. Cummins P., Perry S. V. Chemical and immunochemical characteristics of tropomyosins from striated and smooth muscle. Biochem J. 1974 Jul;141(1):43–49. doi: 10.1042/bj1410043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cummins P., Perry S. V. The subunits and biological activity of polymorphic forms of tropomyosin. Biochem J. 1973 Aug;133(4):765–777. doi: 10.1042/bj1330765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Côté G. P., Smillie L. B. The interaction of equine platelet tropomyosin with skeletal muscle actin. J Biol Chem. 1981 Jul 25;256(14):7257–7261. [PubMed] [Google Scholar]
  9. Côté G. P. Structural and functional properties of the non-muscle tropomyosins. Mol Cell Biochem. 1983;57(2):127–146. doi: 10.1007/BF00849190. [DOI] [PubMed] [Google Scholar]
  10. Côté G., Lewis W. G., Smillie L. B. Non-polymerizability of platelet tropomyosin and its NH2- and COOH-terminal sequences. FEBS Lett. 1978 Jul 15;91(2):237–241. doi: 10.1016/0014-5793(78)81181-8. [DOI] [PubMed] [Google Scholar]
  11. Ebashi S., Endo M., Otsuki I. Control of muscle contraction. Q Rev Biophys. 1969 Nov;2(4):351–384. doi: 10.1017/s0033583500001190. [DOI] [PubMed] [Google Scholar]
  12. Flach J., Lindquester G., Berish S., Hickman K., Devlin R. Analysis of tropomyosin cDNAs isolated from skeletal and smooth muscle mRNA. Nucleic Acids Res. 1986 Nov 25;14(22):9193–9211. doi: 10.1093/nar/14.22.9193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gooding C., Reinach F. C., Macleod A. R. Complete nucleotide sequence of the fast-twitch isoform of chicken skeletal muscle alpha-tropomyosin. Nucleic Acids Res. 1987 Oct 12;15(19):8105–8105. doi: 10.1093/nar/15.19.8105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hallauer P. L., Hastings K. E., Baldwin A. S., Pearson-White S., Merrifield P. A., Emerson C. P., Jr Closely related alpha-tropomyosin mRNAs in quail fibroblasts and skeletal muscle cells. J Biol Chem. 1987 Mar 15;262(8):3590–3596. [PubMed] [Google Scholar]
  15. Heald R. W., Hitchcock-DeGregori S. E. The structure of the amino terminus of tropomyosin is critical for binding to actin in the absence and presence of troponin. J Biol Chem. 1988 Apr 15;263(11):5254–5259. [PubMed] [Google Scholar]
  16. Helfman D. M., Cheley S., Kuismanen E., Finn L. A., Yamawaki-Kataoka Y. Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation. Mol Cell Biol. 1986 Nov;6(11):3582–3595. doi: 10.1128/mcb.6.11.3582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Helfman D. M., Feramisco J. R., Fiddes J. C., Thomas G. P., Hughes S. H. Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library. Proc Natl Acad Sci U S A. 1983 Jan;80(1):31–35. doi: 10.1073/pnas.80.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Helfman D. M., Feramisco J. R., Ricci W. M., Hughes S. H. Isolation and sequence of a cDNA clone that contains the entire coding region for chicken smooth-muscle alpha-tropomyosin. J Biol Chem. 1984 Nov 25;259(22):14136–14143. [PubMed] [Google Scholar]
  19. Hendricks M., Weintraub H. Multiple tropomyosin polypeptides in chicken embryo fibroblasts: differential repression of transcription by Rous sarcoma virus transformation. Mol Cell Biol. 1984 Sep;4(9):1823–1833. doi: 10.1128/mcb.4.9.1823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hitchcock S. E., Huxley H. E., Szent-Györgyi A. G. Calcium sensitive binding of troponin to actin-tropomyosin: a two-site model for troponin action. J Mol Biol. 1973 Nov 15;80(4):825–836. doi: 10.1016/0022-2836(73)90212-x. [DOI] [PubMed] [Google Scholar]
  21. Johnson P., Smillie L. B. Polymerizability of rabbit skeletal tropomyosin: effects of enzymic and chemical modifications. Biochemistry. 1977 May 17;16(10):2264–2269. doi: 10.1021/bi00629a035. [DOI] [PubMed] [Google Scholar]
  22. Krieg P. A., Melton D. A. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 1984 Sep 25;12(18):7057–7070. doi: 10.1093/nar/12.18.7057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lewis W. G., Cote G. P., Mak A. S., Smillie L. B. Amino acid sequence of equine platelet tropomyosin. Correlation with interaction properties. FEBS Lett. 1983 Jun 13;156(2):269–273. doi: 10.1016/0014-5793(83)80511-0. [DOI] [PubMed] [Google Scholar]
  24. Lewis W. G., Smillie L. B. The amino acid sequence of rabbit cardiac tropomyosin. J Biol Chem. 1980 Jul 25;255(14):6854–6859. [PubMed] [Google Scholar]
  25. Lin C. S., Leavitt J. Cloning and characterization of a cDNA encoding transformation-sensitive tropomyosin isoform 3 from tumorigenic human fibroblasts. Mol Cell Biol. 1988 Jan;8(1):160–168. doi: 10.1128/mcb.8.1.160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lin J. J., Helfman D. M., Hughes S. H., Chou C. S. Tropomyosin isoforms in chicken embryo fibroblasts: purification, characterization, and changes in Rous sarcoma virus-transformed cells. J Cell Biol. 1985 Mar;100(3):692–703. doi: 10.1083/jcb.100.3.692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lin J. J., Matsumura F., Yamashiro-Matsumura S. Tropomyosin-enriched and alpha-actinin-enriched microfilaments isolated from chicken embryo fibroblasts by monoclonal antibodies. J Cell Biol. 1984 Jan;98(1):116–127. doi: 10.1083/jcb.98.1.116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. MacLeod A. R. Distinct alpha-tropomyosin mRNA sequences in chicken skeletal muscle. Eur J Biochem. 1982 Aug;126(2):293–297. doi: 10.1111/j.1432-1033.1982.tb06778.x. [DOI] [PubMed] [Google Scholar]
  29. MacLeod A. R., Gooding C. Human hTM alpha gene: expression in muscle and nonmuscle tissue. Mol Cell Biol. 1988 Jan;8(1):433–440. doi: 10.1128/mcb.8.1.433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. MacLeod A. R., Houlker C., Reinach F. C., Smillie L. B., Talbot K., Modi G., Walsh F. S. A muscle-type tropomyosin in human fibroblasts: evidence for expression by an alternative RNA splicing mechanism. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7835–7839. doi: 10.1073/pnas.82.23.7835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. MacLeod A. R., Houlker C., Reinach F. C., Talbot K. The mRNA and RNA-copy pseudogenes encoding TM30nm, a human cytoskeletal tropomyosin. Nucleic Acids Res. 1986 Nov 11;14(21):8413–8426. doi: 10.1093/nar/14.21.8413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. MacLeod A. R., Talbot K., Smillie L. B., Houlker C. Characterization of a cDNA defining a gene family encoding TM30p1, a human fibroblast tropomyosin. J Mol Biol. 1987 Mar 5;194(1):1–10. doi: 10.1016/0022-2836(87)90710-8. [DOI] [PubMed] [Google Scholar]
  33. Mak A. S., Smillie L. B., Stewart G. R. A comparison of the amino acid sequences of rabbit skeletal muscle alpha- and beta-tropomyosins. J Biol Chem. 1980 Apr 25;255(8):3647–3655. [PubMed] [Google Scholar]
  34. Mak A. S., Smillie L. B. Structural interpretation of the two-site binding of troponin on the muscle thin filament. J Mol Biol. 1981 Jul 5;149(3):541–550. doi: 10.1016/0022-2836(81)90486-1. [DOI] [PubMed] [Google Scholar]
  35. Matsumura F., Lin J. J., Yamashiro-Matsumura S., Thomas G. P., Topp W. C. Differential expression of tropomyosin forms in the microfilaments isolated from normal and transformed rat cultured cells. J Biol Chem. 1983 Nov 25;258(22):13954–13964. [PubMed] [Google Scholar]
  36. Matsumura F., Yamashiro-Matsumura S., Lin J. J. Isolation and characterization of tropomyosin-containing microfilaments from cultured cells. J Biol Chem. 1983 May 25;258(10):6636–6644. [PubMed] [Google Scholar]
  37. McLachlan A. D., Stewart M. Tropomyosin coiled-coil interactions: evidence for an unstaggered structure. J Mol Biol. 1975 Oct 25;98(2):293–304. doi: 10.1016/s0022-2836(75)80119-7. [DOI] [PubMed] [Google Scholar]
  38. Ngai P. K., Walsh M. P. Inhibition of smooth muscle actin-activated myosin Mg2+-ATPase activity by caldesmon. J Biol Chem. 1984 Nov 25;259(22):13656–13659. [PubMed] [Google Scholar]
  39. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  40. Ohtsuki I. Molecular arrangement of troponin-T in the thin filament. J Biochem. 1979 Aug;86(2):491–497. doi: 10.1093/oxfordjournals.jbchem.a132549. [DOI] [PubMed] [Google Scholar]
  41. Pearlstone J. R., Smillie L. B. Binding of troponin-T fragments to several types of tropomyosin. Sensitivity to Ca2+ in the presence of troponin-C. J Biol Chem. 1982 Sep 25;257(18):10587–10592. [PubMed] [Google Scholar]
  42. Pearlstone J. R., Smillie L. B. Identification of a second binding region on rabbit skeletal troponin-T for alpha-tropomyosin. FEBS Lett. 1981 Jun 1;128(1):119–122. doi: 10.1016/0014-5793(81)81095-2. [DOI] [PubMed] [Google Scholar]
  43. Pearson-White S. H., Emerson C. P., Jr A novel hybrid alpha-tropomyosin in fibroblasts is produced by alternative splicing of transcripts from the skeletal muscle alpha-tropomyosin gene. J Biol Chem. 1987 Nov 25;262(33):15998–16010. [PubMed] [Google Scholar]
  44. Reinach F. C., MacLeod A. R. Tissue-specific expression of the human tropomyosin gene involved in the generation of the trk oncogene. Nature. 1986 Aug 14;322(6080):648–650. doi: 10.1038/322648a0. [DOI] [PubMed] [Google Scholar]
  45. Ruiz-Opazo N., Nadal-Ginard B. Alpha-tropomyosin gene organization. Alternative splicing of duplicated isotype-specific exons accounts for the production of smooth and striated muscle isoforms. J Biol Chem. 1987 Apr 5;262(10):4755–4765. [PubMed] [Google Scholar]
  46. Ruiz-Opazo N., Weinberger J., Nadal-Ginard B. Comparison of alpha-tropomyosin sequences from smooth and striated muscle. Nature. 1985 May 2;315(6014):67–70. doi: 10.1038/315067a0. [DOI] [PubMed] [Google Scholar]
  47. Sanders C., Smillie L. B. Amino acid sequence of chicken gizzard beta-tropomyosin. Comparison of the chicken gizzard, rabbit skeletal, and equine platelet tropomyosins. J Biol Chem. 1985 Jun 25;260(12):7264–7275. [PubMed] [Google Scholar]
  48. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sobieszek A., Small J. V. Myosin-linked calcium regulation in vertebrate smooth muscle. J Mol Biol. 1976 Mar 25;102(1):75–92. doi: 10.1016/0022-2836(76)90074-7. [DOI] [PubMed] [Google Scholar]
  50. Sobue K., Muramoto Y., Fujita M., Kakiuchi S. Purification of a calmodulin-binding protein from chicken gizzard that interacts with F-actin. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5652–5655. doi: 10.1073/pnas.78.9.5652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sodek J., Hodges R. S., Smillie L. B. Amino acid sequence of rabbit skeletal muscle alpha-tropomyosin. The COOH-terminal half (residues 142 to 284). J Biol Chem. 1978 Feb 25;253(4):1129–1136. [PubMed] [Google Scholar]
  52. Stone D., Smillie L. B. The amino acid sequence of rabbit skeletal alpha-tropomyosin. The NH2-terminal half and complete sequence. J Biol Chem. 1978 Feb 25;253(4):1137–1148. [PubMed] [Google Scholar]
  53. Talbot K., MacLeod A. R. Novel form of non-muscle tropomyosin in human fibroblasts. J Mol Biol. 1983 Feb 15;164(1):159–174. doi: 10.1016/0022-2836(83)90091-8. [DOI] [PubMed] [Google Scholar]
  54. Taylor E. W. Mechanism of actomyosin ATPase and the problem of muscle contraction. CRC Crit Rev Biochem. 1979;6(2):103–164. doi: 10.3109/10409237909102562. [DOI] [PubMed] [Google Scholar]
  55. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  56. Walsh M. P., Bridenbaugh R., Kerrick W. G., Hartshorne D. J. Gizzard Ca2+-independent myosin light chain kinase: evidence in favor of the phosphorylation theory. Fed Proc. 1983 Jan;42(1):45–50. [PubMed] [Google Scholar]
  57. Walsh T. P., Wegner A. Effect of the state of oxidation of cysteine 190 of tropomyosin on the assembly of the actin-tropomyosin complex. Biochim Biophys Acta. 1980 Nov 20;626(1):79–87. doi: 10.1016/0005-2795(80)90199-3. [DOI] [PubMed] [Google Scholar]
  58. Wegner A. Equilibrium of the actin-tropomyosin interaction. J Mol Biol. 1979 Jul 15;131(4):839–853. doi: 10.1016/0022-2836(79)90204-3. [DOI] [PubMed] [Google Scholar]
  59. Yamashiro-Matsumura S., Matsumura F. Characterization of 83-kilodalton nonmuscle caldesmon from cultured rat cells: stimulation of actin binding of nonmuscle tropomyosin and periodic localization along microfilaments like tropomyosin. J Cell Biol. 1988 Jun;106(6):1973–1983. doi: 10.1083/jcb.106.6.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yamawaki-Kataoka Y., Helfman D. M. Isolation and characterization of cDNA clones encoding a low molecular weight nonmuscle tropomyosin isoform. J Biol Chem. 1987 Aug 5;262(22):10791–10800. [PubMed] [Google Scholar]
  61. Yamawaki-Kataoka Y., Helfman D. M. Rat embryonic fibroblast tropomyosin 1. cDNA and complete primary amino acid sequence. J Biol Chem. 1985 Nov 25;260(27):14440–14445. [PubMed] [Google Scholar]
  62. Yang Y. Z., Korn E. D., Eisenberg E. Cooperative binding of tropomyosin to muscle and Acanthamoeba actin. J Biol Chem. 1979 Aug 10;254(15):7137–7140. [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES