Abstract

The long-term goal of this research is to learn about the structure, expression and function of tropomyosin in normal and transformed cells. Full-length cDNA clones encoding all five isoforms of tropomyosin from rat embryonic fibroblasts will be isolated, and their DNA sequences determined to deduce their primary structure. This information will be used to prepare synthetic peptides to be used as immunogens for making isoform specific antibodies. Such antibodies will be useful in determining the subcellular distribution of each form of tropomyosin and if this localization is altered upon transformation. Bacteriophage lambda recombinants which carry the genes for rat tropomyosin will be isolated and the general structure and organization of the genes determined. These studies will determine if all five forms of fibroblast tropomyosin arise from separate but related genes or if some arise from a single gene via differential processing of the message. The cloned DNAs will be used to study tropomyosin expression to determine if there are alterations in transcription, processing and translation of tropomyosin messages in transformed cells. To study the functional significance of the altered pattern of tropomyosins in transformed fibroblasts, tropomysoin will be introduced into living cells by microinjection of purified mRNA or by the expression of cloned DNAs encoding tropomyosin. These studies will determine if expression of tropomyosin in transformed cells will result in changes in cell shape or organization of microfilaments. (L)

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM043049-05
Application #
3301963
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1985-09-01
Project End
1994-03-31
Budget Start
1990-04-01
Budget End
1991-03-31
Support Year
5
Fiscal Year
1990
Total Cost
Indirect Cost

  Institution

Name
Cold Spring Harbor Laboratory
Department
Type
DUNS #
065968786
City
Cold Spring Harbor
State
NY
Country
United States
Zip Code
11724

  Publications

Chen, C D; Kobayashi, R; Helfman, D M (1999) Binding of hnRNP H to an exonic splicing silencer is involved in the regulation of alternative splicing of the rat beta-tropomyosin gene. Genes Dev 13:593-606
Selvakumar, M; Helfman, D M (1999) Exonic splicing enhancers contribute to the use of both 3' and 5' splice site usage of rat beta-tropomyosin pre-mRNA. RNA 5:378-94
Lou, H; Helfman, D M; Gagel, R F et al. (1999) Polypyrimidine tract-binding protein positively regulates inclusion of an alternative 3'-terminal exon. Mol Cell Biol 19:78-85
Chen, C D; Helfman, D M (1999) Donor site competition is involved in the regulation of alternative splicing of the rat beta-tropomyosin pre-mRNA. RNA 5:290-301
Grossman, J S; Meyer, M I; Wang, Y C et al. (1998) The use of antibodies to the polypyrimidine tract binding protein (PTB) to analyze the protein components that assemble on alternatively spliced pre-mRNAs that use distant branch points. RNA 4:613-25
Tsukahara, T; Casciato, C; Helfman, D M (1994) Alternative splicing of beta-tropomyosin pre-mRNA: multiple cis-elements can contribute to the use of the 5'- and 3'-splice sites of the nonmuscle/smooth muscle exon 6. Nucleic Acids Res 22:2318-25
Stamm, S; Zhang, M Q; Marr, T G et al. (1994) A sequence compilation and comparison of exons that are alternatively spliced in neurons. Nucleic Acids Res 22:1515-26
Stamm, S; Mulligan, G J; Krainer, A R et al. (1994) Non-radioactive detection of m3G capped RNAs by immunoblotting. Nucleic Acids Res 22:252-3
Helfman, D M (1994) The generation of protein isoform diversity by alternative RNA splicing. Soc Gen Physiol Ser 49:105-15
Pittenger, M F; Kazzaz, J A; Helfman, D M (1994) Functional properties of non-muscle tropomyosin isoforms. Curr Opin Cell Biol 6:96-104

Showing the most recent 10 out of 22 publications