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)
| Erster, S H; Finn, L A; Frendewey, D A et al. (1988) Use of RNase H and primer extension to analyze RNA splicing. Nucleic Acids Res 16:5999-6014 |
| Helfman, D M; Ricci, W M; Finn, L A (1988) Alternative splicing of tropomyosin pre-mRNAs in vitro and in vivo. Genes Dev 2:1627-38 |
| Yamawaki-Kataoka, Y; Helfman, D M (1987) Isolation and characterization of cDNA clones encoding a low molecular weight nonmuscle tropomyosin isoform. J Biol Chem 262:10791-800 |
| Scott, J D; Glaccum, M B; Zoller, M J et al. (1987) The molecular cloning of a type II regulatory subunit of the cAMP-dependent protein kinase from rat skeletal muscle and mouse brain. Proc Natl Acad Sci U S A 84:5192-6 |
| Helfman, D M; Cheley, S; Kuismanen, E et al. (1986) Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation. Mol Cell Biol 6:3582-95 |
