GENE ORGANIZATION AND EXPRESSION IN EUKARYOTES

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Studies are planned to continue to investigate 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. For example, 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, tropomyosin will be introduced into living cells by (1) microinjection of purified mRNA or (2) the expression of cloned DNAs encoding tropomyosin, for example, via the metallothionein promoter. These studies will determine if expression of tropomyosin in transformed cells will result in changes in cell shape or organization of microfilaments. (V)