GENETIC AND BIOCHEMICAL ANALYSIS OF CELL BEHAVIOR

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We are utilizing the Chinese hamster ovary (CHO) fibroblast to study the genetics and biochemistry of some aspects of the behavior of cultured cells. Our work has emphasized morphology and its relationship to growth control, and the manner in which cyclic AMP regulates cell growth and gene expression. The mechanism of cAMP action on CHO cells has been studied by isolating CHO mutants resistant to growth inhibition by cAMP. These mutants have defective cAMP dependent protein kinases with either altered regulatory (RI) or catalytic (C) subunits. DNA from cells carrying dominant cAMP-resistant defects can be used to transfer the cAMP- resistance phenotype to sensitive cells. Cosmid clones carrying the CHO RI subunit from cAMP resistant cells have been isolated. Sequence analysis indicates that one of these clones, pcosRI has the coding sequence for the CHO RI subunit. The exon-intron structure of the RI gene is complex; the region encoding the cAMP- binding site at the 3' end of the gene is composed of several different exons. The regulation of expression of cAMP-responsive genes is being studied using wild type, cAMP dependent protein kinase mutants, and transferent CHO cell lines expressing the mutant kinase phenotype. We have demonstrated that for the rat tyrosine amino transferase (TAT) gene promoter an intact cAMP dependent protein kinase system is necessary for the regulation of cAMP responsive gene expression.