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. We have used DNA from cells carrying dominant cAMP-resistant defects to transfer the cAMP-resistance phenotype to sensitive cells. The mechanism of mutation in these cell lines is being studied with cloned RI and C subunit probes. Cloned segments of RI have been sequenced from cosmid libraries prepared from our mutant cell lines. The intent of these studies is to isolate cloned mutant cAMP dependent protein kinase subunits for use as movable genetic elements capable of inactivating the protein kinase system in a variety of differentiated cells and transgenic animals. We have also used the CHO protein kinase mutants to demonstrate that the expression of many promoters linked to the reporter gene encoding chloramphenicol acetyl transferase is positively regulated by cAMP in a manner which depends on cAMP dependent protein kinase activity.