GENETIC AND BIOCHEMICAL ANALYSIS OF CELL BEHAVIOR

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The Chinese hamster ovary (CHO) fibroblast is an excellent model system for the study of the genetics and biochemistry of some aspects of the behavior of cultured cells. We are using this system to analyze the manner in which cyclic AMP regulates cell growth and gene expression in mammalian cells. The mechanism of cAMP action of CHO cells has been studied by generating cell lines resistant to growth inhibition by cAMP. One way to develop resistance to cAMP is through mutations in cAMP-dependent protein kinases which result in either altered regulatory (RI) or catalytic (C) subunits. Defects in cAMP-dependent protein kinase block cAMP-stimulated transcription and reduce amounts of mRNA for the multidrug transporter in CHO cells and in adrenal Y-1 cells. We have constructed several mutant forms of the CHO RI subunit and a mutant C subunit and expressed these in E.coli. Bacterially expressed mutant RI's affecting sites for recognition of RI by C and for binding of cAMP to RI have defects in associating with the C subunit and in cAMP-stimulated dissociation of RI and C subunits. An improved system for the inactivation of cAMP dependent protein kinase in mammalian cells has been developed. PCR-directed mutagenesis of the CHO RI cDNA cloned into a mammalian expression system is done to create a RI mutant library which can be electroporated into CHO cells followed by selection of dominant negative mutants. This vector system promises to be useful for analysis of cAMP effects in mammalian cells.