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 recent work has emphasized the manner in which cyclic AMP regulates cell growth and gene expression in CHO cells. 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 as can cDNA expression vectors encoding mutant regulatory subunits. Defects in cAMP-dependent protein kinase block cAMP-stimulated transcription and reduce amounts of mRNA for the multidrug transporter in CHO cells. We have constructed several mutant forms of the CHO RI subunit and expressed these in E.coli. Bacterially expressed mutant RI's have defects in associating with the C subunit and in cAMP-stimulated dissociation of RI and C subunits. A yeast phosphodiesterase (PDE) has also been expressed in CHO cells. Expression of the PDE gene results in insensitivity to cAMP. To test the effect of ablating cAMP responses in pancreatic islet cells in mice, transgenic mice carrying the yeast PDE gene under control of an insulin promoter have been constructed.