GENETIC AND BIOCHEMICAL ANALYSIS OF CELL BEHAVIOR

我们正在利用中国仓鼠卵巢（CHO）成纤维细胞研究 遗传学和生物化学的某些方面的行为培养 细胞我们最近的工作强调了环磷酸腺苷 调节CHO细胞中的细胞生长和基因表达。的机理 通过分离CHO突变体研究了cAMP对CHO细胞的作用 对cAMP的生长抑制有抗性。这些变种人有缺陷 cAMP依赖性蛋白激酶具有改变的调节（RI）或 催化（C）亚基。来自携带显性cAMP抗性的细胞的DNA 缺陷可用于将cAMP抗性表型转移到敏感的表型。 细胞，以及编码突变调节亚基的cDNA表达载体。 cAMP依赖性蛋白激酶缺陷阻断cAMP刺激 转录和减少的mRNA量的多药转运蛋白， CHO细胞。我们构建了CHO RI亚基的几种突变形式 并在大肠杆菌中表达。细菌表达的突变RI具有 与C亚基结合的缺陷和cAMP刺激的 RI和C亚基的解离。酵母磷酸二酯酶（PDE）也具有 在CHO细胞中表达。PDE基因的表达导致 对cAMP不敏感。为了测试消融cAMP反应在 小鼠胰岛细胞，携带酵母PDE基因的转基因小鼠 在胰岛素启动子的控制下，

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.