GENETIC AND BIOCHEMICAL ANALYSIS OF CELL BEHAVIOR

中国仓鼠卵巢(CHO)成纤维细胞是一个很好的模型系统 对某些方面的遗传和生物化学进行研究 培养细胞的行为。我们正在使用这个系统来分析 环磷酸腺苷调节细胞生长和基因表达的方式 哺乳动物细胞。中国仓鼠卵巢细胞cAMP作用的机制是 通过建立抗cAMP抑制生长的细胞系进行研究。 对cAMP产生抗药性的一种方式是通过突变 CAMP依赖的蛋白激酶，导致调控改变 (RI)或催化(C)亚基。CAMP依赖的蛋白激酶缺陷 阻断cAMP刺激的转录并减少血管紧张素转换酶基因表达 CHO细胞和肾上腺Y-1细胞的多药转运蛋白。我们有 构建了CHO RI亚基的几种突变形式和一个突变体C 亚基，并在大肠杆菌中进行了表达。细菌表达的突变体RI‘s C识别RI和cAMP与RI结合的影响部位 在结合C亚基和cAMP刺激方面有缺陷 RI和C亚基的解离。一种改进的系统，用于 哺乳动物细胞中cAMP依赖的蛋白激酶失活 发展起来的。克隆到A株中的CHO RI基因的PCR诱变 哺乳动物表达系统用于创建RI突变体文库 可通过电穿孔进入CHO细胞后进行显性选择 阴性变种人。这个矢量系统有望对分析有用。 在哺乳动物细胞中的cAMP效应。

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.