GENETIC AND BIOCHEMICAL ANALYSIS OF CELL BEHAVIOR

细胞行为的遗传和生化分析

• 批准号: 3796482
• 负责人: M M GOTTESMAN
• 金额: --
• 依托单位: DIVISION OF CANCER BIOLOGY AND DIAGNOSIS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3796482
• 关键词: CHO cells; Escherichia coli; Pseudomonas; cell growth regulation; complementary DNA; cyclic AMP; cytogenetics; drug resistance; electroporation; enzyme activity; enzyme complex; fibroblasts; gel electrophoresis; gene expression; gene mutation; genetic library; genetic regulation; genetic transcription; messenger RNA; molecular cloning; mutant; neoplasm /cancer genetics; nucleic acid sequence; phosphodiesterases; protein kinase; protein kinase A; tissue /cell culture; transforming growth factors; transposon /insertion element; yeasts

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.

中国仓鼠卵巢（CHO）成纤维细胞是一个很好的模型系统 用于研究某些方面的遗传学和生物化学， 培养细胞的行为。 我们用这个系统来分析 环腺苷酸调节细胞生长和基因表达的方式， 哺乳动物细胞 CHO细胞的cAMP作用机制已被证实。 通过产生对cAMP的生长抑制具有抗性的细胞系来研究。 产生对cAMP抗性的一种方式是通过以下突变： cAMP依赖性蛋白激酶，其导致改变的调节 (RI)或催化（C）亚基。 cAMP依赖性蛋白激酶缺陷 阻断cAMP刺激的转录并减少mRNA的量， CHO细胞和肾上腺Y-1细胞中的多药物转运蛋白。 我们有 构建了CHO RI亚基的几种突变形式和突变C 亚基并在大肠杆菌中表达。细菌表达的突变RI 影响C识别RI和cAMP与RI结合的位点 在与C亚基的结合和cAMP刺激的 RI和C亚基的解离。 一种改进的系统， 哺乳动物细胞中cAMP依赖性蛋白激酶的失活已经被 开发 将CHO RI cDNA克隆到一个 进行哺乳动物表达系统以创建RI突变体文库， 可以电穿孔到CHO细胞中，然后选择显性的 阴性突变体 这个矢量系统有望用于分析 cAMP在哺乳动物细胞中的作用。