GENETIC AND BIOCHEMICAL ANALYSIS OF CELL BEHAVIOR

细胞行为的遗传和生化分析

• 批准号: 3939315
• 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/3939315
• 关键词: Rous sarcoma virus; acyltransferase; cell growth regulation; chloramphenicol; cyclic AMP; cytogenetics; drug resistance; fibroblasts; gel electrophoresis; gene expression; genetic library; genetic manipulation; interferons; linkage mapping; molecular biology; molecular cloning; molecular oncology; morphology; mutant; natural gene amplification; neoplasm /cancer genetics; nucleic acid sequence; nucleoside inhibitor; protein kinase; tissue /cell culture; transposon /insertion element

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 work has emphasized morphology and its relationship to growth control, and the manner in which cyclic AMP regulates cell growth and gene expression. 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. We have used DNA from cells carrying dominant cAMP-resistant defects to transfer the cAMP-resistance phenotype to sensitive cells. The mechanism of mutation in these cell lines is being studied with cloned RI and C subunit probes. Cloned segments of RI have been sequenced from cosmid libraries prepared from our mutant cell lines. The intent of these studies is to isolate cloned mutant cAMP dependent protein kinase subunits for use as movable genetic elements capable of inactivating the protein kinase system in a variety of differentiated cells and transgenic animals. We have also used the CHO protein kinase mutants to demonstrate that the expression of many promoters linked to the reporter gene encoding chloramphenicol acetyl transferase is positively regulated by cAMP in a manner which depends on cAMP dependent protein kinase activity.

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， 培养细胞的行为。 我们的工作强调形态学 及其与生长控制的关系，以及 环AMP调节细胞生长和基因表达。 的 cAMP对CHO细胞的作用机制已通过以下方法研究： 分离抗cAMP生长抑制的CHO突变体。 这些突变体具有缺陷的cAMP依赖性蛋白激酶 具有改变的调节（RI）或催化（C）亚基。 我们 使用了来自携带显性cAMP抗性细胞的DNA 将cAMP抗性表型转移到敏感表型的缺陷 细胞 这些细胞系中的突变机制正在研究中。 用克隆的RI和C亚基探针研究。 克隆片段 RI已经从我们的制备的粘粒文库中测序。 突变细胞系 这些研究的目的是分离克隆的 突变型cAMP依赖性蛋白激酶亚基用作 能够使蛋白质失活的可移动遗传元件 激酶系统在各种分化细胞和转基因 动物 我们还使用CHO蛋白激酶突变体， 证明了许多启动子的表达与 编码氯霉素乙酰转移酶的报告基因， 以依赖于cAMP的方式被cAMP正向调节 依赖性蛋白激酶活性。