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 从遗传学和生物化学的角度研究银屑病的某些方面 培养细胞的行为。我们的工作强调了形态 以及它与增长控制的关系，以及 环磷酸腺苷调节细胞生长和基因表达。这个 CAMP对CHO细胞作用机制的研究 筛选抗cAMP抑制生长的CHO突变体。 这些突变体的cAMP依赖蛋白激酶有缺陷。 具有改变的调节(RI)或催化(C)亚基。我们 使用了携带显性cAMP抗性的细胞的DNA 将cAMP抗性表型转化为敏感表型的缺陷 细胞。这些细胞系的突变机制是 用克隆的RI和C亚基探针进行研究。克隆的片段 RI是从我们的COSMID文库中得到的 突变的细胞系。这些研究的目的是分离克隆的 突变型cAMP依赖蛋白激酶亚基用作 能够使蛋白质失活的可移动的遗传元件 多种分化细胞中的激酶系统与转基因 动物。我们还利用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 由cAMP以依赖于cAMP的方式进行正向调节 依赖蛋白激酶活性。

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.