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 研究行为的某些方面的遗传学和生物化学 培养的细胞。我们的工作强调了形态及其 与生长控制的关系，以及环状AMP 调节细胞生长和基因表达。CAMP的作用机制 分离CHO突变株研究其对CHO细胞的作用 能抵抗cAMP的生长抑制。这些变种人有 有缺陷的cAMP依赖蛋白激酶 调节性(RI)或催化(C)亚基。携带DNA的细胞的DNA 显性抗cAMP缺陷可以用来转移cAMP- 对敏感细胞的耐药表型。科斯米德克隆人携带 CAMP抗性细胞的CHO RI亚基已被分离。 序列分析表明，其中一个克隆pCosRI具有 Cho RI亚基的编码序列。外显子-内含子 RI基因结构复杂；编码cAMP的区域- 该基因3‘端的结合部位由几个 不同的外显子。CAMP反应蛋白表达的调控 正在使用野生型cAMP依赖蛋白来研究基因 激酶突变体和转移性CHO细胞株表达 突变的激酶表型。我们已经为老鼠证明了这一点 酪氨酸氨基转移酶(TAT)基因启动子完整的cAMP 依赖的蛋白激酶系统对细胞周期的调节是必需的 CAMP反应基因表达。

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. DNA from cells carrying dominant cAMP-resistant defects can be used to transfer the cAMP- resistance phenotype to sensitive cells. Cosmid clones carrying the CHO RI subunit from cAMP resistant cells have been isolated. Sequence analysis indicates that one of these clones, pcosRI has the coding sequence for the CHO RI subunit. The exon-intron structure of the RI gene is complex; the region encoding the cAMP- binding site at the 3' end of the gene is composed of several different exons. The regulation of expression of cAMP-responsive genes is being studied using wild type, cAMP dependent protein kinase mutants, and transferent CHO cell lines expressing the mutant kinase phenotype. We have demonstrated that for the rat tyrosine amino transferase (TAT) gene promoter an intact cAMP dependent protein kinase system is necessary for the regulation of cAMP responsive gene expression.