Abraham I, Brill S, Hyde J, Fleischmann R, Chapman M, Gottesman M M
J Biol Chem. 1985 Nov 15;260(26):13934-40.
We have used DNA-mediated gene transfer of genomic DNA to introduce into wild-type Chinese hamster ovary (CHO) cells a mutant gene that confers resistance to the growth inhibitory effect of cAMP. This dominant mutation in CHO cell line 10248 is responsible for an alteration in the RI subunit (RI*) of the type I cAMP-dependent protein kinase (Singh, T. J., Hochman, J., Verna, R., Chapman, M., Abraham, I., Pastan, I.H., and Gottesman, M.M. (1985) J. Biol. Chem. 260, 13927-13933). The transformant 11564 which was studied in detail, has the same characteristics as the original mutant 10248 including continued growth in medium containing 8-Br-cAMP, an increase in the Ka for cAMP activation of the kinase, a greatly reduced amount of type II protein kinase activity, an altered incorporation of the photoaffinity label 8-N3[32P]cAMP into the RI* subunit of PKI, and an absence of cAMP-dependent phosphorylation of a Mr = 52,000 protein in intact cells. In addition, analysis of the DNA of the transformant indicates the presence of an increased amount of DNA for the RI gene. These results are consistent with the transfer of a mutant gene for the RI* subunit of the cAMP-dependent protein kinase and its phenotypic expression in the transformant and also support the hypothesis that the mutation responsible for the defect in cell line 10248 is due to an alteration in the gene for RI.
我们已利用基因组DNA的DNA介导基因转移，将一个赋予对cAMP生长抑制作用抗性的突变基因导入野生型中国仓鼠卵巢（CHO）细胞。CHO细胞系10248中的这种显性突变导致I型cAMP依赖性蛋白激酶的RI亚基（RI*）发生改变（辛格，T.J.，霍克曼，J.，韦尔纳，R.，查普曼，M.，亚伯拉罕，I.，帕斯坦，I.H.，和戈特斯曼，M.M.（1985年）《生物化学杂志》260，13927 - 13933）。对详细研究的转化体11564进行分析，发现其具有与原始突变体10248相同的特征，包括在含有8 - Br - cAMP的培养基中持续生长、激酶对cAMP激活的Ka增加、II型蛋白激酶活性大幅降低、光亲和标记8 - N3[32P]cAMP掺入PKI的RI亚基发生改变，以及完整细胞中Mr = 52,000蛋白不存在cAMP依赖性磷酸化。此外，对转化体DNA的分析表明RI基因的DNA量有所增加。这些结果与cAMP依赖性蛋白激酶RI亚基的突变基因转移及其在转化体中的表型表达一致，也支持了细胞系10248缺陷所对应的突变是由于RI基因改变这一假说。
