Caras I W, Martin D W
Genentech, Inc., South San Francisco, California 94080.
Mol Cell Biol. 1988 Jul;8(7):2698-704. doi: 10.1128/mcb.8.7.2698-2704.1988.
Mammalian ribonucleotide reductase is regulated by the binding of dATP and other nucleotide effectors to allosteric sites on subunit M1. Using mRNA from a mutant mouse T-lymphoma (S49) cell line, we have isolated a cDNA which encodes an altered, dATP feedback-resistant subunit M1. The mutant cDNA contains a single point mutation (a G-to-A transition) at codon 57, converting aspartic acid to asparagine. Proof that this mutation is responsible for the phenotype of dATP feedback resistance is provided by the following evidence. (i) The mutation was detected only in mutant S49 cells containing dATP feedback-resistant ribonucleotide reductase and not in wild-type or other mutant S49 cells. (ii) Transfection of Chinese hamster ovary cells with an expression plasmid containing the mutant M1 cDNA resulted in the production of dATP feedback-resistant ribonucleotide reductase. Transfected CHO cells expressing the mutant M1 cDNA exhibited a 15- to 25-fold increase in the frequency of spontaneous mutation to 6-thioguanine resistance, confirming that dATP feedback-resistant ribonucleotide reductase produces a mutator phenotype in mammalian cells. The availability of a cDNA which encodes dATP feedback-resistant subunit M1 thus provides a means of manipulating by transfection the frequency of spontaneous mutation in mammalian cells.
哺乳动物核糖核苷酸还原酶受dATP和其他核苷酸效应物与亚基M1变构位点结合的调控。利用来自突变小鼠T淋巴瘤（S49）细胞系的mRNA，我们分离出了一个cDNA，它编码一种改变的、对dATP反馈有抗性的亚基M1。该突变cDNA在密码子57处有一个单点突变（G到A的转换），将天冬氨酸转变为天冬酰胺。以下证据证明了这种突变导致了对dATP反馈抗性的表型。（i）仅在含有对dATP反馈有抗性的核糖核苷酸还原酶的突变S49细胞中检测到该突变，而在野生型或其他突变S49细胞中未检测到。（ii）用含有突变M1 cDNA的表达质粒转染中国仓鼠卵巢细胞，导致产生对dATP反馈有抗性的核糖核苷酸还原酶。表达突变M1 cDNA的转染CHO细胞对6-硫鸟嘌呤抗性的自发突变频率增加了15至25倍，证实对dATP反馈有抗性的核糖核苷酸还原酶在哺乳动物细胞中产生诱变表型。因此，编码对dATP反馈有抗性的亚基M1的cDNA的可用性提供了一种通过转染来操纵哺乳动物细胞中自发突变频率的方法。
