MOLECULAR-CLONING OF THE CDNA FOR A MUTANT MOUSE RIBONUCLEOTIDE REDUCTASE M1 THAT PRODUCES A DOMINANT MUTATOR PHENOTYPE IN MAMMALIAN-CELLS
MOLECULAR-CLONING OF THE CDNA FOR A MUTANT MOUSE RIBONUCLEOTIDE REDUCTASE M1 THAT PRODUCES A DOMINANT MUTATOR PHENOTYPE IN MAMMALIAN-CELLS
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DOI：
10.1128/mcb.8.7.2698
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发表时间：
1988-07-01
影响因子：
5.3
通讯作者：
MARTIN, DW
中科院分区：
文献类型：
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作者：
CARAS, IW;MARTIN, DW
哺乳动物核糖核苷酸还原酶受dATP和其他核苷酸效应物与M1亚基上的变构位点结合的调节。使用突变的小鼠T淋巴瘤（S49）细胞系的mRNA，我们已经分离出一个cDNA编码改变，dATP反馈抗性亚基M1。突变体cDNA在密码子57处含有单点突变（G到A转变），将天冬氨酸转化为天冬酰胺。以下证据提供了该突变导致dATP反馈抗性表型的证据。(i)该突变仅在含有dATP反馈抗性核糖核苷酸还原酶的突变体S49细胞中检测到，而在野生型或其他突变体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dATP反馈抗性核糖核苷酸还原酶。表达突变体M1 cDNA的转染CHO细胞表现出自发突变为6-硫代鸟嘌呤抗性的频率增加15- 25倍，证实了dATP反馈抗性核糖核苷酸还原酶在哺乳动物细胞中产生突变子表型。因此，编码dATP反馈抗性亚基M1的cDNA的可用性提供了通过转染操纵哺乳动物细胞中自发突变频率的手段。
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.