Simonsen C C, Levinson A D
Proc Natl Acad Sci U S A. 1983 May;80(9):2495-9. doi: 10.1073/pnas.80.9.2495.
We have constructed a cDNA library from a murine cell line expressing high levels of a dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) that displays an abnormally low affinity for methotrexate. From this library we have isolated a cDNA clone similar to, but distinguishable from, a cDNA clone previously demonstrated to encode the wild-type enzyme. Analysis of the nucleotide sequence of this cDNA clone allows us to predict that the altered dihydrofolate reductase differs from the wild-type enzyme at a single amino acid, reflecting the substitution of an arginine for a leucine residue in a region of the polypeptide thought to form a hydrophobic pocket essential for inhibitor binding. To confirm that this substitution was responsible for the altered properties of the enzyme, we genetically localized the region of the cDNA that specified resistance to methotrexate by in vitro recombination. These results reveal that a single nucleotide change in the codon specifying amino acid 22 of the enzyme was sufficient to alter the methotrexate sensitivity of the enzyme. We demonstrate that this altered gene can be employed as a dominant selectable marker in cultured cells expressing normal levels of wild-type dihydrofolate reductase.
我们从一个鼠细胞系构建了一个互补DNA（cDNA）文库，该细胞系表达高水平的二氢叶酸还原酶（四氢叶酸脱氢酶；5,6,7,8-四氢叶酸：NADP+氧化还原酶，EC 1.5.1.3），其对甲氨蝶呤的亲和力异常低。从这个文库中，我们分离出了一个cDNA克隆，它与先前证明编码野生型酶的cDNA克隆相似，但又有所不同。对这个cDNA克隆的核苷酸序列分析使我们能够预测，改变后的二氢叶酸还原酶与野生型酶在单个氨基酸上存在差异，这反映在多肽的一个区域中，一个精氨酸取代了一个亮氨酸残基，该区域被认为形成了对抑制剂结合至关重要的疏水口袋。为了证实这种取代是酶性质改变的原因，我们通过体外重组在基因上定位了cDNA中赋予对甲氨蝶呤抗性的区域。这些结果表明，在编码该酶第22位氨基酸的密码子中的单个核苷酸变化足以改变该酶对甲氨蝶呤的敏感性。我们证明，这个改变的基因可以用作在表达正常水平野生型二氢叶酸还原酶的培养细胞中的显性选择标记。
