Isolation and expression of an altered mouse dihydrofolate reductase cDNA.

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Abstract
A c[complementary]DNA library was constructed 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 a cDNA clone was isolated similar to, but distinquishable from, a cDNA clone previously demonstrated to encode the wild-type enzyme. Analysis of the nucleotide sequence of this cDNA clone predicts that the altered dihydrofolate reductase differs from the wild-type enzyme at a single amino acid, reflecting the substitution of an Arg for a Leu 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, the region of the cDNA that specified resistance to methotrexate was genetically localized by in vitro recombination. A single nucleotide change in the codon specifying amino acid 22 of the enzyme was sufficient to alter the methotrexate sensitivity of the enzyme. This altered gene can be employed as a dominant selectable marker in cultured cells expressing normal levels of wild-type dihydrofolate reductase.