| First Author | Simonsen CC | Year | 1983 |
| Journal | Proc Natl Acad Sci U S A | Volume | 80 |
| Issue | 9 | Pages | 2495-9 |
| PubMed ID | 6573667 | Mgi Jnum | J:25022 |
| Mgi Id | MGI:72738 | Doi | 10.1073/pnas.80.9.2495 |
| Citation | Simonsen CC, et al. (1983) Isolation and expression of an altered mouse dihydrofolate reductase cDNA. Proc Natl Acad Sci U S A 80(9):2495-9 |
| abstractText | 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. |
