Abstract
Dihydrofolate reductase (DHFR) cDNA sequences were isolated from a methotrexate-resistant mouse L5178Y cell line previously shown to contain methotrexate resistant dihydrofolate reductase enzyme activity. Specifically-primed reverse transcription products were amplified using the polymerase chain reaction and then cloned into a mammalian expression plasmid. Candidate clones were identified by restriction analysis and then functionally tested by transfection into mouse 3T3 fibroblasts, selecting for methotrexate-resistant colonies. Sequence analysis of the cDNA clones demonstrated the substitution of tryptophan (TGG) in place of the wild-type phenylalanlne (TTC) at codon 31. Sequencing of PCR-amplified genomic DNA extracted from the drug-resistant L5178Y cells confirmed the tryptophan codon at position 31. Transfection of mammalian tissue culture cells with expression plasmids containing the trp31 DHFR sequence resulted in substantial methotrexate-resistant colony formation. Recombinant trp31 DHFR enzyme activity expressed in stably-transfected Chinese hamster ovary cells wasapproximately 20-fold less sensitive to methotrexateinhibition than wild-type mouse DHFR enzyme activity.We conclude that the cloned Trp31 DHFR sequence encodes an enzyme substantially resistant to methotrexate which confers a drug-resistance phenotype to cells in which it is expressed.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 7025-7032 |
| Number of pages | 8 |
| Journal | Nucleic acids research |
| Volume | 18 |
| Issue number | 23 |
| DOIs | |
| State | Published - 1990 |
Bibliographical note
Funding Information:We are grateful to Dr. J. Goldie for providing the methotrexate-resistant L5178Y-R4 cells and to Marne Power, Jackie Kennedy, Kirstin Thompson and Fran Dunn for excellent technical assistance. This work was supported in part by grants (to R.S.M.) from the National Cancer Institute (CA46878), the American Cancer Society (IN-13 and JFRA247) and the Leukemia Research Foundation (University of Minnesota). Portions of this material were presented in brief at the 1990 UCLA Symposium, Manipulating the Mammalian Genome (J. Cell. Biochem., 14A, D417).