Isolation and characterization of a variant dihydrofolate reductase cDNA from methotrexate-resistant murine L5178Y cells.
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 phenylalanine (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 was approximately 20-fold less sensitive to methotrexate inhibition 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.
McIvor
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RS
Institute of Human Genetics, University of Minnesota, Minneapolis 55455.
Simonsen
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004351:Drug Resistance / Q000235:genetics; D005786:Gene Expression Regulation; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014364:Tryptophan / Q000378:metabolism
D010649:Phenylalanine; D014364:Tryptophan; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate