Identification and characterization of a mutation in the dihydrofolate reductase gene from the methotrexate-resistant Chinese hamster ovary cell line Pro-3 MtxRIII

Adam P. Dicker, Matthias Volkenandt, Barry I. Schweitzer, Debabrata Banerjee, Joseph Bertino

Research output: Contribution to journalArticle

45 Scopus citations

Abstract

A methotrexate-resistant Chinese hamster ovary cell line (Pro-3 MtxRIII), resistant due to a low-level amplified, altered target enzyme, dihydrofolate reductase (DHFR), has been characterized on the molecular level. The cDNA and coding regions of all six DHFR exons were amplified in vitro using Taq polymerase and directly sequenced. Analysis of the Pro-3 MtxRIII DHFR cDNA demonstrated a C → T base transition at nucleotide 67 that results in the substitution of phenylalanine for leucine at residue 22 and the loss of a BsaI site in the Pro-3 MtxRIII cDNA. This mutation results in a decreased binding of methotrexate to the altered enzyme. Molecular modeling of Leu22 → Phe supports the concept of the importance of Leu22 in the active site of the enzyme and indicates that replacement with phenylalanine will decrease the binding of methotrexate.

Original languageEnglish (US)
Pages (from-to)8317-8321
Number of pages5
JournalJournal of Biological Chemistry
Volume265
Issue number14
StatePublished - May 15 1990
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry

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