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 language||English (US)|
|Number of pages||5|
|Journal||Journal of Biological Chemistry|
|State||Published - May 15 1990|
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