Authors: | Dicker, A. P.; Volkenandt, M.; Schweitzer, B. I.; Banerjee, D.; Bertino, J. R. |
Article Title: | Identification and characterization of a mutation in the dihydrofolate reductase gene from the methotrexate-resistant Chinese hamster ovary cell line pro-3 Mtx(RIII) |
Abstract: | A methotrexate-resistant Chinese hamster ovary cell line (Pro-3 Mtx(RIII)), 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 Mtx(RIII) 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 Mtx(RIII) 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. |
Keywords: | gene mutation; mutation; nonhuman; methotrexate; animal cell; animal; gene amplification; cell line; drug resistance; animalia; ovary; dna; amino acid sequence; molecular sequence data; kinetics; cell culture; base sequence; binding sites; molecular structure; leucine; dihydrofolate reductase; tetrahydrofolate dehydrogenase; phenylalanine; cho cell; cricetinae; cricetulus griseus; dna-directed dna polymerase; hamster; female; priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s.; hamsters; taq polymerase |
Journal Title: | Journal of Biological Chemistry |
Volume: | 265 |
Issue: | 14 |
ISSN: | 0021-9258 |
Publisher: | American Society for Biochemistry and Molecular Biology |
Date Published: | 1990-05-15 |
Start Page: | 8317 |
End Page: | 8321 |
Language: | English |
PUBMED: | 2335526 |
PROVIDER: | scopus |
DOI/URL: | |
Notes: | Source: Scopus |