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.
Highlights
Chinese hamster ovary cell line (Pro-3 MtxR”‘), resistant due to a low-level amplified, altered target enzyme, dihydrofolate reductase (DHFR), has been characterized on the molecular level
We report the identification of a mutation in the DHFR gene from a MTX-resistant CHO cell line, Pro-3 MtxR”’, originally described by Flintoff et al [14,15]
In Vitro Amplification and DNA Sequencing of DHFR Gene and Transcript-In uitro amplification of the entire peptide coding region of the CHO DHFR cDNA for both cell lines is shown in Fig. 1
Summary
Materials-Reagents for mRNA isolation and cDNA synthesis were obtained from Invitrogen (San Diego, CA). RNase-free siliconized tubes were obtained from National Scientific (San Rafael, CA). Instruments (Norwalk, CT) or Stratagene (San Diego, CA). Agarose was obtained from FMC (Rockland, ME). Centricon 100 microconcentrators were obtained from Amicon Corp. Reagents for DNA sequencing were obtained from United States Bio-. The restriction enzyme BsaI was a gift from New England Biolabs, Inc. MTX was a gift from Dr Harriet Kiltie (Lederle Laboratories)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
