Molecular cloning of Chinese hamster dihydrofolate reductase-specific cDNA and the identification of multiple dihydrofolate reductase mRNAs in antifolate-resistant Chinese hamster lung fibroblasts.
ds cDNA from antifolate-resistant Chinese hamster lung fibroblast subline DC-3F/MQ19 was ligated to Eco RI and Sal I oligonucleotide linkers and cloned into Eco RI and Sal I digested pBR322. Transformed colonies containing dihydrofolate reductase (DHFR)-specific recombinant plasmid were identified by Grunstein Hogness assay using a Chinese hamster DHFR-specific cDNA probe. A recombinant plasmid, pDHFR6, containing a 650 bp HFR insert was isolated and analyzed. This plasmid was used as a molecular probe in a Northern blot analysis of both cytoplasmic and polysomal DHFR, poly A+ mRNAs of the DC-3F/MQ19 subline, which over-produces a 20,000d DHFR 150-fold, and DC-3F/A3 subline, which over-produces a 21,000d DHFR 170-fold. This analysis revealed the presence of three DHFR mRNA species of 1350, 2200, and 3300 nucleotides in both independently-derived cell lines. The relative abundance of each species however varied strikingly between the two cell lines.
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism; D004351:Drug Resistance; D005493:Folic Acid Antagonists / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics*; D008168:Lung; D008970:Molecular Weight; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
D004274:DNA, Recombinant; D005493:Folic Acid Antagonists; D007527:Isoenzymes; D012333:RNA, Messenger; D013762:Tetrahydrofolate Dehydrogenase; D004262:DNA Restriction Enzymes