Selective amplification in methotrexate-resistant mouse cells of an artificial dihydrofolate reductase transcription unit making use of cryptic splicing and polyadenylation sites.
We have constructed a recombinant ( pMOP ) which is based on the bacterial plasmid pML2 and contains bovine papilloma virus type 1 (BPV 1) sequences linked to an artificial mouse dihydrofolate reductase (DHFR) transcription unit. This unit consists of the SV40 early gene promoter, a complete DHFR coding sequence and splice and polyadenylation sites from a rabbit beta-globin gene. Intact pMOP or a fragment thereof devoid of pML2 sequences will transform mouse cells to methotrexate resistance. The lines obtained contain approximately 200 copies of the DHFR transcription unit. In no case, however, did we find evidence of extrachromosomal maintenance of BPV 1 or DHFR sequences. One line, when selected for resistance to elevated levels of methotrexate, contained amplified copies of a 'novel' DHFR transcription unit resulting from fusion of two normal units. This line contained the DHFR RNA species present in the parent line plus some additional species. The structure of these various RNA species was determined and evidence found for the use of cryptic splice and polyadenylation sites.
D000818:Animals; D052688:Bovine papillomavirus 1 / Q000235:genetics*; D002460:Cell Line; D002472:Cell Transformation, Viral; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant; D004351:Drug Resistance; D005347:Fibroblasts / Q000201:enzymology; D005784:Gene Amplification; D005914:Globins / Q000235:genetics; D008727:Methotrexate / Q000494:pharmacology*; D051379:Mice; D027383:Papillomaviridae / Q000235:genetics*; D010957:Plasmids; D011061:Poly A / Q000378:metabolism; D012313:RNA; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014158:Transcription, Genetic
D004274:DNA, Recombinant; D011061:Poly A; D012313:RNA; D005914:Globins; D013762:Tetrahydrofolate Dehydrogenase; D004262:DNA Restriction Enzymes; D008727:Methotrexate