New host cell system for regulated simian virus 40 DNA replication.
Transformed monkey cell lines (CMT and BMT) that inducible express simian virus 40 (SV40) T antigen from the metallothionein promoter have been isolated and characterized. Immunoprecipitation of pulse-labeled T antigen demonstrates a 5- to 12-fold increase in the rate of synthesis on addition of heavy-metal inducers to the culture medium. Radioimmunoassay of cell extracts indicates the accumulation of three- to fourfold more total T antigen after 2 days of induction by comparison with uninduced controls. A direct correlation was found between the level of T-antigen synthesis and the extent of SV40 DNA replication in inducible cells. Inducible BMT cells expressing a low basal level of T antigen were efficiently transformed by a vector carrying the neomycin resistance marker and an SV40 origin of replication. These vector sequences were maintained in an episomal form in most G418-resistant cell lines examined and persisted even in the absence of biochemical selection. Extensive rearrangements were observed only if the vector contained bacterial plasmid sequences. Expression of a protein product under the control of the SV40 late promoter in such vectors was increased after heavy-metal-dependent amplification of the template. These results demonstrate the ability of BMT cells to maintain a cloned eucaryotic gene in an amplifiable episomal state.
D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor / Q000235:genetics; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004273:DNA, Neoplasm / Q000235:genetics; D005796:Genes; D006367:HeLa Cells; D006801:Humans; D007668:Kidney; D008668:Metallothionein / Q000235:genetics; D009856:Oncogene Proteins, Viral / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor; D004273:DNA, Neoplasm; D009856:Oncogene Proteins, Viral; D008668:Metallothionein; D004262:DNA Restriction Enzymes