New host cell system for regulated simian virus 40 DNA replication.

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RESUMO

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.

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