New host cell system for regulated simian virus 40 DNA replication

R. D. Gerard, Y. Gluzman

Research output: Contribution to journalArticle

91 Scopus citations

Abstract

Transformed monkey cell lines (CMT and BMT) that inducibly express simian virus 40 (SV40) T antigen from the metallothionein promoter have been isolated and characterized. Immunoprecipitaition of pulse-labeled T antigen demonstrates a 5- to 12-fold increase in the rate of syntheses on addition of heavy-metal inducers to the culture medium. Radioimmunoassay of cell extracts indicates the accumulation of three- to four-fold 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.

Original languageEnglish (US)
Pages (from-to)3231-3240
Number of pages10
JournalMolecular and cellular biology
Volume5
Issue number11
DOIs
StatePublished - Jan 1 1985

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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