Co-regulation of two gene activities by tetracycline via a bidirectional promoter

Recently, we have described a regulatory system that allows the stringent control of individual gene activities in higher eukaryotic cell lines, in plants and in animals. The essential components of this system are (i) an RNA polymerase II minimal promoter placed downstream of multiple operator sequences (tetO) of the Escherichia coli Tn10 tetracycline resistance operon and (ii) a fusion between the Tet repressor (TetR) and the herpes simplex virus protein 16 (VP16), named tTA. In the absence of tetracycline (Tc), tTA binds to the tet operators to activate transcription from the minimal promoter, whereas in the presence of Tc its association and consequently its transcription activation is prevented. After the binding of tTA, minimal promoters derived from the cytomegalovirus IE promoter (P sub(hCMV)) and fused to seven tetO sequences reach the remarkable strength of the parent promoter in HeLa cells when compared in transient expression assays. This high activation potential of tTA and the arrangement of its binding sites within P sub(hCMV*-1) suggested the design of a bidirectional promoter which would allow the simultaneous regulation of two transcriptional units from centrally located multiple tetO sequences. Such a promoter should be useful for a number of experimental approaches. First, it may allow the co-regulation of the synthesis of two gene products in stoichiometric amounts, frequently a prerequisite for the production of heterodimeric (or hetero-oligomeric) proteins. Second, by fusing minimal promoters of differing efficiencies to the centrally located tetO sequences, two gene products may be co-regulated at different but defined levels. Third, by integrating an appropriate reporter gene at one side of the bidirectional promoter, the regulation of a not-readily-assayable gene of interest may be monitored via the reporter function. This latter possibility may also facilitate-at the cellular as well as the organismal level-the screening for properly integrated expression units controlling the gene of interest. We report the construction of a bidirectional promoter P sub(bi-1) and show that two reporter genes encoding beta -galactosidase and luciferase are indeed co-regulated by this promoter in a quantitative manner. Moreover, we describe a family of vectors which readily allows the use of P sub(bi-1) for various purposes.