A PHAGE T7 CLASS-III PROMOTER FUNCTIONS AS A POLYMERASE-II PROMOTER IN MAMMALIAN-CELLS

A phage T7 class-III promoter (pT7), which is highly specific for T7 R NA polymerase in bacteria, was tested in mammalian cells for its speci ficity. After having shown that T7 RNA polymerase can transcribe from pT7 in the nucleus of stably transformed cells [Lieber et al., Nucleic Acids Res. 17 (1989) 8485-8493], we describe here that pT7 could also direct efficient intracellular gene expression in the absence of T7 R NA polymerase. Using the genomic human growth hormone-encoding gene an d the firefly luciferase-encoding gene as reporters, we found expressi on levels comparable with those obtained with the Rous sarcoma viral p romoter. Inhibition of expression with alpha-amanitin suggests that tr anscription is by RNA polymerase 11. Binding studies with HeLa cell ex tracts clearly show that synthetic pT7 sequences are specifically boun d (gel retardation) and that the promoter region is protected from DNa se degradation. The experimental data, as well as the nucleotide seque nce, suggest that pT7 has properties of an initiator element. Indeed, the activity of pT7 can be stimulated by the presence of an upstream e lement or an enhancer. These results have practical implications for t he use of pT7 in mammalian expression vectors. Commercial pT7 plasmids can be used for both prokaryotic and eukaryotic expression systems.