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 RNA polymerase in bacteria, was tested in mammalian cells for its specificity. 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 RNA polymerase. Using the genomic human growth hormone-encoding gene and the firefly luciferase-encoding gene as reporters, we found expression levels comparable with those obtained with the Rous sarcoma viral promoter. Inhibition of expression with alpha-amanitin suggests that transcription is by RNA polymerase II. Binding studies with HeLa cell extracts clearly show that synthetic pT7 sequences are specifically bound (gel retardation) and that the promoter region is protected from DNase degradation. The experimental data, as well as the nucleotide sequence, suggest that pT7 has properties of an initiator element. Indeed, the activity of pT7 can be stimulated by the presence of an upstream element or an enhancer. These results have practical implications for the use of pT7 in mammalian expression vectors. Commercial pT7 plasmids can be used for both prokaryotic and eukaryotic expression systems.
Sandig
V
V
Max-Planck-Gesellschaft, Arbeitsgruppe Zellteilungsregulation and Gensubstitution, Berlin-Buch, Germany.
Lieber
A
A
Bähring
S
S
Strauss
M
M
D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D001517:Coleoptera / Q000201:enzymology; D004257:DNA Polymerase II / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D013006:Growth Hormone / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007739:L Cells; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins
D009838:Oligodeoxyribonucleotides; D011506:Proteins; D014764:Viral Proteins; D013006:Growth Hormone; D008156:Luciferases; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004257:DNA Polymerase II