A bacteriophage T3 promoter can be linked to a lethal gene without detectable toxicity for eukaryotic cells. Interest for inducible transgenes.
The bacteriophage T3 promoter can be selectively transcribed by the corresponding RNA polymerase in eukaryotic cells. A toxic gene can in principle be linked to this promoter in a "dormant" and innocuous transgene in a transgenic animal. In this scheme, the activating strain expresses the RNA polymerase. When expression of the gene is needed in the progeny, the 2 lines are crossed. However, when a single molecule is sufficient to kill the cell--as with the diphtheria toxin--transcriptional "leakage" from the promoter may not be tolerated by the cell, even when extremely weak. Therefore, prior to more elaborate studies, diphtheria toxin, as a prototype of a gene toxic to the organism, has been linked to the bacteriophage T3 promoter in a T3-E-DTA construct. The T3-E-DTA plasmid has been transiently transfected into human embryonic kidney derived cells together with a lacZ plasmid. By co-transfection, the T3-E-DTA cells can be readily identified as lacZ positive, and their fate followed by the production of beta-galactosidase at the single cell or overall population level. In spite of the extreme toxicity of the toxin, the cells tolerate the presence of the T3-E-DTA construct, and are only killed--with a high efficiency--when the T3 RNA polymerase is present. Transactivation is usually restricted to the auxiliary factors of transcription. With this study, the promoter and the polymerase are revealed as potential and efficient inducible and activating elements of a very simple binary system.
Amouyal
M
M
URA 1149 CNRS, Département de biologie moléculaire, Institut Pasteur, Paris, France.
D017903:Bacteriophage T3 / Q000201:enzymology / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004167:Diphtheria Toxin / Q000235:genetics; D005804:Genes, Lethal / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000166:cytology*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014162:Transfection
D004167:Diphtheria Toxin; D012321:DNA-Directed RNA Polymerases