Human cell lines expressing hormone regulated T7 RNA polymerase localized at distinct intranuclear sites.
Although several systems are now available for the controlled expression of eukaryotic genes transcribed by RNA polymerase II, regulated expression has been more difficult to achieve in the case of genes transcribed by RNA polymerase III. In the present study the gene for bacteriophage T7 RNA polymerase, implanted with a eukaryotic nuclear localization signal, was linked to a 5'-flanking ecdysone-responsive promoter and stably transformed human cell lines were constructed in which the ecdysone promoter-T7 RNA polymerase gene had been integrated intact, as demonstrated by a polymerase chain reaction assay. Exposure of these cells to the ecdysone analog ponasterone A resulted in the appearance of a single protein having the expected size of T7 RNA polymerase in immunoblots of cell extracts probed with an affinity purified antibody raised against the C-terminus of T7 RNA polymerase. The induced T7 RNA polymerase was exclusively localized in the nucleus of induced cells and was undetectable in uninduced cells either by immunoblotting or immunofluorescence. The induced T7 RNA polymerase was present at numerous punctate foci dispersed throughout the nucleoplasmic regions of the nucleus and was also present in the nucleoli. Both of these observed intranuclear localizations have relevance to the potential applications of this system.
Yarovoi
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SV
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 377 Plantation Street, Worcester, MA 01605, USA.
Pederson
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D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D004440:Ecdysone / Q000235:genetics / Q000494:pharmacology; D004441:Ecdysterone / Q000031:analogs & derivatives* / Q000235:genetics / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006728:Hormones / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D008858:Microscopy, Phase-Contrast; D019913:Nuclear Localization Signals / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014764:Viral Proteins
D006728:Hormones; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D004440:Ecdysone; D004441:Ecdysterone; C008999:ponasterone A; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases