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  Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity

Urlinger, S., Baron, U., Thellmann, M., Hasan, M. T., Bujard, H., & Hillen, W. (2000). Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proceedings of the National Academy of Sciences of the United States of America, 97(14), 7963-7968. doi:10.1073/pnas.130192197.

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Item Permalink: http://hdl.handle.net/21.11116/0000-0002-4679-8 Version Permalink: http://hdl.handle.net/21.11116/0000-0002-467A-7
Genre: Journal Article

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 Creators:
Urlinger, Stefanie, Author
Baron, Udo, Author
Thellmann, Marion, Author
Hasan, Mazahir T.1, 2, Author              
Bujard, Hermann, Author
Hillen, Wolfgang, Author
Affiliations:
1Department of Molecular Neurobiology, Max Planck Institute for Medical Research, Max Planck Society, ou_1497704              
2Department of Biomedical Optics, Max Planck Institute for Medical Research, Max Planck Society, ou_1497699              

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 Abstract: Regulatory elements that control tetracycline resistance in Escherichia coli were previously converted into highly specific transcription regulation systems that function in a wide variety of eukaryotic cells. One tetracycline repressor (TetR) mutant gave rise to rtTA, a tetracycline-controlled transactivator that requires doxycycline (Dox) for binding to tet operators and thus for the activation of P(tet) promoters. Despite the intriguing properties of rtTA, its use was limited, particularly in transgenic animals, because of its relatively inefficient inducibility by doxycycline in some organs, its instability, and its residual affinity to tetO in absence of Dox, leading to elevated background activities of the target promoter. To remove these limitations, we have mutagenized tTA DNA and selected in Saccharomyces cerevisiae for rtTA mutants with reduced basal activity and increased Dox sensitivity. Five new rtTAs were identified, of which two have greatly improved properties. The most promising new transactivator, rtTA2(S)-M2, functions at a 10-fold lower Dox concentration than rtTA, is more stable in eukaryotic cells, and causes no background expression in the absence of Dox. The coding sequences of the new reverse TetR mutants fused to minimal activation domains were optimized for expression in human cells and synthesized. The resulting transactivators allow stringent regulation of target genes over a range of 4 to 5 orders of magnitude in stably transfected HeLa cells. These rtTA versions combine tightness of expression control with a broad regulatory range, as previously shown for the widely applied tTA.

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Language(s): eng - English
 Dates: 2000-02-042000-04-282000-07-05
 Publication Status: Published in print
 Pages: 6
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 Rev. Type: Peer
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Title: Proceedings of the National Academy of Sciences of the United States of America
  Other : Proc. Acad. Sci. USA
  Other : Proc. Acad. Sci. U.S.A.
  Other : Proceedings of the National Academy of Sciences of the USA
  Abbreviation : PNAS
Source Genre: Journal
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Publ. Info: Washington, D.C. : National Academy of Sciences
Pages: - Volume / Issue: 97 (14) Sequence Number: - Start / End Page: 7963 - 7968 Identifier: ISSN: 0027-8424
CoNE: https://pure.mpg.de/cone/journals/resource/954925427230