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. 1996 Aug 1;24(15):2900–2904. doi: 10.1093/nar/24.15.2900

Bi-directional gene switching with the tetracycline repressor and a novel tetracycline antagonist.

J Chrast-Balz 1, R Hooft van Huijsduijnen 1
PMCID: PMC146027  PMID: 8760871

Abstract

We have screened a panel of tetracycline (tc)-like compounds for their potential use with tc-repressor (tetR) based gene switches. The interaction between tc and tetR appears quite specific, as only tc itself and its close homologues anhydro-tc and doxycycline strongly inhibited DNA binding. However, a single tc-like compound, GR33076X, increased DNA binding of the tetR-VP16 fusion protein, both in eukaryotic cells and in bacteria. We provide evidence that this antagonist of tetracycline is potentially useful for accelerated gene switching, especially in whole animals.

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Selected References

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  1. Altschmied L., Baumeister R., Pfleiderer K., Hillen W. A threonine to alanine exchange at position 40 of Tet repressor alters the recognition of the sixth base pair of tet operator from GC to AT. EMBO J. 1988 Dec 1;7(12):4011–4017. doi: 10.1002/j.1460-2075.1988.tb03290.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baumeister R., Helbl V., Hillen W. Contacts between Tet repressor and tet operator revealed by new recognition specificities of single amino acid replacement mutants. J Mol Biol. 1992 Aug 20;226(4):1257–1270. doi: 10.1016/0022-2836(92)91065-w. [DOI] [PubMed] [Google Scholar]
  3. Baumeister R., Müller G., Hecht B., Hillen W. Functional roles of amino acid residues involved in forming the alpha-helix-turn-alpha-helix operator DNA binding motif of Tet repressor from Tn10. Proteins. 1992 Oct;14(2):168–177. doi: 10.1002/prot.340140204. [DOI] [PubMed] [Google Scholar]
  4. Berens C., Altschmied L., Hillen W. The role of the N terminus in Tet repressor for tet operator binding determined by a mutational analysis. J Biol Chem. 1992 Jan 25;267(3):1945–1952. [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Damke H., Baba T., Warnock D. E., Schmid S. L. Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J Cell Biol. 1994 Nov;127(4):915–934. doi: 10.1083/jcb.127.4.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Deuschle U., Meyer W. K., Thiesen H. J. Tetracycline-reversible silencing of eukaryotic promoters. Mol Cell Biol. 1995 Apr;15(4):1907–1914. doi: 10.1128/mcb.15.4.1907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Efrat S., Fusco-DeMane D., Lemberg H., al Emran O., Wang X. Conditional transformation of a pancreatic beta-cell line derived from transgenic mice expressing a tetracycline-regulated oncogene. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3576–3580. doi: 10.1073/pnas.92.8.3576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ettner N., Metzger J. W., Lederer T., Hulmes J. D., Kisker C., Hinrichs W., Ellestad G. A., Hillen W. Proximity mapping of the Tet repressor-tetracycline-Fe2+ complex by hydrogen peroxide mediated protein cleavage. Biochemistry. 1995 Jan 10;34(1):22–31. doi: 10.1021/bi00001a004. [DOI] [PubMed] [Google Scholar]
  10. Fishman G. I., Kaplan M. L., Buttrick P. M. Tetracycline-regulated cardiac gene expression in vivo. J Clin Invest. 1994 Apr;93(4):1864–1868. doi: 10.1172/JCI117174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Furth P. A., St Onge L., Böger H., Gruss P., Gossen M., Kistner A., Bujard H., Hennighausen L. Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9302–9306. doi: 10.1073/pnas.91.20.9302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gatz C., Kaiser A., Wendenburg R. Regulation of a modified CaMV 35S promoter by the Tn10-encoded Tet repressor in transgenic tobacco. Mol Gen Genet. 1991 Jun;227(2):229–237. doi: 10.1007/BF00259675. [DOI] [PubMed] [Google Scholar]
  13. Ghersa P., Whelan J., Pescini R., DeLamarter J. F., Hooft van Huijsduijnen R. Commonly used cat reporter vectors contain a cAMP-inducible, cryptic enhancer that co-operates with NF-kappa B-sites. Gene. 1994 Dec 30;151(1-2):331–332. doi: 10.1016/0378-1119(94)90681-5. [DOI] [PubMed] [Google Scholar]
  14. Gossen M., Bonin A. L., Bujard H. Control of gene activity in higher eukaryotic cells by prokaryotic regulatory elements. Trends Biochem Sci. 1993 Dec;18(12):471–475. doi: 10.1016/0968-0004(93)90009-c. [DOI] [PubMed] [Google Scholar]
  15. Gossen M., Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5547–5551. doi: 10.1073/pnas.89.12.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gossen M., Freundlieb S., Bender G., Müller G., Hillen W., Bujard H. Transcriptional activation by tetracyclines in mammalian cells. Science. 1995 Jun 23;268(5218):1766–1769. doi: 10.1126/science.7792603. [DOI] [PubMed] [Google Scholar]
  17. Hansen D., Altschmied L., Hillen W. Engineered Tet repressor mutants with single tryptophan residues as fluorescent probes. Solvent accessibilities of DNA and inducer binding sites and interaction with tetracycline. J Biol Chem. 1987 Oct 15;262(29):14030–14035. [PubMed] [Google Scholar]
  18. Hinrichs W., Kisker C., Düvel M., Müller A., Tovar K., Hillen W., Saenger W. Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance. Science. 1994 Apr 15;264(5157):418–420. doi: 10.1126/science.8153629. [DOI] [PubMed] [Google Scholar]
  19. Hooft van Huijsduijnen R. A., Bollekens J., Dorn A., Benoist C., Mathis D. Properties of a CCAAT box-binding protein. Nucleic Acids Res. 1987 Sep 25;15(18):7265–7282. doi: 10.1093/nar/15.18.7265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lang Z., Feingold J. M. An autonomously replicating eukaryotic expression vector with a tetracycline-responsive promoter. Gene. 1996 Feb 12;168(2):169–171. doi: 10.1016/0378-1119(95)00788-1. [DOI] [PubMed] [Google Scholar]
  21. Lederer T., Takahashi M., Hillen W. Thermodynamic analysis of tetracycline-mediated induction of Tet repressor by a quantitative methylation protection assay. Anal Biochem. 1995 Dec 10;232(2):190–196. doi: 10.1006/abio.1995.0006. [DOI] [PubMed] [Google Scholar]
  22. Lieber A., Sandig V., Sommer W., Bähring S., Strauss M. Stable high-level gene expression in mammalian cells by T7 phage RNA polymerase. Methods Enzymol. 1993;217:47–66. doi: 10.1016/0076-6879(93)17055-a. [DOI] [PubMed] [Google Scholar]
  23. Passman R. S., Fishman G. I. Regulated expression of foreign genes in vivo after germline transfer. J Clin Invest. 1994 Dec;94(6):2421–2425. doi: 10.1172/JCI117609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Paulus W., Baur I., Boyce F. M., Breakefield X. O., Reeves S. A. Self-contained, tetracycline-regulated retroviral vector system for gene delivery to mammalian cells. J Virol. 1996 Jan;70(1):62–67. doi: 10.1128/jvi.70.1.62-67.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pescini R., Alouani S., Proudfoot A., Power C., Mermod J. J., DeLamarter J. F., Hooft van Huijsduijnen R. Inducible inhibition of eukaryotic gene expression. Biochem Biophys Res Commun. 1994 Aug 15;202(3):1664–1667. doi: 10.1006/bbrc.1994.2125. [DOI] [PubMed] [Google Scholar]
  26. Resnitzky D., Hengst L., Reed S. I. Cyclin A-associated kinase activity is rate limiting for entrance into S phase and is negatively regulated in G1 by p27Kip1. Mol Cell Biol. 1995 Aug;15(8):4347–4352. doi: 10.1128/mcb.15.8.4347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Röder F. T., Schmülling T., Gatz C. Efficiency of the tetracycline-dependent gene expression system: complete suppression and efficient induction of the rolB phenotype in transgenic plants. Mol Gen Genet. 1994 Apr;243(1):32–38. doi: 10.1007/BF00283873. [DOI] [PubMed] [Google Scholar]
  28. Schmid S. L. Tet-à-tet: a call for cells expressing the tetracycline-controllable transactivator. Trends Cell Biol. 1995 Jul;5(7):267–268. doi: 10.1016/s0962-8924(00)89031-5. [DOI] [PubMed] [Google Scholar]
  29. Skerra A. Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli. Gene. 1994 Dec 30;151(1-2):131–135. doi: 10.1016/0378-1119(94)90643-2. [DOI] [PubMed] [Google Scholar]
  30. Smith L. D., Bertrand K. P. Mutations in the Tn10 tet repressor that interfere with induction. Location of the tetracycline-binding domain. J Mol Biol. 1988 Oct 20;203(4):949–959. doi: 10.1016/0022-2836(88)90120-9. [DOI] [PubMed] [Google Scholar]
  31. Takahashi M., Altschmied L., Hillen W. Kinetic and equilibrium characterization of the Tet repressor-tetracycline complex by fluorescence measurements. Evidence for divalent metal ion requirement and energy transfer. J Mol Biol. 1986 Feb 5;187(3):341–348. doi: 10.1016/0022-2836(86)90437-7. [DOI] [PubMed] [Google Scholar]
  32. Van Meir E. G., Polverini P. J., Chazin V. R., Su Huang H. J., de Tribolet N., Cavenee W. K. Release of an inhibitor of angiogenesis upon induction of wild type p53 expression in glioblastoma cells. Nat Genet. 1994 Oct;8(2):171–176. doi: 10.1038/ng1094-171. [DOI] [PubMed] [Google Scholar]
  33. Weinmann P., Gossen M., Hillen W., Bujard H., Gatz C. A chimeric transactivator allows tetracycline-responsive gene expression in whole plants. Plant J. 1994 Apr;5(4):559–569. doi: 10.1046/j.1365-313x.1994.5040559.x. [DOI] [PubMed] [Google Scholar]
  34. Wimmel A., Lucibello F. C., Sewing A., Adolph S., Müller R. Inducible acceleration of G1 progression through tetracycline-regulated expression of human cyclin E. Oncogene. 1994 Mar;9(3):995–997. [PubMed] [Google Scholar]
  35. Wirtz E., Clayton C. Inducible gene expression in trypanosomes mediated by a prokaryotic repressor. Science. 1995 May 26;268(5214):1179–1183. doi: 10.1126/science.7761835. [DOI] [PubMed] [Google Scholar]
  36. Wissmann A., Baumeister R., Müller G., Hecht B., Helbl V., Pfleiderer K., Hillen W. Amino acids determining operator binding specificity in the helix-turn-helix motif of Tn10 Tet repressor. EMBO J. 1991 Dec;10(13):4145–4152. doi: 10.1002/j.1460-2075.1991.tb04992.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wissmann A., Wray L. V., Jr, Somaggio U., Baumeister R., Geissendörfer M., Hillen W. Selection for Tn10 tet repressor binding to tet operator in Escherichia coli: isolation of temperature-sensitive mutants and combinatorial mutagenesis in the DNA binding motif. Genetics. 1991 Jun;128(2):225–232. doi: 10.1093/genetics/128.2.225. [DOI] [PMC free article] [PubMed] [Google Scholar]

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