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Regulation of a modified CaMV 35S promoter by the Tn10-encoded Tet repressor in transgenic tobacco

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Summary

We have investigated the use of the Tn10-encoded tet repressor-operator system to regulate the expression of a suitably engineered cauliflower mosaic virus (CaMV) 35S promoter in transgenic tobacco plants. First, a transgenic plant was generated which constitutively synthesizes 600000 Tet repressor monomers per cell. In a second transformation step, the β-glucuronidase (gus) gene under the control of a modified CaMV 35S promoter, containing two tet operators, was stably integrated into the plant genome of a tetR + plant. Expression of the gus gene is repressed 5-fold, if the operators are located flanking the TATA box, and 50- to 80-fold when both operators are positioned downstream of the TATA box. This indicates that Tet repressor-operator complexes can form on plant chromosomes and interfere with transcription. Maximal induction is achieved after 0.5 h upon application of only 0.1 mg/l tetracycline. This fast and efficient induction makes the system useful for specifically inducing expression of transferred genes at different stages of plant development.

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References

  • Abel PP, Nelson RS, De B, Hoffmann N, Rogers SG, Fraley RT, Beachy RN (1986) Delay of disease development in transgenic plants that express the Tobacco Mosaic Virus coat protein gene. Science 232:738–743

    Google Scholar 

  • Ainley WM, Key JL (1990) Development of a heat shock inducible expression cassette for plants: characterization of parameters for its use in transient expression assays. Plant Mol Biol 14:949–966

    Google Scholar 

  • Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD (1983) Molecular Biology of the Cell. Garland Publishing, New York, p 321

    Google Scholar 

  • Alton KN, Vapnek D (1979) Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9. Nature 282:864–869

    Google Scholar 

  • Amasino R (1986) Acceleration of nucleic acid hybridization rate of polyethylene glycol. Anal Biochem 152:304–307

    Google Scholar 

  • Benfey PN, Ren L, Chua N-H (1989) The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue specific expression patterns. EMBO J 8:2195–2202

    Google Scholar 

  • Benfey PN, Ren L, Chua N-H (1990a) Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development. EMBO J 9:1677–1684

    CAS  PubMed  Google Scholar 

  • Benfey PN, Ren L, Chua N-H (1990b) Combinatorial and synergistic properties of CaMV 35S enhancer subdomains. EMBO J 9:1685–1696

    Google Scholar 

  • Bertrand KP, Postle K, Wray LV, Reznikoff WS (1983) Overlapping divergent promoters control expression of Tn10 tetracycline resistance. Gene 23:149–156

    Google Scholar 

  • Bertrand KP, Postle K, Wray LV, Reznikoff WS (1984) Construction of a single copy promoter vector and its use in analysis of regulation of the transposon Tn10 tetracycline resistance determinant. J Bacteriol 158:910–919

    Google Scholar 

  • Bevan M (1984) Binary Agrobacterium vectors for plant transformantion. Nucleic Acids Res 12:8711–8721

    Google Scholar 

  • Brent R, Ptashne M (1984) A bacterial repressor protein or a yeast transcriptional terminator can block upstream activation of a yeast gene. Nature 312:612–615

    Google Scholar 

  • Brown M, Figge J, Hansen U, Wright C, Jeang K-T, Khoury G, Livingstone DM, Roberts TM (1987) lac repressor can regulate expression from a hybrid SV40 early promoter containing a lac operator in animal cells. Cell 49:603–612

    Google Scholar 

  • Covey SN, Hull R (1985) Advances in Cauliflower Mosaic Virus Research. Oxford Surveys of Plant Molecular and Cell Biology, vol 2. pp 339–346

    Google Scholar 

  • De Block M, Botterman J, Vandewiele M, Dockx J, Thoen C, Gossele V, Rao Movva N, Thompson C, Van Montagu M, Leemans J (1987) Engineering herbicide resistance in plants by expression of a detoxifying enzyme. EMBO J 6:2513–2518

    Google Scholar 

  • Fang R-X, Nagy F, Sivasubranamiam S, Chua N-H (1989) Multiple cis regulatory elements for maximal expression of the Cauliflower Mosaic Virus 35S promoter in transgenic plants. Plant Cell 1:141–150

    Google Scholar 

  • Figge J, Wright C, Collins CJ, Roberts TM, Livingston DM (1988) Stringent regulation of stably integrated chloramphenicol acetyl transferase genes by E. coli lac repressor in monkey cells. Cell 52:713–722

    Google Scholar 

  • Freeling M, Bennett DC (1985) Maize adhl. Annu Rev Genet 19:297–332

    Google Scholar 

  • Fried M, Crothers DM (1981) Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res 9:6505–6525

    Google Scholar 

  • Gatz C, Quail PH (1988) Tn10-encoded tet repressor can regulate an operator-containing plant promoter. Proc Natl Acad Sci USA 85:1394–1397

    Google Scholar 

  • Geissendörfer M, Hillen W (1990) Regulated expression of heterologous genes in Bacillus subtilis using the Tn10-encoded tet regulatory elements. Appl Microbiol Biotechnol 33:657–663

    Google Scholar 

  • Heuer C, Hillen W (1988) Tet repressor-tet operator contacts probed by operator DNA-modification interference studies. J Mol Biol 202:407–415

    Google Scholar 

  • Hillen W, Gatz C, Altschmied L, Schollmeier K, Meier I (1983) Control of expression of the Tn10-encoded tetracycline resistance genes. Equilibrium and kinetic investigation of the regulatory reactions. J Mol Biol 169:707–721

    Google Scholar 

  • Hillen W, Schollmeier K, Gatz C (1984) Control of expression of the Tn10-encoded tetracycline resistance operon. II. Interaction of RNA polymerase and Tet repressor with the tet operon regulatory region. J Mol Biol 172:185–201

    Google Scholar 

  • Höfgen R, Willmitzer L (1990) Biochemical and genetic analysis of different patatin isoforms expressed in various organs of potato (Salanum tuberosum). Plant Sci 66:221–230

    Google Scholar 

  • Hu MC-T, Davidson N (1987) The inducible lac operator-repressor system is functional in mammalian cells. Cell 48:555–566

    Google Scholar 

  • Jefferson RA, Kavanagh RH, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    CAS  PubMed  Google Scholar 

  • Keil M, Sanchez-Serrano JJ, Willmitzer L (1989) Both wound inducible and tuber specific expression are mediated by the promoter of a single member of the potato proteinase inhibitor II gene family. EMBO J 8:1323–1330

    Google Scholar 

  • Klein M, Frederick RJ, Maramorosch K (1972) Chemotherapy of aster yellows: tetracycline-hydrochloride uptake by healthy and diseased plants. Phytopathology 62:111–115

    Google Scholar 

  • Kleinschmidt C, Tovar K, Hillen W, Poerschke D (1988) Dynamics of repressor-operator recognition: the Tn10-encoded tetracycline resistance control. Biochemistry 27:1094–1104

    Google Scholar 

  • Köster-Töpfer M, Frommer WB, Rocha-Sosa M, Rosahl S, Schell J, Willmitzer L (1989) A class II patatin promoter is under developmental control in both transgenic potato and tobacco plants. Mol Gen Genet 219:390–396

    Google Scholar 

  • Kuhlemeier C, Green PJ, Chua N-H (1987) Regulation of gene expression in higher plants. Annu Rev Plant Physiol 38:221–257

    Google Scholar 

  • Lang I, Scholz M, Peter R (1986) Molecular mobility and nucleocytoplasmic flux in hepatoma cells. J Cell Biol 102:1183–1190

    Google Scholar 

  • Lanzer M, Bujard H (1988) Promoters largely determine the efficiency of repressor action. Proc Natl Acad Sci USA 85:8973–8977

    Google Scholar 

  • Lin S, Riggs AD (1975) The general affinity of lac repressor for E. coli DNA: implications for gene regulation in procaryotes and eucaryotes. Cell 4:107–111

    Google Scholar 

  • Logemann J, Schell J, Willmitzer L (1987) Improved method for the isolation of RNA from plant tissues. Anal Biochem 163:16–20

    CAS  PubMed  Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Mitchell PJ, Tijan R (1989) Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245:371–378

    Google Scholar 

  • Nelson WG, Pientak ER, Barrak ET, Coffey DS (1986) The role of nuclear matrix in the organization and function of DNA. Annu Rev Biophys Chem 15:457–475

    Google Scholar 

  • Odell GT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of the Cauliflower Mosaic Virus 35S promoter. Nature 313:810–881

    CAS  PubMed  Google Scholar 

  • Ochmichen R, Klock G, Alltschmied L, Hillen W (1984) Construction of an E. coli strain overproducing the Tn10-encoded Tet repressor and its use for large scale purification. EMBO J 3:539–543

    Google Scholar 

  • Ow DW, Jacobs JD, Howell SH (1987) Functional regions of the cauliflower mosaic virus 35S RNA promoter determined by use of the firefly luciferase gene as a reporter activity. Proc Natl Acad Sci USA 84:4870–4874

    Google Scholar 

  • Paine P, Moore L, Horowitz S (1975) Nuclear envelope permeability. Nature 254:109–112

    Google Scholar 

  • Reznikoff WS, Abelson JN (1980) The lac promoter. In: Miller JH, Reznikoff WS (eds) The operon. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp 221–243

    Google Scholar 

  • Rogers SO, Bendich AJ (1985) Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol Biol 5:69–76

    Google Scholar 

  • Sanders PR, Winter JA, Harnason AR, Rogers SG, Fraley RT (1987) Comparison of cauliflower mosaic virus 35S and nopaline synthase promoters in transgenic plants. Nucleic Acids Res 15:1543–1558

    Google Scholar 

  • Stockhaus J, Schell J, Willmitzer L (1989) Correlation of the expression of the nuclear photosynthetic gene ST-LS1 with the presence of chloroplasts. EMBO J 8:2445–2451

    Google Scholar 

  • Takahashi M, Altschmied L, Hillen W (1986) Kinetic and equilibrium characterization of the Tet repressor-tetracycline complex by flourescence measurements. Evidence for divalent metal ion requirement and energy transfer. J Mol Biol 187:341–348

    Google Scholar 

  • Vaeck M, Reynaerts R, Hofte H, Janssens S, De Beuckeleer M, Dean C, Zabeau M, Van Montagu M, Leemans J (1987) Transgenic plants protected from insect attack. Nature 328:33–37

    Google Scholar 

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  1. Institut fur Genbiologische Forschung, GmbH, Ihnestrasse 63, W-1000, Berlin 33, Germany

    Christiane Gatz, Astrid Kaiser & Regina Wendenburg

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  1. Christiane Gatz
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  2. Astrid Kaiser
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  3. Regina Wendenburg
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Gatz, C., Kaiser, A. & Wendenburg, R. Regulation of a modified CaMV 35S promoter by the Tn10-encoded Tet repressor in transgenic tobacco. Molec. Gen. Genet. 227, 229–237 (1991). https://doi.org/10.1007/BF00259675

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  • DOI: https://doi.org/10.1007/BF00259675

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