Transgenic Research

, Volume 12, Issue 1, pp 83–99 | Cite as

Molecular Structure and Regulatory Potential of a T-DNA Integration Site in Petunia

  • Antje Dietz-Pfeilstetter
  • Nicola Arndt
  • Volker Kay
  • Jürgen Bode


The genomic structure surrounding a T-DNA integration site in a transgenic petunia plant, which shows deregulation of a root-specific promoter, was investigated. We have already demonstrated that T-DNA integration in this transformant (P13) had occurred close to a scaffold/matrix attachment region (S/MAR). A major question regarding the observed promoter leakiness was whether the T-DNA had integrated into the centre or at the border of the Petun-SAR and whether other regulatory elements are located within this genomic region. While small rearrangements were shown to occur during T-DNA integration in agreement with other reports, we find indications of the presence of a SINE retroposon – an apparent landmark for recombinogenic targets – at the integration site. Binding assays to both plant and animal nuclear scaffolds, supported by biomathematical analyses, reveal that the T-DNA is definitely located at the border of a strong S/MAR, which is in agreement with current models on the structure of integration sites. These results, together with a developmentally regulated leaf-specific enhancer effect of the Petun-SAR on gene expression in transgenic tobacco plants, indicate that the Petun-SAR demarcates the right border of a chromatin domain with genes predominantly active in leaves.

gene expression Petunia hybrida S/MAR T-DNA integration transgenic plants 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Antes TJ, Namciu SJ, Fournier REK and Levy-Wilson B (2001) The 5′ boundary of the human apolipoprotein B chromatin domain in intestinal cells. Biochemistry 23: 6731–6742.Google Scholar
  2. Avramova Z and Bennetzen JL (1993) Isolation of matrices from maize leaf nuclei: identification of a matrix-binding site adjacent to the Adh1 gene. Plant Mol Biol 22: 1135–1143.Google Scholar
  3. Benham C, Kohwi-Shigematsu T and Bode J (1997) Stress-induced duplex DNA destabilization in scaffold/matrix attachment regions. J Mol Biol 274: 181–196.Google Scholar
  4. Bennetzen JL (1996) The contributions of retroelements to plant genome organization, function and evolution. Trends Microbiol 4: 347–353.Google Scholar
  5. Bode J and Maass K (1988) Chromatin domain surrounding the human interferon-′ gene as defined by scaffold attached regions. Biochemistry 27: 4706–4711.Google Scholar
  6. Bode J, Pucher HJ and Maass K (1986) Chromatin structure and induction-dependent conformational changes of human interferon-′ genes in a mouse host cell. Eur J Biochem 158: 393–401.Google Scholar
  7. Bode J, Schlake T, Rios-Ramirez M, Mielke C, Stengert M, Kay V et al. (1995) Scaffold/matrix-attached regions: structural properties creating transcriptionally active loci. Int Rev Cytol 162A: 389–454.Google Scholar
  8. Bode J, Stengert-Iber M, Schlake T, Kay V and Dietz-Pfeilstetter A (1996) Scaffold/matrix-attached regions: topological switches with multiple regulatory functions. Crit Rev Eukaryot Gene Expression 6: 115–138.Google Scholar
  9. Bode J, Benham C, Ernst E, Knopp A, Marschalek R, Strick R et al. (2000) Fatal connections: when DNA ends meet on the nuclear matrix. J Cell Biochem Suppl 35: 3–22; issuetoc?ID=82002725.Google Scholar
  10. Bogusz D, Llewellyn DL, Craig S, Dennis ES, Appleby CA and Peacock WJ (1990) Nonlegume hemoglobin genes retain organspecific expression in heterologous transgenic plants. Plant Cell 2: 633–641.Google Scholar
  11. Bonifer C, Hecht A, Saueressig H, Winter DM and Sippel AE (1991) Dynamic chromatin: the regulatory domain organization of eukaryotic gene loci. J Cell Biochem 47: 99–108.Google Scholar
  12. Breyne P, van Montague M, Depicker A and Gheysen G (1992) Characterization of a plant scaffold attachment region in a DNA 98 fragment that normalizes transgene expression in tobacco. Plant Cell 4: 463–471.Google Scholar
  13. Chinn AM and Comai L (1996) The heat shock cognate 80 gene of tomato is flanked by matrix attachment regions. Plant Mol Biol 32: 959–968.Google Scholar
  14. Cockerill (1990) Nuclear matrix attachment occurs in several regions of the IgH locus. Nucl Acids Res 18: 2643–2648.Google Scholar
  15. Dellaporta SL, Wood J and Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1: 19–21.Google Scholar
  16. De Neve M, De Buck S, Jacobs A, Van Montagu M and Depicker A (1997) T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs. The Plant J 11: 15–29.Google Scholar
  17. Deroles SC and Gardner RC (1988) Analysis of the T-DNA structure in a large number of transgenic petunias generated by Agrobacterium-mediated transformation. Plant Mol Biol 11: 365–377.Google Scholar
  18. Dietz A, Kay V, Schlake T, Landsmann J and Bode J (1994) A plant scaffold attached region detected close to a T-DNA integration site is active in mammalian cells. Nucl Acids Res 22: 2744–2751.Google Scholar
  19. Ditta G, Stanfield S, Corbin D and Helinski DR (1980) Broad host range DNA cloning system for Gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77: 7347–7351.Google Scholar
  20. Fackelmayer FO, Dahm K, Renz A, Ramsperger U and Richter A (1994) Nucleic-acid-binding properties of hnRNP-U/SAF-A, a nuclear-matrix protein which binds DNA and RNA in vivo and in vitro. Eur J Biochem 221: 749–757.Google Scholar
  21. Feinberg AP and Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13.Google Scholar
  22. Frisch M, Frech K, Klingenhoff A, Quandt K, Liebich I and Werner T (2002) In silico prediction of matrix attachment regions in large genomic sequences. Genome Res 12: 349–354.Google Scholar
  23. Garrard WT (1990) Chromosomal loop organization in eukaryotic genomes. Nucl Acids Mol Biol 4: 163–175.Google Scholar
  24. Gasser SM and Laemmli UK (1986) Cohabitation of scaffold binding regions with upstream-enhancer elements of three developmentally regulated genes of D. melanogaster. Cell 46: 521–530.Google Scholar
  25. Geyer PK (1997) The role of insulator elements in defining domains of gene expression. Curr Opin Genet Dev 7: 242–248.Google Scholar
  26. Gheysen G, Van Montagu M and Zambryski P (1987) Integration of Agrobacterium tumefaciens transfer DNA (T-DNA) involves rearrangements of target plant DNA sequences. Proc Natl Acad Sci USA 84: 6169–6173.Google Scholar
  27. Goldman MA (1988) The chromatin domain as a unit of gene regulation. BioEssays 9: 50–55.Google Scholar
  28. Grevelding C, Fantes V, Kemper E, Schell J and Masterson R (1993) Single copy T-DNA insertions in Arabidopsis are the predominant form of integration in root-derived transgenics, whereas multiple insertions are found in leaf discs. Plant Mol Biol 23: 847–860.Google Scholar
  29. Hall G, Allen GC, Loer DS, Thompson WF and Spiker S (1991) Nuclear scaffolds and scaffold-attachment regions in higher plants. Proc Natl Acad Sci USA 88: 9320–9324.Google Scholar
  30. Hamilton RH, Künsch U and Tempoli A (1972) Simple rapid procedures for isolation of tobacco leaf nuclei. Anal Biochem 49: 48–57.Google Scholar
  31. Hobbs SLA, Kpodar P and Delong CMO (1990) The effect of TDNA copy number, position and methylation on reporter gene expression in tobacco transformants. Pl Mol Biol 15: 851–864.Google Scholar
  32. Hoekema A, Hirsch PR, Hooykaas PJJ and Schilperoort RA (1983) A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 179–180.Google Scholar
  33. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG and Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227: 1229–1231.Google Scholar
  34. Iglesias VA, Moscone EA, Papp I, Neuhuber F, Michalowski S, Phelan T et al. (1997) Molecular and cytogenetic analyses of stably and unstably expressed transgene loci in tobacco. Plant Cell 9: 1251–1264.Google Scholar
  35. Jones JDG, Gilbert DE, Grady KL and Jorgensen RA (1987) TDNA structure and gene expression in petunia plants transformed by Agrobacterium tumefaciens C58 derivatives. Mol Gen Genet 207: 478–485.Google Scholar
  36. Jorgensen R, Snyder C and Jones JDG (1987) T-DNA is organized predominantly in inverted repeat structures in plants transformed with Agrobacterium tumefaciens C58 derivatives. Mol Gen Genet 207: 471–477.Google Scholar
  37. Kay V and Bode J (1994) Binding specificity of a nuclear scaffold: supercoiled, single-stranded, and scaffold-attached region DNA. Biochemistry 33: 367–374.Google Scholar
  38. Kertbundit S, De Greve H, Deboeck F, van Montagu M and Hernalsteens J-P (1991) In vivo random α-glucuronidase gene fusions in Arabidopsis thaliana. Proc Natl Acad Sci USA 88: 5212–5216.Google Scholar
  39. Koncz C, Martini N, Mayerhofer R, Koncz-Kalman Z, Körber H, Redei GP et al. (1989) High-frequency T-DNA-mediated gene tagging in plants. Proc Natl Acad Sci USA 86: 8467–8471.Google Scholar
  40. Krizkova L and Hrouda M (1998) Direct repeats of T-DNA integrated in tobacco chromosome: characterization of junction regions. The Plant J 16: 673–680.Google Scholar
  41. Landsmann J, Llewellyn D, Dennis ES and Peacock WJ (1988) Organ regulated expression of the Parasponia andersonii haemoglobin gene in transgenic tobacco plants. Mol Gen Genet 214: 68–73.Google Scholar
  42. Leisy DJ, Hnilo J, Zhao Y and Okita TW (1989) Expression of a rice glutelin promoter in transgenic tobacco. Plant Mol Biol 14: 41–50.Google Scholar
  43. Lindsey K, Wei W, Clarke MC, McArdle HF, Rooke LM and Topping JF (1993) Tagging genomic sequences that direct transgene expression by activation of a promoter trap in plants. Transgenic Res 2: 33–47.Google Scholar
  44. Matsumoto S, Ito Y, Hosoi T, Takahashi Y and Machida Y (1990) Integration of Agrobacterium T-DNA into a tobacco chromosome: possible involvement of DNA homology between DNA and plant DNA. Mol Gen Genet 224: 309–316.Google Scholar
  45. Meyer P and Saedler H (1996) Homology-dependent gene silencing in plants. Annu Rev Plant Physiol Plant Mol Biol 47: 23–48.Google Scholar
  46. Mielke C, Kohwi Y, Kohwi-Shigematsu T and Bode J (1990) Hierarchical binding of DNA fragments derived from scaffoldattached regions: correlations of properties in vitro and function in vivo. Biochemistry 29: 7475–7485.Google Scholar
  47. Mielke C, Maass K, Tümmler M and Bode J (1996) Anatomy of highly expressing chromosomal sites targeted by retroviral vectors. Biochemistry 35: 2239–2252.Google Scholar
  48. Mirkovitch J, Mirault ME and Laemmli UK (1984) Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold. Cell 39: 223–232.Google Scholar
  49. Neuhuber F, Park YD, Matzke AJM and Matzke A (1994) Susceptibility of transgene loci to homology-dependent gene silencing. Mol Gen Genet 244: 230–241.Google Scholar
  50. Paulson JR and Laemmli UK (1977) The structure of histonedepleted metaphase chromosomes. Cell 12: 817–828.Google Scholar
  51. Peach C and Velten J (1991) Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters. Plant Mol Biol 17: 49–60.Google Scholar
  52. Razin SV, Kekelidze MG, Scherrer K and Georgiev GP (1986) Replication origins are attached to the nuclear skeleton. Nucl Acids Res 14: 8189–8207.Google Scholar
  53. Razin SV, Shen K, Ioudinkova E and Scherrer K (1999) Functional analysis of DNA sequences located within a cluster of DNase I hypersensitive sites colocalizing with a MAR element at the upstream border of the chicken α-globin gene domain. J Cell Biochem 74: 38–49.Google Scholar
  54. Roberge M and Gasser SM (1992) DNA loops: structural and functional properties of scaffold-attached regions. Mol Microbiol 6: 419–423.Google Scholar
  55. Romig H, Fackelmayer FO, Renz A, Ramsperger U and Richter A (1992) Characterization of SAF-A, a novel nuclear DNAbinding protein from HeLa cells with high affinity for nuclear matrix/scaffold attachment DNA elements. EMBO J 11: 3431–3440.Google Scholar
  56. Sanger F, Nicklen S and Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 560–564.Google Scholar
  57. Sawasaki T, Takahashi M, Goshima N and Morikawa H (1998) Structures of transgene loci in transgenic Arabidopsis plants obtained by particle bombardment: junction regions can bind to nuclear matrices. Gene 218: 27–35.Google Scholar
  58. Schöffl F, Schröder G, Kliem M and Rieping M (1993) An SAR sequence containing 395 bp DNA fragment mediates enhanced, gene-dosage-correlated expression of a chimeric heat shock gene in transgenic tobacco plants. Transgenic Res 2: 93–100.Google Scholar
  59. Singh GB, Kramer JA and Krawetz SA (1997) Mathematical model to predict regions of chromatin attachment to the nuclear matrix. Nucleic Acids Res 25: 1419–1425.Google Scholar
  60. Slatter RE, Dupree P and Gray JC (1991) A scaffold-associated DNA region is located downstream of the pea plastocyanin gene. Plant Cell 3: 1239–1250.Google Scholar
  61. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98: 503–517.Google Scholar
  62. Spielmann A and Simpson RB (1986) T-DNA structure in transgenic tobacco plants with multiple independent integration sites. Mol Gen Genet 205: 34–41.Google Scholar
  63. Stam M, de Bruin R, Kenter S, Van der Hoorn RAL, Van Blokland R, Mol JNM et al. (1997) Post-transcriptional silencing of chalcone synthase in Petunia by inverted transgene repeats. Plant J 12: 63–82.Google Scholar
  64. Tinker NA, Fortin MG and Mather DE (1993) Random amplified polymorphic DNA and pedigree relationships in spring barley. Theor Appl Genet 85: 976–984.Google Scholar
  65. Ueng P, Galili G, Sapanara V, Goldsbrough PB, Dube P, Beachy RN et al. (1988) Expression of a maize storage protein gene in petunia plants is not restricted to seeds. Plant Physiol 86: 1281–1285.Google Scholar
  66. van der Geest AHM and Hall TC (1997) The β-phaseolin 5′ matrix attachment region acts as an enhancer facilitator. Plant Mol Biol 33: 553–557.Google Scholar
  67. van der Geest AHM, Hall Jr GE, Spiker S and Hall TC (1994) The β-phaseolin gene is flanked by matrix attachment regions. Plant J 6: 413–423.Google Scholar
  68. Van der Graaff E, den Dulk-Ras A and Hooykaas PJJ (1996) Deviating T-DNA transfer from Agrobacterium tumefaciens to plants. Plant Mol Biol 31: 677–681.Google Scholar
  69. van der Hoeven C, Dietz A and Landsmann J (1994) Variability of organ-specific gene expression in transgenic tobacco plants. Transgenic Res 3: 159–165.Google Scholar
  70. Watson JC and Thompson WF (1988) Purification and restriction endonuclease analysis of plant nuclear DNA. In: Weissbach A and Weissbach H (eds), Methods for Plant Molecular Biology. (pp. 57–75) Academic Press, San Diego.Google Scholar
  71. Weising K, Bohn H and Kahl G (1990) Chromatin structure of transferred genes in transgenic plants. Developm Genet 11: 233–247.Google Scholar
  72. Zambryski P (1989) Agrobacterium-plant cell DNA transfer. In: Berg DE and Howe MM (eds), Mobile DNA. (pp. 309–333) American Society for Microbiology, Washington, DC.Google Scholar
  73. Zambryski P, Depicker A, Kruger K and Goodman HM (1982) Tumor induction by Agrobacterium tumefaciens. J Mol Appl Genet 1: 361–370.Google Scholar

Copyright information

© Kluwer Academic Publishers 2003

Authors and Affiliations

  • Antje Dietz-Pfeilstetter
    • 1
  • Nicola Arndt
    • 1
  • Volker Kay
    • 2
  • Jürgen Bode
    • 2
  1. 1.Federal Biological Research Centre for Agriculture and ForestryInstitute for Plant Virology, Microbiology and BiosafetyBraunschweigGermany
  2. 2.German Research Center for Biotechnology (GBF), Epigenetic RegulationBraunschweigGermany

Personalised recommendations