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Termini and telomeres in T-DNA transformation

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Abstract

A T-DNA vector for plant transformation has been constructed in which the cloning site is located 9 bp from the right-border (RB) end and 27 bp from the left-border (LB) end. In this vector cloned DNA homologous to plant chromosomal sequences is located at the T-DNA termini, and will thus be exposed by even limited exonucleolysis in planta. The arabidopsis ADH (alcohol dehydrogenase) locus was mobilized from Agrobacterium, and integration into the recipient genome was studied. Despite the terminal location of ADH homology in this vector, the T-DNA integrated essentially at random in the Arabidopsis genome rather than at the endogenous ADH locus. T-DNA integration was blocked, however, when Arabidopsis telomeric sequences were added to the construct at each end of the ADH homology. Thus the predominant mode by which incoming T-DNA is integrated into the continuity of chromosomal DNA involves free DNA ends, but, in contrast to modes of recombination such as gap repair, does not involve extensive terminal DNA sequence homology.

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References

  1. Assaad, FA, Tucker, KL, Signer, ER: Epigenetic repeat-induced gene silencing (RIGS) in Arabidopsis. Plant Mol Biol 22: 1067–1085 (1993).

    PubMed  Google Scholar 

  2. Ausubel, FM, Brent, R, Kingston, RE, Moore, DD, Seidman, JG, Smith, JA, Struhl, K: Current Protocols in Molecular Biology. John Wiley, New York (1987).

    Google Scholar 

  3. Barker, RF, Idler, KB, Thompson, DV, Kemp, JD: Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTil5955. Plant Mol Biol 2: 335–350 (1983).

    Google Scholar 

  4. Bakkeren, G, Koukolikova-Nicola, Z, Grimsley, N, Hohn B: Recovery of Agrobacterium tumefaciens T-DNA molecules from whole plants early after transfer. Cell 57: 847–857 (1989).

    Article  PubMed  Google Scholar 

  5. Barnett, MA, Buckle, VJ, Evans, EP, Porter, ACG, Rout, D, Smith, AG, Brown, WRA: Telomere directed fragmentation of mammalian chromosomes. Nucl Acids Res 21: 27–36 (1993).

    PubMed  Google Scholar 

  6. Bevan, M: Binary Agrobacterium vectors for plant transformation. Nucl Acids Res 12: 8711–8721 (1984).

    PubMed  Google Scholar 

  7. Bourgain, FM, Katinka, MD: Telomeres inhibit end to end fusion and enhance maintenance of linear DNA molecules injected into the Paramecium primaurelia macronucleus. Nucl Acids Res 19: 1541–1547 (1991).

    PubMed  Google Scholar 

  8. Chang, C, Meyerowitz, EM: Molecular cloning and DNA sequence of the Arabidopsis thaliana alcohol dehydrogenase gene. Proc Natl Acad Sci USA 83: 1408–1412 (1986).

    PubMed  Google Scholar 

  9. Church, GM, Gilbert, W: Genomic sequencing. Proc Natl Acad Sci USA 18: 1991–18995 (1984).

    Google Scholar 

  10. Deng, C, Capecchi, MR: Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol Cell Biol 12: 3365–3371 (1992).

    PubMed  Google Scholar 

  11. Ditta, G, Schmidhauser, T, Yacobson, E, Lu, P, Liang, XW, Finlay, DR, Guiney, D, Helinski, DR: Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid 13: 149–153 (1985).

    PubMed  Google Scholar 

  12. Dolferus, R, Van derBossche, D, Jacobs, M: Sequence analysis of two null-mutant alleles of the single Arabidopsis Adh locus. Mol Gen Genet 224: 297–302 (1990).

    Article  PubMed  Google Scholar 

  13. Farr, C, Fantes, J, Goodfellow, P, Cooke, H: Functional reintroduction of human telomeres into mammalian cells. Proc Natl Acad Sci USA 88: 7006–7010 (1991).

    PubMed  Google Scholar 

  14. Freeling, M, Birchler, JA: Mutants and variants of the alcohol dehydrogenase-1 gene in maize. In: Setlow, JK, Hollaender, A (eds) Genetic Engineering: Principles and Methods, pp. 223–264. Plenum Press, New York (1981).

    Google Scholar 

  15. Ganal, MW, Lapitan, NLV, Tanksley, SD: Macrostructure of the tomato telomeres. Plant Cell 3: 87–94 (1991).

    Article  PubMed  Google Scholar 

  16. Gheisen, G, Villarroel, R, VanMontagu, M: Illegitimate recombination in plants: a model for T-DNA integration. Genes Devel 5: 287–297 (1991).

    PubMed  Google Scholar 

  17. Gottschling, DE, Aparicio, OM, Billington, BL, Zakian VA: Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell 63: 751–762 (1990).

    Article  PubMed  Google Scholar 

  18. Halfter, U, Morris, PC, Willmitzer, L: Gene targeting in Arabidopsis thaliana. Mol Gen Genet 231: 186–193 (1992).

    PubMed  Google Scholar 

  19. Hasty, P, Rivera-Perez, J, Chang, C, Bradley, A: Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells. Mol Cell Biol 11: 4509–4517 (1991).

    PubMed  Google Scholar 

  20. Hasty, P, Rivera-Perez, J, Bradley, A: The role and fate of DNA ends for homologous recombination in embryonic stem cells. Mol Cell Biol 12: 2462–2474 (1992).

    Google Scholar 

  21. Henikoff, S, Dreesen, TD: Trans-inactivation of the Drosophila brown gene: evidence for transcriptional repression and somatic pairing dependence. Proc Natl Acad Sci USA 86: 6704–6708 (1989).

    PubMed  Google Scholar 

  22. Howard, EA, Zupan, JR, Citovsky, V, Zambryski, PC: The VirD2 Protein of A. tumefaciens contains a C-terminal bipartite nuclear localization signal: implications for nuclear uptake of DNA in plant cells. Cell 68: 109–118 (1992).

    Article  PubMed  Google Scholar 

  23. Huang, H, Ma, H: An improved procedure for transforming Arabidopsis thaliana (Landsberg erecta) root explants. Plant Mol Biol Rep 10: 372–383 (1992).

    Google Scholar 

  24. Janssen, BJ, Gardner, RC: Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium. Plant Mol Biol 14: 61–72 (1990).

    Article  PubMed  Google Scholar 

  25. Köhler, F, Cardon, G, Pöhlman, M, Gill, R, Schieder, O: Enhancement of transformation rates in higher plants by low-dose irradiation: are DNA repair systems involved in the incorporation of exogenous DNA into the plant genome? Plant Mol Biol 12: 189–199 (1989).

    Google Scholar 

  26. Lazo, GR, Stein, PA, Ludwig, RA: A DNA transformation competent Arabidopsis genomic library in Agrobacterium. Bio/technology 9: 963–967 (1991).

    Article  PubMed  Google Scholar 

  27. Lee, KY, Lund, P, Lowe, K, Dunsmuir, P: Homologous recombination in plant cells after Agrobacterium-mediated transformation. Plant Cell 2: 415–425 (1990).

    Article  PubMed  Google Scholar 

  28. Lilley, DMJ: In vivo consequences of plasmid topology. Nature 292: 380–382 (1981).

    PubMed  Google Scholar 

  29. Marton, L, Browse, J: Facile transformation of Arabidopsis. Plant Cell Rep 10: 235–239 (1991).

    Google Scholar 

  30. Matsumoto, S, Yukihiro, I, Hosoi, T, Takahashi, Y, Machida, Y: Integration of Agrobacterium T-DNA into a tobacco chromosome: possible involvement of DNA homology between T-DNA and plant DNA. Mol Gen Genet 224: 309–316 (1990).

    Article  PubMed  Google Scholar 

  31. Mayerhofer, R, Koncz-Kalman, Z, Nawrath, C, Bakkeren G, Krameri, A, Angelis, K, Redei, GP, Shell, J, Hohn, B, Koncz, C: T-DNA integration: a mode of illegitimate recombination in plants. EMBO J 10: 697–704 (1991).

    PubMed  Google Scholar 

  32. McGill, C, Shafer, B, Strathern, J: Coconversion of flanking sequences with homothallic switching. Cell 57: 459–467 (1989).

    Article  PubMed  Google Scholar 

  33. McLennan, AG: DNA damage, repair, and mutagenesis. In: Bryant, JA, Valgene, LD (eds) DNA Replication in Plants, pp. 135–186. CRC Press, Boca Raton, FL (1988).

    Google Scholar 

  34. Nagel, R, Elliott, A, Masel, A, Birch, RG, Manners, JM: Electroporation of a binary Ti plasmid vector into Agrobacterium tumefaciens and Agrobacterium rhizogenes. FEMS Microbiol Lett 67: 325–328 (1990).

    Article  Google Scholar 

  35. Offringa, R, deGroot, MJA, Haagsman, HJ, Does, MP, Van denElzen, PJM, Hooykaas, PJJ: Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation. EMBO J 9: 3077–3085 (1990).

    PubMed  Google Scholar 

  36. Olszewski, NE, Martin, FB, Ausubel, FM: Specialized binary vector for plant transformation: expression of the Arabidopsis thaliana AHAS gene in Nicotiana tabacum. Nucl Acids Res 16: 10765–10781 (1988).

    PubMed  Google Scholar 

  37. Paszkowski, J, Baur, M, Bogucki, A, Potrykus, I: Gene targeting in plants. EMBO J 7: 4021–4026 (1988).

    Google Scholar 

  38. Peralta, EG, Hellmiss, R, Ream, W: Overdrive, a T-DNA transmission enhancer on the A. tumefaciens tumorinducing plasmid. EMBO J 5: 1137–1142 (1986).

    Google Scholar 

  39. Perrot, M, Barreau, C, Begueret, J: Nonintegrative transformation in the filamentous fungus Podospora anserina: stabilization of a linear vector by the chromosomal ends of Tetrahymena thermophila. Mol Cell Biol 7: 1735–1750 (1987).

    Google Scholar 

  40. Richards, EJ, Ausubel, F: Isolation of a higher eukaryotic telomere from Arabidopsis thaliana. Cell 53: 127–136 (1988).

    Article  PubMed  Google Scholar 

  41. Richards, EJ, Chao, S, Vongs, A, Yang, J: Characterization of Arabidopsis thaliana telomeres isolated in yeast. Nucl Acids Res 20: 4039–4046 (1992).

    PubMed  Google Scholar 

  42. Roth, DB, Wilson, JH: Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction. Mol Cell Biol 6: 4295–4304 (1986).

    PubMed  Google Scholar 

  43. Sambrook, J, Fritsch, EF, Maniatis, T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

    Google Scholar 

  44. Sandell, LL, Zakian, VA: Loss of a yeast telomere: arrest, recovery, and chromosome loss. Cell 75: 729–739 (1993).

    Article  PubMed  Google Scholar 

  45. Te Riele, H, Maandag, ER, Berns, A: Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA construct. Proc Natl Acad Sci USA 89: 5128–5132 (1992).

    PubMed  Google Scholar 

  46. Thaler, DS, Stahl, FW: DNA double-chain breaks in recombination of phage λ and of yeast. Annu Rev Genet 22: 169–197 (1988).

    Article  PubMed  Google Scholar 

  47. Tinland, B, Koukolikova-Nicola, Z., Hall, MN, Hohn, B: The T-DNA-linked VirD2 protein contains two distinct functional nuclear localization signals. Proc Natl Acad Sci USA 89: 7442–7446 (1992).

    PubMed  Google Scholar 

  48. Van derVeen, JS, Wirtz, P: EMS-induced genic male sterility in Arabidopsis thaliana: a model selection experiment. Euphytica 17: 371–377 (1968).

    Google Scholar 

  49. Vollrath, D, Davis, RW, Connelly, C, Mieter, P: Physical mapping of large DNA by chromosome fragmentation. Proc Natl Acad Sci USA 85: 6027–6031 (1988).

    PubMed  Google Scholar 

  50. Woods, JP, Goldman, WE: Autonomous replication of foreign DNA in histoplasma capsulatum: role of native telomeric sequences. J Bact 175: 636–641 (1993).

    PubMed  Google Scholar 

  51. Zakian, VA: Structure and function of telomeres. Annu Rev Genet 23: 579–604 (1989).

    Article  PubMed  Google Scholar 

  52. Zambryski, P: Chronicles from the Agrobacterium-plant cell DNA transfer story. Annu Rev Plant Physiol Plant Mol Biol 43: 465–490 (1992).

    Article  Google Scholar 

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  1. Maurizio Chiurazzi

    Present address: Istituto Internationale di Genetica e Biofisica, 80125, Napoli, Italy

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  1. Department of Biology, Massachusetts Institute of Technology, 02139-4307, Cambridge, MA, USA

    Maurizio Chiurazzi & Ethan R. Signer

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  1. Maurizio Chiurazzi
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Chiurazzi, M., Signer, E.R. Termini and telomeres in T-DNA transformation. Plant Mol Biol 26, 923–934 (1994). https://doi.org/10.1007/BF00028859

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