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Complete sequence analysis of transgene loci from plants transformed via microprojectile bombardment

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Abstract

A substantial literature exists characterizing transgene locus structure from plants transformed via Agrobacterium and direct DNA delivery. However, there is little comprehensive sequence analysis of transgene loci available, especially from plants transformed by direct delivery methods. The goal of this study was to completely sequence transgene loci from two oat lines transformed via microprojectile bombardment that were shown to have simple transgene loci by Southern analysis. In line 3830, transformed with a single plasmid, one major and one of two minor loci were completely sequenced. Both loci exhibited rearranged delivered DNA and flanking genomic sequences. The minor locus contained only 296 bp of two non-contiguous fragments of the delivered DNA flanked by genomic (filler) DNA that did not originate from the integration target site. Predicted recognition sites for topoisomerase II and a MAR region were observed in the transgene integration target site for this non-functional minor locus. Line 11929, co-transformed with two different plasmids, had a single relatively simple transgene locus composed of truncated and rearranged sequences from both delivered DNAs. The transgene loci in both lines exhibited multiple transgene and genomic DNA rearrangements and regions of scrambling characteristic of complex transgene loci. The similar characteristics of recombined fragments and junctions in both transgenic oat lines implicate similar mechanisms of transgene integration and rearrangement regardless of the number of co-transformed plasmids and the level of transgene locus complexity.

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References

  • Allen, G.S., Spiker, S. and Thompson, W.F. 2000. Use of matrix attachment regions (MARs) to minimize transgene silencing. Plant Mol. Biol. 43: 361–376.

    Google Scholar 

  • Ananiev, E.V., Riera-Lizarazu, O., Rines, H.W. and Phillips, R.L. 1997. Oat-maize chromosome addition lines: a new system for mapping the maize genome. Proc Natl. Acad. Sci. USA 94: 3524–3529.

    Google Scholar 

  • Chen, L., Marmey, P., Taylor, N.J., Brizard, J.P., Espinoza, C., D'Cruz, P., Huet, H., Zhang, S., de Kochko, A., Beachy, R.N. and Fauquet, C.M. 1998. Expression and inheritance of multiple transgenes in rice plants. Nature Biotechnol. 16: 1060–1064.

    Google Scholar 

  • Christou, P. 1992. Genetic transformation of crop plants using microprojectile bombardment. Plant J. 2: 275–281.

    Google Scholar 

  • Fu, X., Duc, L.T., Fontana, S., Bong, B.B., Tinjuangjun, P., Sudhakar, D., Twyman, R.M., Christou, P. and Kohli, A. 2000. Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns. Transgenic Res. 9: 11–19.

    Google Scholar 

  • Gelvin, S.B. 1998. Multigene plant transformation: more is better! Nature Biotechnol. 16: 1009–1010.

    Google Scholar 

  • Gelvin, S.B. 2000. Agrobacterium and plant genes involved in TDNA transfer and integration. Annu. Rev. Plant. Physiol. Plant Mol. Biol. 51: 223–256.

    Google Scholar 

  • Gheysen, G., Villarroel, R. and Van Montagu, M. 1991. Illegitimate recombination in plants: a model for T-DNA integration. Genes Dev. 5: 287–297.

    Google Scholar 

  • Gorbunova, V. and Levy, A.A. 1997. Non-homologous DNA end joining in plant cells is associated with deletions and filler DNA insertions. Nucl. Acids Res. 25: 4650–4657.

    Google Scholar 

  • Gorbunova, V., and Levy, A.A. 1999. How plants make ends meet: DNA double-strand break repair. Trends Plant Sci 4: 263–269.

    Google Scholar 

  • Klein, T.M. and Jones, T.J. 1999. Methods of genetic transformation: the gene gun. In: I.K. Vasil (Ed.) Molecular Improvement of Cereal Crops. Advances in Cellular and Molecular Biology of Plants, vol. 5, Kluwer Academic Publishers, Dordrecht, Netherlands, pp. 21–42.

    Google Scholar 

  • Kohli, A., Griffiths, S., Palacios, N., Twyman, R.M., Vain, P., Laurie, D.A. and Christou, P. 1999. Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination. Plant J. 17: 591–601.

    Google Scholar 

  • Kumar, S. and Fladung, M. 2001. Gene stability in transgenic aspen (Populus). II. Molecular characterization of variable expression of transgene in wild and hybrid aspen. Planta 213: 731–740.

    Google Scholar 

  • Kumpatla, S.P. and Hall, T.C. 1999. Organizational complexity of a rice transgene locus susceptible to methylation-based silencing. Life 48: 459–467.

    Google Scholar 

  • Leggett, J.M., Perret, S.J., Harper, J. and Morris, P. 2000. Chromosomal localization of co-transformed transgenes in the hexaploid cultivated oat Avena sativa L. using fluorescence in situ hybridization. Heredity 84: 46–53.

    Google Scholar 

  • McFarlane, M. and Wilson, J.B. 1996. A model for the mechanism of precise integration of a microinjected transgene. Transgenic Res. 5: 171–177.

    Google Scholar 

  • Muller, A.E., Kamisugi, Y., Gruneberg, R., Niedenhof, I., Horold, R.J. and Meyer, P. 1999. Palindromic sequences and A+T-rich DNA elements promote illegitimate recombination in Nicotiana tabacum. J. Mol. Biol. 291: 29–46.

    Google Scholar 

  • Ohba, T., Yoshioka, Y., Machida, C. and Machida, Y. 1995. DNA rearrangement associated with the integration of T-DNA in tobacco: an example for multiple duplications of DNA around the integration target. Plant J. 7: 157–164.

    Google Scholar 

  • Palin, A.H., Critcher, R., Fitzgerald, D.J., Anderson, J.N. and Farr, C.J. 1998. Direct cloning and analysis of DNA sequences from a region of the Chinese hamster genome associated with aphidicolin-sensitive fragility. J Cell Sci. 111: 1623–1634.

    Google Scholar 

  • Pawlowski, W.P. and Somers, D.A. 1996. Transgene inheritance in plants genetically engineered by microprojectile bombardment. Mol. Biotechnol. 6: 17–30.

    Google Scholar 

  • Pawlowski, W.P. and Somers, D.A. 1998. Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA. Proc. Natl. Acad. Sci USA 95: 12106–12110.

    Google Scholar 

  • Petersen, K., Leah, R., Knudsen, S. and Cameron-Mills, V. 2002. Matrix attachment regions (MARs) enhance transformation requencies and reduce variance of transgene expression in barley. Plant Mol. Biol. 49: 45–58.

    Google Scholar 

  • Salomon, S. and Puchta, H. 1998. Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells. EMBO J. 17: 6086–6095.

    Google Scholar 

  • 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 

  • Shera, K.A., Shera, C.A. and McDougall, J.K. 2001. Small tumor virus genomes are integrated near nuclear matrix attachment regions in transformed cells. J. Virol. 75: 12339–12346.

    Google Scholar 

  • Shimizu, K., Takahashi, M., Goshima, N., Kawakami, S., Irifune, K. and Morikawa, H. 2001. Presence of an SAR-like sequence in junction regions between an introduced transgene and genomic DNA of cultured tobacco cells: its effect on transformation frequency. Plant J. 26: 375–384.

    Google Scholar 

  • Singh, G.B., Kramer, J.A. and Krawetz, S.A. 1997. Mathematical model to predict regions of chromatin attachment to the nuclear matrix. Nucl. Acids Res. 25: 1419–1425.

    Google Scholar 

  • Svitashev, S.K. and Somers, D.A. 2001. Genomic interspersions determine the size and complexity of transgene loci in transgenic plants produced by microprojectile bombardment. Genome 44: 691–697.

    Google Scholar 

  • Svitashev, S.K. and Somers, D.A. 2002. Characterization of transgene loci in plants using FISH: A picture is worth a thousand words. Plant Cell Tissue Organ Cult. 69: 205–214.

    Google Scholar 

  • Svitashev, S., Ananiev, E., Pawlowski, W.P. and Somers, D.A. 2000. Association of transgene integration sites with chromosome rearrangements in hexaploid oat. Theor. Appl. Genet. 100: 872–880.

    Google Scholar 

  • Svitashev, S.K., Pawlowski, W.P., Makarevitch, I., Plank, W. and Somers, D.A. 2002. Complex structures of transgene loci in plants genetically engineered by microprojectile bombardment implicate multiple mechanisms of transgene locus formation. Plant J. 32: 33–446.

    Google Scholar 

  • Takano, M., Egawa, H., Ikeda, J.-E. and Wakasa, K. 1997. The structures of integration sites in transgenic rice. Plant J. 11: 353–361.

    Google Scholar 

  • Tax, F.E. and Vernon, D.M. 2001. T-DNA-associated duplication/translocations in Arabidopsis. Implications for mutant analysis and functional genomics. Plant Physiol. 126: 1527–1538.

    Google Scholar 

  • Torbert, K.A., Rines, H.W. and Somers, D.A. 1995. Use of paromomycin as a selective agent for oat transformation. Plant Cell Rep. 14: 635–640.

    Google Scholar 

  • Tzafrir, I., Torbert, K.A., Lockhart, B.E., Somers, D.A. and Olszewski, N.E. 1998. The sugarcane bacilliform badnavirus promoter is active in both monocots and dicots. Plant Mol. Biol. 38: 347–356.

    Google Scholar 

  • van Attikum, H., Bundock, and P., Hooykaas, P.J. 2001. Nonhomologous end-joining proteins are required for Agrobacterium T-DNA integration. EMBO J. 20: 6550–6558.

    Google Scholar 

  • Vergunst, A.C. and Hooykaas, P.J.J. 1999. Recombination in the plant genome and its application in biotechnology. Crit. Rev. Plant Sci. 18: 1–31.

    Google Scholar 

  • Windels, P., Taverniers, I., Depicker, A., Van Bockstaele, E. and De Loose, M. 2001. Characterization of the Roundup Ready soybean insert. Eur. Food Res. Technol. 213: 107–112.

    Google Scholar 

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

    Google Scholar 

  • Zheng, S.J., Henken, B., Sofiari, E., Jacobsen, E., Krens, F.A. and Kik, C. 2001. Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders. Transgenic Res. 10: 237–245.

    Google Scholar 

  • Zoraqi, G. and Spadafora, C. 1997. Integration of foreign DNA sequences into mouse sperm genome. DNA Cell Biol. 16: 291–300.

    Google Scholar 

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  1. S.K. Svitashev

    Present address: Department of Agronomy and Plant Genetics, University of Minnesota, 411 Borlaug Hall, 1991 Buford Circle, St. Paul, MN, 55108, USA

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  1. Department of Agronomy and Plant Genetics, University of Minnesota, 411 Borlaug Hall, 1991 Buford Circle, St. Paul, MN, 55108, USA

    I. Makarevitch & D.A. Somers

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  1. I. Makarevitch
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  2. S.K. Svitashev
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  3. D.A. Somers
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Correspondence to D.A. Somers.

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Makarevitch, I., Svitashev, S. & Somers, D. Complete sequence analysis of transgene loci from plants transformed via microprojectile bombardment. Plant Mol Biol 52, 421–432 (2003). https://doi.org/10.1023/A:1023968920830

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