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Non-recombinant background in gene targeting: illegitimate recombination between a hpt gene and a defective 5′ deleted nptII gene can restore a Kmr phenotype in tobacco

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Abstract

Previously we have demonstrated gene targeting in plants after Agrobacterium-mediated transformation. In these initial experiments a transgenic tobacco line 104 containing a T-DNA insertion with a defective neomycin phosphotransferase (nptII) gene was transformed with a repair construct containing an otherwise defective nptII gene. Homologous recombination between the chromosomally located target and the incoming complementary defective nptII construct generated an intact nptII gene and led to a kanamycin-resistant (Kmr) phenotype. The gene targeting frequency was 1×10−5. In order to compare direct gene transfer and Agrobacterium-mediated transformation with respect to gene targeting we transformed the same transgenic tobacco line 104 via electroporation. A total of 1.35×108 protoplasts were transformed with the repair construct. Out of nearly 221 000 transformed cells 477 Kmr calli were selected. Screening the Kmr calli via PCR for recombination events revealed that in none of these calli gene targeting had occurred. To establish the origin of the high number of Kmr calli in which gene targeting had not occurred we analysed plants regenerated from 24 Kmr calli via PCR and sequence analysis. This revealed that in 21 out of 24 plants analysed the 5′-deleted nptII gene was fused to the hygromycin phosphotransferase (hpt) gene that was also present on the repair construct. Sequence analysis of 7 hpt/nptII gene fusions showed that they all contained a continuous open reading frame. The absence of significant homology at the fusion site indicated that fusion occurred via a process of illegitimate recombination. Therefore, illegitimate recombination between an introduced defective gene and another gene present on the repair construct or the chromosome has to be taken into account as a standard byproduct in gene targeting experiments.

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References

  1. Baur M, Potrykus I, Paszkowski J: Intermolecular homologous recombination in plants. Mol Cell Biol 10: 492–500 (1990).

    Google Scholar 

  2. Beck E, Ludwig G, Auerswald EA, Reiss B, Schaller H: Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19: 327–336 (1982).

    Google Scholar 

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

    Google Scholar 

  4. Bilang R, Peterhans A, Bogucki A, Paszkowski J: Single-stranded DNA as a recombination substrate in plants assessed by stable and transient recombination assays. Mol Cell Biol 12: 329–336 (1992).

    Google Scholar 

  5. Blázques J, Davies J, Moreno F: Mutations in the aphA-2 gene of transposon Tn5 mapping within the regions highly conserved in aminoglycoside phosphotransferases strongly reduce aminoglycoside resistance. Mol Microbiol 5: 1511–1518 (1991).

    Google Scholar 

  6. Bradley A: Modifying the mammalian genome by gene targeting. Curr Opin Biotechnol 2: 823–829 (1991).

    Google Scholar 

  7. Capecchi MR: Altering the genome by homologous recombination. Science 244: 1288–1292 (1989).

    Google Scholar 

  8. Champoux JJ, Bullock PA: Chapter In: Kucherlapati R, Smith GR (eds) Genetic Recombination, pp. 549–574, American Society for Microbiology, Washington DC (1988).

    Google Scholar 

  9. Czernilofsky AP, Hain R, Herrera-Estrella L, Lözz N, Goyvaerts E, Baker BJ, Schell J. Fate of selectable marker DNA integrated into the genome of N. tabacum DNA. DNA 5: 101–113 (1986).

    Google Scholar 

  10. Czernilofsky AP, Hain R, Baker B, Wirtz U: Studies of the structure and functional organization of foreign DNA integrated into the genome of Nicotiana tabacum. DNA 5: 473–482 (1986).

    Google Scholar 

  11. Datla RSS, Hammerlindl JK, Pelcher LE, Crosby WL, Selvaraj G: A bifunctional fusion between β-glucuronidase and neomycin phospostransferase: a broad-spectrum marker enzyme for plants. Gene 101: 239–246 (1991).

    Google Scholar 

  12. de Groot MJA, Offringa R, Does MP, Hooykaas PN, van den Elzen PJM: Mechanisms of intermolecular homologous recombination in plants as studied with single and double-stranded DNA molecules. Nucl Acids Res 20: 2785–2794 (1992).

    Google Scholar 

  13. Deroles SC, Gardner RC: Analysis of the T-DNA structure in a large number of transgenic petunias generated by Agrobacterium-mediated transformation. Plant Mol Biol 11: 365–377 (1988).

    Google Scholar 

  14. Does MP, Dekker BMM, de Groot MJA, Offringa R: A quick method to estimate the T-DNA copy number in transgenic plants at an early stage after transformation, using inverse PCR. Plant Mol Biol 17: 151–153 (1991).

    Google Scholar 

  15. Gasser CS, Fraley RT. Genetically engineering plants for crop improvement. Science 244: 1293–1299 (1989).

    Google Scholar 

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

    Google Scholar 

  17. Hain R, Stabel P, Czernilofsky AP, Steinbiss HH, Herrera-Estrella L, Schell J. Uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant protoplasts. Mol Gen Genet 199: 161–118 (1985).

    Google Scholar 

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

    Google Scholar 

  19. Hasty P, Rivera-Perez J, Bradley A: The length of homology required for gene targeting in embryonic stem cells Mol Cell Biol 11: 5586–5591 (1991).

    Google Scholar 

  20. 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).

    Google Scholar 

  21. Herman L, Jacobs A, Van Montagu M, Depicker A: Plant chromosome/marker gene fusion assay to study normal and truncated T-DNA integration events. Mol Gen Genet 244: 248–256 (1990).

    Google Scholar 

  22. Hinnen A, Hicks JB, Fink GR: Transformation of yeast. Proc Natl Acad Sci USA 75: 1929–1933 (1978).

    Google Scholar 

  23. Hyrien O, Debatisse M, Buttin G, de Saint Vincent BR: A hospot for novel amplification joints in a mosaic of Alu-like repeats and palindromic A+T rich DNA. EMBO J 6: 2401–2408 (1987).

    Google Scholar 

  24. Keohavong P, Thilly WG: Fidelity of DNA polymerase in DNA amplification. Proc Natl Acad Sci USA 86: 9253–9257 (1989).

    Google Scholar 

  25. Koncz C, Martini N, Mayerhofer R, Koncz-Kalman Z, Körber H, Redei GP, Schell J: High-frequency T-DNA mediated gene tagging in plants. Proc Natl Acad Sci USA 86: 8467–8471 (1989).

    Google Scholar 

  26. Konopka AK: Compilation of DNA strand exchange sites for non-homologous recombination in somatic cells. Nucl Acids Res 16: 1739–1758 (1988).

    Google Scholar 

  27. Krogh S, Mortensen UH, Westergaard O, Bonven BJ: Eukaryotic topoisomerase I-DNA interaction is stabilized by helix curvature. Nucl Acids Res 19: 1235–1241 (1991).

    Google Scholar 

  28. Lassner MW, Peterson P, Yoder JI: Simultaneous amplification of multiple DNA fragments by polymerase chain reaction in the analysis of transgenic plants and their progeny. Plant Mol Biol Rep 7: 116–128 (1989).

    Google Scholar 

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

    Google Scholar 

  30. Marchuk D, Drumm M, Saulino A, Collins FS: Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucl Acids Res 19: 1154 (1991).

    Google Scholar 

  31. Marsh JL, Erfle M, Wijkes EJ: The pIC plasmid and phage vectors with versatile cloning sites for recombination selection by insertional inactivation. Gene 32: 481–485 (1984).

    Google Scholar 

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

    Google Scholar 

  33. Mettler IJ: A simple and rapid method for minipreperation of DNA from tissue cultured plant cells. Plant Mol Biol Rep 5: 346–349 (1987).

    Google Scholar 

  34. Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497 (1962).

    Google Scholar 

  35. Offringa R, de Groot MJA, Haagsman HJ, Does MP, van den Elzen PJM, Hooykaas PJJ: Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation. EMBO J 9: 3077–3084 (1990).

    Google Scholar 

  36. Offringa R: Gene targeting in plants using the Agrobacterium vector system. Doctoral thesis, Leiden University (1992).

  37. Offringa R, Franke-van Dijk MEI, de Groot MJA, vanden Elzen PJM, Hooykaas PJJ. Nonreciprocal homologous recombination between Agrobacterium transferred DNA and a plant chromosomal locus. Proc Natl Acad Sci USA 90: 7346–7350 (1993).

    Google Scholar 

  38. Paszkowski J, Shillito RD, Saul M, Mandak V, Hohn T, Hohn B, Potrykus I: Direct gene transfer to plants. EMBO J 3: 2717–2722 (1984).

    Google Scholar 

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

    Google Scholar 

  40. Potrykus I: Gene transfer to plants: Assessment of published approaches and results. Annu Rev Plant Physiol Plant Mol Biol 42: 205–225 (1991).

    Google Scholar 

  41. 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).

    Google Scholar 

  42. 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 

  43. Saul MW, Shillito RD, Negrutiu I: Direct gene transfer to protoplasts with and without electroporation. In: Gelvin SB, Schilperoort RA (eds) Plant Molecular Biology Manual, pp. A1/1-A1:16. Kluwer Academic Publishers, Dordrecht (1988).

    Google Scholar 

  44. Schulman MJ, Nissen L, Collins C: Homologous recombination in hybridoma cells: Dependence on time and fragment length. Mol Cell Biol 10: 4466–4472 (1990).

    Google Scholar 

  45. Stary A, Sarasin A: Molecular analysis of DNA junctions produced by illegitimate recombination in human cells. Nucl Acids Res 20: 4269–4274 (1992).

    Google Scholar 

  46. Thomas KR, Capecchi MR: Site-directed mutagenesis by gene targeting in mouse embryo derived stem cells. Cell 51: 503–511 (1987).

    Google Scholar 

  47. Van den Broeck G, Timko MP, Kaush AP, Cashmore AR, Van Montagu M, Herrera-Estrella L. Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose-1,5-bisphosphate carboxylase. Nature 313: 358–363 (1985).

    Google Scholar 

  48. Winship PR. An improved method for directly sequencing PCR amplified material using dimethyl sulphoxide. Nucl Acids Res 17: 1266 (1989).

    Google Scholar 

  49. Yenofsky RL, Fine M, Pellow JW: A mutant neomycin phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure. Proc Natl Acad Sci USA 87: 3435–3439 (1990).

    Google Scholar 

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Author notes

  1. Marcel J. A. de Groot

    Present address: Calgene Pacific Pty Ltd, 16 Gipps Street, 3066, Collingwood, Victoria, Australia

Authors and Affiliations

  1. Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University, Wassenaarseweg 64, 2333 AL, Leiden, Netherlands

    Remko Offringa, Jürgen Groet & Paul J. J. Hooykaas

  2. MOGEN International nv, Einsteinweg 97, 2333 CB, Leiden, Netherlands

    Marcel J. A. de Groot, Mirjam P. Does & Peter J. M. van den Elzen

Authors
  1. Marcel J. A. de Groot
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  2. Remko Offringa
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  3. Jürgen Groet
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  5. Paul J. J. Hooykaas
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  6. Peter J. M. van den Elzen
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de Groot, M.J.A., Offringa, R., Groet, J. et al. Non-recombinant background in gene targeting: illegitimate recombination between a hpt gene and a defective 5′ deleted nptII gene can restore a Kmr phenotype in tobacco. Plant Mol Biol 25, 721–733 (1994). https://doi.org/10.1007/BF00029609

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  • DOI: https://doi.org/10.1007/BF00029609

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