Abstract
Previously, we demonstrated the occurrence of gene targeting in tobacco cells after Agrobacterium-mediated transformation. In these experiments a defective kanamycin resistance (Kmr) gene residing at a chromosomal location was restored via homologous recombination with an incoming transferred DNA (T-DNA) repair construct (pSDM101) containing a different defective Kmr gene. In this article we describe gene targeting experiments with the same target line, but using an improved repair construct, pSDM321. In one of the Kmr calli obtained after transformation with pSDM321 (line A) the product of homologous recombination was detected using PCR. Further molecular analysis revealed that the defective Kmr gene present on the incoming T-DNA had been restored via homologous recombination with the target locus. The target locus was left unchanged and the corrected T-DNA was found to be inserted on the same chromosome but not close to the target locus. This paper presents molecular evidence in plants for the conversion of an introduced DNA molecule (in this case, T-DNA) by a homologous chromosomal locus.
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- Adair G. M., Nairn R. S., Wilson J. H., Seidman M. M., Brotherman K. A., MacKinnon C., Scheerer J. B. Targeted homologous recombination at the endogenous adenine phosphoribosyltransferase locus in Chinese hamster cells. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4574–4578. doi: 10.1073/pnas.86.12.4574. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Aratani Y., Okazaki R., Koyama H. End extension repair of introduced targeting vectors mediated by homologous recombination in mammalian cells. Nucleic Acids Res. 1992 Sep 25;20(18):4795–4801. doi: 10.1093/nar/20.18.4795. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Capecchi M. R. Altering the genome by homologous recombination. Science. 1989 Jun 16;244(4910):1288–1292. doi: 10.1126/science.2660260. [DOI] [PubMed] [Google Scholar]
- Deblaere R., Bytebier B., De Greve H., Deboeck F., Schell J., Van Montagu M., Leemans J. Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants. Nucleic Acids Res. 1985 Jul 11;13(13):4777–4788. doi: 10.1093/nar/13.13.4777. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Does M. P., Dekker B. M., de Groot M. J., 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. 1991 Jul;17(1):151–153. doi: 10.1007/BF00036819. [DOI] [PubMed] [Google Scholar]
- Gheysen G., Villarroel R., Van Montagu M. Illegitimate recombination in plants: a model for T-DNA integration. Genes Dev. 1991 Feb;5(2):287–297. doi: 10.1101/gad.5.2.287. [DOI] [PubMed] [Google Scholar]
- Marsh J. L., Erfle M., Wykes E. J. The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene. 1984 Dec;32(3):481–485. doi: 10.1016/0378-1119(84)90022-2. [DOI] [PubMed] [Google Scholar]
- Mayerhofer R., Koncz-Kalman Z., Nawrath C., Bakkeren G., Crameri A., Angelis K., Redei G. P., Schell J., Hohn B., Koncz C. T-DNA integration: a mode of illegitimate recombination in plants. EMBO J. 1991 Mar;10(3):697–704. doi: 10.1002/j.1460-2075.1991.tb07999.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Offringa R., de Groot M. J., Haagsman H. J., Does M. P., van den Elzen P. J., Hooykaas P. J. Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation. EMBO J. 1990 Oct;9(10):3077–3084. doi: 10.1002/j.1460-2075.1990.tb07504.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Paszkowski J., Baur M., Bogucki A., Potrykus I. Gene targeting in plants. EMBO J. 1988 Dec 20;7(13):4021–4026. doi: 10.1002/j.1460-2075.1988.tb03295.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Snyder J. G., Virta R. L., Segreti J. M. Evaluation of the phase contrast microscopy method for the detection of fibrous and other elongated mineral particulates by comparison with a STEM technique. Am Ind Hyg Assoc J. 1987 May;48(5):471–477. doi: 10.1080/15298668791385066. [DOI] [PubMed] [Google Scholar]
- Song K. Y., Schwartz F., Maeda N., Smithies O., Kucherlapati R. Accurate modification of a chromosomal plasmid by homologous recombination in human cells. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6820–6824. doi: 10.1073/pnas.84.19.6820. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stachel S. E., Timmerman B., Zambryski P. Activation of Agrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirement for 5' virD gene products. EMBO J. 1987 Apr;6(4):857–863. doi: 10.1002/j.1460-2075.1987.tb04831.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thomas K. R., Folger K. R., Capecchi M. R. High frequency targeting of genes to specific sites in the mammalian genome. Cell. 1986 Feb 14;44(3):419–428. doi: 10.1016/0092-8674(86)90463-0. [DOI] [PubMed] [Google Scholar]
- Yenofsky R. L., Fine M., Pellow J. W. A mutant neomycin phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure. Proc Natl Acad Sci U S A. 1990 May;87(9):3435–3439. doi: 10.1073/pnas.87.9.3435. [DOI] [PMC free article] [PubMed] [Google Scholar]