Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1987 Sep;84(17):6169–6173. doi: 10.1073/pnas.84.17.6169

Integration of Agrobacterium tumefaciens transfer DNA (T-DNA) involves rearrangements of target plant DNA sequences

Godelieve Gheysen 1, Marc Van Montagu 1,*, Patricia Zambryski 1,†
PMCID: PMC299031  PMID: 16578815

Abstract

The transfer DNA (T-DNA) mobilized into plant cells by Agrobacterium tumefaciens seems to integrate rather randomly into the plant genome. We analyzed a target site in the genome of Nicotiana tabacum before and after integration of a T-DNA. Clones presenting right and left T-DNA/plant DNA junctions were used as probes to identify and isolate a unique 1.8-kilobase EcoRI fragment corresponding to the plant DNA target site for a T-DNA insertion event. Comparison of the nucleotide sequences of the plant DNA portions of the T-DNA junction clones with the original plant DNA target revealed that several types of rearrangements resulted from insertion of the T-DNA. The most dramatic alteration was a 158-base-pair direct repeat of target plant sequences at the left and right T-DNA junctions. In addition, there were deletion and insertion events at the ends of the right and left copies of the 158-base-pair repeat. The variety of target-site rearrangements suggests that T-DNA insertion is a multistep process of recombination accompanied by local replicative and repair activities mediated by host-cell enzymes.

Keywords: Nicotiana tabacum

Full text

PDF
6169

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ambros P. F., Matzke A. J. M., Matzke M. A. Localization of Agrobacterium rhizogenes T-DNA in plant chromosomes by in situ hybridization. EMBO J. 1986 Sep;5(9):2073–2077. doi: 10.1002/j.1460-2075.1986.tb04468.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Capecchi M. R. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell. 1980 Nov;22(2 Pt 2):479–488. doi: 10.1016/0092-8674(80)90358-x. [DOI] [PubMed] [Google Scholar]
  3. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  4. Daniels G. R., Deininger P. L. Integration site preferences of the Alu family and similar repetitive DNA sequences. Nucleic Acids Res. 1985 Dec 20;13(24):8939–8954. doi: 10.1093/nar/13.24.8939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dejean A., Bougueleret L., Grzeschik K. H., Tiollais P. Hepatitis B virus DNA integration in a sequence homologous to v-erb-A and steroid receptor genes in a hepatocellular carcinoma. Nature. 1986 Jul 3;322(6074):70–72. doi: 10.1038/322070a0. [DOI] [PubMed] [Google Scholar]
  6. Della Valle G., Fenton R. G., Basilico C. Polyoma large T antigen regulates the integration of viral DNA sequences into the genome of transformed cells. Cell. 1981 Feb;23(2):347–355. doi: 10.1016/0092-8674(81)90130-6. [DOI] [PubMed] [Google Scholar]
  7. Douglas C. J., Staneloni R. J., Rubin R. A., Nester E. W. Identification and genetic analysis of an Agrobacterium tumefaciens chromosomal virulence region. J Bacteriol. 1985 Mar;161(3):850–860. doi: 10.1128/jb.161.3.850-860.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fischer R. L., Goldberg R. B. Structure and flanking regions of soybean seed protein genes. Cell. 1982 Jun;29(2):651–660. doi: 10.1016/0092-8674(82)90181-7. [DOI] [PubMed] [Google Scholar]
  9. Gerondakis S., Cory S., Adams J. M. Translocation of the myc cellular oncogene to the immunoglobulin heavy chain locus in murine plasmacytomas is an imprecise reciprocal exchange. Cell. 1984 Apr;36(4):973–982. doi: 10.1016/0092-8674(84)90047-3. [DOI] [PubMed] [Google Scholar]
  10. Kwok W. W., Nester E. W., Gordon M. P. Unusual plasmid DNA organization in an octopine crown gall tumor. Nucleic Acids Res. 1985 Jan 25;13(2):459–471. doi: 10.1093/nar/13.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Leemans J., Deblaere R., Willmitzer L., De Greve H., Hernalsteens J. P., Van Montagu M., Schell J. Genetic Identification of functions of TL-DNA transcripts in octopine crown galls. EMBO J. 1982;1(1):147–152. doi: 10.1002/j.1460-2075.1982.tb01138.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lemmers M., De Beuckeleer M., Holsters M., Zambryski P., Depicker A., Hernalsteens J. P., Van Montagu M., Schell J. Internal organization, boundaries and integration of Ti-plasmid DNA in nopaline grown gall tumours. J Mol Biol. 1980 Dec 15;144(3):353–376. doi: 10.1016/0022-2836(80)90095-9. [DOI] [PubMed] [Google Scholar]
  13. Loenen W. A., Blattner F. R. Lambda Charon vectors (Ch32, 33, 34 and 35) adapted for DNA cloning in recombination-deficient hosts. Gene. 1983 Dec;26(2-3):171–179. doi: 10.1016/0378-1119(83)90187-7. [DOI] [PubMed] [Google Scholar]
  14. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Peralta E. G., Ream L. W. T-DNA border sequences required for crown gall tumorigenesis. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5112–5116. doi: 10.1073/pnas.82.15.5112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rauth S., Song K. Y., Ayares D., Wallace L., Moore P. D., Kucherlapati R. Transfection and homologous recombination involving single-stranded DNA substrates in mammalian cells and nuclear extracts. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5587–5591. doi: 10.1073/pnas.83.15.5587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ruley H. E., Fried M. Clustered illegitimate recombination events in mammalian cells involving very short sequence homologies. Nature. 1983 Jul 14;304(5922):181–184. doi: 10.1038/304181a0. [DOI] [PubMed] [Google Scholar]
  18. Stachel S. E., Nester E. W. The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens. EMBO J. 1986 Jul;5(7):1445–1454. doi: 10.1002/j.1460-2075.1986.tb04381.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Stachel S. E., Zambryski P. C. Agrobacterium tumefaciens and the susceptible plant cell: a novel adaptation of extracellular recognition and DNA conjugation. Cell. 1986 Oct 24;47(2):155–157. doi: 10.1016/0092-8674(86)90437-x. [DOI] [PubMed] [Google Scholar]
  20. Van Arsdell S. W., Weiner A. M. Pseudogenes for human U2 small nuclear RNA do not have a fixed site of 3' truncation. Nucleic Acids Res. 1984 Feb 10;12(3):1463–1471. doi: 10.1093/nar/12.3.1463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wang K., Herrera-Estrella L., Van Montagu M., Zambryski P. Right 25 bp terminus sequence of the nopaline T-DNA is essential for and determines direction of DNA transfer from agrobacterium to the plant genome. Cell. 1984 Sep;38(2):455–462. doi: 10.1016/0092-8674(84)90500-2. [DOI] [PubMed] [Google Scholar]
  22. Wang K., Stachel S. E., Timmerman B., VAN Montagu M., Zambryski P. C. Site-Specific Nick in the T-DNA Border Sequence as a Result of Agrobacterium vir Gene Expression. Science. 1987 Jan 30;235(4788):587–591. doi: 10.1126/science.235.4788.587. [DOI] [PubMed] [Google Scholar]
  23. Weinberg R. A. Integrated genomes of animal viruses. Annu Rev Biochem. 1980;49:197–226. doi: 10.1146/annurev.bi.49.070180.001213. [DOI] [PubMed] [Google Scholar]
  24. Williams T. J., Fried M. Inverted duplication-transposition event in mammalian cells at an illegitimate recombination join. Mol Cell Biol. 1986 Jun;6(6):2179–2184. doi: 10.1128/mcb.6.6.2179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wood W. B. Host specificity of DNA produced by Escherichia coli: bacterial mutations affecting the restriction and modification of DNA. J Mol Biol. 1966 Mar;16(1):118–133. doi: 10.1016/s0022-2836(66)80267-x. [DOI] [PubMed] [Google Scholar]
  26. Yanofsky M. F., Porter S. G., Young C., Albright L. M., Gordon M. P., Nester E. W. The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease. Cell. 1986 Nov 7;47(3):471–477. doi: 10.1016/0092-8674(86)90604-5. [DOI] [PubMed] [Google Scholar]
  27. Zambryski P., Depicker A., Kruger K., Goodman H. M. Tumor induction by Agrobacterium tumefaciens: analysis of the boundaries of T-DNA. J Mol Appl Genet. 1982;1(4):361–370. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES