Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1989 Oct;9(10):4298–4303. doi: 10.1128/mcb.9.10.4298

Extensive changes in DNA methylation patterns accompany activation of a silent T-DNA ipt gene in Agrobacterium tumefaciens-transformed plant cells.

M C John 1, R M Amasino 1
PMCID: PMC362510  PMID: 2479825

Abstract

We crossed a male-sterile, Agrobacterium-transformed Nicotiana tabacum plant that contains a silent, hypermethylated T-DNA ipt oncogene with a normal tobacco plant and found that the methylated state of the ipt gene was stably inherited through meiosis in the offspring. However, when tissues of these plants were placed in cell culture, the ipt gene was spontaneously reactivated in a very small fraction of the cells; if 5-azacytidine was added to the culture medium, ipt gene reactivation occurred at high frequency. We analyzed the pattern of DNA methylation in a region spanning the ipt gene in a line that does not express the ipt gene, in five derivatives of this line that reexpressed the ipt gene either spontaneously or after 5-azacytidine treatment, and in tissues of a sibling of this line that reexpressed ipt spontaneously. We found that the ipt locus was highly methylated in the unexpressed state but that spontaneous or 5-azacytidine-induced reexpression always resulted in extensive demethylation of a region including 5' upstream, coding, and 3' downstream regions of the ipt gene. The role of DNA methylation in gene regulation in this system is discussed.

Full text

PDF
4301

Images in this article

4300Image
on p.4300
4300Image
on p.4300
4302Image
on p.4302

Selected References

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

  1. Akiyoshi D. E., Klee H., Amasino R. M., Nester E. W., Gordon M. P. T-DNA of Agrobacterium tumefaciens encodes an enzyme of cytokinin biosynthesis. Proc Natl Acad Sci U S A. 1984 Oct;81(19):5994–5998. doi: 10.1073/pnas.81.19.5994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amasino R. M., Powell A. L., Gordon M. P. Changes in T-DNA methylation and expression are associated with phenotypic variation and plant regeneration in a crown gall tumor line. Mol Gen Genet. 1984;197(3):437–446. doi: 10.1007/BF00329940. [DOI] [PubMed] [Google Scholar]
  3. Barry G. F., Rogers S. G., Fraley R. T., Brand L. Identification of a cloned cytokinin biosynthetic gene. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4776–4780. doi: 10.1073/pnas.81.15.4776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Becker P. B., Ruppert S., Schütz G. Genomic footprinting reveals cell type-specific DNA binding of ubiquitous factors. Cell. 1987 Nov 6;51(3):435–443. doi: 10.1016/0092-8674(87)90639-8. [DOI] [PubMed] [Google Scholar]
  5. Busslinger M., Hurst J., Flavell R. A. DNA methylation and the regulation of globin gene expression. Cell. 1983 Aug;34(1):197–206. doi: 10.1016/0092-8674(83)90150-2. [DOI] [PubMed] [Google Scholar]
  6. Cedar H. DNA methylation and gene activity. Cell. 1988 Apr 8;53(1):3–4. doi: 10.1016/0092-8674(88)90479-5. [DOI] [PubMed] [Google Scholar]
  7. Chandler V. L., Walbot V. DNA modification of a maize transposable element correlates with loss of activity. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1767–1771. doi: 10.1073/pnas.83.6.1767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gierl A., Lütticke S., Saedler H. TnpA product encoded by the transposable element En-1 of Zea mays is a DNA binding protein. EMBO J. 1988 Dec 20;7(13):4045–4053. doi: 10.1002/j.1460-2075.1988.tb03298.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gruenbaum Y., Naveh-Many T., Cedar H., Razin A. Sequence specificity of methylation in higher plant DNA. Nature. 1981 Aug 27;292(5826):860–862. doi: 10.1038/292860a0. [DOI] [PubMed] [Google Scholar]
  10. Hepburn A. G., Clarke L. E., Pearson L., White J. The role of cytosine methylation in the control of nopaline synthase gene expression in a plant tumor. J Mol Appl Genet. 1983;2(3):315–329. [PubMed] [Google Scholar]
  11. Jones P. A. Altering gene expression with 5-azacytidine. Cell. 1985 Mar;40(3):485–486. doi: 10.1016/0092-8674(85)90192-8. [DOI] [PubMed] [Google Scholar]
  12. Keshet I., Lieman-Hurwitz J., Cedar H. DNA methylation affects the formation of active chromatin. Cell. 1986 Feb 28;44(4):535–543. doi: 10.1016/0092-8674(86)90263-1. [DOI] [PubMed] [Google Scholar]
  13. Keshet I., Yisraeli J., Cedar H. Effect of regional DNA methylation on gene expression. Proc Natl Acad Sci U S A. 1985 May;82(9):2560–2564. doi: 10.1073/pnas.82.9.2560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lichtenstein C., Klee H., Montoya A., Garfinkel D., Fuller S., Flores C., Nester E., Gordon M. Nucleotide sequence and transcript mapping of the tmr gene of the pTiA6NC octopine Ti-plasmid: a bacterial gene involved in plant tumorigenesis. J Mol Appl Genet. 1984;2(4):354–362. [PubMed] [Google Scholar]
  15. Reid R. A., John M. C., Amasino R. M. Deoxyribonuclease I sensitivity of the T-DNA ipt gene is associated with gene expression. Biochemistry. 1988 Jul 26;27(15):5748–5754. doi: 10.1021/bi00415a053. [DOI] [PubMed] [Google Scholar]
  16. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  17. Watson J. C., Kaufman L. S., Thompson W. F. Developmental regulation of cytosine methylation in the nuclear ribosomal RNA genes of Pisum sativum. J Mol Biol. 1987 Jan 5;193(1):15–26. doi: 10.1016/0022-2836(87)90622-x. [DOI] [PubMed] [Google Scholar]
  18. Watt F., Molloy P. L. Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter. Genes Dev. 1988 Sep;2(9):1136–1143. doi: 10.1101/gad.2.9.1136. [DOI] [PubMed] [Google Scholar]
  19. Yisraeli J., Frank D., Razin A., Cedar H. Effect of in vitro DNA methylation on beta-globin gene expression. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4638–4642. doi: 10.1073/pnas.85.13.4638. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES