Summary
Plasmid rescue can provide an efficient way of cloning T-DNA-tagged genomic DNA of plants. However, rescue has often been hampered by extensive rearrangements in the cloned DNA. We have demonstrated using a transgenic line ofArabidopsis thaliana that the plant DNA flanking the T-DNA tag was heavily cytosine methylated. This methylation could be completely inhibited by growing the plants in the presence of azacytidine. Rescue of the T-DNA tag together with the flanking plant genomic DNA sequences from nontreated control plants into an modified cytosine restriction (mcr) proficient strain ofEscherichia coli resulted in rearrangements of the majority of the rescued plasmids. These rearrangements could be avoided if the methylation was inhibited in the transgenic plants by azacytidine treatment or by cloning into anmcr-deficient strain ofE. coli. The results indicate that cytosine methylation of the DNA in the transgenic plants is the main cause of the DNA rearrangements observed during plasmid rescue and suggest efficient strategies to eliminate such artifacts.
Similar content being viewed by others
References
Amasino, R. M.; Powell, A. L. T.; 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. 197:437–446; 1984.
Andre, D.; Colau, D.; Schell, J., et al. Gene tagging in plants by a T-DNA insertion mutagen that generates APH(3′)II-plant gene fusions. Mol. Gen. Genet. 204:512–518; 1986.
Behringer, F. J.; Medford, J. I. A plasmid rescue technique for the recovery of plant DNA disrupted by T-DNA insertion. Plant Mol. Biol. Rep. 10:190–198; 1992.
Bochardt, A.; Hodal, L.; Palmgren, G., et al. DNA methylation is involved in maintainance of an unusual expression pattern of an introduced gene. Plant. Physiol. 99:409–414; 1992.
Cherdshwasart, W. Assessing methylation of inserted DNA by restriction with isoschizomeric enzymes and inducing demethylation with 5-azacytidine. In: Negrutiu, I.; Gharti-Cchetri, G. B., eds. Structural and functional analysis of genomes and genes: a laboratory guide for cellular and molecular plant biology, 4. Basel: Birkhäuser Verlag; 1991:227–286.
Creusot, F.; Acs, G.; Christman, J. K. Inhibition of DNA methyltransferase and induction of Freind erythroleukemia cell differentiation by 5-azacytidine and 5-aza-2′-deoxycytidine. J. Biol. Chem. 257:2041–2074; 1982.
Dower, W. J.; Miller, J. F.; Ragsdale, C. W. High efficiency transformation ofE. coli by high voltage electroporation. Nucleic Acids Res. 16:6127–6145; 1988.
Feldmann, K. A. T-DNA insertion mutagenesis inArabidopsis: mutational spectrum. Plant J. 1:71–82; 1991.
Feldmann, K. A.; Marks, M. D.; Christianson, M. L., et al. A dwarf mutant ofArabidopsis generated by T-DNA insertion mutagenesis. Science 243:1351–1354; 1989.
Garfin, D. E.; Goodman, H. M. Nucleotide sequences at the cleavage sites of two restriction endonucleases fromHemophilus parainfluenzae. Biochem. Biophys. Res. Comm. 59:108–116; 1974.
Gelvin, S. B.; Karcher, S. J.; DiRita, V. J. Methylation of T-DNA inAgrobacterium tumefaciens and in several crown gall tumors. Nucleic Acids Res. 11:159–174; 1983.
Goldsborough, A.; Bevan, M. New patterns of gene activity in plants detected using anAgrobacterium vector. Plant Mol. Biol. 16:263–269; 1991.
Gruenbaum, Y.; Naveh-Many, T.; Cedar, H., et al. Sequence specificity of methylation in higher plant DNA. Nature 292:860–862; 1981.
Heitman, J.; Model, P. Site-specific methylases induce the SOS DNA repair response inEscherichia coli. J. Bacteriol. 169:3243–3250; 1987.
Hepburn A. G.; Clarke, L. E.; Pearson, L., et al. The role of cytosine methylation in the control of nopaline synthase gene expression in a plant tumor. J. Mol. Appl. Genet. 2:315–329; 1983.
Hsiao, W. L.; Gattoni-Celli S.; Weinstein, I. B.. Effect of 5-azacytidine on the progressive nature of cell transformation. Mol. Cell Biol. 5:1800–1803; 1985.
Jones, P.; Alterating gene expression with 5-azacytidine. Cell 40:485–486; 1985.
Kertbundit, S.; De Greve, H.; Deboeck, F., et al.In vivo random β-glucuronidase gene fusions inArabidopsis thaliana. Proc. Natl. Acad. Sci. USA 88:5212–5216; 1991.
Koncz, C.; Martini, N.; Mayerhofer, R., et al. High-frequency T-DNA-mediated gene tagging in plants. Proc. Natl. Acad. Sci. USA 86:8467–8471; 1989.
Koncz, C.; Mayerhofer, R.; Koncz-Kalman, Z., et al. Isolation of a gene encoding a novel chloroplast protein by T-DNA tagging inArabidopsis thaliana. EMBO J., 9:1337–1346; 1990.
Koncz, C.; Schell, J. The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type ofAgrobacterium binary vector. Mol. Gen. Genet. 204:383–396; 1986.
Mandal, A.; Lang, V.; Orczyk, W., et al. Improved efficiency for T-DNA-mediated transformation and plasmid rescue inArabidopsis thaliana. Theor. Appl. Genet. 86:621–628; 1993.
Matzke, M. A.; Primig, M.; Trnovsky, J., et al. Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J. 8:643–649; 1989.
Mayerhofer, R.; Koncz-Kalman, Z.; Nawrath, C., et al. T-DNA integration: a mode of illegitimate recombination in plants. EMBO J. 10:697–704; 1991.
Meyer, P.; Heidman, I.; Niedendof, I. Differences in DNA-methylation are associated with a paramutation phenomenon in transgenic petunia. Plant J. 4:89–100; 1993.
Mohandas, T.; Sparkes, R. S.; Shapiro, L. J. Reactivation of an inactive human X chromosome: evidence for X inactivation by DNA methylation. Science 211:393–396; 1981.
Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473–497; 1962.
Nick, H.; Boven, B.; Ferl, R. J., et al. Detection of cytosine methylation in the maize alcohol dehydrogenase gene by genomic sequencing. Nature 319:243–246; 1986.
Ott, R. W.; Chua, N. H. Enhancer sequences fromArabidopsis thaliana obtained by library transformation ofNicotiana tabacum. Mol. Gen. Genet. 223:169–179; 1990.
Palmgren, G.; Mattsson, O.; Okkels, F. T.. Treatment ofAgrobacterium or leaf disks with 5-azacytidine increases transgene expression in tobacco. Plant Mol. Biol. 21:429–435; 1993.
Raleigh, E. A. Organization and function of themcrBC genes ofEscherichia coli K-12. Mol. Microbiol. 6:1079–1086; 1992.
Raleigh, E. A.; Murray, N. E.; Revel, H., et al. McrA and McrB restriction phenotypes implications for gene cloning. Nucleic Acids Res. 16:1563–1575; 1988.
Raleigh, E. A.; Wilson, G.Escherichia coli K-12 restricts DNA containing 5-methylcytosine. Proc. Natl. Acad. Sci. USA 83:9070–9074; 1986.
Razin, A.; Riggs, A. D. DNA methylation and gene function. Science 210:604–610; 1980.
Ross, T. K.; Achberger, E. C.; Braymer, H. D. Identification of a second polypeptide required formcrB restriction of 5-methylocytosine-containing DNA inEscherichia coli K12. Mol. Gen. Genet. 216:402–407; 1989.
Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning: a laboratory manula, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
Sano, H.; Kamada, I.; Youssefian, S. et al. A single treatment of rice seedlings with 5-azacytidine induces heritable dwarfism and under-methylation of genomic DNA. Mol. Gen. Genet. 220:441–447; 1990.
Sano, H.; Kamada, I.; Youssefian, S., et al. Correlation between DNA undermethylation and dwarfism in maize. Biochem. Biophys. Acta 1009;35–38; 1989.
Southern, E. Detection of specific DNA sequences among fragments separated by gel electrophoresis. J. Mol. Biol. 98:503–517; 1975.
Teeri, T. H.; Herrera-Estrella, L.; Depicker, A., et al. Identification of plant promotersin situ by T-DNA-mediated transcriptional fusions to thenptII gene. EMBO J. 5:1755–1760; 1986.
Topping, J. F.; Wei, W.; Lindsay, K. Functional tagging of regulatory elements in the plant genome. Development 112:1009–1019; 1991.
Van Lijsebettens, M.; Vanderhaeghen, R.; Van Montagu, M. Insertional mutagenesis inArabidopsis thaliana: isolation of a T-DNA-linked mutation that alters leaf morphology. Theor. Appl. Genet. 81:277–284; 1991.
Waalwijk, C.; Flavell, R. A.Mspl, an isoschizomer ofHpaII which cleaves both unmethylated and methylatedHpaII sites. Nucleic Acids Res. 5:3231–3236; 1978.
Waite-Rees, P. A.; Keating, C. J.; Moran, L. S., et al. Characterization and expression of theEscherichia coli Mrr restriction system. J. Bacteriol. 173:5207–5219; 1991.
Weber, H.; Ziechman, C.; Graessmann, A.,In vitro DNA methylation inhibits gene expression in transgenic tobacco. EMBO J. 9:4409–4415; 1990.
Woodcock, D. M.; Crowther, P. J.; Doherty, J., et al. Quantitative evaluation ofEscherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res. 17:3469–3478; 1989.
Yanofsky, M. F.; Ma, H.; Bowman, J. L., et al. The protein encoded by theArabidopsis homeotic geneagamous resembles transcription factors. Nature 346:35–39; 1990.
Zhu, Z.; Huges, K. W.; Huang, L. Effects of 5-azacytidine on transformation and gene expression inNicotiana tabacum. In Vitro Cell. Dev. Biol. 27P:77–83; 1991.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Mandal, A., Sandgren, M. & Palva, E.T. Inhibition of cytosine methylation allows efficient cloning of T-DNA tagged plant DNA ofArabidopsis thaliana by plasmid rescue. In Vitro Cell.Dev.Biol.–Plant 30, 204–209 (1994). https://doi.org/10.1007/BF02823033
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02823033