Abstract
The methylation status and 5-azacytidine-induced hypomethylation of CCGG sites within a family of tandemly organized, highly repeated DNA sequences of the Nicotiana tabacum L. nuclear genome (HRS60 family) were studied. As shown by in-situ hybridization experiments, the HRS60 family is clustered in a few regions of some tobacco chromosomes. The DNAs of leaf-derived calli, leaf-derived calli cultured on media with 5-azacytidine, and leaves were cleaved with restriction endonucleases differing in the sensitivity to the methylation of cytosine. After electrophoresis and Southern blotting they were hybridized with the HRS60 probe. We show that (i) CpG dinucleotides, and partially also CpCpG trinucleotides, of the HRS60 family are methylated in DNAs of the non-treated calli and leaves, and (ii) that these DNA repeats are sensitive to the action of a hypomethylating drug, 5-azacytidine.
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Abbreviations
- azaC:
-
azacytidine
- bp:
-
basepairs
- mC:
-
methylated cytosine
- CpG:
-
deoxycytidine-phosphate-deoxyguanosine
- X (in CpXpG):
-
any deoxyribonucleoside
References
Adams, R.L.P., Burdon, R.H. (1985) Molecular biology of DNA methylation. Springer, New York
Amasino, R.M., Powell, A.L.T., Gordon, M.P. (1984) Changes in T-DNA methylation and expression are associated with variation and plant regeneration in a crown gall tumour line. Mol. Gen. Genet. 197, 437–446
Ambros, P.F., Matzke, M.A., Matzke, A.J.M. (1986) Detection of a 17kb unique sequence (T-DNA) in plant chromosomes by in situ hybridization. Chromosoma 94, 11–18
Blundy, K.S., Cullis, C.A., Hepburn, A.G. (1987) Ribosomal DNA methylation in a flax genotroph and a crown gall tumour. Plant Mol. Biol. 8, 217–225
Bostock, C. (1980) A function for the satellite DNA? Trends Biochem. Sci. 5, 117–119
Brown, P.T.H. (1989) DNA methylation in plants and its role in tissue culture. Genome 31, 717–729
Brown, P.T.H., Yoneyama, K., Lörz, H. (1989) An investigation into the role of 5-azacytidine in tissue culture. Theor. Appl. Genet. 78, 321–328
Cedar, H. (1988) DNA methylation and gene activity. Cell 53, 3–4
Dellaporta, S.L., Wood, J., Hicks, J.B. (1983) A plant DNA minipreparation: version II. Plant Mol. Biol. Rep. 1, 19–21
Ellis, T.H.N., Goldbrough, P.B., Castleton, J.A. (1983) Transcription and methylation of flax rDNA. Nucleic Acids Res. 11, 3047–3064
Gruenbaum, Y., Naveh-Many, T., Cedar, H., Razin, A. (1981) Sequence specificity of methylation in higher plant DNA. Nature 292, 860–862
Hepburn, A.G., Belanger, F.C., Mattheis, J.R. (1987) DNA methylation in plants. Dev. Genet. 8, 475–493
Jupe, E.R., Zimmer, E.A. (1990) Undermethylated regions in the intergenic spacer of maize and teosinte ribosomal RNA genes. Plant Mol. Biol. 14, 333–347
Koukalová, B., Reich, J., Matyášek, R., Kuhrová, V., Bezděk, M. (1989) A BamHI family of highly repeated DNA sequences of Nicotiana tabacum. Theor. Appl. Genet. 78, 77–80
Koukalová, B., Reich, J., Bezděk, M. (1990) A BamHI family of tobacco highly repeated DNA: A study about its species-specificity. Biol. Plant. (Praha) 32, 445–449
LoSchiavo, F., Pitto, L., Giuliano, G., Torti, G., Nutti-Ronchi, V., Marazziti, D., Vergara, R., Orselli, S., Terzi, M. (1989) DNA methylation of embryonic carrot cell cultures and its variations as caused by mutation, differentiation, hormones and hypomethylating drugs. Theor. Appl. Genet. 77, 325–331
Maniatis, T., Fritsch, E.F., Sambrook, J. (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Matyášek, R., Koukalová, B., Reich, J. (1989) Isolation and sequencing of HRS60dim1, a dimeric member of the HRS60family of a tobacco DNA repeat. Nucleic Acids Res. 17, 4377
Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 15, 473–497
Reddy, A.S., Guha-Mukherjee, S., Upadyaya, K.C. (1988) Demethylation of satellite DNA sequences in the cell cultures of Brassica juncea (L.) Czern. Cell Biol. Int. Rep. 12, 989
Riggs, C.D., Chrispeels, M.J. (1990) The expression of phytohemagglutinin genes in Phaseolus vulgaris is associated with organ-specific DNA methylation patterns. Plant Mol. Biol. 14, 629–632
Saghai-Maroof, M.A., Soliman, K.M., Jorgensen, R.A., Allard, R.W. (1984) Ribosomal DNA spacer-length polymorphisms in barley, Mendelian inheritance, chromosomal location, and population dynamics. Proc. Natl. Acad. Sci. USA 81, 8014–8018
Scott, N.S., Kavanagh, T.A., Timmis, J.N. (1984) Methylation of rRNA genes in some higher plants. Plant Sci. Lett. 35, 213–217
Van Slogteren, G.M.S., Hooykaas, P.J.J., Schilperoort, R.A. (1984) Silent T-DNA genes in plant lines transformed by Agrobacterium tumefaciens are activated by grafting and by 5-azacytidine treatment. Plant Mol. Biol. 3, 333–336
Vergara, R., Verde, F., Pitto, L., LoSchiavo, F., Terzi, M. (1990) Reversible variations in the methylation pattern of carrot DNA during somatic embryogenesis. Plant Cell Rep. 8, 697–700
Vogt, P. (1990) Potential genetic functions of tandem repeated DNA sequence blocks in the human genome are based on a highly conserved “chromatin coding code”. Human Genet. 84, 301–366
von Kalm, L., Vize, P.D., Smyth, D.R. (1986) An undermethylated region in the spacer of ribosomal RNA genes of Lilium henryi. Plant Mol. Biol. 6, 33–39
Vyskot, B., Brzobohatý, B., Karlovská, L., Bezděk, M. (1989) Structural and functional stability of foreign genes in transgenic tobacco plants. Folia Biol. (Praha) 35, 360–372
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We would like to thank Dr. J. Fajkus and Dr. J. Reich (Institute of Biophysics, Brno) for their suggestion of a method of checking the completeness of plant DNA digestion, Professor J. Schell (Max-Planck Institute, Cologne, FRG), Dr. J. Soška and Dr. J. Kypr (Institute of Biophysics, Brno) for critical reading of the manuscript, and H. Suchánková and L. Jedličková for technical assistance.
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Bezděk, M., Koukalová, B., Brzobohatý, B. et al. 5-Azacytidine-induced hypomethylation of tobacco HRS60 tandem DNA repeats in tissue culture. Planta 184, 487–490 (1991). https://doi.org/10.1007/BF00197896
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DOI: https://doi.org/10.1007/BF00197896