In Vitro Methylation of Predetermined Regions in Recombinant DNA Constructs

  • Ilse Van den Wyngaert
  • Roger L. P. Adams
  • Stefan U. Kass
Part of the Methods in Molecular Biology™ book series (MIMB, volume 181)


DNA methylation at position 5 in the cytosine ring in the sequence CpG can be detrimental to the transcription of a variety of genes in higher eukaryotes (1,2). Although the significance of this transcriptional repression is currently under debate (3,4), there is little disagreement that it plays an important role in genomic imprinting and X-chromosome inactivation (5,6). To study the effects of DNA methylation on transcription in an experimental system, bacterial DNA methyltransferases have been used widely in order to mimic the DNA methylation pattern of eukaryotic genes. However, usually every target site in a given recombinant DNA molecule will be subject to DNA methylation by making use of those enzymes. This might result in an exaggeration of the effects of DNA methylation, as most recombinant DNA molecules contain a high degree of prokaryotic DNA, which is rich in CpGs. This methylated CpGrich DNA can contribute to the effects of DNA methylation by formation of a repressive chromatin structure (7,8). In addition, selective DNA methylation is required to distinguish the effects of DNA methylation on transcription initiation and transcript elongation (8,9). Thus, there is a requirement for a method to generate recombinant DNA molecules that are methylated in a predetermined region. The chapter following this one will describe a method that makes use of ligation of methylated DNA fragments into unmethylated vector DNA. This method relies on the availability of suitable restriction sites


dsDNA Fragment Cytosine Ring Repressive Chromatin Structure Transcript Elongation HpaII Digestion 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Kass, S. U., Pruss, D., and Wolffe, A. P. (1997) How does DNA methylation inhibit transcription? Trends Genet. 13, 444–449.PubMedCrossRefGoogle Scholar
  2. 2.
    Ng, H. H. and Bird, A. (1999) DNA methylation and chromatin modification. Curr. Opin. Genet. Develop. 9, 158–163.CrossRefGoogle Scholar
  3. 3.
    Walsh, C. P. and Bestor, T. H. (1999) Cytosine methylation and mammalian development. Genes Dev. 13, 26–34.PubMedCrossRefGoogle Scholar
  4. 4.
    Simmen, M. W., Leitgeb, S. Charlton, J., Jones, S. J. M., Harris, B. R., Clark, V. H., and Bird, A. (1999) Nonmethylated transposable elements and methylated genes in a chordate genome. Science 283, 1164–1167.PubMedCrossRefGoogle Scholar
  5. 5.
    Brannan, C. I. and Bartolomei, M. S. (1999) Mechanisms of genomic imprinting. Curr. Opp. Genet. Dev. 9, 164–170.CrossRefGoogle Scholar
  6. 6.
    Lyon, M. F. (1999) X-chromosome inactivation. Curr. Biol. 9, R235–R237.PubMedCrossRefGoogle Scholar
  7. 7.
    Kass, S. U., Goddard, J. P., and Adams, R. L. P. (1993). Inactive chromatin spreads from a focus of methylation. Mol. Cell Biol. 13, 7372–7379.PubMedGoogle Scholar
  8. 8.
    Hsieh, C. L. (1997) Stability of patch methylation and its impact in regions of transcriptional initiation and elongation. Mol. Cell Biol. 17, 5897–5904.PubMedGoogle Scholar
  9. 9.
    Kass, S. U., Landsberger, N., and Wolffe, A. P. (1997). DNA methylation directs a time-dependent repression of transcription initiation. Curr. Biol. 7, 157–165.PubMedCrossRefGoogle Scholar
  10. 10.
    Renbaum, P., Abrahamove, D., Fainsod, A., Wilson, G. G., Rottem, S., and Razin, A. (1990) Cloning, characterization, and expression in Escherichia coli of the gene CpG-Methylated Regions 249 coding for the CpG DNA methylase from Spiroplasma sp. Strain MQ1 (M.SssI). Nucl. Acids Res. 18, 1145–1152.PubMedCrossRefGoogle Scholar
  11. 11.
    Sambrook, J., Fritsch, E. F., and Maniatis, T., eds. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Google Scholar
  12. 12.
    Blondel, A. and Thillet, J. (1991) A fast and convenient way to produce singlestranded DNA from a phagemid. Nucleic Acids Res. 19, 181.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Ilse Van den Wyngaert
    • 1
  • Roger L. P. Adams
    • 2
  • Stefan U. Kass
    • 1
  1. 1.Department of Genomic TechnologiesJanssen Research FoundationBeerseBelgium
  2. 2.IBLS, University of GlasgowScotland, UK

Personalised recommendations