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
. 1986 Oct;83(20):7713–7717. doi: 10.1073/pnas.83.20.7713

Introduction and expression of the bacterial PaeR7 methylase gene in mammalian cells.

T J Kwoh, D Y Kwoh, A W McCue, G R Davis, D Patrick, T R Gingeras
PMCID: PMC386791  PMID: 3463994

Abstract

An approach is devised for studying the role of DNA methylation in eukaryotic gene expression. The approach is based on the expression of site-specific bacterial methylase genes in animal cells. A model system using the cloned PaeR7 (an isoschizomer of Xho I) methylase gene was constructed to test the feasibility of this approach. Expression plasmids for the PaeR7 methylase gene were introduced into mouse Ltk- cells by cotransfection with the cloned chicken thymidine kinase (tk) gene. Several of the cell strains derived from Tk+ colonies were found to express the PaeR7 gene as judged by four criteria: the cellular DNA of these strains showed increased resistance to cleavage by Xho I; these strains contained cellular proteins that comigrated with pure PaeR7 methylase protein, as visualized by immunoblotting; PaeR7 methylase activity was found in vitro in crude extracts of total cellular protein from these strains; and murine adenovirus genomes grown on cells expressing PaeR7 methylase showed resistance to cleavage to PaeR7 endonuclease. The potential applications of this approach for the study of cellular and viral gene regulation, DNA repair, and restriction modification are discussed.

Full text

PDF
7713

Images in this article

7715Image
on p.7715
7715Image
on p.7715
7715Image
on p.7715
7715Image
on p.7715
7715Image
on p.7715
7715Image
on p.7715
7715Image
on p.7715
7716Image
on p.7716
7716Image
on p.7716

Selected References

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

  1. Adams R. L., Fulton J., Kirk D. The effect of 5-azadeoxycytidine on cell growth and DNA methylation. Biochim Biophys Acta. 1982 Jun 30;697(3):286–294. doi: 10.1016/0167-4781(82)90091-4. [DOI] [PubMed] [Google Scholar]
  2. Banerjee A., Benedict W. F. Production of sister chromatid exchanges by various cancer chemotherapeutic agents. Cancer Res. 1979 Mar;39(3):797–799. [PubMed] [Google Scholar]
  3. Benedict W. F., Banerjee A., Gardner A., Jones P. A. Induction of morphological transformation in mouse C3H/10T1/2 clone 8 cells and chromosomal damage in hamster A(T1)C1-3 cells by cancer chemotherapeutic agents. Cancer Res. 1977 Jul;37(7 Pt 1):2202–2208. [PubMed] [Google Scholar]
  4. Bird A. P., Southern E. M. Use of restriction enzymes to study eukaryotic DNA methylation: I. The methylation pattern in ribosomal DNA from Xenopus laevis. J Mol Biol. 1978 Jan 5;118(1):27–47. doi: 10.1016/0022-2836(78)90242-5. [DOI] [PubMed] [Google Scholar]
  5. Brooks J. E., Blumenthal R. M., Gingeras T. R. The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene. Nucleic Acids Res. 1983 Feb 11;11(3):837–851. doi: 10.1093/nar/11.3.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cabilio P., Robbins H. Sequential Estimation of p with Squared Relative Error Loss. Proc Natl Acad Sci U S A. 1975 Jan;72(1):191–193. doi: 10.1073/pnas.72.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doerfler W., Kruczek I., Eick D., Vardimon L., Kron B. DNA methylation and gene activity: the adenovirus system as a model. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):593–603. doi: 10.1101/sqb.1983.047.01.070. [DOI] [PubMed] [Google Scholar]
  9. Dretzen G., Bellard M., Sassone-Corsi P., Chambon P. A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal Biochem. 1981 Apr;112(2):295–298. doi: 10.1016/0003-2697(81)90296-7. [DOI] [PubMed] [Google Scholar]
  10. Gingeras T. R., Brooks J. E. Cloned restriction/modification system from Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 1983 Jan;80(2):402–406. doi: 10.1073/pnas.80.2.402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Glickman B. W., Radman M. Escherichia coli mutator mutants deficient in methylation-instructed DNA mismatch correction. Proc Natl Acad Sci U S A. 1980 Feb;77(2):1063–1067. doi: 10.1073/pnas.77.2.1063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gruenbaum Y., Cedar H., Razin A. Substrate and sequence specificity of a eukaryotic DNA methylase. Nature. 1982 Feb 18;295(5850):620–622. doi: 10.1038/295620a0. [DOI] [PubMed] [Google Scholar]
  13. Guerry P., LeBlanc D. J., Falkow S. General method for the isolation of plasmid deoxyribonucleic acid. J Bacteriol. 1973 Nov;116(2):1064–1066. doi: 10.1128/jb.116.2.1064-1066.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hare J. T., Taylor J. H. One role for DNA methylation in vertebrate cells is strand discrimination in mismatch repair. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7350–7354. doi: 10.1073/pnas.82.21.7350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Harland R. M. Inheritance of DNA methylation in microinjected eggs of Xenopus laevis. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2323–2327. doi: 10.1073/pnas.79.7.2323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jones P. A., Taylor S. M. Hemimethylated duplex DNAs prepared from 5-azacytidine-treated cells. Nucleic Acids Res. 1981 Jun 25;9(12):2933–2947. doi: 10.1093/nar/9.12.2933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Klein R. D., Selsing E., Wells R. D. A rapid microscale technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis. Plasmid. 1980 Jan;3(1):88–91. doi: 10.1016/s0147-619x(80)90037-2. [DOI] [PubMed] [Google Scholar]
  18. Kwoh T. J., Zipser D., Wigler M. Mutational analysis of the cloned chicken thymidine kinase gene. J Mol Appl Genet. 1983;2(2):191–200. [PubMed] [Google Scholar]
  19. Larsen S. H., Nathans D. Mouse adenovirus: growth of plaque-purified FL virus in cell lines and characterization of viral DNA. Virology. 1977 Oct 1;82(1):182–195. doi: 10.1016/0042-6822(77)90041-1. [DOI] [PubMed] [Google Scholar]
  20. Pollack Y., Stein R., Razin A., Cedar H. Methylation of foreign DNA sequences in eukaryotic cells. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6463–6467. doi: 10.1073/pnas.77.11.6463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pukkila P. J., Peterson J., Herman G., Modrich P., Meselson M. Effects of high levels of DNA adenine methylation on methyl-directed mismatch repair in Escherichia coli. Genetics. 1983 Aug;104(4):571–582. doi: 10.1093/genetics/104.4.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  23. Stein R., Gruenbaum Y., Pollack Y., Razin A., Cedar H. Clonal inheritance of the pattern of DNA methylation in mouse cells. Proc Natl Acad Sci U S A. 1982 Jan;79(1):61–65. doi: 10.1073/pnas.79.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Taylor S. M., Jones P. A. Mechanism of action of eukaryotic DNA methyltransferase. Use of 5-azacytosine-containing DNA. J Mol Biol. 1982 Dec 15;162(3):679–692. doi: 10.1016/0022-2836(82)90395-3. [DOI] [PubMed] [Google Scholar]
  25. Theriault G., Roy P. H., Howard K. A., Benner J. S., Brooks J. E., Waters A. F., Gingeras T. R. Nucleotide sequence of the PaeR7 restriction/modification system and partial characterization of its protein products. Nucleic Acids Res. 1985 Dec 9;13(23):8441–8461. doi: 10.1093/nar/13.23.8441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wagner R., Jr, Meselson M. Repair tracts in mismatched DNA heteroduplexes. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4135–4139. doi: 10.1073/pnas.73.11.4135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wigler M., Levy D., Perucho M. The somatic replication of DNA methylation. Cell. 1981 Apr;24(1):33–40. doi: 10.1016/0092-8674(81)90498-0. [DOI] [PubMed] [Google Scholar]
  29. Wigler M., Sweet R., Sim G. K., Wold B., Pellicer A., Lacy E., Maniatis T., Silverstein S., Axel R. Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell. 1979 Apr;16(4):777–785. doi: 10.1016/0092-8674(79)90093-x. [DOI] [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