Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1985 Aug 12;13(15):5503–5513. doi: 10.1093/nar/13.15.5503

Inhibition of herpes simplex thymidine kinase gene expression by DNA methylation is an indirect effect.

G Buschhausen, M Graessmann, A Graessmann
PMCID: PMC321886  PMID: 2994001

Abstract

The biological activity of in vitro methylated HSV-TK DNA was analysed after microinjection into thymidine kinase negative rat 2 cells. It was found that the fully methylated DNA (Hpa II methylase) was as active as the non methylated control DNA for about 48 hours after injection. DNA reextraction experiments and blot analysis showed that DNA demethylation was not the reason for the observed TK activity. With prolonged cultivation time the methylated DNA becomes rapidly inactive and 100 hrs after injection thymidine incorporation was no longer detectable in the recipient cells. In transformed cells, obtained after coinjection with SV40 DNA, the HSV-DNA was partially demethylated and inactive. Addition of 5-azacytidine to the culture medium induced further demethylation and reactivation of the thymidine kinase gene.

Full text

PDF
5507

Images in this article

Selected References

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

  1. Botchan M., Topp W., Sambrook J. The arrangement of simian virus 40 sequences in the DNA of transformed cells. Cell. 1976 Oct;9(2):269–287. doi: 10.1016/0092-8674(76)90118-5. [DOI] [PubMed] [Google Scholar]
  2. Doerfler W. DNA methylation and gene activity. Annu Rev Biochem. 1983;52:93–124. doi: 10.1146/annurev.bi.52.070183.000521. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Fradin A., Manley J. L., Prives C. L. Methylation of simian virus 40 Hpa II site affects late, but not early, viral gene expression. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5142–5146. doi: 10.1073/pnas.79.17.5142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Graessmann A., Graessmann M., Topp W. C., Botchan M. Retransformation of a simian virus 40 revertant cell line, which is resistant to viral and DNA infections, by microinjection of viral DNA. J Virol. 1979 Dec;32(3):989–994. doi: 10.1128/jvi.32.3.989-994.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Graessmann M., Graessman A. "Early" simian-virus-40-specific RNA contains information for tumor antigen formation and chromatin replication. Proc Natl Acad Sci U S A. 1976 Feb;73(2):366–370. doi: 10.1073/pnas.73.2.366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Graessmann M., Graessmann A. Microinjection of tissue culture cells. Methods Enzymol. 1983;101:482–492. doi: 10.1016/0076-6879(83)01033-2. [DOI] [PubMed] [Google Scholar]
  8. Graessmann M., Graessmann A., Wagner H., Werner E., Simon D. Complete DNA methylation does not prevent polyoma and simian virus 40 virus early gene expression. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6470–6474. doi: 10.1073/pnas.80.21.6470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  10. Lusky M., Berg L., Weiher H., Botchan M. Bovine papilloma virus contains an activator of gene expression at the distal end of the early transcription unit. Mol Cell Biol. 1983 Jun;3(6):1108–1122. doi: 10.1128/mcb.3.6.1108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. McGeady M. L., Jhappan C., Ascione R., Vande Woude G. F. In vitro methylation of specific regions of the cloned Moloney sarcoma virus genome inhibits its transforming activity. Mol Cell Biol. 1983 Mar;3(3):305–314. doi: 10.1128/mcb.3.3.305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. McKnight S. L. Functional relationships between transcriptional control signals of the thymidine kinase gene of herpes simplex virus. Cell. 1982 Dec;31(2 Pt 1):355–365. doi: 10.1016/0092-8674(82)90129-5. [DOI] [PubMed] [Google Scholar]
  13. McKnight S. L. The nucleotide sequence and transcript map of the herpes simplex virus thymidine kinase gene. Nucleic Acids Res. 1980 Dec 20;8(24):5949–5964. doi: 10.1093/nar/8.24.5949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Stein R., Razin A., Cedar H. In vitro methylation of the hamster adenine phosphoribosyltransferase gene inhibits its expression in mouse L cells. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3418–3422. doi: 10.1073/pnas.79.11.3418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Stuhlmann H., Jähner D., Jaenisch R. Infectivity and methylation of retroviral genomes is correlated with expression in the animal. Cell. 1981 Oct;26(2 Pt 2):221–232. doi: 10.1016/0092-8674(81)90305-6. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. van der Ploeg L. H., Flavell R. A. DNA methylation in the human gamma delta beta-globin locus in erythroid and nonerythroid tissues. Cell. 1980 Apr;19(4):947–958. doi: 10.1016/0092-8674(80)90086-0. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES