Gene reactivation: a tool for the isolation of mammalian DNA methylation mutants.

F. Gounari, G. R. Banks, Khashayarsha Khazaie, P. A. Jeggo, R. Holliday

Research output: Contribution to journalArticle

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Abstract

We report the isolation and characterization of a mammalian strain (tsm) that has a temperature-sensitive mutation in DNA methylation. The isolation procedure was based on the observation that treatment of a CHO TK- MT- cell line with demethylating agents introduces up to 46% demethylation, resulting in phenotypic reversion and transcriptional activation of the thymidine kinase (TK) and metallothionein (MT) genes at frequencies ranging from 1% to 59%. Seven thousand individual colonies from an EMS-mutagenized CHO TK- MT- population were screened for spontaneous reversion to TK+ phenotype after treatment at 39 degrees C. Successful isolates were subsequently examined for MT+ reversion. A single clone (tsm) was obtained that showed temperature-dependent reactivation of both TK and MT genes at frequencies of 7.2 X 10(-4) and 6 X 10(-4), respectively. The tsm cells were viable at 39 degrees C and showed no increased mutation frequency. Reactivation correlated with transcriptional activation of the respective genes, whereas backreversion to the TK- phenotype was associated with transcriptional inactivation. TK- backrevertants were reactivable again with demethylating agents. Although demethylation in tsm cells was not detectable by HPLC, Southern blot analysis revealed that reactivants, irrespective of their mode of generation, showed specific demethylation of both TK and MT genes. Also, after about 150 cell generations after treatment, reactivants from both temperature-induced tsm and cells exposed to demethylating agents gained 60% and 23%, respectively, in 5-methylcytosine (5mC). It is proposed that the phenotype of tsm cells is due to a mutation involved in the regulation of DNA methylation. The further characterization of this and other mammalian mutants should help to clarify the physiological role of DNA methylation, as well as its regulation.

Original languageEnglish (US)
Pages (from-to)899-912
Number of pages14
JournalGenes and Development
Volume1
Issue number9
StatePublished - Nov 1987
Externally publishedYes

Fingerprint

Thymidine Kinase
DNA Methylation
Metallothionein
Genes
Phenotype
Gene Frequency
Transcriptional Activation
Temperature
5-Methylcytosine
Mutation
Mutation Rate
Southern Blotting
Clone Cells
High Pressure Liquid Chromatography
Cell Line

ASJC Scopus subject areas

  • Developmental Biology
  • Genetics

Cite this

Gounari, F., Banks, G. R., Khazaie, K., Jeggo, P. A., & Holliday, R. (1987). Gene reactivation: a tool for the isolation of mammalian DNA methylation mutants. Genes and Development, 1(9), 899-912.

Gene reactivation : a tool for the isolation of mammalian DNA methylation mutants. / Gounari, F.; Banks, G. R.; Khazaie, Khashayarsha; Jeggo, P. A.; Holliday, R.

In: Genes and Development, Vol. 1, No. 9, 11.1987, p. 899-912.

Research output: Contribution to journalArticle

Gounari, F, Banks, GR, Khazaie, K, Jeggo, PA & Holliday, R 1987, 'Gene reactivation: a tool for the isolation of mammalian DNA methylation mutants.', Genes and Development, vol. 1, no. 9, pp. 899-912.
Gounari, F. ; Banks, G. R. ; Khazaie, Khashayarsha ; Jeggo, P. A. ; Holliday, R. / Gene reactivation : a tool for the isolation of mammalian DNA methylation mutants. In: Genes and Development. 1987 ; Vol. 1, No. 9. pp. 899-912.
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