Gene reactivation: a tool for the isolation of mammalian DNA methylation mutants.
TLDR
It is proposed that the phenotype of tsm cells is due to a mutation involved in the regulation of DNA methylation, and the further characterization of this and other mammalian mutants should help to clarify the physiological role of DNAmethylation, as well as its regulation.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.read more
Citations
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The inheritance of epigenetic defects.
TL;DR: It is proposed that epigenetic defects in germline cells due to loss of methylation can be repaired by recombination at meiosis but that some are transmitted to offspring.
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High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines.
TL;DR: It is suggested that mutation-like gene inactivation due to CpG island methylation is widespread in many cell lines and could explain the loss of cell type-specific functions in culture.
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DNA methylation and cancer.
TL;DR: The possibility that the ‘histone code’ and the DNA cytosine methylation pattern are closely linked is suggested, suggesting ways in which DNA methylation patterns may be established during normal development.
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Epigenetics of host-pathogen interactions: the road ahead and the road behind.
TL;DR: The evidence available for the role epigenetics on host- Pathogen interactions, and the utility and versatility of the epigenetic technologies available that can be cross-applied to host-pathogen studies are reviewed are reviewed.
References
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Induction of thymidine kinase in enzyme-deficient chinese hamster cells
TL;DR: The data support the view that induction in Chinese hamster cells results from changes in DNA methylation patterns, and suggests studies to define the process in molecular terms.
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Hemimethylated duplex DNAs prepared from 5-azacytidine-treated cells
Peter A. Jones,Shirley M. Taylor +1 more
TL;DR: In this paper, duplex DNA was used as an efficient substrate for a crude DNA methyltransferase preparation which transferred the methyl group from S-adenosylmethionine specifically into cytosine residues within the hypomethylated H strand.
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Doublet frequency analysis of fractionated vertebrate nuclear DNA.
TL;DR: It is concluded that the highly characteristic general design of bulk nDNA, and of the majority of individual sub-fractions of n DNA, is shared by those fractions of nDNA that code for proteins which in turn implies that they have all evolved in response to the same major selection pressures.
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Ultraviolet radiation-induced metallothionein-I gene activation is associated with extensive DNA demethylation
TL;DR: Activation of a quiescent gene by UV has implications for understanding the initiation of carcinogenesis.
Journal ArticleDOI
5-Azacytidine-induced reactivation of a herpes simplex thymidine kinase gene
TL;DR: Mouse cells transformed with herpes simplex virus and containing the viral thymidine kinase (TK) gene in an inactive state were treated with 5-azacytidine and the result was the reexpression of the viral TK gene.