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Journal ArticleDOI

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

01 Nov 1987-Genes & Development (Cold Spring Harbor Lab)-Vol. 1, Iss: 9, pp 899-912

TL;DR: 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.

AbstractWe 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.

Topics: DNA methylation (55%), Mutation frequency (53%), RNA-Directed DNA Methylation (53%), Methylation (53%), Epigenetics (52%)

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Citations
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Journal ArticleDOI
09 Oct 1987-Science
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.
Abstract: Evidence from many sources shows that the control of gene expression in higher organisms is related to the methylation of cytosine in DNA, and that the pattern of methylation is inherited. Loss of methylation, which can result from DNA damage, will lead to heritable abnormalities in gene expression, and these may be important in oncogenesis and aging. Transformed permanent lines often lose gene activity through de novo methylation. 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.

995 citations


Journal ArticleDOI
10 Jul 1992-Cell

912 citations


Journal ArticleDOI
10 Aug 1990-Cell
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.
Abstract: CpG islands are normally methylation free in cells of the animal, even when the associated gene is transcriptionally silent. In mouse NIH 3T3 and L cells, however, over half of the islands are heavily methylated. Near identity of the methylated subset in the two cell lines suggested that methylation is confined to genes that are nonessential in culture. In agreement with this, islands at several tissue-specific genes, but not at housekeeping genes, have become methylated in many human and mouse cell lines. At the chromatin level, methylated islands are Mspl resistant compared with their nonmethylated counterparts. We suggest 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.

678 citations


Journal ArticleDOI
01 Jan 2000-Oncogene
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.
Abstract: There is tremendous ferment in the field of epigenetics as the relationships between chromatin structure and DNA methylation patterns become clearer. Central to this activity is the realization that the 'histone code', which involves the post-translational modification of histones and which has important ramifications for chromatin structure, may be linked to the DNA cytosine methylation pattern. New discoveries have suggested that histone lysine 9 methylation is implicated in the spread of heterochromatin in Drosophila and other organisms. Very recently it has been found that histone lysine 9 methylation is also necessary for some DNA methylation in Neurospora and plants. There is therefore the possibility that these two processes are closely linked, suggesting ways in which DNA methylation patterns may be established during normal development. Understanding these processes is fundamental to understanding what goes awry during the process of aging and carcinogenesis where DNA methylation patterns become substantially altered and contribute to the malignant phenotype.

283 citations


Journal ArticleDOI
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.
Abstract: A growing body of evidence points towards epigenetic mechanisms being responsible for a wide range of biological phenomena, from the plasticity of plant growth and development to the nutritional control of caste determination in honeybees and the etiology of human disease (e.g., cancer). With the (partial) elucidation of the molecular basis of epigenetic variation and the heritability of certain of these changes, the field of evolutionary epigenetics is flourishing. Despite this, the role of epigenetics in shaping host–pathogen interactions has received comparatively little attention. Yet there is plenty of evidence supporting the implication of epigenetic mechanisms in the modulation of the biological interaction between hosts and pathogens. The phenotypic plasticity of many key parasite life-history traits appears to be under epigenetic control. Moreover, pathogen-induced effects in host phenotype may have transgenerational consequences, and the bases of these changes and their heritability probably have an epigenetic component. The significance of epigenetic modifications may, however, go beyond providing a mechanistic basis for host and pathogen plasticity. Epigenetic epidemiology has recently emerged as a promising area for future research on infectious diseases. In addition, the incorporation of epigenetic inheritance and epigenetic plasticity mechanisms to evolutionary models and empirical studies of host–pathogen interactions will provide new insights into the evolution and coevolution of these associations. Here, we review 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. We conclude with recommendations and directions for future research on the burgeoning field of epigenetics as applied to host–pathogen interactions.

191 citations


References
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Journal ArticleDOI
TL;DR: A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described, and these "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.
Abstract: A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.

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Journal ArticleDOI
20 Nov 1943-Genetics
TL;DR: This article reported Luria and Delbruck's breakthrough study in which they established that viruses do not induce mutations in bacteria, but that virus-resisting mutations are spontaneous.
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Journal ArticleDOI
01 May 1980-Cell
TL;DR: Results provide experimental evidence for a role for DNA modification in differentiation, and suggest that cytidine analogs containing an altered 5 position perturb previously established methylation patterns to yield new cellular phenotypes.
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1,660 citations


Journal ArticleDOI
TL;DR: Statistical calculations are made of the distribution numbers of mutants in a culture of bacteria in which the number of mutants increases on account of new mutations and of division of old mutants, which enable the mutation rate to be inferred from experiments with parallel cultures.
Abstract: Statistical calculations are made of the distribution numbers of mutants in a culture of bacteria in which the number of mutants increases on account both of new mutations and of division of old mutants. In this way the largely qualitative conclusions of Luria and Delbruck are extended and placed on a firm quantitative basis. The results of these calculations, which enable the mutation rate to be inferred from experiments with parallel cultures, are presented in the form of tables. Statistically efficient methods of using these tables are discussed.

1,386 citations


Journal ArticleDOI
TL;DR: A model based on DNA methylation is proposed to explain the initiation and maintenance of mammalian X inactivation and certain aspects of other permanent events in eukaryotic cell differentiation using sequence-specific DNA methylases that methylate unmethylated sites with great difficulty but easily methylate half-methylated sites.
Abstract: A model based on DNA methylation is proposed to explain the initiation and maintenance of mammalian X inactivation and certain aspects of other permanent events in eukaryotic cell differentiation. A key feature of the model is the proposal of sequence-specific DNA methylases that methylate unmethylated sites with great difficulty but easily methylate half-methylated sites. Although such enzymes have not yet been detected in eukaryotes, they are known in bacteria. An argument is presented, based on recent data on DNA-binding proteins, that DNA methylation should affect the binding of regulatory proteins. In support of the model, short reviews are included covering both mammalian X inactivation and bacterial restriction and modification enzymes.

1,045 citations