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1.
《Epigenetics》2013,8(1):38-42
Human tumor development is often associated with a DNA demethylation process. This results in the activation of germline-specific genes, such as MAGE-A1, which rely on DNA methylation for repression in somatic tissues. Here, we searched to identify a cell line possessing ongoing DNA demethylation activity targeted to MAGE-A1. We first assessed MAGE-A1-expressing human tumor cell lines, by evaluating their ability to induce demethylation of MAGE-A1 transgenes that were methylated in vitro before transfection. All cell lines lacked such activity, suggesting that MAGE-A1 hypomethylation in tumors results from a past demethylation event. We then turned to mouse embryonic stem (mES) cells, which are characterized by a high level of methylation plasticity. Interestingly, in vitro methylated MAGE-A1 transgenes became demethylated after transfection into mES cells. Demethylation was targeted to the 5’-region of MAGE-A1, and was strongly reduced at mutated MAGE-A1 transgenes exhibiting impaired promoter activity. Our results indicate that mES cells induce demethylation of MAGE-A1, and represent therefore a valuable system to study this tumor-related process. 相似文献
2.
Demethylation and expression of methylated plasmid DNA stably transfected into HeLa cells.
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In vitro methylation at CG dinucleotides (CpGs) in a transfecting plasmid usually greatly inhibits gene expression in mammalian cells. However, we found that in vitro methylation of all CpGs in episomal or non-episomal plasmids containing the SV40 early promoter/enhancer (SV40 Pr/E) driving expression of an antibiotic-resistance gene decreased the formation of antibiotic-resistant colonies by only approximately 30-45% upon stable transfection of HeLa cells. In contrast, when expression of the antibiotic-resistance gene was driven by the Rous sarcoma virus long terminal repeat or the herpes simplex virus thymidine kinase promoter, this methylation decreased the yield of antibiotic-resistant HeLa transfectant colonies approximately 100-fold. The low sensitivity of the SV40 Pr/E to silencing by in vitro methylation was probably due to demethylation upon stable transfection. This demethylation may be targeted to the promoter and extend into the gene. By genomic sequencing, we showed that four out of six of the transfected SV40 Pr/E's adjacent Sp1 sites were hotspots for demethylation in the HeLa transfectants. High frequency demethylation at Sp1 sites was unexpected for a non-embryonal cell line and suggests that DNA demethylation targeted to certain aberrantly methylated regions may function as a repair system for epigenetic mistakes. 相似文献
3.
Loss of chloroplast DNA methylation during dedifferentiation of Chlamydomonas reinhardi gametes. 总被引:2,自引:0,他引:2
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In Chlamydomonas reinhardi the chloroplast DNA (ch;DNA) of mating type plus cells undergoes cyclical methylation and demethylation during the life cycle. Methylation occurs during gametogenesis, and fully differentiated gametes can be dedifferentiated back to vegetative cells which contain nonmethylated chlDNA by the addition of a nitrogen source for growth. We examined the dedifferentiation process and found that the mating ability of gametes was lost rapidly after the start of dedifferentiation at a time when the chlDNA was still methylated. The enzymatic activity of the 200-kilodalton DNA methyltransferase was lost at a rate consistent with the rate of dilution during cell division. Methylation of chlDNA decreased at a slower rate than was expected from cell division alone but was consistent with the continuing activity of the preexisting methyltransferase so long as it was present. These results support the hypothesis that demethylation of chlDNA occurs by dilution out of enzymatic methylating activity rather than by enzymatic demethylation. 相似文献
4.
Effects of beryllium on deoxyribonucleic acid-synthesizing enzymes in regenerating rat liver 总被引:4,自引:3,他引:1
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H. P. Witschi 《The Biochemical journal》1970,120(3):623-634
5.
Valproate induces replication-independent active DNA demethylation 总被引:19,自引:0,他引:19
In this report, we demonstrate that valproic acid (VPA), a drug that has been used for decades in the treatment of epilepsy and as a mood stabilizer, triggers replication-independent active demethylation of DNA. Thus, this drug can potentially reverse DNA methylation patterns and erase stable methylation imprints on DNA in non-dividing cells. Recent discoveries support a role for VPA in the regulation of methylated genes; however, the mechanism has been unclear because it is difficult to dissociate active demethylation from the absence of DNA methylation during DNA synthesis. We therefore took advantage of an assay that measures active DNA demethylation independently from other DNA methylation and DNA replication activities in human embryonal kidney 293 cells. We show that VPA induces histone acetylation, DNA demethylation, and expression of an ectopically methylated CMV-GFP plasmid in a dose-dependent manner. In contrast, valpromide, an analogue of VPA that does not induce histone acetylation, does not induce demethylation or expression of CMV-GFP. Furthermore, we illustrate that methylated DNA-binding protein 2/DNA demethylase (MBD2/dMTase) participates in this reaction since antisense knockdown of MBD2/dMTase attenuates VPA-induced demethylation. Taken together, our data support a new mechanism of action for VPA as enhancing intracellular demethylase activity through its effects on histone acetylation and raises the possibility that DNA methylation is reversible independent of DNA replication by commonly prescribed drugs. 相似文献
6.
S-Adenosylmethionine (AdoMet) is the methyl donor of numerous methylation reactions. The current model is that an increased concentration of AdoMet stimulates DNA methyltransferase reactions, triggering hypermethylation and protecting the genome against global hypomethylation, a hallmark of cancer. Using an assay of active demethylation in HEK 293 cells, we show that AdoMet inhibits active demethylation and expression of an ectopically methylated CMV-GFP (green fluorescent protein) plasmid in a dose-dependent manner. The inhibition of GFP expression is specific to methylated GFP; AdoMet does not inhibit an identical but unmethylated CMV-GFP plasmid. S-Adenosylhomocysteine (AdoHcy), the product of methyltransferase reactions utilizing AdoMet does not inhibit demethylation or expression of CMV-GFP. In vitro, AdoMet but not AdoHcy inhibits methylated DNA-binding protein 2/DNA demethylase as well as endogenous demethylase activity extracted from HEK 293, suggesting that AdoMet directly inhibits demethylase activity, and that the methyl residue on AdoMet is required for its interaction with demethylase. Taken together, our data support an alternative mechanism of action for AdoMet as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA. 相似文献
7.
Isolation of mutants of bacteriophage T4 unable to induce thymidine kinase activity. II. Location of the structural gene for thymidine kinase.
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Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme. 相似文献
8.
9.
Characterization of new regulatory mutants of bacteriophage T4. II. New class of mutants. 总被引:15,自引:8,他引:7
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Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme. 相似文献
10.
Yang L Wang H Luo X Mao P Tian W Shi Y Huang G Zhang J Ma D 《Acta biochimica et biophysica Sinica》2012,44(2):154-161
DNA methylation and demethylation play important roles in mediating epigenetic regulation. So far, the mechanism of DNA demethylation remains elusive and controversial. Here, we constructed a plasmid, named with pCBS-luc, that contained an artificial CpG island, eight Gal4 DNA-binding domain binding site, an SV40 promoter, and a firefly luciferase reporter gene. The linearized pCBS-luc plasmid was methylated in vitro by DNA methyltransferase, and transfected into the HEK293 cells. The stable HEK293 transfectants with methylated pCBS-luc (me-pCBS-luc) were selected and obtained. The methylation status of the selected stable cell lines were confirmed by bisulfite sequencing polymerase chain reaction amplification. The methylation status could be maintained even after 15 passages. The virion protein 16 (VP16) was reported to enhance DNA demethylation around its binding sites of the promoter region in Xenopus fertilized eggs. Using our me-pCBS-luc model, we found that VP16 also had the ability to activate the expression of methylated luciferase reporter gene and induce DNA demethylation in chromatin DNA in mammalian cells. Altogether, we constructed a cell model stably integrated with the me-pCBS-luc reporter plasmid, and in this model we found that VP16 could lead to DNA demethylation. We believe that this cell model will have many potential applications in the future research on DNA demethylation and dynamic process of chromatin modification. 相似文献
11.
12.
Inhibition of Thymidine Kinase Activity and Deoxyribonucleic Acid Synthesis in L Cells Infected with the Meningopneumonitis Agent 总被引:15,自引:6,他引:9
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Hsiu-San Lin 《Journal of bacteriology》1968,96(6):2054-2065
The activities of enzymes related to deoxyribonucleic acid (DNA) synthesis were studied in uninfected L cells and in L cells infected with Chlamydia psittaci (strain meningopneumonitis). The meningopneumonitis agent multiplied normally but failed to induce the synthesis of thymidine kinase in LM (TK(-)) cells which contain no thymidine kinase in the uninfected state. It was concluded that this microorganism has no thymidine kinase of its own and that it does not depend on the functioning of the host enzyme for synthesizing its DNA. Exposure of clone 5b L cells to the meningopneumonitis agent was followed by a decline in their thymidine kinase activity to nearly zero levels, whereas the levels of uridine kinase and thymidylate synthetase remained unchanged. Inhibition of thymidine kinase activity in L cells occurred soon after infection and required new protein synthesis by the meningopneumonitis agent. This inhibition occurred before inhibition of host DNA synthesis, but it was not an essential prelude to the latter inhibition. On the basis of this and previous investigations and in light of present knowledge of the mammalian cell cycle, it was postulated that the meningopneumonitis agent inhibits macromolecular synthesis in L cells by preventing the initiation of a new cell cycle. 相似文献
13.
Utilization of exogenous thymidine by Chlamydia psittaci growing in the thymidine kinase-containing and thymidine kinase-deficient L cells. 总被引:12,自引:5,他引:7
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T P Hatch 《Journal of bacteriology》1976,125(2):706-712
The incorporation of [3H]thymidine into the deoxyribonucleic acid (DNA) of Chlamydia psittaci (strain 6BC) growing in thymidine kinase (adenosine 5'-triphosphate-thymidine 5'-phosphotransferase, EC 1.7.1.21)-containing L cells, L(TK+), and thymidine kinase-deficient L cells, LM(TK-), was examined by autoradiography. Label was detected over C. psittaci inclusions in L(TK+) but not LM(TK-) cells. No evidence for a chlamydia-specific thymidine kinase activity in either L(TK+) or LM(TK-) cells was obtained. Entry of [3H]thymidine into the DNA of C. psittaci growing in L(TK+) cells was quantitated by measuring label in purified C. psittaci. It was 265 times less efficient than entry into infected host cell DNA. It is concluded that low levels of exogenous thymidine are incorporated into the DNA of C. psittaci and that this incorporation is dependent on a fully competent host thymidine kinase activity. Evidence also is presented that L cells possess at least two thymidine kinase activities, both of which are capable of supplying thymidylate precursors for nuclear DNA synthesis. 相似文献
14.
15.
Treatment with the base analogue, 5azaC, increases SCEs in CHO but not in mosquito cells. On the other hand, both types of cells show equivalent increases in exchanges when treated with other compounds, such as mitomycin C. Vertebrate DNA is heavily methylated while diptera DNA is heavily demethylated. The sequence of events leading to an increase in SCEs in CHO cells is as follows: first of all, Cs are replaced by 5azaC; in the next cell cycle, CG palindromic dinucleotides exhibit an asymmetric configuration, the Cs in the parental DNA strand being methylated and the Cs in the daughter DNA strand demethylated; after one more cycle, half of the chromosomes show symmetric methylation and the other half symmetric demethylation of both Cs in CG palindromes. The increase of SCEs occurs in the second cell cycle when the hemimethylated DNA enters replication. DNA hemimethylation is believed to be an intermediate stage in the process of demethylation that accompanies gene expression. If so, gene demethylation would be a cause of SCE increase in normal vertebrate cells. 相似文献
16.
17.
Genomic hypomethylation and far-5'' sequence alterations are associated with carcinogen-induced activation of the hamster thymidine kinase gene. 总被引:5,自引:0,他引:5
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F G Barr S Rajagopalan C A MacArthur M W Lieberman 《Molecular and cellular biology》1986,6(9):3023-3033
We have investigated the mechanism of activation of an inactive but functionally intact hamster thymidine kinase (TK) gene by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Following carcinogen treatment of TK- RJK92 Chinese hamster cells, aminopterin-resistant (HATr) colonies appeared at a frequency 50-fold higher than in untreated controls. More than 80% of these HATr variants expressed TK enzymatic activity and were divided into high- and low-activity classes. In all TK+ variants, TK expression was correlated with demethylation in the 5' region of the TK gene and the appearance a 1,400-nucleotide TK mRNA. Using high-performance liquid chromatography to measure the level of genomic methylation, we found that four of five high-activity lines demonstrated extensive genomic hypomethylation (approximately 25% of normal level) that was associated with demethylation of all TK gene copies. Restriction endonuclease analysis of 15 low-activity lines revealed four instances of sequence alterations in the far-5' region of the TK gene and one instance of a tandem low-copy amplification. In these lines, the structurally altered gene copy was demethylated. Thus, we propose that a chemical carcinogen can activate TK expression by several different mechanisms. Focal demethylation with or without gene rearrangement was associated with low TK activity, whereas demethylation throughout the genome was associated with high TK activity. 相似文献
18.
Thymidine uptake, thymidine incorporation, and thymidine kinase activity in marine bacterium isolates. 总被引:5,自引:4,他引:1
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One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibrio sp. strain D19, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into the cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems. 相似文献
19.
One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibrio sp. strain D19, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into the cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems. 相似文献
20.
Variation of DNA polymerase activities and DNA synthesis in mouse mammary gland during pregnancy and early lactation 总被引:2,自引:0,他引:2
Lech Zwierzchowski Danuta Kleczkowska Wieslaw Niedbalski Izabela Grochowska 《Differentiation; research in biological diversity》1984,28(2):179-185
The rate of DNA synthesis and the activity of DNA polymerases and thymidine kinase were measured during the endocrine-regulated cellular growth and differentiation of mouse mammary gland. Using specific assays, the activity of the DNA polymerases, alpha, beta and gamma, was determined in tissue extracts of mammary glands of mice at various stages of pregnancy and early lactation. In addition, extracts of the mammary tissue of virgin, mid-pregnant and early lactating mice were fractionated on sucrose density gradients, and the activity of DNA polymerase alpha and beta was assayed in the gradient fractions. It was demonstrated that the activity of DNA polymerase alpha varied considerably during pregnancy and after parturition, showing peaks on day 12 of pregnancy and days 3-4 of lactation. In pregnancy, there was an apparently parallel correlation between the amount of DNA-polymerase-alpha activity and the rate at which the cells incorporated labelled thymidine into DNA, but the relationship was less clearly expressed during early lactation. The activity of the DNA polymerases, beta and gamma, as well as that of thymidine kinase showed little variation during these periods. Thus, in the developing mammary gland, no correlation was found between DNA synthesis and the activity of the DNA polymerases, beta and gamma, or thymidine kinase. 相似文献