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Results for “DNA Modification Methylases”

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PubMed1989

[Studies on DNA methylation in transformed mouse liver cells].

Activity of DNA methylase and DNA methylation level were measured from normal mouse liver, mouse liver charged with H22a ascitic hepatoma and H22a ascitic hepatoma cell by measuring incorporation of H3-methyl. S-Adenosyl-3H-methyl-methionine (3H-SAM) was used as methyl donor. DNA methylation level of different cells were measured by HP-LC. DNA methylase activity and DNA methylation level of H22a ascitic hepatoma, mouse liver charged with H22a ascitic hepatoma are lower than normal mouse liver. Treatments of antitumor drugs lead to a rising of DNA methylase activity of tumor cell, however, the DNA methylation level of tumor cell has not rised after such treatments.

Animals↗
PubMed2006

DNA methyltransferases messenger RNA expression and aberrant methylation of CpG islands in non-small-cell lung cancer: association and prognostic value.

BACKGROUND: A significant association between aberrant methylation in regulatory regions of tumor suppressor genes and clinical outcome in various different cancer types has been described. The molecular events for this epigenetic alteration still remain unknown. Evidence suggests that overexpression of DNA methyltransferases (DNMTs) is one potential mechanism for hypermethylation. PATIENTS AND METHODS: Therefore, we investigated the influence of gene expression levels of the 3 DNMT isoforms (DNMT1, DNMT3a, and DNMT3b) and the hypermethylation of adenomatous polyposis coli (APC), the death-associated protein kinase (DAPK), glutathione S-transferase Pi (GSTPI), and the DNA repair gene O6-methylguanine DNA transferase (MGMT) in the pathogenesis and prognosis of patients with non-small cell lung cancer and determined their association to each other. Using a quantitative real-time reverse-transcriptase polymerase chain reaction, we measured messenger RNA expression of DNMT1, DNMT3a, and DNMT3b and DNA hypermethylation of APC, DAPK, GSTPI, and MGMT in 91 matching tumor and nonmalignant lung tissue samples from patients with curatively resected non-small-cell lung cancer. RESULTS: In tumor tissue, the expression of all 3 DNMT isoforms was significantly higher compared with matched normal-appearing tissue (P < 0.001). Hypermethylation in tumor tissue was found in 95% for APC, in 92% for DAPK, in 18% for GSTPI, and in 38% for MGMT. CONCLUSION: No correlation was found between the DNMT messenger RNA expression and DNA hypermethylation status in tumor tissues. Multivariate analysis revealed DNA hypermethylation status and TNM stage as independent prognostic factors.

Adult↗
PubMed1989

Granulocytic differentiation of HL-60 cells is not regulated by DNA de novo methylation.

DNA cytosine methylation and transcription of specific genes are inversely correlated. In granulocytic differentiation of HL-60 cells there is a distinct down regulation of the c-myc proto-oncogene expression, which is probably a causal mechanism. With differentiation of HL-60 cells we found no restriction enzyme fragment length polymorphism (RFLP) within the c-myc proto-oncogene, which indicates that there is no loss of regulatory elements (e.g., TATAA boxes within the first exon). Furthermore, we found no de novo methylation in this region. Methylation of other DNA regions, which could influence c-myc expression, is also not necessary for differentiation, as was shown by inhibition of DNA methylase. L-Ethionine and S-adenosyl-L-homocysteine are both potent inhibitors of DNA methylase and do not influence proliferation of HL-60 cells, as shown by FACS analysis.

Cell Cycle↗
PubMed1993

[Human thyroid gland DNA methylase in nodular and diffuse goiter].

The heterogeneity and some properties of DNA-methylases isolated from nonmalignant human thyroid formations--nodular and diffuse goiters--have been studied. Isoelectrofocusing of methylase preparations produced 6-7 distinct activity peaks distinguished by pI, activity towards Ca2+ and Mg2+, sensitivity towards dithiothreitol and capacity to methylase cytosine into mono-, di- and tripyrimidine blocks in vitro. The degree of DNA methylation in vivo depended on the origin of of the goiter. Analysis of several methylase fractions by two-dimensional electrophoresis performed according to O'Farrell revealed the presence of major polypeptides having similar molecular masses (approximately 36 kDa). Besides the major component, all the preparations under study contained 6-10 characteristic minor components. The data obtained are suggestive of a structural and functional heterogeneity of human methylases.

DNA Modification Methylases↗
PubMed2001

Evolutionary role of restriction/modification systems as revealed by comparative genome analysis.

Type II restriction modification systems (RMSs) have been regarded either as defense tools or as molecular parasites of bacteria. We extensively analyzed their evolutionary role from the study of their impact in the complete genomes of 26 bacteria and 35 phages in terms of palindrome avoidance. This analysis reveals that palindrome avoidance is not universally spread among bacterial species and that it does not correlate with taxonomic proximity. Palindrome avoidance is also not universal among bacteriophage, even when their hosts code for RMSs, and depends strongly on the genetic material of the phage. Interestingly, palindrome avoidance is intimately correlated with the infective behavior of the phage. We observe that the degree of palindrome and restriction site avoidance is significantly and consistently less important in phages than in their bacterial hosts. This result brings to the fore a larger selective load for palindrome and restriction site avoidance on the bacterial hosts than on their infecting phages. It is then consistent with a view where type II RMSs are considered as parasites possibly at the verge of mutualism. As a consequence, RMSs constitute a nontrivial third player in the host-parasite relationship between bacteria and phages.

AT Rich Sequence↗
PubMed1989

Purification, cloning and sequence analysis of RsrI DNA methyltransferase: lack of homology between two enzymes, RsrI and EcoRI, that methylate the same nucleotide in identical recognition sequences.

RsrI DNA methyltransferase (M-RsrI) from Rhodobacter sphaeroides has been purified to homogeneity, and its gene cloned and sequenced. This enzyme catalyzes methylation of the same central adenine residue in the duplex recognition sequence d(GAATTC) as does M-EcoRI. The reduced and denatured molecular weight of the RsrI methyltransferase (MTase) is 33,600 Da. A fragment of R. sphaeroides chromosomal DNA exhibited M.RsrI activity in E. coli and was used to sequence the rsrIM gene. The deduced amino acid sequence of M.RsrI shows partial homology to those of the type II adenine MTases HinfI and DpnA and N4-cytosine MTases BamHI and PvuII, and to the type III adenine MTases EcoP1 and EcoP15. In contrast to their corresponding isoschizomeric endonucleases, the deduced amino acid sequences of the RsrI and EcoRI MTases show very little homology. Either the EcoRI and RsrI restriction-modification systems assembled independently from closely related endonuclease and more distantly related MTase genes, or the MTase genes diverged more than their partner endonuclease genes. The rsrIM gene sequence has also been determined by Stephenson and Greene (Nucl. Acids Res. (1989) 17, this issue).

Amino Acid Sequence↗
PubMed1991

Molecular cloning and characterization of the gene encoding the adenine methyltransferase M.CviRI from Chlorella virus XZ-6E.

The gene encoding the DNA methyltransferase M.CviRI from Chlorella virus XZ-6E was cloned and expressed in Escherichia coli. M.CviRI methylates adenine in TGCA sequences. DNA containing the M.CviRI gene was sequenced and a single open reading frame of 1137 bp was identified which could code for a polypeptide of 379 amino acids with a predicted molecular weight of 42,814. Comparison of the M.CviRI predicted amino acid sequence with another Chlorella virus and 14 bacterial adenine methyltransferases revealed extensive similarity to the other Chlorella virus enzyme.

Amino Acid Sequence↗
PubMed1995

DMI-1, a new DNA methyltransferase inhibitor produced by Streptomyces sp. strain No. 560.

A new inhibitor of DNA methyltransferase named DMI-1 has been discovered in the culture filtrate of Streptomyces sp. strain No. 560. DMI-1 was purified by extraction with ethyl acetate followed by Diaion HP-20SS and silica gel column chromatography. The structure of DMI-1 was determined to be 8-methylpentadecanoic acid (C16H32O2). DMI-1 is a novel inhibitor of methyltransferase isolated from microorganisms and is structurally different from sinefungin and A9145C which are structural analogs of S-adenosylmethionine (methyl donor). DMI-1 was a strong inhibitor of N6-methyladenine-DNA methyltransferase (M. Eco RI, EC 2.1.1.72) in a noncompetitive manner and its inhibition depended on the pH and temperature in the assay media.

DNA Modification Methylases↗
PubMed1988

Immunocytochemical staining of different cell types in vaginal smears by monoclonal anti-DNA methylase antibodies.

Enzymatic methylation of mammalian DNA is closely related to the replication process; also, the synthesis of DNA methylase, an enzyme that is responsible for this process, is cell cycle related. A monoclonal antibody against DNA methylase recognizes proliferatively active cells in the heterogeneous population. We used this antibody to identify the proliferative state of different cell types in normal vaginal smears and in smears of patients with precancerous lesions and cervical cancer. In all preparations, the normal epithelial cells remained unstained. The majority of cancer cells gave a positive immunocytochemical signal indicating the presence of DNA methylase antigen and the proliferative state. Staining was also observed in dyskaryotic cells, particularly in nuclei of parabasal-type cells.

Antibodies, Monoclonal↗
PubMed1994

Crystal structure of catechol O-methyltransferase.

Catechol O-methyltransferase (COMT, EC 2.1.1.6) is important in the central nervous system because it metabolizes catecholamine neurotransmitters such as dopamine. The enzyme catalyses the transfer of the methyl group from S-adenosyl-L-methionine (AdoMet) to one hydroxyl group of catechols. COMT also inactivates catechol-type compounds such as L-DOPA. With selective inhibitors of COMT in combination with L-DOPA, a new principle has been realized in the therapy of Parkinson's disease. Here we solve the atomic structure of COMT to 2.0 A resolution, which provides new insights into the mechanism of the methyl transfer reaction. The co-enzyme-binding domain is strikingly similar to that of an AdoMet-dependent DNA methylase, indicating that all AdoMet methylases may have a common structure.

Adenosine↗
PubMed1994

[Effect of anti-aging drug on the activity of DNA methylase in rat liver].

An obvious anti-aging effect was found by Bushen Shengxue drug to treat Wistar rats. Using a method about incorporation of 3H-labeled methyl group of S-adenosyl-methionine (SAM) into DNA bases, the DNA methylase activity was assayed. The specific activity of DNA methylase was increased by treatment of this drug. The chromatographic behaviour of DNA methylase from rat liver was also changed. These results showed that the mechanism of anti-aging effect of this drug is probably related to DNA methylation.

Aging↗
PubMed1996

[Effect of anti-aging drug on DNA methylase of brain in rats].

DNA methylase activity of brain in Wistar rats treated with Shenfang Bushen Shengxue Drug (SBSD) was observed using 3H-labelled methyl group of S-adenosyl-methionine incorporated into DNA. It was found that the SBSD has a marked effect on anti-aging. After SBSD treatment the specific activity of DNA methylase increased, the thermostability and salt-tolerance of it improved slightly, its optional pH changed from 7.5 to 8.0. A and a difference was found between the electrophoresgram of partially purified product of DNA methylase in SBSD treated rats and that in normal rats, suggesting SBSD could change the activity and characteristics of DNA methylase so as to affect the level of DNA methylation, which might be one pathway of SBSD effects on anti-aging.

Aging↗
PubMed2003

Gene transfer into Clostridium difficile CD630 and characterisation of its methylase genes.

Ignorance of pathogenesis in Clostridium difficile may be attributable to a lack of effective genetic tools. We have now shown that oriT-based shuttle vectors may be conjugated from Escherichia coli donors to the C. difficile strain CD630, at frequencies of around 10(-6) transconjugants per donor cell. Transfer is unaffected by either sequences present on the vector or its methylation status. Whilst the genome of this strain carries five methylase genes, there is no in silico or experimental evidence for cognate restriction enzymes. It would seem that the identified methylases do not participate in restriction-modification, and must, therefore, fulfil another role. A similar situation most likely applies to other clostridia.

Cloning, Molecular↗
PubMed2005

CpG methylation of the PAI-1 gene 5'-flanking region is inversely correlated with PAI-1 mRNA levels in human cell lines.

The physiological and pathophysiological functions of PAI-1 are related to its expression by specific cell types in normal and diseased tissues. We analysed the contribution of DNA methylation to the variation in PAI-1 mRNA levels in five cell lines. We found varying frequencies of methylation of 25 CpGs in the -805/+152 region of the PAI-1 gene in Bowes, MCF-7 and U937 cells, while little or no methylation was detected in Hep2 and HT- 1080 cells. The methylation frequency was inversely correlated with PAI-1 mRNA level within its 20-fold range in Bowes, MCF-7, U937,and Hep2 cells, while the lack of methylation in both Hep2 and HT-1080 cells suggested another mechanism behind the 150-fold higher level in HT- 1080 cells than in Hep2 cells. However, all cell lines exhibited a high frequency of methylation of 10 CpGs in a CpG island at about--1800. Treatment with 5-aza-2'-deoxycytidine led up to circa a 40-fold increase in the PAI-1 mRNA level and a strong decrease in the frequency of methylation in the -805/+152 region in Bowes, MCF-7 and U937. The histone deacetylase inhibitor trichostatin A induced a several fold increase of the PAI-I mRNA level in cells with a high methylation frequency of the -805/+152 region. As compared with matched normal tissue, three samples of oral squamous cell carcinomas displayed decreased frequencies of methylation of the PAI-1 5' flanking region and increased levels of PAI-1 mRNA. These results for the first time implicate DNA methylation and histone acetylation in regulation of the PAI-1 gene, and indicate that without proper CpG islands in 5'-flanking region,trancription may be regulated by methylation of less dense CpGs in the 5'-flanking region rather than methylation of upstream CpG island.

5' Flanking Region↗
PubMed2002

Quantitative analysis of DNA demethylation and transcriptional reactivation of the FMR1 gene in fragile X cells treated with 5-azadeoxycytidine.

In fragile X syndrome, hypermethylation of the expanded CGG repeat and of the upstream promoter leads to transcriptional silencing of the FMR1 gene. Absence of the FMR1 protein results in mental retardation. We previously proved that treatment with 5-azadeoxycytidine (5-azadC) of fragile X cell lines results in reactivation of the FMR1 gene. We now show that this treatment causes passive demethylation of the FMR1 gene promoter. We employed the bisulfite-sequencing technique to detect the methylation status of individual CpG sites in the entire promoter region, upstream of the CGG repeat. Lymphoblastoid cell lines of fragile X males with full mutations of different sizes were tested before and after treatment with 5-azadC at various time points. We observed that individual cells are either completely unmethylated or not, with few relevant exceptions. We also investigated the extent of methylation in the full mutation (CGG repeat) itself by Southern blot analysis after digestion with methylation-sensitive enzymes Fnu4HI and McrBC and found that the CGG repeat remains at least partially methylated in many cells with a demethylated promoter. This may explain the quantitative discrepancy between the large extent of promoter demethylation and the limited levels of FMR1 transcriptional reactivation estimated by quantitative real-time fluorescent RT-PCR analysis.

Azacitidine↗
PubMed1992

Methylation of DNA of the brain and liver of young and old rats.

In vitro methylation of purified DNA and chromatin-DNA in nuclei of the liver and brain of young (18 week) and old (120 week) female rats has been studied using 3H-SAM as the -CH3 group donor. Incorporation of -CH3 group is higher in old liver and brain, but it is far higher in the latter. 5 mC is 11% lower in the old brain, but there is no difference in the liver. Methylation by Hpa II methylase does not show any difference in the incorporation of -CH3 group into DNA of the liver of the two ages. However, its incorporation is lower in the old brain. Methylation by Msp I methylase causes slightly higher incorporation of -CH3 groups in the old brain. This shows a higher percentage of unmethylated external cytosines in the 5'-CCGG-3' sequences. On the contrary, methylation by Eco RI methylase is considerably higher in the old brain. These studies show alterations in the methylation status of the DNA during ageing which may cause changes in the expression of genes.

Aging↗
PubMed1989

[Use of bacterial DNA-methylases for structuro-functional analysis of the eukaryotic genome].

Bacterial DNA-methylases with known recognition sites (RS) were used as probes for structural-and-functional analysis of eukaryotic genome. Adenine and cytosine DNA-methylases recognizing 4 to 6-member unique and degenerative nucleotide sequences having a symmetrical and asymmetrical structure were used for probing. The use of a set of methylases enabled the selection of a probe that was the most sensitive for the given pathology. Thus, severe hypothyrosis was found to be associated with changes in the acceptor capacity of liver DNA in the heterologous++ methylation reaction as could be evidenced from testing by two probes, CCCC and GAATGC. In the cells of chicken liver hepatoma MC29, the acceptor capacity of DNA during GGA methylation appeared to be altered in the greatest degree. DNA-methylases with degenerative SR are weakly specific probes for the study of structural changes (methylation) of the animal genome.

Animals↗