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  • 1
    Keywords: Germany ; IN-VIVO ; SUPPORT ; GENE ; GENOME ; PROTEIN ; PROTEINS ; RNA ; TIME ; DNA ; FAMILY ; SUFFICIENT ; STAGE ; Drosophila ; MELANOGASTER ; DNA methylation ; EMBRYO ; FISSION YEAST ; METHYLTRANSFERASE ACTIVITY ; OVEREXPRESSION ; METHYLATION ; CATALYTIC ACTIVITY ; CYTOSINE-5 METHYLTRANSFERASES ; DE-NOVO METHYLATION ; DNA methylation,Drosophila,DNA methyltransferase,Dnmt2,Su(var)3-9 ; DNA methyltransferase ; EMBRYONIC STEM-CELLS ; EMBRYOS ; HISTONE H3 METHYLTRANSFERASE ; HYPERMETHYLATION ; LETHALITY ; MAMMALIAN DEVELOPMENT ; METHYLTRANSFERASE GENE ; SEQUENCE SPECIFICITY
    Abstract: The methylation status of Drosophila DNA has been discussed controversially over a long time. Recent evidence has provided strong support for the existence of 5-methylcytosine in DNA preparations from embryonic stages of fly development. The Drosophila genome contains a single candidate DNA methyltransferase gene that has been termed Dnmt2. This gene belongs to a widely conserved family of putative DNA methyltransferases. However, no catalytic activity has been demonstrated for any Dnmt2-like protein yet. We have now established a protocol for the immunological detection of methylated cytosine in fly embryos. Confocal analysis of immunostained embryos provided direct evidence for the methylation of embryonic DNA. In order to analyse the function of Dnmt2 in DNA methylation, we depleted the protein by RNA interference. Depletion of Dnmt2 had no detectable effect on embryonic development and resulted in a complete loss of DNA methylation. Consistently, overexpression of Dnmt2 from an inducible transgene resulted in significant genomic hypermethylation at CpT and CpA dinucleotides. These results demonstrate that Dnmt2 is both necessary and sufficient for DNA methylation in Drosophila and suggest a novel CpT/A-specific DNA methyltransferase activity for Dnmt2 proteins
    Type of Publication: Journal article published
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  • 2
    Keywords: CANCER ; Germany ; QUANTIFICATION ; TOOL ; RISK ; GENE ; SAMPLE ; SAMPLES ; TUMORS ; PATIENT ; DNA ; RISK-FACTORS ; MUTATION ; risk factors ; leukemia ; DNA methylation ; RISK FACTOR ; DERIVATIVES ; PARAMETERS ; FLUORESCENCE ; CYTOSINE METHYLATION ; CD38 EXPRESSION ; HYPERMETHYLATION ; 5-METHYLCYTOSINE CONTENT ; TUMORIGENESIS ; HEAVY ; chronic lymphocytic leukemia ; MUTATION STATUS ; 5-methylcytosine ; CPG ISLANDS ; capillary electrophoresis - laser induced fluorescence ; CAPILLARY-ELECTROPHORESIS ; GENOMIC HYPOMETHYLATION ; immunoglobulin variable heavy chain gene homology
    Abstract: Changes in the genomic DNA methylation level have been found to be closely associated with tumorigenesis. In order to analyze the relation of aberrant DNA methylation to clinical and biological risk factors, we have determined the cytosine methylation level of 81 patients diagnosed with chronic lymphocytic leukemia (CLL). The analysis was based on DNA hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with BODIPY FL EDA. Derivatives were separated by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. We analyzed potential correlations between DNA methylation levels and numerous patient parameters, including clinical observations and biological data. As a result, we observed a significant correlation with the immunoglobulin variable heavy chain gene (VH) mutation status. This factor has been repeatedly proposed as a reliable prognostic marker for CLL, which suggests that the methylation level might be a valuable factor in determining the prognostic outcome of CLL. We are now in the process of refining our method to broaden its application potential. In this context, we show here that the oxidation of the fluorescence marker in the samples and the evaporation of methanol in the electrolytes can be prevented by a film of paraffin oil. In summary, our results thus establish capillary electrophoresis as a valuable tool for analyzing the DNA methylation status of clinical samples
    Type of Publication: Journal article published
    PubMed ID: 15188237
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  • 3
    Keywords: EXPRESSION ; tumor ; Germany ; QUANTIFICATION ; SAMPLE ; SAMPLES ; TUMORS ; ACCURACY ; PATIENT ; DNA ; TISSUES ; PATTERNS ; MUTATION ; REQUIRES ; leukemia ; DNA methylation ; METHYLATION ; CALIBRATION ; CHRONIC LYMPHOCYTIC-LEUKEMIA ; HYPERMETHYLATION ; 5-METHYLCYTOSINE CONTENT ; HYPOMETHYLATION
    Abstract: Changes in DNA methylation have been found in the large majority of tumors. This phenomenon includes both genome-wide hypomethylation and gene-specific hypermethylation. However, the clinical relevance of either mechanism has remained contentious. In order to determine DNA methylation levels from a large number of clinical samples, we have established a method for accurate high-throughput quantification of 5- methylcytosine in genomic DNA. Our protocol requires a small amount (〈1 μg) of DNA that is enzymatically hydrolyzed to single nucleotides. Single nucleotides are then derivatized with a fluorescent marker and separated by capillary electrophoresis. After calibration of the method, we have determined cytosine methylation levels from tumor samples of 81 patients that had been diagnosed with chronic lymphocytic leukemia (CLL). These patients showed a high variability in their methylation levels with a general trend towards hypomethylation. Because of its high accuracy and throughput our method will be useful in determining the role of genomic DNA methylation levels in tumorigenesis
    Type of Publication: Journal article published
    PubMed ID: 12527791
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  • 4
    Keywords: Germany ; SYSTEM ; DISEASE ; GENE ; DNA ; cell cycle ; CELL-CYCLE ; CYCLE ; PHOSPHORYLATION ; MOUSE ; PROGRESSION ; MUTATION ; RATES ; DNA methylation ; FISSION YEAST ; DE-NOVO ; H3 METHYLTRANSFERASE ; METHYLATION ; EMBRYONIC LETHALITY ; METHYLTRANSFERASE GENE ; SEQUENCE SPECIFICITY ; DNMT3B ; IMMUNODEFICIENCY SYNDROME ; LYSINE 9
    Abstract: The level of genomic DNA methylation plays an important role in development and disease. In order to establish an experimental system for the functional analysis of genome-wide hypermethylation, we overexpressed the mouse de novo methyltransferase Dnmt3a in Drosophila melanogaster. These flies showed severe developmental defects that could be linked to reduced rates of cell cycle progression and irregular chromosome condensation. In addition, hypermethylated chromosomes revealed elevated rates of histone H3-K9 methylation and a more restricted pattern of H3-S10 phosphorylation. The developmental and chromosomal defects induced by DNA hypermethylation could be rescued by mutant alleles of the histone H3-K9 methyltransferase gene Su(var)3-9. This mutation also resulted in a significantly decreased level of genomic DNA methylation. Our results thus uncover the molecular consequences of genomic hypermethylation and demonstrate a mutual interaction between DNA methylation and histone methylation
    Type of Publication: Journal article published
    PubMed ID: 12640138
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  • 5
    Keywords: measurement ; CANCER ; CELLS ; tumor ; TUMOR-CELLS ; CELL ; Germany ; QUANTIFICATION ; SAMPLE ; SAMPLES ; DNA ; NEOPLASIA ; ASSAY ; INSTABILITY ; FLUORESCENCE ; isolation ; 5-METHYLCYTOSINE CONTENT ; HYPOMETHYLATION ; CAPILLARY-ELECTROPHORESIS ; cancer,capillary electrophoresis,laser-induced fluorescence,DNA adducts,DNA methylation,5-methylcyto ; POTENTIALS
    Abstract: An analytical method to determine the genome-wide DNA methylation in only 100 ng DNA is presented. The analysis is based on DNA isolation and hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with a fluorescence dye (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyI ethylene diamine hydrochloride, Bodipy FL EDA). The separation of the derivatives was carried out by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. To calculate the methylation level, the derivatization factor and the quantum yields of the Bodipy conjugates of 2'-desoxycytidine-3'-monophosphate (dCMP) and 2'-desoxy-5-methylcytidine-3'-monophosphate (5m-dCMP) were determined by measurement of methylated Lambda DNA. The assignment was made by cochromatography with the synthesized and characterized standard compound 5mdCMP After optimization of the method it was possible to determine the methylation level in 100-ng DNA samples with a standard deviation of less than 5%
    Type of Publication: Journal article published
    PubMed ID: 15004844
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  • 6
    Keywords: CELLS ; EXPRESSION ; GROWTH ; CELL ; Germany ; SITE ; GENE ; GENE-EXPRESSION ; GENOME ; PROTEIN ; RNA ; DIFFERENTIATION ; ACTIVATION ; CYCLE ; REPAIR ; STABILITY ; OVEREXPRESSION ; METHYLATION ; NUCLEOTIDE EXCISION-REPAIR ; RE ; HUMAN CANCER ; ARABIDOPSIS ; PROMOTES
    Abstract: DNA methylation is an epigenetic modification that is essential for gene silencing and genome stability in many organisms. Although methyltransferases that promote DNA methylation are well characterized, the molecular mechanism underlying active DNA demethylation is poorly understood and controversial(1,2). Here we show that Gadd45a ( growth arrest and DNA-damage-inducible protein 45 alpha), a nuclear protein involved in maintenance of genomic stability, DNA repair and suppression of cell growth 3,4, has a key role in active DNA demethylation. Gadd45a overexpression activates methylation-silenced reporter plasmids and promotes global DNA demethylation. Gadd45a knockdown silences gene expression and leads to DNA hypermethylation. During active demethylation of oct4 in Xenopus laevis oocytes(5), Gadd45a is specifically recruited to the site of demethylation. Active demethylation occurs by DNA repair and Gadd45a interacts with and requires the DNA repair endonuclease XPG. We conclude that Gadd45a relieves epigenetic gene silencing by promoting DNA repair, which erases methylation marks
    Type of Publication: Journal article published
    PubMed ID: 17268471
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  • 7
    Keywords: EXPRESSION ; Germany ; PATHWAY ; SITE ; SITES ; GENE ; GENES ; RNA ; transcription ; COMPONENTS ; DNA ; MECHANISM ; FAMILY ; CONTRAST ; mechanisms ; CYCLE ; SEQUENCE ; DISRUPTION ; Drosophila ; NUMBER ; DNA methylation ; METHYLATION ; DE-NOVO METHYLATION ; EMBRYONIC STEM-CELLS ; MAMMALIAN DEVELOPMENT ; SEQUENCE SPECIFICITY ; STANDARD ; RESIDUES ; INTERFERENCE ; METHYLTRANSFERASE ; regulation ; C-ELEGANS ; STRAINS ; RNAi ; sequencing ; CAENORHABDITIS-ELEGANS/ ; DISCOIDEUM ; METHYLTRANSFERASES ; POLYMERASE-IV
    Abstract: We have identified a DNA methyltransferase of the Dnmt2 family in Dictyostelium that was denominated DnmA. Expression of the dnmA gene is downregulated during the developmental cycle. Overall DNA methylation in Dictyostelium is similar to 0.2% of the cytosine residues, which indicates its restriction to a limited set of genomic loci. Bisulfite sequencing of specific sites revealed that DnmA is responsible for methylation of mostly asymmetric C-residues in the retrotransposons DIRS-1 and Skipper. Disruption of the gene resulted in a loss of methylation and in increased transcription and mobilization of Skipper. Skipper transcription was also upregulated in strains that had genes encoding components of the RNA interference pathway disrupted. In contrast, DIRS-1 expression was not affected by a loss of DnmA but was strongly increased in strains that had the RNA-directed RNA polymerase gene rrpC disrupted. A large number of siRNAs were found that corresponded to the DIRS-1 sequence, suggesting concerted regulation of DIRS-1 expression by RNAi and DNA modification. No siRNAs corresponding to the standard Skipper element were found. The data show that DNA methylation plays a crucial role in epigenetic gene silencing in Dictyostelium but that different, partially overlapping mechanisms control transposon silencing
    Type of Publication: Journal article published
    PubMed ID: 16282589
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  • 8
    Keywords: CANCER ; IN-VITRO ; VITRO ; DISEASE ; DISEASES ; ENZYMES ; DIFFERENTIATION ; ACCURACY ; LINES ; TIME ; DNA ; CELL-LINES ; treatment ; CLEAVAGE ; PATTERNS ; ASSAY ; CELL-LINE ; LINE ; DNA methylation ; SWEDEN ; cell lines ; METHYLATION ; 5-azacytidine ; METHYLTRANSFERASE ; TUMORIGENESIS ; CPG ISLANDS ; LEVEL ; ENZYME ; HIGH-THROUGHPUT ; EVENTS ; EPIGENETICS ; genomic ; VARIETIES ; CYTOSINE ; EcoRI ; HpaII ; LUMA ; MspI ; pyrosequencing
    Abstract: Changes in genomic DNA methylation are recognized as important events in normal and pathological cellular processes, contributing both to normal development and differentiation as well as cancer and other diseases. Here, we report a novel method to estimate genome-wide DNA methylation, referred to as LUminometric Methylation Assay (LUMA). The method is based on combined DNA cleavage by methylation-sensitive restriction enzymes and polymerase extension assay by Pyrosequencing (TM). The method is quantitative, highly reproducible and easy to scale up. Since no primary modification of genomic DNA, such as bisulfite treatment, is needed, the total assay time is only 6 h. In addition, the assay requires only 200-500 ng of genomic DNA and incorporates an internal control to eliminate the problem of varying amounts of starting DNA. The accuracy and linearity of LUMA were verified by in vitro methylated lambda DNA. In addition, DNA methylation levels were assessed by LUMA in DNA methyltransferase knock-out cell lines and after treatment with the DNA methyltransferase inhibitor (5-AzaCytidine). The LUMA assay may provide a useful method to analyze genome-wide DNA methylation for a variety of physiological and pathological conditions including etiologic, diagnostic and prognostic aspects of cancer. (c) 2006 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16624287
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  • 9
    ISSN: 0020-1693
    Keywords: Alkyl-transition metal reagents ; Chemoselectivity ; Cross-coupling ; Neighbouring-group effect ; Vinylbromides
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1432-1904
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Natural Sciences in General
    Type of Medium: Electronic Resource
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