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D Drahovsky

Publications and source records attributed to D Drahovsky.

At least 37 records · Page 2Linked to original sources
PubMed1986

Preferential binding of DNA methyltransferase and increased de novo methylation of deoxyinosine containing DNA.

Mammalian DNA-cytosine 5-methyltransferases methylate cytosines in deoxyinosine containing DNA polymers more rapidly than in other synthetic or naturally occurring DNAs. The initial methylation rate of poly(dI-dC) X poly(dI-dC) is about 10-times higher than that of poly-(dG-dC) X poly(dG-dC) or of the native Micrococcus luteus DNA. In competitive binding experiments, DNA methyltransferase has about 10-fold higher affinity for the dI-containing alternating DNA polymer than for poly(dG-dC) X poly(dG-dC). The observed high methyl accepting capacity of poly(dI-dC) X poly(dI-dC) may be a useful methodological advance to determine de novo DNA methyltransferase activity in extracts of mammalian cells.

Animals↗
PubMed1986

Use of a biotinylated DNA probe specific for the human Y chromosome for rapid antenatal sex determination.

We have cloned a male-specific 3.4 kb human DNA sequence which showed only little crosshybridisation to autosomal sequences. To further enhance the specificity of the probe, we subcloned an internal TaqI-fragment resulting in clone pH343T33. This clone was used to determine the presence of Y-chromosomal sequences in DNA extracted from amniotic cells and from chorionic villi by Southern and dot hybridisation assays, respectively. Using this clone, we correctly predicted fetal sex in all of 148 cases analysed. To facilitate the use of this clone in clinical practice, we simplified the dot hybridisation procedure so that it can be performed in less than 48 h. The procedure with 32P-labeled DNA probes requires less than 0.5 mL of amniotic fluid; when biotinylated DNA probes are used, 3-5 mL of amniotic fluid usually suffice. We have used this probe in genetic counseling of families at risk for X-linked disorders.

Biotin↗
PubMed1985

Purification and characterization of mammalian DNA methyltransferases by use of monoclonal antibodies.

Previously, we have derived murine hybridomas producing monoclonal antibodies against DNA methyltransferase from human placenta (Kaul, S., Pfeifer, G. P., and Drahovsky, D. (1984) Eur. J. Cell Biol. 34, 330-335). One of these monoclonal antibodies, M2B10, which undergoes immune complex formation also with DNA methyltransferase from P815 mouse mastocytoma cells, was used for the immunoaffinity purification of mouse and human DNA methyltransferases. In sodium dodecyl sulfate-polyacrylamide gels and in immunoblotting studies, the immunoaffinity-purified mouse DNA methyltransferase revealed 5-6 polypeptides of molecular masses 150-190 kDa. The immunoaffinity-purified human placental DNA methyltransferase was characterized by a polypeptide of 158 kDa, presumably representing the native enzyme molecule and by polypeptides of 105-108 kDa and 50-68 kDa, probably generated by a limited proteolysis of the native enzyme molecule. The immunoaffinity-purified DNA methyltransferases preferred hemimethylated DNA substrates over unmethylated ones, and among all unmethylated substrates tested, poly[(dG-dC).(dG-dC)] had the highest methyl-accepting activity. DNA polymers of at least 90 base pairs in length were required for the binding reaction of the immunoaffinity-purified human DNA methyltransferase, and this initial binding was apparently independent of the nucleotide composition of the DNA polymer and of the presence of S-adenosyl-L-methionine.

Animals↗
PubMed1985

Synthesis and characterization of poly[d(G-z5C)]. B-Z transition and inhibition of DNA methylase.

Deoxy-5-azacytidine 5'-triphosphate was synthesized and used as a substrate for the enzymatic synthesis of the polynucleotide poly[d(G-z5C)]. Whereas the triphosphate decomposes in solution, the azacytosine analogue incorporated into DNA is stable under conditions preserving the double-helical structure. Poly[d(G-z5C)] undergoes the transition to the left-handed Z conformation at salt (NaCl and MgCl2) concentrations approximately 30% higher than those required for unsubstituted poly[d(G-C)]. However, the incorporation of azacytidine potentiates the formation at room temperature of the Z helix stabilized by the transition metal Mn2+; in the case of poly[d(G-C)], a heating step is required. The spectral properties of the two polymers in the B and Z forms are similar. Both left-handed forms are recognized by anti-Z DNA immunoglobulins, indicating that the DNAs bear common antigenic features. Poly[d(G-z5C)] is not a substrate for the DNA cytosine 5-methyltransferase from human placenta. It is a potent inhibitor of the enzyme when tested in a competitive binding assay. These results are compatible with a very strong, possibly covalent, mode of interaction between methyltransferases and DNA containing 5-azacytosine.

Azacitidine↗
PubMed1985

Mouse DNA-cytosine-5-methyltransferase: sequence specificity of the methylation reaction and electron microscopy of enzyme-DNA complexes.

Monoclonal antibodies prepared against DNA methyltransferase from human placenta undergo immune complex formation also with DNA methyltransferase from P815 mouse mastocytoma cells. One of these monoclonal antibodies, M2B10, was used for the immunoaffinity purification of this enzyme. Complexes of the immunoaffinity-purified mouse DNA methyltransferase with DNA were visualized by electron microscopy. DNA methyltransferase was found to be distributed along linearized plasmid DNA with a higher incidence of enzyme molecules at the terminal segments. This binding to strand ends was significantly increased after dG- or dGdC-tailing of the DNA, which is compatible with a preferred binding of the enzyme to single-stranded DNA. Sequence specificity analysis using methyl-sensitive restriction enzymes showed that the mouse DNA methyltransferase transferred methyl groups to the internal cytosines in 5'CCGG and 5'GCGC sequences, however, the external cytosine in 5'CCGG sequences was also methylated.

Animals↗
PubMed1984

Rapid three-step purification procedure for isolation of the C1q component of human complement and its use in a solid-phase C1q binding assay.

A new procedure for the isolation of the C1q subcomponent of complement from human sera has been devised. The 3-step protocol employs DEAE Sephadex A-50, hydroxyapatite and Sephacryl S-200 chromatographies and can be performed within 9 h. It yields immunoglobulin-free homogeneous C1q protein with about 80% recovery. The isolated C1q protein is biologically active and may be used for the detection of circulating immune complexes in sera by the solid-phase C1q binding assay.

Antigen-Antibody Complex↗
PubMed1984

DNA-cytosine-5-methyltransferase from P815 mouse mastocytoma cells: "maintenance" and "de novo" activities are carried out by the same enzyme molecule.

DNA-5-methyltransferase has been purified (about 1400-fold) from rapidly proliferating mouse P815 mastocytoma cells by chromatographies on DEAE cellulose, hydroxyapatite and a heparine-agarose affinity step. The isolated enzyme has an isoelectric point of 7.3 and in neutral 10-30% glycerol gradient it bands in an area corresponding to molecular weight of 135,000 dalton. During the enzymatic reaction, the enzyme first interacts with DNA and then accomplishes a series of methyl group transfers without being detached. The formation of the initial DNA-enzyme complexes is probably random and independent of the cofactor, S-adenosyl-L-methionine, as well as the sequences recognized as methylation sites. The "maintenance" and "de novo" types of activity have been monitored using hemimethylated and completely unmethylated DNA as methyl group accepting polymers. Both these activities copurify in three different chromatographic procedures. This, together with the fact that the enzyme purified near to homogeneity possesses both types of activities suggests that "de novo" and "maintenance" DNA methyltransferase activities are exercised by the same enzyme molecule.

Animals↗
PubMed1984

Two-dimensional restriction mapping by digestion with restriction endonucleases of DNA in agarose and polyacrylamide gels.

We have studied with a number of bacterial restriction enzymes the conditions for digestion of DNA in agarose and polyacrylamide gels. The restriction endonucleases HpaII, MspI, HaeIII, HindIII, TaqI, HhaI, AluI, BamHI, EcoRI and SalI are capable of digesting DNA in agarose gels of low electroendosmosis and low sulfate concentration. All enzymes, except BamHI, are also capable of digesting DNA in polyacrylamide gels. With this method, rapid two-dimensional restriction mapping of genomes with low and high sequence complexity is possible.

Animals↗
PubMed1984

Impaired enzymatic methylation of BPDE-modified DNA.

Highly purified DNA methyltransferase from human placenta methylates hemimethylated and 5-methylcytosine-free DNA substrates suggesting that one enzyme molecule may exercise both, the maintenance and de novo activities. Modification of these methyl accepting polymers with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, anti (BPDE) interferes with the methylation reaction, and this inhibition is proportional to the degree of BPDE-modification. This indicates that BPDE - DNA adducts affect both the maintenance and the de novo DNA methyltransferase activities. The mechanism responsible for such inhibition is related neither to interference of BPDE - DNA adducts with the initial binding of the enzyme to DNA nor with the processive mode of action of the enzyme on the modified DNA template. More likely, the BPDE - DNA adducts inhibit the transmethylation reaction directly at the sites of modification.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1984

Preparation of monoclonal antibodies against DNA-cytosine-5-methyltransferase from human placenta.

Mouse monoclonal antibodies were prepared against DNA-cytosine-5-methyltransferase (EC 2.1.1.37) from human placenta by conventional hybridoma technology. Spleen cells from BALB/c mice immunized with highly purified enzyme were fused to X-63 Ag8.653 mouse myeloma cells. After the hybrid selection in HAT medium individual clones were screened for production of antibodies directed against the enzyme by use of solid-phase ELISA or RIA in which highly purified DNA methyltransferase was either immobilized on microtiter plates or 125I-labeled enzyme was used as a tracer. Positive clones were subcloned, re-screened in the same system and the presence of antibodies directed against DNA methyltransferase was definitively proved in a test system in which the enzyme activity was removed from the solution in immune complexes precipitated by anti mouse immunoglobulin antibodies. From more than 3800 constructed clones 8 were selected which produced antibodies against DNA methyltransferase from human placenta. These antibodies may serve as a useful tool for analysis of DNA methyltransferase structure, intracellular localization and molecular heterogeneity of this enzyme.

Antibodies, Monoclonal↗
PubMed1983

Isolation and characterization of DNA cytosine 5-methyltransferase from human placenta.

DNA cytosine 5-methyltransferase has been extensively purified (about 2600-fold) from the soft tissue of human placenta by chromatography on DEAE-cellulose and hydroxyapatite, and by an affinity step on agarose-immobilized S-adenosylhomocysteine. The isolated enzyme has a molecular weight of 135,000 and methylates DNA from various sources in native and heat-denatured forms. The synthetic copolymer poly(dG-dC) . poly(dG-dC) is methylated in B- and Z-conformation to about the same extent. DNA containing hemimethylated sites was isolated from P815 cells grown in the presence of 5-azacytidine. This P815 DNA was used to measure the "maintenance' DNA methylase activity, whereas 5-methylcytosine-free procaryotic DNA served as a substrate for the "de novo' DNA methylase activity in our enzyme preparation. The crude extract as well as the highly purified DNA methylase are capable of transferring methyl groups to these two types of substrate. The fact that both types of activity co-chromatograph during the isolation procedure suggests that one enzyme molecule may exercise both the "maintenance' and "de novo' activity.

Animals↗
PubMed1983

Alteration of enzymatic DNA methylation by chemical carcinogens.

It is generally accepted that the DNA methylation pattern in a particular cell type is stable over the cell generations and is clonally inherited by a semiconservative mechanism in which the methylation of the progeny strand is determined by the 5-methylcytosine residue present in the parental strand. In our experiments, we have been able to show that the chemical carcinogens MNU, MNNG, and L-ethionine affect the methylation process either by modification of the DNA substrate or by interference at the cofactor level. The changes introduced into the cellular methylation pattern persist and are inherited in the progeny cells regardless of whether the carcinogens or their adducts in the DNA persist in the cell or not. The carcinogen-induced hypomethylation correlates with the increased transcriptional complexity of the nRNA of treated cells, indicating the initiation of transcription at sites previously inactive. This means that chemical carcinogens can cause, by interference with the process of DNA methylation, stable changes in the program of genetic expression; the possibility that such changes are related to the process of initiation of carcinogenesis should not be overlooked.

Animals↗
PubMed1983

Aberrant de novo methylation of DNA after treatment of murine cells with N-acetoxy-N-2-acetylaminofluorene.

The ultimate chemical carcinogen N-acetoxy-N-2-acetylaminofluorene inhibits the enzymatic methylation of newly replicated DNA in cultured mouse P815 cells in a dose-dependent manner. After removal of the carcinogen, a significant de novo methylation of newly replicated DNA takes place, the level of methylation being higher than in control cultures. This aberrant methylation persists in the absence of N-acetoxy-N-2-acetylaminofluorene in subsequent cell cycles. Cellular cloning experiments suggest that N-acetoxy-N-2-acetylaminofluorene treatment leads to two distinct sets of cells, one with a higher and another with a lower extent of enzymatic methylation of DNA, contrasting to the apparent uniform methylation pattern in control clones.

2-Acetylaminofluorene↗