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At least 1,657 records · Page 92Linked to original sources
PubMed2007

DNA methylation and histone modification regulate silencing of epithelial cell adhesion molecule for tumor invasion and progression.

Epithelial cell adhesion molecule (Ep-CAM) is believed to have a critical role in carcinogenesis and cell proliferation. However, the association of Ep-CAM with cancer invasion and progression is less clear. We found that Ep-CAM was highly expressed on low-invasive cells compared with highly invasive cells. Forced expression of Ep-CAM decreased cancer invasiveness, and silencing Ep-CAM expression elevated cancer invasiveness. Ep-CAM expression was associated with promoter methylation. Treatment with a demethylating agent, and/or the histone deacetylase inhibitor reactivated Ep-CAM expression in Ep-CAM-negative cells and inhibited cancer invasiveness. Using a promoter-reporter construct, we demonstrated methylation of the promoter fragment drive Ep-CAM-silenced transcription. Additionally, silenced Ep-CAM gene in cancer cells was enriched for hypermethylated histone 3 lysine 9. When unmethylated and active, this promoter was associated with acetylated histone 3 lysine 9. Furthermore, we observed an increased association of Ep-CAM promoter with repression components as tumor invasiveness increased. In cancer tissues, Ep-CAM expression significantly correlated with tumor progression and associated with promoter methylation. Our data support the idea that modulation of Ep-CAM plays a pivotal role in tumor invasion and progression. Moreover, aberrant DNA methylation of Ep-CAM is implicated in enhancing invasive/metastatic proclivity of tumors.

Antigens, Neoplasm↗
PubMed1991

A new method for the rapid identification of genes encoding restriction and modification enzymes.

We have constructed derivatives of Escherichia coli that can be used for the rapid identification of recombinant plasmids encoding DNA restriction enzymes and methyltransferases. The induction of the DNA-damage inducible SOS response by the Mcr and Mrr systems, in the presence of methylated DNA, is used to select plasmids encoding DNA methyltransferases. The strains of E. coli that we have constructed are temperature-sensitive for the Mcr and Mrr systems and have been further modified to include a lacZ gene fused to the damage-inducible dinD locus of E. coli. The detection of recombinant plasmids encoding DNA methyltransferases and restriction enzymes is a simple, one step procedure that is based on the induction at the restrictive temperature of the lacZ gene. Transformants encoding DNA methyltransferase genes are detected on LB agar plates supplemented with X-gal as blue colonies. Using this method, we have cloned a variety of DNA methyltransferase genes from diverse species such as Neisseria, Haemophilus, Treponema, Pseudomonas, Xanthomonas and Saccharopolyspora.

Cloning, Molecular↗
PubMedDate not supplied

Conformational transition of restriction endonuclease MvaI-substrate complex under the influence of Mg2+ probed by DNA-protein cross-linking studies.

The method of protein affinity modification by DNA analogues was used to study the characteristic features of restriction endonuclease MvaI interaction with DNA. Oligonucleotide duplexes containing a monosubstituted pyrophosphate internucleotide bond were used for cross-linking to the enzyme. The conditions of the reaction of MvaI endonuclease with these reagents were investigated. On the basis of data obtained, the model of successive inclusion of two Mg2+ ions into MvaI endonuclease-substrate complex was proposed and confirmed by the kinetic scheme of the process.

Algorithms↗
PubMed1992

DNA polymerase, RNA polymerase and exonuclease activities on a DNA sequence modified by benzo[a]pyrene diolepoxide.

Adducts produced by modification of DNA with benzo[a]pyrene diolepoxide (BPDE) are known to inhibit both DNA and RNA synthesis. This phenomenon has been used as a method for determining the distribution of carcinogen binding within defined DNA sequences. A critical comparison of different enzyme activities on adducted DNA is needed, since different enzymes may process adducted DNA differently. Thus, we compared blocks in DNA polymerase activity with that of an RNA polymerase and with an exonuclease at single base resolution. BPDE adducts blocked the progression of cloned T7 DNA polymerase (Sequenase) in a dose-dependent manner. Although the majority of these blocks were at one base prior to adducted guanines, we also observed some blocks opposite specific guanines, suggesting that in some sequences the polymerase inserted a base opposite the modified guanine. Digestion with T4 DNA polymerase (3'----5') exonuclease activity was also blocked in BPDE-adducted DNA; however, fragments produced by blocks in T4 exonuclease migrated two or more bases longer than the corresponding guanine. Mapping of adduct distributions using both Sequenase and T4 exonuclease gave similar results, demonstrating that a long tract of guanines was preferentially modified, and within a polyguanine sequence, the 5' guanines were more heavily modified than the 3' guanines. Transcription of adducted DNA by SP6 RNA polymerase was also inhibited in a dose-dependent manner. However, adducted bases which posed strong blocks to the DNA polymerase were not always strong blocks to the RNA polymerase. Thus, in terms of adduct distribution, Sequenase and T4 exonuclease provided more consistent results than the RNA polymerase, since blockage of the RNA polymerase correlated poorly with guanines.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1981

Changes in chromatin structure at the replication fork. II The DNPs containing nascent DNA and a transient chromatin modification detected by DNAase I.

Discrete deoxyribonucleoproteins (DNPs) containing nascent and/or bulk DNA, were obtained by fractionating micrococcal nuclease digests of nuclei form 3H-thymidine pulse (15-20 sec) and 14C-thymidine long (16 h) labeled sea urchin embryos in polyacrylamide gels. One of these DNPs was shown to contain the micrococcal nuclease resistant 300 bp "large nascent DNA" described in Cell 14, 259-267, 1978. The bulk and nascent mononucleosome fractions provided evidence for the preferential digestion by micrococcal nuclease of nascent over bulk linker regions to yield mononucleosome cores with nascent DNA. DNAase I was used to probe whether any nascent DNA is in nucleosomes. Nascent as well as bulk single-stranded DNA fragments occurred in multiples of 10.4 bases with higher than random frequencies of certain fragment sizes (for instance 83 bases) as expected from a nucleosome structure. However, a striking background of nascent DNA between nascent DNA peaks was observed. This was eliminated by a pulse-chase treatment or by digestion of pulse-labeled nuclei with micrococcal nuclease together with DNAase I. One of several possible interpretations of these results suggests that a transient change in nucleosome structure may have created additional sites for the nicking of nascent DNA by DNAase I; the micrococcal nuclease sensitivity of the interpeak radioactivity suggest its origin from the linker region. Endogenous nuclease of sea urchin embryos cleaves chromatin DNA in a manner similar to that of DNAase I.

Animals↗
PubMedDate not supplied

[Effective method of DNA purification from contaminated matters and optimization of amplification].

A new modification of DNA purification has been developed. It includes: 1) standard treatment of biological material with proteinase K followed by phenol-chlorophorm extraction; 2) subsequent sample purification using micro-columns packed with Dowex-50 and Sephadex G-50. Oligonucleotide primers often used for DNA typing in man by means of polymerase chain reaction have also been modified. These are VNTR (variable number of tandem repeats) loci of apoB and D17S5. The increase of stability and specificity of amplification of VNTR loci of apoB and D17S5 was achieved by increase of primer length and amplification cycle. The sensitivity of this mode of amplification is 2-4 ng DNA-template. Employment of the nested amplification for apoB locus increased sensitivity of this method up to a few copies of DNA.

Apolipoproteins B↗
PubMed1994

Improvement of the strain for the rapid identification of genes encoding restriction and modification enzymes.

The E. coli AP1-200-9 strain for rapid identification of genes encoding restriction and modification enzymes carries a temperature sensitive lacZ gene fused to the damage-inducible dinD locus. A derivative of this strain was constructed that has a wild-type form of this locus which allows for a more efficient identification of recombinant plasmids encoding restriction and modifications enzymes.

Bacterial Proteins↗
PubMed1983

Poly(ADP-ribose) polymerase auto-modification and interaction with DNA: electron microscopic visualization.

The interaction between purified calf thymus poly(ADP-ribose) polymerase and its activating co-purified DNA (sDNA) was investigated by electron microscopy. We have shown that the enzyme-DNA complex possesses a nucleosome-like structure. The enzyme-bound DNA (sDNA) was found to be enriched in single-stranded regions and branched structures, presumed to be replication forks. The auto-ribosylated polymerase as well as the branched poly(ADP-ribose) formed were visualized by dark field electron microscopy during the auto-ADP-ribosylation reaction and the possible mechanism of this phenomenon is discussed.

Animals↗
PubMed1986

Modification of T-DNA of nopaline Ti plasmid by intermediary vector and utilization of agrocin 84 sensitivity as simple criterion of conjugation transfer of modified Ti plasmid.

The chloramphenicol resistance gene from pSa was introduced into T-DNA of pTi T37 of Agrobacterium tumefaciens by cointegration with intermediary plasmid based on pBR322. The resulting intermediary vector was mobilized to A. tumefaciens T37 by conjugative plasmid pRK2. The RK2 plasmid also forms contegrates with pTi due to the Tn3 transposon which was used for the mobilization of modified pTi into plasmid-less A. tumefaciens strain. Transconjugants were selected on the basis of their antibiotic resistance markers and tested for agrocin sensitivity as proof of Ti plasmid transfer. Agrocin sensitivity of tranconjugants together with chloramphenicol resistance was shown to be a sufficient and simple criterion of transfer of modified Ti plasmids. Agrobacterium strains with modified Ti plasmids showed decreased virulence in consequence of the presence of additional borderline sequence inside their T-DNA.

Adenine Nucleotides↗
PubMed1984

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. EcoRII endonuclease cleavage of substrates with repeated natural and modified recognition sites.

Interaction of EcoRII restriction endonuclease with a set of synthetic concatemer DNA duplexes with natural and modified sites for this enzyme has been studied. DNA duplexes with repeated natural sites are cleaved by EcoRII. Substitution of central AT-pair in the recognition site for a non-complementary TT-or AA-pair reduces the rate of cleavage, this effect being much more pronounced in the last case. Absence of site flanking in one strand from the 5'-terminus also results in very slow cleavage. The results obtained testify to the interaction of EcoRII with both strands of the substrate.

Base Sequence↗
PubMed1993

Kinetic study of the addressed modification by hemin derivatives of oligonucleotides.

Kinetics of oligonucleotide pd(TGAATGGGAAGA) modification by a hemin derivative of the complementary oligonucleotide pd(TTCCCATT) in the presence of hydrogen peroxide was investigated. The treatment of experimental data permitted to evaluate the association and rate constants at 25 degrees C: Kx = (3.40 +/- 0.38) x 10(5) M-1 (association constant of the reagent with the target), kd = 152 +/- 6 M-1 min-1 (degradation constant of the hemin group of the reagent in a parallel reaction), ko = 51.0 +/- 1.7 M-1 min-1 (target modification constant in the reactive duplex). The modification of DNA is incomplete due to competition of the modification reaction with the degradation of the hemin group of the reagent in a parallel reaction.

Base Sequence↗
PubMed1987

Estrous cycle modification of rat uterine DNA alkylation by N-methyl-N-nitrosourea.

The present study was designed to determine whether the stage of the estrous cycle at the time of N-nitroso-N-methylurea (NMU) presentation altered DNA adduct formation and repair in the rat uterus. In uterus the rate of O6-methylguanine (O6-meGua) and 7-methylguanine (7-meGua) formation and the total yield of adducts was estrous cycle dependent. Uterine DNA from rats injected with NMU on diestrus formed O6-meGua and 7-meGua more rapidly and had significantly higher adduct levels than those rats injected on proestrus or estrus. Repair of O6-meGua and 7-meGua was also significantly faster between 1 and 24 h post-NMU in uterine DNA isolated from rats injected on diestrus compared to those injected on proestrus or estrus.

Alkylation↗
PubMed2004

A column method for determination of DNA cytosine-C5-methyltransferase activity.

DNA methylation at the 5th position of cytosine has been found to be correlated with tumorigenesis. An inhibitor of DNA methylase could, therefore, be used as an anticancer drug. However, only a few inhibitory compounds have been discovered due to the limitations for assaying the DNA methylation. In this study, we describe a modification of DNA cytosine-C5-methyltransferase assay system utilizing [(3)H]-labeled S-adenosyl-methionine (SAM) and Sephadex G-25 column. Pre-treatment of either lambda DNA or the promoter region of human telomerase (hTERT) with HaeIII methylase greatly reduced the digestion of the DNAs with the corresponding restriction enzyme HaeIII endonuclease (over 100-fold), and the result was further confirmed by agarose gel electrophoresis. Application of this column method to another modification/restriction system, EcoRI methylase/endonuclease, gave rise to the similar results. Our data suggest that the newly developed column method could be effective for rapid screening of large number of cytosine methylase inhibitors and could also be applicable to other DNA methylases.

Bacteriophage lambda↗