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At least 685 records · Page 38Linked to original sources
PubMed1996

Expression and localization of DNA topoisomerase II during rat spermatogenesis.

The potential role(s) of DNA topoisomerase II (topo II) during chromatin changes that characterize different stages of spermatogenesis was investigated in the rat by an analysis of the expression and localization of topo II mRNA and protein in individual spermatogenic cells. Expression of topo II was restricted to spermatogonia, spermatocytes, and round and early-elongating spermatids. Two protein bands of 177 and 170 kDa were detected in immunoblots of spermatocytes and round spermatids, while bands of 148 and 142 kDa were prominent in preparations of elongating spermatids. Topo II levels and distribution patterns, as observed by immuno-fluorescent microscopy, exhibited cell type-specific variations. Differences in topo II staining patterns were also apparent when nuclear matrices of spermatogenic cells were prepared with different extraction conditions. In addition to its possible function as a structural component, topo II, associated with nuclear matrix preparations from spermatogenic cells, possessed catalytic activity. These observations indicate that both the 177 and 170 kDa and the 148 and 142 kDa forms of topo II share similar structural and functional properties. Topo II beta mRNA was transcribed in rat spermatogenic cells at 6.2 kb. Relative levels of topo II beta mRNA were high in spermatogonia and spermatocytes, and decreased in both round and early-elongating spermatids. Changes in topo II expression levels and localization patterns represent distinct stage-specific markers for the maturation of spermatogenic cells, and are consistent with the involvement of topo II in mediating DNA modifications and chromatin changes during spermatogenesis.

Animals↗
PubMed1991

Induction of gamma delta transposition in response to elevated glucose-6-phosphate levels.

The nonenzymatic glycosylation of nucleic acids in vitro by the reducing sugars, glucose or glucose-6-phosphate, alters both physical and biological properties. Recent investigations have demonstrated that elevated intracellular levels of glucose-6-phosphate in glycolytic mutants of E. coli resulted in a concentration-associated increase in mutations of a target plasmid. The majority of the plasmid mutations were due to large (greater than 1 kb) insertions or deletions. We describe here the further analysis of mutant plasmids isolated from bacteria grown under conditions which were conducive to the intracellular accumulation of glucose-6-phosphate. We have found that a number of the insertional plasmid mutations were the result of the movement of the transposable element gamma delta from the host genome into the plasmid. The frequency of gamma delta transposition was also associated with the amount of glucose-6-phosphate accumulated in the bacterial cells. Furthermore, the presence of another transposable element, either Tn 5 or Tn 10 in the host genome increased the rate of gamma delta transposition without affecting its own movement. The observed increase in gamma delta transposition suggests a novel mechanism of induction by reducing sugars which may be the result of DNA modifications by reducing sugars.

Base Sequence↗
PubMed1984

Individual and evolutionary variation of primate ribosomal DNA transcription initiation regions.

A 16-kilobase region surrounding the transcription initiation site for ribosomal DNA and including the entire ribosomal DNA transcription unit has been characterized in man and compared in higher primates. Restriction analysis of ribosomal DNA from the pygmy chimpanzee (Pan paniscus), the common chimpanzee (Pan troglodytes), the gorilla (Gorilla gorilla), the orangutan (Pongo pygmaeus), the gibbon (Hylobates lar), and the rhesus monkey (Macaca mulatta) allows a primate phylogeny to be constructed based on ribosomal DNA structure. Individual variation and methylation are demonstrated in the ribosomal DNA repeats of all primates examined. Restriction analysis with HincII endonuclease suggests cleavage at sites containing methylated CpG and adds the SalI/HincII pair to those enzymes useful for studying DNA modification.

Animals↗
PubMed2005

Reversal of the silencing of tetracycline-controlled genes requires the coordinate action of distinctly acting transcription factors.

BACKGROUND: Regulation of genes transferred to eukaryotic organisms is often limited by the lack of consistent expression levels in all transduced cells, which may result in part from epigenetic gene silencing effects. This reduces the efficacy of ligand-controlled gene switches designed for somatic gene transfers such as gene therapy. METHODS: A doxycycline-controlled transgene was stably introduced in human cells, and clones were screened for epigenetic silencing of the transgene. Various regulatory proteins were targeted to the silent transgene, to identify those that would mediate regulation by doxycycline. RESULTS: A doxycycline-controlled minimal promoter was found to be prone to gene silencing, which prevents activation by a fusion of the bacterial TetR DNA-binding domain with the VP16 activator. DNA modification studies indicated that the silenced transgene adopts a poorly accessible chromatin structure. Several cellular transcriptional activators were found to restore an accessible DNA structure when targeted to the silent transgene, and they cooperated with Tet-VP16 to mediate regulation by doxycycline. CONCLUSIONS: Reversal of the silencing of a tetracycline-regulated minimal promoter requires a chromatin-remodeling activity for subsequent promoter activation by the Tet-VP16 fusion protein. Thus, distinct regulatory elements may be combined to obtain long-term regulation and persistent expression of exogenous genes in eukaryotic cells.

Cell Line↗
PubMed1988

DNA methylation in plants.

Both replicative and postreplicative nuclear DNA (nDNA) methylation, with the formation of 5-methylcytosine (mC) residues, occurs in plants. These two types of enzymatic DNA modification are different in amount and nucleotide sequence of methylatable sites, as well as in sensitivity to phytohormones, temperature and various inhibitors of DNA methylation, transcription and replication. The role of DNA methylation in regulation of replication, gene expression and cell differentiation is discussed.

5-Methylcytosine↗
PubMed2003

C-terminal deletion of AID uncouples class switch recombination from somatic hypermutation and gene conversion.

Class-switch recombination (CSR), somatic hypermutation (SHM), and antibody gene conversion are distinct DNA modification reactions, but all are initiated by activation-induced cytidine deaminase (AID), an enzyme that deaminates cytidine residues in single-stranded DNA. Here we describe a mutant form of AID that catalyzes SHM and gene conversion but not CSR. When expressed in E. coli, AID(delta189-198) is more active in catalyzing cytidine deamination than wild-type AID. AID(delta189-198) also promotes high levels of gene conversion and SHM when expressed in eukaryotic cells, but fails to induce CSR. These results underscore an essential role for the C-terminal domain of AID in CSR that is independent of its cytidine deaminase activity and that is not required for either gene conversion or SHM.

Animals↗
PubMed1981

Mechanism of inhibition of enzymatic deoxyribonucleic acid methylation by 2-(acetylamino)fluorene bound to deoxyribonucleic acid.

Binding of 2-(acetylamino)fluorene (AAF) to C-8 of guanine induces a local destabilization of the DNA helix. A relationship was observed where the degree of DNA modification by AAF was inversely proportional to its methyl acceptor capacity from S-adenosyl-L-methionine in the presence of rat brain DNA cytosine 5-methyltransferase. Moreover, substituted DNA (DNA-AAF) behaves as a methylation inhibitor of native DNA. This inhibition is of the mixed type. The substituted DNAs have higher affinities for the enzyme than native DNA. The inhibition is irreversible. Addition of DNA-AAF to the enzyme preincubated with native DNA inhibits methylation, but only after a lag period. This agrees with the model in which the methylase "walks" along the strand to methylate cytosine residues before being detached from the DNA. AAF bound to guanine residues may block the movement of the enzyme along the helix. Single-stranded DNA has an affinity for the methylase 1.6 times lower than that of native double-stranded DNA. On the other hand, single-stranded DNA-AAF is more methylated than double-stranded DNA-AAF. A tentative model taking into account these observations is presented under Discussion. The in vitro hypomethylation of DNA-AAF could explain the in vivo observations made by several authors.

2-Acetylaminofluorene↗
PubMed1990

Species and tissue specificities of I-compounds as contrasted with carcinogen adducts in liver, kidney and skin DNA of Sprague-Dawley rats, ICR mice and Syrian hamsters.

I-compounds are age-related bulky DNA modifications that are detected in untreated animals by 32P-postlabeling. To characterize their properties, I-compounds were compared with carcinogen-DNA adducts in liver, kidney and skin of three rodent species. Weanling female Sprague-Dawley rats, ICR mice and Syrian hamsters were fed Teklad LM485 chow diet for 3 months and raised concurrently and strictly under the same environmental conditions. Animals of each species were treated topically with 24 mumol/kg dibenz[a,j]acridine per day for 3 days, then by gavage once with a mixture of safrole and 7,12-dimethylbenz[a]anthracene (60 and 80 mumol/kg respectively), or with one of the individual carcinogens. Liver, kidney and skin DNA from carcinogen-exposed (24 h after treatment) and unexposed animals was analyzed by the monophosphate version of the 32P-postlabeling assay. While each of the three carcinogens produced qualitatively identical major adduct patterns in all samples examined, I-compounds in untreated animals showed distinct species- and tissue-dependent profiles. Rats displayed the highest I-compound levels but the lowest adduct levels in both liver and kidney among the three species. These findings demonstrate fundamental differences between I-compounds and carcinogen-DNA adducts, and support the hypothesis that I-compound formation is primarily related to species-specific, i.e. genetically determined, normal metabolic activities rather than exposure to environmental genotoxic carcinogens.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2001

Site-directed inhibition of DNA replication by triple helix formation.

Sequence-specific DNA recognition can be achieved by the use of triplex-forming molecules, namely, oligonucleotides (TFO) and peptide nucleic acids (PNAs). They have been used to regulate transcription or induce genomic DNA modifications at a selected site in cells and, recently, in vivo. We have determined the conditions under which a triplex structure can inhibit DNA replication in cells. An oligopyrimidine.oligopurine sequence suitable for triplex formation was inserted in a plasmid on both sides of the SV40 origin of replication. This insert-containing plasmid was replicated in COS-1 cells together with the parent plasmid, and the ratio between the corresponding replicated DNAs was quantitated. Selective inhibition of replication of the insert-containing plasmid can be ascribed to ligand binding to the oligopyrimidine.oligopurine sequence. Inhibition of DNA replication was observed using triplex-forming molecules that induce either covalent binding at the double-stranded target sequence (with TFO-psoralen conjugate and irradiation) or noncovalent triplex formation after strand displacement (with bis-PNA). In contrast, in the absence of covalent cross-linking, TFOs (which have been shown to arrest transcription elongation) did not act on replication. These results open new perspectives for future design and use of specific inhibitors of intracellular DNA information processing.

Animals↗
PubMed1977

Different restriction of bacteriophages T3 and T7 by P1-lysogenic cells and the role of the T3-coded SAMase.

The intracellular growth of the phages T3 and T7 is restricted in the presence of the Escherichia coli prophage P1. Phage T3 has a higher ability to express its genome and to damage the host cell than T7. This partial protection of T3 against P1 restriction is due to the T3-coded SAMase, an enzyme which degrades S-adenosylmethionine, the cofactor of the P1 restriction endonuclease. Since we did not observe DNA cleavage in vivo, we conclude that the in vivo action of the P1 nuclease is limited to a SAM-dependent repressor-like binding to T3 and T7 DNA, while further reactions with the DNA (modification vs cleavage) are blocked.

Coliphages↗
PubMed1991

Short-term effects of the tumor promoting polychlorinated biphenyl mixture, Aroclor 1254, on I-compounds in liver, kidney and lung DNA of male Sprague-Dawley rats.

The effects of a tumor promoting polychlorinated biphenyl mixture, Aroclor 1254, on I-compounds (tissue, species and sex dependent DNA modifications that increase with age in untreated rodents) were studied by 32P-postlabeling in male Sprague-Dawley rat liver, kidney, and lung DNA. Aroclor 1254 was dissolved in corn oil and intraperitoneally (i.p.) injected (2 x 500 mg/kg, 2 weeks apart) into 3-month-old rats. Control rats were given corn oil. Groups of 3 animals were sacrificed at 2 and 6 weeks after the second injection of corn oil or Aroclor 1254. At both time points Aroclor 1254-treated rats had significantly lower body weights and higher liver weights while kidney and lung weights were unaffected. Thymidine incorporation into liver and lung DNA was significantly increased at both time points, while kidney DNA showed a small decrease at 2 weeks. Treatment resulted in significant reductions (ranging from 29 to 100%) of each of nine liver I-spots at 2 and 6 weeks. In treated rats there was no decrease in kidney I-spots at 2 weeks, while the levels of only two out of ten kidney spots were reduced by 42-91% at 6 weeks. At 2 weeks three out of seven and at 6 weeks four out of seven lung I-spots were lowered by 51-100% in the Aroclor 1254-treated rats. Thus the effects decreased in the order liver greater than lung greater than kidney. Since Aroclor 1254 has been reported to be a tumor promoter in liver and lung but not kidney, these results suggest a correlation between organ specific promotion of carcinogenesis by Aroclor 1254 and the reduction of DNA I-compounds.

Animals↗
PubMed2005

Prediction of methylated CpGs in DNA sequences using a support vector machine.

DNA methylation plays a key role in the regulation of gene expression. The most common type of DNA modification consists of the methylation of cytosine in the CpG dinucleotide. At the present time, there is no method available for the prediction of DNA methylation sites. Therefore, in this study we have developed a support vector machine (SVM)-based method for the prediction of cytosine methylation in CpG dinucleotides. Initially a SVM module was developed from human data for the prediction of human-specific methylation sites. This module achieved a MCC and AUC of 0.501 and 0.814, respectively, when evaluated using a 5-fold cross-validation. The performance of this SVM-based module was better than the classifiers built using alternative machine learning and statistical algorithms including artificial neural networks, Bayesian statistics, and decision trees. Additional SVM modules were also developed based on mammalian- and vertebrate-specific methylation patterns. The SVM module based on human methylation patterns was used for genome-wide analysis of methylation sites. This analysis demonstrated that the percentage of methylated CpGs is higher in UTRs as compared to exonic and intronic regions of human genes. This method is available on line for public use under the name of Methylator at http://bio.dfci.harvard.edu/Methylator/.

Algorithms↗
PubMed1977

Loss of DNA repair capacity during successive subcultures of primary rat fibroblasts.

Cultures of fibroblasts from newborn rats and successive subcultures of these cells were treated with 4-nitroquinoline-1-oxide to induce DNA repair. DNA from the cultures was examined by velocity sedimentation in alkaline sucrose gradients immediately after drug treatment and after a post-treatment incubation period of 3 h. Early passage cells were able to repair the damage that appeared as single strand breaks, however, by the seventh subculture this activity was not apparent. Measurements of repair synthesis showed a partial loss of this capacity with successive subculture. The results fit a model in which 4NQO causes two kinds of DNA modification, one of which is alkali labile and appears as a single-strand break. Both modifications are subject to excision repair, but each is recognized initially by a specific endonuclease. In the late passage cells, the endonuclease specific for the alkali labile modification is absent.

4-Nitroquinoline-1-oxide↗
PubMed1995

Mechanisms of DNA damage recognition in mammalian nucleotide excision repair.

The ability of nucleotide excision repair (NER) to process multiple forms of DNA damage is highly dependent on the precision by which DNA modifications are located in the genome. Studies of mammalian NER have shown that this system eliminates a wide range of chemically and structurally distinct DNA lesions whereby some types of damage are repaired at higher rates than others. Although the biochemical basis for this broad but heterogeneous response to DNA damage is poorly understood, recent discoveries in closely related areas of DNA metabolism indicate that selectivity for specific sites is achieved through the assembly of nucleoprotein complexes, in which DNA is frequently bent and unwound. In many cases, selectivity may be further enhanced by the action of specialized DNA helicases. These principles in protein-DNA recognition suggest a hypothetical mechanism of damage recognition that accounts for the wide substrate range of mammalian NER and also accommodates its preference for specific DNA lesions.

Animals↗
PubMed1999

Role of base excision repair in protecting cells from the toxicity of chloroethylnitrosoureas.

The chloroethylnitrosoureas react extensively with cellular DNA to produce a variety of DNA adducts, including a deoxycytidine-deoxyguanosine (dC-dG) cross-link that is clearly cytotoxic. It is now well established that O6-alkylguanine-DNA-alkyltransferase can prevent formation of this dC-dG cross-link and thereby diminish the toxicity of the chloroethylnitrosoureas. Besides alkyltransferase, DNA glycosylases from various species can also contribute to cellular resistance to the chloroethylnitrosoureas, but the mechanism for this increased resistance has not been established. It is known, however, that several chloroethylnitrosoureas-modified DNA bases, including the exocyclic adduct, N2,3-ethanoguanine, are released by Escherichia coli 3-methyladenine DNA glycosylase II. In the study described here, we examined the possibility that this enzyme might act on the exocyclic intermediate in dC-dG formation, 1,O6-ethanodeoxyguanosine, and prevent-dC-dG cross-linking in this way. However, the presence of E. coli 3-methyladenine DNA glycosylase II does not decrease the amount of dC-dG cross-link formed when chloroethylnitrosourea reacts with DNA, and we conclude that this enzyme does not recognize 1,O6-ethanodeoxyguanosine. Therefore, its contribution to resistance probably resides in its action on other nitrosourea-induced DNA modifications.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

[Levels of chromatin condensation and activity].

The main progresses which occurred last years in the knowledge of chromatin, in its biochemical components, structure and organization levels, are briefly outlined. Inhibition of transcriptional activity is examined at the molecular, cellular and chromosomal levels. The different states of chromatin condensation according to physiopathological conditions are more particularly reviewed together with recent data on transcriptional regulation depending on histone and DNA modifications.

Animals↗
PubMed1984

Unscheduled DNA synthesis in human bronchial epithelium treated with various chemical carcinogens in vitro.

A system was developed in which organ culture of human bronchial epithelium was used in combination with autoradiography for quantitative measurement of unscheduled DNA synthesis (UDS) in bronchial epithelial cells. Human bronchi obtained at surgery were cut into small sections and treated with various carcinogens plus [methyl-3H]thymidine in short-term organ culture. Significant numbers of silver grains, indicating UDS, were detected on the nuclei of epithelial cells of human bronchi treated with carcinogens, and the numbers were proportional to the concentrations of carcinogens. In this system seven representative carcinogens induced UDS. Four active metabolites of benzo[a]pyrene, and benz[a]anthracene also were found to induce very active UDS in human bronchial epithelium. These findings suggest that human bronchial epithelial cells can repair different types of DNA modification induced by chemical carcinogens.

Autoradiography↗
PubMed2000

Identification of type II restriction and modification systems in Helicobacter pylori reveals their substantial diversity among strains.

A total of 22 type II restriction endonucleases with 18 distinct specificities have been identified in six Helicobacter pylori strains. Among these 18 specificities are three completely new endonucleases, Hpy178III, Hpy99I, and Hpy188I, that specifically cleave DNA at TCNNGA, CGWCG, and TCNGA sites, respectively. The set of endonucleases identified in each strain varies, but all have four- or five-base recognition sequences. Among 16 H. pylori strains, examination of the DNA modification status at the recognition sites of 15 restriction endonucleases reveals that each strain has a substantially different complement of type II modification systems. We conclude that the type II restriction-modification systems in H. pylori are highly diverse between strains, a unique characteristic of H. pylori. The diverse methylation status of H. pylori chromosomal DNA may serve as a new typing system to discriminate H. pylori isolates for epidemiological and clinical purposes. This study also demonstrates that H. pylori is a rich source of type II restriction endonucleases.

Adult↗