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PubMed1986

trans activation of the latent Epstein-Barr virus (EBV) genome after transfection of the EBV DNA fragment.

Transfection of Epstein-Barr virus (EBV)-nonproducer Raji cells with the BamHI Z fragment of EBV DNA induced antigens that were detected with human antiserum against EBV-specific early antigens. Northern blot analysis of transfected cells revealed that one intense RNA band hybridized with the BamHI H and F fragments but not with the BamHI Z fragment. Cooperation between the BamHI H, F, and BamHI Z regions was also confirmed in baby hamster kidney cells that were cotransfected with both fragments. These results indicate that the transfected BamHI Z fragment of EBV DNA induces a trans-acting factor which activates the gene expression of the BamHI H and F region and that the BamHI Z region possibly plays an important role in the latency of EBV.

Animals↗

PubMed1990

Bacteriophage P1 cloning system for the isolation, amplification, and recovery of DNA fragments as large as 100 kilobase pairs.

The development of a bacteriophage P1 cloning system capable of accepting DNA fragments as large as 100 kilobase pairs (kbp) is described. The vectors used in this system contain a P1 packaging site (pac) to package vector and cloned DNA into phage particles, two P1 loxP recombination sites to cyclize the packaged DNA once it has been injected into a strain of Escherichia coli containing the P1 Cre recombinase, a kanr gene to select bacterial clones containing the cyclized DNA, a P1 plasmid replicon to stably maintain that DNA in E. coli at one copy per cell chromosome, and a lac promoter-regulated P1 lytic replicon to amplify the DNA before it is reisolated. An essential feature of the cloning system is a two-stage in vitro packaging reaction that packages vector DNA containing cloned inserts into phage particles that can deliver their DNA to E. coli with near unit efficiency. The packaging reaction can generate 10(5) clones with high molecular weight DNA inserts per microgram of vector DNA. Using NotI fragments from E. coli DNA, it was shown that the system can clone 95- and 100-kbp fragments but not a 106-kbp fragment. Presumably, the combined size of the latter fragment and the vector DNA (13 kbp) exceeds the headful capacity of P1.

Base Composition↗

PubMed1992

Tumor necrosis factor alpha induces apoptosis in mammary adenocarcinoma cells by an increase in intranuclear free Ca2+ concentration and DNA fragmentation.

The incubation of human mammary adenocarcinoma cells (BT-20) with tumor necrosis factor alpha in the absence or presence of cycloheximide resulted in progressive DNA fragmentation. This was preceded by a sustained increase in intracellular free Ca2+ concentration and was not detected in cells pretreated with intracellular Ca2+ chelators, calmodulin antagonists, or activators of protein kinase C. Image analysis of fura-2-loaded BT-20 cells treated with tumor necrosis factor alpha revealed that, in many cells, the initial increase in Ca2+ level occurred in a cellular region that corresponded to the localization of the nucleus. Our findings suggest that tumor necrosis factor alpha can promote an increase in intranuclear free Ca2+ which, in turn, may stimulate Ca(2+)-dependent endonuclease activity, resulting in DNA fragmentation and apoptosis.

Adenocarcinoma↗

PubMed1994

Naturally occurring cell death in the developing cerebral cortex of the rat. Evidence of apoptosis-associated internucleosomal DNA fragmentation.

Naturally occurring dead cells in the developing rat neocortex, subcortical white matter and hippocampus, which increase in number during the first postnatal week and decrease thereafter to disappear by the end of the first month, were examined by in situ labeling of nuclear DNA fragmentation. These cells showed peripheral chromatin condensation or extremely dark, often fragmented, nuclei. Southern hybridization following agarose gel electrophoresis of DNA extracted from the developing cortex, but not from adult brain, showed a 'ladder' pattern which is typical of internucleosomal DNA fragmentation. Taken together these results show that naturally occurring cell death (programmed cell death) in the developing cerebral cortex has the morphology of apoptosis and is associated with endonuclease activation.

Aging↗

PubMed1996

An evaluation of new gel matrices for the separation of PCR-amplified DNA fragments.

It has been shown that the novel gel matrix, PCR Purity Plus which consists of a vinyl-polymer of polyacrylamide, provides a superior and rapid means of separating DNA fragments. As this product has been discontinued, the two new commercial versions of this matrix (MDE and GeneAmp) with PCR Purity Plus were compared. Optimal conditions for resolving DNA fingerprint profiles for both matrices were defined. Both MDE and GeneAmp gels provided a clear separation of DNA fragments. However, the profiles obtained on GeneAmp gel were closest to that of PCR Purity Plus. These results should be useful to DNA fingerprinting studies where it is critical to obtain a clear resolution of complex DNA profiles.

DNA↗

PubMed2003

Homology of primate DNA fragments for estrous-associated oviductal glycoprotein.

In this study, the DNA fragments encoded for oviduct EGP were amplified by PCR method. Corresponding sequences from chimpanzee, orangutan and human were similarly amplified, but we failed to amplify the corresponding DNA sequences from: spider monkey, salmon and several different species of rodents, bandicoot, sheep and pig. Through analyzing the restriction enzyme map and the DNA sequence of the amplified fragments from primates, the data suggest that monkey and human DNA encoded for EGP are genetically more closely related to one another than each of them to other species.

Animals↗

PubMed2003

High-sensitivity capillary gel electrophoretic analysis of DNA fragments on an electrophoresis microchip using electrokinetic injection with transient isotachophoretic preconcentration.

The research adopted a single-channel microchip as the probe, and focused electrokinetic injection combined with transient isotachophoresis preconcentration technique on capillary electrophoresis microchip to improve the analytical sensitivity of DNA fragments. The channel length, channel width and channel depth of the used microchip were 40.5 mm, and 110 and 50 microm, respectively. The separation was detected by CCD (charge-coupled device) (effective length=25 mm, 260 nm). A 1/100 diluted sample (0.2 mg/l of each DNA fragment) of commercially available stepladder DNA sample could be baseline separated in 120 s with S/N=2-5. Compared with conventional chip gel electrophoresis, the proposed method is ideally suited to improve the sensitivity of DNA analysis by chip electrophoresis.

Buffers↗

PubMed1996

The thermal stability of DNA fragments with tandem mismatches at a d(CXYG).d(CY'X'G) site.

Temperature-Gradient Gel Electrophoresis (TGGE) was employed to determine the thermal stabilities of 28 DNA fragments, 373 bp long, with two adjacent mismatched base pairs, and eight DNAs with Watson-Crick base pairs at the same positions. Heteroduplex DNAs containing two adjacent mismatches were formed by melting and reannealing pairs of homologous 373 bp DNA fragments differing by two adjacent base pairs. Product DNAs were separated based on their thermal stability by parallel and perpendicular TGGE. The polyacrylamide gel contained 3.36 M urea and 19.2 % formamide to lower the DNA melting temperatures. The order of stability was determined in the sequence context d(CXYG).d(CY'X'G) where X.X' and Y.Y" represent the mismatched or Watson-Crick base pairs. The identity of the mismatched bases and their stacking interactions influence DNA stability. Mobility transition melting temperatures (T u) of the DNAs with adjacent mismatches were 1.0-3.6 degrees C (+/-0.2 degree C) lower than the homoduplex DNA with the d(CCAG).d(CTGG) sequence. Two adjacent G.A pairs, d(CGAG).d(CGAG), created a more stable DNA than DNAs with Watson-Crick A.T pairs at the same sites. The d(GA).d(GA) sequence is estimated to be 0.4 (+/-30%) kcal/mol more stable in free energy than d(AA).d(TT) base pairs. This result confirms the unusual stability of the d(GA).d(GA) sequence previously observed in DNA oligomers. All other DNAs with adjacent mismatched base pairs were less stable than Watson-Crick homoduplex DNAs. Their relative stabilities followed an order expected from previous results on single mismatches. Two homoduplex DNAs with identical nearest neighbor sequences but different next-nearest neighbor sequences had a small but reproducible difference in T u value. This result indicates that sequence dependent next neighbor stacking interactions influence DNA stability.

Base Composition↗

PubMed1992

Diphtheria toxin- and Pseudomonas A toxin-mediated apoptosis. ADP ribosylation of elongation factor-2 is required for DNA fragmentation and cell lysis and synergy with tumor necrosis factor-alpha.

We have reported that diphtheria toxin (DTX) mediates target cell lysis and intranucleosomal DNA fragmentation (apoptosis) and also synergizes with TNF-alpha. In this paper, we examined which step in the pathway of DTX-mediated inhibition of protein synthesis was important for induction of cytolytic activity and for synergy. Using a DTX-sensitive tumor cell line, we first examined the activity of the mutant CRM 197, which does not catalyze the ADP ribosylation of elongation factor-2 (EF-2). CRM 197 was not cytolytic for target cells and did not mediate intranucleosomal DNA fragmentation of viable cells. The failure of CRM 197 to mediate target cell lysis suggested that the catalytic activity of DTX is prerequisite for target cell lysis. This was corroborated by demonstrating that MeSAdo, which blocks the biosynthesis of diphthamide, inhibited DTX-mediated protein synthesis inhibition and also blocked target cell lysis. Furthermore, the addition of nicotinamide, which competes with NAD+ on the DTX action site of EF-2, also blocked DTX-mediated lysis. These findings suggest that ADP-ribosylation of EF-2 may be a necessary step in the pathway leading to target cell lysis. In contrast to the sensitive line, the SKOV-3 tumor cell line is sensitive to protein synthesis inhibition by DTX but is not susceptible to cytolysis and apoptosis by DTX. Thus, protein synthesis inhibition by DTX is not sufficient to mediate target cell lysis. The synergy in cytotoxicity obtained with the combination of DTX and TNF-alpha was examined in order to determine the pathway mediated by DTX in synergy. Like the direct lysis by DTX, synergy was significantly reduced by MeSAdo and by nicotinamide. Furthermore, synergy was not observed with combination of CRM 197 and TNF-alpha. These results demonstrate that, in synergy, DTX may utilize the same pathway required for its cytolytic activity. Pseudomonas aeruginosa exotoxin shared most the properties shown for DTX. Altogether, these findings demonstrate that DTX-mediated apoptosis is initiated at a step beyond the ADP ribosylation of EF-2.

ADP Ribose Transferases↗

PubMed1996

Minor groove binding ligands alter the rotational positioning of DNA fragments on nucleosome core particles.

We have used hydroxyl radical and DNase I footprinting to examine the interaction of four AT-selective minor groove binding ligands (Hoechst 33258, distamycin, netropsin and berenil) with DNA fragments which have been reconstituted with nucleosome core particles. Hydroxyl radical footprints of reconstituted tyrT DNA show that all four ligands induce changes in the phased cleavage pattern, consistent with the suggestion that they cause the DNA to rotate by 180 degrees on the nucleosome surface. This observation was confirmed by a series of hydroxyl radical and DNase I footprinting experiments on a synthetic DNA fragment containing five different (A/T)4 sites spaced ten bases apart, in phase with the nucleosomal repeat. This fragment produces a phased cleavage pattern when bound to the nucleosome cores, with minima in the AT regions, suggesting that these regions are positioned with their narrow minor grooves facing towards the protein surface. In the presence of the minor groove binding ligands the hydroxyl radical cleavage maxima are shifted by about five base-pairs. It appears that the ligands have caused the DNA to rotate by about 180 degrees on the protein surface; those DNA regions which were facing out are turned in and vice versa. Regions to which the ligands are bound are turned away from the protein surface, thereby minimising electrostatic repulsion between the cationic charges on the ligand and protein. The absence of any observable footprints in the AT-regions suggests that these changes are induced at low levels of occupancy.

Base Composition↗

PubMed2006

DNA fragments induction in human fibroblasts by radiations of different qualities.

Experimental data on DNA double strand break (DSB) induction in human fibroblasts (AG1522), following irradiation with several radiation qualities, namely gamma rays, 0.84 MeV protons, 58.9 MeV u(-1) carbon ions, iron ions of 115 MeV u(-1), 414 MeV u(-1), 1 GeV u(-1), and 5 GeV u(-1), are presented. DSB yields were measured by calibrated Pulsed Field Gel Electrophoresis in the DNA fragment size range 0.023-5.7 Mbp. The DSB yields show little LET dependence, in spite of the large variation of the latter among the beams, and are slightly higher than that obtained using gamma rays. The highest yield was found for the 5 GeV u(-1) iron beam, that gave a value 30% higher than the 1 GeV u(-1) iron beam. A phenomenological method is used to parametrise deviation from randomness in fragment size spectra.

Cells, Cultured↗

PubMed1995

Menadione-induced DNA fragmentation without 8-oxo-2'-deoxyguanosine formation in isolated rat hepatocytes.

Menadione (2-methyl-1,4-naphthoquinone) induces oxidative stress in cells causing perturbations in the cytoplasm as well as nicking of DNA. The mechanisms by which DNA damage occurs are still unclear, but a widely discussed issue is whether menadione-generated reactive oxygen species (ROS) directly damage DNA. In the present study, we measured the effect of menadione on formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), an index of oxidative DNA base modifications, and on DNA fragmentation. Isolated hepatocytes from phenobarbital-pretreated rats were exposed to menadione, 25-400 microM, for 15, 90 or 180 min with or without prior depletion of reduced glutathione (GSH) by diethyl maleate. Menadione caused profound GSH depletion and internucleosomal DNA fragmentation, which was demonstrated by a prominent fragmentation ladder on agarose gel electrophoresis. We found no oxidative modification of DNA in terms of increased 8-oxodG formation. In contrast, the positive control of sunlamp light increased 8-oxodG 5-fold in rat hepatocytes. We conclude that oxidative modification of DNA bases is unlikely to be important in menadione-induced DNA damage.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2003

Separation and recovery of DNA fragments by electrophoresis through a thermoreversible hydrogel composed of poly(ethylene oxide) and poly(propylene oxide).

We have synthesized and characterized a thermoreversible hydrogel of multiplied block copolymers, composed of poly(ethylene oxide) and poly(propylene oxide), for DNA electrophoresis. The aqueous solution of block copolymers turned into a hydrogel upon heating at temperatures above 10-11 degrees C, whereas it reverted into a solution upon cooling. Linear double-stranded DNA molecules migrated through the gel matrices at a rate that was inversely proportional to the logarithm of the DNA length. The hydrogel is most effective for separating DNA fragments in the 10- to 2000-bp range. The resolving range lay in-between the effective ranges of polyacrylamide and agarose gel electrophoreses of DNA. The gel slices containing DNA fragments were liquefied by cooling on ice, and the DNA was precipitated with ethanol. No contaminants that inhibit enzymatic reactions were found in the DNA recovered from the hydrogel. Plasmid DNA recovered from the hydrogel was recircularized with T4 DNA ligase and yielded highly efficient Escherichia coli transformation. Therefore, thermoreversible gel electrophoresis will be a useful method for DNA separation and isolation in recombinant DNA technology.

DNA↗

PubMed1992

Human cytotoxic T lymphocytes and activated peripheral blood lymphocytes enhance cytochalasin B-induced DNA fragmentation of indicator bystander cells.

The cytochalasins are known to have multiple effects on cellular function. Not only do they induce secretion from granule compartments but they can induce DNA fragmentation in numerous cells. Evidence is presented which shows that treatment of human cytotoxic T lymphocytes and activated peripheral blood lymphocytes with cytochalasin B induces release of a factor capable of enhancing DNA fragmentation in cytochalasin-susceptible target cells. This activity can be transferred in the supernatants of cytochalasin B-activated CTL.

Cytochalasin B↗

PubMed1987

Cloning of DNA fragments containing Streptomyces promoter activity.

The thiostrepton-resistance gene is expressed in Streptomyces jumonjinensis [16]-8 SANK 61185 carrying the plasmid pIJ702 but not the tyrosinase gene. We have isolated DNA fragments from various streptomycetes that restore expression of the tyrosinase gene on pIJ702 in this organism. The nucleotide sequences of two of these DNA fragments show that they contain putative ribosome binding sites and- 10 regions of possible promoters.

Base Sequence↗

PubMed1995

DNA fragmentation induced in lymphocytes by gamma irradiation or dexamethasone: inhibition by diethyldithiocarbamate (DTC), potentiated by zinc.

Apoptosis is a process of physiological cell death characterized by DNA fragmentation, chromatin condensation, loss of membrane asymmetry and cell lethality. In the present study, apoptosis induced in thymocytes by dexamethasone or gamma irradiation is evaluated by flow cytometry, gel electrophoresis and other techniques. Treatment of thymocytes with DTC or zinc shows that these products can inhibit radiation- or dexamethasone-induced apoptosis. Moreover, a synergistic effect is observed by using associations of both products (5 microM DTC + 50 microM ZnSO4): ZnSO4 potentiates the effect of DTC at concentrations for which the molecules used separately show a low efficacy. These results indicate that DNA fragmentation induced by dexamethasone or irradiation in thymocytes share some identical mechanisms.

Animals↗

PubMed1998

[Optimization of non-gel sieving capillary electrophoretic separation of DNA fragments of hundreds of base pairs].

Non-gel sieving capillary electrophoresis has been employed in the biological sciences for the size-based separation of macromolecules such as nucleic acids. In this paper, four factors i.e. electric field strength, capillary length, capillary diameter and hydroxy-propylmethylcellulose (HPMC) concentration were integratively evaluated to select the optimal condition of separating DNA fragments of a few hundreds of base pairs through orthogonal analysis. Conclusion was made through comprehensive analysis: better separation could be achieved in longer capillary, smaller inside diameter of capillary and less field strength. In practical application, effective separation in short time is important. We preferred to employ 8 g/L HPMC in coated capillary (37 cm x 75 microns i.d.) and electric field strength of 324 V/cm in separating DNA fragments of hundreds of base pairs. From sampling to getting results only a few more than ten minutes were needed in capillary electrophoresis. It needs less amount of sample (a few nanoliters) and shows higher sensitivity (0.1 pmol in UV detection) than polyacrylamide gel electrophoresis (PAGE). pBR322/Msp I fragments were completely resolved in both capillary electrophoresis and PAGE, and four-bases resolution was obtained. When the concentration of the PCR product is too low (< 20 mg/L), PCR reaction system is used as a negative control to make sure of the peak generated by the unpurified PCR products, avoiding interference made by PCR buffer and polymerase.

Apolipoprotein B-100↗

PubMed1990

The structure and hydration of the A-DNA fragment d(GGGTACCC) at room temperature and low temperature.

The DNA fragment d(GGGTACCC) was crystallized as an A-DNA duplex in the hexagonal space group P6(1). The structure was analyzed at room temperature and low temperature (100K) at a resolution of 2.5 A. The helical conformations at the two temperatures are similar but the low-temperature structure is more economically hydrated than the room-temperature one. The structure of d(GGGTACCC) is compared to those of d(GGGTGCCC) and d(GGGCGCCC). This series of molecules, which consists of a mismatched duplex and its two Watson-Crick analogues, exhibits three conformational variants of the A-form of DNA, which are correlated with the specific intermolecular interactions observed in the various crystals. The largest differences in local conformation are displayed by the stacking geometries of the central pyrimidine-purine and the flanking purine-pyrimidine sites in each of the three duplexes. Stacking energy calculations performed on the crystal structures show that the mismatched duplex is destabilized with respect to each of the error-free duplexes, in accordance with helix-coil transition measurements.

Base Sequence↗