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At least 1,063 records · Page 59Linked to original sources
PubMed1991

Purification of DNA fragments containing excision-repair patches from human cells using streptavidin-biotin.

We have developed a method for purifying DNA fragments containing excision-repair patches which involves incorporation of biotinated deoxyuridine monophosphate into repair patches followed by isolation of biotin-containing DNA fragments using streptavidin and either isopycnic density gradient centrifugation or gel electrophoresis. Normal human fibroblasts were damaged with UV radiation, rendered permeable and allowed to perform repair synthesis in the presence of ATP, dATP, dGTP, [3H]dCTP and biotinated deoxyuridine triphosphate. The DNA was purified, sonicated to a number-average molecular weight of 150 bp, then incubated with streptavidin, a protein with a high affinity for biotin and with a density of 1.3 g/ml in cesium trifluoroacetate compared to 1.6 g/ml for DNA. Isopycnic centrifugation in cesium trifluoroacetate resulted in the separation of the streptavidin-DNA complex with little or no dissociation. The streptavidin-DNA complex was also separated from free DNA by electrophoresis in 2% agarose. This method is applicable to any type DNA damage repaired by the excision repair pathways in which thymine is present in the repair patches, including damage from chemical carcinogens and ionizing radiation.

Bacterial Proteins↗
PubMed1982

Size fractionation of DNA fragments ranging from 20 to 30 000 base pairs by liquid/liquid chromatography.

A chromatographic procedure for the size fractionation of DNA fragments ranging from 20 to 30 000 base pairs within a single chromatographic run is described. The procedure was developed from a method communicated about two years ago [Müller, W. et al. (1979) Nucleic Acids Res. 7, 2483-2499] which applies the aqueous two-phase system formed by poly(ethylene glycol) and dextran using cellulose as support for the lower, dextran rich stationary phase. The parameters found to exert a marked influence on the resolution and the capacity of a column include the ratio of mobile phase to bound phase in the column (phase ratio), the temperature, and the ionic strength in the phases. Concerning the support, no better material than microgranular cellulose has yet been found. A slight preference of dA + dT-rich fragments for the mobile phase manifests itself by elution anomalies comparable in extent to the migration anomalies of the same fragments in polyacrylamide gels. The resolution observed approaches that of polyacrylamide and agarose gels, but the capacity is 200-500 times higher. The DNA fragments passed through the fractionation procedure seem to be fully susceptible to the enzymatic restriction and modification reactions.

Animals↗
PubMed1999

Nonradioactive labeling of large DNA fragments for genome walking, RFLP and northern blot analysis.

In this report, we present a simple nonradioactive labeling procedure for DNA fragments of high specific labeling density that can be used for a variety of applications. The protocol is based on the universal mono-functional platinum reagent for chemical digoxigenin (DIG) labeling of nucleic acids. The labeling protocol was optimized for large DNA templates as complete bacterial artificial chromosomes (BAC). Variations of incubation time and temperature improved the labeling density such that about 30% of the nucleotides were DIG-modified within 30 min. Furthermore, the refined procedure generates in a single-tube reaction and without prior digestion-labeled DNA fragments of 0.5-4.0 kb from a 130-kb template. Hybridization experiments were performed on Southern and northern blots and allowed the detection of single copy genes in 2.5 micrograms genomic DNA from Arabidopsis thaliana, which has a haploid genome size of 0.13 pg (ca. 120 Mb) and medium expressed transcripts from 0.8 microgram poly(A)+ RNA, respectively. The extremely high specific labeling density, the stability and the universal application of the probe generated with the platinum reagent makes this method a useful alternative to classical radioactive nuclei acids labeling techniques.

Alleles↗
PubMed2001

Dantrolene ameliorates delayed cell death and concomitant DNA fragmentation in the rat hippocampal CA1 neurons subjected to mild ischemia.

The present study investigated whether dantrolene, which inhibits the Ca(2+) release from the intracellular Ca(2+) store sites, reduced nuclear DNA fragmentation and produced neuronal protection in a model of global forebrain ischemia. Male Wistar rats were subjected to four-vessel occlusion (4VO) for 5 min and then infused continuously with dantrolene or vehicle into the cerebral ventricle for 3 days. The intact rats did not undergo any intervention. The number of viable and DNA nick-end-labeled neurons in the hippocampal CA1 were evaluated 4 days after the ischemia. The number of viable neurons in the dantrolene-treated rats was significantly higher than that in the vehicle-treated rats and lower than that in the intact animals (P<0.01 and <0.05, respectively). The number of DNA nick-end-labeled nuclei was significantly lower in dantrolene-treated rats compared with the vehicle-treated animals (P<0.0001). No nick-end labeling was observed in the intact animals. A linear correlation was found between the number of viable cells and nick-end labeled nuclei in the CA1 (r=0.91, P<0.0001). These results suggest that the postischemic intraventricular dantrolene is effective in precluding neuronal death and concomitant nuclear DNA fragmentation following transient global ischemia.

Animals↗
PubMed1988

Do mitochondrial DNA fragments promote cancer and aging?

Reactive oxygen species are important in carcinogenesis, diseases, and aging, probably through oxidative damage of DNA. Our understanding of this relationship at the molecular level is very sketchy. It has recently been found that in mitochondria oxidative DNA damage is particularly high and may not be repaired efficiently. I propose that oxidatively generated DNA fragments escape from mitochondria and become integrated into the nuclear genome. This may transform cells to a cancerous state. Time-dependent nuclear accumulation of mitochondrial DNA fragments may progressively change the nuclear information content and thereby cause aging. This proposal can be tested experimentally.

Aging↗
PubMed1990

An improved strategy for generating a family of unidirectional deletions on large DNA fragments.

A modification of the Barnes "kilo-sequencing" method is described. The procedure presented here makes it possible to obtain a series of nested deletions on large DNA fragments in only two days. It applies to double-stranded DNA, and thus can be used with plasmids as well as the M13mp series of bacteriophages. The main improvements are the use of a second restriction enzyme, which makes it possible to begin the deletions at any site on the DNA fragment, and the use of mung bean nuclease for trimming the DNA edges so that any restriction enzyme can be used. This method, using a pUC vector and sequencing on double-stranded DNA, would make it possible to read a DNA nucleotide sequence on both strands starting with only one construction.

Base Sequence↗
PubMed1996

Role of endonuclease activity and DNA fragmentation in Ca2+ ionophore A23187-mediated injury to rabbit isolated gastric mucosal cells.

In the current study, the role of endonuclease activity in calcium ionophore A23187-induced gastric mucosal cellular disruption was examined using rabbit gastric mucosal cells. Cell integrity was assessed using trypan blue dye exclusion and Alamar blue dye absorbance. Ionophore A23187 (1.6-25 microM) induced a concentration-dependent decrease in dye exclusion and cell metabolism in cells suspended in a medium containing Ca2+ (2 mM), while no such effect was observed in cells incubated in the absence of extracellular Ca2+. Cells that were pretreated with the endonuclease inhibitors aurintricarboxylic acid (ATCA; 0.2 or 0.5 mM or Zn2+; 0.01 and 0.1 mM) exhibited significant reduction in the total extent of cell injury when incubated with A23187 in the presence of Ca2+. DNA fragmentation as assessed by measurement of [3H]thymidine liberation or gel electrophoresis was increased in response to ionophore A23187 (12.5 or 25 microM) treatment. A minimal degree of fragmentation was observed when cells were suspended in a Ca(2+)-free medium or incubated in the presence of ATCA or Zn2+. Addition of ethanol (8% w/v) induced a significant increase in cell injury, which was not affected by either removal of extracellular Ca2+ or ATCA pretreatment. Furthermore, treatment with the antioxidants catalase (50 micrograms/ml) or 2',2'-dipyridyl (2 mM) reduced ionophore-induced cell injury but did not reduce the extent of DNA fragmentation. These data suggest that sustained increases in intracellular Ca2+ result in increased endonuclease activity in gastric mucosal cells, leading to extensive DNA lysis and cell damage. Ethanol-induced cell damage does not involve Ca2+ influx and therefore is not mediated by endonuclease activation. Furthermore, sustained increases in cellular Ca2+ may also mediate their effects via formation of reactive oxygen metabolites, but this mechanism of cell damage does not appear to involve DNA fragmentation.

Animals↗
PubMed1994

Induction of differentiation and growth arrest associated with nascent (nonoligosomal) DNA fragmentation and reduced c-myc expression in MCF-7 human breast tumor cells after continuous exposure to a sublethal concentration of doxorubicin.

The effects on DNA integrity of continuous (72-h) exposure of human MCF-7 breast adenocarcinoma cells to 50 nM doxorubicin (a concentration which can be maintained in the plasma by continuous infusion) were characterized by bisbenzimide spectrofluorophotometry, cell flow cytometry, agarose gel electrophoresis, and neutral elution. Spectrofluorophotometry and cell flow cytometry indicated the presence of DNA fragmentation, which was maximal at 24 h. Resolution of these fragments on agarose gels failed to demonstrate "laddered" oligosomal profiles. Neutral elution analysis at 24 h indicated that doxorubicin induced fragmentation of nascent, but not mature, double-stranded DNA. Drug-treated cells exhibited endoreduplication and significant shifts in cell cycle distribution, (i.e., increased G0/G1 and G2/M fractions and a markedly reduced S-phase fraction). These alterations occurred without inhibiting the incorporation of [3H]dThd into cellular DNA; in fact, both the rate and magnitude of [3H]dThd incorporation increased progressively. Doxorubicin also produced a sustained decline in c-myc mRNA levels that paralleled both growth arrest and induction of DNA fragmentation. Ultrastructural examination revealed morphological alterations consistent with the induction of differentiation (e.g., increased lipid content and mitochondrial density, appearance of tight junctions, and secretory ducts) and further suggested the possibility of autocatalysis (e.g., lipofuschin-containing vacuoles). A gradual decline in cell number was observed, with loss of approximately 35% of the cell population after 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗
PubMed1998

The intrinsic curvature of a 51 bp K-DNA fragment of Leishmania tarentolae: a molecular model.

DNA intrinsic structure and curvature is a subject of debate because of the importance of these attributes in processes such as DNA packaging, transcription, and gene regulation. X-ray crystallography of DNA single crystals has provided a wealth of information about the local, short range conformational features of DNA. On the other hand, gel electrophoresis analysis of DNA has not only uncovered the macroscopic curvature of DNA but it also provides most of the available data on DNA intrinsic curvature. However, gel electrophoresis can not identify features of DNA structure at the nucleotide or atomic level. In order to address the problem of DNA intrinsic curvature in an attempt to bridge the gap between X-ray crystallography and gel electrophoresis, we use the computational method of molecular dynamics (MD). In this study, we report the results of 2.0 ns MD simulations on a 51 bp fragment of the K-DNA of Leishmania tarentolae containing several A-tracts. The K-DNA double helix is very stable and remains in an intermediate state between the canonical A and B forms of the duplex. The magnitude of global curvature (75 degrees) agrees well with the experimental estimate (72 degrees) available. Analysis of local (every base triplet) and sublocal (every helix turn) curvature shows that the 51 bp K-DNA fragment has curvature features also present in the Wedge, Junction and Calladine's models of DNA intrinsic curvature. We further characterize the flexibility of individual nucleotides in the molecule and find the sugar flexibility within the A-tracts to be strongly correlated with the pattern of A-tract cleavage by the hydroxyl radical. Differential curvature and flexibility at the 5' and 3'junctions between A-tracts and general-sequence DNA are found to modulate the global curvature of the K-DNA fragment.

Animals↗
PubMed1999

Molecular characterization of a Brucella species large DNA fragment deleted in Brucella abortus strains: evidence for a locus involved in the synthesis of a polysaccharide.

A Brucella melitensis 16M DNA fragment of 17,119 bp, which contains a large region deleted in B. abortus strains and DNA flanking one side of the deletion, has been characterized. In addition to the previously identified omp31 gene, 14 hypothetical genes have been identified in the B. melitensis fragment, most of them showing homology to genes involved in the synthesis of a polysaccharide. Considering that 10 of the 15 genes are missing in B. abortus and that all the polysaccharides described in the Brucella genus (lipopolysaccharide, native hapten, and polysaccharide B) have been detected in all the species, it seems likely that the genes described here might be part of a cluster for the synthesis of a novel Brucella polysaccharide. Several polysaccharides have been identified as important virulence factors, and the discovery of a novel polysaccharide in the brucellae which is probably not synthesized in B. abortus might be interesting for a better understanding of the pathogenicity and host preference differences observed between the Brucella species. However, the possibility that the genes described in this paper no longer encode the synthesis of a polysaccharide cannot be excluded. Brucellae belong to the alpha-2 subdivision of the class Proteobacteria, which includes other microorganisms living in association with eucaryotic cells, some of them synthesizing extracellular polysaccharides involved in the interaction with the host cell. The genes described in this paper might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, and the brucellae might have lost such extracellular polysaccharide during evolution if it was not necessary for survival or for establishment of the infectious process. Nevertheless, further studies are necessary to identify the entire DNA fragment missing in B. abortus strains and to elucidate the mechanism responsible for such deletion, since only 9,948 bp of the deletion was present in the sequenced B. melitensis DNA fragment.

Amino Acid Sequence↗
PubMed1994

Efficient recovery of cloned human cytomegalovirus DNA fragments from agarose gels.

A simple and efficient method for the recovery of DNA fragments from agarose gels is described. After electrophoresis, bands of interest are cut out of the gel and agarose slices pushed through the opening of a syringe needle. The resulting gel slurry is frozen and thawed three times and then centrifuged. DNA in the supernatant is precipitated, resuspended in a small volume of buffer and, finally, desalted. A recombinant pAT153 plasmid carrying the BamHI-P fragment of the human cytomegalovirus (HCMV) genome was detected using purified viral HindIII-E DNA fragment as a probe. Restriction endonuclease analysis was used to confirm the identity of the cloned fragment. Experiments performed with the recombinant pLCR127 plasmid indicate that our freeze/thaw method, with about 80% recovery and very little DNA degradation, is more advantageous than the well known electroelution and NaI/glass methods.

Adsorption↗
PubMed1990

Inhibition of both etoposide-induced DNA fragmentation and activation of poly(ADP-ribose) synthesis by zinc ion.

Treatment of a human promyelocytic leukemia cell line (HL-60) with etoposide for 3-4 hrs produced an extensive degradation of DNA. Agarose gel electrophoresis showed DNA fragmentation in a nucleosomal ladder pattern. Simultaneous addition of zinc ion (ZnSO4, 1 mM) inhibited DNA fragmentation, although the amount of DNA strand breakage introduced initially by etoposide did not change significantly as measured by the DNA unwinding assay. Furthermore, zinc ion abrogated both the activation of poly(ADP-ribose) synthesis and the morphologic changes characteristic of apoptosis by etoposide. These results suggest that zinc ion inhibits a metabolic process somewhere between initial DNA cleavage through an interference with type II topoisomerase and delayed degradation of cellular DNA to a nucleosome-like pattern.

Cell Line↗
PubMed1997

Nitric oxide co-operates with hydrogen peroxide in inducing DNA fragmentation and cell lysis in murine lymphoma cells.

We examined whether NO and H2O2 could interact in inducing DNA fragmentation and cell death. H2O2 and the NO-releasing compounds sodium nitroprusside (SNP) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) by themselves elicited lysis of YAC-1 murine lymphoma cells in a concentration-dependent manner. Exposure of the cells to a combination of sublytic concentrations of SNP (0.78 mM) plus H2O2 (7.8 microM) or SNAP (0.18 mM) plus H2O2 (7.8 microM) resulted in cell death which is mediated, in part, through apoptosis. Evidence for this direction is provided by fluorescence microscopic evaluation of the cells, which revealed the presence of changes in nuclear morphology characteristic of apoptosis in 30-40% of lymphoma cells and by the specific pattern of internucleosomal DNA fragmentation detected by gel electrophoresis. The cytotoxic effect of SNP plus H2O2 could be effectively inhibited by either oxyhaemoglobin, which binds NO, or catalase, which eliminates H2O2. Partial protection from SNP-plus-H2O2-induced cell lysis was observed with the poly(ADP-ribose) polymerase inhibitors, nicotinamide and 3-aminobenzamide, parallelling their ability to reverse depletion of cellular NAD+ pools. These results indicate an interaction between NO and H2O2 which leads to a markedly enhanced cytotoxic activity, in part, via induction of apoptosis and suggest that poly(ADP-ribosylation) and subsequent NAD+ depletion mediate, at least in part, this cytotoxic activity.

Animals↗
PubMed1991

Rapid electroblotting of small DNA fragments from polyacrylamide gels.

With the recent advances in PCR technology, the need for a simplified analysis of small double-stranded DNA fragments (less than 1.5 kb) has increased dramatically. An easy and rapid procedure has been developed for the separation, transfer and probe analysis of small double-stranded DNA fragments from polyacrylamide gels.

DNA↗
PubMed1989

Calcium-activated DNA fragmentation in rat liver nuclei.

Incubation of isolated rat liver nuclei with ATP, NAD+, and submicromolar Ca2+ concentrations resulted in extensive DNA hydrolysis. Half-maximal activity occurred with 200 nM Ca2+, and saturation of the process was observed with 1 microM Ca2+. ATP stimulated a calmodulin-dependent nuclear Ca2+ uptake system which apparently mediated endonuclease activation. Ca2+-activated DNA fragmentation was inhibited by the inhibitor of poly(ADP-ribose) synthetase, 3-aminobenzamide, and was associated with poly(ADP-ribosyl)ation of nuclear protein. The characteristics of this endonuclease activity indicate that it may be responsible for the Ca2+-dependent fragmentation of DNA involved in programmed cell death (apoptosis) and in certain forms of chemically induced cell killing.

Adenosine Triphosphate↗
PubMed1990

Linear contour length dependence of electrical polarizability of nucleosomal DNA fragments: implications for the flexibility of DNA.

The transient electric birefringence of monodisperse oligonucleosomal DNA ranging from 145 to 990 base pairs has been studied. The orientation of fragments can be described in terms of an induced dipole moment with a small contribution of a permanent dipole. The electrical polarizability delta alpha was found to increase linearly with the DNA contour length. This unexpected dependence might result from a bent structure of DNA already considerable for very short segments. The observed delta alpha values agree with a segmental orientation of rigid subunits of length 13-18 nm as estimated in the elastic model of DNA with a kink angle of about 41 degrees.

Animals↗
PubMed2005

Influence of PMMA shielding on DNA fragmentation induced in human fibroblasts by iron and titanium ions.

In the framework of a collaborative project on the influence of the shielding on the biological effectiveness of space radiation, we studied DNA fragmentation induced by 1 GeV/nucleon iron ions and titanium ions with and without a 197-mm-thick polymethylmethacrylate (PMMA) shield in AG1522 human fibroblasts. Pulsed- and constant-field gel electrophoresis were used to analyze DNA fragmentation in the size range 1-5700 kbp. The results show that, mainly owing to a higher production of small fragments (1-23 kbp), titanium ions are more effective than iron ions at inducing DNA double-strand breaks (DSBs), their RBE being 2.4 and 1.5, respectively. The insertion of a PMMA shield decreases DNA breakage, with shielding protection factors (ratio of the unshielded/shielded cross sections for DSB production) of about 1.6 for iron ions and 2.1 for titanium ions. However, the DSB yield (no. of DSBs per unit mass per unit dose) is almost unaffected by the presence of the shield, and the relative contributions of the fragments in the different size ranges are almost the same with or without shielding. This indicates that, under our conditions, the effect of shielding is mainly to reduce the dose per unit incident fluence, leaving radiation quality practically unaffected.

Cells, Cultured↗
PubMed1997

Bcl-2 expression and DNA fragmentation in breast carcinoma, pathologic and steroid hormone receptors correlates.

B-cell leukemia/lymphoma (bcl-2) expression can override the apoptosis development in lymphoid and hormonally regulated tissue-like breast. The presence of estrogen receptor (ER), progesterone receptor (PR), and androgen receptor (AR) have revealed in breast carcinomas, but they have not been correlated to the bcl-2 protein expression and DNA fragmentation markers. We evaluated the immunohistochemical expression of bcl-2 protein and hormonal receptors (ER, PR, AR) and differentiation grade in 37 infiltrating ductal carcinomas of the breast for which frozen tissues were available for DNA extraction. The immunohistochemical reaction for bcl-2 was considered positive if more than 50% of neoplastic cells had intense cytoplasmic staining, whereas for steroid receptor evaluation Battifora's criteria were used. The DNA was extracted according to the phenol-chloroform procedure and used for bcl-2 gene rearrangement study of the major breakpoint region (Southern blot) and for membrane-based end-labeling using digoxigenin-labeled nucleotides and E. coli DNA polymerase I (Klenow fragment). The results were quantified by three different observers. Low-grade carcinomas were positive for bcl-2 protein (27/28, 96.4%) and ER (15/28, 53.6%), whereas the remaining neoplasms were negative for bcl-2 (9/9, 100.0%) and ER (8/9, 53.6%) (p < 0.001). No statistically significant differences were revealed at the bcl-2, PR and AR comparisons. The Southern blot analysis for bcl-2 major breakpoint region showed neither rearrangement nor genetic amplification (densitometric study). Only the membrane-based end-labeling of DNA fragments showed correlation with bcl-2 protein and ER expressions: all except one bcl-2-negative tumor and two bcl-2-positive tumors had positive labeling using 7 pg of DNA at dot blot analysis (p < 0.002). The bcl-2 protein expression would allow both proliferation and cell progression by blocking apoptosis in well-differentiated, ER-positive breast carcinomas. In these neoplasms, DNA fragmentation as a molecular marker of apoptosis was prevented by bcl-2 expression.

Apoptosis↗