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PubMedDate not supplied

Study of large DNA fragments in agarose gels by transient electric birefringence.

The pulsed-field gel electrophoresis (PFG) is a newly developing technique used in the fractionation of large DNA fragments. Advances in PFG demand a better understanding in the corresponding mechanisms of DNA dynamics in the gel network. Detailed experiments are needed to verify and to extend existing theoretical predictions as well as to find optimum conditions for efficient separation of large DNA fragments. In the present study, deformation of large DNA fragments (40-70 kilobase pairs) imbedded in agarose gels were investigated by using the transient electric birefringence (TEB) technique under both singular polarity and bipolarity electric pulses at low applied electric field strengths (E less than or equal to 5 V/cm). The steady-state optical retardation (delta s) of DNA molecules is linearly proportional to E2. At a given E, the amplitude of optical retardation [delta(t)] increases monotonically with the pulse width (PW) and then reaches a plateau value [delta(t = 0) = delta s] where t = 0 denotes the time when the applied field is turned off or reversed. The field-free decay time (tau-a few minutes) is several orders of magnitudes slower than that from previous TEB observations using high electric field strengths (E-kV/cm) and short pulse widths (PW-ms). The degree of deformation (stretching and orientation) and the time of restoration to the equilibrium conformation of overall DNA chains have been related to delta and tau. In field inversion measurements, exponentially rising and linearly falling of birefringence signals in the presence of forward/inverse applied fields were observed. The rising and falling of birefringence signals were reproducible under a sequence of alternating pulses. Comparison of our results with literature findings and discussions with theories are presented.

DNA↗
PubMed2006

Universal interpolating function for the dispersion coefficient of DNA fragments in sieving matrices.

The separation of DNA fragments by (slab or capillary) gel electrophoresis has been studied extensively. To characterize the separation achieved by such systems, one needs to understand the impact (and their dependency upon the experimental quantities) of two physical parameters: the electrophoretic mobility mu and the diffusion coefficient D. Three different regimes have been shown to exist for both mu and D: the Ogston regime, the reptation regime and the reptation with orientation regime (note that separation is only possible for the first two regimes). In the small electric field limit, both mu and D are apparently well described by theories for all three regimes. Unfortunately this results in disjointed scaling laws and no theory-based general equations can apply to all regimes. Recently, an empirical interpolating formula has been proposed that adequately fits the low electric field mobility mu of dsDNA fragments across all three regimes and is compatible with accepted theories. In this article we review and clarify the current state of knowledge regarding the size dependence of the mobility and the diffusion coefficient and propose an interpolating formula for molecular size dependence of the low field diffusion coefficient D. With formulas for both the mobility and the diffusion coefficient as a function of the experimental conditions one could, in principle, optimize any gel/polymer matrix-based electrophoresis system for a wide range of DNA molecular sizes.

DNA↗
PubMed1980

Isolation and characterization of DNA fragments containing the dihydrofolate-reductase gene of coliphage T4.

DNA of a mutant of the bacteriophage T4, which contains cytosine instead of glucosylated hydroxymethylcytosine, was shown to direct the synthesis of enzymatically active dihydrofolate reductase in a coupled in vitro transcription-translation system. The DNA-directed synthesis of the enzyme was used to localize the dihydrofolate-reductase gene frd on a 2300 bp long restriction-nuclease-generated DNA fragment. Fine structure mapping showed that the gene is encoded on a segment of less than 1850 bp but more than 700 bp length. The enzyme, which is synthesized in vitro from the DNA fragment, has a molecular weight of 18 500 to 19 500. A restriction map was constructed which extends about 10 kb to both sides of the reductase gene and which covers the T4 genome between the genes 55 and 63. The two genes which flank the frd gene, genes 32 and td (thymidylate synthetase), were mapped in detail. A correlation between the physical and genetic maps was established.

Chromosome Mapping↗
PubMed2005

[Effect of the DNA isolation method on the results of multiple fluorescent PCR and DNA fragment analysis].

INTRODUCTION: The quality of the nucleic acids has tremendous effects on the results of molecular diagnostic tests. METHODS: The authors have isolated DNA from amniotic fluid samples by using rezin-binding and silica adsorption methods. In the case of the rezin-binding technique they used both frozen and fresh samples. The quality of the isolated DNA was compared by fluorescent PCR and DNA fragment analysis. RESULTS: With the use of the rezin-binding method on frozen samples the authors obtained more PCR fragments following the fluorescent PCR amplification. The gender and the aneuploidy screening is easier from the electrophoretogramms using better quality DNA. Out of the three studied DNA isolation method the silica adsorption seemed to be the best for the fluorescent-PCR and DNA fragment analysis. DISCUSSION: The authors found the silica adsorption method is more suitable for DNA isolation from amniotic fluids for fluorescent PCR and DNA fragment analysis amplification. They suppose silica adsorption DNA isolation results in a total removal of the PCR inhibitors.

Animals↗
PubMed1994

Stability of genomic DNA fragment patterns in methicillin resistant Staphylococcus aureus (MRSA) during the course of intra- and interhospital spread.

The analysis of genomic DNA fragment patterns has revealed as a powerful tool for strain discrimination in Staphylococcus aureus; for use as an epidemiological marker, stability during the course of an outbreak is an essential prerequisite. Genomic DNA fragment patterns (SmaI restriction, pulsed-field electrophoresis) of four different epidemic MRSA strains were compared along with intra- and interhospital and country-wide spread over more than 12 months in Germany. Strain I was isolated from infections in 8 hospitals. In one hospital a subclone arised which differed from the original strain by 4 fragments. Strain II was spread among 4 hospitals, isolates from three of these hospitals exhibited a variability of one to three fragments in the 150-200 kb range. Two hospitals in the Hannover-area were affected by strain III; in 17 isolates of this strain a variability up to three fragments was found in the 170-200 kb range. Strain IV was isolated from 19 cases of infections in 3 hospitals in Berlin. The fragment patterns were completely stable. When S. aureus strains are typed by genomic DNA fragment patterns, a variability in a definite range of molecular masses during the course of an epidemic should be taken into consideration.

Cross Infection↗
PubMed2003

Equine estrogens differentially inhibit DNA fragmentation induced by glutamate in neuronal cells by modulation of regulatory proteins involved in programmed cell death.

BACKGROUND: Recent data indicate that excitotoxicity of high levels of neurotransmitter glutamate may be mediated via programmed cell death (apoptosis) and that it can be prevented in HT22 mouse hippocampal cells by various equine estrogens with Delta8,17beta-estradiol (Delta8,17beta-E2) being the most potent. In order to delineate the mechanism(s), glutamate-induced cell death of HT22 cells was assessed by measuring (a) DNA fragmentation in the presence or absence of 11 equine estrogens (components of the drug CEE); (b) cell death and (c) levels of anti-apoptotic (Bcl-2) and proapoptotic (Bax) proteins in the presence or absence of two equine estrogens, Delta8,17beta-E2 and 17beta-estradiol (17beta-E2) by LDH release assay and Western blot analysis respectively. RESULTS: Glutamate treatment induced cell death was time and dose-dependent. After 18 to 24 h, glutamate induced DNA fragmentation and morphological characteristics of apoptotic cell death. DNA fragmentation and morphological changes induced by 10 mM glutamate were completely inhibited by some equine estrogens. Exposure of cells to various concentrations of glutamate, resulted in a significant increase in cell death associated LDH release that was time-dependent. Both Delta8,17beta-E2 and 17beta-E2 inhibited the glutamate-induced LDH release and cell death in a dose-dependent manner with Delta8,17beta-E2 being 10 times more potent than 17beta-E2. Western blot analysis indicated that glutamate also significantly decreased the levels of Bcl-2 and increased Bax levels. This glutamate-induced change in the ratio of Bcl-2 to Bax was reversed by estrogens with Delta8,17beta-E2 being more potent. CONCLUSIONS: In HT22 mouse hippocampal cells, glutamate induced apoptosis that was associated with DNA fragmentation, morphological changes and up-regulation of the pro-apoptotic protein Bax and down-regulation of the anti-apoptotic protein Bcl-2. This apoptotic process was differentially prevented by some equine estrogens with Delta8,17beta-E2 being more potent than 17beta-E2. Since HT22 cells lacked both glutamate and estrogen receptors, the neuroprotective effects of estrogens most likely involve both genomic and non-genomic mechanisms. Since Delta8-estrogens are less feminizing estrogens than 17beta-E2, further chemical modifications of these Delta8-estrogens may provide more selective estrogens that will be useful in the prevention of neurodegenerative diseases such as Alzheimer's and Parkinson's in both aging men and women.

Animals↗
PubMed2006

Involvement of endonuclease G in nucleosomal DNA fragmentation under sustained endogenous oxidative stress.

We have previously shown that inhibition of catalase and glutathione peroxidase activities by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS), respectively, in rat primary hepatocytes caused sustained endogenous oxidative stress and apoptotic cell death without caspase-3 activation. In this study, we investigated the mechanism of this apoptotic cell death in terms of nucleosomal DNA fragmentation. Treatment with ATZ+MS time-dependently increased the number of deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-positive nuclei from 12 h, resulting in clear DNA laddering at 24 h. The deoxyribonuclease (DNase) inhibitor, aurintricarboxylic acid (ATA), completely inhibited nucleosomal DNA fragmentation but the pan-caspase inhibitor, z-VAD-fmk was without effects; furthermore, the cleavage of inhibitor of caspase-activated DNase was not detected, indicating the involvement of DNase(s) other than caspase-activated DNase. Considering that endonuclease G (EndoG) reportedly acts in a caspase-independent manner, we cloned rat EndoG cDNA for the first time. Recombinant EndoG alone digested plasmid DNA and induced nucleosomal DNA fragmentation in isolated hepatocyte nuclei. Recombinant EndoG activity was inhibited by ATA but not by hydrogen peroxide, even at 10 mm. ATZ+MS stimulation elicited decreases in mitochondrial membrane potential and EndoG translocation from mitochondria to nuclei. By applying RNA interference, the mRNA levels of EndoG were almost completely suppressed and the amount of EndoG protein was decreased to approximately half the level of untreated cells. Under these conditions, decreases in TUNEL-positive nuclei were significantly suppressed. These results indicate that EndoG is responsible, at least in part, for nucleosomal DNA fragmentation under endogenous oxidative stress conditions induced by ATZ+MS.

Active Transport, Cell Nucleus↗
PubMed2001

BAPTA blocks DNA fragmentation and chromatin condensation downstream of caspase-3 and DFF activation in HT-induced apoptosis in HL-60 cells.

DFF ((DNA Fragmentation Factor) is a heterodimer composed of 40 kDa (DFF40, CAD) and 45 kDa (DFF45, ICAD) subunits. During apoptosis, activated caspase-3 cleaves DFF45 and activates DFF40, a DNase that targets nucleosomal linker region and cleaves chromatin DNA into nucleosomal fragments. We have previously reported that HT induced apoptosis in HL-60 cells, and intracellular Ca(2+) chelator BAPTA blocked apoptosis-associated DNA fragmentation induced by HT. We report here that HT also induced activation of caspase-3 and cleavage of DFF45. BAPTA prevented neither the caspase-3 activation nor the cleavage of DFF45. Mitochondrial membrane potential was disrupted in BAPTA-AM treated cells. However, BAPTA did prevent DNA fragmentation and chromatin condensation in HT-treated cells. These data suggest a novel role for intracellular calcium in regulating apoptotic nuclease that causes DNA fragmentation and chromatin condensation.

Antineoplastic Agents↗
PubMed1996

DNA fragmentation of oocytes in aged mice.

To investigate whether female fertility decreases with age due to poor oocyte quality, we examined the presence of DNA fragmentation in ovulated oocytes from young, mature and aged mice. Oocytes from three age groups of female mice (7-8, 20-24 and 40-48 weeks) were retrieved from the oviducts 15 h after human chorionic gonadotrophin (HCG) injection. Oocytes from each mouse were incubated in a CO2 incubator for 0-60 h in human tubal fluid (HTF). After incubation, each oocyte was stained with the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) method. The rate of DNA fragmentation (interpreted as apoptotic changes) was significantly higher for oocytes from aged mice, and the fertilization rate was significantly lower, compared with oocytes from young and mature mice. Our results suggest that DNA fragmentation of oocytes might be one of the reasons for poor oocyte quality and lower fertility in the aged group.

Aging↗
PubMed1999

Methods for rapid cloning and detection for sequencing of cloned inverse PCR-generated DNA fragments adjacent to known sequences in bacterial chromosomes.

Since the invention of PCR, many adaptation techniques have been developed for sequencing DNA fragments flanking known sequences. Of them, inverse PCR is a matter of interest because of the simplicity of its principle. However, the protocols for inverse PCR introduced so far consist of some time-consuming procedures, and with them, we cannot "walk" chromosomes too far since the number of suitable restriction enzymes is limited. Our experiments led to confirming simpler technical approaches applicable to the case of bacterial chromosomes, that is, designing two end-specific "contextual" sequences with which we can quickly detect the desired clones of targeted DNA fragments by simply analyzing PCR products, employing "the minimum value of the desired fragments" as a "discriminating minimum" value to decrease contaminant DNA fragments, and creating a new tandem of two cleaved end fragments of a known sequence ("reordering") for PCR amplification in combination with cloning of the inverse PCR-generated DNA. With the improvements, we could both simplify the procedures and broaden the capacity of the inverse PCR in "walking" chromosomes.

Chromosomes, Bacterial↗
PubMed1986

A method for constructing multiple tandem repeats of specific DNA fragments.

We describe a new method for constructing tandem repeats of DNA fragments that allows one to control the number of tandem copies appearing in a final recombinant DNA clone. The principle of the method is to prepare DNA fragments with predetermined cohesive ends and to ligate them together in such a way that unique multimers are generated. First, the fragment of interest is inserted into a vector with a multisite cloning region. Clones are picked with the fragment in both orientations, and the fragments are excised using different pairs of restriction enzymes. The resulting fragments with preprogrammed cohesive ends are mixed, ligated, and digested with one enzyme whose site appears in the cloning region of the vector. From this material multimeric fragments containing specific ends can be isolated and recloned into appropriate vectors. As an example of the method, we show the construction of clones containing either three or six tandem copies of the origin of DNA replication of the mouse polyoma virus. The prospect of using this technique to construct inverted repeat structures is discussed. As part of this work we have constructed a modified M13mp8 vector which contains a unique Xho I cloning site.

DNA Replication↗
PubMed1990

Calcium-dependent DNA fragmentation in human synovial cells exposed to cold shock.

Exposure of confluent human synovial McCoy's cells to near-freezing temperatures followed by rewarming at 37 degrees C resulted in endonuclease activation and cell death characteristic of a suicide process known as apoptosis. Both DNA fragmentation and cell killing were dependent on a sustained increase in the cytosolic Ca2+ concentration. Sensitivity to cold shock-induced endonuclease activation was critically dependent on the cell cycle (proliferative) status and limited to confluent cells, whereas cells in the logarithmic growth phase were completely resistant. However, DNA fragmentation was promoted in the proliferating McCoy's cells pretreated with H-7 or sphingosine, inhibitors of protein kinase C. In addition, phorbol ester, known to activate PKC, inhibited DNA fragmentation in the confluent cells. Our findings indicate that cold shock-induced DNA fragmentation in McCoy's cells is dependent on a sustained Ca2+ increase, and sensitivity to the process appears to be regulated by the status of protein kinase C.

Aminoquinolines↗
PubMedDate not supplied

Glutathione depletion-induced chromosomal DNA fragmentation associated with apoptosis and necrosis.

Chromosomal DNA and mitochondrial dysfunctions play a role on mammalian cell death induced by oxidative stress. The major biochemical dysfunction of chromosome is the presence of an ordered cleavage of the DNA backborn, which is separated and visualized as an electrophoretic pattern of fragments. Oxidative stress provides chromatin dysfunction such as single strand and double strand DNA fragmentation leading to cell death. More than 1 Mb of giant DNA, 200-800 kb or 50-300 kb high molecular weight (HMW) DNA and internucleosomal DNA fragments are produced during apoptosis or necrosis induced by oxidative stress such as glutathione (GSH) depletion in several types of mammalian cells. Reactive oxygen species (ROS)-mediated DNA fragmentation is enhanced by polyunsaturated fatty acids including arachidonic acid or their hydroperoxides, leading to necrosis. Mitochondrial dysfunction on decrease of trans membrane potential, accumulation of ROS, membrane permeability transition and release of apoptotic factors during apoptosis or necrosis has been implicated. This review refers to the correlation of chromosomal DNA fragmentation and apoptosis or necrosis induced by GSH depletion, and the possible mechanisms of oxidative stress-induced cell death.

Animals↗
PubMedDate not supplied

[Use of a nylon membrane for purifying DNA fragments from agarose gels].

A new and effective technique was proposed for rapid 30-40 minisolation of pure double-stranded DNA fragments from agarose gels and their use for nick-translation, for restriction with cutting enzymes and ligation with vector DNA. The technique developed involved an electrophoretic transferring of a DNA fragment from the gel into a strip of nylon membrane with the pore size of 0.2 mm followed by elution with buffer containing 0.1% SDS. Yield of the DNA fragment with a length up to 8.0 Kbp was at least 50%. Advantages of the technique developed as compared with standard methods are discussed.

DNA↗
PubMed1982

The purification of human DNA fragments containing benzpyrene adducts.

DNA was isolated from human diploid lung epithelial cells treated in culture with [3H]benzpyrene. The DNA contained one covalently bound benzpyrene group per 38 kb and it was digested with a series of restriction endonucleases giving decreasing fragment sizes, and also with DNAase I to 96% acid solubility. The digests were applied to octyl-Sepharose columns under conditions which promote hydrophobic interaction of the benzpyrene groups on the DNA with the octyl groups in the column matrix. Separation of fragments without and with benzpyrene groups was achieved with successive high salt and ethanediol washes. As DNA fragment size is decreased, more DNA-associated benzpyrene is eluted with ethanediol. Under these conditions, DNA from untreated cells is totally removed in the high salt wash and free benzpyrene metabolites are retained on the column. The separation of DNA fragments with covalently-bound hydrophobic benzpyrene groups, from less modified or unmodified DNA will facilitate examination of the distribution of benzpyrene adducts in defined regions of the human genome.

Animals↗
PubMed2003

Characterization of the rat A2A adenosine receptor gene: a 4.8-kb promoter-proximal DNA fragment confers selective expression in the central nervous system.

We isolated and characterized a 4.8-kb 5' flanking region of the rat A2A adenosine receptor (A2A-R) gene in the present study. Promoter activity was observed with this DNA fragment in PC12 cells and C6 cells which contain endogenous A2A-Rs. A fusion fragment consisting of the 4.8-kb promoter-proximal DNA fragment of the A2A-R gene, and the coding region of lacZ was utilized to produce mice harbouring the fusion gene. In three independent founder lines, proteins and transcripts of the transgene were found in many areas of the central nervous system (CNS), but not in three peripheral tissues examined. Double immunohistochemical analyses revealed that the transgene was coexpressed with endogenous A2A-R and proper neuronal markers in the brain. Specifically, the transgene in the striatum was found in the enkephalin-containing GABAergic neurons and in the cholinergic neurons as was found for the endogenous A2A-R. However, a selectively enriched striatal expression of the transgene was not found as was observed for the endogenous A2A-R. Collectively, the 4.8-kb promoter-proximal DNA fragment of the rat A2A-R gene contains important element(s) to direct its expression in the CNS where functional A2A-R are found, but were not sufficient to confer the highly concentrated expression of the striatal A2A-R. Furthermore, expressions of A2A-R and the transgene were found in both neurons and astrocytes, suggesting that adenosine might mediate its function through A2A-R in both cell types.

5' Flanking Region↗
PubMed1999

S-nitrosoglutathione enhances neutrophil DNA fragmentation and cell death.

Enhancing the clearance of neutrophils by enhancing apoptotic cell death and macrophage recognition may be beneficial in acute lung injury. Exogenous nitric oxide gas depresses neutrophil oxidative functions and accelerates cell death (A. H. Daher, J. D. Fortenberry, M. L. Owens, and L. A. Brown. Am. J. Respir. Cell Mol. Biol. 16: 407-412, 1997). We hypothesized that S-nitrosoglutathione (GSNO), a physiologically relevant nitric oxide donor, could also enhance neutrophil DNA fragmentation. Neutrophils were incubated for 2-24 h in the absence and presence of GSNO (dose range 0.1-5 mM) and evaluated for cell death by a fluorescent viability/cytotoxicity assay. Neutrophil DNA fragmentation was assessed by cell death detection ELISA and by terminal deoxynucleotidyltransferase-mediated fluorescence-labeled dUTP nick end labeling assay. Neutrophil oxidative function was also determined. Incubation with GSNO increased cell death at 2, 4, and 24 h. GSNO incubation for 24 h significantly increased DNA fragmentation in a dose-dependent fashion at 0.5 (median 126% of control value; P = 0.002) and 5 mM (185% of control value; P = 0.002) by terminal deoxynucleotidyltransferase-mediated fluorescence-labeled dUTP nick end labeling and at 0.5 mM by ELISA (164% of control value; P = 0.03). The apoptosis-to-total cell death ratio increased with increasing GSNO concentration (P < 0.05). Effects were mitigated by coincubation with superoxide dismutase. Five millimolar GSNO decreased overall superoxide generation and O2 consumption but not when adjusted for dead neutrophils. GSNO significantly enhances cell death and neutrophil DNA fragmentation in a dose-dependent fashion.

Adult↗