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PubMed1997

The effect of column length, applied voltage, gel type, and concentration on the capillary electrophoresis separation of DNA fragments and polymerase chain reaction products.

This work examines the effect of different parameters on migration time, resolution, and speed of analysis of DNA fragments and PCR products. These parameters include column length, applied voltage, gel type and concentration, and buffer ionic strength. Our results indicate that 1 cm capillary at an applied voltage of 185 V/cm, filled with commercial gel, was adequate for the separation of small DNA fragments in under 1 min. Resolution of large fragments is directly proportional to column length at the same field strength. Also, resolution of large fragments is higher (better) at lower field strength at constant column length. Analysis is fastest (high throughput) using a short capillary and moderate field strength (200 v/cm). CE using a single short capillary (2-7 cm) is comparable to slab gel in throughput, but more economical. The Sigma DNA buffer and hydroxyethyl cellulose liquid gel gave equivalent results in terms of resolution and reproducibility. The Sigma DNA replaceable gel gave reproducible results when used as received or diluted at 60%. In our hands hydroxyethyl cellulose gave more reproducible results than polyacrylamide gel.

Buffers↗
PubMed1990

Footprinting studies on the effect of echinomycin on the structure of a bent DNA fragment.

The interaction of echinomycin with a kinetoplast DNA fragment which contains phased runs of adenine residues has been examined by various footprinting techniques. DNAase I footprinting confirms that all drug-binding sites contain the dinucleotide CpG. However, not all such sequences are protected. Three sites, each of which is located between two adenine tracks in the sequence GCGA, are not protected from DNAase I attack. Enhanced cleavage by DNAase I, DNAase II and micrococcal nuclease is observed in regions surrounding drug-binding sites. The results suggest that echinomycin alters the conformation of the AT tracks, making them more like an average DNA structure. Echinomycin renders adenine residues in the sequence CGA hyper-reactive to diethyl pyrocarbonate.

Adenine↗
PubMed1995

Characterization of a DNA fragment carrying the raw starch-digesting alpha-amylase and salt-dependent alpha-amylase genes from Bacillus circulans F-2.

A 5.4 kb HindIII DNA fragment carrying the gene encoding raw starch-digesting alpha-amylase (RSDA), has been previously cloned from Bacillus circulans F-2 and expressed in Escherichia coli [Kim et al. (1990) Biochim. Biophys. Acta 1048, 2233-2238]. Interestingly, when the cell extract of E. coli harboring a plasmid carrying this fragment was incubated with 1 M NaCl, it exhibited about 10 times higher enzyme activity than when assayed without NaCl. Differential zymograms showed two different amylase activities: one for RSDA and the other for a salt-dependent alpha-amylase (SDA). Even though RSDA activity was detected without NaCl, SDA activity was detected only in high concentrations of NaCl. SDA activity was fully detected at above 1 M NaCl. Results from subcloning of the genes, fractionation analysis of cell extracts, and immunological assays clearly suggested that the two amylases are genetically distinct and that genes for both enzymes are closely linked on the 5.4 kb DNA fragment.

Bacillus↗
PubMed1986

DNA fragments of 300 base pairs released from metaphase chromosomes by digestion with deoxyribonuclease I.

DNase I digestion of metaphase chromosomes, that have been extensively digested with Hae III, further released chromosomal DNA and proteins; 3.3% and 10.8% of the chromosomal DNA and proteins, respectively, remained insoluble. However, digestion of chromosomes first with DNase I followed by Hae III caused most of the proteins to remain in the insoluble fraction. DNase I released DNA fragments of 300 base pairs long which were not released by Hae III digestion. These DNA fragments may be protected by protein components from further fragmentation by DNase I.

Animals↗
PubMed1993

A comparison of the DNA fragment patterns of the mouse-virulent challenge strains and clinical isolates of Bordetella pertussis.

The DNA fragment patterns of two mouse-virulent strains of Bordetella pertussis, commonly used in animal protection tests, were compared to those of clinical isolates. Strain W.18-323 and six variants were examined together with strain 353/Z and two variants. Chromosomal DNA was digested with the rare-cutting enzyme Xba I in order to produce relatively large fragments resolved by pulsed-field gel electrophoresis. The resulting DNA fragment patterns of strain 353/Z and those of its variants were indistinguishable from those of the commonest DNA type among clinical isolates. In contrast, strain W.18-323 and its variants showed a distinct genetic type, different from clinical isolates of B. pertussis, B. parapertussis or B. bronchiseptica. This finding, together with some phenotypic anomalies of this strain, raises doubts as to the suitability of W.18-323 as a challenge strain for assessing the efficacy of vaccines in the mouse model.

Animals↗
PubMed1998

[Role of ryanodine receptors in hydrogen peroxide-induced DNA fragmentation and thymidine incorporation in cultured rat astrocytes].

In the CNS, reactive oxygen species (ROS) have been implicated in a wide range of degenerative processes including amyotrophic lateral sclerosis, ischemia-reperfusion injury, Alzheimer disease, Parkinson disease and aging. However, the exact mechanism is unknown, and there is little information on possible roles of ROS in cell injury and the process on recovery of astrocytes, the most abundant glial cells in the brain. We examined hydrogen peroxide (H2O2)-induced DNA fragmentation and thymidine incorporation into cultured astrocytes as an indicator of the process of recovery from astrocytic DNA injury. Astrocytes were isolated from cerebral cortices of 0-day-old rats and treated with 1 mM dibutyryl cyclic AMP for 4 days. H2O2 of 100 microM stimulated thymidine incorporation into astrocytes. Caffeine, ryanodine, cyclic ADP-ribose (endogenous ryanodine receptor agonist) and beta-NAD+ (precursor of cyclic ADP-ribose) suppressed partially the stimulatory effect of H2O2. Ruthenium red (ryanodine receptor antagonist) facilitated further the stimulatory effect of H2O2. The facilitated effect of ruthenium red on H2O2-induced thymidine incorporation was suppressed by caffeine, ryanodine, cyclic ADP-ribose and beta-NAD+. H2O2-induced DNA fragmentation and astrocytic death were suppressed by ruthenium red. These findings suggest that the process of recovery from astrocytic DNA injury by H2O2 may be regulated by Ca2+ efflux from ryanodine-sensitive intracellular Ca2+ stores.

Adenosine Diphosphate Ribose↗
PubMed1984

DNA of 21 clinical cytomegalovirus strains detected by hybridization to cloned DNA fragments of laboratory strain AD-169.

Nine distinct DNA probes prepared from cloned DNA fragments of the cytomegalovirus (CMV) strain AD-169, each representing a relatively small portion of the total genome, were able to detect all 21 epidemiologically distinct clinical isolates of CMV that had been passaged in tissue culture. All probes were of comparable sensitivity in the detection of CMV, and under the hybridization conditions used, no probe gave an unusually high background with uninfected host cells.

Cells, Cultured↗
PubMed2000

Rapid detection of homologously integrated DNA fragments and accurate quantitation of their copy number in transgenic Aspergillus oryzae by PCR.

A simple and rapid method for the analysis of artificially introduced DNA fragments has been developed using competitive PCR and long and accurate PCR. The locus and the copy number of the DNA fragments in each Aspergillus oryzae transformant could be detected more rapidly and accurately by this method than by the conventional Southern hybridization method.

Journal Article↗
PubMed2002

Stable homogeneous gel for molecular-sieving of DNA fragments in capillary electrophoresis.

A polyacrylamide gel crosslinked with allyl-beta-cyclodextrin can be used repeatedly for several weeks for the separation of DNA fragments, since bubbles are not generated during a run. Allyl-beta-cyclodextrin can easily be synthesized in one step from allylglycidylether and beta-cyclodextrin. The plate numbers for DNA fragments, up to about 1500 bp, are high: for the separation of pBR322/HaeIII fragments they were in the range 450,000-1,600,000 m(-1). The resolution was almost independent of the concentration of the crosslinker (allyl-beta-cyclodextrin)--in sharp contrast to gels crosslinked with N,N'-methylenebisacrylamide.

Base Pairing↗
PubMed1989

A 125-base-pair CEN6 DNA fragment is sufficient for complete meiotic and mitotic centromere functions in Saccharomyces cerevisiae.

Saccharomyces cerevisiae centromeres contain a conserved region ranging from 111 to 119 base pairs (bp) in length, which is characterized by the three conserved DNA elements CDEI, CDEII, and CDEIII. We isolated a 125-bp CEN6 DNA fragment (named ML CEN6) containing only these conserved elements and assayed it completely separated from its chromosomal context on circular minichromosomes and on a large linear chromosome fragment. The results show that this 125-bp CEN6 DNA fragment is by itself sufficient for complete mitotic and meiotic centromere functions.

Base Sequence↗
PubMed1989

Identifying protein-binding sites from unaligned DNA fragments.

The ability to determine important features within DNA sequences from the sequences alone is becoming essential as large-scale sequencing projects are being undertaken. We present a method that can be applied to the problem of identifying the recognition pattern for a DNA-binding protein given only a collection of sequenced DNA fragments, each known to contain somewhere within it a binding site for that protein. Information about the position or orientation of the binding sites within those fragments is not needed. The method compares the "information content" of a large number of possible binding site alignments to arrive at a matrix representation of the binding site pattern. The specificity of the protein is represented as a matrix, rather than a consensus sequence, allowing patterns that are typical of regulatory protein-binding sites to be identified. The reliability of the method improves as the number of sequences increases, but the time required increases only linearly with the number of sequences. An example, using known cAMP receptor protein-binding sites, illustrates the method.

Algorithms↗
PubMed2000

Effect of temperature on the separation of DNA fragments by high-performance liquid chromatography and capillary electrophoresis: a comparative study.

This study investigates the effect of experimental temperature on the separation of DNA fragments, 21-587 bp, by both high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). The results show that the temperature plays an important role in the HPLC separation of DNA fragments. The optimum temperature was found to be between 40 and 50 degrees C for HPLC, while 25 degrees C was the optimum temperature for the CE separation. Also, although CE migration times became shorter, efficiency and resolution decreased with an increase in temperature from 25 to 50 degrees C, but the separation was not significantly affected. Also, the optimum HPLC temperature might be different depending on the fragment sizes to be resolved.

Chromatography, High Pressure Liquid↗
PubMed2007

Beneficial effects of dithiothreitol on relative levels of glutathione S-transferase activity and thiols in oocytes, and cell number, DNA fragmentation and allocation at the blastocyst stage in the mouse.

We analyzed the effect of in vitro aging of mouse oocytes in the presence of dithiothreitol (DTT) on relative levels of glutathione S-transferase (GST) activity and thiols in oocytes, and cell number, DNA fragmentation and cellular allocation to the inner cell mass (ICM) and trophectoderm (TE) lineage at the blastocyst stage. Ovulated oocytes from gonadotropin primed hybrid female mice of 6-8 weeks of age were aged in vitro in the presence of 0, 5, 50, or 500 microM DTT for 6 hr prior to insemination. Relative levels of GST activity and thiols in oocytes were determined by confocal laser scanning microscopy, DNA fragmentation using a single-step TUNEL method, and cell allocation to the ICM and TE lineage by blastocyst staining with propidium iodide and Hoechst 33258. Non-aged oocytes exhibited higher relative levels of GST activity and thiols when compared to oocytes aged in the presence of 0, 5, and 50 microM DTT. Day 5 blastocysts from the 5, 50, and 500 microM DTT groups exhibited higher total number of cells, number of ICM cells, and ICM/TE ratio, but lower percentage of number of nuclei with DNA fragmentation/number of ICM cells than blastocyst from the 0 microM DTT group. These data show that DTT counteracts the negative effects of a post-ovulatory aging of mouse oocytes in vitro on relative levels of GST activity and thiols in oocytes, and percentage of number of nuclei with DNA fragmentation/number of ICM cells, total number of cells, number of ICM cells and ICM/TE ratio in Day 5 blastocysts.

Animals↗
PubMed1993

Spontaneous nucleosomal DNA fragmentation in murine leukemic L1210 cells.

Spontaneous nucleosomal DNA fragmentation (SNDF), characteristic of apoptotic cell death was observed in a murine leukemic cell line grown to high density. This effect was eliminated by permitting free diffusional exchange with a large amount of complete medium. Cells seeded at low density did not show SNDF in a conditioned medium that had been used to culture cells with severe SNDF. Cells seeded at high density in the same conditioned medium showed pronounced SNDF, but addition of 10% fetal bovine serum to such a medium significantly reduced SNDF. These observations indicate that neither physical contact among cells in high density culture per se nor secretion of a death factor by the high density culture could account for the induction of SNDF. The results support a model in which the gradual depletion of a serum factor(s) leads to the activation of a cell death program that is manifested as SNDF.

Animals↗
PubMed1994

Photochemical brain injury in rats triggers DNA fragmentation, p53 and HSP72.

The aim of the study was to examine whether apoptosis, apoptosis-related protein p53 and heat-shock protein (HSP) 72 participate in the response of the brain to focal injury. Male Sprague-Dawley rats received intravenously a photosensitive dye rose bengal. Unilateral cortical thrombosis was induced by illuminating the skull of rose bengal-treated rats for 10 min with a focused beam of light. Animals were killed and brains were processed for immunohistochemical detection of DNA fragmentation, p53, and HSP72 kD. DNA fragmentation and p53 were increased only in the perifocal area in the cortex ipsilateral to the thrombotic focus, while HSP72 increased throughout the ipsilateral cortex, except in the immediate perifocal area. The results suggest that in response to focal brain injury, some cells die through an apoptotic process that might involve an accumulation of p53.

Animals↗
PubMed2001

Electrophoretic migration behavior of DNA fragments in polymer solution.

A newly developed polymer coil shrinking theory is described and compared with the existing entangled solution theory to explain electrophoretic migration behaviour of DNA in hydroxypropylmethylcellulose (HPMC) polymer solution in buffer containing 100 mM tris(hydroxymethyl)aminomethane 100 mM boric acid, 2 mM ethylenediaminetetraacetic acid at pH 8.3. The polymer coil shrinking theory gave a better model to explain the results obtained. The polymer coil shrinking concentration, Cs, was found to be 0.305% and the uniform entangled concentration, C+, 0.806%. The existence of three regions (the dilute, semidilute, and concentrated solution) at different polymer concentrations enables a better understanding of the system to guide the selection of the best conditions to separate DNA fragments. For separating large fragments (700/ 800 bp), dilute solutions (HPMC < 0.3%) should be used to achieve a short migration time (10 min). For small fragments (200/300 bp), concentrated solutions are preferred to obtain constant resolution and uniform separation. The best resolution is 0.6% HPMC due to a combined interaction of the polymer coils and the entangled structure. The possibility of DNA separation in semidilute solution is often neglected and the present results indicate that this region has a promising potential for analytical separation of DNA fragments.

Buffers↗
PubMed2000

mu-Calpain activation, DNA fragmentation, and synergistic effects of caspase and calpain inhibitors in protecting hippocampal neurons from ischemic damage.

The differentiated cells seem to share the ability to induce their own death by the activation of an internally encoded suicide program. When activated, this suicide program initiates a characteristic form of cell death called apoptosis. A central challenge in apoptosis research is understanding the mechanisms by which apoptotic cascades are initiated and affected. We tested a potential role for calpain in the programmed cell death under ischemic conditions and found that calpain is (1) activated at a time preceding morphological changes, DNA fragmentation and death, (2) that calpain is translocated to the nucleus before DNA laddering, (3) pretreatment with caspase inhibitors and/or calpain inhibitors block not only the proteolytic actions of the enzyme, but also the cell death process itself in the CA1 subfield after transient global ischemia in a synergistic manner. In conclusion, the present results contribute additional evidence that proteases may play a functional role in apoptotic cell death and extend them to include the possibility that endogenous proteases are capable of inducing the striking DNA fragmentation and chromatin condensation, which are the principle criteria currently used to define apoptotic death. Moreover, the synergistic effect of caspase and calpain inhibitors in protecting neurons form ischemic damage suggests that there is a cross-talk between caspase and calpain during apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed1993

DR-78, a novel Drosophila melanogaster genomic DNA fragment highly homologous to the DNA-binding domain of thyroid hormone-retinoic acid-vitamin D receptor subfamily.

Degenerate oligodeoxyribonucleotides were designed for both ends of the DNA-binding domain of members of the nuclear receptor superfamily. PCR amplified Drosophila melanogaster DNA was purified and cloned (DR plasmids). Genomic lambda DASH clones were identified at high stringency with an amplified DR-78 plasmid DNA and isolated. The partial sequence shows a very probable open reading frame which would encode a peptide highly homologous to members of the thyroid hormone-retinoic acid-vitamin D receptor subfamily. The fragment corresponds to a single copy gene and was mapped at position 78D of chromosome three by in situ hybridization.

Amino Acid Sequence↗