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PubMed1996

One-tube post-PCR fluorescent labeling of DNA fragments.

A method for fluorescent postlabeling of PCR products has been developed. The method uses Klenow fragment of DNA polymerase I that exchanges the 3'-terminal residue of PCR-amplified DNA fragment for fluorescent nucleotides. All reactions, including PCR, are performed in one tube simply by successive addition of reagents. The products can be applied directly to fluorescence-based automated DNA sequencers without purification for either length determination in denaturing electrophoresis or mutation detection in SSCP electrophoresis.

Automation↗
PubMed1994

Non-cloning amplification of specific DNA fragments from whole genomic DNA digests using DNA 'indexers'.

A highly systematic, non-cloning method of distinguishing and isolating every fragment in a class-IIS or interrupted palindrome restriction digest has been developed in our laboratory. These enzymes produce informative, non-identical cohesive ends which can be selectively modified by ligation to individual synthetic oligodeoxyribonucleotides with the corresponding complementary ends. In this way, polymerase chain reaction and sequencing primer sites and labels can be introduced specifically into a single fragment in a total genomic digest. Known and unknown fragments from genomes of the complexity of Escherichia coli can be isolated directly in sequencable form without the necessity of synthesizing unique primers. Human DNA has also been assessed in this way. Problems intrinsic to cloning (selective fragment loss, mutation and sequence rearrangement) are avoided. Systematic characterization of DNA fragments by their cohesive ends and length provides tremendous power and flexibility for analysis of any DNA molecule without specific clones, probes or libraries. We report proof of principle of this remarkable system and indicate potential applications in DNA sequence tagged site and restriction mapping, sequencing, restriction-fragment-length polymorphism analysis and DNA diagnostics.

Base Sequence↗
PubMed1988

A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions.

Specific, end-labeled DNA fragments can be simply and rapidly prepared using the polymerase chain reaction (PCR). Such fragments are suitable for use in DNase I protection footprint assays, chemical sequencing reactions, and for the production and analysis of paused RNA polymerase transcription complexes. Moreover, a general means of introducing a specific mutation at any position along the length of such PCR-generated fragments is described. These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specific interactions of proteins and DNA. A rapid means of removing excess oligonucleotide primers from completed PCRs is also described.

Base Sequence↗
PubMed1993

Activation-driven death of human T cell clones: time course kinetics of the induction of cell shrinkage, DNA fragmentation, and cell death.

Signaling via the CD3/T cell receptor complex induces programmed cell death (apoptosis) in IL-2-dependent human T lymphocytes. We have investigated the time course kinetics of the induction of programmed cell death in cloned CD4+ T cells. Morphological changes (cell shrinkage) were noted by flow cytometry as early as 3 hr after stimulation of clone cells with ionomycin, PHA, or anti-T cell receptor antibody BMA 031. Fragmentation of DNA became visible 3 hr after ionomycin stimulation, and 5 hr after PHA or BMA 031 stimulation, and peaked after 8 to 24 hr. Significant cell death (as revealed by flow cytometry analysis of propidium iodide-positive cells) was detected 5 hr after ionomycin treatment and 10 hr after PHA or BMA 031 treatment. With all three stimuli, maximal cell death was recorded after 16 to 18 hr. Taken together, our data indicate that the activation-induced death of mature human T cells is a rapid event which is completed within 18 hr. Induction of DNA fragmentation is preceded by the reduction of cell size which can be readily examined by flow cytometry.

Apoptosis↗
PubMed1988

[Cloning and insertion mutagenesis of DNA fragment coding for the luminescent system of Photobacterium leiognathi].

Fragments of DNA, obtained from the luminescent bacterium Photobacterium leiognathi and inserted into the plasmid pBR322, were found to code for the luminescence expressed in E. coli cells. The genetic functions necessary for light production in E. coli are localized on a DNA fragment of about 7 kbp. The insertion mutagenesis was used to define the luminescence functions encoded by the hybrid plasmid.

Cloning, Molecular↗
PubMed1994

DNA fragmentation in rat brain after intraperitoneal administration of kainate.

Cell death occurs in many neuropathological conditions. However, the mechanisms governing this process(es) remain generally unknown. In this report we studied whether excitotoxic neuronal death evoked by kainic acid (KA) in rat brain is associated with ladder-like DNA fragmentation. DNA was isolated from hippocampi, entorhinal and sensory cortices at various times following intraperitoneal KA (10 mg kg-1) injections. Typical oligonucleosome-sized DNA fragmentation was observed in all three structures at 18 h and 72 h following KA administration. These findings were further confirmed by in situ nick-translation. DNA fragmentation is believed to be diagnostic for apoptosis. The clear ladders of DNA fragmentation appeared after 18 h, although slight degradation was observed as early as 12 h after KA administration.

Animals↗
PubMed1982

DNA fragmentation and NAD depletion. Their relation to the turnover of endogenous mono(ADP-ribosyl) and poly(ADP-ribosyl) proteins.

Treatment of Ehrlich ascites tumor cells with the trifunctional alkylating agent 2,3-5-tris(ethyleneimino)benzoquinone-1,4 (triaziquonum) led to rapid fragmentation of DNA and depletion of NAD while poly(ADP-ribose) synthetase activity showed a retarded increase. Poly(ADP-ribosyl) residues in treated cells increased 4- to 30-fold, but transiently, and in a dose-dependent manner, exhibiting the same initial kinetics as the loss of NAD and the appearance of DNA strand breaks when determined by the nucleoid method. Although the amounts of "activated ADP-ribosyl" groups present in the substrate NAD (80 nmol/10(8) cells) exceeded by far basal and triaziquonum-induced poly(ADP-ribosyl) groups (up to 250 pmol/10(8) cells), accelerated formation of the polymer, nevertheless, may explain at least partially the loss of NAD seen under these conditions. Addition of benzamide, a potent inhibitor of poly(ADP-ribose) synthetase, to triaziquonum-treated cells effected an immediate drop of poly(ADP-ribose) to basal values. The data indicate a biphasic decay, the half-life of greater than 85% of the polymeric ADP-ribosyl groups exhibiting a t1/2 less than 1 min under these conditions, while the residual fraction died away with t1/2 approximately 6 min. Treatment with the DNA fragmenting agent also led to a 9-fold increase of nuclear mono(ADP-ribosyl) groups, while cytoplasmic mono(ADP-ribosyl) protein conjugates were not significantly affected. The apparent half-life of nuclear mono (ADP-ribosyl) protein conjugates (8-10 min) at peak elevation was definitely longer than that of poly(ADP-ribosyl) residues. This result is consistent with the interpretation that accumulation of mono(ADP-ribosyl) groups is due to a retarded removal of the primary ADP-ribosyl group from the acceptor protein by a separate mono(ADP-ribosyl) protein glycohydrolase, being the rate-limiting step in the overall turnover of poly(ADP-ribosyl) residues.

ADP Ribose Transferases↗
PubMed2004

Preparation and characterization of an anti-DNA monoclonal antibody showing size selectivity toward DNA fragments.

Anti-DNA monoclonal antibodies were prepared using an in vitro immunization method. Balb/c mouse splenocytes were immunized with HeLa cell nuclear extract in the presence of N-acetylmuramyl-L-alanyl-D-isoglutamine and fused with P3U1 myeloma cells using PEG 4000. After HAT selection and ELISA using fragmented HeLa genomic DNA, an anti-DNA monoclonal antibody was obtained. The monoclonal antibody D-1-1, whose isotype was IgM, interacted with a variety of double-stranded DNA. The antibody reacted only with DNA fragments longer than 0.8 kbp, and its apparent dissociation constant for a 1.0-kbp DNA fragment was 34 nM. This antibody will be a helpful tool for the detection of DNA structures.

Antibodies, Monoclonal↗
PubMed1989

Purified perforin induces target cell lysis but not DNA fragmentation.

Rapid and extensive target cell DNA fragmentation is a unique characteristic of CTL-mediated killing. We studied the role of the granule pore-forming protein (PFP/perforin/cytolysin) of CTL in mediating lysis and DNA fragmentation of target cells. Perforin was isolated from murine CTL by sequential application of perforin-enriched granule fractions to four chromatographic columns: DEAE-Sepharose, Q-Sepharose, Polyanion SI, and Superose 12. Purified perforin was eluted as a single band of 70 kD in SDS-PAGE. While purified perforin produced potent lysis of a variety of target cells tested, it did not induce any measurable amount of DNA fragmentation. In parallel experiments, intact CTL produced marked DNA fragmentation of the same target cell populations. Our results suggest that perforin alone is not responsible for the DNA fragmentation observed during CTL-mediated killing and that other, as yet unknown, mediators or mechanisms are likely to be involved in the induction of target cell nuclear damage.

Animals↗
PubMed1975

Enzymatic multiplication of a chemically synthesized DNA fragment.

A synthetic DNA fragment of 19 residues was enlarged by the enzymatic addition of deoxyadenylate residues to its 3'-end with calf thymus terminal deoxynucleotidyl transferase. The 3'-terminus of this elongated DNA strand was blocked with 2', 3'-dideoxyadenylate to prevent hydrolysis by the 3'-exonuclease function of E. coli DNA polymerase I. This elongated and 3'-blocked fragment was annealed to an oligomeric primer and used as a template for the synthesis of a complementary copy of the synthetic 19-mer. The product of such a repair synthesis was separated by gel filtration and analyzed by nearest neighbor techniques. All template strands were copied with complete repair in over 90% of the chains. Facile recovery of the elongated template by virtue of its size permitted repetition of the copy process, thus allowing accumulation of the desired strand.

Animals↗
PubMed1995

Differentiation of Campylobacter jejuni and Campylobacter coli strains by using restriction endonuclease DNA profiles and DNA fragment polymorphisms.

The chromosomal DNA fragment patterns from a total of 169 Campylobacter jejuni and Campylobacter coli isolates from poultry and humans were analyzed by using DNA restriction endonucleases ClaI and EcoRV. The DNA restriction patterns produced by ClaI and EcoRV consisted of unique DNA fragments of 9 to 9.5 kb and 3.5 kb generated with ClaI and a single unique fragment of 3.0 kb produced by EcoRV. These patterns were obtained with all strains of C. jejuni tested. The DNA restriction patterns were further examined by Southern blot analysis with a previously constructed DNA probe, pMO2005, which is also able to distinguish between C. jejuni and C. coli spp. (5). Two types of patterns were produced by hybridization with the ClaI-cleaved DNA of C. jejuni strains, one of a single 18.5-kb genomic fragment and the other of 14.5- and 4.0-kb fragments. This indicated the presence of an extra ClaI site in this genomic fragment in the strains with the duplex pattern. The Southern blot analysis of 169 C. jejuni and C. coli isolates from poultry and from humans with DNA probe pMO2005 demonstrated that 78% of C. jejuni strains isolated from chickens hybridized with DNA probe pMO2005 with a characteristic 14.5- and 4.0-kb banding pattern and 22% hybridized with a single 18.5-kb fragment, whereas 71% of human isolates hybridized with the single 18.5-kb fragment and only 29% hybridized with 14.5- and 4.0-kb fragments. These findings suggest that only a small proportion of C. jejuni strains that colonize chickens may cause disease in humans.

Animals↗
PubMed1994

A new method for quantitative estimation of the degree of DNA fragmentation utilizing agarose gel electrophoresis.

We designed a new method for quantitative analysis of the degree of DNA fragmentation, a characteristic feature of apoptosis. A photograph of an agarose gel electrophoresis of fragmented DNA was incorporated by an optical density scanner or equivalent equipment, and the integrations of middle molecular size area (10,000 to 300 bp), single nucleosomal size area (smaller than 300 bp) and total lane area were calculated. We defined fragmentation rate or (%)FR as the amount of fragmented DNA expressed as the percentage of the total amount of DNA, assigning the coefficients of 1 and 0.5, respectively, to the fragments completely digested into single nucleosomal length and to those partially digested (between 10 k to 300 bp). A standard calibration curve was constructed from triplicate experiments of target nuclei digestion by micrococcal nuclease, which revealed that this method covered a wide range of nuclease activity. We also confirmed that our method was applicable to an autodigestion assay of isolated nuclei which represented endogenous endonuclease activities. This method may be a useful tool for quantitative analysis of endonuclease activities capable of producing nucleosomal-size DNA fragmentation.

Apoptosis↗
PubMed2006

Region-specific interrelations between apoptotic proteins expression and DNA fragmentation in the neonatal rat brain.

DNA fragmentation, mRNA and protein levels of Bcl-XL, Bax and caspase-3 were determined to characterize interrelations between expression of these apoptotic markers in the neonatal brain regions. High DNA fragmentation intensity in the cortex was in consonance with the lowest Bcl-XL/Bax expression ratio, the highest procaspase-3 and active caspase-3 levels. Low and intermediate DNA fragmentation levels in the cerebellum and hippocampus respectively were also in a good agreement with apoptotic proteins expression in these structures. In the cortex, hippocampus and cerebellum DNA fragmentation intensity was proportional to the active caspase-3 level. In contrast to these structures, in the brainstem, the lowest level of this protease was accompanied by the highest intensity of DNA fragmentation among the brain regions studied. The data suggest that cell death normally occurring during early postnatal life could be realized in the developing brainstem via caspase-3-independent pathways in animals that express this protease.

Animals↗
PubMed2001

Improvement of separation method of fragmented DNA from an apoptotic cell DNA sample for the quantitation using agarose gel electrophoresis.

In order to quantify fragmented DNA extracted from apoptotic cells, we devised a separation method which condenses fragmented DNA into a small band, separating it from larger-size DNA with agarose gel electrophoresis. Calf thymus DNA and standard fragmented DNA were loaded onto 1.0% gel for 0.5, 1.0, 1.5 and 2.0 cm length, and onto 0.7, 1.0, 1.5 and 2.0% of gels for 1 cm length. DNA was then extracted from gel slices with the UltraClean 15 DNA Purification Kit, and estimated by measuring fluorescence intensity using Hoechst No.33258 dye. DNA recovery from the gel showed constant values regardless of the amount of loaded DNA up to 1 microg/assay, and a plot of loaded DNA amounts vs. the DNA amount yielded resulted in a strait line in any gel concentration used. Our results show the best conditions to estimate DNA fragmentation rates in apoptotic cells in which fragmented DNA was separated from thymus DNA by loading on 1.0% gel for 1.0 cm length. We used our method to estimate fragmentation rates in DNA fractions extracted from apoptotic human cervical fibroblast, amnion epithelial and chorion laeve trophoblast cells by stimulation with actinomycin D. The results show that DNA fragmentation rates in these cells were consistent with the electrophoretic patterns of the DNA samples shown by their photographs.

Apoptosis↗
PubMed2000

DNA fragmentation in central nervous system vascular malformations.

Recent studies have shown that apoptosis plays an important role in vascular remodeling. We examined central nervous system vascular malformations for the presence of DNA fragmentation which is the evidence of apoptosis. We hypothesize that vascular remodeling through apoptosis may be responsible for recurrence or hemorrhage in these lesions. We examined the specimens of central nervous system vascular malformations by in situ end labeling (ISEL) of fragmented DNA. Moreover, we examined the expression of Caspase-3 which is apoptosis-related proteins in these lesions by immunohistochemistry. DNA fragmentation was observed in all 15 arteriovenous malformation (AVM) specimens. ISEL-positive cells were mainly distributed in the endothelium, media and perivascular tissue. In cavernous hemangioma (CH), DNA fragmentation was also observed in all 5 specimens. ISEL-positive cells were distributed in the endothelium, subendothelium and intercavernous matrix. Thirteen out of 15 AVM lesions stained positive for Caspase-3. Caspase-3 immunoreactivity was mainly distributed in the endothelium, media and perivascular tissue. This distribution was similar to that of ISEL positive cells. As for CHs, all 5 lesions stained positive for Caspase-3. Caspase-3 immunoreactivity was distributed in the endothelium, subendothelium and intercavernous matrix. Our findings indicate that apoptotic cell death and vascular remodeling play a role in the development and maintenance of vascular malformations.

Adolescent↗
PubMed1986

Differences in target cell DNA fragmentation induced by mouse cytotoxic T lymphocytes and natural killer cells.

Fragmentation of YAC-1 target cell DNA during cytolysis mediated by mouse natural killer (NK) cells and cytotoxic T lymphocytes (CTL) was compared. Cleavage of nuclear chromatin was always an extensive and early event in CTL-mediated cytolysis, whereas with NK cell-mediated killing the degree of DNA fragmentation showed an unexpected relationship to the effector:target (E:T) ratio. At low NK:YAC-1 ratios, DNA fragmentation and 51Cr release were equivalent and increased proportionately until a ratio of about 50:1 was reached; at higher ratios, 51Cr release increased as expected but DNA fragmentation decreased dramatically. Comparison of time course data at E:T ratios producing similar rates of 51Cr release showed that the target cell DNA fragmentation observed in NK killing was not nearly as rapid nor as extensive as that observed with CTL effectors. These results suggest that NK cells induce target cell injury via two different mechanisms. One mechanism would involve lysis mediated by cell-to-cell contact, while the other may induce DNA fragmentation via a soluble mediator. In support of this notion, cell-free culture supernatants containing NK cytotoxic factor (NKCF) induced DNA fragmentation in YAC-1 cells. The DNA fragments induced by NK cells and NKCF-containing supernatants consisted of oligonucleosomes indistinguishable from those induced by CTL. The results presented here show distinct differences in target cell DNA fragmentation induced by CTL and NK cells, and suggest that these two effectors use different mechanisms to achieve the same end. CTL seem to induce DNA fragmentation in their targets by direct signaling, whereas NK cells may do so by means of a soluble factor.

Animals↗
PubMed2006

Reconstructing DNA replication kinetics from small DNA fragments.

In higher organisms, DNA replicates simultaneously from many origins. Recent in vitro experiments have yielded large amounts of data on the state of replication of DNA fragments. From measurements of the time dependence of the average size of replicated and nonreplicated domains, one can estimate the rate of initiation of DNA replication origins, as well as the average rate at which DNA bases are copied. One problem in making such estimates is that, in the experiments, the DNA is broken up into small fragments, whose finite size can bias downward the measured averages. Here, we present a systematic way of accounting for this bias by deriving theoretical relationships between the original domain-length distributions and fragment-domain length distributions. We also derive unbiased average-domain-length estimators that yield accurate results, even in cases where the replicated (or nonreplicated) domains are larger than the average DNA fragment. Then we apply these estimators to previously obtained experimental data to extract improved estimates of replication kinetics parameters.

Animals↗
PubMed1999

Relationship between seizure-induced transcription of the DNA damage-inducible gene GADD45, DNA fragmentation, and neuronal death in focally evoked limbic epilepsy.

We investigated the temporal and spatial profile of mRNA transcription for the growth arrest and DNA damage-inducible gene GADD45, DNA fragmentation, and neuronal death in rat brain following focally evoked limbic seizures. GADD45 mRNA was detected by in situ hybridization, whereas fragmented DNA was detected using in situ nick end-labeling by the large (Klenow) fragment of DNA polymerase I. Kainic acid (0.1 microg) was injected into the right amygdala of rats to induce seizures for 45 min, after which diazepam (30 mg/kg) was administered. GADD45 mRNA, DNA fragmentation, and cell death were quantified bilaterally within six limbic brain regions 0-96 h following seizure cessation. All animals underwent seizures of equivalent severity and duration as determined electrographically. In situ hybridization detected bilateral up-regulation of GADD45 mRNA throughout the CA1, CA3, and dentate gyrus of the hippocampus, the piriform and retrosplenial cortices, and the thalamus within 1 h of seizure termination. GADD45 mRNA levels remained elevated for up to 6 h, declining to baseline within all structures by 16 h. Klenow-positive cells were only found within the CA3 pyramidal layer of the ipsilateral hippocampus and appeared 16-72 h following seizure cessation. Morphologic cell death was also restricted to the CA3 subfield. These data demonstrate that focally evoked limbic seizures trigger early bihemispheric GADD45 mRNA transcription within connected limbic structures, whereas subsequent DNA fragmentation and cell death are restricted to selectively vulnerable brain regions.

Amygdala↗