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PubMedDate not supplied

DNA fragmentation of spermatozoa and assisted reproduction technology.

Despite the ever-increasing knowledge of the fertilization process, there is still a need for better understanding of the causes of sperm DNA fragmentation and its impact on fertilization and pregnancy. For this reason, human sperm DNA fragmentation was investigated by means of the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and the production of reactive oxygen species (ROS) in the ejaculate and in the spermatozoa themselves. These data were correlated with fertilization and pregnancy data from IVF and intracytoplasmic sperm injection (ICSI) patients. Sperm DNA fragmentation did not correlate with fertilization rate, but there was a significantly reduced pregnancy rate in IVF patients inseminated with TUNEL-positive spermatozoa. ICSI patients exhibited the same tendency. This implies that spermatozoa with damaged DNA are able to fertilize an oocyte, but at the time the paternal genome is switched on, further development stops. The determination of ROS in the ejaculate and the percentage of ROS-producing spermatozoa revealed markedly stronger correlations between sperm functions (i.e. motility) and the percentage of ROS-producing spermatozoa. The influence of seminal leukocytes, known to produce large amounts of oxidants, on sperm DNA fragmentation should not be neglected.

DNA Damage↗
PubMed1987

Temperature and salt dependence of the gel migration anomaly of curved DNA fragments.

A series of oligonucleotides of different sequences have been cloned to study DNA curvature. Several DNA fragments containing these oligonucleotides in various numbers of repeats were analyzed in 10% polyacrylamide gels. A strong gel migration anomaly was found for dA4 sequences; a comparably very small but clearly detectable anomaly was observed for dA3 (both in a repeat length of 10 base-pairs). The temperature and salt (NaCl, MgCl2) dependence of the gel migration anomaly of these DNA fragments was measured. While a similar behaviour of all sequences is observed for the addition of NaCl, the temperature and MgCl2 dependence of the anomaly varies with the oligonucleotide sequence. These data are interpreted in terms of local DNA structure changes induced by changes in the temperature and the MgCl2 concentration which affect the planarity of the curved DNA fragments.

Base Sequence↗
PubMed1997

Apoptotic DNA fragmentation is detected by a semi-quantitative ligation-mediated PCR of blunt DNA ends.

Apoptosis is a form of programmed cell death (PCD) characterized by morphological changes and stereotypical DNA degradation described as a nucleosomal ;ladder'. However, nucleosomal ladders have only been clearly demonstrated in vertebrate tissues when large numbers of cells die in synchrony. Their absence may be explained by asynchronous death under physiological conditions, or by distinct molecular mechanisms. In this study, nucleosomal ladders were revealed by a ligation-mediated polymerase chain reaction (LMPCR), that amplifies DNA fragments with blunt, 5' phosphorylated ends. Numerous tissues from different organisms were examined which demonstrated that nucleosomal ladders (a) accompany physiological cell death in mammalian tissues where previously DNA fragmentation has not been detected; (b) are produced during invertebrate cell death; (c) are invariably generated via the production of blunt, 5' phosphorylated double strand breaks. These results suggest that PCD in multicellular organisms consistently involves apoptotic mechanisms and that the endonuclease activity is evolutionarily conserved.

Journal Article↗
PubMed2000

Effect of different gravity environments on DNA fragmentation and cell death in Kalanchoe leaves.

Different gravity environments have been shown to significantly affect leaf-plantlet formation and asexual reproduction in Kalanchoë daigremontiana Ham. and Perr. In the present work, we investigated the effect of gravity at tissue and cell levels. Leaves and leaf-plantlets were cultured for different periods of time (min to 15 d) in different levels of gravity stimulation: simulated hypogravity (1 rpm clinostats; 2 x 10(-4) g), 1 g (control) and hypergravity (centrifugation; 20 and 150 g). Both simulated hypogravity and hypergravity affected cell death (apoptosis) in this species, and variations in the number of cells showing DNA fragmentation directly correlated with nitric oxide (NO) formation. Apoptosis in leaves was more common as gravity increased. Apoptotic cells were localized in the epidermis, mainly guard cells, in leaf parenchyma, and in tracheary elements undergoing terminal differentiation. Exposures to acute hypergravity (up to 60 min) showed that chloroplast DNA fragmentation occurred prior to nuclear DNA fragmentation, marginalization of chromatin, nuclear condensation, and nuclear blebbing. Addition of sodium nitroprusside (NO donor) mimicked centrifugation. NO and DNA fragmentation decreased with N(G)-monomethyl-L-arginine (NO-synthase inhibitor). The variations in NO levels, nucleoid DNA fragmentation, and cell death show how chloroplasts, cells and leaves may respond (and adapt) to gravity changes.

Apoptosis↗
PubMed2006

Differences in electrostatic potential around DNA fragments containing adenine and 8-oxo-adenine. An analysis based on regular cylindrical projection.

Changes of electrostatic potential (EP) around the DNA molecule resulting from chemical modifications of nucleotides may play a role in enzymatic recognition of damaged sites. Effects of chemical modifications of nucleotides on the structure of DNA have been characterized through large scale density functional theory computations. Quantum mechanical structural optimizations of DNA fragments with three pairs of nucleotides and accompanying counteractions were performed with a B3LYP exchange-correlation functional and 6-31G(d,p) basis sets. The "intact" DNA fragment contained adenine in the middle layer, while the "damaged" fragment had the adenine replaced with 8-oxo-adenine. The electrostatic potential around these DNA fragments was projected on a cylindrical surface around the double helix. The two-dimensional maps of EP of the intact and damaged DNA fragments were analyzed to identify these modifications of EP that result from the occurrence of 8-oxo-adenine (8oA). It was found that distortions of a phosphate group neighboring 8oA and displacements of the accompanying countercation are clearly reflected in the EP maps.

Adenine↗
PubMed1985

Lack of lymphocyte-induced DNA fragmentation in human targets during lysis represents a species-specific difference between human and murine cells.

Significant cytotoxic lymphocyte-induced DNA fragmentation does not occur in four human target cells lysed by human natural killer lymphocytes, killer lymphocytes, or cytotoxic T lymphocytes. These results contrast with the extensive DNA fragmentation that occurs in murine target cells lysed by the analogous effector lymphocytes. Thus, DNA fragmentation, or the lack thereof, represents a species-specific difference between human and murine cells responding to lysis by cytotoxic lymphocytes. The DNase activity observed in murine cells is probably internally activated rather than delivered by the cytotoxic effector cells, since human killer lymphocytes selectively caused DNA fragmentation in murine but not in human target cells lysed by antibody-dependent cellular cytotoxicity.

Animals↗
PubMed2002

Comparison of the separation of large DNA fragments in the presence and absence of electroosmotic flow at high pH.

This paper describes the analysis of large DNA fragments at pH > 10.0 by capillary electrophoresis (CE) in the presence of electroosmotic flow (EOF) using hydroxyethylcellulose (HEC) solution. HEC solution in the anodic reservoir enters the capillaries filled with high-pH buffer by EOF after sample injection. With respect to resolution, sensitivity, and speed, separation conducted under discontinuous conditions (different pH values of HEC solutions and buffer filling the capillary) is appropriate. Using HEC solution at concentrations higher than its entanglement threshold ensures a good separation of large DNA fragments in the presence of EOF at high pH. In addition to pH and HEC, the electrolyte species, dimethylamine, methylamine, and piperidine, play different roles in determining the resolution. The separation of DNA fragments ranging in size from 5 to 40 kilo base pairs was completed in 6 min using 1.5% HEC prepared in 20 mM methylamine-borate, pH 12.0, and the capillary filled with 40 mM dimethylamine-borate, pH 10.0. In comparison, this method allows faster separations of large DNA fragments compared with that conducted in the absence of EOF using dilute HEC solutions.

Cellulose↗
PubMed1996

Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones.

A flow cytometry-based, ultrasensitive fluorescence detection technique is used to size individual DNA fragments up to 167 kb in length. Application of this technology to the sizing of P1 artificial chromosomes (PACs) in both linear and supercoiled forms is described. It is demonstrated that this method is well suited to characterizing PAC/BAC clones and will be very useful for the analysis of large insert libraries. Fluorescence bursts are recorded as individual, dye stained DNA fragments pass through a low power, focused, continuous laser beam. The magnitudes of the fluorescence bursts are linearly proportional to the lengths of the DNA fragments. The histograms of the burst sizes are generated in <3 min with <1 pg of DNA. Results on linear fragments are consistent with those obtained by pulsed-field gel electrophoresis. In comparison with pulsed-field gel electrophoresis, sizing of large DNA fragments by this approach is more accurate, much faster, requires much less DNA, and is independent of the DNA conformation.

Chromosomes↗
PubMed1995

Association of high molecular weight DNA fragmentation with apoptotic or non-apoptotic cell death induced by calcium ionophore.

Calcium ionophore (A23187)-induced high molecular weight (HMW) and internucleosomal DNA fragmentation were investigated in human leukemia cell lines. An apoptosis-sensitive cell line, HL-60, showed HMW, internucleosomal DNA fragmentation and morphological changes of apoptosis by A23187. MOLT-4, which is resistant to apoptosis, exhibited only HMW DNA fragmentation and died of necrosis under the same conditions. Autodigestion experiments suggested the endonucleolytic activity to cause HMW fragmentation in the cytoplasm of both cell lines. The activity was more dependent on Mg2+ than Ca2+ in HL-60, whereas it was Ca(2+)-dependent in MOLT-4. These results suggest that HMW DNA fragmentation is not specific to apoptosis.

Apoptosis↗
PubMed1997

Role of peroxynitrite in the protein oxidation and apoptotic DNA fragmentation in vascular smooth muscle cells stimulated with bacterial lipopolysaccharide and interferon-gamma.

In the present study, we investigated the role of endogenous and exogenous peroxynitrite in the process of DNA fragmentation and protein oxidation in cultured rat aortic smooth muscle cells. Peroxynitrite induced DNA fragmentation over a 24 hr period. The effect of peroxynitrite was unaffected by pretreatment with 3-aminobenzamide, an inhibitor of the nuclear enzyme poly (ADP-ribose) synthetase (PARS). Stimulation of the smooth muscle cells with bacterial lipopolysaccharide and interferon-gamma produced nitric oxide and peroxynitrite, and resulted in a significant degree of apoptotic DNA fragmentation. The nitric oxide synthase inhibitor NG-methyl-L-arginine (3 mM), but not the PARS inhibitor 3-aminobenzamide (1 mM), reduced the DNA fragmentation. Stimulation with bacterial lipopolysaccharide and interferon-gamma also caused a marked oxidation of proteins in the smooth muscle cells, which was inhibited by NG-methyl-L-arginine, as well as by the superoxide dismutase mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin. Based on these data, we propose a role for peroxynitrite-mediated, PARS-independent pathways in the apoptotic process and in the protein oxidation in bacterial lipopolysaccharide and interferon-gamma-stimulated smooth muscle cells.

Animals↗
PubMed1999

Detection of DNA fragmentation and endonucleases in apoptosis.

DNA degradation during apoptosis is endonuclease mediated and proceeds through an ordered series of stages commencing with the production of large DNA pieces of 300 kb which are then degraded to fragments of 50 kb. The 50-kb fragments are further degraded, in some but not all cells, to smaller pieces (10-40 kb) releasing the small oligonucleosome fragments that are detected as a characteristic DNA ladder on conventional agarose gels. Methodology is presented for the detection of both DNA ladders and the initial stages of DNA fragmentation using pulsed-field gel electrophoresis. We have developed electrophoresis conditions that resolve large fragments of DNA and also retain the smaller fragments on the same gel. Methods for the detection of endonuclease activities responsible for the cleavage of DNA during apoptosis are also presented.

Animals↗
PubMed1991

Differential induction of secondary DNA fragmentation by topoisomerase II inhibitors in human tumor cell lines with amplified c-myc expression.

In order to understand the cellular events associated with cell death after the formation of topoisomerase II-DNA cleavable complexes, we compared the induction of endonucleolytic DNA fragmentation by etoposide and its more potent analog, teniposide (VM-26) in the human cell lines HT-29 and HL-60. A new filter-binding assay is described, which allows rapid quantification of nonprotein-linked DNA fragmentation involved in apoptosis. Both cell lines showed similar loss of colony formation ability following 30 min of treatment with various VM-26 concentrations even though the initial topoisomerase II-mediated DNA single-strand break frequency was higher in HL-60 cells. DNA repair studies following drug removal indicated that VM-26-induced DNA breaks reversed rapidly and completely in HT-29 cells, while in HL-60 cells, the initial lesions persisted at and above 5 microM VM-26. In both cell lines, topoisomerase II cleavage complexes, as measured by DNA-protein cross-links by alkaline elution, reversed rapidly and completely within 2-3 h. Secondary DNA fragmentation resembling chromatin endonucleolytic cleavage by apoptosis could be detected in HL-60 cells 3 h after VM-26 or etoposide treatment but not in HT-29 cells. Secondary DNA fragmentation was also induced in the human colon cancer cell lines COLO 320, which have c-myc amplification. Since HL-60 cells also have c-myc amplification and HT-29 do not, it is possible that c-myc overexpression may be involved in secondary DNA fragmentation. Finally, our results indicate heterogeneity of cell death mechanisms after exposure to topoisomerase II inhibitors among human cancer cell lines.

Adenocarcinoma↗
PubMed1995

DNA repair synthesis and DNA fragmentation in primary cultures of human and rat hepatocytes exposed to cyproterone acetate.

Cyproterone acetate (CPA), a synthetic steroid used in human therapy, has recently been shown to cause DNA damage in cultured rat hepatocytes and in rat liver. In the present study we have investigated whether CPA also induces genotoxic effects in human hepatocytes. Genotoxicity of CPA was determined by measuring the capability of the compound for inducing DNA repair synthesis and for causing the formation of DNA single-strand breaks. Autoradiography and alkaline elution were used to quantitate DNA repair and DNA fragmentation, respectively. Exposure of hepatocytes to CPA for 20 h induced DNA repair synthesis in two hepatocyte preparations derived from males and in four of the five preparations derived from females. In cultures from some donors, induction of repair was detected at 1 microM CPA, the lowest concentration tested. The maximum effect generally occurred at 10-20 microM. Only a very slight increase in the frequency of DNA single-strand breaks was found following exposure of the hepatocytes to 50 microM CPA for 20 h. For comparative purposes, the effects of CPA on DNA repair and DNA fragmentation were also determined in cultured rat hepatocytes. A strong induction of DNA repair synthesis, but only a slight enhancement in DNA fragmentation was observed in CPA-treated hepatocytes derived from female rats. These results indicate that the measurement of repair is a more sensitive indicator for the genotoxicity of CPA than the measurement of DNA fragmentation. No genotoxic effects of CPA were detectable in hepatocyte cultures derived from male rats. The present findings show that CPA is genotoxic in human hepatocytes and that the striking sex difference in the genotoxicity of CPA in rat cells is not observed with human cells.

Aged↗
PubMed1996

1,10-Phenanthroline stimulates internucleosomal DNA fragmentation in isolated rat-liver nuclei by promoting the redox activity of endogenous copper ions.

Isolated rat-liver nuclei were incubated with a series of membrane-permeable metal-ion-complexing agents and examined for DNA damage. Of the reagents tested, only 1,10-phenanthroline (OP) and neocuproine (NC) were found to induce DNA fragmentation. Agarose-gel electrophoresis of the DNA fragments generated in the presence of OP revealed internucleosomal cleavage, which is widely considered to be a hallmark for the enzymic DNA digestion that occurs during apoptosis. Ascorbate, particularly in the presence of hydrogen peroxide, increased the levels of fragmentation induced by OP. As well as undergoing fragmentation, the DNA from nuclei was also found to contain 8-hydroxydeoxyguanosine, which indicates attack (oxidation) by the hydroxyl radical. Complementary experiments in vitro involving ESR determinations of hydroxyl radical formation and measurements of DNA oxidation under biomimetic conditions demonstrated that Cu2+, but not Fe3+, forms a complex with either OP or NC (but not the other complexing agents tested) that stimulates hydroxyl radical formation and DNA damage in the presence of hydrogen peroxide and ascorbate. It is therefore proposed that OP in the nuclei incubations binds to Cu2+, which exists naturally in chromosomes, forming a complex that promotes hydroxyl-radical-dependent DNA fragmentation. These findings demonstrate the promotion of hydroxyl-radical-mediated DNA damage by endogenous Cu2+ and, perhaps more significantly, demonstrate that the internucleosomal DNA 'laddering' that is often used as an indicator of apoptosis may also result from DNA fragmentation by non-enzymic processes.

Animals↗
PubMed1991

Comparison of DNA fragment patterns between the phenolic glycolipid-Tb producers and non-producers of Mycobacterium tuberculosis.

Differences in ability to produce the specific phenolic glycolipid-Tb (PGL-Tb) antigen among Mycobacterium tuberculosis strains have been reported. One of the explanations would be the genotypic variation between the strains. In this study, we compared the DNA fragment patterns after digestion of DNA with various restriction enzymes between the PGL-Tb producing and non-producing strains of M. tuberculosis. Three clinical isolates of M. tuberculosis producing the PGL-Tb antigen detectable by thin-layer chromatography, and M. tuberculosis H37Rv and M. bovis BCG not producing the antigen were grown in Sauton medium. The chromosomal DNA was digested with the restriction endonucleases, Eco RI, Sau3A I, BamH I, Xho I, Sma I, Pst I, Hinc II, and Bst EII. Most of the restriction enzymes used gave no clear DNA bands or no DNA fragment common just to the PGL-Tb producing strains. When DNAs were digested with Bst EII, however, there was a 13 kb DNA fragment common to the PGL-Tb producing isolates of M. tuberculosis and not present in the H37Rv strain and M. bovis BCG. This study thus suggests that there might be differences in DNA fragment patterns between the PLG-Tb producing and non-producing strains of M. tuberculosis.

Antigens, Bacterial↗
PubMed1995

SARs, ARSs, and A,T-rich regions evidenced by restriction mapping on an 835-kb DNA fragment from Drosophila.

In Drosophila, sequences anchoring the DNA molecule to the scaffold (SARs) and sequences able to replicate autonomously (ARSs) had been shown to comap on an 835-kb DNA fragment (Brun et al. (1990) Mol. Cell. Biol. 10, 5455-5463). To investigate the question of whether this comapping results from the coincidental recruitment of SARs and ARSs in A,T-rich regions, A,T-rich regions of the 835-kb DNA fragment have been identified by restriction analysis with enzymes recognizing motifs made exclusively of A and T. Within the limits of sensitivity of this approach, the obtained data favor the idea of a noncoincidental recruitment: obviously a SAR and an ARS subpopulation are preferentially localized in the A,T-rich regions, but not every A,T-rich region displays a SAR activity, or an ARS activity, or both, nor are all SARs or ARSs localized in the A,T-rich regions. In addition, the data support the idea that a statistical assessment of base composition using restriction analysis might be developed into a general useful approach to genome organization.

Adenine↗
PubMed2002

Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase-1 in sensitization of fibroblasts to tumor necrosis factor-induced apoptosis.

DNA fragmentation factor (DFF) comprises DFF45 and DFF40 subunits, the former of which acts as an inhibitor of the latter (the catalytic subunit) and whose cleavage by caspase-3 results in DFF activation. Disruption of the DFF45 gene blocks the generation of 50-kb DNA fragments and confers resistance to apoptosis. We recently suggested that the early fragmentation of DNA by DFF and the consequent activation of poly(ADP-ribose) polymerase-1 (PARP-1), mitochondrial dysfunction, and activation of caspase-3 contribute to an amplification loop in the apoptotic process. To verify the existence of such a loop, we have now examined the effects of restoring DFF expression in DFF45-deficient fibroblasts. Co-transfection of mouse DFF45(-/-) fibroblasts with plasmids encoding human DFF40 and DFF45 reversed the apoptosis resistance normally observed in these cells. The DFF45(-/-) cells regained the ability to fragment their DNA into 50-kb pieces in response to TNF, which resulted in a marked activation of PARP-1 and a concomitant depletion of intracellular NAD. DFF expression also resulted in an increase both in cytochrome c release into the cytosol and in caspase-3 activation triggered by TNF. These results support the importance of DFF, PARP-1, mitochondria, and caspase-3 in an amplification phase of TNF-induced apoptosis.

Adenosine Triphosphate↗
PubMed1978

The expression of tetracycline resistance after insertion of foreign DNA fragments between the EcoRI and HindIII sites of the plasmid cloning vector pBR 322.

In vitro recombination techniques were used to construct hybrid plasmids between pBR 322 and different DNA fragments derived from pML 21 and the E. coli chromosome. Some of the resulting hybrid plasmids express the tetracycline resistance gene of pBR 322, depending on the DNA fragment which has been ligated into the HindIII-site of pBR 322. From these studies we could conclude the direction of transcription of the kanamycin resistance gene in plasmid pML 21 with respect to the SalI, SmaI, HincII, KpnI, EcoRI and HindIII restriction sites. The replacement of the small HindIII-EcoRI-fragment in pBR 322 by other DNA fragments from the E. coli chromosome and selection for tcr-phenotypes showed that this system may be very useful for screening and analysis of promotor-containing DNA fragments.

DNA, Bacterial↗