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PubMed2001

Long-range intermolecular interaction between broken DNA fragments.

We analyzed the long-range intermolecular interaction between fragments of broken DNA. We considered two constituents of long-range intermolecular interaction. The first is a net electrostatic Coulomb interaction between charges, involved in a structure of opposite nucleotides, which we evaluate using Debye-Huckel theory. The second one is the Van der Waals interaction between the nucleotides. The general Lifshitz theory of Van der Waals forces was used to evaluate this interaction. Numerical calculations showed that a repulsive force between broken DNA fragments can arise in specific cases. This repulsion can prevent DNA from repairing itself after a double-strand break. The height of the barrier decreases with an increase of the ionic strength of the intracellular milieu, or with a reduction of its viscosity.

Base Pairing↗
PubMedDate not supplied

Separate pools of endonuclease activity are responsible for internucleosomal and high molecular mass DNA fragmentation during apoptosis.

The oligonucleosomal pattern of DNA fragmentation is the best-characterized biochemical marker of apoptosis and believed to be generated by a, as yet unidentified, Ca2+, Mg(2+)-dependent endonuclease. All apoptotic cells fragment their genome. However, not every cell type undergoing apoptosis is capable of internucleosomal DNA cleavage. We have analyzed the endonuclease activities and patterns of DNA fragmentation in four established cell lines undergoing apoptosis following serum deprivation, i.e., rat 5123tc hepatoma and PC12 pheochromocytoma, as well as human MCF7 breast and DU145 prostatic carcinoma cells. Whereas apoptotic 5123tc and PC12 cells degraded their DNA into oligonucleosomes, the MCF7 and DU145 cells generated only > 50 kilobase pairs (kbp) DNA fragments. However, when isolated nuclei from all four cell lines were incubated with both Ca2+ and Mg2+ ions, their DNA was cleaved into internucleosomal fragments. Following washing with a low ionic strength buffer, the nuclei could only degrade DNA to > 50-kbp fragments. DNA ladders were produced again in these washed nuclei after reconstitution with the nuclear wash, which contained an endonucleolytic activity of approximately 97 kilodaltons. These experiments showed that cells maintain separate pools of endonucleolytic activities responsible for the high and low molecular mass DNA fragmentation, and depending on the cell type, one or both enzymatic pools become activated during apoptosis.

Animals↗
PubMed1983

Use of randomly cloned DNA fragments for identification of Bacteroides thetaiotaomicron.

Randomly cloned fragments of DNA from Bacteroides thetaiotaomicron were used as hybridization probes for differentiation of B. thetaiotaomicron from closely related Bacteroides species. HindIII digestion fragments of DNA from B. thetaiotaomicron (type strain) were inserted into plasmid pBR322 and labeled with [alpha-32P]dCTP by nick translation. These labeled plasmids were screened for hybridization to HindIII digests of chromosomal DNA from type strains of the following human colonic Bacteroides species: B. thetaiotaomicron, Bacteroides ovatus, reference strain 3452-A (formerly part of B. distasonis), Bacteroides uniformis, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis, Bacteroides eggerthii, and reference strain B5-21 (formerly B. fragilis subsp. a). Two of the five cloned fragments hybridized only to DNA from B. thetaiotaomicron. Each of these two fragments hybridized to the same DNA restriction fragment in five strains of B. thetaiotaomicron other than the strain from which the DNA was cloned. One of the cloned fragments (pBT2) was further tested for specificity by determining its ability to hybridize to DNA from 65 additional strains of colonic Bacteroides.

Bacteroides↗
PubMed1995

Antigen-induced death of alloreactive human T-lymphocytes occurs in the absence of low molecular weight DNA fragmentation.

Stimulation via the CD3/TCR molecular complex induces proliferation of resting T cells, but triggers programmed cell death (apoptosis) in immature thymocytes and preactivated mature T cells. Activation-induced cell death (AICD) triggered by anti-CD3/TCR mAb or by staphylococcus enterotoxin superantigen is associated with fragmentation of genomic DNA into oligonucleosomal fragments of 200 bp length, thus displaying the characteristic features of apoptosis. Here, we show that a fraction (20-50%) of cells in alloreactive CD8 human short-term T cell lines, generated by repeated restimulation with EBV-transformed B cell lines, undergo AICD when restimulated with the appropriate (but not with third party) stimulator cells. AICD of responder T cells is inhibited when stimulator cells are preincubated with anti-HLA class I mAb but not with anti-HLA class II mAb, indicating that T cell death is dependent on alloantigen (HLA class I) recognition by responding CD8 T cells. Importantly, alloantigen-induced T cell death occurs in the absence of detectable DNA fragmentation. Thus, several independent assay systems all failed to reveal low molecular weight DNA fragmentation, even though DNA fragmentation was readily detected in T cell lines exposed to PHA or gamma-irradiation. Alloantigen-induced T cell death was prevented by aurintricarboxylic acid, which has previously been shown to inhibit apoptosis in experimental systems where no DNA fragmentation occurs. Taken together, these results demonstrate that alloantigen can trigger AICD in mature responding T cells in the absence of low molecular weight DNA fragmentation.

Cell Death↗
PubMed1992

Reactive oxygen species modified DNA fragments of varying size are the preferred antigen for human anti-DNA autoantibodies.

In vitro studies were carried out to determine if reactive oxygen species modified DNA molecules are the preferred antigen for anti-DNA antibodies found in SLE sera. Reactive oxygen species were generated by 254 nm irradiation of hydrogen peroxide. Single stranded breaks, decrease in Tm and modification of adenine (21.7%) and thymine (48%) were the major effects observed on native DNA fragments of 300 bp in length. The ROS-modified DNA showed increased binding with naturally occurring anti-DNA autoantibodies as compared to unmodified DNA fragments. These results were substantiated by competition ELISA. Measurement of binding with DNA fragments of varying size revealed considerably increased binding as the fragment size increased from 50 bp to 800 bp. The relative affinity of anti-DNA IgG for ROS-modified and native DNA fragments of 300 bp were in the order of 6.26 x 10(-8) M and 4.07 x 10(-8) M, respectively.

Animals↗
PubMed2006

Origin and biological significance of DNA fragmentation in human spermatozoa.

The occurrence of DNA fragmentation in mammalian spermatozoa was identified in 1993. In human, sperm DNA fragmentation is particularly relevant in subfertile patients (i.e, those subjects more likely to be treated by assisted reproductive techniques). Thus, concerns have been raised about the possibility that sperm with DNA fragmentation may be involved in the process of fertilization, in particular when invasive techniques (such as intracytoplasmatic sperm injection) are applied. Knowledge of the mechanisms responsible for generation of DNA strand breaks may thus help in disclosing and possibly identifying new therapies for the treatment of male infertility. However, the mechanisms involved in generating sperm DNA anomalies are far from being clarified. In this review, we summarize and critically analyze the main current theories that explain generation of DNA fragmentation in spermatozoa: abortive apoptosis (anomalies in apoptosis that occur normally during spermatogenesis), problems in packaging of chromatin (mainly anomalies in histone to protamine substitution) and generation of reactive oxygen species (that may occur at any level during spermatogenesis, sperm maturation and transit in the male genital tract).

Animals↗
PubMed1991

Isolation of DNA fragments from a human chromosomal subregion by Alu PCR differential hybridization.

The recent advent of Alu element-mediated PCR (Alu PCR) allows the rapid isolation of human-specific fragments from mixed DNA sources. This technique greatly facilitates the isolation of DNA fragments from specific regions of the human genome. We report a novel technique utilizing Alu PCR products as differential hybridization probes to isolate human DNA fragments from a chromosomal subregion. We used the Alu PCR products from a pair of somatic cell hybrids in which the human DNA content differs only in the 5q11.2-q13.3 region as differential hybridization probes. One hybrid (GM10114) retains an intact chromosome 5, while the other (HHW1064) contains a chromosome 5 deleted for the q11.2-q13.3 region. Phage from a flow-sorted chromosome 5 library were hybridized with the Alu PCR synthesis product from the chromosome 5 hybrid. Positively hybridizing phage were then screened with the Alu PCR product from the deletion 5 hybrid. Phage that hybridized to the Alu PCR product of the chromosome 5 hybrid but did not hybridize to the Alu PCR product of the deletion 5 hybrid were further characterized. We isolated five phage from 5q11.2-q13.3 using this differential hybridization procedure. Only one of these phage corresponded to a detectable difference between the ethidium bromide-stained Alu PCR products of the two somatic cell hybrids. This technique should be applicable to any somatic cell hybrid-deletion hybrid pair.

Animals↗
PubMed1997

[Flow cytometry measurement of DNA fragmentation in the course of cell death via apoptosis. New techniques for evaluation of DNA status for the pathologist].

Cell death by apoptosis is characterized by DNA fragmentation in 200-250 and/or 30-50 kb followed or not by internucleosomal DNA fragmentation in 180-200 pb. Such characteristics have been used to distinguish between necrotic and apoptotic cells, and also to identify and quantify apoptotic cells by flow cytometry. In the case of internucleosomal DNA fragmentation, the analysis of DNA content constitutes the easiest method to identify apoptotic cells giving an hypoploid cell population commonly called "Sub G1". The identification of the "Sub G1" does not depend on the dyes used; however according to the method of cell fixation and permeabilization, of the divalent cations (Ca2+, Mg2+) present in the staining buffers and of the use of trypsin, the "Sub G1" population may be more or less difficult to identify. To detect apoptotic cells whatever the pattern of DNA fragmentation, the most commonly used methods are either in situ nick-translation or TUNEL (TdT dUTP Nick End Labelling). Thus, flow cytometry offers a wide range of attractive techniques to characterize apoptotic cells but it requires the use of methodological controls for validating results.

Apoptosis↗
PubMed2002

Quantitative evaluation of DNA fragmentation.

A new method for evaluation of DNA fragmentation in tissue is based on computer-aided densitometric standardization of the sum of optical densities of DNA fragments visualized by electrophoresis (180-2500 b. p.) in comparison with the density of high molecular weight DNA in the same sample. The method allows evaluation of DNA fragmentation, an acknowledged marker of apoptosis, in the fetal rat brain and detection of increased DNA fragmentation during stress and under the effect of glucocorticoids.

Animals↗
PubMed1999

Colocalization of BAX and BCL-2 in small intestine and kidney biopsies with different degrees of DNA fragmentation.

Morphological changes associated with apoptosis are closely correlated with the expression of specific proteins. However, the cause-effect relationships between the expression of these proteins and DNA degradation are barely known. For studying expression of apoptosis-related proteins in relation to different degrees of DNA fragmentation, the small intestine with its spatially organized continuum of proliferation, differentiation and death is a very useful preparation. Enterocytes towards the apex of the villi become increasingly susceptible to apoptosis. Here, this "apoptotic gradient" is used to demonstrate the presence of BAX and BCL-2 proteins in the cytoplasm of cells at the onset of apoptosis. In semithin serial sections of the small intestine, BAX, BCL-2 and DNA fragmentation were demonstrated. BAX and BCL-2 are always colocalized and only in cells with fragmented DNA. The gradient of BAX or BCL-2 staining is similar to the gradient of DNA fragmentation. Immunoreactivity for BCL-2 or BAX is most intense in cells that are prone to become apoptotic next in the course of cellular turnover but not in cells in an advanced apoptotic state, showing strongly condensed chromatin. When using the same technique on semithin sections of kidney biopsies, containing epithelia with low cellular turnover, we found DNA fragmentation mainly in the epithelial cells of the distal tubules. Similar to the situation in the enterocytes, BAX staining was confined to the cytoplasm of epithelial cells with a moderate degree of DNA fragmentation and reduced in epithelial cells with a high degree of DNA fragmentation. In contrast to the situation in the small intestine, very low levels of BCL-2 were found. The results suggest that expression of BCL-2 and BAX is related to cell damage as indicated by DNA fragmentation but not to advanced stages of cellular death, as indicated by chromatin condensation and cellular shrinkage.

Animals↗
PubMed1977

Transcription and translation of cloned Drosophila DNA fragments in Escherichia coli.

The expression of three unique DNA fragments from Drosophila melanogaster which have been inserted into Escherichia coli (E. coli) via the plasmid, pSC 101, was studied. The hybrid plasmid DNA molecules containing Drosophila DNA were transformed into the minicell producing strain of E. coli, X1411. Drosophila DNA-directed RNA synthesis was studied by hybridizing newly synthesized RNA isolated from the minicells with various DNA fragments which were immobilized on nitrocellulose filters. RNA was synthesized as readily from the inserted Drosophila DNA as from the original bacterial plasmid, pSC 101. In one case, transcription appeared to be initiated preferentially on one of the two strands of a Drosophila DNA fragment regardless of the orientation of that Drosophila DNA fragment with respect to the pSC 101 sequences. Two of the three Drosophila DNA fragments did not induce the synthesis of new polypeptides in minicells as detected by autoradiography of [35S]methionine-labeled polypeptides on polyacrylamide gels. The third Drosophila DNA fragment caused the synthesis of one additional polypeptide of 29 000 daltons. When an 8200 base pair portion of the third inserted Drosophila DNA (63% OF THE TOTAL Drosophila insertion) was removed by digestion with the restriction enzyme, Eco R1, this new polypeptide was no longer synthesized by minicells containing the remaining Drosophila DNA. When the 8200 base pair fragment was placed back into its parent plasmid as an inversion, the new polypeptide did not reappear. In addition, the presence of some, but not all, of the Drosophila DNA insertions affected the relative synthesis of the six polypeptides coded for by the parent plasmid, pSC 101.

Animals↗
PubMed2000

Effect of hypoxia on DNA fragmentation in different brain regions of the newborn piglet.

DNA fragmentation has been studied in different regions of the newborn piglet brain following different times of normobaric hypoxia (5% O(2), 95% N(2)). After 1 hr of hypoxia, fragmented DNA was observed in cerebellum, cortex, hippocampus, and striatum but not in hypothalamus. More fragmentation occurred in these areas of the brain when the animals were kept under hypoxia for times up to 8 hr 45 min. When the animals were submitted to hypoxia for two and a half hours, integrity of DNA was recovered respectively after 3 hr of exposure to the ambient atmosphere in hippocampus and striatum, but 4 hr of recovery were necessary for cerebellum and cortex. These results are discussed in terms of the consequences of neonatal hypoxia and apnea for newborn infants and economical impact for farm animals.

Animals↗
PubMed2006

Sperm nuclear DNA fragmentation in adolescents with varicocele.

OBJECTIVE: To verify if sperm from adolescents with varicocele have an increased rate of DNA fragmentation when compared with adolescents without varicocele. DESIGN: Controlled prospective study. SETTING: Patients in an academic research environment. PATIENT(S): Adolescent patients with a clinical diagnosed bilateral varicocele, grades II and III, and adolescent patients without a varicocele. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Rate of sperm DNA fragmentation as assessed by the Comet assay, graded as class I (no DNA fragmentation), II (little DNA fragmentation), III (meaningful DNA fragmentation), or IV (high DNA fragmentation). RESULT(S): A higher percentage of cells with no DNA fragmentation (class I) was found in the nonvaricocele group (47.62 +/- 7.69) when compared with the varicocele group (27.52 +/- 10.73). A higher percentage of sperm with class III and class IV DNA fragmentation was found in the varicocele group (20.43 +/- 8.97, and 19.57 +/- 10.68) when compared with the nonvaricocele group (11.38 +/- 5.55, and 5.71 +/- 2.35). CONCLUSION(S): Although standard semen analysis showed no difference between the groups, adolescents with varicocele have an increase in sperm nuclear DNA fragmentation. Thus, DNA fragmentation evaluation could be important in deciding treatment options for adolescent patients.

Adolescent↗
PubMed2003

Co-localized expression of FasL, Fas, Caspase-3 and apoptotic DNA fragmentation in mouse testis after oral exposure to di(2-ethylhexyl)phthalate.

Expression of apoptosis-related proteins FasL, Fas and Caspase-3, as well as DNA fragmentation were examined in mouse testis 12 h after exposure to 4-0.004 mg/g di(2-ethylhexyl)phthalate (DEHP). Immunocytochemical examination of the highest dose (4 mg/g DEHP) mouse revealed a distribution of FasL in Sertoli cell and Fas in nearby spermatocyte, and Fas and Caspase-3 in the same spermatocyte. Fas-positive spermatocytes had a DNA-fragmented nucleus detectable by terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL) method. After exposure to 4, 0.4, 0.04 or 0.004 mg/g DEHP, the maximum number of nuclei with fragmented DNA per 0.5 microm testis section was 22, 7, 5 and 3, respectively. In unexposed control the maximum number was 3. To further estimate total amount of the fragmented DNA in testis of the exposed mouse, the extracted DNA fragments were analyzed by agarose gel electrophoresis. The amount of fragments in the first three steps of the DNA ladder was estimated by a photo-densitometry. In the highest dose mouse (4 mg/g DEHP), the fragmented DNA was 2.2 times as much in the control. In lower dose mouse (0.4, 0.04 or 0.004 mg/g DEHP), it was 1.1 times as much in the control. Taken together, these observations suggest that a single oral exposure to DEHP as low as 0.04 mg/g might be effective to testicular DNA fragmentation and apoptosis.

Administration, Oral↗
PubMed2004

Expression of DNase gamma during Fas-independent apoptotic DNA fragmentation in rodent hepatocytes.

Endonuclease-induced DNA fragmentation is a hallmark of apoptosis. DNase gamma (DNase gamma) was recently identified as one of the endonucleases responsible for apoptotic DNA fragmentation. In this study, immunohistochemistry for DNase gamma was performed on paraffin sections of rodent liver in well-defined models of hepatocyte apoptosis induced by Fas antibody (Fas) or cycloheximide (CHX), and necrosis induced by lipopolysaccharide (LPS) or carbon tetrachloride (CCl4). DNase gamma immunoreactivity was compared with TdT-mediated dUTP nick-end labeling (TUNEL) reactivity. Our results showed TUNEL reactivity in both apoptotic and necrotic hepatocytes. DNase gamma immunoreactivity was not detected during LPS-induced or CCl4-induced hepatocyte necrosis. In contrast, it was evident during CHX-induced, but not Fas-induced, apoptotic DNA fragmentation. These findings suggest that DNase gamma plays an important role in Fas-independent apoptotic DNA fragmentation in hepatocytes.

Animals↗
PubMed1993

Sequence analysis of a Papaver somniferum L. mitochondrial DNA fragment promoting autonomous plasmid replication in Saccharomyces cerevisiae and Kluyveromyces lactis.

The minimal fragment of mitochondrial DNA from Papaver somniferum L. (poppy) able to promote autonomous plasmid replication in the yeast Saccharomyces cerevisiae was sequenced. Sequence analysis of the 917-bp MK4/8 DNA fragment revealed a high AT content, and the presence of two 12-bp sequences differing from the ARS core consensus of S. cerevisiae only by a T and C insertion, respectively. The mitochondrial insert contains a further six 11-bp sequences with one mismatch to the S. cerevisiae core consensus, more then 20 related sequences with two base pair exchanges, numerous direct and inverted repeats, and many copies of a sequence motif called the ARS box. The original 4.2-kb mitochondrial DNA fragment, as well as the minimal 917-bp subfragment in vector pFL1-E (a variant of YIP5, lacking an origin of replication in yeast), were then tested for their ability to replicate autonomously in another fungus, Kluyveromyces lactis.

Base Sequence↗
PubMed1997

DNA fragmentation follows delayed neuronal death in CA1 neurons exposed to transient global ischemia in the rat.

Apoptosis is an active, gene-directed process of cell death in which early fragmentation of nuclear DNA precedes morphological changes in the nucleus and, later, in the cytoplasm. In ischemia, biochemical studies have detected oligonucleosomes of apoptosis whereas sequential morphological studies show changes consistent with necrosis rather than apoptosis. To resolve this apparent discrepancy, we subjected rats to 10 minutes of transient forebrain ischemia followed by 1 to 14 days of reperfusion. Parameters evaluated in the CA1 region of the hippocampus included morphology, in situ end labeling (ISEL) of fragmented DNA, and expression of p53. Neurons were indistinguishable from controls at postischemic day 1 but displayed cytoplasmic basophilia or focal condensations at day 2; some neurons were slightly swollen and a few appeared normal. In situ end labeling was absent. At days 3 and 5, approximately 40 to 60% of CA1 neurons had shrunken eosinophilic cytoplasm and pyknotic nuclei, but only half of these were ISEL. By day 14, many of the necrotic neurons had been removed by phagocytes; those remaining retained mild ISEL. Neither p53 protein nor mRNA were identified in control or postischemic brain by in situ hybridization with riboprobes or by northern blot analysis. These results show that DNA fragmentation occurs after the development of delayed neuronal death in CA1 neurons subjected to 10 minutes of global ischemia. They suggest that mechanisms other than apoptosis may mediate the irreversible changes in the CA1 neurons in this model.

Animals↗
PubMed2006

Apoptotic DNA fragmentation factor maintains chromosome stability in a P53-independent manner.

DNA fragmentation factor (DFF)/caspase-activated DNase (CAD) is responsible for DNA fragmentation, a hallmark event during apoptosis. Although DNA fragmentation is an evolutionarily conserved process across species, its biological function is not clearly understood. In this study, we constructed cell lines expressing a mutant ICAD (inhibitor of CAD) protein that is resistant to caspase cleavage and therefore constantly binds to DFF/CAD and inhibits DNA fragmentation. We found that irradiation of these cells led to increased chromosome aberrations and aneuploidy when compared with their parental controls. The increased chromosome instability is observed irrespective of cellular P53 status, suggesting that the effect of DFF/CAD is independent of P53. Inhibition of apoptotic DNA fragmentation resulted in increased clonogenic survival of irradiated cells and a delay in removal of cells with DNA damages induced by radiation, an effect similar to that in cells with p53 mutations. Consistent with DFF/CAD's effect on clonogenic survival, tumors established from cells deficient in DNA fragmentation showed enhanced growth in nude mice. Therefore, our results suggest that DFF/CAD plays an important and P53-independent role in maintaining chromosome stability and suppressing tumor development.

Aneuploidy↗