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At least 1,333 records · Page 74Linked to original sources
PubMed1991

Solid-phase synthesis of oligo(2'-deoxyxylonucleotides) and PCR amplification of base-modified DNA fragments.

1-(2'-Deoxy-beta-D-threo-pentofuranosyl)thymine (xTd) and -adenine (xAd) were converted into their appropriately protected 3'-phosphonates 1a, 2a as well as their 2-cyanoethyl phosphoramidites 1b, 2b. These compounds were used for solid-phase syntheses of the oligo(2'-deoxy-beta-D-xylonucleotides) 5-8. Structural properties and behavior against nucleases is described. Apart from oligo(2'-deoxyxylonucleotides) the PCR-amplification of a pUC18 DNA fragment with Taq polymerase was studied in the presence of the 7-deazapurine derivatives of dGTP, dATP, and dITP. The incorporation efficiency of the modified compounds was compared with those of the parent nucleotides. 7-Deaza-2'-deoxyguanosine protected the DNA-fragment from hydrolysis by the restriction endodeoxyribonuclease Eco RI, Pst I, Bam HI, and Sma I if the nucleoside was located within the recognition site.

Adenosine↗
PubMed1983

Construction of a lambda packageable ColE1 vector which permits cloning of large DNA fragments: cloning of thyA gene of Escherichia coli.

A small packageable plasmid depending on the ColE1 replicon was constructed from ColE1-gal-cos lambda:: Tn3, pKY2113 (T. Nakamura et al. (1981) J. Biochem. 90, 1013), and named pKY2662. This plasmid carries ampicillin resistance and colicin E1 immunity genes as selective markers, and has neither mobilization function nor movable transposon. The molecular size of pKY2662 is 8.7 kb, and it has a single cleavage site each for BamHI, Bg/II, ClaI, EcoRI, HpaI, PstI, and Tth111I. By using pKY2662 as a vector, a 32 kb Escherichia coli DNA fragment covering thyA, recC, recB, and argA genes was cloned. This new small cosmid is among the most efficient vectors hitherto found for in vitro cloning of large DNA fragments.

Bacteriophage lambda↗
PubMed1985

On the sequence determinants and flexibility of the kinetoplast DNA fragment with abnormal gel electrophoretic mobilities.

A 410 base-pair (bp) Sau3A restriction fragment derived from a Leishmania tarentolae kinetoplast DNA minicircle, which is known to have slower than expected electrophoretic mobilities in polyacrylamide gels, has been cloned in a plasmid and deletions from one end of the cloned segment have been constructed. Analysis of the gel electrophoretic mobility data of a large number of restriction fragments derived from the kinetoplast DNA clone and its deletion subclones has led to the conclusion that two sequences, one in the region bp 100 to 170 and the other bp 190 to 250, both numbered from one end of the 410 bp kinetoplast DNA segment, are important for the abnormal gel electrophoretic behavior of the kinetoplast DNA fragment. One common feature of these sequences is the periodic presence of short runs of A residues (3 to 6 As in each); auto-correlation analysis of these runs of A residues shows a strong harmonic component with a period around 11 bp. These results support and extend the previous analysis of Wu & Crothers (1984). The abnormal electrophoretic behavior is accentuated at low temperature and by the addition of Mg2+ to the electrophoresis buffer; addition of Na+ has the opposite effect. Insertion of sequences derived from the kinetoplast DNA fragment into nicked circular DNA causes no unexpected change in its electrophoretic mobility in agarose gel, suggesting that the 410 bp sequence, or segments of it, has no significant spatial writhe. Abnormal shifts in agarose gel mobilities are observed, however, when certain segments of the kinetoplast DNA are inserted into positively or negatively supercoiled DNA topoisomers. These results are consistent with a bent structure of the kinetoplast DNA in which the bend has zero writhe in its undistorted form but is easily distorted.

Animals↗
PubMed1988

Calcium ion concentrations and DNA fragmentation in target cell destruction by murine cloned cytotoxic T lymphocytes.

To investigate the destruction of target cells by murine CTLs, we examined intracellular Ca2+ concentrations ([Ca2+]i) and DNA fragmentation in target cells. Changes in [Ca2+]i were followed by flow cytometry by loading the cells with indo-1, a Ca2+-binding fluorescent dye, and determining the ration of fluorescence intensities at 405 nm (emission maximum for Ca2+-bound dye) over 480 nm (emission maximum for the free dye). Within minutes after interacting with the cytolytic granule fraction that had been isolated from CTLs, [Ca2+]i in target cells was strikingly increased. A pronounced increase in [Ca2+]i was also observed in target cells when they were specifically recognized by intact CTLs. Since ionomycin, a Ca2+ ionophore, caused a similar increase in [Ca2+]i and lysed cells (provided that extracellular Ca2+ was present), it appears that a sustained high level of [Ca2+]i is cytolytic. In contrast with other cells, CTLs, which have been shown to be refractory to granule-mediated lysis and to be poor targets for other CTLs, did not manifest an elevation in [Ca2+]i when they were similarly loaded with indo-1 and treated with isolated granules. The characteristic cleavage of target cell DNA into nucleosome-sized fragments was also induced by isolated granules as well as by valinomycin, a K+ ionophore, but not by ionomycin. The results support the view that lysis of most target cells by cloned CTLs is due primarily to target cell membrane changes that are fundamentally equivalent to the formation of nonspecific ion channels. The resulting large increase in [Ca2+]i is probably responsible for target cell lysis; and changes in intracellular ion concentrations also appear to be responsible for DNA fragmentation, probably by activating endogenous target cell endonucleases.

Animals↗
PubMed1997

DNA fragmentation characteristic of apoptosis and cell loss induced by kainic acid in rabbit retinas.

We have examined whether in vivo exposure to the glutamate analogue, kainic acid, induces cell loss through apoptosis and/or through necrosis. The vulnerability of rabbit retinal cells was evaluated by routine histopathology. The DNA fragmentation was examined using an in situ method (TUNEL: TdT-mediated biotin-dUTP nick-end labelling) and agarose gel electrophoresis of extracted retinal DNA. Retinas were examined at 30 min, and 4, 16, 24 and 36 h, and 2-5 days following the intraocular administration of 140 nmol kainic acid. Although pyknotic cells could be seen already at 30 min post-injection, TUNEL-labelled nuclei were first observed 4 h after the injection. A relatively large number of pyknotic cells and of TUNEL-labelled nuclei were still seen at 5 days post-injection. Pyknotic cells were seen throughout the inner nuclear layer (mostly in the proximal half of the layer) and in the ganglion cell layer. The TUNEL-labelled nuclei were almost only seen in the proximal inner nuclear layer. Analysis of DNA by electrophoresis revealed the presence of large molecular weight fragments 4 h after the injection, and of oligonucleosome-size fragments between 16 h and 2 days after the injection. The present study thus presents evidence that, in our model, the retinal cell loss induced by kainic acid is preceded, probably in most cells, by a fragmentation of DNA characteristic of apoptotic cell death. The process of cell loss following kainic acid administration was found to be relatively slow, further suggesting that a programmed type of cell death, which eventually induces apoptosis, is involved. No indication that cells were lost also through necrosis was obtained.

Animals↗
PubMed1980

Transformation of rat embryo fibroblasts by cloned polyoma virus DNA fragments containing only part of the early region.

Recombinant plasmids containing either the entire polyoma viral genome or one or the other of the two HindIII fragments of polyoma virus DNA were constructed and cloned in Escherichia coli X1776, and their DNAs were individually tested for the capacity to transform an established line of rat cells. The recombinant plasmids containing the entire polyoma genome and those containing the HindIII-1 fragment of polyoma DNA (45-1.4 map units) efficiently transform rat cells, whereas the plasmids containing the HindIII-2 fragment (1.4-45.0 map units) do not. The properties of many independent transformed cell lines established by infection with the cloned HindIII-1 fragment were determined. In contrast to the parent cell line, rat cells transformed with the cloned HindIII-1 fragment grow to high saturation densities, form colonies with high efficiency in dilute agar suspension, produce high levels of plasminogen activator, and display a disorganized arrangement of actin cables. By all criteria examined, these cells transformed by fragments are indistinguishable from cells transformed by whole polyoma viral DNA. Cellular DNA prepared from many HindIII-1 fragment-transformed cell lines was analyzed for the presence and arrangement of polyoma viral sequences by Southern blot-hybridization. In all cases examined, only those viral sequences contained within the HindIII-1 fragment of polyoma DNA were detected. These data establish a strong correlation between polyoma DNA sequences mapping within a restricted portion of the early region and the induction and maintenance of the transformed phenotype.

Animals↗
PubMed1990

[Amplification of mitochondrial DNA fragments from ancient human teeth and bones].

We extracted and visualized DNA from ancient human teeth and bones of 150 to 5,500 years B.P. from three deposits from the south of France. The DNA extracted was used as template for PCR with specific primers corresponding to a portion of the human mitochondrial genome. In our samples, we have amplified a specific DNA fragment of 121 bp which, in the case of one bone of 150 years B.P. has been cloned and sequenced. We show that this sequence is identical to the homologous region of human mitochondrial DNA. The striking implications of this new method for archaeological and paleontological studies are exposed.

Base Sequence↗
PubMed1995

1-methyl-4-phenyl-pyridinium ion (MPP+) causes DNA fragmentation and increases the Bcl-2 expression in human neuroblastoma, SH-SY5Y cells, through different mechanisms.

Apoptosis has been shown to be induced by some pathological stimuli. MPP+ is a neurotoxin and an inducer of parkinsonism. When SH-SY5Y cells, human neuroblastoma cell line, were treated with MPP+, cell death estimated by lactate dehydrogenase (LDH) leakage assay occurred. The cell death was associated with the DNA fragmentation into nucleosomal fragments at 180 bp, suggesting that MPP(+)-induced cell death of SH-SY5Y cells occurs through apoptosis. Although SH-SY5Y cells natively express Bcl-2 protein, which inhibits apoptosis, the level of Bcl-2 protein in SH-SY5Y cells increased with increases in the treatment periods of MPP+. MPP+ inhibits the mitochondrial respiratory chain. The other inhibitors of the mitochondrial respiratory chain, antimycin A and oligomycin, also caused cell death associated with DNA fragmentation, but did not increase the Bcl-2 protein level, suggesting that an MPP(+)-induced apoptosis may be due to the inhibition of the mitochondrial respiratory chain but the MPP(+)-induced increase in the Bcl-2 protein level is not due to it. A protein kinase inhibitor, staurosporine, inhibited the MPP(+)-induced increase in the Bcl-2 protein level, but not the MPP(+)-induced cell death. These results also suggest that the mechanism by which MPP+ increases the Bcl-2 protein level is different from that of MPP(+)-induced cell death.

1-Methyl-4-phenylpyridinium↗
PubMed2003

Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay.

OBJECTIVE: To investigate how moderate and/or high levels of DNA fragmentation (DFI), as measured by the sperm chromatin structure assay (SCSA), affect either IVF or IVF with intracytoplasmic sperm injection (ICSI) fertilization, cleavage, blastulation, implantation, and pregnancy. DESIGN: Retrospective clinical study. SETTING: Academic human reproduction laboratory. PATIENT(S): Eighty-nine couples undergoing IVF with conventional fertilization or ICSI. INTERVENTION(S): Sperm chromatin structure assay testing (SCSA) of semen aliquot taken from ejaculate used for assisted reproductive technology (ART). MAIN OUTCOME MEASURE(S): Related DFI to conventional semen parameters and cycle-specific outcomes after ART. RESULT(S): No patients achieved clinical pregnancy if SCSA values exceeded the DFI (27%, P<.01), moderate DFI (15%, P<.01), or high DFI (15%, P<.05) thresholds. Dividing the DFI sperm population into moderate-fragmentation and high-fragmentation categories did not improve the prognostic value of the SCSA. No coefficient of determination (r(2)) between SCSA parameters and conventional parameters exceeded 0.29. CONCLUSION(S): Sperm chromatin structure assay identified thresholds for negative pregnancy outcome after ART not identified using conventional semen parameters. This is the first study analyzing the clinical value of sperm DFI to [1] include a large number of ART patients (n = 89), [2] perform SCSA analysis on a semen aliquot from the ejaculate used for ART, and [3] examine how the extent (moderate and high DFI) of DFI influenced ART outcomes.

Chromatin↗
PubMed1995

DNA fragmentation and activation of c-Jun in the cerebellum of mutant mice (weaver, Purkinje cell degeneration).

Weaver and Purkinje cell degeneration (pcd) are autosomal recessive mutations in the mouse characterized by an almost complete loss of cerebellar Purkinje neurones and granule cells, respectively. Developmental neuronal death occurs by activation of an apoptotic pathway and chromatin condensation has been observed in degenerating granule cells of weaver mutants. In the present study we demonstrate nuclear DNA fragmentation in Purkinje cells of pcd mice and in granule cells of weaver mutants during the period of neuronal degeneration using in situ end labelling by terminal transferase and fluorescein-dUTP. Furthermore, activation of candidate cell death effector gene c-jun has been detected exclusively within the affected cell populations by immunohistochemistry. Both labelled DNA fragments and nuclear c-Jun immunoreactivity were virtually absent in wild-type animals. Thus, genetically determined cell death in pcd and weaver mutant mice has features of apoptosis and may require activation of cell death effector genes.

Animals↗
PubMed1995

Microglial turnover in the injured CNS: activated microglia undergo delayed DNA fragmentation following peripheral nerve injury.

Microglial proliferation and activation are common events in the injured CNS. The mechanisms, however, by which activated microglia are eliminated following a pathological stimulus are still poorly understood. The present study has therefore examined microglial proliferation by 3H-thymidine autoradiography and programmed cell death by terminal transferase-mediated nick end labeling (TUNEL) and in situ end labeling (ISEL) of nuclear DNA fragments in two models of peripheral nerve injury, i.e. sciatic and hypoglossal nerve transection in the rat. In these models, microglial activation and proliferation occur in CNS projection areas, i.e. in the ventral and dorsal gray matter of lumbar spinal cord and in the nucleus gracilis after sciatic nerve transection as well as in the axotomized hypoglossal nucleus. At these sites, microglial proliferation had a relatively sharp peak between days 2 and 3 post-lesion and then rapidly declined. DNA fragmentation was detected in lectin (GSI-B4)-positive microglia from day 6 after axotomy onward, reached an apparent peak at day 21 and was downregulated by day 60, i.e. the latest time point investigated. However, the expression of bcl-2 and c-myc, i.e. genes potentially controlling programmed cell death, was found to be unchanged during this period. Programmed cell death thus appears to be one mechanism by which activated microglia are gradually eliminated following CNS injury and steady state of microglial cell numbers is achieved in vivo. Expression of microglial growth factors may be instrumental in controlling these processes.

Animals↗
PubMed2002

Germ cells with nuclear DNA fragmentation related to apoptotic cells in rat testis in experimental hyperprolactinemia induced by metoclopramide.

The cells with nuclear DNA fragmentation related to apoptosis were detected by TUNEL technique in the seminiferous epithelium of control rats and of rats with experimental hyperprolactinemia induced by metoclopramide. The percentage of convoluted tubules with apoptotic cells and the number of apoptotic cells (predominantly spermatogonia and spermatocytes) was increased in the experimental group. The results indicated stage-specific germ cell apoptosis. In the experimental group, apoptotic cells were most evident at early (I-IV), middle (VII-VIII) and late (XII-XIV) stages of the seminiferous epithelium cycle, as revealed by light and electron microscopy. We suggest that a decreased concentration of testosterone and an increased concentration of prolactin could disturb spermatogenesis and contribute to the intensive apoptosis of germ cells in rats with hyperprolactinemia. Sertoli cells which have receptors for testosterone and prolactin and play an important role in spermatogenesis and in the initiation of apoptosis in seminiferous epithelium, could mediate such an influence of both hormones.

Animals↗
PubMed2004

Direct visualisation of heavy ion induced DNA fragmentation using atomic force microscopy.

Atomic Force Microscopy of phiX174 plasmids irradiated in vitro was used to visualise the DNA fragmentation induced by heavy ions and to compare it to the fragmentation pattern obtained after X-irradiation. Fragment distributions induced by low ion fuences were found to be much more shifted towards small fragment sizes than the distributions obtained after corresponding doses of X-rays. The average fragment length was found to be significantly smaller than the full plasmid length even for single ion traversals.

DNA↗
PubMed2003

Expansion during PCR of short single-stranded DNA fragments carrying nonselfcomplementary dinucleotide or trinucleotide repeats.

We performed PCR of many DNA fragments of 6-32 nucleotides in length. Some of the fragments expanded into kilobase lengths even in the absence of the complementary strand. The dramatic expansion was observed for (CA)8, (TG)8, (CA)4, (CA)6, (CA)12, (TG)4, (TG)6, (TG)12, (TC)10, (GA)10 and other single strands. Similar expansions were exhibited by related trinucleotide repeats (TTG)5, (CAA)5, (TGG)5, and (CCA)5 as well. However even small perturbations of the strict repetitive nature of the DNA primary structure substantially reduced the expansions. The expansion products had properties characteristic for normal Watson-Crick duplexes. Hence either the Taq polymerase and/or other components of the PCR buffer promote homoduplex formation of the nonselfcomplementary fragments, which is necessary to prime the synthesis of the complementary DNA strand, or the Taq polymerase is able to copy the single-stranded DNA template without any priming effect. The present observations have implications for the evolution of genomic DNA, microsatellite length polymorphism as well as the pathological expansions of trinucleotide repeats in the human genome.

Base Pairing↗
PubMed1994

[Sequencing of hepatitis B virus DNA fragment coding major HBsAg of escape mutant].

We amplificated a HBV DNA fragment coding the major HBsAg from a chronic HBsAg carrier's serum with high titer of HBsAg and anti-HBs using polymerase chain reaction (PCR). The direct sequencing of PCR products revealed a single mutation from adnosine to guanosine at the nucleotide position 532 of HBV DNA of the escape mutant. This mutation might result in an amino acid substitution from threonine to alanime at the amino acid position 126 of HBsAg. Since this amino acid is located in the first loop of the "a" determinant of HBsAg, the mutation might result in an alteration of the structural antigenicity of the "a" determinant and induce escape mutant of HBV.

Antigenic Variation↗
PubMed1998

Translocation of profilaggrin N-terminal domain into keratinocyte nuclei with fragmented DNA in normal human skin and loricrin keratoderma.

The terminal differentiation of epidermal keratinocytes from the granular to enucleated cornified layer involves drastic changes both in morphology and biochemistry. Profilaggrin is a keratinocyte-specific phosphoprotein expressed in the granular layer. Although keratinization has been regarded as a specialized form of apoptosis or programmed cell death, the mechanism for this cellular transition at the molecular level is not yet well understood. In this study, we used light and electron microscopic immunohistochemistry to investigate the localization of the profilaggrin domains during this process. Antibodies specific for the amino-terminal domains of profilaggrin showed localization in keratohyalin granules in the granular cells, but stained the nucleus in transition cells. In contrast, an antibody to filaggrin domains stained the cytoplasm in the transition cells. Nuclei that were positive to amino-terminal profilaggrin contained fragmented DNA, characteristic of apoptosis. In the epidermis of patients with progressive symmetric erythrokeratoderma carrying a mutation in the loricrin gene (loricrin keratoderma), the profilaggrin amino-terminal domains were packed within apoptotic nuclei together with loricrin aggregates and this persisted up to the parakeratotic superficial layer. The present study indicates that the amino-terminal profilaggrin domains are cleaved from the filaggrin repeats and transiently localized to the apoptotic nuclei just before the formation of enucleated stratum corneum in normal epidermis. This suggests a significant role for the profilaggrin amino-terminus in nuclear events associated with keratinocyte terminal differentiation. Disrupted apoptosis found in loricrin keratoderma might explain the marked parakeratotic hyperkeratosis characteristic of this disease.

Biopsy↗
PubMed1993

High-resolution liquid chromatography of DNA fragments on non-porous poly(styrene-divinylbenzene) particles.

DNA restriction fragments and PCR products were separated by means of ion-pair reversed-phase high-performance liquid chromatography on alkylated non-porous poly(styrene-divinylbenzene) particles with a mean diameter of 2.1 microns. Optimum resolution was obtained by using an acetonitrile gradient in 100 mM of triethylammonium acetate and a column temperature of 50 degrees C. This allowed the separation of DNA fragments differing in chain length by 1-5% up to a size of 500 base pairs. PCR products could be analyzed directly in less than two minutes with a concentration sensitivity of at least 300 ng/ml. Compared with anion-exchange chromatography or gel electrophoresis no desaltation of the purified DNA molecules is required because the volatile buffer system can be readily evaporated. Subsequently, the method was used for the semiquantitative evaluation of the expression of multidrug resistance genes in mononuclear white blood cells.

Base Sequence↗