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PubMed1991

Lipopolysaccharide induces double-stranded DNA fragmentation in mouse thymus: protective effect of zinc pretreatment.

Intraperitoneal injection of female NAW/W1 mice with 5 mg of Salmonella typhimurium lipopolysaccharide/kg results in decreased body and thymus weight. Reduced thymic weight is accompanied by fragmentation of DNA into multimers of about 200 bp size. This effect is consistent with the induction of intranucleosomal cleavage of double-stranded DNA in thymus. Maximal fragmentation of DNA occurs between 18 and 24 h after treatment; by 48 h post lipopolysaccharide treatment, there is little evidence of thymic DNA fragmentation. Pretreatment of mice with Zn protects against lipopolysaccharide-induced DNA fragmentation. This effect is maximal at about 72 h after Zn treatment (24 h after lipopolysaccharide treatment) and persists until about 96 h after Zn treatment. At 72 h after pretreatment, the antagonism of thymic DNA fragmentation by Zn is dose-dependent. To examine the role of the acute phase inflammatory response elicited by lipopolysaccharide treatment in the production of changes in thymic weight and DNA integrity, the effects of treatment with casein, a well-characterized inducer of the acute phase inflammatory response in mice, were examined. In contrast to the effect of lipopolysaccharide, casein treatment did not produce a similar pattern of DNA fragmentation in thymus. Taken together, these data suggest that lipopolysaccharide induces DNA fragmentation in thymus by a mechanism which does not occur during the pathophysiological changes which accompany the casein-induced acute phase response. Further, the antagonism by Zn of lipopolysaccharide-induced fragmentation of thymic DNA is consistent with earlier findings that Zn can prevent dexamethasone-induced DNA fragmentation in vitro.

Acute-Phase Reaction↗
PubMed1980

A cloned chicken DNA fragment includes two repeated DNA sequences with remarkably different genomic organizations.

The presence of both single copy sequences and repeated DNA sequences with a broad range of repetition frequency is a hallmark of the eukaryotic genome. The advent of recombinant DNA technology has made it possible to isolate cloned DNA fragments that encompass two or more DNA sequences with different repetition frequencies. This provides the first opportunity to investigate the structural relationships in the genome of DNA of the different repetition frequency classes in a thorough and systematic way. A cloned fragment of the chicken genome containing two different repeated DNA sequences has been used for this purpose. By hybridization of radioactive restriction fragments of the cloned DNA to filter-bound restriction fragments of total chicken DNA, it has been determined that the 5.5-kilobase inserted DNA in this clone contains a copy of each of two long, repeated DNA sequence elements with different repetition frequencies. These two repeated DNA sequences have very diverse arrangements in the chicken genome. The results establish that, in addition to the interspersion of single copy and repeated DNA sequences in the chicken genome, repeated DNA sequences with very different structural characteristics can be interspersed with one another. Thus, the chicken genome is a complex network of related sequences in which some members of a family of repeated DNA sequences are associated with other, nonhomologous repeated DNA sequences, while other members of the same family are flanked by single copy DNA.

Animals↗
PubMed2005

Exploration of factors driving incorporation of unnatural dNTPS into DNA by Klenow fragment (DNA polymerase I) and DNA polymerase alpha.

In order to further understand how DNA polymerases discriminate against incorrect dNTPs, we synthesized two sets of dNTP analogues and tested them as substrates for DNA polymerase alpha (pol alpha) and Klenow fragment (exo-) of DNA polymerase I (Escherichia coli). One set of analogues was designed to test the importance of the electronic nature of the base. The bases consisted of a benzimidazole ring with one or two exocyclic substituent(s) that are either electron-donating (methyl and methoxy) or electron-withdrawing (trifluoromethyl and dinitro). Both pol alpha and Klenow fragment exhibit a remarkable inability to discriminate against these analogues as compared to their ability to discriminate against incorrect natural dNTPs. Neither polymerase shows any distinct electronic or steric preferences for analogue incorporation. The other set of analogues, designed to examine the importance of hydrophobicity in dNTP incorporation, consists of a set of four regioisomers of trifluoromethyl benzimidazole. Whereas pol alpha and Klenow fragment exhibited minimal discrimination against the 5- and 6-regioisomers, they discriminated much more effectively against the 4- and 7-regioisomers. Since all four of these analogues will have similar hydrophobicity and stacking ability, these data indicate that hydrophobicity and stacking ability alone cannot account for the inability of pol alpha and Klenow fragment to discriminate against unnatural bases. After incorporation, however, both sets of analogues were not efficiently elongated. These results suggest that factors other than hydrophobicity, sterics and electronics govern the incorporation of dNTPs into DNA by pol alpha and Klenow fragment.

Benzimidazoles↗
PubMed1998

Rescue of polyomavirus DNA after co-transfection of recombinant plasmids with viral DNA fragments.

Plasmid DNA bearing a single copy of the mouse polyomavirus (Py) genome (template A) was transfected into murine cells together with another DNA (template B) carrying intact the viral sequence interrupted in template A. Rescue of unit-length Py DNA including markers from both templates was observed as long as the viral DNA in B overlapped that split in A by one kbp or more. Such rescue was not detectably enhanced by linearizing either or both template(s), and occurred in the absence of template replication. These findings are suggestive of an intermolecular recombination process taking place soon after transfection and starting with homologous pairing between A and B. Such pairing would facilitate removal of vector DNA from one template (A), followed by closure of the resulting break or gap through recombination with the other template (B). Since B may consist of a PCR-synthesized DNA fragment, these observations could conceivably serve as the basis for a method of generating mutant viral genomes.

Animals↗
PubMed1991

Caesium fluoride-induced changes in the c.d. spectra of synthetic DNA fragments.

Ten DNA fragments containing self-complementary alternating sequences of adenine and thymine differing in length and the starting nucleotide were studied by c.d. spectroscopy. It was found that d(TATATATA) but not d(ATATATAT), d(TATATA), d(CTATATAG) or (dT-dA)20 isomerized into the unusual X-DNA double helix at molar concentrations of CsF in solution. But in contrast to poly(dA-dT), the octamer (dT-dA)4, isomerized very slowly, at relatively low CsF concentrations and the isomerization was strongly dependent on the octamer concentration. A model is proposed to account for the observed properties of the B-to-X isomerization on the oligomer level.

Adenine Nucleotides↗
PubMed1999

Detection of genomic DNA fragmentation during apoptosis (DNA ladder) and the simultaneous isolation of RNA from low cell numbers.

In the present paper we describe a rapid and sensitive method for the simultaneous isolation of total RNA and genomic plus low-molecular-weight DNA from apoptotic cells. Using this method, we were able to detect a DNA ladder from as low as 30,000 apoptotic cells in only 45 min including gel electrophoresis. In addition, RNA can be readily obtained from the same specimen to assess gene expression during apoptosis. This method therefore appears to be advantageous when sensitivity and low amounts of sample material are a limiting factor.

Adsorption↗
PubMed2002

PCR-generated linear DNA fragments utilized as a hantavirus DNA vaccine.

The field of DNA vaccines has grown rapidly, and since most such vaccines involve the inoculation of large circular DNA molecules previously propagated in bacteria, several inconveniences (e.g. the presence of antibiotic resistance genes, impurities from bacterial cultures or inefficient uptake of the large and bulky plasmid DNA molecules to the nucleus) are debated. In this study, we have explored the possibility of using smaller and more flexible PCR-generated linear DNA fragments instead. Phosphorothioate (PTO)-modified primers were used successfully to protect the PCR-generated DNA fragments from exonuclease degradation, and by using a nuclear localization signal-peptide to target the linear DNA to the nucleus the immune response against the encoded antigen was further improved. This approach was tested in cell culture using a sensitive reporter system and in vivo with DNA encoding the amino-terminus of the Puumala hantavirus nucleocapsid protein. Our results indicate that linear DNA fragments have a great potential as a genetic vaccine and phosphorothioate modification in combination with a nuclear localization signal peptide increase the stability and targets the linear DNA molecules to the nucleus resulting in an improved biological response examined both in vitro and in vivo.

Animals↗
PubMedDate not supplied

Cytotoxic manifestations of the interaction between hyperthermia and TNF: DNA fragmentation.

The relationship of DNA fragmentation to the greatly enhanced cytotoxicity seen in vitro against tumour cells when recombinant human tumour necrosis factor-alpha (TNF-alpha) is combined with hyperthermia was investigated. The TNF-alpha-sensitive L929 and -resistant EMT6 cells were treated with 8.8 and 16 ng of TNF-alpha per ml, respectively, and then heated at 40.5 degrees C for 24 h (L929) or at 43 degrees C for 1 h (L929) or 1.5 h (EMT-6) beginning 1 h later. For both cell lines at both temperatures, the addition of heating to the TNF-alpha treatment significantly decreased viability and increased DNA fragmentation at earlier time points than seen with either TNF-alpha or heat alone. DNA fragmentation was further studied using agarose gel electrophoresis to examine the size distribution of the DNA fragments and the ability of intracellular calcium buffering agents BAPTA and quin-2 to inhibit fragmentation. At 4.5 h after L929 cells were treated with TNF-alpha at 43 degrees C, the size distribution of DNA fragments more closely resembled the oligonucleosome sized apoptotic DNA fragmentation, as seen in irradiated rat thymocytes, than the spectrum of DNA fragments seen in necrotic fragmentation. However, while BAPTA and quin-2 inhibited the calcium-dependent apoptotic fragmentation seen in thymocytes they did not inhibit the DNA fragmentation in L929 cells. In addition, the loss of membrane integrity in both L929 and EMT-6 cells preceded or approximated the appearance of DNA fragmentation, whereas loss of membrane integrity usually follows DNA fragmentation in apoptosis. However, morphological studies showed that apoptotic bodies were present in L929 cell cultures treated with TNF-alpha and heat, and were distinguishable from necrosing cells. We conclude that both types of DNA fragmentation are operant in some cell lines exhibiting a cytotoxic response to TNF-alpha and heat treatments, and that increased fragmentation reflects the greatly enhanced cytotoxic interactions seen with combination treatments in those cells.

Animals↗
PubMed1981

Sequencing long DNA fragments cloned in bacteriophage M13 by using internal primers. The sequence analysis of a yeast DNA fragment containing a replication origin.

In the ;shotgun' procedure for sequencing DNA, DNA fragments are cloned into a phage M13 vector and sequenced by using a flanking primer. In a variation of this procedure a longer DNA sequence is cloned into M13, the two single-stranded recombinants identified and sequenced by using a set of internal primers prepared by exonuclease III digestion of restriction fragments.

Base Sequence↗
PubMed2000

In vitro DNA fragmentation of mitochondrial DNA caused by single-stranded breakage related to macroplasmodial senescence of the true slime mold, Physarum polycephalum.

We found that mitochondrial DNA (mtDNA) isolated from Physarum polycephalum fragmented itself in weak ionic solutions. The mtDNA was dissolved in STE (saline Tris-EDTA: 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA), TE (10 mM Tris-HCl, 1 mM EDTA) and DW, and then electrophoresed in an agarose gel. The intact 86-kbp mtDNA band was seen in STE, but several novel bands appeared in TE and DW. In TE, two discrete bands appeared at 6.7-kbp (alpha-band) and 5.0-kbp (beta-band), whereas at least 17 discrete bands were observed in distilled water (DW). These fragmentation patterns were not stoichiometric, as seen when using restriction endonucleases, but were clearly different from the degradation of DNA caused by a physical shearing force or a contaminating nuclease. In this paper, we characterize this in vitro fragmentation of mtDNA from P. polycephalum. We located 19 fragments, including the alpha and beta fragments, on a mtDNA restriction map, and demonstrated that these cleavage sites were S1 nuclease-sensitive regions, which are single-stranded DNA regions such as nicks and gaps in the mtDNA. The alpha and beta fragments are derived from the region encoding ribosomal RNAs (rRNAs) and the ATP synthase (atpA) gene, while the other 17 fragments are not derived from any specific region, but the cleavage sites are located throughout the mtDNA molecule. In P. polycephalum, it is well known that the growth rate of macroplasmodia decreases with aging. Equal amounts of mtDNA from juvenile and aged macroplasmodia were electrophoresed and the frequency of the beta fragment in each sample was measured. The ratio of the beta band to the total signal including background was estimated to be 3.3-4.0% in juvenile macroplasmodia, whereas it increased to 8.3-28.2% in aged macroplasmodia. This result suggests that the in vitro fragmentation of mtDNA is associated with macroplasmodial senescence. The single-stranded breakage of mtDNA of P. polycephalum may accumulate with age.

Aging↗
PubMed1990

CD4+CD8+ thymocytes are susceptible to DNA fragmentation induced by phorbol ester, calcium ionophore and anti-CD3 antibody.

Stimulation of murine thymocytes with phorbol ester or calcium ionophore for 18-24 h resulted in 70%-80% fragmentation of DNA into 180-200-bp multiples, followed by cell death. Experiments with fractionated subpopulations by panning or flow cytometry revealed that DNA fragmentation was selectively observed in CD4+CD8+ cells and in a portion of CD4-CD8+ cells. To investigate whether DNA cleavage is also inducible via antigen-specific receptors, thymocytes were incubated in wells precoated with anti-CD3 antibody. An approximately 20% increase of DNA fragmentation was constantly observed when unseparated thymocytes were stimulated with anti-CD3 antibody. In this anti-CD3-induced DNA degradation, CD4+CD8+ cells are probably the target cells, since (a) fetal thymocytes at day 18 of gestation were found vulnerable to anti-CD3-induced DNA cleavage and (b) flow cytometry analysis of viable cells recovered after cultivation in the anti-CD3-coated wells revealed that CD4+CD8+ cells were preferentially decreased. Further experiments with purified CD4+CD8+ cells, however, could not define a clear-cut increase of DNA fragmentation when isolated CD4+CD8+ cells were stimulated with anti-CD3 antibody. Addition of interleukin (IL) 1, IL 2, IL 3, IL 4 or interferon-gamma to the CD4+CD8+ cell cultures failed to yield a DNA cleavage similar to that of unseparated thymocytes.

Animals↗
PubMedDate not supplied

A simple and sensitive pulsed field gel electrophoresis protocol to study 50 kb apoptotic DNA fragmentation in human lymphocytes.

DNA fragmentation of 50 kb is observed in apoptotic human lymphocytes as measured with pulsed field gel electrophoresis (PFGE). Standard PFGE assay involves embedding of cells into agarose blocks followed by lysis in the presence of proteinase K. In this study, we modified the PFGE protocol by omitting the proteinase K. In this study, we modified the PFGE assay by omitting the proteinase K and changing lysis solution according to the method of anomalous viscosity time dependence (AVTD). The conditions of PFGE were adjusted aiming to compress apoptotic fragments, increasing sensitivity and the number of samples that could be loaded on the same gel. Lymphocytes were irradiated with gamma-rays and apoptotic fragmentation of DNA was determined by PFGE using standard lysis with proteinase K and lysis protocol from AVTD method. Both protocols of lysis resulted in the same pattern of DNA fragments. The yield of radiation-induced apoptotic fragmentation was higher with the AVTD protocol of lysis. The novel PFGE protocol is simple and relatively non-expensive, requires only 7 h running time and gives a possibility to analyse simultaneously up to 69 samples in the same gel. The sensitivity of our protocol provides reproducible detection of 50 kb fragmentation after irradiation of human lymphocytes with 5 cGy of gamma-rays. In 2 of 6 donors tested, this DNA fragmentation was detected at dose on 2 cGy. The novel protocol can be used for quantification of 50 kb apoptotic fragments induced by different agents including low dose ionising radiations, chemicals and electromagnetic fields.

DNA Fragmentation↗
PubMed2001

Caspase-activated DNase/DNA fragmentation factor 40 mediates apoptotic DNA fragmentation in transient cerebral ischemia and in neuronal cultures.

Nuclear changes, including internucleosomal DNA fragmentation, are characteristic features of neuronal apoptosis resulting from transient cerebral ischemia and related brain insults for which the molecular mechanism has not been elucidated. Recent studies suggest that a caspase-3-mediated mechanism may be involved in the process of nuclear degradation in ischemic neurons. In this study, we cloned from rat brain a homolog cDNA encoding caspase-activated deoxyribonuclease (CAD)/DNA fragmentation factor 40 (DFF40), a 40 kDa nuclear enzyme that is activated by caspase-3 and promotes apoptotic DNA degradation. Subsequently, we investigated the role of CAD/DFF40 in the induction of internucleosomal DNA fragmentation in the hippocampus in a rat model of transient global ischemia and in primary neuronal cultures under ischemia-like conditions. At 8-72 hr after ischemia, CAD/DFF40 mRNA and protein were induced in the degenerating hippocampal CA1 neurons. CAD/DFF40 formed a heterodimeric complex in the nucleus with its natural inhibitor CAD (ICAD) and was activated after ischemia in a delayed manner (>24 hr) by caspase-3, which translocated into the nucleus and cleaved ICAD. Furthermore, an induced CAD/DFF40 activity was detected in nuclear extracts in both in vivo and in vitro models, and the DNA degradation activity of CAD/DFF40 was inhibited by purified ICAD protein. These results strongly suggest that CAD/DFF40 is the endogenous endonuclease that mediates caspase-3-dependent internucleosomal DNA degradation and related nuclear alterations in ischemic neurons.

Animals↗
PubMedDate not supplied

Functional correction of episomal mutations with short DNA fragments and RNA-DNA oligonucleotides.

BACKGROUND: Gene correction is an alternative approach to replacement gene therapy. By correcting mutations within the genome, some of the barriers to effective gene therapy are avoided. Homologous nucleic acid sequences can correct mutations by inducing recombination or mismatch repair. Recently, encouraging data have been presented using both short DNA fragments (SDFs) and RNA-DNA oligonucleotides (RDOs) in experimental strategies to realize clinical gene correction. METHODS: The delivery of labelled SDFs and RDOs to a variety of cell lines was tested using both FACS analysis and confocal microscopy. A GFP-based reporter system was constructed, containing a nonsense mutation, to allow quantitation of gene correction in living cells. This reporter was used to compare efficiencies of functional gene correction using SDFs and RDOs in arange of mammalian cell lines. RESULTS: The delivery experiments highlight the inefficient delivery of SDFs and RDOs to the nucleus using polyethylenimine (PEI) transfection. This study compared the episomal correction efficiency of the reporter plasmid mediated by SDFs and RDOs within different cell types; low levels of functional correction were detected in cell culture. CONCLUSIONS: Whilst delivery of PEI-complexed SDFs or RDOs to the cell is highly effective, nuclear entry appears to be a limiting factor. SDFs elicited episomal GFP correction across a range of cell lines, whereas RDOs only corrected the reporter in a cell line that overexpresses RAD51.

Animals↗
PubMedDate not supplied

[Study of the kinetics of the reaction reuniting DNA fragments catalyzed by DNA-ligase. Case of homogeneous molecules].

The kinetics of different DNA forms formation were studied during the ligation reaction. Reaction was carried out using as a model BamH1 DNA fragments of plasmid pBR322 and DNA-ligase of bacterophage T4. The products of the reaction were separated with 1% agarose gel, followed by DNA quantitation in the corresponding bands by scanning fluorimeter. The kinetics of the ligation reaction were measured in the range of DNA concentrations from 1 to 100 micrograms/ml. The system of differential equations, that described the kinetics of the ligation reaction, was obtained. This has no analytical solution, but may be approximately calculated with a computer. The analysis of kinetic equations was made in order to optimize the ligation reaction. The optimal DNA concentrations were found allowing the maximal yield of multimer circular DNA forms. The simple formula for calculatng these concentrations were developed. The yield of circular DNA multimers might be increased if the reaction is carried out at two stages: initially at high DNA concentration for linear multimers formation followed then a lower concentration after appropriated dilution. The formula for determiantion of the dilution time is presented. The data obtained allow to optimize the reaction conditions providing the recombinant DNA molecules formation.

Catalysis↗
PubMed2005

Conversion of nucleotide sequence with single-stranded DNA fragment prepared from phagemid DNA.

The correction of a mutated gene is a highly attractive approach for gene therapy. This in vivo mutagenesis method will also be an effective tool in biotechnology. However, the current small fragment homologous replacement (SFHR) method with a heat-denatured double-stranded PCR fragment yielded the low correction efficiency. Single-stranded DNA fragments were prepared from single-stranded phagemid DNAs and tested in a gene correction assay with a Hyg-EGFP fusion gene inactivated by a substitution mutation, as a model target. A 606-nt sense, single-stranded DNA fragment dramatically (12-fold) improved the gene correction efficiency, although the antisense strand showed only minimal correction efficiency. On the other hand, correction of frameshift mutations with the sense single-stranded DNA fragment were 2-3-fold as efficient as that with the PCR fragment. These results suggest that the use of a sense, single-stranded DNA fragment is useful in the SFHR method for the correction of mutated genes.

Bacteriophages↗
PubMed1991

Precise localization of the alpha-globin gene cluster within one of the 20- to 300-kilobase DNA fragments released by cleavage of chicken chromosomal DNA at topoisomerase II sites in vivo: evidence that the fragments are DNA loops or domains.

We have mapped the position of the alpha-globin gene cluster in the 20- to 300-kilobase fragments of chromosomal DNA isolated from growing chicken HD3 erythroblastoid cells exposed to 4'-demethylepipodophyllotoxinthenylidene beta-D-glucoside. This epipodophyllotoxin traps functioning topoisomerase II molecules, the denaturation of which cleaves DNA and reveals their reaction sites. The DNA fragments, prepared by centrifugation in sucrose gradients, bind selectively to glass-fiber filters and are protected from lambda 5'-exonuclease, properties compatible with the presence of a topoisomerase II subunit bound to their 5' ends. Restriction enzyme cleavage of the fragments and hybridization with cloned alpha-globin-region probes reveal additional distinctive bands not seen in control DNA, allowing the localization of fragment ends near this gene cluster. The terminal regions of fragments from sucrose gradients or from field-inversion electrophoresis gels were also used to probe cloned regions of the gene cluster. Both approaches show that this cluster of three genes, which is not expressed in these cells, is located at a specific position in a approximately 20-kilobase DNA fragment. The upstream end of this fragment lies in a region that contains a site of DNA attachment to the nuclear matrix mapped by both in vivo and in vitro methods, and its downstream end is flanked by approximately 80% A + T sequences characteristic of matrix-attachment regions. These observations suggest that the DNA fragments are formed because topoisomerase II molecules can specifically and readily integrate into DNA at matrix-attachment regions and that the fragments represent entire DNA loops or domains.

Animals↗
PubMed1998

Photodynamic treatment with benzoporphyrin derivative monoacid ring A produces protein tyrosine phosphorylation events and DNA fragmentation in murine P815 cells.

Treatment with benozopophyrin derivative monoacid ring A (BPD-MA, verteporfin) and broad-spectrum fluorescent light rapidly produced apoptosis in murine P815 mastocytoma cells. Fragmentation of DNA, a fundamental characteristic of cells undergoing apoptosis, was evident within 3 h following the photodynamic treatment. Western immunoblot analysis using the specific antiphosphotyrosine monoclonal antibody 4G10 indicated that molecular species of > 200 kDa were phosphorylated on tyrosine residues during or immediately following the irradiation of cells loaded with BPD-MA. Increased tyrosine phosphorylation of a 15 kDa protein was evident by 15 min postirradiation. In the absence of light, BPD-MA did not affect the status of cellular protein tyrosine phosphorylation or cause DNA fragmentation. The protein kinase inhibitor staurosporine prevented tyrosine phosphorylation of the > 200 kDa species but did not affect tyrosine phosphorylation of the 15 kDa protein or the level of DNA fragmentation produced by the photodynamic treatment. The protein tyrosine phosphorylation events observed for P815 cells treated with cytotoxic levels of BPD-MA and light may not be directly related to the induction of the apoptotic cell death pathway.

Animals↗