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PubMed1984

Quantitative predictivity of carcinogenicity for four short-term parameters, evaluated in rat liver: alkaline DNA fragmentation, autoradiographic repair, DNA adducts, preneoplastic nodules.

The possibility of the study of a quantitative correlation between short-term tests and carcinogenicity, instead of a qualitative one, is discussed. Four tests related to the target organ, rat liver, were considered: alkaline DNA fragmentation, DNA repair, DNA adducts and the formation of preneoplastic nodules. All the four tests showed a similar level of correlation with carcinogenic potency (r approximately equal to 0.4). With this level of correlation, the dispersion of the data appeared too large to offer a meaningful degree of quantitative predictivity of carcinogenicity, in reference to a single test. It appeared however, that the use of a battery of two or three independent short-term tests, with the above level of simple correlation, could generate a multiple correlation high enough to be potentially useful for some degree of quantitative predictivity of carcinogenic potency.

Amines↗
PubMedDate not supplied

Site-directed insertion of long single-stranded DNA fragments into plasmid DNA.

A new site-directed method for inserting long single-stranded DNA fragments into any region of a duplex vector is described. Its major advantage is independence of the location of the restriction sites. The method involves the assembly of single-stranded DNA fragments by ligation to both ends of the inserted fragments of two cohesive flanks that are complementary to the target region. Short oligonucleotide templates are used to direct the ligation. The resulting fragments, designated further as omega fragments with cohesive flanks, are hybridized with a gapped DNA vector. The heteroduplexes are transformed into Escherichia coli cells without enzymatic filling and sealing of gapped DNA. As a consequence of intracellular repair and heteroduplex resolution, insertion mutants are recovered. To demonstrate the method's efficiency, we inserted a 51-nucleotide synthetic DNA fragment containing a modified glucocorticoid receptor binding site into the region of pBR322, near the transcription starting point of the tet gene. The method we developed makes possible site-directed insertion of synthetic and genome-derived DNA fragments at least 200 nucleotides long.

Base Sequence↗
PubMed1982

Rapid method for purification of plasmid DNA and DNA fragments from DNA linkers using high-performance liquid chromatography on TSK-PW gel.

High-performance size exclusion chromatography (HPSEC) using TSK-G5000 PW in Tri buffer has been found to be a reliable method for the rapid fractionation of DNA ligation products. Plasmid and fragmented phage DNAs were found to elute in less than 2 min with recoveries greater than 98%. Escherichia coli transfection studies, using plasmid DNA that had been subjected to HPSEC column fractionation, showed high transformation efficiencies. MgCl2, a component of the DNA ligation reaction, was found to produce DNA-column support interactions, which resulted in low DNA recoveries. Such interactions were eliminated by chelation with ethylenediaminetetraacetate prior to chromatography.

Bacteriophages↗
PubMed1995

Microwave-enhanced in situ end-labeling of fragmented DNA: parametric studies in relation to postmortem delay and fixation of rat and human brain.

In situ end-labeling (ISEL) identifies DNA fragmentation in apoptotic or necrotic nuclei in tissue sections. However, application of ISEL on human brain requires conservation of DNA integrity during the postmortem delay (PMD) and good accessibility of fragmented DNA after (prolonged) tissue fixation. We therefore investigated ISEL in relation to PMD and fixation in rat and human brain. Application on a unilateral lesion model in perfused rat brain revealed that prolonged post-fixation strongly diminished ISEL results. However, microwave pre-treatment can counteract these masking effects without inducing nonspecific labeling contralaterally. On the other hand, in briefly post-fixed, perfused brain or immersion-fixed rat and human PMD brain, microwave pre-treatment was deleterious and induced strong nonspecific labeling. In young rat brain, PMD did not influence the low numbers of apoptotic nuclei until 24 hr PMD, when massive nuclear labeling occurred. In human cortex, DNA fragmentation patterns were independent of duration of fixation or PMD and were already present from 4.25 hr PMD onwards. Our data suggest that ISEL on human brain represents antemortem DNA damage rather than PMD artifacts. Furthermore, microwave pre-treatment appears beneficial only in particular fixation conditions.

Aged↗
PubMed1991

Streptozocin- and alloxan-induced H2O2 generation and DNA fragmentation in pancreatic islets. H2O2 as mediator for DNA fragmentation.

Streptozocin (STZ) and alloxan (ALX) exhibit the most potent diabetogenicity and are used for induction of experimental diabetes mellitus. An understanding of the mechanisms of action of the typical diabetogenic agents is important for elucidating the causes of diabetes. Okamoto proposed a model in which DNA fragmentation plays an important role in the development of diabetes. DNA fragmentation supposedly results from the accumulation of superoxide or hydroxyl radicals. However, direct evidence for this accumulation is lacking. With isolated rat pancreatic islets in vitro, we demonstrated that STZ and ALX stimulated H2O2 generation and caused DNA fragmentation. Addition of STZ or ALX resulted in an increase in H2O2 generation. On DNA analysis, when incubated without STZ or ALX, DNA sedimented as a single peak; when incubated with STZ or ALX, DNA sedimented slower as a broad peak and was fragmented. Graded doses of STZ and ALX stimulated H2O2 generation and induced DNA fragmentation; their effects on H2O2 generation and DNA fragmentation were evident at a concentration of 0.1 mM and were maximal at 1 mM. Administration of STX or ALX to rats in vivo stimulated H2O2 generation and caused DNA fragmentation in pancreatic islets. H2O2 itself also induced DNA fragmentation. These findings may support Okamoto's proposal that STZ and ALX induce diabetes through the following biochemical events: STZ and ALX----H2O2 generation----DNA fragmentation----beta-cell destruction. This study may constitute the first demonstration of STZ- and ALX-stimulated H2O2 generation, which probably acts as a mediator of STZ- and ALX-induced DNA fragmentation.

Alloxan↗
PubMed1990

Cytochalasin B induces cellular DNA fragmentation.

Cellular DNA fragmentation can be induced in many biological instances without plasma membrane damage. The fungal metabolite, cytochalasin B, is capable of modifying numerous cellular functions related to DNA synthesis. In this work it is demonstrated that cytochalasin B is capable of inducing DNA fragmentation in a number of cells lines. This DNA fragmentation occurs before plasma membrane lysis and over a period of hours. Cytochalasin E and villin, agents that act on the microfilaments, also induce DNA fragmentation. Phorbol dibutyrate, a diacylglyceral analog, is able to inhibit cytochalasin B-induced DNA fragmentation in a dose-dependent fashion. These findings support the interpretation that cytochalasin B is inducing DNA fragmentation via its effect on the actin filaments.

Actins↗
PubMed1995

In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note.

Detection of DNA fragments in situ using the terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay is increasingly applied to investigate active cell death (apoptosis). We studied the specificity of the assay in well-defined models of apoptosis and necrosis as well as in postmortem autolysis in rat liver. During involution of liver hyperplasia, which follows stopping treatment with the hepatomitogens cyproterone acetate (CPA) or nafenopin (NAF), numerous apoptotic hepatocytes could be observed with TUNEL-positive chromatin residues. A similar TUNEL-positive reaction appeared in necrotic hepatocytes after a cytotoxic dose of carbon tetrachloride (CCl4) or N-nitrosomorpholine (NNM). Also, in insufficiently fixed, autolytic livers TUNEL-positive nuclei were observed. Thus, DNA fragmentation is common to different kinds of cell death; its detection in situ should not be considered a specific marker of apoptosis.

Animals↗
PubMedDate not supplied

[Interaction of EcoRII restriction and modification enzyme with synthetic DNA fragments. IV. DNA duplexes with phosphoamide and pyrophosphate internucleotide bonds--the substrates for the study of single-strand breaks].

A set of DNA duplexes with repeated EcoRII, EcoRI and AluI restriction endonuclease recognition sites in which EcoRII scissile phosphodiester bonds were replaced by phosphoramide or uncleavable pyrophosphate bonds have been synthesized. Endonuclease EcoRII was found not to cleave the substrate at the phosphoramide bond. The substrates containing non-nydrolysable pyrophosphate or phosphoramide bonds in one of the chains of EcoRII recognition sites were used to show that this enzyme is able to catalyze single-strand scissions. These scissions occur both in dA- and dT-containing chains of the recognition site. Endonuclease EcoRII interacts with both strands of the DNA recognition site, each of them being cleaved independently on the cleavage of the other. Synthesized DNA-duplexes are cleaved specifically by EcoRI and AluI endonucleases, this cleavage being retarded if the modified bonds are in the recognition site (EcoRI) or flank it (AluI). For EcoRII and AluI this effect is more pronounced in the case of substrates with pyrophosphate bonds than with the phosphoramide ones.

Autoradiography↗
PubMed1985

The irreversible binding of azacytosine-containing DNA fragments to bacterial DNA(cytosine-5)methyltransferases.

DNA containing 5-azacytosine is an irreversible inhibitor of DNA(cytosine-5)methyltransferase. This paper describes the binding of DNA methyltransferase to 32P-labeled fragments of DNA containing 5-azacytosine. The complexes were identified by gel electrophoresis. The EcoRII methyltransferase specified by the R15 plasmid was purified from Escherichia coli B(R15). This enzyme methylates the second C in the sequence CCAGG and has a molecular mass of 60,000 Da. Specific binding of enzyme to DNA fragments could be detected if either excess unlabeled DNA or 0.8% sodium dodecyl sulfate was added to the reaction mixture prior to electrophoresis. Binding was dependent upon the presence of both the CCAGG sequence and azacytosine in the DNA fragment. S-Adenosylmethionine stimulated the formation of the complex. The complex was stable to 6 M urea but could be digested with pronase. These DNA fragments could be used to detect the presence of several different methyltransferases in crude extracts of E. coli. No DNA protein complexes could be detected in E. coli B extracts, a strain that contains no DNA(cytosine-5)methyltransferases. The chromosomally determined methylase with the same specificity as the purified EcoRII methylase could be detected in crude extracts of E. coli K12 strains. The MspI methylase cloned in E. coli HB101 could also be detected in crude extracts. These enzymes are the only proteins that bind azacytosine-containing DNA in crude extracts of E. coli.

Cytosine↗
PubMed1991

Induction of differentiation by tumour necrosis factor in HL-60 cells is associated with the formation of large DNA fragments.

The DNA fragmentation induced by tumor necrosis factor (TNF) of differentiable human myeloid leukemic HL-60 cells has been further characterized. TNF increased the appearance of very high molecular weight DNA fragments detected by agarose gel electrophoresis. The use of pulsed-field gel electrophoresis (PFGE) revealed these fragments to be as high as 200-400 kilobase pairs. The pattern of HL-60 DNA fragmentation contrasted with that of U937 cells, which exhibited lower molecular weight, nucleosome multiple sized fragments, and greater cytotoxicity in response to TNF. The peak increase of fragments from HL-60 occurred between one and two hours of incubation, with TNF concentrations of 10 U/ml or higher, and was inhibitable by 1 mM Zn2+. Southern blotting of these fragments disclosed enrichment for c-myc related sequences compared with control probes including beta-actin and kappa and lambda light chains. Treatment of DNA with NotI or gamma-irradiation, followed by PFGE, disclosed a class of still higher molecular weight DNA, which decreased following TNF treatment, and which was apparently the precursor of the TNF-induced fragments. TNF thus rapidly increases a class of high molecular weight DNA fragments which are enriched for c-myc related sequences and may arise preferentially from higher molecular weight structures which are detectable following linearization by NotI or gamma-irradiation. Such major but non-random alterations in chromatin structure may contribute to TNF-induced monocytoid differentiation of HL-60.

Blotting, Southern↗
PubMed1991

Homologous recombination of exogenous DNA fragments with genomic DNA in somatic cells of mice.

We compared liposomes and empty viral capsids for their use as vehicles for DNA transfer into cells and animals. DNA binding capacity was high for liposomes, but DNase I protection of DNA bound to liposomes was only moderate in comparison to DNA incorporated into viral capsids. Cellular uptake of radiolabeled and physiologically active DNA was also compared. For animal studies we chose an endogenous retroposon as target gene. To identify recombinational events we replaced a part of this gene with an artificial sequence not present in the mouse genome. The recombination rate for DNA fragments transfected in Polyoma capsids in live mice was higher than for liposome mediated transfection. Homologous recombination could be observed for both DNA transfer methods, mediated by positively charged liposomes (DOTMA) and by empty Polyoma viral capsids.

Animals↗
PubMed1986

High resolution preparative gel electrophoresis of DNA fragments and plasmid DNA using a continuous elution apparatus.

An apparatus designed for preparative gel electrophoretic separation of proteins (M. A. Hediger, (1984) Anal. Biochem. 142, 445-454) has been used successfully for separating DNA restriction fragments. The apparatus displayed yields and resolutions that are higher than those obtainable with commercially available devices. The amounts of DNA applied to the column range from a few micrograms to milligram quantities. Restriction DNA fragments very similar in size were isolated in pure form with the apparatus. After ethanol precipitation, these fragments were successfully used for restriction enzyme cleavages, ligation, or chemical sequencing. Furthermore the apparatus provides a convenient method for the large-scale isolation of plasmid DNA. The method requires only 4 h of electrophoresis and therefore greatly reduces the preparation time compared with the conventional equilibrium centrifugation method which requires centrifugation times of up to 60 h. In contrast to the centrifugation method, contaminants such as RNA, proteins, and chromosomal DNA are efficiently removed by this technique.

DNA↗
PubMed1994

Nondetectability of restriction fragments and independence of DNA fragment sizes within and between loci in RFLP typing of DNA.

We provide experimental evidence showing that, during the restriction-enzyme digestion of DNA samples, some of the HaeIII-digested DNA fragments are small enough to prevent their reliable sizing on a Southern gel. As a result of such nondetectability of DNA fragments, individuals who show a single-band DNA profile at a VNTR locus may not necessarily be true homozygotes. In a population database, when the presence of such nondetectable alleles is ignored, we show that a pseudodependence of alleles within as well as across loci may occur. Using a known statistical method, under the hypothesis of independence of alleles within loci, we derive an efficient estimate of null allele frequency, which may be subsequently used for testing allelic independence within and across loci. The estimates of null allele frequencies, thus derived, are shown to agree with direct experimental data on the frequencies of HaeIII-null alleles. Incorporation of null alleles into the analysis of the forensic VNTR database suggests that the assumptions of allelic independence within and between loci are appropriate. In contrast, a failure to incorporate the occurrence of null alleles would provide a wrong inference regarding the independence of alleles within and between loci.

Alleles↗
PubMed1989

[Species identification by fragmented DNA].

The species of raw meat as well as heat-processed meat can be determined by DNA dot-blot hybridization-techniques. We investigated on artificially degraded DNA whether various DNA qualities could influence the validity of the experiment results. We demonstrated that degraded DNA could also react according to the species. The shorter the DNA fragments, the discreter was the reaction when high molecular weight DNA was used as a probe. This effect is not observed with degraded DNA as a probe.

Animals↗