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At least 271 records · Page 15Linked to original sources
PubMed1993

A concatenated form of Epstein-Barr viral DNA in lymphoblastoid cell lines induced by transfection with BZLF1.

The replicative form of Epstein-Barr virus (EBV) DNA was studied using two lymphoblastoid cell lines, X50-7 and 6F11, which are latently infected by Epstein-Barr virus. The lytic cycle of EBV infection was induced by transfection of the cells with the BRLF1/BZLF1 coding region of the P3HR-1 defective genome. We combined two techniques to identify the productive replicative form of Epstein-Barr viral DNA in the lytic cycle-induced cells. Restriction enzyme analysis followed by Southern blot hybridization identified a significant increase in the fused fragment encompassing both ends of EBV DNA. This indicates an increase in either episomal DNA or concatameric linear DNA. Southern blot analysis of in situ lysing gels revealed that the cellular content of linear EBV DNA was also increased significantly after the initiation of the viral lytic cycle, while the amount of circular DNA remained approximately constant. We propose from these results that the source of the fused fragment encompassing both ends of EBV DNA is a concatenated linear EBV DNA molecule, and that such a concatenated molecule most likely represents a replicative form of EBV DNA in productively infected cells.

Cell Line↗
PubMed1992

Stable amplification of a linear extrachromosomal DNA in mycophenolic acid-resistant Leishmania donovani.

Pulsed field gel electrophoretic analysis of chromosomes of MPA100 cells, a strain of Leishmania donovani that possesses an approx. 15-fold amplified IMP dehydrogenase (IMPDH) gene copy number, revealed a new 280-kb extrachromosomal DNA, IMPDH-280, that was not present in wild type parental cells. Southern blots of these pulsed field gels revealed that the vast majority of the amplified impdh genes were localized on IMPDH-280. In addition to the 700-kb wild type chromosome, the impdh probe also recognized a 740-kb chromosome in the MPA100 genome. The pulse time-dependent relative mobility of IMPDH-280 in pulsed field gels, the failure of limited gamma-irradiation to generate a new discrete DNA fragment, and the susceptibility of IMPDH-280 to lambda-exonuclease digestion, demonstrated that IMPDH-280 was a linear molecule. IMPDH-280 was also recognized by a telomere probe but not by fragments derived from amplified DNAs found in other drug-resistant Leishmania. IMPDH-280 and the drug resistance phenotype remained stable when MPA100 cells were propagated in the absence of drug for 2 years. The appearance of IMPDH-280 in MPA100 cells represents one of the first examples of an amplification of a linear extrachromosomal DNA element mediating drug resistance in Leishmania and the first instance of a linear DNA amplification that is stable in the absence of selective pressure.

Animals↗
PubMed1987

Salt effects on internal motions of superhelical and linear pUC8 DNA. Dynamic light scattering studies.

The plasmid pUC8 (2717 bp) has been studied in its native superhelical and Eco RI-linearized forms by dynamic light scattering at NaCl concentrations from 1.1 mM to 1 M. The data were analyzed using the biexponential model for the dynamic structure factor described by us in a previous paper (J. Langowski, U. Giesen and C. Lehmann, Biophys. Chem. 25 (1986) 191). As before, we could identify two decay components corresponding to the center-of-mass diffusion and to internal motions of the DNA, where the fast component could be identified as a rotational diffusion contribution in the case for superhelical, but not for linear DNA. We found that the conformation of superhelical pUC8 is not affected by changing the ionic strength, while the amplitude of the internal relaxation increases approx. 2-fold when [NaCl] is raised from 1.1 mM to 1 M. The linearized DNA shows an increase of the diffusion coefficient with ionic strength which is, however, not quite as pronounced as that found by others (Z. Kam, N. Borochov and H. Eisenberg, Biopolymers 20 (1981) 2671), and, together with the unchanged conformation of the superhelical DNA, suggests a persistence length which is not strongly dependent on ionic strength. In contrast to the increasing amplitude of internal relaxation for the superhelical DNA, this amplitude remains constant or decreases slightly for linear DNA on going from 1.1 mM to 1 M salt. Our findings are further discussed with respect to possible models of the interwound form of superhelical DNA.

DNA, Superhelical↗
PubMed2001

Interaction of linear homologous DNA duplexes via Holliday junction formation.

Interaction of linear homologous DNA duplexes by formation of Holliday junctions was revealed by electrophoresis and confirmed by electron microscopy. The phenomenon was demonstrated using a model of five purified PCR products of different size and sequence. The double-stranded structure of interacting DNA fragments was confirmed using several consecutive purifications, S1-nuclease analysis, and electron microscopy. Formation of Holliday junctions depends on DNA concentration. A thermodynamic equilibrium between duplexes and Holliday junctions was shown. We propose that homologous duplex interaction is initiated by nucleation of several dissociated terminal base pairs of two fragments. This process is followed by branch migration creating a population of Holliday junctions with the branch point at different sites. Finally, Holliday junctions are resolved via branch migration to new or previously existing duplexes. The phenomenon is a new property of DNA. This type of DNA-DNA interaction may contribute to the process of Holliday junction formation in vivo controlled by DNA conformation and DNA-protein interactions. It is of practical significance for optimization of different PCR-based methods of gene analysis, especially those involving heteroduplex formation.

Animals↗
PubMedDate not supplied

[Induction of single- and double-strand breaks in linear and superhelical DNA by phleomycin].

Phleomycin induced DNA breakage was investigated with superhelical Col. E1 DNA and linear T2 DNA as well. In both DNA-forms besides single-strand breaks direct double-strand breaks were produced by the drug. The double-strand breakage rate obtained after treatment with phleomycin, however, was considerably smaller than that found after bleomycin treatment. Whereas the single- and double-strand breakage rate in Col. E1 DNA showed a linear and nearly quadratic dependence on the phleomycin concentration, respectively, in T2 DNA the breakage rates increased faster than the first or second power of the concentration. This indicates various modes of drug-DNA interaction. Under nondegrading conditions a strong retardation of electrophoretic mobility was observed for all three topological isomers of Col. E1 DNA whereas the sedimentation behaviour remained unchanged. The in vitro effects (strand breakage) of phleomycin and bleomycin are compared with induction of chromosomal aberrations in human lymphocytes and oocytes of Drosophila melanogaster.

Bleomycin↗
PubMed1986

Mechanism of intramolecular recyclization and deletion formation following transformation of Escherichia coli with linearized plasmid DNA.

The deletion end-points of a number of type I (less than monomeric) plasmid deletants obtained by transforming recA+ or recA- E. coli with linear pBR322 DNA were determined by DNA sequencing. In both monodirectional and bidirectional deletions the recyclization point was normally characterized by recombination between directly repeated sequences of between 4 and 10 bp present on each arm of the linearized pBR322 molecule. Frequently, short tracts of uninterrupted homology involved in recombinational recircularization were embedded in regions of relative non-homology. A model predicting the probability of matching sequences in either end of a linear plasmid molecule is presented. It is proposed that exonucleolytic processing of the exposed termini of linear plasmid molecules generates substrates for subsequent recombinational recyclization and deletion. The activity of host recombination and repair functions in recircularizing linear DNA molecules explains the generation of many of the aberrant recombinant DNA constructs obtained during gene cloning procedures.

Base Sequence↗
PubMed1992

Mutations caused by gamma-radiation-induced double-strand breaks in a shuttle plasmid replicated in human lymphoblasts.

The mutagenicity of open-circular DNA (containing base damage and single-strand breaks) and linear DNA (containing base damage, single-strand breaks, and one double-strand break) produced in vitro by gamma-irradiation of shuttle vector pZ189, was analysed after the plasmid's repair and replication in the human lymphoblast line, GM606. By comparing the survival, mutation frequency, and types of mutations in descendants from the two DNA forms, the effects of the double-strand break were determined. The percentage of viable plasmids from linear DNA was two-fold lower than that from open-circular DNA, 7.8 versus 14.0 (compared with unirradiated, control DNA). The mutation frequency in progenies of the open-circular plasmid was 4.2 +/- 1.7 x 10(-3), compared with 7.8 +/- 0.1 x 10(-3) in progenies of the linear DNA, again, nearly a two-fold difference. Approximately 59% of the mutations from the linear DNA were deletions and 34% were base substitutions. In contrast, only 13% of mutations from open-circular DNA were deletions, but 87% were base substitutions. All recoverable deletions were small, ranging from 1 to 205 base pairs, and the majority contained direct repeats at the deletion junctions, indicating non-homologous recombinations. Thus, mutations found among descendants from the linear and open-circular DNAs were qualitatively similar but quantitatively different. The data suggests that producing one double-strand break in DNA by ionizing radiation causes a two-fold increase in both lethality and mutation frequency.

Base Sequence↗
PubMed1985

The linear extrachromosomal DNA of Physarum polycephalum replicates and is maintained under non-selective conditions in two different lower eukaryotes.

The slime mould Physarum polycephalum contains 100 to 200 molecules of extrachromosomal linear DNA (PeDNA). Two sets of the 19S and 26S ribosomal genes are located on each molecule of PeDNA. In the nonmitotic phase of the cell cycle PeDNA is localised in the nucleolus. The molecules are maintained throughout vegetative growth. In order to study the signals responsible for its maintenance, PeDNA was purified and introduced into the two distantly related yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Surprisingly, intact PeDNA transforms both yeasts with high frequency and PeDNA sequences are maintained in the absence of selective pressure.

Chromosomes↗
PubMed1981

Nonspecific cleavage of phi X174 RFI deoxyribonucleic acid by bleomycin.

The covalently closed circular duplex deoxyribonucleic acid (DNA) of phi X174 underwent progressive conversion to nicked and linear DNA with increasing bleomycin/phi X174 RFI DNA molecule ratios. The formation of linear DNA (a double-strand break) occurred under limited reaction conditions as low as an average of 0.2 single-strand break/phi X174 RFI DNA molecule. As bleomycin-produced linear DNA was further fragmented by bleomycin, a broad distribution of DNA fragments without notable concentrations of unique size was formed. Restriction enzymes PstI and SstII did not generate discrete fragments from bleomycin-produced full-length linear phi X174 DNA, nor did bleomycin cleavage generate discrete fragments from HpaII or PstI digests of phi X174 RFI. These findings suggest that bleomycin does not act at a few specific sites on phi X174 RFI DNA. The single-strand nick appeared to be the preferred site for bleomycin action for a second cleavage in a phi X174 molecule.

Bacteriophage phi X 174↗
PubMed1984

Integration and expression of several molecular forms of Rous sarcoma virus DNA used for transfection of mouse cells.

To assess the factors required for integration and expression of retroviral DNA, we have examined viral DNA, RNA, and protein in NIH/3T3 mouse cells transformed by transfection with various forms of cloned Rous sarcoma virus (RSV) DNA. Linear RSV DNA molecules, derived from circular DNA containing two long terminal repeats (LTRs) and permuted by cleavage at the SacI restriction endonuclease site in the leader sequence, were integrated near the ends of the linear molecule, with the LTRs on the 3' side of the src gene. Integration of a subgenomic RSV DNA fragment containing the viral src gene without intact LTRs also occurred near the ends of the linear molecule. Head-to-tail tandem arrays of RSV DNA species were observed in some transformed cell lines that received fully digested DNA and in all cell lines that received DNA ligated to produce oligomers before transfection. Closed circular RSV DNA, with one or two LTRs, integrated without apparent specificity within several regions of the viral genome. After transfection with SacI-permuted RSV DNA still linked to arms of the lambda bacteriophage vector DNA, bacteriophage sequences were joined to host DNA. Transformed cell lines produced by transfection with the various forms of RSV DNA produced similar levels of viral src protein, although the efficiency of successful transformation varied by at least two orders of magnitude. Analyses of viral polyadenylated RNA, together with the patterns of viral DNA in transformed cells, indicated that viral DNA can be integrated and expressed without regard to LTR sequences, with adjacent host DNA presumably supplying signals required for the promotion and processing of functional src mRNA.

Animals↗
PubMed1986

Adriamycin-mediated introduction of a limited number of single-strand breaks into supercoiled DNA.

It was reported previously that Adriamycin converts form I covalently closed circular, supercoiled bacteriophage PM2 DNA to the relaxed circular form II DNA; no form III linear DNA was produced as a result of the extracellular action of Adriamycin in the presence of NADH-dehydrogenase. When form II DNA, produced by the action of Adriamycin, was treated with the BAL 31 nuclease, a single sharp DNA band after agarose gel electrophoresis indicated the presence of only full-length linear form III DNA. As one of its activities, the BAL 31 nuclease introduces a single-strand break in the complementary strand opposite a preexisting single-strand break. When form II DNA, produced by the action of gamma irradiation, was reacted with the BAL enzyme, the resulting linear DNA molecules exhibited a broad range of molecular weights, indicating the presence of many single-strand breaks in the substrate form II DNA. When the Adriamycin-produced form II DNA was treated with restriction endonucleases that cleave PM2 DNA at a single site, either with or without pretreatment with the BAL enzyme, the formation of only full-length linear DNA was observed. Thus, the drug is capable of introducing one or only a very limited number of single-strand breaks into supercoiled DNA; furthermore, these breaks are introduced at random sites along the DNA molecules.

DNA Damage↗
PubMed2001

Structural alterations of gene complexes by cystic fibrosis sputum.

Gene complexes with optimal physicochemical characteristics for cystic fibrosis (CF) gene therapy in vitro may become inactive in vivo as a result of destruction upon interaction with CF mucus. Therefore, we examined in this study to what extent main sputum components (linear DNA, mucin, phosphatidylcholine, phosphatidylglycerol, and albumin) may disintegrate lipoplexes. We found that mixing linear DNA with lipoplexes, in concentration ratios as occurs in the mucus of patients with CF in clinical studies with lipoplexes, drastically altered the surface charge and size of our lipoplexes and resulted in the liberation of plasmid DNA from the lipoplexes. These concentration ratios occur in vivo when the DNA concentration in the sputum becomes > 2.7 mg/ml, a quite realistic concentration even in patients without acute exacerbations. Lipoplexes brought in contact with native CF sputa at clinically relevant concentration ratios dissociated when the DNA concentration in the sputa was > 2.7 mg/ml. However, when the linear DNA was degraded by recombinant human deoxyribonuclease I before lipoplexes were added, the linear DNA did not cause any dissociation of the lipoplexes. Addition of albumin and mucin to the lipoplexes in a clinically relevant concentration ratio changed the surface charge of the lipoplexes to negative, however, without release of plasmid DNA. Mucin, dipalmitoylglycerophosphocholine, and dipalmitoylglycerophosphoglycerol did not cause any change in lipoplex properties at clinically relevant concentration ratios.

Albumins↗
PubMed1981

Replication of linear mitochondrial DNA from Paramecium: sequence and structure of the initiation-end crosslink.

Replication of the 14-micrometer linear Paramecium mitochondrial DNA is initiated by a crosslinking of the duplex strands at the initiation end of the molecule. As a consequence of the crosslink, a head-to-head dimer molecule, or palindrome, is a replicative intermediate. The central region of the dimer molecules of two species was cloned and sequenced, In the distal regions, the sequence is palindromic as expected; however, in the central region, there is a nonpalindromic sequence that is rich in A + T and contains direct tandem repeats. It is proposed that the nonpalindromic sequence constitutes the crosslink. Implications for the replication scheme are discussed. The model is unlike any other for a linear DNA.

Animals↗
PubMed1978

Structural evidence on DNA carcinogen interactions. N-acetoxy-N-2acetylaminofluorene binding to DNA.

Linear dichroism (LD) gives useful information on the interaction between DNA and the directly acting carcinogen N-acetoxy-N-2acetylaminofluorene (AAAF). In 50% methanol solvent with low ionic strength only a weak complex (van der Waals) appears. However, above 40 degrees C strand separation takes place and a covalent aminofluorene complex forms. After renaturation a characteristic positive LD band is observed at 306 nm. The average angular orientation of the long-axis of the fluorene moiety (47 degrees to the local helix axis) is inconsistent with intercalation. It can be explained for instance by a free rotation around a C(DNA)-N(aminofluorene) bond or by a major groove site. The occupation density was 1--2 aminofluorene residues per 100 bases. With native DNA, AAAF slowly forms a covalent complex which has a negative LD at 307 nm. The orientation (70--90 degrees) is consistent with steric direction by the strand.

Acetoxyacetylaminofluorene↗
PubMed2002

Laterally stabilized complexes of DNA with linear reducible polycations: strategy for triggered intracellular activation of DNA delivery vectors.

Target-specific DNA delivery requires vectors that combine stability in the biological milieu, receptor-mediated uptake into target cells, and intracellular activation to mediate transgene expression. This is achieved here using polymer-coated vectors based on plasmid DNA complexed with a reductively degradable polycation (RPC), designed for intercellular degradation. The RPC were prepared by oxidation of the terminal cysteinyl thiol groups of Cys(Lys)10Cys. The complexes were coated and surface-cross-linked using multivalent reactive copolymers of N-(2-hydroxypropyl)methacrylamide (PHPMA), providing a unique combination of steric and reversible lateral stabilization, known to promote extended circulation in the bloodstream. Coated complexes containing RPC exhibited lateral stabilization that was reversible by treatment with 2.5 mM dithiothreitol, releasing free DNA after incubation with a polyanion. In contrast, coated complexes containing nonreducible poly(l-lysine) (PLL) were not destabilized by reduction. The biological usefulness of this trigger mechanism was examined by measuring transfection activity in human retinoblast 911 cells of coated complexes, based on PLL or RPC, targeted to cell surface receptors by covalent linkage of basic fibroblast growth factor. The levels of transgene expression observed for RPC-based targeted vectors indicated efficient intracellular activation, authenticating the concept that lateral stabilization introduced by surface coating with PHPMA can be reversed by intracellular reduction.

Biotransformation↗
PubMed1998

Basic and acidic regions flanking the HMG domain of maize HMGa modulate the interactions with DNA and the self-association of the protein.

The maize HMGa protein is a typical member of the family of plant chromosomal HMG1-like proteins. The HMG domain of HMGa is flanked by a basic N-terminal domain characteristic for plant HMG1-like proteins, and is linked to the acidic C-terminal domain by a short basic region. Various derivatives of the HMGa protein were expressed in Escherichia coli and purified. The individual HMG domain can functionally complement the defect of the HU-like chromatin-associated Hbsu protein in Bacillus subtilis. The basic N-terminal domain which contacts DNA enhances the affinity of the protein for linear DNA, whereas it has little effect on the structure-specific binding to DNA minicircles. The acidic C-terminal domain reduces the affinity of HMGa for linear DNA, but does not affect to the same extent the recognition of DNA structure which is an intrinsic property of the HMG domain. The efficiency of the HMGa constructs to facilitate circularization of short DNA fragments in the presence of DNA ligase is like the binding to linear DNA altered by the basic and acidic domains flanking the HMG domain, while the supercoiling activity of HMGa is only slightly influenced by the same regions. Both the basic N-terminal and the acidic C-terminal domains contribute directly to the self-association of HMGa in the presence of DNA. Collectively, these findings suggest that the intrinsic properties of the HMG domain can be modulated within the HMGa protein by the basic and acidic domains.

Animals↗
PubMed1979

DNase induced after infection of KB cells by herpes simplex virus type 1 or type 2. II. Characterization of an associated endonuclease activity.

Purified preparations of the "exonuclease" specified by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) possess an endonuclease activity. The exonuclease and endonuclease activities copurify and cosediment in a sucrose density gradient. Endonuclease activity is only observed in the presence of a divalent cation, and Mg(2+) or Mn(2+) is equally effective as a cofactor with an optimal concentration of 2 mM. A slight amount of endonuclease activity is observed in the presence of Ca(2+), whereas no activity occurs in the presence of Zn(2+). In the presence of Mg(2+), Ca(2+) and Zn(2+) are inhibitory. Comparison of exonuclease and endonuclease activity in the presence of various divalent cations revealed that, at concentrations of Mn(2+) greater than 1 mM, only endonuclease activity occurs whereas endonuclease and exonuclease activity occur at all concentrations of Mg(2+). The endonuclease was affected by putrescine and spermidine to the same extent as the exonuclease activity, but in marked contrast the endonuclease was inhibited by a 10-fold-lower concentration of spermine compared to the exonuclease. The activity specified by HSV-1 and HSV-2 has very similar properties. HSV-1 and HSV-2 endonuclease cleave covalently closed circular DNA to yield, firstly, nicked circles and then linear DNA which is subsequently hydrolyzed to small oligonucleotides. Cleavage does not appear to be base sequence specific. Conversion of nicked circles to linear DNA and subsequent degradation of linear DNA occurs more rapidly in the presence of Mg(2+) than Mn(2+) presumably by virtue of the presence of the exonuclease activity. Nonsuperhelical covalently closed circular duplex DNA is cleaved by the endonucleases at a rate 60 times slower than the rate observed on the supercoiled form. These data indicate that the HSV-1 and HSV-2 endonuclease preferentially recognize single-stranded DNA regions.

Cations, Divalent↗
PubMed2006

Directed evolution of extradiol dioxygenase by a novel in vivo DNA shuffling.

RecA-dependent homologous recombination in Escherichia coli is a very effective way to construct chimeras between two homologous genes. The disadvantage of in vivo method is a small library size of chimeric genes in comparison with in vitro DNA shuffling. In order to overcome the disadvantage, we have developed novel in vivo DNA shuffling methods with successive homologous recombinations. Linearized DNA molecules with two homologous genes were made with ligation rather than the conventional restriction enzyme cleavage between two genes. The three-way ligation of a vector and two homologous bphC genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenases or the two-way ligation of the donor bphC gene and an acceptor plasmid carrying the homologous bphC gene generated a variety of linearized DNA molecules. The homologous recombination between the genes on the linearized DNA molecules created the large chimeric bphC gene libraries in a recBC sbcA E. coli strain. After three rounds of recombinations, chimeric bphC genes with four-part gene fragments by triple-crossover were easily obtained. By employing a 96-well microtiter plate high-throughput screening, thermally stable chimeric 2,3-dihydroxybiphenyl 1,2-dioxygenases were selected from chimeric bphC gene libraries. This opens up a new way for directed evolution of proteins in vivo.

DNA Shuffling↗