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At least 1,135 records · Page 63Linked to original sources
PubMed1979

Heteroduplexes of phiX174 and G4 DNAs: orientation to genetic map and comparison with predictions from nucleotide sequences.

Heteroduplexes between the viral DNA of phiX174 and DNA from the replicative form (RF) of phage G4 were examined by electron microscopy. The single Eco RI site of G4-RF was utilized as a physical marker by preparing the heteroduplexes from the denatured, linear DNA obtained by restricting G4-RF with Eco RI endonuclease. Restriction fragments of phiX were used in a separate series of heteroduplexes to align the heteroduplex map and the G4 Eco RI site with the similar genetic maps of the two phages. The positions of the branch migrating junctions of recombinant phiX-G4 figure-8s, previously located only with respect to the G4-Eco RI site, have now been located with high proability within the gene A region of the two genomes. The degree of mismatch between the known nucleotide sequences of phi X and G4 accounts for positions of all of the regions of single-strandedness in the observed heteroduplexes, but unexplained discrepancies were also found.

Base Sequence↗
PubMed1990

The monomer covalently closed linear replicative form DNA is an intermediate of Aleutian disease parvovirus DNA replication.

In this report we present data indicating that the recently detected monomer covalently closed linear replicative form DNA (Mccl RF DNA) is an intermediate of Aleutian disease virus (ADV) DNA replication. This DNA molecule is characterized by covalently closed terminal hairpins on either end of the linear ds genomic molecule. Its first detection early after infection in vitro, the association with ADV-specific replication complexes and the de novo synthesis of Mccl RF DNA by isolated replication complexes point to an important role in parvoviral DNA replication. The presence of the Mccl RF DNA in extracts of virus-producing bone marrow cells of naturally infected mink rules out the possibility that this DNA form represents a mere cell culture artefact. The detection of the ADV non-structural protein 1 (NS1) in replication complexes supports the view that this protein is involved in ADV DNA replication.

Aleutian Mink Disease Virus↗
PubMed2006

Large-scale, nonchromatographic purification of plasmid DNA.

A large-scale approach to the purification of plasmid DNA has been developed that overcomes many of the limitations of current chromatography-based processes. The process consists of a scaleable lysis using recombinant lysozyme and a rapid heating and cooling step followed by a selective precipitation with cetyltrimethylammonium bromide (CTAB). Calcium silicate batch adsorption is then utilized to remove residual genomic DNA, linear plasmid, open circular plasmid, endotoxin, detergents, and proteins. Finally, a concentration and diafiltration step utilizing ultrafiltration and a terminal sterile filtration complete the process. The final product exceeds the requirements for clinical-grade plasmid DNA, and the process has been scaled up to yield an average of 18 +/- 4 g (over five lots) of pharmaceutically pure plasmid DNA per 140 L of lysate (from approx 1.3 kg Escherichia coli dry cell weight).

Calcium Compounds↗
PubMed1993

DNA topology and a minimal set of basal factors for transcription by RNA polymerase II.

Immunoglobulin heavy chain (IgH) gene transcription in vitro can be reconstituted with a minimal reaction containing only TATA-binding protein (TBP), TFIIB, and RNA polymerase II (pol II) when the template is negatively supercoiled. Transcription from linear DNA templates containing either the IgH or the adenovirus major late promoters (MLPs) requires in addition TFIIF, TFIIE, TFIIH, and a fraction containing TFIIA and TFIIJ. Promoters vary in their activities in the minimal reaction. Initiation at the adenovirus MLP site was not observed in this reaction, even with templates containing negative superhelical density. When only TBP, TFIIB, and pol II were present in the reaction, the more negatively supercoiled the IgH template DNA was, the more active the transcription. It is suggested that the free energy of supercoiling promotes the formation of an open complex for initiation of transcription by the minimal set of transcription factors.

Cloning, Molecular↗
PubMed2000

[Glucose isomerase gene knock-out by denatured double-stranded DNA].

After Genetic background analysis of Streptomyces diastaticus No. 7 strain M1033, the modified conditions of M1033 protoplasts and transformation were established. Replacement plasmid for homologous recombination was also constructed by inserting tsr gene into glucose isomerase gene. The homologous recombination of GI gene in M1033 chromosomes was achieved by using denatured linearized DNA fragments and glucose isomerase deficient strain M1033LJ was obtained. It is basic for introducing mutation into M1033 chromosome and realizing site-directed molecular reformation.

Aldose-Ketose Isomerases↗
PubMed2005

Formation of linear inverted repeat amplicons following targeting of an essential gene in Leishmania.

Attempts to inactivate an essential gene in the protozoan parasite Leishmania have often led to the generation of extra copies of the wild-type alleles of the gene. In experiments with Leishmania tarentolae set up to disrupt the gene encoding the J-binding protein 1 (JBP1), a protein binding to the unusual base beta-D-glucosyl-hydroxymethyluracil (J) of Leishmania, we obtained JBP1 mutants containing linear DNA elements (amplicons) of approximately 100 kb. These amplicons consist of a long inverted repeat with telomeric repeats at both ends and contain either the two different targeting cassettes used to inactivate JBP1, or one cassette and one JBP1 gene. Each long repeat within the linear amplicons corresponds to sequences covering the JBP1 locus, starting at the telomeres upstream of JBP1 and ending in a approximately 220 bp sequence repeated in an inverted (palindromic) orientation downstream of the JBP1 locus. We propose that these amplicons have arisen by a template switch inside a DNA replication fork involving the inverted DNA repeats and helped by the gene targeting.

Alleles↗
PubMed2000

Ku entry into DNA inhibits inward DNA transactions in vitro.

Association of the DNA end-binding Ku70/Ku80 heterodimer with the 460-kDa serine/threonine kinase catalytic subunit forms the DNA-dependent protein kinase (DNA-PK) that is required for double-strand break repair by non-homologous recombination in mammalian cells. Recently, we have proposed a model in which the kinase activity is required for translocation of the DNA end-binding subunit Ku along the DNA helix when DNA-PK assembles on DNA ends. Here, we have questioned the consequences of Ku entry into DNA on local DNA processes by using human nuclear cell extracts incubated in the presence of linearized plasmid DNA. As two model processes, we have chosen nucleotide excision repair (NER) of UVC DNA lesions and transcription from viral promoters. We show that although NER efficiency is strongly reduced on linear DNA, it can be fully restored in the presence of DNA-PK inhibitors. Simultaneously, the amount of NER proteins bound to the UVC-damaged linear DNA is increased and the amount of Ku bound to the same DNA molecules is decreased. Similarly, the poor transcription efficiency exhibited by viral promoters on linear DNA is enhanced in the presence of DNA-PK inhibitor concentrations that prevent Ku entry into the DNA substrate molecule. The present results show that DNA-PK catalytic activity can regulate DNA transactions including transcription in the vicinity of double-strand breaks by controlling Ku entry into DNA.

Androstadienes↗
PubMed1993

SSV1-encoded site-specific recombination system in Sulfolobus shibatae.

We present evidence for the existence of a conservative site-specific recombination system in Archaea by demonstrating integrative recombination of Sulfolobus shibatae virus SSV1 DNA with the host chromosome, catalysed by the SSV1-encoded integrase in vitro. The putative int gene of SSV1 was expressed in Escherichia coli yielding a protein of about 39 kDa. This protein alone efficiently recombined linear DNA substrates containing chromosomal (attA) and viral (attP) attachment sites; recombination with either negatively or positively supercoiled SSV1 DNA was less efficient. Intermolecular attA x attA and attP x attP recombination was also promoted by the SSV integrase. The invariant 44 bp "common attachment core" present in all att sites contained sufficient information to allow recombination, whilst the flanking sequences effected the efficiency. These features clearly distinguish the SSV1--encoded site--specific recombination system from others and make it suitable for the study of regulatory mechanisms of SSV1 genome--host chromosome interaction and investigations of the evolution of the recombination machinery.

Bacteriophages↗
PubMed1998

Episomal and integrated maintenance of foreign DNA in Giardia lamblia.

Giardia lamblia is an early diverging eukaryote which causes gastrointestinal disease throughout the world. Different subgroups of Giardia have been defined based on several biochemical and genetic criteria. We have developed a method for stably introducing DNA into the nuclei of the parasite using puromycin acetyltransferase (pac) as a dominant selectable marker. Transfected circular DNAs were maintained as episomes in the isolate WB, a representative of one Giardia subgroup. When input DNAs were linearized, integration was observed to occur by homologous recombination producing gene replacements in this isolate. In isolate GS, which represents a different subgroup, both linear and circular transfected DNAs were integrated into the genome by homologous recombination. In GS, linear DNA again produced gene replacements, while circular DNA produced duplicative integration events. The failure of GS to replicate episomes may reflect differences in the structure or recognition of DNA replication origins between these subgroups. A plasmid shuttle vector was also developed for expression of other genes in Giardia lamblia. Utilizing the green fluorescent protein as a reporter gene in the WB isolate, we show that gene expression from this vector correlated with plasmid copy number over a range of two orders of magnitude. Together these tools should greatly enhance our ability to study both the basic biology and the pathogenesis of this ubiquitous parasite.

Acetyltransferases↗
PubMed2003

Generation of an Escherichia coli lysA targeted deletion mutant by double cross-over recombination for potential use in a bacterial growth-based lysine assay.

AIMS: To generate a stable Escherichia coli lysine auxotroph for the lysine bioavailability assay. METHODS AND RESULTS: An E. coli lysine auxotrophic strain was constructed by deleting the entire lysA gene and replacing it with a gene that confers resistance to ampicillin (bla). The linear DNA contained 50 bp homologous sequence of upstream of lysA in one end and 50 bp of downstream of lysA in the other end. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The deltalysA::bla strain exhibited a linear response to lysine supplementation and can be used for quantifying lysine.

Ampicillin Resistance↗
PubMed1998

Identification of an endonuclease secreted by human B lymphoblastic IM9 cells.

We have identified a Mg(2+)-dependent endonuclease from IM9 cell lysates and culture medium using DNA-native-polyacrylamide gel electrophoresis (DNA-native-PAGE) nuclease assay system. This particular endonuclease activity was not detectable in conventional DNA-SDS-polyacrylamide gel electrophoresis assay system which is similar to the method originally described by Rosenthal and Lacks (A.L. Rosenthal and S.A. Lacks, Anal. Biochem. 80 (1977) 76-90). Experimental results clearly demonstrated that the endonuclease activity was not derived from the fetal calf serum in which the cells were grown, but synthesized in the cell and secreted into the culture medium by IM9 cells. Biosynthesis and subsequent release of the endonuclease into the culture medium were significantly decreased by pretreatment of the cells with actinomycin D. Using supercoiled plasmid DNA as a substrate, the endonuclease activity was determined with the enzyme isolated from the cell culture medium by native-PAGE electroelution. The endonuclease, with Mg2+ alone, was able to catalyze the conversion of the plasmid into linear DNA followed by further degradation. This is the first report demonstrating that a distinct Mg(2+)-dependent endonuclease is secreted by a human immune cell line.

Burkitt Lymphoma↗
PubMed2006

Chromatin structure can strongly facilitate enhancer action over a distance.

Numerous DNA transactions in eukaryotic nuclei are regulated by elements (enhancers) that can directly interact with their targets over large regions of DNA organized into chromatin. The mechanisms allowing communication over a distance in chromatin are unknown. We have established an experimental system allowing quantitative analysis of the impact of chromatin structure on distant transcriptional regulation. Assembly of relaxed or linear DNA templates into subsaturated chromatin results in a strong increase of the efficiency of distant enhancer-promoter E-P communication and activation of transcription. The effect is directly proportional to the efficiency of chromatin assembly and cannot be explained only by DNA compaction. Transcription activation on chromatin templates is enhancer- and activator-dependent, and must be accompanied by direct E-P interaction and formation of a chromatin loop. Previously we have shown that DNA supercoiling can strongly facilitate E-P communication on histone-free DNA. The effects of chromatin assembly and DNA supercoiling on the communication are quantitatively similar, but the efficiency of enhancer action in subsaturated chromatin does not depend on the level of unconstrained DNA supercoiling. Thus chromatin structure per se can support highly efficient communication over a distance and functionally mimic the supercoiled state characteristic for prokaryotic DNA.

Animals↗
PubMed1969

Electron microscopy of the circular kinetoplastic DNA from Trypanosoma cruzi: occurrence of catenated forms.

Circular kinetoplastic DNA was fractionated from Trypanosoma cruzi DNA by Hg-Cs(2)SO(4) gradient. Covalantly closed circular DNA was isolated by buoyant density centrifugation in ethidium bromide CsCl density gradients. Electron microscope studies have shown that kinetoplastic DNA molecules are in the form of open and closed circles of contour length 0.45 mu, the smallest known microbial DNA. DNA molecules are also in the form of catenanes consisting of two or more topologically interlocked circular units of the monomer size 0.45 mu. Associations of numerous circles have also been described and confirmed by band sedimentation. Long linear DNA molecules were also seen, their size ranging between 2 and 12 mu. These molecules are free or are attached to associations of circles.

DNA↗
PubMed1984

PMAP, PMAPS: DNA physical map constructing programs.

Computer programs are described, which facilitate the construction of the restriction site (physical) map of DNA molecules. By knowing the length of each fragment and its degree of error in the single and the double restriction enzyme digestions, the programs give all the possibilities for the physical map. This method is applicable to linear DNA molecules. Several examples are presented which indicate the high efficiency of the programs in constructing restriction site maps for the 62 Kb chromosome of bacteriophage 16-3. We have constructed complex maps (i.e. EcoRI map with 16 and EcoRV with 11 fragments).

Base Sequence↗
PubMed2002

Effects of torsional strain on thermal denaturation of DNA.

A class of simple statistical mechanical models for DNA melting, first proposed by Poland and Scheraga, has been demonstrated to exhibit a first or second order thermodynamic singularity, notwithstanding the intrinsic one-dimensional nature of the problem. In the present paper we consider the case of circular DNA and show that the inclusion of twist elastic energy in the Poland-Scheraga models leads either to suppression of the thermodynamic singularity or to a weak, third order singularity. Such behavior may also be present in linear DNA under mechanical influences that preclude the release of torsional strain.

Journal Article↗
PubMed2004

Investigation of a novel DNase of Streptococcus suis serotype 2.

A secreted nuclease, SsnA, was identified in the virulent Streptococcus suis isolate SX332 and subsequently in each of the type strains of capsular serotypes 1 through 9. Screening of 258 porcine clinical isolates from surface (nasal mucosa or palatine tonsil) or internal (joint, brain or other internal organ) locations revealed a significant relationship (P < 0.001) between expression of nuclease and isolation from an internal site. A 3,126-bp gene, ssnA, was identified from a phenotypically nuclease-negative pGh9:ISS1 insertion mutant, and analysis of the predicted SsnA sequence revealed a 35-amino-acid (aa) secretion signal sequence, a 22-aa DNA-binding domain, and a typical gram-positive cell wall sorting motif. A requirement of Ca2+ and Mg2+ for SsnA activity was determined, and the substrate specificity was found to be for single- and double-stranded linear DNA. Reverse transcription-PCR experiments revealed that ssnA is expressed throughout all stages of S. suis growth, and Western blots with porcine anti-S. suis immune sera against a recombinant, truncated SsnA derivative (rSsnADelta) confirmed that SsnA is expressed in vivo. Furthermore, anti-rSsnADelta antibodies were sufficient to neutralize SsnA activity. Analyses of subcellular fractions of SX332 and derived mutants, on DNA-containing polyacrylamide gels and by Western blotting, suggest that SsnA is cell wall located.

Amino Acid Sequence↗
PubMed2006

Single-molecule measurements of trapped and migrating circular DNA during electrophoresis in agarose gels.

The effect of agarose gel concentration and field strength on the electrophoretic trapping of open (relaxed) circular DNA was investigated using microscopic measurements of individual molecules stained with a fluorescent dye. Three open circles with sizes of 52.5, 115, and 220 kbp were trapped by the electric field (6 V/cm) and found to be predominately fixed and stretched at a single point in the gel. The length of the stretched circles did not significantly change with agarose concentration of the gels (mass fractions of 0.0025, 0.01, and 0.02). The relaxation kinetics of the trapped circles was also measured in the gels. The relaxation of the large open circles was found to be a slow process, taking several seconds. The velocity and average length of the 52.5 kbp open circles and 48.5 kbp linear DNA were measured during electrophoresis in the agarose gels. The velocity increased when the agarose concentrations were lowered, but the average length of the open-circle DNA (during electrophoresis) did not significantly change with agarose gel concentrations. The circles move through the gels by cycles of stretching and relaxation during electrophoresis. Linear dichroism was also used to investigate the trapping and alignment of the 52.5 kbp open circles. The results in this study provide information that can be used to improve electrophoretic separations of circular DNA, an important form of genetic material and commonly used to clone DNA.

DNA, Circular↗
PubMed2006

Simple model for DNA adsorption onto a mica surface in 1:1 and 2:1 electrolyte solutions.

We propose a simple theory of interactions between like-charged polyelectrolyte and a surface based on a mean-field Derjaguin-Landau-Verwey-Overbeek approach. It predicts that the van der Waals attractive interactions are responsible for irreversible physisorption of polyelectrolytes onto charged surfaces. We show that monovalent salts contribute significantly to repulsive interactions, while enhancing the attraction very slightly. The effect of the divalent counterions is reverse. Therefore, to achieve the adsorption, the overall repulsion due to 1:1 electrolyte should be counterbalanced by the stronger van der Waals attraction due to the presence of doubly charged counterions in solution. The theory has been validated experimentally against its ability to predict the minimum polymer/surface interaction energy required for the adsorption using DNA/mica in NaCl, MgCl2, and NiCl2 solutions as a test system. The theory explains the mechanism of linear DNA adsorption to a mica surface for different solvent compositions and can be used as a tool for predicting the optimum conditions for AFM experiments on linear polymer systems. The model can also be used to make general conclusions on the conformation of polymer molecules on a surface. We have shown for the DNA/mica surface system that when the adsorption of DNA is mostly governed by long-range van der Waals forces the molecule adopts an ideal 2D conformation. When the adsorption is mostly due to short-range ion-correlation forces, DNA will appear 3D --> 2D projected in agreement with experimental data.

Adsorption↗