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PubMedDate not supplied

[DNA compact form. VI. Changes of DNA secondary structure under conditions preceding its compaction in a solution].

Optical and thermochemical properties of E. coli DNA molecules are compared in solutions containing poly(ethyleneglycol) (PEG) in concentrations at which compactization is not yet observed. It is shown that under conditions preceding DNA compactization (CPEG less than 60 mg/ml) changes in CD spectra occur which suggest that the secondary structure of some DNA fragments is altered. These changes of the secondary structure result from dehydration of DNA molecules in PEG-containing solutions. Electron micrographs of DNA molecules obtained under conditions preceding compactization suggest that under these conditions linear DNA molecules may form "four-stranded" fragments as well as double-stranded "loops".

Circular Dichroism↗
PubMed1987

The anomalous gel migration of a stable cruciform: temperature and salt dependence, and some comparisons with curved DNA.

We have made an analysis of the gel electrophoretic properties of a pseudo-cruciform fragment, a linear DNA molecule containing a stable cruciform. The migration of this construct was analysed in polyacrylamide gels at a various temperatures in the range 5 degrees to 55 degrees C, and in the presence of NaCl, MgCl2 or ethidium bromide. The magnitude of the anomalous migration (retardation) was almost temperature independent up to 40 degrees C, but decreased strongly beyond this point, extrapolating to normal migration at 70 degrees C. Addition of salts reduced the anomaly. This took the form of a continuous reduction in anomalous migration with the addition of NaCl up to 60 mM, while with MgCl2 there was a sharp reduction in the anomaly to a constant value which is reached by 10 mM. Under these conditions, moreover, the migration of the fragment became almost temperature-independent over the entire range. These results have been interpreted to reflect the influence of ion binding at the four-way junction on the relative disposition of the cruciform arms. The detailed electrophoretic properties of the pseudo-cruciform are in marked contrast to those of sequence-directed curved DNA fragments. In particular, the response to the addition of 1 microgram/ml ethidium bromide offers a convenient method for distinguishing between anomalous retardation arising from curvature (greatly reduced anomaly) or a cruciform junction (enhanced anomaly).

Coliphages↗
PubMed1998

Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation.

We have developed a novel system for the sensitive detection of nptII genes (kanamycin resistance determinants) including those present in transgenic plant genomes. The assay is based on the recombinational repair of an nptII gene with an internal 10-bp deletion located on a plasmid downstream of a bacterial promoter. Uptake of an nptII gene by transformation restores kanamycin resistance. In Escherichia coli, promoterless nptII genes provided by electroporation were rescued with high efficiency in a RecA-dependent recombinational process. For the rescue of nptII genes present in chromosomal plant DNA, the system was adapted to natural transformation, which favours the uptake of linear DNA. When competent Acinetobacter sp. BD413 (formerly A. calcoaceticus) cells containing the mutant nptII gene on a plasmid were transformed with DNA from various transgenic plants carrying nptII as a marker gene (Solanum tuberosum, Nicotiana tabacum, Beta vulgaris, Brassica napus, Lycopersicon esculentum), kanamycin-resistant transformants were obtained roughly in proportion to the concentration of nptII genes in the plant DNA. The rescue of nptII genes occurred in the presence of a more than 6 x 10(6)-fold excess of plant DNA. Only 18 ng of potato DNA (2.5 x 10(3) genome equivalents, each with one copy of nptII) was required to produce one kanamycin-resistant transformant. These experiments and others employing DNA isolated from soil samples demonstrate that the system allows reliable and highly sensitive monitoring of nptII genes in transgenic plant DNA and in DNA from environmental sources, such as soil, without the need for prior DNA amplification (e.g. by PCR).

Acinetobacter↗
PubMed1994

Cleavage of supercoiled double-stranded DNA by several ribosome-inactivating proteins in vitro.

Several ribosome-inactivating proteins (RIPs), such as ricin (including its A-chain), luffin, cinnamomin and camphorin, were found to express enzymatic activity to cleave supercoiled double-stranded DNA. In particular, alpha-sarcin, a RIP with a novel ribonuclease activity, was first proved to have this activity. They convert supercoiled DNA into a nicked circular conformation at low concentrations and further into a linear form at high concentrations: they have no effect on linear DNA. Although intact type II RIPs exhibited no RNA N-glucosidase activity, they were detected to cleave supercoiled DNA. Even if ricin A-chain was treated by boiling, its activity on supercoiled DNA was largely retained.

Animals↗
PubMed1976

A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome.

Using the poly(dA-dT) "connector" method (Lobbanand Kaiser, 1973), a population of annealed hybrid circular DNAs was constructed in vitro; each hybrid DNA circle contained one molecule of poly(dT)-tailed Col El-DNA (LRI) annealed to any one of a collection of poly(dA)-tailed linear DNA fragments, produced originally by shearing total E. coli DNA to an average size of 8.5 x 10(6) daltons. This annealed DNA preparation (12 mug) was used to transform an F+ recA E. coli strain (JA200), selecting transformants by their resistance to colicin El. A collection or "bank" pf pver 2000 colicin El-resistant clones was thereby obtained, 70% of which were shown to contain hybrid Col El DNA (E. coli) plasmids. This colony bank is large enough to include hybrid plasmids representative of the entire E. coli genome. Individual plasmids have been readily identified by replica mating the collection onto plates seeded with cultures of various F- auxotrophic recipients, selecting for complementation of the auxotrophic markers by F-mediated transfer of hybrid plasmids to the F- recipients. In this manner, over 80 hybrid Col El-DNA (E. coli), plasmid-bearing clones have been identified in the colony bank, and about 40 known E. coli genes have been tentatively assigned to these various plasmids. The hybrid plasmids are transferred efficiently from F+ donors to appropriate F- recipients. The use of this method to establish similar colony banks in E. coli containing hybrid plasmids representative of various simple eucaryotic genomes is discussed.

Chromosome Mapping↗
PubMed1989

The proximity of DNA sequences in interphase cell nuclei is correlated to genomic distance and permits ordering of cosmids spanning 250 kilobase pairs.

The physical distance between DNA sequences in interphase nuclei was determined using eight cosmids containing fragments of the Chinese hamster genome that span 273 kb surrounding the dihydrofolate reductase (DHFR) gene. The distance between these sequences at the molecular level has been determined previously by restriction enzyme mapping (J.E. Looney and J.L. Hamlin, 1987, Mol. Cell Biol. 7: 569-577; C. Ma et al., 1988, Mol. Cell Biol. 8: 2316-2327). Fluorescence in situ hybridization was used to localize the DNA sequences in interphase nuclei of cells bearing only one copy of this genomic region. The distance between DNA sequences in interphase nuclei was correlated to molecular distance over a range of 25 to at least 250 kb. The observed relationship was such that genomic distance could be predicted to within 40 kb from interphase distance. The correct order of seven probes was derived from interphase distances measured for 19 pair-wise combinations of the probes. Measured distances between sequences approximately 200 kb apart indicate that the DNA is condensed 70- to 100-fold in hybridized nuclei relative to a linear DNA helix molecule. Cell lines with chromosome inversions were used to show that interphase distance increases with genomic distance in the 50-90 Mb range, but less steeply than in the 25-250 kb range.

Animals↗
PubMed2004

Scanning force microscopy of DNA translocation by the Type III restriction enzyme EcoP15I.

Type III restriction enzymes are multifunctional heterooligomeric enzymes that cleave DNA at a fixed position downstream of a non-symmetric recognition site. For effective DNA cleavage these restriction enzymes need the presence of two unmethylated, inversely oriented recognition sites in the DNA molecule. DNA cleavage was proposed to result from ATP-dependent DNA translocation, which is expected to induce DNA loop formation, and collision of two enzyme-DNA complexes. We used scanning force microscopy to visualise the protein interaction with linear DNA molecules containing two EcoP15I recognition sites in inverse orientation. In the presence of the cofactors ATP and Mg(2+), EcoP15I molecules were shown to bind specifically to the recognition sites and to form DNA loop structures. One of the origins of the protein-clipped DNA loops was shown to be located at an EcoP15I recognition site, the other origin had an unspecific position in between the two EcoP15I recognition sites. The data demonstrate for the first time DNA translocation by the Type III restriction enzyme EcoP15I using scanning force microscopy. Moreover, our study revealed differences in the DNA-translocation processes mediated by Type I and Type III restriction enzymes.

Adenosine Triphosphatases↗
PubMed1977

Mapping of sequences with 2-fold symmetry on the simian virus 40 genome: a photochemical crosslinking approach.

Sequences with 2-fold axes of symmetry have been detected and mapped on the simian virus 40 (SV40) genome by their ability to form hairpin turns in single-stranded SV40 DNA. Supercoiled SV40 DNA (SV40 I) was digested with restriction enzymes EcoRI and HpaII. The resulting linear DNA molecules with lengths of the complete SV40 genome were then denatured and photochemically reacted with 4,5',8-trimethylpsoralen (trioxsalen) at 16.0 +/- 0.5 degrees and different ionic strengths. Secondary structures on the single-stranded SV40 DNA were crosslinked and their positions analyzed by electron microscopy. There were no observable hairpin turns on the denatured SV40 DNA when it was photoreacted in 1 mM Tris-HCl/0.1 mM EDTA at pH 7.0. In 20 mM NaCl, one specific hairpin turn was detected at 0.17 +/- 0.02 map units on the map of EcoRI-digested SV40 DNA, where the 3' ends of early 19S mRNA, late 19S mRNA, and 16S mRNA of SV40 have been mapped. In 30 mM NaCl there are five more major hairpin turns besides the most stable one. The centers of four of these specific hairpin turns were mapped at 0.26 +/- 0.02, 0.68 +/- 0.03, 0.84 +/- 0.02, and 0.94 +/- 0.01 units on the map of EcoRI-digested SV40. The fifth one is at or near the unique EcoRI cleavage site on SV40 DNA. The possible functions of these sequences are discussed in terms of the nature of the promoter sites, the replication origin, the processing of RNA precursors, and regulation at the translational level.

Base Sequence↗
PubMed1999

The structure of a chromosomal high mobility group protein-DNA complex reveals sequence-neutral mechanisms important for non-sequence-specific DNA recognition.

The high mobility group (HMG) chromosomal proteins, which are common to all eukaryotes, bind DNA in a non-sequence-specific fashion to promote chromatin function and gene regulation. They interact directly with nucleosomes and are believed to be modulators of chromatin structure. They are also important in V(D)J recombination and in activating a number of regulators of gene expression, including p53, Hox transcription factors and steroid hormone receptors, by increasing their affinity for DNA. The X-ray crystal structure, at 2.2 A resolution, of the HMG domain of the Drosophila melanogaster protein, HMG-D, bound to DNA provides the first detailed view of a chromosomal HMG domain interacting with linear DNA and reveals the molecular basis of non-sequence-specific DNA recognition. Ser10 forms water-mediated hydrogen bonds to DNA bases, and Val32 with Thr33 partially intercalates the DNA. These two 'sequence-neutral' mechanisms of DNA binding substitute for base-specific hydrogen bonds made by equivalent residues of the sequence-specific HMG domain protein, lymphoid enhancer factor-1. The use of multiple intercalations and water-mediated DNA contacts may prove to be generally important mechanisms by which chromosomal proteins bind to DNA in the minor groove.

Amino Acid Sequence↗
PubMed1992

Evidence for cross-linking DNA by bis-intercalators with rigid and extended linkers is provided by knotting and catenation.

A new series of DNA bis-intercalators is reported in which acridine moieties are connected by rigid and extended pyridine-based linkers of varied length. Cross-linking of DNA by bis-intercalation is inferred from the unwinding and folding of linear DNA induced by the compounds; after ligation and removal of the bis-intercalator, superhelical circles, catenanes and knots that bear a residual imprint of the bis-intercalator are observed. These novel bis-intercalators are of interest because they can be used to probe the spatial organization of DNA, especially near sites of replication, recombination or topoisomerase action where two duplexes must be in close proximity. Preliminary results on the effects of the various compounds on the cloning efficiency of bacteria and replication by permeabilized human cells are also presented.

Acridine Orange↗
PubMed1999

Identification of an essential proximal sequence element in the promoter of the telomerase RNA gene of Tetrahymena thermophila.

Telomerase is a ribonucleoprotein reverse transcriptase that synthesizes and maintains telomeric DNA. Studies of telomeres and telomerase are facilitated by the large number of linear DNA molecules found in ciliated protozoa, such as Tetrahymena thermophila. To examine the expression of telomerase, we investigated the transcription of the RNA polymerase III-directed gene encoding the RNA subunit (TER1) of this enzyme. A chimeric gene containing the Glaucoma chattoni TER1 transcribed region flanked by 5' and 3' Tetrahymena regions was used to identify promoter elements following transformation of Tetrahymena cells. Disruption of a conserved proximal sequence element (PSE) located at -55 in the Tetrahymena TER1 5' flanking region eliminated expression of the chimeric gene. In addition, mutation of an A/T-rich element at -25 decreased expression markedly. A gel mobility shift assay and protein-DNA cross-linking identified a PSE-binding polypeptide of 50-60 kDa in Tetrahymena extracts. Gel filtration analysis revealed a native molecular mass of approximately 160 kDa for this binding activity. Our results point to a similar architecture between ciliate telomerase RNA and metazoan U6 small nuclear RNA promoters.

Animals↗
PubMed1988

Repair of DNA-containing pyrimidine dimers.

Ultraviolet light-induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. The most direct biochemical mechanism responding to this kind of genotoxicity involves direct photoreversal by flavin enzymes that specifically monomerize pyrimidine:pyrimidine dimers monophotonically in the presence of visible light. Incision reactions are catalyzed by a combined pyrimidine dimer DNA-glycosylase:apyrimidinic endonuclease found in some highly UV-resistant organisms. At a higher level of complexity, Escherichia coli has a uvr DNA repair system comprising the UvrA, UvrB, and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway, which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding is associated with localized topological unwinding of DNA. This same protein complex can catalyze an ATPase-dependent 5'----3'-directed strand displacement of D-loop DNA or short single strands annealed to a single-stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled excision-resynthesis step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress-induced protease that also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA, it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.

DNA↗
PubMed2000

Vesicle-mediated transfer of virulence genes from Escherichia coli O157:H7 to other enteric bacteria.

Membrane vesicles are released from the surfaces of many gram-negative bacteria during growth. Vesicles consist of proteins, lipopolysaccharide, phospholipids, RNA, and DNA. Results of the present study demonstrate that membrane vesicles isolated from the food-borne pathogen Escherichia coli O157:H7 facilitate the transfer of genes, which are then expressed by recipient Salmonella enterica serovar Enteritidis or E. coli JM109. Electron micrographs of purified DNA from E. coli O157:H7 vesicles showed large rosette-like structures, linear DNA fragments, and small open-circle plasmids. PCR analysis of vesicle DNA demonstrated the presence of specific genes from host and recombinant plasmids (hly, L7095, mobA, and gfp), chromosomal DNA (uidA and eaeA), and phage DNA (stx1 and stx2). The results of PCR and the Vero cell assay demonstrate that genetic material, including virulence genes, is transferred to recipient bacteria and subsequently expressed. The cytotoxicity of the transformed enteric bacteria was sixfold higher than that of the parent isolate (E. coli JM109). Utilization of the nonhost plasmid (pGFP) permitted the evaluation of transformation efficiency (ca. 10(3) transformants microg of DNA(-1)) and demonstrated that vesicles can deliver antibiotic resistance. Transformed E. coli JM109 cells were resistant to ampicillin and fluoresced a brilliant green. The role vesicles play in genetic exchange between different species in the environment or host has yet to be defined.

Animals↗
PubMed1999

Comparative characterization of rep proteins from the helper-dependent adeno-associated virus type 2 and the autonomous goose parvovirus.

Adeno-associated viruses (AAVs) are nonautonomous human parvoviruses in that they are dependent on helper functions supplied by other viruses or on genotoxic stimuli for conditions permissive for replication. In the absence of helper, AAV type 2 enters latency by integration into a specific site on human chromosome 19. This feature of AAV, in combination with a lack of pathogenicity, makes AAV an attractive candidate vector for human gene therapy. Goose parvovirus (GPV) is both autonomous and pathogenic yet is highly homologous to AAV. To address the molecular bases for the different viral lifestyles, we compare the AAV and GPV nonstructural proteins, Rep78 and Rep1, respectively. We find that Rep78 and Rep1 possess several biochemical activities in common, including (i) high-affinity DNA binding for sequences that constitute the minimal DNA replication origin; (ii) nucleoside triphosphate-dependent DNA helicase activity; and (iii) origin-specific replication of double-stranded linear DNA. These experiments also establish a specific 38-bp DNA sequence as the minimal GPV DNA replication origin. It is noteworthy that although the proposed Rep binding sites of GPV and AAV are highly similar, Rep1 and Rep78 show a high degree of specificity for their respective origins, in both binding and replication assays. One significant difference was observed; with the minimal replication origin in adenovirus-uninfected extracts, Rep78-mediated replication exhibited low processivity, as previously reported. In contrast, Rep1 efficiently replicated full-length template. Overall, our studies indicate that GPV Rep1 and AAV Rep78 support a comparable mode of replication. Thus, a comparison of the two proteins provides a model system with which to determine the contribution of Rep in the regulation of dependence and autonomy at the level of DNA replication.

Animals↗
PubMed1988

[Specificity of fragmentation of DNA from pBR322 plasmid by Ca,Mg-dependent endonuclease from cell nuclei of human lymphocytes].

Fragmentation of the plasmid pBR322 DNA by a purified preparation of Ca/Mg-dependent endonuclease has been studied. It was shown that on the first steps of reaction the double-stranded cuts are introduced into the superhelical DNA independent of singlestranded ones. The doublestranded cuts are introduced into superhelical and linear DNA in 12 sites enriched with GC-pairs, 9 of them include pentanucleotide CGCGG(CCGCC) that is functionally significant. Relaxation of the plasmid DNA by topoisomerase I blocks the sitespecific action of the enzyme. Ca/Mg-dependent endonuclease is concluded to be topologically dependent enzyme, possibly, participating in the recombination processes.

Base Sequence↗
PubMed1998

Infection of ducklings with virus particles containing linear double-stranded duck hepatitis B virus DNA: illegitimate replication and reversion.

Double-stranded linear DNA is synthesized as a minor viral DNA species by all hepadnaviruses. In a previous study (W. Yang and J. Summers, J. Virol. 69:4029-4036, 1995) we showed that virus particles containing linear DNA of the duck hepatitis B virus (DHBV) could initiate an infection of primary duck hepatocytes. In cells infected by linear DNA containing viruses the transcriptional template, covalently closed circular DNA, was formed by circularization of linear DNA by nonhomologous recombination between the two ends. This process was shown to result in viral DNA replication through multiple generations of linear DNA intermediates, a process we called illegitimate replication. In this study we showed that viruses containing linear DHBV DNA produced by engineered insertions in the r sequence, which encodes the 5' end of the pregenome, could infect hepatocytes in vivo, and these hepatocytes proceeded to carry out illegitimate replication. Nonhomologous recombination quickly produced revertants and partial revertants in which all or part of the insertion was deleted. One such partial revertant that replicated primarily through circular DNA intermediates, but which synthesized elevated levels of linear DNA, could be sustained for several days as the predominant genotype in vivo, but this mutant was eventually displaced by variants showing full reversion to legitimate replication and that synthesized normal low levels of linear DNA. Full revertants did not necessarily contain the wild-type r sequence. The results suggest that the linear DNA produced during DHBV infection initiates cycles of illegitimate replication by generating mutants with altered r sequences. Some r sequence mutants carry out a mixture of legitimate and illegitimate replication that can contribute to elevated production of linear DNA in individual cells.

Animals↗
PubMed1993

Multiple tandem integrations of transforming DNA sequences in yeast chromosomes suggest a mechanism for integrative transformation by homologous recombination.

In yeast, the fate of linear DNA molecules upon transformation is determined by the existence of sequence homology between chromosomes and the ends of the transforming molecule. To understand the mechanism of integration of transforming DNA, we have studied the influence of DNA concentration on the frequency and type of transformants obtained, using either non-replicative or replicative plasmids. In both cases, increasing DNA concentration results in multiple tandem repeats integrated into the chromosome containing the homologous target sequence. When a diploid strain is transformed, multiple tandem repeats occur in only one of the two homologous chromosomes at a time. The frequency distribution of the different types of integrants observed indicates non-independent integration events likely to result from plasmid-plasmid interaction prior to chromosome integration. In addition, our results define the proper conditions for optimized gene targetting or gene rescue experiments.

Base Sequence↗
PubMed1987

Organization of terminal reiterations in the virion DNA of herpesvirus saimiri.

The population of herpesvirus saimiri (HVS) genomes extracted from extracellular virions are double-stranded, linear DNA molecules of about 160 kilobase pairs (kbp) each composed of a central segment of 110 to 112 kbp and 36% (G + C) (i.e. 'light' or L-DNA) linked to direct reiterations of a 1.44 kbp repeat unit of 71% (G + C) (i.e. 'heavy' or H-DNA) at each terminus. In this paper, we show that the population of HVS DNA molecules contains approximately equal concentrations of genomes with all possible integral numbers of complete repeat units (i.e. from greater than 30 to 1) at either 'left' or 'right' ends but that all molecular ends are derived by a unique cleavage at a site close to the single ApaI restriction endonuclease site of the H-DNA repeat unit. Junctions of proximal H-DNA repeat units with L-DNA occur at, or very close to, the sequence present at the molecular ends. The transition from L- to H-DNA occurs abruptly at this site at the 'right' end of the L-DNA component but some rearranged restriction enzyme cleavage sites typical of H-DNA are found within the first 0.8 kbp of the L-DNA sequences at the 'left' H-L DNA junction. HVS appears to provide an extreme example of the general process whereby herpesvirus DNAs are matured from concatemeric intermediates by a site-specific cleavage/recombination process involving random choice between equivalent sites for the initiation of the process and with choices between alternative termination sites being limited by a headful packaging mechanism.

Base Composition↗