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Results for “DNA, Circular”

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At least 217 records · Page 12Linked to original sources

PubMed1997

The ParB protein encoded by the RP4 par region is a Ca(2+)-dependent nuclease linearizing circular DNA substrates.

The parCBA operon, which together with the parDE operon constitutes an efficient stabilization system of the broad-host-range plasmid RP4, encodes a 20 kDa polypeptide (ParB), which exhibits sequence homology to nucleases. The ParB protein was overexpressed by means of an inducible tac-promoter system. Plate assays with herring sperm DNA as substrate provided evidence for nuclease activity. The ParB nuclease shows specificity for circular DNA substrates and linearizes them regardless of the presence in cis of parts of the RP4 partitioning region. The nuclease activity in vitro is stimulated by the presence of Ca2+ ions. EDTA (5 mM) completely inhibits nuclease activity. By restriction analysis of the ParB-linearized products, cleavage of circular DNA substrates taking place preferentially at specific sites was demonstrated. Run-off sequencing and primer extension analysis of ParB-linearized plasmid DNA revealed a specific target for ParB action adjacent to an AT-rich region containing palindromic sequence elements on a pBR322-derived plasmid.

Amino Acid Sequence↗

PubMed1990

Immunoglobulin switch circular DNA in the mouse infected with Nippostrongylus brasiliensis: evidence for successive class switching from mu to epsilon via gamma 1.

We have characterized immunoglobulin switch circular DNA in mice infected with the nematode parasite Nippostrongylus brasiliensis. Two kinds of circular DNA were identified in the lymph nodes as excision products of switch recombination of immunoglobulin heavy-chain constant region (CH) genes. One is a recombinant between C mu and C gamma 1 (gamma 1 circle), and the other is a recombinant between C gamma 1 and C epsilon (epsilon circle). In the epsilon circle, a short piece of switch mu (S mu) sequence was inserted between S epsilon and S gamma 1 sequences. The inserted S mu sequence could be a trace of the preceding switch from C mu to C gamma 1. These findings indicate that parasitic infection can induce class switch recombinations in a successive manner, first from C mu to C gamma 1, and then from C gamma 1 to C epsilon.

Animals↗

PubMed1982

Yeast RNA polymerase II transcription of circular DNA at different degrees of supercoiling.

Purified yeast RNA polymerase II was tested for transcriptional activity as a function of the degree of circular DNA supercoiling. Chimaeric plasmids P30 and P31 both containing inserts from the yeast transposable element TY1 cloned in pBR322 and the vector pBR322 were used as templates. For pBR322 the transcriptional activity increases about 4 fold from the fully relaxed covalently closed circles to the native supercoiled forms, further supercoiling having no effect on transcription. P30 shows a 5 fold increase of transcriptional activity reaching a plateau at the native supercoiled conformation. However, at an intermediate degree of supercoiling (sigma = 0.024), transcription decreases to a value close to zero. P31 too exhibits a conformation (sigma = 0.014) in which there is a drop of transcriptional activity. Furthermore, a 10 fold increase of transcription is obtained at the higher values of superhelix density. Both kinetic and autoradiographic experiments confirm the existence of DNA conformations that can inhibit "in vitro" transcription.

DNA Transposable Elements↗

PubMed1996

n-Butyrate, a cell cycle blocker, inhibits early amplification of duck hepatitis B virus covalently closed circular DNA after in vitro infection of duck hepatocytes.

During chronic hepadnavirus infection, virus persistence depends on the regulation of the pool of covalently closed circular DNA (cccDNA), which is the template for transcription of viral RNA species. The development of in vitro infection of duck hepatocyte primary cultures by duck hepatitis B virus (DHBV) provides a unique opportunity to study the regulation of cccDNA synthesis. After DHBV in vitro infection, cccDNA is detected 1 day later and is amplified to a high copy number after 1 week in culture. We studied whether this amplification occurs during cell cycle progression of duckling hepatocytes. By using [3H]thymidine incorporation, we found that hepatocytes obtained from 3-week-old ducklings spontaneously entered the S phase of the cell cycle when cultured in serum-free medium without added growth factors. Bromodeoxyuridine labeling confirmed that cellular DNA synthesis took place in more than 50% of parenchymal cells. Cytofluorometry analysis revealed the presence of asynchronous populations and polyploidization processes. The addition of a cell cycle blocker, n-butyrate, completely inhibited [3H]thymidine incorporation and blocked duckling hepatocytes in the G1 phase of the cell cycle. Simultaneously, butyrate inhibited cccDNA amplification and allowed the establishment of DHBV infection, as demonstrated by the detection of a basal level of cccDNA in treated hepatocytes. Both effects were reversible since active cell DNA synthesis was restored and cccDNA accumulated after drug withdrawal.

Animals↗

PubMed1978

[Density gradient ultracentrifugation method of studying sibiromycin interaction with linear and circular DNA].

Sibiromycin added to linear chromosomal E. coli DNA in vitro leads to the decrease of bouyant density in neutral CsCl density gradient. This decrease is a linear function of sibiromycin/DNA ratio and amounts to about 32 mg/ml at the ratio equal to 0.1. Binding sibiromycin does not change the degree of hydration of DNA as revealed by centrifugation in metrizamide density gradients. When added to the covalently closed or open circular DNA of PM-2 phage, sibiromycin decreased the bouyant density of these DNA species to a similiar extent. The antibiotic does not induce single-strand breaks in DNA in vitro as follows from the results of ethidium bromide-CsCl density gradient centrifugation of covalently closed PM-2 DNA.

Antibiotics, Antineoplastic↗

PubMed1985

The twist, writhe, and linking number distributions in closed circular DNA.

We discuss the predictions which follow from the assumption of statistically independent twist and writhe distributions of given variances in circular DNA with single-strand nicks. The nature of the topoisomer distribution produced upon covalent closure of the nicks is described, as well as the nature of the twist and writhe distributions in the fully-closed molecules. In particular, we show how the distributions depend on the magnitudes of the given variances, and how the relative magnitudes of the variances can be deduced from experiment. One additional consequence of the theory is the prediction of a necessary difference between the temperature coefficient of the twist in nicked versus fully-closed circular DNA. The ratio of the two twist coefficients turns out to depend only on the ratio of the twist and writhe variances in nicked DNA.

DNA, Circular↗

PubMed1997

The effect of intrinsic curvature on conformational properties of circular DNA.

Both thermal fluctuations and the intrinsic curvature of DNA contribute to conformations of the DNA axis. We looked for a way to estimate the relative contributions of these two components of the double-helix curvature for DNA with a typical sequence. We developed a model and Monte Carlo procedure to simulate the Boltzmann distribution of DNA conformations with a specific intrinsic curvature. Two steps were used to construct the equilibrium conformation of the model chain. We first specified the equilibrium DNA conformation at the base pair level of resolution, using a set of the equilibrium dinucleotide angles and DNA sequence. This conformation was then approximated by the conformation of the model chain consisting of a reduced number of longer, straight cylindrical segments. Each segment of the chain corresponded to a certain number of DNA base pairs. We simulated conformational properties of nicked circular DNA for different sets of equilibrium dinucleotide angles, different random DNA sequences, and lengths. Only random sequences of DNA generated with equal probability of appearance for all types of bases at any site of the sequence were used. The results showed that for a broad range of intrinsic curvature parameters, the radius of gyration of DNA circles should be nearly independent of DNA sequence for all DNA lengths studied. We found, however, a DNA properly that should strongly depend on DNA sequence if the double helix has essential intrinsic curvature. This property is the equilibrium distribution of the linking number for DNA circles that are 300-1000 bp in length. We found that a large fraction of the distributions corresponding to random DNA sequences should have two separate maxima. The physical nature of this unexpected effect is discussed. This finding opens new opportunities for joined experimental and theoretical studies of DNA intrinsic curvature.

Computer Simulation↗

PubMed1975

Replicating circular DNA molecules in yeast.

The yeast Saccharomyces cerevisiae contains a class of small circular DNA molecules, approximately 2 mum in contour length (Sinclair et al., 1967). In this report, it is shown that these molecules replicate as double-branched circles, similar to those observed during replication of the bacteriophage lambda and Escherichia coli chromosomes. A normal rate of replication of these DNA circles requires the function of a nuclear gene, cdc 8.

Centrifugation, Density Gradient↗

PubMed1978

Specific site of action for single-strand specific nuclease on the double-stranded circular DNA intermediates of an avian RNA tumor virus.

Circular viral DNA intermediates obtained from the quail tumor line, QT6, at 1 day after infection, were opened at one specific location by the single-strand specific nuclease, S1, of Aspergillus oryzae. This site was no longer accessible to the S1 nuclease when circles were first opened at another location with a restriction endonuclease.

Alpharetrovirus↗

PubMed1974

Isolation of heterogeneous circular DNA from induced lysogens of bacteriophage Mu-1.

Covalently-closed circular DNA molecules are formed after induction of phage Mu cts4 and after infection with phage Mu cts4. The circular molecules obtained after induction have a molecular length range from 36.5 to 156.7 kilobases as measured by electron microscopic techniques. These heterogeneous molecules have no consistent correlation to exact multiples of a Mu genome equivalent (37.3 +/- 1.2 kilobases). Direct evidence is given that these molecules contain phage Mu DNA that is covalently linked to other DNA sequences.

Centrifugation, Density Gradient↗

PubMed1994

Characterization of the structural conformation adopted by (TTAGGG)n telomeric DNA repeats of different length in closed circular DNA.

Telomeric DNA sequences are known to adopt unusual DNA structures upon protonation when contained into negatively supercoiled DNA. In this paper, the structural properties of (T2AG3)n telomeric sequences of different length is analyzed in detail. Transition to the protonated form is observed at very low pH for (T2AG3)n > or = 8 sequences. Formation of the protonated form is facilitated by negative supercoiling. The patterns of chemical modification obtained with different chemical reagents indicate that protonation induces denaturation of the (T2AG3)n telomeric sequences. Upon denaturation, the "C-rich" strand becomes structured forming, most likely, hairpin-like conformations stabilized by the formation of C+.C pairs and, probably, of A+.A pairs. The "G-rich" strand of the (T2AG3)8 sequence shows also signs of becoming structured giving rise to various structural conformers which might include triple- and tetra-stranded conformations. However, in the case of shorter sequences, the "G-rich" strand remains basically single-stranded.

Base Sequence↗

PubMed1980

Degradation of linear and circular DNA with gaps by the recBC enzyme of Escherichia coli. Effects of gap length and the presence of cell-free extracts.

It is shown that circular PM2 DNA with two gaps of 13 nucleotides per molecule is degraded by purified recBC enzyme from Escherichia coli to acid-soluble material at a rate which is less than one tenth of the rate of solubilization of linear duplex DNA. Increasing the gap length in the circular DNA to 40-650 nucleotides does not affect the breakdown of the molecules by the recBC enzyme, nor does it change the proportions of the products formed (acid-soluble material, acid-insoluble fragments and non-degraded molecules). On the other hand, terminal gaps in linear duplex DNA produced by limited digestion with either exonuclease III or lambda exonuclease significantly reduce the rate of the degradation by the recBC enzyme, particularly when the gaps exceed 100 nucleotides. The results suggest that the recBC enzyme does not cleave gaps in circular DNA at random positions, but possibly at the junction between single-stranded and duplex DNA or close to it. The degradation of gapped circular DNA by purified recBC enzyme was used to search for an inhibitor of the recBC enzyme in extracts from ultraviolet-irradiated cells. No such inhibitor has been observed but rather a weak stimulatory factor for the solubilization of gapped circular DNA by the recBC enzyme. Thus, the experimental system appears not to be suited as a test in vitro for an ultraviolet-induced inhibitor of the recBC enzyme which has been postulated to be produced in recA+ lexA+ cells of E. coli after ultraviolet irradiation.

DNA, Bacterial↗

PubMed2005

Evaluation of transcriptional efficiency of hepatitis B virus covalently closed circular DNA by reverse transcription-PCR combined with the restriction enzyme digestion method.

Virus persistence in chronic hepatitis B patients is due to the sustaining level of covalently closed circular DNA (cccDNA) within the nuclei of infected hepatocytes. In this study, we used a modified 1.3-fold hepatitis B virus (HBV) genome, with a BclI genetic marker embedded in the redundancy region, to examine the transcriptional activity of cccDNA and the effect of the HBx protein on transcriptional regulation. After harvesting total RNA from transfected cells or stable lines, we specifically identified and monitored the transcripts from cccDNA by using reverse transcription-PCR (RT-PCR) combined with the restriction enzyme digestion method. In this approach, we have found that (i) RT-PCR combined with detection of the BclI marker is a highly specific method for distinguishing cccDNA-derived transcripts from the original integrated viral genome, (ii) the transcriptional ability of cccDNA was less efficient than that from the integrated viral genome, and (iii) the transcriptional activity of cccDNA was significantly regulated by the HBx protein, a potential transcription activator. In conclusion, we provided a tool with which to elucidate the transcriptional regulation of cccDNA and clarified the transcriptional regulation mechanism of HBx on cccDNA. The results obtained may be helpful in the development of a clinical intervention for patients with chronic HBV infections.

Cell Line, Tumor↗

PubMed2003

Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver.

Hepatitis B virus (hepadnavirus) infections are maintained by the presence of a small and regulated number of episomal viral genomes [covalently closed circular DNA (cccDNA)] in the nuclei of infected cells. Although a number of studies have measured the mean copy number of cccDNA molecules in hepadnaviral-infected cells, the distribution of individual copy numbers have not been reported. Using a PCR-based assay, we examined the number of cccDNA molecules of the duck hepatitis B virus in single nuclei isolated from the liver of a chronically infected duck over the course of 131 days of infection. Nuclei were isolated from frozen serial biopsies and individually deposited into PCR microplates by flow sorting. Each nucleus was assayed by nested PCR for cccDNA and for cellular IFN-alpha genes as an internal control. We found that 90% of the nuclei assayed contained between 1 and 17 cccDNA molecules, with the remaining 10% containing more (90% confidence), and that differences in the mean number of copies and distribution of copy numbers occurred within the same animal at different times postinfection. Overall, the data suggest (i) that the number of cccDNA molecules per cell may fluctuate over time, and (ii) that, according to these fluctuations, a substantial fraction of cells may contain only one or a few copies. We infer from the results that infected hepatocytes express virus at different levels and that during cell division it is possible to segregate cells containing no cccDNA.

Animals↗

PubMed1984

Molecular cloning of covalently closed circular DNA of bovine leukemia virus.

The two species of covalently closed circular DNA molecules of bovine leukemia virus were cloned in the lambda phage vector lambda gtWES X lambda B. Of the nine independent recombinant lambda-bovine leukemia virus clones that were analyzed, three were derived from the small and six were derived from the large circular molecules carrying, respectively, one and two copies of the long terminal repeat sequences. Comprehensive restriction endonuclease mapping of the unintegrated bovine leukemia virus and the cloned DNA molecules showed that eight of the nine clones carried viral information without any detectable deletions or insertions of more than ca. 50 base pairs. One of the nine clones, which carries a retroviral insert with one copy of the long terminal repeat, had a deletion of ca. 150 base pairs.

Animals↗

PubMed2000

Increased hepatocyte turnover and inhibition of woodchuck hepatitis B virus replication by adefovir in vitro do not lead to reduction of the closed circular DNA.

The aim of this study was to evaluate the inhibitory effect of the nucleotide analogue adefovir on woodchuck hepatitis B virus (WHV) replication and, in particular, to determine whether the pool of covalently closed circular DNA (cccDNA) could be reduced by adefovir treatment in primary cultures of woodchuck hepatocytes isolated from a chronic carrier. Strong reduction of WHV-DNA synthesis (90%) and secretion (up to 98%) was observed with all 3 doses of adefovir used (1, 10, and 100 micromol/L), whereas in the absence of the drug, high amounts of viral particles were continuously secreted in the culture medium until the end of the study (27 days). Secretion of envelope proteins and viral RNA levels remained constant both in the adefovir-treated and -untreated cultures for the entire course of the study. Intracellular core protein levels declined by approximately 50% in all the cultures, independent of adefovir treatment. There was no indication of cccDNA loss in the adefovir-treated hepatocyte cultures even when cell turnover was induced for 14 days by the addition of epidermal growth factor (EGF) to the culture medium. Our data show that adefovir has a very strong inhibitory effect on WHV-DNA synthesis in chronically infected primary hepatocyte cultures and indicate that cccDNA is a very stable molecule that appears to be efficiently transmitted to the dividing hepatocytes.

Adenine↗

PubMed1988

Thymocyte circular DNA excised from T cell receptor alpha-delta gene complex.

We have characterized thymocyte circular DNA excised from the T cell receptor alpha-delta gene complex. Some delta gene clones contained unusual recombinant structures derived from V-(D)-J joining: (i) a reciprocal joint of direct V to J delta joining, skipping the D delta segment; (ii) a V-D delta coding joint lacking an adjacent D delta-J delta coding joint; (iii) a V- D structure containing two D delta segments. Many of the alpha gen clones contained both coding and reciprocal joints of V alpha-to-J alpha joining on the same structure. Most of these coding joints were out of phase; however, in one clone there was an in-phase V-J alpha structure. Interestingly, some alpha gene clones contained the same V gene sequence as rearranged in the delta gene clone, indicating that the same V gene family, at least in part, could be utilized for both the alpha and delta gene systems.

Amino Acid Sequence↗

PubMed1977

[The topology of circular DNA].

A non-orientable structure, said Möbius stripe, is proposed for certain types of circular DNA. This structure could account for particular forms, such as dimers, double length molecules, or catenans which are molecules topologically interwomen. On the other hand, it is suggested that a second structure derived from the same principal of non-orientability could have gendered the dynamics of DNA replication at the origin of life: this is hypothesis of archetype Möbius strip.

Biological Evolution↗