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Results for “Extrachromosomal DNA”

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PubMed1979

[Nuclear structures in the telotrophic ovarioles of nocturnal ground beetles (Tenebrionidae, Polyphaga). III. The oocyte nucleus. Electron microscopic data].

The fine structural organization of nuclei was studied in the growing oocytes of Blaps lethifera, B. mortisaga and Gnaptor spinimanus. In the beginning of diplotene the nuclei contain primary fibrillar nucleoli and numerous electron dense globules dispersed all over the nucleus; the loose chromosome material (lampbrush chromosomes) is distributed all over the nucleus. With the oocyte growth the chromosomes are spiralized and join into the karyosphere. A capsule of fibrous material forms around the karyosphere. The karyosphere nucleoli appear on the chromosomes and, then, move to the capsule region and outside its limits, to the nuclear envelope. They are fibrillar and non-active with respect to RNA synthesis. The fibrous material of the capsule is represented by strands which consist of bundles of cross-striated filaments. These latter contact directly with the chromosomes in the karyosphere and with the surface of the karyosphere nucleoli. The fibrillar-granular bodies are distributed along the strands in the capsule; they contain both RNA and DNA. The nature of extrachromosomal DNA in the karyosphere capsule and its participation in the formation of the capsule material are discussed. A suggestion is put forward on the similarity of the capsule strands with the modified central elements of synaptinemal complex.

Animals↗

PubMed1981

[Plasmid SCP2 transfer by Streptomyces coelicolor A3(2) actinophages].

The ability of VP5 and phi c31 actinophages of Streptomyces coelicolor A3(2) to transfer plasmid SCP2 genes was tested. Infection with both phages led to formation of variants having the traits characteristics of plasmid bearing strains. The fertility properties of the variants suggested the presence of SCP2 in the autonomous state. Analysis of extrachromosomal DNA isolated from these variants demonstrated its identity with the plasmid DNA from the donor strain. Based on characteristics of the variants obtained, it was suggested that the whole SCP2 plasmid had been transferred by actinophages via general transduction.

Bacteriophages↗

PubMed2005

Tracking of microinjected DNA in live cells reveals the intracellular behavior and elimination of extrachromosomal genetic material.

We here addressed the basic question, how does extrachromosomal DNA behave when it is placed in the nuclear or the cytoplasmic environment and how is it eliminated? To do this, we tracked microinjected DNA molecules in live cells. In the cytoplasm, the diffusion of microinjected DNA was inhibited in a size- and linearity-dependent manner, probably by the intermediate filament. This was followed by the rapid disappearance of the DNA fluorescent signal. In the nucleus, the diffusion was also dependent on the size of the molecule and was accompanied by the aggregation of the DNA. The aggregation may be due to a putative DNA-binding molecule, whose level is high during the G1 phase. Surprisingly, the injected DNA could move across the nuclear membrane and appeared in the cytoplasm, which suggests the presence of a transport system. The intracytoplasmic behavior and the elimination of such DNA was obviously different from the DNA that was directly injected at the cytoplasm. The DNA remaining in the nucleus appeared to be stable and persisted in the nucleus or, after cell division, in the cytoplasm, for more than one cell cycle. These findings provide a novel and basic understanding of the behavior and elimination of a wide variety of extrachromosomal genetic material.

Active Transport, Cell Nucleus↗

PubMed2027

Identification of Genome-Wide Chromatin Structural Aberration in Cancer by Hi-C Analysis.

Aberrant three-dimensional genome organization is a hallmark of cancer, often driving oncogene activation through mechanisms such as enhancer hijacking. High-throughput chromosome conformation capture (Hi-C) maps these interactions on a genome-wide scale. Unlike earlier dilution-based methods, in situ Hi-C performs proximity ligation within intact nuclei, minimizing random ligation noise and enabling fine-scale structure detection. This chapter describes an optimized in situ Hi-C protocol tailored for cancer cell lines using MboI digestion and biotin-mediated pull-down to generate high-complexity libraries. We further outline a computational workflow that extends beyond standard topological mapping of compartments and topologically associating domains to identify cancer-specific aberrations. Specifically, we focus on detecting chromosomal rearrangements (structural variants) and characterizing the distinct circular topology of extrachromosomal DNA. This integrated experimental and analytical framework provides the necessary tools to dissect the spatial dysregulation underlying tumor evolution.

Humans↗

PubMed1995

Chimeric BK virus DNA episomes in a papillary urothelial bladder carcinoma.

BK virus (BKV) DNA sequences were identified in a papillary urothelial bladder carcinoma by Southern blot hybridization. The carcinoma contained both integrated and extrachromosomal DNA. Integrated sequences had a clonal restriction pattern, suggesting that BKV was integrated at some early stage of neoplastic initiation or progression. Viral episomes consisted of a population of covalent polymers based on a high-molecular-weight DNA unit, about 11-12 kb in size. DNA sequences non-homologous to the BKV genome were encompassed within DNA episomes, suggestive of acquisition of cellular sequences by viral DNA replication at the integration site. Extrachromosomal, chimeric DNA molecules were present at an average level of about 50 copies per cell, but their size, apparently incompatible with viral assembly, showed that BKV productive infection was impaired. The data suggest that infected cells underwent reversible changes affecting autonomous BKV DNA replication.

BK Virus↗

PubMed1985

Physical properties of a plasmid-like DNA from Euglena gracilis.

A small circular extrachromosomal DNA of the flagellate protozoan Euglena gracilis has been characterized as having a contour length of 11.3 kb, with a consistent restriction map. The buoyant density (rho = 1.717) and melting temperature (tm = 89 degrees C) both indicate a base content of 59% G + C. The DNA is found in both wild-type cells and those lacking plastids. The copy number is estimated to be about 1000.

DNA Restriction Enzymes↗

PubMed1980

Expression of thymidylate synthetase activity in Bacillus subtilis upon integration of a cloned gene from Escherichia coli.

The gene from Escherichia coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid, pER2, was effective in transforming both E. coli and Bacillus subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine-requiring strains of B. subtilis to thymine independence. Linearization of the chimeric plasmid, pER2, with restriction enzymes markedly diminished its ability to transform B. subtilis auxotrophs. The Thy+ transformants derived from the transformation of B. subtilis with pER2 DNA did not contain detectable extrachromosomal DNA as demonstrated by Southern hybridization patterns and centrifugation in CsCl gradients of DNA isolated from B. subtilis colonies transformed with the chimeric plasmid. We conclude that the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis, demonstrating that extensive homology is not required for the integration of foreign DNA. This is the first reported case of a gene from a Gram-negative bacterium functioning in a Gram-positive organism.

Bacillus subtilis↗

PubMed2003

Changes in the human mitochondrial genome after treatment of malignant disease.

Mitochondrial DNA (mtDNA) is the only extrachromosomal DNA in human cells. The mitochondrial genome encodes essential information for the synthesis of the mitochondrial respiratory chain. Inherited defects of this genome are an important cause of human disease. In addition, the mitochondrial genome seems to be particularly prone to DNA damage and acquired mutations may have a role in ageing, cancer and neurodegeneration. We wished to determine if radiotherapy and chemotherapy used in the treatment of cancer could induce changes in the mitochondrial genome. Such changes would be an important genetic marker of DNA damage and may explain some of the adverse effects of treatment. We studied samples from patients who had received radiotherapy and chemotherapy for point mutations within the mtDNA control region, and for large-scale deletions. In blood samples from patients, we found a significantly increased number of point mutations compared to the control subjects. In muscle biopsies from 7 of 8 patients whom had received whole body irradiation as well as chemotherapy, the level of a specific mtDNA deletion was significantly greater than in control subjects. Our studies have shown that in patients who have been treated for cancer there is an increased level of mtDNA damage.

Adolescent↗

PubMed1997

Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids.

Mitochondrila DNA (mtDNA) is the only extrachromosomal DNA in humans. It is a small (16.5 kb) genome which encodes 13 essential peptides of the respiratory chain, two rRNAs and 22 tRNAs. Defects of this genome are now recognized as important causes of disease and may take the form of point mutations or rearrangements. There is no effective treatment for patients with mtDNA mutations. In the majority of patients with mtDNA defects, both mutant and wild-type molecules are present in the same cell-a phenomenon known as intracellular heteroplasmy. In addition, in the presence of heteroplasmy there is a threshold whereby a certain level of mutant mtDNA is necessary before the disease becomes biochemically and clinically apparent. Based on the presence of heteroplasmy and the recessive nature of these mutations, we believe it will be possible to treat patients by selectively inhibiting the replication of the mutant mtDNA, thereby allowing propagation of only the wild-type molecule. To confirm the validity of such an approach we synthesised peptide nucleic acids (PNAs) complementary to human mtDNA templates containing a deletion breakpoint or single base mutation, both mutations well documented to cause disease. Using an in vitro replication run-off assay under physiological conditions, the antigenomic PNAs specifically inhibited replication of mutant but not wild-type mtDNA templates. Furthermore, we have shown uptake of these PNAs into cultured human myoblasts. We believe that we have therefore established the potential value of antigenomic PNA therapy for patients with heteroplasmic mtDNA disorders.

Cell Line↗

PubMed1977

The organisation of genes for transfer RNA and ribosomal RNA in amoebae and plasmodia of Physarum polycephalum.

1. Using hybridisation techniques nuclei from both amoebae and plasmodia of Physarum polycephalum were found to contain 275 genes each coding for 5.8-S, 19-S and 26-S rRNA, 685 genes for 5-S rRNA and 1050 genes for tRNA. 2. Hybridisation of these RNA species to both amoebal and plasmodial DNA fractionated on CsCl gradients reveal that the 5.8-S, 19-S and 26-S rRNA genes are located at a satellite position (formula: see text) with respect to the main band of DNA, whereas 4-S RNA genes are located exclusively in the main band of DNA (formula: see text). 3. This result was confirmed by demonstrating that only the 5.8-S, 19-S and 26-S rRNA species hybridise to purified plasmodial ribosomal DNA. 4. The 19-S and 26-S rRNA genes of amoebae are located on extrachromosomal DNA molecules of a discrete size (Mr = 38 X 10(6)) with identical properties to plasmodial ribosomal DNA.

Cell Nucleus↗

PubMed1989

A study of drug-induced topoisomerase II-mediated DNA lesions on episomal chromatin.

A well defined extrachromosomal DNA element, referred to as an episome (Ostrowski, M., Richard-Foy, H., Wolford, R., Berard, D., and Hager, G. (1983) Mol. Cell. Biol. 3, 2045-2057), was employed as a target for the topoisomerase II inhibitors amsacrine and teniposide. Both drugs have distinct mechanisms of action in cleaving the episome, as defined by topological forms conversion assays. The concentration ranges required to measure episomal cleavage are similar. The onset of damage induced by amsacrine begins within 1 min and is maintained at that level for at least 1 h. Teniposide induces damage that peaks between 30 and 60 min. The amsacrine-induced damage is only partially reversible, whereas teniposide-induced damage is almost completely reversible. Sites of specific cleavage are quite dissimilar. Multiple cleavage sites are formed in the episomal regulatory regions after amsacrine treatment, whereas a single cleavage in the regulatory region and one outside this region are found after teniposide treatment. Transcriptional activation using dexamethasone does not change the amount or site preference of episomal cleavage induced by either agent. Damage to the episome was quantitatively compared with damage produced in genomic DNA between 500 and 24,000 rad equivalents. The study showed that amsacrine has a significant (33-38-fold) preference for episomal DNA over genomic DNA.

Amsacrine↗

PubMed1995

Extrachromosomal circular forms of the tobacco retrotransposon Tto1.

Extrachromosomal DNA forms of Drosophila retrotransposons (RTn) and retroviruses have been extensively analyzed. However, no such analysis with plant RTn has been reported. Here, we report the analysis of extrachromosomal forms of the tobacco RTn Tto1. Tto1 is one of a few active RTn of plants and has been shown to be activated in tissue culture. Extrachromosomal circular DNA forms of Tto1, with one or two long terminal repeats (LTR), were found in cultured cells. Sequence analysis of the sites of circularization through joining two LTR showed that the junction between the LTR contains small deletions and/or insertions. The insertions are heterogeneous and do not show any homology to the Tto1 sequence. Similar insertions have been detected in the extrachromosomal circular forms of the copia element of Drosophila and suggested to be the result of excision of genomic copia. The structural features of the junctions found in Tto1 suggest that the insertions are produced by a mechanism other than excision. The potential mechanism of production of the extrachromosomal circular forms of Tto1 is discussed.

Animals↗

PubMed2001

Creation of low-copy integrated transgenic lines in Caenorhabditis elegans.

In Caenorhabditis elegans, transgenic lines are typically created by injecting DNA into the hermaphrodite germline to form multicopy extrachromosomal DNA arrays. This technique is a reliable means of expressing transgenes in C. elegans, but its use has limitations. Because extrachromosomal arrays are semistable, only a fraction of the animals in a transgenic extrachromosomal array line are transformed. In addition, because extrachromosomal arrays can contain hundreds of copies of the transforming DNA, transgenes may be overexpressed, misexpressed, or silenced. We have developed an alternative method for C. elegans transformation, using microparticle bombardment, that produces single- and low-copy chromosomal insertions. Using this method, we find that it is possible to create integrated transgenic lines that reproducibly express GFP reporter constructs without the variations in expression level and pattern frequently exhibited by extrachromosomal array lines. In addition, we find that low-copy integrated lines can also be used to express transgenes in the C. elegans germline, where conventional extrachromosomal arrays typically fail to express due to germline silencing.

Animals↗

PubMed1998

A plasmid isolated from phytopathogenic onion yellows phytoplasma and its heterogeneity in the pathogenic phytoplasma mutant.

A 3.6-kbp DNA fragment was cloned from the extrachromosomal DNA of a pathogenic plant mollicute, onion yellows phytoplasma (OY-W). Sequence analysis of the fragment revealed an open reading frame (ORF) encoding the replication (Rep) protein of rolling-circle replication (RCR)-type plasmids. This result suggests the existence of a plasmid (pOYW1) in OY-W that uses the RCR mechanism. This assumption was confirmed by detecting the single-stranded DNA (ssDNA) of a replication intermediate that is specifically produced by the RCR mechanism. This is the first report on the identification of the replication system of this plasmid and the genes encoded in it. With a DNA fragment including the Rep gene region of pOYW1 used as a probe, Southern and Northern (RNA) blot hybridizations were employed to examine the heterogeneity between the plasmids found in OY-W and a pathogenic mutant (OY-M) isolated from OY-W. Multiple bands were detected in the DNA and RNA extracted from both OY-W and OY-M infected plants, although the banding patterns were different. Moreover, the copy number of plasmids from OY-W was about 4.2 times greater than that from OY-M. These results indicate constructive heterogeneity between OY-W and OY-M plasmids, and the possibility of a relationship between the plasmid-encoded genes and the pathogenicity of the phytoplasma was suggested.

Amino Acid Sequence↗

PubMed1988

Isolation and characterization of simian immunodeficiency virus from mandrills in Africa and its relationship to other human and simian immunodeficiency viruses.

Two isolates of simian retrovirus related to the human immunodeficiency virus (HIV) were obtained from apparently healthy mandrills, Papio (Mandrillus) sphinx, in western equatorial Africa. This virus, designated SIVMND (simian immunodeficiency virus from mandrills), appeared morphologically similar to HIV by electron microscopy, showed Mg2+-dependent reverse transcriptase activity, and induced cytopathic effect in human CD4-positive cells. Western blotting (immunoblotting) analyses revealed that the gag and pol products of SIVMND showed cross-reactivity with those of known HIVs and SIVs. Molecular clones covering full-length viral DNA were obtained from closed circular extrachromosomal DNA of SIVMND-infected cells. By clone-on-clone hybridization with known retroviruses of the HIV and SIV groups, SIVMND showed similar cross-hybridization with HIV-1, HIV-2, SIVAGM (African green monkey-derived SIV), and SIVMAC (rhesus macaque-derived SIV) in the gag and pol regions only at low stringency but not at high stringency, a result indicating that SIVMND is a new member of the HIV-SIV group. The existence of distinct SIVs in different monkey species suggest that recent interspecies transfer of HIV-SIV is unlikely in nature.

Animals↗

PubMed1989

Amplified N-myc in human neuroblastoma cells is often arranged as clustered tandem repeats of differently recombined DNA.

Human neuroblastoma cells often carry amplified DNA encompassing the gene N-myc. Amplified N-myc has been found localized in "double minutes" in direct tumor cell preparations. In contrast, later passages carried amplified N-myc almost exclusively within a single homogeneously staining chromosomal region located at a chromosomal site different from the normal location of N-myc. We used pulsed field gel electrophoresis to define the structural arrangement of the amplified DNA. Long-range mapping was facilitated by the presence of several sites for rare cutting restriction endonucleases in the 5' region of N-myc. Amplified DNAs of different neuroblastoma cell lines were heterogeneous in size and had undergone recombination at various distances from N-myc. N-myc occupied a central position within the amplified DNA, and in no case was the coding region affected by recombination. Among neuroblastoma cells, varying proportions of amplified DNA (in some instances close to 100%) consisted of multiple tandem arrays of DNA segments ranging in size from 100 to 700 kilobase pairs. Tumor cells with low degrees of amplification revealed regions of amplified DNA in excess of 1,500 kilobase pairs without apparent rearrangement. Our observations, in concert with the cytogenetic findings, suggest a model of gene amplification which involves unscheduled DNA replication, recombination, and formation of extrachromosomal DNA followed by integration into a chromosome and subsequent in situ multiplication. The central position which N-myc occupies within the amplified sequences and the lack of recombination within the coding region of N-mc indicate that N-myc rather than other genetic information provides the selective advantage for retention of the amplified DNA.

DNA Replication↗

PubMed2026

Genomic instability, postoperative recurrence and therapeutic vulnerabilities in resectable non‑small cell lung cancer (Review).

Resectable non‑small cell lung cancer (NSCLC) is managed largely according to anatomical stage, pathological risk and actionable driver alterations, yet these factors do not fully explain postoperative recurrence. Genomic instability may contribute to recurrence by promoting clonal diversification, intratumoral heterogeneity, occult dissemination, persistence of residual tumor cells, and immune escape. In the present review, chromosomal instability (CIN), copy‑number complexity, whole‑genome doubling, DNA repair defects, replication stress, and extrachromosomal DNA (ecDNA) were critically evaluated using a three‑axis translational framework encompassing biological consequences, potential clinical roles, and strength of evidence. Current evidence suggests that clonal diversity and copy‑number complexity have the clearest near‑term prognostic rationale. By contrast, CIN and whole‑genome doubling are supported more strongly by evolutionary and mechanistic rather than prospective clinical evidence. Defects in DNA repair, replication stress, and ecDNA represent potential therapeutic vulnerabilities, but their clinical relevance remains to be established. To date, no treatment‑predictive biomarkers based on genomic instability have been identified for resectable NSCLC. Direct clinical evidence linking any specific genomic instability feature to the presence or longitudinal dynamics of postoperative molecular residual disease (MRD) remains limited. Postoperative circulating tumor DNA‑defined MRD provides prognostic information more directly related to residual disease but remains assay‑dependent and should not be considered a genomic‑instability phenotype. Therefore, features of genomic instability should remain investigational and should not replace established clinical, pathological, or molecular decision‑making. Their near‑term value lies in refining biological risk models and generating testable hypotheses for biomarker‑defined perioperative trials.

Humans↗

PubMed1991

The Entamoeba histolytica rDNA episome: nuclear localization, DNAase I sensitivity map, and specific DNA-protein interactions.

Structural and functional features of the extrachromosomal DNA element that contains the ribosomal RNA genes of Entamoeba histolytica were studied using a variety of techniques. Using in situ hybridization, the element was found to be distributed along the inner phase of the nuclear membrane in the trophozoite stage; it appears to be part of the so-called peripheral chromatin. DNAase I-sensitive regions on the episome were mapped and found to correspond to the borders of the ribosomal RNA coding region. Other DNAase I-sensitive regions were found to correspond to DNA containing a 145bp sequence that exists in the episome as tandem repeats. Electrophoretic shift assays and footprinting experiments demonstrate the existence of specific nuclear factors that bind specifically to the 145bp repeat. Preliminary analysis of the binding factors showed that a 28 kDa polypeptide is a likely candidate for a specific DNA:protein interaction involving the repetitive element. These results suggest that a protein-binding domain within the 145bp repeat may have a specific function in the episome.

Animals↗