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Results for “Extrachromosomal DNA”

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At least 19 recordsLinked to original sources
PubMed2025

Enhancer activation from transposable elements in extrachromosomal DNA.

Extrachromosomal DNA (ecDNA) drives oncogene amplification and intratumoral heterogeneity in aggressive cancers. While transposable element (TE) reactivation is common in cancer, its role on ecDNA remains unexplored. Here, we map the 3D architecture of MYC-amplified ecDNA in colorectal cancer cells and identify 68 ecDNA-interacting elements (EIEs)-genomic loci enriched for TEs that are frequently integrated onto ecDNA. We focus on an L1M4a1#LINE/L1 fragment co-amplified with MYC, which functions only in the ecDNA amplified context. Using CRISPR-CATCH, CRISPR interference, and reporter assays, we confirm its presence on ecDNA, enhancer activity, and essentiality for cancer cell fitness. These findings reveal that repetitive elements can be reactivated and co-opted as functional rather than inactive sequences on ecDNA, potentially driving oncogene expression and tumor evolution. Our study uncovers a mechanism by which ecDNA harnesses repetitive elements to shape cancer phenotypes, with implications for diagnosis and therapy.

Journal Article↗
PubMed2026

DNA-FISH Metaphase Spreads to Distinguish Extrachromosomal DNA from Homogeneously Staining Regions in Human Cancer Cell Lines.

UNLABELLED: Whole-genome sequencing identifies focal DNA amplifications with base-pair resolution but cannot determine whether amplified sequences reside on extrachromosomal DNA (ecDNA, also known as double minutes) or within chromosomally integrated homogeneously staining regions (HSRs). DNA fluorescence in situ hybridization (DNA-FISH) metaphase spreads remain the gold standard for distinguishing these amplification states at single-cell resolution. Here, we present a detailed protocol for DNA-FISH metaphase spreads using human cancer cell lines, encompassing cell culture, metaphase arrest, hypotonic treatment, fixation, chromosome spreading, fluorescent probe hybridization, and fluorescence imaging. The protocol incorporates intermediate quality-control steps to verify successful chromosome dispersion and optimize metaphase spread quality, making the workflow accessible to laboratories without specialized cytogenetics expertise. Results demonstrate clear visualization of ecDNA and HSR amplification states using locus-specific probes and illustrate common technical artifacts that can affect interpretation. This protocol provides a robust and reproducible approach for studying the structural organization of oncogene amplification in cancer cells. SUMMARY: We report a DNA-FISH metaphase spread protocol that visually detects locus copy number and location within the genome. This approach enables single-cell resolution of amplification states, specifically in cancer cell lines containing extrachromosomal DNA and homogeneously staining regions.

Journal Article↗
PubMed2026

DNA-FISH Metaphase Spreads to Distinguish Extrachromosomal DNA from Homogeneously Staining Regions in Human Cancer Cell Lines.

Whole-genome sequencing identifies focal DNA amplifications with base-pair resolution but cannot determine whether amplified sequences reside on extrachromosomal DNA (ecDNA, also known as double minutes) or within chromosomally integrated homogeneously staining regions (HSRs). DNA fluorescence in situ hybridization (DNA-FISH) metaphase spreads remain the gold standard for distinguishing these amplification states at single-cell resolution. Here, we present a detailed protocol for DNA-FISH metaphase spreads using human cancer cell lines, encompassing cell culture, metaphase arrest, hypotonic treatment, fixation, chromosome spreading, fluorescent probe hybridization, and fluorescence imaging. The protocol incorporates intermediate quality-control steps to verify successful chromosome dispersion and optimize metaphase spread quality, making the workflow accessible to laboratories without specialized cytogenetics expertise. Results demonstrate clear visualization of ecDNA and HSR amplification states using locus-specific probes and illustrate common technical artifacts that can affect interpretation. This protocol provides a robust and reproducible approach for studying the structural organization of oncogene amplification in cancer cells.

Humans↗
PubMed1976

Extrachromosomal DNA in chloramphenicol resistant myxococcus strains.

The presence of extrachromosomal DNA in strains of Myxococcus xanthus and M fulvus was examined by rate-zonal centrifugation of radioactively-labelled DNA in 'cleared lysates'. All the strains examined contained extrachromosomal DNA, with the exception of M. xanthus FBt. Chloramphenicol resistance is inducible in M. xanthus FBt. A peak of extrachromosomal DNA, containing covalently closed molecules, was found in one of the induced strains, implying that induction of chloramphenicol resistance is associated with the production of a plasmid. By incubating R+ strains of Escherichia coli with myxococci, R factor-mediated chloramphenicol resistance can be introduced into the latter. Evidence of extra chromosomal DNA in a derivative of M. xanthus with chloramphenicol resistance from R factor RI. 19 unique to the chloramphenicol strain, was obtained. By using a double-labelling technique, several chloramphenicol-resistant strains of M. fulvus M were examined. Evidence for a peak, unique for the chloramphenicol-resistant strain, was found in a strain with resistance derived from the R factor, S-a, but not from comparable strains with resistance derived from R factors R57b, R1. 19 and R478.

Chloramphenicol↗
PubMed2022

The evolutionary dynamics of extrachromosomal DNA in human cancers.

Oncogene amplification on extrachromosomal DNA (ecDNA) is a common event, driving aggressive tumor growth, drug resistance and shorter survival. Currently, the impact of nonchromosomal oncogene inheritance-random identity by descent-is poorly understood. Also unclear is the impact of ecDNA on somatic variation and selection. Here integrating theoretical models of random segregation, unbiased image analysis, CRISPR-based ecDNA tagging with live-cell imaging and CRISPR-C, we demonstrate that random ecDNA inheritance results in extensive intratumoral ecDNA copy number heterogeneity and rapid adaptation to metabolic stress and targeted treatment. Observed ecDNAs benefit host cell survival or growth and can change within a single cell cycle. ecDNA inheritance can predict, a priori, some of the aggressive features of ecDNA-containing cancers. These properties are facilitated by the ability of ecDNA to rapidly adapt genomes in a way that is not possible through chromosomal oncogene amplification. These results show how the nonchromosomal random inheritance pattern of ecDNA contributes to poor outcomes for patients with cancer.

Biological Evolution↗
PubMed1978

Detection and preliminary characterization of extrachromosomal DNA in clinical isolates of Staphylococcus aureus and Staphylococcus epidermidis.

Isolates of Staphylococcus aureus and Staphylococcus epidermidis frequently harboured extrachromosomal DNA. From the 20 Staphylococcus aureus strains examined 85% contained extrachromosomal DNA and 65% carried more than one plasmid DNA species. The results obtained with the 19 Staphylococcus epidermidis strains analyzed were similar, 84% of the strains had extrachromosomal DNA and 68% contained several DNA species. The agarose gel electrophoresis of the DNA obtained from cleared lysates of the staphylococci proved to be efficient for the detection of plasmids and the determination of their molecular weights. The gel method appeared therefore to be suitable for the establishment of the role of plasmids in the phage typability of staphylococci. Using a Staphylococcus epidermidis strain as an example it was shown by electron microscopy that the extrachromosomal DNA demonstrated by the agarose gel electrophoresis represented covalently closed circular plasmid DNA. This plasmid DNA could be sufficiently labeled with 3H-thymidine to undertake studies on the relationship and molecular characterization of staphylococcal plasmids.

DNA, Bacterial↗
PubMed1978

A rapid method for the purification of extrachromosomal DNA from eukaryotic cells.

A simple and efficient procedure to purify the low molecular weight extrachromosomal DNA from eukaryotic cells is described. Gentle lysis of cells with urea and sodium dodecyl sulfate in 0.24 M phosphate buffer (pH 6.8) is followed by the removal of high molecular weight bulk DNA by centrifugation. Protein and RNA are removed from the supernatant by hydroxyapatite chromatography in urea/phosphate buffer. Urea is then removed with 0.15 M phosphate buffer and the extrachromosomal DNA, virtually free from protein and RNA, is finally eluted in 0.5 M phosphate buffer. The procedure allows the recovery of about 99% simian virus 40 (SV40) DNA from infected monkey kidney cells in the extrachromosomal fraction. In normal mouse, monkey, andhuman cells, approximately 1% of total cell DNA appears to be extrachromosomal.

Cell Line↗
PubMed2026

Plasticity of extrachromosomal DNA segregation during drug adaptation.

Uneven segregation during mitosis is a striking feature of extrachromosomal DNA (ecDNA). Because ecDNA lacks a centromere, it is thought to segregate stochastically, generating intratumoral heterogeneity in genomic copy number. Drug treatment can readily change ecDNA copy number, enabling cells to acquire drug resistance, yet whether these changes reflect static selection of pre-existing clones or active reconfiguration under stress remains unresolved. To address this, we develop a high-throughput framework combining single-cell DNA sequencing with cellular barcoding for clonal tracking. Single-cell cloning reveals that not all clones exhibit identical segregation modes even under drug-free conditions. Under treatment, resistant populations do not simply arise from pre-existing clones with favorable ecDNA states; instead, some clones actively reconfigure their segregation behavior to generate resistant cells. Thus, although ecDNA generally segregates stochastically, it can undergo nonrandom, actively regulated segregation under drug stress, raising the possibility of therapeutically targeting ecDNA segregation mechanisms to counteract adaptive resistance.

Extrachromosomal DNA↗
PubMed2025

Spatial-Temporal Diversity of Extrachromosomal DNA Shapes Urothelial Carcinoma Evolution and Tumor-Immune Microenvironment.

Extrachromosomal DNA (ecDNA) presents a promising target for cancer therapy; however, its spatial-temporal diversity and influence on tumor evolution and the immune microenvironment remain largely unclear. We apply computational methods to analyze ecDNA from whole-genome sequencing data of 595 urothelial carcinoma (UC) patients. We demonstrate that ecDNA drives clonal evolution through structural rearrangements during malignant transformation and recurrence of UC. This supports a model wherein tumors evolve via the selective expansion of ecDNA-bearing cells. Through multi-regional sampling of tumors, we demonstrate that ecDNA contributes to the evolution of multifocality and increased intratumoral heterogeneity. EcDNA is present in 36% of UC tumors and correlates with an immunosuppressive phenotype and poor prognosis. Single-cell RNA sequencing analyses reveal that ecDNA+ malignant cells exhibit diminished expression of major histocompatibility complex class I molecules, enabling them to evade T-cell immunity. Finally, we show that sequencing of urinary sediment-derived DNA has excellent specificity in detecting ecDNA.

Journal Article↗
PubMed1978

Survey of extrachromosomal DNA found in the filamentous cyanobacteria.

Cleared lysates of 13 species of filamentous cyanobacteria were examined for the presence of extrachromosomal DNA by using agarose gel electrophoresis and ethidium bromide staining. Seven of the 13 species contained extrachromosomal covalently closed circular DNA, and all but 1 species contained multiple elements. There was no correlation between the presence of extrachomosomal DNA and either the range of metabolic activities found in the cyanobacteria or the differentiated cell types or structures elaborated by the morphologically complex filamentous cyanobacteria.

Cyanobacteria↗
PubMed1978

[Demonstration of extrachromosomal DNA in Rhizobium meliloti].

Seven effective (nitrogen-fixing) strains of Rhizobium meliloti have been studied. By sedimentation analysis of their alkaline lysates in alkaline sucrose gradients, a plasmid was found in four strains. In a strain (2011 str 3) which gave no result with this method, supercoiled DNA was detected by CsCl-dye buoyant density gradient centrifugation. That result was confirmed by analytical Cs2SO4-Ag+ density gradients, which showed a heterogeneity in the average base composition of the DNA extracted from three strains, including the 2011 str 3 strain. Two of those last strains seemed to contain an extrachromosomal DNA of very high molecular weight.

Centrifugation, Density Gradient↗
PubMed1979

Characterization of extrachromosomal DNA in the flesh fly Sarcophaga bullata.

The polytene pupal foot pad cells of the flesh fly Sarcophaga bullata contain numerous extrachromosomal DNA containing granules. We have determined both the origin and the nature of the DNA sequences present in these granules. Studies done with quinacrine staining of seven day old pupal foot-pad polytene nuclei showed that the granules fluoresced very brightly while the chromosomal bands to which the granules were attached did not. The only other highly fluorescent regions of the polytene karyotype were the centromeric heterochromatin of chromosomes C and E and several bands associated with the nucleolus of Chromsome A. When polytene nuclei were hybridized in situ with cRNA made from highly repetitive DNA, many of the granules positively labeled. Most of the label on these slides was concentrated on the centromeric heterochromatin of chromosomes C and E. Quinacrine staining of the foot-pad cells at very early stages of pupal development showed that when granules were present, they were always closely associated with the same two centromeric regions, those of chromosomes C and E. Since the highly repetitive DNA located in these centromeric regions is underreplicated, we conclude that the granules result from an extrusion process which takes place early during the polytenization of these cells. The chromosomal integrity of the centromeric heterochromatin of chromosomes C and E is apparently disrupted and repetitive sequences are dissociated from the chromosomes as DNA granules which then secondarily become associated with chromosomal bands throughout the nucleus.

Animals↗
PubMed2025

eccDNABase: A Comprehensive and High-Quality Database for Extrachromosomal Circular DNA.

Extrachromosomal circular DNA (eccDNA) refers to small, circular DNA molecules that originate from chromosomal sequences and are prevalent across nearly all eukaryotic organisms. In humans, eccDNAs are widely distributed in normal tissues, cancerous tissues, and body fluids, where they play important roles in tumorigenesis and are often associated with poor clinical outcomes. Given their biological and clinical significance, a well-integrated and high-quality database is essential for advancing eccDNA-related research. To address this need, we developed eccDNABase, a comprehensive and curated resource for browsing, searching, and analyzing eccDNAs across multiple species. The database systematically catalogs eccDNA-disease associations from diverse tissues and organisms. Currently, eccDNABase contains 1,875,452 eccDNA-disease associations, encompassing 8,398 ecDNA entries across nine species, 63 diseases, and healthy individuals. Each entry provides detailed information, including eccDNA ID, type, chromosomal localization, species, tissue or cell line source, disease name and Disease Ontology ID, overlap length and percentage with genes, oncogene overlap, detection method, and links to literature and source databases. Given its extensive and curated datasets, eccDNABase serves as a valuable resource for both basic and translational research, offering deeper insights into the role of eccDNA in health and disease. The database is publicly accessible at http://cgga.org.cn/eccDNABase/.

Humans↗
PubMed1978

Induction of polygalacturonase production in legume roots as a consequence of extrachromosomal DNA carried by Rhizobium meliloti.

The ability of Rhizobium meliloti to induce polygalacturonase production in legume roots decreased during culture under laboratory conditions but was inducible with mitomycin C. This character was irreversibly lost after treatment with acridine orange. Extrachromosomal DNA of molecular weight 5.9 x 10(6) daltons was detectable in neutral sucrose gradient but was absent from cells 'cured' with acridine orange. Therefore, the ability to induce the enzyme production may be controlled by a plasmid.

Acridine Orange↗
PubMed1978

Quality and rate of extracellular polysaccharides produced by Rhizobium meliloti and their inducing effect on polygalacturonase production in legume roots as derived from the presence of extrachromosomal DNA.

The ability of extracellular polysaccharides of different strains of Rhizobium meliloti to induce the production of polygalacturonase by roots of Medicago sativa seedlings has been studied. Some strains showed a high inducing activity while those derived from them, after treatment with acridine orange and in which extrachromosomal DNA was absent, did not show this character. A comparative study of polysaccharide production and preliminary studies on the chemical composition of the active fractions obtained after Sephadex G-25 filtration indicated that the monomers which form the active fractions are qualitatively and quantitatively different according to their origin.

Acridine Orange↗