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Results for “Extrachromosomal DNA”

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At least 127 records · Page 7Linked to original sources
PubMed1987

Extrachromosomal DNA substrates in pre-B cells undergo inversion or deletion at immunoglobulin V-(D)-J joining signals.

Sequences encoding immunoglobulin variable domains are known to be assembled from variable (V), diversity (D), and joining (J) segments by site-specific recombination. We present a sensitive and rapid assay for V-(D)-J recombination that uses plasmid DNA transiently introduced into transformed pre-B cells, and demonstrates that the recombination is independent of any unique chromosomal context. Sequences sufficient to constitute recombination sites are contained within the 84 and 42 bp flanking, respectively, the murine J kappa 1 and V kappa L8 segments, which include the known heptamer-nonamer V-(D)-J joining signals. Deletion and inversion occur at comparable frequencies. Thus, V-(D)-J recombination may be relatively insensitive to the topological arrangement of sites, and events at the two novel junctions produced by the reaction may be coupled.

Animals↗
PubMed1992

A multicopy, extrachromosomal DNA in Leishmania infantum contains two inverted repeats of the 27.5-kilobase LD1 sequence and encodes numerous transcripts.

Leishmania DNA 1 (LD1) is a 27.5-kb sequence that occurs as an inverted repeat in a 55-kb multicopy, circular DNA in Leishmania infantum ITMAP263. The sequence is also found with a different genomic organization, possibly a tandem array, within a 1.5-Mb chromosome in all Leishmania isolates. About 26 stable transcripts of LD1 sequence, ranging from 0.6 to 15 kb, are found in ITMAP263. Transcripts were detected from both strands of the entire LD1 sequence, but the inverted repeat nature of the circular molecule prevented determination of whether transcription proceeded in one or both directions. Nine abundant transcripts (0.6-8.4 kb) from adjacent regions on the same strand of the repeat unit may represent mature mRNAs. One of these transcripts was shown to contain the 39-nucleotide spliced leader sequence characteristic of the 5' termini of trypanosomatid mRNAs. Several transcripts from the other strand of the repeat unit are also abundant and contain sequence complementary to some of the putative mRNAs. Less abundant, larger transcripts that span sequences encoding abundant mRNAs are also present, suggesting that transcription of LD1 is polycistronic.

Animals↗
PubMed2005

Characterization of ABCG2 gene amplification manifesting as extrachromosomal DNA in mitoxantrone-selected SF295 human glioblastoma cells.

The human ABCG2 gene, located on chromosome 4, encodes an ATP-binding cassette half-transporter that has been shown to confer resistance to chemotherapeutic agents. Relatively little is known about the mechanisms controlling expression of ABCG2. In previous studies, we had shown that overexpression of ABCG2 can result from rearrangement or gene amplification involving chromosome 4. To better characterize the mechanisms of ABCG2 overexpression, SF295 glioblastoma cells were exposed to increasing amounts of mitoxantrone to generate the SF295 MX50, MX100, MX250, and MX500 sublines, maintained in mitoxantrone concentrations ranging from 50 to 500 nmol/L. Northern blot analysis confirmed overexpression of ABCG2 mRNA, and immunoblot analysis demonstrated increased protein expression in the selected cell lines. Efflux of BODIPY-prazosin confirmed a functional protein. ABCG2 gene amplification was observed in all resistant sublines, as determined by Southern blot analysis. Fluorescence in situ hybridization (FISH) revealed amplification of ABCG2 via double minute chromosomes (dmins) detected in metaphase chromosome spreads in the SF295 MX50 and MX100 sublines. At higher levels of drug selection, in the MX250 and MX500 sublines, fewer dmins were observed but homogeneously staining regions (hsr) were visible with FISH analysis, revealing reintegration of the ABCG2 gene into multiple chromosomes. Spectral karyotyping (SKY) demonstrated multiple clonal and nonclonal rearrangements of chromosome 4, including hsrs. These results suggest that amplification of ABCG2 occurred initially in the form of dmins, followed by chromosomal reintegration of the amplicon at multiple sites. This occurred with increasing drug-selection pressure, generating a more stable genotype.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed1996

Ku80-deficient cells exhibit excess degradation of extrachromosomal DNA.

Mammalian cells possess a protein complex, termed DNA-PK, which binds to DNA double strand breaks in vitro. The complex consists of the heterodimeric Ku autoantigen and a DNA-dependent protein kinase, DNA-PKcs. Cell lines that are deficient for components of this complex are sensitive to ionizing radiation and have impaired V(D)J recombination, a site-specific recombination process. We have tested these cell lines for their ability to repair double strand breaks in transfected DNA. The xrs-6 cell line, which is deficient for the 80-kDa subunit of the Ku autoantigen, exhibited reduced stability of transfected DNA. Prior to obvious reductions in DNA stability, the levels of homologous recombination and DNA end joining were unaffected. However, the recovery of end joining products with precisely joined ends was reduced, with a concomitant increase in products containing deletions. Unlike the Ku80-deficient cells, no reduction in DNA stability was detected in DNA-PKcs-deficient scid cells. Scid cells also exhibited normal levels of homologous recombination and DNA end joining. These experiments implicate the Ku autoantigen, but not DNA-PKcs, in a direct role in protecting DNA ends from degradation.

Animals↗
PubMed1997

Extrachromosomal DNA in the Apicomplexa.

Malaria and related apicomplexan parasites have two highly conserved organellar genomes: one is of plastid (pl) origin, and the other is mitochondrial (mt). The organization of both organellar DNA molecules from the human malaria parasite Plasmodium falciparum has been determined, and they have been shown to be tightly packed with genes. The 35-kb circular DNA is the smallest known vestigial plastid genome and is presumed to be functional. All but two of its recognized genes are involved with genetic expression: one of the two encodes a member of the clp family of molecular chaperones, and the other encodes a conserved protein of unknown function found both in algal plastids and in eubacterial genomes. The possible evolutionary source and intracellular location of the plDNA are discussed. The 6-kb tandemly repeated mt genome is the smallest known and codes for only three proteins (cytochrome b and two subunits of cytochrome oxidase) as well as two bizarrely fragmented rRNAs. The organization of the mt genome differs somewhat among genera. The mtDNA sequence provides information not otherwise available about the structure of apicomplexan cytochrome b as well as the unusually fragmented rRNAs. The malarial mtDNA has a phage-like replication mechanism and undergoes extensive recombination like the mtDNA of some other lower eukaryotes.

Animals↗
PubMed2001

Effects of hydroxyurea on extrachromosomal DNA in patients with advanced ovarian carcinomas.

PURPOSE: In vitro low concentrations of hydroxyurea eliminate double-minute chromosomes (dmins) containing amplified drug-resistance genes and oncogenes from cancer cells. This clinical trial investigated whether a noncytotoxic dose of oral hydroxyurea could reduce the number of dmins in cancer cells in patients with advanced ovarian carcinomas. EXPERIMENTAL DESIGN: The high frequency of ascites associated with ovarian cancer facilitated the monitoring of cytogenetic variations with minimal discomfort in patients who required frequent abdominal paracentesis. Sixteen patients with advanced ovarian carcinomas resistant to conventional cisplatin-based and/or paclitaxel chemotherapy and with ascites requiring frequent abdominal paracentesis were entered in this study. A course of treatment consisted of a single oral dose of 80 mg/kg hydroxyurea every 3 days for 6 weeks. Blood and i.p. levels of hydroxyurea were determined. We monitored the variations of dmins in tumor cells taken from serial abdominal paracenteses. RESULTS: The median number of courses administered to the patients was 1 (range, 1--9). In ascites, hydroxyurea concentrations were 610.3 +/- 76.3, 219.8 +/- 85.6, and 86.1 micromol/liter at 4, 24, and 30 h after oral administration, respectively. Eleven (78.6%) of 14 patient specimens contained dmins before therapy. The number of spreads with tumor cells containing dmins were reduced by more than 50% in 5 (45%) of 11 and 3 (60%) of 5 patients at the completion of the first and second course of chemotherapy, respectively. Using tumor cells taken directly from the patients and grown in soft agar, we documented that concentrations of hydroxyurea in ascites were too low to have any cytotoxic effects. No grade 3--4 hydroxyurea-related toxicities nor any objective responses were observed. However, despite the utilization of a low noncytotoxic dose of hydroxyurea, two patients had prolonged stabilization of their disease for 6 and 10 months, respectively, with concomitant decreases in the number of dmins that remained until progression. CONCLUSIONS: This study showed that, in some circumstances, a noncytotoxic dose of hydroxyurea given to patients with ovarian cancer can decrease the number of metaphase spreads containing dmins in cancer cells.

Administration, Oral↗
PubMedDate not supplied

[Extrachromosomal DNA in yeast-Saccharomyces].

The recombinant plasmids containing autonomously replicating sequence (ARS) of yeast rDNA repeat are characterized by a high instability in transformed yeast cells. The instability of chimaric plasmids in yeast may result from improper replication and/or irregular mitotic segregation. To study the replication properties alone we have constructed series of hybrid plasmids containing centromeric DNA (CEN3), a selective marker (leu2) and ARS of rDNA. Each of these plasmids with the functional centromere should exhibit chromosomal i. e. regular type of mitotic segregation. The study of mitotic segregation of constructed plasmids has shown that the ARS rDNA from yeast is distinguished from other ARSs described in literature: ARS1, ARS2, ARS o-micron DNA. 1. The activation of replication of ARS rDNA is accidental, i. e. probability of ARS rDNA in the cell cycle is much less than one. 2. Some nuclear mutations as well as rho- mutation result in the increase of replicative activity of ARS rDNA. In some yeast strains the activity of ARS rDNA can reach the activity of ARS1, i. e. was close to one. The features of ARS rDNA may account for the phenomenon of amplification of rDNA genes.

DNA↗
PubMed1996

[Metabolic and structural properties of extrachromosomal DNA in mammalian cells].

Discrete bands of free DNAs of approximately 25 kbp were detected in human cell cultures. According to electrophoretic shifts induced by single and double strand breaks, they from topological isoforms (supercoiled, open, and linear). Long-term labeling (24 h) of growing and quiescent cultured cells by [3H]thymidine indicates differences of free versus chromosomal DNAs including (i) significantly lower specific radioactivity in growing cells, (ii) higher specific radioactivity in quiescent cells, and (iii) resistance to fluorodeoxyuridine labeling. During apoptosis of cultured Namalwa cells, heterogeneous fragments are formed which differ from free DNAs. Crosslinking of nascent RNA with DNA template by 8-methoxypsoralen indicated slight transcriptional activity of free DNAs.

Apoptosis↗
PubMed1997

New insight on the role of extrachromosomal retroviral DNA.

During infection with different retroviruses, high levels of unintegrated extrachromosomal DNA accumulate in infected cells. While extrachromosomal linear DNA is the immediate precursor of the integrated provirus, the function, if any, of extrachromosomal circular DNA has been unclear. Several groups have attempted to address the possible function, activity, and importance of this unintegrated DNA during the life cycle of retroviruses and the course of retroviral-associated diseases. This review summarizes recent work in this field and tries to analyze some aspects of extrachromosomal forms of retroviral DNA and their possible application as a molecular biological tool.

Animals↗
PubMed1991

Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection.

To better understand the basis for human immunodeficiency virus type 1 (HIV-1) persistence and latency, the form in which viral DNA exists in the peripheral T lymphocyte reservoir of infected individuals was investigated. In asymptomatic individuals, HIV-1 was harbored predominantly as full-length, unintegrated complementary DNA. These extrachromosomal DNA forms retained the ability to integrate upon T cell activation in vitro. In patients with acquired immunodeficiency syndrome (AIDS), there was an increase in integrated relative to extrachromosomal DNA forms. By analysis of DNA from patient lymphocyte subpopulations depleted of human lymphocyte antigen-Dr receptor-positive cells, quiescent T cells were identified as the source of extrachromosomal HIV-1 DNA. Thus quiescent T lymphocytes may be a major and inducible HIV-1 reservoir in infected individuals.

DNA, Viral↗