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Results for “Extrachromosomal DNA”

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At least 163 records · Page 9Linked to original sources

PubMed1987

Cloned extrachromosomal circular DNA copies of the human transposable element THE-1 are related predominantly to a single type of family member.

The 2300 base-pair transposon-like human element, THE-1, has been identified in the extrachromosomal circular DNA of the established human cell line HeLa as a relatively homogeneous population of covalently closed 1900 base-pair molecules. THE-1, which has been classified tentatively as a retroviral-like transposable element (a retrotransposon), is present in the extrachromosomal circular DNA of African green monkey (BSC-1) and human lymphoblastoid (Jurkat) cell lines. The 1900 base-pair extrachromosomal elements isolated and cloned from HeLa cells (1) appear to contain only THE-1-specific nucleotide sequences, (2) are circularized versions of the linear chromosomal sequence, and (3) are related predominantly to a single, or single type of, family member.

Autoradiography↗

PubMed1993

Reduced purine accumulation is encoded on an amplified DNA in Leishmania mexicana amazonensis resistant to toxic nucleosides.

Nucleoside analogs are potential anti-Leishmania agents. To better understand how these compounds might lose their effectiveness, Leishmania were independently selected for resistance to inosine dialdehyde or tubercidin. Each of the resistant cells exhibited resistance to inosine dialdehyde and tubercidin as well as to formycin B and allopurinol ribonucleoside. Resistant cells had a greatly reduced capability of accumulating exogenous adenosine, guanosine, thymidine and guanine. This decreased ability to accumulate nucleosides and at least one nucleobase appeared to be due to reduced activity of a number of distinct purine transporters, as the differences between purine metabolizing enzymes were not sufficiently different to account for the decreased accumulation capability. The resistance to toxic nucleosides and the decreased ability to accumulate purines were due to the presence in the resistant cells of an extrachromosomal DNA approximately 55 kb in size. The extrachromosomal DNA was not detected in wild-type cells or revertants which have lost resistance to toxic nucleosides. Except for a 1.2-kb difference, the extrachromosomal DNA from both independently selected resistant cells appeared to be identical. The resistant cells contained 2-4 times as much DNA homologous to the extrachromosomal DNA as compared to wild type cells. When cloned into an E. coli/Leishmania shuttle vector, a portion of the amplified DNA had the ability to confer upon wild-type cells resistance to the toxic purine nucleoside analogs tubercidin and inosine dialdehyde. These transformed cells also exhibited a decreased ability to accumulate non-toxic purine nucleosides.

Animals↗

PubMed1999

Elimination of extrachromosomal c-myc genes by hydroxyurea induces apoptosis.

Hydroxyurea (HU) increases extrachromosomal DNA elimination in tumor cell lines. The c-myc oncogene is one of the many relevant amplified genes contained within the extrachromosomal DNA compartment. Spontaneous loss of amplified copies of c-myc induces terminal differentiation and apoptosis in the human HL-60 leukemia cell lines. In the present study, we evaluate HU's ability to induce apoptosis by eliminating extrachromosomally located c-myc oncogene in human tumor cell lines. The consequences of eliminating extrachromosomal DNA by HU were explored in two different cell lines using the TdT assay and acridine orange/ethidium bromide labeling. COLO 320 clone 3 and COLO 320 clone 21 cell lines contain the same number of amplified copies of c-myc oncogene, but located respectively on extrachromosomal DNA, and intrachromosomally in homogeneously staining regions. HU induced apoptosis in the COLO 320 clone 3 cell line by a time and concentration dependent mechanism but could not induce apoptosis in the COLO 320 clone 21 cell line. These results suggested that HU-induced apoptosis in COLO 320 cell lines depends on elimination of extrachromosomal amplified copies of the c-myc oncogene. The ability of HU to eliminate extrachromosomally amplified copies of the c-myc oncogene and to induce apoptosis should be considered when targeting malignancies with amplification of the c-myc oncogene in an extrachromosomal site.

Journal Article↗

PubMed1991

Double minute chromosomes and homogeneously staining regions in tumors taken directly from patients versus in human tumor cell lines.

There is increasing evidence that copies of amplified oncogenes or drug-resistant genes located on extrachromosomal DNA (e.g. double minutes and/or episomes) can be eliminated from mammalian tumor cell lines by treatment of the cells with low concentrations of hydroxyurea. However, amplified oncogenes or drug-resistant genes located in an intrachromosomal site (such as in a homogeneously staining region (HSR)) cannot be eliminated from the cells. A question which arises is do primary human tumors have extrachromosomal DNA present often enough to make elimination of that extrachromosomal DNA a potentially important therapeutic strategy? To address that question we have reviewed published cytogenetic analyses of 200 tumors taken directly from patients to determine the percentage of primary human tumors which have amplified genes present on extrachromosomal DNA (present in the form of double minutes [DMs]) vs the percentage of tumors which have amplified genes located on an intrachromosomal site (in the form of HSRs). Of the 200 primary human tumors reviewed, 91% contained DMs only, 6.5% contained HSRs, and 2.5% contained both. Of interest, in a parallel review of 109 cell lines with cytogenetic and/or molecular evidence of gene amplification, 60.6% contained DMs, 26.6% contained HSRs, and 12.8% contained both. These data indicate that DMs are the predominant cytogenetic marker for gene amplification in patients, but are present less frequently in established cell lines. These findings indicate that ongoing efforts to eliminate amplified drug-resistant genes or oncogenes contained on DMs (or precursors of DMs) from tumor cells may be relevant for in vivo situations.

Chromosomes↗

PubMed2003

Extrachromosomal circular DNA of tandemly repeated genomic sequences in Drosophila.

One characteristic of genomic plasticity is the presence of extrachromosomal circular DNA (eccDNA). This DNA is found in various eukaryotes from yeast to humans, and its levels are elevated by exposure to carcinogens. eccDNA is heterogeneous in size and composed of chromosomal sequences. In this study we used two-dimensional gel electrophoresis to detect and characterize eccDNA in Drosophila. We found eccDNA throughout the fly's life cycle. These molecules comprise up to 10% of the total repetitive DNA content, and their size ranges from <1 kb to >20 kb. The eccDNA population contains circular multimers of tandemly repeated genes such as histones, rDNA, Stellate, and the Suppressor of Stellate. Multimers of centromeric heterochromatin sequences are included in eccDNA as well. Our findings are consistent with the hypothesis that intramolecular homologous recombination between direct tandem repeats is a favorite mechanism for eccDNA formation. The level of eccDNA increased following MMS treatment of wild-type larvae, consistent with phenomena observed in cultured mammalian cells. This shows mutagen-induced eccDNA formation in the context of the whole organism for the first time. Mutations in the genes okra, mus309, and mei41 did not affect eccDNA under normal conditions or following mutagen treatment, implying that eccDNA formation is different from known pathways of DNA repair.

Animals↗

PubMed1989

Recombination mediates production of an extrachromosomal circular DNA containing a transposon-like human element, THE-1.

An abundant class of HeLa extrachromosomal circular DNA containing the transposon-like element, THE-1, is shown to arise via site specific recombination. The chromosomal locus from which these circles are derived, however, is single-copy. Northern blot analysis detects homology to two polyadenylated RNAs in HeLa cells. The possible presence of an origin of replication and its role in generating these small polydisperse circles is discussed.

Base Sequence↗

PubMed1997

Generation and maintenance of tandemly repeated extrachromosomal plasmid DNA in Chlamydomonas chloroplasts.

Unusual chloroplast transformants of Chlamydomonas reinhardtii that contain 2000 copies of a mutant version of the chloroplast atpB gene, maintained as an extrachromosomal tandem repeat, have recently been described. In this paper studies have been undertaken to (i) address possible mechanisms for generating and maintaining the amplified DNA and (ii) determine whether it is possible to use chloroplast gene amplification to overexpress chloroplast or foreign genes. Data presented here indicate that high copy number transformants harbor characteristic rearrangements in both copies of the chloroplast genome large inverted repeat. These rearrangements appear to be a consequence of, or required for, maintenance of the amplified DNA. In an attempt to mimic the apparently autonomous replication of extrachromosomal DNA in the chloroplast, transformation was carried out with a plasmid that lacked homology with the chloroplast genome or with the same plasmid carrying a putative chloroplast DNA replication origin (oriA). Transformants were recovered only with the plasmid containing oriA, and all transformants contained an integrated plasmid copy at oriA, suggesting that establishment or maintenance of the extrachromosomal tandem repeat requires conditions that were not replicated in this experiment. To determine whether other genes could be maintained at high copy number in the chloroplast, plasmids carrying the wild-type atpB gene or the bacterial aadA gene were introduced into a high copy number transformant. Surprisingly, the copy number of the plasmid tandem repeat declined rapidly after the secondary transformation events, even when strong selective pressure for the introduced gene was applied. Thus, chloroplast transformation can either create or destabilize high copy number tandem repeats.

Animals↗

PubMed1996

Induction of circles of heterogeneous sizes in carcinogen-treated cells: two-dimensional gel analysis of circular DNA molecules.

Extrachromosomal circular DNA molecules are associated with genomic instability, and circles containing inverted repeats were suggested to be the early amplification products. Here we present for the first time the use of neutral-neutral two-dimensional (2D) gel electrophoresis as a technique for the identification, isolation, and characterization of heterogeneous populations of circular molecules. Using this technique, we demonstrated that in N-methyl-N'-nitro-N-nitrosoguanidine-treated simian virus 40-transformed Chinese hamster cells (CO60 cells), the viral sequences are amplified as circular molecules of various sizes. The supercoiled circular fraction was isolated and was shown to contain molecules with inverted repeats. 2D gel analysis of extrachromosomal DNA from CHO cells revealed circular molecules containing highly repetitive DNA which are similar in size to the simian virus 40-amplified molecules. Moreover, enhancement of the amount of circular DNA was observed upon N-methyl-N'-nitro-N-nitrosoguanidine treatment of CHO cells. The implications of these findings regarding the processes of gene amplification and genomic instability and the possible use of the 2D gel technique to study these phenomena are discussed.

Animals↗

PubMed1989

Increase of extrachromosomal circular DNA in mouse 3T6 cells on perturbation of DNA synthesis: implications for gene amplification.

We have analyzed the amount of extrachromosomal double-stranded covalently closed circular nonmitochondrial DNA in mouse 3T6 cells by Southern blotting and electron microscopy. Treatment with 7,1-dimethylbenz[a]anthracene, known to promote amplification of integrated SV40 genomes, elevated the amount of circular DNA. Inhibition of DNA synthesis with hydroxyurea, earlier shown to enhance amplification of the cellular dihydrofolate reductase gene, resulted in yet higher levels. Thus, elevation of the frequency of gene amplification and generation of extrachromosomal circular DNA seem to accompany each other in the situations studied in this paper. Two other DNA synthesis inhibitors, aphidicolin and thymidine, had markedly lesser effects on circular DNA. The significance of these findings for the mechanism of gene amplification is discussed.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1997

Direct PCR amplification and sequence analysis of extrachromosomal Plasmodium DNA from dried blood spots.

The Plasmodium parasite possesses two extrachromosomal genomes; the mitochondrial genetic element and the extrachromosomal plastid-like DNA. The latter has only been fully described for one culture strain of P. falciparum. In this study, a rapid procedure for amplifying plastid DNA from dried blood spots of blood infected with different malaria species was developed. PCR amplification of a 595 bp fragment within the plastid-like large subunit ribosomal-RNA (LSU-rRNA) gene was achieved using primers derived from the P. falciparum sequence. The PCR product was observed in all Plasmodium species examined. Sequence analysis of amplified products homologous to an LSU-rRNA fragment of the plastid-like extrachromosomal circle revealed extensive conservation between Plasmodium species including P. falciparum, P. vivax, P. malariae and P. berghei.

Animals↗

PubMed1996

HIV-1 protein expression from synthetic circles of DNA mimicking the extrachromosomal forms of viral DNA.

We have constructed circular forms of human immunodeficiency virus type 1 viral DNA in vitro that closely resemble the single and double long terminal repeat circular forms of unintegrated viral DNA formed in the nuclei of infected cells. We have analyzed viral protein expression after transient transfection of these circular DNAs into HeLa cells and compared it with expression from a transfected linearized plasmid containing an integrated provirus. Both circular forms are expressed, as judged by the appearance of extracellular p24, and expression is trans-activated by human immunodeficiency virus type 1 Tat. Viral p24 production, however, is approximately an order of magnitude lower than that obtained with transfected integrated viral DNA. Similar data were obtained when a luciferase reporter gene was substituted for the coding regions of the viral DNA. Positional effects of the transcriptional initiation and termination signals in the long terminal repeat appear to account for some of the low expression levels. These data suggest that unintegrated circular viral DNAs are transcriptionally active, although at low levels, and may contribute to overall viral replication in infected people under some conditions.

Base Sequence↗

PubMed1992

Extrachromosomal homologous DNA recombination in plant cells is fast and is not affected by CpG methylation.

Using a sensitive transient assay, we investigated extrachromosomal homologous DNA recombination (ECR) in plant cells. As the plant genome is highly C methylated, we addressed the question of whether CpG methylation has an influence on DNA recombination efficiencies. Whereas the expression level of the fully CpG-methylated DNA molecules was reduced drastically, we found no significant changes in ECR efficiencies between two partly CpG-methylated plasmids or between one fully CpG-methylated and one nonmethylated plasmid. Using a modified polymerase chain reaction analysis, we were able to detect recombination between two fully CpG-methylated plasmids. Furthermore, we characterized the kinetics of the ECR reaction. Cotransfection of plasmids carrying truncated copies of the beta-glucuronidase (GUS) gene resulted in enzyme activity with a delay of only half an hour compared with that of the plasmid carrying the functional marker gene. This indicates that the ECR reaction itself requires no more than 30 min. By polymerase chain reaction, we were able to detect the recombined GUS gene as early as 2 h after transfection. This result and the time course of the transient GUS activity indicate that ECR occurs mainly early after transfection. The biological significance of this finding is discussed, and properties of ECR and intrachromosomal recombination are compared.

Chromosomes↗

PubMed1998

Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase.

Acentric, autonomously replicating extrachromosomal structures called double-minute chromosomes (DMs) frequently mediate oncogene amplification in human tumors. We show that DMs can be removed from the nucleus by a novel micronucleation mechanism that is initiated by budding of the nuclear membrane during S phase. DMs containing c-myc oncogenes in a colon cancer cell line localized to and replicated at the nuclear periphery. Replication inhibitors increased micronucleation; cell synchronization and bromodeoxyuridine-pulse labeling demonstrated de novo formation of buds and micronuclei during S phase. The frequencies of S-phase nuclear budding and micronucleation were increased dramatically in normal human cells by inactivating p53, suggesting that an S-phase function of p53 minimizes the probability of producing the broken chromosome fragments that induce budding and micronucleation. These data have implications for understanding the behavior of acentric DNA in interphase nuclei and for developing chemotherapeutic strategies based on this new mechanism for DM elimination.

Antimetabolites↗

PubMed2002

Developmentally regulated extrachromosomal circular DNA formation in the mesozoan Dicyema japonicum.

The dicyemid mesozoans are simple multicellular parasites with a long cylindrical axial cell surrounded by a single outer layer of 20 to 30 ciliated peripheral somatic cells. Their larval development proceeds within the axial cell. Here we demonstrate the appearance of extrachromosomal circular DNAs and their fate during early embryogenesis in Dicyema japonicum. These DNAs are highly heterogeneous in sequence, suggesting that they consist of unique--not repetitive--elements. Potential open reading frames were not evident in the elements, so these DNAs are unlikely to have a protein-encoding function. In situ hybridization revealed that the circular DNA elements were restricted to the early embryonic larvae and gradually faded out as larvae approached maturity. Furthermore Southern blot analysis and polymerase chain reaction analysis using a high molecular weight DNA as a template provided evidence that the extrachromosomal DNA circles are originally present in chromosomes. These observations suggest DNA elimination--or selective replication--of the elements from chromosomes during early embryogenesis in dicyemid mesozoans.

Animals↗

PubMed1985

L1 sequences in HeLa extrachromosomal circular DNA: evidence for circularization by homologous recombination.

Subcloned probes of the L1 family of repetitive elements were used to isolate L1-carrying clones from a plasmid library of HeLa cell extrachromosomal circular DNA. One clone was analyzed in detail by restriction mapping, cross-hybridization to L1 probes, and base sequence analysis. In addition to approximately the 3' half of a full-sized L1 element, this clone carried 390 base pairs of non-L1 sequence that is single copy in the HeLa genome. A HeLa genomic clone of this unique chromosomal region was isolated and the sequence organization of the circle clone was compared with the linear chromosomal region from which it was ultimately derived. We discuss possible mechanisms of circular DNA formation and propose homologous intrachromosomal recombination between 9-base-pair direct repeats to be most likely in this case.

Chromosome Deletion↗

PubMed1976

Absence of rDNA amplification in the uninucleolate oocyte of the cockroach Blattella germanica (Oorthoptera: Blattidae).

Amplification of the genes coding for ribosomal RNA oocurs in the oocytes of a wide variety of organisms. In oocytes of various species of crickets (Orthoptera: Gryllidae) the amplified DNA is contained in a large extrachromosomal DNA body. Multiple nucleoli form about the periphery of the DNA body during the diplotene stage of meiosis I. In contrast to the general pattern of orthopteran oocytes, oocytes of the cockroach Blattella germanica demonstrate a single large nucleolus instead of many nucleoli. In order to determine whether the genes coding for rRNA are amplified in the oocytes of B. germanica, the relative amount of rDNA in oocytes was compared with the rDNA content of spermatocytes and somatic cells. An extrachromosomal DNA body similar to that present in crickets is not present in B. germanica. A satellite DNA band which contains nucleotide sequences complementary to rRNA accounts for approximately 3-5% of the total DNA in somatic and in male and female gametogenic tissues. Female cells contain approximately twice as much rDNA as do male cells. An XX-XO sex-determining mechanism is operative in B. germanica. In situ hybridization with rRNA indicates that the nucleolar organizer is located on one end of the X chromosome and that oocytes do not contain more than twice the amount of rDNA found in spermato cytes. The data indicate that rDNA is not amplified in the uninucleolate oocyte of B germanica.

Animals↗

PubMed1995

Ribosomal gene amplification in the oocytes of Creophilus maxillosus (Staphylinidae, Coleoptera-polyphaga)--an insect with telotrophic ovaries.

We have shown that the extrachromosomal DNA body present in the oocyte nucleus of Creophilus maxillosus contains amplified copies of ribosomal DNA and that multiple nucleoli associated with extrachromosomal DNA body contain ribosomal RNA. In addition, we assessed the level of rDNA amplification in Creophilus oocytes. The amount of DNA in single extra DNA body corresponds to 86% of total nuclear DNA content or 617% of chromosomal DNA content. In Creophilus, there are two phases of transcriptional activity of extrachromosomal rDNA, first in oogonia, and second in growing oocytes, separated by a short period of transcriptional quiescence in the ovary of newly hatched imago. We have described the organization and activity of extra DNA body in RNA synthesis and production of multiple nucleoli in previtellogenic stages of oocyte growth in imaginal ovariole of Creophilus.

Animals↗

PubMed2005

Structure of the germinal vesicle during oogenesis in leech Glossiphonia heteroclita (Annelida, Hirudinea, Rhynchobdellida).

Oogenesis in the glossiphoniid leech Glossiphonia heteroclita (Hirudinea, Rhynchobdellida) is nutrimental, i.e., the growing oocyte is supported by specialized germline cells, the nurse cells. The main function of the nurse cells is to provide oocytes with cell organelles and RNAs (mainly rRNA). However, in studied leech species, irrespective of the nutrimental mode of oogenesis, the germinal vesicle (GV = oocyte nucleus) seems to be very active in rRNA production. As shown in the present study, during early previtellogenesis in the GV the meiotic chromosomes and prominent primary nucleoli occur. In late previtellogenesis the chromosomes condense and occupy a limited space of nucleoplasm in close vicinity to primary nucleolus, forming a karyosome. At the onset of vitellogenesis several prominent extrachromosomal DNA bodies appear in close association with the karyosome. At the same time, the primary nucleolus is no longer visible in the GV. As vitellogenesis proceeds the extrachromosomal DNA bodies undergo fragmentation and numerous spherical, RNA- and AgNOR-positive inclusions occur in the nucleoplasm. They are regarded as multiple nucleoli. Finally, in late oogenesis numerous accessory nuclei are formed in close proximity to the nuclear envelope. They usually contain one dense body, morphologically similar to multiple nucleoli. The amplification of rDNA genes, the occurrence of extrachromosomal DNA bodies, as well as the presence of multiple nucleoli and accessory nuclei are described for the first time in the phylum Annelida.

Animals↗