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At least 19 recordsLinked to original sources
PubMed1982

Variable-sized free episomes of Shope papilloma virus DNA are present in all non-virus-producing neoplasms and integrated episomes are detected in some.

The state of rabbit (Shope) papilloma virus DNA in virus-induced nonproducing tumors on domestic rabbits was investigated. Virus-specific sequences were resolved into many distinct bands by one-dimensional agarose gel electrophoresis. Two-dimensional gel electrophoresis, CsCl/propidium iodide density equilibrium centrifugation, partial digestion with a restriction endonuclease, and S1 nuclease digestion permitted us to identify the bands as free viral episomes representing circular molecules of increasing size. In some tumors (both papillomas and carcinomas), up to 25% of the virus-specific DNA was linear and comigrated with cellular DNA. Integration of at least some of these sequences was suggested by the detection of viral-cellular junction bands in one tumor after digestion of DNA with EcoRI and Sal I, enzymes that cut Shope DNA once. Finally, the physical states of viral DNA in papillomas and carcinomas were found to be similar, although free episomes were generally larger in carcinomas.

Animals↗
PubMed1993

Efficient episomal expression vector for human transitional carcinoma cells.

To develop an efficient expression vector for human transitional carcinoma cells, we evaluated the replication activity of episomal vectors derived from the BK virus (BKV) and the Epstein-Barr virus (EBV) in HT-1376 bladder carcinoma cells. Southern blot analysis of transient transfectants indicated that the BKV-derived episome replicated extrachromosomally whereas the EBV replicon did not appear to be functional in these cells. HT-1376 cells were stably transfected with BKV-derived episomes containing the neomycin resistance gene as a selectable marker. Southern analysis demonstrated that these stable transfectants contained approximately 150 copies of the BKV episome per cell. There was no evidence of integration of the BKV episome into genomic DNA following selection with G418 for 10 weeks. These stable episomal transfectants had approximately 20-fold higher levels of expression of neomycin resistance gene mRNA than clones of HT-1376 cells transfected with pSV2NEO which contained five integrated copies of this gene. In soft agar cloning experiments, BKV episomes were efficiently transferred to the progeny of these transfectants during cell division over multiple generations. Additionally, BKV episomal copy number is maintained in stable transfectants after withdrawal of selection pressure for over 2 months. These data demonstrate that BKV-derived episomes replicate efficiently in bladder carcinoma cells, yielding stable transfectants having a high episomal copy number and expressing encoded genes at high levels. BKV-derived episomes may be useful in gene therapy strategies to modulate the growth of bladder carcinoma cells.

BK Virus↗
PubMed1994

Damage induced in episomal EBV DNA in Raji cells by antitumor drugs as measured by pulsed field gel electrophoresis.

The studies described below were carried out to analyze the damage induced by DNA active drugs to episomal (Epstein-Barr virus, EBV) DNA in the Raji Burkitt's lymphoma cell line. This work: (i) applies pulsed-field gel electrophoresis (PFGE) techniques to quantify DNA damage on a large (approximately 180 kbp), circular target, (ii) investigates the DNA strand-scission behavior of different classes of drugs on the EBV episome, and (iii) compares EBV episomal damage to that generated in genomic DNA in the Raji cell line. Cells were treated with ionizing radiation to induce random strand scission, and the migration of topological forms of EBV was measured using PFGE. DNA damage induced in the episome by DNA active drugs was then assayed. Three drugs, acting by different types of DNA interactive mechanisms, were used: bleomycin, an intercalative DNA strand-scission agent; and amsacrine (mAMSA) and teniposide (VM26), intercalative and nonintercalative topoisomerase II active drugs, respectively. Rad equivalency of damage was determined by comparing the drug-induced change in percentage of Forms I and III to that generated by ionizing radiation. Additionally, single- and double-strand scission induced in genomic (total cellular) DNA by X-rays, bleomycin, amsacrine, and teniposide were assayed by high-sensitivity alkaline and neutral filter elution techniques. We demonstrate that pulsed-field gel electrophoresis is a useful technique for measuring form conversion in large episomal DNA. While all three drugs effect both episomal and genomic DNA strand scission, bleomycin appears to preferentially damage the EBV episome. The topoisomerase II active drugs mAMSA and VM26 show no evidence of episome-directed damage in this system and, in fact, damage genomic DNA at somewhat higher rates.

Amsacrine↗
PubMed1992

Reconstitution of an episomal mouse aprt gene as a consequence of recombination.

When a functional murine adenine phosphoribosyltransferase (aprt) gene linked to bovine papilloma virus (BPV) DNA is transfected into Aprt- L cells, the cells are rendered Aprt+ and the aprt gene persists as an episome. Cotransfection with two BPV vectors, one containing the 5' half of the aprt gene and the other the 3' half of the gene, that share about 300 bp of common sequence in intron 2, produces Aprt+ cells with functional aprt as an episome. Southern blot analysis of low molecular weight DNA derived from Hirt extracts revealed the regeneration of a diagnostic SmaI fragment, consistent with establishment of an episome with functional aprt that was reconstituted as a consequence of recombination. To establish cells with an episomal target for recombination, BPV vectors containing a G418 resistance marker and either the 5' half or 3' half of aprt were transfected into Aprt- L cells. Stably transfected cells, selected by their growth in G418, were in turn transfected with DNA containing the other half of the aprt gene. Following selection of Aprt+ cells, Southern blot and polymerase chain reaction (PCR) analysis of low molecular weight DNA confirmed the presence of a complete episomal aprt gene. The region of DNA shared by the episomal aprt fragment and the transfected aprt half was sequenced after PCR amplification of the reconstituted, episomal gene and was found to be wild type. The region of overlap that serves as the substrate for recombination lies entirely within an intron and can, therefore, tolerate nucleotide substitutions and deletions. The absence of such errors in the sequences examined is consistent with recombination events that are not error prone.

Adenine Phosphoribosyltransferase↗
PubMed1989

Both episomal and integrated forms of human papillomavirus type 16 are involved in invasive cervical cancers.

The form of human papillomavirus type 16 (HPV 16)DNA in specimens of invasive cervical cancer was investigated. High molecular, tandem repeats of viral sequences were detected as several distinct bands, using a low concentration (0.5%) agarose gel and a no-cut enzyme (HindIII) for HPV 16. Two-dimensional agarose gel electrophoresis allowed us to differentiate between the episomal multimeric and the integrated forms of viral DNA. All 34 cervical cancer specimens showed the characteristic PstI cleavage pattern of HPV 16 DNA, indicating that a full viral genome was present in these specimens, and 24 specimens (70%) showed only episomal monomeric or multimeric forms without the integrated form of HPV 16 DNA. The remaining 10 specimens (30%) showed integrated multimeric forms of viral DNA, either without the episomal form (8 specimens) or with the concomitant episomal form (2 specimens). In addition, a metastatic tumor in a pelvic lymph node showed only the episomal form of viral DNA, whereas its primary cervical cancer showed both episomal and integrated forms of viral DNA. There was no correlation between the forms of viral DNA and the clinical stages of tumors. The result indicates that both episomal and integrated forms of a complete HPV 16 DNA are involved in invasive cervical cancers.

Blotting, Southern↗
PubMed1997

Safety-modified episomal vectors for human gene therapy.

The effectiveness of ongoing gene therapy trials may be limited by the expression characteristics of viral and plasmid-based vectors. To enhance levels of heterologous gene expression, we have developed a safety-modified episomal expression vector that replicates extrachromosomally in human cells. This vector system employs a simian virus 40 (SV40) large T antigen mutant (107/402-T) that is deficient in binding to human tumor suppressor gene products, including p53, retinoblastoma, and p107, yet retains replication competence. These SV40-based episomes replicate to thousands of copies by 2-4 days after gene transfer in multiple types of human cell lines, with lower activity in hamster cells, and no detectable activity in dog, rat, and murine cell lines. Importantly, 107/402-T has enhanced replication activity compared with wild-type T antigen; this finding may be due, in part, to the inability of p53 and retinoblastoma to inactivate 107/402-T function. We demonstrate that the level and duration of 107/402-T expression regulates the observed episomal copy number per cell. Compared with standard plasmid constructs, episomes encoding 107/402-T yield approximately 10- to 100-fold enhanced levels of gene expression in unselected populations of transient transfectants. To determine if 107/402-T-based episomes replicate extrachromosomally in vivo, tumor explants in nude mice were directly injected with liposome/DNA complexes. Using a PCR-based assay, we demonstrate that SV40-based episomes replicate in human cells after direct in vivo gene transfer. These data suggest that safety-modified SV40-based episomes will be effective for cancer gene therapy because high level expression of therapeutic genes in transient transfectants should yield enhanced tumor elimination.

Animals↗
PubMed1989

Epstein-Barr virus episome-based promoter function in human myeloid cells.

Epstein-Barr virus (EBV) episomal replicons offer an expeditious means for amplifying transfected genes in human cells. A panel of EBV episomes was constructed to assess the relative utility of five distinct eukaryotic promoter elements for high level and inducible gene expression in stably transfected human myeloid leukemia cells. The Rous sarcoma virus 3' long terminal repeat (LTR) was most highly suited for EBV episome-based gene expression, whereas the lymphopapilloma virus and the SV40 early regulatory elements exhibited substantially lower activities. Chemically responsive promoter elements, such as the SV40 early, human metallothionein IIA and rat GRP78 gene promoters, retained their inducibility when EBV episome-based. Differences in gene expression obtained with the episomes reflected differential promoter activity rather than significant variations in episome copy numbers per cell. These observations provide guidelines for the optimal design of EBV episomal expression vectors for human expression work.

Cell Differentiation↗
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↗
PubMed1993

Episomal and integrated copies of Epstein-Barr virus coexist in Burkitt lymphoma cell lines.

The Epstein-Barr virus genome is present in more than 95% of the African cases of Burkitt lymphoma. In this tumor, the viral genome is usually maintained in multiple episomal copies. Viral integration has been described only for Namalwa, a cell line lacking episomes. In this study, we have addressed the question of whether integrated and episomal copies can coexist in Burkitt lymphoma cells. Gel electrophoresis was used to demonstrate the presence of episomal as well as free linear DNA in three Burkitt lymphoma cell lines. The numbers of episomal copies per cell were estimated to be 5 to 10 in BL36 and BL137 cells and below 1 in BL60 cells, indicating that BL60 does not represent a homogeneous cell population. Fluorescence in situ hybridization was combined with chromosomal banding to study the association of the viral DNA with metaphase chromosomes. A symmetrical pattern of signals at both chromatids located at the same chromosomal sites in many if not all metaphases was taken as evidence for viral integration. In each of the three cell lines, one site of integration was identified: at chromosome 11p15 in BL36 cells, at chromosome 1p34 in BL137 cells, and at the site of a reciprocal t(11;19) translocation in BL60 cells. Integrated, episomal and linear copies of Epstein-Barr virus DNA thus coexist in Burkitt lymphoma cells. The biological significance of viral integration in Burkitt lymphoma cells remains to be elucidated.

Africa↗
PubMed1987

Characterization of an episome produced in hamster cells that amplify a transfected CAD gene at high frequency: functional evidence for a mammalian replication origin.

In a previous study (G. M. Wahl, B. Robert de Saint Vincent, and M. L. De Rose, Nature (London) 307:516-520, 1984), we used gene transfer of a CAD cosmid to demonstrate that gene position profoundly affects amplification frequency. One transformant, T5, amplified the donated CAD genes at a frequency at least 100-fold higher than did the other transformants analyzed. The CAD genes in T5 and two drug-resistant derivatives were chromosomally located. In this report, we show that a subclone of T5 gives rise to an extrachromosomal molecule (CAD episome) containing the donated CAD genes. Gel electrophoresis indicated that the CAD episome is approximately 250 to 300 kilobase pairs, and a variety of methods showed that it is a covalently closed circle. We show that the CAD episome replicates semiconservatively and approximately once per cell cycle. Since the CAD cosmid, which comprises most of the CAD episome, does not replicate autonomously when transfected into cells, our results indicate that either the process which generated the episome resulted in a cellular origin of DNA replication being linked to the CAD sequences or specific rearrangements within the episome generated a functional origin. The implications of these results for mechanisms of gene amplification and the genesis of minute chromosomes are discussed.

Animals↗
PubMed1991

Episomal and integrated human papillomavirus in cervical neoplasia shown by non-isotopic in situ hybridisation.

It was postulated that non-isotopic in situ hybridisation (NISH) signal types 1-3 for human papillomavirus in cervical biopsy specimens represent episomal or integrated virus. The aim of this study was to validate this hypothesis by independent molecular techniques. Fresh cervical intraepithelial neoplasia (CIN) and squamous cell cancer (SCC) tissue were examined for NISH signal pattern by hybridising with digoxigenin labelled HPV 16. DNA was extracted from the same samples and analysed by restriction endonuclease digestion and Southern blotting to determine the physical state of the viral genome. Six CIN biopsy specimens showed a type 1 NISH signal for HPV 16. On Southern analysis these biopsy specimens contained only episomal HPV 16. Three SCC with a type 2 NISH signal contained integrated HPV 16 by Southern analysis. Two specimens, a CIN 3 and an SCC with a type 3 NISH signal for HPV 16, showed the presence of both episomal and integrated HPV 16 with conventional Southern analysis and two dimensional gel electrophoresis. These results show that episomal HPV can be reliably determined by NISH type 1 signal, integrated HPV by type 2, and a combination of both episomal and integrated HPV, by a type 3 signal in archival paraffin wax embedded cervical biopsy specimens. This will add another variable to the epidemiological studies of HPV infection. In particular, it will now allow retrospective studies to be done to define the role of episomal and integrated HPV in the evolution of cervical intraepithelial neoplasia and other cervical disease associated with this virus.

Blotting, Southern↗
PubMed1994

Clonal analysis of posttransplant lymphoproliferative disorders, using both episomal Epstein-Barr virus and immunoglobulin genes as markers.

The authors analyzed the clonality of 15 samples of B-cell lymphoproliferations arising in eight organ allograft recipients, using immunoglobulin (Ig) gene rearrangement and the fused terminal fragment of episomal Epstein-Barr virus (EBV) DNA as independent clonal markers. The tumors arose from 1 month to 4 years following transplantation. All tumors were monomorphous, high-grade lymphomas of immunoblastic (6 cases), large-cell noncleaved (centroblastic, 1 case), or small noncleaved (1 case) type. All tumors were highly aggressive and failed to respond to decreased immunosuppression alone. Each tumor had clonal Ig gene rearrangements, including those that were negative for surface Ig. In 13 of 15 specimens (seven of the eight cases), the tumors also contained latent, circularized EBV genome. In 10 specimens from six patients, the tumors contained a single predominant form of episomal EBV DNA, indicating clonal cellular proliferation of an EBV-infected progenitor cell. Three specimens from one patient showed more than one band of episomal EBV DNA, suggesting oligoclonal expansion, despite the detection of only a single clone by Ig gene rearrangement. Linear replicating EBV DNA was not detected in any of the cases. Synchronous or metachronous specimens from multiple sites were studied in five patients, four of which were EBV-positive cases. Two patients had identical Ig gene arrangements in each specimen, indicating a single neoplastic clone at all sites; one case was EBV-negative, and the other had identical EBV episomes in each specimen. In the other three cases, apparently different Ig gene rearrangements were found at different sites. In two of these, however, the same predominant EBV episome was present at each site, indicating a common clonal origin. The third case had oligoclonal EBV bands in each specimen, with distinct patterns in at least two different sites, suggesting true multiclonality. Analysis of EBV genomes is a useful adjunct to Ig gene analysis in assessing the clonality of these lesions.

Adult↗
PubMed1974

T7 protein synthesis in F' episome-containing cells: assignment of specific proteins to three translational groups.

Synthesis of many T7 proteins is prevented in F' episome-containing cells. In order to quantitate the degree of inhibition, we measured the activity of several T7 proteins in extracts prepared from T7-infected F(-) and F' cells and cells containing F factors mutant in phage inhibition [F'(PIF(-)2A) and F'(PIF(-)2A,2B)]. In addition, we were able to assign specific T7 proteins to the three translational units previously defined by polyacrylamide gel analysis of T7 proteins made in F(-) and episome-containing cells. After T7 infection, the presence of the wild-type F' (PIF(+)) episome led to greater than 90% inhibition of T7 DNA polymerase (product of gene 5), T7 lysozyme (gene 3.5), and gene 10 capsid protein synthesis. Nearly normal amounts of T7 RNA polymerase (gene 1) were made in these cells. T7 infection of cells containing the mutant F' (PIF(-)2A) episome led to normal synthesis of T7 RNA polymerase and T7 DNA polymerase; T7 lysozyme was synthesized at 30% of the maximal level in these cells; T7 gene 10 capsid protein synthesis was inhibited by 90%, and T7 DNA synthesis was arrested in these cells. T7 infection of cells containing the mutant F' (PIF(-)2A,2B) episome led to synthesis of normal levels of the enzymes assayed.

Cell-Free System↗
PubMed1990

Replication of chromosomal and episomal DNA in X-ray-damaged human cells: a cis- or trans-acting mechanism?

Episomal plasmids and viruses in mammalian cells present small targets for X-ray-induced DNA damage. At doses up to 100 Gy, DNA strand breaks or endonuclease III-sensitive sites were not discernible in 10.3-kb Epstein-Barr virus-based plasmid DNA or in 4.9-kb defective simian virus 40 DNA. DNA replication in these small molecules, however, was inhibited strongly by X-ray doses of greater than or equal to 20 Gy, decreasing to only 20 to 40% of control values. Inhibition was relieved slightly by growth in caffeine but was increased by growth in 3-aminobenzamide. Inhibition of DNA replication in episomal DNA molecules that are too small to sustain significant damage directly to their DNA may be due to either (a) a trans-acting diffusible factor that transfers the consequences of DNA breakage to episomes and to other replicating molecules, (b) a cis-acting mechanism in which episomes are structurally linked to genomic chromatin, and replication of both episomal and chromosomal replicons is under common control, or (c) radiation damage on other cellular structures unrelated to DNA. The resolution of these cellular mechanisms may shed light on the X-ray-resistant replication in ataxia-telangiectasia and may suggest strategies for molecular characterization of potential trans- or cis-acting factors.

Benzamides↗
PubMed1978

Episomal viral DNA in herpesvirus saimiri-transformed lymphoid cell lines.

Structural analysis of episomal viral genomes from two herpesvirus saimiri (HVS)-transformed tumour cell lines (No. 1670 and 70N2) showed that both types of episomes have a higher molecular weight than linear virion DNA. The arrangement of unique (L) and repetitive (H) DNA in No. 1670 episomes was studied by partial denaturation mapping. Part of the L-sequences present in linear virion DNA was found to be missing, part was found to be duplicated in the episomes. The episomal L-DNA regions were correlated with the known physical gene maps of linear HVS DNA.

Animals↗
PubMed1982

[Episome stability in Escherichia coli K-12].

Arg+ and trp+ carrying episomes which have markedly different spontaneous efficiency of elimination, were obtained in experiments with AB673 and KL99 Escherichia coli donor strains. The level of episome maintenance is the result of mutation (deletion, insertion) that occurred in episomes during their formation. Differences in the efficiency of spontaneous elimination are independent of the origin of the donor strain used. The rate of loss of stably maintained episomes is not enhanced significantly by exposure of cells to AO. According to molecular characteristics, the episome demonstrating stable maintenance has a lower molecular mass than that of unstable maintenance.

Acridine Orange↗
PubMed1993

Autonomously replicating single-copy episomes in Trypanosoma brucei show unusual stability.

We have obtained several autonomously replicating plasmids in procyclic Trypanosoma brucei. Two of these have been analyzed in detail. Both exist as monomeric single-copy episomes, which nevertheless demonstrate significant stability. These episomes transform procyclics at frequencies that are at least 60-fold higher than those achieved earlier with non-replicating molecules. A modified version of one of these episomes is able to replicate autonomously in bloodstream form T. brucei but it is unstable in this developmental stage of the organism. These episomes will prove useful as shuttle vectors, in functional complementation studies, in the construction of promoter-traps for the isolation of transcriptional promoters in T. brucei, and importantly, as models for the analysis of DNA replication in this ancient eukaryotic lineage.

Animals↗
PubMed1981

Genetic analysis of mu or mini-mu containing F' pro lac episomes after prophage induction.

We have investigated the fate of different F pro lac episomes carrying a Mu or mini-Mu, after induction of the Mu or mini-Mu prophage, by looking at the frequencies of transfer of the episome and of one chromosomal marker. During the first 10 min after induction the frequency of chromosome mobilization increases while the frequency of episome transfer decreases. This suggests that the F interacts with the chromosome through some kind of Mu mediated process. Later the transfer of both the episome and chromosomal markers is inhibited. Possible reasons for this inhibition are discussed.

Bacteriophage mu↗