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PubMed2002

Epstein-Barr virus (EBV)-positive pyothorax-associated lymphoma (PAL): chromosomal integration of EBV in a novel CD2-positive PAL B-cell line.

Pyothorax-associated lymphoma (PAL) is a clinico-pathological entity arising in the pleural cavity of patients with long-standing inflammatory pyothorax. PAL is closely associated with Epstein-Barr virus (EBV), but how this virus contributes to the development of the lymphoma is unknown. We have successfully obtained a novel EBV-infected PAL cell line, designated Pal-1. The cell line and its source coexpressed CD2 and CD20 molecules, but other representative B- and T-cell markers such as CD1, CD3, CD5, CD7, CD10 and CD19 were not found. The B-cell origin of Pal-1 cells was proven by rearrangement of the immunoglobulin heavy- and light-chain genes without rearranged T-cell receptor genes. Both the cell line and primary tumour cells carried monoclonal EBV genome. Although EBV genome is known to be maintained as circular extrachromosomal DNA, neither circular nor linear extrachromosomal EBV DNA was detectable in Pal-1 cells by in situ lysis gel analysis. Fluorescence in situ hybridization demonstrated viral integration at a marker chromosome mostly consisting of the centromere region of chromosome 1. The viral integration event may enhance a chromosomal instability at the insertion site. This cell line represents the first example of EBV integration in PAL and could enable the study of the potential role of integrated viral infection in the development of PAL as well as mechanism of the aberrant phenotype expression.

Aged↗

PubMed1995

V-D-J rearrangements at the T cell receptor delta locus in mouse thymocytes of the alpha beta lineage.

The T cell receptor (TCR) delta locus lies within the TCR alpha locus and is excised from the chromosome by V alpha-J alpha rearrangement. We show here that delta sequences persist in a large fraction of the DNA from mature CD4+CD8- alpha beta+ mouse thymocytes. Virtually all delta loci in these cells are rearranged and present in extrachromosomal DNA. In immature alpha beta lineage thymocytes (CD3-/loCD4+CD8+) and in CD4+CD8- alpha beta+ thymocytes expressing a transgene-encoded alpha beta receptor, rearranged delta genes are present both in chromosomal and extrachromosomal DNA. Thus, contrary to earlier proposals, commitment to the alpha beta lineage does not require recombinational silencing of the delta locus or its deletion by a site-specific mechanism prior to V alpha-J alpha rearrangement.

Animals↗

PubMed1975

[Ultrastructure of trophocyte nuclei in polytrophic ovarioles of Chrysopa perla (Neuroptera)].

The light and electron microscope and autoradiographic studies (H3-uridin incorporation) were carried out on the trophocyte nuclei of imago polytrophic ovarioles of Chrysopa perla (Neuroptera), from the trophocyte differentiation up to their degeneration. Like the oocytes, one of the seven nurse cells o every ovariole chamber contains extrachromosomal DNA bodies. This nurse cell is formed during differential mitoses in the germarium as one of two prooocytes. In contrast to extrachromosomal DNA of oocytes the trophocyte DNA bodies are less active structures. Several (2--4) complex nucleoli develop in the trophocytes of Chrysopa in the early stages of oogenesis. They consist of three main components: the chromatin mass, fibrillar bodies and granular strands. Such nucleoli grow, through increasing in number of fibrillar bodies and granular strands. They are most developed by the start of the vitellogenesis. At the middle vitellogenesis the general nucleolar structure modify due to the beginning of trophocyte degeneration. The consecutive stages of nuclear degeneration are described. The trophocyte nucleoli synthesize RNA still in germarium. The most intensive RNA synthesis is observed at the beginning of the vitellogenesis to decrease by the beginning of trophocyte degeneration.

Animals↗

PubMed1991

The mechanism of extrachromosomal homologous DNA recombination in plant cells.

By cotransfecting plasmids carrying particular mutations in the beta-glucuronidase (GUS) gene into Nicotiana plumbaginifolia protoplasts and by monitoring the recombination rates using a recently developed transient assay, we were able to obtain insights into the mechanism of extrachromosomal recombination operating in plant cells. An exchange of flanking markers takes place in over 90% of the recombination events. In most of the remaining cases two consecutive, independent single crossover events occur. These events involve the same DNA substrate and lead to two successive exchanges of flanking markers, thus mimicking a presumed double crossover intermediate. A comparison of the outcome of our experiments with the predictions of two recombination models originally proposed for mammalian cells indicates that extrachromosomal recombination in plant cells is best described by the single strand annealing model. According to this model all recombination events result in an exchange of flanking markers. Our results rule out the double strand break repair model which predicts that flanking markers are exchanged in only half of all events.

Base Sequence↗

PubMed1994

Pharmacokinetics of hydroxyurea in nude mice.

Extrachromosomal DNA is the predominant form of gene amplification in human tumors. Hydroxyurea (HU) concentrations of 100-150 microM have been promising in vitro for extrachromosomal DNA elimination. The study objective was to determine the HU dose-concentration relationship in nude mice with HU doses from 0 to 200 mg/kg. For HU t1/2 determination, mice were injected with HU 100 mg/kg. A plasma concentration of 159 microM was achieved and a t1/2 of 11.3 min determined. Based on these findings, In vivo elimination studies will require frequent administration of HU to maintain plasma concentrations from 100 to 150 microM.

Animals↗

PubMed2002

Extrachromosomal plasmid vectors for gene therapy.

Extrachromosomal DNA is becoming widely utilized as a gene therapy vector. Plasmid DNA offers multiple advantages over viral gene therapy vectors, including large packaging capacity, stability without integration and reduced toxicity. Furthermore, plasmid DNA can be delivered to many different tissues, using a variety of delivery techniques currently being developed. This review will discuss the advantages of extrachromosomal DNA as a gene therapy vector, highlighting recent advances and successes in its use in vivo.

Animals↗

PubMed1994

Extrachromosomal eukaryotic DNA substrates for switch recombination: analysis of isotype and cell specificity.

Switch recombination in B lymphocytes is a complex process directed by signals provided by cytokines and/or TH cells. To analyze these signals in an in vitro system, we have developed extrachromosomal eukaryotic substrates for switch recombination that replicate autonomously in murine cells and present pairs of switch (S) regions in an accessible mode. Switch recombination within the S regions results in the expression of the selectable neo gene. The results presented here indicate that substrates containing either S mu and S gamma 2b, S mu and S gamma 2a, or S mu and S alpha undergo switch recombination with similar frequencies in the pre-B-cell line 18-81, which has been previously reported to specifically switch to IgG2b. This indicates that, rather than expressing a gamma 2b isotype-specific recombinase, the 18-81 cells express a switch recombinase capable of acting on any accessible S region, supporting the accessibility model. The extrachromosomal substrates were rearranged in the 18-81 cells, but not in murine myeloma, T-cell, or fibroblast cell lines, supporting the idea that switch recombination is indeed regulated in a cell- and developmentally specific manner. Restriction enzyme analysis of the plasmid DNA recovered from the selected cell lines suggested multiple recombinational events, with most patterns in agreement with deletions within one or both switch regions.

3T3 Cells↗

PubMed1989

[The effect of thiocyanate on bacterial plasmids using the plasmid screening test].

A plasmid screening test (alkaline lysis and agarose gel electrophoresis) was used to assess the effect of thiocyanate on the plasmid replication on two bacterial strains with plasmids of different size, Klebsiella pneumoniae and Acinetobacter calcoaceticus. At concentrations physiologic for mammals (5-50 mg SCN-/l) no effect on the replication of extrachromosomal DNA was noted. This reaffirms the concept that a physiologic anion may be responsible for biological regulatory processes but will not affect the synthesis of extrachromosomal DNA when applied in vitro in physiologic concentrations.

Acinetobacter↗

PubMed1998

Amplification of the translocated c-myc genes in three Burkitt lymphoma cell lines.

Translocations of the coding exons of the human c-myc gene are consistent features of human Burkitt lymphomas (BL). In the BL cell lines CA46, JD40, and ST486, the second and third c-myc exons have been translocated into the immunoglobulin heavy chain locus. In addition to this rearrangement, in all three cell lines, we have found that the translocated c-myc exons show low-level amplification relative to restriction fragments from the germ-line c-myc gene. The patterns of hybridization of an IgM switch region probe suggest that immunoglobulin heavy chain sequences have been co-amplified with the translocated c-myc sequences. Differential sedimentation was used to determine whether the amplified sequences reside in high-molecular-weight chromosomes or low-molecular-weight extrachromosomal DNA. In JD40 and ST486 cells, the amplified c-myc sequences were found on high-molecular-weight chromosomes ST486 cells also contained translocated C-myc sequences in low-molecular-weight, extrachromosomal DNA, as did CA46 cells. These conclusions were corroborated by fluorescence in-situ hybridization (FISH) of HeLa, CA46, ST486 and JD40 metaphase chromosomes. These results suggest that there is ongoing selection for cells containing amplified copies of the expressed c-myc sequences. and that there is continuous generation of extrachromosomal copies of the translocated c-myc sequences in ST486 and CA46 cells.

Burkitt Lymphoma↗

PubMed1972

The timing of meiosis and DNA synthesis during early oogenesis in the toad, Xenopus laevis.

Recently metamorphosed female Xenopus laevis toads were injected with tritiated thymidine. Animals were kept at 20 degrees C and were sacrificed 1-23 days after isotope injection. Radio-autographs of squash preparations of the ovaries were made. The progress of labeled germ cell nuclei was followed to obtain information on the time course of early meiosis and extra-chromosomal DNA synthesis. Premeiotic S was estimated to take not more than 7 days. Leptotene takes 4 days, zygotene takes 5 days, and pachytene was estimated to be completed in about 18 days. The major period of amplification of the extrachromosomal DNA occurs in pachytene and takes about 13 days. A low level of synthesis was observed before and after this period, in zygotene and late pachytene-early diplotene, extending the total time for extrachromosomal DNA synthesis during meiosis to about 18 days. These data allowed the calculation to be made that one round of replication of the amplified DNA takes between 1.2 and 3.0 days. It was also found that in both oogonial and premeiotic interphases, the nucleolus-associated DNA shows asynchronous (probably late) labeling with respect to the chromosomes.

Animals↗

PubMed1984

Characterization of non-beta-lactamase-mediated ampicillin resistance in Haemophilus influenzae.

Ampicillin resistance in Haemophilus influenzae is most often due to the plasmid-mediated production of TEM beta-lactamase. We studied four strains with high-level ampicillin resistance (MIC of 32 micrograms/ml with an inoculum of 10(5) CFU on solid media) which did not produce detectable beta-lactamase activity with two different detection methods. Two of the four strains contained extrachromosomal DNA by agarose gel electrophoresis. Conjugation failed to transfer ampicillin resistance; in contrast, transformation yielded ampicillin-resistant transformants in three of the four strains. These transformants did not contain detectable extrachromosomal DNA. In addition, mobilization of the resistance determinant by transformation to, or conjugation with, recombination-deficient strains was unsuccessful. DNA-DNA hybridization experiments revealed no homology of the DNA of these strains with two R plasmids (one coding for ampicillin resistance, the other for chloramphenicol and tetracycline resistance). We conclude that the genetic basis of the non-beta-lactamase ampicillin resistance in these strains appears to be chromosomally mediated. We investigated the mechanism of resistance in these strains. Enzymatic modification of penicillin was not detected by autoradiography of a thin-layer chromatogram of cell sonic extracts of three ampicillin-resistant transformant strains incubated with [14C]penicillin. To assess changes in permeability of the cell envelope, a plasmid coding for beta-lactamase was conjugated into these strains, and the hydrolysis of penicillin by intact cells and cell sonic extracts was compared. Only one of three transformant strains had significantly diminished permeability. Outer membrane proteins of these strains analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed apparent differences in comparison with the isogenic ampicillin-susceptible recipient strain. Autofluorography of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Sarkosyl-solubilized crude membrane (the putative inner membranes) from these ampicillin-resistant transformant strains incubated with [3H]penicillin compared with the isogenic ampicillin-susceptible recipient strain revealed reduced binding to PBP 3 and 6, 3 and 4, or 4. In addition, affinity binding studies revealed decreased affinity of PBP 4 for ampicillin of all four transformants tested. We conclude that the major mechanism of resistance in these strains is altered penicillin-binding proteins; however, other mechanisms, including permeability, may also play a role.

Ampicillin↗

PubMed2002

Formation of extrachromosomal circles from telomeric DNA in Xenopus laevis.

Instability and plasticity of telomeric DNA, which includes extrachromosomal DNA, are usually correlated with the absence of telomerase and with abnormal growth of mammalian cells. Here, we show the formation of extrachromosomal circular DNA of telomeric repeats (tel-eccDNA) during the development of Xenopus laevis. Tel-eccDNA is double-stranded relaxed circles composed of the vertebrate consensus telomeric repeats [TTAGGG](n). Its size varies from <2 to >20 kb and it comprises up to 10% of the total cellular telomere content of the early embryo (pre-MBT stage). The amount of tel-eccDNA is reduced in later developmental stages and in adult tissues. Using a cell-free system derived from Xenopus egg extracts, we show that tel-eccDNA can be formed de novo from the telomere chromosomal tracts of sperm nuclei and naked DNA in a replication-independent manner. These results reveal an unusual plasticity of telomeric DNA during normal development of Xenopus.

Animals↗

PubMed2000

Isolation and analysis of a circular form of the IncJ conjugative transposon-like elements, R391 and R997: implications for IncJ incompatibility.

The incompatibility between the chromosomally integrating, conjugative transposon-like, IncJ elements R997 (ampicillin resistant) and R391 (kanamycin resistant) was examined by constructing strains harbouring both elements. Unusually, recA(+) strains harbouring the resistance determinants of both elements could be isolated but all strains lacked detectable extrachromosomal DNA. The phenotypic characteristics and transfer patterns observed suggested the formation of recombinant hybrids rather than strains harbouring both elements independently. Formation of strains harbouring two IncJ elements in a recA background was thus examined and resulted in the visualisation of extrachromosomal DNA. When R391 was transferred to a recA strain containing integrated R997, both elements co-existed stably and resulted in the isolation of a plasmid of 93.9 kb. When R997 was transferred to a recA strain harbouring an integrated R391, a plasmid of 85 kb was isolated. Comparison of restriction patterns for both elements revealed many common and several distinct fragments indicating a close physical relationship. These data suggest that although IncJ elements normally integrate at a unique site in the Escherichia coli chromosome, they possess the ability for autonomous replication which becomes manifest in a recA background when this site is occupied. This observation has implications for the nature of the incompatibility associated with IncJ elements and also provides a reliable method for isolating IncJ elements for molecular characterisation.

Ampicillin Resistance↗

PubMed2026

Whole-genome sequencing reveals hidden antimicrobial resistance genes in phenotypically susceptible probiotic candidate lactic acid bacteria.

Phenotypic assays commonly used to evaluate probiotic safety may fail to detect clinically relevant antimicrobial resistance (AMR), potentially allowing genetically concerning strains to appear acceptable based on MIC testing alone. To explore this issue, we applied whole-genome sequencing (WGS) to three lactic acid bacteria (LAB) isolates previously identified as probiotic candidates based on acid and bile tolerance, antagonism against enteric pathogens, and biofilm formation in vitro: Lactiplantibacillus plantarum L25F and L22F (from pigs) and Ligilactobacillus salivarius AF2319 (from a chicken). Genome annotation identified extensive repertoires of probiotic-associated genes (46-47 per strain) linked to stress tolerance, adhesion, immunomodulation, and quorum sensing, supporting functional potential. The two L. plantarum strains exhibited broader predicted metabolic capacities than L. salivarius AF2319. However, genomic analysis revealed acquired AMR genes with complex genotype-phenotype relationships not fully apparent from phenotypic testing. The L. plantarum strains harbored lnu(A) (99.79% identity) on extrachromosomal DNA, conferring the L-phenotype (lincomycin resistance, clindamycin susceptibility); clindamycin MICs (1 mg/L) were concordant with this genotype, though lincomycin MICs were not determined. L. salivarius AF2319 carried tet(M), tet(L), and erm(C) (99.48%, 99.49%, and 99.45% identity by ResFinder, respectively) on extrachromosomal DNA; notably, the erythromycin MIC (1 mg/L) was precisely at the EFSA breakpoint (≤ 1 mg/L), representing borderline genotype-phenotype discordance potentially due to silent gene expression. Under current EFSA QPS criteria, these acquired ARGs would preclude all three strains from approval as probiotic feed additives despite favorable functional profiles, underscoring the indispensable role of WGS-based AMR gene detection in modern probiotic safety evaluation.

Probiotics↗

PubMed2003

Radiation therapy depletes extrachromosomally amplified drug resistance genes and oncogenes from tumor cells via micronuclear capture of episomes and double minute chromosomes.

PURPOSE: To determine if clinically relevant doses of ionizing radiation are capable of inducing extrachromosomal DNA loss in transformed human cell lines. MATERIALS AND METHODS: The multidrug-resistant (MDR) human epidermoid KB-C1 cell line and the human neuroendocrine colon carcinoma line COLO320, which contain extrachromosomally amplified MDR1 drug resistance genes and MYCC oncogenes, were irradiated with 2 Gy fractions up to a total dose of 28 Gy. To track the fate of extrachromosomally amplified genes, cells surviving radiation therapy and unirradiated control cells were analyzed by fluorescent in situ hybridization of chromosomes using MDR1 and MYCC-specific cosmid DNA probes. In addition, total DNA and protein isolated from irradiated and control cells was subjected to Southern and Western blotting procedures, respectively, to determine amplified gene copy number and protein expression levels. Dose-response assays to follow loss of function of the MDR1 gene from KB-C1 cells were also performed. RESULTS: A significant reduction in extrachromosomal DNA, amplified gene copy number, and expression was detected in surviving cells after relatively low doses of radiation. Entrapment of extrachromosomal DNA into micronuclei was a consistent feature of irradiated cells. CONCLUSIONS: Clinically relevant doses of radiation can deplete extrachromosomal DNA in viable human malignant cells and alter their phenotype. Depletion of extrachromosomally amplified genes from tumor cells occurs via entrapment in radiation-induced micronuclei.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1983

Extent of BrdUrd-induced prolactin gene amplification in GH cells.

The thymidine analog, 5-bromodeoxyuridine (BrdUrd), induces prolactin (Prl) synthesis and Prl gene amplification in a subclone of GH cells (rat pituitary tumor cells in culture). Withdrawal of drug treatment reverses both processes. Our previous results show that Prl gene amplification is associated with an event involving extrachromosomal DNA. The results presented in this report show that a 20-kb length of DNA, including all the coding sequences of the Prl gene, is amplified following BrdUrd treatment of this strain of GH cells. Amplification of DNA sequences extends about 3 kb upstream from the 5' end and about 7 kb downstream from the 3' end of the Prl gene. The DNA sequences immediately adjacent to these 5'-and 3'-flanking regions of the Prl gene are not affected by BrdUrd treatment. These results suggest that BrdUrd-induced amplification of Prl gene coding and neighboring sequences is restricted to within 3 kb of the 5' end and within 7 kb of the 3' end of the structural gene Analysis of low-molecular-weight extrachromosomal DNA isolated by Hirt's procedure from drug-treated cells reveals a similar pattern of amplification of the Prl gene and its flanking sequences.

Animals↗

PubMed2003

Megaplasmids in Gram-negative, moderately halophilic bacteria.

We have discovered that many Halomonas species harbour large extrachromosomal DNA elements. Using currently available protocols it is technically very difficult to identify large plasmids in bacteria, and even more so when they are coated in mucous polysaccharide. We used culture conditions suitable for both halophilic and halophilic exopolysaccharide-producing bacteria and applied a modified gel electrophoresis method to locate and visualise the megaplasmids. Almost all the species of Halomonas studied harbour two plasmids of about 70 kb and 600 kb and some species carry other smaller extrachromosomal DNA elements. The common presence of these megaplasmids may well be related to the survival strategies of the bacteria in their special surroundings.

DNA, Bacterial↗

PubMed1983

Evidence for a chromosomal location of the genes coding for chloramphenicol production in Streptomyces venezuelae.

Of seven chloramphenicol-producing actinomycetes examined, only Streptomyces venezuelae strain 13s contained extrachromosomal DNA detectable by agarose gel electrophoresis and cesium chloride-ethidium bromide density gradient centrifugation. The single 17-megadalton plasmid present in this strain was indistinguishable from plasmid pUC3 previously isolated from mutagenized cultures. Strains selected for their inability to produce chloramphenicol after treatment with acriflavine or ethidium bromide still contained a plasmid that had the same electrophoretic mobility as plasmid pUC3 and yielded similar fragments when digested with restriction endonucleases. By regenerating protoplasts of strain 13s and screening for isolates lacking extrachromosomal DNA, strain PC51-5 was obtained. The absence of plasmid pUC3 sequences in this strain was confirmed by Southern hybridization using 32P-labeled plasmid as a probe. Since the plasmidless strain produced as much chloramphenicol as did the parent strain, pUC3 contains neither structural nor regulatory genes for antibiotic production. Evidence from electrophoretic analysis of BamHI digests of total cellular DNA from wild-type and dye-treated nonproducing progeny indicated that acriflavine caused structural changes in the chromosome.

Chloramphenicol↗