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Results for “Extrachromosomal DNA”

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PubMed1999

The prokaryotic beta-recombinase catalyzes site-specific recombination in mammalian cells.

The development of new strategies for the in vivo modification of eukaryotic genomes has become an important objective of current research. Site-specific recombination has proven useful, as it allows controlled manipulation of murine, plant, and yeast genomes. Here we provide the first evidence that the prokaryotic site-specific recombinase (beta-recombinase), which catalyzes only intramolecular recombination, is active in eukaryotic environments. beta-Recombinase, encoded by the beta gene of the Gram-positive broad host range plasmid pSM19035, has been functionally expressed in eukaryotic cell lines, demonstrating high avidity for the nuclear compartment and forming a clear speckled pattern when assayed by indirect immunofluorescence. In simian COS-1 cells, transient beta-recombinase expression promoted deletion of a DNA fragment lying between two directly oriented specific recognition/crossing over sequences (six sites) located as an extrachromosomal DNA substrate. The same result was obtained in a recombination-dependent lacZ activation system tested in a cell line that stably expresses the beta-recombinase protein. In stable NIH/3T3 clones bearing different number of copies of the target sequences integrated at distinct chromosomal locations, transient beta-recombinase expression also promoted deletion of the intervening DNA, independently of the insertion position of the target sequences. The utility of this new recombination tool for the manipulation of eukaryotic genomes, used either alone or in combination with the other recombination systems currently in use, is discussed.

Animals↗

PubMed2006

Transposition-based plant transformation.

Agrobacterium T-DNAs were used to deliver transposable Dissociation (Ds) elements into the nuclei of potato (Solanum tuberosum) cells. A double-selection system was applied to enrich for plants that only contained a transposed Ds element. This system consisted of a positive selection for the neomycin phosphotransferase (nptII) gene positioned within Ds followed by a negative selection against stable integration of the cytosine deaminase (codA) gene-containing T-DNA. Sixteen of 29 transgenic plants were found to contain a transposed element while lacking any superfluous T-DNA sequences. The occurrence of this genotype indicates that Ds elements can transpose from relatively short extrachromosomal DNA molecules into the plant genome. The frequency of single-copy Ds transformation was determined at 0.3%, which is only about 2.5-fold lower than the potato transformation frequency for backbone-free and single-copy T-DNAs. Because of the generally high expression levels of genes positioned within transposed elements, the new transformation method may find broad applicability to crops that are accessible to Agrobacterium T-DNA transfer.

DNA Transposable Elements↗

PubMed1997

Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.

We report the fist genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B. methanolicus with both integrative and multicopy plasmids. For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efficiency from 7- to 11-fold. Two low-copy-number Escherichia coli-B, methanolicus shuttle plasmids, pDQ507 and pDQ508, are described. pDQ508 caries the replication origin cloned from a 17-kb endogenous B. methanolicus plasmid, pBM1. pDQ507 carries a cloned B. methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule. Deletion analysis of pDQ507 indicated two regions required for replication, i.e., a 90-bp AT-rich segment containing a 46-bp imperfect, inverted repeat sequence and a second region 65% homologous to the B. subtilis dpp operon. We also evaluated two E. coli-B. subtilis vectors, pEN1 and pHP13, for use as E. coli-B. methanolicus shuttle vectors. The plasmids pHP13, pDQ507, and pDQ508 were segregationally and structurally stable in B. methanolicus for greater than 60 generations of growth under nonselective conditions; pEN1 was segregationally unstable. Single-stranded plasmid DNA was detected in B. methanolicus transformants carrying either pEN1, pHP13, or pDQ508, suggesting that pDQ508, like the B. subtilis plasmids, is replicated by a rolling-circle mechanism. These studies provide the basic tools for the genetic manipulation of B. methanolicus.

Amino Acid Sequence↗

PubMedDate not supplied

Structural DNA and genetically active DNA in dinoflagellate chromosomes.

High resolution electron microscope autoradiographs of [3H]adenine incorporation in the dinoflagellate Prorocentrum micans suggest that RNA transcription occurs on extrachromosomal DNA filaments, but not on DNA in the main body of the chromosome. This genetically inactive DNA has an important role, however, in stabilising chromosome structure by its association with protein matrix. Evidence for the importance of this molecular association (which is probably cation-mediated) is provided by alkaline buffer extraction of the protein matrix in chromosomes of Amphidinium carterae , leading to complete destabilisation of the DNA framework. The clear distinction between structural DNA and genetically active DNA in these chromosomes provides a marked contrast to normal eucaryote chromosomes. This distinction is related to the occurrence of high DNA values in these organisms, and the evolutionary status of the dinoflagellate chromosome.

Adenine↗

PubMed1985

State of hepatitis B virus DNA in leucocytes of hepatitis B patients.

Hepatitis B virus (HBV) DNA in leucocytes from 50 hepatitis patients with various patterns of HBV serological markers and serum HBV DNA and 13 normal controls were examined by Southern blot hybridization with 32P-labeled 3.2 Kb HBV DNA. A free form of HBV DNA was observed in leucocytes of 8 patients, 7 of whom were positive for serum HBeAg, and in 6 patients an integrated form of HBV DNA was identified. HBV DNA was not identified in leucocytes from 13 normal controls. The free form of HBV DNA in leucocytes existed as a heterogeneous smear from 2.0 to 3.2 Kb, similar to the pattern in liver and hepatocellular carcinoma cells but different from serum HBV DNA in which the 3.2 Kb band was absent. The banding pattern of the integrated form of HBV DNA in leucocytes varied among different patients. During preparation of white blood cells and purification of HBV DNA probes, it was important to remove plasma contamination and traces of pBR322, respectively. The presence of extrachromosomal DNA sequences partially homologous to pBR322 could cause false results. The presence of a free and integrated form of HBV DNA in leucocytes is important for explaining the biology of HBV, the harbouring and replication sites of extrahepatic origin, the mechanism of recurrent infection, and the rationale of the treatment of hepatitis B.

Carrier State↗

PubMed1996

Double minutes in the papillary thyroid cancer cell line PTC-1113A.

The cell line PTC-1113A was established from a metastasizing recurrent papillary thyroid cancer. The cell line was growing as monolayer and showed a complex karyotype with chromosome numbers ranging from 30 to 140/metaphase. A proportion of metaphases contained double minutes and/or pulverized chromosomes. Extrachromosomal DNA seemed to originate from a B-group chromosome. A chromosome 4 painting probe hybridized to extrachromosomal material, representing double minutes (dmin) and possibly minutes. In addition, fluorescence in situ hybridization (FISH) with the chromosome 4 library detected a translocation chromosome and a pulverized chromosome originating from chromosome 4. PTC-1113A is, to our knowledge, the single papillary thyroid cancer cell line demonstrating evidence of gene amplification.

Aged↗

PubMed2001

Genomic interspersions determine the size and complexity of transgene loci in transgenic plants produced by microprojectile bombardment.

The structure of transgene loci in six transgenic allohexaploid oat (Avena sativa L.) lines produced using microprojectile bombardment was characterized using fluorescence in situ hybridization (FISH) on extended DNA fibers (fiber-FISH). The transgene loci in five lines were composed of multiple copies of delivered DNA interspersed with genomic DNA fragments ranging in size from ca. 3 kb to at least several hundred kilobases, and in greater numbers than detected using Southern blot analysis. Although Southern analysis predicted that the transgene locus in one line consisted of long tandem repeats of the delivered DNA, fiber-FISH revealed that the locus actually contained multiple genomic interspersions. These observations indicated that transgene locus size and structure were determined by the number of transgene copies and, possibly to a greater extent, the number and the length of interspersing genomic DNA sequences within the locus. Large genomic interspersions detected in several lines were most likely the products of chromosomal breakage induced either by tissue culture conditions or, more likely, by DNA delivery into the nucleus using microprojectile bombardment. We propose that copies of transgene along with other extrachromosomal DNA fragments are used as patches to repair double-strand breaks (DSBs) in the plant genome resulting in the formation of transgene loci.

Avena↗

PubMed1991

Hydroxyurea accelerates loss of extrachromosomally amplified genes from tumor cells.

Gene amplification is one mechanism mediating the resistance of tumor cells to antineoplastic agents and the overexpression of a variety of oncogenes in diverse tumor types. There is mounting evidence that acentric extrachromosomal elements such as double minute chromosomes are common intermediates in the amplification process. These acentric structures partition unequally at mitosis and can be lost in the absence of selection. In the present study, we used human and hamster cell lines documented to contain extrachromosomally amplified drug resistance genes to investigate the feasibility of enhancing the loss rate of the extrachromosomally amplified genes to make the cells more sensitive to drug treatment. The results show that treatment of each of these lines with hydroxyurea accelerates the loss of the extrachromosomally amplified drug resistance genes. Loss of these extrachromosomal genes was associated with a corresponding increase in the drug sensitivity in the cases examined. The mechanism of accelerated loss does not appear to involve a differential effect on the replication of extrachromosomal DNA sequences. The results suggest that hydroxyurea treatment may provide a valuable tool for the general accelerated elimination of extrachromosomally amplified genes.

Aspartate Carbamoyltransferase↗

PubMed1967

The loss of kinetoplastic DNA in two species of Trypanosomatidae treated with acriflavine.

The effects of acriflavine on two species of Trypanosomatidae, Crithidia luciliae and Trypanosoma mega, have been investigated. It has been observed that kinetoplastic (i.e. mitochondrial) DNA is lost in a high percentage of acriflavine-treated cells. Resting flagellates, from stationary-phase or hemin-deficient cultures, are considerably more resistant to the acridine than are flagellates from a log-phase culture. When the kinetoplast has retained some DNA and still remains visible in stained smears, it appears reduced in size, and its ultrastructure is extremely abnormal: the DNA fibrils, clearly visible in normal kinetoplasts, are condensed; they appear as an electron-opaque, apparently homogeneous mass, separated from the membranes by a space of low electron-opacity. Analyses of DNA extracts, with high speed centrifugation in CsCl density gradients, revealed that the satellite band, presumably kinetoplastic DNA, is lost by trypanosomes grown for 5 days in the presence of acriflavine. Radioautography was used to study the effects of acriflavine on thymidine-(3)H incorporation in C. luciliae. At the concentration which affects the kinetoplast specifically, the dye produces an 87% inhibition of thymidine incorporation in this organelle. The kinetics of this inhibition suggest a direct effect on replication. No decrease in incorporation occurs in the nucleus. These results lead to the conclusion that loss of kinetoplastic DNA is due to continued growth and cell division in the absence of kinetoplastic DNA replication. Several hypotheses are discussed concerning the specificity of the dye's action upon the replication of extrachromosomal DNA.

Acridines↗

PubMed1987

[DNA isolated from Streptococcus sp. Thom-1066--the producer of thomicide].

Thomicide is a complex preparation including a bacteriocin-like substance. To localize the determinant responsible for synthesis of the bacteriocin-like substance, DNA of the streptococcus producing thomicide was isolated and studied. Equilibrium centrifugation of the total DNA preparation in the gradient of cesium chloride-ethidium bromide yielded DNA of one density. The total DNA preparation was obtained with alkaline and neutral lysis. Restriction analysis of the streptococcal DNA followed by electrophoretic separation in agarose gel in comparison to the DNA standards of lambda phage and plasmid pBR 322 confirmed the absence of the plasmid or any other extrachromosomal DNA. Chromosomal localization of the determinant encoding biosynthesis of the thomicide bacteriocin-like substance was shown.

Anti-Bacterial Agents↗

PubMed1979

Loss of plasmid-mediated resistance after conversion of a group B streptococcus strain to a stable cell wall deficient variant.

A group B streptococcus strain carrying plasmid DNA determining resistance to several drugs was converted by penicillin to cell wall (CW) defective and then to CW deficient variants (L-forms). The stable CW deficient variants became susceptible to antibiotics in study. Dye-buoyant density analysis of the DNA of CW deficient variants showed that the loss of antibiotic resistance was associated with the loss of extrachromosomal DNA.

Anti-Bacterial Agents↗

PubMed1986

DNA supercoiling of recombinant plasmids in mammalian cells.

We have used chloroquine/agarose gel electrophoresis and a blot-hybridization technique to study the modulation of superhelicity of extrachromosomal DNA in mammalian cells. The high sensitivity of the procedure has allowed us to measure the change in the specific linking difference or superhelical density (sigma) of a plasmid, psvo alpha 1p3d, after its introduction into COS-7 cells by DNA transfection. Because the molecular weight of psvo alpha 1p3d is approximately the same as that of simian virus 40 (SV40) DNA, the latter can be used as a standard for estimating the average linking difference or number of superhelical turns (tau) of psvo alpha 1p3d after separation of the different supercoiled species on chloroquine/agarose gels. It was found that transfection of monkey cells with either fully supercoiled psvo alpha 1p3d isolated from bacteria (tau = -27 +/- 1, sigma congruent to -0.051) or its relaxed form after treatment with DNA topoisomerase I yields psvo alpha 1p3d samples of the same tau and sigma values of -20 +/- 1 and -0.038, respectively. The difference between the tau values of psvo alpha 1p3d and SV40 in COS-7 cells, in which both plasmids undergo rounds of replication, corresponds to an average difference of 5 +/- 1 superhelical turns. Plasmid psvo alpha 1p3d remains at this lower level of superhelicity for at least 72 hr. The distribution in linking numbers of the topoisomers of psvo alpha 1p3d isolated from transfected COS cells is also more heterogeneous than that of SV40 DNA. These results suggest that the regulation of DNA supercoiling and chromatin assembly may be closely associated with specific DNA sequences. The approach presented here should have a wide application in the study of the regulation and functional role(s) of DNA supercoiling of plasmids in mammalian cells.

Animals↗

PubMed1985

Synthesis in cell culture of the gapped linear duplex DNA of the slow virus visna.

Visna virus is a nontransforming retrovirus that causes slow infections in animals and a rapidly progressive-lytic infection in cell culture. The results of an analysis of the synthesis of viral DNA in cell culture are reported. Region- and strand-specific probes cloned in M13 have been used to define the dynamics of DNA synthesis and the major nucleic acid species formed. It is shown that (i) within the first hours of infection, a full-length copy of the viral RNA genome is synthesized by reverse transcription, (ii) early in infection a major species of DNA is formed that extends from a site near the center of the molecule to the 3' end, (iii) somewhat later a second major species of plus-strand DNA is generated that extends from the 5' end to the middle of the genome. As a consequence, most viral DNA molecules consist of a full-length minus strand, and two plus strands separated by a gap or nick in the center of the molecule (J. D. Harris, J. V. Scott, B. Traynor, M. Brahic, L. Stowring, P. Ventura, A. T. Haase, and R. Peluso (1981). Virology 113, 573-583). The implications of this viral DNA structure for one unusual aspect of the lentivirus life cycle, the production of viral RNA, and virions from extrachromosomal DNA are discussed (J. D. Harris, H. Blum, J. Scott, B. Traynor, P. Ventura, and A. T. Haase (1984). Proc. Natl. Acad. Sci. USA 81, 7212-7215).

Animals↗

PubMed1999

Efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system.

Lentiviruses infect both dividing and nondividing cells. In this study we characterized a lentiviral vector system consisting of a packaging vector (pHP) and a transducing vector (pTV) derived from a recombinant human immunodeficiency virus type 1 (HIV-1). In pHP, the long terminal repeats (LTRs), the 5' untranslated leader and portions of the env and nef genes were deleted. The leader sequence of pHP was substituted with a modified Rous sarcoma virus (RSV) 59 bp leader containing a mutated RSV gag AUG and a functional 5' splice site. The pHP construct was found to direct Gag-Pol synthesis as efficiently as wild-type HIV-1. The pTV construct contains sequences required for RNA packaging, reverse transcription and integration, but lacks viral genes. Co-transfection of pHP, pTV and a vesicular stomatitis virus G (VSV-G) envelope plasmid produced vectors at titers of 10(5)-10(6) transducing units per milliliter in 48 h. Replication-competent virus (RCV) was not detected when deletions were made in the env gene in pHP. The ability of this vector system to transduce dividing and nondividing cell in vitro and in vivo was also demonstrated. Compared with a Moloney murine leukemia virus (MLV) vector, the HP/TV vectors transduced human muscle-, kidney-, liver-derived cell lines and CD34+ primary hematopoietic progenitor cells more efficiently. Although the levels of the pTV transgene expression were high soon after transduction, the expression tended to decrease with time due either to the loss of proviral DNA or to the inactivation of promoter activity, which was found to be cell type-dependent. Analyses of extrachromosomal DNA showed that the unintegrated proviral DNA of lentiviral vectors survived much longer than that of the retroviral vectors. We demonstrate that the HP/TV vector is capable of high efficiency transduction and that long-term expression of lentiviral vectors is dependent on target cell type, the internal promoter and the transgene itself in the transducing vector.

Animals↗

PubMed1992

New methods for the diagnosis of Babesia bigemina infection.

Accurate diagnosis of Babesia bigemina infection, an economically important tick-transmitted protozoan parasite of cattle, is essential in the management of disease control and in epidemiological studies. The currently used methods of diagnosis are blood smear examination and serological tests which include agglutination and immunofluorescence tests. These tests have been used in the fild but because they lack sensitivity and specificity, newer and improved methods of diagnosis are being developed. The quantitative buffy coat (QBC) method, using microhaematocrit tubes and acridine orange staining allows rapid and quicker diagnosis of B. bigemina and other blood parasites compared to light microscopic examination of stained smears. Parasite specific monoclonal antibodies have been used in antigen/antibody capture enzymelinked immunosorbent assays with greater sensitivity and specificity than previously described serological tests. Similarly, DNA probes, derived from a repetitive sequence of the B. bigemina genome, offer a method of detecting very small numbers of parasites which are undetectable by conventional microscopy. An extrachromosomal DNA element, present in all the tick-borne protozoan parasites so far tested, provides an accurate means of differentiating mixed parasite populations in infected animals. These improved methods will greatly facilitate epidemiological studies.

Acridine Orange↗

PubMed2001

The hamster polyomavirus--a brief review of recent knowledge.

The hamster polyomavirus (HaPV) was first described in 1967 as a virus associated with skin epithelioma of the Syrian hamster. The tumors appear spontaneously in a hamster colony bred in Berlin-Buch (HaB). Virus particles isolated from skin epitheliomas cause lymphoma and leukemia when injected into newborn hamsters from a distinct colony bred in Potsdam, Germany (HaP). The viral genome has been totally sequenced and the overall genetic organization establishes HaPV as a member of the polyomaviruses. HaPV is a second example of an middle T (MT) antigen encoding polyomavirus and nucleotide sequence homologies designates the mouse polyomavirus (Py) as the closest relative. Lymphomas induced by HaPV in HaP hamsters do not contain virus particles but instead accumulate different amounts of nonrandomly deleted free and/or integrated viral genomes. Transgenic mice produced by microinjection of HaPV DNA into the pronucleus of fertilized eggs of Gat: NMRI mice developed both, epitheliomas and lymphomas. Both tumor types contain extrachromosomal DNA. HaPV DNA was found to replicate in hamster lymphoid and fibroblast cell lines. Fully reproductive cycles could be detected only in GD36 lymphoblastic leukemia cells. HaPV carries the full transforming properties of a polyomavirus in vitro. Immortalization of primary rat cells is essentially carried out by the HaPV large T (LT) antigen and coexpression of HaPV MT and HaPV small T (ST) antigen is required for full transformation of rat fibroblasts. The preferential binding of HaPV MT to c-Fyn, a Src family kinase, has been proposed as a mechanism leading to lymphoid malignancies. Heterologous expression of HaPV-VP1 allowed the formation of virus like particles (VLPs) resembling HaPV particles. The high flexibility of HaPV-VP1 for insertion of foreign peptides offers a broad range of potential applications, especially in vaccine development.

Animals↗

PubMed1990

Nonradioactive screening method for isolation of disease-specific probes to diagnose plant diseases caused by mycoplasmalike organisms.

DNA fragments of tomato big bud (BB) mycoplasmalike organism (MLO) in diseased periwinkle plants (Catharanthus roseus L.) were cloned to pSP6 plasmid vectors and amplified in Escherichia coli JM83. A nonradioactive method was developed and used to screen for MLO-specific recombinants. Cloned DNA probes were prepared by nick translation of the MLO recombinant plasmids by using biotinylated nucleotides. The probes all hybridized with nucleic acid from BB MLO-infected, but not healthy, plants. Results from dot hybridization analyses indicated that several MLOs, e.g., those of Italian tomato big bud, periwinkle little leaf, and clover phyllody, are closely related to BB MLO. The Maryland strain of aster yellows and maize bushy stunt MLOs are also related to BB MLO. Among the remaining MLOs used in this study, Vinca virescence and elm yellows MLOs may be very distantly related, if at all, to BB MLO. Potato witches' broom, clover proliferation, ash yellows, western X, and Canada X MLOs are distantly related to BB MLO. Southern hybridization analyses revealed that BB MLO contains extrachromosomal DNA that shares sequence homologies with extrachromosomal DNAs from aster yellows and periwinkle little leaf MLOs.

Journal Article↗

PubMed1995

Leishmania amazonensis: multidrug resistance in vinblastine-resistant promastigotes is associated with rhodamine 123 efflux, DNA amplification, and RNA overexpression of a Leishmania mdr1 gene.

A vinblastine-resistant Leishmania amazonensis cell line (RV100) which exhibits cross-resistance to the unrelated drug adriamycin, and thus is considered to be multidrug resistant (MDR), was isolated after stepwise selection with increasing concentrations of vinblastine. This phenotype was partially reverted by the calcium channel antagonist verapamil. Drug transport studies using the hydrophobic fluorescent dye rhodamine 123 demonstrated that the MDR cell line has a reduced dye accumulation due to an increased efflux. Furthermore, DNA and RNA hybridization studies demonstrated that a gene (lamdr1), homologous to ldmdr1 and lemdr1, was overexpressed and amplified within 27 kb extrachromosomal DNA circles (V-circles) in these cells. An independent cell line, RA5000, which was selected for resistance to adriamycin and was not cross-resistant to vinblastine, accumulated normal levels of rhodamine 123 and did not contain amplified DNA or overexpressed RNA of mdr-related sequences.

ATP Binding Cassette Transporter, Subfamily B, Mem↗