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Results for “Extrachromosomal DNA”

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PubMed1994

A homologous recombination strategy to analyze the vinblastine resistance property of the V-circle in Leishmania.

The generation of extrachromosomal DNA elements in Leishmania sp. can occur naturally or during in vitro selection with drugs. Previously, we had established a strong association between V-circle amplification and drug resistance in L. enriettii stepwise selected with increasing concentrations of vinblastine. Further, we demonstrated the presence of the lemdr1 gene in the amplified V-circle and subsequent functional analysis by transfection of this gene alone confirmed its role in conferring a drug-resistant phenotype, but the level of resistance was significantly lower than in cell lines obtained from stepwise drug selection. The aim of this work was to determine if other genes either on the V-circle or elsewhere in the genome were necessary for expression of vinblastine resistance. We report here the development of a homologous recombination method to convert the entire V-circle from the LeV160 cell line into a shuttle vector and further the targeted disruption of specific sites within the V-circle. Our results clearly demonstrate that the V-circle alone is sufficient to confer full vinblastine resistance and the disruption of the lemdr1 locus destroys the ability of the V-circle to confer vinblastine resistance.

Animals↗

PubMed1986

Intranuclear localization of herpes simplex virus immediate-early and delayed-early proteins: evidence that ICP 4 is associated with progeny virus DNA.

The localization of ICP 4, ICP 8, DNA polymerase and alkaline exonuclease within herpes simplex virus type 1 (HSV)-infected cells has been examined by immunofluorescence using specific antibodies to these proteins. Cells were simultaneously counterstained with the DNA-binding fluorochrome 4,6-diamidino-2-phenylindole (DAPI) to reveal the intranuclear distribution of DNA. These studies showed that in the absence of virus DNA replication ICP 4, ICP 8 and DNA polymerase were diffusely distributed throughout the nucleus but during virus DNA replication these proteins accumulated at specific foci within the nucleus. Initially these foci were near the nuclear membrane but with continuing virus DNA replication they increased in size until the whole of the nucleus became affected. The increase in size of these foci was coincident with a redistribution of nuclear DNA and margination of chromatin at the nuclear membrane, as revealed by DAPI staining. The number of foci initially present in an infected cell was dependent on the multiplicity of infection. The distribution of ICP 4, ICP 8 and DNA polymerase within the nucleus was altered by treating the cells with DNase. The majority of alkaline exonuclease was diffusely distributed throughout the nucleus during virus DNA replication and did not localize at specific foci within the nucleus. Autoradiographic examination of the incorporation of [3H]thymidine in cells infected with HSV showed that viral DNA replication occurred in restricted areas within the nucleus that were similar, in terms of number, location and size, to the foci where ICP 4, ICP 8 and DNA polymerase accumulated. Furthermore, in cells blocked in mitosis following infection with HSV, ICP 4, ICP 8 and DNA polymerase, but not alkaline exonuclease, localized in areas outside the condensed chromatin structures. DAPI staining revealed the presence of DNA in these areas and, as such structures were never seen when uninfected cells had entered mitosis, it is suggested that this extrachromosomal DNA is of viral origin. These studies therefore suggest that ICP 4 is associated with progeny virus DNA and that while its intranuclear localization is initially at non-viral sites, as DNA replication proceeds so ICP 4 is recruited into areas of virus DNA transcription and replication.

Animals↗

PubMed1982

Attempts to detect by physicochemical methods plasmid DNA in mycoplasmas of human origin before and after transformation to tetracycline resistance.

Physicochemical methods have been used to compare mycoplasma DNA capable of the genetic transformation of tetracycline resistance with DNA from tetracycline-sensitive mycoplasmas and their transformants. These mycoplasmas were isolated from human patients. The DNA extracted from Mycoplasma hominis tetr resistant to 100 microgram/mL tetracycline transforms tetracycline resistance to sensitive strains of Mycoplasma salivarium tets and Mycoplasma hominis tets but not Mycoplasma fermentans tets. Bulk DNA and DNA extracted by methods which increase the yield of circular DNA moieties were analyzed by cesium chloride and cesium chloride--ethidium bromide buoyant density ultracentrifugation and by horizontal and vertical agarose gel electrophoresis. Extrachromosomal DNA was not detected, which suggests that transformation was mediated by the recombination of chromosomal genes for tetracycline resistance and not by R factors. Moreover, no significant differences were detected in the DNA from the resistant and sensitive species or from their transformants and Mycoplasma fermentans tets which could not be transformed to resistance to 10 micrograms tetracycline/mL medium.

Centrifugation, Density Gradient↗

PubMed1979

'Cells' and 'organisms' as a habitat for DNA.

Although the bulk of the hereditary information in bacteria is organized as a single chromosome, it has been known for some years now that bacteria may also carry pieces of self-replicating extrachromosomal DNA. These units are known as plasmids. Sometimes such plasmids carry the information necessary to give rise to mature bacterial viruses under appropriate conditions, but in other cases they specify the production of enzymes and other proteins which alter the bacterial phenotype. Plasmids are often inessential for survival of bacteria, although they may widen the range of environmental conditions under which they flourish. Thus plasmids may be thought of as adventitious additions to the genetic content of bacterial cells. Recently it has become clear that furthur organizational units of DNA are to be found in bacterial cells. These units are called insertion sequences and transposons. Unlike plasmids and the chromosome, however, these DNA units do not carry enough genetic information to specify their own independent replication: they must rely on plasmids or the chromosome for that purpose. Nevertheless they behave in many respects as independent functional units. Although it is possible to think of the chromosome, plasmids and transposons/insertion sequences as three distinct hierarchies of bacterial DNA, genes may move from one hierarchy to another; and such transitions have important implications for the evolution of bacterial populations. Moreover, their study in bacteria may throw much light on the type of DNA interactions occurring in higher cells.

Base Sequence↗

PubMed2004

Infectious plasmid resistance and efflux pump mediated resistance.

Various bacterial plasmids can be eliminated from bacterial species cultured as pure or mixed bacterial cultures by non-mutagenic heterocyclic compounds at subinhibitory concentrations. For plasmid curing, the replication should be inhibited at three different levels simultaneously: the intracellular replication of plasmid DNA, partition and intercellular transconjugal transfer. The antiplasmid action of the compounds depends on the chemical structure. The targets for antiplasmid compounds were analysed in detail. It was found that amplified extrachromosomal DNA in the superhelical state binds more drug molecules than does the linear or open-circular form of the plasmid or the chromosome, without stereospecificity which leads to functional inactivation of the extrachromosomal genetic code. Plasmid elimination also occurs in ecosystems containing numerous bacterial species simultaneously, but the elimination of antibiotic resistance-encoding plasmids from all individual cells of the population is never complete. The medical significance of plasmid elimination in vitro is, it provides a method to isolate plasmid-free bacteria for biotechnology without any risk of mutations, and it opens up a new perspective in rational drug design against bacterial plasmids. Hypothetically, the combination of antiplasmid drugs and antibiotics may improve the effectivity of antibiotics against resistant bacteria; therefore, the results cannot be exploited until the curing efficiency reaches 100%. Inhibition of the conjugational transfer of antibiotic resistance plasmids can be exploited to reduce the spreading of these plasmids in ecosystems.

Anti-Bacterial Agents↗

PubMed1978

Chromosome age and segregation during sporulation of Bacillus megaterium.

The effect of chromosome age on segregation during sporulation was investigated. Vegetative cells of Bacillus megaterium were labeled with [Me-3H]thymine and then were grown at 30 degrees C in nonradioactive medium for various times before being allowed to sporulate. The ratio of the amount of label in sporal DNA to that in sporangial DNA, obtained after minor correction for the sporulation frequency, remained essentially constant as the postlabeling growth period was increased from one to seven generations. The spores were preferentially located at the older poles of sporangia, i.e. the poles formed by divisions occurring prior to those forming the sporangia. Therefore, it seems that old (labeled) chromosomes segregate randomly with respect to both the morphological and genealogical polarities of sporangia. Examination of total cell lysates by dye-buoyant density gradient centrifugation revealed the presence of covalently closed circular DNA from cells grown at 37 degrees C, but none was obtained from cells grown at 30 degrees C. Thus, possible interference by large amounts of extrachromosomal DNA in the determination of the chromosomal segregation pattern is unlikely.

Bacillus megaterium↗

PubMed2004

Novel integrase-defective lentiviral episomal vectors for gene transfer.

High levels of circular viral extrachromosomal DNA (E-DNA) are normally produced after infection with integration-competent and -incompetent lentiviruses. Although E-DNA has been shown to be transcriptionally active, it lasts for only a short time in replicating cells. Here, we report an integrase (IN)-defective lentiviral episomal vector in which insertion of the simian virus 40 (SV40) promoter, containing the origin of replication (ori), is associated with long-term expression and persistence of E-DNA in the presence of SV40 large T antigen (TAg) from 293T cells. 293 and 293T cell lines transduced with IN-competent lentiviral vectors expressing green fluorescent protein (GFP) or luciferase from the cytomegalovirus (CMV) or SV40 promoter gave similar levels of transduction and expression. In contrast, only transient reporter expression occurred when using the CMV IN-defective control vector in both 293 and 293T cells. However, reporter gene expression was maintained for more than 8 weeks in 293T, but not 293, cells transduced with the IN-defective lentiviral vector containing the SV40-ori promoter. Polymerase chain reaction for two-long terminal repeat (2LTR) extrachromosomal circular forms, a marker of lentiviral E-DNA, and fluorescence in situ hybridization analysis confirmed the persistence and episomal nature of circular E-DNA up to 60 days after transduction. Taken together, these results indicate that insertion of the SV40-ori promoter in a lentiviral vector contributes to long-term expression by promoting episomal replication when TAg is provided in trans. Lentiviral episomal vectors may serve as specific tools for therapeutic approaches to diseases, particularly those associated with episomal replication of DNA viruses including papillomaviruses, polyomaviruses, and herpesviruses.

Cells, Cultured↗

PubMed1992

Molecular typing of Helicobacter pylori by chromosomal and plasmid DNA organization.

Diverse strains of Helicobacter pylori were examined in order to initiate a molecular epidemiological typing scheme for this agent of human gastritis. Twelve differently-sized plasmids from 1.8 to 63 kbp were identified in those strains harbouring extrachromosomal DNA. Recombinant DNA probes were cloned randomly from the chromosome of the (plasmid-free) type strain (NCTC 11637), and used to probe genomic Southern blots for restriction site variation in and around homologous loci. Genus-specific probe DNAs were obtained which grouped strains on the bases of DNA base substitution or rearrangements. On the basis of the four probes examined, all strains exhibited intraspecific chromosomal divergence, indicating that H. pylori is highly diverse genetically, but nonetheless susceptible to chromosome and plasmid molecular typing.

Bacterial Typing Techniques↗

PubMed1976

The elimination of urease activity in Streptococcus faecium as evidence for plasmid-coded urease.

A strain of Streptococcus faecium from the sheep rumen showed spontaneous loss of urease activity when subcultured at the normal rumen temperature of 38 degrees C, although in mixed cultures in vivo or in vitro loss of urease was not apparent. The rate of loss of urease in pure cultures was increased at incubation temperatures above 38 degrees C, but loss was never complete. However, at temperatures below 38 degrees C loss was greater, and at 22 or 18 degrees C the urease was completely eliminated. Incubation with sodium dodecyl sulphate (0-002%) or ethidium bromide (2-5 X 10(-5)M) caused complete loss of urease activity. The urease activity was also eliminated when the streptococcus was grown aerobically, and this loss of activity was irreversible. It is suggested that the urease activity is controlled by a plasmid gene and that aeration, low growth temperature and chemical agents 'cure' the streptococcus of the plasmid. Attempts to demonstrate the presence of covalently closed circular extrachromosomal DNA by caesium chloride-ethidium bromide equilibrium density-gradient centrifugation were unsuccessful.

Aerobiosis↗

PubMed1987

Characterization of the new Chlamydia agent, TWAR, as a unique organism by restriction endonuclease analysis and DNA-DNA hybridization.

Several molecular techniques were used for comparison of the novel Chlamydia agent, TWAR, with Chlamydia trachomatis and Chlamydia psittaci. Unlike all serotypes of C. trachomatis and most strains of C. psittaci, the eight TWAR isolates examined did not contain extrachromosomal DNA. TWAR was readily distinguished from C. trachomatis or C. psittaci by restriction endonuclease analysis, whereas identical or nearly identical restriction patterns were observed among the TWAR isolates. Southern blot analysis with a gene encoding a portion of the C. trachomatis serovar L2 major outer membrane protein as the probe showed that TWAR, like C. psittaci, contained sequences homologous to this gene. However, while the hybridization patterns were identical for all TWAR isolates, they differed from those of any of the other Chlamydia species tested. A PstI gene bank containing TWAR DNA was constructed in pUC19. Random fragments were purified and used for probing Chlamydia chromosomal digests. All of the five probes tested were TWAR specific, with the TWAR isolates showing identical patterns of homology. Qualitative studies of the DNA homology revealed that TWAR did not have significant homology to any of the Chlamydia strains assayed. Collectively, these results demonstrate that the TWAR isolates represent a single strain or closely allied genotypes and are clearly distinct from any of the other chlamydiae tested.

Animals↗

PubMed1990

N-methyl-N-nitrosourea-induced mutations in a shuttle plasmid replicated in human cells.

The supF gene of the recombinant shuttle plasmid pZ190 (modified pZ189) was used as a target to study the nature of mutations induced by N-methyl-N-nitrosourea (MNU) in human cells. Treatment of the intact plasmid with MNU followed by its replication in human lymphoblastoid cells led to extensive inactivation and no detectable mutations of the plasmid. However, exposure of the supF DNA fragment alone, followed by its ligation into the vector, caused a ten-fold increase in mutant frequency when replicated in O6-methylguanine-DNA methyltransferase-deficient cells (from 0.54 x 10(-3) to 5.8 x 10(-3)) and an 80-fold increase when replicated in cells containing normal levels of the enzyme (from 0.047 x 10(-3) to 3.8 x 10(-3)). About 45% of the mutant plasmid molecules recovered from human cells contained deletions and insertions. Sixty to 70% of the mutant molecules of wild-type size contained a single-base substitution. Most of these changes were of the G.C----A.T type, consistent with the hypothesis that O6-methylguanine is the primary mutagenic adduct induced by MNU. However, the distribution of mutation sites was highly nonrandom; more than half of all mutations were localized at the G.C position 123, and the rest were distributed in about a dozen sites. The high yield of mutations induced in the supF DNA in a host cell whose capacity for the removal of O6-methylguanine far exceeded the amount present in the supF suggests that the repair of damages in extrachromosomal DNA may be inefficient. This is supported by the observation that the yield of mutations in supF transfected into lymphoblastoid cells devoid of repair activity for O6-methylguanine was comparable to that observed with repair-proficient host cells. The present data, together with results of mutations induced in pZ189 by other agents, strongly suggest that one major determinant of mutational hot spots is the structure of the target DNA itself.

Bacterial Proteins↗

PubMed1988

Recombinant DNA related to hepatitis B and human immunodeficiency viruses in mononuclear cells of patients with AIDS.

Sixty-eight of 73 patients with human immunodeficiency virus (HIV) infection were positive when tested for the presence of hepatitis B virus (HBV)-related DNA sequences in peripheral blood mononuclear cells (PBMCs) by the dot blot method. Twenty-two of the positive DNAs were examined by Southern hybridization and all exhibited a 3.2 kb extrachromosomal DNA fragment that hybridized to HBV DNA. This DNA was isolated from agarose gels and cloned into the EcoRI site of pBR322 DNA. The cloned DNA (pHBI) hybridized to both HBV DNA and HIV cDNA; HBV DNA did not hybridize to HIV cDNA under the same conditions. The results of restriction enzyme analyses indicated that pHBI contains: 1) a large deletion of HBV sequences spanning the 3' end of the HBV surface antigen gene; 2) a small deletion near the 5' end of the HBV core antigen gene; and 3) a region of homology to a one kb central section of the HIV pol gene. These data suggest that the 3.2 kb DNA found in the PBMCs is a natural recombinant between HBV and HIV DNAs raising the possibility not only that this DNA plays a role in the pathogenesis of AIDS but also that other viral recombinant DNAs may be pathogenic in human disease.

Acquired Immunodeficiency Syndrome↗

PubMed1988

Transfer of the Ti plasmid from Agrobacterium tumefaciens into Escherichia coli cells.

We have screened strains of Agrobacterium tumefaciens for spontaneous mutants showing constitutive transfer of the nopaline Ti plasmid pTiC58 during conjugation. The Ti plasmid derivatives obtained could be transferred not only to A. tumefaciens but also to E. coli cells. The Ti plasmid cannot survive as a freely replicating plasmid in E. coli, but it can occasionally integrate into the E. coli chromosome. However, insertion in tandem of plasmids carrying fd replication origins (pfd plasmids) into the T-DNA provides an indicator for all transfer events into E. coli cells, providing fd gene 2 protein is present in these cells. This viral protein causes the excision of one copy of the pfd plasmid and allows its propagation in the host cell. By using this specially designed Ti plasmid, which was also made constitutive in transfer functions, we found plasmid exchange among A. tumefaciens strains and between A. tumefaciens and E. coli cells to be equally efficient. A Ti plasmid with repressed transfer functions was transferred to E. coli with a rate similar to the low frequency at which it was transferred to A. tumefaciens. The expression of transfer functions of plasmid RP4 either in A. tumefaciens or in E. coli did not increase the transfer of the Ti plasmid into E. coli cells, nor did the addition of acetosyringone, an inducer of T-DNA transfer to plant cells. The results show that A. tumefaciens can transfer the Ti plasmid to E. coli with the same efficiency as within its own species. Conjugational transmission of extrachromosomal DNA like the narrow-host-range Ti plasmid may often not only occur among partners allowing propagation of the plasmid, but also on a 'try-all' basis including hosts which do not replicate the transferred DNA.

Chromosome Mapping↗

PubMed1975

The fate of a bacterial plasmid in mammalian cells.

When hamster cells are infected with the bacterial plasmid colicinogenic factor E1 (ColE1), as much as 5-8% of the input plasmid radioactivity is found in the recipient cell, mainly in the nuclear fraction. Density shift experiments with bromodeoxyuridine labeled ColE1 DNA indicate that part of the input DNA may be replicated in the nucleus. ColE1 specific RNA but no colicin E1, can be detected during the first two generations after the uptake of ColE1 DNA. However, extrachromosomal ColE1 DNA is unstable in the mammalian cells and is degraded to acid soluble fragments after a few generations.

Animals↗

PubMed1983

Deletion and amplification of DNA sequences in melanin-negative variants of Streptomyces reticuli.

The sporulating wild type of Streptomyces reticuli produces the pigment melanin. Though the ability to synthesize tyrosinase is frequently lost, it was demonstrated, that the structural gene coding for this enzyme is not located on the extrachromosomal DNA of the wild type strain or melanin-positive variants. Melanin negative variants were found to have lost this gene and to contain amplified nucleotide sequences within their genomes.

Catechol Oxidase↗

PubMed1989

Cellular and adenovirus dl312 DNA metabolism in cycling or mitotic human cultures exposed to supralethal gamma radiation.

Cellular repair of DNA damage due to lethal gamma irradiation was studied to reveal differences between strains and cell cycle stages that are otherwise difficult to detect. Cycling and metaphase-blocked cultures of normal fibroblasts and carcinoma cells were compared for repair of gamma sites (gamma radiation-induced nicks, breaks, and alkalilabile sites in DNA) at supralethal exposures ranging from 7 to 150 krad 137Cs radiation and at postirradiation incubations of 20-180 min. Fibroblasts from normal human skin or lung repaired gamma sites efficiently when cycling but did not repair them when blocked at mitosis. Bladder (253J) or lung (A549) carcinoma cells, unlike normal fibroblasts, repaired gamma sites efficiently even when blocked at mitosis. HeLa cells degraded their DNA soon after exposure at all doses tested, regardless of mitotic arrest. Whether the above differences in DNA repair between cell cycle stages and between strains result from differences in chromatin structure (cis effects) or from differences in the nuclear enzymatic environment (trans effects) could be resolved by placing an inert, extrachromosomal DNA molecule in the cell nucleus. Specifically, cis effects should be confined to the host chromosomes and would not be detected in the inert probe whereas trans effects should be detected in host chromosomes and inert probe DNA alike. Indeed, we found a suitable DNA molecule in the adenovirus deletion mutant dl312, which does not proliferate in the absence of E1A complementation. Gamma sites in 32P-labeled adenovirus dl312 DNA were repaired efficiently in all hosts, regardless of mitotic arrest. Failure of mitosis-arrested fibroblasts to repair gamma sites was therefore due to a cis effect of chromatin organization rather than to a trans effect such as repair enzyme insufficiency. In sharp contrast, chromosomes of mitotic carcinoma cells remained accessible to repair enzymes and nucleases alike. By means of these new tools, we should get a better understanding of higher-order chromatin management in normal and cancer cells.

Adenoviruses, Human↗

PubMedDate not supplied

[Method for studying horizontal transfer of plasmids in Erwinia carotovora].

A simple method to determine frequency characteristics of the horizontal transfer of antibiotic resistance by transconjugative plasmids of various incompatibility groups into Erwinia carotovora strains has been developed. The proposed method for selection of E. carotovora transconjugates provides for the use of selective medium with pectin and corresponding antibiotic, which permits carrying out reliable counterselection of the donor (Escherichia, Salmonella). The proposed method helped to study horizontal transfer of plasmids RP4 (Inc P), R 391 (Inc J) and pKM101 (Inc N) to some E. carotovora strains and to demonstrate their inheritance as extrachromosomal DNA in Erwinia cells.

Anti-Bacterial Agents↗

PubMed1991

A novel extrachromosomally maintained transformation vector for the lignin-degrading basidiomycete Phanerochaete chrysosporium.

A stable extrachromosomally maintained transformation vector (pG12-1) for the lignin-degrading filamentous fungus Phanerochaete chrysosporium is described. The vector is 6.3 kb and contains a Kanr marker, pBR322 ori, and a 2.2-kb fragment (ME-1) derived from an endogenous extrachromosomal DNA element of P. chrysosporium. Vector pG12-1 was able to transform P. chrysosporium to G418 resistance and was readily and consistently recoverable from the total DNA of transformants via Escherichia coli transformation. Southern blot analyses indicated that pG12-1 is maintained at a low copy number in the fungal transformants. The vector is demonstrable in the total DNA of individual G418-resistant basidiospore progeny of the transformants only after amplification by polymerase chain reaction. Exonuclease III and dam methylation analyses, respectively, indicated that pG12-I undergoes replication in P. chrysosporium and that it is maintained extrachromosomally in a circular form. The vector is stably maintained in the transformants even after long-term nonselective growth. There is no evidence for integration of the vector into the chromosome at any stage.

Base Sequence↗