WorldWideScience

Sample records for circular supercoiled plasmid

  1. The writhe distribution in DNA plasmids as derived from the free energy of supercoiling

    Science.gov (United States)

    Tobias, Irwin

    2000-10-01

    In theoretical work on the molecule, DNA is often treated, approximately, as a naturally straight, inextensible, isotropic elastic rod of circular cross section. It is shown that, consistent with this level of approximation, there exists a general connection between the free energy of supercoiling of plasmids formed by the DNA, and the writhe distribution in plasmids having a given value of the linking number difference, ΔLk. In particular, the writhe distribution in a collection of torsionally relaxed (ΔLk=0), but non-nicked, plasmids is completely determined once the free energy of supercoiling as a function of ΔLk is known. The writhe distribution in the supercoiled plasmids characterized by any other value of ΔLk, we shall also show, is simply related to the distribution in the relaxed plasmid, and, therefore, it, too, is completely determined. These general results are illustrated for two cases: Large plasmids for which the measured free energy of supercoiling, a quadratic function of ΔLk, implies a normal writhe distribution, and miniplasmids for which a theoretical expression for the free energy of supercoiling involving the frequencies of the normal modes of vibration of a circular elastic ring has recently become available. In this latter case, the writhe distribution for supercoiled plasmids is not normal, but shows a skewness related to a property of elastic rings, namely, the loss of stability of the circular equilibrium configuration of the rings when they are twisted beyond a critical value. Such a skewed writhe distribution for miniplasmids is, according to the model, associated with a free energy of supercoiling which is not, as has been assumed, a rigorously quadratic function of ΔLk.

  2. Denaturation of circular DNA: Supercoils and overtwist

    OpenAIRE

    Kabakçıoğlu, Alkan; Bar, Amir ; Mukamel, David

    2012-01-01

    PHYSICAL REVIEW E 86, 061904 (2012) Denaturation of circular DNA: Supercoils and overtwist Amir Bar,1,2 Alkan Kabakc¸ıo˘glu,3 and David Mukamel1 1Department of Physics of Complex Systems, The Weizmann Institute of Science, Rehovot 76100, Israel 2Department of Computer Science and Applied Mathematics, The Weizmann Institute of Science, Rehovot 76100, Israel 3Department of Physics, Koc¸ University, Sarıyer 34450 ˙Istanbul, Turkey (Received 30 July 2012; published 11 December 2...

  3. Enhancer-activated plasmid transcription complexes contain constrained supercoiling.

    OpenAIRE

    Bonilla, P J; Freytag, S O; Lutter, L C

    1991-01-01

    It has been proposed that transcriptionally active chromatin contains totally unconstrained supercoiling. The results of recent studies have raised the possibility that this topological state is the property of highly transcribed genes. Since the transcription rate of RNA polymerase II genes can be dramatically increased by the presence of an enhancer, we have determined if the transcription complex of an enhancer-activated plasmid contains totally unconstrained supercoils. Following transfec...

  4. A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy

    OpenAIRE

    Hassan, S.; Keshavarz-Moore, E.; J. Ward

    2016-01-01

    With the recent revival of the promise of plasmid DNA vectors in gene therapy, a novel synthetic biology approach was used to enhance the quantity, (yield), and quality of the plasmid DNA. Quality was measured by percentage supercoiling and supercoiling density, as well as improving segregational stability in fermentation. We examined the hypothesis that adding a Strong Gyrase binding Site (SGS) would increase DNA gyrase-mediated plasmid supercoiling. SGS from 3 different replicons, (the Mu b...

  5. Visualization of alkali-denatured supercoiled plasmid DNA by atomic force microscopy

    International Nuclear Information System (INIS)

    To study the alkali denaturation of supercoiled DNA, plasmid pBR322 was treated with gradient concentrations of NaOH solution. The results of gel electrophoresis showed that the alkali denaturation of the supercoiled DNA occurred in a narrow range of pH value (12.88-12.90). The alkali-denatured supercoiled DNA ran, as a sharp band, faster than the supercoiled DNA. The supercoiled plasmid DNA of pBR322, pACYC184 and pJGX15A were denatured by NaOH, and then visualized by atomic force microscopy. Compared with the supercoiled DNA, the atomic force microscopy images of the alkali-denatured supercoiled DNA showed rough surface with many kinks, bulges on double strands with inhomogeneous diameters. The apparent contour lengths of the denatured DNA were shortened by 16%, 16% and 50% for pBR322, pACYC184 and pJGX15A, respectively. All evidence suggested that the alkali-denatured supercoiled DNA had a stable conformation with unregistered, topologically constrained double strands and intrastrand secondary structure

  6. A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy.

    Science.gov (United States)

    Hassan, Sally; Keshavarz-Moore, Eli; Ward, John

    2016-09-01

    With the recent revival of the promise of plasmid DNA vectors in gene therapy, a novel synthetic biology approach was used to enhance the quantity, (yield), and quality of the plasmid DNA. Quality was measured by percentage supercoiling and supercoiling density, as well as improving segregational stability in fermentation. We examined the hypothesis that adding a Strong Gyrase binding Site (SGS) would increase DNA gyrase-mediated plasmid supercoiling. SGS from three different replicons, (the Mu bacteriophage and two plasmids, pSC101 and pBR322) were inserted into the plasmid, pUC57. Different sizes of these variants were transformed into E. coli DH5α, and their supercoiling properties and segregational stability measured. A 36% increase in supercoiling density was found in pUC57-SGS, but only when SGS was derived from the Mu phage and was the larger sized version of this fragment. These results were also confirmed at fermentation scale. Total percentage supercoiled monomer was maintained to 85-90%. A twofold increase in plasmid yield was also observed for pUC57-SGS in comparison to pUC57. pUC57-SGS displayed greater segregational stability than pUC57-cer and pUC57, demonstrating a further potential advantage of the SGS site. These findings should augment the potential of plasmid DNA vectors in plasmid DNA manufacture. Biotechnol. Bioeng. 2016;113: 2064-2071. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26928284

  7. l-tryptophan and dipeptide derivatives for supercoiled plasmid DNA purification.

    Science.gov (United States)

    Santos, Tiago; Carvalho, Josué; Corvo, Marta C; Cabrita, Eurico J; Queiroz, J A; Cruz, C

    2016-06-01

    The present study focus on the preparation of chromatography supports for affinity-based chromatography of supercoiled plasmid purification. Three l-tryptophan based supports are prepared through immobilization on epoxy-activated Sepharose and characterized by HR-MAS NMR. The SPR is employed for a fast screening of l-tryptophan derivatives, as potential ligands for the biorecognition of supercoiled isoform, as well as, to establish the suitable experimental conditions for the chromatography. The results reveal that the overall affinity is high (KD=10(-9) and 10(-8)M) and the conditions tested show that the use of HEPES 100mM enables the separation and purification of supercoiled at T=10°C. The STD-NMR is performed to accomplish the epitope mapping of the 5'-mononucleotides bound to l-tryptophan derivatives supports. The data shows that the interactions between the three supports and the 5'-mononucleotides are mainly hydrophobic and π-π stacking. The chromatography experiments are performed with l-tryptophan support and plasmids pVAX-LacZ and pPH600. The supercoiled isoform separation is achieved at T=10°C by decreasing the concentration of (NH4)2SO4 from 2.7 to 0M in HEPES for pVAX-LacZ and 2.65M to 0M in HEPES for pPH600. Overall, l-tryptophan derivatives can be a promising strategy to purify supercoiled for pharmaceutical applications. PMID:26952704

  8. Optimum range of plasmid supercoiled DNA for preparation of ccompetent Top 10 E. coli

    Directory of Open Access Journals (Sweden)

    Muhammad Tahir Majeed

    2011-05-01

    Full Text Available Objectives: In-house preparation of chemically competent andelectrocompetent Top 10 E. coli is not only economical butmeets the needs for most of the molecular cloning work. Forsuch transformations an optimum range of plasmidsupercoiled DNA is needed. Therefore, the present studydescribes the modification of two protocols for the preparationof such cells, and optimization of the amount of plasmidsupercoiled DNA required for better efficiency.Materials and methods: As most of the available protocols torender bacterial cells competent need special media orchemicals and are time consuming, the methods from HelenDonis-Keller Laboratory Manual of Washington University inSt. Louis and Goldberg Laboratory Standard Protocols of theUnited States Department of Agriculture have been used aftermeticulous selection and with few modifications for preparingchemically competent and electrocompetent Top 10 E. coli,respectively. The transformation was carried out using pUC19supercoiled plasmid DNA.Results: The transformation efficiencies of chemicallycompetent and electrocompetent Top 10 E. coli were found tobe 1.1 x 106 and 7.88 x 107 tranformants/μg of DNA,respectively. Such efficiencies are slightly higher than therequired (105-106 transformants/μg DNA for most of thecloning experimentation.Conclusion: The results of the present study indicatethat for sufficient transformation competence rates theoptimum range of plasmid supercoiled DNA is 10 ng forchemically competent and 0.1 ng for electrocompetentTop 10 E. coli.

  9. Negative supercoiling increases the sensitivity of plasmid DNA to single-strand break induction by X-rays

    International Nuclear Information System (INIS)

    Negatively supercoiled topoisomers of the plasmid pIBI 30 were irradiated with 250 kV X-rays and assayed for strand scission by agarose gel electrophoresis. The survival of supercoiled molecules (Form I) decreased exponentially with increasing X-ray exposure and the dose required to reduce the fraction of DNA in Form I to 37% of its value in unirradiated controls (D37) decreased with increasing negative superhelicity. This enhanced radiation sensitivity of underwound DNA is tentatively attributed to the transient denaturation of the double helix that increases the susceptibility of individual strands to free radical attack. (author)

  10. Z DNA and loop structures by immunoelectronmicroscopy of supercoiled pRW751, a plasmid containing left-handed helices.

    OpenAIRE

    Castleman, H; Hanau, L H; Zacharias, W; Erlanger, B. F.

    1988-01-01

    Single and multiple loops were seen when the plasmid pRW751 was allowed to react with anti-Z-DNA or with a Z-specific cross-linking agent. Loop formation was dependent upon negative supercoiling and the presence of Z-specific antibody or cross-linking agent. Restriction enzyme mapping located 18 sites at the bottoms of loops, in addition to the two (dG-dC)n inserts of pRW751. No more than 5 loops were seen in any of the measured molecules; thus, not all potential Z-sites assume the Z conforma...

  11. Linear and Circular Plasmid Content in Borrelia burgdorferi Clinical Isolates

    OpenAIRE

    Iyer, Radha; Kalu, Ogori; Purser, Joye; Norris, Steven; Stevenson, Brian; Schwartz, Ira

    2003-01-01

    The genome of Borrelia burgdorferi, the etiologic agent of Lyme disease, is composed of a linear chromosome and more than 20 linear and circular plasmids. Typically, plasmid content analysis has been carried out by pulsed-field gel electrophoresis and confirmed by Southern hybridization. However, multiple plasmids of virtually identical sizes (e.g., lp28 and cp32) complicate the interpretation of such data. The present study was undertaken to investigate the complete plasmid complements of B....

  12. [The effect of DNA supercoiling DNA on nucleosome structure].

    Science.gov (United States)

    Sivolob, A V; Khrapunov, S N

    1991-01-01

    The circular DNA which contains nucleosomes and additional supercoils has been considered theoretically. The different possible effect of increased negative supercoiling on the nucleosome structure have been studied. According to the model proposed all supercoils in the nucleosome-containing circular DNA are realized as torsional deformations of the double helix. The free energy of both supercoiling (torsional deformations) and nucleosome stabilization have been taken into consideration to obtain the equation for free energy of nucleosome-containing circular DNA. The analysis of this equation and the experimental data by Garner et al. (II Psoc. Natl. Acad. Sci. USA. 1987. P. 2620-2623) about the maximum amount of supercoiling obtained by DNA-topoisomerase II treatment of nucleosome-containing pBR322 plasmid has been performed. It has been shown that two possibilities are consistent with both the equation and experimental data. These are: (1) the increased supercoiling induces the torsional strains not only in linker regions but also in nucleosome DNA and thus supercoiling causes an instability on nucleosome structure; (2) increased supercoiling induces a structural change of nucleosome which is accompanied by nucleosome DNA unwinding and its transition into form with approximately 11 base pairs per turn of double helix. It has been evaluated that in the first case the average torsional rigidity of nucleosome DNA should be approximately 2.5 times as much and in the second case--much more than the rigidity of naked DNA. Both types of nucleosome structural changes may cause its transition to a potentially active state for transcription. It is suggested that increased supercoiling can be a switch mechanism of chromatin activation. PMID:1654518

  13. G-quadruplex formation between G-rich PNA and homologous sequences in oligonucleotides and supercoiled plasmid DNA.

    Science.gov (United States)

    Gaynutdinov, Timur I; Englund, Ethan A; Appella, Daniel H; Onyshchenko, Mykola I; Neumann, Ronald D; Panyutin, Igor G

    2015-04-01

    Guanine (G)-rich DNA sequences can adopt four-stranded quadruplex conformations that may play a role in the regulation of genetic processes. To explore the possibility of targeted molecular recognition of DNA sequences with short G-rich peptide nucleic acids (PNA) and to assess the strand arrangement in such complexes, we used PNA and DNA with the Oxytricha nova telomeric sequence d(G4T4G4) as a model. PNA probes were complexed with DNA targets in the following forms: single-stranded oligonucleotides, a loop of DNA in a hairpin conformation, and as supercoiled plasmid with the (G4T4G4)/(C4A4C4) insert. Gel-shift mobility assays demonstrated formation of stable hybrid complexes between the homologous G4T4G4 PNA and DNA with multiple modes of binding. Chemical and enzymatic probing revealed sequence-specific and G-quadruplex dependent binding of G4T4G4 PNA to dsDNA. Spectroscopic and electrophoretic analysis of the complex formed between PNA and the synthetic DNA hairpin containing the G4T4G4 loop showed that the stoichiometry of a prevailing complex is three PNA strands per one DNA strand. We speculate how this new PNA-DNA complex architecture can help to design more selective, quadruplex-specific PNA probes. PMID:25650982

  14. Tethered particle analysis of supercoiled circular DNA using peptide nucleic acid handles

    DEFF Research Database (Denmark)

    Norregaard, Kamilla; Andersson, Magnus; Nielsen, Peter Eigil;

    2014-01-01

    -digoxigenin-coated glass cover slide, and one conjugated with biotin for attachment to a submicron-sized streptavidin-coated polystyrene bead. Plasmids are constructed, purified and incubated with the PNA handles. The dynamics of the construct is analyzed by tracking the tethered bead using video microscopy: less...

  15. Detection of surface free radical activity of respirable industrial fibres using supercoiled phi X174 RF1 plasmid DNA.

    Science.gov (United States)

    Gilmour, P S; Beswick, P H; Brown, D M; Donaldson, K

    1995-12-01

    The ability of a number of respirable industrial fibres, amosite and crocidolite asbestos, refractory ceramic fibres (RCFs) and man-made vitreous fibres (MMVFs) to cause free radical injury to plasmid phi X174 RFI DNA was assessed. The oxidative DNA damage was observed as depletion of supercoiled DNA after fibre treatment was quantified by scanning laser densitometry. The mechanism of fibre-mediated damage was determined by the use of the specific hydroxyl radical scavenger mannitol and the iron chelator desferrioxamine-B. The amosite and crocidolite asbestos caused substantial damage to DNA that was dose-related. The free radicals responsible for the asbestos-mediated DNA damage were hydroxyl radicals, as determined by inhibition with mannitol. Asbestos fibre-mediated damage to DNA was completely ameliorated by the chelation of fibre-associated iron with desferrioxamine-B. The amount of Fe(II) and Fe(III) released by equal numbers of the different fibre types at equal fibre number was determined. The fibres released very small amounts of Fe(II) and there were no significant differences between the fibre types. The fibres released substantial amounts of Fe(III); MMVF 21 released significantly more Fe(III) than any of the other fibres and short fibre amosite also released more Fe(III) than three of the MMVFs and two of the RCFs. When ability to release Fe(II) and Fe(III) was compared with ability to cause DNA damage there was not a good correlation, because only the long amosite and crocidolite caused substantial free radical injury to DNA; this contrasts with MMVF 21 and short amosite being the two fibres that released the greatest amounts of iron. The loss of ability to damage DNA in DSF-B-treated asbestos fibres shows that iron at the surface of asbestos fibres definitely has a role in generating hydroxyl radicals. However, it is clear that some fibres, such as short amosite and MMVF 21, release large quantities of iron without causing free radical damage, whilst

  16. Hundreds of circular novel plasmids and DNA elements identified in a rat cecum metamobilome.

    Directory of Open Access Journals (Sweden)

    Tue Sparholt Jørgensen

    Full Text Available Metagenomic approaches are widespread in microbiological research, but so far, the knowledge on extrachromosomal DNA diversity and composition has largely remained dependant on cultivating host organisms. Even with the emergence of metagenomics, complete circular sequences are rarely identified, and have required manual curation. We propose a robust in silico procedure for identifying complete small plasmids in metagenomic datasets from whole genome shotgun sequencing. From one very pure and exhaustively sequenced metamobilome from rat cecum, we identified a total of 616 circular sequences, 160 of which were carrying a gene with plasmid replication domain. Further homology analyses indicated that the majority of these plasmid sequences are novel. We confirmed the circularity of the complete plasmid candidates using an inverse-type PCR approach on a subset of sequences with 95% success, confirming the existence and length of discrete sequences. The implication of these findings is a broadened understanding of the traits of circular elements in nature and the possibility of massive data mining in existing metagenomic datasets to discover novel pools of complete plasmids thus vastly expanding the current plasmid database.

  17. Differential Salt-Induced Dissociation of the p53 Protein Complexes with Circular and Linear Plasmid DNA Substrates Suggest Involvement of a Sliding Mechanism

    Directory of Open Access Journals (Sweden)

    Peter Šebest

    2015-01-01

    Full Text Available A study of the effects of salt conditions on the association and dissociation of wild type p53 with different ~3 kbp long plasmid DNA substrates (supercoiled, relaxed circular and linear, containing or lacking a specific p53 binding site, p53CON using immunoprecipitation at magnetic beads is presented. Salt concentrations above 200 mM strongly affected association of the p53 protein to any plasmid DNA substrate. Strikingly different behavior was observed when dissociation of pre-formed p53-DNA complexes in increased salt concentrations was studied. While contribution from the p53CON to the stability of the p53-DNA complexes was detected between 100 and 170 mM KCl, p53 complexes with circular DNAs (but not linear exhibited considerable resistance towards salt treatment for KCl concentrations as high as 2 M provided that the p53 basic C-terminal DNA binding site (CTDBS was available for DNA binding. On the contrary, when the CTDBS was blocked by antibody used for immunoprecipitation, all p53-DNA complexes were completely dissociated from the p53 protein in KCl concentrations ≥200 mM under the same conditions. These observations suggest: (a different ways for association and dissociation of the p53-DNA complexes in the presence of the CTDBS; and (b a critical role for a sliding mechanism, mediated by the C-terminal domain, in the dissociation process.

  18. Liquid supercoiling

    Science.gov (United States)

    Ribe, Neil; Habibi, Mehdi; Hosseini, Hossein; Hassan Khatami, Mohammad

    2011-11-01

    Supercoiling is defined as the large-scale secondary coiling of a slender body that is already coiled at a smaller scale (e.g., telephone cords and DNA strands). We demonstrate experimentally a novel fluid-mechanical form of supercoiling that occurs in the context of the familiar ``liquid rope coiling'' instability of a thin thread of viscous fluid falling onto a rigid surface. Under appropriate conditions, the coiling instability generates a tall pile of coils in the form of a hollow cylindrical column, which in turn becomes unstable to a secondary coiling instability with a frequency ~ 10 % of the primary one. To place this phenomenon in a broader context, we determine experimentally the phase diagram for the different possible behaviors of the thread (stagnation flow, simple coiling, rotatory folding, periodic column collapse, supercoiling) in the space of the fluid viscosity, the flow rate, and the fall height. We formulate a mathematical model for supercoiling by combining a thin-shell description of the column wall with a slender-thread description of the column as a whole. This leads to a set of coupled ordinary differential equations in one space dimension (the arclength along the axis of the coiling column) that we solve numerically using a continuation method. A comparison of the predicted and observed frequencies of secondary coiling will be shown.

  19. Conversion of a linear to a circular plasmid in the relapsing fever agent Borrelia hermsii.

    OpenAIRE

    Ferdows, M S; Serwer, P; Griess, G A; Norris, S. J.; Barbour, A G

    1996-01-01

    Spirochetes of the genus Borrelia have genomes composed of both linear and circular replicons. We characterized the genomic organization of B. burgdorferi, B. hermsii, B. turicatae, and B. anserina with pulsed-field gel electrophoresis. All four species contained a linear chromosome approximately 1 Mb in size and multiple linear plasmids in the 16- to 200-kb size range. Plasmids 180 and 170 kb in size, present in the relapsing fever agents B. hermsii and B. turicatae but not in the other two ...

  20. Photosensitized damage to supercoiled plasmid DNA induced by 334-nm radiation in the presence of 2-thiouracil consists of alkali- and piperidine-labile sites as well as frank strand breaks

    International Nuclear Information System (INIS)

    A covalently closed, supercoiled plasmid was irradiated with 334-nm ultraviolet radiation in the presence of the naturally occurring photosensitizer 2-thiouracil (s2Ura). Irradiation of the plasmid in the absence of s2Ura induced small numbers of frank DNA strand breaks (FSB), alkali-labile sites (ALS), and piperidine-labile sites (PLS). The induction of each of these lesions was enhanced 30 times when s2Ura was present during aerobic irradiation. Anoxia, as well as the hydroxyl radical scavengers acetate and formate, inhibited the formation of all three lesion types. Hydrogen peroxide, γ-irradiation, and s2Ura photosensitization produced nearly identical damage proportions, with PLS: FSB ratios of 1.25:l, 0.78:1, and 0.84:1, respectively. Treatment with singlet oxygen produced a PLS:FSB ratio of 4.1:1. (author)

  1. DNA supercoiling and its role in DNA decatenation and unknotting

    OpenAIRE

    Witz, Guillaume; Stasiak, Andrzej

    2009-01-01

    Chromosomal and plasmid DNA molecules in bacterial cells are maintained under torsional tension and are therefore supercoiled. With the exception of extreme thermophiles, supercoiling has a negative sign, which means that the torsional tension diminishes the DNA helicity and facilitates strand separation. In consequence, negative supercoiling aids such processes as DNA replication or transcription that require global- or local-strand separation. In extreme thermophiles, DNA is positively supe...

  2. Draft Genome Sequence of Polychlorinated Biphenyl-Dechlorinating Dehalococcoides mccartyi Strain SG1, Which Carries a Circular Putative Plasmid

    OpenAIRE

    Wang, Shanquan; Chng, Kern Rei; Wu, Chen; Wilm, Andreas; Nagarajan, Niranjan; HE, Jianzhong

    2014-01-01

    Dehalococcoides mccartyi strain SG1, isolated from digester sludge, dechlorinates polychlorinated biphenyls (PCBs) to lower congeners. Here we report the draft genome sequence of SG1, which carries a 22.65 kbp circular putative plasmid.

  3. Effect of supercoiling on the λ switch.

    Science.gov (United States)

    Norregaard, Kamilla; Andersson, Magnus; Sneppen, Kim; Nielsen, Peter Eigil; Brown, Stanley; Oddershede, Lene B

    2014-01-01

    The lysogenic state of the λ switch is exceptionally stable, still, it is capable of responding to DNA-damage and rapidly enter the lytic state. We invented an assay where PNA mediated tethering of a plasmid allowed for single molecule investigations of the effect of supercoiling on the efficiency of the epigenetic λ switch. Compared with non-supercoiled DNA, the presence of supercoils enhances the CI-mediated DNA looping probability and renders the transition between the looped and unlooped states steeper, thus increasing the Hill coefficient. Interestingly, the transition occurs exactly at the CI concentration corresponding to the minimum number of CI molecules capable of maintaining the pRM-repressed state. Based on these results we propose that supercoiling maintains the pRM-repressible state as CI concentration decline during induction and thus prevent autoregulation of cI from interfering with induction. PMID:24386605

  4. Supercoil formation in DNA denaturation

    OpenAIRE

    Kabakcioglu, A.; Orlandini, E.; Mukamel, D.

    2008-01-01

    We generalize the Poland-Scheraga (PS) model to the case of a circular DNA, taking into account the twisting of the two strains around each other. Guided by recent single-molecule experiments on DNA strands, we assume that the torsional stress induced by denaturation enforces formation of supercoils whose writhe absorbs the linking number expelled by the loops. Our model predicts that, when the entropy parameter of a loop satisfies $c \\le 2$, denaturation transition does not take place. On th...

  5. Separation of plasmid DNA topoisomers by multimodal chromatography.

    Science.gov (United States)

    Silva-Santos, A Rita; Alves, Cláudia P A; Prazeres, Duarte Miguel F; Azevedo, Ana M

    2016-06-15

    The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases. Here, we describe the separation of supercoiled (sc) and open circular (oc) topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid. The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample. PMID:27033004

  6. The Bacillus subtilis Primosomal Protein DnaD Untwists Supercoiled DNA

    OpenAIRE

    Zhang, Wenke; Allen, Stephanie; Roberts, Clive J.; Soultanas, Panos

    2006-01-01

    The essential Bacillus subtilis DnaD and DnaB proteins have been implicated in the initiation of DNA replication. Recently, DNA remodeling activities associated with both proteins were discovered that could provide a link between global or local nucleoid remodeling and initiation of replication. DnaD forms scaffolds and opens up supercoiled plasmids without nicking to form open circular complexes, while DnaB acts as a lateral compaction protein. Here we show that DnaD-mediated opening of supe...

  7. Artificial plasmid labeled with 5-bromo-2'-deoxyuridine: a universal molecular system for strand break detection.

    Science.gov (United States)

    Zylicz-Stachula, Agnieszka; Polska, Katarzyna; Skowron, Piotr; Rak, Janusz

    2014-07-01

    DNA strand breaks (SBs) are among the most cytotoxic forms of DNA damage, and their residual levels correlate directly with cell death. Hence, the type and amount of SBs is directly related to the efficacy of a given anticancer therapy. In this study, we describe a molecular tool that can differentiate between single (SSBs) and double (DSBs) strand breaks and also assess them quantitatively. Our method involves PCR amplification of a linear DNA fragment labeled with a sensitizing nucleotide, circularization of that fragment, and enzymatic introduction of supercoils to transform the circular relaxed form of the synthesized plasmid into a supercoiled one. After exposure of the molecule to a damaging factor, SSB and DSB levels can be easily assayed with gel electrophoresis. We applied this method to prepare an artificial plasmid labeled with 5-bromo-2'-deoxyuridine and to assay SBs photoinduced in the synthesized plasmid. PMID:24850054

  8. Single primer-mediated circular polymerase chain reaction for hairpin DNA cloning and plasmid editing.

    Science.gov (United States)

    Huang, Jiansheng; Khan, Inamullah; Liu, Rui; Yang, Yan; Zhu, Naishuo

    2016-05-01

    We developed and validated a universal polymerase chain reaction (PCR) method, single primer circular (SPC)-PCR, using single primer to simultaneously insert and amplify a short hairpin sequence into a vector with a high success rate. In this method, the hairpin structure is divided into two parts and fused into a vector by PCR. Then, a single primer is used to cyclize the chimera into a mature short hairpin RNA (shRNA) expression vector. It is not biased by loop length or palindromic structures. Six hairpin DNAs with short 4-nucleotide loops were successfully cloned. Moreover, SPC-PCR was also applied to plasmid editing within 3 h with a success rate higher than 95%. PMID:26792375

  9. Isolation of bacterial plasmid-related replication-associated circular DNA from a serum sample of a multiple sclerosis patient.

    Science.gov (United States)

    Gunst, Karin; Zur Hausen, Harald; de Villiers, Ethel-Michele

    2014-01-01

    Psychrobacter species are considered to be opportunistic human pathogens. We report here the isolation of a circular DNA molecule, MSSI1.162, from a serum sample taken from a multiple sclerosis patient during relapse. This isolate is distantly related to known Psychrobacter species and their plasmids. PMID:25169857

  10. Single -and double-strand breaks induced in plasmid DNA irradiated by ultra -soft X-ray

    International Nuclear Information System (INIS)

    In order to investigate the molecular consequences of a carbon K photo-ionization located on DNA, dry pBS plasmid samples were irradiated with ultra-soft X-rays at energies below and above the carbon K-threshold (Ek=278 eV). Single- and double-strand breaks (ssb and dsb) were quantified after resolution of the three plasmid forms (supercoiled, relaxed circular, linear) by gel electrophoresis. A factor of 1.2 was found between the doses required at 250 eV and 380 eV to induce the same number of dsb per plasmid. (authors)

  11. Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA

    OpenAIRE

    Daisuke Mashiko; Yoshitaka Fujihara; Yuhkoh Satouh; Haruhiko Miyata; Ayako Isotani; Masahito Ikawa

    2013-01-01

    CRISPR/Cas mediated genome editing has been successfully demonstrated in mammalian cells and further applications for generating mutant mice were reported by injecting humanized Cas9 (hCas) mRNA and single guide RNA into fertilized eggs. Here we inject the circular plasmids expressing hCas9 and sgRNA into mouse zygotes and obtained mutant mice within a month. When we targeted the Cetn1 locus, 58.8% (10/17) of the pups carried the mutations and six of them were homozygously mutated. Co-injecti...

  12. Effect of the atmospheric pressure nonequilibrium plasmas on the conformational changes of plasmid DNA

    International Nuclear Information System (INIS)

    The cold atmospheric pressure plasma, which has been widely used for biomedical applications, may potentially affect the conformation of DNA. In this letter, an atmospheric pressure plasma plume is used to investigate its effects on the conformational changes of DNA of plasmid pAHC25. It is found that the plasma plume could cause plasmid DNA topology alteration, resulting in the percentage of the supercoiled plasmid DNA form decreased while that of the open circular and linearized form of plasmid DNA increased as detected by agrose gel electrophoresis. On the other hand, further investigation by using polymerase chain reaction method shows that the atmospheric pressure plasma jet treatments under proper conditions does not affect the genes of the plasmid DNA, which may have potential application in increasing the transformation frequency by genetic engineering.

  13. Site-specific recombination of the circular 2 microns-like plasmid pKD1 requires integrity of the recombinase gene A and of the partitioning genes B and C.

    OpenAIRE

    Bianchi, M. M.

    1992-01-01

    In the circular plasmid pKD1, which stably replicates in Kluyveromyces lactis, the three open reading frames encode a site-specific recombinase (gene A) and two proteins involved in mitotic stability (genes B and C). A recombination analysis of plasmids in which gene B or C is inactivated reveals that unlike the 2 microns plasmid of Saccharomyces cerevisiae, these genes are also required for the site specificity of plasmid recombination.

  14. Separation of closed circular DNA from linear DNA by electrophoresis in two dimensions in agarose gels.

    OpenAIRE

    Oppenheim, A

    1981-01-01

    A method that provides an easy, rapid, and reproducible way for separating closed circular DNA species from linear DNA and nicked circles is described. The method is based on the difference in mobility of form I (supercoiled) DNA and form II (nicked circles), and the differential mobility of relaxed circular DNA in the presence and absence of ethidium bromide (EtdBr). It can be used for detection or for purification of plasmid, episomal, or viral DNA from the bulk of cellular DNA, or from oth...

  15. Effects of physiological self-crowding of DNA on shape and biological properties of DNA molecules with various levels of supercoiling

    Science.gov (United States)

    Benedetti, Fabrizio; Japaridze, Aleksandre; Dorier, Julien; Racko, Dusan; Kwapich, Robert; Burnier, Yannis; Dietler, Giovanni; Stasiak, Andrzej

    2015-01-01

    DNA in bacterial chromosomes and bacterial plasmids is supercoiled. DNA supercoiling is essential for DNA replication and gene regulation. However, the density of supercoiling in vivo is circa twice smaller than in deproteinized DNA molecules isolated from bacteria. What are then the specific advantages of reduced supercoiling density that is maintained in vivo? Using Brownian dynamics simulations and atomic force microscopy we show here that thanks to physiological DNA–DNA crowding DNA molecules with reduced supercoiling density are still sufficiently supercoiled to stimulate interaction between cis-regulatory elements. On the other hand, weak supercoiling permits DNA molecules to modulate their overall shape in response to physiological changes in DNA crowding. This plasticity of DNA shapes may have regulatory role and be important for the postreplicative spontaneous segregation of bacterial chromosomes. PMID:25653164

  16. Stannous chloride mediates single strand breaks in plasmid DNA through reactive oxygen species formation

    International Nuclear Information System (INIS)

    Full text. Stannous ion (Sn) has been employed in many activities related to human beings, as in nuclear medicine, or in food industry. The effect of Sn on supercoiled plasmid DNA was studied by the alteration of plasmid topology in neutral and acidic pH by gel electrophoresis; the transformation efficiency of a wild type Escherichia coli (E. coli) strain; and the patterns of electrophoretic mobility with the digested plasmid. Exposure of plasmid 9.1 DNA to Sn ion caused DNA single-strand breaks (ssb), revealed by agarose gel electrophoresis as an increase in the proportion of the open circular form at the expense of the supercoiled form. Addition of hydroxyl (OH) scavenger (sodium benzoate, 100 mM) inhibited the DNA damage (58%) showing the involvement of such reactive oxygen species (R O S) like -OH Decrease of transformation efficiency and alteration in the patterns of electrophoretic mobility with the digested plasmid is also observed. Our results are consistent with a mechanism involving the generation of reactive oxygen species, such as -OH by a Fenton-like reaction, and also a complexation of stannous ions to DNA, that could explain the decrease of its electrophoretic mobility and protection effect exerted during Sn Cl2 treatment at acidic pH

  17. Adsorption of plasmid DNA to mineral surfaces and protection against DNase I

    International Nuclear Information System (INIS)

    The adsorption of [3H]thymidine-labeled plasmid DNA (pHC314; 2.4 kb) of different conformations to chemically pure sand was studied in a flowthrough microenvironment. The extent of adsorption was affected by the concentration and valency of cations, indicating a charge-dependent process. Bivalent cations (Mg2+, Ca2+) were 100-fold more effective than monovalent cations (Na+, K+, NH4+). Quantitative adsorption of up to 1 μg of negatively supercoiled or linearized plasmid DNA to 0.7 g of sand was observed in the presence of 5 mm MgCl2 at pH 7. Under these conditions, more than 85% of DNA adsorbed within 60 s. Maximum adsorption was 4 μg of DNA to 0.7 g of sand. Supercoil molecules adsorbed slightly less than linearized or open circular plasmids. An increase of the pH from 5 to 9 decreased adsorption at 0.5 mM MgCl2 about eightfold. It is concluded that adsorption of plasmid DNA to sand depends on the neutralization of negative charges on the DNA molecules and the mineral surfaces by cations. The results are discussed on the grounds of the polyelectrolyte adsorption model. Sand-adsorbed DNA was 100 times more resistant against DNase I than was DNA free in solution. The data support the idea that plasmid DNA can enter the extracellular bacterial gene pool which is located at mineral surfaces in natural bacterial habitats

  18. Centromeric nucleosomes induce positive supercoils

    OpenAIRE

    Furuyama, Takehito; Henikoff, Steven

    2009-01-01

    Centromeres of higher eukaryotes are epigenetically maintained, however, the mechanism that underlies centromere inheritance is unknown. Centromere identity and inheritance require the assembly of nucleosomes containing the CenH3 histone variant in place of canonical H3. Whereas H3 nucleosomes wrap DNA in a left-handed manner and induce negative supercoils, we show here that CenH3 nucleosomes that are reconstituted from Drosophila histones induce positive supercoils. Furthermore, we show that...

  19. Rates of gyrase supercoiling and transcription elongation control supercoil density in a bacterial chromosome.

    Directory of Open Access Journals (Sweden)

    Nikolay Rovinskiy

    Full Text Available Gyrase catalyzes negative supercoiling of DNA in an ATP-dependent reaction that helps condense bacterial chromosomes into a compact interwound "nucleoid." The supercoil density (σ of prokaryotic DNA occurs in two forms. Diffusible supercoil density (σ(D moves freely around the chromosome in 10 kb domains, and constrained supercoil density (σ(C results from binding abundant proteins that bend, loop, or unwind DNA at many sites. Diffusible and constrained supercoils contribute roughly equally to the total in vivo negative supercoil density of WT cells, so σ = σ(C+σ(D. Unexpectedly, Escherichia coli chromosomes have a 15% higher level of σ compared to Salmonella enterica. To decipher critical mechanisms that can change diffusible supercoil density of chromosomes, we analyzed strains of Salmonella using a 9 kb "supercoil sensor" inserted at ten positions around the genome. The sensor contains a complete Lac operon flanked by directly repeated resolvase binding sites, and the sensor can monitor both supercoil density and transcription elongation rates in WT and mutant strains. RNA transcription caused (- supercoiling to increase upstream and decrease downstream of highly expressed genes. Excess upstream supercoiling was relaxed by Topo I, and gyrase replenished downstream supercoil losses to maintain an equilibrium state. Strains with TS gyrase mutations growing at permissive temperature exhibited significant supercoil losses varying from 30% of WT levels to a total loss of σ(D at most chromosome locations. Supercoil losses were influenced by transcription because addition of rifampicin (Rif caused supercoil density to rebound throughout the chromosome. Gyrase mutants that caused dramatic supercoil losses also reduced the transcription elongation rates throughout the genome. The observed link between RNA polymerase elongation speed and gyrase turnover suggests that bacteria with fast growth rates may generate higher supercoil densities

  20. Strand breaks in plasmid DNA following positional changes of Auger-electron-emitting radionuclides

    International Nuclear Information System (INIS)

    The purpose of our studies is to elucidate the kinetics of DNA strand breaks caused by low-energy Auger electron emitters in close proximity to DNA. Previously we have studied the DNA break yields in plasmids after the decay of indium-111 bound to DNA or free in solution. In this work, we compare the DNA break yields in supercoiled DNA of iodine-125 decaying close to DNA following DNA intercalation, minor-groove binding, or surface binding, and at a distance form DNA. Supercoiled DNA, stored at 4 C to accumulate radiation dose from the decay of 125I, was then resolved by gel electrophoresis into supercoiled, nicked circular, and linear forms, representing undamaged DNA, single-strand breaks, and double-strand breaks respectively. DNA-intercalated or groove-bound 125I is more effective than surface-bound radionuclide or 125I free in solution. The hydroxyl radical scavenger DMSO protects against damage by 125I free in solution but has minimal effect on damage by groove-bound 125I. (orig.)

  1. A fluoroquinolone resistance associated mutation in gyrA affects DNA supercoiling in Campylobacter jejuni

    Directory of Open Access Journals (Sweden)

    Jing eHan

    2012-03-01

    Full Text Available The prevalence of fluoroquinolone (FQ-resistant Campylobacter has become a concern for public health. To facilitate the control of FQ-resistant Campylobacter, it is necessary to understand the impact of FQ resistance on the fitness of Campylobacter in its natural hosts as understanding fitness will help to determine and predict the persistence of FQ-resistant Campylobacter. Previously it was shown that acquisition of resistance to FQ antimicrobials enhanced the in vivo fitness of FQ-resistant Campylobacter. In this study, we confirmed the role of the Thr-86-Ile mutation in GyrA in modulating Campylobacter fitness by reverting the mutation to the wild-type allele, which resulted in the loss of the fitness advantage. Additionally, we determined if the resistance-conferring GyrA mutations alter the enzymatic function of the DNA gyrase. Recombinant wild-type gyrase and mutant gyrases with three different types of mutations (Thr-86-Ile, Thr-86-Lys, and Asp-90-Asn, which are associated with FQ resistance in Campylobacter, were generated in E. coli and compared for their supercoiling activities using an in vitro assay. The mutant gyrase with the Thr-86-Ile change showed a greatly reduced supercoiling activity compared with the wild-type gyrase, while other mutant gyrases did not show an altered supercoiling. Furthermore, we measured DNA supercoiling within Campylobacter cells using a reporter plasmid. Consistent with the results from the in vitro supercoiling assay, the FQ-resistant mutant carrying the Thr-86-Ile change in GyrA showed much less DNA supercoiling than the wild-type strain and the mutant strains carrying other mutations. Together, these results indicate that the Thr-86-Ile mutation, which is predominant in clinical FQ-resistant Campylobacter, modulates DNA supercoiling homeostasis in FQ-resistant Campylobacter.

  2. Temperate membrane-containing halophilic archaeal virus SNJ1 has a circular dsDNA genome identical to that of plasmid pHH205.

    Science.gov (United States)

    Zhang, Ziqian; Liu, Ying; Wang, Shuai; Yang, Di; Cheng, Yichen; Hu, Jiani; Chen, Jin; Mei, Yunjun; Shen, Ping; Bamford, Dennis H; Chen, Xiangdong

    2012-12-20

    A temperate haloarchaeal virus, SNJ1, was induced from the lysogenic host, Natrinema sp. J7-1, with mitomycin C, and the virus produced plaques on lawns of Natrinema sp. J7-2. Optimization of the induction conditions allowed us to increase the titer from ~10(4) PFU/ml to ~10(11) PFU/ml. Single-step growth curves exhibited a burst size of ~100 PFU/cell. The genome of SNJ1 was observed to be a circular, double-stranded DNA (dsDNA) molecule (16,341 bp). Surprisingly, the sequence of SNJ1 was identical to that of a previously described plasmid, pHH205, indicating that this plasmid is the provirus of SNJ1. Several structural protein-encoding genes were identified in the viral genome. In addition, the comparison of putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses, as well as the presence of lipid constituents from the host phospholipid pool, strongly suggest that SNJ1 belongs to the PRD1-type lineage of dsDNA viruses, which have an internal membrane. PMID:22784791

  3. Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA and lengthen linear DNA

    Energy Technology Data Exchange (ETDEWEB)

    Verebová, Valéria [Institute of Biophysics, University of Veterinary Medicine and Pharmacy, Komenského 73, 041 81 Košice (Slovakia); Adamcik, Jozef [Food and Soft Materials Science, Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 9, CH-8092 Zürich (Switzerland); Danko, Patrik; Podhradský, Dušan [Department of Biochemistry, Institute of Chemistry, Faculty of Sciences, P.J. Šafárik University, Moyzesova 11, 041 54 Košice (Slovakia); Miškovský, Pavol [Department of Biophysics, Faculty of Sciences, P.J. Šafárik University, Jesenná 5, 041 54 Košice (Slovakia); Center for Interdisciplinary Biosciences, Faculty of Sciences, P.J. Šafárik University, Jesenná 5, 041 54 Košice (Slovakia); Staničová, Jana, E-mail: jana.stanicova@uvlf.sk [Institute of Biophysics, University of Veterinary Medicine and Pharmacy, Komenského 73, 041 81 Košice (Slovakia)

    2014-01-31

    Highlights: • Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA. • Anthraquinones quinizarin and danthron lengthen linear DNA. • Anthraquinones quinizarin and danthron possess middle binding affinity to DNA. • Anthraquinones quinizarin and danthron interact with DNA by intercalating mode. - Abstract: The intercalating drugs possess a planar aromatic chromophore unit by which they insert between DNA bases causing the distortion of classical B-DNA form. The planar tricyclic structure of anthraquinones belongs to the group of chromophore units and enables anthraquinones to bind to DNA by intercalating mode. The interactions of simple derivatives of anthraquinone, quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8-dihydroxyanthraquinone), with negatively supercoiled and linear DNA were investigated using a combination of the electrophoretic methods, fluorescence spectrophotometry and single molecule technique an atomic force microscopy. The detection of the topological change of negatively supercoiled plasmid DNA, unwinding of negatively supercoiled DNA, corresponding to appearance of DNA topoisomers with the low superhelicity and an increase of the contour length of linear DNA in the presence of quinizarin and danthron indicate the binding of both anthraquinones to DNA by intercalating mode.

  4. Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA and lengthen linear DNA

    International Nuclear Information System (INIS)

    Highlights: • Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA. • Anthraquinones quinizarin and danthron lengthen linear DNA. • Anthraquinones quinizarin and danthron possess middle binding affinity to DNA. • Anthraquinones quinizarin and danthron interact with DNA by intercalating mode. - Abstract: The intercalating drugs possess a planar aromatic chromophore unit by which they insert between DNA bases causing the distortion of classical B-DNA form. The planar tricyclic structure of anthraquinones belongs to the group of chromophore units and enables anthraquinones to bind to DNA by intercalating mode. The interactions of simple derivatives of anthraquinone, quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8-dihydroxyanthraquinone), with negatively supercoiled and linear DNA were investigated using a combination of the electrophoretic methods, fluorescence spectrophotometry and single molecule technique an atomic force microscopy. The detection of the topological change of negatively supercoiled plasmid DNA, unwinding of negatively supercoiled DNA, corresponding to appearance of DNA topoisomers with the low superhelicity and an increase of the contour length of linear DNA in the presence of quinizarin and danthron indicate the binding of both anthraquinones to DNA by intercalating mode

  5. Differential Salt-Induced Dissociation of the p53 Protein Complexes with Circular and Linear Plasmid DNA Substrates Suggest Involvement of a Sliding Mechanism

    Czech Academy of Sciences Publication Activity Database

    Šebest, Peter; Brázdová, Marie; Fojta, Miroslav; Pivoňková, Hana

    2015-01-01

    Roč. 16, č. 2 (2015), s. 3163-3177. E-ISSN 1422-0067 R&D Projects: GA ČR(CZ) GAP301/11/2076; GA ČR(CZ) GBP206/12/G151 Institutional support: RVO:68081707 Keywords : TUMOR-SUPPRESSOR P53 * CISPLATIN-DAMAGED DNA * SUPERCOILED DNA Subject RIV: BO - Biophysics Impact factor: 2.862, year: 2014

  6. Replication of a low-copy-number plasmid by a plasmid DNA-membrane complex extracted from minicells of Escherichia coli.

    OpenAIRE

    Firshein, W; Strumph, P; Benjamin, P; Burnstein, K; Kornacki, J

    1982-01-01

    A DNA-membrane complex was extracted from minicells of an Escherichia coli mutant harboring a "miniplasmid" derivative (11.2 kilobases) of the low-copynumber plasmid RK2 (56 kilobases). The complex contained various species of supercoiled and intermediate forms of plasmid DNA, of which approximately 20% was bound firmly to the membrane after centrifugation in a CsCl density gradient. The plasmid DNA-membrane complex synthesized new plasmid DNA without the addition of exogenous template, enzym...

  7. Depletion of RNase HI activity in Escherichia coli lacking DNA topoisomerase I leads to defects in DNA supercoiling and segregation.

    Science.gov (United States)

    Usongo, Valentine; Nolent, Flora; Sanscartier, Patrick; Tanguay, Cynthia; Broccoli, Sonia; Baaklini, Imad; Drlica, Karl; Drolet, Marc

    2008-08-01

    Gyrase-mediated hypernegative supercoiling is one manifestation of R-loop formation, a phenomenon that is normally suppressed by topoisomerase I (topA) in Escherichia coli. Overproduction of RNase HI (rnhA), an enzyme that removes the RNA moiety of R-loops, prevents hypernegative supercoiling and allows growth of topA null mutants. We previously showed that topA and rnhA null mutations are incompatible. We now report that such mutants were viable when RNase HI or topoisomerase III was expressed from a plasmid-borne gene. Surprisingly, DNA of topA null mutants became relaxed rather than hypernegatively supercoiled following depletion of RNase HI activity. This result failed to correlate with the cellular concentration of gyrase or topoisomerase IV (the other relaxing enzyme in the cell) or with transcription-induced supercoiling. Rather, intracellular DNA relaxation in the absence of RNase HI was related to inhibition of gyrase activity both in vivo and in extracts. Cells lacking topA and rnhA also exhibited properties consistent with segregation defects. Overproduction of topoisomerase III, an enzyme that can carry out DNA decatenation, corrected the segregation defects without restoring supercoiling activity. Collectively these data reveal (i) the existence of a cellular response to loss of RNase HI that counters the supercoiling activity of gyrase, and (ii) supercoiling-independent segregation defects due to loss of RNase HI from topA null mutants. Thus RNase HI plays a more central role in DNA topology than previously thought. PMID:18554330

  8. DNA supercoiling and bacterial gene expression.

    Science.gov (United States)

    Dorman, Charles J

    2006-01-01

    DNA in bacterial cells is maintained in a negatively supercoiled state. This contributes to the organization of the bacterial nucleoid and also influences the global gene expression pattern in the cell through modulatory effects on transcription. Supercoiling arises as a result of changes to the linking number of the relaxed double-stranded DNA molecule and is set and reset by the action of DNA topoisomerases. This process is subject to a multitude of influences that are usually summarized as environmental stress. Responsiveness of linking number change to stress offers the promise of a mechanism for the wholesale adjustment of the transcription programme of the cell as the bacterium experiences different environments. Recent data from DNA microarray experiments support this proposition. The emerging picture is one of DNA supercoiling acting at or near the apex of a regulatory hierarchy where it collaborates with nucleoid-associated proteins and transcription factors to determine the gene expression profile of the cell. PMID:17338437

  9. PcrA function in plasmid replication

    OpenAIRE

    Chisty, L. T.

    2014-01-01

    PcrA is a DNA helicase involved in unwinding plasmi ds as a part of a complex in asymmetric rolling - circle replication of certain plasmids carrying antibiotic resistance genes. PcrA translocates on single stranded DNA by coupling ATP hydrolysis to movement on DNA. Initiator protein, RepD is required to nick supercoiled plasmid site - specifically and open an ssDNA stretch that PcrA can bind. The presence of RepD is needed throughout plasmid unwinding to maintain processivity. Using fluo...

  10. Surface charges effects on the 2D conformation of supercoiled DNA

    CERN Document Server

    Schmatko, Tatiana; Maaloum, Mounir

    2012-01-01

    We have adsorbed plasmid PuC19 DNA on a supported bilayer. The mobility of the lipids within the bilayer ensured a 2D equilibrium of the DNA molecule. By varying the fraction of cationic lipids in the membrane, we have tuned the surface charge. Plasmids conformations were imaged by Atomic Force Microscopy (AFM).We performed two sets of experiments: deposition from salt free solution on charged bilayers and deposition from salty solutions on neutral bilayers. Plasmids can be seen as rings, completely opened structures, or tightly supercoiled plectonemes, depending on the experimental conditions. The plectonemic conformation is observed either on charged surfaces (in the absence of salt) or at 30 mM salt concentration on a neutral bilayer. We demonstrate the equivalence of surface screening by mobile interfacial charges and bulk screening from salt ions.

  11. Exploring writhe in supercoiled minicircle DNA

    Energy Technology Data Exchange (ETDEWEB)

    Fogg, Jonathan M [Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030 (United States); Kolmakova, Natalia [Department of Physics, University of California at Santa Barbara, Santa Barbara, CA 93106 (United States); Rees, Ian [Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX 77030 (United States); Magonov, Sergei [VEECO Instruments, 112 Robin Hill Road, Goleta, CA 93117 (United States); Hansma, Helen [Department of Physics, University of California at Santa Barbara, Santa Barbara, CA 93106 (United States); Perona, John J [Department of Chemistry and Biochemistry, University of California at Santa Barbara, Santa Barbara, CA 93106 (United States); Zechiedrich, E Lynn [Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030 (United States)

    2006-04-12

    Using {lambda}-Int recombination in E. coli, we have generated milligram quantities of supercoiled minicircle DNA. Intramolecular Int recombination was efficient down to lengths {approx}254 bp. When nicked and religated in the presence of ethidium bromide, 339 bp minicircles adopted at least seven unique topoisomers that presumably correspond to {delta}Lk ranging from 0 to -6, which we purified individually. We used these minicircles, with unique {delta}Lk, to address the partition into twist and writhe as a function of {delta}Lk. Gel electrophoresis and atomic force microscopy revealed progressively higher writhe conformations in the presence of 10 mM CaCl{sub 2} or MgCl{sub 2}. From simplistic calculations of the bending and twisting energies, we predict the elastic free energy of supercoiling for these minicircles to be lower than if the supercoiling was partitioned mainly into twist. The predicted writhe corresponds closely with that which we observed experimentally in the presence of divalent metal ions. However, in the absence of divalent metal ions only limited writhe was observed, demonstrating the importance of electrostatic effects on DNA structure, when the screening of charges on the DNA is weak. This study represents a unique insight into the supercoiling of minicircle DNA, with implications for DNA structure in general.

  12. DNA supercoiling enables the Type IIS restriction enzyme BspMI to recognise the relative orientation of two DNA sequences

    OpenAIRE

    Kingston, Isabel J.; Gormley, Niall A.; Halford, Stephen E.

    2003-01-01

    Many proteins can sense the relative orientations of two sequences at distant locations in DNA: some require sites in inverted (head-to-head) orientation, others in repeat (head-to-tail) orientation. Like many restriction enzymes, the BspMI endonuclease binds two copies of its target site before cleaving DNA. Its target is an asymmetric sequence so two sites in repeat orientation differ from sites in inverted orientation. When tested against supercoiled plasmids with two sites 700 bp apart in...

  13. A stochastic model of supercoiling-dependent transcription

    OpenAIRE

    Brackley, C. A.; Johnson, J; Bentivoglio, A; Corless, S.; Gilbert, N.; Gonnella, G.; Marenduzzo, D.

    2016-01-01

    We propose a stochastic model for gene transcription coupled to DNA supercoiling, where we incorporate the experimental observation that polymerases create supercoiling as they unwind the DNA helix, and that these enzymes bind more favourably to regions where the genome is unwound. Within this model, we show that when the transcriptionally induced flux of supercoiling increases, there is a sharp crossover from a regime where torsional stresses relax quickly and gene transcription is random, t...

  14. Inducible Escherichia coli fermentation for increased plasmid DNA production.

    Science.gov (United States)

    Carnes, Aaron E; Hodgson, Clague P; Williams, James A

    2006-11-01

    Bacterial plasmids are the vectors of choice for DNA vaccines and short-term gene therapeutics. Growing plasmid DNA by microbial (Escherichia coli) fermentation is usually combined with alkaline lysis/chromatography methods of purification. To date, typical plasmid fermentation media and processes result in yields of 100-250 mg of plasmid DNA/l of culture medium, using standard high-copy pUC origin-containing plasmids. In order to address this initial and yield-limiting upstream step, we identified novel fermentation control parameters for fed-batch fermentation. The resulting fermentation strategies significantly increased specific plasmid yield with respect to cell mass while enhancing plasmid integrity and maintaining supercoiled DNA content. Fed-batch fermentation yield exceeding 1000 mg of plasmid DNA/l was obtained after reduction of plasmid-mediated metabolic burden during growth, and yields up to 1500 mg of plasmid DNA/l have been achieved with optimized plasmid backbones. Interestingly, by inducing high plasmid levels after sufficient biomass accumulation at low temperature and restricted growth, cells were able to tolerate significantly higher plasmid quantities than cells grown by conventional processes. This 5-10-fold increase in plasmid yield dramatically decreases plasmid manufacturing costs and improves the effectiveness of downstream purification by reducing the fraction of impurities. PMID:16819941

  15. Plasmid purification by phenol extraction from guanidinium thiocyanate solution: development of an automated protocol.

    Science.gov (United States)

    Fisher, J A; Favreau, M B

    1991-05-01

    We have developed a novel plasmid isolation procedure and have adapted it for use on an automated nucleic acid extraction instrument. The protocol is based on the finding that phenol extraction of a 1 M guanidinium thiocyanate solution at pH 4.5 efficiently removes genomic DNA from the aqueous phase, while supercoiled plasmid DNA is retained in the aqueous phase. S1 nuclease digestion of the removed genomic DNA shows that it has been denatured, which presumably confers solubility in the organic phase. The complete automated protocol for plasmid isolation involves pretreatment of bacterial cells successively with lysozyme, RNase A, and proteinase K. Following these digestions, the solution is extracted twice with a phenol/chloroform/water mixture and once with chloroform. Purified plasmid is then collected by isopropanol precipitation. The purified plasmid is essentially free of genomic DNA, RNA, and protein and is a suitable substrate for DNA sequencing and other applications requiring highly pure supercoiled plasmid. PMID:1713749

  16. Quantification Bias Caused by Plasmid DNA Conformation in Quantitative Real-Time PCR Assay

    OpenAIRE

    Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

    2011-01-01

    Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute qu...

  17. Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA

    OpenAIRE

    Hertoghs, Kirsten M. L.; Ellis, Jonathan H.; Catchpole, Ian R.

    2003-01-01

    The available reagents for the attachment of functional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non-sequence- specific manner, with undefined stoichiometry, and affect DNA charge and delivery properties or involve chemical modifications that abolish gene expression. The design and ability of oligonucleotides (ODNs) containing locked nucleic acids (LNAs) to bind supercoiled, double-stranded plasmid DNA in a sequence-specific manner are described for the first ...

  18. In vitro transcription of eukaryotic genes is affected differently by the degree of DNA supercoiling.

    OpenAIRE

    Hirose, S; Suzuki, Y

    1988-01-01

    In a posterior silk gland extract, covalently closed circular (ccc) DNA is in a superhelical state that supports more transcription of fibroin gene than does linear DNA. A HeLa cell extract showed neither the supercoiling activity nor the preference for the transcription of ccc DNA over linear DNA. These activities could be added to the HeLa cell extract. Phosphocellulose fractionation of the posterior silk gland extract yielded a flow-through fraction and a 0.6 M KCl eluate fraction that wer...

  19. Uptake of plasmid deoxyribonucleic acid by Haemophilus.

    OpenAIRE

    Gromkova, R; Goodgal, S

    1981-01-01

    The uptake of circular and linear plasmid RSF0885 deoxyribonucleic acids, (DNAs) obtained from Haemophilus parainfluenzae 14, in both homologous and heterologous recipients was studied and compared with that of chromosomal DNA. High concentrations of divalent cations stimulated the uptake of either circular or linear plasmid DNA in H. parainfluenzae 14 competent cells but did not affect the uptake of chromosomal DNA. The biological activity of linear plasmid DNA was similar to that of circula...

  20. Coarse-grained modelling of supercoiled RNA

    Science.gov (United States)

    Matek, Christian; Šulc, Petr; Randisi, Ferdinando; Doye, Jonathan P. K.; Louis, Ard A.

    2015-12-01

    We study the behaviour of double-stranded RNA under twist and tension using oxRNA, a recently developed coarse-grained model of RNA. Introducing explicit salt-dependence into the model allows us to directly compare our results to data from recent single-molecule experiments. The model reproduces extension curves as a function of twist and stretching force, including the buckling transition and the behaviour of plectoneme structures. For negative supercoiling, we predict denaturation bubble formation in plectoneme end-loops, suggesting preferential plectoneme localisation in weak base sequences. OxRNA exhibits a positive twist-stretch coupling constant, in agreement with recent experimental observations.

  1. A stochastic model of supercoiling-dependent transcription

    CERN Document Server

    Brackley, C A; Bentivogli, A; Corles, S; Gilber, N; Gonnella, G; Marenduzzo, D

    2016-01-01

    We propose a stochastic model for gene transcription coupled to DNA supercoiling, where we incorporate the experimental observation that polymerases create supercoiling as they unwind the DNA helix, and that these enzymes bind more favourably to regions where the genome is unwound. Within this model, we show that when the transcriptionally induced flux of supercoiling increases, there is a sharp crossover from a regime where torsional stresses relax quickly and gene transcription is random, to one where gene expression is highly correlated and tightly regulated by supercoiling. In the latter regime, the model displays transcriptional bursts, waves of supercoiling, and up-regulation of divergent or bidirectional genes. It also predicts that topological enzymes which relax twist and writhe should provide a pathway to down-regulate transcription. This article has been accepted for publication in Physical Review Letters, May 2016.

  2. Studies of DNA supercoiling in vivo and in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Cook, D.N.

    1990-10-01

    This thesis describes a number of diverse experiments whose common theme is to elaborate some aspect of DNA supercoiling. The torsion elastic constant of DNA is measure as a function of superhelix density using the technique of picosecond Time Resolved Fluorescence Polarization Anisotropy (FPA) of intercalated ethidium bromide. The results agree with theories which predict that the anisotropy decay should vary with the square root of the relative viscosity. This experiment furthermore demonstrates a sensitivity of FPA to a change in torsion elastic constant of less than 10%. A number of covalently closed DNA samples, ranging in superhelix density from = {minus}0.123 to {plus}0.042, are then examined. A novel method for measuring changes in local supercoiling on a large PNA molecule which is sensitive to changes in supercoiling of regions of chromosomal DNA as short as 1 kilobase in length is presented. Study of chromosomal supercoiling regulating anaerobic gene expression in the facultative photosynthetic bacterium, Rhodobacter capsulatus showed that no stable change in chromosomal supercoiling upon a shift from aerobic respiratory growth to anaerobic photosynthetic conditions. Studies to detect transient changes in DNA supercoiling indicate that DNA downstream from heavily transcribed genes for the photosynthetic reaction center are relaxed or perhaps overwound upon the induction of photosynthetic metabolism. These results are interpreted in terms of the twin domain model of transcriptional supercoiling.

  3. Studies of DNA supercoiling in vivo and in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Cook, D.N.

    1990-10-01

    This thesis describes a number of diverse experiments whose common theme is to elaborate some aspect of DNA supercoiling. The torsion elastic constant of DNA is measure as a function of superhelix density using the technique of picosecond Time Resolved Fluorescence Polarization Anisotropy (FPA) of intercalated ethidium bromide. The results agree with theories which predict that the anisotropy decay should vary with the square root of the relative viscosity. This experiment furthermore demonstrates a sensitivity of FPA to a change in torsion elastic constant of less than 10%. A number of covalently closed DNA samples, ranging in superhelix density from = [minus]0.123 to [plus]0.042, are then examined. A novel method for measuring changes in local supercoiling on a large PNA molecule which is sensitive to changes in supercoiling of regions of chromosomal DNA as short as 1 kilobase in length is presented. Study of chromosomal supercoiling regulating anaerobic gene expression in the facultative photosynthetic bacterium, Rhodobacter capsulatus showed that no stable change in chromosomal supercoiling upon a shift from aerobic respiratory growth to anaerobic photosynthetic conditions. Studies to detect transient changes in DNA supercoiling indicate that DNA downstream from heavily transcribed genes for the photosynthetic reaction center are relaxed or perhaps overwound upon the induction of photosynthetic metabolism. These results are interpreted in terms of the twin domain model of transcriptional supercoiling.

  4. A Histone-Like Protein Induces Plasmid DNA to Form Liquid Crystals in Vitro and Gene Compaction in Vivo

    Directory of Open Access Journals (Sweden)

    Shiyong Sun

    2013-12-01

    Full Text Available The liquid crystalline state is a universal phenomenon involving the formation of an ordered structure via a self-assembly process that has attracted attention from numerous scientists. In this study, the dinoflagellate histone-like protein HCcp3 is shown to induce super-coiled pUC18 plasmid DNA to enter a liquid crystalline state in vitro, and the role of HCcp3 in gene condensation in vivo is also presented. The plasmid DNA (pDNA-HCcp3 complex formed birefringent spherical particles with a semi-crystalline selected area electronic diffraction (SAED pattern. Circular dichroism (CD titrations of pDNA and HCcp3 were performed. Without HCcp3, pUC18 showed the characteristic B conformation. As the HCcp3 concentration increased, the 273 nm band sharply shifted to 282 nm. When the HCcp3 concentration became high, the base pair (bp/dimer ratio fell below 42/1, and the CD spectra of the pDNA-HCcp3 complexes became similar to that of dehydrated A-form DNA. Microscopy results showed that HCcp3 compacted the super-coiled gene into a condensed state and that inclusion bodies were formed. Our results indicated that HCcp3 has significant roles in gene condensation both in vitro and in histone-less eukaryotes in vivo. The present study indicates that HCcp3 has great potential for applications in non-viral gene delivery systems, where HCcp3 may compact genetic material to form liquid crystals.

  5. Total positive curvature of circular DNA

    DEFF Research Database (Denmark)

    Bohr, Jakob; Olsen, Kasper Wibeck

    2013-01-01

    micro-DNAs longer than the minimum length must be concave, a result that is consistent with typical atomic force microscopy images of plasmids. Predictions for the total positive curvature of circular micro-DNAs are given as a function of length, and comparisons with circular DNAs from the literature...... molecules, e.g., plasmids, it is shown to have implications for the total positive curvature integral. For small circular micro-DNAs it follows as a consequence of Fenchel's inequality that there must exist a minimum length for the circular plasmids to be double stranded. It also follows that all circular...

  6. Nucleation at the DNA supercoiling transition

    CERN Document Server

    Daniels, Bryan C

    2010-01-01

    Twisting DNA under a constant applied force reveals a thermally activated transition into a state with a supercoiled structure known as a plectoneme. Using transition state theory, we predict the rate of this plectoneme nucleation to be of order 10^4 Hz. We reconcile this with experiments that have measured hopping rates of order 10 Hz by noting that the viscosity of the bead used to manipulate the DNA limits the measured rate. We find that the intrinsic bending caused by disorder in the base-pair sequence is important for understanding the free energy barrier that governs the transition. Both analytic and numerical methods are used in the calculations. We provide extensive details on the numerical methods for simulating the elastic rod model with and without disorder.

  7. IHF redistributes bound initiator protein, DnaA, on supercoiled oriC of Escherichia coli.

    Science.gov (United States)

    Grimwade, J E; Ryan, V T; Leonard, A C

    2000-02-01

    In Escherichia coli, initiation of chromosome replication requires that DnaA binds to R boxes (9-mer repeats) in oriC, the unique chromosomal replication origin. At the time of initiation, integration host factor (IHF) also binds to a specific site in oriC. IHF stimulates open complex formation by DnaA on supercoiled oriC in cell-free replication systems, but it is unclear whether this stimulation involves specific changes in the oriC nucleoprotein complex. Using dimethylsulphate (DMS) footprinting on supercoiled oriC plasmids, we observed that IHF redistributed prebound DnaA, stimulating binding to sites R2, R3 and R5(M), as well as to three previously unidentified non-R sites with consensus sequence (A/T)G(G/C) (A/T)N(G/C)G(A/T)(A/T)(T/C)A. Redistribution was dependent on IHF binding to its cognate site and also required a functional R4 box. By reducing the DnaA level required to separate DNA strands and trigger initiation of DNA replication at each origin, IHF eliminates competition between strong and weak sites for free DnaA and enhances the precision of initiation synchrony during the cell cycle. PMID:10692160

  8. Hypernegative Supercoiling Inhibits Growth by Causing RNA Degradation▿

    OpenAIRE

    Baaklini, Imad; Usongo, Valentine; Nolent, Flora; Sanscartier, Patrick; Hraiky, Chadi; Drlica, Karl; Drolet, Marc

    2008-01-01

    Transcription-induced hypernegative supercoiling is a hallmark of Escherichia coli topoisomerase I (topA) mutants. However, its physiological significance has remained unclear. Temperature downshift of a mutant yielded transient growth arrest and a parallel increase in hypernegative supercoiling that was more severe with lower temperature. Both properties were alleviated by overexpression of RNase HI. While ribosomes in extracts showed normal activity when obtained during growth arrest, mRNA ...

  9. Hypernegative Supercoiling Inhibits Growth by Causing RNA Degradation▿

    Science.gov (United States)

    Baaklini, Imad; Usongo, Valentine; Nolent, Flora; Sanscartier, Patrick; Hraiky, Chadi; Drlica, Karl; Drolet, Marc

    2008-01-01

    Transcription-induced hypernegative supercoiling is a hallmark of Escherichia coli topoisomerase I (topA) mutants. However, its physiological significance has remained unclear. Temperature downshift of a mutant yielded transient growth arrest and a parallel increase in hypernegative supercoiling that was more severe with lower temperature. Both properties were alleviated by overexpression of RNase HI. While ribosomes in extracts showed normal activity when obtained during growth arrest, mRNA on ribosomes was reduced for fis and shorter for crp, polysomes were much less abundant relative to monosomes, and protein synthesis rate dropped, as did the ratio of large to small proteins. Altered processing and degradation of lacA and fis mRNA was also observed. These data are consistent with truncation of mRNA during growth arrest. These effects were not affected by a mutation in the gene encoding RNase E, indicating that this endonuclease is not involved in the abnormal mRNA processing. They were also unaffected by spectinomycin, an inhibitor of protein synthesis, which argued against induction of RNase activity. In vitro transcription revealed that R-loop formation is more extensive on hypernegatively supercoiled templates. These results allow us, for the first time, to present a model by which hypernegative supercoiling inhibits growth. In this model, the introduction of hypernegative supercoiling by gyrase facilitates degradation of nascent RNA; overproduction of RNase HI limits the accumulation of hypernegative supercoiling, thereby preventing extensive RNA degradation. PMID:18790862

  10. Hypernegative supercoiling inhibits growth by causing RNA degradation.

    Science.gov (United States)

    Baaklini, Imad; Usongo, Valentine; Nolent, Flora; Sanscartier, Patrick; Hraiky, Chadi; Drlica, Karl; Drolet, Marc

    2008-11-01

    Transcription-induced hypernegative supercoiling is a hallmark of Escherichia coli topoisomerase I (topA) mutants. However, its physiological significance has remained unclear. Temperature downshift of a mutant yielded transient growth arrest and a parallel increase in hypernegative supercoiling that was more severe with lower temperature. Both properties were alleviated by overexpression of RNase HI. While ribosomes in extracts showed normal activity when obtained during growth arrest, mRNA on ribosomes was reduced for fis and shorter for crp, polysomes were much less abundant relative to monosomes, and protein synthesis rate dropped, as did the ratio of large to small proteins. Altered processing and degradation of lacA and fis mRNA was also observed. These data are consistent with truncation of mRNA during growth arrest. These effects were not affected by a mutation in the gene encoding RNase E, indicating that this endonuclease is not involved in the abnormal mRNA processing. They were also unaffected by spectinomycin, an inhibitor of protein synthesis, which argued against induction of RNase activity. In vitro transcription revealed that R-loop formation is more extensive on hypernegatively supercoiled templates. These results allow us, for the first time, to present a model by which hypernegative supercoiling inhibits growth. In this model, the introduction of hypernegative supercoiling by gyrase facilitates degradation of nascent RNA; overproduction of RNase HI limits the accumulation of hypernegative supercoiling, thereby preventing extensive RNA degradation. PMID:18790862

  11. High-frequency transformation of Brevibacterium lactofermentum protoplasts by plasmid DNA.

    OpenAIRE

    Santamaria, R I; Gil, J.A.; Martin, J. F.

    1985-01-01

    An efficient polyethylene glycol-assisted method for transformation of Brevibacterium lactofermentum protoplasts that uses plasmid vectors has been developed. Two small plasmids, pUL330 (5.2 kilobases) and pUL340 (5.8 kilobases), both containing the kanamycin resistance gene from transposon Tn5 and the replication origin of the natural plasmid pBL1 of B. lactofermentum, were selected as vectors. Supercoiled forms of the plasmids yielded a 100-fold higher transformation frequency than did line...

  12. A Superhelical Spiral in the Escherichia coli DNA Gyrase A C-terminal Domain Imparts Unidirectional Supercoiling Bias

    Energy Technology Data Exchange (ETDEWEB)

    Ruthenburg,A.; Graybosch, D.; Huetsch, J.; Verdine, G.

    2005-01-01

    DNA gyrase is unique among type II topoisomerases in that its DNA supercoiling activity is unidirectional. The C-terminal domain of the gyrase A subunit (GyrA-CTD) is required for this supercoiling bias. We report here the x-ray structure of the Escherichia coli GyrA-CTD (Protein Data Bank code 1ZI0). The E. coli GyrA-CTD adopts a circular-shaped {beta}-pinwheel fold first seen in the Borrelia burgdorferi GyrA-CTD. However, whereas the B. burgdorferi GyrA-CTD is flat, the E. coli GyrA-CTD is spiral. DNA relaxation assays reveal that the E. coli GyrA-CTD wraps DNA inducing substantial (+) superhelicity, while the B. burgdorferi GyrA-CTD introduces a more modest (+) superhelicity. The observation of a superhelical spiral in the present structure and that of the Bacillus stearothermophilus ParC-CTD structure suggests unexpected similarities in substrate selectivity between gyrase and Topo IV enzymes. We propose a model wherein the right-handed ((+) solenoidal) wrapping of DNA around the E. coli GyrA-CTD enforces unidirectional (-) DNA supercoiling.

  13. DNA supercoiling enhances cooperativity and efficiency of an epigenetic switch.

    Science.gov (United States)

    Norregaard, Kamilla; Andersson, Magnus; Sneppen, Kim; Nielsen, Peter Eigil; Brown, Stanley; Oddershede, Lene B

    2013-10-22

    Bacteriophage λ stably maintains its dormant prophage state but efficiently enters lytic development in response to DNA damage. The mediator of these processes is the λ repressor protein, CI, and its interactions with λ operator DNA. This λ switch is a model on the basis of which epigenetic switch regulation is understood. Using single molecule analysis, we directly examined the stability of the CI-operator structure in its natural, supercoiled state. We marked positions adjacent to the λ operators with peptide nucleic acids and monitored their movement by tethered particle tracking. Compared with relaxed DNA, the presence of supercoils greatly enhances juxtaposition probability. Also, the efficiency and cooperativity of the λ switch is significantly increased in the supercoiled system compared with a linear assay, increasing the Hill coefficient. PMID:24101469

  14. DNA supercoiling depends on the phosphorylation potential in Escherichia coli

    DEFF Research Database (Denmark)

    Van Workum, M.; van Dooren, S.J.M; Oldenburg, N;

    1996-01-01

    ATP/ADP ratios were varied in different ways and the degree of negative supercoiling was determined in Escherichia coli. Independent of whether the ATP/ADP ratio was reduced by a shift to anaerobic conditions, by addition of protonophore (dinitrophenol) or by potassium cyanide addition, DNA...

  15. Evaluating changes of writhe in computer simulations of supercoiled DNA

    NARCIS (Netherlands)

    Vries, de R.J.

    2005-01-01

    We compute changes in the writhe of a polygonal space curve when one of the vertices is displaced. The resulting expressions can be used in simulations of supercoiled DNA. For Brownian dynamics simulations, the expressions can be used to eliminate the explicit twisting degree of freedom. For Monte C

  16. Purification of large plasmids with methacrylate monolithic columns.

    Science.gov (United States)

    Krajnc, Nika Lendero; Smrekar, Franci; Cerne, Jasmina; Raspor, Peter; Modic, Martina; Krgovic, Danijela; Strancar, Ales; Podgornik, Ales

    2009-08-01

    The rapid evolution of gene therapy and DNA vaccines results in an increasing interest in producing large quantities of pharmaceutical grade plasmid DNA. Most current clinical trials involve plasmids of 10 kb or smaller in size, however, future requirements for multigene vectors including extensive control regions may require the production of larger plasmids, e. g., 20 kb and bigger. The objective of this study was to examine certain process conditions for purification of large plasmids with the size of up to 93 kb. Since there is a lack of knowledge about production and purification of bigger plasmid DNA, cell lysis and storage conditions were investigated. The impact of chromatographic system and methacrylate monolithic column on the degradation of plasmid molecules under nonbinding conditions at different flow rates was studied. Furthermore, capacity measurements varying salt concentration in loading buffer were performed and the capacities up to 13 mg of plasmid per mL of the monolithic column were obtained. The capacity flow independence in the range from 130 to 370 cm/h was observed. Using high resolution monolithic column the separation of linear and supercoiled isoforms of large plasmids was obtained. Last but not least, since the baseline separation of RNA and pDNA was achieved, the one step purification on larger CIM DEAE 8 mL tube monolithic column was performed and the fractions were analyzed by CIM analytical monolithic columns. PMID:19598166

  17. Damage to plasmid DNA induced by low energy carbon ions

    International Nuclear Information System (INIS)

    The damage induced in supercoiled plasmid DNA molecules by 1-6 keV carbon ions has been investigated as a function of ion exposure, energy and charge state. The production of short linear fragments through multiple double strand breaks has been demonstrated and exponential exposure responses for each of the topoisomers have been found. The cross section for the loss of supercoiling was calculated to be (2.2 ± 0.5) x 10-14 cm2 for 2 keV C+ ions. For singly charged carbon ions, increased damage was observed with increasing ion energy. In the case of 2 keV doubly charged ions, the damage was greater than for singly charged ions of the same energy. These observations demonstrate that ion induced damage is a function of both the kinetic and potential energies of the ion

  18. Administrative Circulars

    CERN Multimedia

    Département des Ressources humaines

    2004-01-01

    Administrative Circular N° 2 (Rev. 2) - May 2004 Guidelines and procedures concerning recruitment and probation period of staff members This circular has been revised. It cancels and replaces Administrative Circular N° 2 (Rev. 1) - March 2000. Administrative Circular N° 9 (Rev. 3) - May 2004 Staff members contracts This circular has been revised. It cancels and replaces Administrative Circular N° 9 (Rev. 2) - March 2000. Administrative Circular N° 26 (Rev. 4) - May 2004 Procedure governing the career evolution of staff members This circular has also been revised. It Administrative Circulars Administrative Circular N° 26 (Rev. 3) - December 2001 and brings up to date the French version (Rev. 4) published on the HR Department Web site in January 2004. Operational Circular N° 7 - May 2004 Work from home This circular has been drawn up. Operational Circular N° 8 - May 2004 Dealing with alcohol-related problems...

  19. Partition locus-based classification of selected plasmids in Klebsiella pneumoniae, Escherichia coli and Salmonella enterica spp.: An additional tool

    OpenAIRE

    Bousquet, A.; Henquet, S.; Compain, F.; Genel, N.; Arlet, G.; Decré, D

    2015-01-01

    The dissemination of antimicrobial resistance genes in Enterobacteriaceae has been largely attributed to plasmids, circular DNA molecules capable of autonomous replication. Whereas high-copy-number plasmids primarily rely on passive diffusion for plasmid maintenance, low-copy-number plasmids utilize so-called partition (par) systems. Plasmid partition relies on three structures, i.e. a centromere like DNA site, a centromere-binding protein and an ATPase or a GTPase motor protein for plasmid p...

  20. Homology of plasmids in strains of unicellular cyanobacteria

    NARCIS (Netherlands)

    Hondel, C.A.M.J.J. van den; Keegstra, W.; Borrias, W.E.; Arkel, G.A. van

    1979-01-01

    Six strains of unicellular cyanobacteria were examined for the presence of plasmids. Analysis of lysates of these strains by CsCl-ethidium bromide density centrifugation yielded a major chromosomal DNA band and a minor band containing covalently closed circular plasmid DNA, as shown by electron micr

  1. Improvement of the SUPERCOIL system code. Pt. 1

    International Nuclear Information System (INIS)

    This report describes the areas of the NET contract NET/84-042/PH which relate to problems of cooling and heat deposition in superconducting coils. These areas have to be integrated as extensions into the SUPERCOIL computer program; they are analytical model for force cooling of superconducting magnets by supercritical helium, analytical model of the a.c. losses in the TF and PF coil systems, analytical model for the nuclear heat deposition at the inner edge of the TF coil, and extension of the bath-cooling concept for additional heating sources (besides the ohmic heating losses). (orig.)

  2. Rapid purification of circular DNA by triplex-mediated affinity capture

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Huamin (4817 Sheboygan Ave., Madison, WI 53705); Smith, Lloyd M. (1115 Amherst Dr., Madison, WI 53705)

    1997-01-01

    A single-step capture of a target supercoiled double-stranded DNA molecule is accomplished by forming a local triple-helix among two strands of the supercoiled circular DNA and an oligonucleotide probe. The oligonucleotide is bound to an immobilizing support which facilitates the immobilization and purification of target DNA molecules. Non-target DNA molecules and other contaminating cellular material are easily removed by washing. The triple-helical structure is destabilized by raising the pH, leaving purified target DNA in the supernatant and reusable affinity capture oligonucleotide secured to the immobilizing support.

  3. Rapid purification of circular DNA by triplex-mediated affinity capture

    Energy Technology Data Exchange (ETDEWEB)

    Ji, H.; Smith, L.M.

    1997-01-07

    A single-step capture of a target supercoiled double-stranded DNA molecule is accomplished by forming a local triple-helix among two strands of the supercoiled circular DNA and an oligonucleotide probe. The oligonucleotide is bound to an immobilizing support which facilitates the immobilization and purification of target DNA molecules. Non-target DNA molecules and other contaminating cellular material are easily removed by washing. The triple-helical structure is destabilized by raising the pH, leaving purified target DNA in the supernatant and reusable affinity capture oligonucleotide secured to the immobilizing support. 3 figs.

  4. Information Circulars

    International Nuclear Information System (INIS)

    Information circulars are published from time to time under the symbol INFCIRC/. . . . for the purpose of bringing matters of general interest to the attention of all Members of the Agency. A list of the circulars which were of current interest on 15 January 1969 is given below, followed by an index to their subject matter. Other circulars can be traced by reference to earlier issues of the present document.

  5. Effect of DNA binding protein Ssh12 from hyperthermophilic archaeon Sulfolobus shibatae on DNA supercoiling

    Institute of Scientific and Technical Information of China (English)

    楼慧强; 黄力; VietQ.Mai

    1999-01-01

    An 11.5-ku DNA binding protein, designated as Sshl2, was purified from the hyperthermophilic archaeon Sulfolobus shibatae by column chromatography in SP Sepharose, DNA cellulose and phosphocellulose. Sshl2 accounts for about 4 % of the total cellular protein. The protein is capable of binding to both negatively supercoiled and relaxed DNAs. Nick closure analysis revealed that Sshl2 constrains negative supercoils upon binding to DNA. While the ability of the protein to constrain supercoils is weak at 22℃ , it is enhanced substantially at temperatures higher than 37℃ . Both the cellular content and supercoil-constraining ability of Sshl2 suggest that the protein may play an important role in the organization and stabilization of the chromosome of S. shibatae.

  6. Comparison of DNA loop size and super-coiled domain size in human cells

    International Nuclear Information System (INIS)

    DNA loop size and super-coiled domain size in HeLa cells were investigated by the nucleoid sedimentation technique as well as by analysis of the DNA-nuclearmatrix complex. After X-irradiation, the relaxation of super-coils was not correlated with the release of DNA from the nuclear matrix. The super-coiled domain size was much larger than the average DNA loop size. To explain the results, we propose that unwinding of super-coils in a DNA loop also causes unwinding in adjacent DNA loops in spite of existing attachment sites. Alternatively, the results may be explained by assuming that only very large DNA loops determine the sedimentation behaviour of nucleoids. (orig.)

  7. Information circulars

    International Nuclear Information System (INIS)

    Information circulars are published from time to time under the symbol INFCIRC/... for the purpose of bringing matters of general interest to the attention of all Members of the Agency. The present revision contains INFCIRCs published up to mid-August 1994. A complete numerical list of information circulars is reproduced with their titles in the Annex

  8. Information circulars

    International Nuclear Information System (INIS)

    The document summarizes the Information Circulars published by the IAEA for the purpose of bringing matters of general interest to the attention of all Members of the Agency. This revision contains INFCIRCs published up to mid-August 1992. A complete numerical lift of Information Circulars with their titles is reproduced in an Annex

  9. Information circulars

    International Nuclear Information System (INIS)

    The document summarizes the Information Circulars published by the IAEA for the purpose of bringing matters of general interest to the attention of all Member States. This revision contains INFCIRCs published up to the end of May 1999, grouped by field of activity. A complete list of information circulars in numerical order is given in an annex

  10. Information Circulars

    International Nuclear Information System (INIS)

    Information circulars are published from time to time under the symbol INFCIRC/. for the purpose of bringing matters of general interest to the attention of all Members of the Agency. A list of the circulars that were current on 31 December 1964 is given, followed by an index to their subject matter.

  11. Information circulars

    International Nuclear Information System (INIS)

    The document summarizes the Information Circulars published by the IAEA for the purpose of bringing matters of general interest to the attention of all Member States. This revision contains INFCIRCs published up to February 1997, grouped by field of activity. A complete list of information circulars in numerical order is given in an annex

  12. Information circulars

    International Nuclear Information System (INIS)

    Information circulars are published from time to time under the symbol INFCIRC/... for the purpose of bringing matters of general interest to the attention of all Members of the Agency. The present revision contains INFCIRCs published up to the end of April 2002. A complete numerical list of information circulars is reproduced with their titles in the Annex

  13. nifH Promoter Activity Is Regulated by DNA Supercoiling in Sinorhizobium meliloti

    Institute of Scientific and Technical Information of China (English)

    Yan-Jie LIU; Biao HU; Jia-Bi ZHU; Shan-Jiong SHEN; Guan-Qiao YU

    2005-01-01

    In prokaryotes, DNA supercoiling regulates the expression of many genes; for example, the expression of Klebsiella pneumoniae nifLA operon depends on DNA negative supercoiling in anaerobically grown cells, which indicates that DNA supercoiling might play a role in gene regulation of the anaerobic response. Since the expression of the nifH promoter in Sinorhizobium meliloti is not repressed by oxygen, it is proposed that the status of DNA supercoiling may not affect the expression of the nifH promoter. We tested this hypothesis by analyzing nifH promoter activity in wild-type and gyr- Escherichia coli in the presence and absence of DNA gyrase inhibitors. Our results show that gene expression driven by the S.meliloti nifH promoter requires the presence of active DNA gyrase. Because DNA gyrase increases the number of negative superhelical turns in DNA in the presence of ATP, our data indicate that negative supercoiling is also important for nifH promoter activity. Our study also shows that the DNA supercoilingdependent S. meliloti nifH promoter activity is related to the trans-acting factors NtrC and NifA that activate it. DNA supercoiling appeared to have a stronger effect on NtrC-activated nifH promoter activity than on NifA-activated promoter activity. Collectively, these results from the S. meliloti nifH promoter model system seem to indicate that, in addition to regulating gene expression during anaerobic signaling, DNA supercoiling may also provide a favorable topology for trans-acting factor binding and promoter activation regardless of oxygen status.

  14. Circular Updates

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Circular Updates are periodic sequentially numbered instructions to debriefing staff and observers informing them of changes or additions to scientific and specimen...

  15. Molecular Characterization of Borrelia burgdorferi Linear Plasmids by DNA Hybridization, PCR, Two-Dimensional Gel Electrophoresis, and Electron Microscopy

    OpenAIRE

    Karami, Ali; HOSSEYNI, Seyed Mohammad Javad; KIARUDI, Yaser

    2007-01-01

    Borrelia burgdorferi sensu stricto is the predominant cause of Lyme disease. B. burgdorferi strains carry several linear and circular plasmids and this may be also used as a distinguishing property of the bacteria. The presence of a mixed population of linear and circular plasmids makes separation and characterization of each kind very difficult. In this study, 7 clinical strains of skin and CSF isolates were determined by clonal selection and plasmid profile of B. burgdorferi by electron mic...

  16. Information circulars

    International Nuclear Information System (INIS)

    The document summarizes the information circulars published by the IAEA for the purpose of bringing matters of general interest to the attention of all Members of the Agency. In the main body of the document only those documents which are regarded as likely to be of current interest are listed. A complete numerical list of information circulars with their titles is reproduced in the Annex

  17. Plasmid segregation mechanisms

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Gerdes, Kenn; Charbon, Gitte Ebersbach

    2005-01-01

    Bacterial plasmids encode partitioning (par) loci that ensure ordered plasmid segregation prior to cell division. par loci come in two types: those that encode actin-like ATPases and those that encode deviant Walker-type ATPases. ParM, the actin-like ATPase of plasmid R1, forms dynamic filaments...... that segregate plasmids paired at mid-cell to daughter cells. Like microtubules, ParM filaments exhibit dynamic instability (i.e., catastrophic decay) whose regulation is an important component of the DNA segregation process. The Walker box ParA ATPases are related to MinD and form highly dynamic, oscillating...... filaments that are required for the subcellular movement and positioning of plasmids. The role of the observed ATPase oscillation is not yet understood. However, we propose a simple model that couples plasmid segregation to ParA oscillation. The model is consistent with the observed movement...

  18. Linearization of donor DNA during plasmid transformation in Neisseria gonorrhoeae.

    OpenAIRE

    Biswas, G D; Burnstein, K. L.; Sparling, P F

    1986-01-01

    We examined the fate of plasmid DNA after uptake during transformation in Neisseria gonorrhoeae. An 11.5-kilobase plasmid, pFA10, was processed to linear double-stranded DNA during uptake by competent cells, but cleavage of pFA10 was not site specific. A minority of pFA10 entered as open circles. A 42-kilobase plasmid, pFA14, was degraded into small fragments during uptake; no intracellular circular forms of pFA14 were evident. Since pFA10 DNA linearized by a restriction enzyme was not furthe...

  19. An Improved Method for Including Upper Size Range Plasmids in Metamobilomes

    DEFF Research Database (Denmark)

    Norman, Anders; Riber, Leise; Luo, Wenting; Li, Li Li; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    2014-01-01

    Two recently developed isolation methods have shown promise when recovering pure community plasmid DNA (metamobilomes/plasmidomes), which is useful in conducting culture-independent investigations into plasmid ecology. However, both methods employ multiple displacement amplification (MDA) to ensure...... suitable quantities of plasmid DNA for high-throughput sequencing. This study demonstrates that MDA greatly favors smaller circular DNA elements (<10 Kbp), which, in turn, leads to stark underrepresentation of upper size range plasmids (>10 Kbp). Throughout the study, we used two model plasmids, a 4.4 Kbp...

  20. Operational Circulars

    CERN Document Server

    2003-01-01

    Operational Circular N° 4 - April 2003 Conditions for use by members of the CERN personnel of vehicles belonging to or rented by CERN - This circular has been drawn up. Operational Circular N° 5 - October 2000 Use of CERN computing facilities - Further details on the personal use of CERN computing facilities Operational Circular N° 5 and its Subsidiary Rules http://cern.ch/ComputingRules defines the rules for the use of CERN computing facilities. One of the basic principles governing such use is that it must come within the professional duties of the user concerned, as defined by the user's divisional hierarchy. However, personal use of the computing facilities is tolerated or allowed provided : a) It is in compliance with Operational Circular N° 5 and not detrimental to official duties, including those of other users; b) the frequency and duration is limited and there is a negligible use of CERN resources; c) it does not constitute a political, commercial and/or profit-making activity; d) it is not...

  1. SPP1-mediated plasmid transduction.

    OpenAIRE

    Canosi, U; Lüder, G; Trautner, T A

    1982-01-01

    The virulent Bacillus subtilis phage SPP1 transduces plasmid DNA. Plasmid-transducing phages contain only plasmid DNA. Such DNA represents a concatemer of monomeric plasmid molecules with the molecular weight of mature SPP1 DNA. Biological parameters of plasmid transduction are described.

  2. Entwicklung und Untersuchung eines Verfahrens zur integrierten Aufreinigung von Plasmid DNA mittels wässriger Zweiphasenextraktion

    OpenAIRE

    Frerix, Andreas

    2006-01-01

    Plasmids are double stranded circular DNA molecules, mainly known as vectors for transferring genes. Therefore, plasmids have a high importance for molecular biology and gene technology respectively. Presently, there is a high interest to utilize plasmids in gene therapy and DNA vaccination due to their ability to replace or supplement genes. The manufacturing of plasmid DNA is performed using biotechnological processes by cultivation of optimized E. coli strains. The developed three-step pur...

  3. Supercoiled DNA; plectonemic structure and liquid crystal formation

    CERN Document Server

    Maarel, J R C; Jesse, W; Backendorf, C; Egelhaaf, S U; Lapp, A

    2003-01-01

    We have investigated the phase behaviour of pUC18 plasmid solutions with phase separation experiments and polarized light microscopy. Furthermore, the configuration of the superhelix is monitored with small-angle neutron scattering. The phase diagram is interpreted with liquid crystal theory including the effects of charge, orientation entropy, excluded volume, as well as the elastic, entropic and electrostatic contributions to the molecular free energy.

  4. Enhanced peptide nucleic acid binding to supercoiled DNA: possible implications for DNA "breathing" dynamics

    DEFF Research Database (Denmark)

    Bentin, T; Nielsen, Peter E.

    1996-01-01

    efficient with supercoiled than with linear DNA. In the presence of 140 mM KCI, the PNA binding rate was reduced but, notably, highly dependent on template topology. Negative supercoiling (mean superhelix density, sigma approximately -0.051) increased the rate of binding by 2 orders of magnitude compared...... to that of relaxed DNA. The pseudo-first-order rate constant [k psi (sigma)] obeys an exponential function, k psi (sigma) = k psi (lin)e-sigma delta, where delta is a constant of 105 and k psi lin is the rate of PNA binding to linear DNA (sigma = 0). The activation energy [Ea(sigma)] was determined as approximately...... 93 and approximately 48 kJ mol-1 for PNA binding to linear and supercoiled DNA, respectively. The results are discussed in relation to the possible future use of PNA as an antigene agent and in the framework of DNA "breathing" dynamics....

  5. A modified alkaline lysis method for the preparation of highly purified plasmid DNA from Escherichia coli.

    Science.gov (United States)

    Feliciello, I; Chinali, G

    1993-08-01

    We have developed a very efficient and rapid method for the preparation on a small or large scale of highly purified plasmid DNA from Escherichia coli. The procedure consists of five steps: (1) cell lysis by NaOH-SDS, (2) precipitation of cell lysate with 2 M potassium acetate-1 M acetic acid, (3) precipitation of the resulting supernatant with isopropanol, (4) treatment of the precipitate with RNase, and (5) a second isopropanol precipitation. The new procedure yields a plasmid DNA that is more than 90% in the supercoiled form and virtually free from proteins, RNA, and chromosomal DNA. We have thoroughly tested the method in the preparation of several thousand samples of different plasmids from various E. coli strains. We found that it consistently produced samples of plasmid DNA suitable for all routine uses such as restriction analysis, sequencing, and preparation of DNA probes for cloning and hybridization experiments. Moreover, plasmids purified by this procedure could fully replace plasmids purified on CsCl gradients for more demanding tasks such as the in vitro synthesis of RNA probes by phage RNA polymerases, the generation of deletion mutants with exonuclease III, and the transfection of mammalian cells by the calcium phosphate coprecipitation method, as tested on human fibroblasts and on CV-1 cells. PMID:8214582

  6. Interactions of Drosophila DNA topoisomerase II with left-handed Z-DNA in supercoiled minicircles.

    OpenAIRE

    Glikin, G C; Jovin, T M; Arndt-jovin, D.J.

    1991-01-01

    The native form of Drosophila melanogaster DNA topoisomerase II was purified from Schneider's S3 tissue culture cells and studied with two supercoiled minicircle preparations, mini and mini-CG, 354 bp and 370 bp in length, respectively. Mini-CG contains a d(CG)7 insert which assumes a left-handed Z-DNA conformation in negative supercoiled topoisomers with a negative linking number difference - delta Lk greater than or equal to 2. The interactions of topoisomerase II with topoisomer families o...

  7. Extension, Torque and Supercoiling in Single, Stretched and Twisted DNA Molecules

    CERN Document Server

    Lam, Pui-Man

    2016-01-01

    We reinvestigate the model originally studied by Neukirch and Marko that describes the extension, torque and supercoiling in single, stretched and twisted DNA molecules, which consists of a mixture of extended and supercoiled state, using now a more accurate form of the free energy for the untwisted but stretched DNA. The original model uses an approximate form of this free energy and the agreement with experiment is only qualitative. We find that this more accurate free energy significantly improves the results, bring them into quantitative agreement with experiment, throughout the entire force regime. This is rather surprising, considering that the theory is completely parameter-free.

  8. Information circulars

    International Nuclear Information System (INIS)

    The document summarizes the Information Circulars published by the IAEA under the symbol INFCIRC/ for the purpose of bringing matters of general interest to the attention of all Members of the Agency. A complete list of INFCIRCs in numerical order with their titles is given in the Annex

  9. ADMINISTRATIVE CIRCULARS

    CERN Multimedia

    Division des ressources humaines

    2000-01-01

    N° 2 (Rev. 1) - March 2000Guidelines and procedures concerning recruitment and probation period of staff membersN° 9 (Rev. 2) - March 2000Staff members contractsN° 16 (Rev. 2) - January 2000TrainingN° 30 (Rev. 1) - January 2000Indemnities and reimbursements upon taking up appointment and termination of contractN° 32 - February 2000Principles and procedures governing complaints of harassmentThese circular have been amended (No 2, N° 9, N° 16 and N° 30) or drawn up (N° 32).Copies are available in the Divisional Secretariats.Note:\tAdministrative and operational circulars, as well as the lists of those in force, are available for consultation in the server SRV4_Home in the Appletalk zone NOVELL (as GUEST or using your Novell username and password), volume PE Division Data Disk.The Word files are available in the folder COM, folder Public, folder ADM.CIRC.docHuman Resources DivisionTel. 74128

  10. DNA supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia coli.

    Science.gov (United States)

    Fulcrand, Geraldine; Dages, Samantha; Zhi, Xiaoduo; Chapagain, Prem; Gerstman, Bernard S; Dunlap, David; Leng, Fenfei

    2016-01-01

    Escherichia coli lac repressor (LacI) is a paradigmatic transcriptional factor that controls the expression of lacZYA in the lac operon. This tetrameric protein specifically binds to the O1, O2 and O3 operators of the lac operon and forms a DNA loop to repress transcription from the adjacent lac promoter. In this article, we demonstrate that upon binding to the O1 and O2 operators at their native positions LacI constrains three (-) supercoils within the 401-bp DNA loop of the lac promoter and forms a topological barrier. The stability of LacI-mediated DNA topological barriers is directly proportional to its DNA binding affinity. However, we find that DNA supercoiling modulates the basal expression from the lac operon in E. coli. Our results are consistent with the hypothesis that LacI functions as a topological barrier to constrain free, unconstrained (-) supercoils within the 401-bp DNA loop of the lac promoter. These constrained (-) supercoils enhance LacI's DNA-binding affinity and thereby the repression of the promoter. Thus, LacI binding is superhelically modulated to control the expression of lacZYA in the lac operon under varying growth conditions. PMID:26763930

  11. Binding of wild type and mutant p53 proteins to supercoiled DNA

    Czech Academy of Sciences Publication Activity Database

    Němcová, Kateřina; Pivoňková, Hana; Brázdová, Marie; Fojta, Miroslav

    Istanbul, 2006. s. 20-20. ISSN 1742-464X. [31st FEBS Congress, Molecules in Health and Disease. 24.06.2006-29.06.2006, Istanbul] Institutional research plan: CEZ:AV0Z50040507 Keywords : wtp53 and mutp53 Gly245Ser proteins * proteins modification * supercoil-selective (SCS) DNA binding Subject RIV: BO - Biophysics

  12. Regulation of p53 binding to linear and supercoiled DNAs by monoclonal antibodies

    Czech Academy of Sciences Publication Activity Database

    Brázda, Václav; Jagelská, Eva; Karlovská, Lenka; Paleček, Emil

    Oeiras : Instituto de Tecnologia Química e Biológica, 2001. s. 14. [Protein Structure-Function Trafficking and Signaling. Satellite meeting of the FEBS/PABMB /27./. 28.06.2001-30.06.2001, Oeiras] Institutional research plan: CEZ:AV0Z5004920 Keywords : p53 * supercoiled DNA * monoclonal antibodies Subject RIV: BO - Biophysics

  13. The problem of hypernegative supercoiling and R-loop formation in transcription.

    Science.gov (United States)

    Drolet, Marc; Broccoli, Sonia; Rallu, Fabien; Hraiky, Chadi; Fortin, Charles; Massé, Eric; Baaklini, Imad

    2003-01-01

    DNA supercoiling and topoisomerases have long been known to affect transcription initiation. In many studies, topA mutants were used to perturb chromosomal supercoiling. Although such studies clearly revealed that supercoiling could significantly affect gene expression, they did not tell much about the essential function(s) of DNA topoisomerase I, encoded by topA. Indeed, the topA mutants used in these studies were growing relatively well, although this gene is normally essential for growth. These mutants were either carrying a topA allele with enough residual activity to permit growth, or if deleted for the topA gene, they were carrying a compensatory mutation allowing them to grow. We have recently used a set of isogenic strains carrying a conditional gyrB mutation that allowed us to study the real effects of losing topoisomerase I activity on cell physiology. The results of our work show that an essential function of topoisomerase I is related to transcription, more precisely to inhibit R-loop formation. This is in agreement with a series of biochemical studies that revealed a role for topoisomerase I in inhibiting R-loop formation during transcription in the presence of DNA gyrase. In addition, our studies may have revealed an important role for DNA supercoiling in modulating gene expression, not only at the level of transcription initiation but also during elongation. In this paper, we will first discuss global and local supercoiling, then we will address the topic of R-loop formation and finally, we will review the subject of hypersupercoiling and R-loop formation in gene expression. Whenever possible, we will try to make correlations with growth phenotypes, since such correlations reveal the essential function of DNA topoisomerase I. PMID:12456359

  14. Characterization of Nocardia Plasmid pXT107

    Institute of Scientific and Technical Information of China (English)

    Hai-Yang XIA; Yong-Qiang TIAN; Ran ZHANG; Kai-Chun LIN; Zhong-Jun QIN

    2006-01-01

    Nocardia, Rhodococcus and Streptornyces, all members of the actinomycetes family, are Gram-positive eubacteria with high G+C content and able to form mycelium. We report here a newly identified plasmid pXT107 of Nocardia sp. 107, one of the smallest circular plasmids found in Nocardia.The complete nucleotide sequence of pXT107 consisted of 4335 bp with 65% G+C content, and encoded one replication extragenic palindromic (Rep) and six hypothetical proteins. The Rep, double-strand origin and single-strand origin of pXT107 resembled those of typical rolling-circle-replication plasmids, such as pNI100 of Nocardia, pRE8424 of Rhodococcus and plJ101 of Streptomyces. The Escherichia coli-Nocardia shuttle plasmid pHAQ22, containing thc rep gene of pXT107, is able to propagate in Nocardia but not in Streptomyces.

  15. A procedure for large-scale plasmid isolation without using ultracentrifugation.

    Science.gov (United States)

    Chakrabarti, A; Sitaric, S; Ohi, S

    1992-10-01

    An expedient procedure for large-scale plasmid isolation from Escherichia coli strains without using ultracentrifugation or special setups or reagents is described. The protocol, which utilizes a modified alkaline extraction procedure as well as differential precipitations by isopropanol and lithium chloride, is simple and rapid and yet produces plasmid DNA with a yield of about 2 mg/liter culture. The isolated plasmids consisted of mostly monomeric and dimeric covalently closed circular DNA. The plasmids could be digested by various restriction endonucleases and were compatible with gene cloning, transfection-gene expression, and viral production. PMID:1333773

  16. Plasmids encoding therapeutic agents

    Science.gov (United States)

    Keener, William K.

    2007-08-07

    Plasmids encoding anti-HIV and anti-anthrax therapeutic agents are disclosed. Plasmid pWKK-500 encodes a fusion protein containing DP178 as a targeting moiety, the ricin A chain, an HIV protease cleavable linker, and a truncated ricin B chain. N-terminal extensions of the fusion protein include the maltose binding protein and a Factor Xa protease site. C-terminal extensions include a hydrophobic linker, an L domain motif peptide, a KDEL ER retention signal, another Factor Xa protease site, an out-of-frame buforin II coding sequence, the lacZ.alpha. peptide, and a polyhistidine tag. More than twenty derivatives of plasmid pWKK-500 are described. Plasmids pWKK-700 and pWKK-800 are similar to pWKK-500 wherein the DP178-encoding sequence is substituted by RANTES- and SDF-1-encoding sequences, respectively. Plasmid pWKK-900 is similar to pWKK-500 wherein the HIV protease cleavable linker is substituted by a lethal factor (LF) peptide-cleavable linker.

  17. How topoisomerase IV can efficiently unknot and decatenate negatively supercoiled DNA molecules without causing their torsional relaxation.

    Science.gov (United States)

    Rawdon, Eric J; Dorier, Julien; Racko, Dusan; Millett, Kenneth C; Stasiak, Andrzej

    2016-06-01

    Freshly replicated DNA molecules initially form multiply interlinked right-handed catenanes. In bacteria, these catenated molecules become supercoiled by DNA gyrase before they undergo a complete decatenation by topoisomerase IV (Topo IV). Topo IV is also involved in the unknotting of supercoiled DNA molecules. Using Metropolis Monte Carlo simulations, we investigate the shapes of supercoiled DNA molecules that are either knotted or catenated. We are especially interested in understanding how Topo IV can unknot right-handed knots and decatenate right-handed catenanes without acting on right-handed plectonemes in negatively supercoiled DNA molecules. To this end, we investigate how the topological consequences of intersegmental passages depend on the geometry of the DNA-DNA juxtapositions at which these passages occur. We observe that there are interesting differences between the geometries of DNA-DNA juxtapositions in the interwound portions and in the knotted or catenated portions of the studied molecules. In particular, in negatively supercoiled, multiply interlinked, right-handed catenanes, we detect specific regions where DNA segments belonging to two freshly replicated sister DNA molecules form left-handed crossings. We propose that, due to its geometrical preference to act on left-handed crossings, Topo IV can specifically unknot supercoiled DNA, as well as decatenate postreplicative catenanes, without causing their torsional relaxation. PMID:27106058

  18. How topoisomerase IV can efficiently unknot and decatenate negatively supercoiled DNA molecules without causing their torsional relaxation

    Science.gov (United States)

    Rawdon, Eric J.; Dorier, Julien; Racko, Dusan; Millett, Kenneth C.; Stasiak, Andrzej

    2016-01-01

    Freshly replicated DNA molecules initially form multiply interlinked right-handed catenanes. In bacteria, these catenated molecules become supercoiled by DNA gyrase before they undergo a complete decatenation by topoisomerase IV (Topo IV). Topo IV is also involved in the unknotting of supercoiled DNA molecules. Using Metropolis Monte Carlo simulations, we investigate the shapes of supercoiled DNA molecules that are either knotted or catenated. We are especially interested in understanding how Topo IV can unknot right-handed knots and decatenate right-handed catenanes without acting on right-handed plectonemes in negatively supercoiled DNA molecules. To this end, we investigate how the topological consequences of intersegmental passages depend on the geometry of the DNA-DNA juxtapositions at which these passages occur. We observe that there are interesting differences between the geometries of DNA-DNA juxtapositions in the interwound portions and in the knotted or catenated portions of the studied molecules. In particular, in negatively supercoiled, multiply interlinked, right-handed catenanes, we detect specific regions where DNA segments belonging to two freshly replicated sister DNA molecules form left-handed crossings. We propose that, due to its geometrical preference to act on left-handed crossings, Topo IV can specifically unknot supercoiled DNA, as well as decatenate postreplicative catenanes, without causing their torsional relaxation. PMID:27106058

  19. Structural insight into negative DNA supercoiling by DNA gyrase, a bacterial type 2A DNA topoisomerase

    OpenAIRE

    Papillon, Julie; Ménétret, Jean-François; Batisse, Claire; Hélye, Reynald; Schultz, Patrick; Potier, Noëlle; Lamour, Valérie

    2013-01-01

    Type 2A DNA topoisomerases (Topo2A) remodel DNA topology during replication, transcription and chromosome segregation. These multisubunit enzymes catalyze the transport of a double-stranded DNA through a transient break formed in another duplex. The bacterial DNA gyrase, a target for broad-spectrum antibiotics, is the sole Topo2A enzyme able to introduce negative supercoils. We reveal here for the first time the architecture of the full-length Thermus thermophilus DNA gyrase alone and in a cl...

  20. Molecular analysis of plasmid DNA repair within ultraviolet-irradiated Escherichia coli. I. T4 endonuclease V-initiated excision repair

    International Nuclear Information System (INIS)

    The process by which DNA-interactive proteins locate specific sequences or target sites on cellular DNA within Escherichia coli is a poorly understood phenomenon. In this study, we present the first direct in vivo analysis of the interaction of a DNA repair enzyme, T4 endonuclease V, and its substrate, pyrimidine dimer-containing plasmid DNA, within UV-irradiated E. coli. A pyrimidine dimer represents a small target site within large domains of DNA. There are two possible paradigms by which endonuclease V could locate these small target sites: a processive mechanism in which the enzyme scans DNA for dimer sites or a distributive process in which dimers are located by random three-dimensional diffusion. In order to discriminate between these two possibilities in E. coli, an in vivo DNA repair assay was developed to study the kinetics of plasmid DNA repair and the dimer frequency (i.e. the number of dimer sites on a given plasmid molecule) in plasmid DNA as a function of time during repair. Our results demonstrate that the overall process of plasmid DNA repair initiated by T4 endonuclease V (expressed from a recombinant plasmid within repair-deficient E. coli) occurs by a processive mechanism. Furthermore, by reducing the temperature of the repair incubation, the endonuclease V-catalyzed incision step has been effectively decoupled from the subsequent steps including repair patch synthesis, ligation, and supercoiling. By this manipulation, it was determined that the overall processive mechanism is composed of two phases: a rapid processive endonuclease V-catalyzed incision reaction, followed by a slower processive mechanism, the ultimate product of which is the dimer-free supercoiled plasmid molecule

  1. Toxin plasmids of Clostridium perfringens.

    Science.gov (United States)

    Li, Jihong; Adams, Vicki; Bannam, Trudi L; Miyamoto, Kazuaki; Garcia, Jorge P; Uzal, Francisco A; Rood, Julian I; McClane, Bruce A

    2013-06-01

    In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  2. Efficient non-enzymatic cleavage of Staphylococcus aureus plasmid DNAs mediated by neodymium ions.

    Science.gov (United States)

    Zovčáková, Monika; Španová, Alena; Pantůček, Roman; Doškař, Jiří; Rittich, Bohuslav

    2016-08-15

    Staphylococcus aureus plasmids are the main factor in the spreading of antibacterial resistance among bacterial strains that has emerged on a worldwide scale. Plasmids recovered from 12 clinical and food isolates of S. aureus were treated with 10 mM free lanthanide Nd(3+) ions (non-enzymatic cleavage agent) in Hepes buffer (pH 7.5) at 70 °C. Topological forms of plasmids-closed circular (ccc), open circular (oc), and linear (lin)-produced by cleavage at different times were separated using pulsed-field agarose gel electrophoresis. The method is proposed to detect and differentiate several plasmids in the same bacterial strain according to their size. PMID:27237372

  3. Membrane-bound fractions of R6K plasmid DNA in Escherichia coli.

    OpenAIRE

    Archibold, E R; Wilson, J. D.; Allison, D. P.; Sheehy, R J

    1983-01-01

    The intracellular location of plasmid DNA has been of interest in an effort to understand the maintenance of these molecules. We have employed a simple procedure which enables us to isolate from exponentially grown cells on sucrose gradients membrane-complexed forms of R6K plasmid DNA. Electron micrographs identified the complexing of membrane fractions to circular forms of R6K DNA. Biochemical studies of the complexed R6K molecules showed the presence of membrane-specific proteins and sugges...

  4. DNA supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia coli

    OpenAIRE

    Geraldine Fulcrand; Samantha Dages; Xiaoduo Zhi; Prem Chapagain; Bernard S. Gerstman; David Dunlap; Fenfei Leng

    2016-01-01

    Escherichia coli lac repressor (LacI) is a paradigmatic transcriptional factor that controls the expression of lacZYA in the lac operon. This tetrameric protein specifically binds to the O1, O2 and O3 operators of the lac operon and forms a DNA loop to repress transcription from the adjacent lac promoter. In this article, we demonstrate that upon binding to the O1 and O2 operators at their native positions LacI constrains three (−) supercoils within the 401-bp DNA loop of the lac promoter and...

  5. Interaction of mutant p53 with supercoiled DNA in context of chromatin

    Czech Academy of Sciences Publication Activity Database

    Brázdová, Marie; Němcová, Kateřina; Fojta, Miroslav; Paleček, Emil; Deppert, W.

    Lyon, 2007. s. 77. [International p53 Marathon . Deregulating the p53 Network: Origin and Consequences of TP53 Mutations. 12.11.2007-16.11.2007, Lyon] R&D Projects: GA MŠk(CZ) 1K04119; GA ČR(CZ) GP204/06/P369; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : p53 mutation * gain of function * supercoiled DNA Subject RIV: BO - Biophysics

  6. Persistence Mechanisms of Conjugative Plasmids

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Hansen, Lars H.; Sørensen, Søren Johannes

    2009-01-01

    maintenance in the host cell. These importantly include the ability to self-mobilize in a process termed conjugative transfer, which may occur across species barriers. Other plasmid stabilizing mechanisms include the multimer resolution system, active partitioning, and post-segregational-killing of plasmid......Are plasmids selfish parasitic DNA molecules or an integrated part of the bacterial genome? This chapter reviews the current understanding of the persistence mechanisms of conjugative plasmids harbored by bacterial cells and populations. The diversity and intricacy of mechanisms affecting the...... successful propagation and long-term continued existence of these extra-chromosomal elements is extensive. Apart from the accessory genetic elements that may provide plasmid-harboring cells a selective advantage, special focus is placed on the mechanisms conjugative plasmids employ to ensure their stable...

  7. Single-stranded plasmid DNA in Bacillus subtilis and Staphylococcus aureus.

    OpenAIRE

    te Riele, H; Michel, B.; Ehrlich, S D

    1986-01-01

    Plasmid pC194 was found to exist in a double-stranded and a single-stranded DNA form in Bacillus subtilis and Staphylococcus aureus. This single-stranded DNA was found as a circular molecule of the same size as the parental monomer and corresponded to only one of the two DNA strands. It represented one-third of plasmid copies. Single- and double-stranded DNA copies in similar proportions to the above were detected for five other S. aureus plasmids (pC221, pC223, pE194, pT127, and pT181) and o...

  8. Strand breaks in plasmid DNA, natural and brominated, by low-energy X-rays

    International Nuclear Information System (INIS)

    Single- and double-strand breaks were measured in Col E1 plasmid DNA, natural and partially brominated, irradiated with monoenergetic X-rays (from a synchrotron radiation source) on both sides of the K-absorption edge for Br. The fraction of the undamaged supercoiled form decreased exponentially with the photon dose; its yield in the brominated DNA did not exhibit any energy-dependence. This result is consistent with the calculated relative contributions of photoelectric interactions with individual component atoms which show that an electron flux originating in light atoms outweighs that from bromine. However, X-rays of energy above the Br K-edge appear slightly more efficient in producing double-strand breaks. This result seems to suggest that the positive charge and submolecular effects associated with the photo-absorption in bromine play some role in damaging processes, besides the initial distribution of deposited electron energy. (author)

  9. Effect of intense, ultrashort laser pulses on DNA plasmids in their native state: strand breakages induced by {\\it in-situ} electrons

    CERN Document Server

    D'Souza, J S; Dharmdhikair, A K; Rao, B J; Mathur, D

    2010-01-01

    Single strand breaks are induced in DNA plasmids, pBR322 and pUC19, in aqueous media by intense ultrashort laser pulses (820 nm wavelength, 45 fs pulse duration, 1 kHz repetition rate) at intensities of 1-12 TW cm$^{-2}$. The intense laser radiation generates, {\\it in situ}, electrons that induce transformation of supercoiled DNA into relaxed DNA. The extent of electron-mediated relaxation of DNA structure is quantified. Introduction of electron and radical scavengers inhibits DNA damage.

  10. Effect of intense, ultrashort laser pulses on DNA plasmids in their native state: strand breakages induced by {\\it in-situ} electrons and radicals

    CERN Document Server

    D'Souza, J S; Dharmadhikari, A K; Rao, B J; Mathur, D

    2011-01-01

    Single strand breaks are induced in DNA plasmids, pBR322 and pUC19, in aqueous media exposed to strong fields generated using ultrashort laser pulses (820 nm wavelength, 45 fs pulse duration, 1 kHz repetition rate) at intensities of 1-12 TW cm$^{-2}$. The strong fields generate, {\\it in situ}, electrons and radicals that induce transformation of supercoiled DNA into relaxed DNA, the extent of which is quantified. Introduction of electron and radical scavengers inhibits DNA damage; results indicate that OH radicals are the primary (but not sole) cause of DNA damage.

  11. Depletion of RNase HI activity in Escherichia coli lacking DNA topoisomerase I leads to defects in DNA supercoiling and segregation

    OpenAIRE

    Usongo, Valentine; Nolent, Flora; Sanscartier, Patrick; Tanguay, Cynthia; Broccoli, Sonia; Baaklini, Imad; Drlica, Karl; Drolet, Marc

    2008-01-01

    Gyrase-mediated hypernegative supercoiling is one manifestation of R-loop formation, a phenomenon that is normally suppressed by topoisomerase I (topA) in Escherichia coli. Overproduction of RNase HI (rnhA), an enzyme that removes the RNA moiety of R-loops, prevents hypernegative supercoiling and allows growth of topA null mutants. We previously showed that topA and rnhA null mutations are incompatible. We now report that such mutants were viable when RNase HI or topoisomerase III was express...

  12. Circularity and Lambda Abstraction

    DEFF Research Database (Denmark)

    Danvy, Olivier; Thiemann, Peter; Zerny, Ian

    2013-01-01

    In this tribute to Doaitse Swierstra, we present the rst transformation between lazy circular programs a la Bird and strict cir- cular programs a la Pettorossi. Circular programs a la Bird rely on lazy recursive binding: they involve circular unknowns and make sense equa- tionally. Circular...... unknowns from what is done to them, which we lambda-abstract with functions. The circular unknowns then become dead variables, which we eliminate. The result is a strict circu- lar program a la Pettorossi. This transformation is reversible: given a strict circular program a la Pettorossi, we introduce...

  13. Plasmids in Mycoplasma species isolated from goats and sheep and their preliminary typing

    Directory of Open Access Journals (Sweden)

    Nascimento Elmiro R.

    1999-01-01

    Full Text Available One-hundred-five (105 clinical isolates of mycoplasma from caprine origin and one isolate from ovine were surveyed for plasmids, which were present in thirty-three (31% of them. These mycoplasmas originated from 13 herds. Ten of them were symptomatic for mycoplasmal disease (mastitis, polyarthritis, septicemia and three herds were asymptomatic, i.e., clinically normal. Twenty-eight isolates were Mycoplasma mycoides subspecies mycoides LC (large colony or caprine biotype, four were Mycoplasma capricolum subsp. capricolum and one was Mycoplasma cottewii. The isolated plasmids were linearized by EcoRI, EcoRV, EcoRI and EcoRV or BamHI and EcoRV, and were of five sizes (1.1, 1.6, 1.7, 1.8, and 1.9 Kbp. Based on restriction enzyme digestion and size of the linearized supercoiled extrachromosomal DNA, five plasmid types were recovered (p1II, p2III, p2V, p3I, and p4IV. The small size of these DNA elements probably exclude replicative forms of DNA virus, which are equal or larger than 8.0 Kbp.

  14. Developing strategies to increase plasmid DNA production in Escherichia coli DH5α using batch culture.

    Science.gov (United States)

    Islas-Lugo, Fabiola; Vega-Estrada, Jesús; Alvis, Christian Ariel; Ortega-López, Jaime; Del Carmen Montes-Horcasitas, María

    2016-09-10

    Plasmid DNA (pDNA) production has recently increased as a result of advances in DNA vaccines. The practical development of pDNA vaccines requires high yield and productivity of supercoiled plasmid DNA (sc-pDNA). The yield and productivity are influenced by the host strain, the plasmid, the production process, and especially by growth conditions, such as the culture type and medium. We evaluated different strategies to increase pDNA production by Escherichia coli DH5α in batch culture. The strategies were driven by the development of a four single-factor experimental design and were based on the change of culture media composition in terms of carbon and nitrogen and the modification of the pH control by using NaOH or NH4OH. The results revealed the carbon (50g/L of glycerol) and nitrogen (8.34g/L of YESP) concentration in the culture medium and starting pH control with NH4OH when most of the organic nitrogen was consumed. Under these conditions, we obtained a volumetric yield of 213mg pDNA/L, a specific yield of 10mg pDNA/g DCW (dry cell weight), 92% of sc-pDNA and a productivity of 17.6mg pDNA/(Lh). The pDNA productivities reached were 42% higher than the productivities reported by other authors applying similar conditions. PMID:27374404

  15. In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing

    DEFF Research Database (Denmark)

    Carattoli, Alessandra; Zankari, Ea; García-Fernández, Aurora; Larsen, Mette Voldby; Lund, Ole; Villa, Laura; Aarestrup, Frank Møller; Hasman, Henrik

    2014-01-01

    genomes of multidrug-resistant Enterobacteriaceae species by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the family Enterobacteriaceae in the NCBI nucleotide database were collected to build a consensus database for integration into a...... plasmid sequence types (STs) and new alleles and ST variants. In conclusion, testing of the two Web tools using both fully assembled plasmid sequences and WGS-generated draft genomes showed them to be able to detect a broad variety of plasmids that are often associated with antimicrobial resistance in......In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft...

  16. A novel plasmid pEA68 of Erwinia amylovora and the description of a new family of plasmids.

    Science.gov (United States)

    Ismail, Emadeldeen; Blom, Jochen; Bultreys, Alain; Ivanović, Milan; Obradović, Aleksa; van Doorn, Joop; Bergsma-Vlami, Maria; Maes, Martine; Willems, Anne; Duffy, Brion; Stockwell, Virginia O; Smits, Theo H M; Puławska, Joanna

    2014-12-01

    Recent genome analysis of Erwinia amylovora, the causal agent of fire blight disease on Rosaceae, has shown that the chromosome is highly conserved among strains and that plasmids are the principal source of genomic diversity. A new circular plasmid, pEA68, was found in E. amylovora strain 692 (LMG 28361), isolated in Poland from Sorbus (mountain ash) with fire blight symptoms. Annotation of the 68,763-bp IncFIIa-type plasmid revealed that it contains 79 predicted CDS, among which two operons (tra, pil) are associated with mobility. The plasmid is maintained stably in E. amylovora and does not possess genes associated with antibiotic resistance or known virulence genes. Curing E. amylovora strain 692 of pEA68 did not influence its virulence in apple shoots nor amylovoran synthesis. Of 488 strains of E. amylovora from seventeen countries, pEA68 was only found in two additional strains from Belgium. Although the spread of pEA68 is currently limited to Europe, pEA68 comprises, together with pEA72 and pEA78 both found in North America, a new plasmid family that spans two continents. PMID:25178659

  17. Genome stability of Lyme disease spirochetes: comparative genomics of Borrelia burgdorferi plasmids.

    Directory of Open Access Journals (Sweden)

    Sherwood R Casjens

    Full Text Available Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33-40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi ∼900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short ≤20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant.

  18. Genome Stability of Lyme Disease Spirochetes: Comparative Genomics of Borrelia burgdorferi Plasmids

    Energy Technology Data Exchange (ETDEWEB)

    Casjens S. R.; Dunn J.; Mongodin, E. F.; Qiu, W.-G.; Luft, B. J.; Schutzer, S. E.; Gilcrease, E. B.; Huang, W. M.; Vujadinovic, M.; Aron, J. K.; Vargas, L. C.; Freeman, S.; Radune, D.; Weidman, J. F.; Dimitrov, G. I.; Khouri, H. M.; Sosa, J. E.; Halpin, R. A.; Fraser, C. M.

    2012-03-14

    Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33-40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi {approx}900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short {le}20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant.

  19. Conjugative plasmids of Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Emilia Pachulec

    Full Text Available Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones and with and without different tetM determinants (Dutch and American type tetM determinants have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233 or containing Dutch (pEP5289 or American (pEP5050 type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1alpha, beta, gamma, delta and epsilon subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids

  20. Circular-rubbing Manipulation

    Institute of Scientific and Technical Information of China (English)

    SHEN Guo-quan; XIAO Yuan-chun

    2003-01-01

    @@ "Mo" literally means "rubbing between two things"and "eliminating". Circular-rubbing is one of the earliest manipulations used in clinical practice. Circular-rubbing differs from pressing actually. Pressing is a static manipulation and acts to inhibit motion; circular-rubbing is a movable manipulation and serves to eliminate stationary. Circular-rubbing can be performed by either the palm or the finger.

  1. A plasmid in the archaebacterium Sulfolobus acidocaldarius

    OpenAIRE

    Yeats, Siobhán; McWilliam, Peter; Zillig, Wolfram

    1982-01-01

    A plasmid of mol. wt. ∼9 × 106 has been isolated from the archaebacterium Sulfolobus acidocaldarius strain B12. Plasmid production is induced by u.v. radiation. A copy of the plasmid is probably carried by the chromosome, integrated at a specific site. The entire plasmid, and also restriction fragments of it, has been cloned into Escherichia coli plasmid vectors, and the cleavage sites on the plasmid DNA of three restriction endonucleases have been mapped.

  2. Plasmids of Xylella fastidiosa Mulberry-Infecting Strains Share Extensive Sequence Identity and Gene Complement with pVEIS01 From the Earthworm Symbiont Verminephrobacter Eiseniae

    Science.gov (United States)

    A ~25 kbp plasmid was present in each of four Californian strains of Xylella fastidiosa from mulberry affected with leaf scorch disease. Fragments of each plasmid were cloned into E. coli, sequenced, and assembled into circular contigs of 25,105 bp (pXF-RIV11 and pXF-RIV16) or 24,372 bp (pXF-RIV19 a...

  3. Plasmid acquisition in microgravity

    Science.gov (United States)

    Juergensmeyer, Margaret A.; Juergensmeyer, Elizabeth A.; Guikema, James A.

    1995-01-01

    In microgravity, bacteria often show an increased resistance to antibiotics. Bacteria can develop resistance to an antibiotic after transformation, the acquisition of DNA, usually in the form of a plasmid containing a gene for resistance to one or more antibiotics. In order to study the capacity of bacteria to become resistant to antibiotics in microgravity, we have modified the standard protocol for transformation of Escherichia coli for use in the NASA-flight-certified hardware package, The Fluid Processing Apparatus (FPA). Here we report on the ability of E. coli to remain competent for long periods of time at temperatures that are readily available on the Space Shuttle, and present some preliminary flight results.

  4. Evolved plasmid-host interactions reduce plasmid interference cost.

    Science.gov (United States)

    Yano, Hirokazu; Wegrzyn, Katarznya; Loftie-Eaton, Wesley; Johnson, Jenny; Deckert, Gail E; Rogers, Linda M; Konieczny, Igor; Top, Eva M

    2016-09-01

    Antibiotic selection drives adaptation of antibiotic resistance plasmids to new bacterial hosts, but the molecular mechanisms are still poorly understood. We previously showed that a broad-host-range plasmid was poorly maintained in Shewanella oneidensis, but rapidly adapted through mutations in the replication initiation gene trfA1. Here we examined if these mutations reduced the fitness cost of TrfA1, and whether this was due to changes in interaction with the host's DNA helicase DnaB. The strains expressing evolved TrfA1 variants showed a higher growth rate than those expressing ancestral TrfA1. The evolved TrfA1 variants showed a lower affinity to the helicase than ancestral TrfA1 and were no longer able to activate the helicase at the oriV without host DnaA. Moreover, persistence of the ancestral plasmid was increased upon overexpression of DnaB. Finally, the evolved TrfA1 variants generated higher plasmid copy numbers than ancestral TrfA1. The findings suggest that ancestral plasmid instability can at least partly be explained by titration of DnaB by TrfA1. Thus under antibiotic selection resistance plasmids can adapt to a novel bacterial host through partial loss of function mutations that simultaneously increase plasmid copy number and decrease unfavorably high affinity to one of the hosts' essential proteins. PMID:27121483

  5. Axial distortion as a sensor of supercoil changes: a molecular model for the homeostatic regulation of DNA gyrase

    Indian Academy of Sciences (India)

    Shyam Unniraman; Valakunja Nagaraja

    2001-12-01

    Negative supercoiling stimulates transcription of many genes. In contrast, transcription of the genes coding for DNA gyrase is subject to a novel mechanism of autoregulation, wherein relaxation of the template DNA stimulates their transcription. Since DNA gyrase is the sole supercoiling activity in the eubacterial cell, relaxation-stimulated transcription (RST) could reflect an autoregulatory mechanism to maintain supercoil levels within the cell. Extensive deletion and mutational analyses of Escherichia coli gyrA promoter have shown that the $-10$ region is essential for RST; however, a molecular model has proved to be elusive. We find a strong bend centre immediately downstream of the $-10$ region in the gyrA promoter. On the basis of analysis of various mutants in the $-10$ region, we propose a model where axial distortion acts as a sensor of topological changes in DNA. Our model is consistent with earlier data with E. coli gyrA and gyrB promoters. We also extrapolate the model to explain the phenomenon of RST of gyr promoters in other organisms and contrast it with promoters induced by supercoiling.

  6. Generation of supercoils in nicked and gapped DNA drives DNA unknotting and postreplicative decatenation

    Science.gov (United States)

    Racko, Dusan; Benedetti, Fabrizio; Dorier, Julien; Burnier, Yannis; Stasiak, Andrzej

    2015-01-01

    Due to the helical structure of DNA the process of DNA replication is topologically complex. Freshly replicated DNA molecules are catenated with each other and are frequently knotted. For proper functioning of DNA it is necessary to remove all of these entanglements. This is done by DNA topoisomerases that pass DNA segments through each other. However, it has been a riddle how DNA topoisomerases select the sites of their action. In highly crowded DNA in living cells random passages between contacting segments would only increase the extent of entanglement. Using molecular dynamics simulations we observed that in actively supercoiled DNA molecules the entanglements resulting from DNA knotting or catenation spontaneously approach sites of nicks and gaps in the DNA. Type I topoisomerases, that preferentially act at sites of nick and gaps, are thus naturally provided with DNA–DNA juxtapositions where a passage results in an error-free DNA unknotting or DNA decatenation. PMID:26150424

  7. Analysis of gamma-ray damage to supercoiled DNA from electrooptical birefringence relaxation times

    International Nuclear Information System (INIS)

    Electrooptical birefringence (EO) measurements were performed on covalently closed, supercoiled Simian Virus 40 (SV40) DNA both in its native form, and after graded doses of 60Co γ-irradiation ranging from 2.5 to 104.6 Gy. Characteristic relaxation times are unchanged by γ-ray doses up to 105 Gy, and the subpopulation of molecules exhibiting the relaxation time tau1 also undergoes little change as a function of the absorbed radiation dose. However, the population parameter a2 decreases from approximately 50% to 40%, and a3 increases from 17% to 27% as the γ-ray dose is increased up to 50 Gy. The increase in the fraction of DNA molecules exhibiting the longest relaxation time would be expected insofar as the accumulation of radiation damage produces a progressively larger number of double-strand breaks, with a resultant increase in the number of linear molecules. 2 references, 1 figure

  8. On the stability of plasmid DNA vectors during cell culture and purification.

    Science.gov (United States)

    Freitas, S S; Azzoni, A R; Santos, J A L; Monteiro, G A; Prazeres, D M F

    2007-06-01

    Gene therapy and DNA vaccination applications have increased the demand for highly purified plasmid DNA (pDNA) in the last years. One of the main problems related to the scale-up of pDNA purification is the degradation of the supercoiled (sc) isoforms during cell culture and multi-stage purification. In this work, a systematic study of the stability of two model plasmids (3,697 and 6,050 bp) during a mid-scale production process, which includes fermentation, alkaline lysis, isopropanol and ammonium sulphate precipitation and hydrophobic interaction chromatography, was performed. Results indicate that by extending cell culture (up to 26 h) and cell lysis (up to 2 h) it is possible to significantly reduce the amounts of RNA, without significantly compromising the yields of the sc pDNA isoform, a feature that could be conveniently exploited for downstream processing purposes. The stability of pDNA upon storage of E. coli pellets at different temperatures indicates that, differently from RNA, pDNA is remarkably stable when stored in cell pellets (>3 weeks at 4 degrees C, >12 weeks at -20 degrees C) prior to processing. With alkaline lysates, however, storage at -20 degrees C is mandatory to avoid sc pDNA degradation within the first 8 weeks. Furthermore, the subsequent purification steps could be carried out at room temperature without significant pDNA degradation. Since the unit operations and process conditions studied in this work are similar to those generally used for plasmid DNA production, the results presented here may contribute to improve the current knowledge on plasmid stability and process optimization. PMID:17914194

  9. Plasmid-mediated transformation in Bacillus megaterium.

    OpenAIRE

    Brown, B. J.; Carlton, B C

    1980-01-01

    A transformation system was developed for Bacillus megaterium by using antibiotic resistance plasmid deoxyribonucleic acid molecules derived from Staphylococcus aureus and Bacillus cereus. Lysozyme-generated protoplasts of B. megaterium allowed uptake of plasmid deoxyribonucleic acid in the presence of polyethylene glycol. Transformants expressed the antibiotic resistance determinants present on the plasmid deoxyribonucleic acid, and reisolated plasmid deoxyribonucleic acid yielded restrictio...

  10. Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids.

    Science.gov (United States)

    Bryksin, Anton V; Matsumura, Ichiro

    2010-06-01

    Here we describe a straightforward, efficient, and reliable way to clone an insert of choice into a plasmid of choice without restriction endonucleases or T4 DNA ligase. Chimeric primers containing plasmid sequence at the 5' ends and insert sequence at the 3' ends were used to PCR-amplify insertion sequences of various sizes, namely the genes for GFP (gfp), beta-d-glucuronidase (gusA), and beta-galactosidase (lacZ), as well as the entire luxABCDE operon. These inserts were employed as mega-primers in a second PCR with a circular plasmid template. The original plasmid templates were then destroyed in restriction digests with DpnI, and the overlap extension PCR products were used to transform competent Escherichia coli cells. Phusion DNA polymerase was used for the amplification and fusion reactions, so both reactions were easy to monitor and optimize. PMID:20569222

  11. PUBLICATION OF ADMINISTRATIVE CIRCULAR

    CERN Multimedia

    HR Department

    2008-01-01

    ADMINISTRATIVE CIRCULAR NO. 23 (REV. 2) – SPECIAL WORKING HOURS Administrative Circular No. 23 (Rev. 2) entitled "Special working hours", approved following discussion in the Standing Concertation Committee meeting of 9 December 2008, will be available on the intranet site of the Human Resources Department as from 19 December 2008: http://cern.ch/hr-docs/admincirc/admincirc.asp It cancels and replaces Administrative Circular No. 23 (Rev. 1) entitled "Stand-by duty" of April 1988. A "Frequently Asked Questions" information document on special working hours will also be available on this site. Paper copies of this circular will shortly be available in departmental secretariats. Human Resources Department Tel. 78003

  12. Publication of administrative circular

    CERN Multimedia

    HR Department

    2009-01-01

    ADMINISTRATIVE CIRCULAR NO. 23 (REV. 2) – SPECIAL WORKING HOURS Administrative Circular No. 23 (Rev. 2) entitled "Special working hours", approved following discussion in the Standing Concertation Committee on 9 December 2008, will be available on the intranet site of the Human Resources Department as from 19 December 2008: http://cern.ch/hr-docs/admincirc/admincirc.asp It cancels and replaces Administrative Circular No. 23 (Rev. 1) entitled "Stand-by duty" of April 1988. A "Frequently Asked Questions" information document on special working hours will also be available on this site. Paper copies of this circular will shortly be available in Departmental Secretariats. Human Resources Department Tel. 78003

  13. Toroidal circular dichroism

    Science.gov (United States)

    Raybould, T. A.; Fedotov, V. A.; Papasimakis, N.; Kuprov, I.; Youngs, I. J.; Chen, W. T.; Tsai, D. P.; Zheludev, N. I.

    2016-07-01

    We demonstrate that the induced toroidal dipole, represented by currents flowing on the surface of a torus, makes a distinct and indispensable contribution to circular dichroism. We show that toroidal circular dichroism supplements the well-known mechanism involving electric dipole and magnetic dipole transitions. We illustrate this with rigorous analysis of the experimentally measured polarization-sensitive transmission spectra of an artificial metamaterial, constructed from elements of toroidal symmetry. We argue that toroidal circular dichroism will be found in large biomolecules with elements of toroidal symmetry and should be taken into account in the interpretation of circular dichroism spectra of organics.

  14. [Plasmid characteristics of naphthalene and salicylate biodegradation in Pseudomonas putida].

    Science.gov (United States)

    Zakharian, R A; Bakunin, K A; Gasparian, N S; Kocharian, Sh M; Arakelov, G M

    1980-01-01

    The object of this work was to study the physico-chemical and biological properties of DNAs of the biodegradation plasmids NAH and SAL. A comparative analysis of the physico-chemical parameters for these DNAs made it possible to detect a number of identical properties in them: the same sedimentation profile for covalently-closed circular DNA forms, 68--70 S; the molecular weight of ca. 50 MD; a roughly equal number of fragments (up to 23) was found when the DNAs of NAH and SAL were restricted by EcoRI endonuclease. The transformation of the plasmidless strain PpGI was done. PMID:6259498

  15. Very low-energy and low-fluence ion beam bombardment of naked plasmid DNA

    International Nuclear Information System (INIS)

    Ion beam bombardment of biological organisms has been recently applied to mutation breeding of both agricultural and horticultural plants. In order to explore relevant mechanisms, this study employed low-energy ion beams to bombard naked plasmid DNA. The study aimed at simulation of the final stage of the process of the ion beam bombardment of real cells to check whether and how very low-energy and low-fluence of ions can induce mutation. Argon and nitrogen ions at 5 keV and 2.5 keV respectively bombarded naked plasmid DNA pGFP to very low-fluences, an order of 1013 ions/cm2. Subsequently, DNA states were analyzed using electrophoresis. Results provided evidences that the very low-energy and low-fluence ion bombardment indeed altered the DNA structure from supercoil to short linear fragments through multiple double strand breaks and thus induced mutation, which was confirmed by transfer of the bombarded DNA into bacteria Escherichia coli and subsequent expression of the marker gene.

  16. Low energy electron induced damage to plasmid DNA pQE30

    Science.gov (United States)

    Kumar, S. V. K.; Pota, Tasneem; Peri, Dinakar; Dongre, Anushka D.; Rao, Basuthkar J.

    2012-07-01

    Low energy electrons (LEEs) are produced in copious amounts by the primary radiation used in radiation therapy. The damage caused to the DNA by these secondary electrons in the energy range 5-22 eV has been studied to understand their possible role in radiation induced damage. Electrons are irradiated on dried films of plasmid DNA (pQE30) and analysed using agarose gel electrophoresis. Single strand breaks (SSBs) induced by LEE to supercoiled plasmid DNA show resonance structures at 7, 12, and 15 eV for low doses and 6, 10, and ˜18 eV at saturation doses. The present measurements have an overall agreement with the literature that LEEs resonantly induce SSBs in DNA. Resonant peaks in the SSBs induced by LEEs at 7, 12, and 15 eV with the lowest employed dose in the current study are somewhat different from those reported earlier by two groups. The observed differences are perhaps related to the irradiation dose, conditions and the nature of DNA employed, which is further elaborated.

  17. Low energy electron induced damage to plasmid DNA pQE30

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, S. V. K.; Pota, Tasneem; Peri, Dinakar; Dongre, Anushka D.; Rao, Basuthkar J. [Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005 (India)

    2012-07-28

    Low energy electrons (LEEs) are produced in copious amounts by the primary radiation used in radiation therapy. The damage caused to the DNA by these secondary electrons in the energy range 5-22 eV has been studied to understand their possible role in radiation induced damage. Electrons are irradiated on dried films of plasmid DNA (pQE30) and analysed using agarose gel electrophoresis. Single strand breaks (SSBs) induced by LEE to supercoiled plasmid DNA show resonance structures at 7, 12, and 15 eV for low doses and 6, 10, and {approx}18 eV at saturation doses. The present measurements have an overall agreement with the literature that LEEs resonantly induce SSBs in DNA. Resonant peaks in the SSBs induced by LEEs at 7, 12, and 15 eV with the lowest employed dose in the current study are somewhat different from those reported earlier by two groups. The observed differences are perhaps related to the irradiation dose, conditions and the nature of DNA employed, which is further elaborated.

  18. Low energy electron induced damage to plasmid DNA pQE30

    International Nuclear Information System (INIS)

    Low energy electrons (LEEs) are produced in copious amounts by the primary radiation used in radiation therapy. The damage caused to the DNA by these secondary electrons in the energy range 5–22 eV has been studied to understand their possible role in radiation induced damage. Electrons are irradiated on dried films of plasmid DNA (pQE30) and analysed using agarose gel electrophoresis. Single strand breaks (SSBs) induced by LEE to supercoiled plasmid DNA show resonance structures at 7, 12, and 15 eV for low doses and 6, 10, and ∼18 eV at saturation doses. The present measurements have an overall agreement with the literature that LEEs resonantly induce SSBs in DNA. Resonant peaks in the SSBs induced by LEEs at 7, 12, and 15 eV with the lowest employed dose in the current study are somewhat different from those reported earlier by two groups. The observed differences are perhaps related to the irradiation dose, conditions and the nature of DNA employed, which is further elaborated.

  19. The Circular Camera Movement

    DEFF Research Database (Denmark)

    Hansen, Lennard Højbjerg

    2014-01-01

    It has been an accepted precept in film theory that specific stylistic features do not express specific content. Nevertheless, it is possible to find many examples in the history of film in which stylistic features do express specific content: for instance, the circular camera movement is used...... circular camera movement. Keywords: embodied perception, embodied style, explicit narration, interpretation, style pattern, television style...

  20. Characterization of a small cryptic plasmid from endophytic Pantoea agglomerans and its use in the construction of an expression vector

    OpenAIRE

    Procópio Rudi Emerson de Lima; Welington Luiz Araújo; Fernando Dini Andreote; João Lúcio de Azevedo

    2011-01-01

    A circular cryptic plasmid named pPAGA (2,734 bp) was isolated from Pantoea agglomerans strain EGE6 (an endophytic bacterial isolate from eucalyptus). Sequence analysis revealed that the plasmid has a G+C content of 51% and contains four potential ORFs, 238(A), 250(B), 131(C), and 129(D) amino acids in length without homology to known proteins. The shuttle vector pLGM1 was constructed by combining the pPAGA plasmid with pGFPmut3.0 (which harbors a gene encoding green fluorescent protein, GFP)...

  1. Characterization of a novel plasmid-like element in Neurospora crassa derived mostly from the mitochondrial DNA.

    OpenAIRE

    Almasan, A.; Mishra, N. C.

    1990-01-01

    We have identified a plasmid-like element within mitochondria of Neurospora crassa strain stp-B1. It is derived from the EcoRI-4 and EcoRI-6 regions of the mitochondrial DNA, and an additional 124 bp DNA segment of unknown origin. The plasmid DNA consists of an oligomeric series of circular molecules of monomer length 2.2 kbp. The abundance of the plasmid suggests its autonomous replication and the presence of an efficient origin of replication. An unusually large number of palindromes capabl...

  2. Protocol for Evaluating the Permissiveness of Bacterial Communities Toward Conjugal Plasmids by Quantification and Isolation of Transconjugants

    DEFF Research Database (Denmark)

    Klümper, Uli; Dechesne, Arnaud; Smets, Barth F.

    2014-01-01

    a gfp-tagged plasmid in a mCherry red fluorescently tagged donor strain repressing gfp expression. We take advantage of fluorescent marker genes to microscopically detect plasmid transfer events and use subsequent high-throughput fluorescence-activated cell sorting (FACS) to isolate......The transfer of conjugal plasmids is the main bacterial process of horizontal gene transfer to potentially distantly related bacteria. These extrachromosomal, circular DNA molecules host genes that code for their own replication and transfer to other organisms. Because additional accessory genes...... may encode catabolic pathways, virulence factors, and antibiotic or metal resistances, it is of environmental, evolutionary, and medical relevance to track and monitor the fate of plasmids in mixed microbial community. When assessing the short-term and long-term implications of conjugal plasmid...

  3. Perfect Circular Dichroic Metamirrors

    CERN Document Server

    Wang, Zuojia; Liu, Yongmin

    2015-01-01

    In nature, the beetle Chrysina gloriosa derives its iridescence by selectively reflecting left-handed circularly polarized light only. Here, for the first time, we introduce and demonstrate the optical analogue based on an ultrathin metamaterial, which we term circular dichroic metamirror. A general method to design the circular dichroic metasmirror is presented under the framework of Jones calculus. It is analytically shown that the metamirror can be realized by two layers of anisotropic metamaterial structures, in order to satisfy the required simultaneous breakings of n-fold rotational (n>2) and mirror symmetries. We design an infrared metamirror, which shows perfect reflectance for left-handed circularly polarized light without reversing its handedness, while almost completely absorbs right-handed circularly polarized light. These findings offer new methodology to realize novel chiral optical devices for a variety of applications, including polarimetric imaging, molecular spectroscopy, as well as quantum ...

  4. DNA supercoiling changes and nucleoid protein composition in a group of L5178Y cells of varying radiosensitivity

    International Nuclear Information System (INIS)

    Cells of the radioresistant L5178Y-R, -S35, -SR and M10(neo 5)-l and radiosensitive L5178Y-S, M10 and LX830 cell lines were used to investigate the relationship between radiosensitivity and DNA supercoiling ability mediated by the nuclear matrix within chromatin loops containing DNA damage. The ability of DNA loops to undergo changes in supercoiling in the presence of radiation-induced damage revealed that in all cases the degree of inhibition of supercoil rewinding was greater in the radiosensitive cells. Since the amount of DNA damage induced per unit dose is known to be equal in all these cell lines, the same number of DNA lesions produced a greater loss of topological constraint in the radiosensitive cells. The differential loss of DNA supercoiling ability could be due to differences in DNA-nuclear matrix anchor points. High-resolution two-dimensional gel electrophoresis of nucleoid proteins showed numerous reproducible differences in nuclear matrix protein between the cell lines studied. A total of nine proteins were associated with nucleoids from L5178Y-R cells and absent from L5178Y-S nucleoids. None of them, however, correlated absolutely with radioresistance. Thus, unlike previous studies in CHO cells, no candidates for the conveyance of cellular radiosensitivity that were single proteins were detected. However, these results are consistent with the hypothesis that stability of DNA loop domains in the presence of DNA damage is a determinant of the outcome of radiation-induced DNA damage. 21 refs., 3 figs., 1 tab

  5. Interactions between octopine and nopaline plasmids in Agrobacterium tumefaciens.

    OpenAIRE

    Hooykaas, P J; Den Dulk-Ras, H; Ooms, G.; Schilperoort, R.A.

    1980-01-01

    Transfer of octopine Ti plasmids to strains already carrying an octopine Ti plasmid was found to occur at the same (high) frequency as transfer to Ti plasmid lacking recipients, showing that resident Ti plasmids do not exhibit entry exclusion towards incoming Ti plasmids. The resident octopine Ti plasmid was lost by the recipient after the entrance of the incoming Ti plasmid, which is indicative of the incompatibility between the Ti plasmids. Octopine Ti plasmids were found to become establis...

  6. Effects of genes exerting growth inhibition and plasmid stability on plasmid maintenance.

    OpenAIRE

    Boe, L; Gerdes, K; Molin, S

    1987-01-01

    Plasmid stabilization mediated by the parA+ and parB+ genes of the R1 plasmid and the ccd+ and sop+ genes of the F plasmid was tested on a mini-R1 plasmid and a pBR322 plasmid derivative. The mini-R1 plasmid is thought to be unstably inherited owing to a low copy number and to random segregation of the plasmid at cell division, whereas cells harboring the pBR322 derivative used in this work are lost through competition with plasmid-free cells, mainly as a result of the shorter generation time...

  7. Neutron and gamma-radiation sensitivity of plasmid DNA of varying superhelical density

    International Nuclear Information System (INIS)

    Several families of negatively supercoiled topoisomers of plasmid pIBI30 were prepared by a modification of the procedure of Singleton and Wells. The average superhelical density (σ) was determined by two-dimensional agarose gel electrophoresis and varied from -0.010 to -0.067, corresponding to a change in the number of supercoils from 3 to 19 and an effective volume change from 1.6 x 108 to 4 x 108 angstrom 3. Samples were exposed to either fission-neutron or 60Co γ radiation and assayed for single-strand breaks by agarose gel electrophoresis. Form I DNA for all topoisomers decreased exponentially with increasing dose. The D37 values for both neutron and γ radiation increased monotonically with increasing |σ|. Using a branched plectonemic (interwound) form for DNA over the range of σ studied and standard (single-hit) target theory, a quantitative linear fit to (D37-1) as a function of the effective DNA radius, S(angstrom), was obtained. The model predicts that both the slope (a) and the intercept (b) of (D37)-1 as a function of S(angstrom) are directly proportional to the length of DNA and the radiation fluence. Furthermore, the ratio b/a (= ro) at σ = 0 depends only on the ionic strength of the medium and is independent of the radiation source parameters. Our results support the model and we calculate ro = 13.4 ± 1.4 nm, a value consistent with other investigations. Our results are consistent with studies using 137Cs but disagree with data obtained for X rays. 31 refs., 6 figs., 1 tab

  8. Production of clinical-grade plasmid DNA for human Phase I clinical trials and large animal clinical studies.

    Science.gov (United States)

    Przybylowski, Mark; Bartido, Shirley; Borquez-Ojeda, Oriana; Sadelain, Michel; Rivière, Isabelle

    2007-06-28

    The use of plasmid DNA as vaccines for the treatment of cancer and infectious diseases is on the rise. In order to facilitate the manufacture of clinical-grade plasmid DNA for Phase I clinical trials, we developed a process whereby >200 mg plasmid could be produced in a single production run under Good Manufacturing Practices. A dedicated cleanroom (Class 10,000 with Class 100 biosafety cabinet) is utilized for production of the bacterial cell bank, fermentation, harvest/lysis of the biomass, and downstream purification. Fermentation requires three 16-18 h runs (approximately 12 L each) in shaker-flasks, yielding approximately 60 g bacterial paste following batch centrifugation. The biomass is alkaline-lysed, pooled, and the resulting flocculent precipitate is separated by a novel vacuum step, followed by depth-filtration. Downstream processing includes anion-exchange chromatography, utilizing Qiagen silica-based resin, and precipitation with isopropanol. Following precipitation, the DNA is harvested by centrifugation, dried, formulated, and sterile-filtered using a Sartorius Sartobran 150 filter prior to Final-Filling. All processing steps utilize sterilized, single-use components. This process results in a product manufactured according to regulatory guidelines. The plasmid DNA is sterile with >or=95% supercoiled DNA, an A260/A280 ratio>or=1.9, undetectable or extremely low residual endotoxin, RNA, genomic DNA, protein, and antibiotic. Residual solvent levels are negligible. The product yields the predicted profile upon restriction-enzyme digestion, is biologically active upon transfection and remains stable for several years at -20 degrees C. We have therefore developed a reproducible and cost effective process to manufacture clinical-grade plasmid DNA. This process can be adapted by other academic centers for human or large animal clinical trials. PMID:17537555

  9. Circular statistics in R

    CERN Document Server

    Pewsey, Arthur; Ruxton, Graeme D

    2013-01-01

    Circular Statistics in R provides the most comprehensive guide to the analysis of circular data in over a decade. Circular data arise in many scientific contexts whether it be angular directions such as: observed compass directions of departure of radio-collared migratory birds from a release point; bond angles measured in different molecules; wind directions at different times of year at a wind farm; direction of stress-fractures in concretebridge supports; longitudes of earthquake epicentres or seasonal and daily activity patterns, for example: data on the times of day at which animals are c

  10. Antiparallel plasmid-plasmid pairing may control P1 plasmid replication.

    OpenAIRE

    Abeles, A L; Austin, S J

    1991-01-01

    The copy number of the P1 plasmid replicon is stringently controlled, giving only one or two copies per newborn cell. Control is achieved by the action of the copy-control locus incA, which contains nine repeats of the 19-basepair binding site for the plasmid-encoded initiator protein RepA. A set of five similar repeats are present in the replication origin where RepA acts to trigger initiation. Using an in vitro replication system consisting of an Escherichia coli extract, the P1 origin as a...

  11. A plasmid in Legionella pneumophila.

    OpenAIRE

    Knudson, G B; Mikesell, P

    1980-01-01

    Sixteen strains from the six serogroups of Legionella pneumophila were examined for the presence of extrachromosomal genetic elements by a modified cleared lysate procedure, dye-buoyant centrifugation, and agarose gel electrophoresis. Two strains, Atlanta-1 and Atlanta-2 from serogroup II, each contained a plasmid of cryptic function with a molecular weight of ca. 30 megadaltons.

  12. Bacterial Plasmids in Antarctic Natural Microbial Assemblages

    OpenAIRE

    Kobori, Hiromi; Sullivan, Cornelius W.; Shizuya, Hiroaki

    1984-01-01

    Samples of psychrophilic and psychrotrophic bacteria were collected from sea ice, seawater, sediments, and benthic or ice-associated animals in McMurdo Sound, Antarctica. A total of 155 strains were isolated and tested for the presence of plasmids by DNA agarose gel electrophoresis. Thirty-one percent of the isolates carried at least one kind of plasmid. Bacterial isolates taken from sediments showed the highest plasmid incidence (42%), and isolates from seawater showed the lowest plasmid inc...

  13. A toxicological study of inhalable particulates in an industrial region of Lanzhou City, northwestern China: Results from plasmid scission assay

    Science.gov (United States)

    Xiao, Zhenghui; Shao, Longyi; Zhang, Ning; Wang, Jing; Chuang, Hsiao-Chi; Deng, Zhenzhen; Wang, Zhen; BéruBé, Kelly

    2014-09-01

    The city of Lanzhou in northwestern China experiences serious air pollution episodes in the form of PM10 that is characterized by having high levels of heavy metals. The Xigu District represents the industrial core area of Lanzhou City and is denoted by having the largest petrochemical bases in western China. This study investigates heavy metal compositions and oxidative potential of airborne PM10 (particulate matter with aerodynamic diameter of 10 μm or less) collected in Xigu District in the summer and winter of 2010. An in vitro plasmid scission assay (PSA) was employed to study the oxidative potential of airborne PM10 and inductively coupled plasma-mass spectrometry (ICP-MS) was used to examine heavy metal compositions. Transmission electron microscopy coupled with energy-dispersive X-ray spectrometry (TEM/EDX) was used to investigate elemental compositions and mixing states of PM10. The average mass concentrations of PM10 collected in Xigu District were generally higher than the national standard for daily PM10 (150 μg/m3). Cr, Zn, Pb and Mn were the most abundant metals in the intact whole particles of PM10. Zn, Mn and As was the most abundant metal in the water-soluble fraction, while Cr, Pb, and V existed primarily in insoluble forms. TD20 values (i.e. toxic dosage of PM10 causing 20% of plasmid DNA damage) varied considerably in both winter and summer (from 19 μg/mL to >1000 μg/mL) but were typically higher in summer, suggesting that the winter PM10 exhibited greater bioreactivity. In addition, the PM10 collected during a dust storm episode had a highest TD20 value and thus the least oxidative damage to supercoiled plasmid DNA, while the particles collected on a hazy day had a lowest TD20 value and thus the highest oxidative damage to supercoiled plasmid DNA. The particles collected on the first day after snow fall and on a day of cold air intrusion exhibited minor oxidative potential (i.e. caused limited DNA damage). The water-soluble Zn, Mn, As, and

  14. Successful transfer of plasmid DNA into in vitro cells transfected with an inorganic plasmid-Mg/Al-LDH nanobiocomposite material as a vector for gene expression

    International Nuclear Information System (INIS)

    The delivery of a full plasmid, encoding the green fluorescent protein gene into African monkey kidney (Vero3) cells, was successfully achieved using nanobiocomposites based on layered double hydroxides. This demonstrated the potential of using the system as an alternative DNA delivery vector. Intercalation of the circular plasmid DNA, pEGFP-N2, into Mg/Al-NO3- layered double hydroxides (LDH) was accomplished through anion exchange routes to form the nanobiocomposite material. The host was previously synthesized at the Mg2+ to Al3+ molar ratio Ri = 2 and subsequently intercalated with plasmid DNA. Size expansion of the interlamellae host from 8.8 A in LDH to 42 A was observed in the resulting nanobiocomposite, indicating stable hybridization of the plasmid DNA. The powder x-ray diffraction (PXRD) results, supplemented with Fourier-transform infrared (FTIR) spectroscopy, compositional and electrophoresis studies confirmed the encapsulation episode of the biomaterial. In order to elucidate the use of this resulting nanobiocomposite as a delivery vector, an MTT assay was performed to determine any cytotoxic effects of the host towards cells. The intercalated pEGFP-N2 anion was later successfully recovered through acidification with HNO3 after treatment with DNA-degrading enzymes, thus also showing the ability of the LDH host to protect the intercalated biomaterial from degradation. Cell transfection studies on Vero3 cells were then performed, where cells transfected with the nanobiocomposite exhibited fluorescence as early as 12 h post-treatment compared to naked delivery of the plasmid itself.

  15. Plasmid maintenance functions encoded on Dictyostelium discoideum nuclear plasmid Ddp1.

    OpenAIRE

    Hughes, J E; H. Kiyosawa; Welker, D L

    1994-01-01

    All of the plasmid-carried genes expressed during vegetative growth are essential for long-term maintenance of plasmid Ddp1 in the nucleus of Dictyostelium discoideum. Deletion of Ddp1 genes expressed only during development had no detectable effect on plasmid maintenance. Deletion of vegetatively expressed genes, either singly or in pairs, resulted in (i) a rapid loss of plasmid from cells grown in the absence of selection for plasmid retention, (ii) variation in the proportion of monomer to...

  16. Characterization of plasmids in Erwinia stewartii.

    OpenAIRE

    Coplin, D. L.; Rowan, R G; Chisholm, D A; Whitmoyer, R E

    1981-01-01

    Plasmids in 39 strains of Erwinia stewartii were examined by agarose gel electrophoresis. Most virulent strains had from 11 to 13 plasmids ranging in molecular mass from 2.8 to 210 megadaltons and contained plasmids of 210, 70, 49, 43, 29.5, 16.8, 8.8, and 2.8 megadaltons. Plasmids in strains SW2 and SS104 were characterized by both electron microscopy and agarose gel electrophoresis and may be useful as convenient references for sizing plasmids by electrophoresis. Specific size classes of pl...

  17. Compact waveguide circular polarizer

    Energy Technology Data Exchange (ETDEWEB)

    Tantawi, Sami G.

    2016-08-16

    A multi-port waveguide is provided having a rectangular waveguide that includes a Y-shape structure with first top arm having a first rectangular waveguide port, a second top arm with second rectangular waveguide port, and a base arm with a third rectangular waveguide port for supporting a TE.sub.10 mode and a TE.sub.20 mode, where the end of the third rectangular waveguide port includes rounded edges that are parallel to a z-axis of the waveguide, a circular waveguide having a circular waveguide port for supporting a left hand and a right hand circular polarization TE.sub.11 mode and is coupled to a base arm broad wall, and a matching feature disposed on the base arm broad wall opposite of the circular waveguide for terminating the third rectangular waveguide port, where the first rectangular waveguide port, the second rectangular waveguide port and the circular waveguide port are capable of supporting 4-modes of operation.

  18. Thermodynamics of long supercoiled molecules: insights from highly efficient Monte Carlo simulations.

    Science.gov (United States)

    Lepage, Thibaut; Képès, François; Junier, Ivan

    2015-07-01

    Supercoiled DNA polymer models for which the torsional energy depends on the total twist of molecules (Tw) are a priori well suited for thermodynamic analysis of long molecules. So far, nevertheless, the exact determination of Tw in these models has been based on a computation of the writhe of the molecules (Wr) by exploiting the conservation of the linking number, Lk=Tw+Wr, which reflects topological constraints coming from the helical nature of DNA. Because Wr is equal to the number of times the main axis of a DNA molecule winds around itself, current Monte Carlo algorithms have a quadratic time complexity, O(L(2)), with respect to the contour length (L) of the molecules. Here, we present an efficient method to compute Tw exactly, leading in principle to algorithms with a linear complexity, which in practice is O(L(1.2)). Specifically, we use a discrete wormlike chain that includes the explicit double-helix structure of DNA and where the linking number is conserved by continuously preventing the generation of twist between any two consecutive cylinders of the discretized chain. As an application, we show that long (up to 21 kbp) linear molecules stretched by mechanical forces akin to magnetic tweezers contain, in the buckling regime, multiple and branched plectonemes that often coexist with curls and helices, and whose length and number are in good agreement with experiments. By attaching the ends of the molecules to a reservoir of twists with which these can exchange helix turns, we also show how to compute the torques in these models. As an example, we report values that are in good agreement with experiments and that concern the longest molecules that have been studied so far (16 kbp). PMID:26153710

  19. Characterization of plasmids in a human clinical strain of Lactococcus garvieae.

    Directory of Open Access Journals (Sweden)

    Mónica Aguado-Urda

    Full Text Available The present work describes the molecular characterization of five circular plasmids found in the human clinical strain Lactococcus garvieae 21881. The plasmids were designated pGL1-pGL5, with molecular sizes of 4,536 bp, 4,572 bp, 12,948 bp, 14,006 bp and 68,798 bp, respectively. Based on detailed sequence analysis, some of these plasmids appear to be mosaics composed of DNA obtained by modular exchange between different species of lactic acid bacteria. Based on sequence data and the derived presence of certain genes and proteins, the plasmid pGL2 appears to replicate via a rolling-circle mechanism, while the other four plasmids appear to belong to the group of lactococcal theta-type replicons. The plasmids pGL1, pGL2 and pGL5 encode putative proteins related with bacteriocin synthesis and bacteriocin secretion and immunity. The plasmid pGL5 harbors genes (txn, orf5 and orf25 encoding proteins that could be considered putative virulence factors. The gene txn encodes a protein with an enzymatic domain corresponding to the family actin-ADP-ribosyltransferases toxins, which are known to play a key role in pathogenesis of a variety of bacterial pathogens. The genes orf5 and orf25 encode two putative surface proteins containing the cell wall-sorting motif LPXTG, with mucin-binding and collagen-binding protein domains, respectively. These proteins could be involved in the adherence of L. garvieae to mucus from the intestine, facilitating further interaction with intestinal epithelial cells and to collagenous tissues such as the collagen-rich heart valves. To our knowledge, this is the first report on the characterization of plasmids in a human clinical strain of this pathogen.

  20. Amplified human MYC oncogenes localized to replicating submicroscopic circular DNA molecules

    International Nuclear Information System (INIS)

    Amplification of genes can sometimes be detected by molecular hybridization but not by cytogenetic methods, suggesting that in some cases the units of amplification may be too small to be detected by light microscopy. The experiments reported here investigate whether submicroscopic amplification units are present in early passages of the human tumor cell lines HL-60 and COLO 320. The results show that such cells do contain submicroscopic, extrachromosomal, supercoiled circular molecules harboring MYC genes. The molecules in HL-60 are ∼250 kilobase pairs (kbp), while those in COLO 320 are 120-160 kbp. The extrachromosomal molecules in HL-60 are shown to replicate semiconservatively and approximately once in one cell cycle. The authors propose that these submicroscopic elements are precursors of double-minute chromosomes, the usual extrachromosomal manifestation of gene amplification, since both are structurally similar and replicate autonomously

  1. Direct observation of a 91 bp LacI-mediated, negatively supercoiled DNA loop by atomic force microscope.

    Science.gov (United States)

    Fulcrand, Geraldine; Chapagain, Prem; Dunlap, David; Leng, Fenfei

    2016-03-01

    Escherichia coli lactose repressor (LacI), a tetrameric protein, is a paradigmatic transcriptional factor that controls the expression of lacZYA in the lac operon. It specifically binds to the O1, O2, and O3 operators of the lac promoter, forms DNA loops, and regulates transcription of the lac operon. In this article, utilizing combined techniques of DNA-nicking assay and AFM imaging, we directly observed a 91 bp LacI-mediated, negatively supercoiled DNA loop mimicking the DNA loop between the O1 and O3 operators in the lac promoter. PMID:26878689

  2. Virulence Plasmids of Spore-Forming Bacteria.

    Science.gov (United States)

    Adams, Vicki; Li, Jihong; Wisniewski, Jessica A; Uzal, Francisco A; Moore, Robert J; McClane, Bruce A; Rood, Julian I

    2014-12-01

    Plasmid-encoded virulence factors are important in the pathogenesis of diseases caused by spore-forming bacteria. Unlike many other bacteria, the most common virulence factors encoded by plasmids in Clostridium and Bacillus species are protein toxins. Clostridium perfringens causes several histotoxic and enterotoxin diseases in both humans and animals and produces a broad range of toxins, including many pore-forming toxins such as C. perfringens enterotoxin, epsilon-toxin, beta-toxin, and NetB. Genetic studies have led to the determination of the role of these toxins in disease pathogenesis. The genes for these toxins are generally carried on large conjugative plasmids that have common core replication, maintenance, and conjugation regions. There is considerable functional information available about the unique tcp conjugation locus carried by these plasmids, but less is known about plasmid maintenance. The latter is intriguing because many C. perfringens isolates stably maintain up to four different, but closely related, toxin plasmids. Toxin genes may also be plasmid-encoded in the neurotoxic clostridia. The tetanus toxin gene is located on a plasmid in Clostridium tetani, but the botulinum toxin genes may be chromosomal, plasmid-determined, or located on bacteriophages in Clostridium botulinum. In Bacillus anthracis it is well established that virulence is plasmid determined, with anthrax toxin genes located on pXO1 and capsule genes on a separate plasmid, pXO2. Orthologs of these plasmids are also found in other members of the Bacillus cereus group such as B. cereus and Bacillus thuringiensis. In B. thuringiensis these plasmids may carry genes encoding one or more insecticidal toxins. PMID:26104459

  3. Wiimote Experiments: Circular Motion

    Science.gov (United States)

    Kouh, Minjoon; Holz, Danielle; Kawam, Alae; Lamont, Mary

    2013-01-01

    The advent of new sensor technologies can provide new ways of exploring fundamental physics. In this paper, we show how a Wiimote, which is a handheld remote controller for the Nintendo Wii video game system with an accelerometer, can be used to study the dynamics of circular motion with a very simple setup such as an old record player or a…

  4. Homemade Site Directed Mutagenesis of Whole Plasmids

    Science.gov (United States)

    Laible, Mark; Boonrod, Kajohn

    2009-01-01

    Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers. PMID:19488024

  5. Plasmid and chromosome partitioning: surprises from phylogeny

    DEFF Research Database (Denmark)

    Gerdes, Kenn; Møller-Jensen, Jakob; Bugge Jensen, Rasmus

    2000-01-01

    Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids...... and chromosomes from prokaryotic organisms. All known plasmid-encoded par loci specify three components: a cis-acting centromere-like site and two trans-acting proteins that form a nucleoprotein complex at the centromere (i.e. the partition complex). The proteins are encoded by two genes in an operon...... that is autoregulated by the par-encoded proteins. In all cases, the upstream gene encodes an ATPase that is essential for partitioning. Recent cytological analyses indicate that the ATPases function as adaptors between a host-encoded component and the partition complex and thereby tether plasmids and chromosomal...

  6. In vitro DNA replication of recombinant plasmid DNAs containing the origin of progeny replicative form DNA synthesis of phage phi X174.

    OpenAIRE

    Zipursky, S. L.; Reinberg, D.; Hurwitz, J

    1980-01-01

    The origin of phage phi X174 progeny replicative form (RF) DNA synthesis has been inserted into the plasmid vector pBR322 and cloned. In direct contrast to pBR322, the recombinant superhelical plasmids can substitute for phi X174 RFI DNA as template in phi X174-specific reactions in vitro. We have shown that the recombinant plasmids: (i) are cleaved by the phi X174 A protein; (ii) support net synthesis of unit-length single-stranded circular DNA in the presence of the phi X174 A protein and E...

  7. MEGAWHOP cloning: a method of creating random mutagenesis libraries via megaprimer PCR of whole plasmids.

    Science.gov (United States)

    Miyazaki, Kentaro

    2011-01-01

    MEGAWHOP allows for the cloning of DNA fragments into a vector and is used for conventional restriction digestion/ligation-based procedures. In MEGAWHOP, the DNA fragment to be cloned is used as a set of complementary primers that replace a homologous region in a template vector through whole-plasmid PCR. After synthesis of a nicked circular plasmid, the mixture is treated with DpnI, a dam-methylated DNA-specific restriction enzyme, to digest the template plasmid. The DpnI-treated mixture is then introduced into competent Escherichia coli cells to yield plasmids carrying replaced insert fragments. Plasmids produced by the MEGAWHOP method are virtually free of contamination by species without any inserts or with multiple inserts, and also the parent. Because the fragment is usually long enough to not interfere with hybridization to the template, various types of fragments can be used with mutations at any site (either known or unknown, random, or specific). By using fragments having homologous sequences at the ends (e.g., adaptor sequence), MEGAWHOP can also be used to recombine nonhomologous sequences mediated by the adaptors, allowing rapid creation of novel constructs and chimeric genes. PMID:21601687

  8. Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost.

    Science.gov (United States)

    Benoit, Roger M; Ostermeier, Christian; Geiser, Martin; Li, Julia Su Zhou; Widmer, Hans; Auer, Manfred

    2016-01-01

    Seamless cloning methods, such as co-transformation cloning, sequence- and ligation-independent cloning (SLIC) or the Gibson assembly, are essential tools for the precise construction of plasmids. The efficiency of co-transformation cloning is however low and the Gibson assembly reagents are expensive. With the aim to improve the robustness of seamless cloning experiments while keeping costs low, we examined the importance of complementary single-stranded DNA ends for co-transformation cloning and the influence of single-stranded gaps in circular plasmids on SLIC cloning efficiency. Most importantly, our data show that single-stranded gaps in double-stranded plasmids, which occur in typical SLIC protocols, can drastically decrease the efficiency at which the DNA transforms competent E. coli bacteria. Accordingly, filling-in of single-stranded gaps using DNA polymerase resulted in increased transformation efficiency. Ligation of the remaining nicks did not lead to a further increase in transformation efficiency. These findings demonstrate that highly efficient insert-plasmid assembly can be achieved by using only T5 exonuclease and Phusion DNA polymerase, without Taq DNA ligase from the original Gibson protocol, which significantly reduces the cost of the reactions. We successfully used this modified Gibson assembly protocol with two short insert-plasmid overlap regions, each counting only 15 nucleotides. PMID:27073895

  9. In vitro replication of mitochondrial plasmid mp1 from the higher plant Chenopodium album (L.): a remnant of bacterial rolling circle and conjugative plasmids?

    Science.gov (United States)

    Backert, S; Kunnimalaiyaan, M; Börner, T; Nielsen, B L

    1998-12-11

    According to the endosymbiotic theory, mitochondrial genomes evolved from the chromosome of an alpha-proteobacterium-like ancestor and developed during evolution an extraordinary variation in size, structure and replication. We studied in vitro DNA replication of the mitochondrial circular plasmid mp1 (1309 bp) from the higher plant Chenopodium album (L.) as a model system that replicates in a manner reminiscent of bacterial rolling circle plasmids. Several mp1 subclones were tested for their ability to support DNA replication using a newly developed in vitro system. Neutral/neutral two-dimensional gel electrophoresis of the in vitro products revealed typical simple Y patterns of intermediates consistent with a rolling circle type of replication. Replication activity was very high for a BamHI-restricted total plasmid DNA clone, a 464 bp BamHI/KpnI fragment and a 363 bp BamHI/SmaI fragment. Further subcloning of a 148 bp BamHI/EcoRI fragment resulted in the strongest in vitro DNA replication activity, while a 1161 bp-template outside of this region resulted in a substantial loss of activity. Electron microscopic studies of in vitro DNA replication products from the highly active clones also revealed sigma-shaped molecules. These results support our in vivo data for the presence of a predominant replication origin between positions 628 and 776 on the plasmid map. This sequence shares homology with double-stranded rolling circle origin (dso) or transfer origin (oriT) nicking motifs from bacterial plasmids. mp1 is the first described rolling circle plasmid in eukaryotes. PMID:9837722

  10. Characterization of Atmospheric Pressure Plasma Jet (APPJ) and Its Effect on Plasmid DNA

    Science.gov (United States)

    Adhikari, Ek; Ptasinska, Sylwia

    2015-09-01

    A helium atmospheric pressure plasma jet (APPJ) source was constructed and then characterized by monitoring a deflected current on a high voltage electrode and a potential difference between two electrodes. The deflected current was also monitored for the APPJ source with varied electrical and fed gas composition e.g. admixtures of He and water vapor. The deflected power per cycle for gas admixtures was decreased with the increase in fraction of water vapor. In addition, this APPJ source was used to induce damage to aqueous plasmid DNA. The fraction of supercoiled, single-strand breaks and double-strand breaks in DNA were quantified by using agarose gel electrophoresis. The number of DNA strand breaks increased as a function of plasma irradiation time and decrease as a distance between APPJ and DNA sample increased. The APPJ with the gas admixture, in which the fraction of water vapor was varied, was also used to induce damage to aqueous DNA samples. The damage level decreased with the increase in a fraction of water vapor under specific experimental conditions. The change in numbers of DNA strand breaks irradiated by a pure He plasma and a plasma with a gas admixture is predicted by different physical and chemical process in the APPJ. This material is based upon work supported by the U.S. Department of Energy Office of Science, Office of Basic Energy Sciences under Award Number DE-FC02-04ER15533.

  11. pLS010 plasmid vector

    Science.gov (United States)

    Lacks, Sanford A.; Balganesh, Tanjore S.

    1988-01-01

    Disclosed is recombinant plasmid pLS101, consisting essentially of a 2.0 Kb malM gene fragment ligated to a 4.4 Kb T.sub.c r DNA fragment, which is particularly useful for transforming Gram-positive bacteria. This plasmid contains at least four restriction sites suitable for inserting exogeneous gene sequences. Also disclosed is a method for plasmid isolation by penicillin selection, as well as processes for enrichment of recombinant plasmids in Gram-positive bacterial systems.

  12. Chromate resistance plasmid in Pseudomonas fluorescens.

    OpenAIRE

    Bopp, L H; Chakrabarty, A M; Ehrlich, H. L.

    1983-01-01

    Chromate resistance of Pseudomonas fluorescens LB300, isolated from chromium-contaminated sediment in the upper Hudson River, was found to be plasmid specified. Loss of the plasmid (pLHB1) by spontaneous segregation or mitomycin C curing resulted in a simultaneous loss of chromate resistance. Subsequent transformation of such strains with purified pLHB1 plasmid DNA resulted in a simultaneous re-acquisition of the chromate resistance phenotype and the plasmid. When pLHB1 was transferred by con...

  13. ANALYSIS OF A MODEL OF PLASMID-BEARING, PLASMID-FREE COMPETITION IN A PULSED CHEMOSTAT

    OpenAIRE

    XIANGYUN SHI; XINYU SONG; XUEYONG ZHOU

    2006-01-01

    We introduce and study a chemostat model with plasmid-bearing, plasmid-free competition and impulsive effect. According to the stability analysis of the boundary periodic solution, we obtain the invasion threshold of the plasmid-free organism and plasmid-bearing organism. Furthermore, by using standard techniques of bifurcation theory, we prove the system has a positive τ-periodic solution, which shows that the impulsive effect destroys the equilibria of the unforced continuous system and ini...

  14. Plasmid ColVBtrp maintenance in Erwinia carotovora.

    OpenAIRE

    Schukin, N N

    1981-01-01

    Plasmid ColVBtrp maintenance in Erwinia carotovora cells was followed by measuring kinetics of elimination of plasmid genetic markers and loss of plasmid deoxyribonucleic acid. An E. carotovora mutant stably carrying plasmid ColVBtrp was isolated. Besides stable plasmid maintenance, the mutant showed altered sensitivity to male-specific phage MS2, sensitivity to drugs, and colony morphology.

  15. Circularity-Measuring System

    Science.gov (United States)

    WHIPPO. WALTER B.; Rohrkaste, G. R.; Miller, John E.

    1989-01-01

    Shape gauge and associated computer constitute system measuring deviations of large cylinders from roundness. Shaped and held somewhat like crossbow, measures relative locations of three points on surface of large, round object. By making connected series of measurements around periphery technician using gauge determines deviation of object from perfect circularity. Used to measure straightness, roundness, or complicated shapes of such large geometrical objects as surfaces of aircraft and hulls of ships.

  16. The expression of a plasmid-specified exported protein causes structural plasmid instability in Bacillus subtilis

    NARCIS (Netherlands)

    Cordes, C.; Meima, R; Twiest, B; Kazemier, B; Venema, G; vanDijl, JM; Bron, S

    1996-01-01

    The rolling-circle plasmid pGP1 was used to study the effects of the expression of a plasmid-specified exported protein on structural plasmid stability in Bacillus subtilis. pGP1 contains a fusion between the Bacillus licheniformis penP gene, encoding a C-terminally truncated penicillinase, and the

  17. Circular arc structures

    KAUST Repository

    Bo, Pengbo

    2011-07-01

    The most important guiding principle in computational methods for freeform architecture is the balance between cost efficiency on the one hand, and adherence to the design intent on the other. Key issues are the simplicity of supporting and connecting elements as well as repetition of costly parts. This paper proposes so-called circular arc structures as a means to faithfully realize freeform designs without giving up smooth appearance. In contrast to non-smooth meshes with straight edges where geometric complexity is concentrated in the nodes, we stay with smooth surfaces and rather distribute complexity in a uniform way by allowing edges in the shape of circular arcs. We are able to achieve the simplest possible shape of nodes without interfering with known panel optimization algorithms. We study remarkable special cases of circular arc structures which possess simple supporting elements or repetitive edges, we present the first global approximation method for principal patches, and we show an extension to volumetric structures for truly threedimensional designs. © 2011 ACM.

  18. Switchable circular beam deflectors

    Science.gov (United States)

    Shang, Xiaobing; Joshi, Pankaj; Tan, Jin-Yi; De Smet, Jelle; Cuypers, Dieter; Baghdasaryan, Tigran; Vervaeke, Michael; Thienpont, Hugo; De Smet, Herbert

    2016-04-01

    In this work, we report two types of electrically tunable photonic devices with circularly symmetric polarization independent beam steering performance (beam condensing resp. beam broadening). The devices consist of circular micro grating structures combined with nematic liquid crystal (LC) layers with anti-parallel alignment. A single beam deflector converts a polarized and monochromatic green laser beam (λ =543.5 nm) into a diffraction pattern, with the peak intensity appearing at the third order when 0~{{V}\\text{pp}} is applied and at the zeroth order (no deflection) for voltages above 30~{{V}\\text{pp}} . Depending on the shape of the grating structure (non-inverted or inverted), the deflection is inwards or outwards. Both grating types can be made starting from the same diamond-tooled master mold. A polarized white light beam is symmetrically condensed resp. broadened over 2° in the off state and is passed through unchanged in the on state. By stacking two such devices with mutually orthogonal LC alignment layers, polarization independent switchable circular beam deflectors are realized with a high transmittance (>80%), and with the same beam steering performance as the polarization dependent single devices.

  19. Recollision with circular polarization

    Science.gov (United States)

    Mauger, Francois; Kamor, Adam; Bandrauk, Andre; Chandre, Cristel; Uzer, Turgay

    2013-05-01

    Since its identification in the 90s, the recollision scenario has revealed to be very helpful in explaining many phenomena in atomic and molecular systems subjected to strong and short laser pulses, and it is now at the core of the strong field physics and attosecond science. For linearly polarized laser fields, the recollision scenario has been able to explain nonsequential double ionization (NSDI), high harmonic generation (HHG) and laser induced diffraction (LIED), just to cite them. The same scenario also predicts the absence of recollision when the field is circularly polarized, therefore leading to the absence of NSDI, HHG or LIED. Recently, the influence of the ellipticity of the laser has drawn an increasing level of interest in the strong field community as it is seen as a way to control the electronic dynamics and, for instance, HHG. Using classical models, the common belief of the absence of recollision with circularly polarized laser fields has been proven wrong. In my talk I will present classical and quantum evidence of the presence of recollision with circular polarization. I will discuss the conditions under which such recollisions happen and what they imply.

  20. Isolation and characterization of two novel plasmids from pathogenic Leptospira interrogans serogroup Canicola Serovar Canicola strain Gui44.

    Directory of Open Access Journals (Sweden)

    Wei-Nan Zhu

    2014-08-01

    Full Text Available Previous genomic analysis of pathogenic Leptospira has identified two circular chromosomes but no plasmid. This study aims to investigate potential extrachromosomal elements of L.interrogans serovar Canicola strain Gui44.Two novel plasmids, pGui1 and pGui2, were isolated from the pathogenic strain Gui44, using a modified alkaline lysis method. Southern blotting was performed to determine the presence and size of them. Then, 454 and Hiseq sequencing were applied to obtain and analyze the complete sequences of the two plasmids. Furthermore, real-time quantitative PCR and next-generation sequencing were used to compare relative copy numbers of the two plasmids with that of the chromosomes. Finally, after serial passages in vitro for more than 2 years, the strain Gui44 was subsequently re-sequenced to estimate stability of the two plasmids.The larger plasmid, pGui1, 74,981 base pairs (bp in length with GC content of 34.63%, possesses 62 open reading frames (ORFs. The smaller plasmid, pGui2, is 66,851 bp in length with GC content of 33.33%, and contains 63 ORFs. The replication initiation proteins encoded by pGui1 and pGui2 demonstrate significant sequence similarity with LA1839 (86% and 88%, a well-known replication protein in another pathogenic L.interrogans serovar Lai strain Lai, suggesting the ability for autonomous plasmid replication. Quantitative PCR and next-generation sequencing confirms a single copy of both plasmids and their stable presence in the strain Gui44 with in vitro serial passages after more than 2 years. Interestingly, the two plasmids both contain a significant number of novel genes (35 in pGui1 and 52 in pGui2.This report confirms the presence of two separate circular plasmids in serovar Canicola strain Gui44 and provides a new understanding of genomic organization, adaptation, evolution and pathogenesis of Leptospira, which will aid in the development of in vivo genetic manipulation systems in pathogenic Leptospira species.

  1. Isolation of Plasmid DNA from Butyrivibrio fibrisolvens†

    OpenAIRE

    Teather, Ronald M.

    1982-01-01

    A procedure based on successive precipitation of cell lysates with sodium dodecyl sulfate-NaCl and polyethylene glycol 6000 was developed which allows the isolation of plasmid DNA from Butyrivibrio fibrisolvens. A survey of B. fibrisolvens strains isolated from the bovine rumen showed that plasmids are a common feature of this species.

  2. Plasmid deoxyribonucleic acid replication in Streptomyces griseus.

    OpenAIRE

    Xue, Y.; Zhuang, Z.; Zhu, Y.; Xu, Y.; Dong, K.

    1981-01-01

    A series of electron micrographs showing the presence of different molecular forms representing various replication stages of plasmid deoxyribonucleic acid from Streptomyces griseus was obtained. Based upon an analysis of these electron micrographs, a tentative model for plasmid deoxyribonucleic acid replication in S. griseus is proposed.

  3. n-Nucleotide circular codes in graph theory.

    Science.gov (United States)

    Fimmel, Elena; Michel, Christian J; Strüngmann, Lutz

    2016-03-13

    The circular code theory proposes that genes are constituted of two trinucleotide codes: the classical genetic code with 61 trinucleotides for coding the 20 amino acids (except the three stop codons {TAA,TAG,TGA}) and a circular code based on 20 trinucleotides for retrieving, maintaining and synchronizing the reading frame. It relies on two main results: the identification of a maximal C(3) self-complementary trinucleotide circular code X in genes of bacteria, eukaryotes, plasmids and viruses (Michel 2015 J. Theor. Biol. 380, 156-177. (doi:10.1016/j.jtbi.2015.04.009); Arquès & Michel 1996 J. Theor. Biol. 182, 45-58. (doi:10.1006/jtbi.1996.0142)) and the finding of X circular code motifs in tRNAs and rRNAs, in particular in the ribosome decoding centre (Michel 2012 Comput. Biol. Chem. 37, 24-37. (doi:10.1016/j.compbiolchem.2011.10.002); El Soufi & Michel 2014 Comput. Biol. Chem. 52, 9-17. (doi:10.1016/j.compbiolchem.2014.08.001)). The univerally conserved nucleotides A1492 and A1493 and the conserved nucleotide G530 are included in X circular code motifs. Recently, dinucleotide circular codes were also investigated (Michel & Pirillo 2013 ISRN Biomath. 2013, 538631. (doi:10.1155/2013/538631); Fimmel et al. 2015 J. Theor. Biol. 386, 159-165. (doi:10.1016/j.jtbi.2015.08.034)). As the genetic motifs of different lengths are ubiquitous in genes and genomes, we introduce a new approach based on graph theory to study in full generality n-nucleotide circular codes X, i.e. of length 2 (dinucleotide), 3 (trinucleotide), 4 (tetranucleotide), etc. Indeed, we prove that an n-nucleotide code X is circular if and only if the corresponding graph [Formula: see text] is acyclic. Moreover, the maximal length of a path in [Formula: see text] corresponds to the window of nucleotides in a sequence for detecting the correct reading frame. Finally, the graph theory of tournaments is applied to the study of dinucleotide circular codes. It has full equivalence between the combinatorics

  4. Plasmid-mediated horizontal gene transfer is a coevolutionary process

    OpenAIRE

    Harrison, Ellie; Brockhurst, Michael A

    2012-01-01

    Conjugative plasmids are key agents of horizontal gene transfer (HGT) that accelerate bacterial adaptation by vectoring ecologically important traits between strains and species. However, although many conjugative plasmids carry beneficial traits, all plasmids exert physiological costs-of-carriage on bacteria. The existence of conjugative plasmids, therefore, presents a paradox because non-beneficial plasmids should be lost to purifying selection, whereas beneficial genes carried on plasmids ...

  5. Caulobacter chromosome in vivo configuration matches model predictions for a supercoiled polymer in a cell-like confinement

    DEFF Research Database (Denmark)

    Hong, Sun-Hae; Toro, Esteban; Mortensen, Kim; de la Rosa, Mario A. Díaz; Doniach, Sebastian; Shapiro, Lucy; Spakowitz, Andrew J.; McAdams, Harley H.

    2013-01-01

    the contour length, and cell-to-cell distribution of the interloci distance r is a universal function of r/n0.22 with broad cell-to-cell variability. For DNA segments greater than about 300 kb, the mean interloci distances scale as n, in agreement with previous observations. The 0.22 value of the......We measured the distance between fluorescent-labeled DNA loci of various interloci contour lengths in Caulobacter crescentus swarmer cells to determine the in vivo configuration of the chromosome. For DNA segments less than about 300 kb, the mean interloci distances, 〈r〉, scale as n0.22, where n is...... scaling exponent for short DNA segments is consistent with theoretical predictions for a branched DNA polymer structure. Predictions from Brownian dynamics simulations of the packing of supercoiled DNA polymers in an elongated cell-like confinement are also consistent with a branched DNA structure, and...

  6. Potent catenation of supercoiled and gapped DNA circles by topoisomerase I in the presence of a hydrophilic polymer.

    Science.gov (United States)

    Low, R L; Kaguni, J M; Kornberg, A

    1984-04-10

    An exceptionally potent DNA catenation activity, identified in an extract from Escherichia coli, has been purified and partially characterized. Catenation results from the sequential action of the following two polypeptides: beta, 34 kDa and identical to exonuclease III; and alpha, 101 kDa and identical to DNA topoisomerase I (omega protein). An additional requirement is that a small proportion of the circles be nicked in order to provide the substrate for exonuclease III to generate gaps, estimated to be about 100 nucleotides long. Following exonuclease III digestion, one molecule of topoisomerase I can interlock per minute at 30 degrees C about 20 supercoiled and gapped DNA circles into a massively catenated network. The reaction requires Mg2+ and a hydrophilic polymer (polyvinyl alcohol or polyethylene glycol) at about 7%, but neither ATP nor spermidine. The hydrophilic polymer appears to drive catenation by condensing the DNA; decatenation by topoisomerase I proceeds upon removal of the polymer. PMID:6323479

  7. Circularly polarized antennas

    CERN Document Server

    Gao, Steven; Zhu, Fuguo

    2013-01-01

    This book presents a comprehensive insight into the design techniques for different types of CP antenna elements and arrays In this book, the authors address a broad range of topics on circularly polarized (CP) antennas. Firstly, it introduces to the reader basic principles, design techniques and characteristics of various types of CP antennas, such as CP patch antennas, CP helix antennas, quadrifilar helix antennas (QHA), printed quadrifilar helix antennas (PQHA), spiral antenna, CP slot antennas, CP dielectric resonator antennas, loop antennas, crossed dipoles, monopoles and CP horns. Adva

  8. Antennas on circular cylinders

    DEFF Research Database (Denmark)

    Knudsen, H. L.

    1959-01-01

    antenna in a circular cylinder. By a procedure similar to the one used by Silver and Saunders, expressions have been derived for the field radiated from an arbitrary surface current distribution on a cylinder surface coaxial with a perfectly conducting cylinder. The cases where the space between the two...... cylindrical surfaces have the sane characteristic constants and different constants are treated separately. Extensive numerical computations of the field radiated from the slot antennas described here are being carried out, but no numerical results are yet available...

  9. Bifurcation Analysis of a Chemostat Model of Plasmid-Bearing and Plasmid-Free Competition with Pulsed Input

    OpenAIRE

    Zhong Zhao; Baozhen Wang; Liuyong Pang; Ying Chen

    2014-01-01

    A chemostat model of plasmid-bearing and plasmid-free competition with pulsed input is proposed. The invasion threshold of the plasmid-bearing and plasmid-free organisms is obtained according to the stability of the boundary periodic solution. By use of standard techniques of bifurcation theory, the periodic oscillations in substrate, plasmid-bearing, and plasmid-free organisms are shown when some conditions are satisfied. Our results can be applied to control bioreactor aimed at producing co...

  10. Pi, Archimedes and circular splines

    OpenAIRE

    Sablonnière, Paul

    2013-01-01

    In the present paper, we give approximate values of $\\pi$ deduced from the areas of inscribed and circumscribed quadratic and cubic circular splines. Similar results on circular splines of higher degrees and higher approximation orders can be obtained in the same way. We compare these values to those obtained by computing the {\\em perimeters} of those circular splines. We observe that the former are much easier to compute than the latter and give results of the same order. It also appears tha...

  11. Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli.

    Science.gov (United States)

    Godiska, Ronald; Mead, David; Dhodda, Vinay; Wu, Chengcang; Hochstein, Rebecca; Karsi, Attila; Usdin, Karen; Entezam, Ali; Ravin, Nikolai

    2010-04-01

    Despite recent advances in sequencing, complete finishing of large genomes and analysis of novel proteins they encode typically require cloning of specific regions. However, many of these fragments are extremely difficult to clone in current vectors. Superhelical stress in circular plasmids can generate secondary structures that are substrates for deletion, particularly in regions that contain numerous tandem or inverted repeats. Common vectors also induce transcription and translation of inserted fragments, which can select against recombinant clones containing open reading frames or repetitive DNA. Conversely, transcription from cloned promoters can interfere with plasmid stability. We have therefore developed a novel Escherichia coli cloning vector (termed 'pJAZZ' vector) that is maintained as a linear plasmid. Further, it contains transcriptional terminators on both sides of the cloning site to minimize transcriptional interference between vector and insert. We show that this vector stably maintains a variety of inserts that were unclonable in conventional plasmids. These targets include short nucleotide repeats, such as those of the expanded Fragile X locus, and large AT-rich inserts, such as 20-kb segments of genomic DNA from Pneumocystis, Plasmodium, Oxytricha or Tetrahymena. The pJAZZ vector shows decreased size bias in cloning, allowing more uniform representation of larger fragments in libraries. PMID:20040575

  12. Circular PVDF Airborne Transducer

    Institute of Scientific and Technical Information of China (English)

    JIAO Li-hua; XU Li-mei; HONG Hu

    2007-01-01

    With the required increased audio pressure of the parametric ultrasonic transducer array and the difficulty to theoretically analyse the complex ultrasonic structure in audio beam application, an computafionally efficient model is desired to describe the characteristic of the parametric ultrasonic transducer array for the system design and optimization. By applying the symmetry boundary conditions at the mid-plane in the thickness direction, a finite element model based on the half thickness simplification is presented to analyze the parametric circular transducer which is designed by gluing the poly Vinylidene fluoride film (PVDF). The validity of the proposed model is confirmed by a comparison of finite element aalysis results with the theoretical value and experimental data, which show that they are making a good agreement with each other.

  13. Operational circular No. 1 (Rev. 1) – Operational circulars

    CERN Multimedia

    HR Department

    2011-01-01

    Operational Circular No. 1 (Rev. 1) is applicable to members of the personnel and other persons concerned. Operational Circular No. 1 (Rev. 1) entitled "Operational circulars", approved following discussion at the Standing Concertation Committee meeting on 4 May 2011, is available on the intranet site of the Human Resources Department: https://hr-docs.web.cern.ch/hr-docs/opcirc/opcirc.asp It cancels and replaces Operational Circular No. 1 entitled "Operational Circulars” of December 1996. This new version clarifies, in particular, that operational circulars do not necessarily arise from the Staff Rules and Regulations, and the functional titles have been updated to bring them into line with the current CERN organigram. Department Head Office  

  14. Plasmids as Tools for Containment.

    Science.gov (United States)

    García, José L; Díaz, Eduardo

    2014-10-01

    Active containment systems are a major tool for reducing the uncertainty associated with the introduction of monocultures, genetically engineered or not, into target habitats for a large number of biotechnological applications (e.g., bioremediation, bioleaching, biopesticides, biofuels, biotransformations, live vaccines, etc.). While biological containment reduces the survival of the introduced organism outside the target habitat and/or upon completion of the projected task, gene containment strategies reduce the lateral spread of the key genetic determinants to indigenous microorganisms. In fundamental research, suicide circuits become relevant tools to address the role of gene transfer, mainly plasmid transfer, in evolution and how this transfer contributes to genome plasticity and to the rapid adaptation of microbial communities to environmental changes. Many lethal functions and regulatory circuits have been used and combined to design efficient containment systems. As many new genomes are being sequenced, novel lethal genes and regulatory elements are available, e.g., new toxin-antitoxin modules, and they could be used to increase further the current containment efficiencies and to expand containment to other organisms. Although the current containment systems can increase the predictability of genetically modified organisms in the environment, containment will never be absolute, due to the existence of mutations that lead to the appearance of surviving subpopulations. In this sense, orthogonal systems (xenobiology) appear to be the solution for setting a functional genetic firewall that will allow absolute containment of recombinant organisms. PMID:26104372

  15. Plasmid DNA fermentation strategies: influence on plasmid stability and cell physiology.

    Science.gov (United States)

    Silva, Filomena; Queiroz, João A; Domingues, Fernanda C

    2012-03-01

    In order to provide sufficient pharmaceutical-grade plasmid DNA material, it is essential to gain a comprehensive knowledge of the bioprocesses involved; so, the development of protocols and techniques that allow a fast monitoring of process performance is a valuable tool for bioprocess design. Regarding plasmid DNA production, the metabolic stress of the host strain as well as plasmid stability have been identified as two of the key parameters that greatly influence plasmid DNA yields. The present work describes the impact of batch and fed-batch fermentations using different C/N ratios and different feeding profiles on cell physiology and plasmid stability, investigating the potential of these two monitoring techniques as valuable tools for bioprocess development and design. The results obtained in batch fermentations showed that plasmid copy number values suffered a pronounced increase at the end of almost all fermentation conditions tested. Regarding fed-batch fermentations, the strategies with exponential feeding profiles, in contrast with those with constant feeding, showed higher biomass and plasmid yields, the maximum values obtained for these two parameters being 95.64 OD(600) and 344.3 mg plasmid DNA (pDNA)/L, respectively, when using an exponential feed rate of 0.2 h(-1). Despite the results obtained, cell physiology and plasmid stability monitoring revealed that, although higher pDNA overall yields were obtained, this fermentation exhibited lower plasmid stability and percentage of viable cells. In conclusion, this study allowed clarifying the bioprocess performance based on cell physiology and plasmid stability assessment, allowing improvement of the overall process and not only plasmid DNA yield and cell growth. PMID:22089386

  16. Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration

    OpenAIRE

    Bertoni, Carmen; Jarrahian, Sohail; Wheeler, Thurman M.; LI, YINING; Olivares, Eric C.; Michele P Calos; Rando, Thomas A.

    2005-01-01

    Plasmid-mediated gene therapy can restore dystrophin expression in skeletal muscle in the mdx mouse, a model of Duchenne muscular dystrophy. However, sufficient long-term expression and distribution of dystrophin remain a hurdle for translating this technology into a viable treatment for Duchenne muscular dystrophy. To improve plasmid-mediated gene therapy for muscle diseases, we studied the effects of targeted plasmid integration using a phage integrase (φC31) that can mediate the integratio...

  17. X-ray photoelectron spectroscopic investigations of modifications in plasmid DNA after interaction with Hg nanoparticles

    International Nuclear Information System (INIS)

    The electronic structure of plasmid DNA both prior to and after its conjugation with Hg nanoparticles (NP) has been investigated here. The DNA molecules undergo severe perturbation on their interaction with Hg-NP. The circular plasmid DNA becomes predominantly linearised on interaction with 10 mM Hg salt solution. These perturbations are also reflected in the electronic structure of C1s and O1s suggesting severe modifications in the exocyclic groups, of the nucleic acid bases, participating in the hydrogen bondings. The results reflect the rupturing of the purine-pyrimidine bonds after interaction with the NP. The study also suggests selective and specific interactions of DNA bases with the Hg nanoparticles leading to the formation of metal-purine-pyrimidine complexes through rupturing of G-C base pairs. Hg-NP exclusively interact with the nitrogen bases of the DNA molecule and do not cause any significant modification to the phosphate backbone of DNA.

  18. Turbo cloning: a fast, efficient method for cloning PCR products and other blunt-ended DNA fragments into plasmids.

    OpenAIRE

    Boyd, A C

    1993-01-01

    The method uses a novel plasmid vector, p9lox5, containing a site-specific recombination sequence lox from the lox/Cre recombinase system of bacteriophage P1. There are two distinct stages. Firstly, vector and fragment DNAs are ligated intermolecularly under conditions of macromolecular crowding (15% polyethylene glycol 6000) which accelerate blunt-end joining a thousandfold. Secondly, circular recombinant molecules are efficiently excised from the ligation products by Cre recombinase acting ...

  19. Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification

    OpenAIRE

    Dean, Frank B.; Nelson, John R.; Giesler, Theresa L.; Lasken, Roger S.

    2001-01-01

    We describe a simple method of using rolling circle amplification to amplify vector DNA such as M13 or plasmid DNA from single colonies or plaques. Using random primers and φ29 DNA polymerase, circular DNA templates can be amplified 10,000-fold in a few hours. This procedure removes the need for lengthy growth periods and traditional DNA isolation methods. Reaction products can be used directly for DNA sequencing after phosphatase treatment to inactivate unincorporated nucleotides. Amplified ...

  20. Expression and Immunological Analysis of the Plasmid-Borne mlp Genes of Borrelia burgdorferi Strain B31

    OpenAIRE

    Porcella, Stephen F.; Fitzpatrick, Cecily A.; Bono, James L.

    2000-01-01

    A lipoprotein gene family first identified in Borrelia burgdorferi strain 297, designated 2.9 LP and recently renamed mlp, was found on circular and linear plasmids in the genome sequence of B. burgdorferi strain B31-M1. Sequence analyses of the B31 mlp genes and physically linked variant gene families indicated that mlp gene heterogeneity is unique and unrelated to location or linkage to divergent sequences. Evidence of recombination between B31 mlp alleles was also detected. Northern blot a...

  1. Elimination of multicopy plasmid R6K by bleomycin.

    OpenAIRE

    Attfield, P V; Pinney, R. J.

    1985-01-01

    Bleomycin eliminated multicopy plasmid R6K from growing cells of Escherichia coli AB1157 but failed to cure either of the low-copy plasmids R1 or R46. Measurements of R6K-encoded beta-lactamase and of covalently closed plasmid DNA indicated that the drug causes a progressive reduction in plasmid copy number.

  2. Behavior of IncQ Plasmids in Agrobacterium tumefaciens

    NARCIS (Netherlands)

    Hille, Jacques; Schilperoort, Rob

    1981-01-01

    Inc-Q plasmids were introduced into Agrobacterium tumefuciens, by mobilization from Escherichia coli with an Inc-P plasmid, or by transformation with purified plasmid DNA. It was found that they were stably maintained. The presence of an Inc-Q plasmid did not influence tumorigenicity. These results

  3. Curing of plasmid pXO1 from Bacillus anthracis using plasmid incompatibility.

    Directory of Open Access Journals (Sweden)

    Xiankai Liu

    Full Text Available The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.

  4. Positive epistasis between co-infecting plasmids promotes plasmid survival in bacterial populations

    OpenAIRE

    San Millan, Alvaro; Heilbron, Karl; MacLean, R. Craig

    2014-01-01

    Plasmids have a key role in the horizontal transfer of genes among bacteria. Although plasmids are catalysts for bacterial evolution, it is challenging to understand how they can persist in bacterial populations over the long term because of the burden they impose on their hosts (the ‘plasmid paradox'). This paradox is especially perplexing in the case of ‘small' plasmids, which are unable to self-transfer by conjugation. Here, for the first time, we investigate how interactions between co-in...

  5. Mini-F plasmid genes that couple host cell division to plasmid proliferation.

    OpenAIRE

    Ogura, T; Hiraga, S

    1983-01-01

    A mechanism for stable maintenance of plasmids, besides the replication and partition mechanisms, has been found to be specified by genes of a mini-F plasmid. An oriC plasmid carrying both a mini-F segment necessary for partition [coordinates 46.4-49.4 kilobase pairs (kb) on the F map] and another segment (42.9-43.6 kb), designated ccd (coupled cell division), is more stably maintained than are oriC plasmids carrying only the partition segment; the stability is comparable to that of the paren...

  6. Adaptive Plasmid Evolution Results in Host-Range Expansion of a Broad-Host-Range Plasmid

    OpenAIRE

    De Gelder, Leen; Williams, Julia J.; Ponciano, José M; Sota, Masahiro; Eva M. Top

    2008-01-01

    Little is known about the range of hosts in which broad-host-range (BHR) plasmids can persist in the absence of selection for plasmid-encoded traits, and whether this “long-term host range” can evolve over time. Previously, the BHR multidrug resistance plasmid pB10 was shown to be highly unstable in Stenotrophomonas maltophilia P21 and Pseudomonas putida H2. To investigate whether this plasmid can adapt to such unfavorable hosts, we performed evolution experiments wherein pB10 was maintained ...

  7. Characterization of a Haemophilus ducreyi mobilizing plasmid.

    OpenAIRE

    McNicol, P J; Albritton, W L; Ronald, A R

    1986-01-01

    The OriV site of Haemophilus ducreyi mobilizing plasmid pHD147, determined by replication in Escherichia coli polA, is located close to the OriT site. The OriT site, located by recombination-proficient and -deficient cells, and the OriV site map in a region of pHD147 homologous to the beta-lactamase-specifying plasmids of H. ducreyi and Neisseria gonorrhoeae.

  8. Plasmid and chromosome segregation in prokaryotes

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Bugge Jensen, Rasmus; Gerdes, Kenn

    2000-01-01

    Recent major advances in the understanding of prokaryotic DNA segregation have been achieved by using fluorescence microscopy to visualize the localization of cellular components. Plasmids and bacterial chromosomes are partitioned in a highly dynamic fashion, suggesting the presence of a mitotic......-like apparatus in prokaryotes. The identification of chromosomal homologues of the well-characterized plasmid partitioning genes indicates that there could be a general mechanism of bacterial DNA partitioning. Udgivelsesdato: July 1...

  9. Implementing circularity using partial evaluation

    DEFF Research Database (Denmark)

    Lawall, Julia Laetitia

    2001-01-01

    Complex data dependencies can often be expressed concisely by defining a variable in terms of part of its own value. Such a circular reference can be naturally expressed in a lazy functional language or in an attribute grammar. In this paper, we consider circular references in the context of an...

  10. Plasmid Evolution and Interaction between the Plasmid Addiction Stability Systems of Two Related Broad-Host-Range IncQ-Like Plasmids

    OpenAIRE

    Deane, Shelly M.; Rawlings, Douglas E

    2004-01-01

    Plasmid pTC-F14 contains a plasmid stability system called pas (plasmid addiction system), which consists of two proteins, a PasA antitoxin and a PasB toxin. This system is closely related to the pas of plasmid pTF-FC2 (81 and 72% amino acid identity for PasA and PasB, respectively) except that the pas of pTF-FC2 contains a third protein, PasC. As both pTC-F14 and pTF-FC2 are highly promiscuous broad-host-range plasmids isolated from bacteria that share a similar ecological niche, the plasmid...

  11. Circular chemiresistors for microchemical sensors

    Science.gov (United States)

    Ho, Clifford K.

    2007-03-13

    A circular chemiresistor for use in microchemical sensors. A pair of electrodes is fabricated on an electrically insulating substrate. The pattern of electrodes is arranged in a circle-filling geometry, such as a concentric, dual-track spiral design, or a circular interdigitated design. A drop of a chemically sensitive polymer (i.e., chemiresistive ink) is deposited on the insulating substrate on the electrodes, which spreads out into a thin, circular disk contacting the pair of electrodes. This circularly-shaped electrode geometry maximizes the contact area between the pair of electrodes and the polymer deposit, which provides a lower and more stable baseline resistance than with linear-trace designs. The circularly-shaped electrode pattern also serves to minimize batch-to-batch variations in the baseline resistance due to non-uniform distributions of conductive particles in the chemiresistive polymer film.

  12. Gonadotropin:induced modification of the effect of low-level gamma radiation on DNA supercoiling in sperm of mud loach (Misgurnus fossilis L.)

    International Nuclear Information System (INIS)

    The method of titration of nucleoids with ethidium bromide was used to study the effect of choriogonadotropin on supercoiling of DNA from mud loach sperm. The analysis of the dose-effect curves, obtained after γ-irradiation of DNA in situ, showed that pretreatment with the hormone of the whole organism and an isolated gonad, but not spermia (washed from seminal fluid), modified the primary radiation effect at the level of DNA superhelices

  13. Relationship between plasmid content and auxotype in Neisseria gonorrhoeae isolates.

    OpenAIRE

    Dillon, J R; Pauzé, M

    1981-01-01

    One hundred and forty strains of Neisseria gonorrhoeae, representing 12 different auxotype groups, were examined for differences in plasmid content. Most auxotype groups harbored a phenotypically cryptic 2,6-megadalton plasmid; a few groups also carried a 24.5-megadalton plasmid which has been previously characterized as a transfer plasmid. However, isolates of the proline-, citrulline-, and uracil-requiring (PCU-) auxotype were consistently free of plasmids. The correlation between auxotype ...

  14. Plasmid stability and maintenance of copy number using natural marker

    OpenAIRE

    Hamzah Basil Mohammed; Sudhakar Malla

    2015-01-01

    Present study was conducted to study the plasmid stability with the help of natural plasmid isolated from the bacteria which lodges the ink gland of the sea squid and emits bioluminescence. Isolated bacterial strain was identified by using 16srRNA sequencing and its plasmid DNA was used for the experimental studies. The plasmid is found to be responsible for the bioluminescence. The stability of this plasmid was studied in shake flask method using the different sugar sources (Gluc...

  15. Plasmids spread very fast in heterogeneous bacterial communities.

    OpenAIRE

    Dionisio, Francisco; Matic, Ivan; Radman, Miroslav; Rodrigues, Olivia R; Taddei, François

    2002-01-01

    Conjugative plasmids can mediate gene transfer between bacterial taxa in diverse environments. The ability to donate the F-type conjugative plasmid R1 greatly varies among enteric bacteria due to the interaction of the system that represses sex-pili formations (products of finOP) of plasmids already harbored by a bacterial strain with those of the R1 plasmid. The presence of efficient donors in heterogeneous bacterial populations can accelerate plasmid transfer and can spread by several order...

  16. Plasmid Segregation: Spatial Awareness at the Molecular Level

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Gerdes, Kenn

    2007-01-01

    In bacteria, low-copy number plasmids ensure their stable inheritance by partition loci (par), which actively distribute plasmid replicates to each side of the cell division plane. Using time-lapse fluorescence microscopic tracking of segregating plasmid molecules, a new study provides novel...... insight into the workings of the par system from Escherichia coli plasmid R1. Despite its relative simplicity, the plasmid partition spindle shares characteristics with the mitotic machinery of eukaryotic cells....

  17. Mobilization of Thiobacillus ferrooxidans plasmids among Escherichia coli strains.

    OpenAIRE

    Rawlings, D. E.; Woods, D R

    1985-01-01

    Nonconjugative Thiobacillus ferrooxidans plasmids were mobilized at high frequencies among Escherichia coli strains by the IncP plasmid RP4 and at low frequencies by the IncN plasmid R46, but not by the IncW plasmid pSa. The mobilization region of a nonconjugative T. ferrooxidans plasmid was located on a 5.3-kilobase T. ferrooxidans DNA fragment.

  18. Future Circular Colliders

    CERN Document Server

    AUTHOR|(CDS)2108454; Zimmermann, Frank

    2015-01-01

    In response to a request from the 2013 Update of the European Strategy for Particle Physics, the global Future Circular Collider (FCC) study is preparing the foundation for a next-generation large-scale accelerator infrastructure in the heart of Europe. The FCC study focuses on the design of a 100-TeV hadron collider (FCC-hh), to be accommodated in a new ∼100 km tunnel near Geneva. It also includes the design of a high-luminosity electron-positron collider (FCC-ee), which could be installed in the same tunnel as a potential intermediate step, and a lepton-hadron collider option (FCC-he). The scope of the FCC study comprises accelerators, technology, infrastructure, detectors, physics, concepts for worldwide data services, international governance models, and implementation scenarios. Among the FCC core technologies figure 16-T dipole magnets, based on Nb3Sn superconductor, for the FCC-hh hadron collider, and a highly efficient superconducting radiofrequency system for the FCC-ee lepton collider. The interna...

  19. Future Circular Colliders

    CERN Document Server

    AUTHOR|(CDS)2108454; Zimmermann, Frank

    2015-01-01

    In response to a request from the 2013 Update of the European Strategy for Particle Physics, the global Future Circular Collider (FCC) study is preparing the foundation for a next-generation large-scale accelerator infrastructure in the heart of Europe. The FCC study focuses on the design of a 100-TeV hadron collider (FCC-hh), to be accommodated in a new ∼100 km tunnel near Geneva. It also includes the design of a high-luminosity electron-positron collider (FCC-ee), which could be installed in the same tunnel as a potential intermediate step, and a lepton-hadron collider option (FCC-he). The scope of the FCC study comprises accelerators, technology, infrastructure, detector, physics, concepts for worldwide data services, international governance models, and implementation scenarios. Among the FCC core technologies figure 16-T dipole magnets, based on Nb3Sn superconductor, for the FCC-hh hadron collider, and a highly efficient superconducting radiofrequency system for the FCC-ee lepton collider. The internat...

  20. Towards Future Circular Colliders

    CERN Document Server

    AUTHOR|(CDS)2108454; Zimmermann, Frank

    2015-01-01

    The Large Hadron Collider (LHC) at CERN presently provides proton-proton collisions at a centre-of-mass (c.m.) energy of 13 TeV. The LHC design was started more than 30 years ago, and its physics programme will extend through the second half of the 2030’s. The global Future Circular Collider (FCC) study is now preparing for a post-LHC project. The FCC study focuses on the design of a 100-TeV hadron collider (FCC-hh) in a new ∼100 km tunnel. It also includes the design of a high-luminosity electron-positron collider (FCC-ee) as a potential intermediate step, and a lepton-hadron collider option (FCC-he). The scope of the FCC study comprises accelerators, technology, infrastructure, detectors, physics, concepts for worldwide data services, international governance models, and implementation scenarios. Among the FCC core technologies figure 16-T dipole magnets, based on $Nb_3Sn$ superconductor, for the FCC-hh hadron collider, and a highly efficient superconducting radiofrequency system for the FCC-ee lepton c...

  1. Conservation of Salmonella typhimurium virulence plasmid maintenance regions among Salmonella serovars as a basis for plasmid curing.

    OpenAIRE

    Tinge, S A; Curtiss, R

    1990-01-01

    The association of large plasmids with virulence in invasive Salmonella serovars has led to a number of studies designed to uncover the role of these plasmids in virulence. This study addresses two aspects of virulence-associated plasmids. The first is the distribution of the replication and maintenance regions among the plasmids of different Salmonella serovars, and the second is the use of the conserved virulence plasmid par region to provide a rapid method for eliminating the virulence pla...

  2. Cytoplasmic injection of murine zygotes with Sleeping Beauty transposon plasmids and minicircles results in the efficient generation of germline transgenic mice.

    Science.gov (United States)

    Garrels, Wiebke; Talluri, Thirumala R; Ziegler, Maren; Most, Ilka; Forcato, Diego O; Schmeer, Marco; Schleef, Martin; Ivics, Zoltán; Kues, Wilfried A

    2016-01-01

    Transgenesis in the mouse is an essential tool for the understanding of gene function and genome organization. Here, we describe a simplified microinjection protocol for efficient germline transgenesis and sustained transgene expression in the mouse model employing binary Sleeping Beauty transposon constructs of different topology. The protocol is based on co-injection of supercoiled plasmids or minicircles, encoding the Sleeping Beauty transposase and a transposon construct, into the cytoplasm of murine zygotes. Importantly, this simplified injection avoids the mechanical penetration of the vulnerable pronuclear membrane, resulting in higher survival rates of treated embryos and a more rapid pace of injections. Upon translation of the transposase, transposase-catalyzed transposition into the genome results in stable transgenic animals carrying monomeric transgenes. In summary, cytoplasmic injection of binary transposon constructs is a feasible, plasmid-based, and simplified microinjection method to generate genetically modified mice. The modular design of the components allows the multiplexing of different transposons, and the generation of multi-transposon transgenic mice in a single step. PMID:26470758

  3. Mutations reducing replication from R-loops suppress the defects of growth, chromosome segregation and DNA supercoiling in cells lacking topoisomerase I and RNase HI activity.

    Science.gov (United States)

    Usongo, Valentine; Martel, Makisha; Balleydier, Aurélien; Drolet, Marc

    2016-04-01

    R-loop formation occurs when the nascent RNA hybridizes with the template DNA strand behind the RNA polymerase. R-loops affect a wide range of cellular processes and their use as origins of replication was the first function attributed to them. In Escherichia coli, R-loop formation is promoted by the ATP-dependent negative supercoiling activity of gyrase (gyrA and gyrB) and is inhibited by topoisomerase (topo) I (topA) relaxing transcription-induced negative supercoiling. RNase HI (rnhA) degrades the RNA moiety of R-loops. The depletion of RNase HI activity in topA null mutants was previously shown to lead to extensive DNA relaxation, due to DNA gyrase inhibition, and to severe growth and chromosome segregation defects that were partially corrected by overproducing topo III (topB). Here, DNA gyrase assays in crude cell extracts showed that the ATP-dependent activity (supercoiling) of gyrase but not its ATP-independent activity (relaxation) was inhibited in topA null cells lacking RNase HI. To characterize the cellular event(s) triggered by the absence of RNase HI, we performed a genetic screen for suppressors of the growth defect of topA rnhA null cells. Suppressors affecting genes in replication (holC2::aph and dnaT18::aph) nucleotide metabolism (dcd49::aph), RNA degradation (rne59::aph) and fimbriae synthesis (fimD22::aph) were found to reduce replication from R-loops and to restore supercoiling, thus pointing to a correlation between R-loop-dependent replication in topA rnhA mutants and the inhibition of gyrase activity and growth. Interestingly, the position of fimD on the E. coli chromosome corresponds to the site of one of the five main putative origins of replication from R-loops in rnhA null cells recently identified by next-generation sequencing, thus suggesting that the fimD22::aph mutation inactivated one of these origins. Furthermore, we show that topo III overproduction is unable to complement the growth defect of topA rnhA null mutants at low

  4. Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a

    Institute of Scientific and Technical Information of China (English)

    张继瑜; 刘红; 张笑兵; 杨剑; 杨帆; 杨国威; 沈岩; 侯云德; 金奇

    2003-01-01

    The complete nucleotide sequence and organization of the large virulence plasmid pCP301 (termed by us) of Shigella flexneri 2a strain 301 were determined and analyzed. The result showed that the entire DNA sequence of pCP301 is composed of 221618 bp which form a circular plasmid. Sequence analysis identified 272 open reading frames (ORFs), among which, 194 correspond to the proteins described previously, 61 have low identity (<60%) to known proteins and the rest 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include the genes associated with bacterial virulence, the genes associated with regulation and the genes relating to plasmid maintenance, stability and DNA metabolism. Insertion sequence (IS) elements are 68 kb in length and account for 30 percent of complete sequence of the plasmid which indicates that gene multiple rearrangements of the pCP301 have taken place in Shigella flexneri evolution history. The research result is helpful for interpreting the pathogenesis of Shigella, as well as the genetics and evolution of the plasmid.

  5. Naked plasmid DNA formulation: effect of different disaccharides on stability after lyophilisation.

    Science.gov (United States)

    Quaak, Susanne G L; Haanen, John B A G; Beijnen, Jos H; Nuijen, Bastiaan

    2010-03-01

    Since plasmid DNA (pDNA) is unstable in solution, lyophilisation can be used to increase product shelf life. To prevent stress on pDNA molecules during lyophilisation, cryo- and lyoprotectants have to be added to the formulation. This study assessed the effect of disaccharides on naked pDNA stability after lyophilisation using accelerated stability studies. Naked pDNA was lyophilised with sucrose, trehalose, maltose or lactose in an excipient/DNA w/w ratio of 20. To one part of the vials extra residual moisture was introduced by placing the vials half opened in a 25 degrees C/60% RH climate chamber, before placing all vials in climate chambers (25 degrees C/60% RH and 40 degrees C/75% RH) for stability studies. An ex vivo human skin model was used to assess the effect of disaccharides on transfection efficiency. Lyophilisation resulted in amorphous cakes for all disaccharides with a residual water content of 0.8% w/w. Storage at 40 degrees C/75% RH resulted in decreasing supercoiled (SC) purity levels (sucrose and trehalose maintained approximately 80% SC purity), but not in physical collapse. The addition of residual moisture (values between 7.5% and 10% w/w) resulted in rapid collapse except for trehalose and decreasing SC purity for all formulations. In a separate experiment disaccharide formulation solutions show a slight but significant reduction (<3% with sucrose and maltose) in transfection efficiency when compared to pDNA dissolved in water. We demonstrate that disaccharides, like sucrose and trehalose, are effective lyoprotectants for naked pDNA. PMID:20204715

  6. Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

    DEFF Research Database (Denmark)

    Moreno, Pedro M D; Geny, Sylvain; Pabon, Y Vladimir;

    2013-01-01

    into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON-bisLNA-with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson-Crick binding arm. Optimization was carried...... out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in......In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion...

  7. Tetracycline-resistance encoding plasmids from Paenibacillus larvae, the causal agent of American foulbrood disease, isolated from commercial honeys.

    Science.gov (United States)

    Alippi, Adriana M; León, Ignacio E; López, Ana C

    2014-03-01

    Paenibacillus larvae, the causal agent of American foulbrood disease in honeybees, acquires tetracycline-resistance via native plasmids carrying known tetracycline-resistance determinants. From three P. larvae tetracycline-resistant strains isolated from honeys, 5-kb-circular plasmids with almost identical sequences, designated pPL373 in strain PL373, pPL374 in strain PL374, and pPL395 in strain PL395, were isolated. These plasmids were highly similar (99%) to small tetracycline-encoding plasmids (pMA67, pBHS24, pBSDMV46A, pDMV2, pSU1, pAST4, and pLS55) that replicate by the rolling circle mechanism. Nucleotide sequences comparisons showed that pPL373, pPL374, and pPL395 mainly differed from the previously reported P. larvae plasmid pMA67 in the oriT region and mob genes. These differences suggest alternative mobilization and/or conjugation capacities. Plasmids pPL373, pPL374, and pPL395 were individually transferred by electroporation and stably maintained in tetracycline-susceptible P. larvae NRRL B-14154, in which they autonomously replicated. The presence of nearly identical plasmids in five different genera of gram-positive bacteria, i.e., Bhargavaea, Bacillus, Lactobacillus, Paenibacillus, and Sporosarcina, inhabiting diverse ecological niches provides further evidence of the genetic transfer of tetracycline resistance among environmental bacteria from soils, food, and marine habitats and from pathogenic bacteria such as P. larvae. PMID:25296446

  8. Circularly-Polarized Microstrip Antenna

    Science.gov (United States)

    Stanton, P. H.

    1985-01-01

    Microstrip construction compact for mobile applications. Circularly polarized microstrip antenna made of concentric cylindrical layers of conductive and dielectric materials. Coaxial cable feedlines connected to horizontal and vertical subelements from inside. Vertical subelement acts as ground for horizontal subelement.

  9. Coloring fuzzy circular interval graphs

    OpenAIRE

    Eisenbrand, Friedrich; Niemeier, Martin

    2012-01-01

    Computing the weighted coloring number of graphs is a classical topic in combinatorics and graph theory. Recently these problems have again attracted a lot of attention for the class of quasi-line graphs and more specifically fuzzy circular interval graphs. The problem is NP-complete for quasi-line graphs. For the subclass of fuzzy circular interval graphs however, one can compute the weighted coloring number in polynomial time using recent results of Chudnovsky and Ovetsky and of King and Re...

  10. Energy dissipation in circular tube

    OpenAIRE

    A.D. Girgidov

    2012-01-01

    Energy dissipation distribution along the circular tube radius is important in solving such problems as calculation of heat transfer by the air flow through building envelope; calculation of pressure loss in spiral flows; calculation of cyclones with axial and tangential supply of dust-containing gas.Two types of one-dimensional radially axisymmetric flows in circular tube were considered: axial flow and rotation about the axis (Rankine vortex). Relying on two- and four-layer description of a...

  11. BioShuttle-mediated Plasmid Transfer

    Directory of Open Access Journals (Sweden)

    Klaus Braun, Leonie von Brasch, Ruediger Pipkorn, Volker Ehemann, Juergen Jenne, Herbert Spring, Juergen Debus, Bernd Didinger, Werner Rittgen, Waldemar Waldeck

    2007-01-01

    Full Text Available An efficient gene transfer into target tissues and cells is needed for safe and effective treatment of genetic diseases like cancer. In this paper, we describe the development of a transport system and show its ability for transporting plasmids. This non-viral peptide-based BioShuttle-mediated transfer system consists of a nuclear localization address sequence realizing the delivery of the plasmid phNIS-IRES-EGFP coding for two independent reporter genes into nuclei of HeLa cells. The quantification of the transfer efficiency was achieved by measurements of the sodium iodide symporter activity. EGFP gene expression was measured with Confocal Laser Scanning Microscopy and quantified with biostatistical methods by analysis of the frequency of the amplitude distribution in the CLSM images. The results demonstrate that the “BioShuttle”-Technology is an appropriate tool for an effective transfer of genetic material carried by a plasmid.

  12. BioShuttle-mediated Plasmid Transfer

    Science.gov (United States)

    Braun, Klaus; von Brasch, Leonie; Pipkorn, Ruediger; Ehemann, Volker; Jenne, Juergen; Spring, Herbert; Debus, Juergen; Didinger, Bernd; Rittgen, Werner; Waldeck, Waldemar

    2007-01-01

    An efficient gene transfer into target tissues and cells is needed for safe and effective treatment of genetic diseases like cancer. In this paper, we describe the development of a transport system and show its ability for transporting plasmids. This non-viral peptide-based BioShuttle-mediated transfer system consists of a nuclear localization address sequence realizing the delivery of the plasmid phNIS-IRES-EGFP coding for two independent reporter genes into nuclei of HeLa cells. The quantification of the transfer efficiency was achieved by measurements of the sodium iodide symporter activity. EGFP gene expression was measured with Confocal Laser Scanning Microscopy and quantified with biostatistical methods by analysis of the frequency of the amplitude distribution in the CLSM images. The results demonstrate that the “BioShuttle”-Technology is an appropriate tool for an effective transfer of genetic material carried by a plasmid. PMID:18026568

  13. Electrotransfer of Plasmid Vector DNA into Muscle

    Science.gov (United States)

    Miyazaki, Satsuki; Miyazaki, Jun-Ichi

    Wolff et al. (1990) first reported that plasmid DNA injected into skeletal muscle is taken up by muscle cells and the genes in the plasmid are expressed for more than two months thereafter, although the transfected DNA does not usually undergo chromosomal integration (Wolff et al., 1991, 1992). However, the relatively low expression levels attained by this method have hampered its applications for uses other than as a DNA vaccine (Davis et al., 1995). There are a number of reports analyzing the conditions that affect the efficiency of gene transfer by intramuscular DNA injection and assessing the fine structures of expression plasmid vectors that may affect expression levels (Davis et al., 1993; Liang et al., 1996; Norman et al., 1997). Furthermore, various attempts were done to improve the efficiency of gene transfer by intramus cular DNA injection. Consequently, regenerating muscle was shown to produce 80-fold or more protein than did normal muscle, following injection of an expression plas-mid. Muscle regeneration was induced by treatment with cardiotoxin or bupivacaine (Wells, 1993; Vitadello et al., 1994). We previously demonstrated that by combining a strong promoter and bupivacaine pretreatment intramuscular injection of an IL-5 expression plasmid results in IL-5 production in muscle at a level sufficient to induce marked proliferation of eosinophils in the bone marrow and eosinophil infiltration of various organs (Tokui et al., 1997). It was also reported that a single intramuscular injection of an erythropoietin expression plasmid produced physiologically significant elevations in serum erythropoietin levels and increased hematocrits in adult mice (Tripathy et al., 1996). Hematocrits in these animals remained elevated at >60% for at least 90 days after a single injection. However, improvements to this method have not been sufficient to extend its applications including clinical use.

  14. BioShuttle-mediated Plasmid Transfer

    OpenAIRE

    Braun, Klaus; von Brasch, Leonie; Pipkorn, Ruediger; Ehemann, Volker; Jenne, Juergen; Spring, Herbert; Debus, Juergen; Didinger, Bernd; Rittgen, Werner; Waldeck, Waldemar

    2007-01-01

    An efficient gene transfer into target tissues and cells is needed for safe and effective treatment of genetic diseases like cancer. In this paper, we describe the development of a transport system and show its ability for transporting plasmids. This non-viral peptide-based BioShuttle-mediated transfer system consists of a nuclear localization address sequence realizing the delivery of the plasmid phNIS-IRES-EGFP coding for two independent reporter genes into nuclei of HeLa cells. The quantif...

  15. Competition between Plasmid-Bearing and Plasmid-Free Organisms in a Chemostat with Pulsed Input and Washout

    Directory of Open Access Journals (Sweden)

    Sanling Yuan

    2009-01-01

    Full Text Available We consider a model of competition between plasmid-bearing and plasmid-free organisms in the chemostat with pulsed input and washout. We investigate the subsystem with nutrient and plasmid-free organism and study the stability of the boundary periodic solutions, which are the boundary periodic solutions of the system. The stability analysis of the boundary periodic solution yields the invasion threshold of the plasmid-bearing organism. By using the standard techniques of bifurcation theory, we prove that above this threshold there are periodic oscillations in substrate, plasmid-free, and plasmid-bearing organisms. Numerical simulations are carried out to illustrate our results.

  16. Plasmid maintenance functions of the large virulence plasmid of Shigella flexneri.

    OpenAIRE

    Radnedge, L; Davis, M. A.; Youngren, B; Austin, S. J.

    1997-01-01

    The large virulence plasmid pMYSH6000 of Shigella flexneri contains a replicon and a plasmid maintenance stability determinant (Stb) on adjacent SalI fragments. The presence of a RepFIIA replicon on the SalI C fragment was confirmed, and the complete sequence of the adjacent SalI O fragment was determined. It shows homology to part of the transfer (tra) operon of the F plasmid. Stb stabilizes a partition-defective P1 miniplasmid in Escherichia coli. A 1.1-kb region containing a homolog of the...

  17. Plasmid transfer and plasmid-mediated genetic exchange in Brucella abortus.

    OpenAIRE

    Rigby, C E; Fraser, A.D.

    1989-01-01

    Naturally-occurring plasmids and gene transfer mechanisms have not yet been reported in brucellae. Here we show that Brucella abortus is capable of maintaining and transferring the broad-host-range plasmids pTH10 (IncP), pSa (IncW) and R751 (IncP), and describe pTH10-mediated transfer of B. abortus chromosomal genes to Escherichia coli. All three plasmids transferred by conjugation from E. coli to B. abortus S19, and from B. abortus S19 to B. abortus 292 (biovar 4). They were stably maintaine...

  18. Plasmid transduction by Bacillus subtilis bacteriophage SPP1: effects of DNA homology between plasmid and bacteriophage.

    OpenAIRE

    Deichelbohrer, I; Alonso, J.C.; Lüder, G; Trautner, T A

    1985-01-01

    Any SPP1 DNA restriction fragment cloned into Bacillus subtilis plasmid pC194 or pUB110 increased the transduction frequency of the plasmid by SPP1 100- to 1,000-fold over the transduction level of the plasmid alone. This increment was observed irrespective of whether a fragment contained the SPP1 packaging origin (pac). Furthermore, an SPP1 derivative into whose genome pC194 DNA had been integrated transduced pC194 DNA with a greatly enhanced frequency. Transduction enhancement mediated by D...

  19. Plasmid maintenance and protein overproduction in selective recycle bioreactors.

    Science.gov (United States)

    Ogden, K L; Davis, R H

    1991-02-20

    A new plasmid construct has been used in conjunction with selective recycle to successfully maintain otherwise unstable plasmid-bearing E. coli cells in a continuous bioreactor and to produce significant amounts of the plasmid-encoded protein beta-lactamase. The plasmid is constructed so that pilin expression, which leads to bacterial flocculation, is under control of the tac operon. The plasmid-bearing cells are induced to flocculate in the separator, whereas cell growth and product synthesis occur in the main fermentation vessel without the inhibiting effects of pilin production. Selective recycle allows for the maintenance of the plasmid-bearing cells by separating flocculent, plasmid-bearing cells from nonflocculent, segregant cells in an inclined settler, and recycling only the plasmid-bearing cells to the reactor. As a result, product expression levels are maintained that are more than ten times the level achieved without selective recycle. All experimental data agree well with theoretical predictions. PMID:18597374

  20. Simple method for identification of plasmid-coded proteins

    International Nuclear Information System (INIS)

    Proteins encoded by plasmid DNA are specifically labeled in uv-irradiated cells of Escherichia coli carrying recA and uvrA mutations because extensive degradation of the chromosome DNA occurs concurrently with amplification of plasmid DNA

  1. Protection of vanillin derivative VND3207 on plasmid DNA damage induced by different LET ionizing radiation

    International Nuclear Information System (INIS)

    Objective: To evaluate the radioprotective effect of vanillin derivative VND3207 on DNA damage induced by different LET ionizing radiation. Methods: The plasmid DNA in liquid was irradiated by 60Co γ-rays, proton or 7Li heavy ion with or without VND3207. The conformation changes of plasmid DNA were assessed by agarose gel electrophoresis and the quantification was done using gel imaging system. Results: The DNA damage induced by proton and 7Li heavy ion was much more serious as compared with that by 60Co γ-rays, and the vanillin derivative VND3207 could efficiently decrease the DNA damage induced by all three types of irradiation sources, which was expressed as a significantly reduced ratio of open circular form (OC) of plasmid DNA. The radioprotective effect of VND3207 increased with the increasing of drug concentration. The protective efficiencies of 200 μmol/L VND3207 were 85.3% (t =3.70, P=0.033), 73.3% (t=10.58, P=0.017) and 80.4% (t=8.57, P=0.008) on DNA damage induction by 50 Gy of γ-rays, proton and 7Li heavy ion, respectively. It seemed that the radioprotection of VND3207 was more effective on DNA damage induced by high LET heavy ion than that by proton. Conclusions: VND3207 has a protective effect against the genotoxicity of different LET ionizing radiation, especially for γ-rays and 7 Li heavy ion. (authors)

  2. Protection of plasmid DNA by a Ginkgo biloba extract from the effects of stannous chloride and the action on the labeling of blood elements with technetium-99m

    Directory of Open Access Journals (Sweden)

    Moreno S.R.F.

    2004-01-01

    Full Text Available Ginkgo biloba extract (EGb is a phytotherapeutic agent used for the treatment of ischemic and neurological disorders. Because the action of this important extract is not fully known, assays using different biological systems need to be performed. Red blood cells (RBC are labeled with technetium-99m (Tc-99m and used in nuclear medicine. The labeling depends on a reducing agent, usually stannous chloride (SnCl2. We assessed the effect of different concentrations of EGb on the labeling of blood constituents with Tc-99m, as sodium pertechnetate (3.7 MBq, and on the mobility of a plasmid DNA treated with SnCl2 (1.2 µg/ml at room temperature. Blood was incubated with EGb before the addition of SnCl2 and Tc-99m. Plasma (P and RBC were separated and precipitated with trichloroacetic acid, and soluble (SF-P and SF-RBC and insoluble (IF-P and IF-RBC fractions were isolated. The plasmid was incubated with Egb, SnCl2 or EGb plus SnCl2 and agarose gel electrophoresis was performed. The gel was stained with ethidium bromide and the DNA bands were visualized by fluorescence in an ultraviolet transilluminator system. EGb decreased the labeling of RBC, IF-P and IF-RBC. The supercoiled form of the plasmid was modified by treatment with SnCl2 and protected by 40 mg/ml EGb. The effect of EGb on the tested systems may be due to its chelating action with the stannous ions and/or pertechnetate or to the capability to generate reactive oxygen species that could oxidize the stannous ion.

  3. DISTRIBUTION OF PLASMIDS IN GROUNDWATER BACTERIA

    Science.gov (United States)

    Bacteria isolated from groundwater aquifer core materials of pristine aquifers at Lula and Pickett, Oklahoma, and from a site with a history of aromatic hydrocarbon contamination and natural renovation located at Conroe, Texas, were screened for the presence of plasmid Deoxyribon...

  4. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.

    2014-01-01

    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and veter

  5. Circularly polarized open-loop antenna

    OpenAIRE

    Li, Rong-Lin; Fusco, Vincent F.; Nakano, Hisamatsu

    2003-01-01

    A printed circular open-loop antenna is introduced as a simple structure for producing circular polarization; the antenna is fed with a coaxial probe. By introducing a gap within the circular loop a traveling-wave current is excited and thus circularly polarized radiation can be achieved. An optimized circularly polarized antenna is designed through numerical analysis using a so-called parametric method of moment technique. Experimental verification of the new antenna is presented. The antenn...

  6. Claw-free circular-perfect graphs

    OpenAIRE

    Pecher, Arnaud; Zhu, Xuding

    2007-01-01

    The circular chromatic number of a graph is a well studied refinement of the chromatic number. Circular perfect graphs is a superclass of perfect graphs defined by means of this more general coloring concept. This paper studies claw free circular perfect graphs. A consequence of the strong perfect graph theorem is that minimal circular perfect graphs G. In contrast to this result, it is shown in that minimal circular imperfect graphs G can have arbitrarily large independence number and arbitr...

  7. The influence of biofilms in the biology of plasmids

    OpenAIRE

    Cook, Laura C.C.; Dunny, Gary M.

    2014-01-01

    The field of plasmid biology has historically focused on bacteria growing in liquid culture. Surface attached communities of bacterial biofilms have recently been understood to be the normal environment of bacteria in the natural world. Thus, studies examining plasmid replication, maintenance, and transfer in biofilms are essential for a true understanding of bacterial plasmid biology. This chapter reviews the current knowledge of the interplay between bacterial biofilms and plasmids, focusin...

  8. The 2 micron plasmid purloins the yeast cohesin complex

    OpenAIRE

    Mehta, Shwetal; Yang, Xian Mei; Chan, Clarence S.; Dobson, Melanie J.; Jayaram, Makkuni; Velmurugan, Soundarapandian

    2002-01-01

    The yeast 2 micron plasmid achieves high fidelity segregation by coupling its partitioning pathway to that of the chromosomes. Mutations affecting distinct steps of chromosome segregation cause the plasmid to missegregate in tandem with the chromosomes. In the absence of the plasmid stability system, consisting of the Rep1 and Rep2 proteins and the STB DNA, plasmid and chromosome segregations are uncoupled. The Rep proteins, acting in concert, recruit the yeast cohesin complex to the STB locu...

  9. Plasmid DNA entry into postmitotic nuclei of primary rat myotubes.

    OpenAIRE

    Dowty, M E; Williams, P; Zhang, G.; Hagstrom, J E; Wolff, J A

    1995-01-01

    These studies were initiated to elucidate the mechanism of DNA nuclear transport in mammalian cells. Biotin- or gold-labeled plasmid and plasmid DNA expression vectors for Escherichia coli beta-galactosidase or firefly luciferase were microinjected into the cytoplasm of primary rat myotubes in culture. Plasmid DNA was expressed in up to 70% of the injected myotubes, which indicates that it entered intact, postmitotic nuclei. The nuclear transport of plasmid DNA occurred through the nuclear po...

  10. DNA-binding protein from HeLa cells that binds preferentially to supercoiled DNA damaged by ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene

    International Nuclear Information System (INIS)

    A DNA-binding protein was partially purified from extracts of HeLa cells by high-speed centrifugation and chromatography on DEAE-cellulose, phosphocellulose and ultraviolet light-irradiated DNA-cellulose columns. It eluted from the phosphocellulose column with 0.375 M potassium phosphate and from the ultraviolet light-irradiated DNA-cellulose column between 0.5 M and 1 M NaCl. The protein binds preferentially to supercoiled PM2 DNA treated with ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene, as compared to native supercoiled PM2 DNA. The binding is non-cooperative. Nicked or linear forms of PM2 DNA (damaged or untreated) are not efficient substrates, indicating a requirement of DNA supercoiling for DNA binding. The sedimentation coefficient of the protein estimated by glycerol gradient centrifugation is 2.0-2.5 S, corresponding to a molecular weight of about 20000-25000 if the protein is spherical. The binding to DNA irradiated with ultraviolet light or treated with acetoxyacetylaminofluorene is optimal at around 100-200 mM NaCl and is relatively independent of temperature and pH. MgCl2 and MnCl2 at concentrations between 1 and 5 mM do not markedly affect the binding, but it is inhibited by sucrose, ATP and caffeine. The biological significance of the DNA-binding protein remains to be determined. It does not possess significant glycosylase, endonuclease or exonuclease activities. The dissociation equilibrium constant for the binding reaction of the protein to the ultraviolet light or acetoxyacetylaminofluorene-induced binding sites on DNA is estimated to be 4x10-11 M. There are at least 1x105 DNA-binding protein molecules/HeLa cell. (Auth.)

  11. Postsegregational killing does not increase plasmid stability but acts to mediate the exclusion of competing plasmids

    OpenAIRE

    Cooper, Tim F; Heinemann, Jack A.

    2000-01-01

    Postsegregational killing (PSK) systems consist of a tightly linked toxin–antitoxin pair. Antitoxin must be continually produced to prevent the longer lived toxin from killing the cell. PSK systems on plasmids are widely believed to benefit the plasmid by ensuring its stable vertical inheritance. However, experimental tests of this “stability” hypothesis were not consistent with its predictions. We suggest an alternative hypothesis to explain the evolution of PSK: ...

  12. Plasmid Capture by the Bacillus thuringiensis Conjugative Plasmid pXO16▿

    OpenAIRE

    Timmery, Sophie; Modrie, Pauline; Minet, Olivier; Mahillon, Jacques

    2009-01-01

    Conjugation, mobilization, and retromobilization are three related mechanisms of horizontal gene transfer in bacteria. They have been extensively studied in gram-negative species, where retromobilization, the capture of DNA from a recipient by a donor cell, was shown to result from two successive steps: the transfer of the conjugative plasmid from the donor to the recipient followed by the retrotransfer of the mobilizable plasmid to the donor. This successive model was established for gram-ne...

  13. Conjugative plasmids: Vessels of the communal gene pool

    DEFF Research Database (Denmark)

    Norman, Anders; Hansen, Lars H.; Sørensen, Søren Johannes

    2009-01-01

    over large taxonomic distances. These plasmids are collections of discrete regions of genes that function as ‘backbone modules' to undertake different aspects of overall plasmid maintenance and propagation. Conjugative plasmids often carry suites of ‘accessory elements' that contribute adaptive traits...

  14. Compositional discordance between prokaryotic plasmids and host chromosomes

    Directory of Open Access Journals (Sweden)

    van Kampen Antoine HC

    2006-02-01

    Full Text Available Abstract Background Most plasmids depend on the host replication machinery and possess partitioning genes. These properties confine plasmids to a limited range of hosts, yielding a close and presumably stable relationship between plasmid and host. Hence, it is anticipated that due to amelioration the dinucleotide composition of plasmids is similar to that of the genome of their hosts. However, plasmids are also thought to play a major role in horizontal gene transfer and thus are frequently exchanged between hosts, suggesting dinucleotide composition dissimilarity between plasmid and host genome. We compared the dinucleotide composition of a large collection of plasmids with that of their host genomes to shed more light on this enigma. Results The dinucleotide frequency, coined the genome signature, facilitates the identification of putative horizontally transferred DNA in complete genome sequences, since it was found to be typical for a certain genome, and similar between related species. By comparison of the genome signature of 230 plasmid sequences with that of the genome of each respective host, we found that in general the genome signature of plasmids is dissimilar from that of their host genome. Conclusion Our results show that the genome signature of plasmids does not resemble that of their host genome. This indicates either absence of amelioration or a less stable relationship between plasmids and their host. We propose an indiscriminate lifestyle for plasmids preserving the genome signature discordance between these episomes and host chromosomes.

  15. Engineering Escherichia coli to increase plasmid DNA production in high cell-density cultivations in batch mode

    Directory of Open Access Journals (Sweden)

    Borja Gheorghe M

    2012-09-01

    Full Text Available Abstract Background Plasmid DNA (pDNA is a promising molecule for therapeutic applications. pDNA is produced by Escherichia coli in high cell-density cultivations (HCDC using fed-batch mode. The typical limitations of such cultivations, including metabolic deviations like aerobic acetate production due to the existence of substrate gradients in large-scale bioreactors, remain as serious challenges for fast and effective pDNA production. We have previously demonstrated that the substitution of the phosphotransferase system by the over-expressed galactose permease for glucose uptake in E. coli (strain VH33 allows efficient growth, while strongly decreases acetate production. In the present work, additional genetic modifications were made to VH33 to further improve pDNA production. Several genes were deleted from strain VH33: the recA, deoR, nupG and endA genes were inactivated independently and in combination. The performance of the mutant strains was evaluated in shake flasks for the production of a 6.1 kb plasmid bearing an antigen gene against mumps. The best producer strain was cultivated in lab-scale bioreactors using 100 g/L of glucose to achieve HCDC in batch mode. For comparison, the widely used commercial strain DH5α, carrying the same plasmid, was also cultivated under the same conditions. Results The various mutations tested had different effects on the specific growth rate, glucose uptake rate, and pDNA yields (YP/X. The triple mutant VH33 Δ (recA deoR nupG accumulated low amounts of acetate and resulted in the best YP/X (4.22 mg/g, whereas YP/X of strain VH33 only reached 1.16 mg/g. When cultivated at high glucose concentrations, the triple mutant strain produced 186 mg/L of pDNA, 40 g/L of biomass and only 2.2 g/L of acetate. In contrast, DH5α produced only 70 mg/L of pDNA and accumulated 9.5 g/L of acetate. Furthermore, the supercoiled fraction of the pDNA produced by the triple mutant was nearly constant

  16. Quantification of covalently closed circular DNA of hepatitis B virus in FFPE liver tissues of chronic hepatitis B patients

    OpenAIRE

    Jia-qi HAN; Yan-wei ZHONG; Xiao-qiang REN; Zheng-sheng ZOU; Liu, Shu-Hong; Xue-en LIU; Jing-min ZHAO; Dong-ping XU

    2011-01-01

    Objective To establish a method of detecting HBV covalently closed circular DNA(cccDNA) in micro-formalin fixed paraffin imbedding(FFPE) liver biopsy samples.Methods FFPE liver biopsies from 37 patients with chronic hepatitis B were studied.The intrahepatic HBV DNA was extracted and pre-treated with plasmid-safe ATP-dependent DNAse(PSAD),and then amplified by rolling circular amplification(RCA).The HBV cccDNA was quantitatively detected by Taqman real-time PCR with primers located on both sid...

  17. Guiding strand passage: DNA-induced movement of the gyrase C-terminal domains defines an early step in the supercoiling cycle

    OpenAIRE

    Lanz, Martin A.; Klostermeier, Dagmar

    2011-01-01

    DNA gyrase catalyzes ATP-dependent negative supercoiling of DNA in a strand passage mechanism. A double-stranded segment of DNA, the T-segment, is passed through the gap in a transiently cleaved G-segment by coordinated closing and opening of three protein interfaces in gyrase. T-segment capture is thought to be guided by the C-terminal domains of the GyrA subunit of gyrase that wrap DNA around their perimeter and cause a DNA-crossing with a positive handedness. We show here that the C-termin...

  18. Competition between Plasmid-Bearing and Plasmid-Free Organisms in a Chemostat with Pulsed Input and Washout

    OpenAIRE

    Sanling Yuan; Yu Zhao; Anfeng Xiao

    2009-01-01

    We consider a model of competition between plasmid-bearing and plasmid-free organisms in the chemostat with pulsed input and washout. We investigate the subsystem with nutrient and plasmid-free organism and study the stability of the boundary periodic solutions, which are the boundary periodic solutions of the system. The stability analysis of the boundary periodic solution yields the invasion threshold of the plasmid-bearing organism. By using the standard techniques of bifurcation theory, w...

  19. Co-segregation of yeast plasmid sisters under monopolin-directed mitosis suggests association of plasmid sisters with sister chromatids

    OpenAIRE

    Liu, Yen-Ting; Ma, Chien-Hui; Jayaram, Makkuni

    2013-01-01

    The 2-micron plasmid, a high copy extrachromosomal element in Saccharomyces cerevisiae, propagates itself with nearly the same stability as the chromosomes of its host. Plasmid stability is conferred by a partitioning system consisting of the plasmid-coded proteins Rep1 and Rep2 and a cis-acting locus STB. Circumstantial evidence suggests that the partitioning system couples plasmid segregation to chromosome segregation during mitosis. However, the coupling mechanism has not been elucidated. ...

  20. Podokinetic circular vection: characteristics and interaction with optokinetic circular vection.

    Science.gov (United States)

    Becker, W; Kliegl, K; Kassubek, J; Jürgens, R

    2016-07-01

    Stabilising horizontal body orientation in space without sight on a rotating platform by holding to a stationary structure and circular 'treadmill' stepping in the opposite direction can elicit an illusion of self-turning in space (Bles and Kapteyn in Agressologie 18:325-328, 1977). Because this illusion is analogous to the well-known illusion of optokinetic circular vection (oCV), we call it 'podokinetic circular vection' (pCV) here. Previous studies using eccentric stepping on a path tangential to the rotation found that pCV was always contraversive relative to platform rotation. In contrast, when our subjects stepped at the centre of rotation about their vertical axis, we observed an inverted, ipsiversive pCV as a reproducible trait in many of our subjects. This ipCV occurred at the same latency as the pCV of subjects reporting the actually expected contraversive direction, but had lower gain. In contrast to pCV, the nystagmus accompanying circular treadmill stepping had the same direction in all individuals (slow phase in the direction of platform motion). The direction of an individual's pCV predicted the characteristics of the CV resulting from combined opto- and podokinetic stimulation (circular treadmill stepping while viewing a pattern rotating together with the platform): in individuals with contraversive pCV, latency shortened and both gain and felt naturalness increased in comparison with pure oCV, whereas the opposite (longer latency, reduced gain and naturalness) occurred in individuals with ipCV. Taken together, the reproducibility of ipCV, the constant direction of nystagmus and the fact that pCV direction predicts the outcome of combined stimulation suggest that ipCV is an individual trait of many subjects during compensatory stepping at the centre of rotation. A hypothetical model is presented of how ipCV possibly could arise. PMID:26965438

  1. THE INTEGRATED STATE OF THE ROLLING-CIRCLE PLASMID PTB913 IN THE COMPOSITE BACILLUS PLASMID PTB19

    NARCIS (Netherlands)

    OSKAM, L; HILLENGA, DJ; VENEMA, G; BRON, S

    1992-01-01

    pTB19, a 27 kb plasmid originating from a thermophilic Bacillus species, contains integrated copies of two rolling-circle type plasmids on a 10.6 kb DNA fragment. In the present study we analysed the part of pTB19 that contains the rolling-circle plasmid pTB913 and the region in between the two roll

  2. Stable inheritance of plasmid R1 requires two different loci.

    OpenAIRE

    Gerdes, K; Larsen, J E; Molin, S

    1985-01-01

    The largest EcoRI fragment from plasmid R1 mediates a stability phenotype which is required to ensure the stable inheritance of this low-copy-number plasmid. When covalently linked to small, unstable R1 derivatives, this fragment makes the plasmids as stable as the wild-type R1 plasmid. A genetic analysis showed that two independently acting stabilization functions are encoded by this EcoRI fragment, both of which have the potential of partial stabilization of mini-R1 plasmids. The two loci a...

  3. DNA restriction-modification systems mediate plasmid maintenance.

    OpenAIRE

    Kulakauskas, S; Lubys, A; Ehrlich, S. D.

    1995-01-01

    Two plasmid-carried restriction-modification (R-M) systems, EcoRI (from pMB1 of Escherichia coli) and Bsp6I (from pXH13 of Bacillus sp. strain RFL6), enhance plasmid segregational stability in E. coli and Bacillus subtilis, respectively. Inactivation of the endonuclease or the presence of the methylase in trans abolish the stabilizing activity of the R-M systems. We propose that R-M systems mediate plasmid segregational stability by postsegregational killing of plasmid-free cells. Plasmid-enc...

  4. Circular RNA expands its territory.

    Science.gov (United States)

    Bao, Chunyang; Lyu, Dongbin; Huang, Shenglin

    2016-03-01

    Circular RNAs (circRNAs) represent a novel class of widespread non-coding RNAs in eukaryotes. They are unusually stable RNA molecules with cell type-specific expression patterns, and are predominantly present in the cytoplasm. We recently demonstrated the existence of abundant circRNAs in exosomes and suggest a potential application of exosomal circRNAs for cancer detection. PMID:27308606

  5. High intensity circular proton accelerators

    International Nuclear Information System (INIS)

    Circular machines suitable for the acceleration of high intensity proton beams include cyclotrons, FFAG accelerators, and strong-focusing synchrotrons. This paper discusses considerations affecting the design of such machines for high intensity, especially space charge effects and the role of beam brightness in multistage accelerators. Current plans for building a new generation of high intensity 'kaon factories' are reviewed. 47 refs

  6. Circular polarization observed in bioluminescence

    NARCIS (Netherlands)

    Wijnberg, Hans; Meijer, E.W.; Hummelen, J.C.; Dekkers, H.P.J.M.; Schippers, P.H.; Carlson, A.D.

    1980-01-01

    While investigating circular polarization in luminescence, and having found it in chemiluminescence, we have studied bioluminescence because it is such a widespread and dramatic natural phenomenon. We report here that left and right lanterns of live larvae of the fireflies, Photuris lucicrescens and

  7. Application of circular filter inserts

    International Nuclear Information System (INIS)

    High efficiency particulate air (HEPA) filters are used in the ventilation of nuclear plant as passive clean-up devices. Traditionally, the work-horse of the industry has been the rectangular HEPA filter. An assessment of the problems associated with remote handling, changing, and disposal of these rectangular filters suggested that significant advantages to filtration systems could be obtained by the adoption of HEPA filters with circular geometry for both new and existing ventilation plants. This paper covers the development of circular geometry filters and highlights the advantages of this design over their rectangular counterparts. The work has resulted in a range of commercially available filters for flows from 45 m3/h up to 3400 m3/h. This paper also covers the development of a range of sizes and types of housings that employ simple change techniques which take advantage of the circular geometry. The systems considered here have been designed in response to the requirements for shielded (remote filter change) and for unshielded facilities (potentially for bag changing of filters). Additionally the designs have allowed for the possibility of retrofitting circular geometry HEPA filters in place of the rectangular geometry filter

  8. pEVL: A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly(A) Sequences.

    Science.gov (United States)

    Grier, Alexandra E; Burleigh, Stephen; Sahni, Jaya; Clough, Courtnee A; Cardot, Victoire; Choe, Dongwook C; Krutein, Michelle C; Rawlings, David J; Jensen, Michael C; Scharenberg, Andrew M; Jacoby, Kyle

    2016-01-01

    Increasing demand for large-scale synthesis of in vitro transcribed (IVT) mRNA is being driven by the increasing use of mRNA for transient gene expression in cell engineering and therapeutic applications. An important determinant of IVT mRNA potency is the 3' polyadenosine (poly(A)) tail, the length of which correlates with translational efficiency. However, present methods for generation of IVT mRNA rely on templates derived from circular plasmids or PCR products, in which homopolymeric tracts are unstable, thus limiting encoded poly(A) tail lengths to ~120 base pairs (bp). Here, we have developed a novel method for generation of extended poly(A) tracts using a previously described linear plasmid system, pJazz. We find that linear plasmids can successfully propagate poly(A) tracts up to ~500 bp in length for IVT mRNA production. We then modified pJazz by removing extraneous restriction sites, adding a T7 promoter sequence upstream from an extended multiple cloning site, and adding a unique type-IIS restriction site downstream from the encoded poly(A) tract to facilitate generation of IVT mRNA with precisely defined encoded poly(A) tracts and 3' termini. The resulting plasmid, designated pEVL, can be used to generate IVT mRNA with consistent defined lengths and terminal residue(s). PMID:27093168

  9. Role of Plasmid in Production of Acetobacter Xylinum Biofilms

    Directory of Open Access Journals (Sweden)

    Abbas Rezaee

    2005-01-01

    Full Text Available Acetobacter xylinum has the ability to produce cellulotic biofilms. Bacterial cellulose is expected to be used in many industrial or biomedical materials for its unique characteristics. A. xylinum contains a complex system of plasmid DNA molecules. A 44 kilobases (kb plasmid was isolated in wild type of A. xylinum. To improve the cellulose producing ability of A. xylinum, role of the plasmid in production of cellulose was studied. The comparisons between wild type and cured cells of A. xylinum showed that there is considerably difference in cellulose production. In order to study the relationship between plasmid and the rate of cellulose production, bacteria were screened for plasmid profile by a modified method for preparation of plasmid. This method yields high levels of pure plasmid DNA that can be used for common molecular techniques, such as digestion and transformation, with high efficiency.

  10. Deficient sumoylation of yeast 2-micron plasmid proteins Rep1 and Rep2 associated with their loss from the plasmid-partitioning locus and impaired plasmid inheritance.

    Directory of Open Access Journals (Sweden)

    Jordan B Pinder

    Full Text Available The 2-micron plasmid of the budding yeast Saccharomyces cerevisiae encodes copy-number amplification and partitioning systems that enable the plasmid to persist despite conferring no advantage to its host. Plasmid partitioning requires interaction of the plasmid Rep1 and Rep2 proteins with each other and with the plasmid-partitioning locus STB. Here we demonstrate that Rep1 stability is reduced in the absence of Rep2, and that both Rep proteins are sumoylated. Lysine-to-arginine substitutions in Rep1 and Rep2 that inhibited their sumoylation perturbed plasmid inheritance without affecting Rep protein stability or two-hybrid interaction between Rep1 and Rep2. One-hybrid and chromatin immunoprecipitation assays revealed that Rep1 was required for efficient retention of Rep2 at STB and that sumoylation-deficient mutants of Rep1 and Rep2 were impaired for association with STB. The normal co-localization of both Rep proteins with the punctate nuclear plasmid foci was also lost when Rep1 was sumoylation-deficient. The correlation of Rep protein sumoylation status with plasmid-partitioning locus association suggests a theme common to eukaryotic chromosome segregation proteins, sumoylated forms of which are found enriched at centromeres, and between the yeast 2-micron plasmid and viral episomes that depend on sumoylation of their maintenance proteins for persistence in their hosts.

  11. Modeling sRNA-regulated Plasmid Maintenance

    CERN Document Server

    Gong, Chen Chris

    2016-01-01

    We study a theoretical model for the toxin-antitoxin (hok/sok) mechanism for plasmid maintenance in bacteria. Toxin-antitoxin systems enforce the maintenance of a plasmid through post-segregational killing of cells that have lost the plasmid. Key to their function is the tight regulation of expression of a protein toxin by an sRNA antitoxin. Here, we focus on the nonlinear nature of the regulatory circuit dynamics of the toxin-antitoxin mechanism. The mechanism relies on a transient increase in protein concentration rather than on the steady state of the genetic circuit. Through a systematic analysis of the parameter dependence of this transient increase, we confirm some known design features of this system and identify new ones: for an efficient toxin-antitoxin mechanism, the synthesis rate of the toxin's mRNA template should be lower that of the sRNA antitoxin, the mRNA template should be more stable than the sRNA antitoxin, and the mRNA-sRNA complex should be more stable than the sRNA antitoxin. Moreover, ...

  12. Cu(II) complexes of glyco-imino-aromatic conjugates in DNA binding, plasmid cleavage and cell cytotoxicity

    Indian Academy of Sciences (India)

    Amit Kumar; Atanu Mitra; Amrendra Kumar Ajay; Manoj Kumar Bhat; Chebrolu P Rao

    2012-11-01

    Binding of metal complexes of C2-glucosyl conjugates with DNA has been established by absorption and fluorescence studies. Conformational changes occurred in DNA upon binding have been studied by circular dichroism. All these studies are suggestive that the metal complexes bind to DNA through intercalation. Binding of di-nuclear copper complex 5 was found to be stronger when compared to the other complexes studied. Copper complexes were found to cleave the plasmid DNA in the absence of oxidizing or reducing agent, whereas, zinc complexes do not cleave. Metal complexes have shown toxicity to the HeLa and MCF-7 cell lines.Morphological studies, western blot and FACS analysis are suggestive of apoptotic cell death induced by the metal complexes. Di-nuclear copper complexes were found to be better as compared to the mononuclear ones in binding, plasmid cleavage and also in causing more cell death.

  13. Determination of Plasmid Segregational Stability in a Growing Bacterial Population.

    Science.gov (United States)

    Kramer, M Gabriela

    2016-01-01

    Bacterial plasmids are extensively used as cloning vectors for a number of genes for academic and commercial purposes. Moreover, attenuated bacteria carrying recombinant plasmids expressing genes with anti-tumor activity have shown promising therapeutic results in animal models of cancer. Equitable plasmid distribution between daughter cells during cell division, i.e., plasmid segregational stability, depends on many factors, including the plasmid copy number, its replication mechanism, the levels of recombinant gene expression, the type of bacterial host, and the metabolic burden associated with all these factors. Plasmid vectors usually code for antibiotic-resistant functions, and, in order to enrich the culture with bacteria containing plasmids, antibiotic selective pressure is commonly used to eliminate plasmid-free segregants from the growing population. However, administration of antibiotics can be inconvenient for many industrial and therapeutic applications. Extensive ongoing research is being carried out to develop stably-inherited plasmid vectors. Here, I present an easy and precise method for determining the kinetics of plasmid loss or maintenance for every ten generations of bacterial growth in culture. PMID:26846807

  14. Plasmid transfer between bacteria in soil microcosms and the field

    Directory of Open Access Journals (Sweden)

    Eric Smit

    1997-01-01

    Full Text Available In ibis review factors influencing conjugal plasmid transfer between bacteria and the possible role of naturally occurring selftransmissible plasmide for the dissemination of recombinant DNA in soil will be discussed. In microcosm studies, plasmid transfer between various species of introduced bacteria has been detected. Moreover, plamid transfer to indigenous soil micoorganisms was observed. Soil is an oligotrophic environment and plasmid transfer occurred mainly under conditions which were nutritionally favourable for bacteria, such as in the plant rhizosphere and in the presence of clay minerais or added nutrients. Mobilizable plasmids, lacking the ability to transfer themselves, have been reported to be transferred in the presence of selftransmissible plasmids. A study comparing conjugal transfer in microcosme with those in the field revealed that the transfer rates found in microcosme and in the field were similar. Transfer of chromosomal DNA by plasmid RP4 could only be shown on filters and was not observed in soil. Transfer of plasmids carrying biodegradative genes appeared to be favoured in the presence of the compound that can be degraded. Evidence was found for the presence of naturally-occurring selftransmissible plasmids in bacteria in the rhizosphere which could mobilize recombinant plasmids.

  15. Convex mappings on some circular domains

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    In this paper,we consider some circular domains.And we give an extension theorem for some normalized biholomorphic convex mapping on some circular domains.Especially,we discover the normalized biholomorphic convex mapping on some circular domains have the form f(z) =(f1(z1),...,fn(zn)),where fj:D → C are normalized biholomorphic convex mapping.

  16. Characterization of toxin plasmids in Clostridium perfringens type C isolates.

    Science.gov (United States)

    Gurjar, Abhijit; Li, Jihong; McClane, Bruce A

    2010-11-01

    Clostridium perfringens type C isolates cause enteritis necroticans in humans or necrotizing enteritis and enterotoxemia in domestic animals. Type C isolates always produce alpha toxin and beta toxin but often produce additional toxins, e.g., beta2 toxin or enterotoxin. Since plasmid carriage of toxin-encoding genes has not been systematically investigated for type C isolates, the current study used Southern blot hybridization of pulsed-field gels to test whether several toxin genes are plasmid borne among a collection of type C isolates. Those analyses revealed that the surveyed type C isolates carry their beta toxin-encoding gene (cpb) on plasmids ranging in size from ∼65 to ∼110 kb. When present in these type C isolates, the beta2 toxin gene localized to plasmids distinct from the cpb plasmid. However, some enterotoxin-positive type C isolates appeared to carry their enterotoxin-encoding cpe gene on a cpb plasmid. The tpeL gene encoding the large clostridial cytotoxin was localized to the cpb plasmids of some cpe-negative type C isolates. The cpb plasmids in most surveyed isolates were found to carry both IS1151 sequences and the tcp genes, which can mediate conjugative C. perfringens plasmid transfer. A dcm gene, which is often present near C. perfringens plasmid-borne toxin genes, was identified upstream of the cpb gene in many type C isolates. Overlapping PCR analyses suggested that the toxin-encoding plasmids of the surveyed type C isolates differ from the cpe plasmids of type A isolates. These findings provide new insight into plasmids of proven or potential importance for type C virulence. PMID:20823204

  17. Energy dissipation in circular tube

    Directory of Open Access Journals (Sweden)

    A.D. Girgidov

    2012-01-01

    Full Text Available Energy dissipation distribution along the circular tube radius is important in solving such problems as calculation of heat transfer by the air flow through building envelope; calculation of pressure loss in spiral flows; calculation of cyclones with axial and tangential supply of dust-containing gas.Two types of one-dimensional radially axisymmetric flows in circular tube were considered: axial flow and rotation about the axis (Rankine vortex. Relying on two- and four-layer description of axial turbulent flow energy dissipation was calculated in each layer.Similar calculation for Rankine vortex with viscous sublayer at the tube surface was provided. By employing the dissipation minimum principle the boundary radius between rigid rotation and free vortex is calculated. Approximation of the velocity distribution in Rankine vortex is proposed.

  18. Capacitance of circular patch resonator

    International Nuclear Information System (INIS)

    In this paper the capacitance of the circular microstrip patch resonator is computed. It is shown that the electrostatic problem can be formulated as a system of dual integral equations, and the most interesting techniques of solutions of these systems are reviewed. Some useful approximated formulas for the capacitance are derived and plots of the capacitance are finally given in a wide range of dielectric constants

  19. Capacitance of circular patch resonator

    Energy Technology Data Exchange (ETDEWEB)

    Miano, G.; Verolino, L. [Dip. di Ingegneria Elettrica, Ist. Nazionale di Fisica Nucleare, Naples (Italy); Panariello, G. [Dip. di Ingegneria Elettronica, Naples (Italy); Vaccaro, V.G. [Ist. Nazionale di Fisica Nucleare, Naples (Italy). Dipt. di Scienze Fisiche

    1995-11-01

    In this paper the capacitance of the circular microstrip patch resonator is computed. It is shown that the electrostatic problem can be formulated as a system of dual integral equations, and the most interesting techniques of solutions of these systems are reviewed. Some useful approximated formulas for the capacitance are derived and plots of the capacitance are finally given in a wide range of dielectric constants.

  20. Longitudinal motion in circular accelerators

    International Nuclear Information System (INIS)

    A general description is given of the longitudinal motion of an idealized synchronous particle, exactly in step with the radiofrequency field of a circular accelerator, and a domain in phase and energy, called a bucket, around this particle within which particles are focused around the synchronous particle. This general picture is then made more precise and quantitative. The equations of longitudinal motion and their solutions and the resulting motion are discussed, followed by applications and amplication of the theory. 7 refs., 8 figs

  1. Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells.

    OpenAIRE

    Gerdes, K; Rasmussen, P. B.; Molin, S

    1986-01-01

    The stability locus parB+ of plasmid R1 has been found to specify a unique type of plasmid maintenance function. Two genes, hok (host killing) and sok (suppressor of killing), are required for the stabilizing activity. The hok gene encodes a highly toxic gene product, whose overexpression causes a rapid killing and a concomitant dramatic change in morphology of the host cell. The other gene, sok, was found to encode a product that counteracts the hok gene-mediated killing. The parB+ region wa...

  2. Molecular characterization of "plasmid-free" antibiotic-resistant Haemophilus influenzae.

    OpenAIRE

    Roberts, M C; Smith, A. L.

    1980-01-01

    We examined 14 multiresistant and 8 ampicillin- or tetracycline-resistant Haemophilus influenzae isolates and 4 ampicillin-resistant H. parainfluenzae isolates for plasmid deoxyribonucleic acid. Sixteen strains carried plasmids. Both "plasmid-free" and plasmid-carrying isolates transferred the antibiotic resistance by conjugation. All transconjugants carried plasmid deoxyribonucleic acid, suggesting that the apparent plasmid-free strains contained R plasmids encoding for antibiotic resistance.

  3. Circular Polarization in Pulsar Integrated Profiles: Updates

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    We update the systematic studies of circular polarization in integrated pulse profiles by Han et al. Data of circular polarization profiles are compiled. Sense reversals can occur in core or cone components, or near the intersection between components. The correlation between the sense of circular polarization and the sense of position angle variation for conal-double pulsars is confirmed with a much large database. Circular polarization of some pulsars has clear changes with frequency.Circular polarization of millisecond pulsars is marginally different from that of normal pulsars.

  4. Safety and efficacy of DNA vaccines: Plasmids vs. minicircles

    OpenAIRE

    Stenler, Sofia; Blomberg, Pontus; Smith, Ci Edvard

    2014-01-01

    While DNA vaccination using plasmid vectors is highly attractive, there is a need for further vector optimization regarding safety, stability, and efficiency. In this commentary, we review the minicircle vector (MC), which is an entity devoid of plasmid bacterial sequences, as an alternative to the traditional plasmid construct. The commentary highlights the recent discovery by Stenler et al. (2014) that the small size of an MC enables improved resistance to the shearing forces associated wit...

  5. Pathogenomics of the Virulence Plasmids of Escherichia coli

    OpenAIRE

    Johnson, Timothy J.; Lisa K. Nolan

    2009-01-01

    Summary: Bacterial plasmids are self-replicating, extrachromosomal elements that are key agents of change in microbial populations. They promote the dissemination of a variety of traits, including virulence, enhanced fitness, resistance to antimicrobial agents, and metabolism of rare substances. Escherichia coli, perhaps the most studied of microorganisms, has been found to possess a variety of plasmid types. Included among these are plasmids associated with virulence. Several types of E. col...

  6. Identification of plasmid partition function in coryneform bacteria.

    OpenAIRE

    Kurusu, Y; Satoh, Y.; Inui, M.; Kohama, K; Kobayashi, M.; Terasawa, M.; Yukawa, H

    1991-01-01

    We have identified and characterized a partition function that is required for stable maintenance of plasmids in the coryneform bacteria Brevibacterium flavum MJ233 and Corynebacterium glutamicum ATCC 31831. This function is localized to a HindIII-NspV fragment (673 bp) adjacent to the replication region of the plasmid, named pBY503, from Brevibacterium stationis IFO 12144. The function was independent of copy number control and was not associated directly with plasmid replication functions. ...

  7. Transformation of Bacillus thuringiensis protoplasts by plasmid deoxyribonucleic acid.

    OpenAIRE

    Martin, P A; Lohr, J. R.; Dean, D H

    1981-01-01

    A method has been developed to transform plasmid deoxyribonucleic acid into protoplasts of the insect pathogen Bacillus thuringiensis. Protoplasts were formed by treatment of cells with lysozyme. The efficiency of formation of protoplasts was affected by the strain, the media, and the cell density. Deoxyribonucleic acid uptake was induced by polyethylene glycol. Deoxyribonucleic acid from the Staphylococcus aureus plasmid pC194 was used for transformation. Although this plasmid could not be i...

  8. Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

    OpenAIRE

    Pueschel, Laura; Li, Hongshan; Hymes, Matthew

    2011-01-01

    We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine ...

  9. Comparative genetic organization of incompatibility group P degradative plasmids.

    OpenAIRE

    Burlage, R S; Bemis, L A; Layton, A C; Sayler, G. S.; Larimer, F

    1990-01-01

    Plasmids that encode genes for the degradation of recalcitrant compounds are often examined only for characteristics of the degradative pathways and ignore regions that are necessary for plasmid replication, incompatibility, and conjugation. If these characteristics were known, then the mobility of the catabolic genes between species could be predicted and different catabolic pathways might be combined to alter substrate range. Two catabolic plasmids, pSS50 and pSS60, isolated from chlorobiph...

  10. VUV irradiation studies of plasmid DNA in aqueous solution

    Science.gov (United States)

    Śmialek, M. A.; Hoffmann, S. V.; Folkard, M.; Prise, K. M.; Shuker, D. E. G.; Braithwaite, N. S.; Mason, N. J.

    2008-02-01

    Interactions of VUV light and DNA samples in aqueous solutions are reported. The damage induced by such radiation is quantified by monitoring both loss of supercoiled DNA and formation of single and double strand breaks using agarose gel electrophoresis. Irradiations were performed using synchrotron VUV photons of 130, 150, 170 and 190 nm. VUV irradiation experiments revealed enhanced damage upon irradiation with 170 nm photons as compared with irradiations with photons of 150 nm and 130 nm. Irradiations carried at 190 nm caused the least damage.

  11. VUV irradiation studies of plasmid DNA in aqueous solution

    International Nuclear Information System (INIS)

    Interactions of VUV light and DNA samples in aqueous solutions are reported. The damage induced by such radiation is quantified by monitoring both loss of supercoiled DNA and formation of single and double strand breaks using agarose gel electrophoresis. Irradiations were performed using synchrotron VUV photons of 130, 150, 170 and 190 nm. VUV irradiation experiments revealed enhanced damage upon irradiation with 170 nm photons as compared with irradiations with photons of 150 nm and 130 nm. Irradiations carried at 190 nm caused the least damage

  12. Construction of a eukaryotic expression plasmid of Humanin*

    OpenAIRE

    Luo, Ben-yan; Chen, Xiang-ming; Tang, Min; Chen, Feng; Chen, Zhi

    2004-01-01

    Objective: To construct a eukaryotic expression plasmid pcDNA3.1(-)-Humanin. Methods: The recombinant plasmid pGEMEX-1-Humanin was digested with restriction endonucleases BamH I and Hind III and the Humanin gene fragments, about 100 bp length, were obtained. Then the Humanin gene fragments were inserted into eukaryotic expression vector pcDNA3.1(-) and the recombinant plasmids pcDNA3.1(-)-Humanin were identified by sequencing. Results: Recombinant plasmid DNA successfully produced a band whic...

  13. Characterization of a plasmid from moderately halophilic eubacteria

    OpenAIRE

    Fernández Castillo, Rosario; Vargas, C.; Nieto Gutiérrez, Joaquín José; Ventosa Ucero, Antonio; Ruiz Berraquero, Francisco

    1992-01-01

    A plasmid has been isolated for the first time from moderately halophilic eubacteria. Halomonas elongata, Halomonrrs halmphila, Deleya halophila and Vibrio costkola were found to harbour an 11.5 kbp plasmid (pMH1). The plasmid was isolated and characterized after transformation into Escherichia coh'JM101 cells. A restriction map was constructed, and unique restriction sites for EcoRI, EcoRV and ClaI were detected. The occurrence of such a plasmid in the original halophilic strains was confirm...

  14. Characterization of Chlamydia trachomatis Plasmid-Encoded Open Reading Frames

    OpenAIRE

    Gong, Siqi; Yang, Zhangsheng; Lei, Lei; Shen, Li; Zhong, Guangming

    2013-01-01

    The recent success in transformation of Chlamydia trachomatis represents a major advancement in Chlamydia research. Plasmid-free C. trachomatis serovar L2 organisms can be transformed with chlamydial plasmid-based shuttle vectors pGFP::SW2 and pBRCT. Deletion of plasmid genes coding for Pgp1 to Pgp8 in pBRCT led to the identification of Pgp1, -2, -6, and -8 as plasmid maintenance factors; Pgp4 as a transcriptional regulator of chlamydial virulence-associated gene expression; and Pgp3, -5, and...

  15. Plasmid P1 replication: negative control by repeated DNA sequences.

    OpenAIRE

    Chattoraj, D; Cordes, K.; Abeles, A

    1984-01-01

    The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pi...

  16. Identification of plasmid partition function in coryneform bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Kurusu, Yasurou; Satoh, Yukie; Inui, Masayuki; Kohama, Keiko; Kobayashi, Miki; Terasawa, Masato; Yukawa, Hideaki (Mitsubishi Petrochemical Co., Ltd., Ibaraki (Japan))

    1991-03-01

    The authors have identified and characterized a partition function that is required for stable maintenance of plasmids in the coryneform bacteria Brevibacterium flavum MJ233 and Corynebacterium glutamicum ATCC 31831. This function is localized to a HindIII-NspV fragment (673 bp) adjacent to the replication region of the plasmid, named pBY503, from Brevibacterium stationis IFO 12144. The function was independent of copy number control and was not associated directly with plasmid replication functions. This fragment was able to stabilize the unstable plasmids in cis but not in trans.

  17. Degradative Plasmid and Heavy Metal Resistance Plasmid Naturally Coexist in Phenol and Cyanide Assimilating Bacteria

    OpenAIRE

    Bahig E.  Deeb; Abdullah D. Altalhi

    2009-01-01

    Problem statement: Heavy metals are known to be powerful inhibitors of xenobiotics biodegradation activities. Alleviation the inhibitory effect of these metals on the phenol biodegradation activities in presence of heavy metals resistant plasmid was investigated. Approach: Combination of genetic systems of degradation of xenobiotic compound and heavy metal resistance was one of the approaches to the creation of polyfunctional strains for bioremediation of s...

  18. Photonic plasmid stability of transformed Salmonella Typhimurium: A comparison of three unique plasmids

    Directory of Open Access Journals (Sweden)

    Lay Donald

    2009-07-01

    Full Text Available Abstract Background Acquiring a highly stable photonic plasmid in transformed Salmonella Typhimurium for use in biophotonic studies of bacterial tracking in vivo is critical to experimental paradigm development. The objective of this study was to determine stability of transformed Salmonella Typhimurium (S. typh-lux using three different plasmids and characterize their respective photonic properties. Results In presence of ampicillin (AMP, S. typh-lux with pCGLS-1, pAK1-lux and pXEN-1 plasmids exhibited 100% photon-emitting colonies over a 10-d study period. Photon emitters of S. typh-lux with pCGLS-1, pAK1-lux and pXEN-1 without AMP selection decreased over time (P 7 to 1 × 109 CFU, P 0.05; although photonic emissions across a range of bacterial concentrations were not different (1 × 104 to 1 × 106 CFU, P > 0.05. For very low density bacterial concentrations imaged in 96 well plates photonic emissions were positively correlated with bacterial concentration (P 3 to 1 × 105 CFU low to high were different in the 96-well plate format (P Conclusion These data characterize photon stability properties for S. typh-lux transformed with three different photon generating plasmids that may facilitate real-time Salmonella tracking using in vivo or in situ biophotonic paradigms.

  19. Photonic Plasmid Stability of Transformed Salmonella Typhimurium: A Comparison of Three Unique Plasmids

    Science.gov (United States)

    Background: Acquiring a highly stable photonic plasmid in transformed Salmonella Typhimurium for use in biophotonic studies of bacterial tracking in vivo is critical to experimental paradigm development. The objective of this study was to determine stability of transformed Salmonella Typhimurium (S....

  20. Photonic plasmid stability of transformed Salmonella typhimurium: A comparison of three unique plasmids

    Science.gov (United States)

    Acquiring a highly stable photonic plasmid in transformed Salmonella typhimurium for use in biophotonic studies of bacterial tracking in vivo is critical to experimental paradigm development. The objective of this study was to determine stability of transformed Salmonella typhimurium (S. typh-lux) u...

  1. Bacterial Mitosis: ParM of Plasmid R1 Moves Plasmid DNA by an Actin-like Insertional Polymerization Mechanism

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette;

    2003-01-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system...... act together to generate the force required for plasmid movement during segregation. ParR protein binds cooperatively to the centromeric parC DNA region, thereby forming a complex that interacts with the filament-forming actin-like ParM protein in an ATP-dependent manner, suggesting that plasmid...... movement is powered by insertional polymerization of ParM. Consistently, we find that segregating plasmids are positioned at the ends of extending ParM filaments. Thus, the process of R1 plasmid segregation in E. coli appears to be mechanistically analogous to the actin-based motility operating...

  2. Metagenomic analyses of novel viruses and plasmids from a cultured environmental sample of hyperthermophilic neutrophiles

    DEFF Research Database (Denmark)

    Garrett, Roger Antony; Prangishvili, David; Shah, Shiraz Ali;

    2010-01-01

    Two novel viral genomes and four plasmids were assembled from an environmental sample collected from a hot spring at Yellowstone National Park, USA, and maintained anaerobically in a bioreactor at 85°C and pH 6. The double-stranded DNA viral genomes are linear (22.7 kb) and circular (17.7 kb......), and derive apparently from archaeal viruses HAV1 and HAV2. Genomic DNA was obtained from samples enriched in filamentous and tadpole-shaped virus-like particles respectively. They yielded few significant matches in public sequence databases reinforcing, further, the wide diversity of archaeal viruses....... Several variants of HAV1 exhibit major genomic alterations, presumed to arise from viral adaptation to different hosts. They include insertions up to 350 bp, deletions up to 1.5 kb, and genes with extensively altered sequences. Some result from recombination events occurring at low complexity direct...

  3. Degradative Plasmid and Heavy Metal Resistance Plasmid Naturally Coexist in Phenol and Cyanide Assimilating Bacteria

    Directory of Open Access Journals (Sweden)

    Bahig E.  Deeb

    2009-01-01

    Full Text Available Problem statement: Heavy metals are known to be powerful inhibitors of xenobiotics biodegradation activities. Alleviation the inhibitory effect of these metals on the phenol biodegradation activities in presence of heavy metals resistant plasmid was investigated. Approach: Combination of genetic systems of degradation of xenobiotic compound and heavy metal resistance was one of the approaches to the creation of polyfunctional strains for bioremediation of soil after co-contamination with organic pollutants and heavy metals. Results: A bacterial strain Pseudomonas putida PhCN (pPhCN1, pPhCN2 had been obtained. This bacterium contained two plasmids, a 120 Kb catabolic plasmid that encode for breakdown of phenol (pPhCN1 and pPhCN2 plasmid (100 Kb that code for cadmium and copper resistant. Cyanide assimilation by this bacterium was encoded by chromosomal genes. The inhibitory effect of cadmium (Cd2+ or copper (Cu2+ on the degradation of phenol and cyanide by P. putida strains PhCN and PhCN1 (contained pPhCN1 were investigated. The resistant strain PhCN showed high ability to degrade phenol and cyanide in presence of Cd2+ or Cu2+ comparing with the sensitive strain PhCN1. In addition, Cd2+ or Cu2+ was also found to exert a strong inhibitory effect on the C23O dioxygenase enzyme activity in the presence of cyanide as a nitrogen source. Conclusion: The presence of heavy metal resistance plasmid alleviated the inhibitory effect of metals on the phenol and cyanide assimilation by resistant strain.

  4. Induced mutagenesis of plasmids and chromosomal genes inserted into plasmid DNA 1. Mutagenic effects of irradiations

    International Nuclear Information System (INIS)

    Effect of two physical agents: UV- and γ-radiation has been considered in comparison. DNA of RSF2124 plasmid, determining colcine synthesis and ampicillin resistance, was used as a model. Mutagenous effect is taken into account according to the appearance of Col--mutants, which are not capable of colicine synthesis. Lethal effect is determined according to ampicillin marker inactivation. After reisolation of plasmid DNA from mutant transformant, new traits and antibiotic resistance are preserved during subsequent transformations and reseedings of transformed colonies, which proves mutational nature of the transformations. Under short-wave UV irradiation (lambda=254 nm) of RSF2124 DNA a clear mutagenous effect is detected: relative amount of Col--mutants at the optimum for mutagenesis doses increased by a factor of 10. Under conditions of W-reactivation (additional UV-irradiation of recipient cells of wild C600 type) of lethal injuries an increase in mutagenous effect was observed, which is reliable for 95%. A distinct increase in mutagenesis (approximately by a factor of 4) is observed during UV-irradiation in small doses of only one recipient cell (a so-called indirect UV-mutagenesis). Thus, according to its ability to W- and indirect UV-mutagenesis plasmid DNA behaves as DNA of moderate phages, which can testify to their evolution relationship. Treatment of plasmid DNA with acridine orange before UV-irradiation protected only from lethal injuries. γ-irradiation of 60Co at inactivation approximately 10-2 increased by an order the yield of Col--mutants. The presence of the plasmid in a cell did not affect its UV-resistance

  5. Deterministic Circular Self Test Path

    Institute of Scientific and Technical Information of China (English)

    WEN Ke; HU Yu; LI Xiaowei

    2007-01-01

    Circular self test path (CSTP) is an attractive technique for testing digital integrated circuits(IC) in the nanometer era, because it can easily provide at-speed test with small test data volume and short test application time. However, CSTP cannot reliably attain high fault coverage because of difficulty of testing random-pattern-resistant faults. This paper presents a deterministic CSTP (DCSTP) structure that consists of a DCSTP chain and jumping logic, to attain high fault coverage with low area overhead. Experimental results on ISCAS'89 benchmarks show that 100% fault coverage can be obtained with low area overhead and CPU time, especially for large circuits.

  6. Mutations in an antisense RNA, involved in the replication control of a repABC plasmid, that disrupt plasmid incompatibility and mediate plasmid speciation.

    Science.gov (United States)

    Rivera-Urbalejo, América; Pérez-Oseguera, Ángeles; Carreón-Rodríguez, Ofelia E; Cevallos, Miguel A

    2015-03-01

    The maintenance of large plasmid in a wide variety of alpha-proteobacteria depends on the repABC replication/segregation unit. The intergenic repB-repC region of these plasmids encodes a countertranscribed RNA (ctRNA) that modulates the transcription/translation rate of RepC, the initiator protein. The ctRNA acts as a strong incompatibility factor when expressed in trans. We followed a site directed mutagenesis approach to map those sequences of the ctRNA that are required for plasmid incompatibility and for plasmid replication control. We found that the first three nucleotides of the 5'-end of the ctRNA are essential for interactions with its target RNA. We also found that stretches of 4-5 nucleotides of non-complementarity within the first 10 nucleotides of the left arm of the ctRNA and the target RNA are sufficient to avoid plasmid incompatibility. Additionally, miniplasmid derivatives expressing ctRNAs with mutations in the 5' end or small deletions in the ctRNA are capable of controlling their own replication and coexisting with the parental plasmid. We suggest that a mechanism that could have a crucial role in the speciation process of repABC plasmids is to accumulate enough changes in this small region of the ctRNA gene to disrupt heteroduplex formation between the target RNA of one plasmid and the ctRNA of the other. Plasmids carrying these changes will not have defects in their maintenance. PMID:25644116

  7. Damage to dry plasmid DNA induced by nanosecond XUV-laser pulses

    Science.gov (United States)

    Nováková, Eva; Davídková, Marie; Vyšín, Ludék; Burian, Tomáš; Grisham, Michael E.; Heinbuch, Scott; Rocca, Jorge J.; Juha, Libor

    2011-06-01

    Ionizing radiation induces a variety of DNA damages including single-strand breaks (SSBs), double-strand breaks (DSBs), abasic sites, modified sugar and bases. Most theoretical and experimental studies have been focused on DNA strand scissions, in particular production of DNA double-strand breaks. DSBs have been proven to be a key damage at a molecular level responsible for the formation of chromosomal aberrations, leading often to cell death. The complexity of lesions produced in DNA by ionizing radiations is thought to depend on the amount of energy deposited at the site of each lesion. We have studied the nature of DNA damage induced directly by the pulsed 46.9 nm radiation provided by a capillary-discharge Ne-like Ar laser (CDL). Different surface doses were delivered with a repetition rate of a few Hz and an average pulse energy ~ 1 μJ. A simple model DNA molecule, i.e., dried closed-circular plasmid DNA (pBR322), was irradiated. The agarose gel electrophoresis method was used for determination of both SSB and DSB yields. Results are compared with a previous study of plasmid DNA irradiated with a single sub-nanosecond 1-keV X-ray pulse produced by a large-scale, double-stream gas puff target, illuminated by sub-kJ, near-infrared (NIR) focused laser pulses at the PALS facility (Prague Asterix Laser System).

  8. Effects of 32 P incorporated in plasmid DNA: strand breaks and mutagenesis

    International Nuclear Information System (INIS)

    In order to study the 32 P decay effects in DNA, bacterial plasmid were labeled with different activities of the radioisotope in vivo: 1,2 and 6 x 105 Bk/ml of bacterial culture, leading to 1,2 and 6 x 103 Bk/μg of nucleic acid or in vitro: 0.7, 1.5 and 3.5 x 103 Bk/μg of nucleic acid, stored at -20 deg C and its electroforetic profiles, transformation capacity of wild type and DNA repair. E. coli mutants cells and mutagenesis, were followed during three months. The results achieved in this work suggest that: the decay of the incorporated 32 P in vivo is able to change the pBR322 electroforetic profile, we detected a decrease on the form III (super coiled) and increase on the form II (circular), indicating single strands breaks; the decay incorporated 32 in vitro does not modify the electrophoretic profile of pBR322, suggesting that in some way the effects of the radioactive decay of incorporated 32 P is dependent of the DNA topology, the damages induced by 32 P decay increase mutation frequency in pAC189 plasmids. MRF is increased by a factor of three after 6 t1/2 of storage, indicating direct or indirect action through mismatch DNA repair pathway. (author)

  9. An antiviral protein having deoxyribonuclease and ribonuclease activity from leaves of the post-flowering stage of Celosia cristata.

    Science.gov (United States)

    Begam, M; Narwal, S; Roy, S; Kumar, S; Lodha, M L; Kapoor, H C

    2006-01-01

    An antiviral protein named CCP-27 was purified from the leaves of Celosia cristata at the post-flowering stage by anion-exchange, cation-exchange, and gel-filtration chromatography. It exhibited resistance against sunnhemp rosette virus in its test host Cyamopsis tetragonoloba. It also exhibited deoxyribonuclease activity against supercoiled pBlueScript SK+ plasmid DNA. It was found to nick supercoiled DNA into nicked circular form at lower protein concentration followed by nicked to linear form conversion at higher protein concentration. CCP-27 also possesses strong ribonuclease activity against Torula yeast rRNA. PMID:16487067

  10. Study on transformation of anti-nasopharyngeal carcinoma plasmid pFY and bacterial strains screening%抗鼻咽癌质粒 pFY 转化和菌种筛选的研究

    Institute of Scientific and Technical Information of China (English)

    翁闪凡; 刘娜; 张晓林

    2015-01-01

    目的:筛选携带抗鼻咽癌质粒 pFY 的稳定高产菌株。方法以 CaCl2法制备大肠杆菌 JM109感受态,将抗鼻咽癌质粒 pFY 转化 JM109感受态,对琼脂平板上获得的菌落进行筛选,选出符合标准的单菌落为菌种,进行菌种稳定性实验。用质粒提取试剂盒检测质粒含量。将抗鼻咽癌质粒 pFY 转染到细胞 CNE-2中,四氮唑蓝(MTT)比色法观察转染试剂及质粒载体对细胞生长增殖的影响。结果筛选得到菌株的培养液中质粒 DNA 含量为30 mg/mL,超螺旋 DNA 比例为92%。经电泳和酶切鉴定,该菌株的50子代所携带质粒与原代一致。质粒 pFY 对 CNE-2细胞株生长有明显的抑制作用。结论成功筛选出携带抗鼻咽癌质粒 pFY 的稳定高产菌株,为大批量制备临床应用级质粒奠定了基础。%Objective To screen the stable high-producing strains carrying anti-nasopharyngeal carcinoma(NPC)plasmid pFY.Methods Competent E.coli JM109 was prepared by the CaCl2 method and transformed with anti-NPC plasmid pFY.The bacterial colonies obtained from the agar plate were screened for selecting the single colony conforming to the standards as the bac-terial strain and conducting the stability test.The plasmid content was detected by the plasmid extraction reagent kit.Anti-NPC plasmid pFY was transfected into nasopharynegal carcinoma cell line CNE-2.The influence of transfection reagent and the plasmid vector on the cell proliferation was detected by MTT.Results The DNA concentration of plasmids in the culture solution of bacte-rial strain obtained by screening was 30 mg/mL.The proportion of supercoiled DNA was 92%.The identification of electrophoresis and restriction enzymes showed that the plasmids harbored in the 50th progeny of this strain were same as those in the primary. Plasmid pFY had the evident inhibiting effect on the growth of CNE-3 cell line.Conclusion The stable high-producing strains of E. coli carrying anti-NPC plasmid pFY is

  11. Physics at Future Circular Colliders

    Science.gov (United States)

    Kotwal, Ashutosh

    2016-03-01

    The Large Hadron Collider has been a grand success with the discovery of the Higgs boson, with bright prospects for additional discoveries since the recent increase in collider energy and the anticipated large datasets. Big open questions such as the nature of dark matter, the origin of the matter-antimatter asymmetry in the Universe, and the theoretical puzzle of the finely-tuned parameters in the Higgs sector, demand new physics principles that extend the established Standard Model paradigm. Future circular colliders in a substantially larger tunnel can house both a high luminosity electron-positron collider for precision measurements of Higgs and electroweak parameters, as well as a very high energy proton-proton collider which can directly manifest particles associated with these new physics principles. We discuss the physics goals of these future circular colliders, and the prospects for elucidating fundamental new laws of nature that will significantly extend our understanding of the Universe. Detailed studies of the discovery potential in specific benchmark models will be presented, with implications for detector design.

  12. Construction and Application of R Prime Plasmids, Carrying Different Segments of an Octopine Ti Plasmid from Agrobacterium tumefaciens, for Complementation of vir Genes

    NARCIS (Netherlands)

    Hille, Jacques; Klasen, Ina; Schilperoort, Rob

    1982-01-01

    Several R prime plasmids have been obtained with high efficiency, by enclosing the R plasmid replicator, in an R::Ti cointegrate plasmid, between two copies of the transposon Tn1831, in the same orientation. These R primes carry different segments of an octopine Ti plasmid, and are compatible with T

  13. The Streptomyces leeuwenhoekii genome: de novo sequencing and assembly in single contigs of the chromosome, circular plasmid pSLE1 and linear plasmid pSLE2

    OpenAIRE

    Gomez-Escribano, Juan Pablo; Castro, Jean Franco; Razmilic, Valeria; Chandra, Govind; Andrews, Barbara; Asenjo, Juan A.; Bibb, Mervyn J.

    2015-01-01

    Background Next Generation DNA Sequencing (NGS) and genome mining of actinomycetes and other microorganisms is currently one of the most promising strategies for the discovery of novel bioactive natural products, potentially revealing novel chemistry and enzymology involved in their biosynthesis. This approach also allows rapid insights into the biosynthetic potential of microorganisms isolated from unexploited habitats and ecosystems, which in many cases may prove difficult to culture and ma...

  14. Mixing in Circular and Non-circular Jets in Crossflow

    DEFF Research Database (Denmark)

    Salewski, Mirko; Stankovic, D.; Fuchs, L.

    2008-01-01

    are compared for circular, elliptic, and square nozzles. For the latter configurations, effects of orientation are considered. The computations reveal that the distribution of a passive scalar in a cross-sectional plane can be single- or double-peaked, depending on the nozzle shape and orientation. A proper...... orthogonal decomposition of the transverse velocity indicates that coherent structures may be responsible for this phenomenon. Nozzles which have a single-peaked distribution have stronger modes in transverse direction. The global mixing performance is superior for these nozzle types. This is the case...... for the blunt square nozzle and for the elliptic nozzle with high aspect ratio. It is further demonstrated that the flow field contains large regions in which a passive scalar is transported up the mean gradient (counter-gradient transport) which implies failure of the gradient diffusion hypothesis....

  15. Production and pharmaceutical formulation of plasmid DNA vaccines

    NARCIS (Netherlands)

    van der Heijden, I.

    2013-01-01

    Research leading to the thesis ‘Production and pharmaceutical formulation of plasmid DNA vaccines‘ can be divided into two parts. The first part describes the development of a Good Manufacturing Practice (GMP) compliant plasmid DNA production process of pDNA vaccines for the treatment of Human papil

  16. Plasmid-encoded copper resistance and precipitation by Mycobacterium scrofulaceum.

    OpenAIRE

    Erardi, F X; Failla, M L; Falkinham, J. O.

    1987-01-01

    A copper-tolerant Mycobacterium scrofulaceum strain was able to remove copper from culture medium by sulfate-dependent precipitation as copper sulfide. Such precipitation of copper sulfide was not observed in a derivative that lacks a 173-kilobase plasmid. In addition, the plasmid-carrying strain has a sulfate-independent copper resistance mechanism.

  17. Two novel conjugative plasmids from a single strain of Sulfolobus

    NARCIS (Netherlands)

    Erauso, G.; Stedman, K.M.; Werken, van de H.J.G.; Zillig, W.; Oost, van der J.

    2006-01-01

    Two conjugative plasmids (CPs) were isolated and characterized from the same 'Sulfolobus islandicus' strain, SOG2/4, The plasmids were separated from each other and transferred into Sulfolobus soltataricus. One has a high copy number and is not stable (pSOG1) whereas the other has a low copy number

  18. Inc A/C Plasmids in Multidrug resistant Salmonella enterica

    Science.gov (United States)

    Bacteria plasmids are fragments of extra-chromosomal double stranded deoxyribonucleic acid (DNA) that can contain a variety of genes beneficial to the survival of the host bacteria. Classification and tracking of plasmids is beneficial because they are potentially a medium of horizontal gene transf...

  19. Transformation of Bacillus subtilis by single-stranded plasmid DNA.

    OpenAIRE

    Rudolph, C F; Schmidt, B J; Saunders, C W

    1986-01-01

    The single-stranded form of a pE194-based plasmid transformed Bacillus subtilis protoplasts at least as efficiently as did the double-stranded plasmid, but the single-stranded form did not detectably transform B. subtilis competent cells.

  20. Curing of the 2 mu DNA plasmid from Saccharomyces cerevisiae.

    OpenAIRE

    Toh-e, A; Wickner, R B

    1981-01-01

    The 2 mu DNA plasmid is often eliminated from yeast cells when they are transformed with the 2 mu DNA-LEU2-pMB9 composite plasmid pJDB219. Since pJDB219 is subsequently lost with high frequency, derivatives lacking all 2 mu DNA can be prepared from any strain.

  1. CIRCULAR ECONOMY IN ROMANIA WITHIN EUROPEAN CONTEXT

    OpenAIRE

    Cornelia Marcela Danu; Valentin Nedeff

    2015-01-01

    In the present paper we have approached some conceptual and coordinated marks of the societal reality connected to the circular economy. Generated by „the limits of certainty” regarding the future of the world business, the operationalization of the circular economy has become a part of the EU strategies and started the various stages of implementation as an active process in all countries. We have highlighted the opportunities and the risks related to the circular economy, the European dimen...

  2. A COMPACT CIRCULARLY POLARIZED SLOTTED MICROSTRIP ANTENNA

    OpenAIRE

    V. Jebaraj; K.R.S. Ravi Kumar; D. Mohanageetha

    2014-01-01

    Slot antennas are often used at UHF and microwave frequencies. In slot antenna for RFID reader applications the frequency ranges from 902-923MHz to achieve circular polarization. The shapes and size of the slot, as well as the driving frequency, determine the radiation distribution pattern. The proposed compact size circularly polarized slotted microstrip antenna are summarized with design rules. The circularly polarized radiation in square patch antenna can be obtained by perturbation techni...

  3. Reconfigurable Monopole Antennas With Circular Polarization

    OpenAIRE

    Panahi, Afshin

    2015-01-01

    This thesis presents research on printed circularly-polarized monopole antennas and their application in reconfigurable monopole antennas. The proposed circularly-polarised monopole antennas benefit from advantages such as small size, low-cost, low-profile and simple designs. The first part of this thesis introduces three printed circularly-polarized monopole antennas for global navigation satellite systems and Wi-Fi applications. The primary focus is on the ground plane which is used as a ra...

  4. Plasmid DNA induces dodecyl triethyl ammonium bromide to aggregate into vesicle

    Institute of Scientific and Technical Information of China (English)

    Xiang Mei Ran; Xia Guo; Jia Tong Ding

    2012-01-01

    Single-chained cationic surfactant dodecyl triethyl ammonium bromide and plasmid DNA together can form vesicles once the concentration of plasmid DNA reaches a critical value (Ccvc).Bigger the size of plasmid DNA,higher the value of Ccvc.

  5. Construction of a eukaryotic expression plasmid of Humanin

    Institute of Scientific and Technical Information of China (English)

    LUO Ben-yan; CHEN Xiang-ming; TANG Min; CHEN Feng; CHEN Zhi

    2005-01-01

    Objective: To construct a eukaryotic expression plasmid pcDNA3.1 (-)-Humanin. Methods: The recombinant plasm pGEMEX- 1-Humanin was digested with restriction endonucleases BamH I and Hind Ⅲ and the Humanin gene fragments, abo 100 bp length, were obtained. Then the Humanin gene fragments were inserted into eukaryotic expression vector pcDNA3.1 (-) and the recombinant plasmids pcDNA3. l(-)-Humanin were identified by sequencing. Results: Recombinant plasmid DNA succesfully produced a band which had the same size as that of the Humanin positive control. The sequence of recombinant plasmids accorded with the Humnain gene sequence. Conclusions: A eukaryotic expression plasmid of Humanin was successfully constructed.

  6. Permissiveness of soil microbial communities towards broad host range plasmids

    DEFF Research Database (Denmark)

    Klümper, Uli

    of fluorescently tagged conjugative plasmids into a soil microbial community in solid-surface filter matings under maximized cell-to-cell contact, followed by quantification of transfer events through advanced fluorescent microscopy, isolation of transconjugants through triple-gated fluorescent activated cell...... using model BHR plasmid pKJK5 introduced through the γ-proteobacterial donor E. coli. I found that the permissiveness towards plasmids was modified through stress on a taxon specific basis and cannot be generally predicted for the whole community. The response of the phylogenetic group was specific...... between phylogenetically distant groups. Finally, I extended the high-throughput method to quantify the potential of a microbial community to actively mobilize and transfer exogenous mobilizable plasmids to its indigenous members. I evaluated the transfer frequency of model plasmid RSF1010 by comparing...

  7. Radiosensitivity of plasmid DNA: role of topology and concentration

    International Nuclear Information System (INIS)

    Using the plasmid relaxation assay, the induction of single strand breaks (SSB) by ionizing radiation was investigated in two plasmids of different length, pBS and pSP189. The dose-response was linear for both plasmids but pSP189 exhibited a three times higher sensitivity than mBS. This disparity may be explained by a reduced accessibility to hydroxyl radicals due to a different topology of each plasmid, i.e. degree of compaction, as observed with electron microscopy. pBS plasmid was also exposed at various DNA concentrations to γ rays. The yield of SSB decreased with increasing concentration, suggesting a diminution in the amount of hydroxyl radicals efficient for radiolytic attack. This effect of concentration was also observed with densely ionizing radiation. In conclusion, the accessibility of DNA is a key-parameter in the formation of damage in vitro and in vivo as well. (authors)

  8. Molecular alignment using circularly polarized laser pulses

    CERN Document Server

    Smeenk, C T L

    2013-01-01

    We show that circularly polarized femtosecond laser pulses produce field-free alignment in linear and planar molecules. We study the rotational wavepacket evolution of O$_2$ and benzene created by circularly polarized light. For benzene, we align the molecular plane to the plane of polarization. For O$_2$, we demonstrate that circular polarization yields a net alignment along the laser propagation axis at certain phases of the evolution. Circular polarization gives us the ability to control alignment of linear molecules outside the plane of polarization, providing new capabilities for molecular imaging.

  9. Characterization of a small cryptic plasmid from endophytic Pantoea agglomerans and its use in the construction of an expression vector

    Directory of Open Access Journals (Sweden)

    Procópio Rudi Emerson de Lima

    2011-01-01

    Full Text Available A circular cryptic plasmid named pPAGA (2,734 bp was isolated from Pantoea agglomerans strain EGE6 (an endophytic bacterial isolate from eucalyptus. Sequence analysis revealed that the plasmid has a G+C content of 51% and contains four potential ORFs, 238(A, 250(B, 131(C, and 129(D amino acids in length without homology to known proteins. The shuttle vector pLGM1 was constructed by combining the pPAGA plasmid with pGFPmut3.0 (which harbors a gene encoding green fluorescent protein, GFP, and the resulting construct was used to over-express GFP in E. coli and P. agglomerans cells. GFP production was used to monitor the colonization of strain EGE6gfp in various plant tissues by fluorescence microscopy. Analysis of EGE6gfp colonization showed that 14 days after inoculation, the strain occupied the inner tissue of Eucalyptus grandis roots, preferentially colonizing the xylem vessels of the host plants.

  10. Positive selection and compensatory adaptation interact to stabilize non-transmissible plasmids

    OpenAIRE

    Millan, A. San; Peña-Miller, R.; Toll-Riera, M.; Halbert, Z. V.; McLean, A R; Cooper, B. S.; Maclean, R. C.

    2014-01-01

    Plasmids are important drivers of bacterial evolution, but it is challenging to understand how plasmids persist over the long term because plasmid carriage is costly. Classical models predict that horizontal transfer is necessary for plasmid persistence, but recent work shows that almost half of plasmids are non-transmissible. Here we use a combination of mathematical modelling and experimental evolution to investigate how a costly, non-transmissible plasmid, pNUK73, can be maintained in popu...

  11. Requirements for the formation of plasmid-transducing particles of Bacillus subtilis bacteriophage SPP1.

    OpenAIRE

    Alonso, J.C.; Lüder, G; Trautner, T A

    1986-01-01

    We had previously proposed that the production of concatemeric plasmid DNA in plasmid-transducing SPP1 particles is a consequence of phage-directed rolling-circle-type replication of plasmid DNA. The production of such DNA was greatly enhanced when DNA/DNA homology was provided between phage and plasmid DNAs (facilitation of transduction). Here we present evidence that synthesis of concatemeric plasmid DNA can proceed after phage infection under conditions non-permissive for plasmid replicati...

  12. Construction of Streptococcus lactis subsp. lactis Strains with a Single Plasmid Associated with Mucoid Phenotype

    OpenAIRE

    von Wright, Atte; Tynkkynen, Soile

    1987-01-01

    Lactose-fermenting mucoid (Lac+ Muc+) variants of plasmid-free Streptococcus lactis subsp. lactis MG1614 were obtained by protoplast transformation with total plasmid DNA from Muc+S. lactis subsp. cremoris ARH87. By using plasmid DNA from these variants for further transformations followed by novobiocininduced plasmid curing, Lac− Muc+ MG1614 strains containing only a single 30-megadalton plasmid could be constructed. This plasmid, designated pVS5, appeared to be associated with the Muc+ phen...

  13. Plasmid stability in immobilized and free recombinant Escherichia coli JM105(pKK223-200): importance of oxygen diffusion, growth rate, and plasmid copy number.

    OpenAIRE

    de Taxis du Poët, P; Arcand, Y; Bernier, R.; Barbotin, J N; Thomas, D.

    1987-01-01

    Stability of the plasmid pKK223-200 in Escherichia coli JM105 was studied for both free and immobilized cells during continuous culture. The relationship between plasmid copy number, xylanase activity, which was coded for by the plasmid, and growth rate and culture conditions involved complex interactions which determined the plasmid stability. Generally, the plasmid stability was enhanced in cultured immobilized cells compared with free-cell cultures. This stability was associated with modif...

  14. Bifurcation Analysis of a Chemostat Model of Plasmid-Bearing and Plasmid-Free Competition with Pulsed Input

    Directory of Open Access Journals (Sweden)

    Zhong Zhao

    2014-01-01

    to the stability of the boundary periodic solution. By use of standard techniques of bifurcation theory, the periodic oscillations in substrate, plasmid-bearing, and plasmid-free organisms are shown when some conditions are satisfied. Our results can be applied to control bioreactor aimed at producing commercial producers through genetically altered organisms.

  15. La raó circular

    OpenAIRE

    Pinyol i Bori, Francesc

    2007-01-01

    És un text epistemològic sobre les qüestions cognitives circulars que apareixen: a) Quan el subjecte forma part de l'objecte estudiat; b) En la recerca de les bases del coneixement; c) Al voler justificar la inducció; d) En algunes interpretacions filosòfiques i científiques. S'hi explica l'estat del punt a) el més problemàtic, i s'aclareixen els altres tres punts: els b) i c) pel fet científic que l'evolució cognitiva humana ha anat acoblada a l'evolució biològica, la qual ha generat els a p...

  16. Optimization of kinetic parameters for the degradation of plasmid DNA in rat plasma

    Science.gov (United States)

    Chaudhry, Q. A.

    2014-12-01

    Biotechnology is a rapidly growing area of research work in the field of pharmaceutical sciences. The study of pharmacokinetics of plasmid DNA (pDNA) is an important area of research work. It has been observed that the process of gene delivery faces many troubles on the transport of pDNA towards their target sites. The topoforms of pDNA has been termed as super coiled (S-C), open circular (O-C) and linear (L), the kinetic model of which will be presented in this paper. The kinetic model gives rise to system of ordinary differential equations (ODEs), the exact solution of which has been found. The kinetic parameters, which are responsible for the degradation of super coiled, and the formation of open circular and linear topoforms have a great significance not only in vitro but for modeling of further processes as well, therefore need to be addressed in great detail. For this purpose, global optimization techniques have been adopted, thus finding the optimal results for the said model. The results of the model, while using the optimal parameters, were compared against the measured data, which gives a nice agreement.

  17. Fundamental Plasmid Strings and Black Rings

    CERN Document Server

    Blanco-Pillado, Jose J; Iglesias, Alberto

    2008-01-01

    We construct excited states of fundamental strings that admit a semiclassical description as rotating circular loops of string. We identify them with the supergravity solutions for rotating dipole rings. The identification involves a precise match of the mass, radius and angular momentum of the two systems. Moreover, the degeneracy of the string state reproduces the parametric dependence of the entropy in the supergravity description. When the solutions possess two macroscopic angular momenta, they are better described as toroidal configurations (tubular loops) instead of loops of string. We argue that the decay of the string state can be interpreted as superradiant emission of quanta from the ergoregion of the rotating ring.

  18. Microstrip Antenna Generates Circularly Polarized Beam

    Science.gov (United States)

    Huang, J.

    1986-01-01

    Circular microstrip antenna excited with higher order transverse magnetic (TM) modes generates circularly polarized, conical radiation patterns. Found both theoretically and experimentally that peak direction of radiation pattern is varied within wide angular range by combination of mode selection and loading substrate with materials of different dielectric constants.

  19. Circular Loudspeaker Array with Controllable Directivity

    DEFF Research Database (Denmark)

    Møller, Martin; Olsen, Martin; Agerkvist, Finn T.;

    2010-01-01

    Specific directivity patterns for circular arrays of loudspeakers can be achieved by utilizing the concept of phase-modes, which expands the directivity pattern into a series of circular harmonics. This paper investigates the applicability of this concept applied on a loudspeaker array on a...

  20. Plasmid Copy Number Determination by Quantitative Polymerase Chain Reaction.

    Science.gov (United States)

    Anindyajati; Artarini, A Anita; Riani, Catur; Retnoningrum, Debbie S

    2016-01-01

    Recombinant therapeutic proteins are biopharmaceutical products that develop rapidly for years. Recombinant protein production in certain hosts requires vector expression harboring the gene encoding the corresponding protein. Escherichia coli is the prokaryote organism mostly used in recombinant protein production, commonly using a plasmid as the expression vector. Recombinant protein production is affected by plasmid copy number harboring the encoded gene, hence the determination of plasmid copy number also plays an important role in establishing a recombinant protein production system. On the industrial scale, a low copy number of plasmids are more suitable due to their better stability. In the previous study we constructed pCAD, a plasmid derived from the low copy number pBR322 plasmid. This study was aimed to confirm pCAD's copy number by quantitative polymerase chain reaction (qPCR). Plasmid copy number was determined by comparing the quantification signal from the plasmid to those from the chromosome. Copy number was then calculated by using a known copy number plasmid as a standard. Two pairs of primers, called tdk and ori, were designed for targeting a single gene tdk in the chromosome and a conserved domain in the plasmid's ori, respectively. Primer quality was analyzed in silico using PrimerSelect DNASTAR and PraTo software prior to in vitro evaluation on primer specificity and efficiency as well as optimization of qPCR conditions. Plasmid copy number determination was conducted on E. coli lysates harboring each plasmid, with the number of cells ranging from 10(2)-10(5) cells/μL. Cells were lysed by incubation at 95ºC for 10 minutes, followed by immediate freezing at -4°C. pBR322 plasmid with the copy number of ~19 copies/cell was used as the standard, while pJExpress414-sod plasmid possessing the high copy number pUC ori was also determined to test the method being used. In silico analysis based on primer-primer and primer-template interactions showed

  1. An updated view of plasmid conjugation and mobilization in Staphylococcus.

    Science.gov (United States)

    Ramsay, Joshua P; Kwong, Stephen M; Murphy, Riley J T; Yui Eto, Karina; Price, Karina J; Nguyen, Quang T; O'Brien, Frances G; Grubb, Warren B; Coombs, Geoffrey W; Firth, Neville

    2016-01-01

    The horizontal gene transfer facilitated by mobile genetic elements impacts almost all areas of bacterial evolution, including the accretion and dissemination of antimicrobial-resistance genes in the human and animal pathogen Staphylococcus aureus. Genome surveys of staphylococcal plasmids have revealed an unexpected paucity of conjugation and mobilization loci, perhaps suggesting that conjugation plays only a minor role in the evolution of this genus. In this letter we present the DNA sequences of historically documented staphylococcal conjugative plasmids and highlight that at least 3 distinct and widely distributed families of conjugative plasmids currently contribute to the dissemination of antimicrobial resistance in Staphylococcus. We also review the recently documented "relaxase-in trans" mechanism of conjugative mobilization facilitated by conjugative plasmids pWBG749 and pSK41, and discuss how this may facilitate the horizontal transmission of around 90% of plasmids that were previously considered non-mobilizable. Finally, we enumerate unique sequenced S. aureus plasmids with a potential mechanism of mobilization and predict that at least 80% of all non-conjugative S. aureus plasmids are mobilizable by at least one mechanism. We suggest that a greater research focus on the molecular biology of conjugation is essential if we are to recognize gene-transfer mechanisms from our increasingly in silico analyses. PMID:27583185

  2. Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

    International Nuclear Information System (INIS)

    Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores

  3. Investigation of plasmid-induced growth defect in Pseudomonas putida.

    Science.gov (United States)

    Mi, Jia; Sydow, Anne; Schempp, Florence; Becher, Daniela; Schewe, Hendrik; Schrader, Jens; Buchhaupt, Markus

    2016-08-10

    Genetic engineering in bacteria mainly relies on the use of plasmids. But despite their pervasive use for physiological studies as well as for the design and optimization of industrially used production strains, only limited information about plasmid induced growth defects is available for different replicons and organisms. Here, we present the identification and characterization of such a phenomenon for Pseudomonas putida transformants carrying the pBBR1-derived plasmid pMiS1. We identified the kanamycin resistance gene and the transcription factor encoding rhaR gene to be causal for the growth defect in P. putida. In contrast, this effect was not observed in Escherichia coli. The plasmid-induced growth defect was eliminated after introduction of a mutation in the plasmid-encoded rep gene, thus enabling construction of the non-toxic variant pMiS4. GFP reporters construct analyses and qPCR experiments revealed a distinctly lowered plasmid copy number for pMiS4, which is probably the reason for alleviation of the growth defect by this mutation. Our work expands the knowledge about plasmid-induced growth defects and provides a useful low-copy pBBR1 replicon variant. PMID:27287537

  4. Sociobiological control of plasmid copy number in bacteria.

    Directory of Open Access Journals (Sweden)

    Mukta M Watve

    Full Text Available All genes critical for plasmid replication regulation are located on the plasmid rather than on the host chromosome. It is possible therefore that there can be copy-up "cheater" mutants. In spite of this possibility, low copy number plasmids appear to exist stably in host populations. We examined this paradox using a multilevel selection model. Simulations showed that, a slightly higher copy number mutant could out-compete the wild type. Consequently, another mutant with still higher copy number could invade the first invader. However, the realized benefit of increasing intra-host fitness was saturating whereas that of inter-host fitness was exponential. As a result, above a threshold, intra-host selection was overcompensated by inter-host selection and the low copy number wild type plasmid could back invade a very high copy number plasmid. This led to a rock-paper-scissor (RPS like situation that allowed the coexistence of plasmids with varied copy numbers. Furthermore, another type of cheater that had lost the genes required for conjugation but could hitchhike on a conjugal plasmid, could further reduce the advantage of copy-up mutants. These sociobiological interactions may compliment molecular mechanisms of replication regulation in stabilizing the copy numbers.

  5. Complete Sequence of pABTJ2, A Plasmid from Acinetobacter baumannii MDR-TJ, Carrying Many Phage-like Elements

    Institute of Scientific and Technical Information of China (English)

    He Huang; Yan Dong; Zhi-Liang Yang; Hao Luo; Xi Zhang; Feng Gao

    2014-01-01

    Acinetobacter baumannii is an important opportunistic pathogen in hospital, and the multidrug-resistant isolates of A. baumannii have been increasingly reported in recent years. A num-ber of different mechanisms of resistance have been reported, some of which are associated with plasmid-mediated acquisition of genes. Therefore, studies on plasmids in A. baumannii have been a hot issue lately. We have performed complete genome sequencing of A. baumannii MDR-TJ, which is a multidrug-resistant isolate. Finalizing the remaining large scaffold of the previous assem-bly, we found a new plasmid pABTJ2, which carries many phage-like elements. The plasmid pAB-TJ2 is a circular double-stranded DNA molecule, which is 110,967 bp in length. We annotated 125 CDSs from pABTJ2 using IMG ER and ZCURVE_V, accounting for 88.28%of the whole plasmid sequence. Many phage-like elements and a tRNA-coding gene were detected in pABTJ2, which is rarely reported among A. baumannii. The tRNA gene is specific for asparagine codon GTT, which may be a small chromosomal sequence picked up through incorrect excision during plasmid forma-tion. The phage-like elements may have been acquired during the integration process, as the GC content of the region carrying phage-like elements was higher than that of the adjacent regions. The finding of phage-like elements and tRNA-coding gene in pABTJ2 may provide a novel insight into the study of A. baumannii pan-plasmidome.

  6. CIRCULAR ECONOMY IN ROMANIA WITHIN EUROPEAN CONTEXT

    Directory of Open Access Journals (Sweden)

    Cornelia Marcela Danu

    2015-07-01

    Full Text Available In the present paper we have approached some conceptual and coordinated marks of the societal reality connected to the circular economy. Generated by „the limits of certainty” regarding the future of the world business, the operationalization of the circular economy has become a part of the EU strategies and started the various stages of implementation as an active process in all countries. We have highlighted the opportunities and the risks related to the circular economy, the European dimension and, in particular, the Romanian one of this process, the role of the triad: consumer-company-natural environment, while implementing the circular economy. Circular economy is both a new approach of the societal life, based on changing the mentalities of the individuals having the role of decision makers at the company level and public administration and the decision makers – consumers, as well as a policy meant to be made operational across all entities: governmental, entrepreneurial, individually – human.

  7. Gauge-Invariant Formulation of Circular Dichroism.

    Science.gov (United States)

    Raimbault, Nathaniel; de Boeij, Paul L; Romaniello, Pina; Berger, J A

    2016-07-12

    Standard formulations of magnetic response properties, such as circular dichroism spectra, are plagued by gauge dependencies, which can lead to unphysical results. In this work, we present a general gauge-invariant and numerically efficient approach for the calculation of circular dichroism spectra from the current density. First we show that in this formulation the optical rotation tensor, the response function from which circular dichroism spectra can be obtained, is independent of the origin of the coordinate system. We then demonstrate that its trace is independent of the gauge origin of the vector potential. We also show how gauge invariance can be retained in practical calculations with finite basis sets. As an example, we explain how our method can be applied to time-dependent current-density-functional theory. Finally, we report gauge-invariant circular dichroism spectra obtained using the adiabatic local-density approximation. The circular dichroism spectra we thus obtain are in good agreement with experiment. PMID:27295541

  8. Nanofocusing in circular sector-like nanoantennas

    DEFF Research Database (Denmark)

    Zenin, Volodymyr; Pors, Anders Lambertus; Han, Zhanghua;

    2014-01-01

    Gold circular sector-like nanoantennas (with a radius of 500 nm and a taper angle of 60°, 90°, and 120°) on glass are investigated in a near-infrared wavelength range (900 - 2100 nm). Amplitude- and phase-resolved near-field images of circular sector-like antenna modes at telecom wavelength feature...... a concentric circular line of phase contrast, demonstrating resonant excitation of a standing wave of counter-propagating surface plasmons, travelling between a tip and opposite circular edge of the antenna. Transmission spectra obtained in the range 900 - 2100 nm are in good agreement with...... makes circular sector-like nanoantennas very promising for implementing bowtie antennas and attractive for many applications....

  9. Plasmid pGA1 from Corynebacterium glutamicum codes for a gene product that positively influences plasmid copy number.

    OpenAIRE

    Nesvera, J; Pátek, M; Hochmannová, J; Abrhámová, Z; Becvárová, V; Jelínkova, M; Vohradský, J

    1997-01-01

    The complete nucleotide sequence (4,826 bp) of the cryptic plasmid pGA1 from Corynebacterium glutamicum was determined. DNA sequence analysis revealed four putative coding regions (open reading frame A [ORFA], ORFA2, ORFB, and ORFC). ORFC was identified as a rep gene coding for an initiator of plasmid replication (Rep) according to the high level of homology of its deduced amino acid sequence with the Rep proteins of plasmids pSR1 (from C. glutamicum) and pNG2 (from Corynebacterium diphtheria...

  10. Cloning and Analysis of a Large Plasmid pBMB165 from Bacillus thuringiensis Revealed a Novel Plasmid Organization

    OpenAIRE

    Yueying Wang; Donghai Peng; Zhaoxia Dong; Lei Zhu; Suxia Guo; Ming Sun

    2013-01-01

    In this study, we report a rapid cloning strategy for large native plasmids via a contig linkage map by BAC libraries. Using this method, we cloned a large plasmid pBMB165 from Bacillus thuringiensis serovar tenebrionis strain YBT-1765. Complete sequencing showed that pBMB165 is 77,627 bp long with a GC-content of 35.36%, and contains 103 open reading frames (ORFs). Sequence analysis and comparison reveals that pBMB165 represents a novel plasmid organization: it mainly consists of a pXO2-like...

  11. A series of template plasmids for Escherichia coli genome engineering.

    Science.gov (United States)

    Deb, Shalini S; Reshamwala, Shamlan M S; Lali, Arvind M

    2016-06-01

    Metabolic engineering strategies often employ multi-copy episomal vectors to overexpress genes. However, chromosome-based overexpression is preferred as it avoids the use of selective pressure and reduces metabolic burden on the cell. We have constructed a series of template plasmids for λ Red-mediated Escherichia coli genome engineering. The template plasmids allow construction of genome integrating cassettes that can be used to integrate single copies of DNA sequences at predetermined sites or replace promoter regions. The constructed cassettes provide flexibility in terms of expression levels achieved and antibiotics used for selection, as well as allowing construction of marker-free strains. The modular design of the template plasmids allows replacement of genetic parts to construct new templates. Gene integration and promoter replacement using the template plasmids are illustrated. PMID:27071533

  12. Construction and Identification of Plasmid pTA-TUB2

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    An about 1.40 Kb target gene fragment was yielded by PCR amplification with the plasmid pRB 129,which was identified by restriction enzyme digestion that the PCR product was TU B2 gene.The gene was digested by the restriction enzyme and was linked with pTA plasmid to construct pTA-TU B2 plasmid.The plasmid was transformed into Chaetomium spp.by PEG method and the transformation rate was 27/(2×105) and it is nine times higher than that of pRB 129.The transformants can grow on the PDA containing 1 000 μg*mL-1 carbendazim,which is 1 000 times higher than the original Chaetomium spp.The resistance was stable after 10 times transfer on non-selective medium.

  13. Plasmid DNA labelled with 14C or 3H

    International Nuclear Information System (INIS)

    Plasmid DNA labelled with 14C or 3H in thymine was isolated from the thymine-dependent strain of Escherichia coli 15 SPT bacteria. The specific activity of the plasmid DNA preparations lay in the range from 0.5 to 20 MBq/mg, their relative molecular weight was 1.7 x 106 dalton. Molecular weight, preparation purity, and the degree of damage of the plasmid DNA molecules were examined by UV absorption spectroscopy, by gel electrophoresis, and by electron micrography. The quality of the [thymine-2-14C] plasmid DNA was verified in a diagnostic test for the determination of the anti-dsDNA bonding activity in human serum. (author). 1 tab., 5 figs., 30 refs

  14. Characterization of the Double-Partitioning Modules of R27: Correlating Plasmid Stability with Plasmid Localization

    OpenAIRE

    Trevor D Lawley; Taylor, Diane E.

    2003-01-01

    Plasmid R27 contains two independent partitioning modules, designated Par1 and Par2, within transfer region 2. Par1 is member of the type I partitioning family (Walker-type ATPase), and Par2 is a member of the type II partitioning family (actin-type ATPase). Stability tests of cloned Par1 and Par2 and insertional disruptions of Par1 and Par2 within R27 demonstrated that Par1 is the major stability determinant whereas Par2 is the minor stability determinant. Creation of double-partitioning mut...

  15. The Salmonella typhimurium virulence plasmid encodes a positive regulator of a plasmid-encoded virulence gene.

    OpenAIRE

    Caldwell, A L; Gulig, P A

    1991-01-01

    The 90-kb virulence plasmid of Salmonella typhimurium is necessary for invasion beyond the Peyer's patches to the mesenteric lymph nodes and spleens of orally inoculated mice. Two Tn5 insertions located on the left side of a previously identified 14-kb virulence region (P. A. Gulig and R. Curtiss III, Infect. Immun. 58:3262-3271, 1988) and mapping 272 bp from each other exhibited opposite effects on splenic infection of mice after oral inoculation. spvR23::Tn5 decreased splenic infection by 1...

  16. Comparative analysis of conjugative plasmids mediating gentamicin resistance in Staphylococcus aureus.

    OpenAIRE

    Goering, R. V.; Ruff, E A

    1983-01-01

    Five gentamicin-resistant clinical isolates of Staphylococcus aureus were found to contain self-transmissible plasmids of 32 to 37 megadaltons in size. Restriction endonuclease digests of the plasmids were markedly similar to those of reference plasmids of unrelated geographical origin, thus suggesting the significant contribution of common conjugal plasmids to the emergence of gentamicin resistance in S. aureus populations.

  17. Detection of plasmid deoxyribonucleic acid in an isolate of Lactobacillus acidophilus.

    OpenAIRE

    Klaenhammer, T R; Sutherland, S M

    1980-01-01

    Eight strains of Lactobacillus acidophilus were examined for the presence of plasmid deoxyribonucleic acid, and one, a pig intestinal isolate, showed the presence of a 13.7- and a 6.3-megadalton plasmid. This is the first reported evidence for plasmid deoxyribonucleic acid in Lactobacillus acidophilus. The functions of these plasmids are presently unknown.

  18. Pharmaceutical development of the plasmid DNA vaccine pDERMATT

    OpenAIRE

    Quaak, S.G.L.

    2009-01-01

    The discovery of tumor specific antigens and self tolerance mechanisms against these antigens led to the assumption that antigens circulating at sufficient concentration levels could break this self tolerance mechanism and evoke immunological antitumor effects. pDERMATT (plasmid DNA encoding recombinant MART-1 and tetanus toxin fragment-c) is a plasmid that encodes for MART-1, a melanoma associated antigen that is expressed in a large fraction of melanomas. In animal models administration of ...

  19. A novel chromatographic procedure for purification of bacterial plasmids.

    Science.gov (United States)

    Bywater, M; Bywater, R; Hellman, L

    1983-07-01

    A new, rapid procedure for purifying bacterial plasmids with high recovery is described. The sequence of operations consists essentially of treatment with alkali, ribonuclease, and proteinase K, followed by chisam extraction and gel filtration on Sephacryl S-1000, and finally a precipitation step using isopropanol at room temperature. The method gives rather good yields of plasmid DNA of high purity, and lends itself to scaling up. PMID:6312836

  20. Isolation and properties of plasmids from Deinococcus radiodurans Sark

    International Nuclear Information System (INIS)

    Radioresistant bacterium, Deinococcus radiodurans, can repair completely almost all of DNA damages including double strand breaks induced by gamma-rays up to about 5 kGy. In order to reveal the repair mechanism, it is necessary to develop a cloning vector available for the genetic analysis. We tried to isolate plasmids from D.radiodurans Sark strain. In the present paper the isolation and properties of plasmids were described. (author)

  1. Construction of a bioluminescence reporter plasmid for Francisella tularensis

    OpenAIRE

    Bina, Xiaowen R.; Miller, Mark A.; James E Bina

    2010-01-01

    A Francisella tularensis shuttle vector that constitutively expresses the Photorhabdus luminescens lux operon in type A and type B strains of F. tularensis was constructed. The bioluminescence reporter plasmid was introduced into the live vaccine strain of F. tularensis and used to follow F. tularensis growth in a murine intranasal challenge model in real time by bioluminescence imaging. The results show that the new bioluminescence reporter plasmid represents a useful tool for tularemia rese...

  2. Conjugative plasmid transfer from Enterococcus faecalis to Escherichia coli.

    OpenAIRE

    Trieu-Cuot, P; Carlier, C; Courvalin, P

    1988-01-01

    The possibility of transfer of genetic information by conjugation from gram-positive to gram-negative bacteria was investigated with a pBR322-pAM beta 1 chimeric plasmid, designated pAT191. This shuttle vector, which possesses the tra functions of the streptococcal plasmid pAM beta 1, was conjugatively transferred from Enterococcus faecalis to Escherichia coli with an average frequency of 5 x 10(-9) per donor colony formed after mating.

  3. Breaking the fault tree circular logic

    International Nuclear Information System (INIS)

    Event tree - fault tree approach to model failures of nuclear plants as well as of other complex facilities is noticeably dominant now. This approach implies modeling an object in form of unidirectional logical graph - tree, i.e. graph without circular logic. However, genuine nuclear plants intrinsically demonstrate quite a few logical loops (circular logic), especially where electrical systems are involved. This paper shows the incorrectness of existing practice of circular logic breaking by elimination of part of logical dependencies and puts forward a formal algorithm, which enables the analyst to correctly model the failure of complex object, which involves logical dependencies between system and components, in form of fault tree. (author)

  4. Entanglement of quantum circular states of light

    Science.gov (United States)

    Horoshko, D. B.; De Bièvre, S.; Kolobov, M. I.; Patera, G.

    2016-06-01

    We present a general approach to calculating the entanglement of formation for superpositions of two-mode coherent states, placed equidistantly on a circle in phase space. We show that in the particular case of rotationally invariant circular states the Schmidt decomposition of two modes, and therefore the value of their entanglement, are given by analytical expressions. We analyze the dependence of the entanglement on the radius of the circle and number of components in the superposition. We also show that the set of rotationally invariant circular states creates an orthonormal basis in the state space of the harmonic oscillator, and this basis is advantageous for representation of other circular states of light.

  5. Symmetric Circular Matchings and RNA Folding

    DEFF Research Database (Denmark)

    Hofacker, Ivo L.; Reidys, Christian; Stadler, Peter F.

    2012-01-01

    RNA secondary structures can be computed as optimal solutions of certain circular matching problems. An accurate treatment of this energy minimization problem has to account for the small --- but non-negligible --- entropic destabilization of secondary structures with non-trivial automorphisms....... Such intrinsic symmetries are typically excluded from algorithmic approaches, however, because the effects are small, they play a role only for RNAs with symmetries at sequence level, and they appear only in particular settings that are less frequently used in practical application, such as circular...... asymptotic results for both the circular and the co-folding version are derived....

  6. Circular polarization memory in polydisperse scattering media

    CERN Document Server

    Macdonald, Callum M; Meglinski, Igor

    2015-01-01

    We investigate the survival of circularly polarized light in random scattering media. The surprising persistence of this form of polarization has a known dependence on the size and refractive index of scattering particles, however a general description regarding polydisperse media is lacking. Through analysis of Mie theory, we present a means of calculating the magnitude of circular polarization memory in complex media, with total generality in the distribution of particle sizes and refractive indices. Quantification of this memory effect enables an alternate pathway towards recovering particle size distribution, based on measurements of diffusing circularly polarized light.

  7. Intragenic variation by site-specific recombination in the cryptic plasmid of Neisseria gonorrhoeae.

    OpenAIRE

    Hagblom, P; Korch, C; Jonsson, A B; Normark, S

    1986-01-01

    Cryptic plasmid DNA of Neisseria gonorrhoeae was found integrated into the gonococcal chromosome in both plasmid-bearing strains and plasmid-free strains. At several chromosomal locations only segments of the plasmid were found. However, in at least two strains an intact copy of the plasmid seemed to be present with the joints between the plasmid and the chromosomal DNA being located within the cppB gene of the cryptic plasmid. The cppB gene was shown to undergo a sequence-specific intragenic...

  8. Plasmid vector with temperature-controlled gene expression

    International Nuclear Information System (INIS)

    In plasmid pBR327, a fragment 169 b.p. long including promotor p3 of the bla gene has been deleted. The deletional derivative so obtained (pSP2) has been used to construct a recombinant plasmid bearing a fragment of phage λ DNA with the p/sub R/ promotor and the gene of the temperature-sensitive repressor cI. It has been shown that the plasmid vector so constructed (pCE119) with promotor cR performs repressor-cI-controlled transcription of the bla gene, as a result of which induction for an hour at 420C leads to an almost 100-fold increase in the amount of product of the bla gene as compared with that at 320C. The possibility of the use of plasmid cPE119 for the expression of other genes has been demonstrated for the case of the semisynthetic β-galactosidase gene of E. coli. In this case, on induction of the cells with recombinant plasmid pCEZ12 for 3 hours at 420C, a 300-fold increase in the amount of active β-galactosidase, as compared with that at 320C, was observed. It is important to point out that under these conditions (at 420C), at least 99% of the cells containing the plasmid retain the phenotype lacZ+, which indicates the stability of the proposed vector system

  9. Transfer of conjugative plasmids among bacteria under environmentally relevant conditions

    DEFF Research Database (Denmark)

    Musovic, Sanin

    det oprindelige bakteriesamfund der tager andel i plasmid overførsel blev udviklet. Dyrknings-minimal metode i kombination med reporter gen teknologi og moderne mikroskopi viste en meget høj forekomst af RP4:gfp plasmid overførsel til oprindelige jord bakterier af et bredt værtskab. Der blev også vist......Mobile genetiske elementer (f.eks. plasmider), der ofte bærer ekstra funktioner såsom antibiotikaresistens, eller kataboliske- og xenobiotiske nedbrydnings gener, antages at have en meget vigtigt evolutionær rolle for bakterier. I denne PhD afhandling undersøgte jeg størrelsen af plasmid overførsel...... under de miljørelevante substrat-begrænsede forhold, den del og diversitet af bakteriel samfund der er involveret i overførslen, og effekten af plasmid donor cellens fysiologiske status og de miljørelevante faktorer (selektive tryk) på plasmid spredning. En ny metode til at kvantificere den fraktion af...

  10. The characteristics of micrococcus (deinococcus) radiodurans sark plasmids

    International Nuclear Information System (INIS)

    The characterization of micrococcus (deinococcus) radiodurans sark plasmids. This bacterium has been classified as a new genus deinococcus radiodurans which is resistant to gamma-rays. It can repair itself completely almost all of DNA damages including double strand breaks induced by gamma-rays up to about 5 KGy. To reveal the repair mechanism, several investigations had been done to develop a cloning vector available for the genetic analysis. For this purpose D. radiodurans Sark are to be prepared as a vector by studying the characteristics of its plasmid. Plasmids were isolated by electrophoresis using 0.6% low-melting-temperature agarose in TAE and run for 5.5 hours, followed by the identification. An antibiotic marker was also carried out in this experiment to identify its location in the genetic materials of the cell, beside making a restriction map of the plasmid. Results have shown that D. radiodurans Sark has 4 plasmids (P1, P2, P3, and P4) and the refampicin resistant genes were not found in the plasmid. (authors). 14 refs; 4 figs

  11. Plasmid-free T7-based Escherichia coli expression systems.

    Science.gov (United States)

    Striedner, Gerald; Pfaffenzeller, Irene; Markus, Luchner; Nemecek, Sabine; Grabherr, Reingard; Bayer, Karl

    2010-03-01

    In order to release host cells from plasmid-mediated increases in metabolic load and high gene dosages, we developed a plasmid-free, T7-based E. coli expression system in which the target gene is site-specifically integrated into the genome of the host. With this system, plasmid-loss, a source of instability for conventional expression systems, was eliminated. At the same time, system leakiness, a challenging problem with recombinant systems, was minimized. The efficiency of the T7 RNA polymerase compensates for low gene dosage and provides high rates of recombinant gene expression without fatal consequences to host metabolism. Relative to conventional pET systems, this system permits improved process stability and increases the host cell's capacity for recombinant gene expression, resulting in higher product yields. The stability of the plasmid-free system was proven in chemostat cultivation for 40 generations in a non-induced and for 10 generations in a fully induced state. For this reason plasmid-free systems benefit the development of continuous production processes with E. coli. However, time and effort of the more complex cloning procedure have to be considered in relation to the advantages of plasmid-free systems in upstream-processing. PMID:19891007

  12. Partition-associated incompatibility caused by random assortment of pure plasmid clusters

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Sherratt, David J; Gerdes, Kenn;

    2005-01-01

    Summary Bacterial plasmids and chromosomes encode centromere-like partition loci that actively segregate DNA before cell division. The molecular mechanism behind DNA segregation in bacteria is largely unknown. Here we analyse the mechanism of partition-associated incompatibility for plasmid pB171......, a phenotype associated with all known plasmid-encoded centromere loci. An R1 plasmid carrying par2 from plasmid pB171 was destabilized by the presence of an F plasmid carrying parC1, parC2 or the entire par2 locus of pB171. Strikingly, cytological double-labelling experiments revealed no evidence of long......-lived pairing of plasmids. Instead, pure R1 and F foci were positioned along the length of the cell, and in a random order. Thus, our results raise the possibility that partition-mediated plasmid incompatibility is not caused by pairing of heterologous plasmids but instead by random positioning of pure plasmid...

  13. Identification, Characterization, and Expression of Three New Members of the Borrelia burgdorferi Mlp (2.9) Lipoprotein Gene Family

    OpenAIRE

    Yang, Xiaofeng; Popova, Taissia G.; Hagman, Kayla E.; Wikel, Stephen K.; Schoeler, George B.; Caimano, Melissa J.; Radolf, Justin D.; Norgard, Michael V.

    1999-01-01

    We previously reported on the existence of a family of lipoprotein genes, designated 2.9 lipoprotein genes, encoded in at least seven versions on the circular (supercoiled) cp32 and cp18 plasmids of Borrelia burgdorferi 297. A distinguishing feature of the 2.9 lipoproteins were highly similar signal sequences but variable mature polypeptides that segregated into two antigenic classes. Further screenings of B. burgdorferi 297 genomic libraries led to the identification of three additional 2.9 ...

  14. Tyrosine Partners Coordinate DNA Nicking by the Salmonella typhimurium Plasmid pCU1 Relaxase Enzyme

    OpenAIRE

    Nash, Rebekah P.; Niblock, Franklin C.; Redinbo, Matthew R.

    2011-01-01

    Conjugative plasmid transfer results in the spread of antibiotic resistance genes and virulence factors between bacterial cells. Plasmid transfer is dependent upon the DNA nicking activity of a plasmid-encoded relaxase enzyme. Tyrosine residues within the relaxase cleave the DNA plasmid nic site in a highly sequence-specific manner. The conjugative resistance plasmid pCU1 encodes a relaxase with four tyrosine residues surrounding its active site (Y18,19,26,27). We use activity assays to demon...

  15. Linear Plasmid SLP2 Is Maintained by Partitioning, Intrahyphal Spread, and Conjugal Transfer in Streptomyces▿

    OpenAIRE

    Hsu, Chin-Chen; Chen, Carton W.

    2009-01-01

    Low-copy-number plasmids generally encode a partitioning system to ensure proper segregation after replication. Little is known about partitioning of linear plasmids in Streptomyces. SLP2 is a 50-kb low-copy-number linear plasmid in Streptomyces lividans, which contains a typical parAB partitioning operon. In S. lividans and Streptomyces coelicolor, a parAB deletion resulted in moderate plasmid loss and growth retardation of colonies. The latter was caused by conjugal transfer from plasmid-co...

  16. Transfer of chimeric plasmids among Salmonella typhimurium strains by P22 transduction.

    OpenAIRE

    Orbach, M J; Jackson, E N

    1982-01-01

    Salmonella typhimurium bacteriophage P22 transduced plasmids having P22 sequences inserted in the vector pBR322 with high frequency. Analysis of the structure of the transducing particle DNA and the transduced plasmids indicates that this plasmid transduction involves two homologous recombination events. In the donor cell, a single recombination between the phage and the homologous sequences on the plasmid inserted the plasmid into the phage chromosome, which was then packaged by headfuls int...

  17. Exposing Plasmids as the Achilles’ Heel of Drug-Resistant Bacteria

    OpenAIRE

    Williams, Julia J.; Hergenrother, Paul J.

    2008-01-01

    Many multi-drug resistant bacterial pathogens harbor large plasmids that encode proteins conferring resistance to antibiotics. While the acquisition of these plasmids often enables bacteria to survive in the presence of antibiotics, it is possible that plasmids also represent a vulnerability that can be exploited in tailored antibacterial therapy. This review highlights three recently described strategies designed to specifically combat bacteria harboring such plasmids: Inhibition of plasmid ...

  18. Sequence-nonspecific replication of transfected plasmid DNA in poxvirus-infected cells.

    OpenAIRE

    DeLange, A. M.; McFadden, G

    1986-01-01

    A system in which transfected plasmid DNA replicates in the cytoplasm of poxvirus-infected cells is described. A variety of recombinant plasmids was introduced into poxvirus-infected cells by transfection, and replication of input plasmid DNA was monitored by (i) digestion with restriction enzymes that discriminate between input methylated plasmid DNA and unmethylated DNA produced by replication in mammalian cells; (ii) amplification of intracellular plasmid DNA; and (iii) density shift analy...

  19. Characterization of plasmid burden and copy number in Saccharomyces cerevisiae for optimization of metabolic engineering applications

    OpenAIRE

    Karim, Ashty S.; Curran, Kathleen A.; Alper, Hal S.

    2012-01-01

    Many metabolic engineering and genetic engineering applications in yeast rely on the use of plasmids. Despite their pervasive use and the diverse collections available, there is a fundamental lack of understanding of how commonly used DNA plasmids affect the cell’s ability to grow and how the choice of plasmid components can influence plasmid load and burden. In this study, we characterized the major attributes of the 2μ and centromeric plasmids typically used in yeast by examining the impact...

  20. Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents

    OpenAIRE

    Lezin, George; Kuehn, Michael R.; Brunelli, Luca

    2011-01-01

    Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination...

  1. Comparison of Ti plasmids from three different biotypes of Agrobacterium tumefaciens isolated from grapevines.

    OpenAIRE

    Knauf, V C; Panagopoulos, C G; Nester, E. W.

    1983-01-01

    Twenty-six plasmids from grapevine isolates of Agrobacterium tumefaciens were analyzed by SmaI fingerprinting and by hybridization of nick-translated DNA to DNA of another plasmid. These experiments established that octopine Ti plasmids are not highly conserved, although octopine Ti plasmids from biotype 1 A. tumefaciens strains appeared to be very similar. Octopine Ti plasmids from biotype 3 strains are more variable in terms of host range and SmaI fingerprints, but share extensive DNA homol...

  2. 77 FR 42077 - Environmental Justice: Final Circular

    Science.gov (United States)

    2012-07-17

    ... social service providers to identify transportation challenges and mitigation strategies. FTA is... Register notice (76 FR 60590). II. Chapter-by-Chapter Analysis A. General Comments This section addresses... Circular does not contain any new responsibilities for recipients. Recipients' responsibilities...

  3. Dual frequency launcher for circularly polarized antenna

    Science.gov (United States)

    Chen, Ming H.

    1989-10-01

    A dual frequency antenna feed is formed from a central, circular waveguide connected to the flat boundry of circular, disk-shaped resonant cavity. A second circular waveguide is connected one end of a disk-shaped resonant cavity. Energy of one frequency enters and exits the cavity along the common axis of the waveguides. Energy of the second frequency is introduced to the same resonant cavity by way of a plurality of bandpass filters, also connected to the cavity. This energy enters by way of slots in the cylindrical walls of the cavity. The central circular waveguide is propagating at one frequency but cut off at the second frequency. These bandpass filters are at this pass band for the second frequency, but at the rejection band for the first frequency. Therefore, the isolation between these two input ports are obtained.

  4. Unleashing the Power of the Circular Economy

    Energy Technology Data Exchange (ETDEWEB)

    Kok, L.; Wurpel, G.; Ten Wolde, A. [IMSA Amsterdam, Amsterdam (Netherlands)

    2013-04-15

    The concept of circular economy is an economic and industrial system that focuses on the reusability of products and raw materials, reduces value destruction in the overall system and aims at value creation within each tier of the system. This report for Circle Economy (CE) outlines the general direction and concrete steps that must be taken to accomplish a breakthrough to a circular economy. It also provides a knowledge base behind the concept, connecting it to sustainability.

  5. Construction of Circular Economy Industrial System

    Institute of Scientific and Technical Information of China (English)

    Cao Man; Ye Wenhu

    2007-01-01

    It is difficult to realize the transformation from traditional economy industrial system to circular economy industrial system.Regarding primary raw materials as the indicators,the industrial system has been specified according to the divergence among the indicators and the circular utilization modes.In comparison with the association among industrial systems,the relationship among industrial sub-systems is named as industrial cross-linking in this paper.The industrial system which could completely utilize and recycle the indicators should be increased and strengthened,and the circular economy industrial system with complete industrial association and industrial cross-linking should also be constructed.Taking the development of circular agricultural system basing on the traditional agricultural system as an example,the traditional agricultural products are regarded as the indicators which have been divided into foodstuff and crop straws which are used to produce food and articlesfor use,like fertilizer,energy and papers etc.The way to construct the circular agricultural industrial system is to increase the industrial systems that could utilize the products generated from crop straws,feces and other castoffs and transform the wastewater and other trucks into environmental friendly products.It has also been pointed out that the construction of circular economy industrial system is conducive to the foundation of circular industrial economics and the establishment of the construction layout of circular economy and the application schemes.Suggestions to the theoretical and practical work of the next step have also been brought forward in this paper.

  6. A Conceptual Framework for Circular Design

    Directory of Open Access Journals (Sweden)

    Mariale Moreno

    2016-09-01

    Full Text Available Design has been recognised in the literature as a catalyst to move away from the traditional model of take-make-dispose to achieve a more restorative, regenerative and circular economy. As such, for a circular economy to thrive, products need to be designed for closed loops, as well as be adapted to generate revenues. This should not only be at the point of purchase, but also during use, and be supported by low-cost return chains and reprocessing structures, as well as effective policy and regulation. To date, most academic and grey literature on the circular economy has focused primarily on the development of new business models, with some of the latter studies addressing design strategies for a circular economy, specifically in the area of resource cycles and design for product life extension. However, these studies primarily consider a limited spectrum of the technical and biological cycles where materials are recovered and restored and nutrients (e.g., materials, energy, water are regenerated. This provides little guidance or clarity for designers wishing to design for new circular business models in practice. As such, this paper aims to address this gap by systematically analysing previous literature on Design for Sustainability (DfX (e.g., design for resource conservation, design for slowing resource loops and whole systems design and links these approaches to the current literature on circular business models. A conceptual framework is developed for circular economy design strategies. From this conceptual framework, recommendations are made to enable designers to fully consider the holistic implications for design within a circular economy.

  7. Dual-band Omnidirectional Circularly Polarized Antenna

    OpenAIRE

    Narbudowicz, Adam; Bao, Xiulong; Ammann, Max

    2013-01-01

    A dual-band omnidirectional circularly polarized antenna is proposed. The antenna comprises back-to-back microstrip patches fed by a coplanar waveguide. A very low frequency ratio of 1.182 has been achieved, which can be easily tuned by adjusting four lumped capacitors incorporated into the antenna. An analysis of the omnidirectional circular polarization mechanism as well the dual band operation is provided and confirmed by numerical and experimental data. Key parameters to tune the resonant...

  8. Single particle dynamics in circular accelerators

    Energy Technology Data Exchange (ETDEWEB)

    Ruth, R.D.

    1986-10-01

    The purpose of this paper is to introduce the reader to the theory associated with the transverse dynamics of single particle, in circular accelerators. The discussion begins with a review of Hamiltonian dynamics and canonical transformations. The case of a single particle in a circular accelerator is considered with a discussion of non-linear terms and chromaticity. The canonical perturbation theory is presented and nonlinear resonances are considered. Finally, the concept of renormalization and residue criterion are examined. (FI)

  9. CIRCULAR RIBBON FLARES AND HOMOLOGOUS JETS

    International Nuclear Information System (INIS)

    Solar flare emissions in the chromosphere often appear as elongated ribbons on both sides of the magnetic polarity inversion line (PIL), which has been regarded as evidence of a typical configuration of magnetic reconnection. However, flares having a circular ribbon have rarely been reported, although it is expected in the fan-spine magnetic topology involving reconnection at a three-dimensional (3D) coronal null point. We present five circular ribbon flares with associated surges, using high-resolution and high-cadence Hα blue wing observations obtained from the recently digitized films of Big Bear Solar Observatory. In all the events, a central parasitic magnetic field is encompassed by the opposite polarity, forming a circular PIL traced by filament material. Consequently, a flare kernel at the center is surrounded by a circular flare ribbon. The four homologous jet-related flares on 1991 March 17 and 18 are of particular interest, as (1) the circular ribbons brighten sequentially, with cospatial surges, rather than simultaneously, (2) the central flare kernels show an intriguing 'round-trip' motion and become elongated, and (3) remote brightenings occur at a region with the same magnetic polarity as the central parasitic field and are co-temporal with a separate phase of flare emissions. In another flare on 1991 February 25, the circular flare emission and surge activity occur successively, and the event could be associated with magnetic flux cancellation across the circular PIL. We discuss the implications of these observations combining circular flare ribbons, homologous jets, and remote brightenings for understanding the dynamics of 3D magnetic restructuring.

  10. On the circular polarization of pulsar radiation

    OpenAIRE

    Lyubarskii, Y. E.; Petrova, S. A.

    1999-01-01

    We consider the polarization behaviour of radio waves propagating through an ultrarelativistic highly magnetized electron-positron plasma in a pulsar magnetosphere. The rotation of magnetosphere gives rise to the wave mode coupling in the polarization-limiting region. The process is shown to cause considerable circular polarization in the linearly polarized normal waves. Thus, the circular polarization observed for a number of pulsars, despite the linear polarization of the emitted normal wav...

  11. Angle-resolved x-ray circular and magnetic circular dichroisms: Definitions and applications

    OpenAIRE

    Tong, DSY; X Guo; Tobin, JG; Waddill, GD

    1996-01-01

    We introduce definitions of angle-resolved x-ray circular dichroism (ARXCD) and magnetic x-ray circular dichroism (ARMXCD). As defined, the much larger effect of circular dichroism (ARXCD) is separated from the smaller magnetic (ARMXCD) effect. In all materials, ARXCD is zero along mirror planes while nonzero elsewhere. ARMXCD is nonzero only in magnetic materials. The measurement and analysis of ARMXCD allow element specific surface magnetism and surface structure as well as their inter-rela...

  12. Broadband circularly polarized planar antenna using partially covered circular wide-slot and L-probe

    OpenAIRE

    Fukusako, Takeshi; Sakami, Ryo; Iwata, Kazuki; フクサコ, タケシ; サカミ, リョウ; イワタ, カズキ; 福迫, 武; 酒見, 遼; 岩田, 一樹

    2008-01-01

    The novel structure of a unique circularly polarized broadband antenna that combines an L-shaped probe with a partially covered circular slot has been presented. A principle that can be used to generate CP using the proposed structure has been presented using an L-shaped probe and a modified circular wide slot. Using the above, a 3 -dB AR bandwidth of 58% and matching bandwidth of 57% were obtained at 3.2 GHz.

  13. Conjugative plasmids isolated from bacteria in marine environments show various degrees of homology to each other and are not closely related to well-characterized plasmids.

    OpenAIRE

    Dahlberg, C; Linberg, C; Torsvik, V L; Hermansson, M

    1997-01-01

    Mercury resistance plasmids were exogenously isolated, i.e., recovered after transfer to a model recipient bacterium, from marine air-water interface, bulk water, and biofilm communities during incubation in artificial seawater without added nutrients. Ninety-five plasmids from different environments were classified by restriction endonuclease digestion, and 12 different structural plasmid groups were revealed. The plasmid types isolated from different habitats and from different sampling occ...

  14. Chemical and biological consequences of the radioactive decay of iodine-125 in plasmid DNA

    International Nuclear Information System (INIS)

    The consequences of the decay of iodine-125 incorporated into DNA were studied on a molecular basis. Doubly (14C and 125I) labelled 5-iodo-2'-deoxycytidine 5'-triphosphate (IdCTP) was synthesized and incorporated enzymatically into the SalI-cutting site of the plasmid pBR 322. Part of the radioiodinated DNA was treated with T4-DNA ligase in order to restore the circular structure of the native plasmid molecule. After 4 months of storage under various conditions the stable end products were analyzed by radio GC, radio HPLC and electron microscopy. The experiments were not only carried out with doubly-labelled DNA but also with solutions of 14C-labelled DNA containing Na125I as internal radiation source. The results clearly indicate that radiolysis alone causes only minor damage. Transmutation of the covalently bound iodine, on the other hand, leads to complete destruction of the labelled nucleotide, giving rise to 14CO2 and 14CO as main products. The production of 14CO2 which originates from both the base as well as the sugar component shows a strong solvent effect. The electron microscopy analysis of the DNA reveals that the local effects are always connected with at least one double strand break directly at the site of decay. In addition, one finds DNA double strand breaks in areas which are hundreds of base pairs apart from that site. Under certain circumstances most of the DNA molecules exhibit up to 10 breaks. A comparison between ligase-treated and untreated DNA shows that the configuration of the DNA and the position of the labelled nucleotide play in important role in the extent of the overall damage. It could be demonstrated that there is a linear correlation between gaseous fragmentation products and the number of double strand breaks. (orig./MG)

  15. Exciton circular dichroism in channelrhodopsin.

    Science.gov (United States)

    Pescitelli, Gennaro; Kato, Hideaki E; Oishi, Satomi; Ito, Jumpei; Maturana, Andrés Daniel; Nureki, Osamu; Woody, Robert W

    2014-10-16

    Channelrhodopsins (ChRs) are of great interest currently because of their important applications in optogenetics, the photostimulation of neurons. The absorption and circular dichroism (CD) spectra of C1C2, a chimera of ChR1 and ChR2 of Chlamydomonas reinhardtii, have been studied experimentally and theoretically. The visible absorption spectrum of C1C2 shows vibronic fine structure in the 470 nm band, consistent with the relatively nonpolar binding site. The CD spectrum has a negative band at 492 nm (Δε(max) = -6.17 M(-1) cm(-1)) and a positive band at 434 nm (Δε(max) = +6.65 M(-1) cm(-1)), indicating exciton coupling within the C1C2 dimer. Time-dependent density functional theory (TDDFT) calculations are reported for three models of the C1C2 chromophore: (1) the isolated protonated retinal Schiff base (retPSB); (2) an ion pair, including the retPSB chromophore, two carboxylate side chains (Asp 292, Glu 162), modeled by acetate, and a water molecule; and (3) a hybrid quantum mechanical/molecular mechanical (QM/MM) model depicting the binding pocket, in which the QM part consists of the same ion pair as that in (2) and the MM part consists of the protein residues surrounding the ion pair within 10 Å. For each of these models, the CD of both the monomer and the dimer was calculated with TDDFT. For the dimer, DeVoe polarizability theory and exciton calculations were also performed. The exciton calculations were supplemented by calculations of the coupling of the retinal transition with aromatic and peptide group transitions. For the dimer, all three methods and three models give a long-wavelength C2-axis-polarized band, negative in CD, and a short-wavelength band polarized perpendicular to the C2 axis with positive CD, differing in wavelength by 1-5 nm. Only the retPSB model gives an exciton couplet that agrees qualitatively with experiment. The other two models give a predominantly or solely positive band. We further analyze an N-terminal truncated mutant

  16. PLANAR MOTION OF A SLIGHTLY DISTORTED CIRCULAR CYLINDER AROUND ANOTHER CIRCULAR ONE

    Institute of Scientific and Technical Information of China (English)

    SUN Ren; CHWANG Allen T.

    2004-01-01

    Accurate prediction of the motion of a body moving around another one in an unbounded fluid and determi-nation of the hydrodynamic interaction between them are im-portant in the coastal and offshore engineering. For two-dimensional cases, most of the previous studies were focused on the interaction between circular cylinders without considering the non-circular situation. To break through the limitation of"circular" bodies, in the present paper the boundary perturbation method was employed to investigate the motion of a slightly distorted circular cylinder around a circular one. An approximate complex velocity potential in terms of double infinite series expanded at two singular points was derived using the method of continued fractions. The hydrodynamic interaction between two cylinders was computed by solving the dynamical equations of motion. In a relative coordinate system moving with the uniform stream, the kinetic energy of the fluid was expressed as a function of fifteen added masses. Approximate analytical solutions of added masses in the series form were obtained and applied to determine the trajectories of the slightly distorted circular cylinder around a fixed circular one. Numerical results show that the presence of the circular cylinder affects the planar motion of the slightly distorted cirular cylinder and the initial configuration of the slightly distorted circular cylinder has a decisive influence on the development of its rotational motion.

  17. Plasmids and rickettsial evolution: insight from Rickettsia felis.

    Directory of Open Access Journals (Sweden)

    Joseph J Gillespie

    Full Text Available BACKGROUND: The genome sequence of Rickettsia felis revealed a number of rickettsial genetic anomalies that likely contribute not only to a large genome size relative to other rickettsiae, but also to phenotypic oddities that have confounded the categorization of R. felis as either typhus group (TG or spotted fever group (SFG rickettsiae. Most intriguing was the first report from rickettsiae of a conjugative plasmid (pRF that contains 68 putative open reading frames, several of which are predicted to encode proteins with high similarity to conjugative machinery in other plasmid-containing bacteria. METHODOLOGY/PRINCIPAL FINDINGS: Using phylogeny estimation, we determined the mode of inheritance of pRF genes relative to conserved rickettsial chromosomal genes. Phylogenies of chromosomal genes were in agreement with other published rickettsial trees. However, phylogenies including pRF genes yielded different topologies and suggest a close relationship between pRF and ancestral group (AG rickettsiae, including the recently completed genome of R. bellii str. RML369-C. This relatedness is further supported by the distribution of pRF genes across other rickettsiae, as 10 pRF genes (or inactive derivatives also occur in AG (but not SFG rickettsiae, with five of these genes characteristic of typical plasmids. Detailed characterization of pRF genes resulted in two novel findings: the identification of oriV and replication termination regions, and the likelihood that a second proposed plasmid, pRFdelta, is an artifact of the original genome assembly. CONCLUSION/SIGNIFICANCE: Altogether, we propose a new rickettsial classification scheme with the addition of a fourth lineage, transitional group (TRG rickettsiae, that is unique from TG and SFG rickettsiae and harbors genes from possible exchanges with AG rickettsiae via conjugation. We offer insight into the evolution of a plastic plasmid system in rickettsiae, including the role plasmids may have played in

  18. Characterization of the chromosomal integration of Saccharopolyspora plasmid pCM32 and its application to improve production of spinosyn in Saccharopolyspora spinosa.

    Science.gov (United States)

    Chen, Jian; Xia, Haiyang; Dang, Fujun; Xu, Qingyu; Li, Wenjun; Qin, Zhongjun

    2015-12-01

    Saccharopolyspora spinosa produces tetra-cyclic macrolide spinosyns, a group of highly efficient pesticidal agents. However, this species lacks efficient vectors for genetic manipulation. In this study, the circular plasmid pCM32 was newly isolated from Saccharopolyspora endophytica YIM 61095. The complete nucleotide sequence of pCM32 consists of 14,611 bp and is predicted to encode 17 open reading frames (ORFs). Interestingly, a putative int gene in pCM32 was predicted by homologous alignment to encode an integrase belonging to the tyrosine family of integrases/recombinases. Plasmid pCM238 containing this int locus derived from pCM32 could be transferred by conjugation from Escherichia coli into Sa. spinosa at a high frequency. Integration of pCM238 in the host chromosome was demonstrated as site-specific recombination (at the tRNA (Ser) gene) via a 56-bp core sequence within the attP/attB sites. Plasmid pCM265, a shuttle vector containing the int and attP sequences of pCM32, was constructed to introduce foreign genes into Sa. spinosa. The production of spinosad approximately doubled in Sa. spinosa NRRL18395 after introducing pCM265-derived plasmids carrying the genes for phosphofructokinase (PFK) or anthranilate synthase. These results indicate that plasmid pCM32 is an actinomycete integrative and conjugative element (AICE) and that its derived integrative vectors are useful for efficiently introducing foreign DNA into Sa. spinosa. PMID:26260388

  19. OPERATIONAL CIRCULAR NO 6 - JUNE 2001 'CERN SCIENTIFIC DOCUMENTS'

    CERN Multimedia

    Human Resources Division

    2001-01-01

    This new operational circular has been drawn up. It cancels and replaces Administrative Circular N° 29 entitled "Principles and procedures governing CERN publications and reports and other publications arising from CERN work". Copies are available from Divisional Secretariats. Note : Administrative and operational circulars, as well as the lists of those in force, are available for consultation on WWW : ADMINISTRATIVE CIRCULARSOPERATIONAL CIRCULARS

  20. Plasma-activated air mediates plasmid DNA delivery in vivo.

    Science.gov (United States)

    Edelblute, Chelsea M; Heller, Loree C; Malik, Muhammad A; Bulysheva, Anna; Heller, Richard

    2016-01-01

    Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs was enhanced, resulting in a fourfold increase in luciferase expression over 120 hours compared to injection only (P plasmid DNA encoding green fluorescent protein (GFP) was confirmed in the epidermal layers of the construct. In vivo experiments were performed in BALB/c mice, with skin as the delivery target. PAA exposure significantly enhanced luciferase expression levels 460-fold in exposed sites compared to levels obtained from the injection of plasmid DNA alone (P plasmid DNA encoding GFP to mouse skin was confirmed by immunostaining, where a 3-minute exposure at a 10 mm distance displayed delivery distribution deep within the dermal layers compared to an exposure at 3 mm where GFP expression was localized within the epidermis. Our findings suggest PAA-mediated delivery warrants further exploration as an alternative approach for DNA transfer for skin targets. PMID:27110584

  1. Replicase-based plasmid DNA shows anti-tumor activity

    Directory of Open Access Journals (Sweden)

    Weiss Richard

    2011-03-01

    Full Text Available Abstract Background Double stranded RNA (dsRNA has multiple anti-tumor mechanisms. Over the past several decades, there have been numerous attempts to utilize synthetic dsRNA to control tumor growth in animal models and clinical trials. Recently, it became clear that intracellular dsRNA is more effective than extracellular dsRNA on promoting apoptosis and orchestrating adaptive immune responses. To overcome the difficulty in delivering a large dose of synthetic dsRNA into tumors, we propose to deliver a RNA replicase-based plasmid DNA, hypothesizing that the dsRNA generated by the replicase-based plasmid in tumor cells will inhibit tumor growth. Methods The anti-tumor activity of a plasmid (pSIN-β that encodes the sindbis RNA replicase genes (nsp1-4 was evaluated in mice with model tumors (TC-1 lung cancer cells or B16 melanoma cells and compared to a traditional pCMV-β plasmid. Results In cell culture, transfection of tumor cells with pSIN-β generated dsRNA. In mice with model tumors, pSIN-β more effectively delayed tumor growth than pCMV-β, and in some cases, eradicated the tumors. Conclusion RNA replicase-based plasmid may be exploited to generate intracellular dsRNA to control tumor growth.

  2. Replicase-based plasmid DNA shows anti-tumor activity

    International Nuclear Information System (INIS)

    Double stranded RNA (dsRNA) has multiple anti-tumor mechanisms. Over the past several decades, there have been numerous attempts to utilize synthetic dsRNA to control tumor growth in animal models and clinical trials. Recently, it became clear that intracellular dsRNA is more effective than extracellular dsRNA on promoting apoptosis and orchestrating adaptive immune responses. To overcome the difficulty in delivering a large dose of synthetic dsRNA into tumors, we propose to deliver a RNA replicase-based plasmid DNA, hypothesizing that the dsRNA generated by the replicase-based plasmid in tumor cells will inhibit tumor growth. The anti-tumor activity of a plasmid (pSIN-β) that encodes the sindbis RNA replicase genes (nsp1-4) was evaluated in mice with model tumors (TC-1 lung cancer cells or B16 melanoma cells) and compared to a traditional pCMV-β plasmid. In cell culture, transfection of tumor cells with pSIN-β generated dsRNA. In mice with model tumors, pSIN-β more effectively delayed tumor growth than pCMV-β, and in some cases, eradicated the tumors. RNA replicase-based plasmid may be exploited to generate intracellular dsRNA to control tumor growth

  3. Functional amyloids as inhibitors of plasmid DNA replication.

    Science.gov (United States)

    Molina-García, Laura; Gasset-Rosa, Fátima; Moreno-Del Álamo, María; Fernández-Tresguerres, M Elena; Moreno-Díaz de la Espina, Susana; Lurz, Rudi; Giraldo, Rafael

    2016-01-01

    DNA replication is tightly regulated to constrain the genetic material within strict spatiotemporal boundaries and copy numbers. Bacterial plasmids are autonomously replicating DNA molecules of much clinical, environmental and biotechnological interest. A mechanism used by plasmids to prevent over-replication is 'handcuffing', i.e. inactivating the replication origins in two DNA molecules by holding them together through a bridge built by a plasmid-encoded initiator protein (Rep). Besides being involved in handcuffing, the WH1 domain in the RepA protein assembles as amyloid fibres upon binding to DNA in vitro. The amyloid state in proteins is linked to specific human diseases, but determines selectable and epigenetically transmissible phenotypes in microorganisms. Here we have explored the connection between handcuffing and amyloidogenesis of full-length RepA. Using a monoclonal antibody specific for an amyloidogenic conformation of RepA-WH1, we have found that the handcuffed RepA assemblies, either reconstructed in vitro or in plasmids clustering at the bacterial nucleoid, are amyloidogenic. The replication-inhibitory RepA handcuff assembly is, to our knowledge, the first protein amyloid directly dealing with DNA. Built on an amyloid scaffold, bacterial plasmid handcuffs can bring a novel molecular solution to the universal problem of keeping control on DNA replication initiation. PMID:27147472

  4. Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

    Energy Technology Data Exchange (ETDEWEB)

    Guss, Adam M [ORNL; Olson, Daniel G. [Thayer School of Engineering at Dartmouth; Caiazza, Nicky [Mascoma Corporation; Lynd, Lee R [Thayer School of Engineering at Dartmouth

    2012-01-01

    BACKGROUND: Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. RESULTS: We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+ dcm+ E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAM205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude. CONCLUSION: E. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.

  5. Researches on Relationship between Circular Agriculture and Industrial Diversity

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    First, this paper establishes the conceptual model of circular agriculture, conducts systematic analysis on the circular agriculture on the basis of conceptual model, and discusses the characteristics of closeness and openness of circular agriculture and relationship between closeness and openness of circular agriculture. Second, this paper introduces the industrial diversity related to circular agriculture, defines the concept of industry and the concept of industries related to agriculture, and illustrates the related industries that are conducive to circular agriculture and the related industries that are not conducive to circular agriculture. Finally, this paper analyzes the mutual relationship between circular agriculture and industrial diversity as follows: in the system of circular agriculture, the industrial diversity can transform the wastes in upstream industries into resources in downstream industries; the industrial diversity creates possibility for recycling of agricultural byproducts; the industrial diversity is conducive to the diversification of industries related to circular agriculture.

  6. Mitochondrial pAL2-1 plasmid homologs are senescence factors in Podospora anserina independent of intrinsic senescence

    NARCIS (Netherlands)

    van Diepeningen, Anne D; Debets, Alfons J M; Slakhorst, S Marijke; Hoekstra, Rolf F

    2008-01-01

    Since the first description of a linear mitochondrial plasmid in Podospora anserina, pAL2-1, and homologous plasmids have gone from being considered beneficial longevity plasmids, via neutral genetic elements, toward mutator plasmids causing senescence. The plasmid has an invertron structure, with t

  7. Total and Differential Efficiencies for a Circular Detector Viewing a Circular Radiator of Finite Thickness

    International Nuclear Information System (INIS)

    Total and differential detection efficiencies have been computed for a circular detector viewing a circular radiator of finite thickness. Isotropic, cosines and n-p scattering angular emission distributions of the radiated particles are considered. Tables are given for the total efficiencies as well as for the differential efficiencies in the n-p scattering case

  8. A broadband, circular-polarization selective surface

    Science.gov (United States)

    Momeni Hasan Abadi, Seyed Mohamad Amin; Behdad, Nader

    2016-06-01

    We introduce a new technique for designing wideband circular-polarization selective surfaces (CPSSs) based on anisotropic miniaturized element frequency selective surfaces. The proposed structure is a combination of two linear-to-circular polarization converters sandwiching a linear polarizer. This CPSS consists of a number of metallic layers separated from each other by thin dielectric substrates. The metallic layers are in the form of two-dimensional arrays of subwavelength capacitive patches and inductive wire grids with asymmetric dimensions and a wire grid polarizer with sub-wavelength period. The proposed device is designed to offer a wideband circular-polarization selection capability allowing waves with left-hand circular polarization to pass through while rejecting those having right-hand circular polarization. A synthesis procedure is developed that can be used to design the proposed CPSS based on its desired band of operation. Using this procedure, a prototype of the proposed CPSS operating in the 12-18 GHz is designed. Full-wave electromagnetic simulations are used to predict the response of this structure. These simulation results confirm the validity of the proposed design concept and synthesis procedure and show that proposed CPSS operates within a fractional bandwidth of 40% with a co-polarization transmission discrimination of more than 15 dB. Furthermore, the proposed design is shown to be capable of providing an extremely wide field of view of ±60°.

  9. Environmental issues elimination through circular economy

    Science.gov (United States)

    Špirková, M.; Pokorná, E.; Šujanová, J.; Samáková, J.

    2016-04-01

    Environmental efforts of European Union are currently going towards circular economy. Tools like Extended Producer Responsibility and Eco-design were established. The circular economy deals with resources availability issue on one hand and waste management on the other hand. There are few pioneering companies all over the world with some kind of circular economy practice. Generally the concept is not very wide-spread. The paper aims to evaluate possibility of transition towards circular economy in Slovak industrial companies. They need to have an active approach to material treatment of their products after usage stage. Innovation is another important pre-condition for the transition. Main problem of current cradle to grave system is landfilling of valuable materials after one cycle of usage. Their potential value for next manufacturing cycles is lost. Companies may do not see connection between waste management and material resource prices and volatility of supplies. Municipalities are responsible for municipal waste collection and treatment in Slovakia. The circular economy operates by cradle to cradle principle. Company manages material flow until the material comes back to the beginning of manufacturing process by itself or by another partners. Stable material supplies with quite low costs are provided this way. It is necessary to deal with environmental problems in phase of product design. Questionnaire survey results show on one hand low involvement of industrial companies in waste management area, however on the other hand they are open to environmental innovations in future.

  10. A COMPACT CIRCULARLY POLARIZED SLOTTED MICROSTRIP ANTENNA

    Directory of Open Access Journals (Sweden)

    V. Jebaraj

    2014-12-01

    Full Text Available Slot antennas are often used at UHF and microwave frequencies. In slot antenna for RFID reader applications the frequency ranges from 902-923MHz to achieve circular polarization. The shapes and size of the slot, as well as the driving frequency, determine the radiation distribution pattern. The proposed compact size circularly polarized slotted microstrip antenna are summarized with design rules. The circularly polarized radiation in square patch antenna can be obtained by perturbation technique with different shapes of slot in the orthogonal direction. A single feed configuration based symmetric slotted microstrip antenna is adapted to realize the compact circularly polarized microstrip antennas. Based on the perimeter, the size of the slot on microstrip slot antenna are studied and compared. The Operating frequency of the antenna is 912MHz that can be tuned by varying the perimeter of the slot while the keeping the circularly polarized radiation unchanged. The schematic and layout are configured by using Advanced Design System (ADS. Return loss, Resonant Frequency, Axial Ratio (AR, and Gain were determined for the proposed system using ADS. A measured 3dB Axial Ratio (AR bandwidth around 6MHz with 16MHz impedance bandwidth has been achieved for the antenna on a RO3004C substrate with dielectric constant 3.38.

  11. Circular Ribbon Flares and Homologous Jets

    CERN Document Server

    Wang, Haimin

    2012-01-01

    Solar flare emissions in the chromosphere often appear as elongated ribbons on both sides of the magnetic polarity inversion line (PIL), and this has been regarded as evidence of a typical configuration of magnetic reconnection. However, flares having a closed circular ribbon have rarely been reported, although it is expected in the fan--spine magnetic topology involving reconnection at a three-dimensional (3D) coronal null point. We present five circular ribbon flares with associated surges, using high-resolution and high-cadence \\ha blue wing observations obtained from the recently digitized films of Big Bear Solar Observatory (BBSO). In all the events, a central parasitic magnetic field is encompassed by the opposite magnetic polarity, forming a circular PIL that is also traced by filament material. Consequently, a flare kernel at the center is surrounded by a circular flare ribbon. The four homologous jet-related flares on 1991 March 17 and 18 are of particular interest, as (1) the circular ribbons bright...

  12. Evolution of genes on the Salmonella Virulence plasmid phylogeny revealed from sequencing of the virulence plasmids of S. enterica serotype Dublin and comparative analysis.

    Science.gov (United States)

    Chu, Chishih; Feng, Ye; Chien, An-Chi; Hu, Songnian; Chu, Chi-Hong; Chiu, Cheng-Hsun

    2008-11-01

    Salmonella enterica serotype Dublin harbors an approximately 80-kb virulence plasmid (pSDV), which mediates systemic infection in cattle. There are two types of pSDV: one is pSDVu (pOU1113) in strain OU7025 and the other pSDVr (pOU1115) in OU7409 (SD Lane) and many clinical isolates. Sequence analysis showed that pSDVr was a recombinant plasmid (co-integrate) of pSDVu and a plasmid similar to a 35-kb indigenous plasmid (pOU1114) of S. Dublin. Most of the F-transfer region in pSDVu was replaced by a DNA segment from the pOU1114-like plasmid containing an extra replicon and a pilX operon encoding for a type IV secretion system to form pSDVr. We reconstructed the particular evolutionary history of the seven virulence plasmids of Salmonella by comparative sequence analysis. The whole evolutionary process might begin with two different F-like plasmids (IncFI and IncFII), which then incorporated the spv operon and fimbriae operon from the chromosome to form the primitive virulence plasmids. Subsequently, these plasmids descended by deletion from a relatively large plasmid to smaller ones, with some recombination events occurring over time. Our results suggest that the phylogeny of virulence plasmids as a result of frequent recombination provides the opportunity for rapid evolution of Salmonella in response to the environmental cues. PMID:18718522

  13. Recombinogenic engineering of conjugative plasmids with fluorescent marker cassettes

    DEFF Research Database (Denmark)

    Reisner, A.; Molin, Søren; Zechner, E.L.

    2002-01-01

    An efficient approach for the insertion of fluorescent marker genes with sequence specificity into conjugative plasmids in Escherichia coli is described. For this purpose, homologous recombination of linear double-stranded targeting DNA was mediated by the bacteriophage lambda recombination...... functions using very short regions of homology. Initial manipulation of the IncFII target plasmids R1 and R1drd19 indicated that the linear targeting DNA should be devoid of all extraneous homologies to. the target molecule for optimal insertion specificity. Indeed, a simple recombination assay proved...... resistance genes and fluorescent markers. The choice of 5' non-homologous extensions in primer pairs used for amplifying the marker cassettes determines the site specificity of the targeting DNA. This methodology is applicable to the modification of all plasmids that replicate in E coli and is not restricted...

  14. The development of plasmid-free strains of Agrobacterium tumefaciens by using incompatibility with a Rhizobium meliloti plasmid to eliminate pAtC58.

    Science.gov (United States)

    Hynes, M F; Simon, R; Pühler, A

    1985-03-01

    Agrobacterium tumefaciens strains LBA275 and LBA290 were cured of their cryptic plasmid pAtC58 by the introduction of the Rhizobium meliloti plasmid pRme41a, which is incompatible with pAtC58. pRme41a and pTiC58, the resident Ti plasmid of LBA275, were subsequently eliminated by growth at supraoptimal temperature (40 degrees C). The resulting plasmid-free Agrobacterium strains, UBAPF1 and UBAPF2, have proved extremely useful for the study of Rhizobium plasmids. The loss of the cryptic plasmid pAtC58 has no effect on the tumor-forming ability of the Agrobacterium strains; when the Ti plasmid is present, normal tumors are formed on Kalanchoe daigremontiana. PMID:4001194

  15. Characterization of atypical Aeromonas salmonicida isolates by ribotyping and plasmid profiling

    DEFF Research Database (Denmark)

    Pedersen, Karl; Dalsgaard, Inger; Larsen, J.L.

    1996-01-01

    ) and plasmid profiles. Most epidemiologically unrelated strains had different ribotypes, whereas isolates from the same outbreak were identical. All strains, except one, carried one or more large plasmids (>55 kbp) and all strains, except two, additionally carried one or more smaller plasmids. Many strains...... isolated from the same outbreak showed different plasmid profiles although some plasmids were identical. The results suggest the existence of several atypical Aer, salmonicida. It also seems that ribotypes are stable properties for these bacteria while the plasmids are more labile....

  16. Anion exchange purification of plasmid DNA using expanded bed adsorption.

    Science.gov (United States)

    Ferreira, G N; Cabral, J M; Prazeres, D M

    2000-01-01

    Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity of process streams is of major concern in the purification of plasmids, since it can cause high back pressures in column operations, thus limiting the throughput. In order to avoid these high back pressures, expanded bed anion exchange chromatography was evaluated as an alternative to fixed bed chromatography. A Streamline 25 column filled with 100 ml of Streamline QXL media, was equilibrated with 0.5 M NaCl in TE (10 mM Tris, 1 mM EDTA, pH = 8.0) buffer at an upward flow of 300 cmh-1, E. coli lysates (obtained from up to 3 liters of fermentation broth) were injected in the column. After washing out the unbound material, the media was allowed to sediment and the plasmid was eluted with 1 M NaCl in TE buffer at a downward flow of 120 cmh-1. Purification factors of 36 +/- 1 fold, 26 +/- 0.4 plasmid purity, and close to 100% yields were obtained when less than one settled column volume of plasmid feed was injected. However, both recovery yield and purity abruptly decreased when larger amounts were processed-values of 35 +/- 2 and 5 +/- 0.7 were obtained for the recovery yield and purity, respectively, when 250 ml of feedstock were processed. In these cases, gel clogging and expansion collapse were observed. The processing of larger volumes, thus larger plasmid quantities, was only possible by performing an isopropanol precipitation step prior to the chromatographic step. This step led to an enhancement of the purification step. PMID:10840595

  17. Circularly symmetric light scattering from nanoplasmonic spirals.

    Science.gov (United States)

    Trevino, Jacob; Cao, Hui; Dal Negro, Luca

    2011-05-11

    In this paper, we combine experimental dark-field imaging, scattering, and fluorescence spectroscopy with rigorous electrodynamics calculations in order to investigate light scattering from planar arrays of Au nanoparticles arranged in aperiodic spirals with diffuse, circularly symmetric Fourier space. In particular, by studying the three main types of Vogel's spirals fabricated by electron-beam lithography on quartz substrates, we demonstrate polarization-insensitive planar light diffraction in the visible spectral range. Moreover, by combining dark-field imaging with analytical multiparticle calculations in the framework of the generalized Mie theory, we show that plasmonic spirals support distinctive structural resonances with circular symmetry carrying orbital angular momentum. The engineering of light scattering phenomena in deterministic structures with circular Fourier space provides a novel strategy for the realization of optical devices that fully leverage on enhanced, polarization-insensitive light-matter coupling over planar surfaces, such as thin-film plasmonic solar cells, plasmonic polarization devices, and optical biosensors. PMID:21466155

  18. Analytical Optimization of Piezoelectric Circular Diaphragm Generator

    Directory of Open Access Journals (Sweden)

    S. Mohammadi

    2013-01-01

    Full Text Available This paper presents an analytical study of the piezoelectric circular diaphragm microgenerator using strain energy method. Piezoelectrics are the intelligent materials that can be used as transducer to convert mechanical energy into electrical energy and vice versa. The aim of this paper is to optimize produced electrical energy from mechanical pressure. Therefore, the circular metal plate equipped with piezoelectric circular patch has been considered with simply and clamped supports. A comprehensive modeling, parametrical study and the effect of the boundary conditions on the performance of the microgenerator have been investigated. The system is under variable pressure from an oscillating pressure source. Results are presented for PZT and PMN-PT piezoelectric materials with steel and aluminum substrates. An optimal value for the radius and thickness of the piezoelectric layer with a special support condition has been obtained.

  19. Charged Particle Optics in Circular Higgs Factory

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Yunhai [SLAC National Accelerator Lab., Menlo Park, CA (United States)

    2015-02-26

    Similar to a super B-factory, a circular Higgs factory will require strong focusing systems near the interaction points and a low-emittance lattice in arcs to achieve a factory luminosity. At electron beam energy of 120 GeV, beamstrahlung effects during the collision pose an additional challenge to the collider design. In particular, a large momentum acceptance at 2 percent level is necessary to retain an adequate beam lifetime. This turns out to be the most challenging aspect in the design of circular Higgs factory. In this paper, an example will be provided to illustrate the beam dynamics in circular Higgs factory, emphasizing on the chromatic optics. Basic optical modules and advanced analysis will be presented. Most important, we will show that 2% momentum aperture is achievable

  20. Circular object recognition based on shape parameters

    Institute of Scientific and Technical Information of China (English)

    Chen Aijun; Li Jinzong; Zhu Bing

    2007-01-01

    To recognize circular objects rapidly in satellite remote sensing imagery, an approach using their geometry properties is presented.The original image is segmented to be a binary one by one dimension maximum entropy threshold algorithm and the binary image is labeled with an algorithm based on recursion technique.Then, shape parameters of all labeled regions are calculated and those regions with shape parameters satisfying certain conditions are recognized as circular objects.The algorithm is described in detail, and comparison experiments with the randomized Hough transformation (RHT) are also provided.The experimental results on synthetic images and real images show that the proposed method has the merits of fast recognition rate, high recognition efficiency and the ability of anti-noise and anti-jamming.In addition, the method performs well when some circular objects are little deformed and partly misshapen.

  1. Chaotic Motion of Corrugated Circular Plates

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Large deflection theory of thin anisotropic circular plates was used to analyze the bifurcation behavior and chaotic phenomena of a corrugated thin circular plate with combined transverse periodic excitation and an in-plane static boundary load. The nonlinear dynamic equation for the corrugated plate was derived by employing Galerkin's technique. The critical conditions for occurrence of the homoclinic and subharmonic bifurcations as well as chaos were studied theoretically using the Melnikov function method. The chaotic motion was also simulated numerically using Maple, with the Poincaré map and phase curve used to evaluate when chaotic motion appears. The results indicate some chaotic motion in the corrugated plate. The method is directly applicable to chaotic analysis of an isotropic circular plate.

  2. Sustained plasmid DNA release from dissolving mineral coatings

    OpenAIRE

    Choi, Siyoung; Murphy, William L.

    2010-01-01

    Calcium phosphate (Ca-P) minerals such as hydroxyapatite are able to bind a diverse range of biological molecules due to the presence of anions and cations in their crystal structure. The well-characterized ability of Ca-P minerals to bind and release plasmid DNA, coupled with the ability of biodegradable Ca-P coatings to form on the surface of common biomaterials, provides a potential mechanism for controlled release of plasmid DNA from various biomaterials. In this study we hypothesized tha...

  3. Restriction Fragment Length Polymorphisms of Virulence Plasmids in Rhodococcus equi

    OpenAIRE

    Takai, Shinji; Shoda, Masato; Sasaki, Yukako; Tsubaki, Shiro; Fortier, Guillaume; Pronost, Stephane; Rahal, Karim; Becu, Teotimo; Begg, Angela; Browning, Glenn; Nicholson, Vivian M.; Prescott, John F.

    1999-01-01

    Virulent Rhodococcus equi, which is a well-known cause of pyogranulomatous pneumonia in foals, possesses a large plasmid encoding virulence-associated 15- to 17-kDa antigens. Foal and soil isolates from five countries—Argentina, Australia, Canada, France, and Japan—were investigated for the presence of 15- to 17-kDa antigens by colony blotting, using the monoclonal antibody 10G5, and the gene coding for 15- to 17-kDa antigens by PCR. Plasmid DNAs extracted from positive isolates were digested...

  4. Domain structure of circular and ring magnets

    International Nuclear Information System (INIS)

    We present the results of a study of the domain structure and the magnetization reversal processes in arrays of circular and ring magnets, obtained by magnetic force microscopy and magnetization measurements. The particles, with outer diameter 0.55 and 2.2 μm, were made of Permalloy films of thickness 24 and 66 nm. In the circular elements two domain configurations were observed: a vortex structure with perpendicular magnetization at the core and a 'bud' state. In the ring elements the domain structure was found to depend on the ring width: a flux closed state was formed in wide rings and the 'onion' state was observed in narrow rings

  5. Circular economy in China and recommendations

    Institute of Scientific and Technical Information of China (English)

    Zhou Hongchun

    2006-01-01

    @@ It is an overall, urgent and long-term strategic task for China to vigorously develop the circular economy and build a resource-saving and environmental-friendly society. China is now at the accelerating stage of industrialization and urbanization.Lower per-capita resource amount and tendency of environmental deterioration has not been fundamentally averted. Therefore a circular economy development is an inevitable choice to realize the goal of building a well-off society, ensure sustainable development of national economy, and coordinate the relationship between mankind and nature.

  6. Circularly polarized U-Slot antenna

    OpenAIRE

    Tong, K. F.; Wong, T P

    2007-01-01

    Circularly polarized single-layer U-slot microstrip patch antenna has been proposed. The suggested asymmetrical U-slot can generate the two orthogonal modes for circular polarization without chamfering any corner of the probe-fed square patch microstrip antenna. A parametric study has been carried out to investigate the effects caused by different arm lengths of the U-slot. The thickness of the foam substrate is about 8.5% of the wavelength at the operating frequency. The 3 dB axial ratio ban...

  7. Broadband Suspended Microstrip Antenna For Circular Polarization

    OpenAIRE

    Kasabegoudar, VG; Vinoy, KJ

    2009-01-01

    In this paper we propose a circularly polarized (CP) microstrip antenna on a suspended substrate with a coplanar capacitive feed and a slot within the rectangular patch. The antenna has an axial ratio bandwidth (< 3 dB) of 7.1%. The proposed antenna exhibits a much higher impedance bandwidth of about 49% (S11 < -10 dB) and also yields return loss better than -15 dB in the useful range of circular polarization. Measured characteristics of the antenna are in good agreement with the simulated re...

  8. Free vibrations of circular cylindrical shells

    CERN Document Server

    Armenàkas, Anthony E; Herrmann, George

    2013-01-01

    Free Vibrations of Circular Cylindrical Shells deals with thin-walled structures that undergo dynamic loads application, thereby resulting in some vibrations. Part I discusses the treatment of problems associated with the propagation of plane harmonic waves in a hollow circular cylinder. In such search for solutions, the text employs the framework of the three-dimensional theory of elasticity. The text explains the use of tables of natural frequencies and graphs of representative mode shapes of harmonic elastic waves bounding in an infinitely long isotropic hollow cylinder. The tables are

  9. Circularly Polarized Luminescence from Simple Organic Molecules.

    Science.gov (United States)

    Sánchez-Carnerero, Esther M; Agarrabeitia, Antonia R; Moreno, Florencio; Maroto, Beatriz L; Muller, Gilles; Ortiz, María J; de la Moya, Santiago

    2015-09-21

    This article aims to show the identity of "circularly polarized luminescent active simple organic molecules" as a new concept in organic chemistry due to the potential interest of these molecules, as availed by the exponentially growing number of research articles related to them. In particular, it describes and highlights the interest and difficulty in developing chiral simple (small and non-aggregated) organic molecules able to emit left- or right-circularly polarized light efficiently, the efforts realized up to now to reach this challenging objective, and the most significant milestones achieved to date. General guidelines for the preparation of these interesting molecules are also presented. PMID:26136234

  10. Rectangular-to-circular groove waveguide junction

    Institute of Scientific and Technical Information of China (English)

    CUI; Licheng; (崔立成); YANG; Hongsheng; (杨鸿生)

    2003-01-01

    Mode matching method is used to analyze the scattering characteristics of the rectangular-to-circular groove waveguide junction. Firstly, the scattering matrix equation is obtained by matching the electromagnetic fields at the boundary of the junction. The scattering coefficients can be obtained from the equation. Secondly the scattering characteristics of the iris with rectangular window positioned in circular groove waveguide are briefly analyzed. Thirdly, the convergent problem is discussed and the numerical results are given. At last experiment is made and good agreement is found between the calculated results and the measured results.

  11. DNA sequence analysis of plasmids from multidrug resistant Salmonella enterica serotype Heidelberg isolates.

    Directory of Open Access Journals (Sweden)

    Jing Han

    Full Text Available Salmonella enterica serovar Heidelberg is among the most detected serovars in swine and poultry, ranks among the top five serotypes associated with human salmonellosis and is disproportionately associated with invasive infections and mortality in humans. Salmonella are known to carry plasmids associated with antimicrobial resistance and virulence. To identify plasmid-associated genes in multidrug resistant S. enterica serovar Heidelberg, antimicrobial resistance plasmids from five isolates were sequenced using the 454 LifeSciences pyrosequencing technology. Four of the isolates contained incompatibility group (Inc A/C multidrug resistance plasmids harboring at least eight antimicrobial resistance genes. Each of these strains also carried a second resistance plasmid including two IncFIB, an IncHI2 and a plasmid lacking an identified Inc group. The fifth isolate contained an IncI1 plasmid, encoding resistance to gentamicin, streptomycin and sulfonamides. Some of the IncA/C plasmids lacked the full concert of transfer genes and yet were able to be conjugally transferred, likely due to the transfer genes carried on the companion plasmids in the strains. Several non-IncA/C resistance plasmids also carried putative virulence genes. When the sequences were compared to previously sequenced plasmids, it was found that while all plasmids demonstrated some similarity to other plasmids, they were unique, often due to differences in mobile genetic elements in the plasmids. Our study suggests that Salmonella Heidelberg isolates harbor plasmids that co-select for antimicrobial resistance and virulence, along with genes that can mediate the transfer of plasmids within and among other bacterial isolates. Prevalence of such plasmids can complicate efforts to control the spread of S. enterica serovar Heidelberg in food animal and human populations.

  12. Genetic Characterization of ExPEC-Like Virulence Plasmids among a Subset of NMEC.

    Science.gov (United States)

    Nicholson, Bryon A; West, Aaron C; Mangiamele, Paul; Barbieri, Nicolle; Wannemuehler, Yvonne; Nolan, Lisa K; Logue, Catherine M; Li, Ganwu

    2016-01-01

    Neonatal Meningitis Escherichia coli (NMEC) is one of the most common causes of neonatal bacterial meningitis in the US and elsewhere resulting in mortality or neurologic deficits in survivors. Large plasmids have been shown experimentally to increase the virulence of NMEC in the rat model of neonatal meningitis. Here, 9 ExPEC-like plasmids were isolated from NMEC and sequenced to identify the core and accessory plasmid genes of ExPEC-like virulence plasmids in NMEC and create an expanded plasmid phylogeny. Results showed sequenced virulence plasmids carry a strongly conserved core of genes with predicted functions in five distinct categories including: virulence, metabolism, plasmid stability, mobile elements, and unknown genes. The major functions of virulence-associated and plasmid core genes serve to increase in vivo fitness by adding multiple iron uptake systems to the genetic repertoire to facilitate NMEC's survival in the host's low iron environment, and systems to enhance bacterial resistance to host innate immunity. Phylogenetic analysis based on these core plasmid genes showed that at least two lineages of ExPEC-like plasmids could be discerned. Further, virulence plasmids from Avian Pathogenic E. coli and NMEC plasmids could not be differentiated based solely on the genes of the core plasmid genome. PMID:26800268

  13. Genetic Characterization of ExPEC-Like Virulence Plasmids among a Subset of NMEC.

    Directory of Open Access Journals (Sweden)

    Bryon A Nicholson

    Full Text Available Neonatal Meningitis Escherichia coli (NMEC is one of the most common causes of neonatal bacterial meningitis in the US and elsewhere resulting in mortality or neurologic deficits in survivors. Large plasmids have been shown experimentally to increase the virulence of NMEC in the rat model of neonatal meningitis. Here, 9 ExPEC-like plasmids were isolated from NMEC and sequenced to identify the core and accessory plasmid genes of ExPEC-like virulence plasmids in NMEC and create an expanded plasmid phylogeny. Results showed sequenced virulence plasmids carry a strongly conserved core of genes with predicted functions in five distinct categories including: virulence, metabolism, plasmid stability, mobile elements, and unknown genes. The major functions of virulence-associated and plasmid core genes serve to increase in vivo fitness by adding multiple iron uptake systems to the genetic repertoire to facilitate NMEC's survival in the host's low iron environment, and systems to enhance bacterial resistance to host innate immunity. Phylogenetic analysis based on these core plasmid genes showed that at least two lineages of ExPEC-like plasmids could be discerned. Further, virulence plasmids from Avian Pathogenic E. coli and NMEC plasmids could not be differentiated based solely on the genes of the core plasmid genome.

  14. Yields of strand breaks and base lesions induced by soft X-rays in plasmid DNA

    International Nuclear Information System (INIS)

    The yields of soft-X-ray-induced DNA damages have been measured by using closed-circular plasmid DNA. Several DNA solutions with three kinds of radical scavenger capacity and also fully hydrated DNA samples were irradiated to compare the contribution by indirect reaction of diffusible water radicals, such as OH., with those by direct action of secondary electrons. The yields of prompt single- (SSBs) and double-strand breaks (DSBs) decrease with increasing scavenging capacity. The SSB yields for soft X-rays are approximately midway those between gamma-ray and ultrasoft X-ray data previously reported. Heat labile sites are observed only in the low scavenger condition. The yields of the base lesions revealed by post irradiation treatment with base excision repair enzymes showed a similar value for Nth and Fpg protein except in the hydrated sample. These results indicate that the direct effect of soft X-rays induces the damages with different efficiency from those by indirect effect. (authors)

  15. Circular depolarization ratios of single water droplets and finite ice circular cylinders: a modeling study

    Directory of Open Access Journals (Sweden)

    M. Nicolet

    2011-11-01

    Full Text Available Computations of the phase matrix elements for single water droplets and ice crystals in fixed orientations are presented to determine if circular depolarization δ±C is more accurate than linear depolarization for phase discrimination. T-matrix simulations were performed to calculate right-handed and left-handed circular depolarization ratios δ+C, respectively δ−C and to compare them with linear ones. Ice crystals are assumed to have a circular cylindrical shape where their surface-equivalent diameters range up to 5 μm. The circular depolarization ratios of ice particles were generally higher than linear depolarization and depended mostly on the particle orientation as well as their sizes. The fraction of non-detectable ice crystals (δ < 0.05 was smaller considering a circular polarized light source, reaching 4.5%. However, water droplets also depolarized light circularly for scattering angles smaller than 179° and size parameters smaller than 6 at side- and backscattering regions. Differentiation between ice crystals and water droplets might be difficult for experiments performing at backscattering angles which deviate from 180° unlike lidar applications. If the absence of the liquid phase is confirmed, the use of circular depolarization in single particle detection is more sensitive and less affected by particle orientation.

  16. Determination of G-values for single and double strand break induction in plasmid DNA using agarose gel electrophoresis and a curve-fitting procedure

    International Nuclear Information System (INIS)

    Covalently closed circular double-stranded DNA (CC) of native plasmids was used to determine yields of single strand breaks (ssb) and double strand breaks (dsb) caused by x-radiation. One ssb transforms DNA of the CC form to the nicked circular form (NC); one dsb produced either directly or from random coincidence of single strand breaks transforms DNA of the CC as well as of the NC form to linear DNA molecules (LI form). Plasmids with more than one dsb are cleaved to linear fragments. DNA (30-800 μg/ml) was irradiated in air-saturated buffer. DNA forms were separated by gel electrophoresis and their amounts measured fluorometrically using ethidium bromide. Large linear DNA fragments with the same electrophoretic mobility as the LI form were considered using a curve-fitting procedure. From quantitative changes of each conformation D37 values of ssb and dsb were calculated as a function of the DNA concentration. Finally G-values were calculated by competition plots. The following yields were determined: Gsub(ssb) 3.4 x 10E - 8 mol J-1, and Gsub(dsb) 3.3 x 10E - 10 molJ-1. Gsub(dsb) refers to directly produced dsb. Yields are related to strand breaks without further treatment. (author)

  17. Construction and Use of Flow Cytometry Optimized Plasmid-Sensor Strains

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Oregaard, Gunnar; Sørensen, Søren Johannes;

    2009-01-01

    Determining the stability of plasmids in bacterial populations is traditionally performed by isolating a large number of clones followed by screening for the presence of plasmids by replica transfer to plasmid-selective agar plates. This is often a laborious task, especially when the intrinsic...... stability of the plasmid is high. The method presented here relies on a phenotypic (green fluorescence protein) marker, which is switched on if the host bacteria loses the residing plasmid. The incorporation of flow cytometry for single-cell detection and discrimination between plasmid-free and plasmid......-harboring cells in a bacterial population facilitates a very high throughput of cells and thus provides excellent sensitivity and statistics toward detecting even very low levels of plasmid instability....

  18. Survival and evolution of a large multidrug resistance plasmid in new clinical bacterial hosts

    DEFF Research Database (Denmark)

    Porse, Andreas; Schønning, Kristian; Munck, Christian;

    2016-01-01

    and population sequencing to show that the long-term persistence and molecular integrity of the plasmid is highly influenced by multiple factors within a 25 kb plasmid region constituting a host-dependent burden. In the E. coli hosts investigated here, improved plasmid stability readily evolves via IS......Large conjugative plasmids are important drivers of bacterial evolution and contribute significantly to the dissemination of antibiotic resistance. Although plasmid borne multidrug resistance is recognized as one of the main challenges in modern medicine, the adaptive forces shaping the evolution...... of these plasmids within pathogenic hosts are poorly understood. Here we study plasmid-host adaptations following transfer of a 73 kb conjugative multidrug resistance plasmid to naïve clinical isolates of Klebsiella pneumoniae and Escherichia coli We use experimental evolution, mathematical modelling...

  19. Survival and evolution of a large multidrug resistance plasmid in new clinical bacterial hosts

    DEFF Research Database (Denmark)

    Porse, Andreas; Schønning, Kristian; Munck, Christian;

    2016-01-01

    Large conjugative plasmids are important drivers of bacterial evolution and contribute significantly to the dissemination of antibiotic resistance. Although plasmid borne multidrug resistance is recognized as one of the main challenges in modern medicine, the adaptive forces shaping the evolution...... of these plasmids within pathogenic hosts are poorly understood. Here we study plasmid-host adaptations following transfer of a 73 kb conjugative multidrug resistance plasmid to naïve clinical isolates of Klebsiella pneumoniae and Escherichia coli We use experimental evolution, mathematical modelling and population...... sequencing to show that the long-term persistence and molecular integrity of the plasmid is highly influenced by multiple factors within a 25 kb plasmid region constituting a host-dependent burden. In the E. coli hosts investigated here, improved plasmid stability readily evolves via IS26 mediated deletions...

  20. Conservation of plasmids among plant-pathogenic Pseudomonas syringae isolates of diverse origins.

    Science.gov (United States)

    von Bodman, S B; Shaw, P D

    1987-05-01

    Thirty isolates of Pseudomonas syringae pv. tabaci, pv. angulata (pathogens on tobacco), pv. coronafaciens, and pv. striafaciens (pathogens on oats) were examined for plasmid DNAs. The strains were obtained from plants throughout the world, some over 50 years ago. Of the 22 tobacco pathogens, 16 contain predominantly one type of plasmid, the pJP27.00 type. The remaining six tobacco-specific strains do not harbor detectable plasmids. The oat pathogens contain one, two, or three plasmids. DNA homology studies indicate that the plasmid DNAs are highly conserved. More importantly, the plasmids harbored by strains isolated from one host plant are conserved most stringently; e.g., the plasmids from the tobacco pathogens are, with one exception, indistinguishable by restriction endonuclease digestion and Southern hybridization. There is also extensive homology among plasmids indigenous to the oat-specific P. syringae pv. coronafaciens and pv. striafaciens strains. PMID:3628554

  1. Possible involvement of a plasmid in arginine auxotrophic mutation of Streptomyces kasugaensis.

    OpenAIRE

    Nakano, M M; Ozawa, K; Ogawara, H

    1980-01-01

    Streptomyces kasugaensis gave arginine auxotrophic mutants at high frequency, The coupled loss and reappearance of plasmid deoxyribonucleic acid with arginine auxotrophy suggested that the insertion of the plasmid into chromosomal deoxyribonucleic acid caused the arginine auxotrophy.

  2. A Time-Efficient and User-Friendly Method for Plasmid DNA Restriction Analysis.

    Science.gov (United States)

    LaBanca, Frank; Berg, Claire M.

    1998-01-01

    Describes an experiment in which plasmid DNA is digested with restriction enzymes that cleave the plasmid either once or twice. The DNA is stained, loaded on a gel, electrophoresed, and viewed under normal laboratory conditions during electrophoresis. (DDR)

  3. Transcription of ColE1Ap mbeC induced by conjugative plasmids from twelve different incompatibility groups.

    OpenAIRE

    Selvaratnam, S; Gealt, M A

    1993-01-01

    Although nonconjugative mobilizable plasmids require helping functions of conjugative plasmids in order to be mobilized into recipients, at least some genes from the nonconjugative plasmids may be induced to assist in the DNA transfer process. Conjugative plasmids from 12 different incompatibility groups mobilized the nonconjugative plasmid ColE1Ap between Escherichia coli strains. Introduction of any of the conjugative plasmids into the ColE1Ap-containing strain resulted in an induction of m...

  4. New tetracycline resistance determinant on R plasmids from Vibrio anguillarum.

    OpenAIRE

    Aoki, T.; Satoh, T.; Kitao, T.

    1987-01-01

    Two classes of tetracycline resistance determinants on R plasmids were detected in Vibrio anguillarum strains isolated from ayu (sweat fish; Plecoglossus altivelis) farms in Japan. Tetracycline resistance genes categorized as class B were prevalent from 1973 to 1977; however, a new tetracycline resistance gene, which was not classified into tetracycline resistance determinant class A, B, C, or D, has been prevalent since 1981.

  5. Pharmaceutical development of the plasmid DNA vaccine pDERMATT

    NARCIS (Netherlands)

    Quaak, S.G.L.

    2009-01-01

    The discovery of tumor specific antigens and self tolerance mechanisms against these antigens led to the assumption that antigens circulating at sufficient concentration levels could break this self tolerance mechanism and evoke immunological antitumor effects. pDERMATT (plasmid DNA encoding recombi

  6. Stability of Integrated Plasmids in the Chromosome of Lactococcus lactis

    NARCIS (Netherlands)

    Leenhouts, Kees J.; Kok, Jan; Venema, Gerhardus

    1990-01-01

    Derivatives of plasmids pBR322, pUB110, pSC101, and pTB19, all containing an identical fragment of lactococcal chromosomal DNA, were integrated via a Campbell-like mechanism into the same chromosomal site of Lactococcus lactis MG1363, and the transformants were analyzed for the stability of the inte

  7. Plasmid-determined copper resistance in Pseudomonas syringae from impatiens

    Energy Technology Data Exchange (ETDEWEB)

    Cooksey, D.A. (Univ. of California, Riverside (USA))

    1990-01-01

    A strain of Pseudomonas syringae was recently identified as the cause of a new foliar blight of impatiens. The bacterium was resistant to copper compounds, which are used on a variety of crops for bacterial and fungal disease control. The bacterium contained a single 47-kilobase plasmid (pPSI1) that showed homology to a copper resistance operon previously cloned and characterized from P. syringae pv. tomato plasmid pPT23D (D. Cooksey, Appl. Environ. Microbiol. 53:454-456, 1987). pPSI1 was transformed by electroporation into a copper-sensitive P. syringae strain, and the resulting transformants were copper resistant. A physical map of pPSI1 was constructed, and the extent of homology to pPT23D outside the copper resistance operon was determined in Southern hybridizations. The two plasmids shared approximately 20 kilobases of homologous DNA, with the remainder of each plasmid showing no detectable homology. The homologous regions hybridized strongly, but there was little or no conservation of restriction enzyme recognition sites.

  8. Studying plasmid horizontal transfer in situ: a critical review

    DEFF Research Database (Denmark)

    Sørensen, Søren Johannes; Bailey, Mark; Hansen, Lars Hestbjerg;

    2005-01-01

    This review deals with the prospective, experimental documentation of horizontal gene transfer (HGT) and its role in real-time, local adaptation. We have focused on plasmids and their function as an accessory and/or adaptive gene pool. Studies of the extent of HGT in natural environments have...

  9. Use of plasmid DNA for induction of protective immunity

    DEFF Research Database (Denmark)

    Lorenzen, Niels

    2004-01-01

    Vaccines based on plasmid DNA have been tested for a number of fish pathogens but so far it is only in case of the rhabdoviruses, where the technology has been a real break through in vaccine research. Aspects of dose, time-course and mechanisms of protection, as well as practical use are discussed....

  10. Multilocus sequence typing of IncN plasmids

    DEFF Research Database (Denmark)

    García-Fernández, Aurora; Villa, Laura; Moodley, Arshnee; Hasman, Henrik; Miriagou, Vivi; Guardabassi, Luca; Carattoli, Alessandra

    2011-01-01

    OBJECTIVES: Incompatibility group N (IncN) plasmids have been associated with the dissemination of antimicrobial resistance and are a major vehicle for the spread of blaVIM-1 in humans and blaCTX-M-1 in animals. A plasmid multilocus sequence typing (pMLST) scheme was developed for rapid......, Greece, Denmark, UK and The Netherlands) were classified by DNA sequencing of the amplicons obtained for the repA, traJ and korA loci. RESULTS: Eleven sequence types (STs) were defined on the basis of allele sequences of the three selected loci. Most plasmids carrying blaCTX-M-1 (24/27) isolated in...... spread and persistence of this particular IncN-carrying blaVIM-1 lineage in Greece. CONCLUSIONS: This study proposes the use of pMLST as a suitable and rapid method for identification of IncN epidemic plasmid lineages. The recent spread of blaCTX-M-1 among humans and animals seems to be associated with...

  11. [Transfer of plasmid beta-lactamases in enterobacteria].

    Science.gov (United States)

    Umaran, A; Garaizar, J; Gallego, L; Colom, K; Cisterna, R

    1989-04-01

    The aim of the present study was to determine which types of beta-lactamases codified by plasmids are transferred by conjugation from several species of enterobacteria. To this end, 352 strains of ampicillin-resistant enterobacteria from clinical samples from the Hospital Civil of Bilbao were evaluated. Their beta-lactamase activity and their capacity to transfer this capacity by conjugation were evaluated. The several types of plasmidic beta-lactamases in the strains that conjugated and in their respective transconjugants were characterized by analytic isoelectric approach, and also the sensitivity of these stains to 20 beta-lactamic antibiotics and the size of their plasmids. Twenty different types were detected, with a clear predominance of TEM 1. Type TEM 2 was found in 19% of the strains which conjugated, and much less commonly the types SHV 1, HMS 1 and a beta-lactamase of an approximate pl of 4.9 were found. The transfer of these beta-lactamases is mediated by a great variety of plasmids and is associated with variable levels of resistance to penicillins and unstable cephalosporins. The presence of betalactamases with activity on the more stable cephalosporins has not been detected. PMID:2490696

  12. Beam Dynamics and Beam Losses - Circular Machines

    CERN Document Server

    Kain, V

    2016-01-01

    A basic introduction to transverse and longitudinal beam dynamics as well as the most relevant beam loss mechanisms in circular machines will be presented in this lecture. This lecture is intended for physicists and engineers with little or no knowledge of this subject.

  13. The Global Future Circular Colliders Effort

    CERN Document Server

    Benedikt, Michael

    2013-01-01

    This presentation has been given during the P5 Workshop at BNL Brookhaven (US). It contains - Global Future Circular Collier Studies Overview and Status - Main challenges and R&D areas for hadron collider - Main challenges and R&D areas for lepton collider - Conclusions

  14. Circular Vibration Planing of Inconel 718

    Science.gov (United States)

    Hettiarachchi, Nandita Kalyanakumara; Moriwaki, Toshimichi; Shibasaka, Toshiro; Nakamoto, Keiichi

    Circular vibration milling (CVM) is achieved by vibrating a milling cutter about the machine tool spindle axis in a circular path, in addition to its rotary motion. CVM has been proven capable of producing better surface finishes on difficult to cut materials. However, the CVM process is far slower than conventional milling process. In circular vibration planing (CVP) process, the cutting tool is clamped without rotation and fed at a speed comparable to the feed speed of conventional milling. By superimposing circular vibration motion, necessary cutting speed could be achieved keeping the feed speed at realistic values. Inconel 718 was machined by CVP and conventional milling at a similar feed rate. It was observed that CVP could reduce tool wear and hence produce better surface finishes than conventional milling. A geometric simulation showed a major difference between uncut chip shapes of the two processes. The difference of uncut chip shapes suggests that in CVP process, less rubbing occurs between tool flank face and work before the tool penetrates in to the work to form a chip. The reduced rubbing of the flank face is proposed as the reson for reduced tool wear in CVP when compared with conventional milling.

  15. Numerical simulation of flow past circular duct

    Directory of Open Access Journals (Sweden)

    Ze-gao YIN

    2010-06-01

    Full Text Available On the basis of Fluent software, Renormalization Group (RNGk-ε turbulent model and Volume of Fluid (VOF method are employed to simulate the flow past circular duct to obtain and analyze the hydraulic parameters. According to various upper and bottom gap-ratios, the force on duct is calculated. Firstly, when bottom gap-ratio is 0, drag force coefficient, lift force coefficient and composite force reach the maximum respectively and azimuth reaches the minimum. Secondly, with the increase of bottom gap-ratio from 0 to 1, drag force coefficient and composite force decrease sharply, lift force coefficient decreases a little, but azimuth increases dramatically. Thirdly, with the continuous increase of bottom gap-ratio from 1, drag force coefficient, lift force coefficient, composite force and azimuth vary little. So, bottom gap-ratio is the key factor influencing the force on circular duct. When bottom gap-ratio is less than 1, upper gap-ratio has the remarkable influence on the circular duct force. When bottom gap-ratio is greater than 1, the varation of upper gap-ratio has a little influence on the circular duct force.

  16. Dynamics of the circular bosonic string

    International Nuclear Information System (INIS)

    After a brief discussion about the Nambu-Goto action, we introduce the concept of transverse velocity and find it for a closed circular bosonic string, then we study and solve the string's equations of movement by using the energy conservation law and finally we sketch its corresponding world sheet. (author).

  17. Discrete Fresnel Transform and Its Circular Convolution

    CERN Document Server

    Ouyang, Xing; Gunning, Fatima; Zhang, Hongyu; Guan, Yong Liang

    2015-01-01

    Discrete trigonometric transformations, such as the discrete Fourier and cosine/sine transforms, are important in a variety of applications due to their useful properties. For example, one well-known property is the convolution theorem for Fourier transform. In this letter, we derive a discrete Fresnel transform (DFnT) from the infinitely periodic optical gratings, as a linear trigonometric transform. Compared to the previous formulations of DFnT, the DFnT in this letter has no degeneracy, which hinders its mathematic applications, due to destructive interferences. The circular convolution property of the DFnT is studied for the first time. It is proved that the DFnT of a circular convolution of two sequences equals either one circularly convolving with the DFnT of the other. As circular convolution is a fundamental process in discrete systems, the DFnT not only gives the coefficients of the Talbot image, but can also be useful for optical and digital signal processing and numerical evaluation of the Fresnel ...

  18. Helically corrugated circular waveguides as antenna feeders

    Science.gov (United States)

    Jecko, F.; Papiernik, A.

    1983-07-01

    Rotation of the plane of polarization of the TE(11)-mode is predicted and observed in a helically corrugated circular waveguide. Rotation is suppressed by a longitudinal deformation produced on the corrugation. This modified structure can be used as an antenna feeder with low losses.

  19. Numerical simulation of flow past circular duct

    Institute of Scientific and Technical Information of China (English)

    Ze-gao YIN; Xian-wei Cao; Hong-da SHI; Jian MA

    2010-01-01

    The Renormalization Group(RNG)k-ε turbulence model and Volume of Fluid(VOF)method were employed to simulate the flow past a circular duct in order to obtain and analyze hydraulic parameters.According to various upper and bottom gap ratios,the force on the duct was calculated.When the bottom gap ratio is 0,the drag force coefficient,lift force coefficient,and composite force reach their maximum values,and the azimuth reaches its minimum.With an increase of the bottom gap ratio from 0 to 1,the drag force coefficient and composite force decrease sharply,and the lift force coefficient does not decreases so much,but the azimuth increases dramatically.With a continuous increase of the bottom gap ratio from 1 upward,the drag force coefficient,lift force coefficient,composite force,and azimuth vary little.Thus,the bottom gap ratio is the key factor influencing the force on the circular duct.When the bottom gap ratio is less than 1,the upper gap ratio has a remarkable influence on the force of the circular duct.When the bottom gap ratio is greater than 1,the variation of the upper gap ratio has little influence on the force of the circular duct.

  20. Oblique stability of circularly polarized MHD waves

    International Nuclear Information System (INIS)

    The stability of finite-amplitude weakly dispersive circularly polarized MHD wave trains with respect to oblique modulations is investigated. The mathematical model is a multi-dimensional extension of the DNLS equation. It is found that the right-hand-polarized wave, which is stable with respect to parallel modulations, is unstable with respect to certain oblique modulations for most primary wavenumbers. (author)

  1. Time can't be circular

    CERN Multimedia

    2003-01-01

    "Delivering the Albert Einstein lecture at the Centre for Philosophy and Foundations of Science on Tuesday, Nobel Laureate Professor Gerard Hooft said that any notion of time can not be circular or have loops or multi dimensionality" (1/2 page).

  2. Nuclear spin circular dichroism in fullerenes

    Czech Academy of Sciences Publication Activity Database

    Straka, Michal

    Brno : Masaryk University Press, 2015 - (Sklenář, V.). s. 153 ISBN 978-80-210-7890-1. [EUROMAR 2015. 05.07.2015-10.07.2015, Praha] R&D Projects: GA ČR(CZ) GA14-03564S Institutional support: RVO:61388963 Keywords : nuclear spin circular dichroism * fullerenes Subject RIV: CF - Physical ; Theoretical Chemistry

  3. Vibrational circular dichroism of nucleic acids

    Czech Academy of Sciences Publication Activity Database

    Andrushchenko, Valery; Wieser, H.; Bouř, Petr

    Brescia, 2009. s. 60-60. [International Conference on Circular Dichroism /12./. Interdisciplinary Symposium on Biological Chirality /5./. 30.08.2009-04.09.2009, Brescia] Institutional research plan: CEZ:AV0Z40550506 Keywords : VCD * G-quadruplex Subject RIV: CF - Physical ; Theoretical Chemistry

  4. Self-organization in circular shear layers

    DEFF Research Database (Denmark)

    Bergeron, K.; Coutsias, E.A.; Lynov, Jens-Peter;

    1996-01-01

    Experiments on forced circular shear layers performed in both magnetized plasmas and in rotating fluids reveal qualitatively similar self-organization processes leading to the formation of patterns of coherent vortical structures with varying complexity. In this paper results are presented from...

  5. CIRCULAR CHROMATIC NUMBER AND MYCIELSKI GRAPHS

    Institute of Scientific and Technical Information of China (English)

    Liu Hongmei

    2006-01-01

    For a general graph G, M(G) denotes its Mycielski graph. This article gives a number of new sufficient conditions for G to have the circular chromatic number xc (M(G))equals to the chromatic number x(M(G)), which have improved some best sufficient conditions published up to date.

  6. Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Greulich-Bode, Karin; Wang, Mei; Rhein, Andreas; Weier, Jingly; Weier, Heinz-Ulli

    2008-12-16

    Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-?B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

  7. Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Greulich-Bode, Karin M.; Wang, Mei; Rhein, Andreas P.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-12-04

    Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-{kappa}B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

  8. Plasmid profiles of clinical and environmental isolates of Legionella pneumophila serogroup 1.

    OpenAIRE

    Maher, W E; Plouffe, J F; Para, M F

    1983-01-01

    Clinical and environmental Legionella pneumophila serogroup 1 isolates from a single water source in Columbus, Ohio, exhibited five different plasmid profiles. The multiplicity of plasmid profiles observed within a single geographic area and a skewed distribution of isolates bearing these plasmid profiles within this area suggest that plasmid analyses will be useful in the study of the epidemiology of Legionnaires disease and the environmental distribution of legionellae.

  9. Plasmid marker rescue transformation proceeds by breakage-reunion in Bacillus subtilis.

    OpenAIRE

    Weinrauch, Y; Dubnau, D

    1987-01-01

    Bacillus subtilis carrying a plasmid which replicates with a copy number of about 1 was transformed with linearized homologous plasmid DNA labeled with the heavy isotopes 2H and 15N, in the presence of 32Pi and 6-(p-hydroxyphenylazo)-uracil to inhibit DNA replication. Plasmid DNA was isolated from the transformed culture and fractionated in cesium chloride density gradients. The distribution of total and donor plasmid DNA was examined, using specific hybridization probes. The synthesis of new...

  10. Incidence of Plasmids in Thermus spp. Isolated in Yellowstone National Park

    OpenAIRE

    Munster, Michael J.; Munster, Ann P.; Sharp, Richard J.

    1985-01-01

    Forty-eight strains of Thermus spp. were isolated from thermal sites in Yellowstone National Park, Wyo., and 62.5% showed evidence of plasmid DNA. Attempts to assign function to the plasmid DNA were unsuccessful, and the presence of plasmid DNA could not be correlated with antibiotic or heavy metal resistance. A number of these cryptic plasmids are now being investigated for their potential as vectors for molecular cloning in Thermus spp.

  11. A DNA polymerase mutation that suppresses the segregation bias of an ARS plasmid in Saccharomyces cerevisiae.

    OpenAIRE

    Houtteman, S W; Elder, R T

    1993-01-01

    Yeast autonomously replicating sequence (ARS) plasmids exhibit an unusual segregation pattern during mitosis. While the nucleus divides equally into mother and daughter cells, all copies of the ARS plasmid will often remain in the mother cell. A screen was designed to isolate mutations that suppress this segregation bias. A plasmid with a weak ARS (wARS) that displayed an extremely high segregation bias was constructed. When cells were grown under selection for the wARS plasmid, the resulting...

  12. Modulation of ColE1-like Plasmid Replication for Recombinant Gene Expression

    OpenAIRE

    Camps, Manel

    2010-01-01

    ColE1-like plasmids constitute the most popular vectors for recombinant protein expression. ColE1 plasmid replication is tightly controlled by an antisense RNA mechanism that is highly dynamic, tuning plasmid metabolic burden to the physiological state of the host. Plasmid homeostasis is upset upon induction of recombinant protein expression because of non-physiological levels of expression and because of the frequently biased amino acid composition of recombinant proteins. Disregulation of p...

  13. Participation of the lytic replicon in bacteriophage P1 plasmid maintenance.

    OpenAIRE

    Yarmolinsky, M B; Hansen, E B; Jafri, S; Chattoraj, D K

    1989-01-01

    P1 bacteriophage carries at least two replicons: a plasmid replicon and a viral lytic replicon. Since the isolated plasmid replicon can maintain itself stably at the low copy number characteristic of intact P1 prophage, it has been assumed that this replicon is responsible for driving prophage replication. We provide evidence that when replication from the plasmid replicon is prevented, prophage replication continues, albeit at a reduced rate. The residual plasmid replication is due to incomp...

  14. Stability in Escherichia coli of an antibiotic resistance plasmid from Bacteroides fragilis.

    OpenAIRE

    Rashtchian, A; Booth, S J

    1981-01-01

    A Bacteroides fragilis strain resistant to penicillin G, tetracycline, and clindamycin was screened for the presence of plasmid deoxyribonucleic acid (DNA). Agarose gel electrophoresis of ethanol-precipitated DNA from cleared lysates of this strain revealed two plasmid DNA bands. The molecular weights of the plasmids were estimated by their relative mobility in agarose gel and compared with standard plasmids with known molecular weights. The molecular weights were 3.40 +/- 0.20 x 10(6) and 1....

  15. Properties of R1162, a broad-host-range, high-copy-number plasmid.

    OpenAIRE

    R. MEYER; Hinds, M; Brasch, M.

    1982-01-01

    Regions of plasmid DNA encoding characteristic properties of the IncQ (P-4) group plasmid R1162 were identified by mutagenesis and in vitro cloning. Coding sequences sufficient for expression of incompatibility and efficient conjugal mobilization by plasmid R751 were found to be linked to the origin of DNA replication. In contrast, there was a region remote from the origin, and active in trans, that was required for plasmid maintenance. A derivative that was temperature sensitive for stabilit...

  16. Characterization of different plasmid-borne dihydropteroate synthases mediating bacterial resistance to sulfonamides.

    OpenAIRE

    Swedberg, G; Sköld, O

    1980-01-01

    Plasmid-borne resistance to sulfonamides was studied in both newly isolated and earlier characterized R plasmids. Two different classes of drug-resistant dihydropteroate synthases were found to be responsible for most cases of plasmid-mediated sulfonamide resistance. The plasmid-coded enzymes could be completely separated from their chromosomal counterpart and also showed differences in heat stability and molecular size. The resistant and chromosomal enzymes could bind the normal substrate, p...

  17. Use of agarose gel electrophoresis of plasmid deoxyribonucleic acid to fingerprint gram-negative bacilli.

    OpenAIRE

    Schaberg, D.R.; Tompkins, L S; Falkow, S

    1981-01-01

    Agarose gel electrophoresis of the plasmid deoxyribonucleic acids from 60 gram-negative bacilli recovered during investigations of nosocomial epidemics was used to fingerprint the strains. This method was as specific at differentiating bacterial strains as more conventional phenotyping methods. In all cases, plasmid band fingerprints of epidermic strains isolates were identical whereas coisolate plasmid deoxyribonucleic acid patterns were different. Agarose gel electrophoresis of plasmid deox...

  18. Identification of tetracycline-resistant R-plasmids in Streptococcus agalactiae (group B).

    OpenAIRE

    Burdett, V

    1980-01-01

    In this report, 30 tetracycline-resistant clinical isolates of group B Streptococcus were examined to assess the extent to which tetracycline resistance is plasmid mediated. Of these, 27 showed no physical or genetic evidence of plasmid-mediated resistance; however, one conjugative and two small (3.5 X 10(6)-dalton) multicopy non-self-transmissible tetracycline resistance plasmids were identified. The conjugative plasmid was transmissible to Streptococcus faecalis as well as to Streptococcus ...

  19. The Salmonella virulence plasmid enhances Salmonella-induced lysis of macrophages and influences inflammatory responses.

    OpenAIRE

    Guilloteau, L. A.; Wallis, T S; A.V. Gautier; Macintyre, S.; Platt, D. J.; Lax, A J

    1996-01-01

    The Salmonella dublin virulence plasmid mediates systemic infection in mice and cattle. Here, we analyze the interaction between wild-type and plasmid-cured Salmonella strains with phagocytes in vitro and in vivo. The intracellular recovery of S. dublin from murine peritoneal and bovine alveolar macrophages cultured in the presence of gentamicin in vitro was not related to virulence plasmid carriage. However, the virulence plasmid increased the lytic activity of S. dublin, Salmonella typhimur...

  20. Complete DNA Sequence and Analysis of the Large Virulence Plasmid of Shigella flexneri

    OpenAIRE

    Venkatesan, Malabi M.; Goldberg, Marcia B.; Rose, Debra J.; Grotbeck, Erik J.; Burland, Valerie; Blattner, Frederick R.

    2001-01-01

    The complete sequence analysis of the 210-kb Shigella flexneri 5a virulence plasmid was determined. Shigella spp. cause dysentery and diarrhea by invasion and spread through the colonic mucosa. Most of the known Shigella virulence determinants are encoded on a large plasmid that is unique to virulent strains of Shigella and enteroinvasive Escherichia coli; these known genes account for approximately 30 to 35% of the virulence plasmid. In the complete sequence of the virulence plasmid, 286 ope...