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Sample records for absolute plasmid dna

  1. A BAYESIAN METHOD FOR CALCULATING REAL-TIME QUANTITATIVE PCR CALIBRATION CURVES USING ABSOLUTE PLASMID DNA STANDARDS

    EPA Science Inventory

    In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. However, potential differences in the amplification performance of plasmid DNA compared to genomic DNA standards are often ignore...

  2. Tissue-specific Calibration of Real-time PCR Facilitates Absolute Quantification of Plasmid DNA in Biodistribution Studies

    PubMed Central

    Ho, Joan K; White, Paul J; Pouton, Colin W

    2016-01-01

    Analysis of the tissue distribution of plasmid DNA after administration of nonviral gene delivery systems is best accomplished using quantitative real-time polymerase chain reaction (qPCR), although published strategies do not allow determination of the absolute mass of plasmid delivered to different tissues. Generally, data is expressed as the mass of plasmid relative to the mass of genomic DNA (gDNA) in the sample. This strategy is adequate for comparisons of efficiency of delivery to a single site but it does not allow direct comparison of delivery to multiple tissues, as the mass of gDNA extracted per unit mass of each tissue is different. We show here that by constructing qPCR standard curves for each tissue it is possible to determine the dose of intact plasmid remaining in each tissue, which is a more useful parameter when comparing the fates of different formulations of DNA. We exemplify the use of this tissue-specific qPCR method by comparing the delivery of naked DNA, cationic DNA complexes, and neutral PEGylated DNA complexes after intramuscular injection. Generally, larger masses of intact plasmid were present 24 hours after injection of DNA complexes, and neutral complexes resulted in delivery of a larger mass of intact plasmid to the spleen. PMID:27701400

  3. Plasmid DNA manufacturing technology.

    PubMed

    Carnes, Aaron E; Williams, James A

    2007-01-01

    Today, plasmid DNA is becoming increasingly important as the next generation of biotechnology products (gene medicines and DNA vaccines) make their way into clinical trials, and eventually into the pharmaceutical marketplace. This review summarizes recent patents and patent applications relating to plasmid manufacturing, in the context of a comprehensive description of the plasmid manufacturing intellectual property landscape. Strategies for plasmid manufacturers to develop or in-license key plasmid manufacturing technologies are described with the endpoint of efficiently producing kg quantities of plasmid DNA of a quality that meets anticipated European and FDA quality specifications for commercial plasmid products.

  4. Yeast DNA plasmids.

    PubMed

    Gunge, N

    1983-01-01

    The study of yeast DNA plasmids has been initiated with the discovery of the 2-micron DNA in Saccharomyces cerevisiae. This multiple copy plasmid, organized into chromatin structure in vivo, probably exists in the nucleus and provides a good system to obtain information on eukaryotic DNA replication. Yeast transformation with the 2-micron DNA or artificially constructed chimeric plasmids had contributed significantly to the study of the molecular biology of yeast and eukaryotes, allowing the isolation and characterization of various genes, ars, centromeres, and telomeres, and also serving as a tool to study the expression of various heterologous genes. Encouraged by these fruitful results, new yeast plasmids have been screened among phylogenetically distant yeasts. The linear DNA plasmids (pGKl1 and pGKl2) from Kluyveromyces lactis are the first case of yeast plasmids associated with biological function (killer phenotype). This plasmid system would be ideal as a model to study the structure and function of eukaryotic linear chromosomes. The extracellular secretion of protein toxin suggests the plasmids to be an excellent candidate for a secretion vector. The importance of yeasts as suitable materials for the study of eukaryotic cell biology would be much enhanced by the advent of new transformation systems with diverse host yeasts of genetically and phylogenetically distinct properties.

  5. Phytoplasma plasmid DNA extraction.

    PubMed

    Andersen, Mark T; Liefting, Lia W

    2013-01-01

    Phytoplasma plasmids have generally been detected from DNA extracted from plants and insects using methods designed for the purification of total phytoplasma DNA. Methods include extraction from tissues that are high in phytoplasma titre, such as the phloem of plants, with the use of CsCl-bisbenzimide gradients that exploit the low G+C content of phytoplasma DNA. Many of the methods employed for phytoplasma purification have been described elsewhere in this book. Here we describe in detail two methods that are specifically aimed at isolating plasmid DNA.

  6. Chemical adjuvants for plasmid DNA vaccines.

    PubMed

    Greenland, John R; Letvin, Norman L

    2007-05-10

    Plasmid DNA vaccines are a promising modality for immunization against a variety of human pathogens. Immunization via multiple routes with plasmid DNA can elicit potent cellular immune responses, and these immunogens can be administered repeatedly without inducing anti-vector immunity. Nonetheless, the immunogenicity of plasmid DNA vaccines has been limited by problems associated with delivery. A number of adjuvants have been designed to improve plasmid DNA immunogenicity, either by directly stimulating the immune system or by enhancing plasmid DNA expression. Chemical adjuvants for enhancing plasmid DNA expression include liposomes, polymers, and microparticles, all of which have shown promise for enhancing the expression and immunogenicity of plasmid DNA vaccines in animal models. Micro- and nanoparticles have not been shown to enhance immune responses to plasmid DNA vaccines. However, formulation of plasmid DNA with some non-particulate polymeric adjuvants has led to a statistically significant enhancement of immune responses. Further development of these technologies will significantly improve the utility of plasmid DNA vaccination.

  7. Microwave effects on plasmid DNA.

    PubMed

    Sagripanti, J L; Swicord, M L; Davis, C C

    1987-05-01

    The exposure of purified plasmid DNA to microwave radiation at nonthermal levels in the frequency range from 2.00 to 8.75 GHz produces single- and double-strand breaks that are detected by agarose gel electrophoresis. Microwave-induced damage to DNA depends on the presence of small amounts of copper. This effect is dependent upon both the microwave power and the duration of the exposure. Cuprous, but not cupric, ions were able to mimic the effects produced by microwaves on DNA.

  8. Microwave effects on plasmid DNA

    SciTech Connect

    Sagripanti, J.L.; Swicord, M.L.; Davis, C.C.

    1987-05-01

    The exposure of purified plasmid DNA to microwave radiation at nonthermal levels in the frequency range from 2.00 to 8.75 GHz produces single- and double-strand breaks that are detected by agarose gel electrophoresis. Microwave-induced damage to DNA depends on the presence of small amounts of copper. This effect is dependent upon both the microwave power and the duration of the exposure. Cuprous, but not cupric, ions were able to mimic the effects produced by microwaves on DNA.

  9. Production of Plasmid DNA as Pharmaceutical.

    PubMed

    Schmeer, Marco; Schleef, Martin

    2015-01-01

    Pharmaceutical applications of plasmid DNA require certain quality standards, depending on the intended use of the plasmids. That is, for direct gene transfer into human, GMP Grade is mandatory, however, for GMP production of for example viral vectors (AAV or mRNA etc.), the plasmid DNA used has not to be produced under GMP necessarily. Here we summarize important features of producing plasmid DNA, ensuring the required quality for the intended (pharmaceutical) application.

  10. Topological Behavior of Plasmid DNA.

    PubMed

    Higgins, N Patrick; Vologodskii, Alexander V

    2015-04-01

    The discovery of the B-form structure of DNA by Watson and Crick led to an explosion of research on nucleic acids in the fields of biochemistry, biophysics, and genetics. Powerful techniques were developed to reveal a myriad of different structural conformations that change B-DNA as it is transcribed, replicated, and recombined and as sister chromosomes are moved into new daughter cell compartments during cell division. This article links the original discoveries of superhelical structure and molecular topology to non-B form DNA structure and contemporary biochemical and biophysical techniques. The emphasis is on the power of plasmids for studying DNA structure and function. The conditions that trigger the formation of alternative DNA structures such as left-handed Z-DNA, inter- and intra-molecular triplexes, triple-stranded DNA, and linked catenanes and hemicatenanes are explained. The DNA dynamics and topological issues are detailed for stalled replication forks and for torsional and structural changes on DNA in front of and behind a transcription complex and a replisome. The complex and interconnected roles of topoisomerases and abundant small nucleoid association proteins are explained. And methods are described for comparing in vivo and in vitro reactions to probe and understand the temporal pathways of DNA and chromosome chemistry that occur inside living cells.

  11. Large-scale preparation of plasmid DNA.

    PubMed

    Heilig, J S; Elbing, K L; Brent, R

    2001-05-01

    Although the need for large quantities of plasmid DNA has diminished as techniques for manipulating small quantities of DNA have improved, occasionally large amounts of high-quality plasmid DNA are desired. This unit describes the preparation of milligram quantities of highly purified plasmid DNA. The first part of the unit describes three methods for preparing crude lysates enriched in plasmid DNA from bacterial cells grown in liquid culture: alkaline lysis, boiling, and Triton lysis. The second part describes four methods for purifying plasmid DNA in such lysates away from contaminating RNA and protein: CsCl/ethidium bromide density gradient centrifugation, polyethylene glycol (PEG) precipitation, anion-exchange chromatography, and size-exclusion chromatography.

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

    PubMed Central

    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 quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification. PMID:22194997

  13. Electrotransformation of Yersinia ruckeri by plasmid DNA.

    PubMed

    Cutrín, J M; Conchas, R F; Barja, J L; Toranzo, A E

    1994-01-01

    Yersinia ruckeri, a fish pathogenic bacterium in aquaculture, was used to evaluate the electroporation as a new transformation method for this species. DNA used for the electrotransformation were plasmids of molecular mass ranging from 2.3 kb to 33 kb, and diverse replicons. To optimize this method we used Y. ruckeri 11.29 strain (from serotype 02) and pSU2718 DNA. The best transformation efficiency (6.0 x 10(5) transformants/micrograms DNA) was obtained with 12.5 kV/cm, 25 microF, 400 omega and 2 hours of incubation after pulse. When these conditions were applied to other strains belonging to different serotypes and other plasmids, we obtained transformants in all strains assayed, but only when using low molecular weight plasmids. Plasmid vectors and resident plasmid were not modified in host strains after electrotransformation. In studies of conformation we confirmed that only circular DNA was able for transformation. The utilization of this technique for direct cloning in Y. ruckeri makes possible further studies on recombinant DNA.

  14. Electrotransfer of Plasmid Vector DNA into Muscle

    NASA Astrophysics Data System (ADS)

    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.

  15. Plasmid DNA hydrogels for biomedical applications.

    PubMed

    Costa, Diana; Valente, Artur J M; Miguel, M Graça; Queiroz, João

    2014-03-01

    In the last few years, our research group has focused on the design and development of plasmid DNA (pDNA) based systems as devices to be used therapeutically in the biomedical field. Biocompatible macro and micro plasmid DNA gels were prepared by a cross-linking reaction. For the first time, the pDNA gels have been investigated with respect to their swelling in aqueous solution containing different additives. Furthermore, we clarified the fundamental and basic aspects of the solute release mechanism from pDNA hydrogels and the significance of this information is enormous as a basic tool for the formulation of pDNA carriers for drug/gene delivery applications. The co-delivery of a specific gene and anticancer drugs, combining chemical and gene therapies in the treatment of cancer was the main challenge of our research. Significant progresses have been made with a new p53 encoding pDNA microgel that is suitable for the loading and release of pDNA and doxorubicin. This represents a strong valuable finding in the strategic development of systems to improve cancer cure through the synergetic effect of chemical and gene therapy.

  16. Proteolysis in plasmid DNA stable maintenance in bacterial cells.

    PubMed

    Karlowicz, Anna; Wegrzyn, Katarzyna; Dubiel, Andrzej; Ropelewska, Malgorzata; Konieczny, Igor

    2016-07-01

    Plasmids, as extrachromosomal genetic elements, need to work out strategies that promote independent replication and stable maintenance in host bacterial cells. Their maintenance depends on constant formation and dissociation of nucleoprotein complexes formed on plasmid DNA. Plasmid replication initiation proteins (Rep) form specific complexes on direct repeats (iterons) localized within the plasmid replication origin. Formation of these complexes along with a strict control of Rep protein cellular concentration, quaternary structure, and activity, is essential for plasmid maintenance. Another important mechanism for maintenance of low-copy-number plasmids are the toxin-antitoxin (TA) post-segregational killing (psk) systems, which prevent plasmid loss from the bacterial cell population. In this mini review we discuss the importance of nucleoprotein complex processing by energy-dependent host proteases in plasmid DNA replication and plasmid type II toxin-antitoxin psk systems, and draw attention to the elusive role of DNA in this process.

  17. Therapeutic option of plasmid-DNA based gene transfer.

    PubMed

    Taniyama, Yoshiaki; Azuma, Junya; Kunugiza, Yasuo; Iekushi, Kazuma; Rakugi, Hiromi; Morishita, Ryuichi

    2012-01-01

    Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common method in clinical cases because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid deoxyribonucleic acid (DNA)-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a phase III clinical trial did not show sufficient efficiency. In this situation, more efficient plasmid DNA gene transfer is needed all over the world. This review focuses on plasmid DNA gene transfer and its enhancement, including ultrasound with microbubbles, electroporation, hydrodynamic method, gene gun, jet injection, cationic lipids and cationic polymers.

  18. Evaluating metabolic stress and plasmid stability in plasmid DNA production by Escherichia coli.

    PubMed

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

    2012-01-01

    In the context of recombinant DNA technology, the development of feasible and high-yielding plasmid DNA production processes has regained attention as more evidence for its efficacy as vectors for gene therapy and DNA vaccination arise. When producing plasmid DNA in Escherichia coli, a number of biological restraints, triggered by plasmid maintenance and replication as well as culture conditions are responsible for limiting final biomass and product yields. This termed "metabolic burden" can also cause detrimental effects on plasmid stability and quality, since the cell machinery is no longer capable of maintaining an active metabolism towards plasmid synthesis and the stress responses elicited by plasmid maintenance can also cause increased plasmid instability. The optimization of plasmid DNA production bioprocesses is still hindered by the lack of information on the host metabolic responses as well as information on plasmid instability. Therefore, systematic and on-line approaches are required not only to characterise this "metabolic burden" and plasmid stability but also for the design of appropriate metabolic engineering and culture strategies. The monitoring tools described to date rapidly evolve from laborious, off-line and at-line monitoring to online monitoring, at a time-scale that enables researchers to solve these bioprocessing problems as they occur. This review highlights major E. coli biological alterations caused by plasmid maintenance and replication, possible causes for plasmid instability and discusses the ability of currently employed bioprocess monitoring techniques to provide information in order to circumvent metabolic burden and plasmid instability, pointing out the possible evolution of these methods towards online bioprocess monitoring.

  19. Influenza Plasmid DNA Vaccines: Progress and Prospects.

    PubMed

    Bicho, Diana; Queiroz, João António; Tomaz, Cândida Teixeira

    2015-01-01

    Current influenza vaccines have long been used to fight flu infectious; however, recent advances highlight the importance of produce new alternatives. Even though traditional influenza vaccines are safe and usually effective, they need to be uploaded every year to anticipate circulating flu viruses. This limitation together with the use of embryonated chicken eggs as the substrate for vaccine production, is time-consuming and could involve potential biohazards in growth of new virus strains. Plasmid DNA produced by prokaryote microorganisms and encoding foreign proteins had emerged as a promising therapeutic tool. This technology allows the expression of a gene of interest by eukaryotic cells in order to induce protective immune responses against the pathogen of interest. In this review, we discuss the strategies to choose the best DNA vaccine to be applied in the treatment and prevention of influenza. Specifically, we give an update of influenza DNA vaccines developments, all involved techniques, their main characteristics, applicability and technical features to obtain the best option against influenza infections.

  20. Identifying stabilizers of plasmid DNA for pharmaceutical use.

    PubMed

    Zeng, Yuhong; Ramsey, Joshua D; King, Robert; Leviten, Michael; Mcguire, Ruth; Volkin, David B; Joshi, Sangeeta B; Middaugh, C Russell

    2011-03-01

    To better address the need for developing stable formulations of plasmid DNA-based biopharmaceuticals, 37 compounds from a generally regarded as safe library were examined for their potential use as stabilizers. A plasmid DNA-based therapeutic vaccine, BHT-DNA, was used as a model system. Initial studies were performed to compare the biophysical properties of BHT-DNA plasmid from bulk drug substance and finished drug product. An agarose gel electrophoresis-based assay was then employed in excipient compatibility studies for the drug product by monitoring supercoiled plasmid DNA content in various formulations. After incubation at 40 °C for 30 days, eight out of the 37 excipients tested were able to better retain the supercoil content compared to the control. Sodium citrate appeared to be the most effective stabilizer and its protective capability plateaued at an ionic strength of about 0.4. Several other excipients including malic acid, ethanol, and Pluronic F-68 were also identified as promising stabilizers for BHT-DNA plasmid DNA. Additionally, compounds, including ferrous chloride, ascorbic acid, human serum albumin, and PEG 1000, which significantly destabilized the supercoiled plasmid DNA were identified. These data may also be applicable to other plasmid DNA-based pharmaceuticals for storage stability improvement, due to chemical and structural similarities of these macromolecules.

  1. Intradermal naked plasmid DNA immunization: mechanisms of action.

    PubMed

    Elnekave, Mazal; Furmanov, Karina; Hovav, Avi-Hai

    2011-08-01

    Plasmid DNA is a promising vaccine modality that is regularly examined in prime-boost immunization regimens. Recent advances in skin immunity increased our understanding of the sophisticated cutaneous immune network, which revived scientific interest in delivering vaccines to the skin. Intradermal administration of plasmid DNA via needle injection is a simple and inexpensive procedure that exposes the plasmid and its encoded antigen to the dermal immune surveillance system. This triggers unique mechanisms for eliciting local and systemic immunity that can confer protection against pathogens and tumors. Understanding the mechanisms of intradermal plasmid DNA immunization is essential for enhancing and modulating its immunogenicity. With regard to vaccination, this is of greater importance as this routine injection technique is highly desirable for worldwide immunization. This article will focus on the current understanding of the mechanisms involved in antigen expression and presentation during primary and secondary syringe and needle intradermal plasmid DNA immunization.

  2. Tubular cationized pullulan hydrogels as local reservoirs for plasmid DNA.

    PubMed

    San Juan, Aurélie; Ducrocq, Grégory; Hlawaty, Hanna; Bataille, Isabelle; Guénin, Erwann; Letourneur, Didier; Feldman, Laurent J

    2007-12-01

    In the present study, we measured the ability of various cationized pullulan tubular hydrogels to retain plasmid DNA, and tested the ability of retained plasmid DNA to transfect vascular smooth muscle cells (VSMCs). Cationized pullulans were obtained by grafting at different charge densities ethylamine (EA) or diethylaminoethylamine (DEAE) on the pullulan backbone. Polymers were characterized by elemental analysis, acid-base titration, size exclusion chromatography, Fourier-transform infrared spectroscopy, and proton nuclear magnetic resonance. The complexation of cationized pullulans in solution with plasmid DNA was evidenced by fluorescence quenching with PicoGreen. Cationized pullulans were then chemically crosslinked with phosphorus oxychloride to obtain tubular cationized pullulan hydrogels. Native pullulan tubes did not retain loaded plasmid DNA. In contrast, the ability of cationized pullulan tubes to retain plasmid DNA was dependent on both the amine content and the type of amine. The functional integrity of plasmid DNA in cationized pullulan tubes was demonstrated by in vitro transfection of VSMCs. Hence, cationized pullulan hydrogels can be designed as tubular structures with high affinity for plasmid DNA, which may provide new biomaterials to enhance the efficiency of local arterial gene transfer strategies.

  3. DNA Assembly Tools and Strategies for the Generation of Plasmids.

    PubMed

    Baek, Chang-Ho; Liss, Michael; Clancy, Kevin; Chesnut, Jonathan; Katzen, Federico

    2014-10-01

    Since the discovery of restriction enzymes and the generation of the first recombinant DNA molecule over 40 years ago, molecular biology has evolved into a multidisciplinary field that has democratized the conversion of a digitized DNA sequence stored in a computer into its biological counterpart, usually as a plasmid, stored in a living cell. In this article, we summarize the most relevant tools that allow the swift assembly of DNA sequences into useful plasmids for biotechnological purposes. We cover the main components and stages in a typical DNA assembly workflow, namely in silico design, de novo gene synthesis, and in vitro and in vivo sequence assembly methodologies.

  4. Plasma-activated air mediates plasmid DNA delivery in vivo

    PubMed Central

    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 < 0.05). Delivery of 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 < 0.001). Expression levels were enhanced when the plasma reactor was positioned more distant from the injection site. Delivery of 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

  5. Plasmid DNA-based gene transfer with ultrasound and microbubbles.

    PubMed

    Taniyama, Yoshiaki; Azuma, Junya; Rakugi, Hiromi; Morishita, Ryuichi

    2011-12-01

    Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common option in the real world because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid DNA-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a Phase III clinical trial did not show sufficient efficiency. Recently, a Phase III clinical trial of hepatocyte growth factor gene therapy in peripheral artery disease (PAD) showed improvement of ischemic ulcers, but it could not salvage limbs from amputation. In addition, a Phase I/II clinical study of fibroblast growth factor gene therapy in PAD extended amputation-free survival, but it seemed to fail in Phase III. In this situation, we and others have developed plasmid DNA-based gene transfer using ultrasound with microbubbles to enhance its efficiency while maintaining safety. Ultrasound-mediated gene transfer has been reported to augment the gene transfer efficiency and select the target organ using cationic microbubble phospholipids which bind negatively charged DNA. Ultrasound with microbubblesis likely to create new therapeutic options inavariety of diseases.

  6. Functional amyloids as inhibitors of plasmid DNA replication

    PubMed Central

    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

  7. Plasmid DNA damage induced by helium atmospheric pressure plasma jet

    NASA Astrophysics Data System (ADS)

    Han, Xu; Cantrell, William A.; Escobar, Erika E.; Ptasinska, Sylwia

    2014-03-01

    A helium atmospheric pressure plasma jet (APPJ) is applied to induce damage to aqueous plasmid DNA. The resulting fractions of the DNA conformers, which indicate intact molecules or DNA with single- or double-strand breaks, are determined using agarose gel electrophoresis. The DNA strand breaks increase with a decrease in the distance between the APPJ and DNA samples under two working conditions of the plasma source with different parameters of applied electric pulses. The damage level induced in the plasmid DNA is also enhanced with increased plasma irradiation time. The reactive species generated in the APPJ are characterized by optical emission spectra, and their roles in possible DNA damage processes occurring in an aqueous environment are also discussed.

  8. Controlled release of plasmid DNA from hyaluronan nanoparticles.

    PubMed

    Mahor, Sunil; Collin, Estelle; Dash, Biraja C; Pandit, Abhay

    2011-07-01

    Encapsulation of plasmid DNA (pDNA) in nanoparticulate gene delivery systems offers the possibility of control in dosing, enhanced pDNA uptake, increased resistance to nuclease degradation and sustained release of functionally active pDNA over time. Extracellular matrix based biomaterial i.e. hyaluronan (HA) was used to encapsulate pDNA (pCMV-GLuc, Gaussia Luciferase reporter plasmid DNA having CMV promoter) in submicron size particulate system. Nano size range (~400-600 nm) pDNA loaded hyaluronan nanoparticles were formulated by ionic gelation followed by the cross-linking method with high encapsulation efficiency (~75-85%). The particle preparation process was further optimized for molecular weight, cross-linking method, cross-linking time and plasmid/polymer ratio. The entrapped plasmid maintained its structural and functional integrity as revealed by agarose gel electrophoresis. The pDNA was released from the hyaluronan nanoparticles in a controlled manner over a period of one month. In vitro transfection by one-week released pDNA from nanoparticles with transfecting agent branched polyethyleneimine (bPEI) resulted in significantly higher expression levels than those in pDNA alone which demonstrated the functional bioactivity of released pDNA. For cellular localization studies, the hyaluronan nanoparticles encapsulated with FITC-dextran were incubated with adipose derived stem cells (ADSCs) and localization in the cellular environment were investigated. The results of this study illustrate that hyaluronan nanoparticles were rapidly internalized by the cells through nonspecific endocytosis and remained intact in the cytosol for up to 24 h.

  9. Current trends in separation of plasmid DNA vaccines: a review.

    PubMed

    Ghanem, Ashraf; Healey, Robert; Adly, Frady G

    2013-01-14

    Plasmid DNA (pDNA)-based vaccines offer more rapid avenues for development and production if compared to those of conventional virus-based vaccines. They do not rely on time- or labour-intensive cell culture processes and allow greater flexibility in shipping and storage. Stimulating antibodies and cell-mediated components of the immune system are considered as some of the major advantages associated with the use of pDNA vaccines. This review summarizes the current trends in the purification of pDNA vaccines for practical and analytical applications. Special attention is paid to chromatographic techniques aimed at reducing the steps of final purification, post primary isolation and intermediate recovery, in order to reduce the number of steps necessary to reach a purified end product from the crude plasmid.

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

    NASA Astrophysics Data System (ADS)

    Giustranti, C.; Pérez, C.; Rousset, S.; Balanzat, E.; Sage, E.

    1999-01-01

    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 pBS. 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 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. En utilisant la technique de relaxation de plasmide, l'induction de cassures simple brin (SSB) par les radiations ? a été comparée dans deux plasmides de taille différente, pSP189 et pBS. La relation dose-effet est linéaire pour les deux plasmides, mais il se forme trois fois plus de SSB dans pSP189 que dans pBS. Cette disparité semble pouvoir être reliée au degré de compaction différent des plasmides, observé en microscopie électronique. Elle s'expliquerait en terme d'accessibilité aux espèces radicalaires formées lors de la radiolyse de l'eau. Le plasmide pBS, à différentes concentrations, a été ensuite exposé aux radiations γ. Le taux de cassures décroit lorsque la concentration en ADN croit, suggérant une diminution du nombre de radicaux pouvant efficacement réagir avec l'ADN. Cet effet a également été mis en évidence lors d'une irradiation avec des particules de TEL élevé. En conclusion, l'accessibilité de l'ADN est un paramètre- clé dans la formation des dommages, tant in vitro que in vivo.

  11. A Time-Efficient and User-Friendly Method for Plasmid DNA Restriction Analysis.

    ERIC Educational Resources Information Center

    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)

  12. Using Plasmids as DNA Vaccines for Infectious Diseases.

    PubMed

    Tregoning, John S; Kinnear, Ekaterina

    2014-12-01

    DNA plasmids can be used to induce a protective (or therapeutic) immune response by delivering genes encoding vaccine antigens. That naked DNA (without the refinement of coat proteins or host evasion systems) can cross from outside the cell into the nucleus and be expressed is particularly remarkable given the sophistication of the immune system in preventing infection by pathogens. As a result of the ease, low cost, and speed of custom gene synthesis, DNA vaccines dangle a tantalizing prospect of the next wave of vaccine technology, promising individual designer vaccines for cancer or mass vaccines with a rapid response time to emerging pandemics. There is considerable enthusiasm for the use of DNA vaccination as an approach, but this enthusiasm should be tempered by the successive failures in clinical trials to induce a potent immune response. The technology is evolving with the development of improved delivery systems that increase expression levels, particularly electroporation and the incorporation of genetically encoded adjuvants. This review will introduce some key concepts in the use of DNA plasmids as vaccines, including how the DNA enters the cell and is expressed, how it induces an immune response, and a summary of clinical trials with DNA vaccines. The review also explores the advances being made in vector design, delivery, formulation, and adjuvants to try to realize the promise of this technology for new vaccines. If the immunogenicity and expression barriers can be cracked, then DNA vaccines may offer a step change in mass vaccination.

  13. Genetic immunization with plasmid DNA mediated by electrotransfer.

    PubMed

    Rochard, Alice; Scherman, Daniel; Bigey, Pascal

    2011-07-01

    The concept of DNA immunization was first advanced in the early 1990s, but was not developed because of an initial lack of efficiency. Recent technical advances in plasmid design and gene delivery techniques have allowed renewed interest in the idea. Particularly, a better understanding of genetic immunization has led to construction of optimized plasmids and the use of efficient molecular adjuvants. The field also took great advantage of new delivery techniques such as electrotransfer. This is a simple physical technique consisting of injecting plasmid DNA into a target tissue and applying an electric field, allowing up to a thousandfold more expression of the transgene than naked DNA. DNA immunization mediated by electrotransfer is now effective in a variety of preclinical models against infectious or acquired diseases such as cancer or autoimmune diseases, and is making its way through the clinics in several ongoing phase I human clinical trials. This review will briefly describe genetic immunization mediated by electrotransfer and the main fields of application.

  14. Cold atmospheric pressure plasma jet interactions with plasmid DNA

    SciTech Connect

    O'Connell, D.; Cox, L. J.; Hyland, W. B.; McMahon, S. J.; Reuter, S.; Graham, W. G.; Gans, T.; Currell, F. J.

    2011-01-24

    The effect of a cold (<40 deg. C) radio frequency-driven atmospheric pressure plasma jet on plasmid DNA has been investigated. Gel electrophoresis was used to analyze the DNA forms post-treatment. The experimental data are fitted to a rate equation model that allows for quantitative determination of the rates of single and double strand break formation. The formation of double strand breaks correlates well with the atomic oxygen density. Taken with other measurements, this indicates that neutral components in the jet are effective in inducing double strand breaks.

  15. A new plasmid vector for DNA delivery using lactococci

    PubMed Central

    Guimarães, Valeria; Innocentin, Sylvia; Chatel, Jean-Marc; Lefèvre, François; Langella, Philippe; Azevedo, Vasco; Miyoshi, Anderson

    2009-01-01

    Background The use of food-grade lactococci as bacterial carriers to DNA delivery into epithelial cells is a new strategy to develop live oral DNA vaccine. Our goal was to develop a new plasmid, named pValac, for antigen delivery for use in lactococci. The pValac plasmid was constructed by the fusion of: i) a eukaryotic region, allowing the cloning of an antigen of interest under the control of the pCMV eukaryotic promoter to be expressed by a host cell and ii) a prokaryotic region allowing replication and selection of bacteria. In order to evaluate pValac functionality, the gfp ORF was cloned into pValac (pValac:gfp) and was analysed by transfection in PK15 cells. The applicability of pValac was demonstrated by invasiveness assays of Lactococcus lactis inlA+ strains harbouring pValac:gfp into Caco-2 cells. Results After transfection with pValac:gfp, we observed GFP expression in PK15 cells. L. lactis inlA+ were able to invade Caco-2 cells and delivered a functional expression cassette (pCMV:gfp) into epithelial cells. Conclusion We showed the potential of an invasive L. lactis harbouring pValac to DNA delivery and subsequent triggering DNA expression by epithelial cells. Further work will be to examine whether these strains are able to deliver DNA in intestinal cells in vivo. PMID:19208231

  16. Transformation of vegetative cells of Bacillus thuringiensis by plasmid DNA.

    PubMed

    Heierson, A; Landén, R; Lövgren, A; Dalhammar, G; Boman, H G

    1987-03-01

    Plasmid DNA-mediated transformation of vegetative cells of Bacillus thuringiensis was studied with the following two plasmids: pBC16 coding for tetracycline resistance and pC194 expressing chloramphenicol resistance. A key step was the induction of competence by treatment of the bacteria with 50 mM Tris hydrochloride buffer (pH 8.9) containing 30% sucrose. Transformation frequency was strongly influenced by culture density during the uptake of DNA and required the presence of polyethylene glycol. Growth in a minimal medium supplemented with Casamino Acids gave 35 times more transformants than growth in a rich medium. The highest frequencies were obtained with covalently closed circular DNA. With all parameters optimized, the frequency was 10(-3) transformants per viable cell or 10(4) transformants per microgram of DNA. Cells previously frozen were also used as recipients in transformation experiments; such cells gave frequencies similar to those obtained with freshly grown cells. The procedure was optimized for B. thuringiensis subsp. gelechiae, but B. thuringiensis subsp. kurstaki, B. thuringiensis subsp. galleriae, B. thuringiensis subsp. thuringiensis, and B. thuringiensis subsp. israelensis were also transformed. Compared with protoplast transformation, our method is much faster and 3 orders of magnitude more efficient per microgram of added DNA.

  17. Complexation Between Cationic Diblock Copolymers and Plasmid DNA

    NASA Astrophysics Data System (ADS)

    Jung, Seyoung; Reineke, Theresa; Lodge, Timothy

    Deoxyribonucleic acids (DNA), as polyanions, can spontaneously bind with polycations to form polyelectrolyte complexes. When the polycation is a diblock copolymer with one cationic block and one uncharged hydrophilic block, the polyelectrolyte complexes formed with plasmid DNA (pDNA) are often colloidally stable, and show great promise in the field of polymeric gene therapy. While the resulting properties (size, stability, and toxicity to biological systems) of the complexes have been studied for numerous cationic diblocks, the fundamentals of the pDNA-diblock binding process have not been extensively investigated. Herein, we report how the cationic block content of a diblock influences the pDNA-diblock interactions. pDNA with 7164 base pairs and poly(2-deoxy-2-methacrylamido glucopyranose)-block-poly(N-(2-aminoethyl) methacrylamide) (PMAG-b-PAEMA) are used as the model pDNA and cationic diblock, respectively. To vary the cationic block content, two PMAG-b-PAEMA copolymers with similar PMAG block lengths but distinct PAEMA block lengths and a PAEMA homopolymer are utilized. We show that the enthalpy change from pDNA-diblock interactions is dependent on the cationic diblock composition, and is closely associated with both the binding strength and the pDNA tertiary structure.

  18. Preparation of Plasmid DNA by Alkaline Lysis with Sodium Dodecyl Sulfate: Minipreps.

    PubMed

    Green, Michael R; Sambrook, Joseph

    2016-10-03

    In this protocol, plasmid DNA is isolated from small-scale (1-2 mL) bacterial cultures. Yields vary between 100 and 5 µg of DNA, depending on the copy number of the plasmid. Miniprep DNA is sufficiently pure for use as a substrate or template in many in vitro enzymatic reactions. However, further purification is required if the plasmid DNA is used as the substrate in sequencing reactions.

  19. Adsorption of plasmid DNA on anion exchange chromatography media.

    PubMed

    Tarmann, Christina; Jungbauer, Alois

    2008-08-01

    Anion exchange chromatography (AEC) is a useful and effective tool for DNA purification, but due to average pore sizes between 40 and 100 nm most AEC resins lack truly useful binding capacities for plasmid DNA (pDNA). Equilibrium binding capacities and uptake kinetics of AEC media including conventional media (Source 30 Q, Q Sepharose HP), a polymer grafted medium (Fractogel EMD DEAE (M)), media with large pores (Celbeads DEAE, PL SAX 4000 A 30 microm) and a monolithic medium (CIM-DEAE) were investigated by batch uptake or shallow bed experiments at two salt concentrations. Theoretical and experimental binding capacities suggest that the shape of the pDNA molecule can be described by a rod with a length to diameter ratio of 20:1 and that the molecule binds in upright position. The arrangement of DNA like a brush at the surface can be considered as entropy driven, kind of self-assembly process which is inherent to highly and uniformly charged DNA molecules. The initial phase of adsorption is very fast and levels off, associated with a change in mass transfer mechanism. Feed concentrations higher than 0.1 mg/mL pDNA pronounce this effect. Monolithic media showed the fastest adsorption rate and highest binding capacity with 13 mg pDNA per mL.

  20. Biological consequences of strand breaks in plasmid and viral DNA.

    PubMed Central

    Schulte-Frohlinde, D.

    1987-01-01

    Some biological consequences of strand breakage in biologically active single- and double-stranded plasmid and viral DNA are examined. A double-strand break in DNA produced by restriction-endonucleases in aqueous solution is not a 100% lethal damage. The survival depends strongly on the structure of the end groups. Evidence is presented that survival is the result of a balance between degradation and repair. The enzymatically produced double-strand break (dsb) is a potentially lethal damage similar to the irradiation-produced dsb in cells. Results with double-stranded biologically active DNA treated either with gamma-rays, heat, pancrease nuclease or UV-light in aqueous solution suggest that a single-strand damage is also a potentially lethal damage. Mechanisms for conversion of single-strand damage to lethal events are discussed. PMID:3307866

  1. Processing of plasmid DNA with ColE1-like replication origin.

    PubMed

    Wang, Zhijun; Yuan, Zhenghong; Hengge, Ulrich R

    2004-05-01

    With the increasing utilization of plasmid DNA as a biopharmaceutical drug, there is a rapidly growing need for high quality plasmid DNA for drug applications. Although there are several different kinds of replication origins, ColE1-like replication origin is the most extensively used origin in biotechnology. This review addresses problems in upstream and downstream processing of plasmid DNA with ColE1-like origin as drug applications. In upstream processing of plasmid DNA, regulation of replication of ColE1-like origin was discussed. In downstream processing of plasmid DNA, we analyzed simple, robust, and scalable methods, which can be used in the efficient production of pharmaceutical-grade plasmid DNA.

  2. Scaling-up recombinant plasmid DNA for clinical trial: current concern, solution and status.

    PubMed

    Ismail, Ruzila; Allaudin, Zeenathul Nazariah; Lila, Mohd-Azmi Mohd

    2012-09-07

    Gene therapy and vaccines are rapidly developing field in which recombinant nucleic acids are introduced in mammalian cells for enhancement, restoration, initiation or silencing biochemical function. Beside simplicity in manipulation and rapid manufacture process, plasmid DNA-based vaccines have inherent features that make them promising vaccine candidates in a variety of diseases. This present review focuses on the safety concern of the genetic elements of plasmid such as propagation and expression units as well as their host genome for the production of recombinant plasmid DNA. The highlighted issues will be beneficial in characterizing and manufacturing plasmid DNA for save clinical use. Manipulation of regulatory units of plasmid will have impact towards addressing the safety concerns raised in human vaccine applications. The gene revolution with plasmid DNA by alteration of their plasmid and production host genetics will be promising for safe delivery and obtaining efficient outcomes.

  3. Microneedle-mediated transcutaneous immunization with plasmid DNA coated on cationic PLGA nanoparticles

    PubMed Central

    Kumar, Amit; Wonganan, Piyanuch; Sandoval, Michael A.; Li, Xinran; Zhu, Saijie; Cui, Zhengrong

    2012-01-01

    Previously, it was shown that microneedle-mediated transcutaneous immunization with plasmid DNA can potentially induce a stronger immune response than intramuscular injection of the same plasmid DNA. In the present study, we showed that the immune responses induced by transcutaneous immunization by applying plasmid DNA onto a skin area pretreated with solid microneedles were significantly enhanced by coating the plasmid DNA on the surface of cationic nanoparticles. In addition, the net surface charge of the DNA-coated nanoparticles significantly affected their in vitro skin permeation and their ability to induce immune responses in vivo. Transcutaneous immunization with plasmid DNA-coated net positively charged anoparticles elicited a stronger immune response than with plasmid DNA-coated net negatively charged nanoparticles or by intramuscular immunization with plasmid DNA alone. Transcutaneous immunization with plasmid DNA-coated net positively charged nanoparticles induced comparable immune responses as intramuscular injection of them, but transcutaneous immunization was able to induce specific mucosal immunity and a more balanced T helper type 1 and type 2 response. The ability of the net positively charged DNA-coated nanoparticles to induce a strong immune response through microneedle-mediated transcutaneous immunization may be attributed to their ability to increase the expression of the antigen gene encoded by the plasmid and to more effectively stimulate the maturation of antigen-presenting cells. PMID:22921518

  4. Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents.

    PubMed

    Lezin, George; Kuehn, Michael R; Brunelli, Luca

    2011-08-01

    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 with protein. In addition, large-scale NID/HSS solutions eliminated lipopolysaccharides (LPS) contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high-quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive, and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures.

  5. Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents

    PubMed Central

    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 with protein. In addition, large-scale NID/HSS solutions eliminated LPS contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures. PMID:21351074

  6. Plasmid DNA gene therapy by electroporation: principles and recent advances.

    PubMed

    Murakami, Tatsufumi; Sunada, Yoshihide

    2011-12-01

    Simple plasmid DNA injection is a safe and feasible gene transfer method, but it confers low transfection efficiency and transgene expression. This non-viral gene transfer method is enhanced by physical delivery methods, such as electroporation and the use of a gene gun. In vivo electroporation has been rapidly developed over the last two decades to deliver DNA to various tissues or organs. It is generally considered that membrane permeabilization and DNA electrophoresis play important roles in electro-gene transfer. Skeletal muscle is a well characterized target tissue for electroporation, because it is accessible and allows for long-lasting gene expression ( > one year). Skin is also a target tissue because of its accessibility and immunogenicity. Numerous studies have been performed using in vivo electroporation in animal models of disease. Clinical trials of DNA vaccines and immunotherapy for cancer treatment using in vivo electroporation have been initiated in patients with melanoma and prostate cancer. Furthermore, electroporation has been applied to DNA vaccines for infectious diseases to enhance immunogenicity, and the relevant clinical trials have been initiated. The gene gun approach is also being applied for the delivery of DNA vaccines against infectious diseases to the skin. Here, we review recent advances in the mechanism of in vivo electroporation, and summarize the findings of recent preclinical and clinical studies using this technology.

  7. Low energy electrons and ultra-soft X-rays irradiation of plasmid DNA. Technical innovations

    NASA Astrophysics Data System (ADS)

    Fromm, Michel; Boulanouar, Omar

    2016-11-01

    In this paper we present in a first part the latest results of our group which are in relation with the study of DNA damages inflicted by low energy electrons (0-20 eV) in ultra-high vacuum as well as in air under atmospheric conditions. A short description of the drop-casting technique we developed to produce thin and nanometre-scaled DNA layers onto graphite sheets is given. We provide the absolute cross-section for loss of supercoiled topology of plasmid DNA complexed with 1,3-diaminopropane (Dap) in the vacuum under 10 eV electron impact and suggest a specific pathway for the dissociation of the transient negative ion formed by resonant capture of such a low energy electron (LEE) by the DNA's phosphate group when complexed to Dap. Well-gauged DNA-Dap layers with various nanometre-scaled thicknesses are used to evaluate the effective attenuation length of secondary photo-LEEs in the energy range (0-20 eV). The values of 11-16 nm for DNA kept under atmospheric conditions are in good agreement with the rare literature data available and which are stemming from computer simulations. In a second part, we describe the method we have developed in order to expose liquid samples of plasmid DNA to ultra-soft X-rays (Al Kα line at 1.5 keV) under hydroxyl radical scavenging conditions. We provide an experimentally determined percentage of indirect effects in aqueous medium kept under standard conditions of 94.7±2.1% indirect effects; in satisfactory agreement with the data published by others (i.e. 97.7%) relative to gamma irradiation of frozen solutions (Tomita et al., 1995).

  8. Absolute cross-sections for DNA strand breaks and crosslinks induced by low energy electrons.

    PubMed

    Chen, Wenzhuang; Chen, Shiliang; Dong, Yanfang; Cloutier, Pierre; Zheng, Yi; Sanche, Léon

    2016-12-07

    Absolute cross sections (CSs) for the interaction of low energy electrons with condensed macromolecules are essential parameters to accurately model ionizing radiation induced reactions. To determine CSs for various conformational DNA damage induced by 2-20 eV electrons, we investigated the influence of the attenuation length (AL) and penetration factor (f) using a mathematical model. Solid films of supercoiled plasmid DNA with thicknesses of 10, 15 and 20 nm were irradiated with 4.6, 5.6, 9.6 and 14.6 eV electrons. DNA conformational changes were quantified by gel electrophoresis, and the respective yields were extrapolated from exposure-response curves. The absolute CS, AL and f values were generated by applying the model developed by Rezaee et al. The values of AL were found to lie between 11 and 16 nm with the maximum at 14.6 eV. The absolute CSs for the loss of the supercoiled (LS) configuration and production of crosslinks (CL), single strand breaks (SSB) and double strand breaks (DSB) induced by 4.6, 5.6, 9.6 and 14.6 eV electrons are obtained. The CSs for SSB are smaller, but similar to those for LS, indicating that SSB are the main conformational damage. The CSs for DSB and CL are about one order of magnitude smaller than those of LS and SSB. The value of f is found to be independent of electron energy, which allows extending the absolute CSs for these types of damage within the range 2-20 eV, from previous measurements of effective CSs. When comparison is possible, the absolute CSs are found to be in good agreement with those obtained from previous similar studies with double-stranded DNA. The high values of the absolute CSs of 4.6 and 9.6 eV provide quantitative evidence for the high efficiency of low energy electrons to induce DNA damage via the formation of transient anions.

  9. Ultrasound enhances in vivo tumor expression of plasmid DNA by PEG-introduced cationized dextran.

    PubMed

    Hosseinkhani, Hossein; Tabata, Yasuhiko

    2005-11-28

    This study is an investigation to experimentally confirm whether or not ultrasound (US) irradiation is effective in enhancing the in vivo gene expression of plasmid DNA in tumor. Dextran was cationized by introducing spermine to the hydroxyl groups to allow to polyionically complex with a plasmid DNA. The cationized dextran prepared was additionally modified with poly(ethylene glycol) (PEG) molecules which have an active ester and methoxy groups at each terminal, to obtain cationized dextran with different percentages of PEG introduced. Various cationized dextrans with or without PEG introduction were mixed with a plasmid DNA of LacZ to form cationized dextran-plasmid DNA complexes. Electrophoretical examination revealed that the plasmid DNA was complexed both with the cationized dextran and PEG-introduced cationized dextran, irrespective of the PEG introduction percentage, although the higher N/P ratio was needed for plasmid DNA complexation with the latter. By complexation with the cationized dextran, the zeta potential of plasmid DNA was changed to be positive. The charge of PEG-introduced cationized dextran-plasmid DNA complexes became close to 0 mV as their percentage of PEG introduced increased, although the molecular size was about 250 nm, irrespective of the PEG introduction. When cationized dextran-plasmid DNA complexes with or without PEG introduction were intravenously injected to mice carrying a subcutaneous Meth-AR-1 fibrosarcoma mass and the subsequent US irradiation to the tumor mass percutaneously, the PEG-introduced cationized dextran-plasmid DNA complex plus US irradiation enhanced the tumor level of gene expression to a significantly high extent compared with the cationized dextran-plasmid DNA complex and free plasmid DNA with or without US irradiation. The enhanced level depended on the time period and timing of US irradiation. Fluorescent microscopic studies revealed that the localization of plasmid DNA and the gene expression were observed in

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

    NASA Astrophysics Data System (ADS)

    Yan, Xu; Zou, Fei; Lu, Xin Pei; He, Guangyuan; Shi, Meng Jun; Xiong, Qing; Gao, Xuan; Xiong, Zilan; Li, Yin; Ma, Feng Yun; Yu, Men; Wang, Chang Dong; Wang, Yuesheng; Yang, Guangxiao

    2009-08-01

    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.

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

    SciTech Connect

    Yan Xu; He Guangyuan; Shi Mengjun; Gao Xuan; Li Yin; Ma Fengyun; Yu Men; Wang Changdong; Wang Yuesheng; Yang Guangxiao; Zou Fei; Lu Xinpei; Xiong Qing; Xiong Zilan

    2009-08-24

    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.

  12. RK2 plasmid dynamics in Caulobacter crescentus cells--two modes of DNA replication initiation.

    PubMed

    Wegrzyn, Katarzyna; Witosinska, Monika; Schweiger, Pawel; Bury, Katarzyna; Jenal, Urs; Konieczny, Igor

    2013-06-01

    Undisturbed plasmid dynamics is required for the stable maintenance of plasmid DNA in bacterial cells. In this work, we analysed subcellular localization, DNA synthesis and nucleoprotein complex formation of plasmid RK2 during the cell cycle of Caulobacter crescentus. Our microscopic observations showed asymmetrical distribution of plasmid RK2 foci between the two compartments of Caulobacter predivisional cells, resulting in asymmetrical allocation of plasmids to progeny cells. Moreover, using a quantitative PCR (qPCR) method, we estimated that multiple plasmid particles form a single fluorescent focus and that the number of plasmids per focus is approximately equal in both swarmer and predivisional Caulobacter cells. Analysis of the dynamics of TrfA-oriV complex formation during the Caulobacter cell cycle revealed that TrfA binds oriV primarily during the G1 phase, however, plasmid DNA synthesis occurs during the S and G2 phases of the Caulobacter cell cycle. Both in vitro and in vivo analysis of RK2 replication initiation in C. crescentus cells demonstrated that it is independent of the Caulobacter DnaA protein in the presence of the longer version of TrfA protein, TrfA-44. However, in vivo stability tests of plasmid RK2 derivatives suggested that a DnaA-dependent mode of plasmid replication initiation is also possible.

  13. Biodegradable poly(ethylenimine) for plasmid DNA delivery.

    PubMed

    Ahn, Cheol-Hee; Chae, Su Young; Bae, You Han; Kim, Sung Wan

    2002-04-23

    Poly(ethylenimine) (PEI) has been known as an efficient gene carrier with the highest cationic charge potential. High transfection efficiency of PEI, along with its cytotoxicity, strongly depends on the molecular weight. Synthesis of cationic copolymers derived from the low molecular weight of PEI and hydrophilic poly(ethylene glycol) (PEG), which are water soluble and degradable under physiological conditions, was investigated for plasmid delivery. Hydrophilic PEG is expected to reduce the toxicity of the copolymer, improve the poor solubility of the PEI and DNA complexes, and help to introduce degradable bonds by reaction with the primary amines in the PEI. Considering the dependence of transfection efficiency and cytotoxicity on the molecular weight of the PEI, high transfection efficiency is expected from an increased molecular weight of the copolymer and low cytotoxicity from the introduction of PEG and the degradation of the copolymer into low molecular weight PEIs. Reaction conditions were carefully controlled to produce water soluble copolymers. Results from a gel retardation assay and zetapotentiometer indicated that complete neutralization of the complexes was achieved at the charge ratios of copolymer/pSV-beta-gal plasmid from 0.8 to 1.0 with the mean particle size of the polyplexes ranging from 129.8+/-0.9 to 151.8+/-3.4 nm. In vitro transfection efficiency of the synthesized copolymer increased up to three times higher than that of starting low molecular weight PEI, while the cell viability was maintained over 80%.

  14. Molecular cloning of complementary DNA: preparation of a plasmid vector with low transformation background.

    PubMed

    Leriche, A; Christophe, D; Brocas, H; Vassart, G

    1983-02-15

    A simple method that allows the rapid preparation of oligo dG-tailed plasmid vectors is presented. The procedure involves purification of the tailed molecules by hybridization to oligo dC-cellulose followed by a stepwise thermal elution. The resulting plasmid is virtually devoid of transformation activity in the absence of oligo dC-tailed DNA fragments. It allows construction of cDNA libraries with as low as 1% of colonies harboring wild-type plasmids.

  15. A Simple and Inexpensive Method for Sending Binary Vector Plasmid DNA by Mail

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We describe a simple cost-effective technique for the transport of plasmid DNA by mail. Our results demonstrate that common multipurpose printing paper is a satisfactory substrate and superior to the more absorbent 3MM chromatography paper for the transport of plasmid DNA through the U.S. first clas...

  16. Controlled release of plasmid DNA from hydrogels prepared from gelatin cationized by different amine compounds.

    PubMed

    Kushibiki, Toshihiro; Tomoshige, Ryuji; Iwanaga, Kazunori; Kakemi, Masawo; Tabata, Yasuhiko

    2006-05-15

    This paper is an investigation to compare the in vivo controlled release of a plasmid DNA from biodegradable hydrogels prepared from gelatin cationized by different amine compounds, ethylenediamine, putrescine, spermidine, and spermine and the consequent profile of gene expression. Cationized gelatin prepared through the chemical introduction of each amine compound was crosslinked by various concentrations of glutaraldehyde to obtain cationized gelatin hydrogels for the carrier of plasmid DNA release. When the cationized gelatin hydrogels incorporating 125I-labeled plasmid DNA were implanted into the femoral muscle of mice, the radioactivity remaining decreased with time and the retention period of radioactivity prolonged with a decrease in the water content of hydrogels. When 125I-labeled cationized gelatin hydrogels with the higher water content was implanted, the radioactivity remaining was decreased faster with time. The remaining time profile of plasmid DNA radioactivity was in good accordance with that of hydrogel radioactivity, irrespective of the type of cationized gelatin. Following intramuscular implantation, any cationized gelatin hydrogel incorporating plasmid DNA enhanced the expression level of plasmid DNA to a significantly higher extent than the free plasmid DNA injection. In addition, prolonged time period of gene expression was observed although there was no significant difference in the expressed period between the cationized gelatin hydrogels. It was concluded that plasmid DNA of biological activity was released from every cationized gelatin hydrogel accompanied with the in vivo degradation, resulting in enhanced and prolonged gene expression.

  17. Low-Cost Fabrication of Centimetre-Scale Periodic Arrays of Single Plasmid DNA Molecules

    PubMed Central

    Kirkland, Brett; Wang, Zhibin; Zhang, Peipei; Takebayashi, Shin-ichiro; Lenhert, Steven; Gilbert, David M.

    2013-01-01

    We report development of a low-cost method to generate a centimetre-scale periodic array of single plasmid DNA of 11 kilobase pairs. The arrayed DNA is amenable to enzymatic and physical manipulation. PMID:23824041

  18. Non-DSB clustered DNA lesions induced by ionizing radiation are largely responsible for the loss of plasmid DNA functionality in the presence of cisplatin.

    PubMed

    Kouass Sahbani, S; Rezaee, M; Cloutier, P; Sanche, L; Hunting, D J

    2014-06-25

    The combination of cisplatin and ionizing radiation (IR) increases cell toxicity by both enhancing DNA damage and inhibiting repair mechanisms. Although the formation of cluster DNA lesions, particularly double-strand breaks (DSB) at the site of cisplatin-DNA-adducts has been reported to induce cell death, the contribution of DSB and non-DSB cluster lesions to the cellular toxicity is still unknown. Although both lesions are toxic, it is not always possible to measure their frequency and cell survival in the same model system. To overcome this problem, here, we investigate the effect of cisplatin-adducts on the induction of DSB and non-DSB cluster DNA lesions by IR and determine the impact of such lesions on plasmid functionality. Cluster lesions are two or more lesions on opposite DNA strands with a short distance such that error free repair is difficult or impossible. At a ratio of two cisplatin per plasmid, irradiation of platinated DNA in solution with (137)Cs γ-rays shows enhancements in the formation of DNA DSB and non-DSB cluster lesions by factors of 2.6 and 2.1, respectively, compared to unmodified DNA. However, in absolute terms, the yield for non-DSB cluster lesions is far larger than that for DSB, by a factor of 26. Unmodified and cisplatin-modified DNA were irradiated and subsequently transformed into Escherichia coli to give survival curves representing the functionality of the plasmid DNA as a function of radiation dose. Our results demonstrate that non-DSB cluster lesions are the only toxic lesions present at a sufficient frequency to account for the loss of DNA functionality. Our data also show that Frank-DSB lesions are simply too infrequent to account for the loss of DNA functionality. In conclusion, non-DSB cluster DNA damage is known to be difficult to repair and is probably the lesion responsible for the loss of functionality of DNA modified by cisplatin.

  19. Large-scale purification of pharmaceutical-grade plasmid DNA using tangential flow filtration and multi-step chromatography.

    PubMed

    Sun, Bo; Yu, XiangHui; Yin, Yuhe; Liu, Xintao; Wu, Yongge; Chen, Yan; Zhang, Xizhen; Jiang, Chunlai; Kong, Wei

    2013-09-01

    The demand for pharmaceutical-grade plasmid DNA in vaccine applications and gene therapy has been increasing in recent years. In the present study, a process consisting of alkaline lysis, tangential flow filtration, purification by anion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography was developed. The final product met the requirements for pharmaceutical-grade plasmid DNA. The chromosomal DNA content was <1 μg/mg plasmid DNA, and RNA was not detectable by agarose gel electrophoresis. Moreover, the protein content was <2 μg/mg plasmid DNA, and the endotoxin content was <10 EU/mg plasmid DNA. The process was scaled up to yield 800 mg of pharmaceutical-grade plasmid DNA from approximately 2 kg of bacterial cell paste. The overall yield of the final plasmid DNA reached 48%. Therefore, we have established a rapid and efficient production process for pharmaceutical-grade plasmid DNA.

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

    PubMed

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

    1985-04-01

    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 linear forms. The optimal transformation frequency was achieved with 10 ng of DNA in 1 ml of transformation buffer. Higher concentrations of plasmid DNA resulted in a decrease in transformation frequency per microgram of DNA. Optimal transformation was obtained with 25 to 35% polyethylene glycol 6000. Under optimal conditions, 10(6) transformants per microgram of DNA were obtained.

  1. Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods.

    PubMed

    Taverniers, Isabel; Van Bockstaele, Erik; De Loose, Marc

    2004-03-01

    Analytical real-time PCR technology is a powerful tool for implementation of the GMO labeling regulations enforced in the EU. The quality of analytical measurement data obtained by quantitative real-time PCR depends on the correct use of calibrator and reference materials (RMs). For GMO methods of analysis, the choice of appropriate RMs is currently under debate. So far, genomic DNA solutions from certified reference materials (CRMs) are most often used as calibrators for GMO quantification by means of real-time PCR. However, due to some intrinsic features of these CRMs, errors may be expected in the estimations of DNA sequence quantities. In this paper, two new real-time PCR methods are presented for Roundup Ready soybean, in which two types of plasmid DNA fragments are used as calibrators. Single-target plasmids (STPs) diluted in a background of genomic DNA were used in the first method. Multiple-target plasmids (MTPs) containing both sequences in one molecule were used as calibrators for the second method. Both methods simultaneously detect a promoter 35S sequence as GMO-specific target and a lectin gene sequence as endogenous reference target in a duplex PCR. For the estimation of relative GMO percentages both "delta C(T)" and "standard curve" approaches are tested. Delta C(T) methods are based on direct comparison of measured C(T) values of both the GMO-specific target and the endogenous target. Standard curve methods measure absolute amounts of target copies or haploid genome equivalents. A duplex delta C(T) method with STP calibrators performed at least as well as a similar method with genomic DNA calibrators from commercial CRMs. Besides this, high quality results were obtained with a standard curve method using MTP calibrators. This paper demonstrates that plasmid DNA molecules containing either one or multiple target sequences form perfect alternative calibrators for GMO quantification and are especially suitable for duplex PCR reactions.

  2. Engineering of bacterial strains and vectors for the production of plasmid DNA.

    PubMed

    Bower, Diana M; Prather, Kristala L J

    2009-04-01

    The demand for plasmid DNA (pDNA) is anticipated to increase significantly as DNA vaccines and non-viral gene therapies enter phase 3 clinical trials and are approved for use. This increased demand, along with renewed interest in pDNA as a therapeutic vector, has motivated research targeting the design of high-yield, cost-effective manufacturing processes. An important aspect of this research is engineering bacterial strains and plasmids that are specifically suited to the production of plasmid biopharmaceuticals. This review will survey recent innovations in strain and vector engineering that aim to improve plasmid stability, enhance product safety, increase yield, and facilitate downstream purification. While these innovations all seek to enhance pDNA production, they can vary in complexity from subtle alterations of the host genome or vector backbone to the investigation of non-traditional host strains for higher pDNA yields.

  3. Ultrasound enhancement of in vitro transfection of plasmid DNA by a cationized gelatin.

    PubMed

    Hosseinkhani, Hossein; Aoyama, Teruyoshi; Ogawa, Osamu; Tabata, Yasuhiko

    2002-05-01

    In vitro transfection efficiency of a plasmid DNA for rat gastric mucosal (RGM)-1 cells was enhanced by ultrasound (US) irradiation. Ethylenediamine was introduced to the carboxyl groups of gelatin to prepare a cationized gelatin as the vector of plasmid DNA encoding luciferase. An electrophoresis experiment revealed that the cationized gelatin was mixed with plasmid DNA at the weight ratio of 5.0 to form a cationized gelatin-plasmid DNA complex. The complex obtained was about 200nm in diameter with a positive charge. When incubated with the cationized gelatin-plasmid DNA complex and subsequently exposed to US, RGM-1 cells exhibited a significantly enhanced luciferase activity although the extent increased with an increase in the DNA concentration, in contrast to the cationized gelatin alone with or without US irradiation and US irradiation alone. US irradiation was also effective in enhancing the activity by free plasmid DNA although the extent was less than that of the complex. The US-induced enhancement of luciferase activity was influenced by the exposure time period, frequency, and intensity of US. The activity enhancement became higher to be significant at the irradiation time period of 60 s and thereafter decreased. A series of cytotoxicity experiments revealed that an increase in the irradiation time period and intensity of US decreased the viability of cells themselves. It is possible that US irradiation under an appropriate condition enables cells to accelerate the permeation of the cationized gelatin-plasmid DNA complex through the cell membrane, resulted in enhanced transfection efficiency of plasmid DNA. These findings clearly indicate that US exposure is a simple and promising method to enhance the gene expression of plasmid DNA.

  4. Enhanced recognition of hydroxyl radical modified plasmid DNA by circulating cancer antibodies.

    PubMed

    Khan, F; Ali, A; Ali, R

    2005-06-01

    Reactive oxygen species have been implicated in various human diseases which are also responsible for the elimination of invading pathogens. In disease state and inflammatory responses, the excess of these radicals damage cellular macromolecules. DNA is susceptible to attacks by OH-induced damage. Oxidative DNA damage is an important factor in mutagenesis and carcinogenesis. In the present study, purified plasmid Bluescript DNA was modified by hydroxyl radical. Modifications incurred in DNA were characterized by physico-chemical techniques. Sera from patients of cancer were studied for their binding to native and hydroxyl radical modified plasmid DNA. Direct binding ELISA and competition binding results indicated that autoantibodies in cancer showed higher recognition to ROS-plasmid DNA as compared to the native form. Retarded mobility of the immune complex formation between IgG isolated from cancer sera using native and ROS-plasmid DNA as antigens reiterated preferential recognition of modified plasmid DNA by cancer autoantibodies. Therefore, it can be concluded that circulating autoantibodies in cancer sera bind preferentially to ROS-plasmid DNA as compared to native polymer. The data presented in the present communication suggest a role of ROS in the etiology of cancer.

  5. Plasmid-Chromosome Recombination of Irradiated Shuttle Vector DNA in African Green Monkey Kidney Cells.

    NASA Astrophysics Data System (ADS)

    Mudgett, John Stuart

    1987-09-01

    An autonomously replicating shuttle vector was used to investigate the enhancement of plasmid-chromosome recombination in mammalian host cells by ultraviolet light and gamma radiation. Sequences homologous to the shuttle vector were stably inserted into the genome of African Green Monkey kidney cells to act as the target substrate for these recombination events. The SV40- and pBR322-derived plasmid DNA was irradiated with various doses of radiation before transfection into the transformed mammalian host cells. The successful homologous transfer of the bacterial ampicillin resistance (amp^{rm r}) gene from the inserted sequences to replace a mutant amp^->=ne on the shuttle vector was identified by plasmid extraction and transformation into E. coli host cells. Ultraviolet light (UV) was found not to induce homologous plasmid-chromosome recombination, while gamma radiation increased the frequency of recombinant plasmids detected. The introduction of specific double -strand breaks in the plasmid or prolonging the time of plasmid residence in the mammalian host cells also enhanced plasmid-chromosome recombination. In contrast, plasmid mutagenesis was found to be increased by plasmid UV irradiation, but not to change with time. Plasmid survival, recombination, and mutagenesis were not affected by treating the mammalian host cells with UV light prior to plasmid transfection. The amp^{rm r} recombinant plasmid molecules analyzed were found to be mostly the result of nonconservative exchanges which appeared to involve both homologous and possibly nonhomologous interactions with the host chromosome. The observation that these recombinant structures were obtained from all of the plasmid alterations investigated suggests a common mechanistic origin for plasmid -chromosome recombination in these mammalian cells.

  6. Quantification of plasmid DNA copies in the nucleus after lipoplex and polyplex transfection.

    PubMed

    Cohen, Richard N; van der Aa, Marieke A E M; Macaraeg, Nichole; Lee, Ai Ping; Szoka, Francis C

    2009-04-17

    Nuclear uptake of plasmid DNA is one of the many cellular barriers that limit the efficiency of non-viral gene delivery systems. We have determined the number of plasmids that reach the nucleus of a transfected cell using an internally standardized quantitative PCR (qPCR) assay. We isolated nuclei using two different protocols: a density gradient technique and a detergent-based method. The density gradient procedure yielded nuclei with substantially less adhering plasmids on the outside of the nuclei. Using the density gradient protocol we determined that cells transfected with Lipofectamine lipoplexes or polyethylenimine polyplexes contained between 75 and 50,000 plasmids/nucleus, depending on the applied plasmid dose. Any increase above 3000 plasmids/nucleus resulted in only marginal increases in transgene expression. Furthermore, lipoplex-delivered plasmids were more efficiently expressed, on the basis of protein expression per plasmid number in the nucleus, than polyplex-delivered plasmids. This indicates that polymer may remain bound to some plasmids in the nucleus. Lastly, by sorting transfected cells into high- and low-expressing sub-populations, we observe that a sub-population of cells contain 3x greater plasmids/nucleus but express nearly 100x more transgene than other cells within a single transfection reaction. Taken together these results suggest the importance of considering the processes downstream from nuclear entry for strategies to improve the efficiency of gene transfer reagents.

  7. Upstream processing of plasmid DNA for vaccine and gene therapy applications.

    PubMed

    Tejeda-Mansir, Armando; Montesinos, Rosa M

    2008-01-01

    The demand for plasmid DNA has increased vastly in response to rapid advances in its use in gene therapy and vaccines. These therapies are based on the same principle, i.e. the introduction of nucleic acids in human/non-human cells receptor to restore, cancel, enhance or introduce a biochemical function. Naked plasmid DNA as a vector has attracted a lot of interest since it offers several advantages over a viral vector, especially weak immunogenicity, better safety and easy to manufacture, but low transfection efficacy. Non-viral gene therapy may require considerable amounts (milligram scale) of pharmaceutical-grade pDNA per patient since the efficacy and duration of gene expression is presently relatively low. Reliance on fermentation, which generates large lysate volumes, for producing the needed quantities of pDNA is becoming more widespread. Through optimization of the biological system, growth environment and the growth mode, improvements can be achieved in biomass productivity, plasmid yield, plasmid quality and production costs. The information on large-scale plasmid production is scarce and usually not available to the scientific community. This review summarizes recent patents and patent applications relating to plasmid upstream processing manufacturing, ranging from plasmid design to growth strategies to produce plasmid-bearing E. coli.

  8. Mechanistic basis of plasmid-specific DNA binding of the F plasmid regulatory protein, TraM.

    PubMed

    Peng, Yun; Lu, Jun; Wong, Joyce J W; Edwards, Ross A; Frost, Laura S; Mark Glover, J N

    2014-11-11

    The conjugative transfer of bacterial F plasmids relies on TraM, a plasmid-encoded protein that recognizes multiple DNA sites to recruit the plasmid to the conjugative pore. In spite of the high degree of amino acid sequence conservation between TraM proteins, many of these proteins have markedly different DNA binding specificities that ensure the selective recruitment of a plasmid to its cognate pore. Here we present the structure of F TraM RHH (ribbon-helix-helix) domain bound to its sbmA site. The structure indicates that a pair of TraM tetramers cooperatively binds an underwound sbmA site containing 12 base pairs per turn. The sbmA is composed of 4 copies of a 5-base-pair motif, each of which is recognized by an RHH domain. The structure reveals that a single conservative amino acid difference in the RHH β-ribbon between F and pED208 TraM changes its specificity for its cognate 5-base-pair sequence motif. Specificity is also dictated by the positioning of 2-base-pair spacer elements within sbmA; in F sbmA, the spacers are positioned between motifs 1 and 2 and between motifs 3 and 4, whereas in pED208 sbmA, there is a single spacer between motifs 2 and 3. We also demonstrate that a pair of F TraM tetramers can cooperatively bind its sbmC site with an affinity similar to that of sbmA in spite of a lack of sequence similarity between these DNA elements. These results provide a basis for the prediction of the DNA binding properties of the family of TraM proteins.

  9. Supercoiled plasmid DNA purification by integrating membrane technology with a monolithic chromatography.

    PubMed

    Nunes, Catherine; Sousa, Angela; Nunes, José C; Morão, António M; Sousa, Fani; Queiroz, João A

    2014-06-01

    The present study describes the integration of membrane technology with monolithic chromatography to obtain plasmid DNA with high quality. Isolation and clarification of plasmid DNA lysate were first conducted by a microfiltration step, by using a hydrophilic nylon microfiltration membrane, avoiding the need of centrifugation. For the total elimination of the remaining impurities, a suitable purification step is required. Monolithic stationary phases have been successfully applied as an alternative to conventional supports. Thus, the sample recovered from the membrane process was applied into a nongrafted CarbonylDiImidazole disk. Throughout the global procedure, a reduced level of impurities such as proteins and RNA was obtained, and no genomic DNA was detectable in the plasmid DNA sample. The chromatographic process demonstrated an efficient performance on supercoiled plasmid DNA purity and recovery (100 and 84.44%, respectively). Thereby, combining the membrane technology to eliminate some impurities from lysate sample with an efficient chromatographic strategy to purify the supercoiled plasmid DNA arises as a powerful approach for industrial-scale systems aiming at plasmid DNA purification.

  10. In vitro transfection of plasmid DNA by cationized gelatin prepared from different amine compounds.

    PubMed

    Kushibiki, Toshihiro; Tomoshige, Ryuji; Iwanaga, Kazunori; Kakemi, Masawo; Tabata, Yasuhiko

    2006-01-01

    The objective of this paper is to compare the in vitro transfection efficiency of a luciferase plasmid DNA using cationized gelatin prepared from different amine compounds. The compounds used here were ethylenediamine, putrescine, spermidine and spermine, chemically introduced to the carboxyl group of gelatin for the cationization. Complexation of the cationized gelatin with the plasmid DNA was performed by simply mixing the two materials at various N+/P- mixing ratios (the molar number ratio of amino groups of gelatin to the phosphate groups of DNA) in aqueous solution. Gel retardation studies revealed that the formation of cationized-gelatin-plasmid DNA complexes depended on the N+/P- mixing ratio. The stronger interaction of plasmid DNA with the cationized gelatin of spermine compared to the other cationized gelatins was observed by an ethidium bromide intercalation assay and Scatchard binding analysis. When the transfection efficiency of plasmid DNA complexed with the various cationized gelatins at different N+/P- mixing ratios was evaluated for mouse L929 fibroblasts, the highest transfection efficiency was observed for the complex prepared from the cationized gelatin of spermine at a N+/P- mixing ratio of 2. The present study indicates that there is an optimal N+/P- mixing ratio and a type of amine compound or cationization extent of cationized gelatin to enhance the transfection efficiency of plasmid DNA.

  11. Structure and regulation of gene expression of a Clo DF13 plasmid DNA region involved in plasmid segregation and incompatibility.

    PubMed Central

    van den Elzen, P J; Hakkaart, M J; van Putten, A J; Walters, H H; Veltkamp, E; Nijkamp, H J

    1983-01-01

    The bacteriocinogenic plasmid Clo DF13 contains genetic information involved in the accurate partitioning of the plasmid (parA and parB) as well as in incompatibility phenomena (incA, B, C and D). In this paper we report on the primary structure and regulation of gene expression of the 29% - 50% part of Clo DF13, containing the DNA regions incA, incB and parB as well as genes K and L. According to the results of our DNA sequence analysis, mapping of transposon insertions, RNA blotting and S1 mapping experiments, we conclude that: a) genes K and L are transcribed as one operon; transcription of this operon is initiated at a promoter (P2) located at 32.5% and proceeds in a clockwise direction. b) treatment of cells with mitomycin-C, significantly enhances transcription from P2, although this promoter is probably not directly repressed by lexA protein. c) Termination of transcription of this operon occurs between genes K and L, as well as distal to gene L. The possible role of gene products and/or sites, located within the 29-50% DNA region, in plasmid incompatibility and segregation is discussed. Images PMID:6324101

  12. Reporter gene expression in dendritic cells after gene gun administration of plasmid DNA.

    PubMed

    Watkins, Craig; Hopkins, John; Harkiss, Gordon

    2005-07-21

    Dendritic cells (DC) play an integral role in plasmid DNA vaccination. However, the interaction between plasmid DNA and DC in vivo is incompletely understood. In this report, we utilise the sheep pseudoafferent cannulation model to examine the interaction between plasmid DNA encoding enhanced green fluorescent protein (pEGFP) and afferent lymph DC (ALDC) following gene gun administration. The results show that peaks of fluorescent ALDC tended to appear around days 1-4 and 9-13, then erratically thereafter for up to 2 months. Phenotypic analysis showed that EGFP+ ALDC expressed MHC class II, WC6, CD1b, and SIRPalpha markers. Plasmid, detected by PCR, was found in lymph cells and cell-free plasma on a daily basis, and was present variably for up to 2 months. Plasmid was also detected in purified CD1b+ ALDC, but the presence of plasmid did not correlate with EGFP expression by ALDC. Free EGFP in afferent lymph plasma was detectable by luminometry only after three administrations of the plasmid. The results show that gene gun administered pEGFP persisted for extended periods after a single administration, leeching out of skin on a daily basis. The plasmid was associated with both the cellular and fluid components of afferent lymph. EGFP protein appeared in afferent lymph in a pulsatile manner, but associated only with ALDC.

  13. Enhanced purification of plasmid DNA isoforms by exploiting ionic strength effects during ultrafiltration.

    PubMed

    Li, Ying; Currie, David; Zydney, Andrew L

    2016-04-01

    The solution structure of plasmid DNA is known to be a strong function of solution conditions due to intramolecular electrostatic interactions between the charged phosphate groups along the DNA backbone. The objective of this work was to determine whether it was possible to enhance the use of ultrafiltration for separation of different plasmid isoforms by proper selection of the solution ionic strength and ion type. Experiments were performed with a 3.0 kbp plasmid using composite regenerated cellulose ultrafiltration membranes. The transmission of the linear isoform was nearly independent of solution ionic strength, but increased significantly with increasing filtrate flux due to the elongation of the highly flexible plasmid in the converging flow field into the membrane pores. In contrast, the transmission of the open-circular and supercoiled plasmids both increased with increasing NaCl or MgCl2 concentration due to the change in plasmid size and conformational flexibility. The effect of ionic strength was greatest for the supercoiled plasmid, providing opportunities for enhanced purification of this therapeutically active isoform. This behavior was confirmed using experiments performed with binary mixtures of the different isoforms. These results clearly demonstrate the potential for enhancing the performance of membrane systems for plasmid DNA separations by proper selection of the ionic conditions.

  14. Nucleocytoplasmic transport of plasmid DNA: a perilous journey from the cytoplasm to the nucleus.

    PubMed

    Lechardeur, Delphine; Lukacs, Gergely L

    2006-09-01

    Nonviral vectors represent a promising approach for the safe delivery of therapeutic DNA in genetic and acquired human diseases. Before synthetic vector systems can be used for clinical applications, their limited efficacy must be addressed. At the cellular level, successful gene transfer is dependent on several additional factors including DNA uptake, release from the DNA-vector complex, and nucleocytoplasmic transport. This paper reviews the major metabolic and physical impediments that plasmid DNA vectorized by synthetic vectors encounters between the cytosol and the nucleus. Plasmid DNA that escapes the endolysosomal compartment encounters the diffusional and metabolic barriers of the cytoplasm, reducing the number of intact plasmids that reach the nuclear envelope. Nuclear translocation of DNA requires either the disassembly of the nuclear envelope during cell division or active nuclear transport via the nuclear pore complex. In the nucleus, plasmid DNA is relatively stable, but its transcription and its fate during cell division are still debated. A better understanding of the cellular and molecular basis of nonviral gene transfer during nucleocytoplasmic trafficking may provide strategies to overcome those obstacles that limit the efficiency of nonviral gene delivery. We review some of the current methods of gene transfer mediated by synthetic vectors, highlighting systems that exploit our actual knowledge of the nucleocytoplasmic transport of plasmid DNA.

  15. Purification of transfection-grade plasmid DNA from bacterial cells with superparamagnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Chiang, Chen-Li; Sung, Ching-Shan

    2006-07-01

    The functionalized magnetic nanobeads were used to develop a rapid protocol for extracting and purifying transfection-grade plasmid DNA from bacterial culture. Nanosized superparamagnetic nanoparticles (Fe 3O 4) were prepared by chemical coprecipitation method using Fe 2+, Fe 3+ salt, and ammonium hydroxide under a nitrogen atmosphere. The surface of Fe 3O 4 nanoparticles was modified by coating with the multivalent cationic agent, polyethylenimine (PEI). The PEI-modified magnetic nanobeads were employed to simplify the purification of plasmid DNA from bacterial cells. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of Escherichia coli. The loaded nanobeads are recovered by magnetically driven separation and regenerated by exposure to the elution buffer with optimal ionic strength (1.25 M) and pH (9.0). Up to approximately 819 μg of high-purity (A 260/A 280 ratio=1.86) plasmid DNA was isolated from 100 ml of overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and animal cell transfection applications with success. The PEI-modified magnetic nanobead delivers significant time-savings, overall higher yields and better transfection efficiencies compared to anion-exchange and other methods. The results presented in this report show that PEI-modified magnetic nanobeads are suitable for isolation and purification of transfection-grade plasmid DNA.

  16. Plasmid RSF1010 DNA replication in vitro promoted by purified RSF1010 RepA, RepB and RepC proteins.

    PubMed Central

    Scherzinger, E; Haring, V; Lurz, R; Otto, S

    1991-01-01

    We have constructed and analyzed an in vitro system that will efficiently replicate plasmid RSF1010 and its derivatives. The system contains a partially purified extract from E.coli cells and three purified RSF1010-encoded proteins, the products of genes repA, repB (or mobA/repB), and repC. Replication in this system mimics the in vivo mechanism in that it (i) is initiated at oriV, the origin of vegetative DNA replication, (ii) proceeds in a population of plasmid molecules in both directions from this 396-base-pair origin region, and (iii) is absolutely dependent on the presence of each of the three rep gene products. In addition, we find that E.coli DNA gyrase, DnaZ protein (gamma subunit of poIIII holoenzyme) and SSB are required for in vitro plasmid synthesis. The bacterial RNA polymerase, the initiation protein DnaA, and the primosomal proteins DnaB, DnaC, DnaG and DnaT are not required. Furthermore, the replicative intermediates seen in the electron microscope suggest that replication in vitro begins with the simultaneous or non-simultaneous formation of two displacement loops that expand for a short stretch of DNA toward each other, and form a theta-type structure when the two displacing strands pass each other. Images PMID:1851552

  17. Plasmid segregation: how to survive as an extra piece of DNA.

    PubMed

    Salje, Jeanne

    2010-08-01

    Non-essential extra-chromosomal DNA elements such as plasmids are responsible for their own propagation in dividing host cells, and one means to ensure this is to carry a miniature active segregation system reminiscent of the mitotic spindle. Plasmids that are maintained at low numbers in prokaryotic cells have developed a range of such active partitioning systems, which are characterized by an impressive simplicity and efficiency and which are united by the use of dynamic, nucleotide-driven filaments to separate and position DNA molecules. A comparison of different plasmid segregation systems reveals (i) how unrelated filament-forming and DNA-binding proteins have been adopted and modified to create a range of simple DNA segregating complexes and (ii) how subtle changes in the few components of these DNA segregation machines has led to a remarkable diversity in the molecular mechanisms of closely related segregation systems. Here, our current understanding of plasmid segregation systems is reviewed and compared with other DNA segregation systems, and this is extended by a discussion of basic principles of plasmid segregation systems, evolutionary implications and the relationship between an autonomous DNA element and its host cell.

  18. Affinity chromatography approaches to overcome the challenges of purifying plasmid DNA.

    PubMed

    Sousa, Fani; Prazeres, Duarte M F; Queiroz, João A

    2008-09-01

    The diversity of biomolecules present in plasmid DNA (pDNA)-containing extracts and the structural and chemical similarities between pDNA and impurities are some of the main challenges of improving or establishing novel purification procedures. In view of the unequalled specificity of affinity purification, this technique has recently begun to be applied in downstream processing of plasmids. This paper discusses the progress and importance of affinity chromatography (AC) for the purification of pDNA-based therapeutic products. Several affinity approaches have already been successfully developed for a variety of applications, and we will focus here on highlighting their possible contributions to the pDNA purification challenge. Diverse affinity applications and their advantages and disadvantages are discussed, as well as the most significant results and improvements in the challenging task of purifying plasmids.

  19. First report on vertical transmission of a plasmid DNA in freshwater prawn, Macrobrachium rosenbergii.

    PubMed

    Chowdhury, Labrechai Mog; Gireesh-Babu, P; Pavan-Kumar, A; Suresh Babu, P P; Chaudhari, Aparna

    2014-09-01

    Outbreak of WSSV disease is one of the major stumbling blocks in shrimp aquaculture. DNA vaccines have shown potential for mass scale vaccination owing to their stability, cost effectiveness and easy maintenance. Development of economically feasible delivery strategies remains to be a major challenge. This study demonstrates vertical transmission of a plasmid DNA in a decapod Macrobrachium rosenbergii for the first time. Females at three different maturation stages (immature, matured and berried) and mature males were injected with a plasmid DNA and allowed to spawn with untreated counterparts. Using specific primers the plasmid DNA could be amplified from the offspring of all groups except that of berried females. For this confirmation genomic DNA was isolated from 3 pools of 10 post larvae in each group. This presents an ideal strategy to protect young ones at zero stress.

  20. Conformational transitions of plasmid ds-DNA on ultrathin films of alkylamines on graphite

    NASA Astrophysics Data System (ADS)

    Falk, Caroline; Liang, Hua; Severin, Nikolai; Zhuang, Wei; Zauscher, Stefan; Rabe, Jürgen P.

    2015-03-01

    DNA replication is an important process in the human body. Replication of double-stranded (ds)-DNA requires its local melting into two single strands. DNA, when stretched in solution, overwinds and melts. This was argued to give insight onto the replication mechanism. It is difficult, however, to access the direct conformational changes during stretching in solution. Recent work demonstrated that this transition can be imaged with scanning force microscopy on a graphite surface that is coated with an alkylamine layer. ds-DNA can be controlled by an amphiphilic layer, since the DNA conformation depends on the amphiphile concentration. In particular we analyzed different DNA lengths on the same surface, and we found that at a specific concentration of octadecylamine the ds-DNA pUC19 plasmid ring splits into two single strands at one position. We will discuss methods to mark the DNA to determine the exact location at which the plasmid ring splits.

  1. Differential requirement for SUB1 in chromosomal and plasmid double-strand DNA break repair.

    PubMed

    Yu, Lijian; Volkert, Michael R

    2013-01-01

    Non homologous end joining (NHEJ) is an important process that repairs double strand DNA breaks (DSBs) in eukaryotic cells. Cells defective in NHEJ are unable to join chromosomal breaks. Two different NHEJ assays are typically used to determine the efficiency of NHEJ. One requires NHEJ of linearized plasmid DNA transformed into the test organism; the other requires NHEJ of a single chromosomal break induced either by HO endonuclease or the I-SceI restriction enzyme. These two assays are generally considered equivalent and rely on the same set of NHEJ genes. PC4 is an abundant DNA binding protein that has been suggested to stimulate NHEJ. Here we tested the role of PC4's yeast homolog SUB1 in repair of DNA double strand breaks using different assays. We found SUB1 is required for NHEJ repair of DSBs in plasmid DNA, but not in chromosomal DNA. Our results suggest that these two assays, while similar are not equivalent and that repair of plasmid DNA requires additional factor(s) that are not required for NHEJ repair of chromosomal double-strand DNA breaks. Possible roles for Sub1 proteins in NHEJ of plasmid DNA are discussed.

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

    PubMed Central

    Hassan, Sally; Ward, John

    2016-01-01

    ABSTRACT 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

  3. Identification of putative DnaN-binding motifs in plasmid replication initiation proteins.

    PubMed

    Dalrymple, Brian P; Kongsuwan, Kritaya; Wijffels, Gene

    2007-01-01

    Recently the plasmid RK2 replication initiation protein, TrfA, has been shown to bind to the beta subunit of DNA Polymerase III (DnaN) via a short pentapeptide with the consensus QL[S/D]LF. A second consensus peptide, the hexapeptide QLxLxL, has also been demonstrated to mediate binding to DnaN. Here we describe the results of a comprehensive survey of replication initiation proteins encoded by bacterial plasmids to identify putative DnaN-binding sites. Both pentapeptide and hexapeptide motifs have been identified in a number of families of replication initiation proteins. The distribution of sites is sporadic and closely related families of proteins may differ in the presence, location, or type of putative DnaN-binding motif. Neither motif has been identified in replication initiation proteins encoded by plasmids that replicate via rolling circles or strand displacement. The results suggest that the recruitment of DnaN to the origin of replication of a replisome by plasmid replication initiation proteins is not generally required for plasmid replication, but that in some cases it may be beneficial for efficiency of replication initiation.

  4. Proteins encoded by Agrobacterium tumefaciens Ti plasmid DNA (T-DNA) in crown gall tumors

    PubMed Central

    McPherson, Joan C.; Nester, Eugene W.; Gordon, Milton P.

    1980-01-01

    In order to detect proteins that may be produced in crown gall tumors as a result of expression of incorporated Agrobacterium tumefaciens Ti plasmid DNA (T-DNA), we have isolated mRNA complementary to T-DNA and translated this in a protein-synthesizing system derived from wheat germ. mRNA prepared from cultured E1 tumor from Nicotiana tabacum hybridized with HindIII fragment 1 sequences of T-DNA immobilized on cellulose nitrate filters. Two proteins of 30,000 and 16,500 Mr were produced when this selected RNA was released and translated. Other tumor lines from N. tabacum were investigated, and a protein of slightly less than 30,000 Mr was encoded by HindIII fragment 1 sequences of 15955/01 tumor. No products were observed for 15955/1 tumor line, which differs from E1/B6-806 and 15955/01 in that it does not produce octopine. mRNA species of each of the tumor lines hybridized to Bst I fragment 8 sequences of T-DNA and produced a common protein of 15,000 Mr. Because this protein is derived from the region of the T-DNA that is conserved in octopine- and nopaline-type crown gall tumors, it may play a role in oncogenicity. Images PMID:16592819

  5. Induced mutagenesis of plasmid and chromosomal genes inserted into the plasmid DNA. II. Mutagenic action of chemical factors

    SciTech Connect

    Esipova, V.V.; Vedunova, S.L.; Kriviskii, A.S.

    1986-02-01

    Following the study of the mutagenic action of UV and ..gamma..-radiation on plasmid DNA in vitro, they investigated the induction of mutations under the influence of chemical mutagens on the same DNA of plasmid RSF2124, determining the synthesis of colicine E1 and resistance to ampicillin. The inactivating action of the mutagen was assessed from the yield of transformants resistant to the antibiotic and the mutagenic effect from the loss by colonies of transformants that were capable of releasing colicine into the external medium. In these experiments they mainly used chemical compounds whose mutagenic effect if well known in other systems (transforming and transfecting DNA, microbial viruses). As a result all mutagens tested for their activity were divided into four groups: first group, those exceeding the level of mutagenesis by more than 100-fold above the spontaneous background (hydroxylamine, O-methylhydroxylamine); second group, those exceeding it by a factor of 10 (UV radiation (lambda = 254 nm), W-mutagenesis, ionizing radiation, nitrous acid, mitomycin C); third group, those exceeding it by a factor of <10 (indirect UV mutagenesis, nitrous acid, ..beta..-chloroethyldiethylamine hydrochloride, nitrosoguanidine); fourth group, no mutagenic effect (acridine orange, ethyl methane sulfonate, sodium azide, 0-..beta..-diethylaminoethylhydroxylamine).

  6. Autonomous replication of plasmids bearing monkey DNA origin-enriched sequences

    SciTech Connect

    Frappier, L.; Zannis-Hadjopoulos, M.

    1987-10-01

    Twelve clones of origin-enriched sequences (ORS) isolated from early replicating monkey (CV-1) DNA were examined for transient episomal replication in transfected CV-1, COS-7, and HeLa cells. Plasmid DNA was isolated at time intervals after transfection and screened by the Dpn I resistance assay or by the bromodeoxyuridine substitution assay to differentiate between input and replicated DNA. The authors have identified four monkey ORS (ORS3, -8, -9, and -12) that can support plasmid replication in mammalian cells. This replication is carried out in a controlled and semiconservative manner characteristic of mammalian replicons. ORS replication was most efficient in HeLa cells. Electron microscopy showed ORS8 and ORS12 plasmids of the correct size with replication bubbles. Using a unique restriction site in ORS12, we have mapped the replication bubble within the monkey DNA sequence.

  7. Iteron Plasmids.

    PubMed

    Konieczny, Igor; Bury, Katarzyna; Wawrzycka, Aleksandra; Wegrzyn, Katarzyna

    2014-12-01

    Iteron-containing plasmids are model systems for studying the metabolism of extrachromosomal genetic elements in bacterial cells. Here we describe the current knowledge and understanding of the structure of iteron-containing replicons, the structure of the iteron plasmid encoded replication initiation proteins, and the molecular mechanisms for iteron plasmid DNA replication initiation. We also discuss the current understanding of control mechanisms affecting the plasmid copy number and how host chaperone proteins and proteases can affect plasmid maintenance in bacterial cells.

  8. Fabrication of Size-Tunable Metallic Nanoparticles Using Plasmid DNA as a Biomolecular Reactor.

    PubMed

    Samson, Jacopo; Piscopo, Irene; Yampolski, Alex; Nahirney, Patrick; Parpas, Andrea; Aggarwal, Amit; Saleh, Raihan; Drain, Charles Michael

    2011-10-21

    Plasmid DNA can be used as a template to yield gold, palladium, silver, and chromium nanoparticles of different sizes based on variations in incubation time at 70 °C with gold phosphine complexes, with the acetates of silver or palladium, or chromium acetylacetonate. The employment of mild synthetic conditions, minimal procedural steps, and aqueous solvents makes this method environmentally greener and ensures general feasibility. The use of plasmids exploits the capabilities of the biotechnology industry as a source of nanoreactor materials.

  9. Fabrication of Size-Tunable Metallic Nanoparticles Using Plasmid DNA as a Biomolecular Reactor

    PubMed Central

    Samson, Jacopo; Piscopo, Irene; Yampolski, Alex; Nahirney, Patrick; Parpas, Andrea; Aggarwal, Amit; Saleh, Raihan; Drain, Charles Michael

    2011-01-01

    Plasmid DNA can be used as a template to yield gold, palladium, silver, and chromium nanoparticles of different sizes based on variations in incubation time at 70 °C with gold phosphine complexes, with the acetates of silver or palladium, or chromium acetylacetonate. The employment of mild synthetic conditions, minimal procedural steps, and aqueous solvents makes this method environmentally greener and ensures general feasibility. The use of plasmids exploits the capabilities of the biotechnology industry as a source of nanoreactor materials.

  10. Targeting of plasmid DNA to renal interstitial fibroblasts by cationized gelatin.

    PubMed

    Kushibiki, Toshihiro; Nagata-Nakajima, Natsuki; Sugai, Manabu; Shimizu, Akira; Tabata, Yasuhiko

    2005-10-01

    Renal interstitial fibrosis is the common pathway of chronic renal disease, while it causes end-stage renal failure. A lot of cytokines and biologically active substances are well recognized to be the candidates of primary mediators to induce accumulation of extracelluar matrix (ECM) in the interstitial fibrotic area. Interstitial fibroblasts are played a crucial role in the accumulation of excess ECM during renal interstitial fibrogenesis. Therefore, the targeting of therapeutic drugs and genes to interstitial renal fibroblasts is effective in suppressing the progress of interstitial renal failure. However, despite various approaches and techniques, few successful results have been reported on the in vivo targeting for interstitial fibroblasts. The objective of this study is to deliver an enhanced green fluorescent protein (EGFP) plasmid DNA, as a model plasmid DNA, into renal interstitial space by a cationized gelatin. After the plasmid DNA with or without complexation of the cationized gelatin was injected to the left kidney of mice via the ureter, unilateral ureteral obstruction (UUO) was performed for the mice injected to induce the renal interstitial fibrosis. When the EGFP plasmid DNA complexed with the cationized gelatin was injected, EGFP expression was observed in the fibroblasts in the interstitial area of renal cortex. It is concluded that the retrograde injection of EGFP plasmid DNA complexed with the cationized gelatin is available to target the interstitial renal fibroblasts which are currently considered as the cell source responsible for excessive ECM synthesis.

  11. Short direct repeats flank the T-DNA on a nopaline Ti plasmid

    PubMed Central

    Yadav, Narendra S.; Vanderleyden, Jos; Bennett, Donald R.; Barnes, Wayne M.; Chilton, Mary-Dell

    1982-01-01

    Crown gall disease results from the insertion of a segment of the Agrobacterium Ti plasmid, called T-DNA, into host plant nuclear DNA. We have subjected to sequence analysis the border regions of pTi T37 (ends of T-DNA) and one left T-DNA/plant DNA border fragment isolated from BT37 tobacco teratoma by molecular cloning. These sequence studies, taken together with published sequence of a right T-DNA/plant DNA border fragment, allowed us to identify the positions of left and right borders at the DNA sequence level. Comparison of left and right border regions of the Ti plasmid revealed a “core” direct repeat of 13 of 14 bases (12 contiguous) precisely at the borders of T-DNA. An extended repeat of 21 of 25 bases overlaps this core repeat. T-DNA on the Ti plasmid exhibits no longer direct or inverted repeats in the border regions, based on Southern hybridization studies. The physical structure of T-DNA differs from that of known prokaryotic and eukaryotic transposable elements but bears a structural resemblance to the prophage of bacteriophage λ. Images PMID:16593241

  12. Mode of degradation of plasmid DNA with ozone.

    PubMed

    Sawadaishi, K; Miura, K; Ohthuka, E; Ueda, T; Ishizaki, K; Shinriki, N

    1984-01-01

    The ozonization of pBR322 closed circular DNA showed the conversion to open circular DNA. The damaged site was investigated by restriction mapping. The results showed the damage and subsequent cleavage of the DNA strand of ccDNA by ozonization may occur at the region sensitive to nuclease S1.

  13. An improved method for large-scale preparation of negatively and positively supercoiled plasmid DNA.

    PubMed

    Barth, Marita; Dederich, Debra; Dedon, Peter

    2009-07-01

    A rigorous understanding of the biological function of superhelical tension in cellular DNA requires the development of new tools and model systems for study. To this end, an ethidium bromide[#x02013]free method has been developed to prepare large quantities of either negatively or positively super-coiled plasmid DNA. The method is based upon the known effects of ionic strength on the direction of binding of DNA to an archaeal histone, rHMfB, with low and high salt concentrations leading to positive and negative DNA supercoiling, respectively. In addition to fully optimized conditions for large-scale (>500 microg) supercoiling reactions, the method is advantageous in that it avoids the use of mutagenic ethidium bromide, is applicable to chemically modified plasmid DNA substrates, and produces both positively and negatively supercoiled DNA using a single set of reagents.

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

    SciTech Connect

    Romanowski, G.; Lorenz, M.G.; Wackernagel, W. )

    1991-04-01

    The adsorption of ({sup 3}H)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 (Mg{sup 2+}, Ca{sup 2+}) were 100-fold more effective than monovalent cations (Na{sup +}, K{sup +}, NH{sub 4}{sup +}). Quantitative adsorption of up to 1 {mu}g of negatively supercoiled or linearized plasmid DNA to 0.7 g of sand was observed in the presence of 5 mm MgCl{sub 2} at pH 7. Under these conditions, more than 85% of DNA adsorbed within 60 s. Maximum adsorption was 4 {mu}g 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 MgCl{sub 2} 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.

  15. Advances in chromatographic supports for pharmaceutical-grade plasmid DNA purification.

    PubMed

    Sousa, Angela; Sousa, Fani; Queiroz, João A

    2012-11-01

    Chromatographic technology is undoubtedly one of the most diverse and powerful purification methods for downstream process applications. The diversity and quantity of biomolecules present in crude extracts as well as the similarities between impurities and the target biomolecule are considered the critical challenges in the extraction and purification steps. Thus, it is important to optimize the purification protocol to achieve maximum recovery of the target sample. The structure of chromatographic supports has been continuously developed to afford rapid and efficient separations, as well as, the application of specific ligands to improve the selectivity for the target molecule. The present review discusses the structural progress and evolution of the chromatographic supports that have been used for plasmid DNA purification. Nowadays, the most desirable form of plasmid for gene therapy and DNA vaccination is the supercoiled isoform, due to its stability and higher transfection efficiency over other plasmid topologies. However, the main challenge is not only to produce high quantities of supercoiled plasmid DNA but also to preserve its quality, meeting the strict requirements recommended by the regulatory agencies. Therefore, this review will focus on the chemical and structural classification of the different media and on some of the specific ligands used for plasmid DNA bioseparation.

  16. The effects of a low-intensity red laser on bacterial growth, filamentation and plasmid DNA

    NASA Astrophysics Data System (ADS)

    Roos, C.; Santos, J. N.; Guimarães, O. R.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2013-07-01

    Exposure of nonphotosynthesizing microorganisms to light could increase cell division in cultures, a phenomenon denominated as biostimulation. However, data concerning the importance of the genetic characteristics of cells on this effect are as yet scarce. The aim of this work was to evaluate the effects of a low-intensity red laser on the growth, filamentation and plasmids in Escherichia coli cells proficient and deficient in DNA repair. E. coli cultures were exposed to a laser (658 nm, 10 mW, 1 and 8 J cm-2) to study bacterial growth and filamentation. Also, bacterial cultures hosting pBSK plasmids were exposed to the laser to study DNA topological forms from the electrophoretic profile in agarose gels. Data indicate the low-intensity red laser: (i) had no effect on the growth of E. coli wild type and exonuclease III deficient cells; (ii) induced bacterial filamentation, (iii) led to no alteration in the electrophoretic profile of plasmids from exonuclease III deficient cells, but plasmids from wild type cells were altered. A low-intensity red laser at the low fluences used in phototherapy has no effect on growth, but induces filamentation and alters the topological forms of plasmid DNA in E. coli cultures depending on the DNA repair mechanisms.

  17. Activated air produced by shielded sliding discharge plasma mediates plasmid DNA delivery to mammalian cells.

    PubMed

    Edelblute, Chelsea M; Heller, Loree C; Malik, Muhammad A; Heller, Richard

    2015-12-01

    Cold plasma is emerging as a potential method for medical applications. The current study assessed the efficacy of a novel cold plasma reactor based on shielded sliding discharge producing cathode-directed streamers generated in ambient air for the delivery of plasmid DNA. Experiments were performed with mouse melanoma cells (B16F10) and human keratinocyte cells (HaCaT) inoculated with plasmid DNA encoding luciferase. Quantitative results measured over a 72-h period displayed luciferase expression levels as high as 5-fold greater in cells exposed to plasma-activated air (PAA) than levels obtained from the inoculation of plasmid DNA alone (P < 0.05, P < 0.01). No effect on cell viability was observed. Delivery of plasmid encoding GFP to HaCaT cells seeded on polycaprolactone (PCL) scaffolds was confirmed by immunostaining. The use of cold plasma for DNA delivery is attractive as it provides a non-viral, non-invasive method where the electrode or the plasma itself never directly contacts the exposed site. The current device design provides localized DNA transfer using a novel technology. Our report suggests PAA warrants further exploration as an alternative or supplemental approach for DNA transfer.

  18. Advances in host and vector development for the production of plasmid DNA vaccines.

    PubMed

    Mairhofer, Juergen; Lara, Alvaro R

    2014-01-01

    Recent developments in DNA vaccine research provide a new momentum for this rather young and potentially disruptive technology. Gene-based vaccines are capable of eliciting protective immunity in humans to persistent intracellular pathogens, such as HIV, malaria, and tuberculosis, for which the conventional vaccine technologies have failed so far. The recent identification and characterization of genes coding for tumor antigens has stimulated the development of DNA-based antigen-specific cancer vaccines. Although most academic researchers consider the production of reasonable amounts of plasmid DNA (pDNA) for immunological studies relatively easy to solve, problems often arise during this first phase of production. In this chapter we review the current state of the art of pDNA production at small (shake flasks) and mid-scales (lab-scale bioreactor fermentations) and address new trends in vector design and strain engineering. We will guide the reader through the different stages of process design starting from choosing the most appropriate plasmid backbone, choosing the right Escherichia coli (E. coli) strain for production, and cultivation media and scale-up issues. In addition, we will address some points concerning the safety and potency of the produced plasmids, with special focus on producing antibiotic resistance-free plasmids. The main goal of this chapter is to make immunologists aware of the fact that production of the pDNA vaccine has to be performed with as much as attention and care as the rest of their research.

  19. Reactive oxygen species controllable non-thermal helium plasmas for evaluation of plasmid DNA strand breaks

    NASA Astrophysics Data System (ADS)

    Young Kim, Jae; Lee, Dong-Hoon; Ballato, John; Cao, Weiguo; Kim, Sung-O.

    2012-11-01

    Non-thermal, oxygen-rich helium plasmas were investigated to achieve an enhanced reactive oxygen species concentration at low voltage driving conditions. A non-thermal plasma device was fabricated based on a theta-shaped tube, and its potential was investigated for use in topological alteration of plasmid DNA. The optical emission spectra of the plasma showed that the oxygen flow affected the plasma properties, even though an oxygen plasma was not produced. The plasmid DNA strand breaks became more significant with the addition of oxygen flow to the helium in a single hollow, theta-shaped tube with other experimental conditions being unchanged.

  20. Transfer of Large Contiguous DNA Fragments onto a Low Copy Plasmid or into the Bacterial Chromosome

    PubMed Central

    Reeves, Analise Z; Lesser, Cammie F

    2017-01-01

    Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Here we describe a robust 3-step method to transfer large defined fragments of DNA from virulence plasmids or cosmids onto smaller autonomously replicating plasmids or directly into defined sites in the bacterial chromosome that incorporates endogenous yeast and λ Red homologous recombination systems. This methodology has been successfully used to isolate and integrate at least 31 kb of contiguous DNA and can be readily adapted for the recombineering of E. coli and its close relatives. PMID:28203614

  1. DNA repair in cells sensitive and resistant to cis-diamminedichloroplatinum(II): Host cell reactivation of damaged plasmid DNA

    SciTech Connect

    Sheibani, N.; Jennerwein, M.M.; Eastman, A. )

    1989-04-04

    cis-Diamminedichloroplatinum(II) (cis-DDP) has a broad clinical application as an effective anticancer drug. However, development of resistance to the cytotoxic effects is a limiting factor. In an attempt to understand the mechanism of resistance, the authors have employed a host cell reactivation assay of DNA repair using a cis-DDP-damaged plasmid vector. The efficiency of DNA repair was assayed by measuring the activity of an enzyme coded for by the plasmid vector. The plasmid expression vector pRSV cat contains the bacterial gene coding for chloramphenicol acetyltransferase (CAT) in a configuration which permits expression in mammalian cells. The plasmid was transfected into repair-proficient and -deficient Chinese hamster ovary cells, and CAT activity was subsequently measured in cell lysates. In the repair-deficient cells, one cis-DDP adduct per cat gene was sufficient to eliminate expression. An equivalent inhibition of CAT expression in the repair-proficient cells did not occur until about 8 times the amount of damage was introduced into the plasmid. These results implicate DNA intrastrand cross-links as the lesions responsible for the inhibition of CAT expression. This assay was used to investigate the potential role of DNA repair in mediating cis-DDP resistance in murine leukemia L1210 cells. The assay readily detects the presence or absence of repair and confirms that these resistant L1210 cells have an enhanced capacity for repair of cis-DDP-induced intrastrand cross-links.

  2. Evaluation of plasmid and genomic DNA calibrants used for the quantification of genetically modified organisms.

    PubMed

    Caprioara-Buda, M; Meyer, W; Jeynov, B; Corbisier, P; Trapmann, S; Emons, H

    2012-07-01

    The reliable quantification of genetically modified organisms (GMOs) by real-time PCR requires, besides thoroughly validated quantitative detection methods, sustainable calibration systems. The latter establishes the anchor points for the measured value and the measurement unit, respectively. In this paper, the suitability of two types of DNA calibrants, i.e. plasmid DNA and genomic DNA extracted from plant leaves, for the certification of the GMO content in reference materials as copy number ratio between two targeted DNA sequences was investigated. The PCR efficiencies and coefficients of determination of the calibration curves as well as the measured copy number ratios for three powder certified reference materials (CRMs), namely ERM-BF415e (NK603 maize), ERM-BF425c (356043 soya), and ERM-BF427c (98140 maize), originally certified for their mass fraction of GMO, were compared for both types of calibrants. In all three systems investigated, the PCR efficiencies of plasmid DNA were slightly closer to the PCR efficiencies observed for the genomic DNA extracted from seed powders rather than those of the genomic DNA extracted from leaves. Although the mean DNA copy number ratios for each CRM overlapped within their uncertainties, the DNA copy number ratios were significantly different using the two types of calibrants. Based on these observations, both plasmid and leaf genomic DNA calibrants would be technically suitable as anchor points for the calibration of the real-time PCR methods applied in this study. However, the most suitable approach to establish a sustainable traceability chain is to fix a reference system based on plasmid DNA.

  3. DNA-membrane association is necessary for initiation of chromosomal and plasmid replication in Bacillus subtilis.

    PubMed

    Winston, S; Sueoka, N

    1980-05-01

    We examined the effect of the inhibition of initiation of DNA replication on the membrane association of the chromosomal origin of replication of Bacillus subtilis and the Staphylococcus aureus-Bacillus pumilus chimeric plasmid pSL103, using temperature-sensitive mutants of B. subtilis that have specifically affected initiation. Inhibition of initiation of the chromosome and pSL103 in the initiation mutant dna-1 results in a decrease in the membrane association of both a marker near the chromosomal origin, purA16, and the plasmid pSL103. The membrane association of both purA16 and pSL103 can be recovered by allowing initiation to resume at the permissive temperature. In another initiation mutant, dnaB19, only the initiation and membrane association of the host chromosome are affected at the nonpermissive temperature, whereas both initiation and membrane association are not affected in the plasmid pSL103. In experiments in vitro, DNA containing the purA16 marker and pSL103 DNA molecules are both selectively released during incubation of purified DNA-membrane complexes prepared from dna-1 cells at the nonpermissive temperature. On the other hand, only purA16 DNA is released in vitro from the DNA-membrane complex prepared from dnaB19 cells. This consistent coupling between initiation and membrane association indicates that DNA-membrane association is critical for the initiation of the B. subtilis chromosome and the plasmid pSL103.

  4. The heat-shock DnaK protein is required for plasmid R1 replication and it is dispensable for plasmid ColE1 replication.

    PubMed Central

    Giraldo-Suárez, R; Fernández-Tresguerres, E; Díaz-Orejas, R; Malki, A; Kohiyama, M

    1993-01-01

    Plasmid R1 replication in vitro is inactive in extracts prepared from a dnaK756 strain but is restored to normal levels upon addition of purified DnaK protein. Replication of R1 in extracts of a dnaKwt strain can be specifically inhibited with polyclonal antibodies against DnaK. RepA-dependent replication of R1 in dnaK756 extracts supplemented with DnaKwt protein at maximum concentration is partially inhibited by rifampicin and it is severely inhibited at sub-optimal concentrations of DnaK protein. The copy number of a run-away R1 vector is reduced in a dnaK756 background at 30 degrees C and at 42 degrees C the amplification of the run-away R1 vector is prevented. However a runaway R1 vector containing dnaK gene allows the amplification of the plasmid at high temperature. These data indicate that DnaK is required for both in vitro and in vivo replication of plasmid R1 and show a partial compensation for the low level of DnaK by RNA polymerase. In contrast ColE1 replication is not affected by DnaK as indicated by the fact that ColE1 replicates with the same efficiency in extracts from dnaKwt and dnaK756 strains. Images PMID:8265367

  5. Persistence of Free Plasmid DNA in Soil Monitored by Various Methods, Including a Transformation Assay

    PubMed Central

    Romanowski, Gerd; Lorenz, Michael G.; Sayler, Gary; Wackernagel, Wilfried

    1992-01-01

    The persistence and stability of free plasmid pUC8-ISP DNA introduced into 10-g samples of various soils and kept at 23°C were monitored over a period of 60 days. The soils were sampled at a plant science farm and included a loamy sand soil (no. 1), a clay soil (no. 2), and a silty clay soil (no. 3). Four different methods allowed monitoring of (i) the production of acid-soluble radioactive material from [3H]thymidine-labeled plasmid DNA, (ii) the decrease of hybridizing nucleotide sequences in slot blot analysis, (iii) the loss of plasmid integrity measured by Southern hybridization, and (iv) the decay of the biological activity as determined by transformation of Ca2+-treated Escherichia coli cells with the DNA extracted from soil. Acid-soluble material was not produced within the first 24 h but then increased to 45% (soil no. 1), 27% (soil no. 2), and 77% (soil no. 3) until the end of incubation. A quite parallel loss of material giving a slot blot hybridization signal was observed. Southern hybridization indicated that after 1 h in the soils, plasmid DNA was mostly in the form of circular and full-length linear molecules but that, depending on the soil type, after 2 to 5 days full-length plasmid molecules were hardly detectable. The transforming activity of plasmid DNA reextracted from the soils followed inactivation curves over 2 to 4 orders of magnitude and dropped below the detection limit after 10 days. The inactivation was slower in soil no. 2 (28.2-h half-life time of the transforming activity of a plasmid molecule) than in soils no. 3 (15.1 h) and no. 1 (9.1 h). The studies provide data on the persistence of free DNA molecules in natural bacterial soil habitats. The data suggest that plasmid DNA may persist long enough to be available for uptake by competent recipient cells in situ. Images PMID:16348772

  6. Asbestos fibers mediate transformation of monkey cells by exogenous plasmid DNA

    SciTech Connect

    Appel, J.D.; Fasy, T.M.; Kohtz, D.S.; Kohtz, J.D.; Johnson, E.M. )

    1988-10-01

    The authors have tested the ability of chrysotile asbestos fibers to introduce plasmid DNA into monkey COS-7 cells and the ability of this DNA to function in both replication and gene expression. Chrysotile fibers are at least as effective as calcium phosphate in standard transfection assays at optimal ratios of asbestos to DNA. After transfection with chrysotile, a minor percentage of introduced plasmid DNA bearing a simian virus 40 origin of replication replicates after 24 hr. Fragmentation of entering DNA is more prominent with asbestos than with calcium phosphate, and after 72 hr most DNA introduced by asbestos is associated with chromosomal DNA. Cells transfected with plasmid p11-4, bearing the p53 protooncogene, express this gene. Cells transfected with pSV2-neo express a gene conferring resistance of antibiotic G418, allowing isolation of colonies of transformed cells after 18 days. The introduction of exogenous DNA into eukaryotic cells could cause mutations in several ways and thus contribute to asbestos-induced oncogenesis.

  7. Biomedical application of plasmid DNA in gene therapy: a new challenge for chromatography.

    PubMed

    Sousa, F; Passarinha, L; Queiroz, J A

    2010-01-01

    Gene therapy and DNA vaccination are clinical fields gradually emerging in the last few decades, in particular after the discovery of some gene-related diseases. The increased relevance of biomedical applications of plasmid DNA (pDNA) to induce therapeutic effects has had a great impact on biopharmaceutical research and industry. Although there are several steps involved in the pDNA manufacturing process, the several unit operations must be designed and integrated into a global process. After the plasmid has been designed according to the requirements for clinical administeration to humans, it is biosynthesised mainly by an E. coli host. The overriding priority of the production process is to improve plasmid quantity - the production conditions need to be optimised to guarantee pDNA stability and biological activity. The complexity and diversity of biomolecules present on the pDNA-containing extracts represent the main concern and limitation to achieve pure and biologically active pDNA. There has been a recent intenstification of the improvement of existing purification procedures or the establishment of novel schemes for plasmid purification. This review focuses on the progress and relevance of chromatographic methodologies in the purification of pDNA-based therapeutic products. The review will attempt to assemble their different contributions of the different chromatographic procedures that are being used in the pDNA purification area. The advantages and disadvantages of the different chromatographic techniques, as well as the most significant improvements in response to the challenge of purifying pDNA will be discussed, emphasizing the future directions in this field.

  8. Conjugative DNA Transfer Is Enhanced by Plasmid R1 Partitioning Proteins

    PubMed Central

    Gruber, Christian J.; Lang, Silvia; Rajendra, Vinod K. H.; Nuk, Monika; Raffl, Sandra; Schildbach, Joel F.; Zechner, Ellen L.

    2016-01-01

    Bacterial conjugation is a form of type IV secretion used to transport protein and DNA directly to recipient bacteria. The process is cell contact-dependent, yet the mechanisms enabling extracellular events to trigger plasmid transfer to begin inside the cell remain obscure. In this study of plasmid R1 we investigated the role of plasmid proteins in the initiation of gene transfer. We find that TraI, the central regulator of conjugative DNA processing, interacts physically, and functionally with the plasmid partitioning proteins ParM and ParR. These interactions stimulate TraI catalyzed relaxation of plasmid DNA in vivo and in vitro and increase ParM ATPase activity. ParM also binds the coupling protein TraD and VirB4-like channel ATPase TraC. Together, these protein-protein interactions probably act to co-localize the transfer components intracellularly and promote assembly of the conjugation machinery. Importantly these data also indicate that the continued association of ParM and ParR at the conjugative pore is necessary for plasmid transfer to start efficiently. Moreover, the conjugative pilus and underlying secretion machinery assembled in the absence of Par proteins mediate poor biofilm formation and are completely dysfunctional for pilus specific R17 bacteriophage uptake. Thus, functional integration of Par components at the interface of relaxosome, coupling protein, and channel ATPases appears important for an optimal conformation and effective activation of the transfer machinery. We conclude that low copy plasmid R1 has evolved an active segregation system that optimizes both its vertical and lateral modes of dissemination. PMID:27486582

  9. The immunogenicity of viral haemorragic septicaemia rhabdovirus (VHSV) DNA vaccines can depend on plasmid regulatory sequences.

    PubMed

    Chico, V; Ortega-Villaizan, M; Falco, A; Tafalla, C; Perez, L; Coll, J M; Estepa, A

    2009-03-18

    A plasmid DNA encoding the viral hemorrhagic septicaemia virus (VHSV)-G glycoprotein under the control of 5' sequences (enhancer/promoter sequence plus both non-coding 1st exon and 1st intron sequences) from carp beta-actin gene (pAE6-G(VHSV)) was compared to the vaccine plasmid usually described the gene expression is regulated by the human cytomegalovirus (CMV) immediate-early promoter (pMCV1.4-G(VHSV)). We observed that these two plasmids produced a markedly different profile in the level and time of expression of the encoded-antigen, and this may have a direct effect upon the intensity and suitability of the in vivo immune response. Thus, fish genetic immunisation assays were carried out to study the immune response of both plasmids. A significantly enhanced specific-antibody response against the viral glycoprotein was found in the fish immunised with pAE6-G(VHSV). However, the protective efficacy against VHSV challenge conferred by both plasmids was similar. Later analysis of the transcription profile of a set of representative immune-related genes in the DNA immunized fish suggested that depending on the plasmid-related regulatory sequences controlling its expression, the plasmid might activate distinct patterns of the immune system. All together, the results from this study mainly point out that the selection of a determinate encoded-antigen/vector combination for genetic immunisation is of extraordinary importance in designing optimised DNA vaccines that, when required for inducing protective immune response, could elicit responses biased to antigen-specific antibodies or cytotoxic T cells generation.

  10. DNA fusion product of phage P2 with plasmid pBR322 - A new phasmid

    NASA Technical Reports Server (NTRS)

    Nicoletti, M.; Bertani, G.

    1983-01-01

    The chromosome of the temperate bacteriophage P2 and that of the plasmid pBR322 have been joined in vitro after treatment with restriction endonuclease EcoRI. The fusion product - a phasmid - can behave as a plasmid, as a phage and as a prophage. It can replicate its DNA under the control of either the specific replication mechanism of the parent phage in a polA mutant or that of the parent plasmid in a rep mutant. Several interesting interactions between the two replication modes are indicated. In particular, phage particles may be produced even when the phage mode of DNA replication is blocked, and this throws new light on the involvement of the early gene A in the regulation of late gene expression in phage P2.

  11. In vivo release of plasmid DNA from composites of oligo(poly(ethylene glycol)fumarate) and cationized gelatin microspheres.

    PubMed

    Kasper, F Kurtis; Kushibiki, Toshihiro; Kimura, Yu; Mikos, Antonios G; Tabata, Yasuhiko

    2005-10-20

    Composites of cationized gelatin microspheres (CGMS), crosslinked with either 3 mM or 6 mM glutaraldehyde solution, and a novel hydrogel material, oligo(poly(ethylene glycol)fumarate) (OPF) were fabricated and investigated toward prolonging the release of plasmid DNA in vivo relative to the constituent materials. The composites and constituent materials were investigated in a subcutaneous murine model to assess the release of 125I-labeled plasmid DNA and 125I-labeled cationized gelatin in vivo. The time profiles of the radioactivity remaining were employed to compare the profiles of DNA release and cationized gelatin degradation. Both composite formulations (incorporating either 3 mM or 6 mM CGMS) prolonged the bioavailability of plasmid DNA relative to both injected plasmid DNA solution and the respective non-embedded cationized gelatin microspheres. Injected plasmid DNA solution persisted in the subject for only 7-10 days, whereas the persistence of DNA from composites of OPF and either 3 mM or 6 mM CGMS extended to at least day 42. The 3 mM and 6 mM CGMS each increased the persistence of DNA slightly, relative to injection of DNA solution, to between 28 and 35 days. Interestingly, the release profile of plasmid DNA from composites was not significantly different from the release of DNA from OPF alone. The release of plasmid DNA from the composites was in accord with the degradation of the microspheres within the OPF. These results show that composites of OPF and cationized gelatin microspheres are able to prolong the availability of plasmid DNA in vivo relative to cationized gelatin microspheres alone and provide a promising candidate material for the sustained, controlled release of plasmid DNA.

  12. Ca2+ Promoted the Low Transformation Efficiency of Plasmid DNA Exposed to PAH Contaminants

    PubMed Central

    Gao, Yanzheng; Long, Jian; Wang, Qian

    2013-01-01

    The effects of interactions between genetic materials and polycyclic aromatic hydrocarbons (PAHs) on gene expression in the extracellular environment remain to be elucidated and little information is currently available on the effect of ionic strength on the transformation of plasmid DNA exposed to PAHs. Phenanthrene and pyrene were used as representative PAHs to evaluate the transformation of plasmid DNA after PAH exposure and to determine the role of Ca2+ during the transformation. Plasmid DNA exposed to the test PAHs demonstrated low transformation efficiency. In the absence of PAHs, the transformation efficiency was 4.7 log units; however, the efficiency decreased to 3.72–3.14 log units with phenanthrene/pyrene exposures of 50 µg·L–1. The addition of Ca2+ enhanced the low transformation efficiency of DNA exposed to PAHs. Based on the co-sorption of Ca2+ and phenanthrene/pyrene by DNA, we employed Fourier-transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and mass spectrometry (MS) to determine the mechanisms involved in PAH-induced DNA transformation. The observed low transformation efficiency of DNA exposed to either phenanthrene or pyrene can be attributed to a broken hydrogen bond in the double helix caused by planar PAHs. Added Ca2+ formed strong electrovalent bonds with “–POO––” groups in the DNA, weakening the interaction between PAHs and DNA based on weak molecular forces. This decreased the damage of PAHs to hydrogen bonds in double-stranded DNA by isolating DNA molecules from PAHs and consequently enhanced the transformation efficiency of DNA exposed to PAH contaminants. The findings provide insight into the effects of anthropogenic trace PAHs on DNA transfer in natural environments. PMID:23484001

  13. Evaluation of different buffers on plasmid DNA encapsulation into PLGA microparticles.

    PubMed

    Tse, Man Tsuey; Blatchford, Chris; Oya Alpar, H

    2009-03-31

    Double emulsion solvent evaporation is a widely used method to prepare poly(dl-lactide-co-glycolide) (PLGA) microparticles encapsulating plasmid DNA. There are inherent problems associated with preparing plasmid DNA in this form, in particular the DNA is liable to degrade during manufacture and the resulting powder has low encapsulation efficiencies. This study compares the use of two buffers, 0.1M NaHCO(3) and 0.07M Na(2)HPO(4) and the effect these have on the encapsulation efficiency and other critical parameters associated with these encapsulated DNA materials. Both buffers preserved the conformation of the original plasmid DNA during the homogenization process, but those made with 0.07M Na(2)HPO(4) had higher encapsulation efficiencies, as well as smaller diameters, compared with those made with 0.1M NaHCO(3) (encapsulation efficiencies of 40.72-45.65%, and mean volume diameters of 2.96-4.45microm). Buffers with a range of pH from 5 to 12 were investigated, and it was demonstrated that pH 9 was the point at which the highest amount of supercoiled DNA was balanced with the highest encapsulation efficiency. To simulate in vitro release, it was shown that microparticles made with 0.07M Na(2)HPO(4) had lower DNA release rates than those made with 0.1M NaHCO(3). These results demonstrate that the use of different buffers can aid in retaining the conformation of plasmid DNA, and can also modulate the amount of DNA encapsulated and the release profiles of microparticles.

  14. Production and purification of plasmid DNA vaccines: is there scope for further innovation?

    PubMed

    Xenopoulos, Alex; Pattnaik, Priyabrata

    2014-12-01

    The demand for plasmid DNA (pDNA) has vastly increased over the past decade in response to significant advances that have been made in its application for gene therapy and vaccine development. Plasmid DNA-based vaccines are experiencing a resurgence due to success with prime-boost immunization strategies. The challenge has always been poor productivity and delivery of pDNA. Plasmid DNA-based vaccines have traditionally required milligram scale of GMP-grade product for vaccination due to the relatively low efficacy and duration of gene expression. However, efforts to increase pDNA vaccine effectiveness are evolving in genetic manipulations of bacterial host, improvements in product recovery and innovative delivery methods. This review summarizes recent advances in large-scale pDNA vaccine manufacturing, ranging from upstream processing, downstream processing and formulation, as such information is usually not available to the scientific community. The article will highlight technology gaps and offer insight on further scope of innovation.

  15. Microneedle delivery of plasmid DNA to living human skin: formulation coating, skin insertion and gene expression

    PubMed Central

    Pearton, Marc; Saller, Verena; Coulman, Sion A; Gateley, Chris; Anstey, Alexander V; Zarnitsyn, Vladimir; Birchall, James C

    2012-01-01

    Microneedle delivery of nucleic acids, in particular plasmid DNA (pDNA), to the skin represents a potential new approach for the clinical management of genetic skin diseases and cutaneous cancers, and for intracutaneous genetic immunization. In this study excised human skin explants were used to investigate and optimize key parameters that will determine stable and effective microneedle-facilitated pDNA delivery. These include (i) high dose-loading of pDNA onto microneedle surfaces, (ii) stability and functionality of the coated pDNA, (iii) skin penetration capability of pDNA-coated microneedles, and (iv) efficient gene expression in human skin. Optimization of a dip-coating method enabled significant increases in the loading capacity, up to 100 micrograms of pDNA per 5-microneedle array. Coated microneedles were able to reproducibly perforate human skin at low (<1 Newton) insertion forces. The physical stability of the coated pDNA was partially compromised on storage, although this was improved through the addition of saccharide excipients without detriment to the biological functionality of pDNA. The pDNA-coated microneedles facilitated reporter gene expression in viable human skin. The efficiency of gene expression from coated microneedles will depend upon suitable DNA loading, efficient and reproducible skin puncture and rapid in situ dissolution of the plasmid at the site of delivery. PMID:22516089

  16. PLASMID DNA DAMAGE CAUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    EPA Science Inventory

    PLASMID DNA DAMAGE CAOUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    ABSTRACT

    Both dimethylarsinic acid (DMA(V)) and dimethylarsinous acid (DMA(III)) release iron from human liver ferritin (HLF) with or without the presence of ascorbic acid. ...

  17. Plasmids containing the gene for DNA polymerase I from Streptococcus pneumoniae

    DOEpatents

    Lacks, S.A.; Martinez, S.; Lopez, P.; Espinosa, M.

    1987-08-28

    A method is disclosed for cloning the gene which encodes a DNA polymerase-exonuclease of /und Streptococcus/ /und pneumoniae/. Plasmid pSM22, the vector containing the pneumococcal polA gene, facilitates the expression of 50-fold greater amounts of the PolI enzyme. 1 fig., 1 tab.

  18. Effect of amine type on the expression of plasmid DNA by cationized dextran.

    PubMed

    Jo, Jun-ichiro; Nagane, Kentaro; Yamamoto, Masaya; Tabata, Yasuhiko

    2010-01-01

    The objective of this study is to prepare a non-viral carrier of gene expression from the polysaccharide dextran and evaluate the effect of amine compounds introduced to dextran on the level of gene expression. Dextran with a molecular weight of 74 x 10(3) was cationized by the chemical introduction of different amine compounds. The cationized dextran was complexed with a plasmid DNA and the vitro gene transfection was investigated for HeLa cells. The level of gene expression depended on the amine compound introduced to dextran. The highest level was observed for the complex of spermine-introduced dextran and plasmid DNA. The highest cellular internalization and the best buffering effect were observed among every cationized dextran. Every complex did not show any cytotoxicity. It is concluded that the superior properties of spermine-introduced dextran enabled the plasmid DNA to enhance the expression level to a great extent compared with other cationized dextrans. Cationized dextran is a promising non-viral carrier of plasmid DNA.

  19. PLASMID DNA DAMAGE CAUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    EPA Science Inventory

    Plasmid DNA damage caused by methylated arsenicals, ascorbic acid and human liver ferritin.

    Arsenic causes cancer in human skin, urinary bladder, lung, liver and kidney and is a significant world-wide public health problem. Although the metabolism of inorganic arsenic is ...

  20. Plasmids containing the gene for DNA polymerase I from Streptococcus pneumoniae

    DOEpatents

    Lacks, S.A.; Martinez, S.; Lopez, P.; Espinosa, M.

    1991-03-26

    A method is disclosed for cloning the gene which encodes a DNA polymerase-exonuclease of Streptococcus pneumoniae. Plasmid pSM22, the vector containing the pneumocccal polA gene, facilitates the expression of 50-fold greater amounts of the PolI enzyme. 1 figure.

  1. Plasmid DNA nanogels as photoresponsive materials for multifunctional bio-applications.

    PubMed

    Costa, Diana; Valente, Artur J M; Queiroz, João

    2015-05-20

    This study provides a detailed description on the synthesis and characterization of novel polyamine plasmid DNA nanogels. Ethylene glycol diglycidyl ether was used as cross-linker, in conjugation with polyamines to promote pDNA condensation. The biocompatible nanovectors exhibit a unique swelling behavior in water and salt solutions. These systems are light photodegradable allowing their use in a broad range of biotechnological applications. Different plasmids, pVAX1-LacZ and pcDNA3-FLAG-p53, and anticancer drugs were, thus, efficiently loaded in the nanogels and their controlled release was demonstrated. Furthermore, the dual delivery of pcDNA3-FLAG-p53 gene and anticancer drugs illustrates the possibility of the combination of chemical and gene therapies. This new versatile and easy method of nanohydrogels preparation provides a potential synthetic approach for the design of tunable systems which can display multiple functions, sensitivity to different stimuli and exhibit programmed responses as well.

  2. Hundreds of Circular Novel Plasmids and DNA Elements Identified in a Rat Cecum Metamobilome

    PubMed Central

    Jørgensen, Tue Sparholt; Xu, Zhuofei; Hansen, Martin Asser; Sørensen, Søren Johannes; Hansen, Lars Hestbjerg

    2014-01-01

    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. PMID:24503942

  3. Formation of oligomeric structures from plasmid DNA carrying cos lambda that is packaged into bacteriophage lambda heads.

    PubMed Central

    Miwa, T; Matsubara, K

    1983-01-01

    Plasmids that carry cos lambda, the region necessary for lambda phage packaging and that are as small as four kilobases in size can be packaged into lambda phage heads in head-to-tail tandem oligomeric structures. Multimeric oligomers as large as undecamers have been detected. Oligomer formation depends upon the products of red and gam of lambda, and the general recombination occurs between different plasmids that share homologous DNA regions. The packaging efficiency of plasmids depends on its copy number in cells and its genome size. Upon injection into a cell, the DNA establishes itself as a plasmid in a tandem structure. When such a plasmid in a high oligomeric structure is used as the source of packaging DNA, the packaging efficiency of the plasmids is elevated. The oligomers are stable in recA cells, whereas they drift toward lower oligomers in recA+ cells. Images PMID:6217189

  4. Cholesterol-conjugated supramolecular assemblies of low generations polyamidoamine dendrimers for enhanced EGFP plasmid DNA transfection

    NASA Astrophysics Data System (ADS)

    Golkar, Nasim; Samani, Soliman Mohammadi; Tamaddon, Ali Mohammad

    2016-05-01

    Aimed to prepare an enhanced gene delivery system with low cytotoxicity and high transfection efficiency, various cholesterol-conjugated derivates of low generation polyamidoamine (PAMAM) dendrimers were prepared. The conjugates were characterized by TNBS assay, FTIR, and 1H-NMR spectroscopy. Self-assembly of the dendrimer conjugates (G1-Chol, G2-Chol, and G3-Chol) was investigated by pyrene assay. Following formation of the complexes between enhanced green fluorescence protein plasmid and the dendrimer conjugates at various N (primary amine)/P (phosphate) mole ratios, plasmid condensation, biologic stability, cytotoxicity, and protein expression were investigated. The conjugates self-assembled into micellar dispersions with the critical micelle concentration values (<50 µg/ml) depending on the dendrimer generation and cholesterol/amine mole ratio. Cholesterol conjugation resulted in higher resistance of the condensed plasmid DNA in a competition assay with heparin sulfate. Also, the transfection efficiency was determined higher for the cholesterol conjugates than unmodified dendrimers in HepG2 cells, showing the highest for G2-Chol at 40 % degree of cholesterol modification (G2-Chol40 %) among various dendrimer generations. Interestingly, such conjugate showed a complete protection of plasmid against serum nucleases. Our results confirmed that the cholesterol conjugation to PAMAM dendrimers of low generations bearing little cytotoxicity improves their several physicochemical and biological characteristics required for an enhanced delivery of plasmid DNA into cells.

  5. High-Throughput Plasmid cDNA Library Screening

    SciTech Connect

    Wan, Kenneth H.; Yu, Charles; George, Reed A.; Carlson, JosephW.; Hoskins, Roger A.; Svirskas, Robert; Stapleton, Mark; Celniker, SusanE.

    2006-05-24

    Libraries of cDNA clones are valuable resources foranalysing the expression, structure, and regulation of genes, as well asfor studying protein functions and interactions. Full-length cDNA clonesprovide information about intron and exon structures, splice junctionsand 5'- and 3'-untranslated regions (UTRs). Open reading frames (ORFs)derived from cDNA clones can be used to generate constructs allowingexpression of native proteins and N- or C-terminally tagged proteins.Thus, obtaining full-length cDNA clones and sequences for most or allgenes in an organism is critical for understanding genome functions.Expressed sequence tag (EST) sequencing samples cDNA libraries at random,which is most useful at the beginning of large-scale screening projects.However, as projects progress towards completion, the probability ofidentifying unique cDNAs via EST sequencing diminishes, resulting in poorrecovery of rare transcripts. We describe an adapted, high-throughputprotocol intended for recovery of specific, full-length clones fromplasmid cDNA libraries in five days.

  6. High-frequency intermolecular homologous recombination during herpes simplex virus-mediated plasmid DNA replication.

    PubMed

    Fu, Xinping; Wang, Hua; Zhang, Xiaoliu

    2002-06-01

    Homologous recombination is a prominent feature of herpes simplex virus (HSV) type 1 DNA replication. This has been demonstrated and traditionally studied in experimental settings where repeated sequences are present or are being introduced into a single molecule for subsequent genome isomerization. In the present study, we have designed a pair of unique HSV amplicon plasmids to examine in detail intermolecular homologous recombination (IM-HR) between these amplicon plasmids during HSV-mediated DNA replication. Our data show that IM-HR occurred at a very high frequency: up to 60% of the amplicon concatemers retrieved from virion particles underwent intermolecular homologous recombination. Such a high frequency of IM-HR required that both plasmids be replicated by HSV-mediated replication, as IM-HR events were not detected when either one or both plasmids were replicated by simian virus 40-mediated DNA replication, even with the presence of HSV infection. In addition, the majority of the homologous recombination events resulted in sequence replacement or targeted gene repair, while the minority resulted in sequence insertion. These findings imply that frequent intermolecular homologous recombination may contribute directly to HSV genome isomerization. In addition, HSV-mediated amplicon replication may be an attractive model for studying intermolecular homologous recombination mechanisms in general in a mammalian system. In this regard, the knowledge obtained from such a study may facilitate the development of better strategies for targeted gene correction for gene therapy purposes.

  7. PEGylation enhances tumor targeting of plasmid DNA by an artificial cationized protein with repeated RGD sequences, Pronectin.

    PubMed

    Hosseinkhani, Hossein; Tabata, Yasuhiko

    2004-05-31

    The objective of this study is to investigate feasibility of a non-viral gene carrier with repeated RGD sequences (Pronectin F+) in tumor targeting for gene expression. The Pronectin F+ was cationized by introducing spermine (Sm) to the hydroxyl groups to allow to polyionically complex with plasmid DNA. The cationized Pronectin F+ prepared was additionally modified with poly(ethylene glycol) (PEG) molecules which have active ester and methoxy groups at the terminal, to form various PEG-introduced cationized Pronectin F+. The cationized Pronectin F+ with or without PEGylation at different extents was mixed with a plasmid DNA of LacZ to form respective cationized Pronectin F+-plasmid DNA complexes. The plasmid DNA was electrophoretically complexed with cationized Pronectin F+ and PEG-introduced cationized Pronectin F+, irrespective of the PEGylation extent, although the higher N/P ratio of complexes was needed for complexation with the latter Pronectin F+. The molecular size and zeta potential measurements revealed that the plasmid DNA was reduced in size to about 250 nm and the charge was changed to be positive by the complexation with cationized Pronectin F+. For the complexation with PEG-introduced cationized Pronectin F+, the charge of complex became neutral being almost 0 mV with the increasing PEGylation extents, while the molecular size was similar to that of cationized Pronectin F+. When cationized Pronectin F+-plasmid DNA complexes with or without PEGylation were intravenously injected to mice carrying a subcutaneous Meth-AR-1 fibrosarcoma mass, the PEG-introduced cationized Pronectin F+-plasmid DNA complex specifically enhanced the level of gene expression in the tumor, to a significantly high extent compared with the cationized Pronectin F+-plasmid DNA complexes and free plasmid DNA. The enhanced level of gene expression depended on the percentage of PEG introduced, the N/P ratio, and the plasmid DNA dose. A fluorescent microscopic study revealed that the

  8. Nucleosome organization of the yeast 2-micrometer DNA plasmid: a eukaryotic minichromosome.

    PubMed Central

    Nelson, R G; Fangman, W L

    1979-01-01

    The eukaryotic microorganism Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2-micrometer DNA. The intracellular structure of these molecules, which represent about 4% of the total DNA, was examined by digestion of total cellular chromatin with micrococcal nuclease (nucleate 3'-oligonucleotidohydrolase, EC 3.1.31.1). Nuclease-resistant DNA fragments were fractionated by gel electrophoresis and 2-micrometer DNA sequences were detected by hybridization. The 2-micrometer and chromosomal DNA digestion patterns were very similar indicating that both types of DNA are condensed into nucleosomes. An analysis of these digestion patterns showed that the kinetics of digestion of 2-micrometer chromatin and total chromatin are similar and that both have the same nucleosome repeat length of about 165 base pairs. Native 2-micrometer plasmids were examined by zone sedimentation in sucrose gradients containing 0.15 M NaCl and were found to have a sedimentation constant of 75 S, about 3 times the sedimentation constant of protein-free 2-micrometer DNA. This sedimentation property is what would be expected for a 2-micrometer DNA minichromosome. We conclude that within the cell 2-micrometer DNA molecules are organized in a chromatin structure very similar to that of the yeast chromosomes. Images PMID:392520

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

    SciTech Connect

    Yu Jia; Zhang Zhenfeng; Cao Kou; Huang Xitai

    2008-09-26

    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.

  10. Purification of plasmid DNA from Escherichia coli ferments using anion-exchange membrane and hydrophobic chromatography.

    PubMed

    Guerrero-Germán, Patricia; Montesinos-Cisneros, Rosa Ma; Prazeres, D Miguel F; Tejeda-Mansir, Armando

    2011-01-01

    A novel downstream bioprocess was developed to obtain purified plasmid DNA (pDNA) from Escherichia coli ferments. The intermediate recovery and purification of the pDNA in cell lysate was conducted using hollow-fiber tangential filtration and frontal anion-exchange membrane and elution hydrophobic chromatographies. The purity of the solutions of pDNA obtained during each process stage was investigated. The results show that the pDNA solution purity increased 30-fold and more than 99% of RNA in the lysate was removed during the process operations. The combination of membrane operations and hydrophobic interaction chromatography resulted in an efficient way to recover pDNA from cell lysates. A better understanding of membrane-based technology for the purification of pDNA from clarified E. coli lysate was developed in this research.

  11. Spermine and spermidine protection of plasmid DNA against single-strand breaks induced by singlet oxygen.

    PubMed Central

    Khan, A U; Di Mascio, P; Medeiros, M H; Wilson, T

    1992-01-01

    Oxidative damage to DNA induced by singlet molecular oxygen (1O2*) includes single-strand breaks, which the biologically occurring 1O2* quenchers spermine and spermidine are shown to prevent. These polyamines at a physiological concentration (10 mM) reduce the percentage of the open circular form of pBR322 plasmid DNA, which is generated at the expense of the native supercoiled form when the plasmids are incubated with a chemical source of 1O2*, the water-soluble endoperoxide of 3,3'-(1,4-naphthylidene)dipropionate. Spermine and spermidine can be expected to protect DNA against other damaging effects of 1O2*. Images PMID:1454831

  12. Isothermal titration calorimetric analysis of the interaction between cationic lipids and plasmid DNA.

    PubMed

    Lobo, B A; Davis, A; Koe, G; Smith, J G; Middaugh, C R

    2001-02-01

    The effects of buffer and ionic strength upon the enthalpy of binding between plasmid DNA and a variety of cationic lipids used to enhance cellular transfection were studied using isothermal titration calorimetry at 25.0 degrees C and pH 7.4. The cationic lipids DOTAP (1,2-dioleoyl-3-trimethyl ammonium propane), DDAB (dimethyl dioctadecyl ammonium bromide), DOTAP:cholesterol (1:1), and DDAB:cholesterol (1:1) bound endothermally to plasmid DNA with a negligible proton exchange with buffer. In contrast, DOTAP: DOPE (L-alpha-dioleoyl phosphatidyl ethanolamine) (1:1) and DDAB:DOPE (1:1) liposomes displayed a negative enthalpy and a significant uptake of protons upon binding to plasmid DNA at neutral pH. These findings are most easily explained by a change in the apparent pKa of the amino group of DOPE upon binding. Complexes formed by reverse addition methods (DNA into lipid) produced different thermograms, sizes, zeta potentials, and aggregation behavior, suggesting that structurally different complexes were formed in each titration direction. Titrations performed in both directions in the presence of increasing ionic strength revealed a progressive decrease in the heat of binding and an increase in the lipid to DNA charge ratio at which aggregation occurred. The unfavorable binding enthalpy for the cationic lipids alone and with cholesterol implies an entropy-driven interaction, while the negative enthalpies observed with DOPE-containing lipid mixtures suggest an additional contribution from changes in protonation of DOPE.

  13. Intrathecal Injection of Naked Plasmid DNA Provides Long-term Expression of Secreted Proteins

    PubMed Central

    Hughes, Travis S; Langer, Stephen J; Johnson, Kirk W; Chavez, Raymond A; Watkins, Linda R; Milligan, Erin D; Leinwand, Leslie A

    2008-01-01

    Therapeutic benefit has been reported to result from intrathecal (i.t.) injection of transgene vectors, including naked DNA. However, most studies using naked DNA have measured only the transgene expression of intracellular proteins. Here we demonstrate that i.t. injection of naked DNA can result in long-term expression of secreted proteins. Plasmids expressing either secreted alkaline phosphatase (SEAP) or human interleukin-10 (hIL-10) were injected into the i.t. space in rats, and transgene products were repeatedly measured in the cerebrospinal fluid (CSF). Both SEAP and hIL-10 were maximal at 1 and 2 days after the injection and still detectable at 4 months. The utilization of a plasmid having two features that are hypothesized to increase gene expression (matrix attachment regions (MARs) and lack of CpG dinucleotides) resulted in a significant increase in gene expression. Reinjection of SEAP or hIL-10 plasmids after 4 months significantly increased protein levels at 1 and 14 days after the reinjection. SEAP was uniformly distributed between the DNA delivery site (~vertebral level T13) and the lumbar puncture site (L5/L6 inter-vertebral space), was reduced at the cisterna magna, and was detectable, though at much lower levels, in serum. These data suggest that naked DNA has the potential to be used as a therapeutic tool for applications that require long-term release of transgenes into the CSF. PMID:18941439

  14. Characterization of the endogenous plasmid from Pseudomonas alcaligenes NCIB 9867: DNA sequence and mechanism of transfer.

    PubMed

    Kwong, S M; Yeo, C C; Suwanto, A; Poh, C L

    2000-01-01

    The endogenous plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 was determined to have 32,743 bp with a G+C content of 59.8%. Sequence analysis predicted a total of 29 open reading frames, with approximately half of them contributing towards the functions of plasmid replication, mobilization, and stability. The Pac25I restriction-modification system and two mobile elements, Tn5563 and IS1633, were physically localized. An additional eight open reading frames with unknown functions were also detected. pRA2 was genetically tagged with the OmegaStr(r)/Spc(r) gene cassette by homologous recombination. Intrastrain transfer of pRA2-encoded genetic markers between isogenic mutants of P. alcaligenes NCIB 9867 were observed at high frequencies (2.4 x 10(-4) per donor). This transfer was determined to be mediated by a natural transformation process that required cell-cell contact and was completely sensitive to DNase I (1 mg/ml). Efficient transformation was also observed when pRA2 DNA was applied directly onto the cells, while transformation with foreign plasmid DNAs was not observed. pRA2 could be conjugally transferred into Pseudomonas putida RA713 and KT2440 recipients only when plasmid RK2/RP4 transfer functions were provided in trans. Plasmid stability analysis demonstrated that pRA2 could be stably maintained in its original host, P. alcaligenes NCIB 9867, as well as in P. putida RA713 after 100 generations of nonselective growth. Disruption of the pRA2 pac25I restriction endonuclease gene did not alter plasmid stability, while the pRA2 minireplicon exhibited only partial stability. This indicates that other pRA2-encoded determinants could have significant roles in influencing plasmid stability.

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

    NASA Astrophysics Data System (ADS)

    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).

  16. Phage type and DNA plasmid profile of Salmonella typhimurium isolates in the area of Isernia, Italy.

    PubMed Central

    Fantasia, M.; Ricci, N.; Manuppella, A.; Martini, A.; Filetici, E.; Laurelli, T.

    1990-01-01

    Thirty-eight Salmonella typhimurium strains isolated from December 1987 to March 1988 in Isernia, Central Italy, were characterized on the basis of their phage type, resistance to antimicrobials and plasmid profiles. According to their phage types, the isolates could be assigned to one of six groups, the prevalent one being PT 195 which accounted for 73.6% of isolates. On the basis of their plasmid content, the isolates could be assigned to one of ten groups. The prevalent plasmid profile (60.0; 6.0; 4.3; 4.0; 3.2 megadaltons) was found in 60.4% of isolates. All the isolates from a particular food (salsicce), and as most of isolates from humans who had consumed this food belonged to phage type 195 and were of the same plasmid profile. The combined use of phage typing and DNA plasmid analysis proved to be a useful tool in identifying epidemiologically related isolates in this investigation. Images Fig. 2 PMID:2209736

  17. Construction of a shuttle vector and transformation of Xylella fastidiosa with plasmid DNA.

    PubMed

    Qin, X; Hartung, J S

    2001-09-01

    We have isolated, cloned, and sequenced a 5823-bp cryptic plasmid from a strain of Xylella fastidiosa. This plasmid encodes five open reading frames (ORF) greater than 400 nucleotides each. ORF 2 encodes a protein with 37% amino acid identity to the replication initiator protein of plasmid pECB2 from Pseudomonas alcaligenes. This RepA protein from X. fastidiosa contains both a leucine zipper and helix turn helix motif characteristic of proteins involved in DNA replication. The sequence 5' of ORF 2 has all of the features characteristic of plasmid origins of replication as well as regulatory elements required for transcription of ORF 2. Open reading frame 2, along with the upstream origin of replication, was cloned as an EcoRI fragment into pUC19 to create a shuttle vector. This construct was introduced into Xylella fastidiosa by electroporation, with selection for carbenicillin resistance. Transformation was verified by both PCR and Southern hybridization experiments. Frequency of transformation was low, but increased ten-fold when the plasmid was grown in X. fastidiosa rather than Escherichia coli prior to transformation. This work represents the first step towards the development of a system for genetic analysis of this important plant pathogen of citrus, grapevines, and other horticultural crops.

  18. Model System for DNA Replication of a Plasmid DNA Containing the Autonomously Replicating Sequence from Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Ishimi, Yukio; Matsumoto, Ken

    1993-06-01

    A negatively supercoiled plasmid DNA containing autonomously replicating sequence (ARS) 1 from Saccharomyces cerevisiae was replicated with the proteins required for simian virus 40 DNA replication. The proteins included simian virus 40 large tumor antigen as a DNA helicase, DNA polymerase α^\\cdotprimase, and the multisubunit human single-stranded DNA-binding protein from HeLa cells; DNA gyrase from Escherichia coli, which relaxes positive but not negative supercoils, was included as a "swivelase." DNA replication started from the ARS region, proceeded bidirectionally with the synthesis of leading and lagging strands, and resulted in the synthesis of up to 10% of the input DNA in 1 h. The addition of HeLa DNA topoisomerase I, which relaxes both positive and negative supercoils, to this system inhibited DNA replication, suggesting that negative supercoiling of the template DNA is required for initiation. These results suggest that DNA replication starts from the ARS region where the DNA duplex is unwound by torsional stress; this unwound region can be recognized by a DNA helicase with the assistance of the multisubunit human single-stranded DNA-binding protein.

  19. 99mTc-labeled HYNIC-DAPI causes plasmid DNA damage with high efficiency.

    PubMed

    Kotzerke, Joerg; Punzet, Robert; Runge, Roswitha; Ferl, Sandra; Oehme, Liane; Wunderlich, Gerd; Freudenberg, Robert

    2014-01-01

    (99m)Tc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, (99m)Tc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a (99m)Tc-labeled HYNIC-DAPI compound with that of (99m)Tc pertechnetate ((99m)TcO4(-)). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03) was twice that caused by (99m)TcO4(-) (0.51), and the number of DSBs increased fivefold in the (99m)Tc-HYNIC-DAPI-treated sample compared with the (99m)TcO4(-) treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the (99m)TcO4(-) treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the (99m)Tc-HYNIC-DAPI-treated samples. These results indicated that (99m)Tc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the (99m)Tc-labeled compound with DNA. In contrast to these results, (99m)TcO4(-) induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of (99m)Tc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by

  20. 99mTc-Labeled HYNIC-DAPI Causes Plasmid DNA Damage with High Efficiency

    PubMed Central

    Kotzerke, Joerg; Punzet, Robert; Runge, Roswitha; Ferl, Sandra; Oehme, Liane; Wunderlich, Gerd; Freudenberg, Robert

    2014-01-01

    99mTc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, 99mTc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a 99mTc-labeled HYNIC-DAPI compound with that of 99mTc pertechnetate (99mTcO4−). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03) was twice that caused by 99mTcO4− (0.51), and the number of DSBs increased fivefold in the 99mTc-HYNIC-DAPI-treated sample compared with the 99mTcO4− treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the 99mTcO4– treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the 99mTc-HYNIC-DAPI-treated samples. These results indicated that 99mTc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the 99mTc-labeled compound with DNA. In contrast to these results, 99mTcO4− induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of 99mTc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by approximately 10-fold in terms of

  1. Plasmid DNA replication and topology as visualized by two-dimensional agarose gel electrophoresis.

    PubMed

    Schvartzman, J B; Martínez-Robles, M L; Hernández, P; Krimer, D B

    2010-01-01

    During the last 20 years, two-dimensional agarose gel electrophoresis combined with other techniques such as Polymerase Chain Reaction, helicase assay and electron microscopy, helped to characterize plasmid DNA replication and topology. Here we describe some of the most important findings that were made using this method including the characterization of uni-directional replication, replication origin interference, DNA breakage at the forks, replication fork blockage, replication knotting, replication fork reversal, the interplay of supercoiling and catenation and other changes in DNA topology that take place as replication progresses.

  2. Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

    PubMed

    Fantoni, A; Bozzoni, I; Ullu, E; Farace, M G

    1979-08-10

    Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z.

  3. Reduction of electron deficient guanine radical species in plasmid DNA by tyrosine derivatives.

    PubMed

    Tsoi, Mandi; Do, Trinh T; Tang, Vicky J; Aguilera, Joseph A; Milligan, Jamie R

    2010-06-07

    Guanine bases are the most easily oxidized sites in DNA and therefore electron deficient guanine radical species are major intermediates in the direct effect of ionizing radiation (ionization of the DNA itself) on DNA as a consequence of hole migration to guanine. As a model for this process we have used gamma-irradiation in the presence of thiocyanate ions to generate single electron oxidized guanine radicals in a plasmid target in aqueous solution. The stable species formed from these radicals can be detected and quantified by the formation of strand breaks in the plasmid after a post-irradiation incubation using a suitable enzyme. If a tyrosine derivative is also present during irradiation, the production of guanine oxidation products is decreased by electron transfer from tyrosine to the intermediate guanyl radical species. By using cationic tyrosine containing ligands we are able to observe this process when the tyrosine is electrostatically bound to the plasmid. The driving force dependence of this reaction was determined by comparing the reactivity of tyrosine with its 3-nitro analog. The results imply that the electron transfer reaction is coupled to a proton transfer. The experimental conditions used in this model system provide a reasonable approximation to those involved in the radioprotection of DNA by tightly bound proteins in chromatin.

  4. Formation of AAV single stranded DNA genome from a circular plasmid in Saccharomyces cerevisiae.

    PubMed

    Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

    2011-01-01

    Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3(+) clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway.

  5. Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae

    PubMed Central

    Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

    2011-01-01

    Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3+ clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway. PMID:21853137

  6. Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

    SciTech Connect

    Chowdhury, E.H.

    2011-06-17

    Highlights: {yields} Cytoplasmic stability of plasmid DNA is enhanced by fluoride incorporation into carbonate apatite carrier. {yields} Fluoridated carbonate apatite promotes a robust increase in transgene expression. {yields} Controlled dissolution of fluoridated carbonate apatite in endosomal acidic environment might buffer the endosomes and prevent degradation of the released DNA. -- Abstract: Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.

  7. Hypermutable ligation of plasmid DNA ends in cells from patients with Werner syndrome.

    PubMed

    Rünger, T M; Bauer, C; Dekant, B; Möller, K; Sobotta, P; Czerny, C; Poot, M; Martin, G M

    1994-01-01

    Werner Syndrome is a rare autosomal recessive disorder characterized by an increased cancer risk and by symptoms suggestive of premature aging. Cells from these patients demonstrate a typical pattern of chromosomal instability and a spontaneous hypermutability with a high rate of unusually large deletions. We have studied the in vivo DNA ligation in three lymphoblast cell lines from Werner syndrome patients and three from normal donors. In our host cell ligation assay we transfected linearized plasmid pZ189 and measured the amount of plasmid DNA ends rejoined by these host cells as the ability of the recovered plasmid to transform bacteria. A mutagenesis marker gene close to the ligation site allowed screening for mutations. Subsequent mutation analysis provided information about the accuracy of the ligation process. The cells from Werner syndrome patients were as effective as normal cells in ligating DNA ends. However, mutation analysis revealed that the three Werner syndrome cell lines introduced 2.4-4.6 times more mutations (p < 0.001) than the normal cell lines during ligation of the DNA ends: the mutation rates were 69.4, 97.2, and 58.7%, as compared to 23.6, 21.7, and 24.4% in the normal cell lines. These increased mutation frequencies in plasmids ligated during passage through Werner syndrome cells were mainly due to a significant (p < 0.001) increase in deletions. This error-prone DNA ligation might be responsible for the spontaneous hypermutability and the genomic instability in Werner syndrome cells and related to the apparently accelerated aging and high cancer risk in affected patients.

  8. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation.

    PubMed

    Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J; Fox, Catherine A

    2016-04-07

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

  9. Dichromatic laser radiation effects on DNA of Escherichia coli and plasmids

    NASA Astrophysics Data System (ADS)

    Martins, W. A.; Polignano, G. A. C.; Guimarães, O. R.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2015-04-01

    Dichromatic and consecutive laser radiations have attracted increased attention for clinical applications as offering new tools for the treatment of dysfunctional tissues in situations where monochromatic radiation is not effective. This work evaluated the survival, filamentation and morphology of Escherichia coli cells, and the induction of DNA lesions, in plasmid DNA exposed to low-intensity consecutive dichromatic laser radiation. Exponential and stationary wild type and formamidopyrimidine DNA glycosylase/MutM protein deficient E. coli cultures were exposed to consecutive low-intensity dichromatic laser radiation (infrared laser immediately after red laser) to study the survival, filamentation and morphology of bacterial cells. Plasmid DNA samples were exposed to dichromatic radiation to study DNA lesions by electrophoretic profile. Dichromatic laser radiation affects the survival, filamentation and morphology of E. coli cultures depending on the growth phase and the functional repair mechanism of oxidizing lesions in DNA, but does not induce single/double strands breaks or alkali-labile DNA lesions. Results show that low-intensity consecutive dichromatic laser radiation induces biological effects that differ from those induced by monochromatic laser radiation, suggesting that other therapeutic effects could be obtained using dichromatic radiation.

  10. Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

    PubMed Central

    Coelho-Castelo, AAM; Trombone, AP; Rosada, RS; Santos, RR; Bonato, VLD; Sartori, A; Silva, CL

    2006-01-01

    In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system. PMID:16445866

  11. Simple and efficient oligonucleotide-directed mutagenesis using one primer and circular plasmid DNA template.

    PubMed

    Marotti, K R; Tomich, C S

    1989-01-01

    A rapid and simple procedure for site-directed mutagenesis is described. This method uses only a single oligonucleotide primer with the double-stranded circular plasmid DNA as the template for mutagenesis. The phage T4 gene 32 product is included during primer extension in vitro to increase efficiency. Single and multiple changes as well as deletions have been obtained at an efficiency of 1-2%.

  12. "Curing" of plasmid DNA in acetogen using microwave or applying an electric pulse improves cell growth and metabolite production as compared to the plasmid-harboring strain.

    PubMed

    Berzin, Vel; Kiriukhin, Michael; Tyurin, Michael

    2013-03-01

    Plasmid-free acetogen Clostridium sp. MT962 electrotransformed with a small cryptic plasmid pMT351 was used to develop time- and cost-effective methods for plasmid elimination. Elimination of pMT351 restored production of acetate and ethanol to the levels of the plasmid-free strain with no dry cell weight changes. Destabilizing cell membrane via microwave at 2.45 GHz, or exposure to a single 12 ms square electric pulse at 35 kV cm⁻¹, eliminated pMT351 in 42-47 % of cells. Plasmid elimination with a single square electric pulse required 10 versus 0.1 J needed to introduce the same 3,202-bp plasmid into the cells as calculated per cell sample of Clostridium sp. MT962. Microwave caused visible changes in repPCR pattern and increased ethanol production at the expense of acetate. This is the first report on microwave of microwave ovens, wireless routers, and mobile devices causing chromosomal DNA aberrations in microbes along with carbon flux change.

  13. Dual recombinant Lactococcus lactis for enhanced delivery of DNA vaccine reporter plasmid pPERDBY.

    PubMed

    Yagnik, Bhrugu; Sharma, Drashya; Padh, Harish; Desai, Priti

    2017-03-04

    Food grade Lactococcus lactis (L. lactis) has been widely used as an antigen and DNA delivery vehicle. We had previously reported the use of non-invasive L. lactis for the delivery of newly constructed immunostimulatory DNA vaccine reporter plasmid, pPERDBY. In the present report, we outline the construction of dual recombinant L. lactis expressing Internalin A of Listeria monocytogenes and harbouring pPERDBY (LL InlA+ pPERDBY) to enhance the DNA delivery efficiency of L. lactis. After confirmation and validation of LL InlA+ pPERDBY, its DNA delivery potential was compared with previously developed non-invasive r- L. lactis::pPERDBY. The use of invasive L. lactis resulted in around three fold increase in number of Enhanced Green Fluorescent Protein expressing Caco- cells. Thus, these findings reinforce the prospective application of invasive strain of L. lactis in delivery of DNA/RNA and antigens.

  14. A comparison of plasmid DNA delivery efficiency and cytotoxicity of two cationic diblock polyoxazoline copolymers.

    PubMed

    Lehner, Roman; Liu, Kegang; Wang, Xueya; Wolf, Marc; Hunziker, Patrick

    2017-04-28

    Cationic polymers as non-viral gene delivery carriers are widely used because of their strong condensing properties and long-term safety, but acute cytotoxicity is a persistent challenge. In this study, two types of polyplexes were prepared by co-formulating plasmid DNA and two cationic diblock copolymers PABOXA5-b-PMOXA33-PA (primary amine) and PABOXA5-b-PMOXA33-TA (tertiary amine) to check their transfection efficacies in HeLa cells and HEK293T cells, respectively. The plasmid DNA/PABOXA5-b-PMOXA33-PA polyplex showed higher transfection efficacy compared to the plasmid DNA/PABOXA5-b-PMOXA33-TA polyplex under an N/P ratio of 40. Both polymers exhibited low toxicity, attributed to the shielding effect of a hydrophilic, noncharged block. Mechanistic insight into differential transfection efficiencies of the polymers were gained by visualization and comparison of the condensates via transmission electron and atomic force microscopy. The results provide information suited for further structure optimization of polymers that are aimed for targeted gene delivery.

  15. Enantioselective cleavage of supercoiled plasmid DNA catalyzed by chiral macrocyclic lanthanide(III) complexes.

    PubMed

    Krężel, Artur; Lisowski, Jerzy

    2012-02-01

    The enantiomers of the Sm (III), Eu (III) and Yb (III) complexes [LnL(NO(3))(2)](NO(3)) of a chiral hexaazamacrocycle were tested as catalysts for the hydrolytic cleavage of supercoiled plasmid DNA. The catalytic activity was remarkably enantioselective; while the [LnL(SSSS)(NO(3))(2)](NO(3)) enantiomers promoted the cleavage of plasmid pBR322 from the supercoiled form (SC) to the nicked form (NC), the [LnL(RRRR)(NO(3))(2)](NO(3)) enantiomers were inactive. Kinetics of plasmid DNA hydrolysis was also investigated by agarose electrophoresis and it indicated typical single-exponential cleavage reaction. The hydrolytic mechanism of DNA cleavage was confirmed by the successful ligation of hydrolysis product by T4 ligase. The NMR study of the solutions of the complexes in various buffers indicated that the complexes exist as monomeric cationic complexes [LnL(H(2)O)(3)](3+) in slightly acidic solutions and as dimeric cationic complexes [Ln(2)L(2)(μ-OH)(2)(H(2)O)(2)](4+) in slightly basic 8mM solutions, with the latter form being a possible catalyst for hydrolysis of phosphodiester bonds.

  16. Copper(II) facilitates bleomycin-mediated unwinding of plasmid DNA

    SciTech Connect

    Levy, M.J.; Hecht, S.M.

    1988-04-19

    The unwinding of plasmid DNA by bleomycin A/sub 2/ (BLM A/sub 2/) was investigated by use of two-dimensional gel electrophoresis. It was found that Cu/sup 2 +/ ions greatly facilitated the unwinding of topoisomers of plasmid DNA by BLM A/sub 2/ at concentrations where cupric ions along had no effect on DNA supercoiling. The concentration of BLM A/sub 2/ required for observable unwinding was reduced at least 100-fold in the presence of equimolar Cu/sup 2 +/. A plot of (Cu/sup 2 +/) vs extent of DNA unwinding in the presence of 10/sup -4/ M BLM A/sub 2/ gave a curve consistent with the action of cupric ions on BLM in an allosteric fashion, possibly rearranging the drug into a conformation that facilitates DNA unwinding. The participation of the metal center in enhancing DNA unwinding via direct ionic interaction with one or more negatively charged groups on the DNA duplex also seems possible. Further analysis of the structural factors required for BLM-mediated DNA unwinding was carried out with Cu/sup 2 +/ + BLM demethyl A/sub 2/, the latter of which differs from BLM A/sub 2/ only in that it lacks a methyl group, and associated positive charge, at the C-terminus. Cu(II) x BLM demethyl A/sub 2/ was found to be much less effective than Cu(II) x BLM A/sub 2/ as a DNA unwinding agent, emphasizing the strong dependence of this process on the presence of positively charged groups within the BLM molecule. These findings constitute the first direct evidence that the metal center of BLM can participate in DNA interaction, as well as in the previously recognized role of oxygen binding and activation.

  17. Process considerations related to the microencapsulation of plasmid DNA via ultrasonic atomization.

    PubMed

    Ho, Jenny; Wang, Huanting; Forde, Gareth M

    2008-09-01

    An effective means of facilitating DNA vaccine delivery to antigen presenting cells is through biodegradable microspheres. Microspheres offer distinct advantages over other delivery technologies by providing release of DNA vaccine in its bioactive form in a controlled fashion. In this study, biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres containing polyethylenimine (PEI) condensed plasmid DNA (pDNA) were prepared using a 40 kHz ultrasonic atomization system. Process synthesis parameters, which are important to the scale-up of microspheres that are suitable for nasal delivery (i.e., less than 20 microm), were studied. These parameters include polymer concentration; feed flowrate; volumetric ratio of polymer and pDNA-PEI (plasmid DNA-polyethylenimine) complexes; and nitrogen to phosphorous (N/P) ratio. PDNA encapsulation efficiencies were predominantly in the range 82-96%, and the mean sizes of the particle were between 6 and 15 microm. The ultrasonic synthesis method was shown to have excellent reproducibility. PEI affected morphology of the microspheres, as it induced the formation of porous particles that accelerate the release rate of pDNA. The PLGA microspheres displayed an in vitro release of pDNA of 95-99% within 30 days and demonstrated zero order release kinetics without an initial spike of pDNA. Agarose electrophoresis confirmed conservation of the supercoiled form of pDNA throughout the synthesis and in vitro release stages. It was concluded that ultrasonic atomization is an efficient technique to overcome the key obstacles in scaling-up the manufacture of encapsulated vaccine for clinical trials and ultimately, commercial applications.

  18. Efficient plasmid DNA cleavage by a mononuclear copper(II) complex.

    PubMed

    Sissi, Claudia; Mancin, Fabrizio; Gatos, Maddalena; Palumbo, Manlio; Tecilla, Paolo; Tonellato, Umberto

    2005-04-04

    The Cu(II) complex of the ligand all-cis-2,4,6-triamino-1,3,5-trihydroxycyclohexane (TACI) is a very efficient catalyst of the cleavage of plasmid DNA in the absence of any added cofactor. The maximum rate of degradation of the supercoiled plasmid DNA form, obtained at pH 8.1 and 37 degrees C, in the presence of 48 microM TACI.Cu(II), is 2.3 x 10(-3) s(-1), corresponding to a half-life time of only 5 min for the cleavage of form I (supercoiled) to form II (relaxed circular). The dependence of the rate of plasmid DNA cleavage from the TACI.Cu(II) complex concentration follows an unusual and very narrow bell-like profile, which suggests an high DNA affinity of the complexes but also a great tendency to form unreactive dimers. The reactivity of the TACI.Cu(II) complexes is not affected by the presence of several scavengers for reactive oxygen species or when measured under anaerobic conditions. Moreover, no degradation of the radical reporter Rhodamine B is observed in the presence of such complexes. These results are consistent with the operation of a prevailing hydrolytic pathway under the normal conditions used, although the failure to obtain enzymatic religation of the linearized DNA does not allow one to rule out the occurrence of a nonhydrolytic oxygen-independent cleavage. A concurrent oxidative mechanism becomes competitive upon addition of reductants or in the presence of high levels of molecular oxygen: under such conditions, in fact, a remarkable increase in the rate of DNA cleavage is observed.

  19. Structural biology of plasmid partition: uncovering the molecular mechanisms of DNA segregation.

    PubMed

    Schumacher, Maria A

    2008-05-15

    DNA segregation or partition is an essential process that ensures stable genome transmission. In prokaryotes, partition is best understood for plasmids, which serve as tractable model systems to study the mechanistic underpinnings of DNA segregation at a detailed atomic level owing to their simplicity. Specifically, plasmid partition requires only three elements: a centromere-like DNA site and two proteins: a motor protein, generally an ATPase, and a centromere-binding protein. In the first step of the partition process, multiple centromere-binding proteins bind co-operatively to the centromere, which typically consists of several tandem repeats, to form a higher-order nucleoprotein complex called the partition complex. The partition complex recruits the ATPase to form the segrosome and somehow activates the ATPase for DNA separation. Two major families of plasmid par systems have been delineated based on whether they utilize ATPase proteins with deviant Walker-type motifs or actin-like folds. In contrast, the centromere-binding proteins show little sequence homology even within a given family. Recent structural studies, however, have revealed that these centromere-binding proteins appear to belong to one of two major structural groups: those that employ helix-turn-helix DNA-binding motifs or those with ribbon-helix-helix DNA-binding domains. The first structure of a higher-order partition complex was recently revealed by the structure of pSK41 centromere-binding protein, ParR, bound to its centromere site. This structure showed that multiple ParR ribbon-helix-helix motifs bind symmetrically to the tandem centromere repeats to form a large superhelical structure with dimensions suitable for capture of the filaments formed by the actinlike ATPases. Surprisingly, recent data indicate that the deviant Walker ATPase proteins also form polymer-like structures, suggesting that, although the par families harbour what initially appeared to be structurally and functionally

  20. Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies.

    PubMed

    Rombouts, K; Braeckmans, K; Remaut, K

    2016-02-17

    Live-cell imaging has provided the life sciences with insights into the cell biology and dynamics. Fluorescent labeling of target molecules proves to be indispensable in this regard. In this Review, we focus on the current fluorescent labeling strategies for nucleic acids, and in particular mRNA (mRNA) and plasmid DNA (pDNA), which are of interest to a broad range of scientific fields. By giving a background of the available techniques and an evaluation of the pros and cons, we try to supply scientists with all the information needed to come to an informed choice of nucleic acid labeling strategy aimed at their particular needs.

  1. Implementation of an Automated High-Throughput Plasmid DNA Production Pipeline.

    PubMed

    Billeci, Karen; Suh, Christopher; Di Ioia, Tina; Singh, Lovejit; Abraham, Ryan; Baldwin, Anne; Monteclaro, Stephen

    2016-12-01

    Biologics sample management facilities are often responsible for a diversity of large-molecule reagent types, such as DNA, RNAi, and protein libraries. Historically, the management of large molecules was dispersed into multiple laboratories. As methodologies to support pathway discovery, antibody discovery, and protein production have become high throughput, the implementation of automation and centralized inventory management tools has become important. To this end, to improve sample tracking, throughput, and accuracy, we have implemented a module-based automation system integrated into inventory management software using multiple platforms (Hamilton, Hudson, Dynamic Devices, and Brooks). Here we describe the implementation of these systems with a focus on high-throughput plasmid DNA production management.

  2. The mechanism and control of DNA transfer by the conjugative relaxase of resistance plasmid pCU1

    SciTech Connect

    Nash, Rebekah Potts; Habibi, Sohrab; Cheng, Yuan; Lujan, Scott A.; Redinbo, Matthew

    2010-11-15

    Bacteria expand their genetic diversity, spread antibiotic resistance genes, and obtain virulence factors through the highly coordinated process of conjugative plasmid transfer (CPT). A plasmid-encoded relaxase enzyme initiates and terminates CPT by nicking and religating the transferred plasmid in a sequence-specific manner. We solved the 2.3 {angstrom} crystal structure of the relaxase responsible for the spread of the resistance plasmid pCU1 and determined its DNA binding and nicking capabilities. The overall fold of the pCU1 relaxase is similar to that of the F plasmid and plasmid R388 relaxases. However, in the pCU1 structure, the conserved tyrosine residues (Y18,19,26,27) that are required for DNA nicking and religation were displaced up to 14 {angstrom} out of the relaxase active site, revealing a high degree of mobility in this region of the enzyme. In spite of this flexibility, the tyrosines still cleaved the nic site of the plasmid's origin of transfer, and did so in a sequence-specific, metal-dependent manner. Unexpectedly, the pCU1 relaxase lacked the sequence-specific DNA binding previously reported for the homologous F and R388 relaxase enzymes, despite its high sequence and structural similarity with both proteins. In summary, our work outlines novel structural and functional aspects of the relaxase-mediated conjugative transfer of plasmid pCU1.

  3. Photoinduced interactions of supramolecular ruthenium(II) complexes with plasmid DNA: synthesis and spectroscopic, electrochemical, and DNA photocleavage studies.

    PubMed

    Swavey, Shawn; DeBeer, Madeleine; Li, Kaiyu

    2015-04-06

    Two new bridging ligands have been synthesized by combining substituted benzaldehydes with phenanthrolinopyrrole (php), resulting in new polyazine bridging ligands. The ligands have been characterized by (1)H NMR, mass spectroscopy, and elemental analysis. These new ligands display π-π* transitions above 500 nm with modest molar absorptivities. Upon excitation at the ligand-centered charge-transfer transition, weak emission with a maximum wavelength of 612 nm is observed. When coordinated to two ruthenium(II) bis(bipyridyl) groups, the new bimetallic complexes generated give an overall 4+ charge. The electronic transitions of the bimetallic ruthenium(II) complexes display traditional π-π* transitions at 287 nm and metal-to-ligand charge-transfer transitions at 452 nm with molar absorptivities greater than 30000 M(-1) cm(-1). Oxidation of the ruthenium(II) metal centers to ruthenium(III) occurs at potentials above 1.4 V versus the Ag/AgCl reference electrode. Spectroscopic and electrochemical measurements indicate that the ruthenium(II) moieties behave independently. Both complexes are water-soluble and show the ability to photonick plasmid DNA when irradiated with low-energy light above 550 nm. In addition, one of the complexes, [Ru(bpy)2php]2Van(4+), shows the ability to linearize plasmid DNA and gives evidence, by gel electrophoresis, of photoinduced binding to plasmid DNA.

  4. Poly(hydroxyethyl methacrylate) based affinity cryogel for plasmid DNA purification.

    PubMed

    Perçin, Işık; Sağlar, Emel; Yavuz, Handan; Aksöz, Erol; Denizli, Adil

    2011-05-01

    The aim of this study is to prepare supermacroporous pseudospecific cryogel which can be used for the purification of plasmid DNA (pDNA) from bacterial lysate. N-methacryloyl-(l)-histidine methyl ester (MAH) was chosen as the pseudospecific ligand and/or comonomer. Poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-histidine methyl ester) [PHEMAH] cryogel was produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. Compared with the PHEMA cryogel (50 μg/g polymer), the pDNA adsorption capacity of the PHEMAH cryogel (13,350 μg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The amount of pDNA bound onto the PHEMAH cryogel disks first increased and then reached a saturation value (i.e., 13,350μg/g) at around 300 μg/ml pDNA concentration. pDNA adsorption amount decreased from 1137 μg/g to 160 μg/g with the increasing NaCl concentration. The maximum pDNA adsorption was achieved at 25 °C. The overall recovery of pDNA was calculated as 90%. The PHEMAH cryogel could be used 3 times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH cryogel disks promise high selectivity for pDNA.

  5. Interleukin-12 plasmid DNA delivery using l-thyroxine-conjugated polyethylenimine nanocarriers

    NASA Astrophysics Data System (ADS)

    Dehshahri, Ali; Sadeghpour, Hossein; Kazemi Oskuee, Reza; Fadaei, Mahin; Sabahi, Zahra; Alhashemi, Samira Hossaini; Mohazabieh, Erfaneh

    2014-05-01

    In this study, l-thyroxine was covalently grafted on 25 kDa branched polyethylenimine (PEI), and the ability of the nano-sized polyplexes for transferring plasmid encoding interleukin-12 (IL-12) gene was evaluated. As there are several problems in systemic administration of recombinant IL-12 protein, local expression of the plasmid encoding IL-12 gene inside the tumor tissue has been considered as an effective alternative approach. The l-thyroxine-conjugated PEI polyplexes were prepared using pUMVC3-hIL12 plasmid, and their transfection activity was determined in HepG2 human liver carcinoma and Neuro2A neuroblastoma cell lines. The polyplexes characterized in terms of DNA condensation ability, particle size, zeta potential, and buffering capacity as well as cytotoxicity and resistance to enzyme digestion. The results revealed that l-thyroxine conjugation of PEI increased gene transfer ability by up to two fold relative to unmodified 25 kDa PEI, the gold standard for non-viral gene delivery, with the highest increase occurring at degrees of conjugation around 10 %. pDNA condensation tests and dynamic light scattering measurements exhibited the ability of PEI conjugates to optimally condense the plasmid DNA into polyplexes in the size range around 200 nm. The modified polymers showed remarkable buffering capacity and protection against enzymatic degradation comparable to that of unmodified PEI. These results suggest that l-thyroxine conjugation of PEI is a simple modification strategy for future investigations aimed at developing a targeting gene vehicle.

  6. Multiple factors affect immunogenicity of DNA plasmid HIV vaccines in human clinical trials.

    PubMed

    Jin, Xia; Morgan, Cecilia; Yu, Xuesong; DeRosa, Stephen; Tomaras, Georgia D; Montefiori, David C; Kublin, James; Corey, Larry; Keefer, Michael C

    2015-05-11

    Plasmid DNA vaccines have been licensed for use in domesticated animals because of their excellent immunogenicity, but none have yet been licensed for use in humans. Here we report a retrospective analysis of 1218 healthy human volunteers enrolled in 10 phase I clinical trials in which DNA plasmids encoding HIV antigens were administered. Elicited T-cell immune responses were quantified by validated intracellular cytokine staining (ICS) stimulated with HIV peptide pools. HIV-specific binding and neutralizing antibody activities were also analyzed using validated assays. Results showed that, in the absence of adjuvants and boosting with alternative vaccines, DNA vaccines elicited CD8+ and CD4+ T-cell responses in an average of 13.3% (95% CI: 9.8-17.8%) and 37.7% (95% CI: 31.9-43.8%) of vaccine recipients, respectively. Three vaccinations (vs. 2) improved the proportion of subjects with antigen-specific CD8+ responses (p=0.02), as did increased DNA dosage (p=0.007). Furthermore, female gender and participants having a lower body mass index were independently associated with higher CD4+ T-cell response rate (p=0.001 and p=0.008, respectively). These vaccines elicited minimal neutralizing and binding antibody responses. These findings of the immunogenicity of HIV DNA vaccines in humans can provide guidance for future clinical trials.

  7. Antibacterial effect of cationic porphyrazines and anionic phthalocyanine and their interaction with plasmid DNA

    NASA Astrophysics Data System (ADS)

    Hassani, Leila; Hakimian, Fatemeh; Safaei, Elham; Fazeli, Zahra

    2013-11-01

    Resistance to antibiotics is a public health issue and identification of new antibacterial agents is one of the most important goals of pharmacological research. Among the novel developed antibacterial agents, porphyrin complexes and their derivatives are ideal candidates for use in medical applications. Phthalocyanines differ from porphyrins by having nitrogen atoms link the individual pyrrol units. The aza analogues of the phthalocyanines (azaPcs) such as tetramethylmetalloporphyrazines are heterocyclic Pc analogues. In this investigation, interaction of an anionic phthalocyanine (Cu(PcTs)) and two cationic tetrapyridinoporphyrazines including [Cu(2,3-tmtppa)]4+ and [Cu(3,4-tmtppa)]4+ complexes with plasmid DNA was studied using spectroscopic and gel electrophoresis methods. In addition, antibacterial effect of the complexes against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria was investigated using dilution test method. The results indicated that both porphyrazines have significant antibacterial properties, but Cu(PcTs) has weak antibacterial effect. Compairing the binding of the phthalocyanine and the porphyrazines to DNA demonstrated that the interaction of cationic porphyrazines is stronger than the anionic phthalocyanine remarkably. The extent of hypochromicity and red shift of absorption spectra indicated preferential intercalation of the two porphyrazine into the base pairs of DNA helix. Gel electrophoresis result implied Cu(2,3-tmtppa) and Cu(3,4-tmtppa) are able to perform cleavage of the plasmid DNA. Consequently, DNA binding and cleavage might be one of the antibacterial mechanisms of the complexes.

  8. High-Voltage Electroporation of Bacteria: Genetic Transformation of Campylobacter jejuni with Plasmid DNA

    NASA Astrophysics Data System (ADS)

    Miller, Jeff F.; Dower, William J.; Tompkins, Lucy S.

    1988-02-01

    Electroporation permits the uptake of DNA by mammalian cells and plant protoplasts because it induces transient permeability of the cell membrane. We investigated the utility of high-voltage electroporation as a method for genetic transformation of intact bacterial cells by using the enteric pathogen Campylobacter jejuni as a model system. This report demonstrates that the application of high-voltage discharges to bacterial cells permits genetic transformation. Our method involves exposure of a Campylobacter cell suspension to a high-voltage exponential decay discharge (5-13 kV/cm) for a brief period of time (resistance-capacitance time constant = 2.4-26 msec) in the presence of plasmid DNA. Electrical transformation of C. jejuni results in frequencies as high as 1.2 × 106 transformants per μ g of DNA. We have investigated the effects of pulse amplitude and duration, cell growth conditions, divalent cations, and DNA concentration on the efficiency of transformation. Transformants of C. jejuni obtained by electroporation contained structurally intact plasmid molecules. In addition, evidence is presented that indicates that C. jejuni possesses DNA restriction and modification systems. The use of electroporation as a method for transforming other bacterial species and guidelines for its implementation are also discussed.

  9. Low energy electron induced damage to plasmid DNA pQE30

    SciTech Connect

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

    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.

  10. Low energy electron induced damage to plasmid DNA pQE30

    NASA Astrophysics Data System (ADS)

    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.

  11. Development of safe and effective nonviral gene therapy by eliminating CpG motifs from plasmid DNA vector.

    PubMed

    Takahashi, Yuki; Nishikawa, Makiya; Takakura, Yoshinobu

    2012-01-01

    Nonviral gene therapy is expected to become a regular treatment for a variety of difficult-to-treat diseases, such as cancer and virus infection. Plasmid DNA, which is used in most nonviral gene delivery systems, usually contains, unmethylated cytosine-guanine dinucleotides, so called CpG motifs. CpG motifs are recognized by immune cells as a danger signal, leading to an inflammatory response. Such inflammatory responses could affect the safety and effectiveness of nonviral gene therapy. Therefore, reducing the number of CpG motifs in plasmid DNA has been used to increase the potency of plasmid DNA-based gene therapy. Previous studies have demonstrated that CpG reduction can extend the time period of transgene expression from plasmid DNA after in vivo gene transfer. In this review, the biological functions of the CpG motif are briefly summarized. Then, safety issues of nonviral gene therapy are discussed from the viewpoint of the inflammatory response to the CpG motif in plasmid DNA, and the effects of the CpG motif in plasmid DNA on the transgene expression profile of nonviral gene transfer are reviewed.

  12. From plasmids to protection: a review of DNA vaccines against infectious diseases.

    PubMed

    Laddy, Dominick J; Weiner, David B

    2006-01-01

    The field of DNA vaccine development began over 16 years ago with the observation that plasmid DNA could be injected into and expressed in vivo and drive adaptive immune responses. Since then, there has been great interest in developing this technology to create a new generation of vaccines with the ability to elicit both humoral and cellular immune responses from an inherently innocuous injection. However, DNA vaccines have yet to proceed past phase I/II clinical trials in humans--primarily due to a desire to induce more potent immune responses. This review will examine how DNA vaccines function to induce an immune response and how this information might be useful in future vaccine design.

  13. In vitro reporter gene transfection via plasmid DNA delivered by metered dose inhaler.

    PubMed

    Bains, Baljinder K; Birchall, James C; Toon, Richard; Taylor, Glyn

    2010-07-01

    Aerosolised DNA administration could potentially advance the treatment of inheritable lung diseases, lung malignancies and provide genetic immunisation against infection. Jet nebulisation, the current standard for introducing DNA formulations into the lung, is inherently inefficient. Pressurised metered dose inhalers (pMDIs) offer a potentially more efficacious and convenient alternative, especially for repeat administration. We aim to modify a novel low-energy nanotechnology process to prepare surfactant-coated pDNA nanoparticles for pulmonary gene delivery via a pMDI. Water-in-oil microemulsions containing green fluorescent protein reporter plasmid were snap-frozen and lyophilised. Lyophilised pDNA, in some cases following a surfactant wash, was incorporated into pMDIs with hydrofluoroalkane 134a (HFA134a) propellant and ethanol as cosolvent. To assess biological functionality, A549 human lung epithelial cells were exposed to aerosolised pDNA particles in the presence of dioleoyl-trimethylammonium propane (DOTAP). Transfection studies demonstrated that pDNA biological functionality was maintained following aerosolisation. In vitro toxicity assays (MTT) showed no significant cell viability loss following aerosolised pDNA treatment. We have demonstrated that pDNA particles can be incorporated into an HFA134a formulation and aerosolised using a standard valve and actuator. Particles prepared by this novel process have potential for stable and efficient delivery of pDNA to the lower respiratory tract via standard pMDI technology.

  14. Delivery of plasmid DNA to vascular tissue in vivo using catheter balloons coated with polyelectrolyte multilayers.

    PubMed

    Saurer, Eric M; Yamanouchi, Dai; Liu, Bo; Lynn, David M

    2011-01-01

    We report an approach for the localized delivery of plasmid DNA to vascular tissue from the surfaces of inflatable embolectomy catheter balloons. Using a layer-by-layer approach, ultrathin multilayered polyelectrolyte films were fabricated on embolectomy catheter balloons by alternately adsorbing layers of a hydrolytically degradable poly(β-amino ester) and plasmid DNA. Fluorescence microscopy revealed that the films coated the surfaces of the balloons uniformly. Coated balloons that were incubated in phosphate-buffered saline at 37 °C released ∼25 μg DNA/cm(2) over 24 h. Analysis of the DNA by gel electrophoresis showed that the DNA was released in open-circular ('nicked') and supercoiled conformations, and in vitro cell transfection assays confirmed that the released DNA was transcriptionally active. Arterial injury was induced in the left common, carotid arteries of Sprague-Dawley rats using uncoated balloons, followed by treatment with film-coated balloons for 20 min. X-gal, immunohistochemical, and immunofluorescence staining of sectioned arteries indicated high levels of β-galactosidase or enhanced green fluorescent protein (EGFP) expression in arteries treated with film-coated balloons. β-galactosidase and EGFP expression were observed throughout the medial layers of arterial tissue, and around approximately two-thirds of the circumference of the treated arteries. The layer-by-layer approach reported here provides a general platform for the balloon-mediated delivery of DNA to vascular tissue. Our results suggest the potential of this approach to deliver therapeutically relevant DNA to prevent complications such as intimal hyperplasia that arise after vascular interventions.

  15. Heavy ion induced damage to plasmid DNA: plateau region vs. spread out Bragg-peak

    NASA Astrophysics Data System (ADS)

    Dang, H. M.; van Goethem, M. J.; van der Graaf, E. R.; Brandenburg, S.; Hoekstra, R.; Schlathölter, T.

    2011-08-01

    We have investigated the damage of synthetic plasmid pBR322 DNA in dilute aqueous solutions induced by fast carbon ions. The relative contribution of indirect damage and direct damage to the DNA itself is expected to vary with linear energy transfer along the ion track, with the direct damage contribution increasing towards the Bragg peak. Therefore, 12C ions at the spread-out Bragg peak (dose averaged LET∞ = 189 ± 15 keV/ μm) and in the plateau region of the Bragg curve (LET = 40 keV/ μm) were employed and the radical scavenger concentration in the plasmid solution was varied to quantify the indirect effect. In order to minimize the influence of 12C break-up fragments, a relatively low initial energy of 90 MeV/nucleon was employed for the carbon ions. DNA damage has been quantified by subsequent electrophoresis on agarose gels. We find that strand breaks due to both indirect and direct effects are systematically higher in the plateau region as compared to the Bragg peak region with the difference being smallest at high scavenging capacities. In view of the fact that the relative biological effectiveness for many biological endpoints is maximum at the Bragg peak our findings imply that DNA damage at the Bragg peak is qualitatively most severe.

  16. Plasmid DNA damage by heavy ions at spread-out Bragg peak energies

    NASA Astrophysics Data System (ADS)

    Dang, H. M.; van Goethem, M. J.; van der Graaf, E. R.; Brandenburg, S.; Hoekstra, R.; Schlathölter, T.

    2010-10-01

    Interaction of ionizing radiation with plasmid DNA can lead to formation of single strand breaks, double strand breaks and clustered lesions. We have investigated the response of the synthetic plasmid pBR322 in aqueous solution upon irradiation with 12C ions under spread-out Bragg peak conditions (densely ionizing) and with 137Cs γ-photons (sparsely ionizing) as a function of dose. To evaluate the relevance of indirect effects, i.e. influences of diffusion limited radical induced DNA damage triggered by water radiolysis, the experiments were performed at various concentrations of the radical scavenger mannitol. Agarose gel electrophoresis was employed to quantify the DNA damage. At low scavenger concentration for a given dose DNA damage is higher for γ-photons than for 12C. For the latter, the microscopic dose distribution is inhomogeneous, with very high dose deposited along the few tracks through the solution. This is in agreement with the concept that scavengers efficiently reduce damage for γ-photons, implying that the underlying damage mechanism is single strand break induction by OH radicals. For 12C induced damage, the fraction of SSB and DSB that is unaffected by radical scavengers and thus due to direct effect is quantified.

  17. Using DNA origami nanostructures to determine absolute cross sections for UV photon-induced DNA strand breakage.

    PubMed

    Vogel, Stefanie; Rackwitz, Jenny; Schürman, Robin; Prinz, Julia; Milosavljević, Aleksandar R; Réfrégiers, Matthieu; Giuliani, Alexandre; Bald, Ilko

    2015-11-19

    We have characterized ultraviolet (UV) photon-induced DNA strand break processes by determination of absolute cross sections for photoabsorption and for sequence-specific DNA single strand breakage induced by photons in an energy range from 6.50 to 8.94 eV. These represent the lowest-energy photons able to induce DNA strand breaks. Oligonucleotide targets are immobilized on a UV transparent substrate in controlled quantities through attachment to DNA origami templates. Photon-induced dissociation of single DNA strands is visualized and quantified using atomic force microscopy. The obtained quantum yields for strand breakage vary between 0.06 and 0.5, indicating highly efficient DNA strand breakage by UV photons, which is clearly dependent on the photon energy. Above the ionization threshold strand breakage becomes clearly the dominant form of DNA radiation damage, which is then also dependent on the nucleotide sequence.

  18. Clinical optimization of antigen specific modulation of type 1 diabetes with the plasmid DNA platform.

    PubMed

    Gottlieb, Peter; Utz, Paul J; Robinson, William; Steinman, Lawrence

    2013-12-01

    Some clinical trials in humans have aimed at modulation of type 1 diabetes (T1D) via alteration of the immune response to putative islet cell antigens, particularly proinsulin and insulin, glutamic acid decarboxylase and the peptide, DiaPep 277, derived from heat shock protein 60. The focus here is on development of a specially engineered DNA plasmid encoding proinsulin to treat T1D. The plasmid is engineered to turn off adaptive immunity to proinsulin. This approach yielded exciting results in a randomized placebo controlled trial in 80 adult patients with T1D. The implications of this trial are explored in regards to the potential for sparing inflammation in islets and thus allowing the functioning beta cells to recover and produce more insulin. Strategies to further strengthen the effects seen thus far with the tolerizing DNA plasmid to proinsulin will be elucidated. The DNA platform affords an opportunity for easy modifications. In addition standard exploration of dose levels, route of administration and frequency of dose are practical. Optimization of the effects seen to date on C-peptide and on depletion of proinsulin specific CD8 T cells are feasible, with expected concomitant improvement in other parameters like hemoglobin A1c and reduction in insulin usage. T1D is one of the few autoimmune conditions where antigen specific therapy can be achieved, provided the approach is tested intelligently. Tolerizing DNA vaccines to proinsulin and other islet cell autoantigens is a worthy pursuit to potentially treat, prevent and to perhaps even 'cure' or 'prevent' type 1 diabetes.

  19. Comparative modeling of DNA and RNA polymerases from Moniliophthora perniciosa mitochondrial plasmid

    PubMed Central

    Andrade, Bruno S; Taranto, Alex G; Góes-Neto, Aristóteles; Duarte, Angelo A

    2009-01-01

    Background The filamentous fungus Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora is a hemibiotrophic Basidiomycota that causes witches' broom disease of cocoa (Theobroma cacao L.). This disease has resulted in a severe decrease in Brazilian cocoa production, which changed the position of Brazil in the market from the second largest cocoa exporter to a cocoa importer. Fungal mitochondrial plasmids are usually invertrons encoding DNA and RNA polymerases. Plasmid insertions into host mitochondrial genomes are probably associated with modifications in host generation time, which can be involved in fungal aging. This association suggests activity of polymerases, and these can be used as new targets for drugs against mitochondrial activity of fungi, more specifically against witches' broom disease. Sequencing and modeling: DNA and RNA polymerases of M. perniciosa mitochondrial plasmid were completely sequenced and their models were carried out by Comparative Homology approach. The sequences of DNA and RNA polymerase showed 25% of identity to 1XHX and 1ARO (pdb code) using BLASTp, which were used as templates. The models were constructed using Swiss PDB-Viewer and refined with a set of Molecular Mechanics (MM) and Molecular Dynamics (MD) in water carried out with AMBER 8.0, both working under the ff99 force fields, respectively. Ramachandran plots were generated by Procheck 3.0 and exhibited models with 97% and 98% for DNA and RNA polymerases, respectively. MD simulations in water showed models with thermodynamic stability after 2000 ps and 300 K of simulation. Conclusion This work contributes to the development of new alternatives for controlling the fungal agent of witches' broom disease. PMID:19744344

  20. Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells.

    PubMed

    Pimentel, Belén; Nair, Radhika; Bermejo-Rodríguez, Camino; Preston, Mark A; Agu, Chukwuma A; Wang, Xindan; Bernal, Juan A; Sherratt, David J; de la Cueva-Méndez, Guillermo

    2014-02-18

    Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid-bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.

  1. Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells

    PubMed Central

    Pimentel, Belén; Nair, Radhika; Bermejo-Rodríguez, Camino; Preston, Mark A.; Agu, Chukwuma A.; Wang, Xindan; Bernal, Juan A.; Sherratt, David J.; de la Cueva-Méndez, Guillermo

    2014-01-01

    Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid–bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs. PMID:24449860

  2. Growth medium selection and its economic impact on plasmid DNA production.

    PubMed

    Danquah, Michael K; Forde, Gareth M

    2007-12-01

    Current developments in gene medicine and vaccination studies are utilizing plasmid DNA (pDNA) as the vector. For this reason, there has been an increasing trend towards larger and larger doses of pDNA utilized in human trials: from 100-1000 microg in 2002 to 500-5000 microg in 2005. The increasing demand of pDNA has created the need to revolutionalize current production levels under optimum economy. In this work, different standard media (LB, TB and SOC) for culturing recombinant Escherichia coli DH5alpha harbouring pUC19 were compared to a medium optimised for pDNA production. Lab scale fermentations using the standard media showed that the highest pDNA volumetric and specific yields were for TB (11.4 microg/ml and 6.3 microg/mg dry cell mass respectively) and the lowest was for LB (2.8 microg/ml and 3.3 microg/mg dry cell mass respectively). A fourth medium, PDMR, designed by modifying a stoichiometrically-formulated medium with an optimised carbon source concentration and carbon to nitrogen ratio displayed pDNA volumetric and specific yields of 23.8 microg/ml and 11.2 microg/mg dry cell mass respectively. However, it is the economic advantages of the optimised medium that makes it so attractive. Keeping all variables constant except medium and using LB as a base scenario (100 medium cost [MC] units/mg pDNA), the optimised PDMR medium yielded pDNA at a cost of only 27 MC units/mg pDNA. These results show that greater amounts of pDNA can be obtained more economically with minimal extra effort simply by using a medium optimised for pDNA production.

  3. Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization

    PubMed Central

    2014-01-01

    Background Pulmonary-delivered gene therapy promises to mitigate vaccine safety issues and reduce the need for needles and skilled personnel to use them. While plasmid DNA (pDNA) offers a rapid route to vaccine production without side effects or reliance on cold chain storage, its delivery to the lung has proved challenging. Conventional methods, including jet and ultrasonic nebulizers, fail to deliver large biomolecules like pDNA intact due to the shear and cavitational stresses present during nebulization. Methods In vitro structural analysis followed by in vivo protein expression studies served in assessing the integrity of the pDNA subjected to surface acoustic wave (SAW) nebulisation. In vivo immunization trials were then carried out in rats using SAW nebulized pDNA (influenza A, human hemagglutinin H1N1) condensate delivered via intratracheal instillation. Finally, in vivo pulmonary vaccinations using pDNA for influenza was nebulized and delivered via a respirator to sheep. Results The SAW nebulizer was effective at generating pDNA aerosols with sizes optimal for deep lung delivery. Successful gene expression was observed in mouse lung epithelial cells, when SAW-nebulized pDNA was delivered to male Swiss mice via intratracheal instillation. Effective systemic and mucosal antibody responses were found in rats via post-nebulized, condensed fluid instillation. Significantly, we demonstrated the suitability of the SAW nebulizer to administer unprotected pDNA encoding an influenza A virus surface glycoprotein to respirated sheep via aerosolized inhalation. Conclusion Given the difficulty of inducing functional antibody responses for DNA vaccination in large animals, we report here the first instance of successful aerosolized inhalation delivery of a pDNA vaccine in a large animal model relevant to human lung development, structure, physiology, and disease, using a novel, low-power (<1 W) surface acoustic wave (SAW) hand-held nebulizer to produce droplets of pDNA

  4. Experimental and clinical application of plasmid DNA in the field of central nervous diseases.

    PubMed

    Shimamura, Munehisa; Sato, Naoyuki; Morishita, Ryuichi

    2011-12-01

    Novel therapeutic strategies utilizing plasmid DNA (pDNA) have been sought for non-treatable neurological disorders, such as ischemic stroke, Parkinson disease (PD), Alzheimer disease (AD), and multiple sclerosis (MS). One strategy is to induce overexpression of growth factors, such as vascular endothelial growth factor (VEGF), glial cell-line derived neurotrophic factor (GDNF), and hepatocyte growth factor (HGF), in the brain. Since ischemic stroke, PD, and AD show damage of neurons, the transfer of pDNA encoding these genes has been examined and shown to protect neurons from damage, associated with a better behavioral outcome. These growth factors have also been shown to accelerate angiogenesis, neurite outgrowth, and neurogenesis in the brain, and overexpression of these factors showed therapeutic effects in cerebral ischemia in rodents. Another application of pDNA is as a "DNA vaccine" to induce immunity against amyloid Aβ in AD, which requires a predominantly Th2 response to avoid autoimmune encephalomyelitis evoked by a Th1 response. Since the combination of pDNA and special devices and/or modification of pDNA could induce a predominantly Th2 response to a targeted antigen, a pDNA-based vaccine would be ideal for AD. Interestingly, pDNA could also induce immune tolerance, and pDNA-based vaccines to induce immune tolerance to autoimmune antibodies have been extensively examined in an animal model of MS. Based on the results, a pDNA vaccine has already been tried in MS patients and reported to be safe and partly effective in phase I/II clinical studies. In this review, we discuss the potential and problems of pDNA-mediated medicine in neurological disorders.

  5. Induction of mucosal immunity against herpes simplex virus by plasmid DNA immunization.

    PubMed Central

    Kuklin, N; Daheshia, M; Karem, K; Manickan, E; Rouse, B T

    1997-01-01

    The ability of mucosally delivered plasmid DNA encoding glycoprotein B (gB) of herpes simplex virus type 1 (HSV-1) to generate systemic as well as distal mucosal immunity was evaluated. BALB/c mice were immunized intranasally (i.n.) with gB DNA or DNA expressing beta-galactosidase (beta-Gal). Two days following immunization, gB and beta-Gal gene expression was detected by reverse transcription (RT)-PCR in lungs and cervical lymph nodes (CLN). Histological analysis showed that beta-Gal protein was expressed in vivo in the lungs and the CLN of animals immunized with i.n. administered beta-Gal DNA. The immune responses generated by i.n. administration of gB DNA with or without cholera toxin (CT) were compared to those generated by intramuscular (i.m.) gB DNA and i.n. live HSV administration. Three i.n. doses of gB DNA over a 3-week period resulted in a distal mucosal immunoglobulin A (IgA) response. In addition, the mucosal IgA response was enhanced by coadministration of CT with gB DNA. The i.m. route of immunization induced a strong IgG response in the serum and vagina but was inefficient in generating a mucosal IgA response. Antigen-specific cytokine ELISPOT analyses as well as the serum IgG1/IgG2a ratio indicated induction of stronger Th2 responses following the additional i.n. administration of CT compared to i.n. or i.m. gB DNA or i.n. live HSV immunization. In addition, mucosal immunization with gB DNA induced anti-HSV cell-mediated immunity in vivo as measured by delayed-type hypersensitivity. Although i.n. DNA immunization was an effective means of inducing mucosal antibody, it was inferior to i.m. DNA delivery in providing protection against lethal HSV challenge via the vaginal route. In addition, both i.m. and i.n. plasmid immunizations failed to generate an immune barrier to viral invasion of the mucosa. PMID:9060677

  6. Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA.

    PubMed

    Tullman, Jennifer; Guntas, Gurkan; Dumont, Matthew; Ostermeier, Marc

    2011-11-01

    We demonstrate that S1 nuclease converts supercoiled plasmid DNA to unit-length, linear dsDNA through the creation of a single, double-stranded break in a plasmid molecule. These double-stranded breaks occur not only in the origin of replication near inverted repeats but also at a wide variety of locations throughout the plasmid. S1 nuclease exhibits this activity under conditions typically employed for the nuclease's single-stranded nuclease activity. Thus, S1 nuclease digestion of plasmid DNA, unlike analogous digestion with DNaseI, effectively halts after the first double-stranded break. This property makes easier the construction of large domain insertion libraries in which the goal is to insert linear DNA at a variety of locations throughout a plasmid. We used this property to create a library in which a circularly permuted TEM1 β-lactamase gene was inserted throughout a plasmid containing the gene encoding Escherichia coli ribose binding protein. Gene fusions that encode allosteric switch proteins in which ribose modulates β-lactamase catalytic activity were isolated from this library using a combination of a genetic selection and a screen.

  7. Transformation of heat-treated Clostridium acetobutylicum protoplasts with pUB110 plasmid DNA

    SciTech Connect

    Lin, Y.L.; Blaschek, H.P.

    1984-10-01

    Heat treatment of Clostridium acetobutylicum SA-1 protoplasts at 55/sup 0/C for 15 min before transformation resulted in expression in this microorganism of the kanamycin resistance determinant associated with plasmid pUB110. No heat treatment, or heat treatment at 65 or 44/sup 0/C for various time intervals, resulted in no kanamycin resistance transformants being recovered on selective kanamycin-containing regeneration medium. DNase plate assay indicated that treatment at 55/sup 0/C for 15 min completely inactivated the DNase activity associated with SA-1 protoplasts. Treatment of protoplasts at 65 or 55/sup 0/C for various periods under simulated transformation conditions had an inhibitory effect, although prolonged treatment at 55 or 44/sup 0/C appeared to stimulate DNase activity. Inactivation of protoplast-associated DNase activity by heat treatment at 55/sup 0/C for 15 min correlated with successful expression of kanamycin resistance and suggests that an extremely active, heat-sensitive, protoplast-associated DNase may be a factor in the polyethylene glycol-induced transformation of C. acetobutylicum SA-1 protoplasts. Plasmid pUB110 DNA was isolated from C. acetobutylicum SA-1 kanamycin-resistant (Km/sup r/) transformant cultures by a modification of the procedure used for C. perfringens plasmids. Detection of pUB110 DNA was possible only when diethyl pyrocarbonate was incorporated into isolation protocols to inactivate DNase activity. Restriction studies further verified the presence of pUB110 DNA in C. acetobutylicum SA-1 Km/sup r/ transformants. 36 references, 4 figures, 1 table.

  8. Dose Response in Rodents and Nonhuman Primates After Hydrodynamic Limb Vein Delivery of Naked Plasmid DNA

    PubMed Central

    Hegge, Julia O.; Zhang, Guofeng; Sebestyén, Magdolna G.; Noble, Mark; Griffin, Jacob B.; Pfannes, Loretta V.; Herweijer, Hans; Hagstrom, James E.; Braun, Serge; Huss, Thierry; Wolff, Jon A.

    2011-01-01

    Abstract The efficacy of gene therapy mediated by plasmid DNA (pDNA) depends on the selection of suitable vectors and doses. Using hydrodynamic limb vein (HLV) injection to deliver naked pDNA to skeletal muscles of the limbs, we evaluated key parameters that affect expression in muscle from genes encoded in pDNA. Short-term and long-term promoter comparisons demonstrated that kinetics of expression differed between cytomegalovirus (CMV), muscle creatine kinase, and desmin promoters, but all gave stable expression from 2 to 49 weeks after delivery to mouse muscle. Expression from the CMV promoter was highest. For mice, rats, and rhesus monkeys, the linear range for pDNA dose response could be defined by the mass of pDNA relative to the mass of target muscle. Correlation between pDNA dose and expression was linear between a threshold dose of 75 μg/g and maximal expression at approximately 400 μg/g. One HLV injection into rats of a dose of CMV-LacZ yielding maximal expression resulted in an average transfection of 28% of all hind leg muscle and 40% of the gastrocnemius and soleus. Despite an immune reaction to the reporter gene in monkeys, a single injection transfected an average of 10% of all myofibers in the targeted muscle of the arms and legs and an average of 15% of myofibers in the gastrocnemius and soleus. PMID:21338336

  9. Loss of DNA mismatch repair facilitates reactivation of a reporter plasmid damaged by cisplatin

    PubMed Central

    Cenni, B; Kim, H-K; Bubley, G J; Aebi, S; Fink, D; Teicher, B A; Howell, S B; Christen, R D

    1999-01-01

    In addition to recognizing and repairing mismatched bases in DNA, the mismatch repair (MMR) system also detects cisplatin DNA adducts and loss of MMR results in resistance to cisplatin. A comparison was made of the ability of MMR-proficient and -deficient cells to remove cisplatin adducts from their genome and to reactivate a transiently transfected plasmid that had previously been inactivated by cisplatin to express the firefly luciferase enzyme. MMR deficiency due to loss of hMLH1 function did not change the extent of platinum (Pt) accumulation or kinetics of removal from total cellular DNA. However, MMR-deficient cells, lacking either hMLH1 or hMSH2, generated twofold more luciferase activity from a cisplatin-damaged reporter plasmid than their MMR-proficient counterparts. Thus, detection of the cisplatin adducts by the MMR system reduced the efficiency of reactivation of the damaged luciferase gene compared to cells lacking this detector. The twofold reduction in reactivation efficiency was of the same order of magnitude as the difference in cisplatin sensitivity between the MMR-proficient and -deficient cells. We conclude that although MMR-proficient and -deficient cells remove Pt from their genome at equal rates, the loss of a functional MMR system facilitates the reactivation of a cisplatin-damaged reporter gene. © 1999 Cancer Research Campaign PMID:10360646

  10. Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography.

    PubMed

    Hilbrig, Frank; Freitag, Ruth

    2012-01-01

    Hydroxyapatite and related stationary phases increasingly play a role in the downstream processing of high-value biological materials, such as recombinant proteins, therapeutic antibodies and pharmaceutical-grade plasmid DNA. Chromatographic hydroxyapatite is an inorganic, ceramic material identical in composition, if not in structure, to calcium phosphate found in human bones and teeth. The interaction of hydroxyapatite with biomacromolecules is complex and highly dynamic, which can make predicting performance difficult, but also allows the design of very selective isolation processes. This review discusses the currently commercially available chromatographic materials, different retention mechanisms supported by these materials and differential exploitation for the design of highly specific isolation procedures. The state of the art of antibody purification by hydroxy- and fluoroapatite is reviewed together with tested routines for method development and implementation. Finally, the isolation of plasmid DNA is discussed, since the purification of DNA therapeutics at a sufficiently large scale is an emerging need in bioprocess development and perhaps the area in bioseparation where apatite chromatography can make its most important contribution to date.

  11. Metal chelate affinity precipitation of RNA and purification of plasmid DNA

    NASA Technical Reports Server (NTRS)

    Balan, Sindhu; Murphy, Jason; Galaev, Igor; Kumar, Ashok; Fox, George E.; Mattiasson, Bo; Willson, Richard C.

    2003-01-01

    The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine 'tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.

  12. Immunization with plasmid DNA encoding the hemagglutinin and the nucleoprotein confers robust protection against a lethal canine distemper virus challenge.

    PubMed

    Dahl, Lotte; Jensen, Trine Hammer; Gottschalck, Elisabeth; Karlskov-Mortensen, Peter; Jensen, Tove Dannemann; Nielsen, Line; Andersen, Mads Klindt; Buckland, Robin; Wild, T Fabian; Blixenkrone-Møller, Merete

    2004-09-09

    We have investigated the protective effect of immunization of a highly susceptible natural host of canine distemper virus (CDV) with DNA plasmids encoding the viral nucleoprotein (N) and hemagglutinin (H). The combined intradermal and intramuscular routes of immunization elicited high virus-neutralizing serum antibody titres in mink (Mustela vison). To mimic natural exposure, we also conducted challenge infection by horizontal transmission from infected contact animals. Other groups received a lethal challenge infection by administration to the mucosae of the respiratory tract and into the muscle. One of the mink vaccinated with N plasmid alone developed severe disease after challenge. In contrast, vaccination with the H plasmid together with the N plasmid conferred solid protection against disease and we were unable to detect CDV infection in PBMCs or in different tissues after challenge. Our findings show that DNA immunization by the combined intradermal and intramuscular routes can confer solid protective immunity against naturally transmitted morbillivirus infection and disease.

  13. Dinuclear Zinc (II) Complexes of Macrocyclic Polyamine Ligands Containing an Imidazolium Bridge: Synthesis, Characterization, and Their Interaction with Plasmid DNA

    PubMed Central

    Huang, Jun; Huang, Qing-Dong; Zhang, Ji; Zhou, Li-Hong; Li, Qiang-Lin; Li, Kun; Jiang, Ning; Lin, Hong-Hui; Wu, Jiang; Yu, Xiao-Qi

    2007-01-01

    Two novel macrocyclic polyamine ligands and their dinuclear zinc (II) complexes were synthesized and characterized. Their interaction with plasmid DNA was studied by gel electrophoresis and fluorescence quenching experiment. The result showed that these complexes could bind DNA efficiently under physiological conditions.

  14. Infrared laser effects at fluences used for treatment of dentin hypersensitivity on DNA repair in Escherichia coli and plasmids

    NASA Astrophysics Data System (ADS)

    Rocha Teixeira, Gleica; da Silva Marciano, Roberta; da Silva Sergio, Luiz Philippe; Castanheira Polignano, Giovanni Augusto; Roberto Guimarães, Oscar; Geller, Mauro; de Paoli, Flavia; de Souza da Fonseca, Adenilson

    2014-12-01

    Low-intensity infrared lasers are proposed in clinical protocols based on biostimulative effects, yet dosimetry is inaccurate and their effects on DNA at therapeutic doses are controversial. The aim of this work was to evaluate the effects of low-intensity infrared laser on survival and induction of filamentation of Escherichia coli cells, and induction of DNA lesions in bacterial plasmids. E. coli cultures were exposed to laser (808 nm, 100 mW, 40 and 60 J/cm2) to study bacterial survival and filamentation. Also, bacterial plasmids were exposed to laser to study DNA lesions by electrophoretic profile and action of DNA repair enzymes. Data indicate low-intensity infrared laser has no effect on survival of E. coli wild type and exonuclease III, but decreases the survival of formamidopyrimidine DNA glycosylase/MutM protein and endonuclease III deficient cells in stationary growth phase, induces bacterial filamentation, does not alter the electrophoretic profile of plasmids in agarose gels and does not alter the electrophoretic profile of plasmids incubated with endonuclease III, formamidopyrimidine DNA glycosylase/MutM protein and exonuclease III. Our findings show that low-intensity laser exposure causes DNA lesions at sub-lethal level and induces cellular mechanisms involved in repair of oxidative lesions in DNA. Studies about laser dosimetry and safety strategies are necessary for professionals and patients exposed to low-intensity lasers at therapeutic doses.

  15. Effect of growth rate on plasmid DNA production and metabolic performance of engineered Escherichia coli strains.

    PubMed

    Wunderlich, Martin; Taymaz-Nikerel, Hilal; Gosset, Guillermo; Ramírez, Octavio T; Lara, Alvaro R

    2014-03-01

    Two engineered Escherichia coli strains, designated VH33 and VH34, were compared to their parent strain W3110 in chemostat mode during plasmid DNA (pDNA) production. In strain VH33 the glucose uptake system was modified with the aim of reducing overflow metabolism. The strain VH34 has an additional deletion of the pyruvate kinase A gene (pykA) to increase pDNA formation. pDNA formation rates as well as kinetic and stoichiometric parameters were investigated in dependence of the growth rate within a range from 0.02 to 0.25 h(-1). Differences between strains were found in terms of the biomass yields on nitrogen and oxygen, as well as on the cell maintenance coefficients. The deletion of pykA led to a significantly increased pDNA yield and productivity. At an optimal growth rate of 0.20 h(-1) it was nearly 60% higher than that of W3110 and VH33. Metabolic fluxes calculated by metabolite balance analysis showed differences mainly in reactions catalyzed by pyruvate kinase and glucose 6-phosphate dehydrogenase. The obtained data are useful for the design of cultivation schemes for pDNA production by E. coli.

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

    NASA Astrophysics Data System (ADS)

    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.

  17. Plasmids in diatom species.

    PubMed Central

    Hildebrand, M; Corey, D K; Ludwig, J R; Kukel, A; Feng, T Y; Volcani, B E

    1991-01-01

    We have discovered plasmids in 5 of 18 diatom species surveyed. In several species, more than one type of plasmid is present. Several of the plasmids show similarity by hybridization previously characterized plasmids in Cylindrotheca fusiformis (J. D. Jacobs et al., unpublished data). Additionally, there is similarity between the plasmids found in C. fusiformis and chloroplast DNA in three diatom species. These results add to the evidence that the plasmids have features of mobile genetic elements. Images PMID:1885558

  18. Preparation of Biodegradable Gelatin Nanospheres with a Narrow Size Distribution for Carrier of Cellular Internalization of Plasmid DNA.

    PubMed

    Doi, Norio; Jo, Jun-Ichiro; Tabata, Yasuhiko

    2012-01-01

    The objective of this study is to design biodegradable nanospheres of cationized gelatin as a carrier of cellular internalization of plasmid DNA. Ethylenediamine was chemically introduced into the carboxyl groups of gelatin to obtain cationized gelatin. The gelatin solution was filtered through a glass membrane under high pressure and dropped into 2-butanol, acetone or a mixture of the two to form nanospheres of cationized gelatin. The microspheres of cationized gelatin were prepared by the conventional water-in-oil emulsion method. The resulting nano- and microspheres of cationized gelatin were dehydrothermally treated at 160°C for different time periods to allow them to cross-link chemically. The size of nanospheres, prepared by the filtration method and changed by the type of solvents, was 1.86, 0.83 or 0.24 μm. The in vitro degradation of spheres became faster as the time period of dehydrothermal treatment was shorter. The degradation time of spheres in HCl solution linearly increased with an increase in the cross-linking time, irrespective of the sphere size. However, in the collagenase solution, when compared at the similar cross-linking density, the smaller spheres were degraded more slowly than the larger ones. The plasmid DNA incorporated in the nanospheres was released from the nanospheres with their degradation. The nanospheres incorporating plasmid DNA were internalized into cells, and intracellularly degraded with time to release plasmid DNA. The time period of plasmid DNA release was prolonged by increasing the nanosphere degradation time.

  19. Ultrasound enhances the transfection of plasmid DNA by non-viral vectors.

    PubMed

    Hosseinkhani, Hossein; Aoyama, Teruyoshi; Ogawa, Osamu; Tabata, Yasuhiko

    2003-04-01

    Increasing attention has been paid to technology used for the delivery of genetic materials into cells for gene therapy and the generation of genetically engineered cells. So far, viral vectors have been mainly used because of their inherently high transfection efficiency of gene. However, there are some problems to be resolved for the clinical applications, such as the pathogenicity and immunogenicity of viral vectors themselves. Therefore, many research trials with non-viral vectors have been performed to enhance their efficiency to a level comparable to the viral vector. Two directions of these trials exist: material improvement of non-viral vectors and their combination with various external physical stimuli. This paper reviews the latter research trials, with special attention paid to the enhancement of gene expression by ultrasound (US). The expression level of plasmid DNA by various cationized polymers and liposomes is promoted by US irradiation in vitro as well as in vivo. This US-enhanced expression of plasmid DNA will be discussed to emphasize the technical feasibility of US in gene therapy and biotechnology.

  20. Mapping of bionic array electric field focusing in plasmid DNA-based gene electrotransfer.

    PubMed

    Browne, C J; Pinyon, J L; Housley, D M; Crawford, E N; Lovell, N H; Klugmann, M; Housley, G D

    2016-04-01

    Molecular medicine through gene therapy is challenged to achieve targeted action. This is now possible utilizing bionic electrode arrays for focal delivery of naked (plasmid) DNA via gene electrotransfer. Here, we establish the properties of array-based electroporation affecting targeted gene delivery. An array with eight 300 μm platinum ring electrodes configured as a cochlear implant bionic interface was used to transduce HEK293 cell monolayers with a plasmid-DNA green fluorescent protein (GFP) reporter gene construct. Electroporation parameters were pulse intensity, number, duration, separation and electrode configuration. The latter determined the shape of the electric fields, which were mapped using a voltage probe. Electrode array-based electroporation was found to require ~100 × lower applied voltages for cell transduction than conventional electroporation. This was found to be due to compression of the field lines orthogonal to the array. A circular area of GFP-positive cells was created when the electrodes were ganged together as four adjacent anodes and four cathodes, whereas alternating electrode polarity created a linear area of GFP-positive cells. The refinement of gene delivery parameters was validated in vivo in the guinea pig cochlea. These findings have significant clinical ramifications, where spatiotemporal control of gene expression can be predicted by manipulation of the electric field via current steering at a cellular level.

  1. Synergistic cytotoxicity and DNA strand breaks in cells and plasmid DNA exposed to uranyl acetate and ultraviolet radiation.

    PubMed

    Wilson, Janice; Zuniga, Mary C; Yazzie, Filbert; Stearns, Diane M

    2015-04-01

    Depleted uranium (DU) has a chemical toxicity that is independent of its radioactivity. The purpose of this study was to explore the photoactivation of uranyl ion by ultraviolet (UV) radiation as a chemical mechanism of uranium genotoxicity. The ability of UVB (302 nm) and UVA (368 nm) radiation to photoactivate uranyl ion to produce single strand breaks was measured in pBR322 plasmid DNA, and the presence of adducts and apurinic/apyrimidinic sites that could be converted to single strand breaks by heat and piperidine was analyzed. Results showed that DNA lesions in plasmid DNA exposed to UVB- or UVA-activated DU were only slightly heat reactive, but were piperidine sensitive. The cytotoxicity of UVB-activated uranyl ion was measured in repair-proficient and repair-deficient Chinese hamster ovary cells and human keratinocyte HaCaT cells. The cytotoxicity of co-exposures of uranyl ion and UVB radiation was dependent on the order of exposure and was greater than co-exposures of arsenite and UVB radiation. Uranyl ion and UVB radiation were synergistically cytotoxic in cells, and cells exposed to photoactivated DU required different DNA repair pathways than cells exposed to non-photoactivated DU. This study contributes to our understanding of the DNA lesions formed by DU, as well as their repair. Results suggest that excitation of uranyl ion by UV radiation can provide a pathway for uranyl ion to be chemically genotoxic in populations with dermal exposures to uranium and UV radiation, which would make skin an overlooked target organ for uranium exposures.

  2. Synergistic cytotoxicity and DNA strand breaks in cells and plasmid DNA exposed to uranyl acetate and ultraviolet radiation

    PubMed Central

    Wilson, Janice; Zuniga, Mary C.; Yazzie, Filbert; Stearns, Diane M.

    2015-01-01

    Depleted uranium (DU) has a chemical toxicity that is independent of its radioactivity. The purpose of this study was to explore the photoactivation of uranyl ion by ultraviolet (UV) radiation as a chemical mechanism of uranium genotoxicity. The ability of UVB (302 nm) and UVA (368 nm) radiation to photoactivate uranyl ion to produce single strand breaks was measured in pBR322 plasmid DNA, and the presence of adducts and apurinic/apyrimidinic sites that could be converted to single strand breaks by heat and piperidine was analyzed. Results showed that DNA lesions in plasmid DNA exposed to UVB- or UVA-activated DU were only slightly heat reactive, but were piperidine sensitive. The cytotoxicity of UVB-activated uranyl ion was measured in repair-proficient and repair-deficient Chinese hamster ovary cells and human keratinocyte HaCaT cells. The cytotoxicity of co-exposures of uranyl ion and UVB radiation was dependent on the order of exposure and was greater than co-exposures of arsenite and UVB radiation. Uranyl ion and UVB radiation were synergistically cytotoxic in cells, and cells exposed to photoactivated DU required different DNA repair pathways than cells exposed to non-photoactivated DU. This study contributes to our understanding of the DNA lesions formed by DU, as well as their repair. Results suggest that excitation of uranyl ion by UV radiation can provide a pathway for uranyl ion to be chemically genotoxic in populations with dermal exposures to uranium and UV radiation, which would make skin an overlooked target organ for uranium exposures. PMID:24832689

  3. Plasmid DNA initiates replication of yellow fever vaccine in vitro and elicits virus-specific immune response in mice

    SciTech Connect

    Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat; Jokinen, Jenny; Lukashevich, Igor S.; Pushko, Peter

    2014-11-15

    Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20 μg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF. - Highlights: • The iDNA{sup ®} platform combines advantages of DNA and live attenuated vaccines. • Yellow fever (YF) 17D vaccine was launched from iDNA plasmid in vitro and in vivo. • Safety of iDNA-generated 17D virus was confirmed in AG129 mice. • BALB/c mice seroconverted after a single-dose vaccination with iDNA. • YF virus-neutralizing response was elicited in iDNA-vaccinated mice.

  4. Ca(2+)-mediated anionic lipid-plasmid DNA lipoplexes. Electrochemical, structural, and biochemical studies.

    PubMed

    Barrán-Berdón, Ana L; Yélamos, Belén; Malfois, Marc; Aicart, Emilio; Junquera, Elena

    2014-10-07

    Several experimental methods, such as zeta potential, gel electrophoresis, small-angle X-ray scattering, gene transfection, fluorescence microscopy, flow cytometry, and cell viability/cytotoxicity assays, have been used to analyze the potential of anionic lipids (AL) as effective nontoxic and nonviral DNA vectors, assisted by divalent cations. The lipoplexes studied are those comprised of the green fluorescent protein-encoding plasmid DNA pEGFP-C3, an anionic lipid as 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) or 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and a zwitterionic lipid, the 1,2-dioleoyl-sn -glycero-3-phosphatidylethanolamine (DOPE, not charged at physiological pH). The studies have been carried on at different liposome and lipoplex compositions and in the presence of a variety of [Ca2+]. Electrochemical experiments reveal that DOPG/DOPE and DOPS/DOPE anionic liposomes may compact more effectively pDNA at low molar fractions (with an excess of DOPE) and at AL/pDNA ratios ≈20. Calcium concentrations around 15-20 mM are needed to yield lipoplexes neutral or slightly positive. From a structural standpoint, DOPG/DOPE-Ca2+-pDNA lipoplexes are self-assembled into a HIIc phase (inverted cylindrical micelles in hexagonal ordering with plasmid supercoils inside the cylinders), while DOPS/DOPE-Ca2+-pDNA lipoplexes show two phases in coexistence: one classical HIIc phase which contains pDNA supercoils and one Lα phase without pDNA among the lamellae, i.e., a lamellar stack of lipidic bilayers held together by Ca2+ bridges. Transfection and cell viability studies were done with HEK293T and HeLa cells in the presence of serum. Lipoplexes herein studied show moderate-to-low transfection levels combined with moderate-to-high cell viability, comparable to those yield by Lipofectamine2000*, which is a cationic lipid (CL) standard formulation, but none of them improve the output of typical CL gen vectors, mostly if they are gemini or dendritic

  5. Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection.

    PubMed

    Mao, Mao; Wang, Liangli; Chang, Chun-Chi; Rothenberg, Katheryn E; Huang, Jianyong; Wang, Yingxiao; Hoffman, Brenton D; Liton, Paloma B; Yuan, Fan

    2017-03-01

    Electrotransfection is a widely used method for delivering genes into cells with electric pulses. Although different hypotheses have been proposed, the mechanism of electrotransfection remains controversial. Previous studies have indicated that uptake and intracellular trafficking of plasmid DNA (pDNA) are mediated by endocytic pathways, but it is still unclear which pathways are directly involved in the delivery. To this end, the present study investigated the dependence of electrotransfection on macropinocytosis. Data from the study demonstrated that electric pulses induced cell membrane ruffling and actin cytoskeleton remodeling. Using fluorescently labeled pDNA and a macropinocytosis marker (i.e., dextran), the study showed that electrotransfected pDNA co-localized with dextran in intracellular vesicles. Furthermore, electrotransfection efficiency could be decreased significantly by reducing temperature or treatment of cells with a pharmacological inhibitor of Rac1 and could be altered by changing Rac1 activity. Taken together, the findings suggested that electrotransfection of pDNA involved Rac1-dependent macropinocytosis.

  6. Plasmid DNA is internalized from the apical plasma membrane of the salivary gland epithelium in live animals.

    PubMed

    Sramkova, Monika; Masedunskas, Andrius; Weigert, Roberto

    2012-08-01

    Non-viral-mediated gene delivery represents an alternative way to express the gene of interest without inducing immune responses or other adverse effects. Understanding the mechanisms by which plasmid DNAs are delivered to the proper target in vivo is a fundamental issue that needs to be addressed in order to design more effective strategies for gene therapy. As a model system, we have used the submandibular salivary glands in live rats and we have recently shown that reporter transgenes can be expressed in different cell populations of the glandular epithelium, depending on the modality of administration of plasmid DNA. Here, by using a combination of immunofluorescence and intravital microscopy, we have explored the relationship between the pattern of transgenes expression and the internalization of plasmid DNA. We found that plasmid DNA is internalized: (1) by all the cells in the salivary gland epithelium, when administered alone, (2) by large ducts, when mixed with empty adenoviral particles, and (3) by acinar cells upon stimulation of compensatory endocytosis. Moreover, we showed that plasmid DNA utilizes different routes of internalization, and evades both the lysosomal degradative pathway and the retrograde pathway towards the Golgi apparatus. This study clearly shows that in vivo approaches have the potential to address fundamental questions on the cellular mechanisms regulating gene delivery.

  7. Self-catalyzed site-specific depurination of G residues mediated by cruciform extrusion in closed circular DNA plasmids.

    PubMed

    Amosova, Olga; Kumar, Veena; Deutsch, Aaron; Fresco, Jacques R

    2011-10-21

    A major variety of "spontaneous" genomic damage is endogenous generation of apurinic sites. Depurination rates vary widely across genomes, occurring with higher frequency at "depurination hot spots." Recently, we discovered a site-specific self-catalyzed depurinating activity in short (14-18 nucleotides) DNA stem-loop-forming sequences with a 5'-G(T/A)GG-3' loop and T·A or G·C as the first base pair at the base of the loop; the 5'-G residue of the loop self-depurinates at least 10(5)-fold faster than random "spontaneous" depurination at pH 5. Formation of the catalytic intermediate for self-depurination in double-stranded DNA requires a stem-loop to extrude as part of a cruciform. In this study, evidence is presented for self-catalyzed depurination mediated by cruciform formation in plasmid DNA in vitro. Cruciform extrusion was confirmed, and its extent was quantitated by digestion of the plasmid with single strand-specific mung bean endonuclease, followed by restriction digestion and sequencing of resulting mung bean-generated fragments. Appearance of the apurinic site in the self-depurinating stem-loop was confirmed by digestion of plasmid DNA with apurinic endonuclease IV, followed by primer extension and/or PCR amplification to detect the endonuclease-generated strand break and identify its location. Self-catalyzed depurination was contingent on the plasmid being supercoiled and was not observed in linearized plasmids, consistent with the presence of the extruded cruciform in the supercoiled plasmid and not in the linear one. These results indicate that self-catalyzed depurination is not unique to single-stranded DNA; rather, it can occur in stem-loop structures extruding from double-stranded DNA and therefore could, in principle, occur in vivo.

  8. Enhanced gene expression of systemically administered plasmid DNA in the liver with therapeutic ultrasound and microbubbles.

    PubMed

    Raju, Balasundar I; Leyvi, Evgeniy; Seip, Ralf; Sethuraman, Shriram; Luo, Xiaoyan; Bird, Andrew; Li, Songtao; Koeberl, Dwight

    2013-01-01

    Ultrasound-mediated delivery (USMD) of novel therapeutic agents in the presence of microbubbles is a potentially safe and effective method for gene therapy offering many desired characteristics, such as low toxicity, potential for repeated treatment, and organ specificity. In this study, we tested the capability of USMD to improve gene expression in mice livers using glycogen storage disease Type Ia as a model disease under systemic administration of naked plasmid DNA. Image-guided therapeutic ultrasound was used in two studies to provide therapeutic ultrasound to mice livers. In the first study, involving wild-type mice, control animals received naked plasmid DNA (pG6Pase 150 μg) via the tail vein, followed by an infusion of microbubbles; the treated animals additionally received therapeutic ultrasound (1 MHz). Following the procedure, the animals were left to recover and were subsequently euthanized after 2 d and liver samples were extracted. Reverse transcription polymerase chain reaction (RT-PCR) assays were performed on the samples to quantify mRNA expression. In addition, Western blot assays of FLAG-tagged glucose-6-phosphatase (G6Pase) were performed to evaluate protein expression. Ultrasound-exposed animals showed a 4-fold increase in G6Pase RNA in the liver, in comparison with control animals. Furthermore, results from Western blot analysis demonstrated a 2-fold increased protein expression in ultrasound-exposed animals after two days ( p < 0.05). A second pilot study was performed with G6Pase knockout mice, and the animals were monitored for correction of hypoglycemia over a period of 3 weeks before tissue analysis. The RT-PCR assays of samples from these animals demonstrated increased G6Pase RNA in the liver following ultrasound treatment. These results demonstrate that USMD can increase gene expression of systemically injected naked pDNA in the liver and also provide insight into the development of realistic approaches that can be translated into

  9. Therapeutic plasmid DNA versus siRNA delivery: common and different tasks for synthetic carriers.

    PubMed

    Scholz, Claudia; Wagner, Ernst

    2012-07-20

    Gene therapy offers great opportunities for the treatment of severe diseases including cancer. In recent years the design of synthetic carriers for nucleic acid delivery has become a research field of increasing interest. Studies on the delivery of plasmid DNA (pDNA) have brought up a variety of gene delivery vehicles. The more recently emerged gene silencing strategy by the intracellular delivery of small interfering RNA (siRNA) takes benefit from existing expertise in pDNA transfer. Despite common properties however, delivery of siRNA also faces distinct challenges due to apparent differences in size, stability of the formed nucleic acid complexes, the location and mechanism of action. This review emphasizes the common aspects and main differences between pDNA and siRNA delivery, taking into consideration a wide spectrum of polymer-based, lipidic and peptide carriers. Challenges and opportunities which result from these differences as well as the recent progress made in the optimization of carrier design are presented.

  10. Yields of strand breaks and base lesions induced by soft X-rays in plasmid DNA.

    PubMed

    Yokoya, A; Fujii, K; Ushigome, T; Shikazono, N; Urushibara, A; Watanabe, R

    2006-01-01

    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.

  11. Replication initiates at multiple dispersed sites in the ribosomal DNA plasmid of the protozoan parasite Entamoeba histolytica.

    PubMed Central

    Dhar, S K; Choudhury, N R; Mittal, V; Bhattacharya, A; Bhattacharya, S

    1996-01-01

    In the protozoan parasite Entamoeba histolytica (which causes amoebiasis in humans), the rRNA genes (rDNA) in the nucleus are carried on an extrachromosomal circular plasmid. For strain HM-1:IMSS, the size of the rDNA plasmid is 24.5 kb, and 200 copies per genome are present. Each circle contains two rRNA transcription units as inverted repeats separated by upstream and downstream spacers. We have studied the replication of this molecule by neutral/neutral two-dimensional gel electrophoresis and by electron microscopy. All restriction fragments analyzed by two-dimensional gel electrophoresis gave signals corresponding to simple Y's and bubbles. This showed that replication initiated in this plasmid at multiple, dispersed locations spread throughout the plasmid. On the basis of the intensity of the bubble arcs, initiations from the rRNA transcription units seemed to occur more frequently than those from intergenic spacers. Multiple, dispersed initiation sites were also seen in the rDNA plasmid of strain HK-9 when it was analyzed by two-dimensional gel electrophoresis. Electron microscopic visualization of replicating plasmid molecules in strain HM-1:IMISS showed multiple replication bubbles in the same molecule. The location of bubbles on the rDNA circle was mapped by digesting with PvuI or BsaHI, which linearize the molecule, and with SacII, which cuts the circle twice. The distance of the bubbles from one end of the molecule was measured by electron microscopy. The data corroborated those from two-dimensional gels and showed that replication bubbles were distributed throughout the molecule and that they appeared more frequently in rRNA transcription units. The same interpretation was drawn from electron microscopic analysis of the HK-9 plasmid. Direct demonstration of more than one bubble in the same molecule is clear evidence that replication of this plasmid initiates at multiple sites. Potential replication origins are distributed throughout the plasmid. Such a

  12. Plasmid Biopharmaceuticals.

    PubMed

    Prazeres, Duarte Miguel F; Monteiro, Gabriel A

    2014-12-01

    Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its mission to produce pharmaceutical proteins, antibodies, vaccines, industrial enzymes, and molecular diagnostics, to name a few key products. Furthermore, plasmids have gradually stepped up in the past 20 years as useful biopharmaceuticals in the context of gene therapy and DNA vaccination interventions. This review provides a concise coverage of the scientific progress that has been made since the emergence of what are called today plasmid biopharmaceuticals. The most relevant topics are discussed to provide researchers with an updated overview of the field. A brief outline of the initial breakthroughs and innovations is followed by a discussion of the motivation behind the medical uses of plasmids in the context of therapeutic and prophylactic interventions. The molecular characteristics and rationale underlying the design of plasmid vectors as gene transfer agents are described and a description of the most important methods used to deliver plasmid biopharmaceuticals in vivo (gene gun, electroporation, cationic lipids and polymers, and micro- and nanoparticles) is provided. The major safety issues (integration and autoimmunity) surrounding the use of plasmid biopharmaceuticals is discussed next. Aspects related to the large-scale manufacturing are also covered, and reference is made to the plasmid products that have received marketing authorization as of today.

  13. Open-complex formation by the host initiator, DnaA, at the origin of P1 plasmid replication.

    PubMed Central

    Mukhopadhyay, G; Carr, K M; Kaguni, J M; Chattoraj, D K

    1993-01-01

    Replication of P1 plasmid requires both the plasmid-specific initiator, RepA, and the host initiator, DnaA. Here we show that DnaA can make the P1 origin reactive to the single-strand specific reagents KMnO4 and mung bean nuclease. Addition of RepA further increased the KMnO4 reactivity of the origin, although RepA alone did not influence the reaction. The increased reactivity implies that the two initiators interact in some way to alter the origin conformation. The KMnO4 reactivity was restricted to one strand of the origin. We suggest that the roles of DnaA in P1 plasmid and bacterial replication are similar: origin opening and loading of the DnaB helicase. The strand-bias in chemical reactivity at the P1 origin most likely indicates that only one of the strands is used for the loading of DnaB, a scenario consistent with the unidirectional replication of the plasmid. Images PMID:8223464

  14. How does plasmid DNA penetrate cell membranes in artificial transformation process of Escherichia coli?

    PubMed

    Panja, Subrata; Aich, Pulakesh; Jana, Bimal; Basu, Tarakdas

    2008-08-01

    Artificial transformation of Escherichia coli with plasmid DNA in presence of CaCl2 is a widely used technique in recombinant DNA technology. However, exact mechanism of DNA transfer across cell membranes is largely obscure. In this study, measurements of both steady state and time-resolved anisotropies of fluorescent dye trimethyl ammonium diphenyl hexatriene (TMA-DPH), bound to cellular outer membrane, indicated heat-pulse (0 degrees C42 degrees C) step of the standard transformation procedure had lowered considerably outer membrane fluidity of cells. The decrease in fluidity was caused by release of lipids from cell surface to extra-cellular medium. A subsequent cold-shock (42 degrees C0 degrees C) to the cells raised the fluidity further to its original value and this was caused by release of membrane proteins to extra-cellular medium. When the cycle of heat-pulse and cold-shock steps was repeated, more release of lipids and proteins respectively had taken place, which ultimately enhanced transformation efficiency gradually up to third cycle. Study of competent cell surface by atomic force microscope showed release of lipids had formed pores on cell surface. Moreover, the heat-pulse step almost depolarized cellular inner membrane. In this communication, we propose heat-pulse step had two important roles on DNA entry: (a) Release of lipids and consequent formation of pores on cell surface, which helped DNA to cross outer membrane barrier, and (b) lowering of membrane potential, which facilitated DNA to cross inner membrane of E. coli.

  15. Modified mRNA as an alternative to plasmid DNA (pDNA) for transcript replacement and vaccination therapy

    PubMed Central

    Youn, Hyewon; Chung, June-Key

    2015-01-01

    Introduction: Current gene therapy involves replacement of defective gene by delivery of healthy genetic material to precede normal function. Virus-mediated gene delivery is the most successful and efficient method for gene therapy, but it has been challenged due to serious safety concerns. Conversely, gene delivery using plasmid DNA (pDNA) is considered safer, but its transfection efficiency is much lower than virus-mediated gene transfer. Recently, mRNA has been suggested as an alternative option to avoid undesired insertion of delivered DNA sequences with higher transfection efficiency and stability. Area covered: In this review, we summarize the currently available strategies of mRNA modification to increase the therapeutic efficacy; we also highlight the recent improvements of mRNA delivery for in vivo applications of gene therapy. Expert opinion: The use of mRNA-based gene transfer could indeed be a promising new strategy for gene therapy. Notable advantages include no risk of integration into the genomic DNA, adjustable gene expression and easier modulation of the immune system. By reducing or utilizing the immunogenic properties, mRNA offers a promising tool for gene/or transcript replacement. PMID:26125492

  16. Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity

    PubMed Central

    2011-01-01

    Background Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs) are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare. Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD), FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2), FHD and commercially available AuNPs (Plano-AuNP), and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI%) positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence. Results The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%), whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50%), while the magnet

  17. Co-delivery of drugs and plasmid DNA for cancer therapy.

    PubMed

    Teo, Pei Yun; Cheng, Wei; Hedrick, James L; Yang, Yi Yan

    2016-03-01

    Cancer is an extremely complex disease involving multiple signaling pathways that enable tumor cells to evade programmed cell death, thus making cancer treatment extremely challenging. The use of combination therapy involving both gene therapy and chemotherapy has resulted in enhanced anti-cancer effects and has become an increasingly important strategy in medicine. This review will cover important design parameters that are incorporated into delivery systems for the co-administration of drug and plasmid-based nucleic acids (pDNA and shRNA), with particular emphasis on polymers as delivery materials. The unique challenges faced by co-delivery systems and the strategies to overcome such barriers will be discussed. In addition, the advantages and disadvantages of combination therapy using separate carrier systems versus the use of a single carrier will be evaluated. Finally, future perspectives in the design of novel platforms for the combined delivery of drugs and genes will be presented.

  18. Conjugative transfer of staphylococcal antibiotic resistance markers in the absence of detectable plasmid DNA.

    PubMed Central

    el Solh, N; Allignet, J; Bismuth, R; Buret, B; Fouace, J M

    1986-01-01

    Eleven Staphylococcus aureus clinical isolates were tested for transfer of resistance markers by transduction and filter mating. The resistance markers of six of the strains could be transferred only by transduction; however, the five remaining strains transferred their resistance both by transduction and filter mating. The resistance markers that were cotransferred in filter matings (transfer of resistance to penicillin and streptogramin A was accompanied, in each case, by the transfer of one or more markers, i.e., resistance to aminoglycosides, cadmium, or tetracycline, depending on the donor) were not cotransduced. The filter mating transfers were recA independent and were observed with both Staphylococcus aureus and Staphylococcus epidermidis recipients. Experiments to elucidate the mechanism of transfer by filter mating suggested that conjugation requiring cell-to-cell contact may have been involved. These transfers occurred in the absence of detectable plasmid DNA. PMID:2944478

  19. Photoresponsive Bridged Silsesquioxane Nanoparticles with Tunable Morphology for Light-Triggered Plasmid DNA Delivery.

    PubMed

    Fatieiev, Yevhen; Croissant, Jonas G; Alsaiari, Shahad; Moosa, Basem A; Anjum, Dalaver H; Khashab, Niveen M

    2015-11-18

    Bridged silsesquioxane nanocomposites with tunable morphologies incorporating o-nitrophenylene-ammonium bridges are described. The systematic screening of the sol-gel parameters allowed the material to reach the nanoscale with controlled dense and hollow structures of 100-200 nm. The hybrid composition of silsesquioxanes with 50% organic content homogeneously distributed in the nanomaterials endowed them with photoresponsive properties. Light irradiation was performed to reverse the surface charge of nanoparticles from +46 to -39 mV via a photoreaction of the organic fragments within the particles, as confirmed by spectroscopic monitorings. Furthermore, such nanoparticles were applied for the first time for the on-demand delivery of plasmid DNA in HeLa cancer cells via light actuation.

  20. Promising plasmid DNA vector based on APTES-modified silica nanoparticles.

    PubMed

    Cheang, Tuck-yun; Tang, Bing; Xu, An-wu; Chang, Guang-qi; Hu, Zuo-jun; He, Wei-ling; Xing, Zhou-hao; Xu, Jian-bo; Wang, Mian; Wang, Shen-ming

    2012-01-01

    Nanoparticles have an enormous potential for development in biomedical applications, such as gene or drug delivery. We developed and characterized aminopropyltriethoxysilane-functionalized silicon dioxide nanoparticles (APTES-SiNPs) for gene therapy. Lipofectamine(®) 2000, a commonly used agent, served as a contrast. We showed that APTES-SiNPs had a gene transfection efficiency almost equal to that of Lipofectamine 2000, but with lower cytotoxicity. Thus, these novel APTES-SiNPs can achieve highly efficient transfection of plasmid DNA, and to some extent reduce cytotoxicity, which might overcome the critical drawbacks in vivo of conventional carriers, such as viral vectors, organic polymers, and liposomes, and seem to be a promising nonviral gene therapy vector.

  1. Synthesis of FinP RNA by plasmids F and pSLT is regulated by DNA adenine methylation.

    PubMed Central

    Torreblanca, J; Marqués, S; Casadesús, J

    1999-01-01

    DNA adenine methylase mutants of Salmonella typhimurium contain reduced amounts of FinP, an antisense RNA encoded by the virulence plasmid pSLT. Lowered FinP levels are detected in both Dam- FinO+ and Dam- FinO- backgrounds, suggesting that Dam methylation regulates FinP production rather than FinP half-life. Reduced amounts of F-encoded FinP RNA are likewise found in Dam- mutants of Escherichia coli. A consequence of FinP RNA scarcity in the absence of DNA adenine methylation is that Dam- mutants of both S. typhimurium and E. coli show elevated levels of F plasmid transfer. Inhibition of F fertility by the S. typhimurium virulence plasmid is also impaired in a Dam- background. PMID:10408954

  2. Dead-end hollow-fiber ultrafiltration for concentration and enumeration of Escherichia coli and broad-host-range plasmid DNA from wastewater

    PubMed Central

    Asfahl, Kyle L.; Savin, Mary C.

    2012-01-01

    Broad-host-range plasmids can facilitate dissemination of antibiotic resistance determinants among diverse bacterial populations. We evaluated hollow-fiber ultrafiltration for increases in detection efficiency of broad-host-range plasmids and Escherichia coli DNA in wastewater. Ultrafiltration followed by PCR showed limited increases in DNA detection and quantification in effluent compared with membrane filtration alone. PMID:22251424

  3. Dry Powder Formulation of Plasmid DNA and siRNA for Inhalation.

    PubMed

    Chow, Michael Y T; Lam, Jenny K W

    2015-01-01

    Nucleic acid therapeutics has huge potential for the treatment of a wide range of diseases including respiratory diseases. Plasmid DNA (pDNA) and small interfering RNA (siRNA) are the two most widely investigated nucleic acids for therapeutic development. However, efficient and safe delivery of nucleic acids is still a major hurdle in translating nucleic acid therapy into clinical practice. For the treatment of respiratory diseases, administration via inhalation is the most direct and effective way to deliver therapeutic nucleic acids to the lungs. Although liquid aerosol formulation is investigated in most of the studies, it is not desirable in terms of maintaining the stability of nucleic acid especially during long-term storage. This problem could be circumvented by formulating the therapeutic nucleic acids into dry powder for inhalation, and should be considered as the future direction of developing inhalable nucleic acids. In this review, the three major particle engineering methods investigated for the preparation of inhalable pDNA and siRNA formulations, including spray drying (SD), spray freeze drying (SFD) and supercritical fluid (SFC) drying, are discussed and compared. Moreover, common assessment methods and the challenges of evaluating the biological activities of inhalable nucleic acid powders are also reviewed.

  4. Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker

    PubMed Central

    Heap, John T.; Ehsaan, Muhammad; Cooksley, Clare M.; Ng, Yen-Kuan; Cartman, Stephen T.; Winzer, Klaus; Minton, Nigel P.

    2012-01-01

    Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms. PMID:22259038

  5. Plasmid DNA Initiates Replication of Yellow Fever Vaccine In Vitro and Elicits Virus-Specific Immune Response in Mice

    PubMed Central

    Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat; Jokinen, Jenny; Lukashevich, Igor S.; Pushko, Peter

    2014-01-01

    Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20µg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF. PMID:25129436

  6. Plasmid DNA initiates replication of yellow fever vaccine in vitro and elicits virus-specific immune response in mice.

    PubMed

    Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat; Jokinen, Jenny; Lukashevich, Igor S; Pushko, Peter

    2014-11-01

    Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20 μg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF.

  7. [Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells].

    PubMed

    Misic, V; El-Mogy, M; Geng, S; Haj-Ahmad, Y

    2016-01-01

    Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.

  8. Polyplex-Microbubble Hybrids for Ultrasound-Guided Plasmid DNA Delivery to Solid Tumors

    PubMed Central

    Sirsi, Shashank; Hernandez, Sonia; Zielinski, Lukasz; Blomback, Henning; Koubaa, Adel; Synder, Milo; Homma, Shunichi; Kandel, Jessica J.; Yamashiro, Darrell J.; Borden, Mark A.

    2013-01-01

    Microbubble ultrasound contrast agents are being developed as image-guided gene carriers for targeted delivery in vivo. In this study, novel polyplex-microbubbles were synthesized, characterized and evaluated for systemic circulation and tumor transfection. Branched polyethylenimine (PEI; 25 kDa) was modified with polyethylene glycol (PEG; 5 kDa), thiolated and covalently attached to maleimide groups on lipid-coated microbubbles. The PEI-microbubbles demonstrated increasingly positive surface charge and DNA loading capacity with increasing maleimide content. The in vivo ultrasound contrast persistence of PEI-microbubbles was measured in the healthy mouse kidney, and a two-compartment pharmacokinetic model accounting for free and adherent microbubbles was developed to describe the anomalous time-intensity curves. The model suggested that PEI loading dramatically reduced free circulation and increased nonspecific adhesion to the vasculature. However, DNA loading to form polyplex-microbubbles increased circulation in the bloodstream and decreased nonspecific adhesion. PEI-microbubbles coupled to a luciferase bioluminescence reporter plasmid DNA were shown to transfect tumors implanted in the mouse kidney. Site-specific delivery was achieved using ultrasound applied over the tumor area following bolus injection of the DNA/PEI-microbubbles. In vivo imaging showed over 10-fold higher bioluminescence from the tumor region compared to untreated tissue. Ex vivo analysis of excised tumors showed greater than 40-fold higher expression in tumor tissue than non-sonicated control (heart) tissue. These results suggest that the polyplex-microbubble platform offers improved control of DNA loading and packaging suitable for ultrasound-guided tissue transfection. PMID:21945680

  9. Recombinant goose-type lysozyme in channel catfish: Lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme g (CC-Lys-g) produced in E. coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme g plasmid DNA could be used as an immunostimulant to protect chann...

  10. Recombinant goose-type lysozyme in channel catfish: lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme g (CC-Lys-g) produced in E. coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme g plasmid DNA could be used as an immunostimulant to protect chann...

  11. Fetal Gene Therapy for Ornithine Transcarbamylase Deficiency by Intrahepatic Plasmid DNA-Micro-Bubble Injection Combined with Hepatic Ultrasound Insonation.

    PubMed

    Oishi, Yoshie; Kakimoto, Takashi; Yuan, Wenji; Kuno, Shuichi; Yamashita, Hiromasa; Chiba, Toshio

    2016-06-01

    We evaluated the therapeutic efficacy of hepatic transfection of plasmid DNA using micro-bubbles and ultrasound insonation for fetal correction of ornithine transcarbamylase (OTC) deficiency in mice. Twenty-three sparse-fur heterozygous pregnant mice (day 16 of gestation) were divided into three groups: injection of plasmid-DNA micro-bubble mixture into fetal liver with ultrasound insonation (Tr, n = 11); control group 1 (C1), injection of plasmid-DNA micro-bubble mixture into fetal liver with no insonation (n = 5); and control group 2 (C2), injection of saline-micro-bubble mixture into fetal liver with ultrasound insonation (n = 7). Levels of blood ammonia and urinary orotic acid were significantly lower in the Tr group than in the C1 and C2 groups (p < 0.05, p < 0.01, respectively), whereas OTC activity was not different between groups. Therefore, ultrasound insonation with micro-bubbles enhanced plasmid DNA transfection into fetal mouse liver, leading to one of the therapeutic methods in ammonia metabolism. This might provide more time for OTC-deficient infants until liver transplantation.

  12. Selective ploidy ablation, a high-throughput plasmid transfer protocol, identifies new genes affecting topoisomerase I–induced DNA damage

    PubMed Central

    Reid, Robert J.D.; González-Barrera, Sergio; Sunjevaric, Ivana; Alvaro, David; Ciccone, Samantha; Wagner, Marisa; Rothstein, Rodney

    2011-01-01

    We have streamlined the process of transferring plasmids into any yeast strain library by developing a novel mating-based, high-throughput method called selective ploidy ablation (SPA). SPA uses a universal plasmid donor strain that contains conditional centromeres on every chromosome. The plasmid-bearing donor is mated to a recipient, followed by removal of all donor-strain chromosomes, producing a haploid strain containing the transferred plasmid. As proof of principle, we used SPA to transfer plasmids containing wild-type and mutant alleles of DNA topoisomerase I (TOP1) into the haploid yeast gene-disruption library. Overexpression of Top1 identified only one sensitive mutation, rpa34, while overexpression of top1-T722A allele, a camptothecin mimetic, identified 190 sensitive gene-disruption strains along with rpa34. In addition to known camptothecin-sensitive strains, this set contained mutations in genes involved in the Rpd3 histone deacetylase complex, the kinetochore, and vesicle trafficking. We further show that mutations in several ESCRT vesicle trafficking components increase Top1 levels, which is dependent on SUMO modification. These findings demonstrate the utility of the SPA technique to introduce plasmids into the haploid gene-disruption library to discover new interacting pathways. PMID:21173034

  13. Physiological effects of pH gradients on Escherichia coli during plasmid DNA production.

    PubMed

    Cortés, José T; Flores, Noemí; Bolívar, Francisco; Lara, Alvaro R; Ramírez, Octavio T

    2016-03-01

    A two-compartment scale-down system was used to mimic pH heterogeneities that can occur in large-scale bioreactors. The system consisted of two interconnected stirred tank reactors (STRs) where one of them represented the conditions of the bulk of the fluid and the second one the zone of alkali addition for pH control. The working volumes ratio of the STRs was set to 20:1 in order to simulate the relative sizes of the bulk and alkali addition zones, respectively, in large-scale bioreactors. Residence times (tR ) in the alkali addition STR of 60, 120, 180, and 240 s were simulated during batch cultures of an engineered Escherichia coli strain that produced plasmid DNA (pDNA). pH gradients of up to 0.9 units, between the two compartments, were attained. The kinetic, stoichiometric, and pDNA topological changes due to the pH gradients were studied and compared to cultures at constant pH of 7.2 and 8.0. As the tR increased, the pDNA and biomass yields, as well as pDNA final titer decreased, whereas the accumulation of organic acids increased. Furthermore, the transcriptional response of 10 selected genes to alkaline stress (pH 8.0) and pH gradients was monitored at different stages of the cultures. The selected genes coded for ion transporters, amino acids catabolism enzymes, and transcriptional regulators. The transcriptional response of genes coding for amino acids catabolism, in terms of relative transcription level and stage of maximal expression, was different when the alkaline stress was constant or transient. This suggests the activation of different mechanisms by E. coli to cope with pH fluctuations compared to constant alkaline pH. Moreover, the transcriptional response of genes related to negative control of DNA synthesis did not correlate with the lower pDNA yields. This is the first study that reports the effects of pH gradients on pDNA production by E. coli cultures. The information presented can be useful for the design of better bioreactor scale

  14. Designing plasmid vectors.

    PubMed

    Tolmachov, Oleg

    2009-01-01

    Nonviral gene therapy vectors are commonly based on recombinant bacterial plasmids or their derivatives. The plasmids are propagated in bacteria, so, in addition to their therapeutic cargo, they necessarily contain a bacterial replication origin and a selection marker, usually a gene conferring antibiotic resistance. Structural and maintenance plasmid stability in bacteria is required for the plasmid DNA production and can be achieved by carefully choosing a combination of the therapeutic DNA sequences, replication origin, selection marker, and bacterial strain. The use of appropriate promoters, other regulatory elements, and mammalian maintenance devices ensures that the therapeutic gene or genes are adequately expressed in target human cells. Optimal immune response to the plasmid vectors can be modulated via inclusion or exclusion of DNA sequences containing immunostimulatory CpG sequence motifs. DNA fragments facilitating construction of plasmid vectors should also be considered for inclusion in the design of plasmid vectors. Techniques relying on site-specific or homologous recombination are preferred for construction of large plasmids (>15 kb), while digestion of DNA by restriction enzymes with subsequent ligation of the resulting DNA fragments continues to be the mainstream approach for generation of small- and medium-size plasmids. Rapid selection of a desired recombinant plasmid against a background of other plasmids continues to be a challenge. In this chapter, the emphasis is placed on efficient and flexible versions of DNA cloning protocols using selection of recombinant plasmids by restriction endonucleases directly in the ligation mixture.

  15. Plasmids from Euryarchaeota.

    PubMed

    Forterre, Patrick; Krupovic, Mart; Raymann, Kasie; Soler, Nicolas

    2014-12-01

    Many plasmids have been described in Euryarchaeota, one of the three major archaeal phyla, most of them in salt-loving haloarchaea and hyperthermophilic Thermococcales. These plasmids resemble bacterial plasmids in terms of size (from small plasmids encoding only one gene up to large megaplasmids) and replication mechanisms (rolling circle or theta). Some of them are related to viral genomes and form a more or less continuous sequence space including many integrated elements. Plasmids from Euryarchaeota have been useful for designing efficient genetic tools for these microorganisms. In addition, they have also been used to probe the topological state of plasmids in species with or without DNA gyrase and/or reverse gyrase. Plasmids from Euryarchaeota encode both DNA replication proteins recruited from their hosts and novel families of DNA replication proteins. Euryarchaeota form an interesting playground to test evolutionary hypotheses on the origin and evolution of viruses and plasmids, since a robust phylogeny is available for this phylum. Preliminary studies have shown that for different plasmid families, plasmids share a common gene pool and coevolve with their hosts. They are involved in gene transfer, mostly between plasmids and viruses present in closely related species, but rarely between cells from distantly related archaeal lineages. With few exceptions (e.g., plasmids carrying gas vesicle genes), most archaeal plasmids seem to be cryptic. Interestingly, plasmids and viral genomes have been detected in extracellular membrane vesicles produced by Thermococcales, suggesting that these vesicles could be involved in the transfer of viruses and plasmids between cells.

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

    SciTech Connect

    Swenberg, C.E.; Speicher, J.M.

    1995-12-01

    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 ({sigma}) 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 10{sup 8} to 4 x 10{sup 8} {angstrom}{sup 3}. Samples were exposed to either fission-neutron or {sup 60}Co {gamma} radiation and assayed for single-strand breaks by agarose gel electrophoresis. Form I DNA for all topoisomers decreased exponentially with increasing dose. The D{sub 37} values for both neutron and {gamma} radiation increased monotonically with increasing {vert_bar}{sigma}{vert_bar}. Using a branched plectonemic (interwound) form for DNA over the range of {sigma} studied and standard (single-hit) target theory, a quantitative linear fit to (D{sub 37}{sup -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 (D{sub 37}){sup -1} as a function of S({angstrom}) are directly proportional to the length of DNA and the radiation fluence. Furthermore, the ratio b/a (= r{sub o}) at {sigma} = 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 r{sub o} = 13.4 {+-} 1.4 nm, a value consistent with other investigations. Our results are consistent with studies using {sup 137}Cs but disagree with data obtained for X rays. 31 refs., 6 figs., 1 tab.

  17. Crystal Structure of pi Initiator Protein-iteron Complex of Plasmid R6K: Implications for Initiation of Plasmid DNA Replication

    SciTech Connect

    Swan,M.; Bastia, D.; Davies, C.

    2006-01-01

    We have determined the crystal structure of a monomeric biologically active form of the {pi} initiator protein of plasmid R6K as a complex with a single copy of its cognate DNA-binding site (iteron) at 3.1-{angstrom} resolution. The initiator belongs to the family of winged helix type of proteins. The structure reveals that the protein contacts the iteron DNA at two primary recognition helices, namely the C-terminal {alpha}4' and the N-terminal {alpha}4 helices, that recognize the 5' half and the 3' half of the 22-bp iteron, respectively. The base-amino acid contacts are all located in {alpha}4', whereas the {alpha}4 helix and its vicinity mainly contact the phosphate groups of the iteron. Mutational analyses show that the contacts of both recognition helices with DNA are necessary for iteron binding and replication initiation. Considerations of a large number of site-directed mutations reveal that two distinct regions, namely {alpha}2 and {alpha}5 and its vicinity, are required for DNA looping and initiator dimerization, respectively. Further analysis of mutant forms of {pi} revealed the possible domain that interacts with the DnaB helicase. Thus, the structure-function analysis presented illuminates aspects of initiation mechanism of R6K and its control.

  18. Screening of L-histidine-based ligands to modify monolithic supports and selectively purify the supercoiled plasmid DNA isoform.

    PubMed

    Amorim, Lúcia F A; Sousa, Fani; Queiroz, João A; Cruz, Carla; Sousa, Ângela

    2015-06-01

    The growing demand of pharmaceutical-grade plasmid DNA (pDNA) suitable for biotherapeutic applications fostered the development of new purification strategies. The surface plasmon resonance technique was employed for a fast binding screening of l-histidine and its derivatives, 1-benzyl-L-histidine and 1-methyl-L-histidine, as potential ligands for the biorecognition of three plasmids with different sizes (6.05, 8.70, and 14 kbp). The binding analysis was performed with different isoforms of each plasmid (supercoiled, open circular, and linear) separately. The results revealed that the overall affinity of plasmids to l-histidine and its derivatives was high (KD  > 10(-8)  M), and the highest affinity was found for human papillomavirus 16 E6/E7 (K(D)  = 1.1 × 10(-10)  M and KD  = 3.34 × 10(-10)  M for open circular and linear plasmid isoforms, respectively). L-Histidine and 1-benzyl-L-histidine were immobilized on monolithic matrices. Chromatographic studies of L-histidine and 1-benzyl-L-histidine monoliths were also performed with the aforementioned samples. In general, the supercoiled isoform had strong interactions with both supports. The separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration in the eluent, in both supports, but a lower salt concentration was required in the 1-benzyl-L-histidine monolith because of stronger interactions promoted with pDNA. The efficiency of plasmid isoforms separation remained unchanged with flow rate variations. The binding capacity for pDNA achieved with the l-histidine monolith was 29-fold higher than that obtained with conventional L-histidine agarose. Overall, the combination of either L-histidine or its derivatives with monolithic supports can be a promising strategy to purify the supercoiled isoform from different plasmids with suitable purity degree for pharmaceutical applications.

  19. Transfer of plasmid DNA to clinical coagulase-negative staphylococcal pathogens by using a unique bacteriophage.

    PubMed

    Winstel, Volker; Kühner, Petra; Krismer, Bernhard; Peschel, Andreas; Rohde, Holger

    2015-04-01

    Genetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a unique Staphylococcus aureus strain via a specific S. aureus bacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinical Staphylococcus epidermidis isolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion.

  20. Self-Assembled Functional Nanostructure of Plasmid DNA with Ionic Liquid [Bmim][PF₆]: Enhanced Efficiency in Bacterial Gene Transformation.

    PubMed

    Soni, Sarvesh K; Sarkar, Sampa; Mirzadeh, Nedaossadat; Selvakannan, P R; Bhargava, Suresh K

    2015-04-28

    The electrostatic interaction between the negatively charged phosphate groups of plasmid DNA and the cationic part of hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF6]), initiates spontaneous self-assembly to form the functional nanostructures made up of DNA and ionic liquid (IL). These functional nanostructures were demonstrated as promising synthetic nonviral vectors for the efficient bacterial pGFP gene transformation in cells. In particular, the functional nanostructures that were made up of 1 μL of IL ([Bmim][PF6]) and 1 μg of plasmid DNA can increase the transformation efficiency by 300-400% in microbial systems, without showing any toxicity for E. coli DH5α cells. (31)P nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron (XPS) spectroscopic analysis revealed that the electrostatic interaction between negatively charged phosphate oxygen and cationic Bmim(+) tends to initiate the self-assembly process. Thermogravimetric analysis of the DNA-IL functional nanostructures showed that these nanostructures consist of ∼16 wt % ionic liquid, which is considered to provide the stability to the plasmid DNA that eventually enhanced the transformation efficiency.

  1. Homology mapping of T-DNA regions on three Agrobacterium rhizogenes Ri plasmids by electron microscope heteroduplex studies.

    PubMed

    Brevet, J; Tempé, J

    1988-03-01

    Recombinant plasmids carrying segments of the Agrobacterium rhizogenes T-DNA regions of the three Ri plasmids 1855 (TL-DNA only), 8196, and 2659 were used for establishing homology maps by electron microscope examination of heteroduplexes. Plasmid DNA was linearized by digestion with suitable restriction endonucleases in order to generate large T-DNA segments. Heteroduplexes were prepared in 50% formamide and spread under standard conditions. Measurements of double and single strands allowed the drawing of homology maps. The three T-DNAs share mainly two homologous sequences of respectively about 2.5 and 1.5 kb, bracketing a largely nonhomologous central part which is about 5.5 kb long. The T-DNAs from pRi1855 and pRi2659 appear to be more related to each other than to that of pRi8196. With reference to the published nucleotide sequence of the TL-DNA of pRiA4 (probably identical to that of pRi1855), ORFs 8 and 14 seem to be the most conserved sequences of the three T-DNAs. The significance of these conserved sequences is unclear since the genetic loci involved in rhizogenicity of agropine strains identified previously are located in nonhomologous regions.

  2. Correction of the lack of commutability between plasmid DNA and genomic DNA for quantification of genetically modified organisms using pBSTopas as a model.

    PubMed

    Zhang, Li; Wu, Yuhua; Wu, Gang; Cao, Yinglong; Lu, Changming

    2014-10-01

    Plasmid calibrators are increasingly applied for polymerase chain reaction (PCR) analysis of genetically modified organisms (GMOs). To evaluate the commutability between plasmid DNA (pDNA) and genomic DNA (gDNA) as calibrators, a plasmid molecule, pBSTopas, was constructed, harboring a Topas 19/2 event-specific sequence and a partial sequence of the rapeseed reference gene CruA. Assays of the pDNA showed similar limits of detection (five copies for Topas 19/2 and CruA) and quantification (40 copies for Topas 19/2 and 20 for CruA) as those for the gDNA. Comparisons of plasmid and genomic standard curves indicated that the slopes, intercepts, and PCR efficiency for pBSTopas were significantly different from CRM Topas 19/2 gDNA for quantitative analysis of GMOs. Three correction methods were used to calibrate the quantitative analysis of control samples using pDNA as calibrators: model a, or coefficient value a (Cva); model b, or coefficient value b (Cvb); and the novel model c or coefficient formula (Cf). Cva and Cvb gave similar estimated values for the control samples, and the quantitative bias of the low concentration sample exceeded the acceptable range within ±25% in two of the four repeats. Using Cfs to normalize the Ct values of test samples, the estimated values were very close to the reference values (bias -13.27 to 13.05%). In the validation of control samples, model c was more appropriate than Cva or Cvb. The application of Cf allowed pBSTopas to substitute for Topas 19/2 gDNA as a calibrator to accurately quantify the GMO.

  3. A candidate reference method for quantification of low concentrations of plasmid DNA by exhaustive counting of single DNA molecules in a flow stream

    NASA Astrophysics Data System (ADS)

    Yoo, Hee-Bong; Oh, Donggeun; Song, Jae Yong; Kawaharasaki, Mamoru; Hwang, Jeeseong; Yang, In Chul; Park, Sang-Ryoul

    2014-10-01

    This work demonstrates accurate measurement of the amount of substance concentration of low concentration plasmid DNA by counting individual DNA molecules using a high-sensitivity flow cytometric setup. Plasmid DNA is a widely used form of DNA, and its quantity often needs to be accurately determined. This work establishes a reference analytical method for direct quantification of low concentration plasmid DNA prepared as reference standards for polymerase chain reaction-based DNA quantification. The model plasmid DNA pBR322 (4361 bp) was stained with a fluorescent dye and was detected in a flow stream in a micro-fluidic channel with laser-induced fluorescence detection, for which the DNA flow was electro-hydrodynamically focused at the centre of the channel. 200 to 8000 DNA molecules in a ˜1 µL sample volume were counted within 2 min in an ‘exhaustive counting’ manner, which facilitated quantitation without calibration. The sample volume was measured and validated from the close agreement of the results of two independent measurement methods, gravimetric determination of water filling the capillary and graphical estimation of actual cross sectional area of the capillary tubing with the image of calibrated scanning electron microscopy. Within the given concentration range, an excellent measurement linearity (R2 = 0.999) was achieved with appropriate data processing for the correction of the events of double molecules (detection of double molecules opposed to single molecule detection assumed, which occurs due to their coincidental passing of the detection zone). The validity of the proposed method was confirmed from the close agreement with the results of quantitation of enzymatically released nucleotides using capillary electrophoresis.

  4. Functionalized Multi-Wall Carbon Nanotubes Enhance Transfection and Expression Efficiency of Plasmid DNA in Fish Cells.

    PubMed

    Liu, Guanglu; Wang, Yuan; Hu, Yang; Yu, Xiaobo; Zhu, Bin; Wang, Gaoxue

    2016-03-03

    DNA vaccines are considered to be the most promising method against infectious diseases in the aquaculture industry. In the present study, we investigated the potency of ammonium group-functionalized multi-walled carbon nanotubes (MWCNTs) in enhancing the transfection and expression efficiency of plasmid DNA (pEGFP-vp5) in Ctenopharyngodon idellus kidney (CIK) cells. Agarose gel shift assay results show that ammonium group-functionalized carbon nanotubes are able to condense DNA in varying degrees. Scanning electron microscope (SEM) images shows that CIK cells show a great affinity for MWCNTs-NH₃⁺ and the CNTs covering the cell surface tend to orient their tips perpendicularly to the cell surface, and appear to be "needle-pricking the cells". Transmission electron microscope (TEM) images confirmed that MWCNTs-NH₃⁺ penetrate the cell membranes and are widely dispersed in the CIK cell. Real-time PCR was used to detect the transfection efficiency through the expression of the outer capsid protein (VP5). The results showed that the MWCNTs-NH₃⁺:DNA complexes are able to transfect CIK cells effectively at different charge ratio than naked DNA. Subsequent studies confirmed that both functional groups and charge ratio are important factors that determine the transfection efficiency of plasmid DNA. All these results indicated that MWCNTs-NH₃⁺:DNA complexes could be suitable for developing DNA vaccine for the control of virus infection in the aquaculture industry.

  5. DNA damage produced by exposure of supercoiled plasmid DNA to high- and low-LET ionizing radiation: Effects of hydroxyl radical quenchers. DNA breakage, neutrons, OH radicals

    SciTech Connect

    Peak, J.G.; Ito, T.; Peak, M.J.; Robb, F.T.

    1994-08-01

    A supercoiled plasmid of 7300 base pairs was isolated and exposed in an aqueous environment to {sup 60}Co {gamma} rays and JANUS 0.85 MeV fission-spectrum neutrons. Dose responses for the production of single-strand breaks (SSBs), double-strand breaks (DSBs) and alkali-labile sites (ALSs) were compared with computations made from the conversion of the supercoil to its relaxed and linear forms. The relative biological effectiveness (RBE) for production of SSBs and DSBs was similar to that previously measured in the cellular environment. The RBE for destruction of genetic transforming activity of M13 viral DNA followed that for DNA damage. This is in contrast to the situation for biological effects such as lethality, mutagenesis, and cellular transformation measured in mammalian cells, where the RBE values are reversed. The role of hydroxyl (OH) radical in DNA damage induction by neutrons was investigated by exposure of plasmid in the presence of known quenchers of this species. Of four quenchers tested, all were able to reduce the yields of both SSBs and DSBs. These findings are consistent with a model for SSB and DSB induction by high linear energy transfer that involves OH radical mediation.

  6. Size effect on transfection and cytotoxicity of nanoscale plasmid DNA/polyethyleneimine complexes for aerosol gene delivery

    SciTech Connect

    Hoon Byeon, Jeong; Kim, Jang-Woo

    2014-02-03

    Nanoscale plasmid DNA (pDNA)/polyethyleneimine (PEI) complexes were fabricated in the aerosol state using a nebulization system consisting of a collison atomizer and a cool-walled diffusion dryer. The aerosol fabricated nanoscale complexes were collected and employed to determine fundamental properties of the complexes, such as size, structure, surface charge, and in vitro gene transfection efficiency and cytotoxicity. The results showed that mass ratio between pDNA and PEI should be optimized to enhance gene transfection efficiency without a significant loss of cell viability. These findings may support practical advancements in the field of nonviral gene delivery.

  7. Absolute cross section for loss of supercoiled topology induced by 10 eV electrons in highly uniform /DNA/1,3-diaminopropane films deposited on highly ordered pyrolitic graphite

    NASA Astrophysics Data System (ADS)

    Boulanouar, Omar; Fromm, Michel; Bass, Andrew D.; Cloutier, Pierre; Sanche, Léon

    2013-08-01

    It was recently shown that the affinity of doubly charged, 1-3 diaminopropane (Dap2+) for DNA permits the growth on highly ordered pyrolitic graphite (HOPG) substrates, of plasmid DNA films, of known uniform thickness [O. Boulanouar, A. Khatyr, G. Herlem, F. Palmino, L. Sanche, and M. Fromm, J. Phys. Chem. C 115, 21291-21298 (2011)]. Post-irradiation analysis by electrophoresis of such targets confirms that electron impact at 10 eV produces a maximum in the yield of single strand breaks that can be associated with the formation of a DNA- transient anion. Using a well-adapted deterministic survival model for the variation of electron damage with fluence and film thickness, we have determined an absolute cross section for strand-break damage by 10 eV electrons and inelastic scattering attenuation length in DNA-Dap complex films.

  8. Absolute cross section for loss of supercoiled topology induced by 10 eV electrons in highly uniform /DNA/1,3-diaminopropane films deposited on highly ordered pyrolitic graphite.

    PubMed

    Boulanouar, Omar; Fromm, Michel; Bass, Andrew D; Cloutier, Pierre; Sanche, Léon

    2013-08-07

    It was recently shown that the affinity of doubly charged, 1-3 diaminopropane (Dap(2+)) for DNA permits the growth on highly ordered pyrolitic graphite (HOPG) substrates, of plasmid DNA films, of known uniform thickness [O. Boulanouar, A. Khatyr, G. Herlem, F. Palmino, L. Sanche, and M. Fromm, J. Phys. Chem. C 115, 21291-21298 (2011)]. Post-irradiation analysis by electrophoresis of such targets confirms that electron impact at 10 eV produces a maximum in the yield of single strand breaks that can be associated with the formation of a DNA(-) transient anion. Using a well-adapted deterministic survival model for the variation of electron damage with fluence and film thickness, we have determined an absolute cross section for strand-break damage by 10 eV electrons and inelastic scattering attenuation length in DNA-Dap complex films.

  9. Absolute cross section for loss of supercoiled topology induced by 10 eV electrons in highly uniform /DNA/1,3-diaminopropane films deposited on highly ordered pyrolitic graphite

    SciTech Connect

    Boulanouar, Omar; Fromm, Michel; Bass, Andrew D.; Cloutier, Pierre; Sanche, Léon

    2013-08-07

    It was recently shown that the affinity of doubly charged, 1-3 diaminopropane (Dap{sup 2+}) for DNA permits the growth on highly ordered pyrolitic graphite (HOPG) substrates, of plasmid DNA films, of known uniform thickness [O. Boulanouar, A. Khatyr, G. Herlem, F. Palmino, L. Sanche, and M. Fromm, J. Phys. Chem. C 115, 21291–21298 (2011)]. Post-irradiation analysis by electrophoresis of such targets confirms that electron impact at 10 eV produces a maximum in the yield of single strand breaks that can be associated with the formation of a DNA{sup −} transient anion. Using a well-adapted deterministic survival model for the variation of electron damage with fluence and film thickness, we have determined an absolute cross section for strand-break damage by 10 eV electrons and inelastic scattering attenuation length in DNA-Dap complex films.

  10. Gene therapy using plasmid DNA-encoded anti-HER2 antibody for cancers that overexpress HER2

    PubMed Central

    Kim, H; Danishmalik, S N; Hwang, H; Sin, J-I; Oh, J; Cho, Y; Lee, H; Jeong, M; Kim, S-H; Hong, H J

    2016-01-01

    Plasmid DNA-encoded antibodies, or DNA-based monoclonal antibodies (dMAbs), are delivered by intramuscular injection and in vivo electroporation (EP) and are effective in virus neutralization, although they have not been evaluated for tumor gene therapy. Here we investigated whether a dMAb was appropriate for tumor gene therapy. We constructed the expression plasmids coding for the heavy or light chain of a parental murine antibody of Herceptin with the antibody genes codon- and RNA-optimized and fused to the Kozak-IgE leader sequence in pVax1. Transfection of the plasmids into human muscle RD cells resulted in functional expression of the antibody, and this exhibited the same in vitro antiproliferative activity as Herceptin. A single intramuscular injection and in vivo EP of the plasmids (100 μg per head) resulted in high and sustained antibody expression in the sera of normal mice and in effective inhibition of tumor growth in nude mice bearing HER2-positive human breast carcinoma BT474 xenografts. The antitumor efficacy of the anti-HER2 dMAb was similar to that of four doses of intravenously injected 10 mg kg−1 Herceptin. The results demonstrate that the dMAb is effective in the treatment of HER2-positive breast cancer, suggesting that this dMAb may be applicable for tumor gene therapy. PMID:27632934

  11. Identification of Erwinia amylovora, the Fireblight Pathogen, by Colony Hybridization with DNA from Plasmid pEA29

    PubMed Central

    Falkenstein, Hildegard; Bellemann, Peter; Walter, Sabine; Zeller, Wolfgang; Geider, Klaus

    1988-01-01

    All strains of Erwinia amylovora characterized carry a medium-size plasmid of 29 kilobases (pEA29). We mapped this plasmid with various restriction enzymes, cloned the whole DNA into an Escherichia coli plasmid, and subcloned restriction fragments. These DNA species were used for identification of E. amylovora after handling of strains in the laboratory and also in field isolates. About 70 strains of E. amylovora and 24 strains from nine other species, mainly found in plant habitats, were checked in a colony hybridization test. Virulent and avirulent E. amylovora strains reacted positively, whereas the other species were negative. Apart from the hybridization assay, the positive strains were additionally tested for ooze production on rich agar with 5% sucrose and on immature-pear slices. Unspecific background hybridization of non-E. amylovora strains found for hybridization with the whole E. amylovora plasmid was almost eliminated when a 5-kilobase SalI fragment from pEA29 was used as a probe and when the washes after the hybridization procedure were done with high stringency. Under these conditions, E. amylovora could be readily identified from field isolates. Images PMID:16347778

  12. Optimization of supercoiled HPV-16 E6/E7 plasmid DNA purification with arginine monolith using design of experiments.

    PubMed

    Almeida, A M; Queiroz, J A; Sousa, F; Sousa, A

    2015-01-26

    The progress of DNA vaccines is dependent on the development of suitable chromatographic procedures to successfully purify genetic vectors, such as plasmid DNA. Human Papillomavirus is associated with the development of tumours due to the oncogenic power of E6 and E7 proteins, produced by this virus. The supercoiled HPV-16 E6/E7 plasmid-based vaccine was recently purified with the arginine monolith, with 100% of purity, but only 39% of recovery was achieved. Therefore, the present study describes the application of experimental design tools, a newly explored methodology in preparative chromatography, in order to improve the supercoiled plasmid DNA recovery with the arginine monolith, maintaining the high purity degree. In addition, the importance and influence of pH in the pDNA retention to the arginine ligand was also demonstrated. The Composite Central Face design was validated and the recovery of the target molecule was successfully improved from 39% to 83.5%, with an outstanding increase of more than double, while maintaining 100% of purity.

  13. A combined approach of hollow microneedles and nanocarriers for skin immunization with plasmid DNA encoding ovalbumin.

    PubMed

    Pamornpathomkul, Boonnada; Wongkajornsilp, Adisak; Laiwattanapaisal, Wanida; Rojanarata, Theerasak; Opanasopit, Praneet; Ngawhirunpat, Tanasait

    2017-01-01

    The aim of this study was to investigate the use of different types of microneedles (MNs) and nanocarriers for in vitro skin permeation and in vivo immunization of plasmid DNA encoding ovalbumin (pOVA). In vitro skin permeation studies indicated that hollow MNs had a superior enhancing effect on skin permeation compared with solid MN patches, electroporation (EP) patches, the combination of MN and EP patches, and untreated skin. Upon using hollow MNs combined with nanocarriers for pOVA delivery, the skin permeation was higher than for the delivery of naked pOVA, as evidenced by the increased amount of pOVA in Franz diffusion cells and immunoglobulin G (IgG) antibody responses. When the hollow MNs were used for the delivery of nanocarrier:pOVA complexes into the skin of mice, they induced a stronger IgG immune response than conventional subcutaneous (SC) injections. In addition, immunization of mice with the hollow MNs did not induce signs of skin infection or pinpoint bleeding. Accordingly, the hollow MNs combined with a nanocarrier delivery system is a promising approach for delivering pOVA complexes to the skin for promoting successful immunization.

  14. A combined approach of hollow microneedles and nanocarriers for skin immunization with plasmid DNA encoding ovalbumin

    PubMed Central

    Pamornpathomkul, Boonnada; Wongkajornsilp, Adisak; Laiwattanapaisal, Wanida; Rojanarata, Theerasak; Opanasopit, Praneet; Ngawhirunpat, Tanasait

    2017-01-01

    The aim of this study was to investigate the use of different types of microneedles (MNs) and nanocarriers for in vitro skin permeation and in vivo immunization of plasmid DNA encoding ovalbumin (pOVA). In vitro skin permeation studies indicated that hollow MNs had a superior enhancing effect on skin permeation compared with solid MN patches, electroporation (EP) patches, the combination of MN and EP patches, and untreated skin. Upon using hollow MNs combined with nanocarriers for pOVA delivery, the skin permeation was higher than for the delivery of naked pOVA, as evidenced by the increased amount of pOVA in Franz diffusion cells and immunoglobulin G (IgG) antibody responses. When the hollow MNs were used for the delivery of nanocarrier:pOVA complexes into the skin of mice, they induced a stronger IgG immune response than conventional subcutaneous (SC) injections. In addition, immunization of mice with the hollow MNs did not induce signs of skin infection or pinpoint bleeding. Accordingly, the hollow MNs combined with a nanocarrier delivery system is a promising approach for delivering pOVA complexes to the skin for promoting successful immunization. PMID:28184159

  15. Breakthrough performance of plasmid DNA on ion-exchange membrane columns.

    PubMed

    Montesinos-Cisneros, Rosa Ma; Olivas, Jonathan de la Vega; Ortega, Jaime; Guzmán, Roberto; Tejeda-Mansir, Armando

    2007-01-01

    Breakthrough performance of plasmid DNA adsorption on ion-exchange membrane columns was theoretically and experimentally investigated using batch and fixed-bed systems. System dispersion curves showed the absence of flow non-idealities in the experimental arrangement. Breakthrough curves (BTC) were significantly affected by inlet flow rate and solute concentration. In the theoretical analysis, a model was integrated by the serial coupling of the membrane transport model and the system dispersion model. A transport model that considers finite kinetic rate and column dispersed flow was used in the study. A simplex optimization routine, coupled to the solution of the partial differential model equations, was employed to estimate the maximum adsorption capacity constant, the equilibrium desorption constant, and the forward interaction rate constant, which are the parameters of the membrane transport model. The analysis shows that as inlet concentration or flow rate increases, the deviation of the model from the experimental behavior decreases. The BTCs displacement as inlet concentration increases was explained in terms of a greater degree of column saturation reached and more efficient operation accomplished. The degree of column saturation was not influenced by inlet flow rate. It was necessary to consider in the column model the slight variation in the BTC produced by the axial dispersion, in order to accomplish the experimental curve dispersion. Consequently, the design criteria that for Pe > 40 the column axial dispersion can be neglected should be taken with precaution.

  16. Contactless magneto-permeabilization for intracellular plasmid DNA delivery in-vivo.

    PubMed

    Kardos, Thomas J; Rabussay, Dietmar P

    2012-11-01

    Electroporation, an attractive process for delivering DNA and other molecules into target cells in vivo and in vitro is limited by the necessity of electrodes that need to be in contact with the subject or object to be electroporated. We have used magnetic fields, which do not require material contact with the subject, to temporarily permeabilize cells in guinea pig skin in vivo to enhance uptake and expression of GFP plasmid DNA. The results show for the first time that magnetic fields can trigger a process likely similar to electroporation. In designing the magnetic pulses, our most important criterion was a high rate of change of the magnetic field, based on the principle described by Michael Faraday which is expressed by the formula: E = -dB/dt, (E, electric field, B, magnetic field, t, time). Magnetic fields were generated by a flat electromagnet in a hand-held applicator positioned above the target tissue. The magnetic pulses had a peak magnetic flux density of 4 tesla; 50 pulses were applied in 5 sec. Biphasic magnetic pulses were twice as effective as monophasic pulses and about equally effective as traditional electroporation pulses . Advantages of magnetopermeabilization over electoporation include: No contact between applicator and subject ("contact-less"); no need for invasive, disposable, sterile electrodes ("needle-less"); no pain from needles and reduced overall pain; no known side effects; easier and faster to administer than electroporation; less expensive due to absence of disposables; and, importantly, greater tissue penetration of the magnetic field allowing treatment of anatomical areas inaccessible by electroporation.

  17. The plasmid replicon of EBV consists of multiple cis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element.

    PubMed Central

    Aiyar, A; Tyree, C; Sugden, B

    1998-01-01

    Plasmids containing oriP, the plasmid origin of Epstein-Barr virus (EBV), are replicated stably in human cells that express a single viral trans-acting factor, EBNA-1. Unlike plasmids of other viruses, but akin to human chromosomes, oriP plasmids are synthesized once per cell cycle, and are partitioned faithfully to daughter cells during mitosis. Although EBNA-1 binds multiple sites within oriP, its role in DNA synthesis and partitioning has been obscure. EBNA-1 lacks enzymatic activities that are present in the origin-binding proteins of other mammalian viruses, and does not interact with human cellular proteins that provide equivalent enzymatic functions. We demonstrate that plasmids with oriP or its constituent elements are synthesized efficiently in human cells in the absence of EBNA-1. Further, we show that human cells rapidly eliminate or destroy newly synthesized plasmids, and that both EBNA-1 and the family of repeats of oriP are required for oriP plasmids to escape this catastrophic loss. These findings indicate that EBV's plasmid replicon consists of genetic elements with distinct functions, multiple cis-acting elements that facilitate DNA synthesis and viral cis/trans elements that permit retention of replicated DNA in daughter cells. They also explain historical failures to identify mammalian origins of DNA synthesis as autonomously replicating sequences. PMID:9799247

  18. Polyamine-induced Z-DNA conformation in plasmids containing (dA-dC)n.(dG-dT)n inserts and increased binding of lupus autoantibodies to the Z-DNA form of plasmids.

    PubMed Central

    Thomas, T J; Thomas, T

    1994-01-01

    Blocks of potential Z-DNA-forming (dA-dC)n.(dG-dT)n sequences are ubiquitous in eukaryotic genomes. We examined whether naturally occurring polyamines, putrescine, spermidine and spermine, could provoke the Z-DNA conformation in plasmids pDHf2 and pDHf14 with 23 and 60 bp inserts respectively of (dA-dC)n.(dG-dT)n sequences using an e.l.i.s.a. Spermidine and spermine could provoke Z-DNA conformation in these plasmids, but putrescine was ineffective. For pDHf2 and pDHf14, the concentration of spermidine at the midpoint of B-DNA to Z-DNA transition was 25 microM, whereas that of spermine was 16 microM. Polyamine structural specificity was evident in the ability of spermidine homologues to induce Z-DNA. Inorganic cations, Co(NH3)6(3+) and Ru(NH3)6(3+), were ineffective. Our experiments also showed increased binding of anti-DNA autoantibodies from lupus patients as well as autoimmune MRL-lpr/lpr mice to pDHf2 and pDHf14 in the presence of polyamines. These data demonstrate that small blocks of (dA-dC)n.(dG-dT)n sequences could assume the Z-DNA conformation in the presence of natural polyamines. Increased concentrations of polyamines in the sera of lupus patients might facilitate immune complex-formation involving circulating DNA and anti-Z-DNA antibodies. PMID:8135759

  19. Production of recombinant RNase Ba and its application in downstream processing of plasmid DNA for pharmaceutical use.

    PubMed

    Voss, Carsten; Lindau, Dennis; Flaschel, Erwin

    2006-01-01

    The demand for new strategies in downstream processing of biopharmaceutical plasmid DNA has increased in response to the importance of nucleic acids as active pharmaceutical ingredients (API) in gene therapy and genetic vaccination. Led by the problematic usage of animal-derived proteins for producing reagents of clinical applications, we present an opportunity of removing RNA prior to chromatographic steps by using a recombinant RNase Ba (barnase of Bacillus amyloliquefaciens) as an alternative to bovine RNase A. An expression vector for RNase Ba production was constructed enabling periplasmic localization of the recombinant protein. Cultivation of the RNase-producing clone showed stable activity (3.6 kU mL(-1) during stationary phase) throughout the cultivation process. After purification the RNase activity was tested and compared to that of commercially available RNase A. RNase Ba showed no DNase activity even after prolonged incubation with plasmid DNA. Thus, it is a suitable substitute for bovine RNase A in pharmaceutical purification processes.

  20. New roles of DNA and SopB in polymerization of SopA of Escherichia coli F plasmid.

    PubMed

    Hanai, Ryo; Arai, Yui

    2015-06-01

    Active equi-paritioning of the F plasmid is achieved by its sopABC gene. SopA binds to the sopAB promoter region and SopB binds to sopC. SopA also polymerizes in the presence of ATP and Mg(II), which is stimulated by SopB. Non-specific DNA is known to inhibit SopA polymerization and disassemble SopA polymer. This study followed kinetics of polymerization and de-polymerization of SopA by turbidity measurement and found new effects by DNA and SopB. Plasmid DNA, at low concentrations, shortened the lag (nucleation) phase of SopA polymerization and also caused an initial 'burst' of turbidity. Results with two non-specific 20-bp DNAs indicated sequence/length dependence of these effects. sopAB operator DNA only showed inhibition of SopA polymerization. Results of turbidity decrease of pre-formed SopA polymer in the presence of ethylenediaminetetraacetic acid showed that SopB also accelerates disassembly of the SopA polymer. The steady-state level of turbidity in the presence of SopB and plasmid DNA indicated synergy between SopB and DNA in the disassembly. SopB protein showed no effect on SopA polymerization, when SopB was specifically bound to DNA. This result and others with truncation mutants of SopB suggested that a proper configuration of the domains of SopB is important for SopA-SopB interactions.

  1. Novel synthetic plasmid and Doggybone™ DNA vaccines induce neutralizing antibodies and provide protection from lethal influenza challenge in mice

    PubMed Central

    Scott, Veronica L; Patel, Ami; Villarreal, Daniel O; Hensley, Scott E; Ragwan, Edwin; Yan, Jian; Sardesai, Niranjan Y; Rothwell, Paul J; Extance, Jonathan P; Caproni, Lisa J; Weiner, David B

    2015-01-01

    Nucleic acid-based vaccines (NAVs) are a promising alternative to conventional influenza vaccines with the potential to increase influenza vaccine availability due to their simplicity in design and rapid speed of production. NAVs can also target multiple influenza antigens and control flu variants. Traditionally NAVs have been DNA plasmids however, we are continuing to explore new methods that may enhance vaccine efficacy. Recently new focus has been on RNA cassettes as NAVs. RNA vaccines combine conceptual advantages in that they focus on delivery of only the coding cassette. However, RNA vaccines have a short half-life and cause interferon-induced fevers. Here we describe a new NAV approach where we study delivery of a linear DNA cassette [Doggybone™ linear closed DNA [(dbDNA™)] produced by an enzymatic process that yields an antigen expression cassette comprising a promoter, DNA antigen, poly A tail, and telomeric ends. This focused approach has many of the advantages of plasmid DNA as well as a minimal cassette size similar to RNA strategies. For this study, we characterized the specific CD4+ and CD8+ T cell responses and determined the hemagglutination inhibition (HI) titers induced by dbDNA™ and compared the responses with those of an optimized plasmid DNA (pDNA) vaccine encoding the same H1N1 influenza A/PR/8/34 HA gene. Immunizations with the constructs resulted in similar humoral and cellular immune responses. Both constructs induced high-titer HI antibodies and fully protected animals from lethal viral challenge. The data obtained from this study provides important validation for further development of novel vector approaches. PMID:26091432

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

    NASA Astrophysics Data System (ADS)

    Fayard, B.; Touati, A.; Sage, E.; Abel, F.; Champion, C.; Chetoui, A.

    1999-01-01

    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 (E_K=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. Dans le but d'étudier les conséquences à l'échelle moléculaire d'une photo- ionisation en couche K du carbone de l'ADN, des dépots de plasmides ont été irradiés à sec par des X ultra-mous d'énergies situées de part et d'autre du seuil d'ionisation en couche interne du carbone (E_K=278 eV). Les taux de cassures simple- et double-brin (ssb et dsb) ont été quantifiées après résolution des trois formes de plasmide (surenroulé, circulaire relaché, linéaire) par électrophorèse. Un facteur de 1.2 a été mesuré entre les doses nécessaires à 250 eV et 380 eV pour produire le même nombre de dsb par plasmide.

  3. Thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR.

    PubMed

    Le, Yilin; Chen, Huayou; Zagursky, Robert; Wu, J H David; Shao, Weilan

    2013-08-01

    Polymerase chain reaction (PCR) is a powerful method to produce linear DNA fragments. Here we describe the Tma thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR. In this thermostable DNA ligase-mediated whole-plasmid amplification method, the resultant DNA nick between the 5' end of the PCR primer and the extended newly synthesized DNA 3' end of each PCR cycle is ligated by Tma DNA ligase, resulting in circular plasmid DNA product that can be directly transformed. The template plasmid DNA is eliminated by 'selection marker swapping' upon transformation. When performed under an error-prone condition with Taq DNA polymerase, PPCP allows one-step construction of mutagenesis libraries based on in situ error-prone PCR so that random mutations are introduced into the target gene without altering the expression vector plasmid. A significant difference between PPCP and previously published methods is that PPCP allows exponential amplification of circular DNA. We used this method to create random mutagenesis libraries of a xylanase gene and two cellulase genes. Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PPCP for creating random mutagenesis libraries for directed evolution.

  4. Supercoiled plasmid DNA as a model target for assessing the generation of free radicals at the surface of fibres.

    PubMed

    Donaldson, K; Gilmour, P S; Beswick, P H

    1995-09-01

    The ability of respirable amosite and crocidolite asbestos, refractory ceramic fibres (RCFs) and man made vitreous fibres (MMVFs) to cause free radical injury to plasmid, phiX174 RFI DNA was assessed. The amosite and crocidolite asbestos caused substantial damage to the DNA and, in the main, the free radicals responsible for the asbestos-mediated DNA damage were hydroxyl radicals as determined by inhibition with mannitol. Asbestos fibre-mediated damage to the DNA was completely ameliorated by the chelation of fibre-associated iron by pre-treatment of fibres with desferrioxamine-B, confirming the importance of iron in the production of free radicals. MMVFs and RCFs produced modest free radical damage to the DNA, which was prevented by mannitol but not by iron chelation.

  5. DNA vs. mirror-image DNA: a universal approach to tune the absolute configuration in DNA-based asymmetric catalysis.

    PubMed

    Wang, Jocelyn; Benedetti, Erica; Bethge, Lucas; Vonhoff, Stefan; Klussmann, Sven; Vasseur, Jean-Jacques; Cossy, Janine; Smietana, Michael; Arseniyadis, Stellios

    2013-10-25

    Mirror mirror on the wall: By taking advantage of the unique structural features of L-DNA, the first examples of left-helical enantioselective induction in the field of DNA-based asymmetric catalysis were realized. Most importantly, this approach is the only one that allows a reliable and predictable access to both enantiomers for any given reaction.

  6. Single step plasmid DNA purification using methacrylate monolith bearing combination of ion-exchange and hydrophobic groups.

    PubMed

    Smrekar, Vida; Smrekar, Franc; Strancar, Aleš; Podgornik, Aleš

    2013-02-08

    Purification of high quantities of human grade plasmid DNA is one of the most intensive production steps. Because of that several methods have been proposed, among them also chromatographic purification using methacrylate monoliths. Recently, a process comprising the combination of hydrophobic interaction (HIC) monolith and ion-exchange monolith was developed. In this work both chemistries were tried to be introduced on a single monolith. Methacrylate monoliths bearing octylamine groups, combination of butyl (C4) grafted methacrylate groups and diethylaminoethyl (DEAE) groups as well as grafted chains bearing both C4 and DEAE groups were prepared. All monoliths were investigated for their ionic and protein capacity and compared to conventional epoxy, C4, and DEAE methacrylate monoliths. Octylamine monolith and monolith bearing combination of C4 grafted methacrylate groups and DEAE groups were found to be the most promising candidates and were further tested for plasmid DNA (pDNA) dynamic binding capacity under ion-exchange (IEX) and HIC binding conditions and ability to separate open circular (OC) from supercoiled (SC) pDNA forms and RNA from pDNA. Since monolith bearing combination of grafted C4 methacrylate groups and DEAE groups was superior in all three tested features, exhibiting pDNA dynamic binding capacity of 4.7 mg/ml under IEX conditions and 2.1mg/ml under HIC conditions, it was used for the development of a single step purification method and tested with pure pDNA as well as with cell lysate. Developed method removed over 99% of RNA, host cell proteins (HCP) and genomic DNA (gDNA) demonstrating capacity to purify around 1.5mg of pDNA/ml of monolith from cell lysate.

  7. Isolation and characterization of a plasmid DNA from periodontopathogenic bacterium, Eikenella corrodens 1073, which affects pilus formation and colony morphology.

    PubMed

    Azakami, Hiroyuki; Akimichi, Hiromi; Usui, Masakatsu; Yumoto, Hiromichi; Ebisu, Shigeyuki; Kato, Akio

    2005-05-23

    Eikenella corrodens (Ec) is one of a group of periodontopathogenic bacteria. A plasmid DNA (8.7 kb) isolated from Ec 1073 was designated pMU1. Agarose gel electrophoresis and Southern analysis suggested that pMU1-like plasmids were carried in 2 Ec strains, including 1073, with higher hemagglutination (HA) activity than other strains. We determined the nucleotide sequence of this plasmid and identified 7 ORFs. A homology search revealed that 4 ORFs of pMU1 were homologous to ORFs in pJTPS1, found in a spontaneous avirulent mutant of the phytopathogenic bacterium, Ralstonia solanacearum. pJTPS1 is a putative hypovirulent plasmid, which is thought to control the virulence of R. solanacearum. We also found the ORF to be homologous to the recombinase specific to the type IV pilin gene. We introduced a part of pMU1 into the Ec 23834 strain, which has a pilus structure on its cell surface and forms corroding colonies on solid medium. No pilus structure was observed on the surface of transformants, most of which formed non-corroding colonies. When such transformants (or Ec 1073) were cured of pMU1 with acridine orange, they remained non-foliated and non-corroding. The results suggest that pMU1 might irreversibly affect pilus formation and colony morphology, and might be involved in the pathogenicity and virulence of Ec.

  8. Effect of human polymorphonuclear and mononuclear leukocytes on chromosomal and plasmid DNA of Escherichia coli. Role of acid DNase

    SciTech Connect

    Rozenberg-Arska, M.; van Strijp, J.A.; Hoekstra, W.P.; Verhoef, J.

    1984-05-01

    Phagocytosis and killing by polymorphonuclear and mononuclear leukocytes are important host resistance factors against invading microorganisms. Evidence showing that killing is rapidly followed by degradation of bacterial components is limited. Therefore, we studied the fate of Escherichia coli DNA following phagocytosis of E. coli by polymorphonuclear and mononuclear leukocytes. (/sup 3/H)Thymidine-labeled, unencapsulated E. coli PC2166 and E. coli 048K1 were incubated in serum, washed, and added to leukocytes. Uptake and killing of the bacteria and degradation of DNA were measured. Although phagocytosis and killing by mononuclear leukocytes was less efficient than that by polymorphonuclear leukocytes, only mononuclear leukocytes were able to degrade E. coli PC2166 DNA. Within 2 h, 60% of the radioactivity added to mononuclear leukocytes was released into the supernate, of which 40% was acid soluble. DNA of E. coli 048K1 was not degraded. To further analyze the capacity of mononuclear leukocytes to degrade E. coli DNA, chromosomal and plasmid DNA was isolated from ingested bacteria and subjected to agarose gel-electrophoresis. Only chromosomal DNA was degraded after phagocytosis. Plasmid DNA of E. coli carrying a gene coding for ampicillin resistance remained intact for a 2-h period after ingestion, and was still able to transform recipient E. coli cells after this period. Although we observed no DNA degradation during phagocytosis by polymorphonuclear leukocytes, lysates of both polymorphonuclear and mononuclear leukocytes contained acid-DNase activity with a pH optimum of 4.9. However, the DNase activity of mononuclear leukocytes was 20 times higher than that of polymorphonuclear leukocytes. No difference was observed between DNase activity from polymorphonuclear and mononuclear leukocytes from a chronic granulomatous disease patient with DNase activity from control polymorphonuclear and mononuclear leukocytes.

  9. Inhibition of endonuclease cleavage and DNA replication of E. coli plasmid by the antitumor rhodium(II) complex.

    PubMed

    Rahman, Md Masudur; Yasuda, Hachiro; Katsura, Shinji; Mizuno, Akira

    2007-08-01

    Binding effect of the antitumor complex rhodium(II) acetate [Rh(2)(O(2)CCH(3))(4)] (Rh1) to the plasmid pUC19 DNA has been studied under different molar ratio of Rh1 compound to base pair of pUC19 DNA (R(f)) and reaction time. The Rh1 binding inhibited the activity of restriction enzyme. The binding effect was monitored using gel electrophoresis. The results indicate that at least one Rh1 binds with the recognition sequence and the binding has no preference between A-T and G-C pairs. At high value of R(f)=100, ICP-MS (Inductively Coupled Plasma Mass Spectrometry) measurement confirmed that 46% of Rh1 binds to DNA. PCR amplification of the DNA was also inhibited by the Rh1 binding. The transformation experiment using Escherichia coli suggested that the cell growth was inhibited after binding the Rh1 to the plasmid. These results indicated that DNA synthesis could be inhibited both in vitro and in vivo by the Rh(2)(O(2)CCH(3))(4) binding.

  10. Enhanced suppression of tumor growth using a combination of NK4 plasmid DNA-PEG engrafted cationized dextran complex and ultrasound irradiation.

    PubMed

    Hosseinkhani, H; Kushibiki, T; Matsumoto, K; Nakamura, T; Tabata, Y

    2006-05-01

    This investigation aims to determine experimentally whether or not ultrasound (US) irradiation is effective in enhancing the in vivo gene expression of NK4 plasmid DNA and suppressing tumor growth. NK4, composed of the NH2-terminal hairpin and subsequent four-kringle domains of hepatocyte growth factor (HGF), acts as an HGF-antagonist and angiogenesis inhibitor. Dextran was cationized by introducing spermine to the hydroxyl groups to allow for polyionic complexation with NK4 plasmid DNA. The cationized dextran was additionally modified with poly(ethylene glycol) (PEG) molecules giving PEG engrafted cationized dextran. Significant suppression of tumor growth was observed when PEG engrafted cationized dextran-NK4 plasmid DNA complexes were intravenously injected into mice carrying a subcutaneous Lewis lung carcinoma tumor mass with subsequent US irradiation when compared with the cationized dextran-NK4 plasmid DNA complex and naked NK4 plasmid DNA with or without US irradiation. We conclude that complexation with PEG-engrafted cationized dextran in combination with US irradiation is a promising way to target the NK4 plasmid DNA to the tumor for gene expression.

  11. Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

    PubMed

    Scollo, Francesco; Egea, Leticia A; Gentile, Alessandra; La Malfa, Stefano; Dorado, Gabriel; Hernandez, Pilar

    2016-12-15

    Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number.

  12. DNA and RNA polymerase activity in a Moniliophthora perniciosa mitochondrial plasmid and self-defense against oxidative stress.

    PubMed

    Andrade, B S; Villela-Dias, C; Gomes, D S; Micheli, F; Góes-Neto, A

    2013-06-13

    Moniliophthora perniciosa (Stahel) Aime and Phillips-Mora is a hemibiotrophic basidiomycete (Agaricales, Tricholomataceae) that causes witches' broom disease in cocoa (Theobroma cacao L.). This pathogen carries a stable integrated invertron-type linear plasmid in its mitochondrial genome that encodes viral-like DNA and RNA polymerases related to fungal senescence and longevity. After culturing the fungus and obtaining its various stages of development in triplicate, we carried out total RNA extraction and subsequent complementary DNA synthesis. To analyze DNA and RNA polymerase expression levels, we performed real-time reverse transcriptase polymerase chain reaction for various fungal phases of development. Our results showed that DNA and RNA polymerase gene expression in the primordium phase of M. perniciosa is related to a potential defense mechanism against T. cacao oxidative attack.

  13. Molecular characterization of Syrian date palm cultivars using plasmid-like DNA markers.

    PubMed

    Haider, N; Nabulsi, I

    2012-02-01

    Date palm (Phoenix dactylifera L.) is one of the most important domesticated fruit trees in the Near East and North African countries. This tree has been, for several decades, in serious threat of being completely destroyed by the "Bayoud" disease caused by Fusarium oxysporum f. sp. albedinis. In this study, 18 Syrian date palm cultivars and four male trees were analyzed according to the identity of mitochondrial plasmid-like DNAs. A PCR strategy that employs plasmid-like DNAs-specific primer pair was used. These primers amplify a product of either 373-bp or 265-bp that corresponds to the S-(Bayoud-susceptible) or the R-plasmid (Bayoud-resistant), respectively. Generated data revealed that only six cultivars ('Medjool', 'Ashrasi', 'Gish Rabi', 'Khineze', and yellow- and red-'Kabkab') have the S-plasmid, suggesting their susceptibility to the fusariosis, while the remaining 12 cultivars and the four male trees contain the R-plasmid, suggesting their resistance to the fusariosis. The PCR process applied here has been proved efficient for the rapid screening for the presence of the S and R DNAs in Syrian date palm. PCR markers developed in this study could be useful for the screening of date palm lines growing in the field. The availability of such diagnostic tool for plasmid characterization in date palm would also be of great importance in establishing propagation and breeding programs of date palm in Syria.

  14. Analysis of Heat-Labile Sites Generated by Reactions of Depleted Uranium and Ascorbate in Plasmid DNA

    PubMed Central

    Wilson, Janice; Young, Ashley; Civitello, Edgar R.

    2013-01-01

    The goal of this study was to characterize how depleted uranium (DU) causes DNA damage. Procedures were developed to assess the ability of organic and inorganic DNA adducts to convert to single strand breaks (SSB) in pBR322 plasmid DNA in the presence of heat or piperidine. DNA adducts formed by methyl methanesulfonate (MMS), cis-platin (cis-Pt), and chromic chloride were compared to those formed by reaction of uranyl acetate (UA) and ascorbate (Asc). Uranyl ion in the presence of Asc produced U-DNA adducts that converted to SSB upon heating. Piperidine, which acted on DNA methylated by MMS to convert methyl-DNA adducts to SSB, served in the opposite fashion with U-DNA adducts by decreasing SSB. The observation that piperidine also decreased the gel shift for metal-DNA adducts formed by monofunctional cis-Pt and chromic chloride was interpreted to suggest that piperidine served to remove U-DNA adducts. Radical scavengers did not affect formation of U-induced SSB, suggesting that SSB arose from the presence of U-DNA adducts and not from free radicals. A model is proposed to predict how U-DNA adducts may serve as initial lesions that convert to SSB or AP sites. Results suggest that DU can act as a chemical genotoxin that does not require radiation for its mode of action. Characterizing the DNA lesions formed by DU is necessary to assess the relative importance of different DNA lesions in the formation of DU-induced mutations. Understanding mechanisms of formation of DU-induced mutations may contribute to identification of biomarkers of DU exposures in humans. PMID:24218036

  15. Plasmid diversity in neisseriae.

    PubMed

    van Passel, Mark W J; van der Ende, Arie; Bart, Aldert

    2006-08-01

    Horizontal gene transfer constitutes an important force in prokaryotic genome evolution, and it is well-known that plasmids are vehicles for DNA transfer. Chromosomal DNA is frequently exchanged between pathogenic and commensal neisseriae, but relatively little is known about plasmid diversity and prevalence among these nasopharyngeal inhabitants. We investigated the plasmid contents of 18 Neisseria lactamica isolates and 20 nasopharyngeal Neisseria meningitidis isolates. Of 18 N. lactamica strains, 9 harbored one or more plasmids, whereas only one N. meningitidis isolate contained a plasmid. Twelve plasmids were completely sequenced, while five plasmid sequences from the public databases were also included in the analyses. On the basis of nucleic acid sequences, mobilization, and replicase protein alignments, we distinguish six different plasmid groups (I to VI). Three plasmids from N. lactamica appeared to be highly similar on the nucleotide level to the meningococcal plasmids pJS-A (>99%) and pJS-B (>75%). The genetic organizations of two plasmids show a striking resemblance with that of the recently identified meningococcal disease-associated (MDA) phage, while four putative proteins encoded by these plasmids show 25% to 39% protein identity to those encoded by the MDA phage. The putative promoter of the gene encoding the replicase on these plasmids contains a polycytidine tract, suggesting that replication is subjected to phase variation. In conclusion, extensive plasmid diversity is encountered among commensal neisseriae. Members of three plasmid groups are found in both pathogenic and commensal neisseriae, indicating plasmid exchange between these species. Resemblance between plasmids and MDA phage may be indicative of dissemination of phage-related sequences among pathogenic and commensal neisseriae.

  16. Recombination frequency in plasmid DNA containing direct repeats--predictive correlation with repeat and intervening sequence length.

    PubMed

    Oliveira, Pedro H; Lemos, Francisco; Monteiro, Gabriel A; Prazeres, Duarte M F

    2008-09-01

    In this study, a simple non-linear mathematical function is proposed to accurately predict recombination frequencies in bacterial plasmid DNA harbouring directly repeated sequences. The mathematical function, which was developed on the basis of published data on deletion-formation in multicopy plasmids containing direct-repeats (14-856 bp) and intervening sequences (0-3872 bp), also accounts for the strain genotype in terms of its recA function. A bootstrap resampling technique was used to estimate confidence intervals for the correlation parameters. More than 92% of the predicted values were found to be within a pre-established +/-5-fold interval of deviation from experimental data. The correlation does not only provide a way to predict, with good accuracy, the recombination frequency, but also opens the way to improve insight into these processes.

  17. Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping

    NASA Astrophysics Data System (ADS)

    Müller, Vilhelm; Rajer, Fredrika; Frykholm, Karolin; Nyberg, Lena K.; Quaderi, Saair; Fritzsche, Joachim; Kristiansson, Erik; Ambjörnsson, Tobias; Sandegren, Linus; Westerlund, Fredrik

    2016-12-01

    Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples.

  18. Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping

    PubMed Central

    Müller, Vilhelm; Rajer, Fredrika; Frykholm, Karolin; Nyberg, Lena K.; Quaderi, Saair; Fritzsche, Joachim; Kristiansson, Erik; Ambjörnsson, Tobias; Sandegren, Linus; Westerlund, Fredrik

    2016-01-01

    Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples. PMID:27905467

  19. Swapping single-stranded DNA sequence specificities of relaxases from conjugative plasmids F and R100

    PubMed Central

    Harley, Matthew J.; Schildbach, Joel F.

    2003-01-01

    Conjugative plasmid transfer is an important mechanism for diversifying prokaryotic genomes and disseminating antibiotic resistance. Relaxases are conjugative plasmid-encoded proteins essential for plasmid transfer. Relaxases bind and cleave one plasmid strand site- and sequence-specifically before transfer of the cleaved strand. TraI36, a domain of F plasmid TraI that contains relaxase activity, binds a plasmid sequence in single-stranded form with subnanomolar KD and high sequence specificity. Despite 91% amino acid sequence identity, TraI36 domains from plasmids F and R100 discriminate between binding sites. The binding sites differ by 2 of 11 bases, but both proteins bind their cognate site with three orders of magnitude higher affinity than the other site. To identify specificity determinants, we generated variants having R100 amino acids in the F TraI36 background. Although most retain F specificity, the Q193R/R201Q variant binds the R100 site with 10-fold greater affinity than the F site. The reverse switch (R193Q/Q201R) in R100 TraI36 confers a wild-type F specificity on the variant. Nonadditivity of individual amino acid and base contributions to recognition suggests that the specificity difference derives from multiple interactions. The F TraI36 crystal structure shows positions 193 and 201 form opposite sides of a pocket within the binding cleft, suggesting binding involves knob-into-hole interactions. Specificity is presumably modulated by altering the composition of the pocket. Our results demonstrate that F-like relaxases can switch between highly sequence-specific recognition of different sequences with minimal amino acid substitution. PMID:14504391

  20. Immunogenicity of a plasmid DNA vaccine encoding 42kDa fragment of Plasmodium vivax merozoite surface protein-1.

    PubMed

    Sheikh, Inayat Hussain; Kaushal, Deep C; Chandra, Deepak; Kaushal, Nuzhat A

    2016-10-01

    Plasmodium vivax is the second major human malaria parasite that inflicts debilitating morbidity and consequent economic impact in South-East Asian countries. The relapsing nature of P. vivax along with the emergence of drug-resistant P. vivax strains has emphasized the urgent need for a vaccine. However, the development of an effective vivax vaccine is seriously hampered due to the diversity and variation in parasite antigens and non-availability of suitable animal models. DNA based vaccines represent an alternative approach in inducing immunity to multiple targets from different stages of malaria parasite. DNA prime-boosting strategies induce both antibody mediated and cell-mediated immune responses that are the major mechanisms of protection against malaria parasites. We have earlier studied the immunogenicity and protective efficacy of the soluble and refolded forms of recombinant 42kDa fragment of Plasmodium vivax merozoite surface protein-1 (PvMSP-142) using P. cynomolgi rhesus monkey model. In the present study, we have constructed a recombinant DNA vaccine encoding 42kDa fragment of P. vivax MSP-1 and studied the immunogenicity of PvMSP-142 DNA vaccine construct in mice. The 42kDa gene fragment of PvMSP-1 was PCR amplified using gene specific primers and subcloned into pcDNA 3.1 (+) eukaryotic expression vector. In vitro expression of PvMSP-142 plasmid construct was checked by transfection in COS-1 cell line. Indirect immunofluorescence of transfected COS-1 cells probed with monoclonal antibodies against PvMSP-142 exhibited positive fluorescence. Immunization of BALB/c mice with PvMSP-142-pcDNA vaccine construct revealed the immunogenicity of recombinant vaccine plasmid that can be enhanced by prime boosting with recombinant protein corresponding to the DNA vaccine as evidenced by significant elevation of antibody and the cytokines responses.

  1. Nanospines incorporation into the structure of the hydrophobic cryogels via novel cryogelation method: an alternative sorbent for plasmid DNA purification.

    PubMed

    Üzek, Recep; Uzun, Lokman; Şenel, Serap; Denizli, Adil

    2013-02-01

    In this study, it was aimed to prepare hydrophobic cryogels for plasmid DNA (pDNA) purification from Escherichia coli lysate. The hydrophobicity was achieved by incorporating a hydrophobic ligand, N-methacryloyl-(L)-phenylalanine (MAPA), into the cryogel backbone. In addition to the conventional cryogelation process, freeze-drying step was included to create nanospines. Three different cryogels {poly(2-hydoxyethyl methacrylate-N-methacryloyl-L-phenylalanine)-freeze dried, [P(HEMA-MAPA)-FD]; poly(2-hydoxyethyl methacrylate-N-methacryloyl-L-phenylalanine, [P(HEMA-MAPA)] and poly(2-hydoxyethyl methacrylate)-freeze dried, [P(HEMA)-FD]} were prepared, characterized, and used for DNA (salmon sperm DNA) adsorption studies from aqueous solution. The specific surface areas of cryogels were determined to be 21.4 m(2)/g for P(HEMA)-FD, 17.65 m(2)/g for P(HEMA-MAPA) and 36.0 m(2)/g for P(HEMA-MAPA)-FD. The parameters affecting adsorption such as temperature, initial DNA concentration, salt type and concentration were examined in continuous mode. The maximum adsorption capacities were observed as 45.31 mg DNA/g, 27.08 mg DNA/g and 1.81 mg DNA/g for P(HEMA-MAPA)-FD, P(HEMA-MAPA) and P(HEMA)-FD, respectively. Desorption process was performed using acetate buffer (pH 5.50) without salt. First, pDNA was isolated from E. coli lysate and the purity of pDNA was then determined by agarose gel electrophoresis. Finally, the chromatographic performance of P(HEMA-MAPA)-FD cryogel for pDNA purification was tested in FPLC. The resolution (R(s)) was 2.84, and the specific selectivity for pDNA was 237.5-folds greater than all impurities.

  2. Recombinant goose-type lysozyme in channel catfish: lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection.

    PubMed

    Pridgeon, Julia W; Klesius, Phillip H; Dominowski, Paul J; Yancey, Robert J; Kievit, Michele S

    2013-10-01

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme-g (CC-Lys-g) produced in Escherichia coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme-g plasmid DNA could be used as an immunostimulant to protect channel catfish against Aeromonas hydrophila infection. Recombinant CC-Lys-g produced in E. coli expression system exhibited significant (P < 0.05) lytic activity against Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant channel catfish lysozyme-g (pcDNA-Lys-g) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-g offered significant (P < 0.05) protection to G1B cells against A. hydrophila infection. When channel catfish were intraperitoneally injected with pcDNA-Lys-g along with an adjuvant QCDCR, the transcriptional level of Lys-g was significantly (P < 0.05) increased. When pcDNA-Lys-g injected fish was challenged with a highly virulent A. hydrophila strain AL-09-71, pcDNA-Lys-g offered 100% protection to channel catfish at two days post DNA injection. Macrophages of fish injected with pcDNA-Lys-g produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish injected with pcDNA vector alone at two days post DNA injection. Taken together, our results suggest that pcDNA-Lys-g could be used as a novel immunostimulant to offer immediate protection to channel catfish against A. hydrophila infection.

  3. Arginine-based cationic liposomes for efficient in vitro plasmid DNA delivery with low cytotoxicity

    PubMed Central

    Sarker, Satya Ranjan; Aoshima, Yumiko; Hokama, Ryosuke; Inoue, Takafumi; Sou, Keitaro; Takeoka, Shinji

    2013-01-01

    Background Currently available gene delivery vehicles have many limitations such as low gene delivery efficiency and high cytotoxicity. To overcome these drawbacks, we designed and synthesized two cationic lipids comprised of n-tetradecyl alcohol as the hydrophobic moiety, 3-hydrocarbon chain as the spacer, and different counterions (eg, hydrogen chloride [HCl] salt or trifluoroacetic acid [TFA] salt) in the arginine head group. Methods Cationic lipids were hydrated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer to prepare cationic liposomes and characterized in terms of their size, zeta potential, phase transition temperature, and morphology. Lipoplexes were then prepared and characterized in terms of their size and zeta potential in the absence or presence of serum. The morphology of the lipoplexes was determined using transmission electron microscopy and atomic force microscopy. The gene delivery efficiency was evaluated in neuronal cells and HeLa cells and compared with that of lysine-based cationic assemblies and Lipofectamine™ 2000. The cytotoxicity level of the cationic lipids was investigated and compared with that of Lipofectamine™ 2000. Results We synthesized arginine-based cationic lipids having different counterions (ie, HCl-salt or TFA-salt) that formed cationic liposomes of around 100 nm in size. In the absence of serum, lipoplexes prepared from the arginine-based cationic liposomes and plasmid (p) DNA formed large aggregates and attained a positive zeta potential. However, in the presence of serum, the lipoplexes were smaller in size and negative in zeta potential. The morphology of the lipoplexes was vesicular. Arginine-based cationic liposomes with HCl-salt showed the highest transfection efficiency in PC-12 cells. However, arginine-based cationic liposomes with TFA salt showed the highest transfection efficiency in HeLa cells, regardless of the presence of serum, with very low associated cytotoxicity. Conclusion The gene

  4. Broad host range plasmids.

    PubMed

    Jain, Aayushi; Srivastava, Preeti

    2013-11-01

    Plasmids are and will remain important cloning vehicles for biotechnology. They have also been associated with the spread of a number of diseases and therefore are a subject of environmental concern. With the advent of sequencing technologies, the database of plasmids is increasing. It will be of immense importance to identify the various bacterial hosts in which the plasmid can replicate. The present review article describes the features that confer broad host range to the plasmids, the molecular basis of plasmid host range evolution, and applications in recombinant DNA technology and environment.

  5. Photosensitization of plasmid-DNA loaded with platinum nano-particles and irradiated by low energy X-rays

    NASA Astrophysics Data System (ADS)

    Porcel, E.; Kobayashi, K.; Usami, N.; Remita, H.; Le Sech, C.; Lacombe, S.

    2011-01-01

    Damage in DNA plasmids (pBR322) loaded with platinum nanoparticles (NP-Pt) DNA-NP and irradiated with monochromatic X-rays tuned to the resonant photoabsorption energy of the LIII and MIII electronic inner-shell of platinum - respectively 11556 eV and 2649 eV - and off-resonant X-rays - 11536 eV and 2639 eV- is investigated. In all the experiments, an enhancement of the single and double strand break - SSB and DSB - yields is observed when NP-Pt are present. Amplification effects are almost similar for the irradiations performed at on and off the L or M shell resonance suggesting that a non resonant mechanism is responsible for the major part of the DNA breaks enhancement.The amount of DNA breaks measured in the present work is compared to the results in similar experiments made with complexes of plasmid-DNA containing platinum molecule : chloroterpyridine platinum (PtTC). The average number of PtTC molecules in the solution is the same as in the experiments made with NP-Pt in order to study a possible difference in the radiosensitization efficiency when the high-Z atoms are clustered (NP-Pt) or dispersed in the system (PtTC). A mechanism is suggested involving photoelectrons which can efficiently ionize the platinum atoms. These results are consistent with those observed when DNA-NP complexes are irradiated by fast atomic ions. These findings suggest that any nanoparticle made of high-Z atoms might behaves as radiation enhancer whatever the ionizing radiation is electromagnetic or charged particle source.

  6. Comparative study of the lethal effects of near-UV light (360 nm) and 8-methoxypsoralen plus near-UV on plasmid DNA

    SciTech Connect

    Paramio, J.M.; Bauluz, C.; de Vidania, R. )

    1991-01-01

    The authors have studied the lethality produced on pBR322 by near-UV radiation and by 8-Methoxypsoralen plus near-UV (PUV treatment). Samples of pBR322 DNA were irradiated with increasing fluences of 360 nm-light either in the absence or presence of 400 molecules of 8-Methoxypsoralen (8-MOP) per plasmid molecule. They have estimated to what extent the global lethality of PUVA treatment is due to the presence of psoralen adducts in DNA or to radiation itself. In order to analyse the involvement of DNA repair mechanisms in the removal of plasmid lesions, several strains of E. coli (differing in their repair capacities) they are used as recipients of the treated plasmids. Results showed that excision and recombination participate in the repair of near-UV-induced plasmid lesions. Repair of PUV-induced lesions showed an even greater requirement of the excision pathway. Besides, a slight increase on plasmid mutation frequencies was observed after near-UV or PUV treatment in wild type and uvrA cells. Estimation of the contribution of 8-MOP to the global lethality of PUV treatment showed that only the excision pathway was involved in removing psoralen adducts from plasmid DNA, suggesting the involvement of the recombinational pathway in the repair of near-UV-derived lesions.

  7. Compatibility of plasmids encoding bovine viral diarrhea virus type 1 and type 2 E2 in a single DNA vaccine formulation.

    PubMed

    Liang, Rong; Babiuk, Lorne A; van Drunen Littel-van den Hurk, Sylvia

    2007-08-10

    Type 2 bovine viral diarrhea virus (BVDV) has become increasingly prevalent worldwide, and currently the ratio of type 2 to type 1 strains in the USA approaches 50%. Although there is cross-reactivity between BVDV type 1 and type 2 strains, BVDV1 vaccine strains poorly protect from type 2 infection, so vaccines against BVDV should contain antigens from both BVDV types. Previously we demonstrated efficacy of a BVDV1 E2 DNA vaccine, and in this study we optimized a BVDV2 E2 DNA vaccine. Furthermore, as an approach to vaccinate with a DNA vaccine against both BVDV types, we compared two strategies, mixing of plasmids encoding type 1 and type 2 E2, and co-expression of type 1 and type 2 E2 from one plasmid with an internal ribosomal entry site (IRES). An evaluation of the IRES-containing plasmids demonstrated that the C-terminally expressed protein is produced at lower levels and induces weaker immune responses than the N-terminally expressed protein, regardless of the position of the type 1 and type 2 E2 genes. In contrast, when both plasmids encoding type 1 and type 2 E2 were administered to mice, the immune responses were similar to those induced by the individual plasmids. Thus, a mixture of plasmids encoding type 1 and type 2 E2 could be a potential DNA vaccine candidate against both BVDV1 and BVDV2.

  8. Efficient in vivo gene transfer by intraperitoneal injection of plasmid DNA and calcium carbonate microflowers in mice.

    PubMed

    Fumoto, Shintaro; Nakajima, Sayuri; Mine, Toyoharu; Yoshikawa, Naoki; Kitahara, Takashi; Sasaki, Hitoshi; Miyamoto, Hirotaka; Nishida, Koyo

    2012-07-02

    Gene transfer to intraperitoneal organs is thought to be a promising approach to treat such conditions as peritoneal fibrosis and peritoneal dissemination of cancers. We previously discovered that simple instillation of naked plasmid DNA (pDNA) onto intraperitoneal organs such as the liver and stomach could effectively transfer foreign genes in mice. In this study, we developed a novel nonviral method to enhance transfection efficiency of naked pDNA to intraperitoneal organs using a calcium carbonate suspension containing pDNA. Using commercially available calcium carbonate, we successfully transfected pDNA to the stomach. Handling of commercially available calcium carbonate, however, was troublesome owing to rapid precipitation and caking. To obtain slowly settling particles of calcium carbonate, we tried to synthesize novel versions of such particles and succeeded in creating flower-shaped particles, named calcium carbonate microflowers. Sedimentation of calcium carbonate microflowers was sufficiently slow for in vivo experiments. Moreover, the transfection efficiency of the suspension of calcium carbonate microflowers to the stomach was more effective than that of commercially available calcium carbonate, especially at low concentrations. Intraperitoneal injection of the suspension of calcium carbonate microflowers containing pDNA greatly enhanced naked pDNA transfer to whole intraperitoneal organs in mice. Furthermore, lactate dehydrogenase activities in intraperitoneal fluid and plasma were not raised by the suspension of calcium carbonate microflowers.

  9. Binding and condensation of plasmid DNA onto functionalized carbon nanotubes: toward the construction of nanotube-based gene delivery vectors.

    PubMed

    Singh, Ravi; Pantarotto, Davide; McCarthy, David; Chaloin, Olivier; Hoebeke, Johan; Partidos, Charalambos D; Briand, Jean-Paul; Prato, Maurizio; Bianco, Alberto; Kostarelos, Kostas

    2005-03-30

    Carbon nanotubes (CNTs) constitute a class of nanomaterials that possess characteristics suitable for a variety of possible applications. Their compatibility with aqueous environments has been made possible by the chemical functionalization of their surface, allowing for exploration of their interactions with biological components including mammalian cells. Functionalized CNTs (f-CNTs) are being intensively explored in advanced biotechnological applications ranging from molecular biosensors to cellular growth substrates. We have been exploring the potential of f-CNTs as delivery vehicles of biologically active molecules in view of possible biomedical applications, including vaccination and gene delivery. Recently we reported the capability of ammonium-functionalized single-walled CNTs to penetrate human and murine cells and facilitate the delivery of plasmid DNA leading to expression of marker genes. To optimize f-CNTs as gene delivery vehicles, it is essential to characterize their interactions with DNA. In the present report, we study the interactions of three types of f-CNTs, ammonium-functionalized single-walled and multiwalled carbon nanotubes (SWNT-NH3+; MWNT-NH3+), and lysine-functionalized single-walled carbon nanotubes (SWNT-Lys-NH3+), with plasmid DNA. Nanotube-DNA complexes were analyzed by scanning electron microscopy, surface plasmon resonance, PicoGreen dye exclusion, and agarose gel shift assay. The results indicate that all three types of cationic carbon nanotubes are able to condense DNA to varying degrees, indicating that both nanotube surface area and charge density are critical parameters that determine the interaction and electrostatic complex formation between f-CNTs with DNA. All three different f-CNT types in this study exhibited upregulation of marker gene expression over naked DNA using a mammalian (human) cell line. Differences in the levels of gene expression were correlated with the structural and biophysical data obtained for the f-CNT:DNA

  10. Characterization of a targeted gene carrier, lactose-polyethylene glycol-grafted poly-L-lysine and its complex with plasmid DNA.

    PubMed

    Choi, Y H; Liu, F; Choi, J S; Kim, S W; Park, J S

    1999-11-01

    The physicochemical properties and gene transfer ability of lactose-polyethylene glycol-grafted poly-L-lysine (Lac-PEG-PLL) were investigated. A dye displacement assay showed that plasmid DNA self-assembled with Lac-PEG-PLL, and condensation began at a <1:1 charge ratio of plasmid DNA to polymer. In atomic force microscopy, spontaneously assembled Lac-PEG-PLL/DNA complexes revealed a compact structure, with a size of about 100-200 nm. Circular dichroism spectra of Lac-PEG-PLL/DNA complexes revealed that the secondary structure of DNA was altered by complex formation and was similar to that of the poly-L-lysine/DNA complex. Lac-PEG-PLL was shown to protect DNA against nuclease action in a DNase I protection assay. The cytotoxicity test demonstrated that the complex composed of plasmid DNA and Lac-PEG-PLL had little influence on the viability of HepG2 cells, especially in comparison with that of poly-L-lysine/DNA complexes. In conclusion, our copolymer, Lac-PEG-PLI, formed complexes with plasmid DNA (on average, 150 nm), gave little cytotoxicity, and showed increased efficiency of gene transfer into hepatoma cells in vitro. Lactose-polyethylene glycol was grafted to poly-L-lysine to be used as a gene carrier for hepatoma cell targeting and to improve the solubility of the polyplexes. The average size of the carrier/DNA complexes was about 150 nm. The complexes also proved to have high resistance against nuclease attack and little cytotoxicity. The polymer also delivered plasmid DNA efficiently into a HepG2 cell line. Lac-PEG-PLL was more efficient than Lipofectin or galactose-PEG-PLL in transfection efficiency.

  11. An initiator protein for plasmid R6K DNA replication. Mutations affecting the copy-number control.

    PubMed

    Inuzuka, M; Wada, Y

    1988-02-08

    Two kinds of mutations affecting the copy-number control of plasmid R6K were isolated and identified in an initiator pi protein by DNA sequencing. Firstly, a temperature-sensitive replication mutation, ts22, with decreased copy number results in a substitution of threonine to isoleucine at position 138 of the 305-amino-acid pi protein. Secondly, a high-copy-number (cop21) mutant was isolated from this ts mutant and was identified by an alteration of alanine to serine at position 162. This cop21 mutation suppressed the Ts character and was recessive to the wild-type allele in the copy control.

  12. Use of a plasmid DNA probe to monitor populations of Bacillus pumilus inoculant strains in hay

    SciTech Connect

    Hendrick, C.A.; Smiley, B.K.; Shelley, T.H.; Tomes, N.J. )

    1991-03-01

    The authors are evaluating naturally occurring isolates of Bacillus pumilus for use as microbial hay preservatives. Seven isolates of B, pumilus from hay contained a 42-kb cryptic plasmid (pMGD296). They wished to determine whether pMGD296 could be used as a molecular marker to follow populations of these isolates in hay over time. Southern blots and colony blots of 69 isolates of B. pumilus and other Bacillus spp. were probed with {sup 32}P-labeled pMGD296. Twenty-nine probe-positive isolates were identified; of these, 28 contained a plasmid with a restriction profile identical to that of pMGD296. One isolate from untreated hay contained a 40-kb plasmid (pMGD150) that was homologous to pMGD296 but had a different restriction fragment pattern. Regions of homology between the two plasmids were identified by Southern blotting, and a 1.9-kb HindIII-PstI fragment of pMGD296 lacking strong homology to pMGD150 was cloned in pUC18. The cloned fragment hybridized only with isolates containing pMGD296 and was used to estimate populations of these isolates in treated and untreated hay.

  13. Vaccination with plasmid DNA encoding KMPII, TRYP, LACK and GP63 does not protect dogs against Leishmania infantum experimental challenge.

    PubMed

    Rodríguez-Cortés, Alhelí; Ojeda, Ana; López-Fuertes, Laura; Timón, Marcos; Altet, Laura; Solano-Gallego, Laia; Sánchez-Robert, Elisenda; Francino, Olga; Alberola, Jordi

    2007-11-14

    Vaccination of dogs, the domestic reservoir of Leishmania infantum, is the best method for controlling zoonotic visceral leishmaniasis. This strategy would reduce the incidence of disease in both the canine and, indirectly, the human population. Different vaccination approaches have been investigated against canine leishmaniasis (CaL) but to date there is only one licensed vaccine against this disease in dogs, in Brazil. DNA immunization is a promising method for inducing both humoral and cellular immune responses against this parasitic disease. Here, we report the results of a multiantigenic plasmid DNA vaccine encoding KMPII, TRYP, LACK and GP63 L. infantum antigens against experimentally induced CaL. Twelve dogs were randomly assigned to two groups receiving, at a 15 days interval, either four doses of plasmid DNA or similar injections of PBS. After vaccination, dogs were intravenously challenged with 5 x 10(7) promastigotes of L. infantum. The vaccine showed to be safe and well-tolerated. Neither cellular immune response nor antibodies directed against whole Leishmania antigen were detected after immunization in vaccinated dogs, although anti-LACK-specific antibodies were sporadically detected in two vaccinated dogs before challenge, thus suggesting that antigens were indeed expressed. A delay in the development of detectable specific immune response and parasite multiplication in vaccinated dogs was observed after challenge. Nevertheless, the multiantigenic Leishmania DNA vaccine was unable to induce protection against parasite dissemination or disease. This study emphasizes the need to strengthen DNA vaccines in order to obtain effective immune responses in models other than the murine.

  14. Well-defined star polymers for co-delivery of plasmid DNA and imiquimod to dendritic cells.

    PubMed

    Lin, Wenjing; Hanson, Samuel; Han, Wenqing; Zhang, Xiaofang; Yao, Na; Li, Hongru; Zhang, Lijuan; Wang, Chun

    2017-01-15

    Co-delivery of antigen-encoding plasmid DNA (pDNA) and immune-modulatory molecules has importance in advancing gene-based immunotherapy and vaccines. Here novel star polymer nanocarriers were synthesized for co-delivery of pDNA and imiquimod (IMQ), a poorly soluble small-molecule adjuvant, to dendritic cells. Computational modeling and experimental results revealed that the polymers formed either multimolecular or unimolecular core-shell-type micelles in water, depending on the nature of the outer hydrophilic shell. Micelles loaded with both IMQ and pDNA were able to release IMQ in response to intracellular pH of the endo-lysosome and transfect mouse dendritic cells (DC2.4 line) in vitro. Importantly, IMQ-loaded micelle/pDNA complexes displayed much enhanced transfection efficiency than IMQ-free complexes. These results demonstrate the feasibility of co-delivery of pDNA and IMQ to antigen-presenting cells by multifunctional polymer nanocarriers with potential use in gene-based vaccine approaches.

  15. Plasmid Vector-Linked Maturation of Natural Killer (NK) Cells Is Coupled to Antigen-Dependent NK Cell Activation during DNA-Based Immunization in Mice ▿

    PubMed Central

    Zhu, Ren; Mancini-Bourgine, Maryline; Zhang, Xiao Ming; Bayard, Florence; Deng, Qiang; Michel, Marie-Louise

    2011-01-01

    Plasmid DNA vaccines serve in a wide array of applications ranging from prophylactic vaccines to potential therapeutic tools against infectious diseases and cancer. In this study, we analyzed the mechanisms underlying the activation of natural killer (NK) cells and their potential role in adaptive immunity during DNA-based immunization against hepatitis B virus surface antigen in mice. We observed that the mature Mac-1+ CD27− NK cell subset increased in the liver of mice early after DNA injection, whereas the number of the less mature Mac-1+ CD27+ NK cells in the liver and spleen was significantly reduced. This effect was attributed to bacterial sequences present in the plasmid backbone rather than to the encoded antigen and was not observed in immunized MyD88-deficient mice. The activation of NK cells by plasmid-DNA injection was associated with an increase in their effector functions that depended on the expressed antigen. Maturation of NK cells was abrogated in the absence of T cells, suggesting that cross talk exists between NK cells and antigen-specific T cells. Taken together, our data unravel the mechanics of plasmid vector-induced maturation of NK cells and plasmid-encoded antigen-dependent activation of NK cells required for a crucial role of NK cells in DNA vaccine-induced immunogenicity. PMID:21775455

  16. Electrotransfer of plasmid DNA radiosensitizes B16F10 tumors through activation of immune response

    PubMed Central

    Savarin, Monika; Kamensek, Urska; Cemazar, Maja; Heller, Richard

    2017-01-01

    Abstract Background Tumor irradiation combined with adjuvant treatments, either vascular targeted or immunomodulatory, is under intense investigation. Gene electrotransfer of therapeutic genes is one of these approaches. The aim of this study was to determine, whether gene electrotransfer of plasmid encoding shRNA for silencing endoglin, with vascular targeted effectiveness, can radiosensitize melanoma B16F10 tumors. Materials and methods The murine melanoma B16F10 tumors, growing on the back of C57Bl/6 mice, were treated by triple gene electrotransfer and irradiation. The antitumor effect was evaluated by determination of tumor growth delay and proportion of tumor free mice. Furthermore, histological analysis of tumors (necrosis, apoptosis, proliferation, vascularization, presence of hypoxia and infiltration of immune cells,) was used to evaluate the therapeutic mechanisms. Results Gene electrotransfer of plasmid silencing endoglin predominantly indicated vascular targeted effects of the therapy, since significant tumor growth delay and 44% of tumor free mice were obtained. In addition, irradiation had minor effects on radioresistant melanoma, with 11% of mice tumor free. The combined treatment resulted in excellent effectiveness with 88% of mice tumor free, with more than half resistant to secondary tumor challenge, which was observed also with the plasmid devoid of the therapeutic gene. Histological analysis of tumors in the combined treatment group, demonstrated similar mode of action of the gene electrotransfer of plasmid encoding shRNA for silencing endoglin and devoid of it, both through the induction of an immune response. Conclusions The results of this study indicate that irradiation can in radioresistant melanoma tumors, by release of tumor associated antigens, serve as activator of the immune response, besides directly affecting tumor cells and vasculature. The primed antitumor immune response can be further boosted by gene electrotransfer of plasmid

  17. Novel synthetic (S,S) and (R,R)-secoisolariciresinol diglucosides (SDGs) protect naked plasmid and genomic DNA From gamma radiation damage.

    PubMed

    Mishra, Om P; Pietrofesa, Ralph; Christofidou-Solomidou, Melpo

    2014-07-01

    Secoisolariciresinol diglucoside (SDG) is the major lignan in wholegrain flaxseed. However, extraction methods are complex and are associated with low yield and high costs. Using a novel synthetic pathway, our group succeeded in chemically synthesizing SDG (S,S and R,R enantiomers), which faithfully recapitulates the properties of their natural counterparts, possessing strong antioxidant and free radical scavenging properties. This study further extends initial findings by now investigating the DNA-radioprotective properties of the synthetic SDG enantiomers compared to the commercial SDG. DNA radioprotection was assessed by cell-free systems such as: (a) plasmid relaxation assay to determine the extent of the supercoiled (SC) converted to open-circular (OC) plasmid DNA (pBR322) after exposure of the plasmid to gamma radiation; and (b) determining the extent of genomic DNA fragmentation. Exposure of plasmid DNA to 25 Gy of γ radiation resulted in decreased supercoiled form and increased open-circular form, indicating radiation-induced DNA damage. Synthetic SDG (S,S) and SDG (R,R), and commercial SDG at concentrations of 25-250 μM significantly and equipotently reduced the radiation-induced supercoiled to open-circular plasmid DNA in a dose-dependent conversion. In addition, exposure of calf thymus DNA to 50 Gy of gamma radiation resulted in DNA fragments of low-molecular weight (<6,000 bps), which was prevented in a dose-dependence manner by all synthetic and natural SDG enantomers, at concentrations as low as 0.5 μM. These novel results demonstrated that synthetic SDG (S,S) and SDG (R,R) isomers and commercial SDG possess DNA-radioprotective properties. Such properties along with their antioxidant and free radical scavenging activity, reported earlier, suggest that SDGs are promising candidates for radioprotection for normal tissue damage as a result of accidental exposure during radiation therapy for cancer treatment.

  18. Construction of adiponectin-encoding plasmid DNA and gene therapy of non-obese type 2 diabetes mellitus.

    PubMed

    Nan, Mei Hua; Park, Jeong-Sook; Myung, Chang-Seon

    2010-01-01

    Adiponectin (ADN), an insulin-sensitizing adipokine, stimulates glucose uptake, inhibits gluconeogenesis, and plays an important role in improving insulin sensitivity. Since blood levels of ADN are low in type 2 diabetes mellitus (DM), this study was designed to investigate the therapeutic effectiveness of increasing the ADN level through injection of plasmid DNA encoding ADN in type 2 DM. A non-obese type 2 DM mouse model was established via combined administration of streptozotocin with nicotinamide and exhibited significantly higher plasma glucose concentration and insulin resistance compared with normal controls according to oral glucose tolerance and insulin challenge tests. Plasmid DNA encoding mouse ADN from differentiated NIH3T3 adipocytes was constructed in pVAX1 (pVAX/ADN). Transfection of pVAX/ADN into various cell lines including HeLa, HT22, HEK293, HepG2, and SK-Hep1 cells, increased ADN mRNA expression levels in a dose-dependent manner. The administration of pVAX/ADN into non-obese type 2 DM mice via tail vein significantly increased the blood level of ADN and decreased the plasma glucose concentration. Moreover, the parameters related to insulin resistance (HOMA-IR) and insulin sensitivity (QUICKI) were significantly improved. These results suggest that ADN gene therapy could be a clinically effective tool for the treatment of type 2 DM.

  19. DNA-based methods to prepare helper virus-free herpes amplicon vectors and versatile design of amplicon vector plasmids.

    PubMed

    Kasai, Kazue; Saeki, Yoshinaga

    2006-06-01

    The herpes simplex virus (HSV) amplicon vector is a versatile plasmid-based gene delivery vehicle with a large transgene capacity (up to 150 kb) and the ability to infect a broad range of cell types. The vector system was originally developed by Frenkel and her colleagues in 1980. Ever since, a great deal of effort by various investigators has been directed at minimizing the toxicity associated with the inevitable contamination by helper virus. In 1996, Fraefel and his colleagues successfully devised a cosmid-based packaging system that was free of contamination by helper virus (so-called helper virus-free packaging), which utilized as helper a set of 5 overlapping cosmid clones that covered the entire HSV genome, which lacked the DNA packaging/cleavage signals. With the helper virus-free system, broader applications of the vector became possible. Cloning of the entire HSV genome in bacteria artificial chromosome (BAC) plasmids enabled stable maintenance and propagation of the helper HSV genome in bacteria. It also allowed for the development of BAC-based helper virus-free packaging systems. In this article, we review various versions of DNA-based methods to prepare HSV amplicon vectors free of helper virus contamination. We also examine recent advances in vector design, including methods of vector construction, hybrid amplicon vectors, and the infectious BAC system. Future directions in improving packaging systems and vector designs are discussed.

  20. Fe3O4 nanoparticles engineered for plasmid DNA delivery to Escherichia coli

    NASA Astrophysics Data System (ADS)

    Saei, Amir Ata; Barzegari, Abolfazl; Majd, Mostafa Heidari; Asgari, Davoud; Omidi, Yadollah

    2014-08-01

    Heat shock treatment is the most popular method for transformation of Escherichia coli. We have used 19-nm Fe3O4 nanoparticles for improving heat shock protocol. PGEM- T (3,000 bp) and pCAMBIA (8,428 bp) were used as test plasmids for transformation of competent E. coli cells (strains DH5α and Jm107) obtained from heat shock- and CaCl2-treated bacteria. A combination of heat shock and Fe3O4 nanoparticles led to a significant increase (6-10 fold) in number of transformed colonies in comparison with heat shock alone. The percent increase in transformation efficiency was higher for larger pCAMBIA plasmids compared to PGEM- T. The transformation efficiency decreased in the absence of CaCl2 and increased by addition of glycerol to the bacterial culture.

  1. Studies on the batch adsorption of plasmid DNA onto anion-exchange chromatographic supports.

    PubMed

    Ferreira, G N; Cabral, J M; Prazeres, D M

    2000-01-01

    The adsorption of a supercoiled 4.8 kbp plasmid onto quaternary ammonium anion-exchangers was studied in a finite bath. Equilibrium experiments were performed with pure plasmid, at 25 degrees C, using commercial Q-Sepharose matrices differing in particle diameter (High Performance, 34 microm; Fast Flow, 90 microm; and Big Beads, 200 microm) and a recently commercialized ion-exchanger, Streamline QXL (d(p) = 200 microm) at different salt concentrations (0.5, 0.7, and 1 M NaCl). Plasmid adsorption was found to follow second-order kinetics (Langmuir isotherm) with average association constants K(A) = 0.32+/-0.12 mL microg(-)(1) and K(A) = 0.25+/-0.15 mL microg(-1) at 0.5 and 0.7 M Nacl, respectively. The maximum binding capacities were not dependent on the ionic strength in the range 0.5-0.7 M but decreased with increasing particle diameter, suggesting that adsorption mainly occurs at the surface of the particles. No adsorption was found at 1 M NaCl. A nonporous model was applied to describe the uptake rate of plasmid onto Streamline QXL at 0.5 M NaCl. The overall process rate was controlled by mass transfer in the regions of low relative amounts of adsorbent (initial stages) and kinetically controlled in the later stages of the process for high relative amounts of adsorbent. The forward reaction rate constant (k(1) = 0.09+/-0.01 mL mg(-1) s(-1)) and film mass transfer coefficient (K(f) = (6 +/- 2) x 10(-4) cm s(-1)) were calculated. Simulations were performed to study the effect of the relative amount of adsorbent on the overall process rate, yield, and media capacity utilization.

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

    PubMed Central

    2012-01-01

    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 throughout the cultivation

  3. Transient Depletion of Kupffer Cells Leads to Enhanced Transgene Expression in Rat Liver Following Retrograde Intrabiliary Infusion of Plasmid DNA and DNA Nanoparticles

    PubMed Central

    Dai, Hui; Jiang, Xuan; Leong, Kam W.

    2011-01-01

    Abstract In this report, we have demonstrated that by temporarily removing Kupffer cells (KCs), the transgene expression levels mediated by retrograde intrabiliary infusion (RII) of plasmid DNA, polyethylenimine-DNA, and chitosan nanoparticles were enhanced by 1,927-, 131-, and 23,450-fold, respectively, in comparison with the respective groups without KC removal. KC removal also led to significantly prolonged transgene expression in the liver that received all three carriers. This increased transgene expression was correlated with significantly reduced serum tumor necrosis factor-α level as an indicator for KC activation. These results suggest that KC activation is a significant contributing factor to the lowered transgene expression by polycation-DNA nanoparticles delivered by RII. More importantly, the combination of RII and transient removal of KCs may be adopted as an effective approach to achieving high and persistent transgene expression in the liver mediated by nonviral nanoparticles. PMID:21091274

  4. Auto-assembly of nanometer thick, water soluble layers of plasmid DNA complexed with diamines and basic amino acids on graphite: Greatest DNA protection is obtained with arginine.

    PubMed

    Khalil, T T; Boulanouar, O; Heintz, O; Fromm, M

    2017-02-01

    We have investigated the ability of diamines as well as basic amino acids to condense DNA onto highly ordered pyrolytic graphite with minimum damage after re-dissolution in water. Based on a bibliographic survey we briefly summarize DNA binding properties with diamines as compared to basic amino acids. Thus, solutions of DNA complexed with these linkers were drop-cast in order to deposit ultra-thin layers on the surface of HOPG in the absence or presence of Tris buffer. Atomic Force Microscopy analyses showed that, at a fixed ligand-DNA mixing ratio of 16, the mean thickness of the layers can be statistically predicted to lie in the range 0-50nm with a maximum standard deviation ±6nm, using a simple linear law depending on the DNA concentration. The morphology of the layers appears to be ligand-dependent. While the layers containing diamines present holes, those formed in the presence of basic amino acids, except for lysine, are much more compact and dense. X-ray Photoelectron Spectroscopy measurements provide compositional information indicating that, compared to the maximum number of DNA sites to which the ligands may bind, the basic amino acids Arg and His are present in large excess. Conservation of the supercoiled topology of the DNA plasmids was studied after recovery of the complex layers in water. Remarkably, arginine has the best protection capabilities whether Tris was present or not in the initial solution.

  5. Biochemical transformation of thymidine kinase (TK)-deficient mouse cells by herpes simplex virus type 1 DNA fragments purified from hybrid plasmids.

    PubMed

    Kit, S; Otsuka, H; Qavi, H; Trkula, D; Dubbs, D R; Hazen, M

    1980-11-25

    The thymidine kinase (TK) gene of HSV-1 has been cloned in Escherichia coli K12 plasmids, pMH1, pMH1A, and pMH4. These plasmids contain a 1,92Obp HSV-1 TK DNA sequence, which replaces a 2,067 bp EcoR I to Pvu II sequence of plasmid pBR322 DNA. Superhelical DNAs of plasmids pMH1, pMH1A, and pMH4 as well as plasmid DNAs cleaved by EcoR I, Hinc II, Bg1 II, Sma I, and Pvu II transformed TK-deficient LM(TK-) cells to the TK+ phenotype. A 1,230bp EcoR I-Sma I fragment purified from pMH1 DNA (and from plasmid pAGO, DNA, the parent of pMH1) also transformed LM(TK-) cells. Serological and disc PAGE studies demonstrated that the TK activity expressed in biochemically transformed cells were HSV-1-specific. The experiments suggest that the HSV-1 TK coding region may be contained within a l.1kbp DNA sequence extending from about the Hinc II (or Bgl II) cleavage site to the Sma I site. 35S-methionine labeling experiments carried out on cell lines transformed by Hinc II-cleaved pMH1 DNA and by the EcoR I-Sma I fragment showed that the TKs purified from the transformed cells consisted of about 39-40,000 dalton polypeptides.

  6. Induction of DNA strand breaks, base lesions and clustered damage sites in hydrated plasmid DNA films by ultrasoft X rays around the phosphorus K edge.

    PubMed

    Yokoya, Akinari; Cunniffe, Siobhan M T; Watanabe, Ritsuko; Kobayashi, Katsumi; O'Neill, Peter

    2009-09-01

    To characterize the DNA damage induced by K-shell ionization of phosphorus atom in DNA backbone on the level of hydration, the yields of DNA strand breaks and base lesions arising from the interaction of ultrasoft X rays with energies around the phosphorus K edge were determined using dry and fully hydrated pUC18 plasmid DNA samples. Base lesions and bistranded clustered DNA damage sites were revealed by postirradiation treatment with the base excision repair proteins endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). The yield of prompt single-strand breaks (SSBs) with dry DNA irradiated at the phosphorus K resonance energy (2153 eV) is about one-third that below the phosphorus K edge (2147 eV). The yields of prompt double-strand breaks (DSBs) were found to be less dependent on the X-ray energy, with the yields being about two times lower when irradiated at 2153 eV. Heat-labile sites were not produced in detectable amounts. The yields of base lesions were dependent on the energy of the X rays, especially when the DNA was fully hydrated. Bistranded clustered DNA damage sites, revealed enzymatically as additional DSBs, were produced in dry as well as in hydrated DNA with all three energies of X rays. The yields of these enzyme-sensitive sites were also lower when irradiated at the phosphorus K resonance energy. On the other hand, the yields of prompt SSBs and enzyme-sensitive sites for the two off-resonance energies were, larger than those determined previously for gamma radiation. The results indicate that the photoelectric effect caused by X rays and dense ionization and excitation events along the tracks of low-energy secondary electrons are more effective at inducing SSBs and enzyme-sensitive sites. The complex types of damage, prompt and enzymatically induced DSBs, are preferentially induced by phosphorus K resonance at 2153 eV rather than simple SSBs and isolated base lesions, particularly in hydrated conditions. It is concluded that not

  7. An experimental study on use of 7T MRI for evaluation of myocardial infarction in SD rats transfected with pcDNA 3.1(+)/VEGF121 plasmid

    PubMed Central

    Zhang, Yan; Tian, Ruiqing; Shen, Xiangchun; Chen, Yushu; Chen, Wei; Gan, Lu; Shen, Guiquan; Ju, Haiyue; Yang, Li; Gao, Fabao

    2016-01-01

    This study aims to build the myocardial infarction model in SD rats transfected with pcDNA 3.1(+)/VEGF121 plasmid and study the effect of the transfection using 7T MRI. Twenty-four male SD rats were randomly divided into 2 groups, pcDNA 3.1(+)/VEGF121 plasmid transfection group (with improved coronary perfusion delivery) and myocardial infarction model group. Cardiac cine magnetic resonance imaging (Cine-MRI), T2-mapping and late gadolinium enhancement (LGE) cardiac imaging were performed at 24 h, 48 h, 72 h and 7 d after myocardial infarction, respectively. The signal intensity, area at risk (AAR), myocardium infarction core (MIC) and salvageable myocardial zone (SMZ) were compared. The hearts were harvested for anatomic characterization, which was related to pathological examination (TTC staining, HE staining, Masson staining and immunohistochemical staining). The Cine-MRI results showed that pcDNA 3.1(+)/VEGF121 plasmid transfection group had higher end-diastolic volume (EDV) with a reduction in MIC and SMZ, as compared with the myocardial infarction model group. MIC, SMZ and AAR of the plasmid transfection declined over time. At 7 d, the two groups did not differ significantly in AAR and T2 value. According to Western Blotting, VEGF was up-regulated, while CaSR and caspase-3 were downregulated in the plasmid transfection group, as compared with the model group. In conclusion, a good treatment effect was achieved by coronary perfusion of pcDNA 3.1(+)/VEGF121 plasmid. 7T CMR sequences provide a non-invasive quantification of the treatment efficacy. However, the assessment of myocardial injury using T2 value and AAR in the presence of edema is less accurate. The myocardial protection of the plasmid transfection group may be related to the inhibition of myocardial apoptosis, vascular endothelial cell (VEC) proliferation and collagen proliferation. The CaSR signaling pathway may contribute to reversing the apoptosis. PMID:27648128

  8. Enhanced plasmid DNA utilization in transiently transfected CHO-DG44 cells in the presence of polar solvents.

    PubMed

    Rajendra, Yashas; Balasubramanian, Sowmya; Kiseljak, Divor; Baldi, Lucia; Wurm, Florian M; Hacker, David L

    2015-01-01

    Although the protein yields from transient gene expression (TGE) with Chinese hamster ovary (CHO) cells have recently improved, the amount of plasmid DNA (pDNA) needed for transfection remains relatively high. We describe a strategy to reduce the pDNA amount by transfecting CHO-DG44 cells with 0.06 μg pDNA/10(6) cells (10% of the optimal amount) in the presence of nonspecific (filler) DNA and various polar solvents including dimethylsufoxide, dimethyl formamide, acetonitrile, dimethyl acetamide (DMA), and hexamethyl phosphoramide (HMP). All of the polar solvents with the exception of HMP increased the production of a recombinant antibody in comparison to the untreated control transfection. In the presence of 0.25% DMA, the antibody yield in a 7-day batch culture was 500 mg/L. This was fourfold higher than the yield from the untreated control transfection. Mechanistic studies revealed that the polar solvents did not affect polyethylenimine-mediated pDNA delivery into cells or nuclei. The steady-state transgene mRNA level was elevated in the presence of each of the polar solvents tested, while the transgene mRNA half-life remained the same. These results indicated that the polar solvents enhanced transgene transcription. When screening a panel of recombinant antibodies and Fc-fusion proteins for production in the presence of the polar solvents, the highest increase in yield was observed following DMA addition for 11 of the 12 proteins. These results are expected to enhance the applicability of high-yielding TGE processes with CHO-DG44 cells by decreasing the amount of pDNA required for transfection.

  9. Plasmid DNA topology assayed by two-dimensional agarose gel electrophoresis.

    PubMed

    Schvartzman, Jorge B; Martínez-Robles, María-Luisa; Hernández, Pablo; Krimer, Dora B

    2013-01-01

    Two-dimensional (2D) agarose gel electrophoresis is nowadays one of the best methods available to analyze DNA molecules with different masses and shapes. The possibility to use nicking enzymes and intercalating agents to change the twist of DNA during only one or in both runs, improves the capacity of 2D gels to discern molecules that apparently may look alike. Here we present protocols where 2D gels are used to understand the structure of DNA molecules and its dynamics in living cells. This knowledge is essential to comprehend how DNA topology affects and is affected by all the essential functions that DNA is involved in: replication, transcription, repair and recombination.

  10. Cationized gelatin hydrogels mixed with plasmid DNA induce stronger and more sustained gene expression than atelocollagen at calvarial bone defects in vivo.

    PubMed

    Komatsu, K; Shibata, T; Shimada, A; Ideno, H; Nakashima, K; Tabata, Y; Nifuji, A

    2016-01-01

    Gene transduction of exogenous factors at local sites in vivo is a promising approach to promote regeneration of tissue defects owing to its simplicity and capacity for expression of a variety of genes. Gene transduction by viral vectors is highly efficient; however, there are safety concerns associated with viruses. As a method for nonviral gene transduction, plasmid DNA delivery is safer and simpler, but requires an efficient carrier substance. Here, we aimed to develop a simple, efficient method for bone regeneration by gene transduction and to identify optimal conditions for plasmid DNA delivery at bone defect sites. We focused on carrier substances and compared the efficiencies of two collagen derivatives, atelocollagen, and gelatin hydrogel, as substrates for plasmid DNA delivery in vivo. To assess the efficiencies of these substrates, we examined exogenous expression of green fluorescence protein (GFP) by fluorescence microscopy, polymerase chain reaction, and immunohistochemistry. GFP expression at the bone defect site was higher when gelatin hydrogel was used as a substrate to deliver plasmids than when atelocollagen was used. Moreover, the gelatin hydrogel was almost completely absorbed at the defect site, whereas some atelocollagen remained. When a plasmid harboring bone morphogenic protein 2 was delivered with the substrate to bony defect sites, more new bone formation was observed in the gelatin group than in the atelocollagen group. These results suggested that the gelatin hydrogel was more efficient than atelocollagen as a substrate for local gene delivery and may be a superior material for induction of bone regeneration.

  11. Successful gene transfer into dendritic cells with cationized gelatin and plasmid DNA complexes via a phagocytosis-dependent mechanism.

    PubMed

    Inada, Satoshi; Fujiwara, Hitoshi; Atsuji, Kiyoto; Takashima, Kazuhiro; Araki, Yasunobu; Kubota, Takeshi; Tabata, Yasuhiko; Yamagishi, Hisakazu

    2006-01-01

    The use of gene-modified dendritic cells (DC) is a powerful tool to enhance antitumor immune responses stimulated by these cells in cancer immunotherapy. Cationized gelatin is preferably incorporated via phagocytosis and is gradually degraded by proteolysis while buffering lysosomal activity. This may be appropriate for gene transfer into phagocytic cells, such as immature DC. In the present study, successful transfection into monocyte-derived immature DC was demonstrated using cationized gelatin and plasmid DNA complexes. A high transfection efficiency, approaching 16%, was obtained upon transfection of the enhanced green fluorescent protein (EGFP) gene as evaluated by flow cytometry. Transgene expression of EGFP and murine interleukin 12 were also detected by RT-PCR. The antigen-presenting capacity of the transfected DC was equal to that of untransfected DC as evaluated by the allogeneic mixed lymphocyte reaction. Cationized gelatin has the potential to be a unique non-viral vector for gene transfer into DC.

  12. Vaxfectin (registered trademark) Enhances Both Antibody and In Vitro T Cell Responses to Each Component of a 5-gene Plasmodium falciparum Plasmid DNA Vaccine Mixture Administered at Low Doses

    DTIC Science & Technology

    2010-01-01

    female outbred CD- 1 mice obtained from Charles River Laboratories (Wilmington, MA) were used for antibody response studies. 2.2. Plasmids Plasmid...Vaxfectin®-formulated pDNA stock endotoxin levels were less than 30 EU/mg, while the unformulated pDNA stock endotoxin levels were less than 7.5 EU

  13. Antiangiogenic gene therapy of solid tumor by systemic injection of polyplex micelles loading plasmid DNA encoding soluble flt-1.

    PubMed

    Oba, Makoto; Vachutinsky, Yelena; Miyata, Kanjiro; Kano, Mitsunobu R; Ikeda, Sorato; Nishiyama, Nobuhiro; Itaka, Keiji; Miyazono, Kohei; Koyama, Hiroyuki; Kataoka, Kazunori

    2010-04-05

    In this study, a polyplex micelle was developed as a potential formulation for antiangiogenic gene therapy of subcutaneous pancreatic tumor model. Poly(ethylene glycol)-poly(l-lysine) block copolymers (PEG-PLys) with thiol groups in the side chain of the PLys segment were synthesized and applied for preparation of disulfide cross-linked polyplex micelles through ion complexation with plasmid DNA (pDNA) encoding the soluble form of vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1), which is a potent antiangiogenic molecule. Antitumor activity and gene expression of polyplex micelles with various cross-linking rates were evaluated in mice bearing subcutaneously xenografted BxPC3 cell line, derived from human pancreatic adenocarcinoma, and polyplex micelles with optimal cross-linking rate achieved effective suppression of tumor growth. Significant gene expression of this micelle was detected selectively in tumor tissue, and its antiangiogenic effect was confirmed by decreased vascular density inside the tumor. Therefore, the disulfide cross-linked polyplex micelle loading sFlt-1 pDNA has a great potential for antiangiogenic therapy against subcutaneous pancreatic tumor model by systemic application.

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

    PubMed

    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%.

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

    PubMed

    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. Fractionation of protein, RNA, and plasmid DNA in centrifugal precipitation chromatography using cationic surfactant CTAB containing inorganic salts NaCl and NH(4)Cl.

    PubMed

    Tomanee, Panarat; Hsu, James T; Ito, Yoichiro

    2004-10-05

    Centrifugal precipitation chromatography (CPC) is a separation system that mainly employs a moving concentration gradient of precipitating agent along a channel and solutes of interest undergo repetitive precipitation-dissolution, fractionate at different locations, and elute out from the channel according to their solubility in the precipitating agent solution. We report here for the first time the use of a CPC system for fractionation of protein, RNA, and plasmid DNA in clarified lysate produced from bacterial culture. The cationic surfactant cetyltrimethylammonium bromide (CTAB) was initially used as a precipitating agent; however, all biomolecules showed no differential solubility in the moving concentration gradient of this surfactant and, as a result, no separation of protein, RNA, and plasmid DNA occurred. To overcome this problem, inorganic salts such as NaCl and NH(4)Cl were introduced into solution of CTAB. The protein and RNA were found to have higher solubility with the addition of these salts and separated from the plasmid DNA. Decreasing surface charge density of CTAB upon addition of NaCl and NH(4)Cl was believed to lead to lower surfactant complexation, and therefore caused differential solubility and fractionation of these biomolecules. Addition of CaCl(2) did not improve solubility and separation of RNA from plasmid DNA.

  17. 16S rRNA gene probe quantitates residual host cell DNA in pharmaceutical-grade plasmid DNA.

    PubMed

    Wang, Kai-Yu; Guo, Ying-Jun; Sun, Shu-Han; Shi, Ke; Zhang, Shu; Wang, Kai-Hui; Yi-Zhang; Chen, Zu-Huan

    2006-03-24

    The development and widespread use of DNA-based vaccination against infectious pathogens have been a great triumph of medical science. Quality control of DNA vaccines as biopharmaceutical productions is a problem to solve. Residual genomic DNA of engineering bacteria has been identified as a potential risk factor, so whose level must be controlled under the regulatory standards. We report a dot-blot hybridization method to detect residual host cell DNA in purified DNA vaccines. The assay utilizes PCR amplified and digoxigenin-labeled Escherichia coli 16S rRNA gene as probe. The sensitivity of the dot-blot hybridization assay with E. coli 16S rRNA gene probe was evaluated in comparison with single copy UidR gene probe. The optimized dot-blot hybridization assay had both low background and a suitable sensitivity, detecting 10 pg of residual E. coli DNA. The method is suitable in the routine use of measuring the levels of residual E. coli DNA in the pharmaceutical-grade DNA vaccine.

  18. Rapid Delivery of Foreign Genes into Plants by Direct Rub-Inoculation with Intact Plasmid DNA of a Tomato Bushy Stunt Virus Gene Vector

    PubMed Central

    Scholthof, Herman B.

    1999-01-01

    Tomato bushy stunt virus (TBSV) cDNA, positioned between a modified cauliflower mosaic virus 35S promoter and the hepatitis delta virus antigenomic ribozyme with a downstream nopaline synthase gene polyadenylation signal, established infections upon rub-inoculation of plants with intact plasmids. Application of this methodology produced a TBSV DNA-based gene vector which yielded readily detectable levels of localized foreign gene expression in inoculated leaves. This is the first demonstration of an infectious DNA from a member of the Tombusviridae which permits rapid TBSV-mediated foreign-gene expression upon direct rub-inoculation of miniprep DNA onto a variety of plant species. PMID:10438874

  19. Invertrons, a class of structurally and functionally related genetic elements that includes linear DNA plasmids, transposable elements, and genomes of adeno-type viruses.

    PubMed Central

    Sakaguchi, K

    1990-01-01

    Invertrons are genetic elements composed of DNA with inverted terminal repeats at both ends, covalently bonded to terminal proteins involved in the initiation of DNA replication at both their 5' termini when they exist in the cytoplasm of their host in free form. They function as viruses, linear DNA plasmids, transposable elements, and sometimes combinations of two of these properties. They differ from retroviruses and related retro-type transposons which have direct repeats on both their genomic ends and exploit RNA intermediates for replication of their DNA. A model for replication and integration of invertrons is presented, as well as a model for transposition of transposable elements. PMID:2157134

  20. Immunogenicity and protective efficacy of mycobacterial DNA vaccines incorporating plasmid-encoded cytokines against Mycobacterium bovis.

    PubMed

    Young, Sarah L; Slobbe, Lynn J; Peacey, Matthew; Gilbert, Sarah C; Buddle, Bryce M; de Lisle, Geoffrey W; Buchan, Glenn S

    2010-08-01

    DNA-based vaccines, alone or in combination with other sub-unit vaccination regimes, represent an alternative to live mycobacterial vaccines for protective immunization against tuberculosis. Here, we have used a murine immunization or Mycobacterium bovis aerosol challenge model to assess the immunogenicity and protective efficacy of mycobacterial DNA vaccines. Mice that received immunization with DNA constructs encoding M. bovis antigen 85A (Ag85-A) and arget(ESAT-6) produced measurable interferon-gamma (IFN-gamma) responses to CD4(+) T-cell epitope-peptide recall antigens in vitro. The magnitude of these responses was enhanced by co-delivery of a construct encoding murine cytokines (macrophage inhibitory protein (MIP)-1 alpha or interleukin(IL)-7), although they did not the match responses observed in mice that received Bacille Calmette-Guerin(BCG) immunisation. In contrast, DNA priming followed by boosting with modified vaccinia Ankara (MVA) vaccine (expressing M. tuberculosis Ag85-A) invoked higher IFN-gamma levels, with the most immunogenic regime of Ag85 or ESAT or IL-7 prime followed by MVA boost being of commensurate immunogenicity to BCG. Despite this, neither DNA alone nor DNA-prime or MVA boost regimes conferred measurable protection against aerosol challenge with virulent M. bovis. These data highlight both the promise and the shortcomings of new generation subunit tuberculosis vaccines, with particular emphasis on their potential as vaccines against M. bovis.

  1. On the chemical yield of base lesions, strand breaks, and clustered damage generated in plasmid DNA by the direct effect of X rays.

    PubMed

    Purkayastha, Shubhadeep; Milligan, Jamie R; Bernhard, William A

    2007-09-01

    The purpose of this study was to determine the yield of DNA base damages, deoxyribose damage, and clustered lesions due to the direct effects of ionizing radiation and to compare these with the yield of DNA trapped radicals measured previously in the same pUC18 plasmid. The plasmids were prepared as films hydrated in the range 2.5 < Gamma < 22.5 mol water/mol nucleotide. Single-strand breaks (SSBs) and double-strand breaks (DSBs) were detected by agarose gel electrophoresis. Specific types of base lesions were converted into SSBs and DSBs using the base-excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). The yield of base damage detected by this method displayed a strikingly different dependence on the level of hydration (Gamma) compared with that for the yield of DNA trapped radicals; the former decreased by 3.2 times as Gamma was varied from 2.5 to 22.5 and the later increased by 2.4 times over the same range. To explain this divergence, we propose that SSB yields produced in plasmid DNA by the direct effect cannot be analyzed properly with a Poisson process that assumes an average of one strand break per plasmid and neglects the possibility of a single track producing multiple SSBs within a plasmid. The yields of DSBs, on the other hand, are consistent with changes in free radical trapping as a function of hydration. Consequently, the composition of these clusters could be quantified. Deoxyribose damage on each of the two opposing strands occurs with a yield of 3.5 +/- 0.5 nmol/J for fully hydrated pUC18, comparable to the yield of 4.1 +/- 0.9 nmol/J for DSBs derived from opposed damages in which at least one of the sites is a damaged base.

  2. Excess intracellular concentration of the pSC101 RepA protein interferes with both plasmid DNA replication and partitioning.

    PubMed Central

    Ingmer, H; Cohen, S N

    1993-01-01

    RepA, a plasmid-encoded gene product required for pSC101 replication in Escherichia coli, is shown here to inhibit the replication of pSC101 in vivo when overproduced 4- to 20-fold in trans. Unlike plasmids whose replication is prevented by mutations in the repA gene, plasmids prevented from replicating by overproduction of the RepA protein were lost rapidly from the cell population instead of being partitioned evenly between daughter cells. Removal of the partition (par) locus increased the inhibitory effect of excess RepA on replication, while host and plasmid mutations that compensate for the absence of par, or overproduction of the E. coli DnaA protein, diminished it. A repA mutation (repA46) that elevates pSC101 copy number almost entirely eliminated the inhibitory effect of RepA at high concentration and stimulated replication when the protein was moderately overproduced. As the RepA protein can exist in both monomer and dimer forms, we suggest that overproduction promotes RepA dimerization, reducing the formation of replication initiation complexes that require the RepA monomer and DnaA; we propose that the repA46 mutation alters the ability of the mutant protein to dimerize. Our discovery that an elevated intracellular concentration of RepA specifically impedes plasmid partitioning implies that the RepA-containing complexes initiating pSC101 DNA replication participate also in the distribution of plasmids at cell division. Images PMID:8253672

  3. In vitro excision of adeno-associated virus DNA from recombinant plasmids: Isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences

    SciTech Connect

    Gottlieb, J.; Muzyczka, N.

    1988-06-01

    When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, the authors isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G . C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat and in some cases as the result of cloning the AAV genome by G . C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.

  4. Lipid nanocapsules functionalized with polyethyleneimine for plasmid DNA and drug co-delivery and cell imaging

    NASA Astrophysics Data System (ADS)

    Skandrani, Nadia; Barras, Alexandre; Legrand, Dominique; Gharbi, Tijani; Boulahdour, Hatem; Boukherroub, Rabah

    2014-06-01

    The paper reports on the preparation of lipid nanocapsules (LNCs) functionalized with poly(ethyleneimine) (PEI) moieties and their successful use as drug and gene delivery systems. The cationic LNCs were produced by a phase inversion process with a nominal size of 25 nm and subsequently modified with PEI chains using a transacylation reaction. The functionalization process allowed good control over the nanoscale particle size (26.2 +/- 3.9 nm) with monodisperse size characteristics (PI < 0.2) and positive surface charge up to +18.7 mV. The PEI-modified LNCs (LNC25-T) displayed good buffering capacity. Moreover, the cationic LNC25-T were able to condense DNA and form complexes via electrostatic interactions in a typical weight ratio-dependent relationship. It was found that the mean diameter of LNC25-T/pDNA complexes increased to ~40-50 nm with the LNC25-T/pDNA ratio from 1 to 500. Gel electrophoresis and cell viability experiments showed that the LNC25-T/pDNA complexes had high stability with no cytotoxicity due to the anchored PEI polymers on the surface of LNCs. Finally, the transfection efficiency of the LNC25-T/pDNA complexes was studied and evaluated on HEK cell lines in comparison with free PEI/pDNA polyplexes. The combination of cationic LNCs with pDNA exhibited more than a 2.8-fold increase in transfection efficiency compared to the standard free PEI/pDNA polyplexes at the same PEI concentrations. Moreover, we have demonstrated that LNC25-T/pDNA loaded with a hydrophobic drug, paclitaxel, showed high drug efficacy. The high transfection efficiency combined with the potential of simultaneous co-delivery of hydrophobic drugs, relatively small size of LNC25-T/pDNA complexes, and fluorescence imaging can be crucial for gene therapy, as small particle sizes may be more favorable for in vivo studies.The paper reports on the preparation of lipid nanocapsules (LNCs) functionalized with poly(ethyleneimine) (PEI) moieties and their successful use as drug and gene

  5. Optimized and enhanced DNA plasmid vector based in vivo construction of a neutralizing anti-HIV-1 envelope glycoprotein Fab.

    PubMed

    Muthumani, Kar; Flingai, Seleeke; Wise, Megan; Tingey, Colleen; Ugen, Kenneth E; Weiner, David B

    2013-10-01

    Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an "enhanced and optimized" DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This "enhanced" DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of "adaptive" in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection, against diverse

  6. Vibration and glycerol-mediated plasmid DNA transformation for Escherichia coli.

    PubMed

    Shanehbandi, Dariush; Saei, Amir A; Zarredar, Habib; Barzegari, Abolfazl

    2013-11-01

    Escherichia coli transformation is an essential step in many molecular biology experiments. Despite earlier advances in the field, many studies including shotgun cloning still require more efficient transformation protocols. Chemical transformation has been the most popular method, in which competent cells are transformed following a brief period of heat shock. Here, we report a novel protocol with higher efficiency, in which competent E. coli cells (treated with CaCl2 ) grown in media containing glycerol experience a gentle vibration. Three E. coli strains DH5α, Jm107 and BL21 (DE3) and three plasmids pGEM-T, pET-28a and pCAMBIA with different sizes (3000, 5369 and 8428 bp, respectively) were used to test the protocol. The results indicated a significant increase in number of transformed colonies compared with heat-shock method. Our findings also demonstrated the favourable impacts of glycerol on transformation of E. coli.

  7. Plasmid Partition Mechanisms.

    PubMed

    Baxter, Jamie C; Funnell, Barbara E

    2014-12-01

    The stable maintenance of low-copy-number plasmids in bacteria is actively driven by partition mechanisms that are responsible for the positioning of plasmids inside the cell. Partition systems are ubiquitous in the microbial world and are encoded by many bacterial chromosomes as well as plasmids. These systems, although different in sequence and mechanism, typically consist of two proteins and a DNA partition site, or prokaryotic centromere, on the plasmid or chromosome. One protein binds site-specifically to the centromere to form a partition complex, and the other protein uses the energy of nucleotide binding and hydrolysis to transport the plasmid, via interactions with this partition complex inside the cell. For plasmids, this minimal cassette is sufficient to direct proper segregation in bacterial cells. There has been significant progress in the last several years in our understanding of partition mechanisms. Two general areas that have developed are (i) the structural biology of partition proteins and their interactions with DNA and (ii) the action and dynamics of the partition ATPases that drive the process. In addition, systems that use tubulin-like GTPases to partition plasmids have recently been identified. In this chapter, we concentrate on these recent developments and the molecular details of plasmid partition mechanisms.

  8. Plasmid DNA delivery into MDA-MB-453 cells mediated by recombinant Her-NLS fusion protein.

    PubMed

    Jeyarajan, Sivakumar; Xavier, Jennifer; Rao, N Madhusudhana; Gopal, Vijaya

    2010-10-05

    A major rate-limiting step in nonviral gene delivery is the entry of nucleic acids across various membrane barriers and eventually into the nucleus where it must be transcribed. Cell-penetrating peptides and proteins are employed to generate formulations that overcome these challenges to facilitate DNA delivery into cells efficiently. However, these are limited by their inability to deliver nucleic acids selectively due to lack of specificity because they deliver to both cancer and normal cells. In this study, through modular design, we generated a recombinant fusion protein designated as Her-nuclear localization sequence (Her-NLS), where heregulin-α (Her), a targeting moiety, was cloned in frame with cationic NLS peptide to obtain a cell-specific targeting biomolecule for nucleic acid delivery. The heregulin-α(1) isoform possesses the epidermal growth factor-like domain and binds to HER2/3 heterodimers which are overexpressed in certain breast cancers. Purified recombinant Her-NLS fusion protein binds plasmid DNA and specifically transfects MDA-MB-453 cells overexpressing the epidermal growth factor receptors HER2/3 in vitro. The approach described would also permit replacement of heregulin ligand with other targeting moieties that would be suited to cell-specific nucleic acid delivery mediated via receptor-ligand interactions.

  9. Abnormal ultraviolet mutagenic spectrum in plasmid DNA replicated in cultured fibroblasts from a patient with the skin cancer-prone disease, xeroderma pigmentosum

    SciTech Connect

    Seetharam, S.; Protic-Sabljic, M.; Seidman, M.M.; Kraemer, K.H.

    1987-12-01

    A shuttle vector plasmid, pZ189, was utilized to assess the types of mutations that cells from a patient with xeroderma pigmentosum, complementation group D, introduce into ultraviolet (UV) damaged, replicating DNA. Patients with xeroderma pigmentosum have clinical and cellular UV hypersensitivity, increased frequency of sun-induced skin cancer, and deficient DNA repair. In comparison to UV-treated pZ189 replicated in DNA repair-proficient cells, there were fewer surviving plasmids, a higher frequency of plasmids with mutations, fewer plasmids with two or more mutations in the marker gene, and a new mutagenic hotspot. The major type of base substitution mutation was the G:C to A:T transition with both cell lines. These results, together with similar findings published earlier with cells from a xeroderma pigmentosum patient in complementation group A, suggest that isolated G:C to A:T somatic mutations may be particularly important in generation of human skin cancer by UV radiation.

  10. Optimizing a Method for the Quantification by Quantitative Real-Time Polymerase Chain Reaction of Host Cell DNA in Plasmid Vector Batches Used in Human Gene Therapy.

    PubMed

    Ferro, Serge; Fabre, Isabelle; Chenivesse, Xavier

    2016-08-01

    Gene therapy products are very complex advanced therapy medicinal products produced using different processes that require many chemical and biological reagents and production intermediates, such as producing cells. The quantification of residual impurities in gene therapy vectors is a major quality control step when these vectors are used for therapeutic purposes, whether or not they are derived from viruses. Indeed, in nonviral gene therapy products, particularly plasmid vectors used to transfer genetic material, the presence of host-cell DNA (HCDNA) from the bacterial cells used for the vector production is an important concern because of the risk of immunogenicity and insertional mutagenesis. Several methods have been developed to quantify residual HCDNA, but real-time quantitative polymerase chain reaction (qPCR) seems to be most suitable because it allows detecting traces of "contaminating" DNA. The French National Agency for Medicines and Health Products Safety (ANSM) ensures the quality and safety of gene transfer medicinal products and must be able to quantify, in its own laboratories, the amount of HCDNA present in plasmid vector batches. Therefore, we developed and validated a qPCR method to quantify at the femtogram level the presence of Escherichia coli residual DNA in plasmid vectors. This approach uses the capillary-based LightCycler 1.5 System (Roche) with SYBR Green I, a primer pair against the E. coli 23S ribosomal RNA gene and different concentrations of a linearized plasmid that contains the 23S target sequence, as standard. This qPCR method is linear on an 8-decade logarithmic scale, accurate, reproducible, and sensitive (quantification of up to 10 copies of 23S target sequence per reaction, or 1.4 E. coli genome, or 7 fg of bacterial DNA). This technique allows ensuring that batches of plasmid vectors to be used in clinical trials comply with the specifications on HCDNA content.

  11. Characterization of a linear DNA plasmid from the filamentous fungal plant pathogen Glomerella musae [Anamorph: Colletotrichum musae (Berk. and Curt.) arx.

    USGS Publications Warehouse

    Freeman, S.; Redman, R.S.; Grantham, G.; Rodriguez, R.J.

    1997-01-01

    A 7.4-kilobase (kb) DNA plasmid was isolated from Glomerella musae isolate 927 and designated pGML1. Exonuclease treatments indicated that pGML1 was a linear plasmid with blocked 5' termini. Cell-fractionation experiments combined with sequence-specific PCR amplification revealed that pGML1 resided in mitochondria. The pGML1 plasmid hybridized to cesium chloride-fractionated nuclear DNA but not to A + T-rich mitochondrial DNA. An internal 7.0-kb section of pGML1 was cloned and did not hybridize with either nuclear or mitochondrial DNA from G. musae. Sequence analysis revealed identical terminal inverted repeats (TIR) of 520 bp at the ends of the cloned 7.0-kb section of pGML1. The occurrence of pGML1 did not correspond with the pathogenicity of G. musae on banana fruit. Four additional isolates of G. musae possessed extrachromosomal DNA fragments similar in size and sequence to pGML1.

  12. Optimization of a new non-viral vector for transfection: Eudragit nanoparticles for the delivery of a DNA plasmid.

    PubMed

    Gargouri, M; Sapin, A; Bouli, S; Becuwe, P; Merlin, J L; Maincent, P

    2009-12-01

    The development of new vectors to deliver DNA into cells for therapy of cancers or genetic diseases has been a major area of research for many years. However, the clinical application of this technology requires the development of efficient, reliable and sterile vectors enabling the transfer of genes in vivo. Non viral, polymer or lipid-based vectors offer a new impetus to gene therapy because they are less toxic than viral vectors (no endogenous recombination, fewer immunological reactions, easy production and delivery of large-sized plasmid). The aim of this study is to develop a new tool for DNA delivery composed of methacrylic polymeric (Eudragit RS and RL) nanoparticles. These nanoparticles were prepared by two methods: nanoprecipitation and double emulsion. The nanoparticles were characterized by their size, zeta potential and amount of DNA adsorption. Cytotoxicity tests based on mitochondrial activity (MTT test) revealed that the nanoparticles had limited cytotoxicity and that this depended on both the cell type and the nanoparticle concentration. Transgene expression was observed using the Green Fluorescence Protein gene as reporter gene, and was evaluated by flow cytometry in FaDu, MDA-MB 231 and MCF-7 cell lines. The results showed that transfection rates ranging between 4 and 7% were achieved in FaDu and MDA-MB 231 cells with nanoparticles prepared by the nanoprecipitation method. In MCF-7 cells transfected with nanoparticles prepared by either the double emulsion or the nanoprecipitation method, the transfection efficiency was between 2 and 4%. Nanoparticles prepared by nanoprecipitation were slightly more efficient than nanoparticles prepared from a double emulsion. Particle size was not an important factor for transfection, since no significant difference was observed with size between 50 and 350 nm. We showed that Eudragit RS and RL nanoparticles could introduce the transgene into different types of cells, but were generally less effective than the

  13. Analysis of the Vaccine Potential of Plasmid DNA Encoding Nine Mycolactone Polyketide Synthase Domains in Mycobacterium ulcerans Infected Mice

    PubMed Central

    Roupie, Virginie; Pidot, Sacha J.; Einarsdottir, Tobba; Van Den Poel, Christophe; Jurion, Fabienne; Stinear, Timothy P.; Huygen, Kris

    2014-01-01

    There is no effective vaccine against Buruli ulcer. In experimental footpad infection of C57BL/6 mice with M. ulcerans, a prime-boost vaccination protocol using plasmid DNA encoding mycolyltransferase Ag85A of M. ulcerans and a homologous protein boost has shown significant, albeit transient protection, comparable to the one induced by M. bovis BCG. The mycolactone toxin is an obvious candidate for a vaccine, but by virtue of its chemical structure, this toxin is not immunogenic in itself. However, antibodies against some of the polyketide synthase domains involved in mycolactone synthesis, were found in Buruli ulcer patients and healthy controls from the same endemic region, suggesting that these domains are indeed immunogenic. Here we have analyzed the vaccine potential of nine polyketide synthase domains using a DNA prime/protein boost strategy. C57BL/6 mice were vaccinated against the following domains: acyl carrier protein 1, 2, and 3, acyltransferase (acetate) 1 and 2, acyltransferase (propionate), enoylreductase, ketoreductase A, and ketosynthase load module. As positive controls, mice were vaccinated with DNA encoding Ag85A or with M. bovis BCG. Strongest antigen specific antibodies could be detected in response to acyltransferase (propionate) and enoylreductase. Antigen-specific Th1 type cytokine responses (IL-2 or IFN-γ) were induced by vaccination against all antigens, and were strongest against acyltransferase (propionate). Finally, vaccination against acyltransferase (propionate) and enoylreductase conferred some protection against challenge with virulent M. ulcerans 1615. However, protection was weaker than the one conferred by vaccination with Ag85A or M. bovis BCG. Combinations of these polyketide synthase domains with the vaccine targeting Ag85A, of which the latter is involved in the integrity of the cell wall of the pathogen, and/or with live attenuated M. bovis BCG or mycolactone negative M. ulcerans may eventually lead to the development of an

  14. KEY COMPARISON: CCQM-K61: Quantitation of a linearised plasmid DNA, based on a matched standard in a matrix of non-target DNA

    NASA Astrophysics Data System (ADS)

    Ellison, Stephen L. R.; Holden, Marcia J.; Woolford, Alison; Haynes, Ross J.; Salit, Marc L.; Burns, Malcolm; Parkes, Helen; Cherdchu, Chainarong; Corbisier, Philippe; Emslie, Kerry R.; Gao, Yunhua; Gioria, Sabrina; Griffiths, Kate R.; Kawaharasaki, Mamoru; Konopelko, Leonid A.; Kustikov, Yury A.; Park, Sang-Ryoul; Phunbua, Nittaya; Vonsky, Maxim S.; Wang, Jing; Yang, Inchul

    2009-01-01

    Key comparison CCQM-K61 was performed to demonstrate and document the capability of interested national metrology institutes in the determination of the quantity of specific DNA target in an aqueous solution. The study provides support for the following measurement claim: "Quantitation of a linearised plasmid DNA, based on a matched standard in a matrix of non-target DNA". The comparison was an activity of the Bioanalysis Working Group (BAWG) of the Comité Consultatif pour la Quantité de Matière and was coordinated by NIST (Gaithersburg, USA) and LGC (Teddington, UK). The following laboratories (in alphabetical order) participated in this key comparison. DMSC (Thailand); IRMM (European Union); KRISS (Republic of Korea); LGC (UK); NIM (China); NIST (USA); NMIA (Australia); NMIJ (Japan); VNIIM (Russian Federation) Good agreement was observed between the reported results of all nine of the participants. Uncertainty estimates did not account fully for the dispersion of results even after allowance for possible inhomogeneity in calibration materials. Preliminary studies suggest that the effects of fluorescence threshold setting might contribute to the excess dispersion, and further study of this topic is suggested Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (MRA).

  15. Escherichia coli DNA helicase I catalyzes a sequence-specific cleavage/ligation reaction at the F plasmid origin of transfer.

    PubMed

    Sherman, J A; Matson, S W

    1994-10-21

    Recent studies have shown that the Escherichia coli F plasmid-encoded traI gene product (TraIp), also known as DNA helicase I, catalyzes the formation of the site- and strand-specific nick that initiates F plasmid DNA transfer. Scission of the phosphodiester bond at the nic site within the origin of transfer (oriT) is accompanied by the covalent attachment of TraIp to the 5'-phosphate of the nicked DNA strand. This mechanism suggests that TraIp may also be capable of catalyzing a DNA ligation reaction using the energy stored in the protein-DNA intermediate. To test this possibility, an in vitro assay was designed that utilized short single-stranded DNA oligonucleotides of different lengths derived from the region within oriT that spanned the nic site. Purified TraIp was capable of efficiently cleaving single-stranded DNA that contained a nic site, and upon cleavage, the protein became covalently linked to the 5'-end of the nic site. When TraIp was incubated with two oligonucleotides of different length that contained the nic site, there was formation of novel recombinant products resulting from a TraIp-catalyzed cleavage/ligation reaction. Furthermore, the cleavage and ligation reactions were both sequence-specific. These data suggest that TraIp plays an important role in the initiation and termination of conjugative DNA transfer.

  16. Structural basis for replication origin unwinding by an initiator primase of plasmid ColE2-P9: duplex DNA unwinding by a single protein.

    PubMed

    Itou, Hiroshi; Yagura, Masaru; Shirakihara, Yasuo; Itoh, Tateo

    2015-02-06

    Duplex DNA is generally unwound by protein oligomers prior to replication. The Rep protein of plasmid ColE2-P9 (34 kDa) is an essential initiator for plasmid DNA replication. This protein binds the replication origin (Ori) in a sequence-specific manner as a monomer and unwinds DNA. Here we present the crystal structure of the DNA-binding domain of Rep (E2Rep-DBD) in complex with Ori DNA. The structure unveils the basis for Ori-specific recognition by the E2Rep-DBD and also reveals that it unwinds DNA by the concerted actions of its three contiguous structural modules. The structure also shows that the functionally unknown PriCT domain, which forms a compact module, plays a central role in DNA unwinding. The conservation of the PriCT domain in the C termini of some archaeo-eukaryotic primases indicates that it probably plays a similar role in these proteins. Thus, this is the first report providing the structural basis for the functional importance of the conserved PriCT domain and also reveals a novel mechanism for DNA unwinding by a single protein.

  17. Differential salt-induced dissociation of the p53 protein complexes with circular and linear plasmid DNA substrates suggest involvement of a sliding mechanism.

    PubMed

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

    2015-01-30

    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. Effect of the Plasmid-DNA Vaccination on Macroscopic and Microscopic Damage Caused by the Experimental Chronic Trypanosoma cruzi Infection in the Canine Model

    PubMed Central

    Rodríguez-Morales, Olivia; Carrillo-Sánchez, Silvia C.; García-Mendoza, Humberto; Aranda-Fraustro, Alberto; Ballinas-Verdugo, Martha A.; Alejandre-Aguilar, Ricardo; Rosales-Encina, José Luis; Arce-Fonseca, Minerva

    2013-01-01

    The dog is considered the main domestic reservoir for Trypanosoma cruzi infection and a suitable experimental animal model to study the pathological changes during the course of Chagas disease (CD). Vaccine development is one of CD prevention methods to protect people at risk. Two plasmids containing genes encoding a trans-sialidase protein (TcSP) and an amastigote-specific glycoprotein (TcSSP4) were used as DNA vaccines in a canine model. Splenomegaly was not found in either of the recombinant plasmid-immunized groups; however, cardiomegaly was absent in animals immunized only with the plasmid containing the TcSSP4 gene. The inflammation of subendocardial and myocardial tissues was prevented only with the immunization with TcSSP4 gene. In conclusion, the vaccination with these genes has a partial protective effect on the enlargement of splenic and cardiac tissues during the chronic CD and on microscopic hearth damage, since both plasmids prevented splenomegaly but only one avoided cardiomegaly, and the lesions in heart tissue of dog immunized with plasmid containing the TcSSP4 gene covered only subepicardial tissue. PMID:24163822

  19. An MboII/FokI trimming plasmid allowing consecutive cycles of precise 1- to 12-base-pair deletions in cloned DNA.

    PubMed

    Hasan, N; Kur, J; Szybalski, W

    1989-10-30

    A novel trimming plasmid has been designed which allows, in a preprogrammed fashion, the precise deletion of up to 12 bp per cleavage cycle, from one end of a cloned fragment. The plasmid, which carries the dhfr gene, contains unique recognition sites for two class-IIS restriction enzymes, MboII and FokI, which are arranged in the form of a cassette, so that consecutive cleavages with these endonucleases, followed by blunting with mung bean nuclease (MB), will precisely delete 12 bp of adjacent cloned DNA. When either MboII or FokI is used alone (followed by MB), 1 or 4 bp are removed, respectively. The final step in the trimming cycle is religation of the plasmid with T4 ligase. After required number of cycles, plasmids were transformed into Escherichia coli C600, and transformants selected by resistance to trimethoprim. Since the MboII/FokI cassette remains intact during these operations, one can repeat the cycle, consisting of cleaving, MB blunting and religation, several times, each time removing up to 12 bp from the cloned target DNA. Examples are provided of one-, two- and three-cycle trimmings.

  20. Longevity of rAAV vector and plasmid DNA in blood after intramuscular injection in nonhuman primates: implications for gene doping.

    PubMed

    Ni, W; Le Guiner, C; Gernoux, G; Penaud-Budloo, M; Moullier, P; Snyder, R O

    2011-07-01

    Legitimate uses of gene transfer technology can benefit from sensitive detection methods to determine vector biodistribution in pre-clinical studies and in human clinical trials, and similar methods can detect illegitimate gene transfer to provide sports-governing bodies with the ability to maintain fairness. Real-time PCR assays were developed to detect a performance-enhancing transgene (erythropoietin, EPO) and backbone sequences in the presence of endogenous cellular sequences. In addition to developing real-time PCR assays, the steps involved in DNA extraction, storage and transport were investigated. By real-time PCR, the vector transgene is distinguishable from the genomic DNA sequence because of the absence of introns, and the vector backbone can be identified by heterologous gene expression control elements. After performance of the assays was optimized, cynomolgus macaques received a single dose by intramuscular (IM) injection of plasmid DNA, a recombinant adeno-associated viral vector serotype 1 (rAAV1) or a rAAV8 vector expressing cynomolgus macaque EPO. Macaques received a high plasmid dose intended to achieve a significant, but not life-threatening, increase in hematocrit. rAAV vectors were used at low doses to achieve a small increase in hematocrit and to determine the limit of sensitivity for detecting rAAV sequences by single-step PCR. DNA extracted from white blood cells (WBCs) was tested to determine whether WBCs can be collaterally transfected by plasmid or transduced by rAAV vectors in this context, and can be used as a surrogate marker for gene doping. We demonstrate that IM injection of a conventional plasmid and rAAV vectors results in the presence of DNA that can be detected at high levels in blood before rapid elimination, and that rAAV genomes can persist for several months in WBCs.

  1. Reconciliation of chemical, enzymatic, spectroscopic and computational data to assign the absolute configuration of the DNA base lesion spiroiminodihydantoin.

    PubMed

    Fleming, Aaron M; Orendt, Anita M; He, Yanan; Zhu, Judy; Dukor, Rina K; Burrows, Cynthia J

    2013-12-04

    The diastereomeric spiroiminodihydantoin-2'-deoxyribonucleoside (dSp) lesions resulting from 2'-deoxyguanosine (dG) or 8-oxo-7,8-dihydro-2'-deoxyguanosine (dOG) oxidation have generated much attention due to their highly mutagenic nature. Their propeller-like shape leads these molecules to display mutational profiles in vivo that are stereochemically dependent. However, there exist conflicting absolute configuration assignments arising from electronic circular dichroism (ECD) and NOESY-NMR experiments; thus, providing definitive assignments of the 3D structure of these molecules is of great interest. In the present body of work, we present data inconsistent with the reported ECD assignments for the dSp diastereomers in the nucleoside context, in which the first eluting isomer from a Hypercarb HPLC column was assigned to be the S configuration, and the second was assigned the R configuration. The following experiments were conducted: (1) determination of the diastereomer ratio of dSp products upon one-electron oxidation of dG in chiral hybrid or propeller G-quadruplexes that expose the re or si face to solvent, respectively; (2) absolute configuration analysis using vibrational circular dichroism (VCD) spectroscopy; (3) reinterpretation of the ECD experimental spectra using time-dependent density functional theory (TDDFT) with the inclusion of 12 explicit H-bonding waters around the Sp free bases; and (4) reevaluation of calculated specific rotations for the Sp enantiomers using the hydration model in the TDDFT calculations. These new insights provide a fresh look at the absolute configuration assignments of the dSp diastereomers in which the first eluting from a Hypercarb-HPLC column is (-)-(R)-dSp and the second is (+)-(S)-dSp. These assignments now provide the basis for understanding the biological significance of the stereochemical dependence of enzymes that process this form of DNA damage.

  2. Novel actin filaments from Bacillus thuringiensis form nanotubules for plasmid DNA segregation

    PubMed Central

    Jiang, Shimin; Narita, Akihiro; Popp, David; Ghoshdastider, Umesh; Lee, Lin Jie; Srinivasan, Ramanujam; Balasubramanian, Mohan K.; Oda, Toshiro; Koh, Fujiet; Larsson, Mårten; Robinson, Robert C.

    2016-01-01

    Here we report the discovery of a bacterial DNA-segregating actin-like protein (BtParM) from Bacillus thuringiensis, which forms novel antiparallel, two-stranded, supercoiled, nonpolar helical filaments, as determined by electron microscopy. The BtParM filament features of supercoiling and forming antiparallel double-strands are unique within the actin fold superfamily, and entirely different to the straight, double-stranded, polar helical filaments of all other known ParMs and of eukaryotic F-actin. The BtParM polymers show dynamic assembly and subsequent disassembly in the presence of ATP. BtParR, the DNA-BtParM linking protein, stimulated ATP hydrolysis/phosphate release by BtParM and paired two supercoiled BtParM filaments to form a cylinder, comprised of four strands with inner and outer diameters of 57 Å and 145 Å, respectively. Thus, in this prokaryote, the actin fold has evolved to produce a filament system with comparable features to the eukaryotic chromosome-segregating microtubule. PMID:26873105

  3. Physical Factors Affecting Plasmid DNA Compaction in Stearylamine-Containing Nanoemulsions Intended for Gene Delivery

    PubMed Central

    Silva, André Leandro; Júnior, Francisco Alexandrino; Verissimo, Lourena Mafra; Agnez-Lima, Lucymara Fassarella; Egito, Lucila Carmem Monte; de Oliveira, Anselmo Gomes; do Egito, Eryvaldo Socrates Tabosa

    2012-01-01

    Cationic lipids have been used in the development of non-viral gene delivery systems as lipoplexes. Stearylamine, a cationic lipid that presents a primary amine group when in solution, is able to compact genetic material by electrostatic interactions. In dispersed systems such as nanoemulsions this lipid anchors on the oil/water interface confering a positive charge to them. The aim of this work was to evaluate factors that influence DNA compaction in cationic nanoemulsions containing stearylamine. The influence of the stearylamine incorporation phase (water or oil), time of complexation, and different incubation temperatures were studied. The complexation rate was assessed by electrophoresis migration on agarose gel 0.7%, and nanoemulsion and lipoplex characterization was done by Dynamic Light Scattering (DLS). The results demonstrate that the best DNA compaction process occurs after 120 min of complexation, at low temperature (4 ± 1 °C), and after incorporation of the cationic lipid into the aqueous phase. Although the zeta potential of lipoplexes was lower than the results found for basic nanoemulsions, the granulometry did not change. Moreover, it was demonstrated that lipoplexes are suitable vehicles for gene delivery. PMID:24281666

  4. Physical factors affecting plasmid DNA compaction in stearylamine-containing nanoemulsions intended for gene delivery.

    PubMed

    Silva, André Leandro; Alexandrino, Francisco; Verissimo, Lourena Mafra; Agnez-Lima, Lucymara Fassarella; Egito, Lucila Carmem Monte; de Oliveira, Anselmo Gomes; do Egito, Eryvaldo Socrates Tabosa

    2012-06-18

    Cationic lipids have been used in the development of non-viral gene delivery systems as lipoplexes. Stearylamine, a cationic lipid that presents a primary amine group when in solution, is able to compact genetic material by electrostatic interactions. In dispersed systems such as nanoemulsions this lipid anchors on the oil/water interface confering a positive charge to them. The aim of this work was to evaluate factors that influence DNA compaction in cationic nanoemulsions containing stearylamine. The influence of the stearylamine incorporation phase (water or oil), time of complexation, and different incubation temperatures were studied. The complexation rate was assessed by electrophoresis migration on agarose gel 0.7%, and nanoemulsion and lipoplex characterization was done by Dynamic Light Scattering (DLS). The results demonstrate that the best DNA compaction process occurs after 120 min of complexation, at low temperature (4 ± 1 °C), and after incorporation of the cationic lipid into the aqueous phase. Although the zeta potential of lipoplexes was lower than the results found for basic nanoemulsions, the granulometry did not change. Moreover, it was demonstrated that lipoplexes are suitable vehicles for gene delivery.

  5. Plasmid Detection, Characterization, and Ecology.

    PubMed

    Smalla, Kornelia; Jechalke, Sven; Top, Eva M

    2015-02-01

    Plasmids are important vehicles for rapid adaptation of bacterial populations to changing environmental conditions. It is thought that to reduce the cost of plasmid carriage, only a fraction of a local population carries plasmids or is permissive to plasmid uptake. Plasmids provide various accessory traits which might be beneficial under particular conditions. The genetic variation generated by plasmid carriage within populations ensures the robustness toward environmental changes. Plasmid-mediated gene transfer plays an important role not only in the mobilization and dissemination of antibiotic resistance genes but also in the spread of degradative pathways and pathogenicity determinants of pathogens. Here we summarize the state-of-the-art methods to study the occurrence, abundance, and diversity of plasmids in environmental bacteria. Increasingly, cultivation-independent total-community DNA-based methods are being used to characterize and quantify the diversity and abundance of plasmids in relation to various biotic and abiotic factors. An improved understanding of the ecology of plasmids and their hosts is crucial in the development of intervention strategies for antibiotic-resistance-gene spread. We discuss the potentials and limitations of methods used to determine the host range of plasmids, as the ecology of plasmids is tightly linked to their hosts. The recent advances in sequencing technologies provide an enormous potential for plasmid classification, diversity, and evolution studies, but numerous challenges still exist.

  6. Origin recognition specificity in pT181 plasmids is determined by a functionally asymmetric palindromic DNA element.

    PubMed Central

    Wang, P Z; Projan, S J; Henriquez, V; Novick, R P

    1993-01-01

    The leading strand replication origin of pT181 plasmids consists of two adjacent inverted repeat elements (IR-II and IR-III), which are involved in origin recognition by the initiator (Rep) protein. The conserved core element, IR-II, which contains the initiation nick site, is induced by Rep to form a cruciform structure, probably the primary substrate for the initiation of rolling circle replication. The divergent repeat, IR-III, constitutes the determinant of origin recognition specificity. We show here that the distal arm of IR-III is not required for sequence-specific recognition, whereas the proximal arm and central region are required. Since the initiator is dimeric, we presume that it binds symmetrically to IR-III. A unique type of DNA-protein interaction is proposed, in which the lack of sequence requirement for the distal arm is a consequence of binding to the adjacent IR-II, which thereby polarizes the stringency of binding to the two arms of IR-III. In addition, genetic evidence indicates that both the spacing and the phasing of IR-II to IR-III are crucial for function and that the central segment of IR-III may serve to position the two flanking half-sites for optimal interaction of Rep with IR-III. Images PMID:8428593

  7. DNA Sequence and Comparative Genomics of pAPEC-O2-R, an Avian Pathogenic Escherichia coli Transmissible R Plasmid

    PubMed Central

    Johnson, Timothy J.; Siek, Kylie E.; Johnson, Sara J.; Nolan, Lisa K.

    2005-01-01

    In this study, a 101-kb IncF plasmid from an avian pathogenic Escherichia coli (APEC) strain (APEC O2) was sequenced and analyzed, providing the first completed APEC plasmid sequence. This plasmid, pAPEC-O2-R, has functional transfer and antimicrobial resistance-encoding regions. The resistance-encoding region encodes resistance to eight groups of antimicrobial agents, including silver and other heavy metals, quaternary ammonium compounds, tetracycline, sulfonamides, aminoglycosides, trimethoprim, and beta-lactam antimicrobial agents. This region of the plasmid is unique among previously described IncF plasmids in that it possesses a class 1 integron that harbors three gene cassettes and a heavy metal resistance operon. This region spans 33 kb and is flanked by the RepFII plasmid replicon and an assortment of plasmid maintenance genes. pAPEC-O2-R also contains a 32-kb transfer region that is nearly identical to that found in the E. coli F plasmid, rendering it transferable by conjugation to plasmid-less strains of bacteria, including an APEC strain, a fecal E. coli strain from an apparently healthy bird, a Salmonella enterica serovar Typhimurium strain, and a uropathogenic E. coli strain from humans. Differences in the G+C contents of individual open reading frames suggest that various regions of pAPEC-O2-R had dissimilar origins. The presence of pAPEC-O2-R-like plasmids that encode resistance to multiple antimicrobial agents and that are readily transmissible from APEC to other bacteria suggests the possibility that such plasmids may serve as a reservoir of resistance genes for other bacteria of animal and human health significance. PMID:16251312

  8. Easy Absolute Values? Absolutely

    ERIC Educational Resources Information Center

    Taylor, Sharon E.; Mittag, Kathleen Cage

    2015-01-01

    The authors teach a problem-solving course for preservice middle-grades education majors that includes concepts dealing with absolute-value computations, equations, and inequalities. Many of these students like mathematics and plan to teach it, so they are adept at symbolic manipulations. Getting them to think differently about a concept that they…

  9. Antiangiogenic gene therapy of experimental pancreatic tumor by sFlt-1 plasmid DNA carried by RGD-modified crosslinked polyplex micelles.

    PubMed

    Vachutinsky, Yelena; Oba, Makoto; Miyata, Kanjiro; Hiki, Shigehiro; Kano, Mitsunobu R; Nishiyama, Nobuhiro; Koyama, Hiroyuki; Miyazono, Kohei; Kataoka, Kazunori

    2011-01-05

    Disulfide crosslinked polyplex micelles with RGD peptides were formed through ion complexation of thiolated c(RGDfK)-poly(ethylene glycol)-block-poly(L-lysine) (c(RGDfK)-PEG-P(Lys-SH)) and plasmid DNA encoding sFlt-1 and tested for their therapeutic effect in BxPC3 pancreatic adenocarcinoma tumor bearing mice. These micelles, systemically injected, demonstrated significant inhibition of tumor growth up to day 18, as a result of the antiangiogenic effect that was confirmed by vascular density measurements. Significant therapeutic activity of the 15% crosslinked micelle (c(RGDfK)-PEG-P(Lys-SH15)) was achieved by combined effect of increased tumor accumulation, interaction with endothelial cells and enhanced intracellular uptake through receptor-mediated endocytosis. These results suggest that RGD targeted crosslinked polyplex micelles can be effective plasmid DNA carriers for antiangiogenic gene therapy.

  10. Functional activity of plasmid DNA after entry into the atmosphere of earth investigated by a new biomarker stability assay for ballistic spaceflight experiments.

    PubMed

    Thiel, Cora S; Tauber, Svantje; Schütte, Andreas; Schmitz, Burkhard; Nuesse, Harald; Moeller, Ralf; Ullrich, Oliver

    2014-01-01

    Sounding rockets represent an excellent platform for testing the influence of space conditions during the passage of Earth's atmosphere and re-entry on biological, physical and chemical experiments for astrobiological purposes. We designed a robust functionality biomarker assay to analyze the biological effects of suborbital spaceflights prevailing during ballistic rocket flights. During the TEXUS-49 rocket mission in March 2011, artificial plasmid DNA carrying a fluorescent marker (enhanced green fluorescent protein: EGFP) and an antibiotic resistance cassette (kanamycin/neomycin) was attached on different positions of rocket exterior; (i) circular every 90 degree on the outer surface concentrical of the payload, (ii) in the grooves of screw heads located in between the surface application sites, and (iii) on the surface of the bottom side of the payload. Temperature measurements showed two major peaks at 118 and 130 °C during the 780 seconds lasting flight on the inside of the recovery module, while outer gas temperatures of more than 1000 °C were estimated on the sample application locations. Directly after retrieval and return transport of the payload, the plasmid DNA samples were recovered. Subsequent analyses showed that DNA could be recovered from all application sites with a maximum of 53% in the grooves of the screw heads. We could further show that up to 35% of DNA retained its full biological function, i.e., mediating antibiotic resistance in bacteria and fluorescent marker expression in eukaryotic cells. These experiments show that our plasmid DNA biomarker assay is suitable to characterize the environmental conditions affecting DNA during an atmospheric transit and the re-entry and constitute the first report of the stability of DNA during hypervelocity atmospheric transit indicating that sounding rocket flights can be used to model the high-speed atmospheric entry of organics-laden artificial meteorites.

  11. Functional Activity of Plasmid DNA after Entry into the Atmosphere of Earth Investigated by a New Biomarker Stability Assay for Ballistic Spaceflight Experiments

    PubMed Central

    Thiel, Cora S.; Tauber, Svantje; Schütte, Andreas; Schmitz, Burkhard; Nuesse, Harald; Moeller, Ralf; Ullrich, Oliver

    2014-01-01

    Sounding rockets represent an excellent platform for testing the influence of space conditions during the passage of Earth's atmosphere and re-entry on biological, physical and chemical experiments for astrobiological purposes. We designed a robust functionality biomarker assay to analyze the biological effects of suborbital spaceflights prevailing during ballistic rocket flights. During the TEXUS-49 rocket mission in March 2011, artificial plasmid DNA carrying a fluorescent marker (enhanced green fluorescent protein: EGFP) and an antibiotic resistance cassette (kanamycin/neomycin) was attached on different positions of rocket exterior; (i) circular every 90 degree on the outer surface concentrical of the payload, (ii) in the grooves of screw heads located in between the surface application sites, and (iii) on the surface of the bottom side of the payload. Temperature measurements showed two major peaks at 118 and 130°C during the 780 seconds lasting flight on the inside of the recovery module, while outer gas temperatures of more than 1000°C were estimated on the sample application locations. Directly after retrieval and return transport of the payload, the plasmid DNA samples were recovered. Subsequent analyses showed that DNA could be recovered from all application sites with a maximum of 53% in the grooves of the screw heads. We could further show that up to 35% of DNA retained its full biological function, i.e., mediating antibiotic resistance in bacteria and fluorescent marker expression in eukariotic cells. These experiments show that our plasmid DNA biomarker assay is suitable to characterize the environmental conditions affecting DNA during an atmospheric transit and the re-entry and constitute the first report of the stability of DNA during hypervelocity atmospheric transit indicating that sounding rocket flights can be used to model the high-speed atmospheric entry of organics-laden artificial meteorites. PMID:25426925

  12. Evaluation on the efficacy and immunogenicity of recombinant DNA plasmids expressing spike genes from porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus.

    PubMed

    Meng, Fandan; Ren, Yudong; Suo, Siqingaowa; Sun, Xuejiao; Li, Xunliang; Li, Pengchong; Yang, Wei; Li, Guangxing; Li, Lu; Schwegmann-Wessels, Christel; Herrler, Georg; Ren, Xiaofeng

    2013-01-01

    Porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PDEV) can cause severe diarrhea in pigs. Development of effective vaccines against TGEV and PEDV is one of important prevention measures. The spike (S) protein is the surface glycoprotein of TGEV and PEDV, which can induce specific neutralization antibodies and is a candidate antigen for vaccination attempts. In this study, the open reading frames of the TGEV S1 protein and in addition of the S or S1 proteins of PEDV were inserted into the eukaryotic expression vector, pIRES, resulting in recombinant plasmids, pIRES-(TGEV-S1-PEDV-S1) and pIRES-(TGEV-S1-PEDV-S). Subsequently, 6-8 weeks old Kunming mice were inoculated with both DNA plasmids. Lymphocyte proliferation assay, virus neutralization assay, IFN-γ assay and CTL activity assay were performed. TGEV/PEDV specific antibody responses as well as kinetic changes of T lymphocyte subgroups of the immunized mice were analyzed. The results showed that the recombinant DNA plasmids increased the proliferation of T lymphocytes and the number of CD4+ and CD8+ T lymphocyte subgroups. In addition, the DNA vaccines induced a high level of IFN-γ in the immunized mice. The specific CTL activity in the pIRES-(TGEV-S1-PEDV-S) group became significant at 42 days post-immunization. At 35 days post-immunization, the recombinant DNA plasmids bearing full-length S genes of TGEV and PEDV stimulated higher levels of specific antibodies and neutralizing antibodies in immunized mice.

  13. The intracellular delivery of plasmid DNA using cationic reducible carbon nanotube - Disulfide conjugates of polyethylenimine.

    PubMed

    Nia, Azadeh Hashem; Eshghi, Hossein; Abnous, Kalil; Ramezani, Mohammad

    2017-03-30

    A series of polyethylenimine conjugates of single-walled carbon nanotube (PEI-SWNT) containing bioreducible disulfide bonds was synthesized and evaluated for their transfection efficiency. Different molecular weights of polyethylenimine (PEI) were thiolated with different mole ratio of 2-iminothiolane (2-IT). Single-walled carbon nanotube (SWNT) was first carboxylated and then three different cysteine-functionalized SWNT formulations were synthesized via introduced linkers: a) carbonyl group b) spermidine c) 1,8-diamino 3,6-dioxo octane. The final nanocarriers were fabricated upon conjugation of thiolated PEIs and thiolated SWNT via oxidative disulfide bond formation. All PEI-disulfide-SWNT conjugates were capable of DNA condensation and showed improved viability and transfection efficiency compared to PEI itself. Transfection efficiencies were up to 1500 times greater than PEI 25kDa (C/P=0.8). The results of this study suggest that the synthesized formulations based on SWNT-CO-Cysteine and PEI 1.8kDa were the most efficient carriers. Considering the decreased cytotoxicity and higher transfection levels, the conjugates bear the potential for effective delivery of genetic materials.

  14. Chlamydial plasmids and bacteriophages.

    PubMed

    Pawlikowska-Warych, Małgorzata; Śliwa-Dominiak, Joanna; Deptuła, Wiesław

    2015-01-01

    Chlamydia are absolute pathogens of humans and animals; despite being rather well recognised, they are still open for discovery. One such discovery is the occurrence of extrachromosomal carriers of genetic information. In prokaryotes, such carriers include plasmids and bacteriophages, which are present only among some Chlamydia species. Plasmids were found exclusively in Chlamydia (C.) trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum and C. caviae. In prokaryotic organisms, plasmids usually code for genes that facilitate survival of the bacteria in the environment (although they are not essential). In chlamydia, their role has not been definitely recognised, apart from the fact that they participate in the synthesis of glycogen and encode proteins responsible for their virulence. Furthermore, in C. suis it was evidenced that the plasmid is integrated in a genomic island and contains the tetracycline-resistance gene. Bacteriophages specific for chlamydia (chlamydiaphages) were detected only in six species: C. psittaci, C. abortus, C. felis, C. caviae C. pecorum and C. pneumoniae. These chlamydiaphages cause inhibition of the developmental cycle, and delay transformation of reticulate bodies (RBs) into elementary bodies (EBs), thus reducing the possibility of infecting other cells in time. Plasmids and bacteriophages can be used in the diagnostics of chlamydioses; although especially in the case of plasmids, they are already used for detection of chlamydial infections. In addition, bacteriophages could be used as therapeutic agents to replace antibiotics, potentially addressing the problem of increasing antibiotic-resistance among chlamydia.

  15. Mechanisms of plasmid segregation: have multicopy plasmids been overlooked?

    PubMed

    Million-Weaver, Samuel; Camps, Manel

    2014-09-01

    Plasmids are self-replicating pieces of DNA typically bearing non-essential genes. Given that plasmids represent a metabolic burden to the host, mechanisms ensuring plasmid transmission to daughter cells are critical for their stable maintenance in the population. Here we review these mechanisms, focusing on two active partition strategies common to low-copy plasmids: par systems type I and type II. Both involve three components: an adaptor protein, a motor protein, and a centromere, which is a sequence area in the plasmid that is recognized by the adaptor protein. The centromere-bound adaptor nucleates polymerization of the motor, leading to filament formation, which can pull plasmids apart (par I) or push them towards opposite poles of the cell (par II). No such active partition mechanisms are known to occur in high copy number plasmids. In this case, vertical transmission is generally considered stochastic, due to the random distribution of plasmids in the cytoplasm. We discuss conceptual and experimental lines of evidence questioning the random distribution model and posit the existence of a mechanism for segregation in high copy number plasmids that moves plasmids to cell poles to facilitate transmission to daughter cells. This mechanism would involve chromosomally-encoded proteins and the plasmid origin of replication. Modulation of this proposed mechanism of segregation could provide new ways to enhance plasmid stability in the context of recombinant gene expression, which is limiting for large-scale protein production and for bioremediation.

  16. [Effect of plasmid pKM101 on the expression of bacterial genes not related to DNa metabolism].

    PubMed

    Skavronskaya, A G; Tiganova, I G; Andreeva, I V; Rusina, O Iu

    1999-02-01

    An experimental system ensuring fusion of bacterial genes to the lac operon of the Mu dl(Aplac) phage was used. Fusion operons in which the lac operon was under the control of promoters of the elt gene, responsible for synthesis of the LT toxin, of the tetracyclin-resistance tet gene, and sfiA gene encoding filament production, was studied. Using this experimental system, plasmid pKM101 was shown to be capable of activating the expression of the above Escherichia coli and Salmonella typhimurium genes, which is manifested as the activation of beta-galactosidase synthesis. The activation of the elt gene expression by the pKM101 plasmid was also confirmed in experiments on detecting the LT toxin synthesized by bacteria carrying this plasmid. Effect of the plasmid on the activation of elt operon expression, unlike the effect of this plasmid on mutability, does not depend on the functioning of the lexA and recA genes, i.e., this is not a SOS-regulated process. The mutant plasmid pGW12, a derivative of pKM101, deficient in the mucAB genes responsible for mutagenesis, causes a more pronounced activation of the elt gene than plasmid pKM101.

  17. A single amino acid alteration in the initiation protein is responsible for the DNA overproduction phenotype of copy number mutants of plasmid R6K.

    PubMed Central

    Inuzuka, M; Wada, Y

    1985-01-01

    A novel type of high copy-number (cop) mutants of a mini-R6K plasmid were isolated. The mutations were mapped in the pir gene which encodes the pi initiation protein for plasmid R6K DNA replication. They resulted in an alteration by substitution of a single amino acid: threonine to isoleucine at the 108th position for the cop41, and proline to serine at the 113th position for the cop50, of the 305 amino acid pi protein. The cop41 mutation in the pi protein was found to be trans-dominant over the wild-type allele in the copy control of plasmid R6K. Moreover, it was shown that the altered pi protein was not overproduced in maxicells carrying this mutant plasmid and had a higher affinity to the repeated sequence which is present in the pir promoter region. Most likely the mutated pi protein also interacts more efficiently with the same repeated sequences, a target of pi, in the replication origin region and increases the frequency of the initiation event per cell division. Images Fig. 2. Fig. 5. PMID:3000771

  18. Sodium citrate and potassium phosphate as alternative adsorption buffers in hydrophobic and aromatic thiophilic chromatographic purification of plasmid DNA from neutralized lysate.

    PubMed

    Bonturi, Nemailla; Radke, Vanessa Soraia Cortez Oliveira; Bueno, Sônia Maria Alves; Freitas, Sindélia; Azzoni, Adriano Rodrigues; Miranda, Everson Alves

    2013-03-01

    The number of studies on gene therapy using plasmid vectors (pDNA) has increased in recent years. As a result, the demand for preparations of pDNA in compliance with recommendations of regulatory agencies (EMEA, FDA) has also increased. Plasmid DNA is often obtained through fermentation of transformed Escherichia coli and purification by a series of unit operations, including chromatography. Hydrophobic interaction chromatography (HIC) and thiophilic aromatic chromatography (TAC), both using ammonium sulfate buffers, are commonly employed with success. This work was aimed at studying the feasibility of utilizing alternative salts in the purification of pDNA from neutralized lysate with phenyl-agarose (HIC) and mercaptopyrimidine-agarose (TAC) adsorbents. Their selectivity toward sc pDNA was evaluated through adsorption studies using 1.5 mol/L sodium citrate and 2.0 mol/L potassium phosphate as adsorption buffers. Chromatography with mercaptopyrimidine-agarose adsorbent and 1.5 mol/L sodium citrate was able to recover 91.1% of the pDNA with over 99.0% removal of gDNA and endotoxin. This represents a potential alternative for the primary recovery of sc pDNA. However, the most promising result was obtained using 2.0 mol/L potassium phosphate buffer and a mercaptopyrimidine-agarose column. In a single chromatographic step, this latter buffer/adsorbent system recovered 68.5% of the pDNA with 98.8% purity in accordance with the recommendations of regulatory agencies with regard to RNA and endotoxin impurity.

  19. Effective cross sections for production of single-strand breaks in plasmid DNA by 0.1 to 4.7 eV electrons.

    PubMed

    Panajotovic, Radmila; Martin, Frédéric; Cloutier, Pierre; Hunting, Darel; Sanche, Léon

    2006-04-01

    We determined effective cross sections for production of single-strand breaks (SSBs) in plasmid DNA [pGEM 3Zf(-)] by electrons of 10 eV and energies between 0.1 and 4.7 eV. After purification and lyophilization on a chemically clean tantalum foil, dry plasmid DNA samples were transferred into a high-vacuum chamber and bombarded by a monoenergetic electron beam. The amount of the circular relaxed DNA in the samples was separated from undamaged molecules and quantified using agarose gel electrophoresis. The effective cross sections were derived from the slope of the yield as a function of exposure and had values in the range of 10(-15)- 10(-14) cm2, giving an effective cross section of the order of 10(-18) cm2 per nucleotide. Their strong variation with incident electron energy and the resonant enhancement at 1 eV suggest that considerable damage is inflicted by very low-energy electrons to DNA, and it indicates the important role of pi* shape resonances in the bond-breaking process. Furthermore, the fact that the energy threshold for SSB production is practically zero implies that the sensitivity of DNA to electron impact is universal and is not limited to any particular energy range.

  20. Highly Effective Non-Viral Antitumor Gene Therapy System Comprised of Biocompatible Small Plasmid Complex Particles Consisting of pDNA, Anionic Polysaccharide, and Fully Deprotected Linear Polyethylenimine

    PubMed Central

    Koyama, Yoshiyuki; Sugiura, Kikuya; Yoshihara, Chieko; Inaba, Toshio; Ito, Tomoko

    2015-01-01

    We have reported that ternary complexes of plasmid DNA with conventional linear polyethylenimine (l-PEI) and certain polyanions were very stably dispersed, and, with no cryoprotectant, they could be freeze-dried and re-hydrated without the loss of transfection ability. These properties enabled the preparation of a concentrated suspension of very small pDNA complex, by preparing the complexes at highly diluted conditions, followed by condensation via lyophilization-and-rehydration procedure. Recently, a high potency linear polyethylenimine having no residual protective groups, i.e., Polyethylenimine “Max” (PEI “Max”), is available, which has been reported to induce much higher gene expression than conventional l-PEI. We tried to prepare the small DNA/PEI “Max”/polyanion complexes by a similar freeze-drying method. Small complex particles could be obtained without apparent aggregation, but transfection activity of the rehydrated complexes was severely reduced. Complex-preparation conditions were investigated in details to achieve the freeze-dried DNA/PEI “Max”/polyanion small ternary complexes with high transfection efficiency. DNA/PEI “Max”/polyanion complexes containing cytokine-coding plasmids were then prepared, and their anti-tumor therapeutic efficacy was examined in tumor-bearing mice. PMID:26213961

  1. Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication.

    PubMed

    Allen, Jennifer M; Simcha, David M; Ericson, Nolan G; Alexander, David L; Marquette, Jacob T; Van Biber, Benjamin P; Troll, Chris J; Karchin, Rachel; Bielas, Jason H; Loeb, Lawrence A; Camps, Manel

    2011-09-01

    DNA polymerase I (pol I) processes RNA primers during lagging-strand synthesis and fills small gaps during DNA repair reactions. However, it is unclear how pol I and pol III work together during replication and repair or how extensive pol I processing of Okazaki fragments is in vivo. Here, we address these questions by analyzing pol I mutations generated through error-prone replication of ColE1 plasmids. The data were obtained by direct sequencing, allowing an accurate determination of the mutation spectrum and distribution. Pol I's mutational footprint suggests: (i) during leading-strand replication pol I is gradually replaced by pol III over at least 1.3 kb; (ii) pol I processing of Okazaki fragments is limited to ∼20 nt and (iii) the size of Okazaki fragments is short (∼250 nt). While based on ColE1 plasmid replication, our findings are likely relevant to other pol I replicative processes such as chromosomal replication and DNA repair, which differ from ColE1 replication mostly at the recruitment steps. This mutation footprinting approach should help establish the role of other prokaryotic or eukaryotic polymerases in vivo, and provides a tool to investigate how sequence topology, DNA damage, or interactions with protein partners may affect the function of individual DNA polymerases.

  2. The Agrobacterium Ti Plasmids.

    PubMed

    Christie, Peter J; Gordon, Jay E

    2014-12-01

    Agrobacterium tumefaciens is a plant pathogen with the capacity to deliver a segment of oncogenic DNA carried on a large plasmid called the tumor-inducing or Ti plasmid to susceptible plant cells. A. tumefaciens belongs to the class Alphaproteobacteria, whose members include other plant pathogens (Agrobacterium rhizogenes), plant and insect symbionts (Rhizobium spp. and Wolbachia spp., respectively), human pathogens (Brucella spp., Bartonella spp., Rickettsia spp.), and nonpathogens (Caulobacter crescentus, Rhodobacter sphaeroides). Many species of Alphaproteobacteria carry large plasmids ranging in size from ∼100 kb to nearly 2 Mb. These large replicons typically code for functions essential for cell physiology, pathogenesis, or symbiosis. Most of these elements rely on a conserved gene cassette termed repABC for replication and partitioning, and maintenance at only one or a few copies per cell. The subject of this review is the ∼200-kb Ti plasmids carried by infectious strains of A. tumefaciens. We will summarize the features of this plasmid as a representative of the repABC family of megaplasmids. We will also describe novel features of this plasmid that enable A. tumefaciens cells to incite tumor formation in plants, sense and respond to an array of plant host and bacterial signal molecules, and maintain and disseminate the plasmid among populations of agrobacteria. At the end of this review, we will describe how this natural genetic engineer has been adapted to spawn an entire industry of plant biotechnology and review its potential for use in future therapeutic applications of plant and nonplant species.

  3. ATP-regulated interactions between P1 ParA, ParB and non-specific DNA that are stabilized by the plasmid partition site, parS

    PubMed Central

    Havey, James C.; Vecchiarelli, Anthony G.; Funnell, Barbara E.

    2012-01-01

    Localization of the P1 plasmid requires two proteins, ParA and ParB, which act on the plasmid partition site, parS. ParB is a site-specific DNA-binding protein and ParA is a Walker-type ATPase with non-specific DNA-binding activity. In vivo ParA binds the bacterial nucleoid and forms dynamic patterns that are governed by the ParB–parS partition complex on the plasmid. How these interactions drive plasmid movement and localization is not well understood. Here we have identified a large protein–DNA complex in vitro that requires ParA, ParB and ATP, and have characterized its assembly by sucrose gradient sedimentation and light scattering assays. ATP binding and hydrolysis mediated the assembly and disassembly of this complex, while ADP antagonized complex formation. The complex was not dependent on, but was stabilized by, parS. The properties indicate that ParA and ParB are binding and bridging multiple DNA molecules to create a large meshwork of protein–DNA molecules that involves both specific and non-specific DNA. We propose that this complex represents a dynamic adaptor complex between the plasmid and nucleoid, and further, that this interaction drives the redistribution of partition proteins and the plasmid over the nucleoid during partition. PMID:21965538

  4. Induction of single- and double-strand breaks in plasmid DNA by monoenergetic alpha-particles with energies below the Bragg-maximum.

    PubMed

    Scholz, V; Weidner, J; Köhnlein, W; Frekers, D; Wörtche, H J

    1997-01-01

    The yield of single-strand breaks (ssb) and double-strand breaks (dsb) produced by alpha-particles at the end of their track in DNA-films was determined experimentally. Helium nuclei were accelerated to 600 keV in the 400 kV ion accelerator and scattered at a carbon target. The elastically scattered alpha-particles with energies of 344 keV and 485 keV were used to irradiate supercircular plasmid DNA in vacuo. For the dosimetry of the alpha-particles a surface barrier detector was used and the energy distribution of the alpha-particles determined. The energy loss of the particles in the DNA-layer was calculated. DNA samples were separated into the three conformational isomers using agarose gel electrophoresis. After fluorochromation the number of ssb and dsb per plasmid DNA molecule was established from the band intensities assuming the validity of Poisson statistics. Linear dose effect correlations were found for ssb and dsb per plasmid molecule. In the case of 344 keV-alpha-particles the yield of dsb was (8.6 +/- 0.9) x 10(-11) breaks/Gy x dalton. The ratio of ssb/dsb was 0.5 +/- 0.2. This is at least a factor of six larger than the ratio found in experiments with higher energy alpha-particles and from model calculations. Similar experiments with protons yielded a relative biological effectiveness (rbe) value of 2.8 for the induction of double-strand breaks by track end alpha-particles.

  5. Plasmid Rolling-Circle Replication.

    PubMed

    Ruiz-Masó, J A; MachóN, C; Bordanaba-Ruiseco, L; Espinosa, M; Coll, M; Del Solar, G

    2015-02-01

    Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

  6. A New Broad Range Plasmid for DNA Delivery in Eukaryotic Cells Using Lactic Acid Bacteria: In Vitro and In Vivo Assays.

    PubMed

    Mancha-Agresti, Pamela; Drumond, Mariana Martins; Carmo, Fillipe Luiz Rosa do; Santos, Monica Morais; Santos, Janete Soares Coelho Dos; Venanzi, Franco; Chatel, Jean-Marc; Leclercq, Sophie Yvette; Azevedo, Vasco

    2017-03-17

    Lactococcus lactis is well documented as a promising candidate for development of novel oral live vaccines. It has been broadly engineered for heterologous expression, as well as for plasmid expression vector delivery, directly inside eukaryotic cells, for DNA vaccine, or as therapeutic vehicle. This work describes the characteristics of a new plasmid, pExu (extra chromosomal unit), for DNA delivery using L. lactis and evaluates its functionality both by in vitro and in vivo assays. This plasmid exhibits the following features: (1) a theta origin of replication and (2) an expression cassette containing a multiple cloning site and a eukaryotic promoter, the cytomegalovirus (pCMV). The functionality of pExu:egfp was evaluated by fluorescence microscopy. The L. lactis MG1363 (pExu:egfp) strains were administered by gavage to Balb/C mice and the eGFP expression was monitored by fluorescence microscopy. The pExu vector has demonstrated an excellent stability either in L. lactis or in Escherichia coli. The eGFP expression at different times in in vitro assay showed that 15.8% of CHO cells were able to express the protein after transfection. The enterocytes of mice showed the expression of eGFP protein. Thus, L. lactis carrying the pExu is a good candidate to deliver genes into eukaryotic cells.

  7. Enhancing immune responses of EV71 VP1 DNA vaccine by co-inoculating plasmid IL-12 or GM-CSF expressing vector in mice.

    PubMed

    Peng, X; Fang, X; Li, J; Kong, L; Li, B; Ding, X

    2016-04-30

    Enterovirus 71 (EV71) is a major causative viral agent for large outbreaks of hand, foot, and mouth disease in children and infants, yet there is no vaccine or effective antiviral treatment for severe EV71 infection. The immunogenicity of EV71 VP1 DNA vaccine and the immunoregulatory activity of interleukin-12 (IL-12) or granulocyte-monocyte colony stimulating factor (GM-CSF) were investigated. DNA vaccine plasmids, pcDNA-VP1, pcDNA-IL-12 and pcDNA-GM-CSF were constructed and inoculated into BALB/c mice with or without pcDNA-IL-12 or pcDNA-GM-CSF by intramuscular injection. Cellular and humoral immune responses were assessed by indirect ELISA, lymphocyte proliferation assays, cytokine release assay and FACS. The VP1 DNA vaccine had good immunogenicity and can induce specific humoral and cellular immunity in BALB/c mice, while IL-2 or GM-CSF plays an immunoadjuvant role and enhances specific immune responses. This study provides a frame of reference for the design of DNA vaccines against EV71.

  8. Chitosan-plasmid DNA nanoparticles encoding small hairpin RNA targeting MMP-3 and -13 to inhibit the expression of dedifferentiation related genes in expanded chondrocytes.

    PubMed

    Zhao, Jingxin; Fan, Xiangli; Zhang, Qiang; Sun, Fangfei; Li, Xiaojian; Xiong, Chuan; Zhang, Chunli; Fan, Hongbin

    2014-02-01

    Overexpression of matrix metalloproteinase (MMP)-3 and -13 can lead to the dedifferentiation of expanded chondrocytes. After implanting dedifferentiated cells for cartilage defect repair, graft failure may occur. Short hairpin RNA (shRNA) is a powerful genetic tool to reduce the expression of target genes. This study investigated the effects of chitosan-plasmid DNA (pDNA) nanoparticles encoding shRNA targeting MMP-3 and -13 on the dedifferentiation of expanded chondrocytes. The objective was to optimize the parameters of chitosan-pDNA formulation for achieving higher efficiency of pDNA delivery and gene silencing. The chitosan-pDNA nanoparticles were prepared using a complex coacervation process. Then the characteristics including size, shape, stability, and transfection efficiency were compared in different groups. The results indicated that chitosan of 800 kDa at N/P ratio of 4 and pH 7.0 was optimal to prepare chitosan-pDNA nanoparticles. These nanoparticles showed high DNA loading efficiency (95.8 ± 1.5%) and high gene transfection efficiency (24.5 ± 1.6%). After the expanded chondrocytes were transfected by chitosan-pDNA nanoparticles, MMP-3-610 and MMP-13-2024 groups showed greater suppression in mRNA and protein levels. The results indicated that chitosan-pDNA nanoparticles encoding shRNA targeting MMP-3 and -13 had great potential in silencing the dedifferentiation-related genes for regenerating prolonged and endurable cartilage.

  9. Delivery of plasmid DNA encoding bone morphogenetic protein-2 with a biodegradable branched polycationic polymer in a critical-size rat cranial defect model.

    PubMed

    Chew, Sue Anne; Kretlow, James D; Spicer, Patrick P; Edwards, Austin W; Baggett, L Scott; Tabata, Yasuhiko; Kasper, F Kurtis; Mikos, Antonios G

    2011-03-01

    This study investigated the delivery of plasmid DNA (pDNA) encoding bone morphogenetic protein-2 in the form of polyplexes with a biodegradable branched triacrylate/amine polycationic polymer (TAPP) that were complexed with gelatin microparticles (GMPs) loaded within a porous tissue engineering scaffold. More specifically, the study investigated the interplay between TAPP degradation, gelatin degradation, pDNA release, and bone formation in a critical-size rat cranial defect model. The pDNA release kinetics in vitro were not affected by the crosslinking density of the GMPs but depended, rather, on the degradation rates of the TAPPs. Besides the initial release of polyplexes not bound to the GMPs and the minimal release of polyplexes through diffusion or dissociation from the GMPs, the pDNA was likely released as naked pDNA or as part of an incomplete polyplex, after the degradation of fragments of the polycationic polymer. After 30 days, significantly higher amounts of pDNA were released (93%-98%) from composite scaffolds containing naked pDNA or pDNA complexed with P-AEPZ (synthesized with 1-[2-aminoethyl]piperazine, a faster degrading TAPP) compared with those containing pDNA complexed with P-DED (synthesized with N,N-dimethylethylenediamine, a slower degrading TAPP) (74%-82%). Composite scaffolds containing GMPs complexed with TAPP/pDNA polyplexes did not result in enhanced bone formation, as analyzed by microcomputed tomography and histology, in a critical-size rat cranial defect at 12 weeks postimplantation compared with those loaded with naked pDNA. The results demonstrate that polycationic polymers with a slow degradation rate can prolong the release of pDNA from the composite scaffolds and suggest that a gene delivery system comprising biodegradable polycationic polymers should be designed to release the pDNA in an intact polyplex form.

  10. Effects of DDA, CpG-ODN, and plasmid-encoded chicken IFN-γ on protective immunity by a DNA vaccine against IBDV in chickens

    PubMed Central

    Roh, Ha Jung; Sung, Haan Woo

    2006-01-01

    This study examined the adjuvant effects of dimethyl dioctadecyl ammonium bromide (DDA), CpG oligodeoxynucleotides (CpG-ODN), and chicken interferon-γ (ChIFN-γ) on a DNA vaccine (pcDNA-VP243) against the infectious bursal disease virus (IBDV). A plasmid encoding chicken IFN-ã was constructed. Twice at 2-week intervals, two-week-old chickens were injected intramuscularly and intraperitoneally with either a DNA vaccine alone or a DNA vaccine together with the respective adjuvants. On week 2 after the second immunization, the chickens were orally challenged with the highly virulent IBDV. The groups that received the DNA vaccines plus either DDA or CpG-ODN showed significantly lower survival rates than the group that received the DNA vaccine alone. However, the survival rates for the DNA vaccine alone and for the DNA vaccine plus ChIFN-γ were similar. The chickens had no detectable antibodies to the IBDV before the challenge but all the surviving chickens in all groups except for the normal control group showed the induction of antibodies to the IBDV at day 10 after the challenge. As judged by the lymphocyte proliferation assays using the a WST-8 solution performed on the peripheral blood and splenic lymphocytes, the stimulation indices (SI) of the peripheral blood lymphocytes in all groups except for the normal control group were similar immediately before the challenge. At 10 days post-challenge, the SI for DNA vaccine plus either CpG-ODN or ChIFN-γ was similar to that of the DNA vaccine control group. For splenic lymphocytes, the SI in the DNA vaccine plus CpG-ODN and DNA vaccine plus ChIFN-γ groups were higher than for the DNA vaccine control. These results suggest that DDA actually compromises the protection against the IBDV by DNA vaccine, and CpG-ODN and IFN-γ had no significant effect. PMID:17106228

  11. Combinations of various CpG motifs cloned into plasmid backbone modulate and enhance protective immunity of viral replicon DNA anthrax vaccines.

    PubMed

    Yu, Yun-Zhou; Ma, Yao; Xu, Wen-Hui; Wang, Shuang; Sun, Zhi-Wei

    2015-08-01

    DNA vaccines are generally weak stimulators of the immune system. Fortunately, their efficacy can be improved using a viral replicon vector or by the addition of immunostimulatory CpG motifs, although the design of these engineered DNA vectors requires optimization. Our results clearly suggest that multiple copies of three types of CpG motifs or combinations of various types of CpG motifs cloned into a viral replicon vector backbone with strong immunostimulatory activities on human PBMC are efficient adjuvants for these DNA vaccines to modulate and enhance protective immunity against anthrax, although modifications with these different CpG forms in vivo elicited inconsistent immune response profiles. Modification with more copies of CpG motifs elicited more potent adjuvant effects leading to the generation of enhanced immunity, which indicated a CpG motif dose-dependent enhancement of antigen-specific immune responses. Notably, the enhanced and/or synchronous adjuvant effects were observed in modification with combinations of two different types of CpG motifs, which provides not only a contribution to the knowledge base on the adjuvant activities of CpG motifs combinations but also implications for the rational design of optimal DNA vaccines with combinations of CpG motifs as "built-in" adjuvants. We describe an efficient strategy to design and optimize DNA vaccines by the addition of combined immunostimulatory CpG motifs in a viral replicon DNA plasmid to produce strong immune responses, which indicates that the CpG-modified viral replicon DNA plasmid may be desirable for use as vector of DNA vaccines.

  12. Optimal induction of hepatitis C virus envelope-specific immunity by bicistronic plasmid DNA inoculation with the granulocyte-macrophage colony-stimulating factor gene.

    PubMed

    Lee, S W; Cho, J H; Sung, Y C

    1998-10-01

    In this study, we have constructed various DNA vaccine vectors that carried hepatitis C virus (HCV) envelope genes without and with the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene in several different ways. In Buffalo rats that received plasmids carrying the HCV envelope genes, which encode envelope proteins E1 and E2, both antibody and lymphoproliferative responses against these proteins were induced. These responses were greatly enhanced by the codelivery of the GM-CSF gene. In particular, inoculation with a bicistronic plasmid that independently expressed the GM-CSF gene and the envelope genes in the same construct generated the highest antibody titers and significantly increased lymphoproliferative responses against these proteins. Moreover, strong antibody responses to homologous and heterologous hypervariable region 1 peptides were elicited in the immunized rats.

  13. Absolute binding-free energies between standard RNA/DNA nucleobases and amino-acid sidechain analogs in different environments.

    PubMed

    de Ruiter, Anita; Zagrovic, Bojan

    2015-01-01

    Despite the great importance of nucleic acid-protein interactions in the cell, our understanding of their physico-chemical basis remains incomplete. In order to address this challenge, we have for the first time determined potentials of mean force and the associated absolute binding free energies between all standard RNA/DNA nucleobases and amino-acid sidechain analogs in high- and low-dielectric environments using molecular dynamics simulations and umbrella sampling. A comparison against a limited set of available experimental values for analogous systems attests to the quality of the computational approach and the force field used. Overall, our analysis provides a microscopic picture behind nucleobase/sidechain interaction preferences and creates a unified framework for understanding and sculpting nucleic acid-protein interactions in different contexts. Here, we use this framework to demonstrate a strong relationship between nucleobase density profiles of mRNAs and nucleobase affinity profiles of their cognate proteins and critically analyze a recent hypothesis that the two may be capable of direct, complementary interactions.

  14. Repeated intrathecal administration of plasmid DNA complexed with polyethylene glycol-grafted polyethylenimine led to prolonged transgene expression in the spinal cord.

    PubMed

    Shi, L; Tang, G P; Gao, S J; Ma, Y X; Liu, B H; Li, Y; Zeng, J M; Ng, Y K; Leong, K W; Wang, S

    2003-07-01

    Gene delivery into the spinal cord provides a potential approach to the treatment of spinal cord traumatic injury, amyotrophic lateral sclerosis, and spinal muscular atrophy. These disorders progress over long periods of time, necessitating a stable expression of functional genes at therapeutic levels for months or years. We investigated in this study the feasibility of achieving prolonged transgene expression in the rat spinal cord through repeated intrathecal administration of plasmid DNA complexed with 25 kDa polyethylenimine (PEI) into the lumbar subarachnoid space. With a single injection, DNA/PEI complexes could provide transgene expression in the spinal cord 40-fold higher than naked plasmid DNA. The transgene expression at the initial level persisted for about 5 days, with a low-level expression being detectable for at least 8 weeks. When repeated dosing was tested, a 70% attenuation of gene expression was observed following reinjection at a 2-week interval. This attenuation was associated with apoptotic cell death and detected even using complexes containing a noncoding DNA that did not mediate any gene expression. When each component of the complexes, PEI polymer or naked DNA alone, were tested in the first dosing, no reduction was found. Using polyethylene glycol (PEG)-grafted PEI for DNA complexes, no attenuation of gene expression was detected after repeated intrathecal injections, even in those rats receiving three doses, administered 2 weeks apart. Lumbar puncture is a routine and relatively nontraumatic clinical procedure. Repeated administration of DNA complexed with PEG-grafted PEI through this less invasive route may prolong the time span of transgene expression when needed, providing a viable strategy for the gene therapy of spinal cord disorders.

  15. Chemical repair of base lesions, AP-sites, and strand breaks on plasmid DNA in dilute aqueous solution by ascorbic acid

    SciTech Connect

    Hata, Kuniki; Urushibara, Ayumi; Yamashita, Shinichi; Shikazono, Naoya; Yokoya, Akinari; Katsumura, Yosuke

    2013-05-03

    Highlights: •We report a novel mechanism of radiation protection of DNA by chemical activity of ascorbic acid. •The “chemical repair” of DNA damage was revealed using biochemical assay and chemical kinetics analysis. •We found that ascorbic acid significantly repairs precursors of nucleobase lesions and abasic sites. •However, ascorbic acid seldom repairs precursors of DNA-strand breaks. -- Abstract: We quantified the damage yields produced in plasmid DNA by γ-irradiation in the presence of low concentrations (10–100 μM) of ascorbic acid, which is a major antioxidant in living systems, to clarify whether it chemically repairs radiation damage in DNA. The yield of DNA single strand breaks induced by irradiation was analyzed with agarose gel electrophoresis as conformational changes in closed circular plasmids. Base lesions and abasic sites were also observed as additional conformational changes by treating irradiated samples with glycosylase proteins. By comparing the suppression efficiencies to the induction of each DNA lesion, in addition to scavenging of the OH radicals derived from water radiolysis, it was found that ascorbic acid promotes the chemical repair of precursors of AP-sites and base lesions more effectively than those of single strand breaks. We estimated the efficiency of the chemical repair of each lesion using a kinetic model. Approximately 50–60% of base lesions and AP-sites were repaired by 10 μM ascorbic acid, although strand breaks were largely unrepaired by ascorbic acid at low concentrations. The methods in this study will provide a route to understanding the mechanistic aspects of antioxidant activity in living systems.

  16. The presence of the region on pBR322 that encodes resistance to tetracycline is responsible for high levels of plasmid DNA knotting in Escherichia coli DNA topoisomerase I deletion mutant.

    PubMed Central

    Shishido, K; Ishii, S; Komiyama, N

    1989-01-01

    Plasmid pBR322 DNA isolated from Escherichia coli DNA topoisomerase I deletion mutant DM800 is estimated to contain about 10% of the knotted forms (Shishido et al., 1987). These knotted DNA species were shown to have the same primary structure as usual, unknotted pBR322 DNA. Analysis of the knotting level of deletion, insertion and sequence-rearranged derivatives of pBR322 in DM800 showed that the presence of the region on pBR322 encoding resistance to tetracycline (tet) is required for high levels of plasmid knotting. When the entire tet region is present in a native orientation, the level of knotting is highest. Inactivating the tet promoter is manifested by a middle level of knotting. For deletion derivatives lacking various portions of the tet region, the level of knotting ranges from lowest to high depending on the site and length of the tet gene remaining. Inverting the orientation of tet region on the pBR322 genome results in a middle level of knotting. Deleting the ampicillin-resistance (bla)gene outside of its second promoter does not affect the level of knotting, if the entire tet gene remains. A possible mechanism of regulation of plasmid knotting is discussed. Images PMID:2557587

  17. Inhibition of gamma-radiation induced DNA damage in plasmid pBR322 by TMG, a water-soluble derivative of vitamin E.

    PubMed

    Rajagopalan, Rema; Wani, Khalida; Huilgol, Nagaraj G; Kagiya, Tsutomu V; Nair, Cherupally K Krishnan

    2002-06-01

    Alpha-tocopherol monoglucoside (TMG), a water-soluble derivative of alpha-tocopherol, has been examined for its ability to protect DNA against radiation-induced strand breaks. Gamma radiation, up to a dose of 6 Gy (dose rate, 0.7 Gy/minute), induced a dose-dependent increase in single strand breaks (SSBs) in plasmid pBR322 DNA. TMG inhibited the formation of gamma-radiation induced DNA single strand breaks (SSBs) in a concentration-dependent manner; 500 microM of TMG protected the single strand breaks completely. It also protected thymine glycol formation induced by gamma-radiation in a dose-dependent manner, based on an estimation of thymine glycol by HPLC.

  18. Homology of cryptic plasmid of Neisseria gonorrhoeae with plasmids from Neisseria meningitidis and Neisseria lactamica.

    PubMed

    Ison, C A; Bellinger, C M; Walker, J

    1986-10-01

    DNA probe hybridisation was used to examine the relation between the cryptic plasmid from Neisseria gonorrhoeae and plasmids carried by pharyngeal isolates of Neisseria meningitidis and Neisseria lactamica. The complete gonococcal cryptic plasmid and HinfI derived digestion fragments subcloned into Escherichia coli were used to probe Southern blots of plasmid extracts. Homology was found to a plasmid of approximate molecular weight 4.5 kilobase pairs (Kb) but not to plasmids of less than 3.2 Kb or 6.5 Kb. Eleven of 16 strains of N meningitidis and two of six strains of N lactamica carried plasmids that showed strong hybridisation with the 4.2 Kb gonococcal plasmid. Hybridisation of plasmids from non-gonococcal species of neisseria with the gonococcal cryptic plasmid indicates that caution should be taken when using the cryptic plasmid as a diagnostic probe for gonorrhoea.

  19. Reduction of intimal hyperplasia in injured rat arteries promoted by catheter balloons coated with polyelectrolyte multilayers that contain plasmid DNA encoding PKCδ.

    PubMed

    Bechler, Shane L; Si, Yi; Yu, Yan; Ren, Jun; Liu, Bo; Lynn, David M

    2013-01-01

    New therapeutic approaches that eliminate or reduce the occurrence of intimal hyperplasia following balloon angioplasty could improve the efficacy of vascular interventions and improve the quality of life of patients suffering from vascular diseases. Here, we report that treatment of arteries using catheter balloons coated with thin polyelectrolyte-based films ('polyelectrolyte multilayers', PEMs) can substantially reduce intimal hyperplasia in an in vivo rat model of vascular injury. We used a layer-by-layer (LbL) process to coat the surfaces of inflatable catheter balloons with PEMs composed of nanolayers of a cationic poly(β-amino ester) (polymer 1) and plasmid DNA (pPKCδ) encoding the δ isoform of protein kinase C (PKCδ), a regulator of apoptosis and other cell processes that has been demonstrated to reduce intimal hyperplasia in injured arterial tissue when administered via perfusion using viral vectors. Insertion of balloons coated with polymer 1/pPKCδ multilayers into injured arteries for 20 min resulted in local transfer of DNA and elevated levels of PKCδ expression in the media of treated tissue three days after delivery. IFC and IHC analysis revealed these levels of expression to promote downstream cellular processes associated with up-regulation of apoptosis. Analysis of arterial tissue 14 days after treatment revealed polymer 1/pPKCδ-coated balloons to reduce the occurrence of intimal hyperplasia by ~60% compared to balloons coated with films containing empty plasmid vectors. Our results demonstrate the potential therapeutic value of this nanotechnology-based approach to local gene delivery in the clinically important context of balloon-mediated vascular interventions. These PEM-based methods could also prove useful for other in vivo applications that require short-term, surface-mediated transfer of plasmid DNA.

  20. Recruitment of terminal protein to the ends of Streptomyces linear plasmids and chromosomes by a novel telomere-binding protein essential for linear DNA replication

    PubMed Central

    Bao, Kai; Cohen, Stanley N.

    2003-01-01

    Bidirectional replication of Streptomyces linear plasmids and chromosomes from a central origin produces unpaired 3′-leading-strand overhangs at the telomeres of replication intermediates. Filling in of these overhangs leaves a terminal protein attached covalently to the 5′ DNA ends of mature replicons. We report here the essential role of a novel 80-kD DNA-binding protein (telomere-associated protein, Tap) in this process. Biochemical studies, yeast two-hybrid analysis, and immunoprecipitation/immunodepletion experiments indicate that Tap binds tightly to specific sequences in 3′ overhangs and also interacts with Tpg, bringing Tpg to telomere termini. Using DNA microarrays to analyze the chromosomes of tap mutant bacteria, we demonstrate that survivors of Tap ablation undergo telomere deletion, chromosome circularization, and amplification of subtelomeric DNA. Microarray-based chromosome mapping at single-ORF resolution revealed common endpoints for independent deletions, identified amplified chromosomal ORFs adjacent to these endpoints, and quantified the copy number of these ORFs. Sequence analysis confirmed chromosome circularization and revealed the insertion of adventitious DNA between joined chromosome ends. Our results show that Tap is required for linear DNA replication in Streptomyces and suggest that it functions to recruit and position Tpg at the telomeres of replication intermediates. They also identify hotspots for the telomeric deletions and subtelomeric DNA amplifications that accompany chromosome circularization. PMID:12651895

  1. Enhancement of the immunogenicity of an alphavirus replicon-based DNA vaccine against classical swine fever by electroporation and coinjection with a plasmid expressing porcine interleukin 2.

    PubMed

    Tian, Da-Yong; Sun, Yuan; Wai, Sing Fai; Lee, Fuk Ki; Meng, Qi-Lin; Suen, Kar Man; Wang, Nan; Han, Wen; Li, Su; Li, Yong-Feng; Li, Dan; Ling, Li-Jun; Liao, Ya-Jin; Qiu, Hua-Ji

    2012-05-21

    Alphavirus replicon-based DNA vaccines have emerged as a promising approach to generation of antigen-specific immune responses. However, due to their low immunogenicity, there is a need for other approaches to enhance the vaccine potency. In this study, electroporation (EP) and a plasmid expressing porcine interleukin 2 (IL-2) were used to improve the immunogenicity of an alphavirus replicon-based DNA vaccine pSFV1CS-E2 against classical swine fever (CSF). Pigs were immunized with pSFV1CS-E2 alone or together with IL-2 by EP or by simple intramuscular injection. The results showed that EP combined with IL-2 resulted in marked enhancement of E2-specific antibody responses. Moreover, CSFV-specific lymphocyte proliferation, IFN-γ and IL-4 responses were increased significantly in the pSFV1CS-E2+IL-2/EP group. Pigs immunized with pSFV1CS-E2 plus IL-2 by EP were completely protected from lethal challenge, which is comparable to the sterilizing immunity and full protection offered by the live attenuated vaccine C-strain and in contrast with the incomplete protection conferred by pSFV1CS-E2 without or with IL-2 or EP alone, as demonstrated by the presence of pathological changes or/and viral loads. We conclude that EP in combination with IL-2 can significantly improve the immunogenicity of the plasmid DNA vaccine.

  2. Protective effect of a prime-boost strategy with plasmid DNA followed by recombinant adenovirus expressing TgAMA1 as vaccines against Toxoplasma gondii infection in mice.

    PubMed

    Yu, Longzheng; Yamagishi, Junya; Zhang, Shoufa; Jin, Chunmei; Aboge, Gabriel Oluga; Zhang, Houshuang; Zhang, Guohong; Tanaka, Tetsuya; Fujisaki, Kozo; Nishikawa, Yoshifumi; Xuan, Xuenan

    2012-09-01

    A heterologous prime-boost strategy with priming plasmid DNA followed by recombinant virus expressing relevant antigens is known to stimulate protective immunity against intracellular parasites. In this study, we have evaluated a heterologous prime-boost strategy for immunizing mice against Toxoplasma gondii infection. Our results revealed that the prime-boost strategy using both plasmid DNA and adenoviral vector encoding TgAMA1 may stimulate both humoral and Th1/Th2 cellular immune responses specific for TgAMA1. Moreover, C57BL/6 mice immunized with the pAMA1/Ad5Null, pNull/Ad5AMA1, and pAMA1/Ad5AMA1 constructs showed survival rates of 12.5%, 37.5%, and 50%, respectively. In contrast, all the pNull/Ad5Null immunized mice died after infection with the PLK-GFP strain of T. gondii. Brain cyst burden was reduced by 23% in mice immunized with pAMA1/Ad5AMA1 compared with the pNull/Ad5AMA1 immunized mice. These results demonstrate that the heterologous DNA priming and recombinant adenovirus boost strategy may provide protective immunity against T. gondii infection.

  3. Nonviral Plasmid DNA Carriers Based on N,N'-Dimethylaminoethyl Methacrylate and Di(ethylene glycol) Methyl Ether Methacrylate Star Copolymers.

    PubMed

    Mendrek, Barbara; Sieroń, Łukasz; Żymełka-Miara, Iwona; Binkiewicz, Paulina; Libera, Marcin; Smet, Mario; Trzebicka, Barbara; Sieroń, Aleksander L; Kowalczuk, Agnieszka; Dworak, Andrzej

    2015-10-12

    Star polymers with random and block copolymer arms made of cationic N,N'-dimethylaminoethyl methacrylate (DMAEMA) and nonionic di(ethylene glycol) methyl ether methacrylate (DEGMA) were synthesized via atom transfer radical polymerization (ATRP) and used for the delivery of plasmid DNA in gene therapy. All stars were able to form polyplexes with plasmid DNA. The structure and size of the polyplexes were precisely determined using light scattering and cryo-TEM microscopy. The hydrodynamic radius of a complex of DNA with star was dependent on the architecture of the star arms, the DEGMA content and the number of amino groups in the star compared to the number of phosphate groups of the nucleic acid (N/P ratio). The smallest polyplexes (Rh90°∼50 nm) with positive zeta potentials (∼15 mV) were formed of stars with N/P=6. The introduction of DEGMA into the star structure caused a decrease of polyplex cytotoxicity in comparison to DMAEMA homopolymer stars. The overall transfection efficiency using HT-1080 cells showed that the studied systems are prospective gene delivery agents. The most promising results were obtained for stars with random copolymer arms of high DEGMA content.

  4. Mechanisms of Theta Plasmid Replication.

    PubMed

    Lilly, Joshua; Camps, Manel

    2015-02-01

    Plasmids are autonomously replicating pieces of DNA. This article discusses theta plasmid replication, which is a class of circular plasmid replication that includes ColE1-like origins of replication popular with expression vectors. All modalities of theta plasmid replication initiate synthesis with the leading strand at a predetermined site and complete replication through recruitment of the host's replisome, which extends the leading strand continuously while synthesizing the lagging strand discontinuously. There are clear differences between different modalities of theta plasmid replication in mechanisms of DNA duplex melting and in priming of leading- and lagging-strand synthesis. In some replicons duplex melting depends on transcription, while other replicons rely on plasmid-encoded trans-acting proteins (Reps); primers for leading-strand synthesis can be generated through processing of a transcript or in other replicons by the action of host- or plasmid-encoded primases. None of these processes require DNA breaks. The frequency of replication initiation is tightly regulated to facilitate establishment in permissive hosts and to achieve a steady state. The last section of the article reviews how plasmid copy number is sensed and how this feedback modulates the frequency of replication.

  5. Enhanced anti-fibrotic activity of plasmid DNA expressing small interference RNA for TGF-beta type II receptor for a mouse model of obstructive nephropathy by cationized gelatin prepared from different amine compounds.

    PubMed

    Kushibiki, Toshihiro; Nagata-Nakajima, Natsuki; Sugai, Manabu; Shimizu, Akira; Tabata, Yasuhiko

    2006-02-21

    The objective of this study is to increase the transfection efficiency of a plasmid DNA expressing small interference RNA (siRNA) for transforming growth factor-beta receptor (TGF-betaR) by various cationized gelatins of non-viral carrier and evaluate the anti-fibrotic effect with a mouse model of unilateral ureteral obstruction (UUO). Ethylenediamine, putrescine, spermidine or spermine was chemically introduced to the carboxyl groups of gelatin for the cationization. The plasmid DNA of TGF-betaR siRNA expression vector with or without complexation of each cationized gelatin was injected to the left kidney of mice via the ureter to prevent the progression of renal fibrosis of UUO mice. Irrespective of the type of cationized gelatin, the injection of plasmid DNA-cationized gelatin complex significantly decreased the renal level of TGF-betaR over-expression and the collagen content of mice kidney, in marked contrast to free plasmid DNA injection. It is concluded that retrograde injection of TGF-betaR siRNA expression vector plasmid DNA complexed with the cationized gelatin is available to suppress the progression of renal interstitial fibrosis.

  6. Quantification of DNA by Agarose Gel Electrophoresis and Analysis of the Topoisomers of Plasmid and M13 DNA Following Treatment with a Restriction Endonuclease or DNA Topoisomerase I

    ERIC Educational Resources Information Center

    Tweedie, John W.; Stowell, Kathryn M.

    2005-01-01

    A two-session laboratory exercise for advanced undergraduate students in biochemistry and molecular biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves treatment of various topological forms of…

  7. CD44-Targeted Hyaluronic Acid-Coated Redox-Responsive Hyperbranched Poly(amido amine)/Plasmid DNA Ternary Nanoassemblies for Efficient Gene Delivery.

    PubMed

    Gu, Jijin; Chen, Xinyi; Ren, Xiaoqing; Zhang, Xiulei; Fang, Xiaoling; Sha, Xianyi

    2016-07-20

    Hyaluronic acid (HA), which can specifically bind to CD44 receptor, is a specific ligand for targeting to CD44-overexpressing cancer cells. The current study aimed to develop ternary nanoassemblies based on HA-coating for targeted gene delivery to CD44-positive tumors. A novel reducible hyperbranched poly(amido amine) (RHB) was assembled with plasmid DNA (pDNA) to form RHB/pDNA nanoassemblies. HA/RHB/pDNA nanoassemblies were fabricated by coating HA on the surface of the RHB/pDNA nanoassembly core through electrostatic interaction. After optimization, HA/RHB/pDNA nanoassemblies were spherical, core-shell nanoparticles with nanosize (187.6 ± 11.4 nm) and negative charge (-9.1 ± 0.3 mV). The ternary nanoassemblies could efficiently protect the condensed pDNA from enzymatic degradation by DNase I, and HA could significantly improve the stability of nanoassemblies in the sodium heparin solution or serum in vitro. As expected, HA significantly decreased the cytotoxicity of RHB/pDNA nanoassemblies due to the negative surface charges. Moreover, it revealed that HA/RHB/pDNA nanoassemblies showed higher transfection activity than RHB/pDNA nanoassemblies in B16F10 cells, especially in the presence of serum in vitro. Because of the active recognition between HA and CD44 receptor, there was significantly different transfection efficiency between B16F10 (CD44+) and NIH3T3 (CD44-) cells after treatment with HA/RHB/pDNA nanoassemblies. In addition, the cellular targeting and transfection activity of HA/RHB/pDNA nanoassemblies were further evaluated in vivo. The results indicated that the interaction between HA and CD44 receptor dramatically improved the accumulation of HA/RHB/pDNA nanoassemblies in CD44-positive tumor, leading to higher gene expression than RHB/pDNA nanoassemblies. Therefore, HA/RHB/pDNA ternary nanoassemblies may be a potential gene vector for delivery of therapeutic genes to treat CD44-overexpressing tumors in vivo.

  8. Modulation of Macrophage Functional Polarity towards Anti-Inflammatory Phenotype with Plasmid DNA Delivery in CD44 Targeting Hyaluronic Acid Nanoparticles

    PubMed Central

    Tran, Thanh-Huyen; Rastogi, Ruchir; Shelke, Juili; Amiji, Mansoor M.

    2015-01-01

    The purpose of this study was to modulate macrophage polarity from the pro-inflammatory M1 to anti-inflammatory M2 phenotype using plasmid DNA (pDNA) expressing interleukin-4 (IL4) or interleukin-10 (IL10)-encapsulated in hyaluronic acid-poly(ethyleneimine) (HA-PEI) nanoparticles (NPs). The HA-PEI/pDNA NPs with spherical shape, average size of 186 nm were efficiently internalized by J774A.1 macrophages. Transfection of HA-PEI/pDNA-IL4 and HA-PEI/pDNA-IL10 NPs increased IL4 and IL10 gene expression in J774 macrophages which could re-program the macrophages from M1 to M2 phenotype as evidenced by a significant increase in the Arg/iNOS level, and upregulation of CD206 and CD163 compared to untreated macrophages. Following intraperitoneal (IP) injection to C57BL/6 mice, HA-PEI NPs effectively targeted peritoneal macrophages over-expressing CD44 receptor. In an in vivo model of stimulated peritoneal macrophages, IP administration of HA-PEI/pDNA-IL4 and HA-PEI/pDNA-IL10 to C57BL/6 mice significantly increased the Arg/iNOS ratio and CD163 expression in the cells. Furthermore, HA-PEI/pDNA-IL10 NPs significantly increased peritoneal and serum IL10 levels which effectively suppressed LPS-induced inflammation by reducing level of TNF-α and IL-1β in peritoneal macrophages and in the peritoneal fluid. The results demonstrated that pDNA-IL10-encapsulate HA-PEI NPs skewed macrophage functional polarity from M1 toward an anti-inflammatory M2 phenotype which may be a promising platform for the treatment of inflammatory diseases. PMID:26577684

  9. Electron Resonance Decay into a Biological Function: Decrease in Viability of E. coli Transformed by Plasmid DNA Irradiated with 0.5-18 eV Electrons.

    PubMed

    Kouass Sahbani, S; Cloutier, P; Bass, A D; Hunting, D J; Sanche, L

    2015-10-01

    Transient negative ions (TNIs) are ubiquitous in electron-molecule scattering at low electron impact energies (0-20 eV) and are particularly effective in damaging large biomolecules. Because ionizing radiation generates mostly 0-20 eV electrons, TNIs are expected to play important roles in cell mutagenesis and death during radiotherapeutic cancer treatment, although this hypothesis has never been directly verified. Here, we measure the efficiency of transforming E. coli bacteria by inserting into the cells, pGEM-3ZfL(-) plasmid DNA that confers resistance to the antibiotic ampicillin. Before transformation, plasmids are irradiated with electrons of specific energies between 0.5 and 18 eV. The loss of transformation efficiency plotted as a function of irradiation energy reveals TNIs at 5.5 and 9.5 eV, corresponding to similar states observed in the yields of DNA double strand breaks. We show that TNIs are detectable in the electron-energy dependence of a biological process and can decrease cell viability.

  10. Thioredoxin-like proteins in F and other plasmid systems.

    PubMed

    Hemmis, Casey W; Schildbach, Joel F

    2013-09-01

    Bacterial conjugation is the process by which a conjugative plasmid transfers from donor to recipient bacterium. During this process, single-stranded plasmid DNA is actively and specifically transported from the cytoplasm of the donor, through a large membrane-spanning assembly known as the pore complex, and into the cytoplasm of the recipient. In Gram negative bacteria, construction of the pore requires localization of a subset of structural and catalytically active proteins to the bacterial periplasm. Unlike the cytoplasm, the periplasm contains proteins that promote disulfide bond formation within or between cysteine-containing proteins. To ensure proper protein folding and assembly, bacteria employ periplasmic redox systems for thiol oxidation, disulfide bond/sulfenic acid reduction, and disulfide bond isomerization. Recent data suggest that plasmid-based proteins belonging to the disulfide bond formation family play an integral role in the conjugative process by serving as mediators in folding and/or assembly of pore complex proteins. Here we report the identification of 165 thioredoxin-like family members across 89 different plasmid systems. Using phylogenetic analysis, all but nine family members were categorized into thioredoxin-like subfamilies. In addition, we discuss the diversity, conservation, and putative roles of thioredoxin-like proteins in plasmid systems, which include homologs of DsbA, DsbB, DsbC, DsbD, DsbG, and CcmG from Escherichia coli, TlpA from Bradyrhizobium japonicum, Com1 from Coxiella burnetii, as well as TrbB and TraF from plasmid F, and the absolute conservation of a disulfide isomerase in plasmids containing homologs of the transfer proteins TraH, TraN, and TraU.

  11. Building mosaics of therapeutic plasmid gene vectors.

    PubMed

    Tolmachov, Oleg E

    2011-12-01

    Plasmids are circular or linear DNA molecules propagated extra-chromosomally in bacteria. Evolution shaped plasmids are inherently mosaic structures with individual functional units represented by distinct segments in the plasmid genome. The patchwork of plasmid genetic modules is a convenient template and a model for the generation of artificial plasmids used as vehicles for gene delivery into human cells. Plasmid gene vectors are an important tool in gene therapy and in basic biomedical research, where these vectors offer efficient transgene expression in many settings in vitro and in vivo. Plasmid vectors can be attached to nuclear directing ligands or transferred by electroporation as naked DNA to deliver the payload genes to the nuclei of the target cells. Transgene expression silencing by plasmid sequences of bacterial origin and immune stimulation by bacterial unmethylated CpG motifs can be avoided by the generation of plasmid-based minimized DNA vectors, such as minicircles. Systems of efficient site-specific integration into human chromosomes and stable episomal maintenance in human cells are being developed for further reduction of the chances for transgene silencing. The successful generation of plasmid vectors is governed by a number of vector design rules, some of which are common to all gene vectors, while others are specific to plasmid vectors. This review is focused both on the guiding principles and on the technical know-how of plasmid gene vector design.

  12. Bacterial Genome Partitioning: N-Terminal Domain of IncC Protein Encoded by Broad-Host-Range Plasmid RK2 Modulates Oligomerisation and DNA Binding

    PubMed Central

    Batt, Sarah M.; Bingle, Lewis E.H.; Dafforn, Tim R.; Thomas, Christopher M.

    2009-01-01

    ParA Walker ATPases form part of the machinery that promotes better-than-random segregation of bacterial genomes. ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding. Unusually, and for as yet unknown reasons, parA (incC) of IncP-1 plasmids is translated from alternative start codons producing two forms, IncC1 (364 aa) and IncC2 (259 aa), whose ratio varies between hosts. IncC2 could be detected as an oligomeric form containing dimers, tetramers and octamers, but the N-terminal extension present in IncC1 favours nucleotide-stimulated dimerisation as well as high-affinity and ATP-dependent non-specific DNA binding. The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides. Mixing IncC1 and IncC2 improved polymerisation and DNA binding. Thus, the NTD may modulate the polymerisation interface, facilitating polymerisation/depolymerisation and DNA binding, to promote the cycle that drives partitioning. PMID:19109978

  13. Development of targeted recombinant polymers that can deliver siRNA to the cytoplasm and plasmid DNA to the cell nucleus.

    PubMed

    Canine, Brenda F; Wang, Yuhua; Ouyang, Wenyun; Hatefi, Arash

    2011-04-10

    One of the major limitations to effective siRNA delivery is the lack of a siRNA-specific delivery system. Currently, the same delivery systems that are used for plasmid DNA (pDNA) delivery to the cell nucleus are used for siRNA delivery to the cytoplasm. To fill this gap, the objective of this study was to design a biopolymer that can be programmed via its amino acid sequence to deliver siRNA specifically to cytoplasm. For pDNA delivery, a nuclear localization signal (NLS) was added to the biopolymer structure to facilitate active translocation of the genetic material towards nucleus. The biopolymers were complexed with pEGFP and GFP-siRNA and used to transfect SKOV-3 (HER2+) cells. The intracellular trafficking of the nanoparticles was also monitored in real-time and live cells. The results demonstrated that the biopolymer with NLS is a suitable carrier for pDNA delivery but not siRNA delivery. Conversely, the biopolymer without NLS was suitable for siRNA delivery to the cytoplasm but not pDNA to the cell nucleus. The potential use of the designed biopolymer for combination therapy of cancer cells with gene (thymidine kinase) and siRNA (BCL2) was also examined in SKOV-3 cancer cells.

  14. Variable effects of the co-administration of a GM-CSF-expressing plasmid on the immune response to flavivirus DNA vaccines in mice.

    PubMed

    Chen, Hui; Gao, Na; Wu, Jiangman; Zheng, Xiaoyan; Li, Jieqiong; Fan, Dongying; An, Jing

    2014-11-01

    As a cytokine adjuvant, granulocyte-macrophage colony-stimulating factor (GM-CSF) has been demonstrated to play central roles in the enhancement of the immune response and protection elicited by experimental vaccines. However, in our previous work, the co-administration of GM-CSF produced untoward effects on the immune response induced by a Japanese encephalitis virus DNA vaccine candidate. This study aimed to elucidate the adjuvant roles of GM-CSF in several Flaviviridae virus DNA vaccine candidates. Our results showed that the effects of GM-CSF were diverse: co-inoculated GM-CSF caused significant suppression to the dengue virus type 1 and type 2 prM-E DNA vaccinations and influenced protective efficiency against virus challenge. In contrast, GM-CSF showed little effect or an enhancement on the immune response elicited by hepatitis C virus C or E1 DNA vaccine candidates. Notably, these effects of GM-CSF were highly durable. Our results suggested that the adjuvant roles of the GM-CSF plasmid were complex and diverse, ranging from enhancement to suppression, depending on the immunogen of Flaviviridae virus DNA vaccine candidates. Therefore, the application of GM-CSF as a vaccine adjuvant or a therapeutic agent should be evaluated carefully.

  15. Antibiotic resistance free plasmid DNA expressing LACK protein leads towards a protective Th1 response against Leishmania infantum infection.

    PubMed

    Ramos, I; Alonso, A; Peris, A; Marcen, J M; Abengozar, M A; Alcolea, P J; Castillo, J A; Larraga, V

    2009-11-12

    Canine visceral leishmaniasis is a serious public health concern in the Mediterranean basin since dogs are the main Leishmania infantum reservoir. However, there is not a vaccination method in veterinary use in this area, and therefore the development of a vaccine against this parasite is essential for the possible control of the disease. Previous reports have shown the efficacy of heterologous prime-boost vaccination with the pCIneo plasmid and the poxvirus VV (both Western Reserve and MVA strains) expressing L. infantum LACK antigen against canine leishmaniasis. As pCIneo-LACK plasmid contains antibiotic resistance genes, its use as a profilactic method is not recommended. Hence, the antibiotic resistance gene free pORT-LACK plasmid is a more suitable tool for its use as a vaccine. Here we report the protective and immunostimulatory effect of the prime-boost pORT-LACK/MVA-LACK vaccination tested in a canine experimental model. Vaccination induced a reduction in clinical signs and in parasite burden in the liver, an induction of the Leishmania-specific T cell activation, as well as an increase of the expression of Th1 type cytokines in PBMC and target organs.

  16. Phase IIb trial of in vivo electroporation mediated dual-plasmid hepatitis B virus DNA vaccine in chronic hepatitis B patients under lamivudine therapy

    PubMed Central

    Yang, Fu-Qiang; Rao, Gui-Rong; Wang, Gui-Qiang; Li, Yue-Qi; Xie, Yao; Zhang, Zhan-Qing; Deng, Cun-Liang; Mao, Qing; Li, Jun; Zhao, Wei; Wang, Mao-Rong; Han, Tao; Chen, Shi-Jun; Pan, Chen; Tan, De-Ming; Shang, Jia; Zhang, Ming-Xiang; Zhang, Yue-Xin; Yang, Ji-Ming; Chen, Guang-Ming

    2017-01-01

    AIM To assess the efficacy and safety of in vivo electroporation (EP)-mediated dual-plasmid hepatitis B virus (HBV) DNA vaccine vs placebo for sequential combination therapy with lamivudine (LAM) in patients with chronic hepatitis B. METHODS Two hundred and twenty-five patients were randomized to receive either LAM + vaccine (vaccine group, n = 109) or LAM + placebo (control group, n = 116). LAM treatment lasted 72 wk. Patients received the DNA vaccine or placebo by intramuscular injection mediated by EP at weeks 12 (start of treatment with vaccine or placebo, SOT), 16, 24, and 36 (end of treatment with vaccine or placebo, EOT). RESULTS In the modified intent-to-treat population, more patients had a decrease in HBV DNA > 2 log10 IU/mL in the vaccine group at week 12 after EOT compared with the control group. A trend toward a difference in the number of patients with undetectable HBV DNA at week 28 after EOT was obtained. Adverse events were similar. In the dynamic per-protocol set, which excluded adefovir (ADV) add-on cases at each time point instantly after ADV administration due to LAM antiviral failure, more patients had a decrease in HBV DNA > 2 log10 IU/mL in the vaccine group at week 12 and 28 after EOT compared with the control group. More patients with undetectable HBV DNA at week 28 after EOT in the vaccine group were also observed. Among patients with a viral load < 1000 copies/mL at week 12, more patients achieved HBeAg seroconversion in the vaccine group than among controls at week 36 after EOT, as well as less virological breakthrough and YMDD mutations. CONCLUSION The primary endpoint was not achieved using the HBV DNA vaccine. The HBV DNA vaccine could only be beneficial in subjects that have achieved initial virological response under LAM chemotherapy. PMID:28127204

  17. Protective efficacy of a prime-boost protocol using H5-DNA plasmid as prime and inactivated H5N2 vaccine as the booster against the Egyptian avian influenza challenge virus.

    PubMed

    Hussein, H A; Ahmed, B M; Aly, S M; El-Deeb, A H; El-Sanousi, A A; Rohaim, M A; Arafa, A A; Gadalla, M R

    In this study, a recombinant DNA plasmid was constructed, encoding for HA1 of a selected Egyptian H5N1 virus (isolated during the 2012 outbreaks). In the immunization and challenge experiments, SPF chickens received 1 or 2 doses of H5-DNA plasmid prime, and boosted with the inactivated H5N2 vaccine. Haemagglutination inhibition (HI) titers, protection levels, and the magnitude of virus shedding were compared to that of the chickens that received either DNA plasmid or inactivated H5N2 vaccine alone. H5N1 virus A/chicken/Egypt/128s/2012 (H5N1) highly pathogenic avian influenza (HPAI) clade 2.2.1/C was used for the challenge. Chickens immunized with 1 or 2 doses of H5-DNA vaccine failed to overcome the challenge with 0% and 10% protection, respectively. Quantitative real-time reverse transcription-PCR revealed virus shedding of 2.2 x 104 PCR copies/ml 3 days post challenge (dpc) in the only surviving bird from the group that received 2 doses of plasmid. However, chickens immunized with 1 or 2 doses of H5-DNA plasmid as prime and inactivated H5N2 vaccine as booster, showed 80% protection after challenge, with a viral shedding of 1.2 x 104 PCR copies/ml (1 dose) and 1.6 x 104 PCR copies/ml (2 doses) 3 dpc. The surviving birds in both groups did not shed the virus at 5 and 7 dpc. In H5N2-vaccinated chickens, protection levels were 70% with relatively high virus shedding (1.8 x 104 PCR copies/ml) 3 dpc. HI titers were protective to the surviving chickens. This study reports the efficacy of H5-DNA plasmid to augment reduction in viral shedding and to provide better protection when applied in a prime-boost program with the inactivated AI vaccine.

  18. Gene delivery nanocarriers of bioactive glass with unique potential to load BMP2 plasmid DNA and to internalize into mesenchymal stem cells for osteogenesis and bone regeneration

    NASA Astrophysics Data System (ADS)

    Kim, Tae-Hyun; Singh, Rajendra K.; Kang, Min Sil; Kim, Joong-Hyun; Kim, Hae-Won

    2016-04-01

    The recent development of bioactive glasses with nanoscale morphologies has spurred their specific applications in bone regeneration, for example as drug and gene delivery carriers. Bone engineering with stem cells genetically modified with this unique class of nanocarriers thus holds great promise in this avenue. Here we report the potential of the bioactive glass nanoparticle (BGN) system for the gene delivery of mesenchymal stem cells (MSCs) targeting bone. The composition of 15% Ca-added silica, proven to be bone-bioactive, was formulated into surface aminated mesoporous nanospheres with enlarged pore sizes, to effectively load and deliver bone morphogenetic protein-2 (BMP2) plasmid DNA. The enlarged mesopores were highly effective in loading BMP2-pDNA with an efficiency as high as 3.5 wt% (pDNA w.r.t. BGN), a level more than twice than for small-sized mesopores. The BGN nanocarriers released the genetic molecules in a highly sustained manner (for as long as 2 weeks). The BMP2-pDNA/BGN complexes were effectively internalized to rat MSCs with a cell uptake level of ~73%, and the majority of cells were transfected to express the BMP2 protein. Subsequent osteogenesis of the transfected MSCs was demonstrated by the expression of bone-related genes, including bone sialoprotein, osteopontin, and osteocalcin. The MSCs transfected with BMP2-pDNA/BGN were locally delivered inside a collagen gel to the target calvarium defects. The results showed significantly improved bone regeneration, as evidenced by the micro-computed tomographic, histomorphometric and immunohistochemical analyses. This study supports the excellent capacity of the BGN system as a pDNA-delivery nanocarrier in MSCs, and the engineered system, BMP2-pDNA/BGN with MSCs, may be considered a new promising candidate to advance the therapeutic potential of stem cells through genetic modification, targeting bone defects and diseases.The recent development of bioactive glasses with nanoscale morphologies has

  19. Gene delivery nanocarriers of bioactive glass with unique potential to load BMP2 plasmid DNA and to internalize into mesenchymal stem cells for osteogenesis and bone regeneration.

    PubMed

    Kim, Tae-Hyun; Singh, Rajendra K; Kang, Min Sil; Kim, Joong-Hyun; Kim, Hae-Won

    2016-04-21

    The recent development of bioactive glasses with nanoscale morphologies has spurred their specific applications in bone regeneration, for example as drug and gene delivery carriers. Bone engineering with stem cells genetically modified with this unique class of nanocarriers thus holds great promise in this avenue. Here we report the potential of the bioactive glass nanoparticle (BGN) system for the gene delivery of mesenchymal stem cells (MSCs) targeting bone. The composition of 15% Ca-added silica, proven to be bone-bioactive, was formulated into surface aminated mesoporous nanospheres with enlarged pore sizes, to effectively load and deliver bone morphogenetic protein-2 (BMP2) plasmid DNA. The enlarged mesopores were highly effective in loading BMP2-pDNA with an efficiency as high as 3.5 wt% (pDNA w.r.t. BGN), a level more than twice than for small-sized mesopores. The BGN nanocarriers released the genetic molecules in a highly sustained manner (for as long as 2 weeks). The BMP2-pDNA/BGN complexes were effectively internalized to rat MSCs with a cell uptake level of ∼73%, and the majority of cells were transfected to express the BMP2 protein. Subsequent osteogenesis of the transfected MSCs was demonstrated by the expression of bone-related genes, including bone sialoprotein, osteopontin, and osteocalcin. The MSCs transfected with BMP2-pDNA/BGN were locally delivered inside a collagen gel to the target calvarium defects. The results showed significantly improved bone regeneration, as evidenced by the micro-computed tomographic, histomorphometric and immunohistochemical analyses. This study supports the excellent capacity of the BGN system as a pDNA-delivery nanocarrier in MSCs, and the engineered system, BMP2-pDNA/BGN with MSCs, may be considered a new promising candidate to advance the therapeutic potential of stem cells through genetic modification, targeting bone defects and diseases.

  20. Enhancement of the immunogenicity of a porcine circovirus type 2 DNA vaccine by a recombinant plasmid coexpressing capsid protein and porcine interleukin-6 in mice.

    PubMed

    Guo, Xiao-Qing; Wang, Lin-Qing; Qiao, Han; Yang, Xing-Wu; Yang, Ming-Fan; Chen, Hong-Ying

    2015-03-01

    The development of effective vaccines against porcine circovirus type 2 (PCV2) has been accepted as an important strategy in the prophylaxis of post-weaning multisystemic wasting syndrome; a DNA vaccine expressing the major immunogenic capsid (Cap) protein of PCV2 is considered to be a promising candidate. However, DNA vaccines usually induce weak immune responses. In this study, it was found that the efficacy of a DNA vaccine expressing Cap protein was improved by simultaneous expression of porcine IL-6. A plasmid (pIRES-ORF2/IL6) separately expressing both Cap protein and porcine IL-6 was constructed and compared with another plasmid (pIRES-ORF2) expressing Cap protein for its potential to induce PCV2-specific immune responses. Mice were vaccinated i.m. twice at 3 week intervals and the induced humoral and cellular responses evaluated. All animals vaccinated with pIRES-ORF2/IL6 and pIRES-ORF2 developed specific anti-PCV2 antibodies (according to enzyme-linked immunosorbent assay) and a T lymphocyte proliferation response. The percentages of CD3(+), CD3(+)CD8(+), and CD3(+)CD4(+) subgroups of peripheral blood T-lymphocytes were significantly higher in mice immunized with pIRES-ORF2/IL6 than in those that had received pIRES-ORF2. After challenge with the virulent PCV2 Wuzhi isolate, mice vaccinated with pIRES-ORF2/IL6 had significantly less viral replication than those vaccinated with pIRES-ORF2, suggesting that the protective immunity induced by pIRES-ORF2/IL6 is superior to that induced by pIRES-ORF2.

  1. High levels of gene expression in the hepatocytes of adult mice, neonatal mice and tree shrews via retro-orbital sinus hydrodynamic injections of naked plasmid DNA.

    PubMed

    Yan, Shaoduo; Fu, Qiuxia; Zhou, Yong; Wang, Jidong; Liu, Ying; Duan, Xiangguo; Jia, Shuaizheng; Peng, Jianchun; Gao, Bo; Du, Juan; Zhou, Qianqian; Li, Yuan; Wang, Xiaohui; Zhan, Linsheng

    2012-08-10

    Hydrodynamic-based gene delivery has emerged as an efficient and simple method for the intracellular transfection of naked plasmid DNA (pDNA) in vivo. In this system, a hydrodynamic injection via the tail vein is the most effective non-viral method of liver-targeted gene delivery. However, this injection is often technically challenging when used in animals whose tail veins are difficult to visualize or too small to operate on. To overcome this limitation, an alternative in vivo gene delivery method, the rapid injection of large volume of pDNA solution through retro-orbital sinus, was established. Using this technique, we successfully delivered pDNA to the tissue of adult mice, neonatal mice and tree shrews. The efficient expression of exogenous genes was specifically detected in the liver of test animals treated with this gene delivery method. This study demonstrates for the first time that the hydrodynamic gene delivery via the retro-orbital sinus can not only reach the same transgene efficiency as a tradition hydrodynamic-based intravascular injection but also be used in animals that are difficult to inject via the tail vein. This method could open up new areas in gene function studies and gene therapy disease treatment.

  2. Varying the chain length in N4,N9-diacyl spermines: non-viral lipopolyamine vectors for efficient plasmid DNA formulation.

    PubMed

    Ghonaim, Hassan M; Ahmed, Osama A A; Pourzand, Charareh; Blagbrough, Ian S

    2008-01-01

    The aims of this work are to study the effect of varying the chain length in synthesized N4,N9-diacyl spermines on DNA condensation and then to compare their transfection efficiencies in cell lines. The five novel N4,N9-diacyl lipopolyamines: N4,N9-[didecanoyl, dilauroyl, dimyristoyl, dimyristoleoyl, and dipalmitoyl]-1,12-diamino-4,9-diazadodecane were synthesized from the naturally occurring polyamine spermine. The abilities of these novel compounds to condense DNA and to form nanoparticles were studied using ethidium bromide fluorescence quenching and nanoparticle characterization techniques. Transfection efficiency was studied in FEK4 primary skin cells and in an immortalized cancer cell line (HtTA), and compared with a saturated (distearoyl) analogue and also with the non-liposomal transfection formulation Lipogen, N4,N9-dioleoyl-1,12-diamino-4,9-diazadodecane. By incorporating two aliphatic chains and changing their length in a stepwise manner, we show efficient circular plasmid DNA (pEGFP) formulation and transfection of primary skin and cancer cell lines. Two C14 chains (both saturated or both cis-monounsaturated) were efficient transfecting agents, even in the presence of serum, but they were too toxic. N4,N9-Dioleoyl spermine efficiently condenses pDNA and achieves the highest transfection levels with the highest cell viability among the studied lipopolyamines in cultured cells even in the presence of serum.

  3. Interactions between the RepB initiator protein of plasmid pMV158 and two distant DNA regions within the origin of replication

    PubMed Central

    Ruiz-Masó, José A.; Lurz, Rudi; Espinosa, Manuel; del Solar, Gloria

    2007-01-01

    Plasmids replicating by the rolling circle mode usually possess a single site for binding of the initiator protein at the origin of replication. The origin of pMV158 is different in that it possesses two distant binding regions for the initiator RepB. One region was located close to the site where RepB introduces the replication-initiating nick, within the nic locus; the other, the bind locus, is 84 bp downstream from the nick site. Binding of RepB to the bind locus was of higher affinity and stability than to the nic locus. Contacts of RepB with the bind and nic loci were determined through high-resolution footprinting. Upon binding of RepB, the DNA of the bind locus follows a winding path in its contact with the protein, resulting in local distortion and bending of the double-helix. On supercoiled DNA, simultaneous interaction of RepB with both loci favoured extrusion of the hairpin structure harbouring the nick site while causing a strong DNA distortion around the bind locus. This suggests interplay between the two RepB binding sites, which could facilitate loading of the initiator protein to the nic locus and the acquisition of the appropriate configuration of the supercoiled DNA substrate. PMID:17267412

  4. A Vaxfectin(®)-adjuvanted HSV-2 plasmid DNA vaccine is effective for prophylactic and therapeutic use in the guinea pig model of genital herpes.

    PubMed

    Veselenak, Ronald L; Shlapobersky, Mark; Pyles, Richard B; Wei, Qun; Sullivan, Sean M; Bourne, Nigel

    2012-11-19

    Here we describe studies in the guinea pig model of genital herpes to evaluate a novel plasmid DNA (pDNA) vaccine encoding the HSV-2 glycoprotein D and UL46 and UL47 genes encoding tegument proteins VP11/12 and VP 13/14 (gD2/UL46/UL47), formulated with a cationic lipid-based adjuvant Vaxfectin(®). Prophylactic immunization with Vaxfectin(®)-gD2/UL46/UL47 significantly reduced viral replication in the genital tract, provided complete protection against both primary and recurrent genital skin disease following intravaginal HSV-2 challenge, and significantly reduced latent HSV-2 DNA in the dorsal root ganglia compared to controls. We also examined the impact of therapeutic immunization of HSV-2 infected animals. Here, Vaxfectin(®)-gD2/UL46/UL47 immunization significantly reduced both the frequency of recurrent disease and viral shedding into the genital tract compared to controls. This novel adjuvanted pDNA vaccine has demonstrated both prophylactic and therapeutic efficacy in the guinea pig model of genital herpes and warrants further development.

  5. A Recombinant DNA Plasmid Encoding the sIL-4R-NAP Fusion Protein Suppress Airway Inflammation in an OVA-Induced Mouse Model of Asthma.

    PubMed

    Liu, Xin; Fu, Guo; Ji, Zhenyu; Huang, Xiabing; Ding, Cong; Jiang, Hui; Wang, Xiaolong; Du, Mingxuan; Wang, Ting; Kang, Qiaozhen

    2016-08-01

    Asthma is a chronic inflammatory airway disease. It was prevalently perceived that Th2 cells played the crucial role in asthma pathogenesis, which has been identified as the important target for anti-asthma therapy. The soluble IL-4 receptor (sIL-4R), which is the decoy receptor for Th2 cytokine IL-4, has been reported to be effective in treating asthma in phase I/II clinical trail. To develop more efficacious anti-asthma agent, we attempt to test whether the Helicobacter pylori neutrophil-activating protein (HP-NAP), a novel TLR2 agonist, would enhance the efficacy of sIL-4R in anti-asthma therapy. In our work, we constructed a pcDNA3.1-sIL-4R-NAP plasmid, named PSN, encoding fusion protein of murine sIL-4R and HP-NAP. PSN significantly inhibited airway inflammation, decreased the serum OVA-specific IgE levels and remodeled the Th1/Th2 balance. Notably, PSN is more effective on anti-asthma therapy comparing with plasmid only expressing sIL-4R.

  6. In vitro DNA binding, pBR322 plasmid cleavage and molecular modeling study of chiral benzothiazole Schiff-base-valine Cu(II) and Zn(II) complexes to evaluate their enantiomeric biological disposition for molecular target DNA

    NASA Astrophysics Data System (ADS)

    Alizadeh, Rahman; Afzal, Mohd; Arjmand, Farukh

    2014-10-01

    Bicyclic heterocyclic compounds viz. benzothiazoles are key components of deoxyribonucleic acid (DNA) molecules and participate directly in the encoding of genetic information. Benzothiazoles, therefore, represent a potent and selective class of antitumor compounds. The design and synthesis of chiral antitumor chemotherapeutic agents of Cu(II) and Zn(II), L- and -D benzothiazole Schiff base-valine complexes 1a &b and 2a &b, respectively were carried out and thoroughly characterized by spectroscopic and analytical techniques. Interaction of 1a and b and 2a and b with CT DNA by employing UV-vis, florescence, circular dichroic methods and cleavage studies of 1a with pBR322 plasmid, molecular docking were done in order to demonstrate their enantiomeric disposition toward the molecular drug target DNA. Interestingly, these studies unambiguously demonstrated the greater potency of L-enantiomer in comparison to D-enantiomer.

  7. In vitro DNA binding, pBR322 plasmid cleavage and molecular modeling study of chiral benzothiazole Schiff-base-valine Cu(II) and Zn(II) complexes to evaluate their enantiomeric biological disposition for molecular target DNA.

    PubMed

    Alizadeh, Rahman; Afzal, Mohd; Arjmand, Farukh

    2014-10-15

    Bicyclic heterocyclic compounds viz. benzothiazoles are key components of deoxyribonucleic acid (DNA) molecules and participate directly in the encoding of genetic information. Benzothiazoles, therefore, represent a potent and selective class of antitumor compounds. The design and synthesis of chiral antitumor chemotherapeutic agents of Cu(II) and Zn(II), L- and -D benzothiazole Schiff base-valine complexes 1a &b and 2a &b, respectively were carried out and thoroughly characterized by spectroscopic and analytical techniques. Interaction of 1a and b and 2a and b with CT DNA by employing UV-vis, florescence, circular dichroic methods and cleavage studies of 1a with pBR322 plasmid, molecular docking were done in order to demonstrate their enantiomeric disposition toward the molecular drug target DNA. Interestingly, these studies unambiguously demonstrated the greater potency of L-enantiomer in comparison to D-enantiomer.

  8. Distinct immune responses of recombinant plasmid DNA replicon vaccines expressing two types of antigens with or without signal sequences.

    PubMed

    Yu, Yun-Zhou; Li, Na; Wang, Wen-Bin; Wang, Shuang; Ma, Yao; Yu, Wei-Yuan; Sun, Zhi-Wei

    2010-11-03

    Here, DNA replicon vaccines encoding the Hc domain of botulinum neurotoxin serotype A (AHc) or the receptor binding domain of anthrax protective antigen (PA4) with or without signal sequences were evaluated in mice. Strong antibody and protective responses were elicited only from AHc DNA vaccines with an Ig κ signal sequence or tissue plasminogen activator signal sequence. Meanwhile, there were no differences in total antibody responses or isotypes, lymphocyte proliferative responses, cytokine profiles and protective immune responses with the PA4 DNA vaccines with or without a signal sequence. Therefore, use of targeting sequences in designing DNA replicon vaccines depends on the specific antigen.

  9. Plasmid diversity in Vibrio vulnificus biotypes.

    PubMed

    Roig, Francisco J; Amaro, Carmen

    2009-02-01

    Vibrio vulnificus is a heterogeneous bacterial species that can be virulent for humans and fish. Virulence in fish seems to rely on a recently described plasmid that can be transmitted between strains, aided by a conjugative plasmid. The main objective of this work was to analyse the plasmid content of a wide collection of strains from the three biotypes of the species, as well as to identify putative conjugative and virulence plasmids by means of Southern hybridization with specific probes and sequence analysis of selected gene markers. We found 28 different plasmid profiles in a total of 112 strains, which were relatively biotype- or serovar-specific. Biotype 1 lacked high-molecular-mass plasmids, with the exception of a putative conjugative plasmid of 48 kb that was present in 42.8% of clinical and environmental strains isolated worldwide. All biotype 2 strains possessed the virulence plasmid, whose molecular mass ranged between 68 and 70 kb, and 89.65% of these strains also had a putative conjugative plasmid with a molecular size of 52-56 kb. Finally, a 48 kb putative conjugative plasmid was present in all biotype 3 strains. Data from partial sequencing of traD, traI and the whole vep07 (a recently described plasmid-borne virulence gene) from a selection of strains suggest that the plasmids of 48-56 kb probably belong to the same family of F-plasmids as pYJ016 and that the gene vep07 is absolutely essential for fish virulence. Additional cryptic plasmids of low molecular mass were present in the three biotypes. In conclusion, plasmids are widespread among V. vulnificus species and could contribute substantially to genetic plasticity of the species.

  10. The mechanism of plasmid curing in bacteria.

    PubMed

    Spengler, Gabriella; Molnár, Annamária; Schelz, Zsuzsanna; Amaral, Leonard; Sharples, Derek; Molnár, Joseph

    2006-07-01

    Bacterial plasmids have a major impact on metabolic function. Lactose fermentation of E. coli or hemolysin B transporter expressed by the plasmids that carry these respective genes could be readily obviated by heterocyclic compounds that readily bind to plasmid DNA. These compounds could also reverse the resistance to antibiotics of E. coli, Enterobacter, Proteus, Staphylococcus and Yersinia strains by eliminating plasmids. However, the frequency and extent of this effect was significantly less than might have been expected based on a complex interaction with plasmid DNA. The effects of heterocyclic compounds on the plasmids responsible for the virulence of Yersinia and A. tumefaciens, or on nodulation, nitrogen fixation of Rhizobia accounted for the elimination of 0.1 to 1.0 % of plasmids present in the populations studied. Bacterial plasmids can be eliminated from bacterial species grown as pure or mixed bacterial cultures in the presence of sub-inhibitory concentrations of non-mutagenic heterocyclic compounds. The antiplasmid action of the compounds depends on the chemical structure of amphiphillic compounds having a planar ring system with substitution in the L-molecular region. A symmetrical pi-electron conjugation at the highest occupied molecular orbitals favours the antiplasmid effect. The antiplasmid effect of heterocyclic compounds is expressed differentially in accordance with the structural form of the DNA to which they bind. In this manner "extrachromosomal" plasmid DNA that exists in a superhelical state binds more compound than its linear or open-circular form; and least to the chromosomal DNA of the bacterium, that carries the plasmid. It can also be noted that these compounds are not mutagenic and their antiplasmid effects correlate with the energy of HOMO-orbitals. Plasmid elimination is considered also to take place in ecosystems containing numerous bacterial species. This opens up a new perspective in rational drug design against bacterial

  11. Co-encapsulation of Nigella sativa oil and plasmid DNA for enhanced gene therapy of Alzheimer's disease.

    PubMed

    Doolaanea, Abd Almonem; Mansor, Nur 'Izzati; Mohd Nor, Nurul Hafizah; Mohamed, Farahidah

    2016-01-01

    Alzheimer disease involves genetic and non-genetic factors and hence it is rational to be treated with genetic and non-genetic therapeutic agents. Nigella sativa has multiple therapeutic properties including neuroregeneration. Nigella sativa oil (NSO) was encapsulated in PLGA nanoparticles and pDNA was loaded either by adsorption on chitosan-modified particles or encapsulation within PLGA nanoparticles. The particle size and zeta potential of NSO-pDNA-chitosan-PLGA nanoparticles were highly dependent on the medium and exhibited high burst release. Meanwhile, NSO-pDNA-PLGA nanoparticles were more consistent with lower burst release. The fabricated nanoparticles revealed the expected outcomes of both pDNA and NSO. The pDNA transfected N2a cell while the encapsulated NSO promoted neurite outgrowth that is crucial for neuroregeneration. Results from this study suggest that NSO could be added to the gene delivery carrier to enhance treatment benefits for Alzheimer disease.

  12. 5-Aminolevulinic acid induces single-strand breaks in plasmid pBR322 DNA in the presence of Fe2+ ions.

    PubMed

    Onuki, J; Medeiros, M H; Bechara, E J; Di Mascio, P

    1994-02-22

    5-Aminolevulinic acid (ALA), a heme precursor accumulated in chemical and inborn porphyrias, has been demonstrated to produce reactive oxygen species upon metal-catalyzed aerobic oxidation and to cause oxidative damage to proteins, liposomes and subcellular structures. Exposure of plasmid pBR322 DNA to ALA (0.01-3 mM) in the presence of 10 microM Fe2+ ions causes DNA single-strand breaks (ssb), revealed by agarose gel electrophoresis as an increase in the proportion of the open circular form (75 +/- 7.5% at 3 mM ALA) at the expense of the supercoiled form. Addition of either anti-oxidant enzymes such as superoxide dismutase (10 micrograms/ml) and catalase (20 micrograms/ml), or a metal chelator (DTPA, 2.5 mM), or a HO. scavenger (mannitol, 100 mM) inhibited the damage (by 30, 45, 55, and 81%, respectively), evidencing the involvement of O2-., H2O2 and HO. (by the Haber-Weiss reaction) in this process. Hydrogen peroxide (100 microM) or Fe2+ (10 microM) alone were of little effect on the extent of DNA ssb. The present data may shed light on the correlation reported between primary liver-cell carcinoma and intermittent acute porphyria.

  13. Co-Administration of a Plasmid DNA Encoding IL-15 Improves Long-Term Protection of a Genetic Vaccine against Trypanosoma cruzi

    PubMed Central

    Sullivan, Nicole L.; Blazevic, Azra; Bruna-Romero, Oscar; Rodrigues, Mauricio M.; Hoft, Daniel F.

    2011-01-01

    Background Immunization of mice with the Trypanosoma cruzi trans-sialidase (TS) gene using plasmid DNA, adenoviral vector, and CpG-adjuvanted protein delivery has proven highly immunogenic and provides protection against acute lethal challenge. However, long-term protection induced by TS DNA vaccines has not been reported. The goal of the present work was to test whether the co-administration of a plasmid encoding IL-15 (pIL-15) could improve the duration of protection achieved through genetic vaccination with plasmid encoding TS (pTS) alone. Methodology We immunized BALB/c mice with pTS in the presence or absence of pIL-15 and studied immune responses [with TS-specific IFN-γ ELISPOT, serum IgG ELISAs, intracellular cytokine staining (IFN-γ, TNF-α, and IL-2), tetramer staining, and CFSE dilution assays] and protection against lethal systemic challenge at 1 to 6 months post vaccination. Mice receiving pTS alone developed robust TS-specific IFN-γ responses and survived a lethal challenge given within the first 3 months following immunization. The addition of pIL-15 to pTS vaccination did not significantly alter T cell responses or protection during this early post-vaccination period. However, mice vaccinated with both pTS and pIL-15 challenged 6 months post-vaccination were significantly more protected against lethal T. cruzi challenges than mice vaccinated with pTS alone (P<0.05). Improved protection correlated with significantly higher numbers of TS-specific IFN-γ producing total and CD8+ T cells detected>6 months post immunization. Also, these TS-specific T cells were better able to expand after in vitro re-stimulation. Conclusion Addition of pIL-15 during genetic vaccination greatly improved long-term T cell survival, memory T cell expansion, and long-term protection against the important human parasite, T. cruzi. PMID:21408124

  14. In vivo monitoring of transfected DNA, gene expression kinetics, and cellular immune responses in mice immunized with a human NIS gene-expressing plasmid.

    PubMed

    Son, Hye-Youn; Jeon, Yong-Hyun; Chung, June-Key; Kim, Chul-Woo

    2016-12-01

    In assessing the effectiveness of DNA vaccines, it is important to monitor: (1) the kinetics of target gene expression in vivo; and (2) the movement of cells that become transfected with the plasmid DNA used in the immunization of a subject. In this study, we used, as a visual imaging marker, expression of the transfected human sodium/iodide symporter (hNIS) gene, which enhances intracellular radio-pertechnetate (TcO4-) accumulation. After intradermal (i.d.) and systemic injection of mice with pcDNA-hNIS and radioactive Technetium-99m (Tc-99m), respectively, whole-body images were obtained by nuclear scintigraphy. The migration of mice cells transfected with the hNIS gene was monitored over a 2-week period by gamma-radioactivity counting of isolated cell populations and was demonstrated in peripheral lymphoid tissues, especially in the draining lymph nodes (dLNs). Beginning at 24 h after DNA inoculation and continuing for the 2-week monitoring period, hNIS-expressing cells were observed specifically in the T-cell-rich zones of the paracortical area of the dLNs. Over the same time period, high levels of INF-γ-secreting CD8 T-cells were found in the dLNs of the pcDNA-hNIS immunized mice. Tumor growth was also significantly retarded in the mice that received hNIS DNA immunization followed by inoculation with CT26 colorectal adenocarcinoma cells that had been transfected with the rat NIS gene (rNIS), which is 93% homologous to the hNIS gene. In conclusion, mouse cells transfected with hNIS DNA after i.d. immunization were found to traffic to the dLNs, and hNIS gene expression in these cells continued for at least 2 weeks post immunization. Furthermore, sequential presentation of NIS DNA to T-cells by migratory antigen presenting cells could induce NIS DNA-specific Th1 immune responses and thus retard the growth of NIS-expressing tumors.

  15. Increasing versatility of the DNA vaccines through modification of the subcellular location of plasmid-encoded antigen expression in the in vivo transfected cells.

    PubMed

    Martinez-Lopez, Alicia; García-Valtanen, Pablo; Ortega-Villaizan, María Del Mar; Chico, Verónica; Medina-Gali, Regla María; Perez, Luis; Coll, Julio; Estepa, Amparo

    2013-01-01

    The route of administration of DNA vaccines can play a key role in the magnitude and quality of the immune response triggered after their administration. DNA vaccines containing the gene of the membrane-anchored glycoprotein (gpG) of the fish rhabdoviruses infectious haematopoietic necrosis virus (IHNV) or viral haematopoietic septicaemia virus (VHSV), perhaps the most effective DNA vaccines generated so far, confer maximum protection when injected intramuscularly in contrast to their low efficacy when injected intraperitoneally. In this work, taking as a model the DNA vaccine against VHSV, we focused on developing a more versatile DNA vaccine capable of inducing protective immunity regardless of the administration route used. For that, we designed two alternative constructs to gpG₁₋₅₀₇ (the wild type membrane-anchored gpG of VHSV) encoding either a soluble (gpG₁₋₄₆₂) or a secreted soluble (gpG(LmPle20-462)) form of the VHSV-gpG. In vivo immunisation/challenge assays showed that only gpG(LmPle20-462) (the secreted soluble form) conferred protective immunity against VHSV lethal challenge via both intramuscular and intraperitoneal injection, being this the first description of a fish viral DNA vaccine that confers protection when administered intraperitoneally. Moreover, this new DNA vaccine construct also conferred protection when administered in the presence of an oil adjuvant suggesting that DNA vaccines against rhabdoviruses could be included in the formulation of current multicomponent-intaperitoneally injectable fish vaccines formulated with an oil adjuvant. On the other hand, a strong recruitment of membrane immunoglobulin expressing B cells, mainly membrane IgT, as well as t-bet expressing T cells, at early times post-immunisation, was specifically observed in the fish immunised with the secreted soluble form of the VHSV-gpG protein; this may indicate that the subcellular location of plasmid-encoded antigen expression in the in vivo

  16. DNA-binding domain of the RepE initiator protein of mini-F plasmid: involvement of the carboxyl-terminal region.

    PubMed Central

    Matsunaga, F; Kawasaki, Y; Ishiai, M; Nishikawa, K; Yura, T; Wada, C

    1995-01-01

    The RepE initiator protein (251 residues) is essential for mini-F replication in Escherichia coli and exhibits two major functions: initiation of DNA replication from ori2 and autogenous repression of repE transcription. Whereas the initiation is mediated by RepE monomers that bind to the ori2 iterons (direct repeats), the autogenous repression is mediated by dimers that bind to the repE operator, which contains an inverted repeat sequence related to the iterons. We now report that the binding of RepE to these DNA sites is primarily determined by the C-terminal region of this protein. The mutant RepE proteins lacking either the N-terminal 33 (or more) residues or the C-terminal 7 (or more) residues were first shown to be defective in binding to both the ori2 and the operator DNAs. However, direct screening and analysis of mutant RepEs which are specifically affected in binding to the ori2 iterons revealed that the mutations (mostly amino acid substitutions) occur exclusively in the C-terminal region (residues 168 to 242). These mutant proteins exhibited reduced binding to ori2 and no detectable binding to the operator. Thus, whereas truncation of either end of RepE can destroy the DNA-binding activities, the C-terminal region appears to represent a primary DNA-binding domain of RepE for both ori2 and the operator. Analogous DNA-binding domains seem to be conserved among the initiator proteins of certain related plasmids. PMID:7721691

  17. Combined IL-12 Plasmid and Recombinant SjGST Enhance the Protective and Anti-pathology Effect of SjGST DNA Vaccine Against Schistosoma japonicum.

    PubMed

    Cheng, Po-Ching; Lin, Ching-Nan; Peng, Shih-Yi; Kang, Tsung-Fu; Lee, Kin-Mu

    2016-02-01

    Schistosomiasis is listed as one of most important tropical diseases and more than 200 million people are estimated to be infected. Development of a vaccine is thought to be the most effective way to control this disease. Recombinant 26-kDa glutathione S-transferase (rSjGST) has previously been reported to achieve a worm reduction rate of 42-44%. To improve the efficiency of the vaccine against Schistosoma japonicum, we immunized mice with a combination of pcDNA vector-encoded 26-kDa SjGST (pcDNA/SjGST), IL-12 expressing-plasmid (pIL-12), and rSjGST. Co-vaccination with pcDNA/SjGST, pIL-12, and rSjGST led to a reduction in worm burden, hepatic egg burden, and the size of liver tissue granulomas than that in the untreated infection controls. In addition, we detected high levels of specific IgG, IgG1, and IgG2a against the rSjGST antigen in infected mice vaccinated with this combination of pcDNA/SjGST, pIL-12, and rSjGST. Moreover, high expression levels of Th2 cytokines, including IL-4 and IL-10, were also detected in this group, without diminished levels of IL-12, INF-γ, and TNF-α cytokines that are related to parasite killing. In conclusion, we have developed a new vaccination regimen against S. japonicum infection and shown that co-immunization with pcDNA/SjGST vaccine, pIL-12, and rSjGST has significant anti-parasite, anti-hepatic egg and anti-pathology effects in mice. The efficacy of this vaccination method should be further validated in large animals such as water buffalo. This method may help to reduce the transmission of zoonotic schistosomiasis japonica.

  18. Combined IL-12 Plasmid and Recombinant SjGST Enhance the Protective and Anti-pathology Effect of SjGST DNA Vaccine Against Schistosoma japonicum

    PubMed Central

    Cheng, Po-Ching; Lin, Ching-Nan; Peng, Shih-Yi; Kang, Tsung-Fu; Lee, Kin-Mu

    2016-01-01

    Schistosomiasis is listed as one of most important tropical diseases and more than 200 million people are estimated to be infected. Development of a vaccine is thought to be the most effective way to control this disease. Recombinant 26-kDa glutathione S-transferase (rSjGST) has previously been reported to achieve a worm reduction rate of 42–44%. To improve the efficiency of the vaccine against Schistosoma japonicum, we immunized mice with a combination of pcDNA vector-encoded 26-kDa SjGST (pcDNA/SjGST), IL-12 expressing-plasmid (pIL-12), and rSjGST. Co-vaccination with pcDNA/SjGST, pIL-12, and rSjGST led to a reduction in worm burden, hepatic egg burden, and the size of liver tissue granulomas than that in the untreated infection controls. In addition, we detected high levels of specific IgG, IgG1, and IgG2a against the rSjGST antigen in infected mice vaccinated with this combination of pcDNA/SjGST, pIL-12, and rSjGST. Moreover, high expression levels of Th2 cytokines, including IL-4 and IL-10, were also detected in this group, without diminished levels of IL-12, INF-γ, and TNF-α cytokines that are related to parasite killing. In conclusion, we have developed a new vaccination regimen against S. japonicum infection and shown that co-immunization with pcDNA/SjGST vaccine, pIL-12, and rSjGST has significant anti-parasite, anti-hepatic egg and anti-pathology effects in mice. The efficacy of this vaccination method should be further validated in large animals such as water buffalo. This method may help to reduce the transmission of zoonotic schistosomiasis japonica. PMID:26891172

  19. Coimmunization with an optimized IL15 plasmid adjuvant enhances humoral immunity via stimulating B cells induced by genetically engineered DNA vaccines expressing consensus JEV and WNV E DIII.

    PubMed

    Ramanathan, Mathura P; Kutzler, Michele A; Kuo, Yuan-Chia; Yan, Jian; Liu, Harrison; Shah, Vidhi; Bawa, Amrit; Selling, Bernard; Sardesai, Niranjan Y; Kim, J Joseph; Weiner, David B

    2009-07-09

    The Japanese encephalitis virus (JEV) and West Nile virus (WNV) are responsible for a large proportion of viral encephalitis in humans. Currently, there is no FDA approved specific treatment for either, though there are attempts to develop vaccines against both viruses. In this study, we proposed novel genetically engineered DNA vaccines against these two neurotrophic flaviviruses. The structural domain III (DIII) of E protein from these viruses is reported to carry dominant epitopes that induce neutralizing antibodies. Therefore we created consensus sequence of DIII domain across numerous strains of JEV and WNV. Based on the consensus amino acid sequence, synthetic codon and RNA optimized DIII-expressing DNA vaccine constructs with an efficient leader sequence were synthesized for immunization studies. In addition, we also constructed a genetically engineered IL15 DNA vaccine molecular adjuvant for co-stimulating the immune response against DIII clones. Vaccine constructs were delivered into BALB/C mice intramuscularly followed by electroporation using the CELLECTRA in vivo electroporator. We have observed that the combined delivery of both WNV DIII and IL15-ECRO DNA vaccine constructs resulted in not only the highest level of antibody against DIII, but also enhanced cross reactivity with two other antigens tested. Also, coimmunization with IL15 plasmid further increased the immune response by four- to five-fold. Importantly, we have shown that IL15 coimmunization adjuvanted humoral responses against DIII antigens by elevating the level of antibody secreting B cells. Such a DNA vaccine approach may better help to control potential travel related infectious agents such as JEV.

  20. Plasmid pSM19035, a model to study stable maintenance in Firmicutes.

    PubMed

    Lioy, Virginia S; Pratto, Florencia; de la Hoz, Ana B; Ayora, Silvia; Alonso, Juan C

    2010-07-01

    pSM19035 is a low-copy-number theta-replicating plasmid, which belongs to the Inc18 family. Plasmids of this family, which show a modular organization, are functional in evolutionarily diverse bacterial species of the Firmicutes Phylum. This review summarizes our understanding, accumulated during the last 20 years, on the genetics, biochemistry, cytology and physiology of the five pSM19035 segregation (seg) loci, which map outside of the minimal replicon. The segA locus plays a role both in maximizing plasmid random segregation, and in avoiding replication fork collapses in those plasmids with long inverted repeated regions. The segB1 locus, which acts as the ultimate determinant of plasmid maintenance, encodes a short-lived epsilon(2) antitoxin protein and a long-lived zeta toxin protein, which form a complex that neutralizes zeta toxicity. The cells that do not receive a copy of the plasmid halt their proliferation upon decay of the epsilon(2) antitoxin. The segB2 locus, which encodes two trans-acting, ParA- and ParB-like proteins and six cis-acting parS centromeres, actively ensures equal or roughly equal distribution of plasmid copies to daughter cells. The segC locus includes functions that promote the shift from the use of DNA polymerase I to the replicase (PolC-PolE DNA polymerases). The segD locus, which encodes a trans-acting transcriptional repressor, omega(2), and six cis-acting cognate sites, coordinates the expression of genes that control copy number, better-than-random segregation and partition, and assures the proper balance of these different functions. Working in concert the five different loci achieve almost absolute plasmid maintenance with a minimal growth penalty.

  1. How does the spacer length of cationic gemini lipids influence the lipoplex formation with plasmid DNA? Physicochemical and biochemical characterizations and their relevance in gene therapy.

    PubMed

    Muñoz-Úbeda, Mónica; Misra, Santosh K; Barrán-Berdón, Ana L; Datta, Sougata; Aicart-Ramos, Clara; Castro-Hartmann, Pablo; Kondaiah, Paturu; Junquera, Elena; Bhattacharya, Santanu; Aicart, Emilio

    2012-12-10

    Lipoplexes formed by the pEGFP-C3 plasmid DNA (pDNA) and lipid mixtures containing cationic gemini surfactant of the 1,2-bis(hexadecyl dimethyl ammonium) alkanes family referred to as C16CnC16, where n=2, 3, 5, or 12, and the zwitterionic helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) have been studied from a wide variety of physical, chemical, and biological standpoints. The study has been carried out using several experimental methods, such as zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), cryo-TEM, gene transfection, cell viability/cytotoxicity, and confocal fluorescence microscopy. As reported recently in a communication (J. Am. Chem. Soc. 2011, 133, 18014), the detailed physicochemical and biological studies confirm that, in the presence of the studied series lipid mixtures, plasmid DNA is compacted with a large number of its associated Na+ counterions. This in turn yields a much lower effective negative charge, qpDNA−, a value that has been experimentally obtained for each mixed lipid mixture. Consequently, the cationic lipid (CL) complexes prepared with pDNA and CL/DOPE mixtures to be used in gene transfection require significantly less amount of CL than the one estimated assuming a value of qDNA−=−2. This drives to a considerably lower cytotoxicity of the gene vector. Depending on the CL molar composition, α, of the lipid mixture, and the effective charge ratio of the lipoplex, ρeff, the reported SAXS data indicate the presence of two or three structures in the same lipoplex, one in the DOPE-rich region, other in the CL-rich region, and another one present at any CL composition. Cryo-TEMand SAXS studies with C16CnC16/DOPE-pDNA lipoplexes indicate that pDNA is localized between the mixed lipid bilayers of lamellar structures within a monolayer of ∼2 nm. This is consistent with a highly compacted supercoiled pDNA conformation compared with that of linear DNA. Transfection studies were carried out

  2. Plasmid-encoded trimethoprim resistance in staphylococci.

    PubMed Central

    Archer, G L; Coughter, J P; Johnston, J L

    1986-01-01

    High-level (greater than 1,000 micrograms/ml) resistance to the antimicrobial agent trimethoprim was found in 17 of 101 (17%) coagulase-negative staphylococci and 5 of 51 (10%) Staphylococcus aureus from a number of different hospitals in the United States. Resistance was plasmid encoded and could be transferred by conjugation in 4 of the 17 (24%) Tpr coagulase-negative staphylococci and 3 of the 5 (60%) Tpr S. aureus. A 1.2-kilobase segment of plasmid DNA from one of the plasmids (pG01) was cloned on a high-copy-number vector in Escherichia coli and expressed high-level Tpr (MIC, 1,025 micrograms/ml) in the gram-negative host. In situ filter hybridization demonstrated homology between the cloned Tpr gene probe and plasmid DNA from each conjugative Tpr plasmid, a single nonconjugative plasmid from a United States Staphylococcus epidermidis isolate, a nonconjugative plasmid from an Australian methicillin-resistant S. aureus isolate, and chromosomal DNA from three Tpr S. epidermidis isolates that did not contain any plasmid DNA that was homologous with the probe. No homology was seen between the probe and staphylococcal plasmids not mediating Tpr, plasmid DNA from 12 Tpr S. epidermidis isolates not transferring Tpr by conjugation, or plasmid-encoded Tpr genes derived from gram-negative bacteria. Plasmid-encoded Tpr appears to be a relatively new gene in staphylococci and, because it can be transferred by conjugation, could become more prevalent in nonsocomial isolates. Images PMID:3729338

  3. Plasmid DNA immunization with Trypanosoma cruzi genes induces cardiac and clinical protection against Chagas disease in the canine model

    PubMed Central

    2012-01-01

    The only existing preventive measure against American trypanosomosis, or Chagas disease, is the control of the transmitting insect, which has only been effective in a few South American regions. Currently, there is no vaccine available to prevent this disease. Here, we present the clinical and cardiac levels of protection induced by expression to Trypanosoma cruzi genes encoding the TcSP and TcSSP4 proteins in the canine model. Physical examination, diagnostic chagasic serology, and serial electrocardiograms were performed before and after immunization, as well as after experimental infection. We found that immunization with recombinant plasmids prevented hyperthermia in the acute phase of experimental infection and produced lymphadenomegaly as an immunological response against the parasite and additionally prevented heart rate elevation (tachycardia) in the acute and/or chronic stages of infection. Immunization with T. cruzi genes encoding the TcSP and TcSSP4 antigens diminished the quality and quantity of the electrocardiographic abnormalities, thereby avoiding progression to more severe developments such as right bundle branch block or ventricular premature complexes in a greater number of dogs. PMID:23148870

  4. Safety Profile of the Merck Human Immunodeficiency Virus-1 Clade B gag DNA Plasmid Vaccine With and Without Adjuvants.

    PubMed

    Quirk, Erin K; Brown, Elizabeth L; Leavitt, Randi Y; Mogg, Robin; Mehrotra, Devan V; Evans, Robert K; DiNubile, Mark J; Robertson, Michael N

    2014-03-01

    The immunogenicity results from 3 phase I trials of the Merck DNA human immunodeficiency virus (HIV) vaccine have previously been reported. Because preventive DNA vaccine strategies continue to be leveraged for diverse infections, the safety and tolerability results from these studies can inform the field moving forward, particularly regarding adverse reactions and adjuvants. No serious vaccine-related adverse events were reported during the 3-dose priming phase. Pain at the injection site was more common with adjuvanted formulations than with the phosphate-buffered saline diluent alone. Febrile reactions were usually low grade. Although the AlPO4 or CRL1005 adjuvants used in these studies did not significantly enhance the immunogenicity of the DNA vaccine, adverse events were numerically more common with adjuvanted formulations than without adjuvants.

  5. A novel type of self-assembled nanoparticles as targeted gene carriers: an application for plasmid DNA and antimicroRNA oligonucleotide delivery

    PubMed Central

    Zhu, Yanliang; Liang, Gaofeng; Sun, Bo; Tian, Tian; Hu, Feihu; Xiao, Zhongdang

    2016-01-01

    In this study, a new type of amphiphilic cetylated polyethyleneimine (PEI) was synthesized, and then polylactic-co-glycolic acid (PLGA)/cetylated PEI/hyaluronic acid nanoparticles (PCPH NPs) were developed by self-assembly as a novel type of gene-delivering vehicle. The PCPH NPs showed good DNA-condensation ability by forming polyplexes with small particle size and positive zeta potential. The transfection efficiency and cytotoxicity of PCPH NPs were evaluated as plasmid DNA vectors to transfect HepG2 in vitro. PCPH NPs exhibited much lower cytotoxicity and higher gene-transfection efficiency than PEI (25,000) and commercial transfection reagents. Furthermore, PCPH NPs were used as an anti-miR-221 vector for transfecting HepG2 cells, and anti-miR-221 was effectively transfected into cells and produced a greater inhibitory effect on cancer-cell growth by PCPH NPs. These results demonstrate that PCPH NPs can be a promising nonviral vector for gene-delivery systems. PMID:26869785

  6. Prime-boost vaccination with plasmid DNA followed by recombinant vaccinia virus expressing BgGARP induced a partial protective immunity to inhibit Babesia gibsoni proliferation in dogs.

    PubMed

    Cao, Shinuo; Mousa, Ahmed Abdelmoniem; Aboge, Gabriel Oluga; Kamyingkird, Ketsarin; Zhou, Mo; Moumouni, Paul Franck Adjou; Terkawi, Mohamad Alaa; Masatani, Tatsunori; Nishikawa, Yoshifumi; Suzuki, Hiroshi; Fukumoto, Shinya; Xuan, Xuenan

    2013-12-01

    A heterologous prime-boost vaccination regime with DNA and recombinant vaccinia virus (rvv) vectors expressing relevant antigens has been shown to induce effective immune responses against several infectious pathogens. In this study, we describe the effectiveness of the prime-boost strategy by immunizing dogs with a recombinant plasmid followed by vaccinia virus, both of which expressed the glutamic acid-rich protein (BgGARP) of Babesia gibsoni. The dogs immunized with the prime-boost regime developed a significantly high level of specific antibodies against BgGARP when compared with the control groups. The antibody level was strongly increased after a booster immunization with a recombinant vaccinia virus. Two weeks after the booster immunization with a recombinant vaccinia virus expressing BgGARP, the dogs were challenged with B. gibsoni parasite. The dogs immunized with the prime-boost regime showed partial protection, manifested as a significantly low level of parasitemia. These results indicated that this type of DNA/rvv prime-boost immunization approach may have use against B. gibsoni infection in dogs.

  7. Identification by DNA sequence analysis of a new plasmid-encoded trimethoprim resistance gene in fecal Escherichia coli isolates from children in day-care centers.

    PubMed Central

    Singh, K V; Reves, R R; Pickering, L K; Murray, B E

    1992-01-01

    In our ongoing studies of trimethoprim resistance (Tmpr) in day-care centers (DCC), we have shown a high rate of fecal colonization with Tmpr Escherichia coli and, using total plasmid content analysis, have shown that this is due to a diversity of strains. In the present study, we analyzed 367 highly Tmpr (MIC, greater than or equal to 2,000 micrograms/ml) isolates of E. coli from 72 children over a 5-month period and found at least 83 distinct plasmid patterns, indicating that at least 83 strains were involved. Several strains were particularly common in a given DCC, including one found in 61% of children with Tmpr E. coli; these common strains usually persisted within a DCC for several months. Colony lysates were hybridized with gene probes for dihydrofolate reductases (DHFR) types I, II, III, V, and VII; 21% hybridized under stringent conditions, and all of these were with type I (17%) or type V (4%) probes. Tmpr was cloned from a probe-negative Tmpr transconjugant, and an intragenic probe was prepared from this clone. Approximately 21% of the Tmpr E. coli strains (76 isolates) in the DCC were found to have this new gene, 74 of which were in one DCC. The DNA sequence of this gene was determined, and the predicted amino acid sequence was shown to have between 32% and 39% identity with the amino acid sequences for types I, III, V, VI, and VII and the partial sequence of type IV and approximately 26% identity with types IX and X DHFR. This confirms the uniqueness of this gene, which has tentatively been named dhfrxii, and its translation product, DHFR type XII. Images PMID:1416855

  8. A mouse strain defective in both T cells and NK cells has enhanced sensitivity to tumor induction by plasmid DNA expressing both activated H-Ras and c-Myc.

    PubMed

    Sheng-Fowler, Li; Tu, Wei; Fu, Haiqing; Murata, Haruhiko; Lanning, Lynda; Foseh, Gideon; Macauley, Juliete; Blair, Donald; Hughes, Stephen H; Coffin, John M; Lewis, Andrew M; Peden, Keith

    2014-01-01

    As part of safety studies to evaluate the risk of residual cellular DNA in vaccines manufactured in tumorigenic cells, we have been developing in vivo assays to detect and quantify the oncogenic activity of DNA. We generated a plasmid expressing both an activated human H-ras gene and murine c-myc gene and showed that 1 µg of this plasmid, pMSV-T24-H-ras/MSV-c-myc, was capable of inducing tumors in newborn NIH Swiss mice. However, to be able to detect the oncogenicity of dominant activated oncogenes in cellular DNA, a more sensitive system was needed. In this paper, we demonstrate that the newborn CD3 epsilon transgenic mouse, which is defective in both T-cell and NK-cell functions, can detect the oncogenic activity of 25 ng of the circular form of pMSV-T24-H-ras/MSV-c-myc. When this plasmid was inoculated as linear DNA, amounts of DNA as low as 800 pg were capable of inducing tumors. Animals were found that had multiple tumors, and these tumors were independent and likely clonal. These results demonstrate that the newborn CD3 epsilon mouse is highly sensitive for the detection of oncogenic activity of DNA. To determine whether it can detect the oncogenic activity of cellular DNA derived from four human tumor-cell lines (HeLa, A549, HT-1080, and CEM), DNA (100 µg) was inoculated into newborn CD3 epsilon mice both in the presence of 1 µg of linear pMSV-T24-H-ras/MSV-c-myc as positive control and in its absence. While tumors were induced in 100% of mice with the positive-control plasmid, no tumors were induced in mice receiving any of the tumor DNAs alone. These results demonstrate that detection of oncogenes in cellular DNA derived from four human tumor-derived cell lines in this mouse system was not possible; the results also show the importance of including a positive-control plasmid to detect inhibitory effects of the cellular DNA.

  9. Survey of plasmids in various mycoplasmas.

    PubMed Central

    Harasawa, R.; Barile, M. F.

    1983-01-01

    Thirty-three strains representing 15 distinct Mycoplasma, Acholeplasma, and Spiroplasma species were examined for the presence of plasmid DNA by agarose gel electrophoresis. The electrophoretic patterns of the DNAs of three strains, Mycoplasma sp. strain 747, Spiroplasma mirum strain SMCA, and M. hominis strain 1257, suggested the presence of a plasmid with molecular weights of approximately 70, 10, and 9 megadaltons, respectively. The functions of these plasmids are currently unknown. Images FIG. 1 PMID:6679154

  10. The ratio of single- to double-strand DNA breaks and their absolute values determine cell death pathway

    PubMed Central

    Tounekti, O; Kenani, A; Foray, N; Orlowski, S; Mir, L M

    2001-01-01

    Bleomycin is a cytotoxic antibiotic that generates DNA double-strand breaks (DSB) and DNA single-strand breaks (SSB). It is possible to introduce known quantities of bleomycin molecules into cells. Low amounts kill the cells by a slow process termed mitotic cell death, while high amounts produce a fast process that has been termed pseudoapoptosis. We previously showed that these types of cell death are a direct consequence of the DSB generated by bleomycin. Here, we use deglyco-bleomycin, a bleomycin derivative lacking the carbohydrate moiety. Although this molecule performs the same nucleophilic attacks on DNA as bleomycin, we show that deglyco-bleomycin is at least 100 times less toxic to Chinese hamster fibroblasts than bleomycin. In fact, deglyco-bleomycin treatment results in apoptosis induction. In contrast, however, deglyco-bleomycin was found to generate almost exclusively SSB. Our results suggest that more than 150 000 SSB per cell are required to trigger apoptosis in Chinese hamster fibroblasts and that SSB are 300 times less toxic than DSB. Taken together with previous studies on bleomycin, our data demonstrates that cells can die by apoptosis, mitotic cell death, or pseudoapoptosis, depending on the number of DNA breaks and on the ratio of SSB to DSB. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11336481

  11. Inducing humoral and cellular responses to multiple sporozoite and liver-stage malaria antigens using exogenous plasmid DNA.

    PubMed

    Ferraro, B; Talbott, K T; Balakrishnan, A; Cisper, N; Morrow, M P; Hutnick, N A; Myles, D J; Shedlock, D J; Obeng-Adjei, N; Yan, J; Kayatani, A K K; Richie, N; Cabrera, W; Shiver, R; Khan, A S; Brown, A S; Yang, M; Wille-Reece, U; Birkett, A J; Sardesai, N Y; Weiner, D B

    2013-10-01

    A vaccine candidate that elicits humoral and cellular responses to multiple sporozoite and liver-stage antigens may be able to confer protection against Plasmodium falciparum malaria; however, a technology for formulating and delivering such a vaccine has remained elusive. Here, we report the preclinical assessment of an optimized DNA vaccine approach that targets four P. falciparum antigens: circumsporozoite protein (CSP), liver stage antigen 1 (LSA1), thrombospondin-related anonymous protein (TRAP), and cell-traversal protein for ookinetes and sporozoites (CelTOS). Synthetic DNA sequences were designed for each antigen with modifications to improve expression and were delivered using in vivo electroporation (EP). Immunogenicity was evaluated in mice and nonhuman primates (NHPs) and assessed by enzyme-linked immunosorbent assay (ELISA), gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay, and flow cytometry. In mice, DNA with EP delivery induced antigen-specific IFN-γ production, as measured by ELISpot assay and IgG seroconversion against all antigens. Sustained production of IFN-γ, interleukin-2, and tumor necrosis factor alpha was elicited in both the CD4(+) and CD8(+) T cell compartments. Furthermore, hepatic CD8(+) lymphocytes produced LSA1-specific IFN-γ. The immune responses conferred to mice by this approach translated to the NHP model, which showed cellular responses by ELISpot assay and intracellular cytokine staining. Notably, antigen-specific CD8(+) granzyme B(+) T cells were observed in NHPs. Collectively, the data demonstrate that delivery of gene sequences by DNA/EP encoding malaria parasite antigens is immunogenic in animal models and can harness both the humoral and cellular arms of the immune system.

  12. Cationic Lipid-Nucleic Acid Complexes for Gene Delivery And Silencing: Pathways And Mechanisms for Plasmid Dna And Sirna

    SciTech Connect

    Ewert, K.K.; Zidovska, A.; Ahmad, A.; Bouxsein, N.F.; Evans, H.M.; McAllister, C.S.; Samuel, C.E.; Safinya, C.R.; /SLAC

    2012-07-17

    Motivated by the promises of gene therapy, there is great interest in developing non-viral lipid-based vectors for therapeutic applications due to their low immunogenicity, low toxicity, ease of production, and the potential of transferring large pieces of DNA into cells. In fact, cationic liposome (CL) based vectors are among the prevalent synthetic carriers of nucleic acids (NAs) currently used in gene therapy clinical trials worldwide. These vectors are studied both for gene delivery with CL-DNA complexes and gene silencing with CL-siRNA (short interfering RNA) complexes. However, their transfection efficiencies and silencing efficiencies remain low compared to those of engineered viral vectors. This reflects the currently poor understanding of transfection-related mechanisms at the molecular and self-assembled levels, including a lack of knowledge about interactions between membranes and double stranded NAs and between CL-NA complexes and cellular components. In this review we describe our recent efforts to improve the mechanistic understanding of transfection by CL-NA complexes, which will help to design optimal lipid-based carriers of DNA and siRNA for therapeutic gene delivery and gene silencing.

  13. DNA immunizations with M2 muscarinic and beta1 adrenergic receptor coding plasmids impair cardiac function in mice.

    PubMed

    Giménez, Luis E D; Hernández, Ciria C Q; Mattos, Elisabete C; Brandão, Izaira Tincani; Olivieri, Bianca; Campelo, Roberto P; Araújo-Jorge, Tânia; Silva, Célio Lopes; Campos de Carvalho, Antônio C; Kurtenbach, Eleonora

    2005-05-01

    Autoimmune mediated myocardial damage is likely to be a pathogenic mechanism for acquired dilated cardiomyopathies. Evidence confirms that autoantibodies that bind to M(2) muscarinic (M(2)AChR) and beta(1) adrenergic receptors (beta(1)AR) are present in idiopathic dilated cardiomyopathy and Chagasic patients' sera. To elucidate the role of these antibodies in cardiac functional impairment, we used a murine model immunized with plasmids encoding the M(2)AChR or beta(1)AR via gene-gun bombardment. Anti-M(2)AChR and beta(1)AR antibodies were detected over the course of 37 weeks. These antibodies were directed to the second extracellular loop (el2) of both receptors and the third intracellular loop (il3) of the M(2)AChR. Peak antibody titers from weeks 2 to 5 against M(2)AChR-el2 and beta(1)AR-el2 as well as elevated titers against M(2)AChR-il3 were detected. Anti-M(2)AChR-il3 and anti-beta(1)AR-el2 antibodies were predominant in IgG1 subclass immunoglobulins, suggesting a T-helper-2 biased lymphocyte response. Heart morphology and function was assessed by echocardiography over the course of 42 weeks. Data showed progressive decrease in left ventricular (LV) wall thickness and LV mass that was mostly evident for beta(1)AR-immunized mice albeit a small change in LV dimensions. Fractional shortening was altered and values of 41%, 37% and 48% were observed at week 42 for the M(2)AChR, beta(1)AR and control groups respectively. In support of autonomic deregulation, a twofold increase in M(2)AChR and a similar decrease in beta(1)AR density were observed in radioligand saturation assays for both experimental groups. Histological analysis revealed myofibril disarray and fibrosis, pointing towards remodeling as a consequence of the long-term presence of anti-receptor antibodies.

  14. Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization.

    PubMed Central

    Raz, E; Tighe, H; Sato, Y; Corr, M; Dudler, J A; Roman, M; Swain, S L; Spiegelberg, H L; Carson, D A

    1996-01-01

    We compared the antigen-specific antibody isotypes and lymphokine secretion by CD4+ T cells in BALB/c mice immunized intradermally with either Escherichia coli beta-galactosidase (beta-gal) or plasmid DNA (pDNA) encoding beta-gal in a cytomegalovirus-based expression vector (pCMV-LacZ). pCMV-LacZ induced mainly IgG2a, whereas beta-gal in saline or alum induced IgG1 and IgE beta-gal-specific antibodies. In addition, splenic CD4+ T helper (Th) cells isolated from pDNA-immunized mice secreted interferon-gamma but not interleukin (IL)-4 and IL-5, whereas Th cells from beta-gal-injected mice secreted IL-4 and IL-5 but not interferon-gamma after in vitro stimulation with antigen. Together these data demonstrate that pDNA immunization induced a T helper type 1 (Th1) response, whereas protein immunization induced a T helper type 2 (Th2) response to the same antigen. Interestingly, priming of mice with pCMV-LacZ prevented IgE antibody formation to a subsequent i.p. beta-gal in alum injection. This effect was antigen-specific, because priming with pCMV-LacZ did not inhibit IgE anti-ovalbumin antibody formation. Most importantly, intradermal immunization with pCMV-LacZ (but not pCMV-OVA) of beta-gal in alum-primed mice caused a 66-75% reduction of the IgE anti-beta-gal titer in 6 weeks. Also, pCMV-LacZ induced specific IgG2a antibody titers and interferon-gamma secretion by Th cells in the beta-gal in alum-primed mice. The data demonstrate that gene immunization induces a Th1 response that dominates over an ongoing protein-induced Th2 response in an antigen-specific manner. This suggests that immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases. PMID:8643542

  15. Plasmid DNA linearization in the antibacterial action of a new fluorescent Ag nanoparticle-paracetamol dimer composite

    NASA Astrophysics Data System (ADS)

    Sahoo, Amaresh Kumar; Sk, Md Palashuddin; Ghosh, Siddhartha Sankar; Chattopadhyay, Arun

    2011-10-01

    Herein, we report the generation of a composite comprised of p-hydroxyacetanilide dimer and Ag nanoparticles (NPs) by reaction of AgNO3 and p-hydroxyacetanilide. The formation of the composite was established by UV-vis, FTIR and NMR spectroscopy, transmission electron microscopy and X-ray diffraction along with substantiation by mass spectrometry. Interestingly, the composite exhibited an emission spectrum with a peak at 435 nm when excited by light of wavelength 320 nm. The composite showed superior antimicrobial activity with respect to its individual components against a wide range of Gram positive and Gram negative bacteria at relatively low concentrations of Ag NPs and at which there was no apparent cytotoxicity against mammalian cells. Our results suggest that the composite strongly interacted with the bacterial cell walls leading to cell bursting. Interestingly, enhancement in the reactive oxygen species (ROS) generation in bacteria was observed in the presence of the composite. It is proposed that the ROS generation led to oxidation of the dimer to N-acetyl-p-benzoquinone imine (NAPQI). The generated NAPQI acted as a DNA gyrase inhibitor causing cell death following linearization of DNA.Herein, we report the generation of a composite comprised of p-hydroxyacetanilide dimer and Ag nanoparticles (NPs) by reaction of AgNO3 and p-hydroxyacetanilide. The formation of the composite was established by UV-vis, FTIR and NMR spectroscopy, transmission electron microscopy and X-ray diffraction along with substantiation by mass spectrometry. Interestingly, the composite exhibited an emission spectrum with a peak at 435 nm when excited by light of wavelength 320 nm. The composite showed superior antimicrobial activity with respect to its individual components against a wide range of Gram positive and Gram negative bacteria at relatively low concentrations of Ag NPs and at which there was no apparent cytotoxicity against mammalian cells. Our results suggest that the

  16. Absolute Zero

    NASA Astrophysics Data System (ADS)

    Donnelly, Russell J.; Sheibley, D.; Belloni, M.; Stamper-Kurn, D.; Vinen, W. F.

    2006-12-01

    Absolute Zero is a two hour PBS special attempting to bring to the general public some of the advances made in 400 years of thermodynamics. It is based on the book “Absolute Zero and the Conquest of Cold” by Tom Shachtman. Absolute Zero will call long-overdue attention to the remarkable strides that have been made in low-temperature physics, a field that has produced 27 Nobel Prizes. It will explore the ongoing interplay between science and technology through historical examples including refrigerators, ice machines, frozen foods, liquid oxygen and nitrogen as well as much colder fluids such as liquid hydrogen and liquid helium. A website has been established to promote the series: www.absolutezerocampaign.org. It contains information on the series, aimed primarily at students at the middle school level. There is a wealth of material here and we hope interested teachers will draw their student’s attention to this website and its substantial contents, which have been carefully vetted for accuracy.

  17. Cyclen Grafted with poly[(Aspartic acid)-co-Lysine]: Preparation, Assembly with Plasmid DNA, and in Vitro Transfection Studies.

    PubMed

    Ma, Chunying; Zhang, Jin; Guo, Liwen; Du, Changguo; Song, Ping; Zhao, Baojing; Li, Ling; Li, Chao; Qiao, Renzhong

    2016-01-04

    Development of safe and effective gene carriers is the key to the success of gene therapy. Nowadays, it is still required to develop new methods to improve nonviral gene delivery efficiency. Herein, copolymers of poly[(aspartic acid)-co-lysine] grafted with cyclen (cyclen-pAL) were designed and evaluated for efficient gene delivery. Two copolymers with different Asp/Lys block ratios were prepared and characterized by NMR and gel permeation chromatography analysis. Agarose gel retardation, circular dichroism, and fluorescent quenching assays showed the strong DNA-binding and protection ability for the title compounds. Atomic force microscopy studies clearly delineated uniform DNA globules with a diameter around 100 nm, induced by cyclen-pAL. By grafting cyclen on Asp, relatively high gene delivery efficiency and low cytotoxicity of the modified copolymers were achieved compared with their parent compounds. The present work might help to develop strategies for design and modification of polypeptide copolymers, which may also be applied to favorable gene expression and delivery.

  18. Phase 1/2 open-label dose-escalation study of plasmid DNA expressing two isoforms of hepatocyte growth factor in patients with painful diabetic peripheral neuropathy.

    PubMed

    Ajroud-Driss, Senda; Christiansen, Mark; Allen, Jeffrey A; Kessler, John A

    2013-06-01

    This study aimed to evaluate the safety and preliminary efficacy of intramuscular injections of plasmid DNA (VM202) expressing two isoforms of hepatocyte growth factor (HGF) in subjects with painful diabetic peripheral neuropathy (PDPN). Twelve patients in three cohorts (4, 8, and 16 mg) received two sets of VM202 injections separated by two weeks. Safety and tolerability were evaluated and the visual analog scale (VAS), the short form McGill questionnaire (SF-MPQ), and the brief pain inventory for patients with diabetic peripheral neuropathy (BPI-DPN) measured pain level throughout 12 months after treatment. No serious adverse events (AEs) were observed. The mean VAS was reduced from baseline by 47.2% (P = 0.002) at 6 months and by 44.1% (P = 0.005) at 12 months after treatment. The VAS scores for the 4, 8, and 16 mg dose cohorts at 6 months follow-up decreased in a dose-responsive manner, by 21% (P = 0.971), 53% (P = 0.014), and 62% (P = 0.001), respectively. The results with the BPI-DPN and SF-MPQ showed patterns similar to the VAS scores. In conclusion, VM202 treatment appeared to be safe, well tolerated, and sufficient to provide long term symptomatic relief and improvement in the quality of life in patients with PDPN.

  19. Preparative fractionation of protein, RNA, and plasmid DNA using centrifugal precipitation chromatography with tubular dialysis membrane inside a convoluted tubing as separation channel.

    PubMed

    Tomanee, Panarat; Hsu, James T; Ito, Yoichiro

    2006-01-01

    Fractionation of clarified E. coli lysate components in bench-scale and preparative-scale centrifugal precipitation chromatography (CPC), using a solution of cationic surfactant cetyltrimethylammonium bromide (CTAB) containing 0.5 M NaCl as precipitant, are compared here. Step gradient of CTAB from 0.50% to 0.16% (w/v) gave a successful fractionation in bench-scale CPC; however, a linear gradient of lower CTAB concentration, 0.20-0% (w/v), was used in the preparative scale and resulted in similar fractionation. The preparative-scale CPC has a superior sample loading capacity by the use of tubular dialysis membrane inside convoluted tubing as the separation channel. In this study, the quantity of the sample loaded into the preparative CPC was about 15 times more than that in the bench scale, and in a single run the preparative CPC could prepare approximately 3 mg of plasmid DNA with about 96% of RNA removed. The higher surface area per length of the separation channel in the preparative CPC was believed to benefit mass transfer of CTAB across the membrane, leading to less CTAB being required in the process.

  20. Synthesis, characterization, in vitro antitumoral investigations and interaction with plasmid pBR322 DNA of R2eddp-platinum(IV) complexes (R = Et, n-Pr).

    PubMed

    Kaluderović, Goran N; Kommera, Harish; Schwieger, Sebastian; Paethanom, Anchan; Kunze, Michael; Schmidt, Harry; Paschke, Reinhard; Steinborn, Dirk

    2009-12-28

    The studies on synthetic, spectroscopic and biological properties of platinum(IV) complexes, [PtCl(4)(R(2)eddp)] (R = Et, 1; n-Pr, 2), containing kappa(2)N,N' bidentate ligands, esters of ethylenediamine-N,N'-di-3-propionic acid (HOOCCH(2)CH(2)NHCH(2)CH(2)NHCH(2)CH(2)COOH, H(2)eddp), are reported. Complexes have been characterized by infrared, (1)H and (13)C NMR spectroscopy and elemental analysis and it was concluded that the coordination of the ligands occurs via nitrogen donor atoms of the ester ligands (R(2)eddp). Cytotoxicity studies were performed for ligand precursors and corresponding platinum(IV) complexes. Although the n-Pr(2)eddp.2HCl itself showed no activity (IC(50) values > 125 microM) in selected cell lines, the activity of complex 2, via apoptotic mode of cell death, has increased significantly for a broad range of cancer cell lines tested in vitro (IC(50) = 8.6-49 microM). As it was found that complexes 1 and 2 are able to interact with pBR322 plasmid DNA, platinum(IV) complexes of this type may act as drugs and pro-drugs.

  1. Plasmid-mediated quinolone resistance.

    PubMed

    Jacoby, George A; Strahilevitz, Jacob; Hooper, David C

    2014-10-01

    Three mechanisms for plasmid-mediated quinolone resistance (PMQR) have been discovered since 1998. Plasmid genes qnrA, qnrB, qnrC, qnrD, qnrS, and qnrVC code for proteins of the pentapeptide repeat family that protects DNA gyrase and topoisomerase IV from quinolone inhibition. The qnr genes appear to have been acquired from chromosomal genes in aquatic bacteria, are usually associated with mobilizing or transposable elements on plasmids, and are often incorporated into sul1-type integrons. The second plasmid-mediated mechanism involves acetylation of quinolones with an appropriate amino nitrogen target by a variant of the common aminoglycoside acetyltransferase AAC(6')-Ib. The third mechanism is enhanced efflux produced by plasmid genes for pumps QepAB and OqxAB. PMQR has been found in clinical and environmental isolates around the world and appears to be spreading. The plasmid-mediated mechanisms provide only low-level resistance that by itself does not exceed the clinical breakpoint for susceptibility but nonetheless facilitates selection of higher-level resistance and makes infection by pathogens containing PMQR harder to treat.

  2. Plasmid-mediated quinolone resistance

    PubMed Central

    Jacoby, George A.; Strahilevitz, Jacob; Hooper, David C.

    2014-01-01

    Summary Three mechanisms for plasmid-mediated quinolone resistance (PMQR) have been discovered since 1998. Plasmid genes qnrA, qnrB, qnrC, qnrD, qnrS, and qnrVC code for proteins of the pentapeptide repeat family that protects DNA gyrase and topoisomerase IV from quinolone inhibition. The qnr genes appear to have been acquired from chromosomal genes in aquatic bacteria, are usually associated with mobilizing or transposable elements on plasmids, and are often incorporated into sul1-type integrons. The second plasmid-mediated mechanism involves acetylation of quinolones with an appropriate amino nitrogen target by a variant of the common aminoglycoside acetyltransferase AAC(6′)-Ib. The third mechanism is enhanced efflux produced by plasmid genes for pumps QepAB and OqxAB. PMQR has been found in clinical and environmental isolates around the world and appears to be spreading. The plasmid-mediated mechanisms provide only low-level resistance that by itself does not exceed the clinical breakpoint for susceptibility but nonetheless facilitates selection of higher-level resistance and makes infection by pathogens containing PMQR harder to treat. PMID:25584197

  3. pLS101 plasmid vector

    DOEpatents

    Lacks, S.A.; Balganesh, T.S.

    1985-02-19

    Disclosed is recombinant plasmid pLS101, consisting essentially of a 2.0 Kb ma1M gene fragment ligated to a 4.4 Kb Tcr 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. 5 figs., 2 tabs.

  4. pLS010 plasmid vector

    SciTech Connect

    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.

  5. Absolute Summ

    NASA Astrophysics Data System (ADS)

    Phillips, Alfred, Jr.

    Summ means the entirety of the multiverse. It seems clear, from the inflation theories of A. Guth and others, that the creation of many universes is plausible. We argue that Absolute cosmological ideas, not unlike those of I. Newton, may be consistent with dynamic multiverse creations. As suggested in W. Heisenberg's uncertainty principle, and with the Anthropic Principle defended by S. Hawking, et al., human consciousness, buttressed by findings of neuroscience, may have to be considered in our models. Predictability, as A. Einstein realized with Invariants and General Relativity, may be required for new ideas to be part of physics. We present here a two postulate model geared to an Absolute Summ. The seedbed of this work is part of Akhnaton's philosophy (see S. Freud, Moses and Monotheism). Most important, however, is that the structure of human consciousness, manifest in Kenya's Rift Valley 200,000 years ago as Homo sapiens, who were the culmination of the six million year co-creation process of Hominins and Nature in Africa, allows us to do the physics that we do. .

  6. Cationized gelatin delivery of a plasmid DNA expressing small interference RNA for VEGF inhibits murine squamous cell carcinoma.

    PubMed

    Matsumoto, Goichi; Kushibiki, Toshihiro; Kinoshita, Yukihiko; Lee, Ushaku; Omi, Yasushi; Kubota, Eiro; Tabata, Yasuhiko

    2006-04-01

    Double-stranded RNA (dsRNA) plays a major role in RNA interference (RNAi), a process in which segments of dsRNA are initially cleaved by the Dicer into shorter segments (21-23 nt) called small interfering RNA (siRNA). These siRNA then specifically target homologous mRNA molecules causing them to be degraded by cellular ribonucleases. RNAi down regulates endogenous gene expression in mammalian cells. Vascular endothelial growth factor (VEGF) is a key molecule in vasculogenesis as well as in angiogenesis. Tumor growth is an angiogenesis-dependent process, and therapeutic strategies aimed at inhibiting angiogenesis are theoretically attractive. To investigate the feasibility of using siRNA for VEGF in the specific knockdown of VEGF mRNA, thereby inhibiting angiogenesis, we have performed experiments with a DNA vector based on a siRNA system that targets VEGF (siVEGF). It almost completely inhibited the expression of three different isoforms (VEGF120, VEGF164 and VEGF188) of VEGF mRNA and the secretion of VEGF protein in mouse squamous cell carcinoma NRS-1 cells. The siVEGF released from cationized gelatin microspheres suppressed tumor growth in vivo. A marked reduction in vascularity accompanied the inhibition of a siVEGF-transfected tumor. Fluorescent microscopic study showed that the complex of siVEGF with cationized gelatin microspheres was still present around the tumor 10 days after injection, while free siVEGF had vanished by that time. siVEGF gene therapy increased the fraction of vessels covered by pericytes and induced expression of angiopoietin-1 by pericytes. These data suggest that cationized-gelatin microspheres containing siVEGF can be used to normalize tumor vasculature and inhibit tumor growth in a NRS-1 squamous cell carcinoma xenograft model.

  7. Antibiotic resistance of vibrio cholerae: special considerations of R-plasmids.

    PubMed

    Kuwahara, S

    1978-09-01

    Studies on the transmission of R plasmid by conjugation between enterobacteria and vibrio or related bacteria were reviewed. The majority of the reports confirmed successful transmission from enterobacteria to Vibrio cholerae and related species, although the transmission frequencies were extremely low and the transmitted R plasmid was very unstable except for thermosensitive kanamycin plasmid and usual R plasmid coexisting with P plasmid. Strains of V. cholerae and Aeromonas liquefaciens as well as A. salmonicida bearing R plasmid were detected in nature. R plasmid was relatively unstable in V. cholerae strains with which transmission of R plasmid to enterobacteria was confirmed. At present, only 3 R plasmids have been obtained from naturally occurring strains of V. cholerae. Although the 2 European plasmids belong to the C incompatibility group with 98 megadalton closed covalent circular DNA molecule, one plasmid belongs to the J group with more than 25 megadalton molecular weight, and no CCC of satelite DNA was detected in bacteria harboring this plasmid.

  8. A novel method of plasmid isolation using laundry detergent.

    PubMed

    Yadav, P; Yadav, A; Garg, V; Datta, T K; Goswami, S L; De, S

    2011-07-01

    Since the discovery of plasmid, various methods have been developed to isolate plasmid DNA. All the methods have one common and important target of isolating plasmid DNA of high quality and quantity in less time. These methods are not completely safe because of use of toxic chemicals compounds. The developed protocol for plasmid extraction is based on the alkaline lysis method of plasmid preparation (extraction atpH 8.0) with slight modifications. Cell lysis reagent sodium dodecyl sulfate is replaced by lipase enzyme present in laundry detergent. A good plasmid preparation can be made, which is well suited for subsequent molecular biology applications. By taking safety measures on count, contaminants like, RNA and protein can be completely avoided with maximized plasmid yield. The resultant plasmid quality and quantity can be well comparable to other prevalent methods.

  9. Rolling-circle replication of bacterial plasmids.

    PubMed Central

    Khan, S A

    1997-01-01

    Many bacterial plasmids replicate by a rolling-circle (RC) mechanism. Their replication properties have many similarities to as well as significant differences from those of single-stranded DNA (ssDNA) coliphages, which also replicate by an RC mechanism. Studies on a large number of RC plasmids have revealed that they fall into several families based on homology in their initiator proteins and leading-strand origins. The leading-strand origins contain distinct sequences that are required for binding and nicking by the Rep proteins. Leading-strand origins also contain domains that are required for the initiation and termination of replication. RC plasmids generate ssDNA intermediates during replication, since their lagging-strand synthesis does not usually initiate until the leading strand has been almost fully synthesized. The leading- and lagging-strand origins are distinct, and the displaced leading-strand DNA is converted to the double-stranded form by using solely the host proteins. The Rep proteins encoded by RC plasmids contain specific domains that are involved in their origin binding and nicking activities. The replication and copy number of RC plasmids, in general, are regulated at the level of synthesis of their Rep proteins, which are usually rate limiting for replication. Some RC Rep proteins are known to be inactivated after supporting one round of replication. A number of in vitro replication systems have been developed for RC plasmids and have provided insight into the mechanism of plasmid RC replication. PMID:9409148

  10. Genetic and functional characterization of a yet-unclassified rhizobial Dtr (DNA-transfer-and-replication) region from a ubiquitous plasmid conjugal system present in Sinorhizobium meliloti, in Sinorhizobium medicae, and in other nonrhizobial Gram-negative bacteria.

    PubMed

    Giusti, María de los Ángeles; Pistorio, Mariano; Lozano, Mauricio J; Tejerizo, Gonzalo A Torres; Salas, María Eugenia; Martini, María Carla; López, José Luis; Draghi, Walter O; Del Papa, María Florencia; Pérez-Mendoza, Daniel; Sanjuán, Juan; Lagares, Antonio

    2012-05-01

    Rhizobia are Gram-negative bacteria that live in soils and associate with leguminous plants to establish nitrogen-fixing symbioses. The ability of these bacteria to undergo horizontal gene transfer (HGT) is thought to be one of the main features to explain both the origin of their symbiotic life-style and the plasticity and dynamics of their genomes. In our laboratory we have previously characterized at the species level the non-pSym plasmid mobilome in Sinorhizobium meliloti, the symbiont of Medicago spp., and have found a high incidence of conjugal activity in many plasmids (Pistorio et al., 2008). In this work we characterized the Dtr (DNA-transfer-and-replic