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Sample records for plasmid dna fragments

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

    Science.gov (United States)

    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. DNA fragmentation in apoptosis

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis. Elegant biochemical work identified the DNA fragmentation factor (DFF) as a major apoptotic endonuclease for DNA fragmentation in vitro. Genetic studies in mice support the importance of DFF in DNA fragmentation and possibly in apoptosis in vivo. Recent work also suggests the existence of additional endonucleases for DNA degradation. Understanding the roles of individual endonucleases in apoptosis, and how they might coordinate to degrade DNA in different tissues during normal development and homeostasis, as well as in various diseased states, will be a major research focus in the near future.

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

    OpenAIRE

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

    1984-01-01

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

  4. DNA fragmentation in spermatozoa

    DEFF Research Database (Denmark)

    Rex, A S; Aagaard, J.; Fedder, J

    2017-01-01

    Sperm DNA Fragmentation has been extensively studied for more than a decade. In the 1940s the uniqueness of the spermatozoa protein complex which stabilizes the DNA was discovered. In the fifties and sixties, the association between unstable chromatin structure and subfertility was investigated....... In the seventies, the impact of induced DNA damage was investigated. In the 1980s the concept of sperm DNA fragmentation as related to infertility was introduced as well as the first DNA fragmentation test: the Sperm Chromatin Structure Assay (SCSA). The terminal deoxynucleotidyl transferase nick end labelling...... (TUNEL) test followed by others was introduced in the nineties. The association between DNA fragmentation in spermatozoa and pregnancy loss has been extensively investigated spurring the need for a therapeutic tool for these patients. This gave rise to an increased interest in the aetiology of DNA damage...

  5. Electroeluting DNA fragments.

    Science.gov (United States)

    Zarzosa-Alvarez, Ana L; Sandoval-Cabrera, Antonio; Torres-Huerta, Ana L; Bermudez-Cruz, Rosa M

    2010-09-05

    Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a previous electrophoresis of the DNA fragments in order to separate the band of interest. Then, this band is excised out from an agarose or acrylamide gel and purified by using either: binding and elution from glass or silica particles, DEAE-cellulose membranes, "crush and soak method", electroelution or very often expensive commercial purification kits. Thus, selecting a method will depend mostly of what is available in the laboratory. The electroelution procedure allows one to purify very clean DNA to be used in a large number of applications (sequencing, radiolabeling, enzymatic restriction, enzymatic modification, cloning etc). This procedure consists in placing DNA band-containing agarose or acrylamide slices into sample wells of the electroeluter, then applying current will make the DNA fragment to leave the agarose and thus be trapped in a cushion salt to be recovered later by ethanol precipitation.

  6. Homology and repair of UV-irradiated plasmid DNA in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Cabrea-Juarez, E.; Setlow, J.K.

    1983-02-01

    UV-irradiated plasmid pNov1 containing a cloned fragment of chromosomal DNA could be repaired by excision, but plasmid p2265 without homology to the chromosome could not. Establishment of pNov1 was more UV resistant in Rec/sup -/ than in Rec/sup +/ cells. 19 references, 2 figures.

  7. Rapid isolation of plasmid DNA by LiCl-ethidium bromide treatment and gel filtration.

    Science.gov (United States)

    Kondo, T; Mukai, M; Kondo, Y

    1991-10-01

    We established a simple and rapid plasmid DNA purification method. Crude plasmid DNA preparations are treated with 4 M LiCl in the presence of 0.6 mg/ml ethidium bromide to precipitate RNA and proteins contained in the DNA preparations. After removal of RNA and protein precipitates, the supernatant is filtered through a Sepharose CL6B column to remove low-molecular-weight contaminants. This procedure takes only 30 min and provides pure plasmid DNA preparations that consist mainly of covalently closed circular plasmid DNA but have no detectable RNA and protein. The purified DNA preparations are susceptible to various six- and four-base-recognition restriction endonucleases, T4 DNA ligase, the Klenow fragment of DNA polymerase I, and T7 and Taq DNA polymerase. Since no special equipment is needed for this purification method, 20 or more samples of microgram to milligram levels can be treated in parallel.

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

    OpenAIRE

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

    1995-01-01

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

  9. Sample displacement chromatography of plasmid DNA isoforms.

    Science.gov (United States)

    Černigoj, Urh; Martinuč, Urška; Cardoso, Sara; Sekirnik, Rok; Krajnc, Nika Lendero; Štrancar, Aleš

    2015-10-02

    Sample displacement chromatography (SDC) is a chromatographic technique that utilises different relative binding affinities of components in a sample mixture and has been widely studied in the context of peptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) or linear isoform. Since displacement is more efficient when mass transfer between stationary and mobile chromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobicities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoform separation was shown to be dependent on column selectivity for individual isoform, column efficiency and on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative mode elution often operate in parallel, therefore negative mode elution additionally influences the efficiency of the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNA homogeneity and a dynamic binding capacity of over 1mg/mL at a relatively low concentration of AS. SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes, and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used. This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, which is compatible with continuous, multicolumn chromatography systems, and could therefore be used to increase productivity of pDNA production in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Production and pharmaceutical formulation of plasmid DNA vaccines

    NARCIS (Netherlands)

    van der Heijden, I.

    2013-01-01

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

  11. Production and pharmaceutical formulation of plasmid DNA vaccines

    NARCIS (Netherlands)

    van der Heijden, I.

    2013-01-01

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

  12. Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent.

    Science.gov (United States)

    Wooddell, Christine I; Subbotin, Vladimir M; Sebestyén, Magdolna G; Griffin, Jacob B; Zhang, Guofeng; Schleef, Martin; Braun, Serge; Huss, Thierry; Wolff, Jon A

    2011-02-01

    Various plasmids were delivered into rodent limb muscles by hydrodynamic limb vein (HLV) injection of naked plasmid DNA (pDNA). Some of the pDNA preparations caused significant muscle necrosis and associated muscle regeneration 3 to 4 days after the injection whereas others caused no muscle damage. Occurrence of muscle damage was independent of plasmid sequence, size, and encoded genes. It was batch dependent and correlated with the quantity of bacterial genomic DNA (gDNA) that copurified with the pDNA. To determine whether such an effect was due to bacterial DNA or simply to fragmented DNA, mice were treated by HLV injection with sheared bacterial or murine gDNA. As little as 20 μg of the large fragments of bacterial gDNA caused muscle damage that morphologically resembled damage caused by the toxic pDNA preparations, whereas murine gDNA caused no damage even at a 10-fold higher dose. Toxicity from the bacterial gDNA was not due to endotoxin and was eliminated by DNase digestion. We conclude that pDNA itself does not cause muscle damage and that purification methods for the preparation of therapeutic pDNA should be optimized for removal of bacterial gDNA.

  13. A one-step miniprep for the isolation of plasmid DNA and lambda phage particles.

    Directory of Open Access Journals (Sweden)

    George Lezin

    Full Text Available Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E. coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.

  14. Isolation of T—DNA flanking plant DNA from T—DNA insertional embryo—lethal mutants of Arabidopsis thaliana by plasmid rescue technique

    Institute of Scientific and Technical Information of China (English)

    YAOXIAOLI; JIANGESUN; 等

    1996-01-01

    Three T-DNA insertional embryonic lethal mutants from NASC(The Nottingham Arabidopsis Stock Center) were first checked with their segregation ratio of abortive and normal seeds and the copy number of T-DNA insertion.The N4081 mutant has a segregation ratio of 1:3.04 in average and one T-DNA insertion site according to our assay.It was therefore chosen for further analysis.To isolate the joint fragment of T-DNA and plant DNA,the plasmid rescue technique was used.pEL-7,one of plasmids from left border of T-DNA,which contained pBR322 was selected from ampicillin plate.The T-DNA fragment of pEL-7 was checked by restriction enzyme analysis and Southern Blot.Restriction analysis confirmed the presence of known sites of EcoRI,PstI and PvuII on it.For confirming the presence of flanking plant DNA in this plasmid,pEL-7 DNA was labeled and hybridized with wild type and mutant plant DNA.The Southern Blot indicated the hybridization band in both of them.Furthermore,the junction of T-DNA/plant DNA was subcloned into bluescript SK+ and sequenced by Applied Biosystem 373A sequencer.The results showed the 822 bp fragment contained a 274 bp sequence,which is 99.6%homolog(273bp/274bp) to Ti plasmid pTi 15955,DNA.The bp of left 25 bp border repeat were also found in the juction of T-DNA and Plant DNA. Taken together,pEL-7 should coutain a joint fragment of T-DNA and flanking plant DNA.This plasmid DNA could be used for the isolation of plant gene,which will be helpful to elucidate the relationship between gene function and plant embryo development.

  15. DNA Studies Using Atomic Force Microscopy: Capabilities for Measurement of Short DNA Fragments

    Directory of Open Access Journals (Sweden)

    Dalong ePang

    2015-01-01

    Full Text Available Short DNA fragments, resulting from ionizing radiation induced DNA double strand breaks (DSBs, or released from cells as a result of physiological processes and circulating in the blood stream, may play important roles in cellular function and potentially in disease diagnosis and early intervention. The size distribution of DNA fragments contribute to knowledge of underlining biological processes. Traditional techniques used in radiation biology for DNA fragment size measurements lack the resolution to quantify short DNA fragments. For the measurement of cell-free circulating DNA (ccfDNA, real time quantitative Polymerase Chain Reaction (q-PCR provides quantification of DNA fragment sizes, concentration and specific gene mutation. A complementary approach, the imaging-based technique using Atomic Force Microscopy (AFM provides direct visualization and measurement of individual DNA fragments. In this review, we summarize and discuss the application of AFM-based measurements of DNA fragment sizes. Imaging of broken plasmid DNA, as a result of exposure to ionizing radiation, as well as ccfDNA in clinical specimens offer an innovative approach for studies of short DNA fragments and their biological functions.

  16. Influenza Plasmid DNA Vaccines: Progress and Prospects.

    Science.gov (United States)

    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.

  17. The pPSU Plasmids for Generating DNA Molecular Weight Markers.

    Science.gov (United States)

    Henrici, Ryan C; Pecen, Turner J; Johnston, James L; Tan, Song

    2017-05-26

    Visualizing nucleic acids by gel electrophoresis is one of the most common techniques in molecular biology, and reference molecular weight markers or ladders are commonly used for size estimation. We have created the pPSU1 & pPSU2 pair of molecular weight marker plasmids which produce both 100 bp and 1 kb DNA ladders when digested with two common restriction enzymes. The 100 bp ladder fragments have been optimized to migrate appropriately on both agarose and native polyacrylamide, unlike many currently available DNA ladders. Sufficient plasmid DNA can be isolated from 100 ml E. coli cultures for the two plasmids to produce 100 bp or 1 kb ladders for 1000 gels. As such, the pPSU1 and pPSU2 plasmids provide reference fragments from 50 to 10000 bp at a fraction of the cost of commercial DNA ladders. The pPSU1 and pPSU2 plasmids are available without licensing restrictions to nonprofit academic users, affording freely available high-quality, low-cost molecular weight standards for molecular biology applications.

  18. 99mTc-Labeled HYNIC-DAPI Causes Plasmid DNA Damage with High Efficiency

    OpenAIRE

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

    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 ca...

  19. DNA polymerase beta can substitute for DNA polymerase I in the initiation of plasmid DNA replication.

    OpenAIRE

    1995-01-01

    We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant ...

  20. Low pressure microfluidic-based DNA fragmentation

    NARCIS (Netherlands)

    Shui, Lingling; Sparreboom, Wouter; Bomer, Johan G.; Jin, Mingliang; Carlen, Edwin; van den Berg, Albert

    2011-01-01

    We report a low-pressure microfluidic deoxyribonucleic acid (DNA) fragmentation device based on a combination of me-chanical hydrodynamic shearing and low temperature sample heating. Conventional DNA fragmentation based on hydrody-namic shearing is capable of achieving fragment lengths (FL) < 10k bp

  1. In vitro assembly of multiple DNA fragments using successive hybridization.

    Science.gov (United States)

    Jiang, Xinglin; Yang, Jianming; Zhang, Haibo; Zou, Huibin; Wang, Cong; Xian, Mo

    2012-01-01

    Construction of recombinant DNA from multiple fragments is widely required in molecular biology, especially for synthetic biology purposes. Here we describe a new method, successive hybridization assembling (SHA) which can rapidly do this in a single reaction in vitro. In SHA, DNA fragments are prepared to overlap one after another, so after simple denaturation-renaturation treatment they hybridize in a successive manner and thereby assemble into a recombinant molecule. In contrast to traditional methods, SHA eliminates the need for restriction enzymes, DNA ligases and recombinases, and is sequence-independent. We first demonstrated its feasibility by constructing plasmids from 4, 6 and 8 fragments with high efficiencies, and then applied it to constructing a customized vector and two artificial pathways. As SHA is robust, easy to use and can tolerate repeat sequences, we expect it to be a powerful tool in synthetic biology.

  2. Separation of plasmid DNA topoisomers by multimodal chromatography.

    Science.gov (United States)

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

    2016-06-15

    The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases. Here, we describe the separation of supercoiled (sc) and open circular (oc) topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid. The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Plasmid DNA Manufacturing for Indirect and Direct Clinical Applications.

    Science.gov (United States)

    Schmeer, Marco; Buchholz, Tatjana; Schleef, Martin

    2017-10-01

    Plasmid DNA is currently gaining increasing importance for clinical research applications in gene therapy and genetic vaccination. For direct gene transfer into humans, good manufacturing practice (GMP)-grade plasmid DNA is mandatory. The same holds true if the drug substance contains a genetically modified cell, for example chimeric antigen receptor (CAR) T cells, where these cells as well as the contained plasmids are used. According to the responsible regulatory agencies, they have to be produced under full GMP. On the other hand, for GMP production of, for example, mRNA or viral vectors (lentiviral vectors, adeno-associated virus vectors, etc.), in many cases, High Quality Grade plasmid DNA is accepted as a starting material. The manufacturing process passes through different production steps. To ensure the right conditions are used for the plasmid, a pilot run must be conducted at the beginning. In this step, a followed upscaling with respect to reproducibility and influences on product quality is performed. Subsequently, a cell bank of the transformed productions strain is established and characterized. This cell bank is used for the cultivation process. After cell harvesting and lysis, several chromatography steps are conducted to receive a pure plasmid product. Depending on the respective required quality grade, the plasmid product is subject to several quality controls. The last step consists of formulation and filling of the product.

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

    Science.gov (United States)

    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

  5. Short-fragment DNA-mediated in vivo DNA electroporation delivery.

    Science.gov (United States)

    Peng, Jinliang; Zhao, Yonggang; Xu, Yuhong

    2014-01-01

    Electroporation is an effective physical delivery method. A variety of factors have been shown to affect the electroporation-mediated gene delivery efficiency. Here we report the usefulness of noncoding short-fragment DNA (sf-DNA) for facilitating electroporation-mediated gene transfer. The plasmid pGL3-control encoding firefly luciferase was injected into tissue together with or without sf-DNA in different length or dose. Immediately after injection, the tissues were electroporated and the level of luciferase activity was assessed 24 h later. The results showed that plasmid DNA formulated with sf-DNA resulted in significant improvement in electroporation-mediated gene transfer efficiency. The effect is dose and length dependent, and also found in low-voltage electroporation. These results indicated that sf-DNA can be used as a helper molecule to improve the electroporation-mediated gene transfection efficiency.

  6. Mechanism of DNA Segregation in Prokaryotes: Replicon Pairing by parC of Plasmid R1

    Science.gov (United States)

    Jensen, Rasmus Bugge; Lurz, Rudi; Gerdes, Kenn

    1998-07-01

    Prokaryotic chromosomes and plasmids encode partitioning systems that are required for DNA segregation at cell division. The systems are thought to be functionally analogous to eukaryotic centromeres and to play a general role in DNA segregation. The parA system of plasmid R1 encodes two proteins ParM and ParR, and a cis-acting centromere-like site denoted parC. The ParR protein binds to parC in vivo and in vitro. The ParM protein is an ATPase that interacts with ParR specifically bound to parC. Using electron microscopy, we show here that parC mediates efficient pairing of plasmid molecules. The pairing requires binding of ParR to parC and is stimulated by the ParM ATPase. The ParM mediated stimulation of plasmid pairing is dependent on ATP hydrolysis by ParM. Using a ligation kinetics assay, we find that ParR stimulates ligation of parC-containing DNA fragments. The rate-of-ligation was increased by wild type ParM protein but not by mutant ParM protein deficient in the ATPase activity. Thus, two independent assays show that parC mediates pairing of plasmid molecules in vitro. These results are consistent with the proposal that replicon pairing is part of the mechanism of DNA segregation in prokaryotes.

  7. Functional amyloids as inhibitors of plasmid DNA replication

    Science.gov (United States)

    Molina-García, Laura; Gasset-Rosa, Fátima; Moreno-del Álamo, María; Fernández-Tresguerres, M. Elena; Moreno-Díaz de la Espina, Susana; Lurz, Rudi; Giraldo, Rafael

    2016-01-01

    DNA replication is tightly regulated to constrain the genetic material within strict spatiotemporal boundaries and copy numbers. Bacterial plasmids are autonomously replicating DNA molecules of much clinical, environmental and biotechnological interest. A mechanism used by plasmids to prevent over-replication is ‘handcuffing’, i.e. inactivating the replication origins in two DNA molecules by holding them together through a bridge built by a plasmid-encoded initiator protein (Rep). Besides being involved in handcuffing, the WH1 domain in the RepA protein assembles as amyloid fibres upon binding to DNA in vitro. The amyloid state in proteins is linked to specific human diseases, but determines selectable and epigenetically transmissible phenotypes in microorganisms. Here we have explored the connection between handcuffing and amyloidogenesis of full-length RepA. Using a monoclonal antibody specific for an amyloidogenic conformation of RepA-WH1, we have found that the handcuffed RepA assemblies, either reconstructed in vitro or in plasmids clustering at the bacterial nucleoid, are amyloidogenic. The replication-inhibitory RepA handcuff assembly is, to our knowledge, the first protein amyloid directly dealing with DNA. Built on an amyloid scaffold, bacterial plasmid handcuffs can bring a novel molecular solution to the universal problem of keeping control on DNA replication initiation. PMID:27147472

  8. Efficient transformation of Bacillus thuringiensis requires nonmethylated plasmid DNA.

    OpenAIRE

    Macaluso, A; Mettus, A M

    1991-01-01

    The transformation efficiency of Bacillus thuringiensis depends upon the source of plasmid DNA. DNA isolated from B. thuringiensis, Bacillus megaterium, or a Dam- Dcm- Escherichia coli strain efficiently transformed several B. thuringiensis strains, B. thuringiensis strains were grouped according to which B. thuringiensis backgrounds were suitable sources of DNA for transformation of other B. thuringiensis strains, suggesting that B. thuringiensis strains differ in DNA modification and restri...

  9. Humoral Immune Response Elicited by Plasmid DNA Containing HGV E2 Gene Fragment%含庚型肝炎病毒E2基因片段质粒DNA能诱发相当强烈的体液免疫应答

    Institute of Scientific and Technical Information of China (English)

    Fethia Ben Yebdri; Abderrahmane AAZAZ; 叶凯; 马辉文; 童立恒

    2004-01-01

    研究了庚型肝炎病毒E2(HGV E2)基因片段作为DNA疫苗的可行性.将来自于质粒pThioHis-E2编码HGV E2的基因片段(559bp)亚克隆到质粒pCMV-S中,使之和HBsAg基因位于同一阅读框,形成重组质粒pCMV-S-E2.用纯化的质pCMV-S-E2 DNA注射到昆明小鼠后腿四头肌中来免疫小鼠,同进用pCMV-S作为对照.间隔14天再加强一次免疫.在加强免疫后8天眼眶取血.用E2-GST融合蛋白作为固定化抗原,通过ELISA检测受试小鼠的体液免疫应答.结果表明,用质粒pCMV-S-E DNA免疫的小鼠可以产生很强的体液免疫应答.%In order to study the feasibility of E2 gene fragment of hepatitis virus G(HGV)as a component of DNA vaccine against the hepatitis virus G infection,a 559bp DNA fragment encoding HGV E2 was cloned into plasmid pCMV-S from pThioHis-E2 in the same reading frame with HBsAg gene to form a recombinant plasmid named pCMV-S-E2.BALB/c mice of Kunming strain were immunized with purified plasmid DNA of pCMV-S-E2 by intra-muscularly inoculation.The immunizations were boosted twice at an interval of 14 days.The whole blood was collected from mice orbit on the day-8 after the last boost.Mice sera were screened by ELISA to determine the humoral immune response using E2-GST fusion protein as the immobilized antigen and the sera from mice immunized with pCMVS as control.The result indicated that the immunization with plasmid DNA of pCMV-S-E2 could induce quite strong humoral immune response.

  10. Multiple Pathways of Plasmid DNA Transfer in Helicobacter pylori

    Science.gov (United States)

    Rohrer, Stefanie; Holsten, Lea; Weiss, Evelyn; Benghezal, Mohammed; Fischer, Wolfgang; Haas, Rainer

    2012-01-01

    Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species. PMID:23029142

  11. Cationic lipids delay the transfer of plasmid DNA to lysosomes.

    Science.gov (United States)

    Wattiaux, R; Jadot, M; Laurent, N; Dubois, F; Wattiaux-De Coninck, S

    1996-10-14

    Plasmid 35S DNA, naked or associated with different cationic lipid preparations was injected to rats. Subcellular distribution of radioactivity in the liver one hour after injection, was established by centrifugation methods. Results show that at that time, 35S DNA has reached lysosomes. On the contrary, when 35S DNA was complexed with lipids, radioactivity remains located in organelles whose distribution after differential and isopycnic centrifugation, is clearly distinct from that of arylsulfatase, lysosome marker enzyme. Injection of Triton WR 1339, a specific density perturbant of lysosomes, four days before 35S DNA injection causes a density decrease of radioactivity bearing structures, apparent one hour after naked 35S DNA injection but visible only after more than five hours, when 35S DNA associated with a cationic lipid is injected. These observations show that cationic lipids delay the transfer to lysosomes, of plasmid DNA taken up by the liver.

  12. Liquid-Crystalline Mesophases of Plasmid DNA in Bacteria

    Science.gov (United States)

    Reich, Ziv; Wachtel, Ellen J.; Minsky, Abraham

    1994-06-01

    Bacterial plasmids may often reach a copy number larger than 1000 per cell, corresponding to a total amount of DNA that may exceed the amount of DNA within the bacterial chromosome. This observation highlights the problem of cellular accommodation of large amounts of closed-circular nucleic acids, whose interwound conformation offers negligible DNA compaction. As determined by x-ray scattering experiments conducted on intact bacteria, supercoiled plasmids segregate within the cells into dense clusters characterized by a long-range order. In vitro studies performed at physiological DNA concentrations indicated that interwound DNA spontaneously forms liquid crystalline phases whose macroscopic structural properties are determined by the features of the molecular supercoiling. Because these features respond to cellular factors, DNA supercoiling may provide a sensitive regulatory link between cellular parameters and the packaging modes of interwound DNA in vivo.

  13. Fragment Length of Circulating Tumor DNA.

    Directory of Open Access Journals (Sweden)

    Hunter R Underhill

    2016-07-01

    Full Text Available Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134-144 bp vs. 167 bp, respectively. Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132-145 bp vs. 165 bp, respectively. Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA.

  14. Fragment Length of Circulating Tumor DNA.

    Science.gov (United States)

    Underhill, Hunter R; Kitzman, Jacob O; Hellwig, Sabine; Welker, Noah C; Daza, Riza; Baker, Daniel N; Gligorich, Keith M; Rostomily, Robert C; Bronner, Mary P; Shendure, Jay

    2016-07-01

    Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134-144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132-145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA.

  15. [Chromatographic separation of plasmid DNA by anion-exchange cryogel].

    Science.gov (United States)

    Guo, Yantao; Shen, Shaochuan; Yun, Junxian; Yao, Kejian

    2012-08-01

    Plasmid DNA (pDNA) is used as an important vector for gene therapy, and its wide application is restricted by the purity and yield. To obtain high-purity pDNA, a chromatographic method based on anion-exchange supermacroporous cryogel was explored. The anion-exchange cryogel was prepared by grafting diethylaminoethyl-dextran to the epoxide groups of polyacrylamide-based matrix and pUC19 plasmid was used as a target to test the method. The plasmid was transferred into Escherichia coli DH5alpha, cultivated, harvested and lysed. The obtained culture was centrifuged and the supernatant was used as the plasmid feedstock, which was loaded into the anion-exchange cryogel bed for chromatographic separation. By optimizing the pH of running buffer and the elution conditions, high-purity pDNA was obtained by elution with 0.5 mol/L sodium chloride solution at pH 6.6. Compared to the traditional methods for purification of pDNA, animal source enzymes and toxic reagents were not involved in the present separation process, ensuring the safety of both the purification operations and the obtained pDNA.

  16. Persistence of plasmid DNA in different soils

    African Journals Online (AJOL)

    STORAGESEVER

    2008-08-04

    Aug 4, 2008 ... ... transformation in bacteria (Davison, 1999), binding of DNA from Bacillus subtilis on clay mineral montmorillonite, and the ability of ... soil was taken and soil extract was prepared with sterile water the DNA was isolated and.

  17. Cyclization of short DNA fragments

    Science.gov (United States)

    Lam, Pui-Man; Zhen, Yi

    2017-09-01

    From the per unit length free energy for DNA under tension, we have calculated an effective contour length dependent persistence length for short DNA. This effective persistence length results from the enhanced fluctuations in short DNA. It decreases for shorter DNA, making shorter DNA more flexible. The results of the J-factor calculated using this effective persistence length are in good agreement with experimental data.

  18. Pharmaceutical development of the plasmid DNA vaccine pDERMATT

    NARCIS (Netherlands)

    Quaak, S.G.L.

    2009-01-01

    The discovery of tumor specific antigens and self tolerance mechanisms against these antigens led to the assumption that antigens circulating at sufficient concentration levels could break this self tolerance mechanism and evoke immunological antitumor effects. pDERMATT (plasmid DNA encoding recombi

  19. Pharmaceutical development of the plasmid DNA vaccine pDERMATT

    NARCIS (Netherlands)

    Quaak, S.G.L.

    2009-01-01

    The discovery of tumor specific antigens and self tolerance mechanisms against these antigens led to the assumption that antigens circulating at sufficient concentration levels could break this self tolerance mechanism and evoke immunological antitumor effects. pDERMATT (plasmid DNA encoding

  20. Use of plasmid DNA for induction of protective immunity

    DEFF Research Database (Denmark)

    Lorenzen, Niels

    2004-01-01

    Vaccines based on plasmid DNA have been tested for a number of fish pathogens but so far it is only in case of the rhabdoviruses, where the technology has been a real break through in vaccine research. Aspects of dose, time-course and mechanisms of protection, as well as practical use are discussed....

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

    Science.gov (United States)

    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.

  2. DNA fragmentation status in patients with necrozoospermia.

    Science.gov (United States)

    Brahem, Sonia; Jellad, Sonia; Ibala, Samira; Saad, Ali; Mehdi, Meriem

    2012-12-01

    The aim of this study was to determine if a relationship exists between the levels of sperm DNA fragmentation and necrospermia in infertile men. Semen samples obtained from 70 men consulting for infertility evaluation were analyzed according to World Health Organization (WHO) guidelines. Patients were subdivided into three groups according to the percentage of necrotic spermatozoa: normozoospermia (80%; n = 20). DNA fragmentation was detected by the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL) assay. The sperm DNA fragmentation index (DFI) was 9.28 ± 2.98% in patients with a normal level of necrotic spermatozoa, 20.25 ± 3.21% in patients with moderate necrozoospermia, and 35.31 ± 5.25% in patients with severe necrozoospermia. There was a statistically significant increase of DNA fragmentation in the necrozoospermic group (P DNA fragmentation. We concluded that patients with necrozoospermia showed a high level of DNA fragmentation compared to normozoospermic men. Severe necrozoospermia (>80%) is a predictive factor for increased sperm DNA damage.

  3. DNA fragmentation in spermatozoa: a historical review.

    Science.gov (United States)

    Rex, A S; Aagaard, J; Fedder, J

    2017-07-01

    Sperm DNA Fragmentation has been extensively studied for more than a decade. In the 1940s the uniqueness of the spermatozoa protein complex which stabilizes the DNA was discovered. In the fifties and sixties, the association between unstable chromatin structure and subfertility was investigated. In the seventies, the impact of induced DNA damage was investigated. In the 1980s the concept of sperm DNA fragmentation as related to infertility was introduced as well as the first DNA fragmentation test: the Sperm Chromatin Structure Assay (SCSA). The terminal deoxynucleotidyl transferase nick end labelling (TUNEL) test followed by others was introduced in the nineties. The association between DNA fragmentation in spermatozoa and pregnancy loss has been extensively investigated spurring the need for a therapeutic tool for these patients. This gave rise to an increased interest in the aetiology of DNA damage. The present decade continues within this research area. Some of the more novel methods recently submerging are sorting of cells with increased DNA fragmentation and hyaluronic acid (HA) binding techniques. The clinical value of these tests remains to be elucidated. In spite of half a century of research within the area, this analysis is not routinely implemented into the fertility clinics. The underlying causes are multiple. The abundance of methods has impeded the need for a clinical significant threshold. One of the most promising methods was commercialized in 2005 and has been reserved for larger licensed laboratories. Myriads of reviews and meta-analyses on studies using different assays for analysis of DNA fragmentation, different clinical Artificial Reproductive Treatments (ART), different definitions of successful ART outcome and small patient cohorts have been published. Although the area of DNA fragmentation in spermatozoa is highly relevant in the fertility clinics, the need for further studies focusing on standardization of the methods and clinical

  4. Successfully introduce maize DNA fragments into rice

    Institute of Scientific and Technical Information of China (English)

    WANGKaizhi

    1994-01-01

    The maize DNA fragments was successfully incorporated into rice by Associate Prof WAN Wenju's research team at Hunan Agricultural College, Changsha, China. The new gene transferring rice is named Genetic Engineered Rice (GER) line.

  5. Supramolecular gel electrophoresis of large DNA fragments.

    Science.gov (United States)

    Tazawa, Shohei; Kobayashi, Kazuhiro; Oyoshi, Takanori; Yamanaka, Masamichi

    2017-07-06

    Pulsed-field gel electrophoresis is a frequent technique used to separate exceptionally large DNA fragments. In a typical continuous field electrophoresis, it is challenging to separate DNA fragments larger than 20 kbp because they migrate at a comparable rate. To overcome this challenge, it is necessary to develop a novel matrix for the electrophoresis. Here, we describe the electrophoresis of large DNA fragments up to 166 kbp using a supramolecular gel matrix and a typical continuous field electrophoresis system. C3 -symmetric tris-urea self-assembled into a supramolecular hydrogel in tris-boric acid-EDTA buffer, a typical buffer for DNA electrophoresis, and the supramolecular hydrogel was used as a matrix for electrophoresis to separate large DNA fragments. Three types of DNA marker, the λ-Hind III digest (2 to 23 kbp), Lambda DNA-Mono Cut Mix (10 to 49 kbp), and Marker 7 GT (10 to 165 kbp), were analyzed in this study. Large DNA fragments of greater than 100 kbp showed distinct mobility using a typical continuous field electrophoresis system. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Enhanced brain targeting efficiency of intranasally administered plasmid DNA: an alternative route for brain gene therapy.

    Science.gov (United States)

    Han, In-Kwon; Kim, Mi Young; Byun, Hyang-Min; Hwang, Tae Sun; Kim, Jung Mogg; Hwang, Kwang Woo; Park, Tae Gwan; Jung, Woon-Won; Chun, Taehoon; Jeong, Gil-Jae; Oh, Yu-Kyoung

    2007-01-01

    Recently, nasal administration has been studied as a noninvasive route for delivery of plasmid DNA encoding therapeutic or antigenic genes. Here, we examined the brain targeting efficiency and transport pathways of intranasally administered plasmid DNA. Quantitative polymerase chain reaction (PCR) measurements of plasmid DNA in blood and brain tissues revealed that intranasally administered pCMVbeta (7.2 kb) and pN2/CMVbeta (14.1 kb) showed systemic absorption and brain distribution. Following intranasal administration, the beta-galactosidase protein encoded by these plasmids was significantly expressed in brain tissues. Kinetic studies showed that intranasally administered plasmid DNA reached the brain with a 2,595-fold higher efficiency than intravenously administered plasmid DNA did, 10 min post-dose. Over 1 h post-dose, the brain targeting efficiencies were consistently higher for intranasally administered plasmid DNA than for intravenously administered DNA. To examine how plasmid DNA enters the brain and moves to the various regions, we examined tissues from nine brain regions, at 5 and 10 min after intranasal or intravenous administration of plasmid DNA. Intravenously administered plasmid DNA displayed similar levels of plasmid DNA in the nine different regions, whereas, intranasally administered plasmid DNA exhibited different levels of distribution among the regions, with the highest plasmid DNA levels in the olfactory bulb. Moreover, plasmid DNA was mainly detected in the endothelial cells, but not in glial cells. Our results suggest that intranasally applied plasmid DNA may reach the brain through a direct route, possibly via the olfactory bulb, and that the nasal route might be an alternative method for efficiently delivering plasmid DNA to the brain.

  7. Anion exchange purification of plasmid DNA using expanded bed adsorption.

    Science.gov (United States)

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

    2000-01-01

    Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity of process streams is of major concern in the purification of plasmids, since it can cause high back pressures in column operations, thus limiting the throughput. In order to avoid these high back pressures, expanded bed anion exchange chromatography was evaluated as an alternative to fixed bed chromatography. A Streamline 25 column filled with 100 ml of Streamline QXL media, was equilibrated with 0.5 M NaCl in TE (10 mM Tris, 1 mM EDTA, pH = 8.0) buffer at an upward flow of 300 cmh-1, E. coli lysates (obtained from up to 3 liters of fermentation broth) were injected in the column. After washing out the unbound material, the media was allowed to sediment and the plasmid was eluted with 1 M NaCl in TE buffer at a downward flow of 120 cmh-1. Purification factors of 36 +/- 1 fold, 26 +/- 0.4 plasmid purity, and close to 100% yields were obtained when less than one settled column volume of plasmid feed was injected. However, both recovery yield and purity abruptly decreased when larger amounts were processed-values of 35 +/- 2 and 5 +/- 0.7 were obtained for the recovery yield and purity, respectively, when 250 ml of feedstock were processed. In these cases, gel clogging and expansion collapse were observed. The processing of larger volumes, thus larger plasmid quantities, was only possible by performing an isopropanol precipitation step prior to the chromatographic step. This step led to an enhancement of the purification step.

  8. Using Plasmids as DNA Vaccines for Infectious Diseases.

    Science.gov (United States)

    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.

  9. The effects of 4-MEI on cell proliferation, DNA breaking and DNA fragmentation.

    Science.gov (United States)

    Tazehkand, M Norizadeh; Moridikia, A; Hajipour, O; Valipour, E; Timocin, T; Topaktas, M; Yilmaz, M B

    4-Methylimidazole (4-MEI) is a color widely found in cola drinks, roasted foods, grilled meats, coffee and other foods. This study was aimed to investigate the 4-MEI effects on the cell proliferation, purified circular DNA and DNA from cells of rats treated with the 4-MEI.In this study, mouse 3T3-L1 cell line was treated with 4-MEI at concentrations of 300, 450, 600 and 750 µg/mL for 24 hours and 48 hours periods, after that cytotoxic effect of the 4-MEI was studied by MTT test. Also, the effect of 4-MEI on purified circular DNA (pET22b) was investigated by treating of the DNA with 4-MEI concentrations of 300, 450, 600 and 750 µg/ml. DNA was extracted from liver cells of rats that have been treated with 4-MEI doses of 25 and 50 mg/kg for 10 week and it was subjected to agarose gel electrophoreses analyses.4-MEI significantly inhibited cell proliferation of 3T3-L1 cell line at highest concentration for 24 h and at all concentration for 48 h treatment time. DNA fragmentation assay showed that 4-MEI at 50 mg/kg concentration clearly produced characteristic DNA smear and no DNA laddering (200bp) was observed when mouse was exposed to 4-MEI. The results obtained from plasmid DNA damaging assay showed that 4-MEI has noeffect on the DNA, because the electrophoretic pattern of DNA treated with 4-MEI showed three bands on agarose gel electrophoresis as it was for untreated control. 4-MEI showed cytotoxic effect on 3T3-L1 cells but no effect on plasmid DNA breaking. According to DNA fragmentation assay 4-MEI has necrosis effects on mouse liver cells (Tab. 1, Fig. 4, Ref. 27).

  10. Scientific advice on the suitability of data for the assessment of DNA integration into the fish genome of a genetically modified DNA plasmid-based veterinary vaccine

    Directory of Open Access Journals (Sweden)

    European Food Safety Authority

    2013-05-01

    Full Text Available Pancreas disease caused by salmonid alphavirus in farmed Atlantic salmon (Salmo salar leads to high mortality rates post infection and histopathological lesions in several organs. As protection against pancreas disease, Novartis developed a prophylactic DNA plasmid-based vaccine to be administered to salmon as naked plasmid in a single intramuscular injection. In order to assess the legal status of the fish vaccinated with this new vaccine with regard to the legislation on genetically modified organisms, the European Commission suggested that the company carry out a scientific study on the integration/non-integration of the plasmid DNA into the fish genome. Subsequently, the European Commission requested EFSA to give scientific advice on the study design and the conclusions drawn by the company. PCR based analysis of genomic DNA from muscle samples, taken from at or around the injection site 436 days post vaccination, led the company to conclude that integration of plasmid DNA into the fish genome is extremely unlikely. After an assessment of the study, EFSA considers that the study presented by Novartis Animal Health on the integration/non-integration of DNA plasmid-based vaccine into the salmon genomic DNA provides insufficient information on the potential integration of plasmid DNA fragments into the fish genome due to a limited coverage of the plasmid DNA by the detection method provided, the limited number of samples analysed and an insufficient limit of detection and method validation. Therefore, EFSA is of the opinion that the results from the integration/non-integration study submitted by Novartis Animal Health are not sufficient to support the conclusion of non-integration of plasmid DNA into the fish genome drawn by the company.

  11. Expression and humoral immune response to Hepatitis C virus using a plasmid DNA construct

    Directory of Open Access Journals (Sweden)

    Ray S

    2003-01-01

    Full Text Available PURPOSE: The objective of this study was to clone a c-DNA fragment of hepatitis C virus in a eukaryotic expression vector and to measure the efficacy of humoral immune responses in mice inoculated with this recombinant plasmid. This study was an attempt to lay a foundation for HCV nucleic acid vaccine development in the future. METHODS: A c-DNA fragment of BK146, a clone of HCV type 1b, was sub-cloned into an eukaryotic expression vector pMT3. HepG2 and COS cells were transfected with this construct, named pMT3-BK146. The expression of HCV mRNA and proteins was studied by reverse transcribed polymerase chain reaction, radio Immunoprecipitation (RIPA and immunofluorescence (IFA. The DNA of this construct was injected into the footpad of BALB/c mice and antibody response was tested by enzyme immunoassay and indirect immunofluorescence. RESULTS: COS and HepG2 cells transiently transfected with the recombinant plasmid pMT3-BK146 showed the expression of HCV proteins by RT-PCR, RIPA and immunofluorescence. This DNA clone when injected into Balb/c mice was able to generate specific antibody response to hepatitis C virus by ELISA and IFA. CONCLUSIONS: A c-DNA fragment of HCV cloned in an eukaryotic expression vector was able to express core protein. This DNA clone was also able to elicit antibody response in mice. This can be an initial step towards the development of a potential DNA vaccine for hepatitis C virus infection.

  12. Dataset of plasmid DNA extraction using different magnetic nanoparticles (MNPs

    Directory of Open Access Journals (Sweden)

    H. Rahnama

    2016-12-01

    MNPs were characterized by energy dispersive spectroscopy (EDS and transmission electron microscopy (TEM. Finally, the overall efficiency of different MNPs (Fe3O4, Fe3O4/SiO2, Fe3O4/SiO2/TiO2 in plasmid DNA isolation was compared using gel electrophoresis analysis. The data supplied in this article supports the accompanying publication “Comparative study of three magnetic nano-particles (FeSO4, FeSO4/SiO2, FeSO4/SiO2/TiO2 in plasmid DNA extraction” (H. Rahnama, A. Sattarzadeh, F. Kazemi, N. Ahmadi, F. Sanjarian, Z. Zand, 2016 [1].

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system...

  14. Transformation of Azotobacter vinelandii OP with a broad host range plasmid containing a cloned chromosomal nif-DNA marker.

    Science.gov (United States)

    Bingle, W H

    1988-05-01

    The non-nitrogen-fixing (Nif-) strain UW10 of Azotobacter vinelandii OP (UW) was naturally induced to competence and transformed with broad host range plasmid pKT210 containing the cloned wild-type nif-10 locus from A. vinelandii UW (Nif+); this marker was unable to complement the nif-10 mutation in trans, but could through recombination with the chromosome. The most frequent type of transformation event observed was recombination between the homologous regions of the plasmid and chromosome (producing Nif+ transformants) with loss of the plasmid vector. At a substantially lower frequency, transformants expressing the plasmid-encoded antibiotic resistance determinants were isolated which were phenotypically Nif-. Agarose gel electrophoresis showed that these transformants contained a plasmid migrating with the same mobility as the original donor plasmid. During culture these transformants acquired a Nif+ phenotype without the loss of the plasmid, as judged by the use of a hybridization probe specific for the cloned nif-DNA fragment. These data indicate that plasmids carrying sequences homologous to chromosomal sequences could be maintained in recombination-proficient A. vinelandii UW. The introduction of plasmids containing sequences homologous to chromosomal sequences was facilitated by prelinearization of the plasmid using a restriction endonuclease generating cohesive ends. Because the site of linearization could be chosen outside the region of shared homology, it was unlikely that the route of plasmid establishment occurred via a homology-facilitated transformation mechanism. The data also indicated that A. vinelandii UW could harbor broad host range cloning vectors based on plasmid RSF1010 without significant impairment of its nitrogen-fixation ability.

  15. High yield DNA fragmentation using cyclical hydrodynamic shearing

    NARCIS (Netherlands)

    Shui, Lingling; Sparreboom, Wouter; Spang, Peter; Roeser, Tina; Nieto, Benjamin; Guasch, Francesc; Corbera, Antoni Homs; van den Berg, Albert; Carlen, Edwin

    2013-01-01

    We report a new DNA fragmentation technique that significantly simplifies conventional hydrodynamic shearing fragmentation by eliminating the need for sample recirculation while maintaining high fragmentation yield and low fragment length variation, and therefore, reduces instrument complexity and c

  16. High yield DNA fragmentation using cyclical hydrodynamic shearing

    NARCIS (Netherlands)

    Shui, Lingling; Sparreboom, Wouter; Spang, Peter; Roeser, Tina; Nieto, Benjamin; Guasch, Francesc; Corbera, Antoni Homs; van den Berg, Albert; Carlen, Edwin

    2013-01-01

    We report a new DNA fragmentation technique that significantly simplifies conventional hydrodynamic shearing fragmentation by eliminating the need for sample recirculation while maintaining high fragmentation yield and low fragment length variation, and therefore, reduces instrument complexity and

  17. The relationship between sperm viability and DNA fragmentation rates

    OpenAIRE

    Mary K. Samplaski; Dimitromanolakis, Apostolos; Lo, Kirk C; Grober, Ethan D.; Mullen, Brendan; Garbens, Alaina; Jarvi, Keith A

    2015-01-01

    Background In humans, sperm DNA fragmentation rates have been correlated with sperm viability rates. Reduced sperm viability is associated with high sperm DNA fragmentation, while conversely high sperm viability is associated with low rates of sperm DNA fragmentation. Both elevated DNA fragmentation rates and poor viability are correlated with impaired male fertility, with a DNA fragmentation rate of > 30% indicating subfertility. We postulated that in some men, the sperm viability assay coul...

  18. DNA repair in bacterial cultures and plasmid DNA exposed to infrared laser for treatment of pain

    Science.gov (United States)

    Canuto, K. S.; Sergio, L. P. S.; Marciano, R. S.; Guimarães, O. R.; Polignano, G. A. C.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2013-06-01

    Biostimulation of tissues by low intensity lasers has been described on a photobiological basis and clinical protocols are recommended for treatment of various diseases, but their effects on DNA are controversial. The objective of this work was to evaluate effects of low intensity infrared laser exposure on survival and bacterial filamentation in Escherichia coli cultures, and induction of DNA lesions in bacterial plasmids. In E. coli cultures and plasmids exposed to an infrared laser at fluences used to treat pain, bacterial survival and filamentation and DNA lesions in plasmids were evaluated by electrophoretic profile. Data indicate that the infrared laser (i) increases survival of E. coli wild type in 24 h of stationary growth phase, (ii) induces bacterial filamentation, (iii) does not alter topological forms of plasmids and (iv) does not alter the electrophoretic profile of plasmids incubated with exonuclease III or formamidopyrimidine DNA glycosylase. A low intensity infrared laser at the therapeutic fluences used to treat pain can alter survival of E. coli wild type, induce filamentation in bacterial cells, depending on physiologic conditions and DNA repair, and induce DNA lesions other than single or double DNA strand breaks or alkali-labile sites, which are not targeted by exonuclease III or formamidopyrimidine DNA glycosylase.

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

    Science.gov (United States)

    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.

    Directory of Open Access Journals (Sweden)

    Joerg Kotzerke

    Full Text Available (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

  1. Complexation Between Cationic Diblock Copolymers and Plasmid DNA

    Science.gov (United States)

    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.

  2. Cloning and sequencing of a DNA fragment encoding N37 apoptotic peptide derived from p53

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective It was reported that p53 apoptotic peptide (N37) could inhibit p73 gene through being bound with iASPP,which could induce tumor cell apoptosis. To further explore the function of N37,we constructed the cloning plasmid of DNA fragment encoding p53 (N37) apoptotic peptide by using DNA synthesis and molecular biology methods. Methods According to human p53 sequence from the GenBank database,the primer of p53(N37) gene was designed using Primer V7.0 software. The DNA fragment encoding p53 (N37) apopto...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  5. Multi-antigenic DNA immunization using herpes simplex virus type 2 genomic fragments.

    Science.gov (United States)

    Braun, Ralph P; Dong, Lichun; Jerome, Sarah; Herber, Renee; Roberts, Lee K; Payne, Lendon G

    2008-01-01

    A novel DNA vaccine was generated using genomic fragments of a pathogen as the source of both the antigen coding and regulatory regions. The constructs, termed subgenomic vaccines (SGVs), incorporated genomic DNA sequences up to 45 kbp that encompass 15-20 different genes. The SGVs were developed to generate vaccines capable of expressing multiple genes from a single construct, which could be of great benefit for commercialization. The unique feature of the SGVs is that genes are expressed from their native promoters rather than heterologous promoters typical of DNA vaccines. SGVs composed of genomic fragments from the DS-DNA virus Herpes Simplex Virus Type 2 (HSV-2) induced HSV-2 specific immune responses following particle-mediated epidermal delivery (PMED) in mice and these responses protected animals from lethal infectious challenge. A second generation SGV (SGV-H2), intended as an HSV-2 therapeutic vaccine, was generated that had five HSV-2 genes and was capable of generating multi-antigenic responses in naïve mice, and enhancing responses in infected animals. When compared with standard single plasmid vaccines, immunization with the SGV-H2 was found to be at least as effective as single plasmids or plasmid mixtures. The activity of the SGV-H2 could be greatly enhanced by co-delivering plasmids expressing E. coli heat labile toxin (LT) or cholera toxin CT as adjuvants as has been found previously for standard single-gene DNA vaccines.

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

    Science.gov (United States)

    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. Bacterial mitosis: ParM of plasmid R1 moves plasmid DNA by an actin-like insertional polymerization mechanism.

    Science.gov (United States)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette; Jensen, Rasmus B; Roepstorff, Peter; Gerdes, Kenn

    2003-12-01

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

  8. Determination of plasmid copy number reveals the total plasmid DNA amount is greater than the chromosomal DNA amount in Bacillus thuringiensis YBT-1520.

    Directory of Open Access Journals (Sweden)

    Chunying Zhong

    Full Text Available Bacillus thuringiensis is the most widely used bacterial bio-insecticide, and most insecticidal crystal protein-coding genes are located on plasmids. Most strains of B. thuringiensis harbor numerous diverse plasmids, although the plasmid copy numbers (PCNs of all native plasmids in this host and the corresponding total plasmid DNA amount remains unknown. In this study, we determined the PCNs of 11 plasmids (ranging from 2 kb to 416 kb in a sequenced B. thuringiensis subsp. kurstaki strain YBT-1520 using real-time qPCR. PCNs were found to range from 1.38 to 172, and were negatively correlated to plasmid size. The amount of total plasmid DNA (∼8.7 Mbp was 1.62-fold greater than the amount of chromosomal DNA (∼5.4 Mbp at the mid-exponential growth stage (OD(600 = 2.0 of the organism. Furthermore, we selected three plasmids with different sizes and replication mechanisms to determine the PCNs over the entire life cycle. We found that the PCNs dynamically shifted at different stages, reaching their maximum during the mid-exponential growth or stationary phases and remaining stable and close to their minimum after the prespore formation stage. The PCN of pBMB2062, which is the smallest plasmid (2062 bp and has the highest PCN of those tested, varied in strain YBT-1520, HD-1, and HD-136 (172, 115, and 94, respectively. These findings provide insight into both the total plasmid DNA amount of B. thuringiensis and the strong ability of the species to harbor plasmids.

  9. Adsorption behavior of plasmid DNA onto perfusion chromatographic matrix

    Institute of Scientific and Technical Information of China (English)

    Miladys LIMONTA; Lourdes ZUMALACARREGUI; Dayana SOLER

    2012-01-01

    Anion exchange chromatography is the most popular chromatographic method for plasmid separa-tion.POROS RI 50 is a perfusion chromatographic support which is a reversed phase matrix and is an alterna-tive to conventional ones due to its mass transfer properties.The adsorption and elution of the pIDKE2 plasmidonto reversed phase POROS RI 50 was studied.Langmuir isotherm model was adjusted in order to get the max-imum adsorption capacity and the dissociation constant for POROS RI 50-plasmid DNA (pDNA) system.Break-through curves were obtained for volumetric flows between 0.69-3.33mL/min,given dynamic capacity up to2.3 times higher than those reported for ionic exchange matrix used during the purification process of plasmidswith similar size to that of pIDKE2.The efficiency was less than 45% for the flow conditions and initial concen-tration studied,which means that the support will not be operated under saturation circumstances.

  10. The relationship between sperm viability and DNA fragmentation rates.

    Science.gov (United States)

    Samplaski, Mary K; Dimitromanolakis, Apostolos; Lo, Kirk C; Grober, Ethan D; Mullen, Brendan; Garbens, Alaina; Jarvi, Keith A

    2015-05-14

    In humans, sperm DNA fragmentation rates have been correlated with sperm viability rates. Reduced sperm viability is associated with high sperm DNA fragmentation, while conversely high sperm viability is associated with low rates of sperm DNA fragmentation. Both elevated DNA fragmentation rates and poor viability are correlated with impaired male fertility, with a DNA fragmentation rate of >30% indicating subfertility. We postulated that in some men, the sperm viability assay could predict the sperm DNA fragmentation rates. This in turn could reduce the need for sperm DNA fragmentation assay testing, simplifying the infertility investigation and saving money for infertile couples. All men having semen analyses with both viability and DNA fragmentation testing were identified via a prospectively collected database. Viability was measured by eosin-nigrosin assay. DNA fragmentation was measured using the sperm chromosome structure assay. The relationship between DNA fragmentation and viability was assessed using Pearson's correlation coefficient. From 2008-2013, 3049 semen analyses had both viability and DNA fragmentation testing. A strong inverse relationship was seen between sperm viability and DNA fragmentation rates, with r=-0.83. If viability was ≤50% (n=301) then DNA fragmentation was ≥ 30% for 95% of the samples. If viability was ≥75% (n=1736), then the DNA fragmentation was ≤30% for 95% of the patients. Sperm viability correlates strongly with DNA fragmentation rates. In men with high levels of sperm viability≥75%, or low levels of sperm viability≤ 30%, DFI testing may be not be routinely necessary. Given that DNA fragmentation testing is substantially more expensive than vitality testing, this may represent a valuable cost-saving measure for couples undergoing a fertility evaluation.

  11. Variety of molecular conformation of plasmid pUC18 DNA and solenoidally supercoiled DNA

    Institute of Scientific and Technical Information of China (English)

    黄熙泰; 王照清; 吴永文; 樊廷玉; 王树荣; 王勖焜

    1996-01-01

    The plasmid pUC18 DNA isolated from Escherichia coli HB101 were analyzed by two-dimensional agarose gel electrophoresis and hybridization. The results show that the DNA sample can be separated into six groups of different structural components. The plectonemically and solenoidally supercoiled pUC18 DNA coexist in it. These two different conformations of supercoiled DNA are interchangeable with the circumstances (ionic strength and type, etc.). The amount of solenoidally supercoiled pUC18 DNA in the samples can be changed by treatment of DNA topoisome rases. Under an electron microscope, the solenoidal supercoiling DNA has a round shape with an average diameter of 45 nm. The facts suggest that solenoidaUy supercoiled DNA be a structural entity independent of histones. The polymorphism of DNA structure may be important to packing of DNA in vivo.

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

    Science.gov (United States)

    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. PROTECTION AGAINST LEPTOSPIROSIS BY IMMUNIZATION WITH PLASMID DNA ENCODING 33 kDa ENDOFLAGELLIN OF L.INTERROGANS SEROVAR LAI

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective. To evaluate how the efficacy of DNA inocutation affects the ability to raise protective immunity against Leptospira.Methods. A pair of oligonucleotide primers were designed to amplify the endoflagellar gene of L. interrogans sensu stricto serovar lai. An approximately 840bp fragment was generated with PCR and inserted into VR1012, a plasmid DNA expression vector, after the fragment and VR1012 were digested respectively with EcoRV and Sal I. A recombinant plasmid designated as VR1012+flaB2 was obtained. The vector, VR1012 consits of a pUC18 backbone with the cytomegalovirus(CMV) IE1 enhancer, promoter, and intron A, transcription regulatory elements and the BGH polyadenylation sequences driving the expressing of leptospiral endoflagellar gene of L. interrogans sensu stricto serovar lai. Plasmid encoding leptospiral endoflagellin gene was injected into quadriceps of NZW rabbits.Results.This resulted in the generation of specific leptospiral antibody with high ELISA titer (1:32768) in the rabbits. Immuno/protection was performed in guinea pigs without adjuvant. The group"VR1012+flaB2" showed higher survival rate(90%,9/10 animals),compared with the group "VR1012 lack flaB2" and the group "normal saline".Conclusion.The technique of DNA vaccine has potential advantages over certain other vaccine preparation technologies. However whether DNA vaccine will be useful for vaccine development remains to be tested.

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

    Science.gov (United States)

    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.

  15. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.

    Science.gov (United States)

    Bierman, M; Logan, R; O'Brien, K; Seno, E T; Rao, R N; Schoner, B E

    1992-07-01

    We have constructed cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. All vectors contain the 760-bp oriT fragment from the IncP plasmid, RK2. Transfer functions need to be supplied in trans by the E. coli donor strain. We have incorporated into these vectors selectable antibiotic-resistance markers (AmR, ThR, SpR) that function in Streptomyces spp. and other features that should allow for: (i) integration via homologous recombination between cloned DNA and the Streptomyces spp. chromosome, (ii) autonomous replication, or (iii) site-specific integration at the bacteriophage phi C31 attachment site. Shuttle cosmids for constructing genomic libraries and bacteriophage P1 cloning vector capable of accepting approx. 100-kb fragments are also described. A simple mating procedure has been developed for the conjugal transfer of these vectors from E. coli to Streptomyces spp. that involves plating of the donor strain and either germinated spores or mycelial fragments of the recipient strain. We have shown that several of these vectors can be introduced into Streptomyces fradiae, a strain that is notoriously difficult to transform by PEG-mediated protoplast transformation.

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

    Science.gov (United States)

    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...

  17. [Nucleotide sequence analysis of a species specific probe by an inserted fragment from recombinant plasmid pCX7 of L. interrogans sensu stricto serovar lai].

    Science.gov (United States)

    Dai, B; Xiao, J; Yan, Z; Shen, C; Li, S; Fang, Z

    1998-12-01

    The etiological agents of leptospirosis are the pathogenic leptospires (L. interrogans sensu lato) which can be divided into 223 serovars organized into 23 serogroups. The serovar remains the basic taxon, but serotyping may now be accomplished and recognized by acceptable methods. Complementary molecular approaches are being used extensively to assess genetic relatedness amongst leptospires with restriction endonuclese analysis (REA), pulse field gel electrophoresis (PFGE) and DNA-DNA hybridization as well established tools. However, the method is cumbersome and unsuitable for routine application. To develop a sensitive and specific method for identification of pathogenic leptospires, a genomic library of L. interrogans sensu stricto serovar lai was constructed with the plasmid vector pUC9. A recombinant plasmid, designated pCX7 which has homologous fragment of pathogenic leptospires was screened from the bank. pCX7 could recognize pathogenic leptospiral DNA fragment 1.7 kb of strain 017 without cross hybridization to nonpathogenic leptospiral DNA. Inserted fragment of pCX7 DNA sequencing was performed by Dr. Yan Zhengxin (Max-Plank-Institut fur Biology, Tubingen, Germany). Insert fragment was cloned into pBluescript and sequenced by using ABI(Applied Bio. Systems, Model 373A). Nucleotide sequences were analyzed by Dr. Xiao Jianguo (Texas University Medical School and School of Public Health, Center for Infectious Diseases) using a suit of computer program (NIH). One open reading frame of 306 nucleotids were identified. There were identifiable initiation codons, terminators, pribnow box and sextama box within the sequenced regions. These results further confirmed that the little homology between L. interrogans sensu strito and L. borgpeterseni serovar javanica, L. inadai serovar ranarun and serovar manhao (L. genomospecies 2), L. biflexa serovar patoc, L. illini. pCX7 DNA probe could provide a base for identification and classification of leptospires.

  18. Microfluidic DNA fragmentation for on-chip genomic analysis

    NARCIS (Netherlands)

    Shui, Lingling; Bomer, Johan G.; Jin, Mingliang; Carlen, Edwin T.; Berg, van den Albert

    2011-01-01

    We report a high-throughput clog-free microfluidic deoxyribonucleic acid (DNA) fragmentation chip that is based on hydrodynamic shearing. Salmon sperm DNA has been reproducibly fragmented down to ∼5k bp fragment lengths by applying low hydraulic pressures (≤1 bar) across micromachined constrictions

  19. Microfluidic DNA fragmentation for on-chip genomic analysis

    NARCIS (Netherlands)

    Shui, Lingling; Bomer, Johan G.; Jin, Mingliang; Carlen, Edwin; van den Berg, Albert

    2011-01-01

    We report a high-throughput clog-free microfluidic deoxyribonucleic acid (DNA) fragmentation chip that is based on hydrodynamic shearing. Salmon sperm DNA has been reproducibly fragmented down to ∼5k bp fragment lengths by applying low hydraulic pressures (≤1 bar) across micromachined constrictions

  20. High-throughput on-chip DNA fragmentation

    NARCIS (Netherlands)

    Shui, Lingling; Jin, Mingliang; Bomer, Johan G.; Carlen, Edwin; van den Berg, Albert; Abelmann, Leon; Abelmann, L.; Groenland, J.P.J.; van Honschoten, J.W.

    2010-01-01

    free microfluidic deoxyribonucleic acid (DNA) fragmentation chip that is based on hydrodynamic shearing. Genomic DNA has been reproducibly fragmented with 2-10 kbp fragment lengths by applying hydraulic pressure ΔP across micromachined constrictions in the microfluidic channels. The utilization of a

  1. Menadione-induced DNA fragmentation without 8-hydroxy-2'-deoxyguanosine formation in isolated rat hepatocytes

    DEFF Research Database (Denmark)

    Fischer-Nielsen, Anne; Corcoran, George B.; Poulsen, Henrik E.

    1995-01-01

    Farmakologi, frie iltradikaler, menadion, DNA fragmentering, rotteleverceller, oksidativ DNA skade......Farmakologi, frie iltradikaler, menadion, DNA fragmentering, rotteleverceller, oksidativ DNA skade...

  2. Separation of topological forms of plasmid DNA by anion-exchange HPLC: shifts in elution order of linear DNA.

    Science.gov (United States)

    Smith, Clara R; DePrince, Randolph B; Dackor, Jennifer; Weigl, Debra; Griffith, Jack; Persmark, Magnus

    2007-07-01

    We sought to establish a single anion-exchange HPLC method for the separation of linear, open circular and supercoiled plasmid topoisomers using purified topoisomeric forms of three plasmids (3.0, 5.5 and 7.6 kb). However, finding one condition proved elusive as the topoisomer elution order was determined to depend on salt gradient slope. The observed change in selectivity increased with plasmid size and was most pronounced for the linear form. Indeed, the elution order of the linear 7.6 kb plasmid was reversed relative to the supercoiled form. This observation may have implications for methods used in quality control of plasmid DNA.

  3. Optimizing hyaluronidase dose and plasmid DNA delivery greatly improves gene electrotransfer efficiency in rat skeletal muscle

    DEFF Research Database (Denmark)

    Åkerström, Thorbjörn; Vedel, Kenneth; Needham Andersen, Josefine

    2015-01-01

    Transfection of rat skeletal muscle in vivo is a widely used research model. However, gene electrotransfer protocols have been developed for mice and yield variable results in rats. We investigated whether changes in hyaluronidase pre-treatment and plasmid DNA delivery can improve transfection...... efficiency in rat skeletal muscle. We found that pre-treating the muscle with a hyaluronidase dose suitable for rats (0.56. U/g b.w.) prior to plasmid DNA injection increased transfection efficiency by >200% whereas timing of the pre-treatment did not affect efficiency. Uniformly distributing plasmid DNA...... delivery across the muscle by increasing the number of plasmid DNA injections further enhanced transfection efficiency whereas increasing plasmid dose from 0.2 to 1.6. μg/g b.w. or vehicle volume had no effect. The optimized protocol resulted in ~80% (CI95%: 79-84%) transfected muscle fibers...

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

    KAUST Repository

    Fatieiev, Yevhen

    2015-09-25

    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 –unlike most reported bridged silsesquioxane materials– with controlled dense and hollow structures of 100 to 200 nm. The hybrid composition of silsesquioxanes with 50% of organic content homogenously 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 the photoreaction of the organic fragments within the particles, as confirmed by spectroscopic monitorings. Furthermore, such NPs were ap-plied for the first time for the on-demand delivery of plasmid DNA in HeLa cancer cells via light actuation.

  5. Low-dose plasmid DNA treatment increases plasma vasopressin and regulates blood pressure in experimental endotoxemia

    Directory of Open Access Journals (Sweden)

    Malardo Thiago

    2012-11-01

    Full Text Available Abstract Background Although plasmid DNA encoding an antigen from pathogens or tumor cells has been widely studied as vaccine, the use of plasmid vector (without insert as therapeutic agent requires further investigation. Results Here, we showed that plasmid DNA (pcDNA3 at low doses inhibits the production of IL-6 and TNF-α by lipopolysaccharide (LPS-stimulated macrophage cell line J774. These findings led us to evaluate whether plasmid DNA could act as an anti-inflammatory agent in a Wistar rat endotoxemia model. Rats injected simultaneously with 1.5 mg/kg of LPS and 10 or 20 μg of plasmid DNA had a remarkable attenuation of mean arterial blood pressure (MAP drop at 2 hours after treatment when compared with rats injected with LPS only. The beneficial effect of the plasmid DNA on MAP was associated with decreased expression of IL-6 in liver and increased concentration of plasma vasopressin (AVP, a known vasoconstrictor that has been investigated in hemorrhagic shock management. No difference was observed in relation to nitric oxide (NO production. Conclusion Our results demonstrate for the first time that plasmid DNA vector at low doses presents anti-inflammatory property and constitutes a novel approach with therapeutic potential in inflammatory diseases.

  6. DNA-fragments are transcytosed across CaCo-2 cells by adsorptive endocytosis and vesicular mediated transport.

    Directory of Open Access Journals (Sweden)

    Lene E Johannessen

    Full Text Available Dietary DNA is degraded into shorter DNA-fragments and single nucleosides in the gastrointestinal tract. Dietary DNA is mainly taken up as single nucleosides and bases, but even dietary DNA-fragments of up to a few hundred bp are able to cross the intestinal barrier and enter the blood stream. The molecular mechanisms behind transport of DNA-fragments across the intestine and the effects of this transport on the organism are currently unknown. Here we investigate the transport of DNA-fragments across the intestinal barrier, focusing on transport mechanisms and rates. The human intestinal epithelial cell line CaCo-2 was used as a model. As DNA material a PCR-fragment of 633 bp was used and quantitative real time PCR was used as detection method. DNA-fragments were found to be transported across polarized CaCo-2 cells in the apical to basolateral direction (AB. After 90 min the difference in directionality AB vs. BA was >10(3 fold. Even undegraded DNA-fragments of 633 bp could be detected in the basolateral receiver compartment at this time point. Transport of DNA-fragments was sensitive to low temperature and inhibition of endosomal acidification. DNA-transport across CaCo-2 cells was not competed out with oligodeoxynucleotides, fucoidan, heparin, heparan sulphate and dextrane sulphate, while linearized plasmid DNA, on the other hand, reduced transcytosis of DNA-fragments by a factor of approximately 2. Our findings therefore suggest that vesicular transport is mediating transcytosis of dietary DNA-fragments across intestinal cells and that DNA binding proteins are involved in this process. If we extrapolate our findings to in vivo conditions it could be hypothesized that this transport mechanism has a function in the immune system.

  7. Microcalorimetric Studies on Gene Promoter Function of Cloned DNA Fragements from Halobacterium halobium J7 Plasmid pHH205 in Escherichia coli TG1

    Institute of Scientific and Technical Information of China (English)

    LEI,Ke-Lin; HOU,Han-Na; LIU,Yi; YE,Xue-Cheng; SHEN,Ping

    2007-01-01

    Halobacterium halobium is a typical kind of extremely halophilic bacterium. Combined with the antibiotic resistance assay, the microcalorimetric method was used to study the promoter function of the cloned DNA fragments from Halobacterium halobium J7 plasmid pHH205 in Escherichia coli TG1. The promoter probe vector, plasmid pKK232-8, was used to form the recombinants. The DNA fragment, which is the promoter for the chloramphenicol acetyl transferase (CAT) gene in plasmid pKK232-8, is about 800 bp, and the chloramphenicol resistance level presented by IC50 is about 200 μg·mL-1, which suggests a high promoter activity. The conclusions show that there probably exist double-function or trinary-function gene promoters in Halobacterium halobium, and Archaea may contain rich genetic resources.

  8. Directly Transforming PCR-Amplified DNA Fragments into Plant Cells Is a Versatile System That Facilitates the Transient Expression Assay

    Science.gov (United States)

    Lu, Yuming; Chen, Xi; Wu, Yuxuan; Wang, Yanping; He, Yuqing; Wu, Yan

    2013-01-01

    A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments) based transient expression system (PCR-TES) for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells. PMID:23468926

  9. Directly transforming PCR-amplified DNA fragments into plant cells is a versatile system that facilitates the transient expression assay.

    Directory of Open Access Journals (Sweden)

    Yuming Lu

    Full Text Available A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments based transient expression system (PCR-TES for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells.

  10. A DNA polymerase mutation that suppresses the segregation bias of an ARS plasmid in Saccharomyces cerevisiae.

    Science.gov (United States)

    Houtteman, S W; Elder, R T

    1993-03-01

    Yeast autonomously replicating sequence (ARS) plasmids exhibit an unusual segregation pattern during mitosis. While the nucleus divides equally into mother and daughter cells, all copies of the ARS plasmid will often remain in the mother cell. A screen was designed to isolate mutations that suppress this segregation bias. A plasmid with a weak ARS (wARS) that displayed an extremely high segregation bias was constructed. When cells were grown under selection for the wARS plasmid, the resulting colonies grew slowly and had abnormal morphology. A spontaneous recessive mutation that restored normal colony morphology was identified. This mutation suppressed plasmid segregation bias, as indicated by the increased stability of the wARS plasmid in the mutant cells even though the plasmid was present at a lower copy number. An ARS1 plasmid was also more stable in mutant cells than in wild-type cells. The wild-type allele for this mutant gene was cloned and identified as POL delta (CDC2). This gene encodes DNA polymerase delta, which is essential for DNA replication. These results indicate that DNA polymerase delta plays some role in causing the segregation bias of ARS plasmids.

  11. Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis: isolation of a spontaneous mutant of Bacillus subtilis with enhanced transformability for Escherichia coli-propagated chimeric plasmid DNA.

    OpenAIRE

    Ostroff, G. R.; Pène, J. J.

    1983-01-01

    Hybrid plasmid DNA cloned in Escherichia coli undergoes deletions when returned to competent Bacillus subtilis, even in defined restriction and modification mutants of strain 168. We have isolated a mutant of B. subtilis MI112 which is stably transformed at high frequency by chimeric plasmid DNA propagated in E. coli.

  12. Differential interactions of plasmid DNA, RNA and genomic DNA with amino acid-based affinity matrices.

    Science.gov (United States)

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

    2010-09-01

    The development of a strategy to plasmid DNA (pDNA) purification has become necessary for the development of gene therapy and DNA vaccine production processes in recent years, since this nucleic acid and most of contaminants, such as RNA, genomic DNA and endotoxins, are negatively charged. An ideal separation methodology may be achieved with the use of affinity interactions between immobilized amino acids and nucleic acids. In this study, the binding behaviour of nucleic acids under the influence of different environmental conditions, such as the composition and ionic strength of elution buffer, and the temperature, is compared with various amino acids immobilized on chromatography resins. Supercoiled (sc) plasmid isoform was isolated with all matrices used, but in some cases preferential interactions with other nucleic acids were found. Particularly, lysine chromatography showed to be an ideal technology mainly on RNA purification using low salt concentration. On the other hand, arginine ligands have shown a greater ability to retain the sc isoform comparatively to the other nucleic acids retention, becoming this support more adequate to sc pDNA purification. The temperature variation, competitive elution and oligonucleotides affinity studies also allowed to recognize the dominant interactions inherent to biorecognition of pDNA molecule and the affinity matrices.

  13. Biological and environmental conditionings for a sperm DNA fragmentation.

    Science.gov (United States)

    Bojar, Iwona; Witczak, Mariusz; Wdowiak, Artur

    2013-01-01

    The objective of the presented study was determination of the effect of selected agents on sperm DNA fragmentation--superoxide dismutase in seminal plasma, the patients' age, and burdening with the tobacco smoking habit. An attempt was also undertaken to evaluate the effect of DNA fragmentation on the effectiveness of infertility treatment. The study covered 186 men who received treatment due to infertility. The database and statistical analyses were performed using computer software STATISTICA 7.1. A relationship was observed between sperm DNA fragmentation and superoxide dismutase activity, the higher the SOD activity, the lower the percentage of sperm fragmentation (rs=-0.324; P=0.000; r = -0.2110). A statistical relationship was found between sperm DNA fragmentation and the percentage of pregnancies obtained during the first year of treatment--patients with the lower DFI more frequently became fathers during the first year of trying, compared to the remainder (t=2.51; P=0.013). A statistically significant relationship was confirmed (rs=-0.370; P=0.000) consisting in an increase in the DFI with respondents' age. No significant differences were noted between the DFI and the tobacco smoking habit (Chi2=0.29; P=0.926). The percentage of sperm DNA fragmentation was inversely proportional to superoxide dismutase activity in seminal plasma. DNA fragmentation becomes intensified with patients' age. Cigarette smoking has no effect on sperm DNA fragmentation. DNA fragmentation exerts an effect on the effectiveness of infertility treatment.

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

    Science.gov (United States)

    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 (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.

  15. Characterization of Plasmid DNA Location within Chitosan/PLGA/pDNA Nanoparticle Complexes Designed for Gene Delivery

    Directory of Open Access Journals (Sweden)

    Hali Bordelon

    2011-01-01

    Full Text Available Poly(D,L-lactide-co-glycolide- (PLGA-chitosan nanoparticles are becoming an increasingly common choice for the delivery of nucleic acids to cells for various genetic manipulation techniques. These particles are biocompatible, with tunable size and surface properties, possessing an overall positive charge that promotes complex formation with negatively charged nucleic acids. This study examines properties of the PLGA-chitosan nanoparticle/plasmid DNA complex after formation. Specifically, the study aims to determine the optimal ratio of plasmid DNA:nanoparticles for nucleic acid delivery purposes and to elucidate the location of the pDNA within these complexes. Such characterization will be necessary for the adoption of these formulations in a clinical setting. The ability of PLGA-chitosan nanoparticles to form complexes with pDNA was evaluated by using the fluorescent intercalating due OliGreen to label free plasmid DNA. By monitoring the fluorescence at different plasmid: nanoparticle ratios, the ideal plasmid:nanoparticle ration for complete complexation of plasmid was determined to be 1:50. Surface-Enhanced Raman Spectroscopy and gel digest studies suggested that even at these optimal complexation ratios, a portion of the plasmid DNA was located on the outer complex surface. This knowledge will facilitate future investigations into the functionality of the system in vitro and in vivo.

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

    Science.gov (United States)

    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.

  17. Effects of medium composition on the production of plasmid DNA vector potentially for human gene therapy

    Institute of Scientific and Technical Information of China (English)

    XU Zhi-nan; SHEN Wen-he; CHEN Hao; CEN Pei-lin

    2005-01-01

    Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The effects of different MMBL components on plasmid yield, cell mass and specific plasmid DNA productivity were evaluated on shake-flask scale. The results showed that glucose was the optimal carbon source. High plasmid yield (58.3 mg/L) was obtained when 5.0 g/L glucose was added to MMBL. Glycerol could be chosen as a complementary carbon source because of the highest specific plasmid productivity (37.9 mg DNA/g DCW). After tests of different levels of nitrogen source and inorganic phosphate, a modified MMBL medium was formulated for optimal plasmid production. Further study showed that the initial acetate addition (less than 4.0 g/L) in MMBL improved plasmid production significantly, although it inhibited cell growth. The results will be useful for large-scale plasmid production using recombinant E. coli system.

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

    Science.gov (United States)

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

  19. Effect of Plasmid Incompatibility on DNA Transfer to Streptococcus cremoris

    OpenAIRE

    Van Der Lelie, Daniel; Vossen, Jos M.B.M. van der; Venema, Gerard

    1988-01-01

    Several Streptococcus cremoris strains were used in protoplast transformation and interspecific protoplast fusion experiments with Streptococcus lactis and Bacillus subtilis, with pGKV110, pGKV21, and ΔpAMβ1 as the marker plasmids. ΔpAMβ1 is a 15.9-kilobase nonconjugative, deletion derivative of pAMβ1, which is considerably larger than the pGKV plasmids (approximately 4.5 kilobases). In general, ΔpAMβ1 was transferred more efficiently than the pGKV plasmids. Using electroporation, we were abl...

  20. Preparation and evaluation of nanoparticles loading plasmid DNAs inserted with siRNA fragments targeting c-Myc gene.

    Science.gov (United States)

    Ma, Tao; Jiang, Jin-Ling; Liu, Ying; Ye, Zheng-Bao; Zhang, Jun

    2014-09-01

    c-Myc plays a key role in glioma cancer stem cell maintenance. A drug delivery system, nanoparticles loading plasmid DNAs inserted with siRNA fragments targeting c-Myc gene (NPs-c-Myc-siRNA-pDNAs), for the treatment of glioma, has not previously been reported. NPs-c-Myc-siRNA-pDNAs were prepared and evaluated in vitro. Three kinds of c-Myc-siRNA fragments were separately synthesized and linked with empty siRNA expression vectors in the mole ratio of 3:1 by T4 DNA ligase. The linked products were then separately transfected into Escherichia coli. DH5α followed by extraction with Endofree plasmid Mega kit (Qiagen, Hilden, Germany) obtained c-Myc-siRNA-pDNAs. Finally, the recombinant c-Myc-siRNA3-pDNAs, generating the highest transfection efficiency and the greatest apoptotic ability, were chosen for encapsulation into NPs by the double-emulsion solvent-evaporation procedure, followed by stability, transfection efficiency, as well as qualitative and quantitative apoptosis evaluation. NPs-c-Myc-siRNA3-pDNAs were obtained with spherical shape in uniform size below 150 nm, with the zeta potential about -18 mV, the encapsulation efficiency and loading capacity as 76.3 ± 5.4% and 1.91 ± 0.06%, respectively. The stability results showed that c-Myc-siRNA3-pDNAs remained structurally and functionally stable after encapsulated into NPs, and NPs could prevent the loaded c-Myc-siRNA3-pDNAs from DNase degradation. The transfection efficiency of NPs-c-Myc-siRNA3-pDNAs was proven to be positive. Furthermore, NPs-c-Myc-siRNA3-pDNAs produced significant apoptosis with the apoptotic rate at 24.77 ± 5.39% and early apoptosis cells observed. Methoxy-poly-(ethylene-glycol)-poly-(lactide-co-glycolide) nanoparticles (MPEG-PLGA-NPs) are potential delivery carriers for c-Myc-siRNA3-pDNAs.

  1. Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology

    Directory of Open Access Journals (Sweden)

    Li Li

    2010-01-01

    Full Text Available Abstract Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1 DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2 Miniprep DNA from E. coli can be eluted with as little as 5 μl water, leading to high DNA concentrations (>1 μg/μl for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3 Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4 High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.

  2. Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology.

    Science.gov (United States)

    Li, Jian-Feng; Li, Li; Sheen, Jen

    2010-01-14

    Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2) Miniprep DNA from E. coli can be eluted with as little as 5 mul water, leading to high DNA concentrations (>1 mug/mul) for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG)-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3) Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4) High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.

  3. Plasmid loss and changes within the chromosomal DNA of Streptomyces reticuli.

    OpenAIRE

    Schrempf, H

    1982-01-01

    The sporulating wild-type strain of Streptomyces reticuli, which produces a melanin pigment and the macrolide leucomycin, contains plasmid DNA of 48 to 49 megadaltons. Plasmidless variants had an altered secondary metabolism and a changed antibiotic resistance pattern. By using a new colony hybridization technique developed for streptomycetes, it could be shown that plasmidless variants could be transformed with the wild-type plasmid DNA, which, however, is quickly lost from regenerated mycel...

  4. Efficient encapsulation of plasmid DNA in anionic liposomes by a freeze/thaw extrusion procedure

    NARCIS (Netherlands)

    Schoen, P; Bijl, L; Wilschut, J

    1998-01-01

    In this study we investigated whether intact plasmid DNA can be efficiently encapsulated in anionic liposomes prepared by freeze/thaw and extrusion techniques. There is controversy about this method of DNA encapsulation, especially as to whether DNA remains intact and retains its biological activity

  5. Inheritance of Mitochondrial DNA and Plasmids in the Ascomycetous Fungus, Epichloe Typhina

    OpenAIRE

    Chung, K. R.; Leuchtmann, A.; Schardl, C. L.

    1996-01-01

    We analyzed the inheritance of mitochondrial DNA (mtDNA) species in matings of the grass symbiont Epichloe typhina. Eighty progeny were analyzed from a cross in which the maternal (stromal) parent possessed three linear plasmids, designated Callan-a (7.5 kb), Aubonne-a (2.1 kb) and Bergell (2.0 kb), and the paternal parent had one plasmid, Aubonne-b (2.1 kb). Maternal transmission of all plasmids was observed in 76 progeny; two progeny possessed Bergell and Callan-a, but had the maternal Aubo...

  6. Targeted Collection of Plasmid DNA in Large and Growing Animal Muscles 6 Weeks after DNA Vaccination with and without Electroporation

    Directory of Open Access Journals (Sweden)

    Daniel Dory

    2015-01-01

    Full Text Available DNA vaccination has been developed in the last two decades in human and animal species as a promising alternative to conventional vaccination. It consists in the injection, in the muscle, for example, of plasmid DNA encoding the vaccinating polypeptide. Electroporation which forces the entrance of the plasmid DNA in cells at the injection point has been described as a powerful and promising strategy to enhance DNA vaccine efficacy. Due to the fact that the vaccine is composed of DNA, close attention on the fate of the plasmid DNA upon vaccination has to be taken into account, especially at the injection point. To perform such studies, the muscle injection point has to be precisely recovered and collected several weeks after injection. This is even more difficult for large and growing animals. A technique has been developed to localize precisely and collect efficiently the muscle injection points in growing piglets 6 weeks after DNA vaccination accompanied or not by electroporation. Electroporation did not significantly increase the level of remaining plasmids compared to nonelectroporated piglets, and, in all the cases, the levels were below the limit recommended by the FDA to research integration events of plasmid DNA into the host DNA.

  7. Cloning of 1183 bp Fragment from Rhoptry Protein I (ROPI Gene of Toxoplasma gondii (RH in Expression Prokaryote Plasmid PET32a

    Directory of Open Access Journals (Sweden)

    Zahra Eslamirad

    2013-10-01

    Full Text Available Background: Toxoplasma gondii is an obligatory intracellular protozoan. Considering to high prevalence of this disease the best way to reduce the raised loses is prevention of human and animal infection, rapid diagnosis, differentiation between acute and chronic disease. Rhoptry protein 1 of Toxoplasma gondii is an excretory-secretory antigen that exists in the most stages of life cycle. According to specifications of excretory-secretory antigen that seems this antigen is a suitable candidate to produce recombinant vaccine and diagnostic kit. The main object of the present work was cloning rhoptry protein 1 (ROP1 gene of Toxoplasma gondii (RH in a cloning vector for further production of rhoptry proteins.Materials and Methods: Genomic DNA was extracted by phenol-chloroform method. The ROP1 fragment was amplified by PCR. This product was approved by sequencing and was cloned between the EcoR1 and Sal1 sites of the pTZ57R/T vector. Then transformed into Escherichia coli DH5α strain and screened by IPTG and X-Gal. After isolating of this gene from pTZ57R/T, it was subcloned into pET32a plasmid.Results: The plasmid was purified and approved by electrophoresis, enzyme restriction and PCR. After isolating of this gene from pTZ57R/T, it was subcloned into pET32a plasmid. After enzyme restriction and electrophoresis a fragment about 1183bp was separated from pET32a.Conclusion: Recombinant plasmid of ROP1 gene was constructed and ready for future study. That seems the antigen is a suitable candidate to produce recombinant vaccine and diagnostic kit.

  8. High fragmentation characterizes tumour-derived circulating DNA.

    Directory of Open Access Journals (Sweden)

    Florent Mouliere

    Full Text Available BACKGROUND: Circulating DNA (ctDNA is acknowledged as a potential diagnostic tool for various cancers including colorectal cancer, especially when considering the detection of mutations. Certainly due to lack of normalization of the experimental conditions, previous reports present many discrepancies and contradictory data on the analysis of the concentration of total ctDNA and on the proportion of tumour-derived ctDNA fragments. METHODOLOGY: In order to rigorously analyse ctDNA, we thoroughly investigated ctDNA size distribution. We used a highly specific Q-PCR assay and athymic nude mice xenografted with SW620 or HT29 human colon cancer cells, and we correlated our results by examining plasma from metastatic CRC patients. CONCLUSION/SIGNIFICANCE: Fragmentation and concentration of tumour-derived ctDNA is positively correlated with tumour weight. CtDNA quantification by Q-PCR depends on the amplified target length and is optimal for 60-100 bp fragments. Q-PCR analysis of plasma samples from xenografted mice and cancer patients showed that tumour-derived ctDNA exhibits a specific amount profile based on ctDNA size and significant higher ctDNA fragmentation. Metastatic colorectal patients (n = 12 showed nearly 5-fold higher mean ctDNA fragmentation than healthy individuals (n = 16.

  9. TOL plasmid carriage enhances biofilm formation and increases extracellular DNA content in Pseudomonas putida KT2440

    DEFF Research Database (Denmark)

    D'Alvise, Paul; Sjoholm, O.R.; Yankelevich, T.;

    2010-01-01

    : TOL carriage leads to more extracellular DNA (eDNA) in pellicles and biofilms. Pellicles were dissolved by DNase I treatment. Enhanced cell lysis due to plasmid carriage was ruled out as the mechanism for eDNA release. We report, for the first time, that carriage of a conjugative plasmid leads......Adherent growth of Pseudomonas putida KT2440 with and without the TOL plasmid (pWWO) at the solid-liquid and air-liquid interface was examined. We compared biofilm formation on glass in flow cells, and assayed pellicle (air-liquid interface biofilm) formation in stagnant liquid cultures by confocal...... laser scanning microscopy. The TOL-carrying strains formed pellicles and thick biofilms, whereas the same strains without the plasmid displayed little adherent growth. Microscopy using fluorescent nucleic acid-specific stains revealed differences in the production of extracellular polymeric substances...

  10. Optimizing hyaluronidase dose and plasmid DNA delivery greatly improves gene electrotransfer efficiency in rat skeletal muscle

    DEFF Research Database (Denmark)

    Åkerström, Thorbjörn; Vedel, Kenneth; Needham Andersen, Josefine;

    2015-01-01

    delivery across the muscle by increasing the number of plasmid DNA injections further enhanced transfection efficiency whereas increasing plasmid dose from 0.2 to 1.6. μg/g b.w. or vehicle volume had no effect. The optimized protocol resulted in ~80% (CI95%: 79-84%) transfected muscle fibers......Transfection of rat skeletal muscle in vivo is a widely used research model. However, gene electrotransfer protocols have been developed for mice and yield variable results in rats. We investigated whether changes in hyaluronidase pre-treatment and plasmid DNA delivery can improve transfection...... with a homogenous distribution. We also show that transfection was stable over five weeks of regular exercise or inactivity. Our findings show that species-specific plasmid DNA delivery and hyaluronidase pre-treatment greatly improves transfection efficiency in rat skeletal muscle....

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  13. Sperm DNA fragmentation affects epigenetic feature in human male pronucleus.

    Science.gov (United States)

    Rajabi, H; Mohseni-Kouchesfehani, H; Eslami-Arshaghi, T; Salehi, M

    2017-03-06

    To evaluate whether the sperm DNA fragmentation affects male pronucleus epigenetic factors, semen analysis was performed and DNA fragmentation was assessed by the method of sperm chromatin structure assay (SCSA). Human-mouse interspecies fertilisation was used to create human male pronucleus. Male pronucleus DNA methylation and H4K12 acetylation were evaluated by immunostaining. Results showed a significant positive correlation between the level of sperm DNA fragmentation and DNA methylation in male pronuclei. In other words, an increase in DNA damage caused an upsurge in DNA methylation. In the case of H4K12 acetylation, no correlation was detected between DNA damage and the level of histone acetylation in the normal group, but results for the group in which male pronuclei were derived from sperm cells with DNA fragmentation, increased DNA damage led to a decreased acetylation level. Sperm DNA fragmentation interferes with the active demethylation process and disrupts the insertion of histones into the male chromatin in the male pronucleus, following fertilisation. © 2017 Blackwell Verlag GmbH.

  14. A mechanism of gene amplification driven by small DNA fragments.

    Directory of Open Access Journals (Sweden)

    Kuntal Mukherjee

    Full Text Available DNA amplification is a molecular process that increases the copy number of a chromosomal tract and often causes elevated expression of the amplified gene(s. Although gene amplification is frequently observed in cancer and other degenerative disorders, the molecular mechanisms involved in the process of DNA copy number increase remain largely unknown. We hypothesized that small DNA fragments could be the trigger of DNA amplification events. Following our findings that small fragments of DNA in the form of DNA oligonucleotides can be highly recombinogenic, we have developed a system in the yeast Saccharomyces cerevisiae to capture events of chromosomal DNA amplification initiated by small DNA fragments. Here we demonstrate that small DNAs can amplify a chromosomal region, generating either tandem duplications or acentric extrachromosomal DNA circles. Small fragment-driven DNA amplification (SFDA occurs with a frequency that increases with the length of homology between the small DNAs and the target chromosomal regions. SFDA events are triggered even by small single-stranded molecules with as little as 20-nt homology with the genomic target. A double-strand break (DSB external to the chromosomal amplicon region stimulates the amplification event up to a factor of 20 and favors formation of extrachromosomal circles. SFDA is dependent on Rad52 and Rad59, partially dependent on Rad1, Rad10, and Pol32, and independent of Rad51, suggesting a single-strand annealing mechanism. Our results reveal a novel molecular model for gene amplification, in which small DNA fragments drive DNA amplification and define the boundaries of the amplicon region. As DNA fragments are frequently found both inside cells and in the extracellular environment, such as the serum of patients with cancer or other degenerative disorders, we propose that SFDA may be a common mechanism for DNA amplification in cancer cells, as well as a more general cause of DNA copy number variation

  15. DNA fragmentation in spermatozoa and its relationship with impaired spermatogenesis

    Directory of Open Access Journals (Sweden)

    S. A. Rudneva

    2014-01-01

    Full Text Available Sperm cells DNA fragmentation is one of the factors of male sub-/infertility discovered recently. At present, pathophysiological mechanisms that cause DNA fragmentation have not been studied completely. It is suggested that they may be caused with defects of chromatin remodeling, apoptosis, and oxidative stress. Spermiological examination was performed in 461 infertile men. With 23 % of the patients examined, the frequency of sperm cells DNA fragmentation comprises over 15 %, with that, 18 % of the patients demonstrated its range from 15.1 to 30 %, and with 5 % of patients, it exceeded 30 %. We found that the amount of sperm cells with fragmented DNA with severe forms of pathozoospermia is higher that with less manifested disturbances of spermatogenesis. Negative dynamics was revealed regarding the change in sperm concentration in men that have increased frequency of DNA fragmentation. Obtained results confirm the suggestion of the correlation between some semen parameters (concentration, motility, and morphology and sperm DNA fragmentation. Thus, one can state that the DNA fragmentation parameter of sperm cells has a certain diagnostic and forecasting value for married couples with reproduction disorders.

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

    Science.gov (United States)

    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.

  17. Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies.

    Directory of Open Access Journals (Sweden)

    Maxim Kostylev

    Full Text Available Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six double-stranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. We demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work.

  18. Mega primer-mediated molecular cloning strategy for chimaeragenesis and long DNA fragment insertion.

    Science.gov (United States)

    Zhang, Hui; Liu, Chang-Jun; Jiang, Hui; Zhou, Lu; Li, Wen-Ying; Zhu, Ling-Yun; Wu, Lei; Meng, Er; Zhang, Dong-Yi

    2017-04-30

    Molecular cloning methods based on primer and overlap-extension PCR are widely used due to their simplicity, reliability, low cost and high efficiency. In this article, an efficient mega primer-mediated (MP) cloning strategy for chimaeragenesis and long DNA fragment insertion is presented. MP cloning is a seamless, restriction/ligation-independent method that requires only three steps: (i) the first PCR for mega primer generation; (ii) the second PCR for exponential amplification mediated by the mega primers and (iii) DpnI digestion and transformation. Most importantly, for chimaeragenesis, genes can be assembled and constructed into the plasmid vector in a single PCR step. By employing this strategy, we successfully inserted four DNA fragments (approximately 500 bp each) into the same vector simultaneously. In conclusion, the strategy proved to be a simple and efficient tool for seamless cloning.

  19. Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods

    Directory of Open Access Journals (Sweden)

    Sedlackova Tatiana

    2013-02-01

    Full Text Available Abstract Background Specific applications and modern technologies, like non-invasive prenatal testing, non-invasive cancer diagnostic and next generation sequencing, are currently in the focus of researchers worldwide. These have common characteristics in use of highly fragmented DNA molecules for analysis. Hence, for the performance of molecular methods, DNA concentration is a crucial parameter; we compared the influence of different levels of DNA fragmentation on the accuracy of DNA concentration measurements. Results In our comparison, the performance of the currently most commonly used methods for DNA concentration measurement (spectrophotometric, fluorometric and qPCR based were tested on artificially fragmented DNA samples. In our comparison, unfragmented and three specifically fragmented DNA samples were used. According to our results, the level of fragmentation did not influence the accuracy of spectrophotometric measurements of DNA concentration, while other methods, fluorometric as well as qPCR-based, were significantly influenced and a decrease in measured concentration was observed with more intensive DNA fragmentation. Conclusions Our study has confirmed that the level of fragmentation of DNA has significant impact on accuracy of DNA concentration measurement with two of three mostly used methods (PicoGreen and qPCR. Only spectrophotometric measurement was not influenced by the level of fragmentation, but sensitivity of this method was lowest among the three tested. Therefore if it is possible the DNA quantification should be performed with use of equally fragmented control DNA.

  20. Plasmid containing a DNA ligase gene from Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.; Griffin, K.; Setlow, J.K.

    1984-05-01

    A ligase gene from Haemophilus influenzae was cloned into the shuttle vector pDM2. Although the plasmid did not affect X-ray sensitivity, it caused an increase in UV sensitivity of the wild-type but not excision-defective H. influenzae and a decrease in UV sensitivity of the rec-1 mutant. 14 references, 2 figures.

  1. TOL Plasmid Carriage Enhances Biofilm Formation and Increases Extracellular DNA Content in Pseudomonas Putida KT2440

    DEFF Research Database (Denmark)

    Smets, Barth F.; D'Alvise, Paul; Yankelovich, T.;

    of extracellular polymeric substances: TOL carriage leads to more extracellular DNA (eDNA) in pellicles and biofilms. Pellicles were dissolved by DNAse I treatment. eDNA was observed as ominous fibrous structures. Quantitative analysis of live and dead cells in static cultures was performed by flow cytometry......Adherent growth of Pseudomonas putida KT2440 with and without the TOL plasmid (pWWO) at the solid-liquid and air-liquid interface was examined. We compared biofilm formation on glass in flow cells, and assayed pellicle (air-liquid interface biofilm) formation in stagnant liquid cultures by confocal...... combined with specific cytostains; release of cytoplasmic material was assayed by a β-glucosidase assay. Enhanced cell lysis due to plasmid carriage was ruled out as the mechanism for eDNA release. We report, for the first time, that carriage of a conjugative plasmid leads to increased biofilm formation...

  2. Prolonged incubation of processed human spermatozoa will increase DNA fragmentation.

    Science.gov (United States)

    Nabi, A; Khalili, M A; Halvaei, I; Roodbari, F

    2014-05-01

    One of the causes of failure in ART is sperm DNA fragmentation which may be associated with long period of spermatozoa incubation at 37 °C. The objective was to evaluate the rate of sperm DNA fragmentation using the sperm chromatin dispersion (SCD) test after swim-up at different time intervals prior to use. In this prospective study, 21 normozoospermic specimens were analysed. The samples were incubated at 37 °C after preparation by direct swim-up. DNA fragmentation was assessed at different time intervals (0, 1, 2 and 3 h) using SCD test. Spermatozoa with no DNA fragmentation showed large- or medium-sized halos, and sperm cells with DNA fragmentation showed either a small halo or no halo. The rates of normal morphology and progressive motility after sperm processing were 72.33 ± 2.53% and 90 ± 1.02%, respectively. The rate of sperm DNA fragmentation was significantly higher after 2 h (8.81 ± 0.93%, P = 0.004) and 3 h (10.76 ± 0.89%, P fragmentation. Therefore, sperm samples intended for ART procedures should be used within 2 h of incubation at 37 °C. © 2013 Blackwell Verlag GmbH.

  3. DNA sequence conservation between the Bacillus anthracis pXO2 plasmid and genomic sequence from closely related bacteria

    Directory of Open Access Journals (Sweden)

    Sabin Robert

    2002-12-01

    Full Text Available Abstract Background Complete sequencing and annotation of the 96.2 kb Bacillus anthracis plasmid, pXO2, predicted 85 open reading frames (ORFs. Bacillus cereus and Bacillus thuringiensis isolates that ranged in genomic similarity to B. anthracis, as determined by amplified fragment length polymorphism (AFLP analysis, were examined by PCR for the presence of sequences similar to 47 pXO2 ORFs. Results The two most distantly related isolates examined, B. thuringiensis 33679 and B. thuringiensis AWO6, produced the greatest number of ORF sequences similar to pXO2; 10 detected in 33679 and 16 in AWO6. No more than two of the pXO2 ORFs were detected in any one of the remaining isolates. Dot-blot DNA hybridizations between pXO2 ORF fragments and total genomic DNA from AWO6 were consistent with the PCR assay results for this isolate and also revealed nine additional ORFs shared between these two bacteria. Sequences similar to the B. anthracis cap genes or their regulator, acpA, were not detected among any of the examined isolates. Conclusions The presence of pXO2 sequences in the other Bacillus isolates did not correlate with genomic relatedness established by AFLP analysis. The presence of pXO2 ORF sequences in other Bacillus species suggests the possibility that certain pXO2 plasmid gene functions may also be present in other closely related bacteria.

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

    Science.gov (United States)

    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.

  5. Microfluidic DNA fragmentation for on-chip genomic analysis.

    Science.gov (United States)

    Shui, Lingling; Bomer, Johan G; Jin, Mingliang; Carlen, Edwin T; van den Berg, Albert

    2011-12-09

    We report a high-throughput clog-free microfluidic deoxyribonucleic acid (DNA) fragmentation chip that is based on hydrodynamic shearing. Salmon sperm DNA has been reproducibly fragmented down to ∼ 5k bp fragment lengths by applying low hydraulic pressures (≤1 bar) across micromachined constrictions positioned in larger microfluidic channels that create point-sink flow with large velocity gradients near the constriction entrance. Long constrictions (100 µm) produce shorter fragment lengths compared to shorter constrictions (10 µm), while increasing the hydrodynamic pressure requirement. Sample recirculation (10 ×) in short constrictions reduces the mean fragment length and fragment length variation, and improves yield compared to single-pass experiments without increasing the hydrodynamic pressure.

  6. Bacterial natural transformation by highly fragmented and damaged DNA

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren; Harms, Klaus; Orlando, Ludovic Antoine Alexandre

    2013-01-01

    of DNA from a 43,000-y-old woolly mammoth bone, we further demonstrate that such natural transformation events include ancient DNA molecules. We find that the DNA recombination is RecA recombinase independent and is directly linked to DNA replication. We show that the adjacent nucleotide variations......DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often DNA is recognized as nutrient source...... for microbes, but not as potential substrate for bacterial evolution. Here, we show that fragmented DNA molecules (≥20 bp) that additionally may contain abasic sites, cross-links, or miscoding lesions are acquired by the environmental bacterium Acinetobacter baylyi through natural transformation. With uptake...

  7. [High ligation of the spermatic vein and sperm DNA fragmentation].

    Science.gov (United States)

    Hu, Yang-yang; Lin, Li-zhang; Li, Cheng-di; Cai, Jian

    2011-10-01

    To investigate the effect of high ligation of the spermatic vein (HLSV) on DNA fragmentation in varicocele (VC) patients. Thirty-four VC patients underwent HLSV. Sperm motion indexes and the results of papanicolaou staining and DNA fragmentation detection were analyzed before and 3 months after the operation according to the WHO guidelines. Compared with pre-operation, HLSV achieved a significant increase in the percentage of morphologically normal sperm (P DNA fragmentation, sperm deformity index (SDI) and multiple anomalies index (MAI) (P DNA fragmentation in those with grades I - III VC were markedly lower (P 0.05). The percentage of big-halo sperm was significantly increased (P < 0.01), while those of the medium-, small- and non-halo sperm remarkably decreased (P < 0.01) after HLSV. HLSV can effectively improve the sperm quality of VC patients.

  8. [Fragmentation and/or decondensation of spermatic DNA: which consequences?].

    Science.gov (United States)

    Baldi, E; Muratori, M; Marchiani, S; Tamburrino, L; Fallet, C

    2010-04-01

    Absence of DNA fragmentation and/or decondensation is a marker of sperm quality and is related to outcome of assisted reproductive techniques: new tests have been set up to determine fragmentation rate. Copyright 2010 Elsevier Masson SAS. All rights reserved.

  9. Electromobility of plasmid DNA in tumor tissues during electric field-mediated gene delivery.

    Science.gov (United States)

    Zaharoff, D A; Barr, R C; Li, C-Y; Yuan, F

    2002-10-01

    Interstitial transport is a crucial step in plasmid DNA-based gene therapy. However, interstitial diffusion of large nucleic acids is prohibitively slow. Therefore, we proposed to facilitate interstitial transport of DNA via pulsed electric fields. To test the feasibility of this approach to gene delivery, we developed an ex vivo technique to quantify the magnitude of DNA movement due to pulsed electric fields in two tumor tissues: B16.F10 (a mouse melanoma) and 4T1 (a mouse mammary carcinoma). When the pulse duration and strength were 50 ms and 233 V/cm, respectively, we found that the average plasmid DNA movements per 10 pulses were 1.47 microm and 0.35 microm in B16.F10 and 4T1 tumors, respectively. The average plasmid DNA movements could be approximately tripled, ie to reach 3.69 microm and 1.01 microm, respectively, when the pulse strength was increased to 465 V/cm. The plasmid DNA mobility was correlated with the tumor collagen content, which was approximately eight times greater in 4T1 than in B16.F10 tumors. These data suggest that electric field can be a powerful driving force for improving interstitial transport of DNA during gene delivery.

  10. Sperm DNA fragmentation and oxidation are independent of malondialdheyde.

    Science.gov (United States)

    Zribi, Nassira; Chakroun, Nozha Feki; Elleuch, Henda; Abdallah, Fatma Ben; Ben Hamida, Afifa Sellami; Gargouri, Jalel; Fakhfakh, Faiza; Keskes, Leila Ammar

    2011-04-14

    There is clinical evidence to show that sperm DNA damage could be a marker of sperm quality and extensive data exist on the relationship between DNA damage and male fertility status. Detecting such damage in sperm could provide new elements besides semen parameters in diagnosing male infertility. We aimed to assess sperm DNA fragmentation and oxidation and to study the association between these two markers, routine semen parameters and malondialdehyde formation. Semen samples from 55 men attending the Histology-Embryology Laboratory of Sfax Faculty of Medicine, Tunisia, for semen investigations were analysed for sperm DNA fragmentation and oxidation using flow cytometry. The Sperm was also assessed spectrophotometrically for malondialdehyde formation. Within the studied group, 21 patients were nonasthenozoospermic (sperm motility ≥ 50%) and 34 patients were considered asthenozoospermic (sperm motility DNA fragmentation and oxidation (p = 0.01; r = 0.33). We also found a negative correlation between sperm DNA fragmentation and some sperm parameters: total motility (p = 0.001; r = -0.43), rapid progressive motility (type a motility) (p = 0.04; r = -0.27), slow progressive motility (type b motility) (p = 0.03; r = -0.28), and vitality (p DNA fragmentation was positively correlated with coiled tail (p = 0.01; r = 0.34). The two parameters that were found to be correlated with oxidative DNA damage were leucocytes concentrations (p = 0.01; r = 0.38) and broken neck (p = 0.02; r = 0.29). Sperm MDA levels were negatively correlated with sperm concentration (p DNA fragmentation and DNA oxidation. Our results support the evidence that oxidative stress plays a key role in inducing DNA damage; but nuclear alterations and malondialdehyde don't seem to be synchronous.

  11. Cryopreservation increases DNA fragmentation in spermatozoa of smokers.

    Science.gov (United States)

    Aydin, Mehmet Serif; Senturk, Gozde Erkanli; Ercan, Feriha

    2013-05-01

    Smoking causes subfertility due to deterioration of spermatozoa including decreased concentration and abnormal morphology. Although evidence on the deleterious effects of smoking on spermatozoa parameters is well known, its interference with cryopreservation is not clear. This study aimed to investigate the effects of cryopreservation on sperm parameters and DNA fragmentation in non-smokers and smokers. Semen samples were obtained from 40 normospermic male volunteers of whom 20 were non-smokers and 20 smokers. Samples were analyzed in terms of motility, concentration, morphology, and DNA fragmentation before freezing and 1 and 3 months after freezing and thawing. Ultrastructural alterations were investigated by transmission electron microscopy. Sperm morphology seemed to be more affected after cryopreservation in samples obtained from smokers. Ultrastructural examination showed alterations in the integrity of the membranes and increased subacrosomal swelling. Before freezing, the increase in DNA fragmentation rate in smokers was not statistically significant compared to that of non-smokers. However, after thawing, the DNA fragmentation rates were significantly high in both non-smokers and smokers compared to their respective rates before freezing. The extent of the increase in DNA fragmentation rate was significantly higher in smokers after thawing compared to that of non-smokers. In conclusion, cryopreservation causes alterations in membrane integrity and increases DNA fragmentation, thus triggering relatively negative effects on the sperm samples of smokers compared to that of non-smokers. Copyright © 2012 Elsevier GmbH. All rights reserved.

  12. Short read DNA fragment anchoring algorithm.

    Science.gov (United States)

    Wang, Wendi; Zhang, Peiheng; Liu, Xinchun

    2009-01-30

    The emerging next-generation sequencing method based on PCR technology boosts genome sequencing speed considerably, the expense is also get decreased. It has been utilized to address a broad range of bioinformatics problems. Limited by reliable output sequence length of next-generation sequencing technologies, we are confined to study gene fragments with 30 - 50 bps in general and it is relatively shorter than traditional gene fragment length. Anchoring gene fragments in long reference sequence is an essential and prerequisite step for further assembly and analysis works. Due to the sheer number of fragments produced by next-generation sequencing technologies and the huge size of reference sequences, anchoring would rapidly becoming a computational bottleneck. We compared algorithm efficiency on BLAT, SOAP and EMBF. The efficiency is defined as the count of total output results divided by time consumed to retrieve them. The data show that our algorithm EMBF have 3 - 4 times efficiency advantage over SOAP, and at least 150 times over BLAT. Moreover, when the reference sequence size is increased, the efficiency of SOAP will get degraded as far as 30%, while EMBF have preferable increasing tendency. In conclusion, we deem that EMBF is more suitable for short fragment anchoring problem where result completeness and accuracy is predominant and the reference sequences are relatively large.

  13. Bacterial spores as particulate carriers for gene gun delivery of plasmid DNA.

    Science.gov (United States)

    Aps, Luana R M M; Tavares, Milene B; Rozenfeld, Julio H K; Lamy, M Teresa; Ferreira, Luís C S; Diniz, Mariana O

    2016-06-20

    Bacillus subtilis spores represent a suitable platform for the adsorption of proteins, enzymes and viral particles at physiological conditions. In the present work, we demonstrate that purified spores can also adsorb DNA on their surface after treatment with cationic molecules. In addition, we demonstrate that DNA-coated B. subtilis spores can be used as particulate carriers and act as an alternative to gold microparticles for the biolistic (gene gun) administration of plasmid DNA in mice. Gene gun delivery of spores pre-treated with DODAB (dioctadecyldimethylammonium bromide) allowed efficient plasmid DNA absorption and induced protein expression levels similar to those obtained with gold microparticles. More importantly, we demonstrated that a DNA vaccine adsorbed on spores can be loaded into biolistic cartridges and efficiently delivered into mice, which induced specific cellular and antibody responses. Altogether, these data indicate that B. subtilis spores represent a simple and low cost alternative for the in vivo delivery of DNA vaccines by the gene gun technology.

  14. Immune Responses in Mice Injected with gD Plasmid DNA of Infectious Bovine Rhinotracheitis Virus

    Institute of Scientific and Technical Information of China (English)

    LI Ji-chang; TONG Guang-zhi; QIU Hua-ji

    2004-01-01

    The gene encoding gD of isolate Luojing of infectious bovine rhinotracheitis virus (IBRV)was amplified,sequenced, and cloned into plasmid pcDNA 3.1, resulting in a recombinant pcDNA-gD. Groups of BALB/c mice were injected with 100 μ g of plasmid only or together with liposome. After immunization, serum samples were collected from mice every 2 weeks for a 10-week period and tested for protein-specific antibody with enzyme-linked immunosorbent assay(ELISA). It was showed that the plasmid encoding IBRV glycopretein D developed gene-specific antibody. This report indicates the potential of DNA injection as a method of vaccination.

  15. Transduction of plasmid DNA in Streptomyces spp. and related genera by bacteriophage FP43.

    Science.gov (United States)

    McHenney, M A; Baltz, R H

    1988-05-01

    A segment (hft) of bacteriophage FP43 DNA cloned into plasmid pIJ702 mediated high-frequency transduction of the resulting plasmid (pRHB101) by FP43 in Streptomyces griseofuscus. The transducing particles contained linear concatemers of plasmid DNA. Lysates of FP43 prepared on S. griseofuscus containing pRHB101 also transduced many other Streptomyces species, including several that restrict plaque formation by FP43 and at least two that produce restriction endonucleases that cut pRHB101 DNA. Transduction efficiencies in different species were influenced by the addition of anti-FP43 antiserum to the transduction plates, the temperature for cell growth before transduction, the multiplicity of infection, and the host on which the transducing lysate was prepared. FP43 lysates prepared on S. griseofuscus(pRHB101) also transduced species of Streptoverticillium, Chainia, and Saccharopolyspora.

  16. Molecular mechanism of immune response induced by foreign plasmid DNA after oral administration in mice

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To study immune response induced by foreign plasmid DNA after oral administration in mice.METHODS: Mice were orally administered with 200 μg of plasmid pcDNA3 once and spleen was isolated 4 h and 18 h after administration. Total RNA was extracted from spleen and gene expression profile of BALB/c mice spleen was analyzed by using Affymetrix oligonucleotide GeneChip. Functional cluster analysis was conducted by GenMAPP software.RESULTS: At 4 h and 18 h after oral plasmid pcDNA3 administration, a number of immune-related genes,including cytokine and cytokine receptors, chemokines and chemokine receptor, complement molecule,proteasome, histocompatibility molecule, lymphocyte antigen complex and apoptotic genes, were up-regulated. Moreover, MAPPFinder results also showed that numerous immune response processes were up-regulated. In contrast, the immunoglobulin genes were down-regulated.CONCLUSION: Foreign plasmid DNA can modulate the genes expression related to immune system via the gastrointestinal tract, and further analysis of the related immune process may help understand the molecular mechanisms of immune response induced by foreign plasmid via the gastrointestinal tract.

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

    Science.gov (United States)

    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.

  18. Electronic transport in methylated fragments of DNA

    Energy Technology Data Exchange (ETDEWEB)

    Almeida, M. L. de; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L., E-mail: umbertofulco@gmail.com; Albuquerque, E. L. [Departamento de Biofísica e Farmacologia, Universidade Federal do Rio Grande do Norte, 59072-970 Natal-RN (Brazil); Freire, V. N. [Departamento de Física, Universidade Federal do Ceará, 60455-760 Fortaleza, CE (Brazil); Caetano, E. W. S. [Instituto Federal de Educação, Ciência e Tecnologia do Ceará, 60040-531 Fortaleza, CE (Brazil); Moura, F. A. B. F. de; Lyra, M. L. [Instituto de Física, Universidade Federal de Alagoas, 57072-900 Maceió-AL (Brazil)

    2015-11-16

    We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics.

  19. Sperm DNA fragmentation, recurrent implantation failure and recurrent miscarriage

    Directory of Open Access Journals (Sweden)

    Carol Coughlan

    2015-01-01

    Full Text Available Evidence is increasing that the integrity of sperm DNA may also be related to implantation failure and recurrent miscarriage (RM. To investigate this, the sperm DNA fragmentation in partners of 35 women with recurrent implantation failure (RIF following in vitro fertilization, 16 women diagnosed with RM and seven recent fathers (control were examined. Sperm were examined pre- and post-density centrifugation by the sperm chromatin dispersion (SCD test and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay. There were no significant differences in the age of either partner or sperm concentration, motility or morphology between three groups. Moreover, there were no obvious differences in sperm DNA fragmentation measured by either test. However, whilst on average sperm DNA fragmentation in all groups was statistically lower in prepared sperm when measured by the SCD test, this was not seen with the results from the TUNEL assay. These results do not support the hypothesis that sperm DNA fragmentation is an important cause of RIF or RM, or that sperm DNA integrity testing has value in such patients. It also highlights significant differences between test methodologies and sperm preparation methods in interpreting the data from sperm DNA fragmentation tests.

  20. Sperm DNA fragmentation, recurrent implantation failure and recurrent miscarriage.

    Science.gov (United States)

    Coughlan, Carol; Clarke, Helen; Cutting, Rachel; Saxton, Jane; Waite, Sarah; Ledger, William; Li, Tinchiu; Pacey, Allan A

    2015-01-01

    Evidence is increasing that the integrity of sperm DNA may also be related to implantation failure and recurrent miscarriage (RM). To investigate this, the sperm DNA fragmentation in partners of 35 women with recurrent implantation failure (RIF) following in vitro fertilization, 16 women diagnosed with RM and seven recent fathers (control) were examined. Sperm were examined pre- and post-density centrifugation by the sperm chromatin dispersion (SCD) test and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. There were no significant differences in the age of either partner or sperm concentration, motility or morphology between three groups. Moreover, there were no obvious differences in sperm DNA fragmentation measured by either test. However, whilst on average sperm DNA fragmentation in all groups was statistically lower in prepared sperm when measured by the SCD test, this was not seen with the results from the TUNEL assay. These results do not support the hypothesis that sperm DNA fragmentation is an important cause of RIF or RM, or that sperm DNA integrity testing has value in such patients. It also highlights significant differences between test methodologies and sperm preparation methods in interpreting the data from sperm DNA fragmentation tests.

  1. Human papillomavirus DNA from warts for typing by endonuclease restriction patterns: purification by alkaline plasmid methods.

    Science.gov (United States)

    Chinami, M; Tanikawa, E; Hachisuka, H; Sasai, Y; Shingu, M

    1990-01-01

    The alkaline plasmid DNA extraction method of Birnboim and Doly was applied for the isolation of human papillomavirus (HPV) from warts. Tissue from common and plantar warts was digested with proteinase K, and the extrachromosomal circular covalently-closed form of HPV-DNA was rapidly extracted by alkaline sodium dodecyl sulphate and phenol-chloroform treatment. Recovery of HPV-DNA from the tissue was sufficient for determination of endonuclease restriction patterns by agarose gel electrophoresis.

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

    Science.gov (United States)

    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.

  3. Agarose gel electrophoresis for the separation of DNA fragments.

    Science.gov (United States)

    Lee, Pei Yun; Costumbrado, John; Hsu, Chih-Yuan; Kim, Yong Hoon

    2012-04-20

    Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb(1). Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits(2). During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight(3). The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along(4). The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation(5); 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: Understand the mechanism by which DNA fragments are separated within a gel matrix Understand how conformation of the DNA molecule will determine its mobility through a gel matrix Identify an agarose solution of appropriate

  4. Design of expanded bed supports for the recovery of plasmid DNA by anion exchange adsorption

    DEFF Research Database (Denmark)

    Theodossiou, Irini; Søndergaard, M.; Thomas, Owen R. T.

    2001-01-01

    In this study we detail the rational design of new chromatographic adsorbents tailored for the capture of plasmid DNA. Features present on current chromatographic supports that can significantly enhance plasmid binding capacity have been identified in packed bed chromatography experiments...... and blueprints for improved expanded bed adsorbents have been put forward. The characterisation and testing of small (20-40 mum) high density (>3.7 g cm(-3)) pellicular expanded bed materials functionalised with various anion exchange structures is presented. In studies with calf thymus DNA, dynamic binding...

  5. High Efficiency Hydrodynamic DNA Fragmentation in a Bubbling System

    Science.gov (United States)

    Li, Lanhui; Jin, Mingliang; Sun, Chenglong; Wang, Xiaoxue; Xie, Shuting; Zhou, Guofu; van den Berg, Albert; Eijkel, Jan C. T.; Shui, Lingling

    2017-01-01

    DNA fragmentation down to a precise fragment size is important for biomedical applications, disease determination, gene therapy and shotgun sequencing. In this work, a cheap, easy to operate and high efficiency DNA fragmentation method is demonstrated based on hydrodynamic shearing in a bubbling system. We expect that hydrodynamic forces generated during the bubbling process shear the DNA molecules, extending and breaking them at the points where shearing forces are larger than the strength of the phosphate backbone. Factors of applied pressure, bubbling time and temperature have been investigated. Genomic DNA could be fragmented down to controllable 1-10 Kbp fragment lengths with a yield of 75.30-91.60%. We demonstrate that the ends of the genomic DNAs generated from hydrodynamic shearing can be ligated by T4 ligase and the fragmented DNAs can be used as templates for polymerase chain reaction. Therefore, in the bubbling system, DNAs could be hydrodynamically sheared to achieve smaller pieces in dsDNAs available for further processes. It could potentially serve as a DNA sample pretreatment technique in the future.

  6. Sperm DNA fragmentation in couples with unexplained recurrent spontaneous abortions.

    Science.gov (United States)

    Khadem, N; Poorhoseyni, A; Jalali, M; Akbary, A; Heydari, S T

    2014-03-01

    The aim of the present study was to evaluate the degree of sperm DNA fragmentation in couples with idiopathic recurrent spontaneous abortion (RSA) and in those with no history of infertility or abortion. In this cohort study, 30 couples with RSA and 30 fertile couples as control group completed the demographic data questionnaires, and their semen samples were analysed according to World Health Organization (WHO) standards (September 2009-March 2010) for evaluation of sperm DNA fragmentation, using sperm chromatin dispersion (SCD) technique. The percentage of morphologically normal sperm was significantly lower in RSA patients compared with control group (51.50 ± 11.60 versus 58.00 ± 9.05, P = 0.019), but not in other parameters. Additionally, the level of abnormal DNA fragmentation in the RSA group was significantly higher than in the control group (43.3% versus 16.7%, P = 0.024). Our results indicated a negative correlation between the number of sperm with progressive motility and DNA fragmentation (r = -0.613; P fragmentation and poor motility than those of the control group, indicating a possible relationship between idiopathic RSA and DNA fragmentation. © 2012 Blackwell Verlag GmbH.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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

  11. Regulation of mammalian horizontal gene transfer by apoptotic DNA fragmentation

    Science.gov (United States)

    Yan, B; Wang, H; Li, F; Li, C-Y

    2006-01-01

    Previously it was shown that horizontal DNA transfer between mammalian cells can occur through the uptake of apoptotic bodies, where genes from the apoptotic cells were transferred to neighbouring cells phagocytosing the apoptotic bodies. The regulation of this process is poorly understood. It was shown that the ability of cells as recipient of horizontally transferred DNA was enhanced by deficiency of p53 or p21. However, little is known with regard to the regulation of DNA from donor apoptotic cells. Here we report that the DNA fragmentation factor/caspase-activated DNase (DFF/CAD), which is the endonuclease responsible for DNA fragmentation during apoptosis, plays a significant role in regulation of horizontal DNA transfer. Cells with inhibited DFF/CAD function are poor donors for horizontal gene transfer (HGT) while their ability of being recipients of HGT is not affected. PMID:17146478

  12. [Sperm DNA fragmentation: association with semen parameters in young men].

    Science.gov (United States)

    Osadchuk, L V; Tataru, D A; Kuznetsova, N N; Kleshev, M A; Markova, E V; Svetlakov, A V

    2016-12-01

    Abnormal sperm DNA integrity is an important risk factor for male infertility. The aim of this work was to examine sperm DNA fragmentation in a cohort of young male volunteers (n=111; age 21.0+/-0.2 years) from the general population and establish the association between the level of sperm DNA fragmentation and sperm functional parameters. Sperm DNA fragmentation index (DFI) was determined by SCSA (sperm chromatin structure assay) using flow cytometry. Standard semen parameters (concentration, motility, and morphology) were evaluated according to the WHO guidelines (2010). and conclusions. In the study cohort, 79.0%, 12.4% and 8.6% of men had normal (DFIfragmentation, respectively. Men with impaired spermatogenesis had greater IDF values (14.53+/-1.43%) than men with normal semen parameters (8.88+/-0.77%, pfragmentation using SCSA technique can be employed in epidemiological studies of male fertility.

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

    Science.gov (United States)

    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.

  14. Natural transformation of bacteria by fragmented, damaged and ancient DNA

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren

    Organisms release DNA both when they live and die. Eventually the DNA disintegrates entirely or it is re-metabolized. There is a constant deposition and decomposition that maintains an environmental pool with large quantities of extracellular DNA, some of which can be thousands of years old...... it by damaged short DNA with abasic sites, crosslinks, and miscoding lesions, which are the most common damages in environmental DNA. This is emphasized by successful natural transformation by 43,000-year-old DNA. We find that the process is a simple variant of natural transformation. On top, we illustrate...... acquire functional genetic signatures of the deeper past. Moreover, not only can old DNA revert microbes to past genotypes, but damaged DNA can also produce new variants of already functional sequences. Besides, DNA fragments carry potential to combine functional domains in new ways. The identified novel...

  15. Natural transformation of bacteria by fragmented, damaged and ancient DNA

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren

    it by damaged short DNA with abasic sites, crosslinks, and miscoding lesions, which are the most common damages in environmental DNA. This is emphasized by successful natural transformation by 43,000-year-old DNA. We find that the process is a simple variant of natural transformation. On top, we illustrate......Organisms release DNA both when they live and die. Eventually the DNA disintegrates entirely or it is re-metabolized. There is a constant deposition and decomposition that maintains an environmental pool with large quantities of extracellular DNA, some of which can be thousands of years old....... The degrading DNA is fragmented and damaged, often to less than one hundred base pairs. Such DNA is only recognized as microbial nutrients and is not considered as direct contributors to bacterial evolutionary processes. The main study shows natural transformation by very short DNA (≥20bp). Further we also show...

  16. DNA fragments assembly based on nicking enzyme system.

    Directory of Open Access Journals (Sweden)

    Rui-Yan Wang

    Full Text Available A couple of DNA ligation-independent cloning (LIC methods have been reported to meet various requirements in metabolic engineering and synthetic biology. The principle of LIC is the assembly of multiple overlapping DNA fragments by single-stranded (ss DNA overlaps annealing. Here we present a method to generate single-stranded DNA overlaps based on Nicking Endonucleases (NEases for LIC, the method was termed NE-LIC. Factors related to cloning efficiency were optimized in this study. This NE-LIC allows generating 3'-end or 5'-end ss DNA overlaps of various lengths for fragments assembly. We demonstrated that the 10 bp/15 bp overlaps had the highest DNA fragments assembling efficiency, while 5 bp/10 bp overlaps showed the highest efficiency when T4 DNA ligase was added. Its advantage over Sequence and Ligation Independent Cloning (SLIC and Uracil-Specific Excision Reagent (USER was obvious. The mechanism can be applied to many other LIC strategies. Finally, the NEases based LIC (NE-LIC was successfully applied to assemble a pathway of six gene fragments responsible for synthesizing microbial poly-3-hydroxybutyrate (PHB.

  17. Selective binding of anti-DNA antibodies to native dsDNA fragments of differing sequence.

    Science.gov (United States)

    Uccellini, Melissa B; Busto, Patricia; Debatis, Michelle; Marshak-Rothstein, Ann; Viglianti, Gregory A

    2012-03-30

    Systemic autoimmune diseases are characterized by the development of autoantibodies directed against a limited subset of nuclear antigens, including DNA. DNA-specific B cells take up mammalian DNA through their B cell receptor, and this DNA is subsequently transported to an endosomal compartment where it can potentially engage TLR9. We have previously shown that ssDNA-specific B cells preferentially bind to particular DNA sequences, and antibody specificity for short synthetic oligodeoxynucleotides (ODNs). Since CpG-rich DNA, the ligand for TLR9 is found in low abundance in mammalian DNA, we sought to determine whether antibodies derived from DNA-reactive B cells showed binding preference for CpG-rich native dsDNA, and thereby select immunostimulatory DNA for delivery to TLR9. We examined a panel of anti-DNA antibodies for binding to CpG-rich and CpG-poor DNA fragments. We show that a number of anti-DNA antibodies do show preference for binding to certain native dsDNA fragments of differing sequence, but this does not correlate directly with the presence of CpG dinucleotides. An antibody with preference for binding to a fragment containing optimal CpG motifs was able to promote B cell proliferation to this fragment at 10-fold lower antibody concentrations than an antibody that did not selectively bind to this fragment, indicating that antibody binding preference can influence autoreactive B cell responses.

  18. Development of new plasmid DNA vaccine vectors with R1-based replicons

    Directory of Open Access Journals (Sweden)

    Bower Diana M

    2012-08-01

    Full Text Available Abstract Background There has been renewed interest in biopharmaceuticals based on plasmid DNA (pDNA in recent years due to the approval of several veterinary DNA vaccines, on-going clinical trials of human pDNA-based therapies, and significant advances in adjuvants and delivery vehicles that have helped overcome earlier efficacy deficits. With this interest comes the need for high-yield, cost-effective manufacturing processes. To this end, vector engineering is one promising strategy to improve plasmid production. Results In this work, we have constructed a new DNA vaccine vector, pDMB02-GFP, containing the runaway R1 origin of replication. The runaway replication phenotype should result in plasmid copy number amplification after a temperature shift from 30°C to 42°C. However, using Escherichia coli DH5α as a host, we observed that the highest yields of pDMB02-GFP were achieved during constant-temperature culture at 30°C, with a maximum yield of approximately 19 mg pDNA/g DCW being observed. By measuring mRNA and protein levels of the R1 replication initiator protein, RepA, we determined that RepA may be limiting pDMB02-GFP yield at 42°C. A mutant plasmid, pDMB-ATG, was constructed by changing the repA start codon from the sub-optimal GTG to ATG. In cultures of DH5α[pDMB-ATG], temperature-induced plasmid amplification was more dramatic than that observed with pDMB02-GFP, and RepA protein was detectable for several hours longer than in cultures of pDMB02-GFP at 42°C. Conclusions Overall, we have demonstrated that R1-based plasmids can produce high yields of high-quality pDNA without the need for a temperature shift, and have laid the groundwork for further investigation of this class of vectors in the context of plasmid DNA production.

  19. Ca2+ promoted the low transformation efficiency of plasmid DNA exposed to PAH contaminants.

    Directory of Open Access Journals (Sweden)

    Fuxing Kang

    Full Text Available 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 Ca(2+ 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 Ca(2+ enhanced the low transformation efficiency of DNA exposed to PAHs. Based on the co-sorption of Ca(2+ 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 Ca(2+ 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.

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

    Science.gov (United States)

    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.

  1. Encapsulation of plasmid DNA in calcium phosphate nanoparticles: stem cell uptake and gene transfer efficiency

    Directory of Open Access Journals (Sweden)

    Cao X

    2011-12-01

    Full Text Available Xia Cao*, Wenwen Deng*, Yuan Wei*, Weiyan Su, Yan Yang, Yawei Wei, Jiangnan Yu, Ximing XuDepartment of Pharmaceutics, School of Pharmacy, and Center for Nano Drug/Gene Delivery and Tissue Engineering, Jiangsu University, Jingkou District, Zhenjiang, People's Republic of China*These authors contributed equally to this workBackground: The purpose of this study was to develop calcium phosphate nanocomposite particles encapsulating plasmid DNA (CP-pDNA nanoparticles as a nonviral vector for gene delivery.Methods: CP-pDNA nanoparticles employing plasmid transforming growth factor beta 1 (TGF-β1 were prepared and characterized. The transfection efficiency and cell viability of the CP-pDNA nanoparticles were evaluated in mesenchymal stem cells, which were identified by immunofluorescence staining. Cytotoxicity of plasmid TGF-β1 and calcium phosphate to mesenchymal stem cells were evaluated by MTT assay.Results: The integrity of TGF-β1 encapsulated in the CP-pDNA nanoparticles was maintained. The well dispersed CP-pDNA nanoparticles exhibited an ultralow particle size (20–50 nm and significantly lower cytotoxicity than Lipofectamine™ 2000. Immunofluorescence staining revealed that the cultured cells in this study were probably mesenchymal stem cells. The cellular uptake and transfection efficiency of the CP-pDNA nanoparticles into the mesenchymal stem cells were higher than that of needle-like calcium phosphate nanoparticles and a standard calcium phosphate transfection kit. Furthermore, live cell imaging and confocal laser microscopy vividly showed the transportation process of the CP-pDNA nanoparticles in mesenchymal stem cells. The results of a cytotoxicity assay found that both plasmid TGF-β1 and calcium phosphate were not toxic to mesenchymal stem cells.Conclusion: CP-pDNA nanoparticles can be developed into an effective alternative as a nonviral gene delivery system that is highly efficient and has low cytotoxicity.Keywords: calcium

  2. An auxiliary mode of apoptotic DNA fragmentation provided by phagocytes

    Science.gov (United States)

    McIlroy, Dorian; Tanaka, Masato; Sakahira, Hideki; Fukuyama, Hidehiro; Suzuki, Misao; Yamamura, Ken-ichi; Ohsawa, Yoshiyuki; Uchiyama, Yasuo; Nagata, Shigekazu

    2000-01-01

    CAD (caspase-activated DNase) can cause DNA fragmentation in apoptotic cells. Transgenic mice that ubiquitously express a caspase-resistant form of the CAD inhibitor (ICAD) were generated. Thymocytes prepared from the mice were resistant to DNA fragmentation induced by a variety of stimuli. However, similar numbers of TUNEL-positive cells were present in adult tissues of transgenic and wild-type mice. Exposure to γ-irradiation caused a striking increase in the number of TUNEL-positive cells in the thymus of wild-type, but not transgenic, mice. TUNEL-positive nuclei in transgenic mice were confined to thymic macrophages. When apoptotic thymocytes from the transgenic mice were cocultured with macrophages, the thymocytes underwent phagocytosis and their chromosomal DNA underwent fragmentation. This DNA fragmentation was sensitive to inhibitors that block the acidification of lysosomes. Hence, we conclude that the DNA fragmentation that occurs during apoptosis not only can result cell-autonomously from CAD activity but can also be attributed to a lysosomal acid DNase(s), most likely DNase II, after the apoptotic cells are engulfed. PMID:10716943

  3. DNA fragmentation and sperm head morphometry in cat epididymal spermatozoa.

    Science.gov (United States)

    Vernocchi, Valentina; Morselli, Maria Giorgia; Lange Consiglio, Anna; Faustini, Massimo; Luvoni, Gaia Cecilia

    2014-10-15

    Sperm DNA fragmentation is an important parameter to assess sperm quality and can be a putative fertility predictor. Because the sperm head consists almost entirely of DNA, subtle differences in sperm head morphometry might be related to DNA status. Several techniques are available to analyze sperm DNA fragmentation, but they are labor-intensive and require expensive instrumentations. Recently, a kit (Sperm-Halomax) based on the sperm chromatin dispersion test and developed for spermatozoa of different species, but not for cat spermatozoa, became commercially available. The first aim of the present study was to verify the suitability of Sperm-Halomax assay, specifically developed for canine semen, for the evaluation of DNA fragmentation of epididymal cat spermatozoa. For this purpose, DNA fragmentation indexes (DFIs) obtained with Sperm-Halomax and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) were compared. The second aim was to investigate whether a correlation between DNA status, sperm head morphology, and morphometry assessed by computer-assisted semen analysis exists in cat epididymal spermatozoa. No differences were observed in DFIs obtained with Sperm-Halomax and TUNEL. This result indicates that Sperm-Halomax assay provides a reliable evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be independent from all the measured variables of sperm head morphology and morphometry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to the completion of the standard analysis of fresh or frozen semen used in assisted reproductive technologies. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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.

  5. Levels of FSH, LH and testosterone, and sperm DNA fragmentation.

    Science.gov (United States)

    Wdowiak, Artur; Raczkiewicz, Dorota; Stasiak, Magdalena; Bojar, Iwona

    2014-01-01

    Having an offspring is the most important human biological goal, which is necessary for survival of the human species. Lack of offspring is a phenomenon concerning approximately 15% of married couples in Poland. In a half of the cases, a causative agent is the male factor infertility problem. There is evidence that certain male fertility problems are related with disorders of the process of spermatogenesis. The course of normal spermatogenesis depends on proper pituitary secretion of folliculostimulin (FSH), luteinizing hormone (LH), as well as testicular secretion of testosterone. It is considered that in approximately 20% of patients with idiopathic infertility an elevated level of sperm DNA fragmentation may be the cause of failure in reproduction. The objective of the present study was determination of the relationship between FSH, LH and testosterone levels, and the occurrence of sperm DNA fragmentation. The present study was conducted in the year 2012 in the Non-Public Health Care Unit 'Ovum Reproduction and Andrology' in Lublin, and covered 186 men treated for infertility. For inclusion into the study group we qualified males aged 25-35, who have been treated for infertility for more than 1 year, with no pathological features observed in the female partner. The structure of sperm chromatin was evaluated using the technique of flow cytometry-Sperm Chromatin Structure assay (SCSA). The result of the examination was a sperm DNA Fragmentation Index (DFI), i.e., the percentage of sperm with DNA lesions (DNA fragmentation). A morning blood sample (5 mL volume) was obtained and sent to an authorized laboratory to assess serum levels of testosterone, LH and FSH. An intensified sperm DNA fragmentation co-occurred with both extremely low and extremely high levels of FSH and LH. Sperm DNA fragmentation was negatively correlated with testosterone level.

  6. An easy and versatile 2-step protocol for targeted modification and subcloning of DNA from bacterial artificial chromosomes using non-commercial plasmids

    Directory of Open Access Journals (Sweden)

    Hartwich Heiner

    2012-03-01

    Full Text Available Abstract Background Promoter-specific expression of foreign DNA in transgenic organisms often relies on bacterial artificial chromosomes (BACs. This approach requires modification and subcloning of BAC-DNA by recombineering technologies in Escherichia coli. Most current protocols rely on commercial kits or isolation of BACs, their transfer between different host strains, and their restriction. Findings In this report we present a 2-step protocol for efficient modification and subcloning of DNA from bacterial artificial chromosomes using the non-commercial plasmids pKM208 and pTP223, distributed from addgene.com. A targeting cassette was successfully integrated into a BAC and 42 kb of this construct were subcloned. Both a plasmid-derived substrate with longer homology arms and a PCR-generated substrate with short homology arms (50 bp were used for recombination. pKM208 and pTP223 contain all required genes for recombineering, but differ in their antibiotic resistance genes. This makes the system independent of the selection markers on the DNA molecules targeted for recombination. Conclusions The time and cost saving protocol presented here compares favorably to currently used systems. Using non-commercial plasmids, it allows targeted modification and cloning of large DNA (> 40 kb fragments in vivo without restriction and ligation. Furthermore, both steps are performed in the same host eliminating the need to isolate BAC DNA and to use different bacterial strains.

  7. Improvement of in vivo transfer of plasmid DNA in muscle : Comparison of electroporation versus ultrasound

    NARCIS (Netherlands)

    Kusumanto, Yoka H.; Mulder, Nanno H.; Dam, Wendy A.; Losen, Mario H.; Meijer, Coby; Hospers, Geke A. P.

    2007-01-01

    Plasmid-based gene delivery to muscle is a treatment strategy for many diseases with potential advantages above viral-based gene delivery methods, however, with a relative low transfection efficiency. We compared two physical methods-electroporation and ultrasound-that facilitate DNA uptake into cel

  8. TOL plasmid carriage enhances biofilm formation and increases extracellular DNA content in Pseudomonas putida KT2440.

    Science.gov (United States)

    D'Alvise, Paul W; Sjøholm, Ole R; Yankelevich, Tatiana; Jin, Yujie; Wuertz, Stefan; Smets, Barth F

    2010-11-01

    Adherent growth of Pseudomonas putida KT2440 with and without the TOL plasmid (pWWO) at the solid-liquid and air-liquid interface was examined. We compared biofilm formation on glass in flow cells, and assayed pellicle (air-liquid interface biofilm) formation in stagnant liquid cultures by confocal laser scanning microscopy. The TOL-carrying strains formed pellicles and thick biofilms, whereas the same strains without the plasmid displayed little adherent growth. Microscopy using fluorescent nucleic acid-specific stains revealed differences in the production of extracellular polymeric substances: TOL carriage leads to more extracellular DNA (eDNA) in pellicles and biofilms. Pellicles were dissolved by DNase I treatment. Enhanced cell lysis due to plasmid carriage was ruled out as the mechanism for eDNA release. We report, for the first time, that carriage of a conjugative plasmid leads to increased biofilm formation by production of eDNA. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Tue Sparholt Jørgensen

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

  10. Differential behavior of plasmids containing chromosomal DNA insertions of various sizes during transformation and conjugation in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1985-01-01

    Plasmids with chromosomal insertions were constructed by removal of a 1.1-kilobase-pair piece from the 9.8-kilobase-pair vector plasmid pDM2 by EcoRI digestion and inserting in its place various lengths of chromosomal DNA (1.7, 3.4, and 9.0 kilobase pairs) coding for resistance to novobiocin. A fourth plasmid was constructed by insertion of the largest piece of chromosomal DNA into the SmaI site of pDM2. The plasmids without inserts were taken up poorly by competent cells and thus were considered not to contain specific DNA uptake sites. The presence of even the smallest insert of chromosomal DNA caused a large increase in transformation of Rec/sup +/ and Rec/sup -/ strains. The frequency of plasmid establishment in Rec/sup +/ cells by transformation increased exponentially with increasing insert size, but in Rec/sup -/ cells there was less transformation by the larger plasmids. Conjugal transfer of these plasmids was carried out with the 35-kilobase-pair mobilizing plasmid pHD147. The frequency of establishment of plasmids by this method not only was not markedly affected by the presence of the insertions, but also decreased somewhat with increase in insert size and was independent of rec-1 and rec-2 genes. Recombination between plasmid and chromosome was readily detected after transformation, but could not be detected after transconjugation even when the recipient cells were Rec/sup +/ and made competent. These data suggested that there is a special processing of plasmid DNA that enters the competent cells in transformation that makes possible recombination of homologous regions of the plasmid with the chromosome and pairing with the chromosome that aids plasmid establishment.

  11. Recovery of small DNA fragments from serum using compaction precipitation.

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    Binh V Vu

    Full Text Available BACKGROUND: While most nucleic acids are intracellular, trace amounts of deoxyribonucleic acid (DNA and ribonucleic acid (RNA, including micro RNAs, can also be found in peripheral blood. Many studies have suggested the potential utility of these circulating nucleic acids in prenatal diagnosis, early cancer detection, and the diagnosis of infectious diseases. However, DNA circulating in blood is usually present at very low concentrations (ng/ml, and is in the form of relatively small fragments (<1,000 bp, making its isolation challenging. METHODS: Here we report an improved method for the isolation of small DNA fragments from serum using selective precipitation by quaternary ammonium compaction agents. A 151 bp fragment of double-stranded DNA from the Escherichia coli bacteriophage lambda served as the model DNA in our experiments. DNA was serially diluted in serum until undetectable by conventional polymerase chain reaction (PCR, before being enriched by compaction precipitation. RESULTS: Starting with concentrations two to three orders of magnitude lower than the PCR-detectable level (0.01 ng/ml, we were able to enrich the DNA to a detectable level using a novel compaction precipitation protocol. The isolated DNA product after compaction precipitation was largely free of serum contaminants and was suitable for downstream applications. CONCLUSIONS: Using compaction precipitation, we were able to isolate and concentrate small DNA from serum, and increase the sensitivity of detection by more than four orders of magnitude. We were able to recover and detect very low levels (0.01 ng/ml of a small DNA fragment in serum. In addition to being very sensitive, the method is fast, simple, inexpensive, and avoids the use of toxic chemicals.

  12. High-Throughput Plasmid cDNA Library Screening

    Energy Technology Data Exchange (ETDEWEB)

    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.

  13. Isolation and characterization of yeast DNA repair genes. II. Isolation of plasmids that complement the mutations rad50-1, rad51-1, rad54-3, and rad55-3

    Energy Technology Data Exchange (ETDEWEB)

    Calderon, I.L.; Contopoulou, C.R.; Mortimer, R.K.

    1983-01-01

    Plasmids that complement the yeast mutations rad50-1, rad51-1, rad54-3, and rad55-3 were obtained by transforming strains that carried a leu2 marker and the particular rad mutation, with YEp 13 plasmids containing near random yeast DNA inserts. Integration of these plasmids or of fragments of these plasmids was accomplished. Genetic studies using the integrants established the presence of the genes RAD50, RAD54 and RAD55 in the respective plasmids. However, a BamHI subclone of the rad50-1 complementing plasmid failed to integrate at the RAD50 locus, indicating that no homology exists between this fragment and the RAD50 gene. A BamHI fragment for the RAD54 plasmid was shown to be internal to the RAD54 gene: its integration within a wild type copy of RAD54 causes the cell to become Rad/sup -/; its excision is X-ray inducible and restores the Rad/sup -/ phenotype. Since cells bearing a disrupted copy of RAD54 are able to survive, the author concludes that this is not essential.

  14. Method for the preparation of plasmid DNA suitable for physicochemical measurements.

    Science.gov (United States)

    Vojtísková, M; Lukásová, E; Palecek, E

    1985-01-01

    A method has been developed for the isolation of plasmid DNA suitable for physical and physicochemical measurements. The procedure is based on the deproteinization of the cleared lysate of bacterial cells (after amplification of plasmids by chloramphenicol) by phenol at pH 8.0 and subsequent removal of chromosomal DNA by means of phenol at pH 4.0 and separation of RNA on a hydroxyapatite column at higher temperature. ColE1 DNA sample was compared with samples of the same DNA prepared by three thus far used methods. Samples obtained by means of the latter methods were contaminated with chromosomal DNA, RNA, or ethidium bromide. The presence of ethidium bromide in the DNA sample was a factor interfering in the electrochemical analysis, chromosomal DNA and RNA were disturbing in the use of other methods. DNA separated by the method devised by us was free of any detectable contaminants and fulfilled the high requirements for sample purity of differential pulse polarography. Measurements performed by means of differential pulse polarography showed that the content of single-stranded segments in superhelical ColE1 DNA is less than 0.15% (i.e. less than 20 bases per molecule). This is in keeping with the notion that a cruciform is formed in this DNA (as a result of tension due to supercoiling) in the region of inverted repeat sequence, containing only 5 bases in the single-stranded loop region.

  15. Hole Conductivity in Heterogeneous DNA Fragments

    OpenAIRE

    2013-01-01

    The characteristics of cation radical (hole) migration in heterogeneous DNA were investigated on the basis of Kubo formula, in which correlation functions were obtained from solutions of systems of Bogoliubov hierarchy. The cutting of Bogoliubov hierarchy was carried out by excepting correlations of the third and higher order. The obtained system of non-linear differential equations was investigated both analytically and numerically. The environment polarization, caused by interaction of hole...

  16. Modified Classical Graph Algorithms for the DNA Fragment Assembly Problem

    Directory of Open Access Journals (Sweden)

    Guillermo M. Mallén-Fullerton

    2015-09-01

    Full Text Available DNA fragment assembly represents an important challenge to the development of efficient and practical algorithms due to the large number of elements to be assembled. In this study, we present some graph theoretical linear time algorithms to solve the problem. To achieve linear time complexity, a heap with constant time operations was developed, for the special case where the edge weights are integers and do not depend on the problem size. The experiments presented show that modified classical graph theoretical algorithms can solve the DNA fragment assembly problem efficiently.

  17. Variation of clonal, mesquite-associated rhizobial and bradyrhizobial populations from surface and deep soils by symbiotic gene region restriction fragment length polymorphism and plasmid profile analysis.

    Science.gov (United States)

    Thomas, P M; Golly, K F; Zyskind, J W; Virginia, R A

    1994-04-01

    Genetic characteristics of 14 Rhizobium and 9 Bradyrhizobium mesquite (Prosopis glandulosa)-nodulating strains isolated from surface (0- to 0.5-m) and deep (4- to 6-m) rooting zones were determined in order to examine the hypothesis that surface- and deep-soil symbiont populations were related but had become genetically distinct during adaptation to contrasting soil conditions. To examine genetic diversity, Southern blots of PstI-digested genomic DNA were sequentially hybridized with the nodDABC region of Rhizobium meliloti, the Klebsiella pneumoniae nifHDK region encoding nitrogenase structural genes, and the chromosome-localized ndvB region of R. meliloti. Plasmid profile and host plant nodulation assays were also made. Isolates from mesquite nodulated beans and cowpeas but not alfalfa, clover, or soybeans. Mesquite was nodulated by diverse species of symbionts (R. meliloti, Rhizobium leguminosarum bv. phaseoli, and Parasponia bradyrhizobia). There were no differences within the groups of mesquite-associated rhizobia or bradyrhizobia in cross-inoculation response. The ndvB hybridization results showed the greatest genetic diversity among rhizobial strains. The pattern of ndvB-hybridizing fragments suggested that surface and deep strains were clonally related, but groups of related strains from each soil depth could be distinguished. Less variation was found with nifHDK and nodDABC probes. Large plasmids (>1,500 kb) were observed in all rhizobia and some bradyrhizobia. Profiles of plasmids of less than 1,000 kb were related to the soil depth and the genus of the symbiont. We suggest that interacting selection pressures for symbiotic competence and free-living survival, coupled with soil conditions that restrict genetic exchange between surface and deep-soil populations, led to the observed patterns of genetic diversity.

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

    Directory of Open Access Journals (Sweden)

    Charles Michael Drain

    2011-10-01

    Full Text Available 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.

  19. DNA fragmentation of spermatozoa and assisted reproduction technology.

    Science.gov (United States)

    Henkel, Ralf; Kierspel, Eva; Hajimohammad, Marjam; Stalf, Thomas; Hoogendijk, Christiaan; Mehnert, Claas; Menkveld, Roelof; Schill, Wolf-Bernhard; Kruger, Thinus F

    2003-01-01

    Despite the ever-increasing knowledge of the fertilization process, there is still a need for better understanding of the causes of sperm DNA fragmentation and its impact on fertilization and pregnancy. For this reason, human sperm DNA fragmentation was investigated by means of the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and the production of reactive oxygen species (ROS) in the ejaculate and in the spermatozoa themselves. These data were correlated with fertilization and pregnancy data from IVF and intracytoplasmic sperm injection (ICSI) patients. Sperm DNA fragmentation did not correlate with fertilization rate, but there was a significantly reduced pregnancy rate in IVF patients inseminated with TUNEL-positive spermatozoa. ICSI patients exhibited the same tendency. This implies that spermatozoa with damaged DNA are able to fertilize an oocyte, but at the time the paternal genome is switched on, further development stops. The determination of ROS in the ejaculate and the percentage of ROS-producing spermatozoa revealed markedly stronger correlations between sperm functions (i.e. motility) and the percentage of ROS-producing spermatozoa. The influence of seminal leukocytes, known to produce large amounts of oxidants, on sperm DNA fragmentation should not be neglected.

  20. Putative DNA-dependent RNA polymerase in Mitochondrial Plasmid of Paramecium caudatum Stock GT704

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    Trina Ekawati Tallei

    2015-10-01

    Full Text Available Mitochondria of Paramecium caudatum stock GT704 has a set of four kinds of linear plasmids with sizes of 8.2, 4.1, 2.8 and 1.4 kb. The plasmids of 8.2 and 2.8 kb exist as dimers consisting of 4.1- and 1.4-kb monomers, respectively. The plasmid 2.8 kb, designated as pGT704-2.8, contains an open reading frame encodes for putative DNA-dependent RNA polymerase (RNAP. This study reveals that this RNAP belongs to superfamily of DNA/RNA polymerase and family of T7/T3 single chain RNA polymerase and those of mitochondrial plasmid of fungi belonging to Basidiomycota and Ascomycota. It is suggested that RNAP of pGT704-2.8 can perform transcription without transcription factor as promoter recognition. Given that only two motifs were found, it could not be ascertained whether this RNAP has a full function independently or integrated with mtDNA in carrying out its function.

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

    Science.gov (United States)

    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

  2. Insight into F plasmid DNA segregation revealed by structures of SopB and SopB–DNA complexes

    OpenAIRE

    2010-01-01

    Accurate DNA segregation is essential for genome transmission. Segregation of the prototypical F plasmid requires the centromere-binding protein SopB, the NTPase SopA and the sopC centromere. SopB displays an intriguing range of DNA-binding properties essential for partition; it binds sopC to form a partition complex, which recruits SopA, and it also coats DNA to prevent non-specific SopA–DNA interactions, which inhibits SopA polymerization. To understand the myriad functions of SopB, we dete...

  3. Characterization of a linear DNA plasmid from the filamentous fungal plant pathogen Glomerella musae [Anamorph: Colletotrichum musae (Berk. and Curt.) arx.

    Science.gov (United States)

    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.

  4. Binding and elution strategy for improved performance of arginine affinity chromatography in supercoiled plasmid DNA purification.

    Science.gov (United States)

    Sousa, F; Prazeres, D M F; Queiroz, J A

    2009-02-01

    New interesting strategies for plasmid DNA (pDNA) purification were designed, exploiting affinity interactions between amino acids and nucleic acids. The potential application of arginine-based chromatography to purify pDNA has been recently described in our work; however, to achieve higher efficiency and selectivity in arginine affinity chromatography, it is essential to characterize the behaviour of binding/elution of supercoiled (sc) isoforms. In this study, two different strategies based on increased sodium chloride (225-250 mm) or arginine (20-70 mm) stepwise gradients are described to purify sc isoforms. Thus, it was proved that well-defined binding/elution conditions are crucial to enhance the purification performance, resulting in an improvement of the final plasmids yields and transfection efficiency, as this could represent a significant impact on therapeutic applications of the purified sc isoform. Copyright (c) 2008 John Wiley & Sons, Ltd.

  5. Mutant DNA quantification by digital PCR can be confounded by heating during DNA fragmentation.

    Science.gov (United States)

    Kang, Qing; Parkin, Brian; Giraldez, Maria D; Tewari, Muneesh

    2016-04-01

    Digital PCR (dPCR) is gaining popularity as a DNA mutation quantification method for clinical specimens. Fragmentation prior to dPCR is required for non-fragmented genomic DNA samples; however, the effect of fragmentation on DNA analysis has not been well-studied. Here we evaluated three fragmentation methods for their effects on dPCR point mutation assay performance. Wild-type (WT) human genomic DNA was fragmented by heating, restriction digestion, or acoustic shearing using a Covaris focused-ultrasonicator. dPCR was then used to determine the limit of blank (LoB) by quantifying observed WT and mutant allele counts of the proto-oncogenes KRAS and BRAF in the WT DNA sample. DNA fragmentation by heating to 95°C, while the simplest and least expensive method, produced a high background mutation frequency for certain KRAS mutations relative to the other methods. This was due to heat-induced mutations, specifically affecting dPCR assays designed to interrogate guanine to adenine (G>A) mutations. Moreover, heat-induced fragmentation overestimated gene copy number, potentially due to denaturation and partition of single-stranded DNA into different droplets. Covaris acoustic shearing and restriction enzyme digestion showed similar LoBs and gene copy number estimates to one another. It should be noted that moderate heating, commonly used in genomic DNA extraction protocols, did not significantly increase observed KRAS mutation counts.

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

    Science.gov (United States)

    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.

  7. Molecular characterization of a DNA fragment harboring the replicon of pBMB165 from Bacillus thuringiensis subsp. tenebrionis

    Directory of Open Access Journals (Sweden)

    Yu Ziniu

    2006-10-01

    Full Text Available Abstract Background Bacillus thuringiensis belongs to the Bacillus cereus sensu lato group of Gram-positive and spore-forming bacteria. Most isolates of B. thuringiensis can bear many endogenous plasmids, and the number and size of these plasmids can vary widely among strains or subspecies. As far as we know, the replicon of the plasmid pBMB165 is the first instance of a plasmid replicon being isolated from subsp. tenebrionis and characterized. Results A 20 kb DNA fragment containing a plasmid replicon was isolated from B. thuringiensis subsp. tenebrionis YBT-1765 and characterized. By Southern blot analysis, this replicon region was determined to be located on pBMB165, the largest detected plasmid (about 82 kb of strain YBT-1765. Deletion analysis revealed that a replication initiation protein (Rep165, an origin of replication (ori165 and an iteron region were required for replication. In addition, two overlapping ORFs (orf6 and orf10 were found to be involved in stability control of plasmid. Sequence comparison showed that the replicon of pBMB165 was homologous to the pAMβ1 family replicons, indicating that the pBMB165 replicon belongs to this family. The presence of five transposable elements or remnants thereof in close proximity to and within the replicon control region led us to speculate that genetic exchange and recombination are potentially responsible for the divergence among the replicons of this plasmid family. Conclusion The replication and stability features of the pBMB165 from B. thuringiensis subsp. tenebrionis YBT-1765 were identified. Of particular interest is the homology and divergence shared between the pBMB165 replicon and other pAMβ1 family replicons.

  8. Isolation of cDNA Fragment of Gene Encoding for Actin from Melastoma malabthricum.

    Directory of Open Access Journals (Sweden)

    Suharsono

    2010-11-01

    Full Text Available Isolation of cDNA Fragment of Gene Encoding for Actin from Melastoma malabthricum. M. malabathricumgrows well in acidic soil with high Al solubility, thereby it can be used as a model plant for tolerance to aluminum andacid stresses. Actin is housekeeping gene used as an internal control for gene expression analysis. The objective of thisresearch was to isolate and clone the cDNA fragments of MmACT encoding for actin of M. malabathricum. Total RNAwas isolated and used as the template for cDNA synthesis by reverse transcription. Four cDNA fragments of MmACT,called MmACT1, MmACT2, MmACT3, and MmACT4, had been isolated and inserted into pGEM-T Easy plasmid.Nucleotide sequence analysis showed that the size of MmACT1 and MmACT2 is 617 bp, whereas MmACT3 andMmACT4 is 735 bp. The similarity among these four MmACT is about 78%-99% based on nucleotide sequence andabout 98%-100% based on amino acid sequence. Phylogenetic analysis based on amino acid sequence showed that at1% dissimilarity, the MmACT1, MmACT2, MmACT3 and the ACT5 Populus trichocarpha are clustered in one group,while the MmACT4 is grouped with ACT9 P. trichocarpa and ACT1 Gossypium hirsutum, and these two groups areseparated from actin group of monocotyledonous plants. The sequence of MmACT fragments were registered inGenBank/EMBL/DDBJ database with accession numbers AB500686, AB500687, AB500688, and AB500689.

  9. Construction of Recombinant Plasmids by Random Ligation of Blunt-Ended DNA%平末端DNA随机连接构建重组质粒

    Institute of Scientific and Technical Information of China (English)

    查向东; 刘杨; 吴敌; 卢颖虎; 梁琳; 陈青峰

    2012-01-01

    目的:拼接DNA片段并克隆.方法:用T4 DNA连接酶将DNA片段以平末端随机连接,随后用限制性内切酶切割,琼脂糖电泳分离酶切产物,挑选特定片段纯化回收,与线性化的载体质粒连接,转化大肠杆菌感受态细胞.结果:通过以上步骤,成功拼接了不同DNA片段,构建了含有目的拼接片段的重组质粒.结论:该方法简便、易行、可靠,可作为拼接、克隆DNA的备选方案,在分子生物学研究和基因工程中应用.%Objective: To join different DNA fragments and clone the joint DNA. Methods: We randomly ligated the blunt-ended DNA fragments with T4 DNA ligase,cleaved the ligated molecules with the restriction endonucleas-es,then separated the hydrolysed products by agarose gel electrophoresis. We identified the specific band on the agarose gel,from which the DNA molecules were recovered,integrated into the linearized vector plasmid and trans-formed into the competent E.coli cells. Results: In this way we successfully joined the different DNA fragments and constructed the recombinant plasmids haboring the joint DNA. Conclusion: The stragety is simple,convenient and reliable,providing an alternative procedure for joining and cloning DNA which can be used in molecular biology research and gene engenering.

  10. Avoiding adsorption of DNA to polypropylene tubes and denaturation of short DNA fragments

    OpenAIRE

    Gaillard, Claire; Strauss, Francois

    1998-01-01

    Two problems can arise when working with small quantities of DNA in polypropylene tubes: first, significant amounts of DNA can become lost by sticking to the tube walls; second, short DNA fragments tend to denature when binding to polypropylene. In addition, DNA also tends to denature upon dehydration. We have found that a simple way to solve these problems is by using polyallomer tubes instead of polypropylene and by avoiding certain salts, such as sodium acetate, when drying DNA.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Chowdhury, E.H., E-mail: md.ezharul.hoque@med.monash.edu.my [Jeffrey Cheah School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, Selangor Darul Ehsan (Malaysia)

    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.

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

    Science.gov (United States)

    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.

  14. Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III

    Directory of Open Access Journals (Sweden)

    Hiroshi Arakawa

    2015-06-01

    Full Text Available Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1, DNA ligase 3 (Lig3 and DNA ligase 4 (Lig4. While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at the DNA replication fork and the ligation steps of long-patch base-excision repair (BER, homologous recombination repair (HRR and nucleotide excision repair (NER. Lig4 is responsible for DNA ligation at DNA double strand breaks (DSBs by the classical, DNA-PKcs-dependent pathway of non-homologous end joining (C-NHEJ. Lig3 is implicated in a short-patch base excision repair (BER pathway, in single strand break repair in the nucleus, and in all ligation requirements of the DNA metabolism in mitochondria. In this scenario, Lig1 and Lig4 feature as the major DNA ligases serving the most essential ligation needs of the cell, while Lig3 serves in the cell nucleus only minor repair roles. Notably, recent systematic studies in the chicken B cell line, DT40, involving constitutive and conditional knockouts of all three DNA ligases individually, as well as of combinations thereof, demonstrate that the current view must be revised. Results demonstrate that Lig1 deficient cells proliferate efficiently. Even Lig1/Lig4 double knockout cells show long-term viability and proliferate actively, demonstrating that, at least in DT40, Lig3 can perform all ligation reactions of the cellular DNA metabolism as sole DNA ligase. Indeed, in the absence of Lig1, Lig3 can efficiently support semi-conservative DNA replication via an alternative Okazaki-fragment ligation pathway. In addition, Lig3 can back up NHEJ in the absence of Lig4, and can support NER and HRR in the absence of Lig1. Supporting observations are available in less elaborate genetic models in mouse cells. Collectively, these observations raise Lig3 from a niche

  15. Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III.

    Science.gov (United States)

    Arakawa, Hiroshi; Iliakis, George

    2015-06-23

    Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1), DNA ligase 3 (Lig3) and DNA ligase 4 (Lig4). While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at the DNA replication fork and the ligation steps of long-patch base-excision repair (BER), homologous recombination repair (HRR) and nucleotide excision repair (NER). Lig4 is responsible for DNA ligation at DNA double strand breaks (DSBs) by the classical, DNA-PKcs-dependent pathway of non-homologous end joining (C-NHEJ). Lig3 is implicated in a short-patch base excision repair (BER) pathway, in single strand break repair in the nucleus, and in all ligation requirements of the DNA metabolism in mitochondria. In this scenario, Lig1 and Lig4 feature as the major DNA ligases serving the most essential ligation needs of the cell, while Lig3 serves in the cell nucleus only minor repair roles. Notably, recent systematic studies in the chicken B cell line, DT40, involving constitutive and conditional knockouts of all three DNA ligases individually, as well as of combinations thereof, demonstrate that the current view must be revised. Results demonstrate that Lig1 deficient cells proliferate efficiently. Even Lig1/Lig4 double knockout cells show long-term viability and proliferate actively, demonstrating that, at least in DT40, Lig3 can perform all ligation reactions of the cellular DNA metabolism as sole DNA ligase. Indeed, in the absence of Lig1, Lig3 can efficiently support semi-conservative DNA replication via an alternative Okazaki-fragment ligation pathway. In addition, Lig3 can back up NHEJ in the absence of Lig4, and can support NER and HRR in the absence of Lig1. Supporting observations are available in less elaborate genetic models in mouse cells. Collectively, these observations raise Lig3 from a niche-ligase to a

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

    Science.gov (United States)

    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.

  17. Design of amphiphilic oligopeptides as models for fine tuning peptide assembly with plasmid DNA.

    Science.gov (United States)

    Goparaju, Geetha N; Gupta, Pardeep K

    2014-08-01

    We discuss the design of novel amphiphilic oligopeptides with hydrophobic and cationic amino acids to serve as models to understand peptide-DNA assembly. Biophysical and thermodynamic characterization of interaction of these amphiphilic peptides with plasmid DNA is presented. Peptides with at least +4 charges favor stable complex formation. Surface potential is dependent on the type of hydrophobic amino acid for a certain charge. Thermodynamically it is a spontaneous interaction between most of the peptides and plasmid DNA. Lys(7) and Tyr peptides with +4/+5 charges indicate cooperative binding with pDNA without saturation of interaction while Val(2)-Gly-Lys(4), Val-Gly-Lys(5), and Phe-Gly-Lys(5) lead to saturation of interaction indicating condensed pDNA within the range of N/Ps studied. We show that the biophysical properties of DNA-peptide complexes could be modulated by design and the peptides presented here could be used as building blocks for creating DNA-peptide complexes for various biomedical applications, mainly nucleic acid delivery.

  18. Enhanced transformation efficiency of recalcitrant Bacillus cereus and Bacillus weihenstephanensis isolates upon in vitro methylation of plasmid DNA

    NARCIS (Netherlands)

    Nierop Groot, M.N.; Nieboer, F.; Abee, T.

    2008-01-01

    Digestion patterns of chromosomal DNAs of Bacillus cereus and Bacillus weihenstephanensis strains suggest that Sau3AI-type restriction modification systems are widely present among the isolates tested. In vitro methylation of plasmid DNA was used to enhance poor plasmid transfer upon electroporation

  19. Effects of the presence of ColE1 plasmid DNA in Escherichia coli on the host cell metabolism

    Directory of Open Access Journals (Sweden)

    Węgrzyn Alicja

    2006-11-01

    Full Text Available Abstract Background Although understanding of physiological interactions between plasmid DNA and its host is important for vector design and host optimization in many biotechnological applications, to our knowledge, global studies on plasmid-host interactions have not been performed to date even for well-characterized plasmids. Results Escherichia coli cells, either devoid of plasmid DNA or bearing plasmid pOri1 (with a single ColE1 replication origin or plasmid pOri2 (with double ColE1 replication origins, were cultured in a chemostat. We used a combination of metabolic flux analysis, DNA microarray and enzyme activity analysis methods to explore differences in the metabolism between these strains. We found that the presence of plasmids significantly influenced various metabolic pathways in the host cells, e.g. glycolysis, the tricarboxylic acid (TCA cycle and the pentose phosphate (PP pathway. Expression of rpiA, a gene coding for ribose-5-phosphate isomerase A, was considerably decreased in E. coli carrying a high copy number plasmid relative to E. coli carrying a low copy number plasmid and plasmid-free E. coli. The rpiA gene was cloned into an expression vector to construct plasmid pETrpiA. Following induction of pETrpiA-bearing E. coli, which harbored either pOri1 or pOri2, with isopropyl-β-D-thiogalactopyranoside (IPTG, the copy number of pOri1 and pOri2 was sigificantly higher than that measured in a host devoid of pETrpiA. Conclusion The presence of plasmids can significantly influence some metabolic pathways in the host cell. We believe that the results of detailed metabolic analysis may be useful in optimizing host strains, vectors and cultivation conditions for various biotechnological purposes.

  20. Investigation Into the Effects of Nucleotide Content on the Electrical Characteristics of DNA Plasmid Molecular Wires.

    Science.gov (United States)

    Goshi, Noah; Narenji, Alaleh; Bui, Chris; Mokili, John L; Kassegne, Sam

    2016-09-01

    In this study, we investigate the effect of nucleotide content on the conductivity of plasmid length DNA molecular wires covalently bound to high aspect-ratio gold electrodes. The DNA wires were all between [Formula: see text] in length (>6000bp), and contained either 39%, 53%, or 64% GC base-pairs. We compared the current-voltage (I-V) and frequency-impedance characteristics of the DNA wires with varying GC content, and observed statistically significantly higher conductivity in DNA wires containing higher GC content in both AC and DC measurement methods. Additionally, we noted that the conductivity decreased as a function of time for all DNA wires, with the impedance at 100 Hz nearly doubling over a period of seven days. All readings were taken in humidity and temperature controlled environments on DNA wires suspended above an insulative substrate, thus minimizing the effect of experimental and environmental factors as well as potential for nonlinear alternate DNA confirmations. While other groups have studied the effect of GC content on the conductivity of nanoscale DNA molecules (DNA wires at scales that may be required during the fabrication of DNA-based electronics. Furthermore, our results provide further evidence that many of the charge transfer theories developed from experiments using nanoscale DNA molecules may still be applicable for DNA wires at the micro scale.

  1. Investigation of Effects of Nucleotide Content on Electrical Characteristics of DNA Plasmid Molecular Wires.

    Science.gov (United States)

    Goshi, Noah; Narenji, Alaleh; Bui, Chris; Mokili, John L; Kassegne, Sam

    2016-07-28

    In this study, we investigate the effect of nucleotide content on the conductivity of plasmid length DNA molecular wires covalently bound to high aspect-ratio gold electrodes. The DNA wires were all between 2.20-2.35μm in length (>6000bp), and contained either 39%, 53%, or 64% GC base-pairs. We compared the current-voltage (I-V) and frequency-impedance characteristics of the DNA wires with varying GC content, and observed statistically significantly higher conductivity in DNA wires containing higher GC content in both AC and DC measurement methods. Additionally, we noted that the conductivity decreased as a function of time for all DNA wires, with the impedance at 100Hz nearly doubling over a period of seven days. All readings were taken in humidity and temperature controlled environments on DNA wires suspended above an insulative substrate, thus minimizing the effect of experimental and environmental factors as well as potential for nonlinear alternate DNA confirmations. While other groups have studied the effect of GC content on the conductivity of nano-scale DNA molecules (DNA wires at scales that may be required during the fabrication of DNA-based electronics. Furthermore, our results provide further evidence that many of the charge transfer theories developed from experiments using nano-scale DNA molecules may still be applicable for DNA wires at the micro-scale.

  2. The Cloning of the Human Tumor Supressor Gene INGI: DNA Cloning into Plasmid Vector and DNA Analysis by Restriction Enzymes

    Directory of Open Access Journals (Sweden)

    Elza Ibrahim Auerkari

    2015-11-01

    Full Text Available DNA cloning is one of the most important techniques In the field of molecular biology, with a critical role in analyzing the structure and function of genes and their adjacent regulatory regions. DNA cloning is helpful in learning fundamental molecular biological techniques, since DNA cloning involves a series of them, such as polymerase chain reaction (PCR, DNA ligation, bacterial transformation, bacterial culture, plasmid DNA extraction, DNA digestion with restriction enzymes and agarose gel electrophoresis. In this paper the cloning of the human tumor suppressor gene INGI has been used to illustrate the methodology. The gene was amplified by PCR, cloned into a TA-cloning vectore, and restriction enzyme mapping was used to distinguish the sense INGI construct from the antisense INGI construct.

  3. "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.

    Science.gov (United States)

    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.

  4. Exponential megapriming PCR (EMP) cloning--seamless DNA insertion into any target plasmid without sequence constraints.

    Science.gov (United States)

    Ulrich, Alexander; Andersen, Kasper R; Schwartz, Thomas U

    2012-01-01

    We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.

  5. Analysis of heavy-ion-induced DNA strand breaks in plasmid pUC18

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Plasmid DNA was irradiated or implanted by mixed particle field(CR) or lithium-ion-beam to detect strand breaks.The primary results showed that mixed particle field could induce single and double strand breaks with positive linear-dose-effects;most of sequence changes induced by CR were point mutant.Lithium-ion-beam could induce strand breaks also,but it was only at dose of 20Gy.

  6. Exponential megapriming PCR (EMP cloning--seamless DNA insertion into any target plasmid without sequence constraints.

    Directory of Open Access Journals (Sweden)

    Alexander Ulrich

    Full Text Available We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.

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

    Directory of Open Access Journals (Sweden)

    Muhammad Tahir Majeed

    2011-05-01

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

  8. Encapsulation and delivery of plasmid DNA by virus-like nanoparticles engineered from Macrobrachium rosenbergii nodavirus.

    Science.gov (United States)

    Jariyapong, Pitchanee; Chotwiwatthanakun, Charoonroj; Somrit, Monsicha; Jitrapakdee, Sarawut; Xing, Li; Cheng, Holland R; Weerachatyanukul, Wattana

    2014-01-22

    Virus-like particles (VLPs) are potential candidates in developing biological containers for packaging therapeutic or biologically active agents. Here, we expressed Macrobrachium rosenbergii nodavirus (MrNv) capsid protein (encoding amino acids M1-N371 with 6 histidine residuals) in an Escherichia coli BL21(DE3). These easily purified capsid protein self-assembled into VLPs, and disassembly/reassembly could be controlled in a calcium-dependent manner. Physically, MrNv VLPs resisted to digestive enzymes, a property that should be advantageous for protection of active compounds against harsh conditions. We also proved that MrNv VLPs were capable of encapsulating plasmid DNA in the range of 0.035-0.042 mol ratio (DNA/protein) or 2-3 plasmids/VLP (assuming that MrNV VLPs is T=1, i made up of 60 capsid monomers). These VLPs interacted with cultured insect cells and delivered loaded plasmid DNA into the cells as shown by green fluorescent protein (GFP) reporter. With many advantageous properties including self-encapsulation, MrNv VLPs are good candidates for delivery of therapeutic agents.

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

    Science.gov (United States)

    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.

  10. Lichen metabolites prevent UV light and nitric oxide-mediated plasmid DNA damage and induce apoptosis in human melanoma cells.

    Science.gov (United States)

    Russo, A; Piovano, M; Lombardo, L; Garbarino, J; Cardile, V

    2008-09-26

    In humans both UV-A and UV-B can cause gene mutations and suppress immunity, which leads to skin cancer, including melanoma. Inhibition of reactive oxygen species (ROS) and reactive nitrogen species (RNS) appears particularly promising as ROS and RNS production by both UV-A and UV-B contributes to inflammation, immunosuppression, gene mutation and carcinogenesis. We evaluated the effect of two lichen compounds, sphaerophorin (depside) and pannarin (depsidone) on pBR322 DNA cleavage induced by hydroxyl radicals (()OH), and by nitric oxide (NO), and their superoxide anion (O(2)(-)) scavenging capacity. In addition, we investigated the growth inhibitory activity of these compounds against human melanoma cells (M14 cell line). Sphaerophorin and pannarin showed a protective effect on plasmid DNA and exhibited a superoxide dismutase like effect. The data obtained in cell culture show that these lichen metabolites inhibit the growth of melanoma cells, inducing an apoptotic cell death, demonstrated by the fragmentation of genomic DNA (COMET and TUNEL Assays) and by a significant increase of caspase-3 activity, and correlated, at least in part, to the increase of ROS generation, These results confirm the promising biological properties of sphaerophorin and pannarin and encourage further investigations on their molecular mechanisms.

  11. Vaccination with Trypomastigote Surface Antigen 1-Encoding Plasmid DNA Confers Protection against Lethal Trypanosoma cruzi Infection

    OpenAIRE

    1998-01-01

    DNA vaccination was evaluated with the experimental murine model of Trypanosoma cruzi infection as a means to induce antiparasite protective immunity, and the trypomastigote surface antigen 1 (TSA-1), a target of anti-T. cruzi antibody and major histocompatibility complex (MHC) class I-restricted CD8+ cytotoxic T-lymphocyte (CTL) responses, was used as the model antigen. Following the intramuscular immunization of H-2b and H-2d mice with a plasmid DNA encoding an N-terminally truncated TSA-1 ...

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

    Science.gov (United States)

    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.

  13. Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine.

    Directory of Open Access Journals (Sweden)

    Utut Widyastuti Suharsono

    2008-11-01

    Full Text Available Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine. M. affine can grow well in acid soil with high level of soluble aluminum. One of the important proteins in the detoxifying xenobiotic stress including acid and Al stresses is a multidrug resistance associated protein (MRP encoded by mrp gene. The objective of this research is to isolate and clone the cDNA fragment of MaMrp encoding MRP from M. affine. By reverse transcription, total cDNA had been synthesized from the total RNA as template. The fragment of cDNA MaMrp had been successfully isolated by PCR by using total cDNA as template and mrp primer designed from A. thaliana, yeast, and human. This fragment was successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5α. Nucleotide sequence analysis showed that the lenght of MaMrp fragment is 633 bp encoding 208 amino acids. Local alignment analysis based on nucleotide of mRNA showed that MaMrp fragment is 69% identical to AtMrp1 and 63% to AtMrp from A. thaliana. Based on deduced amino acid sequence, MaMRP is 84% identical to part of AtMRP13, 77% to AtMRP12, and 73% to AtMRP1 from A. thaliana respectively. Alignment analysis with AtMRP1 showed that MaMRP fragment is located in TM1 and NBF1 domains and has a specific amino acid sequence QCKAQLQNMEEE.

  14. Local gene delivery via endovascular stents coated with dodecylated chitosan–plasmid DNA nanoparticles

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    Dunwan Zhu

    2010-12-01

    Full Text Available Dunwan Zhu1*, Xu Jin2*, Xigang Leng1, Hai Wang1, Junbo Bao1, Wenguang Liu3, Kangde Yao3, Cunxian Song11Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China; 2Department of Anesthesia and Pain Therapy, Capital Medical University Affiliated Beijing Tiantan Hospital, Beijing, China; 3Research Institute of Polymeric Materials, Tianjin University, Tianjin, China; *Both investigators contributed equally to this work and are senior authors.Abstract: Development of efficacious therapeutic strategies to prevent and inhibit the occurrences of restenosis after percutaneous transluminal coronary angioplasty is critical for the treatment of cardiovascular diseases. In this study, the feasibility and efficiency of stents coated with dodecylated chitosan–plasmid DNA nanoparticles (DCDNPs were evaluated as scaffolds for localized and prolonged delivery of reporter genes into the diseased blood vessel wall. Dodecylated chitosan–plasmid DNA complexes formed stable positive charged nanospheres with mean diameter of approximately 90–180 nm and zeta potential of +28 ± 3 mV. As prepared DCDNPs were spray-coated on stents, a thin layer of dense DCDNPs was successfully distributed onto the metal struts of the endovascular stents as demonstrated by scanning electron microscopy. The DCDNP stents were characterized for the release kinetics of plasmid DNA, and further evaluated for gene delivery and expression both in vitro and in vivo. In cell culture, DCDNP stents containing plasmid EGFP-C1 exhibited high level of GFP expression in cells grown on the stent surface and along the adjacent area. In animal studies, reporter gene activity was observed in the region of the artery in contact with the DCDNP stents, but not in adjacent arterial segments or distal organs. The DCDNP stent provides a very promising strategy for cardiovascular gene therapy

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  17. Condensation of Plasmid DNA Enhances Mitochondrial Association in Skeletal Muscle Following Hydrodynamic Limb Vein Injection

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    Yukari Yasuzaki

    2014-08-01

    Full Text Available Mitochondrial gene therapy and diagnosis have the potential to provide substantial medical benefits. However, the utility of this approach has not yet been realized because the technology available for mitochondrial gene delivery continues to be a bottleneck. We previously reported on mitochondrial gene delivery in skeletal muscle using hydrodynamic limb vein (HLV injection. HLV injection, a useful method for nuclear transgene expression, involves the rapid injection of a large volume of naked plasmid DNA (pDNA. Moreover, the use of a condensed form of pDNA enhances the nuclear transgene expression by the HLV injection. The purpose of this study was to compare naked pDNA and condensed pDNA for mitochondrial association in skeletal muscle, when used in conjunction with HLV injection. PCR analysis showed that the use of condensed pDNA rather than naked pDNA resulted in a more effective mitochondrial association with pDNA, suggesting that the physicochemical state of pDNA plays a key role. Moreover, no mitochondrial toxicities in skeletal muscle following the HLV injection of condensed pDNA were confirmed, as evidenced by cytochrome c oxidase activity and mitochondrial membrane potential. These findings have the potential to contribute to the development for in vivo mitochondrial gene delivery system.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  20. Synthesis and Characterization of Chitosan-Saponin Nanoparticle for Application in Plasmid DNA Delivery

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    Faruku Bande

    2015-01-01

    Full Text Available Nonviral delivery system receives attention over the last decade. Chitosan (CS is a cationic polymer whereas saponin (SP is classified as glycoside. In this study, a spherically-shaped CS-SP nanoparticle was synthesized and characterized. The ability of the nanoparticle to protect DNA from enzymatic degradation, its thermostability and cytotoxicity were evaluated. The particle size was found below 100 nm as determined by Zetasizer, transmission electron microscopy (TEM, and field scanning electron microscopy (FSEM results. The surface charge ranges from 43.7 mV to 38.5 mV before and after encapsulation with DNA plasmid, respectively. In terms of thermostability, Thermal Gravimetric Analysis (TGA and Differential Scanning Calorimetry (DSC revealed that CS-SP nanoparticle had a melting temperature of 110°C, with rapid decomposition occurring at 120°C. Encapsulation of DNA with the synthesized nanoparticle was evidenced by changes in the FTIR spectra including characteristic peaks at 3267.39 and 1635.58 cm−1, wavenumbers. Additional peak was also observed at 1169.7 cm−1 following encapsulation. Electrophoretic mobility showed that CS-SP nanoparticle protected plasmid DNA from enzymatic degradation, while cell viability assays confirmed that the synthesized nanoparticle exhibited low cytotoxicity at different concentrations in avian cells. Taken together these, CS-SP nanoparticle showed potentials for applications as a DNA delivery system.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Sperm DNA fragmentation and oxidation are independent of malondialdheyde

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    Gargouri Jalel

    2011-04-01

    Full Text Available Abstract Background There is clinical evidence to show that sperm DNA damage could be a marker of sperm quality and extensive data exist on the relationship between DNA damage and male fertility status. Detecting such damage in sperm could provide new elements besides semen parameters in diagnosing male infertility. We aimed to assess sperm DNA fragmentation and oxidation and to study the association between these two markers, routine semen parameters and malondialdehyde formation. Methods Semen samples from 55 men attending the Histology-Embryology Laboratory of Sfax Faculty of Medicine, Tunisia, for semen investigations were analysed for sperm DNA fragmentation and oxidation using flow cytometry. The Sperm was also assessed spectrophotometrically for malondialdehyde formation. Results Within the studied group, 21 patients were nonasthenozoospermic (sperm motility ≥ 50% and 34 patients were considered asthenozoospermic (sperm motility Conclusions Our results support the evidence that oxidative stress plays a key role in inducing DNA damage; but nuclear alterations and malondialdehyde don't seem to be synchronous.

  3. Fed-batch microbioreactor platform for scale down and analysis of a plasmid DNA production process.

    Science.gov (United States)

    Bower, Diana M; Lee, Kevin S; Ram, Rajeev J; Prather, Kristala L J

    2012-08-01

    The rising costs of bioprocess research and development emphasize the need for high-throughput, low-cost alternatives to bench-scale bioreactors for process development. In particular, there is a need for platforms that can go beyond simple batch growth of the organism of interest to include more advanced monitoring, control, and operation schemes such as fed-batch or continuous. We have developed a 1-mL microbioreactor capable of monitoring and control of dissolved oxygen, pH, and temperature. Optical density can also be measured online for continuous monitoring of cell growth. To test our microbioreactor platform, we used production of a plasmid DNA vaccine vector (pVAX1-GFP) in Escherichia coli via a fed-batch temperature-inducible process as a model system. We demonstrated that our platform can accurately predict growth, glycerol and acetate concentrations, as well as plasmid copy number and quality obtained in a bench-scale bioreactor. The predictive abilities of the micro-scale system were robust over a range of feed rates as long as key process parameters, such as dissolved oxygen, were kept constant across scales. We have highlighted plasmid DNA production as a potential application for our microbioreactor, but the device has broad utility for microbial process development in other industries as well. Copyright © 2012 Wiley Periodicals, Inc.

  4. Gene Transfer into the Lung by Nanoparticle Dextran-Spermine/Plasmid DNA Complexes

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    Syahril Abdullah

    2010-01-01

    Full Text Available A novel cationic polymer, dextran-spermine (D-SPM, has been found to mediate gene expression in a wide variety of cell lines and in vivo through systemic delivery. Here, we extended the observations by determining the optimal conditions for gene expression of D-SPM/plasmid DNA (D-SPM/pDNA in cell lines and in the lungs of BALB/c mice via instillation delivery. In vitro studies showed that D-SPM could partially protect pDNA from degradation by nuclease and exhibited optimal gene transfer efficiency at D-SPM to pDNA weight-mixing ratio of 12. In the lungs of mice, the levels of gene expression generated by D-SPM/pDNA are highly dependent on the weight-mixing ratio of D-SPM to pDNA, amount of pDNA in the complex, and the assay time postdelivery. Readministration of the complex at day 1 following the first dosing showed no significant effect on the retention and duration of gene expression. The study also showed that there was a clear trend of increasing size of the complexes as the amount of pDNA was increased, where the sizes of the D-SPM/pDNA complexes were within the nanometer range.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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).

  7. Vaccination with trypomastigote surface antigen 1-encoding plasmid DNA confers protection against lethal Trypanosoma cruzi infection.

    Science.gov (United States)

    Wizel, B; Garg, N; Tarleton, R L

    1998-11-01

    DNA vaccination was evaluated with the experimental murine model of Trypanosoma cruzi infection as a means to induce antiparasite protective immunity, and the trypomastigote surface antigen 1 (TSA-1), a target of anti-T. cruzi antibody and major histocompatibility complex (MHC) class I-restricted CD8(+) cytotoxic T-lymphocyte (CTL) responses, was used as the model antigen. Following the intramuscular immunization of H-2(b) and H-2(d) mice with a plasmid DNA encoding an N-terminally truncated TSA-1 lacking or containing the C-terminal nonapeptide tandem repeats, the antibody level, CTL response, and protection against challenge with T. cruzi were assessed. In H-2(b) mice, antiparasite antibodies were induced only by immunization with the DNA construct encoding TSA-1 containing the C-terminal repeats. However, both DNA constructs were efficient in eliciting long-lasting CTL responses against the protective H-2Kb-restricted TSA-1515-522 epitope. In H-2(d) mice, inoculation with either of the two TSA-1-expressing vectors effectively generated antiparasite antibodies and primed CTLs that lysed T. cruzi-infected cells in an antigen-specific, MHC class I-restricted, and CD8(+)-T-cell-dependent manner. When TSA-1 DNA-vaccinated animals were challenged with T. cruzi, 14 of 22 (64%) H-2(b) and 16 of 18 (89%) H-2(d) mice survived the infection. The ability to induce significant murine anti-T. cruzi protective immunity by immunization with plasmid DNA expressing TSA-1 provides the basis for the application of this technology in the design of optimal DNA multicomponent anti-T. cruzi vaccines which may ultimately be used for the prevention or treatment of Chagas' disease.

  8. Specific recognition of supercoiled plasmid DNA by affinity chromatography using the intercalator DAPP as ligand.

    Science.gov (United States)

    Caramelo-Nunes, C; Almeida, P; Marcos, J C; Tomaz, C T

    2013-06-01

    Small molecules that bind DNA with high specificity present a promising opportunity for application as chromatographic ligands for plasmid DNA (pDNA) purification. This research used the intercalator 3,8-diamino-6-phenylphenanthridine (DAPP) as an immobilized ligand for the specific separation of supercoiled (sc) pDNA by affinity chromatography. The results showed that the protonated DAPP-Sepharose support has a great affinity for sc pDNA isoform, separating it from the less active open circular and linear isoforms. All pDNA isoforms were retained in the column using 10mM acetate buffer pH 5. Selective elution of oc and linear isoforms was achieved with 0.22M of sodium chloride in the same buffer. Finally, increasing the concentration to 0.55M led to the elution of the sc isoform. The binding of pDNA to DAPP-Sepharose varies in function of pH, and the stability of the protonated DAPP-DNA complex decreases with increasing salt concentration. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. A comparison of synthetic oligodeoxynucleotides, DNA fragments and AAV-1 for targeted episomal and chromosomal gene repair

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    Leclerc Xavier

    2009-04-01

    Full Text Available Abstract Background Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. An attractive alternative to these approaches would be to correct the endogenous mutated gene in the affected individual. This study presents a quantitative comparison of the repair efficiency using different forms of donor nucleic acids, including synthetic DNA oligonucleotides, double stranded DNA fragments with sizes ranging from 200 to 2200 bp and sequences carried by a recombinant adeno-associated virus (rAAV-1. Evaluation of each gene repair strategy was carried out using two different reporter systems, a mutated eGFP gene or a dual construct with a functional eGFP and an inactive luciferase gene, in several different cell systems. Gene targeting events were scored either following transient co-transfection of reporter plasmids and donor DNAs, or in a system where a reporter construct was stably integrated into the chromosome. Results In both episomal and chromosomal assays, DNA fragments were more efficient at gene repair than oligonucleotides or rAAV-1. Furthermore, the gene targeting frequency could be significantly increased by using DNA repair stimulating drugs such as doxorubicin and phleomycin. Conclusion Our results show that it is possible to obtain repair frequencies of 1% of the transfected cell population under optimized transfection protocols when cells were pretreated with phleomycin using rAAV-1 and dsDNA fragments.

  10. Small DNA pieces in C. elegans are intermediates of DNA fragmentation during apoptosis.

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    P Joseph Aruscavage

    Full Text Available While studying small noncoding RNA in C. elegans, we discovered that protocols used for isolation of RNA are contaminated with small DNA pieces. After electrophoresis on a denaturing gel, the DNA fragments appear as a ladder of bands, approximately 10 nucleotides apart, mimicking the pattern of nuclease digestion of DNA wrapped around a nucleosome. Here we show that the small DNA pieces are products of the DNA fragmentation that occurs during apoptosis, and correspondingly, are absent in mutant strains incapable of apoptosis. In contrast, the small DNA pieces are present in strains defective for the engulfment process of apoptosis, suggesting they are produced in the dying cell prior to engulfment. While the small DNA pieces are also present in a number of strains with mutations in predicted nucleases, they are undetectable in strains containing mutations in nuc-1, which encodes a DNase II endonuclease. We find that the small DNA pieces can be labeled with terminal deoxynucleotidyltransferase only after phosphatase treatment, as expected if they are products of DNase II cleavage, which generates a 3' phosphate. Our studies reveal a previously unknown intermediate in the process of apoptotic DNA fragmentation and thus bring us closer to defining this important pathway.

  11. Design and preparation of cloned DNA fragment from pac gene of Streptococcus mutans

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    目的:为克隆构建DNA防龋疫苗制备出表达片段.方法: 酶切电泳鉴定含pac基因的重组质粒;用计算机辅助选定pac基因中重要区域并设计引物, 对其进行PCR扩增,酶切电泳鉴定扩增产物.结果:pPC41重组质粒包含pac 基因; 自pac基因中选定的核苷酸序列为模板所扩增的DNA片段为预期目的片段.结论:对含pac基因重要抗原决定簇区域的成功扩增为构建基因重组防龋疫苗完成了重要的准备工作.%Objective:To prepare the expressed DNA fragment for the co nstr uction of anti-caries DNA vaccine. Methods:To confirm that pPC 41,a recombinant plasmid,contains pac gene by way of restriction enzyme digestio n and electrophoresis;To choose important regions in pac gene and to design prim ers with computer, and then to amplify DNA sample. Restriction enzyme digestion and electrophoresis were used to identify PCR products. Results:pPC41 was verified to contain pac gene.The DNA fragment amplified from pac gen e was expected one. Conclusion:The successful PCR amplification to important regions in pac gene is a significant preparation for the construct ion of recombinant anti-caries vaccine.

  12. Plasmid cloning vehicle for Haemophilus influenzae and Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.; Clayton, N.L.; Setlow, J.K.

    1982-09-01

    A new plasmid cloning vehicle (pDM2) was used to introduce a library of Haemophilus influenzae chromosomal fragments into H. influenzae. Transformants of the higly recombination-defective rec-1 mutant were more likely to contain exclusively recombinant plasmids after exposure to ligated DNA mixtures than was the wild type. pDM2 could replicate in Escherichia coli K-12.

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

    Science.gov (United States)

    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.

  14. Aqueous extract of Pinus caribaea inhibits the damage induced by ultraviolet radiations, in plasmid DNA

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    Marioly Vernhes Tamayo

    2017-08-01

    Full Text Available Context: The incidence of solar ultraviolet radiation (UV on Earth has increased due to diminish of the ozone layer. This enviromental agent is highly genotoxic causing numerous damage in DNA molecule. Nowadays there is a growing interest in the search of compounds capable to minimize these effects. In particular, phytocompounds have been tested as excelent candidates for their antigenotoxic properties. Aims: To evaluate the protective effect of the aqueous extract of Pinus caribaea (EPC against the damage induced by the UVB and UVC radiation. Methods: The cell-free plasmid DNA assay was employed. The forms of plasmid were separated electrophoretically in agarose gel. For genotoxic and photoprotective evaluation of P. caribaea, different concentrations of the extract (0.1 – 2.0 mg/mL and exposure times were evaluated. The CPD lesions were detected enzymatically. Additionally, the transmittance of the aqueous extract against 254 nm and 312 nm was measured. Results: None of the concentrations were genotoxic in 30 min of treatment, for superior times a clastogenic effect was observed. The EPC despite inhibiting the activity of the enzyme T4 endo V, impedes photolesions formation in DNA at concentrations ≥ 0.1 mg/mL. Conclusions: The EPC has photoprotective properties, this effect could be related with its antioxidants and absorptives capacities.

  15. Optimization of a lipitoid-based plasmid DNA transfection protocol for bovine trophectoderm CT-1 cells.

    Science.gov (United States)

    Schiffmacher, Andrew T; Keefer, Carol L

    2012-08-01

    Embryo-derived cell lines are important in vitro models for investigating the molecular mechanisms directing embryonic tissue lineage segregation and maintenance. The bovine trophectoderm-derived CT-1 cell line has been widely used to identify regulatory mechanisms of interferon tau gene expression, and it possesses potential as a model for characterizing the gene regulatory network controlling trophoblast lineage differentiation and development. This functional potential, however, is severely limited as CT-1 cells are very recalcitrant to standard transfection methods. The focus of this study was to test the cationic lipitoid reagent as an effective transfection reagent for DNA plasmid delivery. Optimization of liptoid-based transfection of plasmid DNA resulted in 9% transfection efficiency averaged across entire CT-1 colonies, with many subregions of CT-1 colonies achieving transfection rates of 15%. These rates are a substantial improvement over near-zero efficiencies achieved using other standard transfection techniques. CT-1 cells were also successfully adapted to substrate-free culture for over 20 passages, eliminating the need to culture CT-1 colonies on feeder cells or matrix-coated cultureware. Together, these results increase the utility of the CT-1 cell line as an in vitro bovine trophoblast model and provide insight into overcoming DNA delivery difficulties in other cell lines not amenable to genetic manipulation.

  16. A bimetallic nanocomposite electrode for direct and rapid biosensing of p53 DNA plasmid

    Indian Academy of Sciences (India)

    Ezat Hamidi-Asl; Jahan-Bakhsh Raoof; Nahid Naghizadeh; Simin Sharifi; Mohammad Saeid Hejazi

    2015-09-01

    A new label-free electrochemical DNA biosensor is presented based on carbon paste electrode (CPE) modified with gold (Au) and platinum (Pt) nanoparticles to prepare the bimetallic nanocomposite electrode. The proposed sensor was made by immobilization of 15-mer single stranded oligonucleotide probe related to p53 gene for detection of DNA plasmid samples. The hybridization detection relied on the alternation in the guanine oxidation signal following hybridization of the probe with complementary genomic DNA.The technique of differential pulse voltammetry (DPV) was used for monitoring guanine oxidation. To optimize the performance of the modified CPE, different electrodes were prepared in various percentages of Au and Pt nanoparticles. The modified electrode containing 15% Au/Pt bimetallic nanoparticles (15% Au/Pt-MCPE) was selected as the best working electrode. The selectivity of the sensor was investigated using plasmid samples containing non-complementary oligonucleotides. The detection limit of the biosensor was studied and calculated to be 53.10 pg L−1.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  20. [Localization of denitrification genes in plasmid DNA of bacteria Azospirillum brasilense].

    Science.gov (United States)

    Petrova, L P; Varshalomidze, O É; Shelud'ko, A V; Katsy, E I

    2010-07-01

    In 85-Mda plasmid (p85) of plant-associated bacteria Azospirillum brasilense Sp245 model strain, the genes encoding copper-containing nitrite reductase (nirK); heterodimeric NO-reductase (norCB); NorQ and NorD proteins affecting synthesis and (or) activation of NirK and (or) NO-reductase (norQD); catalytic subunit I ofcytochrom c oxidase (CccoN); presumable NO sensor carrying two hemeerythrine domains (orf181); and an enzyme required for synthesis of presumable NO antagonist, homocystein (metC) were identified. In the same region of p85, orf293 encoding transcriptional regulator of LysR type, orf208 whose protein product carries a formylmethanofuran dehydrogenase subunit E domain, and an orf164-encoding conservative secretory protein with unknown function were also found. Localization of a set of denitrification genes in the plasmid DNA A. brasilense Sp245 adjacent to IS elements ISAzba1 and ISAzba2 indicates potential mobility of these genes and high probability of their horizontal transfer among populations of rhizospheric bacteria. A site homologous to p85 nirK-orf208-orf181 genes was detected in the 115 kb plasmid of A. brasilense Sp7 type strain.

  1. CONSECUTIVE IMMUNIZATION WITH RECOMBINANT FOWLPOX VIRUS AND PLASMID DNA FOR ENHANCING CELLULAR AND HUMORAL IMMUNITY

    Institute of Scientific and Technical Information of China (English)

    罗坤; 金宁一; 郭志儒; 秦云龙; 郭炎; 方厚华; 安汝国; 殷震

    2001-01-01

    To investigate the influence of consecutive immunization on cellular and humoral immunity in mice. Methods: We evaluated a consecutive immunization strategy of priming with recombinant fowlpox virus vUTALG and boosting with plasmid DNA pcDNAG encoding HIV-1 capsid protein Gag. Results: In immunized mice, the number of CD4+ T cells from splenic lymphocytes increased significantly and the proliferation response of splenocytes to ConA and LPS elevated markedly and HIV-1-specific antibody response could be induced. Conclusion: Consecutive immunization could increase cellular and humoral immunity responses in mice.

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

    OpenAIRE

    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 s...

  3. Transgenic Crops by Direct Treatment of Exogenous DNA Without Agrobacterium tumefaciens Plasmid and Tissue Culture

    Institute of Scientific and Technical Information of China (English)

    ZhangGuodong

    1995-01-01

    Gene transfter methods are developing quickly recently,but each method has its limitations.We introduce a new gene transfer technique in this paper,which is simple,effective,and easy to operate,but does not get enough attention from scientists.This technique is used to transform plants by injecting exogenous DNA to stigma,style,ovary,young fruit or meristem of the recipient,or soaking the recipient's seeds in exogenous DNA solution.Los of heritable variations were found in many characters of many crops,It may be used to creaste new germplasms or realize gene exchange between different species,gerera,or families,even between animals and plants,A brief discussion was given to the mechanism of exogenous DNA introduction,integration into and expression in the recipient.We also discussed the merits and limitations of the technique.Currently there are two successful approaches that can be used to transform plants genetically,but each method has its limitations that are delaying the application of the techniques to certaincommercially important crops.The first tecnhique exploits a natural genetic engineer,Agrobacterium tumefaciens,which contains a tumor-inducing(Ti) plasmid that transfers a DNA segment(the T-DNA) from the plasmid to the nuclear genome of infected plants(or in vitro to plant tissue).The method is restricted to dicotyledenous plants;monocotyledenous plants are usually not susceptible to agrobacterial infection.The second technique involves direct transfter of DNA to plant protoplast ,prepared by enzymatic digestion of cell walls,for example by chemically stimulated uptake using polyethylene glycol or a high voltage pulse,generating transient'holes'in the protoplast membrane.This technique depends on a tissue culture system that allows regeneration of mature plants from protoplasts,But so far it is impossible to achieve plant regeneration from protoplasts in many crops.Both techniques use dominant selectable markers(for example,kanamycin resistance) to

  4. Poly(hydroxyethyl methacrylate) based magnetic nanoparticles for plasmid DNA purification from Escherichia coli lysate

    Energy Technology Data Exchange (ETDEWEB)

    Percin, Is Latin-Small-Letter-Dotless-I k [Department of Biology, Hacettepe University, Ankara (Turkey); Karakoc, Veyis [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey); Akgoel, Sinan [Department of Biochemistry, Ege University, Izmir (Turkey); Aksoez, Erol [Department of Biology, Hacettepe University, Ankara (Turkey); Denizli, Adil, E-mail: denizli@hacettepe.edu.tr [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey)

    2012-07-01

    The aim of this study is to prepare poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine) [PHEMAH] magnetic nanoparticles for plasmid DNA (pDNA) purification from Escherichia coli (E. coli) cell lysate. Magnetic nanoparticles were produced by surfactant free emulsion polymerization. mPHEMAH nanoparticles were characterized by elemental analysis, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), vibrating sample magnetometer (VSM), electron spin resonance (ESR), thermogravimetric analyses (TGA) and transmission electron microscopy (TEM). Surface area, average particle size and size distribution were also performed. Specific surface area of the mPHEMAH nanoparticles was found to be 1180 m{sup 2}/g. Elemental analysis of MAH for nitrogen was estimated as 0.18 mmol/g polymer. The amount of pDNA adsorbed onto the mPHEMAH nanoparticles first increased and then reached a saturation value at around 1.0 mg/mL of pDNA concentration. Compared with the mPHEMA nanoparticles (50 {mu}g/g polymer), the pDNA adsorption capacity of the mPHEMAH nanoparticles (154 mg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The maximum pDNA adsorption was achieved at 25 Degree-Sign C. The overall recovery of pDNA was calculated as 92%. The mPHEMAH nanoparticles could be used six times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH nanoparticles promise high selectivity for pDNA. - Highlights: Black-Right-Pointing-Pointer Magnetic nanoparticles have several advantages over conventional adsorbents. Black-Right-Pointing-Pointer MAH acted as the pseudospecific ligand, ligand immobilization step was eliminated. Black-Right-Pointing-Pointer pDNA adsorption amount was 154 mg/g. Black-Right-Pointing-Pointer Fifty-fold capacity increase was obtained when compared to conventional matrices.

  5. Cloning of DNA fragments: ligation reactions in agarose gel.

    Science.gov (United States)

    Furtado, Agnelo

    2014-01-01

    Ligation reactions to ligate a desired DNA fragment into a vector can be challenging to beginners and especially if the amount of the insert is limiting. Although additives known as crowding agents, such as PEG 8000, added to the ligation mixes can increase the success one has with ligation reactions, in practice the amount of insert used in the ligation can determine the success or the failure of the ligation reaction. The method described here, which uses insert DNA in gel slice added directly into the ligation reaction, has two benefits: (a) using agarose as the crowding agent and (b) reducing steps of insert purification. The use of rapid ligation buffer and incubation of the ligation reaction at room temperature greatly increase the efficiency of the ligation reaction even for blunt-ended ligation.

  6. Efficient expression of human factor Ⅸ cDNA in livermediated by hydrodynamics-based plasmid administration

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Hydrodynamics-based administration via tail vein was used to deliver naked plasmid with human factor Ⅸ (hFⅨ) cDNA in 2.2 mL Ringer's solution into mice within 7 s. The peak level of expression of hFⅨ was 2921 ng/mL in mouse plasma. The hFⅨ cDNA expression increased with increasing the amount of plasmid DNA injected. The peak level of gene expression declined after repeated injection of plasmid (1459 ng/mL). The hFⅨ cDNA was detected in various organs, but the highest level of gene expression appeared in liver. Transaminase levels and liver histological results showed that rapid intravenous plasmid injection into mice induced transient focal acute liver damage, which was rapidly repaired within 3-10 d. These results suggested that high-level expression of hFⅨ cDNA can be achieved by hydrodynamics-based plasmid transfer and this method is now further used for gene therapy and gene function study in our lab.

  7. Cavitation Enhancing Nanodroplets Mediate Efficient DNA Fragmentation in a Bench Top Ultrasonic Water Bath.

    Directory of Open Access Journals (Sweden)

    Sandeep K Kasoji

    Full Text Available A perfluorocarbon nanodroplet formulation is shown to be an effective cavitation enhancement agent, enabling rapid and consistent fragmentation of genomic DNA in a standard ultrasonic water bath. This nanodroplet-enhanced method produces genomic DNA libraries and next-generation sequencing results indistinguishable from DNA samples fragmented in dedicated commercial acoustic sonication equipment, and with higher throughput. This technique thus enables widespread access to fast bench-top genomic DNA fragmentation.

  8. Cavitation Enhancing Nanodroplets Mediate Efficient DNA Fragmentation in a Bench Top Ultrasonic Water Bath

    Science.gov (United States)

    Malc, Ewa P.; Jayakody, Chatura N.; Tsuruta, James K.; Mieczkowski, Piotr A.; Janzen, William P.; Dayton, Paul A.

    2015-01-01

    A perfluorocarbon nanodroplet formulation is shown to be an effective cavitation enhancement agent, enabling rapid and consistent fragmentation of genomic DNA in a standard ultrasonic water bath. This nanodroplet-enhanced method produces genomic DNA libraries and next-generation sequencing results indistinguishable from DNA samples fragmented in dedicated commercial acoustic sonication equipment, and with higher throughput. This technique thus enables widespread access to fast bench-top genomic DNA fragmentation. PMID:26186461

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Jokinen, Jenny; Lukashevich, Igor S. [Department of Pharmacology and Toxicology, School of Medicine, Center for Predictive Medicine and Emerging Infectious Diseases, University of Louisville, Louisville, KY (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States)

    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.

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

    Science.gov (United States)

    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.

  13. Validation and scale-up of plasmid DNA purification by phenyl-boronic acid chromatography.

    Science.gov (United States)

    Gomes, A Gabriela; Azevedo, Ana M; Aires-Barros, M Raquel; Prazeres, D Miguel F

    2012-11-01

    This study addresses the feasibility of scaling-up the removal of host cell impurities from plasmid DNA (pDNA)-containing Escherichia coli lysates by phenyl-boronic (PB) acid chromatography using columns packed with 7.6 and 15.2 cm(3) of controlled porous glass beads (CPG) derivatized with PB ligands. Equilibration was performed with water at 10 cm(3) /min and no conditioning of the lysate feed was required. At a ratio of lysate feed to adsorbent volume of 1.3, 93-96% of pDNA was recovered in the flow through while 66-71% of impurities remained bound (~2.5-fold purification). The entire sequence of loading, washing, elution, and re-equilibration was completed in 20 min. Run-to-run consistency was observed in terms of chromatogram features and performance (yield, purification factor, agarose electrophoresis) across the different amounts of adsorbent (0.75-15.2 cm(3) ) by performing successive injections of lysates prepared independently and containing 3.7 or 6.1 kbp plasmids. The column productivity at large scale was 4 dm(3) of alkaline lysate per hour per dm(3) of PB-CPG resin. The method is rapid, reproducible, simple, and straightforward to scale-up. Furthermore, it is capable of handling heavily contaminated samples, constituting a good alternative to purification techniques such as isopropanol precipitation, aqueous two-phase systems, and tangential flow filtration. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Charge density and particle size effects on oligonucleotide and plasmid DNA binding to nanosized hydrotalcite.

    Science.gov (United States)

    Sanderson, Brian A; Sowersby, Drew S; Crosby, Sergio; Goss, Marcus; Lewis, L Kevin; Beall, Gary W

    2013-12-01

    Hydrotalcite (HT) and other layered double metal hydroxides are of great interest as gene delivery and timed release drug delivery systems and as enteric vehicles for biologically active molecules that are sensitive to gastric fluids. HT is a naturally occurring double metal hydroxide that can be synthesized as a nanomaterial consisting of a brucite structure with isomorphous substitution of aluminum ions. These positively charged nanoparticles exhibit plate-like morphology with very high aspect ratios. Biomolecules such as nucleic acids and proteins form strong associations with HT because they can associate with the positively charged layers. The binding of nucleic acids with HT and other nanomaterials is currently being investigated for potential use in gene therapy; however, the binding of specific nucleic acid forms, such as single- and double-stranded DNA, has been little explored. In addition, the effects of charge density and particle size on DNA adsorption has not been studied. In this paper, the binding of different forms of DNA to a series of HTs prepared at different temperatures and with different anion exchange capacities has been investigated. Experiments demonstrated that HTs synthesized at higher temperatures associate with both single- and double-stranded oligomers and circular plasmid DNA more tightly than HTs synthesized at room temperature, likely due to the hydrothermal conditions promoting larger particle sizes. HT with an anion exchange capacity of 300 meq/100 g demonstrated the highest binding of DNA, likely due to the closer match of charge densities between the HT and DNA. The details of the interaction of various forms of DNA with HT as a function of charge density, particle size, and concentration are discussed.

  15. Spermatozoal cell death-inducing DNA fragmentation factor-α-like effector A (CIDEA) gene expression and DNA fragmentation in infertile men with metabolic syndrome and normal seminogram.

    Science.gov (United States)

    Elsamanoudy, Ayman Z; Abdalla, Hussein Abdelaziz; Hassanien, Mohammed; Gaballah, Mohammad A

    2016-01-01

    This is the first study to investigate spermatozoal cell death-inducing DNA fragmentation factor-α-like effector A (CIDEA) gene expression and DNA fragmentations in the spermatozoa of men diagnosed with metabolic syndrome (MS) who have normal seminograms with unexplained infertility, and to correlate these parameters with seminal glucose concentration. This study included 120 participants: 75 male subjects with MS (38 fertile and 37 infertile), and a control group of 45 fertile males without MS. HOMA-IR, semen analysis, and biochemical measurement of seminal plasma insulin and glucose levels were carried out. Spermatozoal insulin gene and CIDEA gene expressions were performed by the RT-PCR method. The percentage of spermatozoal DNA fragmentation was also estimated. The spermatozoal insulin and CIDEA gene expression, as well as the DNA fragmentation, were significantly higher in the infertile MS group than in the fertile MS group, and significantly higher in both the MS groups than in the control group. Seminal glucose concentration showed significant positive correlations with seminal insulin level, spermatozoa insulin, CIDEA gene expression, and DNA fragmentation. Moreover, there was a positive correlation between spermatozoa CIDEA gene expression and DNA fragmentation. It can be concluded that MS may affect male fertility at the molecular level, through its possible inducing effect of spermatozoa CIDEA and insulin gene expression, DNA fragmentation, and increased seminal glucose.

  16. Phototransfection of mouse embryonic stem cells with plasmid DNA using femtosecond laser pulses

    Science.gov (United States)

    Thobakgale, Lebogang; Manoto, Sello Lebohang; Ombinda Lemboumba, Saturnin; Maaza, Malik; Mthunzi-Kufa, Patience

    2017-02-01

    Cellular manipulation by delivery of molecules into cells has been applied extensively in tissue engineering research for medical applications . The different molecular delivery techniques used range from viral and chemical agents to physical and electrical methods. Although successful in most studies, these techniques have inherent difficulties such as toxicity, unwanted genetic mutations and low reproducibility respectively. Literature recognizes pulsed lasers at femtosecond level to be most efficient in photonic interactions with biological material. As of late, laser pulses have been used for drug and DNA delivery into cells via transient optical perforation of the cellular membrane. Thus in this study, we design and construct an optical system coupled to a femtosecond laser for the purpose of phototransfection or insertion of plasmid DNA (pDNA) into cells using lasers. We used fluorescent green protein (pGFP) to transfect mouse embryonic stem cells as our model. Secondly, we applied fluorescence imaging to view the extent of DNA delivery using this method. We also assessed the biocompatibility of our system by performing molecular assays of the cells post irradiation using adenosine triphosphate (ATP) and lactate dehydrogenase (LDH).

  17. Bacillus stearothermophilus contains a plasmid-borne gene for alpha-amylase.

    Science.gov (United States)

    Mielenz, J R

    1983-01-01

    The gene for thermostable alpha-amylase from the thermophilic bacterium Bacillus stearothermophilus has been cloned and expressed in Escherichia coli. Each alpha-amylase-producing colony contained at least a 9.7-kilobase-pair (kb) chimeric plasmid composed of the vector pBR322 and a common 5.4-kb HindIII fragment of DNA. B. stearothermophilus contains four plasmids with sizes from 12 kb to over 108 kb. Restriction endonuclease analysis of these naturally occurring plasmids showed they also contain a 5.4-kb HindIII fragment of DNA. Cloning experiments with the four plasmids yielded alpha-amylase-producing E. coli that contained the same 9.7-kb chimeric plasmid. Restriction endonuclease analysis and further recombinant DNA experiments identified a 26-kb plasmid that contains the gene for alpha-amylase. A spontaneous mutant of B. stearothermophilus unable to produce alpha-amylase was missing the 26-kb plasmid but contained a 20-kb plasmid. A 6-kb deletion within the region of the 5.4-kb HindIII fragment yielded the 20-kb plasmid unable to code for alpha-amylase. A nick-translated probe for the alpha-amylase coding region did not hybridize to either plasmid or total cellular DNA from this mutant strain of B. stearothermophilus. These results demonstrate the gene for alpha-amylase is located exclusively on a 26-kb plasmid in B. stearothermophilus with no genetic counterpart present on the chromosome. Images PMID:6193526

  18. Plasmid Biopharmaceuticals.

    Science.gov (United States)

    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.

  19. Linear forms of plasmid DNA are superior to supercoiled structures as active templates for gene expression in plant protoplasts.

    Science.gov (United States)

    Ballas, N; Zakai, N; Friedberg, D; Loyter, A

    1988-07-01

    Introduction of the plasmids pUC8CaMVCAT and pNOSCAT into plant protoplasts is known to result in transient expression of the chloramphenicol acetyl transferase (CAT) gene. Also, transfection with the plasmid pDO432 results in transient appearance of the luciferase enzyme. In the present work we have used these systems to study the effect of DNA topology on the expression of the above recombinant genes. Linear forms of the above plasmids exhibited much higher activity in supporting gene expression than their corresponding super-coiled structures. CAT activity in protoplasts transfected with the linear forms of pUC8CaMVCAT and pNOSCAT was up to ten-fold higher than that observed in protoplasts transfected by the supercoiled template of these plasmids. This effect was observed in protoplasts derived from two different lines of Petunia hybrida and from a Nicotiana tabacum cell line. Transfection with the relaxed form of pUC8CaMVCAT resulted in very low expression of the CAT gene.Northern blot analysis revealed that the amount of poly(A)(+) RNA extracted from protoplasts transformed with the linear forms of the DNA was about 10-fold higher than that found in protoplasts transformed with supercoiled DNA.Southern blot analysis revealed that about the same amounts of supercoiled and linear DNA molecules were present in nuclei of transfected protoplasts. No significant quantitative differences have been observed between the degradation rates of the various DNA templates used.

  20. Vaccination of Plasmid DNA Encoding Somatostatin Gene Fused with GP5 Gene of Porcine Reproductive and Respiratory Syndrome Virus Induces Anti-GP5 Antibodies and Promotes Growth Performance in Immunized Pigs

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Somatostatin (SS) is a hormone that inhibits the secretion of growth hormone. Immunization against SS can promote the growth of animals. This paper described the effects of DNA immunization on the growth and antibody response in mice and pigs immunized with a plasmid DNA encoding SS fused with GP5 of porcine reproductive and respiratory syndrome virus (PRRSV). A fragment of 180 bp encoding partial SS gene was amplified by PCR from the genomic DNA of peripheral blood mononuclear cells of pigs, and cloned as a fusion gene with PRRSV GP5 in plasmid pISGRTK3. Three times of immunization with the resulting plasmid pISG-SS/GP5 induced anti-GP5 antibodies in BALB/c mice and pigs, as demonstrated by GP5-specific ELISA and immunoblotting. Compared with pigs immunized with empty vector pISGRTK3, the growth performance of pigs immunized with pISG-SS/GP5 was increased by 11.1% on the 13th week after the last vaccination. The results indicated the plasmid DNA encoding SS and PRRSV GP5 fusion gene elicited anti-GP5 antibodies and improved the growth performance of immunized pigs.

  1. INVESTIGATION OF POLYDL-LACTIDE-b-POLY(ETHYLENE GLYCOL)-b-POLYDL-LACTIDE MICROSPHERES CONTAINING PLASMID DNA

    Institute of Scientific and Technical Information of China (English)

    Yu Liu; Long Huang; Xiao-rong Qiao; Xian-mo Deng; Wen-xiang Jia; Xiao-hong Li

    2004-01-01

    PolyDL-lactide (PDLLA) and the block copolymer, polyDL-lactide-b-poly(ethylene glycol)-b-polyDL-lactide (PELA) were used as the microsphere matrix to encapsulate plasmid DNA. The PDLLA, PELA, pBR322-1oaded PDLLA and pBR322-1oaded PELA microspheres were prepared by solvent extraction method based on the formation of multiple w1/o/w2 emulsion. The microspheres were characterized by surface morphology, mean particle size, particle size distribution and loading efficiency. The integrity of DNA molecules after being extracted from microspheres was determined by agarose gel electrophoresis. The result suggested that plasmid DNA molecules could retain their integrity after being encapsulated by PELA. The PELA microspheres could prevent plasmid DNA from being digested by DNase. The in vitro degradation and release profiles of plasmid DNA-loaded microspheres were measured in pH = 7.4 buffer solution at 37℃. The in vitro degradation profiles of the microspheres were evaluated by the deterioration in microspheres surface morphology, the molecular weight reduction of polymer, the mass loss of microspheres, the changes of pH values of degradation medium, and the changes of particle size. The in vitro release profiles of the microspheres were assessed by measurement of the amount of DNA presented in the release medium at determined intervals. The release profiles were correlation with the degradation profiles. The release of plasmid DNA from PELA microspheres showed a similar biphasic trend, that is, an initial burst release was followed by a slow, but sustained release.

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

    Directory of Open Access Journals (Sweden)

    Nolte Ingo

    2011-10-01

    Full Text Available Abstract 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

  3. Experimental Study of Plasmid TGF-β1 DNA Gene Transfer with Lipofectamine into Rabbit Corneal Epithelial Cells In Vitro

    Institute of Scientific and Technical Information of China (English)

    黄琼; 胡燕华; 姜发纲; 陈宏

    2002-01-01

    To investigate whether the TGF-β1 plasmid DNA carried by lipofectamine could be introduced into cultured rabbit corneal epithelial cells, specific expression of the plasmid pMAM TGF-β1in the cultured corneal epithelial cells was studied. Two days after 12 h of transfection of pMAMTGF-β1 mediated by lipofectamine into the cultured corneal epithelial cells, the TGF-β1 protein expression specific for pMAMTGF-β1 in the cells was detected by means of immunohistochemical staining and the positive rate was 23. 37 %. The results suggested that foreign plasmid DNA could be effectively delivered into cultured rabbit corneal epithelial cells by means of lipofectamine, and this will provide a promising method of studying TGF-β1 on the mechanism of physiology and pathology concerned with corneal epithelial cells.

  4. DNA sequence analysis of plasmids from multidrug resistant Salmonella enterica serotype Heidelberg isolates.

    Directory of Open Access Journals (Sweden)

    Jing Han

    Full Text Available Salmonella enterica serovar Heidelberg is among the most detected serovars in swine and poultry, ranks among the top five serotypes associated with human salmonellosis and is disproportionately associated with invasive infections and mortality in humans. Salmonella are known to carry plasmids associated with antimicrobial resistance and virulence. To identify plasmid-associated genes in multidrug resistant S. enterica serovar Heidelberg, antimicrobial resistance plasmids from five isolates were sequenced using the 454 LifeSciences pyrosequencing technology. Four of the isolates contained incompatibility group (Inc A/C multidrug resistance plasmids harboring at least eight antimicrobial resistance genes. Each of these strains also carried a second resistance plasmid including two IncFIB, an IncHI2 and a plasmid lacking an identified Inc group. The fifth isolate contained an IncI1 plasmid, encoding resistance to gentamicin, streptomycin and sulfonamides. Some of the IncA/C plasmids lacked the full concert of transfer genes and yet were able to be conjugally transferred, likely due to the transfer genes carried on the companion plasmids in the strains. Several non-IncA/C resistance plasmids also carried putative virulence genes. When the sequences were compared to previously sequenced plasmids, it was found that while all plasmids demonstrated some similarity to other plasmids, they were unique, often due to differences in mobile genetic elements in the plasmids. Our study suggests that Salmonella Heidelberg isolates harbor plasmids that co-select for antimicrobial resistance and virulence, along with genes that can mediate the transfer of plasmids within and among other bacterial isolates. Prevalence of such plasmids can complicate efforts to control the spread of S. enterica serovar Heidelberg in food animal and human populations.

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

    Science.gov (United States)

    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

  6. Preparation and characterization of chitosan/β-cyclodextrin nanoparticles containing plasmid DNA encoding interleukin-12.

    Science.gov (United States)

    Nahaei, M; Valizadeh, H; Baradaran, B; Nahaei, M R; Asgari, D; Hallaj-Nezhadi, S; Dastmalchi, S; Lotfipour, F

    2013-01-01

    Interleukin-12 (IL-12) as a cytokine has been proved to possess antitumor effects via stimulating the immune system. Non-viral gene delivery systems offer several advantages, including easiness in production, low cost, safety; low immunogenicity and can carry higher amounts of genetic material without limitation on their sizes.pUMVC3-hIL12 loaded Low Molecular Weight chitosan/β-cyclodextrin (LMW CS/CD) nanoparticles were prepared using ionotropic gelation method and characterized in terms of size, zeta potential, polydispersity index, morphology, loading efficiency and cytotoxicity against the CT-26 colon carcinoma cell line.All prepared particles were spherical in shape and nano-sized (171.3±2.165 nm, PdI: 0.231±0.014) and exhibited a positive zeta potential (34.3±1.55). The nanoparticles demonstrated good DNA encapsulation efficiencies (83.315%±2.067). Prepared pUMVC3-hIL12 loaded LMW CS/CD nanoparticles showed no cell toxicity in murine CT-26 colon carcinoma cells. At the concentration of 0.1 µg/ml of nanoparticles, the transfection ability was obviously higher than that of the naked DNA.LMW CS/CD-plasmid DNA nanoparticles encoding IL-12 prepared using ionotropic gelation method with no toxic effect on the tested cells can be considered as a basis for further gene delivery studies both in vitro and in vivo to enhance the expression of IL-12.

  7. Sperm DNA fragmentation is related to sperm morphological staining patterns.

    Science.gov (United States)

    Sá, Rosália; Cunha, Mariana; Rocha, Eduardo; Barros, Alberto; Sousa, Mário

    2015-10-01

    In this prospective comparative study, sperm DNA fragmentation (sDNAfrag) was compared at each step of a sequential semen preparation, with semen parameters according to their degree of severity. At each step (fractions) of the sequential procedure, sDNAfrag was determined: fresh (Raw), after gradient centrifugation, washing, and swim-up (SU) for 70 infertile men enrolled in intracytoplasmic sperm injection cycles. sDNAfrag significantly (P = 0.04; P < 0.0001) decreased throughout the steps of semen preparation, with centrifugation and washing not increasing it. A negative correlation to sperm motility was observed in Raw and SU fractions, and a higher sDNAfrag was observed in samples with lower semen quality. Our results confirm that the steps of the sequential procedure do not compromise sperm DNA integrity and progressively decreased sDNAfrag regardless of the sperm abnormality and that semen parameters with lower quality present higher sDNAfrag. Four distinct patterns were observed, of which the entire sperm head staining was the pattern most expressed in all studied fractions. Additionally, the sperm head gene-rich region staining pattern was reduced by the procedure. This suggests that pattern quantification might be a useful adjunct when performing sDNAfrag testing for male infertility. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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.

  9. Gene therapy of experimental autoimmune thyroiditis mice by in vivo administration of plasmid DNA coding for human interleukin-10

    Institute of Scientific and Technical Information of China (English)

    ZHANGZhen-Lin; LINBo; YULu-Yang; SHENShui-Xian; ZHULi-Hua; WANGWui-Ping; GUOLi-He

    2003-01-01

    AIM: To investigate the effect of interleukin-10 (IL-10) gene on experimental autoimmune thyroiditis mice.METHODS: Mice were immunized to induce autoimmune thyroiditis with porcine thyroglobulin (pTg), and thyroids of mice were injected with IL-10 DNA. On d 28 after immunization with pTg, mRNA expression of IL-10 inthyroid glands was detected and thyroid specimens were histopathological studied. RESULTS: The mRNA expression of IL-10 was detected in thyroid glands on d 7 and 14 after injection of IL-10 plasmid DNA or on COS-7 cells48 h after IL-10 plasmid DNA transfection. In addition, hlL-10 levels in culture media significantly increased 48 hand 72 h after IL-10 plasmid DNA transfection. Infiltration index of lymphocytes (1.1±0.4) in thyroids ofIL-10-treated mice was significantly lower than that of pcDNA3-null-treated mice (2.2±0.5) (P<0.01). Comparedwith pcDNA3-null control mice, IL-10-treated mice had lower levels of serum IFN-γ(P<0.01). CONCLUSION:The direct injection of DNA expression vectors encoding IL-10 into thyroid significantly inhibited development oflymphocytic infiltration of thyroid of autoimmune th,yroiditis mice, and alleviated the progression of this disease.

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

    Science.gov (United States)

    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.

  11. Advances in the research of adjuvants for plasmid DNA vaccines%DNA疫苗佐剂的研究进展

    Institute of Scientific and Technical Information of China (English)

    蒋丽明; 叶琳

    2009-01-01

    DNA疫苗是一种很有希望的免疫方法,经多途径接种质粒DNA能引起有效的免疫应答,重复给予不会产生抗载体免疫.然而,质粒DNA疫苗在小型实验动物中诱导的免疫应答远强于在人类和其他非人灵长类动物中.已设计多种佐剂通过直接刺激免疫系统或增强DNA表达来提高疫苗的免疫原性,这些佐剂包括免疫协同刺激分子、细胞因子、补体分子、脂质体、核酸、聚合物、纳米粒和微粒类佐剂.此文对DNA疫苗佐剂的研究进展作一综述.%Plasmid DNA vaccine is a promising modality for immunization. Immunization with plasmid DNA by various routes can trigger effective iimnune responses. The immunogens can be administered repeatedly without inducing anti-vector immunity. However, the immune responses induced by plasmid DNA vaccines are much stronger in small laboratory animal models than in non-human primates and humans. A number of adjuvants, including immune co-stimulatory molecules, cytokines, complement molecules, liposomes, nucleic acids, polymers, micro-and nano-particles, have been designed to improve the immunogenicity of DNA vaccines by directly stimulating the immune system or by enhancing plasmid DNA expression. This review introduces the progress in development of these adjuvants for plasmid DNA vaccines.

  12. [Isolation and characteristics of DNA fragments for the region of the tissue plasminogen activator genes and areas adjacent to it in the human genome].

    Science.gov (United States)

    Sarafanov, A G; Timofeeva, M Ia; Aleshkov, S B; Kupriianova, N S; Bannikov, V M; Zakhar'ev, V M; Baev, A A

    1994-01-01

    Fragments overlapping the tPA gene and its 5'- and 3'-flanking regions were isolated from human liver DNA library cloned in lambda Charon4A vector. A BglII fragment comprising the 3' end and the adjacent genomic region (total length 3.7 kb) was subcloned in plasmid pUC19 and its restriction map was determined. The nucleotide sequence of the 5' region of this fragment was compared with the 3' end region of the tPA gene and the corresponding regions of five published variants of tPA mRNA cDNA from different tissues; discrepancies in seven positions were revealed, which might be caused by intragenomic polymorphism.

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

    Science.gov (United States)

    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. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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...

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

    Indian Academy of Sciences (India)

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

    2012-11-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  17. The deinococcal DdrB protein is involved in an early step of DNA double strand break repair and in plasmid transformation through its single-strand annealing activity.

    Science.gov (United States)

    Bouthier de la Tour, Claire; Boisnard, Stéphanie; Norais, Cédric; Toueille, Magali; Bentchikou, Esma; Vannier, Françoise; Cox, Michael M; Sommer, Suzanne; Servant, Pascale

    2011-12-10

    The Deinococcus radiodurans bacterium exhibits an extreme resistance to ionizing radiation. Here, we investigated the in vivo role of DdrB, a radiation-induced Deinococcus specific protein that was previously shown to exhibit some in vitro properties akin to those of SSB protein from Escherichia coli but also to promote annealing of single stranded DNA. First we report that the deletion of the C-terminal motif of the DdrB protein, which is similar to the SSB C-terminal motif involved in recruitment to DNA of repair proteins, did neither affect cell radioresistance nor DNA binding properties of purified DdrB protein. We show that, in spite of their different quaternary structure, DdrB and SSB occlude the same amount of ssDNA in vitro. We also show that DdrB is recruited early and transiently after irradiation into the nucleoid to form discrete foci. Absence of DdrB increased the lag phase of the extended synthesis-dependent strand annealing (ESDSA) process, affecting neither the rate of DNA synthesis nor the efficiency of fragment reassembly, as indicated by monitoring DNA synthesis and genome reconstitution in cells exposed to a sub-lethal ionizing radiation dose. Moreover, cells devoid of DdrB were affected in the establishment of plasmid DNA during natural transformation, a process that requires pairing of internalized plasmid single stranded DNA fragments, whereas they were proficient in transformation by a chromosomal DNA marker that integrates into the host chromosome through homologous recombination. Our data are consistent with a model in which DdrB participates in an early step of DNA double strand break repair in cells exposed to very high radiation doses. DdrB might facilitate the accurate assembly of the myriad of small fragments generated by extreme radiation exposure through a single strand annealing (SSA) process to generate suitable substrates for subsequent ESDSA-promoted genome reconstitution.

  18. Boldo prevents UV light and nitric oxide-mediated plasmid DNA damage and reduces the expression of Hsp70 protein in melanoma cancer cells.

    Science.gov (United States)

    Russo, Alessandra; Cardile, Venera; Caggia, Silvia; Gunther, Germán; Troncoso, Nicolas; Garbarino, Juan

    2011-09-01

    This study was designed to investigate the potential protective effect of a methanolic extract of Peumus boldus leaves on UV light and nitric oxide (NO)-mediated DNA damage. In addition, we investigated the growth inhibitory activity of this natural product against human melanoma cells (M14). Boldine, catechin, quercetin and rutin were identified using a HPLC method. The extract was incubated with plasmid DNA and, before irradiating the samples with UV-R, H(2) O(2) was added. For analysis of DNA single-strand breaks induced by NO, the experiments were performed by incubating the extract with Angeli's salt. In the study on M14 cell line, cell viability was measured using MTT assay. Release of lactate dehydrogenase, a marker of membrane breakdown, was also measured. For the detection of apoptosis, the evaluation of DNA fragmentation (COMET assay) and caspase-3 activity assay were employed. The expression of heat shock protein 70 (Hsp70) was detected by Western blot analysis. Generation of reactive oxygen species was measured by using a fluorescent probe. The extract (demonstrating the synergistic effect of the constituents boldine and flavonoids), showed a protective effect on plasmid DNA and selectively inhibited the growth of melanoma cells. But a novel finding was that apoptosis evoked by this natural product in M14 cells, appears to be mediated, at least in part, via the inhibition of Hsp70 expression, which may be correlated with a modulation of redox-sensitive mechanisms. These results confirm the promising biological properties of Peumus boldus and encourage in-vivo investigations into its potential anti-cancer activity. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

  19. Plasmid genes required for microcin B17 production.

    Science.gov (United States)

    San Millán, J L; Kolter, R; Moreno, F

    1985-09-01

    The production of the antibiotic substance microcin B17 (Mcc) is determined by a 3.5-kilobase DNA fragment from plasmid pMccB17. Several Mcc- mutations on plasmid pMccB17 were obtained by both transposon insertion and nitrosoguanidine mutagenesis. Plasmids carrying these mutations were tested for their ability to complement Mcc- insertion or deletion mutations on pMM102 (pMM102 is a pBR322 derivative carrying the region encoding microcin B17). Results from these experiments indicate that at least four plasmid genes are required for microcin production.

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

    Science.gov (United States)

    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.

  1. Effect of the caffeine on treated and non-treated plasmid DNA with stannic chloride; Efeito da cafeina em DNA plasmidial tratado e nao tratado com cloreto estanoso

    Energy Technology Data Exchange (ETDEWEB)

    Moreno, Silvana Ramos F. [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Inst. de Biologia. Dept. de Biofisica e Biometria]|[Universidade Federal Fluminense, Niteroi, RJ (Brazil). Faculdade de Ciencias Medicas. Curso de Pos-graduacao em Patologia Experimental; Mattos, Jose C.P. de; Dantas, Flavio; Araujo, Adriano Caldeira de; Bernardo-Filho, Mario [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Inst. de Biologia. Dept. de Biofisica e Biometria]. E-mail: bernardo@uerj.br

    2000-07-01

    Caffeine, a methilxantine drug is a component of coffee, tea, stimulants and other drinks. Caffeine inhibits phosphodiesterase leading to intracellular accumulation of cyclic AMP, blocks adenosine receptors, and increases the release of Ca{sup 2+}. We have studied the possible effect of caffeine in DNA plasmid treated or not with stannous chloride (SnCl{sub 2}). Previous evaluations of the effect of caffeine on the labeling of red blood cells and plasma proteins with technetium-99m have showed a decrease of % ATI in the insoluble fraction of plasma proteins. Samples of DNA were treated with SnCl{sub 2} (0 and 200{mu}g/ml) in 0.8% agarose. SnCl{sub 2} has induced break on DNA and caffeine has not showed effect on the DNA. This indicates that caffeine does not eliminate the oxidant action of SnCl{sub 2} and does not promote break in isolated DNA plasmid. (author)

  2. Structural features and oxidative stress towards plasmid DNA of apramycin copper complex.

    Science.gov (United States)

    Balenci, D; Bonechi, G; D'Amelio, N; Gaggelli, E; Gaggelli, N; Molteni, E; Valensin, G; Szczepanik, W; Dziuba, M; Swiecicki, G; Jezowska-Bojczuk, M

    2009-02-21

    The interaction of apramycin with copper at different pH values was investigated by potentiometric titrations and EPR, UV-vis and CD spectroscopic techniques. The Cu(II)-apramycin complex prevailing at pH 6.5 was further characterized by NMR spectroscopy. Metal-proton distances derived from paramagnetic relaxation enhancements were used as restraints in a conformational search procedure in order to define the structure of the complex. Longitudinal relaxation rates were measured with the IR-COSY pulse sequence, thus solving the problems due to signal overlap. At pH 6.5 apramycin binds copper(II) with a 2 : 1 stoichiometry, through the vicinal hydroxyl and deprotonated amino groups of ring III. Plasmid DNA electrophoresis showed that the Cu(II)-apramycin complex is more active than free Cu(II) in generating strand breakages. Interestingly, this complex in the presence of ascorbic acid damages DNA with a higher yield than in the presence of H(2)O(2).

  3. Spatial and temporal regulation of DNA fragmentation in the aleurone of germinating barley

    NARCIS (Netherlands)

    Wang, M.; Oppedijk, B.J.; Caspers, M.P.M.; Lamers, G.E.M.; Boot, M.J.; Geerlings, D.N.G.; Bakhuizen, B.; Meijer, A.H.; Duijn, B. van

    1998-01-01

    During germination of barley grains, the appearance of DNA fragmentation started in aleurone cells near the embryo and extended to the distal end in a time-dependent manner. DNA fragmentation was demonstrated to occur only after the expression of α-amylase mRNA in the aleurone layer. In addition, ce

  4. A Mini-Library of Sequenced Human DNA Fragments: Linking Bench Experiments with Informatics

    Science.gov (United States)

    Dalgleish, Raymond; Shanks, Morag E.; Monger, Karen; Butler, Nicola J.

    2012-01-01

    We describe the development of a mini-library of human DNA fragments for use in an enquiry-based learning (EBL) undergraduate practical incorporating "wet-lab" and bioinformatics tasks. In spite of the widespread emergence of the polymerase chain reaction (PCR), the cloning and analysis of DNA fragments in "Escherichia coli"…

  5. Solution structure of a short dna fragment studied by neutron scattering

    DEFF Research Database (Denmark)

    Lederer, H.; May, R. P.; Kjems, Jørgen

    1986-01-01

    The solution structure of a DNA fragment of 130 base pairs and known sequence has been investigated by neutron small-angle scattering. In 0.1 M NaCl, the overall structure of the DNA fragment which contains the strong promoter A1 of the Escherichia coli phage T7 agrees with that expected for B...

  6. Spatial and temporal regulation of DNA fragmentation in the aleurone of germinating barley

    NARCIS (Netherlands)

    Wang, M.; Oppedijk, B.J.; Caspers, M.P.M.; Lamers, G.E.M.; Boot, M.J.; Geerlings, D.N.G.; Bakhuizen, B.; Meijer, A.H.; Duijn, B. van

    1998-01-01

    During germination of barley grains, the appearance of DNA fragmentation started in aleurone cells near the embryo and extended to the distal end in a time-dependent manner. DNA fragmentation was demonstrated to occur only after the expression of α-amylase mRNA in the aleurone layer. In addition, ce

  7. A Mini-Library of Sequenced Human DNA Fragments: Linking Bench Experiments with Informatics

    Science.gov (United States)

    Dalgleish, Raymond; Shanks, Morag E.; Monger, Karen; Butler, Nicola J.

    2012-01-01

    We describe the development of a mini-library of human DNA fragments for use in an enquiry-based learning (EBL) undergraduate practical incorporating "wet-lab" and bioinformatics tasks. In spite of the widespread emergence of the polymerase chain reaction (PCR), the cloning and analysis of DNA fragments in "Escherichia coli"…

  8. qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, Christopher B., E-mail: Christopher.jackson@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Gallati, Sabina, E-mail: sabina.gallati@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Schaller, Andre, E-mail: andre.schaller@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Serial qPCR accurately determines fragmentation state of any given DNA sample. Black-Right-Pointing-Pointer Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. Black-Right-Pointing-Pointer Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. Black-Right-Pointing-Pointer Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA ({lambda}{sub nDNA}) and mtDNA ({lambda}{sub mtDNA}) we present an approach to possibly correct measurements in

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 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 DNA injection. Macrophages of fish injected with pcDNA-Lys-g produced significantly (P 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.

  11. Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors

    Directory of Open Access Journals (Sweden)

    Lough John W

    2010-08-01

    Full Text Available Abstract Background The use of lentiviruses to reprogram human somatic cells into induced pluripotent stem (iPS cells could limit their therapeutic usefulness due to the integration of viral DNA sequences into the genome of the recipient cell. Recent work has demonstrated that human iPS cells can be generated using episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, or recombinant proteins. While these approaches offer an advance, the protocols have some drawbacks. Commonly the procedures require either subcloning to identify human iPS cells that are free of exogenous DNA, a knowledge of virology and safe handling procedures, or a detailed understanding of protein biochemistry. Results Here we report a simple approach that facilitates the reprogramming of human somatic cells using standard techniques to transfect expression plasmids that encode OCT4, NANOG, SOX2, and LIN28 without the need for episomal stability or selection. The resulting human iPS cells are free of DNA integration, express pluripotent markers, and form teratomas in immunodeficient animals. These iPS cells were also able to undergo directed differentiation into hepatocyte-like and cardiac myocyte-like cells in culture. Conclusions Simple transient transfection of plasmid DNA encoding reprogramming factors is sufficient to generate human iPS cells from primary fibroblasts that are free of exogenous DNA integrations. This approach is highly accessible and could expand the use of iPS cells in the study of human disease and development.

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

    Science.gov (United States)

    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.

  13. Menadione-induced DNA fragmentation without 8-oxo-2'-deoxyguanosine formation in isolated rat hepatocytes

    DEFF Research Database (Denmark)

    Fischer-Nielsen, A; Corcoran, G B; Poulsen, H E

    1995-01-01

    damage DNA. In the present study, we measured the effect of menadione on formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), an index of oxidative DNA base modifications, and on DNA fragmentation. Isolated hepatocytes from phenobarbital-pretreated rats were exposed to menadione, 25-400 micro......M, for 15, 90 or 180 min with or without prior depletion of reduced glutathione (GSH) by diethyl maleate. Menadione caused profound GSH depletion and internucleosomal DNA fragmentation, which was demonstrated by a prominent fragmentation ladder on agarose gel electrophoresis. We found no oxidative...

  14. The Saccharomyces cerevisiae Dna2 can function as a sole nuclease in the processing of Okazaki fragments in DNA replication.

    Science.gov (United States)

    Levikova, Maryna; Cejka, Petr

    2015-09-18

    During DNA replication, synthesis of the lagging strand occurs in stretches termed Okazaki fragments. Before adjacent fragments are ligated, any flaps resulting from the displacement of the 5' DNA end of the Okazaki fragment must be cleaved. Previously, Dna2 was implicated to function upstream of flap endonuclease 1 (Fen1 or Rad27) in the processing of long flaps bound by the replication protein A (RPA). Here we show that Dna2 efficiently cleaves long DNA flaps exactly at or directly adjacent to the base. A fraction of the flaps cleaved by Dna2 can be immediately ligated. When coupled with DNA replication, the flap processing activity of Dna2 leads to a nearly complete Okazaki fragment maturation at sub-nanomolar Dna2 concentrations. Our results indicate that a subsequent nucleolytic activity of Fen1 is not required in most cases. In contrast Dna2 is completely incapable to cleave short flaps. We show that also Dna2, like Fen1, interacts with proliferating cell nuclear antigen (PCNA). We propose a model where Dna2 alone is responsible for cleaving of RPA-bound long flaps, while Fen1 or exonuclease 1 (Exo1) cleave short flaps. Our results argue that Dna2 can function in a separate, rather than in a Fen1-dependent pathway.

  15. Mechanical processing of hyperviscous semen specimens can negatively affect sperm DNA fragmentation.

    Science.gov (United States)

    Kussler, Ana Paula S; Pimentel, Anita M; Alcoba, Diego D; Liu, Isabella P; Brum, Ilma Simoni; Capp, Edison; Corleta, Helena V E

    2014-04-01

    The present study compared the DNA fragmentation in human sperm samples with reduced, physiological, and increased viscosity in order to evaluate whether the process used to reduce viscosity (expulsion of semen through a needle and syringe) alters significantly sperm DNA fragmentation. The seminal parameters of semen samples from 123 patients were evaluated and classified according to their viscosity. Samples with increased viscosity were submitted to a process of expulsion of semen through a 10-mL syringe and an 18-gauge (18G) needle to reduce the seminal viscosity. The DNA fragmentation of all samples was analysed using TUNEL assay (Terminal deoxynucleotidyl transferase mediated dUTP Nick-end labelling assay); in samples with increased viscosity, the fragmentation was assessed before and after the process of expulsion with syringe and needle. There was no difference in DNA fragmentation between groups with different viscosity (P = 0.857). A significantly increase in sperm DNA fragmentation after expulsion of hyperviscous semen through the syringe was observed (P = 0.035). There was no difference in DNA fragmentation rate between samples with reduced, increased and physiological viscosities; however, the physical process of expulsion of semen through a syringe and needle increased sperm DNA fragmentation.

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

    Science.gov (United States)

    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.

  17. Coupling of importin beta binding peptide on plasmid DNA: transfection efficiency is increased by modification of lipoplex's physico-chemical properties

    Directory of Open Access Journals (Sweden)

    Escriou Virginie

    2003-09-01

    Full Text Available Abstract Background Non-viral vectors for gene transfer are less immunogenic than viral vectors but also less efficient. Significant effort has focused on enhancing non-viral gene transfer efficiency by increasing nuclear import of plasmid DNA, particularly by coupling nuclear localization peptidic sequences to plasmid DNA. Results We have coupled a 62-aminoacid peptide derived from hSRP1α importin beta binding domain, called the IBB peptide to plasmid DNA by using the heterobifunctional linker N-(4-azido-2,3,5,6 tetrafluorobenzyl-6-maleimidyl hexanamide (TFPAM-6. When covalently coupled to plasmid DNA, IBB peptide did not increase the efficiency of cationic lipid mediated transfection. The IBB peptide was still able to interact with its nuclear import receptor, importin β, but non-specifically. However, we observed a 20-fold increase in reporter gene expression with plasmid DNA / IBB peptide complexes under conditions of inefficient transfection. In which case, IBB was associated with plasmid DNA through self assembling ionic interaction. Conclusions The improvement of transfection activity was not due to an improved nuclear import of DNA, but rather by the modification of physicochemical properties of IBB peptide / plasmid complexes. IBB peptide increased lipoplex size and these larger complexes were more efficient for gene transfer.

  18. Toward the identification of a type I toxin-antitoxin system in the plasmid DNA of dairy Lactobacillus rhamnosus.

    Science.gov (United States)

    Folli, Claudia; Levante, Alessia; Percudani, Riccardo; Amidani, Davide; Bottazzi, Stefania; Ferrari, Alberto; Rivetti, Claudio; Neviani, Erasmo; Lazzi, Camilla

    2017-09-21

    Plasmids carry genes that give bacteria beneficial traits and allow them to survive in competitive environments. In many cases, they also harbor toxin-antitoxin (TA) systems necessary for plasmid maintenance. TA systems are generally characterized by a stable "toxin", a protein or peptide capable of killing the cell upon plasmid loss and by an unstable "antitoxin", a protein or a non-coding RNA that inhibits toxin activity. Here we report data toward the identification of a RNA-regulated TA system in the plasmid DNA of L. rhamnosus isolated from cheese. The proposed TA system comprises two convergently transcribed RNAs: a toxin RNA encoding a 29 amino acid peptide named Lpt and an antitoxin non-coding RNA. Both toxin and antitoxin RNAs resulted upregulated under conditions mimicking cheese ripening. The toxicity of the Lpt peptide was demonstrated in E. coli by cloning the Lpt ORF under the control of an inducible promoter. Bioinformatics screening of the bacterial nucleotide database, shows that regions homologous to the Lpt TA locus are widely distributed in the Lactobacillus genus, particularly within the L. casei group, suggesting a relevant role of TA systems in plasmid maintenance of cheese microbiota.

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

    Energy Technology Data Exchange (ETDEWEB)

    Hoon Byeon, Jeong, E-mail: jbyeon@purdue.edu [Department of Chemistry, Purdue University, West Lafayette, Indiana 47907 (United States); Kim, Jang-Woo, E-mail: jwkim@hoseo.edu [Department of Digital Display Engineering, Hoseo University, Asan 336-795 (Korea, Republic of)

    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.

  20. Integral parametrization of the Kinetics of Crosslink production in plasmid DNA as a function of 8-methoxypsoralen concentration

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-07-01

    In this paper we present results of crosslink production in pBR322 DNA along a wide range of 8-methoxypsoralen (8-MOP) concentration. Experimental data were obtained as DNA renaturation percentages, from the shift in hyperchromicity after a temperature-dependent denaturation-renaturation process. the experimental results showed a three-stage profile when represented as a function of the natural logarithms of 8-MOP concentration. an integral parametrization which allows a simultaneous fit of the three observed stages is presented here. the theoretical values of crosslink production determined from the fit are useful to asses the genotoxicity of psoralen-induced crosslinks in plasmid DNA. (Author) 24 refs.

  1. Microfluidic chip for stacking, separation and extraction of multiple DNA fragments.

    Science.gov (United States)

    Wu, Ruige; Seah, Y P; Wang, Zhiping

    2016-03-11

    A disposable integrated microfluidic device was developed for rapid sample stacking, separation and extraction of multiple DNA fragments from a relatively large amount of sample. Isotachophoresis hyphenated gel electrophoresis (ITP-GE) was used to pre-concentrate and separate DNA fragments, followed by extraction of pure DNA fragments with electroelution on-chip. DNA fragments of 200bp, 500bp and 1kbp were successfully separated and collected in the extraction chamber within 25min. The extraction efficiency obtained from the chip was 49.9%, 52.1% and 53.7% for 200bp, 500bp and 1kbp DNA fragments, respectively. The extracted DNA fragments exhibited compatibility with downstream enzymatic reactions, for example PCR. The chip was also used to extract DNA fragments with specific size range from sheared genomic DNA and demonstrated similar performance to that using traditional gel cutting method. The whole assay can finish in 32min, 6 times faster than traditional method. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. [Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment].

    Science.gov (United States)

    Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

    2016-01-01

    Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

  3. Modular construction of plasmids by parallel assembly of linear vector components.

    Science.gov (United States)

    Gao, XinZheng; Yan, Pu; Shen, Wentao; Li, Xiaoying; Zhou, Peng; Li, Yuenan

    2013-06-15

    Construction of plasmids is the basic and pivotal technology in molecular biology. The common method for constructing plasmids is to cut DNA fragments by restriction enzymes and then join the resulting fragments using ligase. We present here a modified Golden Gate cloning method for modular construction of plasmids. Unlike the original Golden Gate cloning system for cloning from entry vector to expression vector, this method can be used to construct plasmids immediately from linear DNA fragments. After polymerase chain reaction (PCR) amplification for flanking with BsaI sites, multiple linear DNA components (modules) can be parallel assembled into a circle plasmid by a single restriction-ligation reaction using the method. This method is flexible to construct different types of plasmids because the modules can be freely selected and assembled in any combination. This method was applied successfully to construct a prokaryotic expression plasmid from four modules and a plant expression plasmid from five modules (fragments). The results suggest that this method provides a simple and flexible platform for modular construction of plasmids.

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

    Science.gov (United States)

    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.

  5. Comparative Studies on Preparation of Large Plant DNA Fragments by Pulse Field Gel Electrophoresis

    Institute of Scientific and Technical Information of China (English)

    Yang Jiliang(杨继良); Wang Qinghua(王庆华); Deng Daiyong; Yang Dianer; Jin Demin; Weng Manli; Zhang Juren; Wang Bin

    2004-01-01

    The preparation of large plant DNA fragments is extremely important to the construction of large insert DNA libraries (YAC, BAC, PAC and TAC). Although several techniques have been developed in each step of large plant DNA fragments preparation, the whole processing remains complicated and difficult. Based on authors research experience and the recent worldwide development in this field, the following aspects are discussed in this paper: techniques of plant high molecular weight (HMW) DNA purification by pre-electrophoresis, the optimal conditions for the partial digestion of the HMW DNA by HindIII, the isolation effects of of large plant DNA fragments (100~400 kb) with different parameters of pulse field gel electrophoresis (PFGE), and the recovery of large DNA fragments. Through comparative studies, the advantages and disadvantages of each technique are discussed and some recommendations are proposed for preparing high quality large plant DNA fragments. The suggested techniques have been used in preparing the large DNA fragments of maize, rice, moss, laver, sea tangle and peach,and similar results are obtained among all the materials. This paper only reports the results using maize as material.

  6. Amplification of plasmid DNA bound on soil colloidal particles and clay minerals by the polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Polymerase chain reaction (PCR) was used to amplify a 600-base pair (bp) sequence of plasmid pGEX-2T DNA bound on soil colloidal particles from Brown soil (Alfisol) and Red soil (Ultisol), and three different minerals (goethite, kaolinite, montmorillonite). DNA bound on soil colloids, kaolinite, and montmorillonite was not amplified when the complexes were used directly but amplification occurred when the soil colloid or kaolinite-DNA complex was diluted, 10- and 20-fold. The montmorillonite-DNA complex required at least 100-fold dilution before amplification could be detected. DNA bound on goethite was amplified irrespective of whether the complex was used directly, or diluted 10- and 20-fold. The amplification of mineral-bound plasmid DNA by PCR is, therefore, markedly influenced by the type and concentration of minerals used. This information is of fundamental importance to soil molecular microbial ecology with particular reference to monitoring the fate of genetically engineered microorganisms and their recombinant DNA in soil environments.

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

    Science.gov (United States)

    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.

  8. Evaluation of Maltose-Based Cationic Liposomes with Different Hydrophobic Tails for Plasmid DNA Delivery

    Directory of Open Access Journals (Sweden)

    Bo Li

    2017-03-01

    Full Text Available In this paper, three cationic glycolipids with different hydrophobic chains Malt-DiC12MA (IX a, Malt-DiC14MA (IX b and Malt-DiC16MA (IX c were constructed by using maltose as starting material via peracetylation, selective 1-O-deacetylation, trichloroacetimidation, glycosylation, azidation, deacetylation, Staudinger reaction, tertiary amination and quaternization. Target compounds and some intermediates were characterized by 1H-NMR, 13C-NMR, 1H-1H COSY and 1H-13C HSQC. The results of gel electrophoresis assay, atomic force microscopy images (AFM and dynamic light scattering (DLS demonstrate that all the liposomes could efficiently bind and compact DNA (N/P ratio less than 2 into nanoparticles with proper size (88 nm–146 nm, PDI < 0.4 and zeta potential (+15 mV–+26 mV. The transfection efficiency and cellular uptake of glycolipids in HEK293 cell were evaluated through the enhanced green fluorescent protein (EGFP expression and Cy3-labeled pEGFP-C1 (Enhanced Green Fluorescent Protein plasmid images, respectively. Importantly, it indicated that Malt-DiC14MA exhibited high gene transfer efficiency and better uptake capability at N/P ratios of 8:1. Additionally, the result of cell viability showed glycolipids exhibited low biotoxicity and good biocompatibility by thiazolyl blue tetrazolium bromide (MTT assay.

  9. A seven-year storage report of good manufacturing practice-grade naked plasmid DNA: stability, topology, and in vitro/in vivo functional analysis.

    Science.gov (United States)

    Walther, Wolfgang; Schmeer, Marco; Kobelt, Dennis; Baier, Ruth; Harder, Alexander; Walhorn, Volker; Anselmetti, Dario; Aumann, Jutta; Fichtner, Iduna; Schleef, Martin

    2013-12-01

    The great interest for naked plasmid DNA in gene therapy studies is reflected by the fact that it is currently used in 18% of all gene therapy trials. Therefore, validation of topology and functionality of DNA resulting from its long-term stability is an essential requirement for safe and effective gene transfer. To this aim, we analyzed the stability of good manufacturing practice-grade pCMVβ reporter plasmid DNA by capillary gel electrophoresis, agarose gel electrophoresis, and atomic force microscopy. The plasmid DNA was produced for a clinical gene transfer study started in 2005 and was stored for meanwhile 7 years under continuously monitored conditions at -20 °C. The stability of plasmid DNA was monitored by LacZ transgene expression functional assays performed in vitro and in vivo on the 7-year-old plasmid DNA samples compared with plasmid batches newly produced in similar experimental conditions and quality standards. The analyses revealed that during the overall storage time and conditions, the proportion of open circular and supercoiled or covalently closed circular forms is conserved without linearization or degradation of the plasmid. The in vitro transfection and the in vivo jet-injection of DNA showed unaltered functionality of the long-stored plasmid. In summary, the 7-year-old and the newly produced plasmid samples showed similar topology and expression performance. Therefore, our stable storage conditions are effective to preserve the integrity of the DNA to be used in clinical studies. This is an important prerequisite for the long-term performance of gene transfer materials used in trials of long duration as well as of the reference material used in standardization procedures and assays.

  10. CASA derived human sperm abnormalities: correlation with chromatin packing and DNA fragmentation.

    Science.gov (United States)

    Sivanarayana, T; Krishna, Ch Ravi; Prakash, G Jaya; Krishna, K Murali; Madan, K; Rani, B Sireesha; Sudhakar, G; Raju, G A Rama

    2012-12-01

    The present study was undertaken to evaluate the effects of morphokinetic abnormalities of human spermatozoa on chromatin packing and DNA integrity and possible beneficial effects of sperm selection in ICSI. Semen samples from 1002 patients were analysed for morphology and motility using CASA. Protamine status and DNA fragmentation were analysed by chromomycin A3 staining and sperm chromatin dispersion assay respectively. Sperms with elongated, thin, round, pyri, amorphous, micro and macro forms were significantly higher in teratozoospermic and oligoasthenoteratozoospermic groups. Significant difference in chromatin packing and DNA fragmentation index was observed in these abnormal groups compared with normal. Similarly significant correlation was also seen between abnormal motility parameters and DNA fragmentation index in asthenozoospermic group compared with normal. Specific abnormal morphological forms have higher incidence of chromatin packing abnormalities and DNA fragmentation. Using these sperms in ICSI might have an impact on fertilization, embryo development and abortion rates. These can be selectively avoided during ICSI procedure to improve ART outcome.

  11. [Influence of Storage Temperature and Cryopreservation Conditions on the Extent of Human Sperm DNA Fragmentation].

    Science.gov (United States)

    Simonenko, E Yu; Garmaeva, S B; Yakovenko, S A; Grigorieva, A A; Tverdislov, V A; Mironova, A G; Aprishko, V P

    2016-01-01

    With the direct labeling procedure for detecting DNA fragmentation we explored the influence of the different storage temperature conditions as well as different methods of cryopreservation on the structure of DNA organization in the human sperm. 19 sperm samples obtained from healthy men with normozoospermia (according to the criteria of the World Health Organization) were used for investigation. A significant increase of human sperm DNA-fragmentation was observed after 8 hours of incubation at +39 degrees C (by 76.7%) and at +37 degrees C (by 68.9%). It was found that sperm cooling with the use of a cryoprotectant immediately after thawing did not produce significant differences in the extent of DNA fragmentation, although samples, containing cryoprotectants, showed a sharp increase of DNA fragmentation after 24 hours of incubation, that could suggest cryoprotectant cytotoxicity.

  12. Quantification of DNA fragmentation in processed foods using real-time PCR.

    Science.gov (United States)

    Mano, Junichi; Nishitsuji, Yasuyuki; Kikuchi, Yosuke; Fukudome, Shin-Ichi; Hayashida, Takuya; Kawakami, Hiroyuki; Kurimoto, Youichi; Noguchi, Akio; Kondo, Kazunari; Teshima, Reiko; Takabatake, Reona; Kitta, Kazumi

    2017-07-01

    DNA analysis of processed foods is performed widely to detect various targets, such as genetically modified organisms (GMOs). Food processing often causes DNA fragmentation, which consequently affects the results of PCR analysis. In order to assess the effects of DNA fragmentation on the reliability of PCR analysis, we investigated a novel methodology to quantify the degree of DNA fragmentation. We designed four real-time PCR assays that amplified 18S ribosomal RNA gene sequences common to various plants at lengths of approximately 100, 200, 400, and 800 base pairs (bp). Then, we created an indicator value, "DNA fragmentation index (DFI)", which is calculated from the Cq values derived from the real-time PCR assays. Finally, we demonstrated the efficacy of this method for the quality control of GMO detection in processed foods by evaluating the relationship between the DFI and the limit of detection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Evidence for plasmid-encoded virulence factors in the phytopathogenic bacterium Clavibacter michiganensis subsp. michiganensis NCPPB382.

    Science.gov (United States)

    Meletzus, D; Bermphol, A; Dreier, J; Eichenlaub, R

    1993-01-01

    The tomato pathogen Clavibacter michiganensis subsp. michiganensis NCPPB382, which causes bacterial wilt, harbors two plasmids pCM1 (27.5 kb) and pCM2 (72 kb). After curing of the plasmids, bacterial derivatives were still proficient in the ability to colonize the host plant and in the production of exopolysaccharides but exhibited a reduced virulence. When one of the two plasmids is lost, there is a significant delay in the development of wilting symptoms after infection and a plasmid-free derivative is not able to induce disease symptoms. By cloning of restriction fragments of both plasmids in the plasmid-free strain CMM100, two DNA fragments which restored the virulent phenotype were identified. Further analysis suggested that a fragment of plasmid pCM1 encodes an endocellulase which is involved in the expression of the pathogenic phenotype. Images PMID:8458855

  14. Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomoea nil)

    NARCIS (Netherlands)

    Yamada, T.; Takatsu, Y.; Kasumi, K.; Ichimura, K.; Doorn, van W.G.

    2006-01-01

    We studied DNA degradation and nuclear fragmentation during programmed cell death (PCD) in petals of Ipomoea nil (L.) Roth flowers. The DNA degradation, as observed on agarose gels, showed a large increase. Using DAPI, which stains DNA, and flow cytometry for DAPI fluorescence, we found that the num

  15. Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomoea nil)

    NARCIS (Netherlands)

    Yamada, T.; Takatsu, Y.; Kasumi, K.; Ichimura, K.; Doorn, van W.G.

    2006-01-01

    We studied DNA degradation and nuclear fragmentation during programmed cell death (PCD) in petals of Ipomoea nil (L.) Roth flowers. The DNA degradation, as observed on agarose gels, showed a large increase. Using DAPI, which stains DNA, and flow cytometry for DAPI fluorescence, we found that the

  16. Rapid assessment of the effect of ciprofloxacin on chromosomal DNA from Escherichia coli using an in situ DNA fragmentation assay

    Directory of Open Access Journals (Sweden)

    Gosalvez Jaime

    2009-04-01

    Full Text Available Abstract Background Fluoroquinolones are extensively used antibiotics that induce DNA double-strand breaks (DSBs by trapping DNA gyrase and topoisomerase IV on DNA. This effect is usually evaluated using biochemical or molecular procedures, but these are not effective at the single-cell level. We assessed ciprofloxacin (CIP-induced chromosomal DNA breakage in single-cell Escherichia coli by direct visualization of the DNA fragments that diffused from the nucleoid obtained after bacterial lysis in an agarose microgel on a slide. Results Exposing the E. coli strain TG1 to CIP starting at a minimum inhibitory concentration (MIC of 0.012 μg/ml and at increasing doses for 40 min increased the DNA fragmentation progressively. DNA damage started to be detectable at the MIC dose. At a dose of 1 μg/ml of CIP, DNA damage was visualized clearly immediately after processing, and the DNA fragmentation increased progressively with the antibiotic incubation time. The level of DNA damage was much higher when the bacteria were taken from liquid LB broth than from solid LB agar. CIP treatment produced a progressively slower rate of DNA damage in bacteria in the stationary phase than in the exponentially growing phase. Removing the antibiotic after the 40 min incubation resulted in progressive DSB repair activity with time. The magnitude of DNA repair was inversely related to CIP dose and was noticeable after incubation with CIP at 0.1 μg/ml but scarce after 10 μg/ml. The repair activity was not strictly related to viability. Four E. coli strains with identified mechanisms of reduced sensitivity to CIP were assessed using this procedure and produced DNA fragmentation levels that were inversely related to MIC dose, except those with very high MIC dose. Conclusion This procedure for determining DNA fragmentation is a simple and rapid test for studying and evaluating the effect of quinolones.

  17. Internucleosomal DNA fragmentation in wild emmer wheat is catalyzed by S1-type endonucleases translocated to the nucleus upon induction of cell death.

    Science.gov (United States)

    Granot, Gila; Morgenstern, Yaakov; Khan, Asif; Rapp, Yemima Givaty; Pesok, Anat; Nevo, Eviatar; Grafi, Gideon

    2015-03-01

    Leaves of cereal plants display nucleosomal fragmentation of DNA attributed to the action of nucleases induced during program cell death (PCD). Yet, the specific nuclease activity responsible for generating double strand DNA breaks (DSBs) that lead to DNA fragmentation has not been fully described. Here, we characterized a Ca2+/Mg2+-dependent S1-type endonuclease activity in leaves of wild emmer wheat (Triticum dicoccoides Köern.) capable of introducing DSBs as demonstrated by the conversion of supercoiled plasmid DNA into a linear duplex DNA. In-gel nuclease assay revealed a nuclease of about 35 kDa capable of degrading both single stranded DNA and RNA. We further showed that the endonuclease activity can be purified on Concanavalin A and treatment with peptide-N-glycosidase F (PNGase F) did not abolish its activity. Furthermore, ConA-associated endonuclease was capable of generating nucleosomal DNA fragmentation in tobacco nuclei. Since S1-type endonucleases lack canonical nuclear localization signal it was necessary to determine their subcellular localization. To this end, a cDNA encoding for a putative 34 kDa S1-type nuclease, designated TaS1-like (TaS1L) was synthesized based on available sequence data of Triticum aestivum and fused with RFP. Introduction into protoplasts showed that TaS1L-RFP is cytoplasmic 24h post transformation but gradually turn nuclear at 48 h concomitantly with induction of cell death. Our results suggest that DNA fragmentation occurring in leaves of wild emmer wheat may be attributed to S1-type endonuclease(s) that reside in the cytoplasm but translocate to the nucleus upon induction of cell death. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. OPTIMIZATION OF ELECTROPORATION PARAMETERS FOR TRANSFECTION OF PLASMID DNA INTO MURINE BONE MARROW-DERIVED DENDRITIC CELL

    Institute of Scientific and Technical Information of China (English)

    KE Shan; CHEN Xue-hua; LI Hao; LI Jian-fang; GU Qin-long; ZHU Zheng-gang; LIU Bing-ya

    2006-01-01

    Objective To explore the optimal electroporation parameters for transfection of plasmid DNA into murine bone marrow-derived dendritic cells. Methods Murine bone marrow-derived dendritic cells (DCs) were electroporated with plasmid DNA in varied conditions, such as electrical voltage, pulse time,pre-electroporation cell condition and serum concentration in electrical buffer. Inverted fluorescence microscope and flow cytometer were used to determine the transfection efficiency. Some of the DCs genetically modified under different conditions were stained with trypan-blue and its viability was observed microscopically 48h after electroporation. Results Highest transfection efficiency (22.10%) could be reached when electrical voltage was 250V and pulse time was 20ms. Refreshing the culture medium pre-electroporation may help the cells recover more easily from gene transfer.Besides, electrical buffer containing serum may benefit the viability of DC after electroporation and temperature may has little influence on transfection efficiency. Conclusion Our observations demonstrated plasmid DNA could be efficiently transferred into murine bone marrow-derived DCs by electroporation. These data may helpful for cancer research related to murine DC transfection.

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

    Science.gov (United States)

    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.

  20. New approach to assess sperm DNA fragmentation dynamics: Fine-tuning mathematical models.

    Science.gov (United States)

    Ortiz, Isabel; Dorado, Jesús; Morrell, Jane; Gosálvez, Jaime; Crespo, Francisco; Jiménez, Juan M; Hidalgo, Manuel

    2017-01-01

    Sperm DNA fragmentation (sDF) has been proved to be an important parameter in order to predict in vitro the potential fertility of a semen sample. Colloid centrifugation could be a suitable technique to select those donkey sperm more resistant to DNA fragmentation after thawing. Previous studies have shown that to elucidate the latent damage of the DNA molecule, sDF should be assessed dynamically, where the rate of fragmentation between treatments indicates how resistant the DNA is to iatrogenic damage. The rate of fragmentation is calculated using the slope of a linear regression equation. However, it has not been studied if sDF dynamics fit this model. The objectives of this study were to evaluate the effect of different after-thawing centrifugation protocols on sperm DNA fragmentation and elucidate the most accurate mathematical model (linear regression, exponential or polynomial) for DNA fragmentation over time in frozen-thawed donkey semen. After submitting post-thaw semen samples to no centrifugation (UDC), sperm washing (SW) or single layer centrifugation (SLC) protocols, sDF values after 6 h of incubation were significantly lower in SLC samples than in SW or UDC. Coefficient of determination (R(2)) values were significantly higher for a second order polynomial model than for linear or exponential. The highest values for acceleration of fragmentation (aSDF) were obtained for SW, followed by SLC and UDC. SLC after thawing seems to preserve longer DNA longevity in comparison to UDC and SW. Moreover, the fine-tuning of models has shown that sDF dynamics in frozen-thawed donkey semen fit a second order polynomial model, which implies that fragmentation rate is not constant and fragmentation acceleration must be taken into account to elucidate hidden damage in the DNA molecule.

  1. The Convenience of Single Homology Arm Donor DNA and CRISPR/Cas9-Nickase for Targeted Insertion of Long DNA Fragment.

    Science.gov (United States)

    Basiri, Mohsen; Behmanesh, Mehrdad; Tahamtani, Yaser; Khalooghi, Keynoosh; Moradmand, Azadeh; Baharvand, Hossein

    2017-01-01

    CRISPR/Cas9 technology provides a powerful tool for targeted modification of genomes. In this system, a donor DNA harboring two flanking homology arms is mostly used for targeted insertion of long exogenous DNA. Here, we introduced an alternative design for the donor DNA by incorporation of a single short homology arm into a circular plasmid. In this experimental study, single homology arm donor was applied along with a single guide RNA (sgRNA) specific to the homology region, and either Cas9 or its mutant nickase variant (Cas9n). Using Pdx1 gene as the target locus the functionality of this system was evaluated in MIN6 cell line and murine embryonic stem cells (ESCs). Both wild type Cas9 and Cas9n could conduct the knock-in process with this system. We successfully applied this strategy with Cas9n for generation of Pdx1(GFP) knock-in mouse ESC lines. Altogether, our results demonstrated that a combination of a single homology arm donor, a single guide RNA and Cas9n is capable of precisely incorporating DNA fragments of multiple kilo base pairs into the targeted genomic locus. While taking advantage of low off-target mutagenesis of the Cas9n, our new design strategy may facilitate the targeting process. Consequently, this strategy can be applied in knock-in or insertional inactivation studies.

  2. The Convenience of Single Homology Arm Donor DNA and CRISPR/Cas9-Nickase for Targeted Insertion of Long DNA Fragment

    Directory of Open Access Journals (Sweden)

    Mohsen Basiri

    2016-10-01

    Full Text Available Objective: CRISPR/Cas9 technology provides a powerful tool for targeted modification of genomes. In this system, a donor DNA harboring two flanking homology arms is mostly used for targeted insertion of long exogenous DNA. Here, we introduced an alternative design for the donor DNA by incorporation of a single short homology arm into a circular plasmid. Materials and Methods: In this experimental study, single homology arm donor was applied along with a single guide RNA (sgRNA specific to the homology region, and either Cas9 or its mutant nickase variant (Cas9n. Using Pdx1 gene as the target locus the functionality of this system was evaluated in MIN6 cell line and murine embryonic stem cells (ESCs. Results: Both wild type Cas9 and Cas9n could conduct the knock-in process with this system. We successfully applied this strategy with Cas9n for generation of Pdx1GFP knock-in mouse ESC lines. Altogether, our results demonstrated that a combination of a single homology arm donor, a single guide RNA and Cas9n is capable of precisely incorporating DNA fragments of multiple kilo base pairs into the targeted genomic locus. Conclusion: While taking advantage of low off-target mutagenesis of the Cas9n, our new design strategy may facilitate the targeting process. Consequently, this strategy can be applied in knock-in or insertional inactivation studies.

  3. Chemotherapy of Bacterial Plasmids

    Science.gov (United States)

    1979-01-29

    render them non-susceptible to K: z plasmid-encoded enzymes. (3) Development of drugs which are selective inhibitor! 1 4, of plasmid DNA replication. (4... Development of drugs which inhibit phenotypic as expression of plasmid genes, and (5) Development of drugs which are inhibitors o, drug-inactivating...Barnes [2] them non-susceptible to plasmid-encoded enzymes, tabulated data on the incidence of Gram-negative 3) development of drugs which are

  4. One abstinence day decreases sperm DNA fragmentation in 90 % of selected patients.

    Science.gov (United States)

    Pons, Isabel; Cercas, Rosa; Villas, Celia; Braña, Cristina; Fernández-Shaw, Sylvia

    2013-09-01

    The aim of this prospective descriptive study was to evaluate the efficacy of reducing sexual abstinence as a strategy to decrease sperm DNA fragmentation. Men with one or more of the following characteristics were included in the study: older than 44, smoking more than 10 cigarettes per day, with a body mass index over 25, diabetes mellitus, varicocele, a previous chemotherapy treatment, severe oligozoospermia, prostatitis, cryptorchidism, having a partner with recurrent miscarriage and/or implantation failure, poor embryo morphology and/or fertilization failure. Patients were asked to produce a semen sample after 3 to 7 abstinence days which was subjected to a sperm DNA fragmentation test. When DNA fragmentation was above or equal to 30 %, it was considered to be altered. Patients with increased DNA fragmentation were asked to produce another semen sample following a "one abstinence day protocol". This protocol required producing up to three semen samples with 1 day of abstinence and measuring sperm DNA fragmentation. Four hundred and sixteen patients produced a first semen sample after a sexual abstinence of 3 to 7 days. Sperm DNA fragmentation was altered in 46 samples (11.1 %). Thirty five patients with increased DNA fragmentation samples completed the "one abstinence day protocol". DNA fragmentation decreased to normal values in one of the three attempts in 91.4 % of the patients: 81.3 % in the first attempt, 12.5 % in the second try and 6.3 % in the third. This approach could be a simple, low-cost and effective way to decrease sperm DNA damage to normal values.

  5. Evaluation of effect of selected trace elements on dynamics of sperm DNA fragmentation.

    Science.gov (United States)

    Wdowiak, Artur; Bakalczuk, Grzegorz; Bakalczuk, Szymon

    2015-12-31

    Lead and cadmium can lead to negative effects on sperm chromatin DNA integrity. Copper, zinc and selenium are essential components of many enzymes which are important for reproduction. The aim of this research was to evaluate the influence of lead, cadmium, zinc, copper and selenium on the dynamics of semen DNA fragmentation. The present study concerned 85 fertile and 131 infertile men aged 25-35. DNA fragmentation in the samples was determined after 3 h, 6 h and 12 h. The Pb, Cd, Cu, Zn, and Se measurements were performed by the electrothermal-atomic absorption spectrometry method. We found that sperm DNA fragmentation was a dynamic process which was intensified with an increase in the level of lead in seminal plasma. The levels of lead and cadmium were higher in seminal plasma of infertile men, compared to fertile men. The levels of zinc, copper and selenium in seminal plasma were higher in men with proven fertility, compared to infertile men, and did not exert a significant effect on the dynamics of sperm DNA fragmentation. The level of cadmium had no significant effect on intensification of sperm DNA fragmentation in time. Reports in the literature which concern the effect of trace elements on human reproduction are equivocal. The present study confirmed an unfavourable effect, especially that of lead, on the dynamics of sperm DNA fragmentation; however, these studies need to be expanded and continued in the future.

  6. Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART

    National Research Council Canada - National Science Library

    Borini, A; Tarozzi, N; Bizzaro, D; Bonu, M A; Fava, L; Flamigni, C; Coticchio, G

    2006-01-01

    ...) and sperm chromatin alterations has not been satisfactorily explained. The aim of this study was to assess the relationship between sperm DNA fragmentation in IVF/ICSI patients, sperm parameters...

  7. Comparison of DNA fragmentation and color thresholding for objective quantitation of apoptotic cells

    Science.gov (United States)

    Plymale, D. R.; Ng Tang, D. S.; Fermin, C. D.; Lewis, D. E.; Martin, D. S.; Garry, R. F.

    1995-01-01

    Apoptosis is a process of cell death characterized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the number of cultured CD4+ T-lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to gamma-irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with apoptosis. These results suggest that substantial DNA fragmentation can precede or occur in the absence of the morphological changes which are associated with apoptosis in gamma-irradiated RH9 cells.

  8. Changes in DNA fragmentation during sperm preparation for intracytoplasmic sperm injection over time.

    Science.gov (United States)

    Rougier, Natalia; Uriondo, Heydy; Papier, Sergio; Checa, Miguel Angel; Sueldo, Carlos; Alvarez Sedó, Cristian

    2013-07-01

    To compare the DNA fragmentation of semen samples established by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) after incubation in polyvinylpyrrolidone (PVP) and hyaluronic acid (HA) for different time periods. Comparative prospective study. Center for reproductive medicine. Twenty-seven semen samples from infertile patients. None. Semen analysis and DNA fragmentation assays (TUNEL) were performed. Two groups were established: A) normal TUNEL (DNA fragmentation significantly decreased after centrifugation gradient, regardless of the initial levels of the sample. Samples with TUNEL ≥ 20% were more susceptible to a significant increase in DNA fragmentation over time, with similar increases being observed over time for samples that were incubated in HA or PVP. These data may be relevant for sperm preparation for intracytoplasmic sperm injection. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  9. Excessive cytosolic DNA fragments as a potential trigger of Graves’ disease: an encrypted message sent by animal models

    Directory of Open Access Journals (Sweden)

    Yuqian Luo

    2016-11-01

    Full Text Available Graves’ hyperthyroidism is caused by autoantibodies directed against the thyroid stimulating hormone receptor (TSHR that mimic the action of TSH. The establishment of Graves’ hyperthyroidism in experimental animals has proven to be an important approach to dissect the mechanisms of self-tolerance breakdown that lead to the production of thyroid-stimulating TSHR autoantibodies (TSAbs. ‘Shimojo’s model was the first successful Graves’ animal model, wherein immunization with fibroblasts cells expressing TSHR and a major histocompatibility complex (MHC class II molecule, but not either alone, induced TSAb production in AKR/N (H-2k mice. This model highlights the importance of coincident MHC class II expression on TSHR-expressing cells in the development of Graves’ hyperthyroidism. These data are also in agreement with the observation that Graves’ thyrocytes often aberrantly express MHC class II antigens via mechanisms that remain unclear. Our group demonstrated that cytosolic self-genomic DNA fragments derived from sterile injured cells can induce aberrant MHC class II expression and production of multiple inflammatory cytokines and chemokines in thyrocytes in vitro, suggesting that severe cell injury may initiate immune responses in a way that is relevant to thyroid autoimmunity mediated by cytosolic DNA signaling. Furthermore, more recent successful Graves’ animal models were primarily established by immunizing mice with TSHR-expressing plasmids or adenovirus. In these models, double-stranded DNA vaccine contents presumably exert similar immune-activating effect in cells at inoculation sites and thus might pave the way toward successful Graves’ animal models. This review focuses on evidence suggesting that cell injury-derived self-DNA fragments could act as Graves’ disease triggers.

  10. No increased sperm DNA fragmentation index in semen containing human papillomavirus or herpesvirus

    DEFF Research Database (Denmark)

    Kaspersen, Maja Døvling; Bungum, Mona; Fedder, Jens

    2013-01-01

    -based hybridization array that identifies all HHVs and 35 of the most common HPVs. Sperm DNA integrity was determined by the sperm chromatin structure assay. HPVs or HHVs, or both, were found in 57% of semen samples; however, sperm DNA fragmentation index was not increased in semen containing these viruses.......It remains unknown whether human papillomaviruses (HPVs) or human herpesviruses (HHVs) in semen affect sperm DNA integrity. We investigated whether the presence of these viruses in semen was associated with an elevated sperm DNA fragmentation index. Semen from 76 sperm donors was examined by a PCR...

  11. In vivo assembly of DNA-fragments in the moss, Physcomitrella patens

    DEFF Research Database (Denmark)

    King, Brian Christopher; Vavitsas, Konstantinos; Ikram, Nur Kusaira Binti Khairul

    2016-01-01

    Direct assembly of multiple linear DNA fragments via homologous recombination, a phenomenon known as in vivo assembly or transformation associated recombination, is used in biotechnology to assemble DNA constructs ranging in size from a few kilobases to full synthetic microbial genomes. It has also...... enabled the complete replacement of eukaryotic chromosomes with heterologous DNA. The moss Physcomitrella patens, a non-vascular and spore producing land plant (Bryophyte), has a well-established capacity for homologous recombination. Here, we demonstrate the in vivo assembly of multiple DNA fragments...

  12. Isolation of 24 novel cDNA fragments from microdis—sected human chromosome band

    Institute of Scientific and Technical Information of China (English)

    ZHANGMIN; LONGYU; 等

    1998-01-01

    The strategy of isolating the band0specific expression fragments from a probe pool generated by human chromosome microdissection was reported.A chromosome 14q 24.3 band-specific single copy DNA pool was constructed based on this probe pool.Using total DNA of the pool as probe to hybridize the human marrow cDNA library,68 primary positive clones were selected from 5×105 cDNA clones.Among these primary clones,32 secondary clones were obtained after second-round screening and designed as cFD14-1-32.Finally,24 band-specific expression fragments were identified from these 32 positive clones by DNA hybridization.Those band-specific clones can hybridize to both 14q24.3 DNA and human genomic DNA but cann't hybridize to 17q11-12 DNA,Partial sequences of 13 fragments of them were sequenced and idenfified as novel cDNA sequences,and these sequences were proved to have some homology with known genes in NCBI database.Analysis of expression spectrum of cFD 14-1 suggested that the cDNA fragments thus obtained should be used to isolate the genes can not been cloned in 14q24.3 region.

  13. Fragment-based discovery of 6-azaindazoles as inhibitors of bacterial DNA ligase.

    Science.gov (United States)

    Howard, Steven; Amin, Nader; Benowitz, Andrew B; Chiarparin, Elisabetta; Cui, Haifeng; Deng, Xiaodong; Heightman, Tom D; Holmes, David J; Hopkins, Anna; Huang, Jianzhong; Jin, Qi; Kreatsoulas, Constantine; Martin, Agnes C L; Massey, Frances; McCloskey, Lynn; Mortenson, Paul N; Pathuri, Puja; Tisi, Dominic; Williams, Pamela A

    2013-12-12

    Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase.

  14. Autocrine DNA fragmentation of intra-epithelial lymphocytes (IELs) in mouse small intestine.

    Science.gov (United States)

    Ogata, Masaki; Ota, Yuta; Nanno, Masanobu; Suzuki, Ryuji; Itoh, Tsunetoshi

    2015-09-01

    Intraepithelial lymphocytes (IELs) are present in the intestinal epithelium. Mechanisms of IELs for the protection of villi from foreign antigens and from infections by micro-organisms have not been sufficiently explained. Although more than 70% of mouse duodenal and jejunal IELs bear γδTCR (γδIELs), the functions of γδIELs are little investigated. We stimulate γδIELs by anti-CD3 monoclonal antibody (mAb) injection. The mAb activates γδIELs to release Granzyme B (GrB) into the spaces surrounding the γδIELs and intestinal villous epithelial cells (IECs). Released GrB induces DNA fragmentation in IECs independently of Perforin (Pfn). IECs immediately repair their fragmented DNA. Activated IELs reduce their cell size, remain for some time in the epithelium after the activation and are ultimately eliminated without leaving the site. We focus our attention on the response of IELs to the released GrB present in the gap surrounding IELs, after activation, in order to examine whether the released GrB has a similar effect on IELs to that observed on IECs in our previous studies. DNA fragmentation is also induced in IELs together with the repair of fragmented DNA thereafter. The time-kinetics of both events were found to be identical to those observed in IECs. DNA fragmentation in IELs is Pfn-independent. Here, we present Pfn-independent "autocrine DNA fragmentation" in IELs and the repair of fragmented DNA in IELs and discuss their biological significance. Autocrine DNA fragmentation has never been reported to date in vivo.

  15. Effect of cryopreservation on the sperm DNA fragmentation dynamics of the bottlenose dolphin (Tursiops truncatus).

    Science.gov (United States)

    Sánchez-Calabuig, M J; López-Fernández, C; Johnston, S D; Blyde, D; Cooper, J; Harrison, K; de la Fuente, J; Gosálvez, J

    2015-04-01

    Sperm DNA fragmentation is one of the major causes of infertility; the sperm chromatin dispersion test (SCDt) evaluates this parameter and offers the advantage of species-specific validated protocol and ease of use under field conditions. The main purpose of this study was to evaluate sperm DNA fragmentation dynamics in both fresh and post-thaw bottlenose dolphin sperm using the SCDt following different cryopreservation protocols to gain new information about the post-thaw differential sperm DNA longevity in this species. Fresh and cryopreserved semen samples from five bottlenose dolphins were examined for sperm DNA fragmentation dynamics using the SCDt (Halomax(®)). Sperm DNA fragmentation was assessed immediately at collection and following cryopreservation (T0) and then after 0.5, 1, 4, 8, 24, 48 and 72 h incubation at 37°C. Serially collected ejaculates from four dolphins were frozen using different cryopreservation protocols in a TES-TRIS-fructose buffer (TTF), an egg-yolk-free vegetable lipid LP1 buffer (LP1) and human sperm preservation medium (HSPM). Fresh ejaculated spermatozoa initially showed low levels of DNA fragmentation for up to 48 h. Lower Sperm DNA fragmentation (SDF) was found in the second fresh ejaculate compared to the first when more than one sample was collected on the same day (p fragmentation after 24- and 48-h incubation than those frozen in TTF or HSPM. No correlation was found between any seminal characteristic and DNA fragmentation in either fresh and/or frozen-thawed samples. © 2015 Blackwell Verlag GmbH.

  16. Characterisation of LMD virus-like nanoparticles self-assembled from cationic liposomes, adenovirus core peptide mu and plasmid DNA.

    Science.gov (United States)

    Tagawa, T; Manvell, M; Brown, N; Keller, M; Perouzel, E; Murray, K D; Harbottle, R P; Tecle, M; Booy, F; Brahimi-Horn, M C; Coutelle, C; Lemoine, N R; Alton, E W F W; Miller, A D

    2002-05-01

    Liposome:mu:DNA (LMD) is a ternary nucleic acid delivery system built around the mu peptide associated with the condensed core complex of the adenovirus. LMD is prepared by precondensing plasmid DNA (D) with mu peptide (M) in a 1:0.6 (w/w) ratio and then combining these mu:DNA (MD) complexes with extruded cationic liposomes (L) resulting in a final lipid:mu:DNA ratio of 12:0.6:1 (w/w/w). Correct buffer conditions, reagent concentrations and rates of mixing are all crucial to success. However, once optimal conditions are established, homogeneous LMD particles (120 +/- 30 nm) will result that each appear to comprise an MD particle encapsulated within a cationic bilammellar liposome. LMD particles can be formulated reproducibly, they are amenable to long-term storage (>1 month) at -80 degrees C and are stable to aggregation at a plasmid DNA concentration up to 5 mg/ml (15 mM nucleotide concentration). Furthermore, LMD transfections are significantly more time and dose efficient in vitro than cationic liposome-plasmid DNA (LD) transfections. Transfection times as short as 10 min and plasmid DNA doses as low as 0.001 microg/well result in significant gene expression. LMD transfections will also take place in the presence of biological fluids (eg up to 100% serum) giving 15-25% the level of gene expression observed in the absence of serum. Results from confocal microscopy experiments using fluorescent-labelled LMD particles suggest that endocytosis is not a significant barrier to LMD transfection, although the nuclear membrane still is. We also confirm that topical lung transfection in vivo by LMD is at least equal in absolute terms with transfection mediated by GL-67:DOPE:DMPE-PEG(5000) (1:2:0.05 m/m/m), an accepted 'gold-standard' non-viral vector system for topical lung transfection, and is in fact at least six-fold more dose efficient. All these features make LMD an important new non-viral vector platform system from which to derive tailor-made non-viral delivery

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

    Directory of Open Access Journals (Sweden)

    Sarker SR

    2013-04-01

    Full Text Available Satya Ranjan Sarker, Yumiko Aoshima, Ryosuke Hokama, Takafumi Inoue, Keitaro Sou, Shinji Takeoka Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University (TWIns, Tokyo, Japan 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

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

    Science.gov (United States)

    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.

  19. Simultaneous vitality and DNA-fragmentation measurement in spermatozoa of smokers and non-smokers.

    Science.gov (United States)

    De Bantel, A; Fleury-Feith, J; Poirot, C; Berthaut, I; Garcin, C; Landais, P; Ravel, C

    2015-03-01

    Because cigarette smoke is a powerful ROS producer, we hypothesized that the spermatozoa of smokers would be more at risk of having increased DNA fragmentation than spermatozoa of non-smoking men. A cross-sectional study was performed on consenting smokers and non-smokers, consulting in an infertility clinic for routine sperm analysis. The application of a novel TUNEL assay coupled to a vitality marker, LIVE/DEAD®, allowed both DNA fragmentation and viability measurement within spermatozoa of participants to be analyzed by flow cytometry. The coupled vitality-DNA fragmentation analysis revealed that non-smokers and smokers, respectively presented medians of 3.6% [0.6-36.8] and 3.3% [0.9-9.6] DNA fragmented spermatozoa among the living spermatozoa population (P > 0.05). No deleterious effect of smoking on spermatozoa was found in our study. More studies concerning potential mutagenic capacities of cigarette smoke on spermatozoa are necessary. In addition, the coupled vitality-DNA fragmentation analysis may orient Assisted Reproductive Technology teams when confronted with patients having a high percentage of DNA-fragmented living spermatozoa. © 2014 International Clinical Cytometry Society.

  20. Sperm nuclear DNA fragmentation and its association with semen quality in Greek men.

    Science.gov (United States)

    Evgeni, E; Lymberopoulos, G; Touloupidis, S; Asimakopoulos, B

    2015-12-01

    Due to the limitations of conventional semen analysis in predicting a man's fertility potential, sperm DNA fragmentation was recently introduced as a novel marker of sperm quality. This prospective study was undertaken to investigate the associations between conventional seminal parameters and DNA fragmentation in Greek men. A total of 669 subject data were evaluated in two groups, normozoospermic (n = 184) and non-normozoospermic (n = 485), according to the WHO 2010 (WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edn. World Health Organization), reference limits. For all the subjects, semen volume, sperm concentration, total count, rapid and total progressive motility and morphology were recorded following the WHO 2010 methods and DNA fragmentation was assessed by the sperm chromatin dispersion assay. An inverse correlation was established between DNA fragmentation and all conventional seminal parameters except semen volume in men with seminal profiles below the reference limits, with statistical significance for rapid and total progressive motility. Normozoospermic men exhibited lower levels of DNA fragmentation than their non-normozoospermic counterparts, even though the values were not always below 30%. DNA fragmentation testing and traditional semen analysis should therefore be considered as complementary diagnostic tools in a comprehensive evaluation of male infertility. © 2015 Blackwell Verlag GmbH.

  1. Sperm DNA fragmentation as a result of ultra-endurance exercise training in male athletes.

    Science.gov (United States)

    Vaamonde, D; Algar-Santacruz, C; Abbasi, A; García-Manso, J M

    2017-03-15

    Intensive sports practice seems to exert negative effects on semen parameters; in order to assess these effects, the objective of this study was to assess semen, including DNA fragmentation, and hormone parameters in elite triathletes. Twelve high-level triathletes preparing for a National Triathlon Championship participated in the study. The qualitative sperm parameters analysed were volume, sperm count, motility, morphology and DNA fragmentation; when needed, additional testing was performed. Assessed hormones were testosterone (T), cortisol (C) and testosterone-cortisol ratio (T/C). Maximum oxygen consumption and training characteristics were also assessed. Hormonal values and physical semen parameters were within normal ranges. DNA fragmentation showed high values (20.4 ± 6.1%). Round cells in semen were higher than normal (2.8 ± 1.5 million/ml), with the presence of macrophages. Correlations were found for several parameters: concentration of round cells positively correlated with progressive sperm motility (p = .01) and sperm morphology (p = .02); contrarily, the correlation found with DNA fragmentation was negative (p = .04). Sperm DNA fragmentation and the T/C ratio, however, were correlated in a positive manner (p = .03). As evidenced by the observed results, sperm DNA fragmentation is affected by high-level sports practice; therefore, high loads of endurance training could potentially interfere with the athlete's fertility potential. © 2017 Blackwell Verlag GmbH.

  2. Sperm DNA fragmentation abnormalities in men from couples with a history of recurrent miscarriage.

    Science.gov (United States)

    Leach, Mikaela; Aitken, Robert J; Sacks, Gavin

    2015-08-01

    Previous studies have described an association between sperm with DNA damage and a history of recurrent miscarriage (RM), although it is not clear whether there is benefit in screening for sperm DNA fragmentation and to what extent DNA fragmentation impacts upon RM. To identify what proportion of couples experiencing RM are affected by DNA fragmentation abnormalities. In this retrospective study, between 2008 and 2013, couples with a history of recurrent miscarriage (≥3 first trimester miscarriages) were investigated comprehensively for known causes (karyotype, uterine, antiphospholipid syndrome, thrombophilia) and also by semen analysis, including DNA fragmentation [sperm chromatin structure analysis (SCSA)]. Statistical analysis was performed on SPSS software with significance taken as P fragmentation index (DFI) of 9.50%. Normal levels were found in 70.5% of men (DFI 30%). Couples with otherwise unexplained recurrent miscarriage had significantly higher DFI than those with other causes identified on routine screening (P = 0.012). In couples experiencing RM, 30% (32/108) of men had sperm with high levels of DNA fragmentation (DFI > 15%). This may be a contributing factor to the clinical syndrome of RM, and future clinical trials of therapies for these couples are warranted. © 2015 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists.

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

    Science.gov (United States)

    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.

  4. Multiple Antibiotic Resistance Plasmids Allow Scalable, PCR-Mediated DNA Manipulation and Near-Zero Background Cloning

    Directory of Open Access Journals (Sweden)

    Remigiusz Arnak

    2016-01-01

    Full Text Available We have constructed two plasmids that can be used for cloning as templates for PCR-based gene disruption, mutagenesis and the construction of DNA chromosome translocation cassettes. To our knowledge, these plasmids are the first vectors that confer resistance to ampicillin, kanamycin and hygromycin B in bacteria, and to geneticin (G418 and hygromycin B in Saccharomyces cerevisiae simultaneously. The option of simultaneously using up to three resistance markers provides a highly stringent control of recombinant selection and the almost complete elimination of background resistance, while unique restriction sites allow easy cloning of chosen genetic material. Moreover, we successfully used these new vectors as PCR templates for the induction of chromosome translocation in budding yeast by the bridge-induced translocation system. Cells in which translocation was induced carried chromosomal rearrangements as expected and exhibited resistance to both, G418 and hygromycin B. These features make our constructs very handy tools for many molecular biology applications.

  5. Lower sperm DNA fragmentation after r-FSH administration in functional hypogonadotropic hypogonadism.

    Science.gov (United States)

    Ruvolo, Giovanni; Roccheri, Maria Carmela; Brucculeri, Anna Maria; Longobardi, Salvatore; Cittadini, Ettore; Bosco, Liana

    2013-04-01

    An observational clinical and molecular study was designed to evaluate the effects of the administration of recombinant human FSH on sperm DNA fragmentation in men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In the study were included 53 men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In all patients, sperm DNA fragmentation index (DFI), assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) in situ DNA nick end-labelling (TUNEL) assay, was evaluated before starting the treatment with 150 IU of recombinant human FSH, given three times a week for at least 3 months. Patients' semen analysis and DNA fragmentation index were re-evaluated after the 3-month treatment period. After recombinant human FSH therapy, we did not find any differences in terms of sperm count, motility and morphology. The average DNA fragmentation index was significantly reduced (21.15 vs 15.2, p15 %), while no significant variation occurred in the patients with DFI values ≤ 15 %. Recombinant human FSH administration improves sperm DNA integrity in hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia men with DNA fragmentation index value >15 % .

  6. Processes involved in assisted reproduction technologies significantly increase sperm DNA fragmentation and phosphatidylserine translocation.

    Science.gov (United States)

    Balasuriya, A; Serhal, P; Doshi, A; Harper, J C

    2014-03-01

    Sperm preparation techniques in assisted reproduction technologies (ART) are potential generators of exogenous stresses that cause additional DNA damage. DNA fragmentation tests, such as the sperm chromatin structure assay, involve freezing sperm samples in the absence of cryoprotectant. Thermal, oxidative stress (OS) and freezing are detrimental to sperm DNA fragmentation and phosphatidylserine (PS) translocation. The primary aim of this study was to subject mature sperm to environmental insults that normally occur during ART. We tested the hypotheses that OS, thermal stress and freeze-thawing caused sperm nuclear and membrane damage and that a positive correlation exists between PS translocation and DNA fragmentation. Sperm DNA integrity deteriorates in semen samples from men with advancing age and a sperm concentration of DNA fragmentation at 37 °C after merely 1 h is important clinically as semen liquefaction and short-term sperm storage in an ART cycle involve incubating samples at this temperature. Freezing without a cryoprotectant significantly increases the level of sperm nuclear damage, so it is important not to freeze neat semen prior to DNA fragmentation testing. This study highlights the importance of minimising the production of exogenous stresses during sperm preparation in ART. © 2012 Blackwell Verlag GmbH.

  7. [A method for determining DNA sequence by labeling the end of the molecule and cleaving at the base. Isolation of DNA fragments, end-labeling, cleavage, electrophoresis in polyacrylamide gel and analysis of results].

    Science.gov (United States)

    Maxam, A M; Gilbert, W

    1986-01-01

    We elaborate basic chemical principles and current laboratory procedures for sequencing end-labeled DNA by partial cleavage and gel electrophoresis (A. M. Maxam and W. Gilbert, Proc. Natl. Acad. Sci. USA, 1977, v. 74, p. 560-564). We provide step-by-step protocols for 32P-labeling DNA ends, segregating the labeled ends by cutting with a second restriction enzyme or separating strands, partially cleaving the DNA at specific bases with reagents, electrophoresing the labeled products of cleavage on sequencing gels, and interpreting sequencing band patterns. Many of these procedures have been condensed, to make them faster and easier, and some are new. We also discuss sequencing strategies, and suggest a technique which will reduce plasmid or viral DNA to a collection of singly-end-labeled fragments in one day, for efficient sequencing of these chromosomes in 250-nucleotide blocks.

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

    Science.gov (United States)

    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.

  9. Unraveling the sperm proteome and post-genomic pathways associated with sperm nuclear DNA fragmentation.

    Science.gov (United States)

    Intasqui, Paula; Camargo, Mariana; Del Giudice, Paula T; Spaine, Deborah M; Carvalho, Valdemir M; Cardozo, Karina H M; Cedenho, Agnaldo P; Bertolla, Ricardo P

    2013-09-01

    Sperm DNA fragmentation has been suggested as a marker for infertility diagnosis and prognosis. Hence, understanding its impact on male physiology and post-genomic pathways would be clinically important. We performed the proteomics and functional enrichment analyses of viable spermatozoa from ejaculates with low and high sperm DNA fragmentation to identify protein expression and pathways altered in association with sperm DNA fragmentation. Sperm DNA fragmentation using the Comet assay and the Komet 6.0.1 software was assessed in raw samples from 89 subjects from a human reproduction service. The Low and High sperm DNA fragmentation groups were formed according to the Olive Tail Moment variable. Spermatozoa proteins from these groups were pooled and analyzed by a shotgun proteomic approach (2D nanoUPLC-ESI-MS(E)). Differentially expressed proteins were used for a functional enrichment study. Two hundred and fifty-seven proteins were identified or quantified in sperm from the Low and High sperm DNA fragmentation groups. Of these, seventy-one proteins were exclusively or overexpressed in the Low group, whereas twenty-three proteins were exclusively or overexpressed in the High group. One hundred and sixty-three proteins were conserved between these groups. We also functionally related the differentially expressed proteins in viable spermatozoa from the groups. Processes such as triacylglycerol metabolism, energy production, protein folding, response to unfolded proteins, and cellular detoxification were found to be altered in these cells. Sperm DNA fragmentation is associated with differential protein expression in viable spermatozoa. These proteins may potentially be used as biomarkers for sperm DNA integrity.

  10. HPLC-an Effective Method of DNA Fragment Determination

    Institute of Scientific and Technical Information of China (English)

    HOU JingGuo; HE TianXi; MAO XueFeng; LIU HuiLing; DU XinZheng; NA PengJun; DENG HuaLing; GAO JingZhang

    2001-01-01

    @@ The role played by DNA in molecular biology is clearly recognized to be vital to life on this planet. 8-oxo-7,8-dihydro-2deoxyguanosine(=8-OHdG), is probably the most important product of "oxidative stress” in DNA. Its concentration in DNA is, in fact. a quantitative analysis of the degree of DNA damage that an organism has undergone. Due to the importance of 8-OHdG of nucleic acidg in mutagenesis, carcinogenesis and aging, numerous chemical and biological investigations have been made on this subject in the past time. Kuchino and co-workers have found that 8-OHdG residue in DNA is misreading during the process of DNA replication. Recently, some reports have been presented on high 8-OHdG levels in patients suffering from various diseases such as chronic hepatitis, Fanconi s anemia, diabetes mellitus and Helicobacter pylori infections. As a result, 8-OHdG is a useful marker for the study of DNA damage arising from reactive oxygen species and is of great significance for cancer research. The 8-OHdG levels in DNA can help understand the mechanism of carcinogens and lead to more effective treatments for many different types of cancer. For these reasons, an analysis of 8-OHdG with quickness, low cost and high accuracy is required.

  11. HPLC-an Effective Method of DNA Fragment Determination

    Institute of Scientific and Technical Information of China (English)

    HOU; JingGuo

    2001-01-01

    The role played by DNA in molecular biology is clearly recognized to be vital to life on this planet. 8-oxo-7,8-dihydro-2deoxyguanosine(=8-OHdG), is probably the most important product of "oxidative stress” in DNA. Its concentration in DNA is, in fact. a quantitative analysis of the degree of DNA damage that an organism has undergone. Due to the importance of 8-OHdG of nucleic acidg in mutagenesis, carcinogenesis and aging, numerous chemical and biological investigations have been made on this subject in the past time. Kuchino and co-workers have found that 8-OHdG residue in DNA is misreading during the process of DNA replication. Recently, some reports have been presented on high 8-OHdG levels in patients suffering from various diseases such as chronic hepatitis, Fanconi s anemia, diabetes mellitus and Helicobacter pylori infections. As a result, 8-OHdG is a useful marker for the study of DNA damage arising from reactive oxygen species and is of great significance for cancer research. The 8-OHdG levels in DNA can help understand the mechanism of carcinogens and lead to more effective treatments for many different types of cancer. For these reasons, an analysis of 8-OHdG with quickness, low cost and high accuracy is required.  ……

  12. Cerebral ischemia produces laddered DNA fragments distinct from cardiac ischemia and archetypal apoptosis.

    Science.gov (United States)

    MacManus, J P; Fliss, H; Preston, E; Rasquinha, I; Tuor, U

    1999-05-01

    The electrophoretic pattern of laddered DNA fragments which has been observed after cerebral ischemia is considered to indicate that neurons are dying by apoptosis. Herein the authors directly demonstrate using ligation-mediated polymerase chain reaction methods that 99% of the DNA fragments produced after either global or focal ischemia in adult rats, or produced after hypoxia-ischemia in neonatal rats, have staggered ends with a 3' recess of approximately 8 to 10 nucleotides. This is in contrast to archetypal apoptosis in which the DNA fragments are blunt ended as seen during developmental programmed cell death in dying cortical neurons, neuroblastoma, or thymic lymphocytes. It is not simply ischemia that results in staggered ends in DNA fragments because ischemic myocardium is similar to archetypal apoptosis with a vast majority of blunt-ended fragments. It is concluded that the endonucleases that produce this staggered fragmentation of the DNA backbone in ischemic brain must be different than those of classic or type I apoptosis.

  13. The study of spermatic DNA fragmentation and sperm motility in infertile subjects.

    Science.gov (United States)

    Peluso, Giuseppina; Palmieri, Alessandro; Cozza, Pietro Paolo; Morrone, Giancarlo; Verze, Paolo; Longo, Nicola; Mirone, Vincenzo

    2013-04-19

    Although the pathophysiology of the testicular damage associated with varicocele remains unclear, sperm DNA damage has been identified as a potential explanation for this cause of male infertility. The current study was designed to determine the extent of sperm nuclear DNA damage in patients with varicocele, and to examine its relationship with parameters of seminal motility. Semen samples from 60 patients with clinical varicocele and 90 infertile men without varicocele were examined. Varicocele sperm samples were classified as normal or pathological according to the 1999 World Health Organizzation guidelines. Sperm DNA damage was evalutated using the Halosperm kit, an improved Sperm Chromatin Dispersion (SCD) test. The DNA fragmentation index (DFI: percentage of sperm with denatured nuclei) values was significantly higher in patients with varicocele, either with normal or abnormal (DFI 25.8 ± 3.2 vs 17.4 ± 2.8 - P spermatic motility and the degree of spermatic DNA fragmentation in patients with clinical varicocele. Varicocele is associated with high levels of DNA-damage in spermatozoa. In addition, in subjects with varicocele, abnormal spermatozoa motility is associated with higher levels of sperm DNA fragmentation. DNA fragmentation may therefore be an essential additional diagnostic test that should be recommended for patients with clinical varicocele.

  14. The study of spermatic DNA fragmentation and sperm motility in infertile subjects

    Directory of Open Access Journals (Sweden)

    Giuseppina Peluso

    2013-04-01

    Full Text Available Introduction: Although the pathophysiology of the testicular damage associated with varicocele remains unclear, sperm DNA damage has been identified as a potential explanation for this cause of male infertility. The current study was designed to determine the extent of sperm nuclear DNA damage in patients with varicocele, and to examine its relationship with parameters of seminal motility. Materials and method: Semen samples from 60 patients with clinical varicocele and 90 infertile men without varicocele were examined. Varicocele sperm samples were classified as normal or pathological according to the 1999 World Health Organizzation guidelines. Sperm DNA damage was evalutated using the Halosperm kit, an improved Sperm Chromatin Dispersion (SCD test. Results: The DNA fragmentation index (DFI: percentage of sperm with denatured nuclei values was significantly higher in patients with varicocele, either with normal or abnormal (DFI 25.8 ± 3.2 vs 17.4 ± 2.8 - P < 0,01 semen profiles. In addition, an inverse correlation was found between spermatic motility and the degree of spermatic DNA fragmentation in patients with clinical varicocele. Conclusions: Varicocele is associated with high levels of DNA-damage in spermatozoa. In addition, in subjects with varicocele, abnormal spermatozoa motility is associated with higher levels of sperm DNA fragmentation. DNA fragmentation may therefore be an essential additional diagnostic test that should be recommended for patients with clinical varicocele.

  15. A Quick, Cost-Free Method of Purification of DNA Fragments from Agarose Gel

    Directory of Open Access Journals (Sweden)

    Yuan Sun, Kannappan Sriramajayam, Dianzhong Luo, D. Joshua Liao

    2012-01-01

    Full Text Available In this short communication we report a quick, cost-free method of purification of DNA fragments from agarose gel. Unlike those procedures that involve commercial kits, this method uses glass wool or absorbent cotton to filter agarose gel during a quick spinning-down of DNA, thus significantly simplifying the routine practice of many molecular biologists and decreasing the cost.

  16. The Restriction Fragment Map of Rat-Liver Mitochondrial DNA : A Reconsideration

    NARCIS (Netherlands)

    Pepe, G.; Bakker, H.; Holtrop, M.; Bollen, J.E.; Bruggen, E.F.J. van; Cantatore, P.; Terpstra, P.; Saccone, C.

    1977-01-01

    1. Rat-liver mitochondrial DNA (mtDNA) contains at least 8 cleavage sites for the restriction endonuclease Eco RI, 6 for the restriction endonuclease Hind III, 2 for the restriction endonuclease Bam HI and 11 for the restriction endonuclease Hap II. 2. The physical map of the restriction fragments o

  17. AN IMAGE-ANALYSIS TECHNIQUE FOR DETECTION OF RADIATION-INDUCED DNA FRAGMENTATION AFTER CHEF ELECTROPHORESIS

    NARCIS (Netherlands)

    ROSEMANN, M; KANON, B; KONINGS, AWT; KAMPINGA, HH

    1993-01-01

    CHEF-electrophoresis was used as a technique to detect radiation-induced DNA breakage with special emphasis to biological relevant X-ray doses (0-10 Gy). Fluorescence detection of DNA-fragments using a sensitive image analysis system was directly compared with conventional scintillation counting of

  18. Sperm DNA fragmentation and its role in wildlife conservation.

    Science.gov (United States)

    Gosálvez, Jaime; Holt, William V; Johnston, Stephen D

    2014-01-01

    Until about 20 years ago, sperm assessment in the laboratory was focused on motility, morphology and acrosomal integrity. Then came the gradual realisation that, because the main objective of a spermatozoon is to deliver an intact genetic payload of DNA to the egg, being able to check DNA quality of spermatozoa would be equally important, if not more so. Research over the last two decades has therefore led to the development of several techniques for reliably detecting DNA strand breaks, and the more recent focus has been directed towards understanding the fertility implications of DNA damage. It is now clear that evolutionary history has played an important role in determining the stability of sperm DNA under stressful conditions, and that the nature of the DNA-protein interactions also influence the extent to which fertility is affected by both technical procedures involved in sperm preservation and the basic biology of the species concerned. Here we present an overview of the principles involved in DNA assessment and also provide some cases studies that illustrate the influences of species diversity.

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

    Science.gov (United States)

    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.

  20. Differential pre-amplification of STR loci for fragmented forensic DNA profiling.

    Science.gov (United States)

    Ham, Seon-Kyu; Kim, Se-Yong; Seo, Bo Young; Woo, Kwang-Man; Lee, Seung-Hwan; Choi, Cheol Yong

    2016-11-01

    DNA profiling of short tandem repeats (STR) has been successfully used for the identification of individuals in forensic samples, accidents and natural disasters. However, STR profiling of DNA isolated from old crime scenes and damaged biological samples is difficult due to DNA degradation and fragmentation. Here, we show that pre-amplification of STR loci using biotinylated primers for the STR loci is an efficient strategy to obtain STR profiling results from fragmented forensic samples. Analysis of STR loci with longer amplicon sizes is generally hampered, since these relatively long loci are vulnerable to DNA fragmentation. This problem was overcome by using reduced or increased primer concentrations for loci with shorter or longer amplicon sizes, respectively, in our pre-amplification strategy. In addition, pre-amplification of STR loci into two groups of short or long amplicon size increases the efficiency of STR profiling from highly fragmented forensic DNA samples. Therefore, differential pre-amplification of STR loci is an effective way to obtain DNA profiling results from fragmented forensic samples. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. In vitro incubation of human spermatozoa promotes reactive oxygen species generation and DNA fragmentation.

    Science.gov (United States)

    Cicaré, J; Caille, A; Zumoffen, C; Ghersevich, S; Bahamondes, L; Munuce, M J

    2015-10-01

    The aim of this study was to investigate the oxidative process associated with sperm capacitation and its impact on DNA fragmentation and sperm function. Redox activity and lipid peroxidation were analysed in human spermatozoa after 3, 6 and 22 h of incubation in Ham's F10 medium plus bovine albumin at 37° and 5% CO2 for capacitation. DNA status, tyrosine phosphorylation pattern and induced acrosome reaction were evaluated after capacitating conditions. At 22 h of incubation, there was a significant (P DNA fragmentation. These results indicate that when spermatozoa are incubated for several hours (22 h), a common practice in assisted reproductive techniques, an increase in oxidative sperm metabolism and in the proportion of fragmented DNA should be expected. However, there was no effect on any of the other functional parameters associated with sperm fertilising capacity. © 2014 Blackwell Verlag GmbH.

  2. Selective binding of specific mouse genomic DNA fragments by mouse vimentin filaments in vitro.

    Science.gov (United States)

    Wang, X; Tolstonog, G; Shoeman, R L; Traub, P

    1996-03-01

    Mouse vimentin intermediate filaments (IFs) reconstituted in vitro were analyzed for their capacity to select certain DNA sequences from a mixture of about 500-bp-long fragments of total mouse genomic DNA. The fragments preferentially bound by the IFs and enriched by several cycles of affinity binding and polymerase chain reaction (PCR) amplification were cloned and sequenced. In general, they were G-rich and highly repetitive in that they often contained Gn, (GT)n, and (GA)n repeat elements. Other, more complex repeat sequences were identified as well. Apart from the capacity to adopt a Z-DNA and triple helix configuration under superhelical tension, many fragments were potentially able to form cruciform structures and contained consensus binding sites for various transcription factors. All of these sequence elements are known to occur in introns and 5'/3'-flanking regions of genes and to play roles in DNA transcription, recombination and replication. A FASTA search of the EMBL data bank indeed revealed that sequences homologous to the mouse repetitive DNA fragments are commonly associated with gene-regulatory elements. Unexpectedly, vimentin IFs also bound a large number of apparently overlapping, AT-rich DNA fragments that could be aligned into a composite sequence highly homologous to the 234-bp consensus centromere repeat sequence of gamma-satellite DNA. Previous experiments have shown a high affinity of vimentin for G-rich, repetitive telomere DNA sequences, superhelical DNA, and core histones. Taken together, these data support the hypothesis that, after penetration of the double nuclear membrane via an as yet unidentified mechanism, vimentin IFs cooperatively fix repetitive DNA sequence elements in a differentiation-specific manner in the nuclear periphery subjacent to the nuclear lamina and thus participate in the organization of chromatin and in the control of transcription, replication, and recombination processes. This includes aspects of global

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

    Directory of Open Access Journals (Sweden)

    Shiyong Sun

    2013-12-01

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

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

    Science.gov (United States)

    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.

  5. Mass Spectrometry Based Ultrasensitive DNA Methylation Profiling Using Target Fragmentation Assay.

    Science.gov (United States)

    Lin, Xiang-Cheng; Zhang, Ting; Liu, Lan; Tang, Hao; Yu, Ru-Qin; Jiang, Jian-Hui

    2016-01-19

    Efficient tools for profiling DNA methylation in specific genes are essential for epigenetics and clinical diagnostics. Current DNA methylation profiling techniques have been limited by inconvenient implementation, requirements of specific reagents, and inferior accuracy in quantifying methylation degree. We develop a novel mass spectrometry method, target fragmentation assay (TFA), which enable to profile methylation in specific sequences. This method combines selective capture of DNA target from restricted cleavage of genomic DNA using magnetic separation with MS detection of the nonenzymatic hydrolysates of target DNA. This method is shown to be highly sensitive with a detection limit as low as 0.056 amol, allowing direct profiling of methylation using genome DNA without preamplification. Moreover, this method offers a unique advantage in accurately determining DNA methylation level. The clinical applicability was demonstrated by DNA methylation analysis using prostate tissue samples, implying the potential of this method as a useful tool for DNA methylation profiling in early detection of related diseases.

  6. A PCR-free cloning method for the targeted φ80 Int-mediated integration of any long DNA fragment, bracketed with meganuclease recognition sites, into the Escherichia coli chromosome.

    Science.gov (United States)

    Ublinskaya, Anna A; Samsonov, Valeriy V; Mashko, Sergey V; Stoynova, Nataliya V

    2012-06-01

    The genetic manipulation of cells is the most promising strategy for designing microorganisms with desired traits. The most widely used approaches for integrating specific DNA-fragments into the Escherichia coli genome are based on bacteriophage site-specific and Red/ET-mediated homologous recombination systems. Specifically, the recently developed Dual In/Out integration strategy enables the integration of DNA fragments directly into specific chromosomal loci (Minaeva et al., 2008). To develop this strategy further, we designed a method for the precise cloning of any long DNA fragments from the E. coli chromosome and their targeted insertion into the genome that does not require PCR. In this method, the region of interest is flanked by I-SceI rare-cutting restriction sites, and the I-SceI-bracketed region is cloned into the unique I-SceI site of an integrative plasmid vector that then enables its targeted insertion into the E. coli chromosome via bacteriophage φ80 Int-mediated specialized recombination. This approach allows any long specific DNA fragment from the E. coli genome to be cloned without a PCR amplification step and reproducibly inserted into any chosen chromosomal locus. The developed method could be particularly useful for the construction of marker-less and plasmid-less recombinant strains in the biotechnology industry.

  7. Effect of intramuscular injection of hepatocyte growth factor plasmid DNA with electroporation on bleomycin-induced lung fibrosis in rats

    Institute of Scientific and Technical Information of China (English)

    LONG Xiang; XIONG Sheng-dao; XIONG Wei-ning; XU Yong-jian

    2007-01-01

    Background So far, there is no efficient treatment for pulmonary fibrosis. The objective of this study was to determine whether intramuscular injection of the hepatocyte growth factor (HGF) plasmid DNA by in vivo electroporation could prevent bleomycin-induced pulmonary fibrosis in rats, and to investigate the possible mechanisms.Methods Twenty male Wistar rats were randomly divided into four groups: control group(group C), model group (group M), early intervention group (group Ⅰ ) and late intervention group (groupⅡ). Groups M, Ⅰ and Ⅱ were intratracheally infused with bleomycin, then injected the plasmid pcDNA3.1-hHGF to group Ⅰ on day 7, 14 and 21. Group Ⅱ received the same treatment like Group Ⅰ on day 14 and 21. All the rats were killed on day 28 after bleomycin injection. We detected Homo HGF expression in the rats with ELISA method and estimated the pathological fibrosis score of lung tissue using hematoxylin eosin (HE) and Massion staining. The mRNA expression of transforming growth factor-β1 (TGF-β1),cycloxygenase-2 (COX-2), and rat HGF in rat pulmonary parenchyma were evaluated by RT-PCR.Immunohistochemistry and Western blotting were performed to determine the protein expression of transforming TGF-β1 and COX-2 in lung parenchyma.Results The plasmid pcDNA3.1-hHGF could express hHGF in NIH3T3 cells and the hHGF protein is secreted into the culture medium. The expression of hHGF protein could be monitored in quadriceps muscle, plasma and lung in Groups Ⅰ and Ⅱ. Pulmonary fibrosis levels of Groups Ⅰ and Ⅱ were obviously lower than that of group M (P<0.05).Expression of TGF-β1 protein and mRNA in lung tissue was markedly decreased in Groups Ⅰ and Ⅱ compared with Group M (P<0.05). The level of expression of HGF and COX-2 mRNA was higher in Groups Ⅰ and Ⅱ than in Group M (P<0.05).Conclusions Injection of the plasmid pcDNA3.1-hHGF into skeletal muscle with electroporation has a potential role in the treatment of bleomycin

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

    Science.gov (United States)

    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.

  9. [THE OPTIMAL CONDITIONS OF STORAGE OF SPERMATOZOA FOR ANALYSIS OF DNA FRAGMENTATION].

    Science.gov (United States)

    Tataru, D A; Markova, E V; Osadchuk, L V; Sheina, E V; Svetlakov, A V

    2015-04-01

    The analysis of fragmentation of DNA of spermatozoons using technique of flow cytometry to evaluate male fertility more and more often begins to be applied in clinical diagnostic. However, development of optimal protocol of storage and preparation of spermatozoons for analysis still is at the stage of experimental elaboration. The studv was carried out to analyse effect of different conditions of preparation of ejaculate for adequate evaluation of index of fragmentation of DNA of spermatozoons using sperm chromatin structure assay technique. The sampling consisted of 20 patients of the Krasnoyarsk center of reproductive medicine. The sperm chromatin structure assay technique was applied to evaluate index of fragmentation of DNA of spermatozoons in fresh native ejaculate and after storage of spermatozoons under different temperature (37, 25 and 4 degrees C) and duration (1-2 and 1-3 days) and conditions of storage (-20 or -70 degrees C) of frozen spermatozoons (as native ejaculate or in TNE-buffer). It is demonstrated that index of fragmentation of DNA of spermatozoons has no significant alterations in ejaculate stored under 4 degrees C during 48 hours. In case of storage of ejaculate under 25 or 37 degrees C index of fragmentation of DNA of spermatozoons significantly increases already after first day of storage. The incubation of ejaculate under 37 degrees C results in increasing of index of fragmentation of DNA of spermatozoons already after first hour. The individual differences are established related to degree of increasing of index of fragmentation of DNA of spermatozoons because of impact of studied temperatures of ejaculate incubation. The storage of spermatozoons under temperature of - 20 and -70 degrees C in native ejaculate or in TNE-buffer has no effect of index of fragmentation of DNA of spermatozoons with measurement during 1-2 hours. Therefore, storage and transportation of native ejaculate under 4 degrees C during 1-2 days or in frozen condition

  10. STUDY REGARDING EFFICIENCY OF INDUCED GENETIC TRANSFORMATION IN BACILLUS LICHENIFORMIS WITH PLASMID DNA

    Directory of Open Access Journals (Sweden)

    VINTILĂ T.

    2007-01-01

    Full Text Available A strain of Bacillus licheniformis was subject to genetic transformation with plasmidvectors (pLC1 and pNC61, using electroporation technique, protoplasttransformation and bivalent cations (CaCl2 mediated transformation. In the case oftransformation by electroporation of Bacillus licheniformis B40, the highest numberof transformed colonies (3 were obtained only after a 1,79 KV electric shock, for 2,2milliseconds. Using this transformation technique we have obtained six kanamycinresistant transformants. The frequency of Bacillus licheniformis B40 protoplaststransformation using pLC1 and pNC61 plasmid vectors is approximately 10% (TF =10%. As a result of pLC1 plasmid integration in Bacillus licheniformis protoplasts,six kanamycin resistant transformants were obtained. The pNC61 plasmid, whichconfers trimethoprim resistance, does not integrate in receiver cells by protoplasttransformation. The direct genetic transformation in the presence of bivalent cations(CaCl2, mediated by pLC1 and pNC61 plasmid vectors, produce a lowtransformation frequency. Using this technique, we have obtained three trimethoprimresistant colonies and four kanamycin resistant colonies. The chemical way oftransformation is the only technique, which realizes the integration of pNC61 in B.licheniformis B40 cells.

  11. TOL Plasmid Carriage Enhances Biofilm Formation and Increases Extracellular DNA Content in Pseudomonas Putida KT2440

    DEFF Research Database (Denmark)

    Smets, Barth F.; D'Alvise, Paul; Yankelovich, T.

    Adherent growth of Pseudomonas putida KT2440 with and without the TOL plasmid (pWWO) at the solid-liquid and air-liquid interface was examined. We compared biofilm formation on glass in flow cells, and assayed pellicle (air-liquid interface biofilm) formation in stagnant liquid cultures by confoc...

  12. Effect of magnesium ions and temperature on the sequence-dependent curvature of DNA restriction fragments

    Energy Technology Data Exchange (ETDEWEB)

    Stellwagen, Nancy C; Lu Yongjun, E-mail: nancy-stellwagen@uiowa.ed [Department of Biochemistry, University of Iowa, Iowa City, IA 52242 (United States)

    2010-12-15

    Transient electric birefringence has been used to quantify the curvature of two DNA restriction fragments, a 199-base-pair fragment taken from the origin of replication of the M13 bacteriophage and a 207-base-pair fragment taken from the VP1 gene in the SV40 minichromosome. Stable curvature in the SV40 and M13 restriction fragments is due to a series of closely spaced A tracts, runs of 4-6 contiguous adenine residues located within 40 or 60 base pair 'curvature modules' near the center of each fragment. The M13 and SV40 restriction fragments exhibit bends of {approx} 45{sup 0} in solutions containing monovalent cations and {approx} 60{sup 0} in solutions containing Mg{sup 2+} ions. The curvature is not localized at a single site but is distributed over the various A tracts in the curvature modules. Thermal denaturation studies indicate that the curvature in the M13 and SV40 restriction fragments remains constant up to 30 {sup 0}C in solutions containing monovalent cations, and up to 40 {sup 0}C in solutions containing Mg{sup 2+} ions, before beginning to decrease slowly with increasing temperature. Hence, stable curvature in these DNA restriction fragments exists at the biologically important temperature of 37 {sup 0}C.

  13. Relationship of spermatozoal DNA fragmentation with semen quality in varicocele-positive men.

    Science.gov (United States)

    Moazzam, A; Sharma, R; Agarwal, A

    2015-10-01

    The aim of the study was to assess the semen quality and levels of spermatozoal nuclear DNA fragmentation in subfertile subjects clinically diagnosed with varicocele, subfertile subjects without varicocele and healthy fertile controls. Semen samples were obtained from 302 subjects. Of them, 115 were healthy fertile controls having normal semen characteristics, 121 subfertile men diagnosed with varicocele, both, clinically and on ultrasonography, while 66 subjects were subfertile with no varicocele. Spermatozoal concentration, percentage motility, morphology and DNA fragmentation were measured. In the study population, deterioration in semen quality-decreased spermatozoal concentration, percentage motility and normal morphology was seen in subfertile subjects, especially with varicocele. Highest spermatozoal DNA fragmentation was observed in varicocele-positive subjects as compared with varicocele-negative subjects and healthy fertile controls. Significant negative correlation was seen between spermatozoal DNA fragmentation and concentration (r = -0.310), motility (r = -0.328) normal morphology, WHO method (r = -0.221) and Tygerberg strict criteria (r = -0.180) in the varicocele-positive subfertile subjects. In conclusion, this study suggests existence of a negative relationship between spermatozoal DNA fragmentation and semen quality in varicocele-positive subfertile subjects. © 2014 Blackwell Verlag GmbH.

  14. Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues

    Directory of Open Access Journals (Sweden)

    Watson Spencer K

    2006-12-01

    Full Text Available Abstract Background Formalin-fixed paraffin-embedded (FFPE tissues represent the largest source of archival biological material available for genomic studies of human cancer. Therefore, it is desirable to develop methods that enable whole genome amplification (WGA using DNA extracted from FFPE tissues. Multiple-strand Displacement Amplification (MDA is an isothermal method for WGA that uses the large fragment of Bst DNA polymerase. To date, MDA has been feasible only for genomic DNA isolated from fresh or snap-frozen tissue, and yields a representational distortion of less than threefold. Results We amplified genomic DNA of five FFPE samples of normal human lung tissue with the large fragment of Bst DNA polymerase. Using quantitative PCR, the copy number of 7 genes was evaluated in both amplified and original DNA samples. Four neuroblastoma xenograft samples derived from cell lines with known N-myc gene copy number were also evaluated, as were 7 samples of non-small cell lung cancer (NSCLC tumors with known Skp2 gene amplification. In addition, we compared the array comparative genomic hybridization (CGH-based genome profiles of two NSCLC samples before and after Bst MDA. A median 990-fold amplification of DNA was achieved. The DNA amplification products had a very high molecular weight (> 23 Kb. When the gene content of the amplified samples was compared to that of the original samples, the representational distortion was limited to threefold. Array CGH genome profiles of amplified and non-amplified FFPE DNA were similar. Conclusion Large fragment Bst DNA polymerase is suitable for WGA of DNA extracted from FFPE tissues, with an expected maximal representational distortion of threefold. Amplified DNA may be used for the detection of gene copy number changes by quantitative realtime PCR and genome profiling by array CGH.

  15. Purification of supercoiled plasmid DNA from clarified bacterial lysate by arginine-affinity chromatography: effects of spacer arms and ligand density.

    Science.gov (United States)

    Bai, Jin-Shan; Bai, Shu; Shi, Qing-Hong; Sun, Yan

    2014-06-01

    Efficient loading on a chromatographic column is the dilemma of the process development faced by engineers in plasmid DNA purification. In this research, novel arginine-affinity chromatographic beads were prepared to investigate the effect of spacer arm and ligand density to their chromatographic performance for the purification of plasmid. The result indicated that dynamic binding capacity for plasmid increased with an increasing ligand density and carbon number of spacer arm, and the highest binding capacity for plasmid of 6.32 mg/mL bead was observed in the column of arginine bead with a ligand density of 47 mmol/L and 10-atom carbon spacer. Furthermore, this arginine bead exhibited better selectivity to supercoiled (sc) plasmid. The evidence of a linear gradient elution suggested further that the binding of plasmid on arginine beads was driven by electrostatic interaction and hydrogen bonding. Hence, sc plasmid could successfully be purified from clarified lysate by two-stepwise elution of salt concentration. By the refinement of the elution scheme and loading volume of clarified lysate, the column of arginine bead with a ligand density of 47 mmol/L exhibited the highest recovery yield and a much higher productivity among arginine-affinity columns. Therefore, reshaped arginine beads provided more feasible and practical application in the preparation of sc plasmid from clarified lysate. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Inter- and intra-laboratory standardization of TUNEL assay for assessment of sperm DNA fragmentation.

    Science.gov (United States)

    Ribeiro, S; Sharma, R; Gupta, S; Cakar, Z; De Geyter, C; Agarwal, A

    2017-05-01

    One of the challenges with the sperm DNA fragmentation results is the inconsistency and the large variability in the results obtained by different techniques. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay quantifies the incorporation of fluoresceinated dUTP into single- and double-strand DNA breaks by labeling the 3'-OH terminal with TdT. The goal of this study was optimize the TUNEL protocol for assessment of sperm DNA fragmentation by standardization of the method and comparison of the data across two reference laboratories (i) at Basel, Switzerland and (ii) Cleveland Clinic, Ohio, USA. Semen samples from 31 subjects grouped into three cohorts. Sperm DNA fragmentation was data measured by two experienced operators at two different laboratories using identical semen samples, assay kit, protocol and acquisition settings using identical flow cytometers (BD Accuri C6). No significant differences were observed between the duplicates in any of the experiments performed. By including an additional washing step after fixation in paraformaldehyde, a high correlation was seen between the two laboratories (r = 0.94). A strong positive correlation was observed between the average sperm DNA fragmentation rates (r = 0.719). The mean sperm DNA fragmentation measured in each laboratory was similar. Both flow cytometers were identical in their settings and performance. This inter- and intra-laboratory study establishes that TUNEL is a reproducible assay when utilizing a standardized staining protocol and flow cytometer acquisition settings. Standardization and consensual guidelines for TUNEL validate the assay and establishes TUNEL as a robust test for measuring sperm DNA fragmentation especially in a multicenter setting. © 2017 American Society of Andrology and European Academy of Andrology.

  17. 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.

    Science.gov (United States)

    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-replication) region of one of those plasmids, pSmeLPU88b. This mobilization region was found to represent a previously unclassified Dtr type in rhizobia (hereafter type-IV), highly ubiquitous in S. meliloti and found in other genera of Gram-negative bacteria as well; including Agrobacterium, Ochrobactrum, and Chelativorans. The oriT of the type-IV Dtr described here could be located by function within a DNA fragment of 278 bp, between the divergent genes parA and mobC. The phylogenetic analysis of the cognate relaxase MobZ indicated that this protein groups close to the previously defined MOB(P3) and MOB(P4) type of enzymes, but is located in a separate and novel cluster that we have designated MOB(P0). Noteworthy, MOB(P0) and MOB(P4) relaxases were frequently associated with plasmids present in rhizospheric soil bacteria. A comparison of the nod-gene locations with the phylogenetic topology of the rhizobial relaxases revealed that the symbiotic genes are found on diverse plasmids bearing any of the four Dtr types, thus indicating that pSym plasmids are not specifically associated with any particular mobilization system. Finally, we demonstrated that the type-IV Dtr promoted the mobilization of plasmids from S. meliloti to Sinorhizobium medicae as well as from these rhizobia to other bacteria by means of their own

  18. Simulating Molecular Interactions of Carbon Nanoparticles with a Double-Stranded DNA Fragment

    Directory of Open Access Journals (Sweden)

    Zhuang Wang

    2015-01-01

    Full Text Available Molecular interactions between carbon nanoparticles (CNPs and a double-stranded deoxyribonucleic acid (dsDNA fragment were investigated using molecular dynamics (MD simulations. Six types of CNPs including fullerenes (C60 and C70, (8,0 single-walled carbon nanotube (SWNT, (8,0 double-walled carbon nanotube (DWNT, graphene quantum dot (GQD, and graphene oxide quantum dot (GOQD were studied. Analysis of the best geometry indicates that the dsDNA fragment can bind to CNPs through pi-stacking and T-shape. Moreover, C60, DWNT, and GOQD bind to the dsDNA molecules at the minor groove of the nucleotide, and C70, SWNT, and GQD bind to the dsDNA molecules at the hydrophobic ends. Estimated interaction energy implies that van der Waals force may mainly contribute to the mechanisms for the dsDNA-C60, dsDNA-C70, and dsDNA-SWNT interactions and electrostatic force may contribute considerably to the dsDNA-DWNT, dsDNA-GQD, and dsDNA-GOQD interactions. On the basis of the results from large-scale MD simulations, it was found that the presence of the dsDNA enhances the dispersion of C60, C70, and SWNT in water and has a slight impact on DWNT, GQD, and GOQD.

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

    Science.gov (United States)

    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.

  20. Accurate phylogenetic classification of DNA fragments based onsequence composition

    Energy Technology Data Exchange (ETDEWEB)

    McHardy, Alice C.; Garcia Martin, Hector; Tsirigos, Aristotelis; Hugenholtz, Philip; Rigoutsos, Isidore

    2006-05-01

    Metagenome studies have retrieved vast amounts of sequenceout of a variety of environments, leading to novel discoveries and greatinsights into the uncultured microbial world. Except for very simplecommunities, diversity makes sequence assembly and analysis a verychallenging problem. To understand the structure a 5 nd function ofmicrobial communities, a taxonomic characterization of the obtainedsequence fragments is highly desirable, yet currently limited mostly tothose sequences that contain phylogenetic marker genes. We show that forclades at the rank of domain down to genus, sequence composition allowsthe very accurate phylogenetic 10 characterization of genomic sequence.We developed a composition-based classifier, PhyloPythia, for de novophylogenetic sequence characterization and have trained it on adata setof 340 genomes. By extensive evaluation experiments we show that themethodis accurate across all taxonomic ranks considered, even forsequences that originate fromnovel organisms and are as short as 1kb.Application to two metagenome datasets 15 obtained from samples ofphosphorus-removing sludge showed that the method allows the accurateclassification at genus level of most sequence fragments from thedominant populations, while at the same time correctly characterizingeven larger parts of the samples at higher taxonomic levels.

  1. Sperm chromatin dispersion test in the assessment of DNA fragmentation and aneuploidy in human spermatozoa.

    Science.gov (United States)

    Balasuriya, A; Speyer, B; Serhal, P; Doshi, A; Harper, J C

    2011-05-01

    Sperm DNA damage is thought to be increased in men with male factor infertility. Previous studies suggest a correlation between sperm DNA fragmentation and aneuploidy. The sperm chromatin dispersion (SCD) test was modified to produce the Halosperm Kit. The SCD-fluorescent in-situ hybridization (FISH) test allows the simultaneous detection of DNA fragmentation and aneuploidy on the same sperm cell. The objectives of this study were to validate the SCD, SCD-FISH and Halosperm tests for the analysis of sperm DNA fragmentation and compare them to the sperm chromatin structure assay (SCSA). Semen samples from 20 males undergoing IVF/intracytoplasmic sperm injection were processed using FISH, SCD-FISH, SCD and Halosperm, and compared with SCSA results. There was a significant difference between FISH and SCD-FISH results in the detection of aneuploidy (P=0.000) and the level of sperm DNA fragmentation in the samples subjected to SCSA and SCD (P=0.001) or SCSA and SCD-FISH (P=0.001). There was no significant correlation between DNA fragmentation and aneuploidy. If sperm aneuploidy is to be determined, more reliable results will be obtained if FISH is performed rather than SCD-FISH. A lack of validation and unknown clinical significance question the value of DNA fragmentation assays. DNA damage in the male germ line may result in adverse clinical outcomes and the pathophysiology and clinical consequences of sperm DNA damage are being actively researched. Many DNA fragmentation assays such as the Halosperm Kit have been developed recently and are now available at a commercial level. Unfortunately, aimed at vulnerable couples with difficulty conceiving, many of these tests have not been clinically validated. Despite its plausible appeal and fervour of its supporters, the benefits of widespread DNA testing that only achieves the distressing of couples with the knowledge that effectual therapeutic strategies are absent are questionable. Commercially, however, it is no doubt

  2. TNF-α is involved in activating DNA fragmentation in skeletal muscle

    Science.gov (United States)

    Carbó, N; Busquets, S; van Royen, M; Alvarez, B; López-Soriano, F J; Argilés, J M

    2002-01-01

    Intraperitoneal administration of 100 μg kg−1 (body weight) of tumour necrosis factor-α to rats for 8 consecutive days resulted in a significant decrease in protein content, which was concomitant with a reduction in DNA content. Interestingly, the protein/DNA ratio was unchanged in the skeletal muscle of the tumour necrosis factor-α-treated animals as compared with the non-treated controls. Analysis of muscle DNA fragmentation clearly showed enhanced laddering in the skeletal muscle of tumour necrosis factor-α-treated animals, suggesting an apoptotic phenomenon. In a different set of experiments, mice bearing a cachexia-inducing tumour (the Lewis lung carcinoma) showed an increase in muscle DNA fragmentation (9.8-fold) as compared with their non-tumour-bearing control counterparts as previously described. When gene-deficient mice for tumour necrosis factor-α receptor protein I were inoculated with Lewis lung carcinoma, they were also affected by DNA fragmentation; however the increase was only 2.1-fold. These results suggest that tumour necrosis factor-α partly mediates DNA fragmentation during experimental cancer-associated cachexia. British Journal of Cancer (2002) 86, 1012–1016. DOI: 10.1038/sj/bjc/6600167 www.bjcancer.com © 2002 Cancer Research UK PMID:11953838

  3. Performance of heuristic methods driven by chaotic dynamics for ATSP and applications to DNA fragment assembly

    Science.gov (United States)

    Kato, Tomohiro; Hasegawa, Mikio

    Chaotic dynamics has been shown to be effective in improving the performance of combinatorial optimization algorithms. In this paper, the performance of chaotic dynamics in the asymmetric traveling salesman problem (ATSP) is investigated by introducing three types of heuristic solution update methods. Numerical simulation has been carried out to compare its performance with simulated annealing and tabu search; thus, the effectiveness of the approach using chaotic dynamics for driving heuristic methods has been shown. The chaotic method is also evaluated in the case of a combinatorial optimization problem in the real world, which can be solved by the same heuristic operation as that for the ATSP. We apply the chaotic method to the DNA fragment assembly problem, which involves building a DNA sequence from several hundred fragments obtained by the genome sequencer. Our simulation results show that the proposed algorithm using chaotic dynamics in a block shift operation exhibits the best performance for the DNA fragment assembly problem.

  4. Synthesis and NMR of {sup 15}N-labeled DNA fragments

    Energy Technology Data Exchange (ETDEWEB)

    Jones, R.A. [Rutgers, The State Univ. of New Jersey, Piscataway, NJ (United States)

    1994-12-01

    DNA fragments labeled with {sup 15}N at the ring nitrogens and at the exocyclic amino groups can be used to obtain novel insight into interactions such as base pairing, hydration, drug binding, and protein binding. A number of synthetic routes to {sup 15}N-labeled pyrimidine nucleosides, purines, and purine nucleosides have been reported. Moreover, many of these labeled bases or monomers have been incorporated into nucleic acids, either by chemical synthesis or by biosynthetic procedures. The focus of this chapter will be on the preparation of {sup 15}N-labeled purine 2{prime}-deoxynucleosides, their incorporation into DNA fragments by chemical synthesis, and the results of NMR studies using these labeled DNA fragments.

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

    Science.gov (United States)

    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.

  6. Construction and expression of recombinant plasmid TAZ-pcDNA3 .1 and TAZ-pEGFP-C2%T AZ 基因重组质粒的构建与表达

    Institute of Scientific and Technical Information of China (English)

    仲念念; 朱伶俐; 王旋; 房娜

    2015-01-01

    Objective Two recombinant plasmids , TAZ‐pcDNA3 .1 and TAZ‐pEGFP‐C2 , were established . The protein expression of TAZ in HEK293 cells was detected by Western Blot and the roles of TAZ in promoting cell proliferation and migration were further explored . Methods AZ gene was amplified by PCR , fragments were recovered followed by connection with glue T carrier , blue‐white screening , transformation and extraction of plasmid DNA . Then the plasmid DNA was digested , connected by T 4 DNA Ligase , and then sub‐cloned into pEGFP‐C2 and pcDNA3 .1 to construct new recombinant plasmids . These plasmids were transfected into HEK293 cells to observe the distribution of TAZ using a fluorescence detector . The protein expression was detected by Western Blot .Results By restriction enzyme identification and sequence analysis , the recombinant plasmids were successfully constructed . Fluorescent photos show that the distribution of TAZ molecule was in the nucleus and cytoplasm . Western Blot test results showed that TAZ molecule could induce over‐expression of specific proteins . Conclusion Two recombinant plasmids were successfully constructed . The effects of TAZ over‐expression were validated , which will lay a foundation for revealing the mechanism of TAZ in promoting cell proliferation and migration .%目的:构建重组质粒TAZ‐pcDNA31.及 TAZ‐pEGFP‐C2,并应用Western Blot检测TAZ蛋白在细胞内的表达情况,初步探索TAZ分子促进细胞增殖和迁移的作用机制。方法通过PCR扩增获得 TAZ基因片段,胶回收后连接T载体,蓝白斑筛选,转化,提质粒,酶切,用T4 DNA Ligase连接,亚克隆进入pEGFP‐C2和pcDNA31.获得新的重组质粒,分别转染 HEK293细胞,智能型荧光细胞监测仪观察TAZ分子在细胞内的分布情况,Western Blot检测其在细胞内的表达情况。结果重组质粒经双酶切鉴定和测序证明构建成功,荧光照片显示 TAZ分子分布在

  7. FragIdent – Automatic identification and characterisation of cDNA-fragments

    Directory of Open Access Journals (Sweden)

    Goehler Heike

    2009-03-01

    Full Text Available Abstract Background Many genetic studies and functional assays are based on cDNA fragments. After the generation of cDNA fragments from an mRNA sample, their content is at first unknown and must be assigned by sequencing reactions or hybridisation experiments. Even in characterised libraries, a considerable number of clones are wrongly annotated. Furthermore, mix-ups can happen in the laboratory. It is therefore essential to the relevance of experimental results to confirm or determine the identity of the employed cDNA fragments. However, the manual approach for the characterisation of these fragments using BLAST web interfaces is not suited for larger number of sequences and so far, no user-friendly software is publicly available. Results Here we present the development of FragIdent, an application for the automatic identification of open reading frames (ORFs within cDNA-fragments. The software performs BLAST analyses to identify the genes represented by the sequences and suggests primers to complete the sequencing of the whole insert. Gene-specific information as well as the protein domains encoded by the cDNA fragment are retrieved from Internet-based databases and included in the output. The application features an intuitive graphical interface and is designed for researchers without any bioinformatics skills. It is suited for projects comprising up to several hundred different clones. Conclusion We used FragIdent to identify 84 cDNA clones from a yeast two-hybrid experiment. Furthermore, we identified 131 protein domains within our analysed clones. The source code is freely available from our homepage at http://compbio.charite.de/genetik/FragIdent/.

  8. Specific sperm defects are differentially correlated with DNA fragmentation in both normozoospermic and teratozoospermic subjects.

    Science.gov (United States)

    Mangiarini, A; Paffoni, A; Restelli, L; Ferrari, S; Guarneri, C; Ragni, G; Somigliana, E

    2013-11-01

    A positive effect of selecting spermatozoa under high magnification during intracytoplasmic sperm injection (ICSI) has been described, but a clear explanation has not been given yet. Previous works have shown that high magnification selected spermatozoa have significantly better chromatin status than unselected cells; on the other hand, it has been reported that spermatozoa with no morphological defects can also be negatively associated with embryo quality and pregnancy outcome attributable to DNA fragmentation. The aim of this study was to investigate whether sperm morphology is correlated with DNA fragmentation, both in normozoospermic and teratozoospermic patients. A prospective cohort study involving 32 subjects was recruited over a 3-month period. Spermatozoa were fixed on a slide for TUNEL assay and evaluated using an epifluorescent light microscope equipped with a video monitor. Single TUNEL-positive or -negative cells were evaluated for morphology at ×4400 magnification. Each spermatozoon was then classified according to morphological normalcy or specific defects. The median percentage of typical forms was 11 and 0%, in the normozoospermic and teratozoospermic groups respectively (p = 0.001). In normozoospermic samples, the percentage of TUNEL-positive morphologically normal spermatozoa was 4%. By comparison, spermatozoa showing a vacuolated head or a small non-oval head had a significantly higher incidence of DNA fragmentation in both groups (12 and 13%, 19 and 13% respectively; p DNA fragmentation rate similar to typical forms (3 and 5%, in normozoospermic and teratozoospermic respectively). This study shows that specific defects evaluated in fixed spermatozoa under high-power magnification are more likely to be associated with DNA fragmentation. High-magnification evaluation of spermatozoa can therefore reduce the probability of selecting cells carrying fragmented DNA during ICSI. © 2013 American Society of Andrology and European Academy of

  9. Genetic alterations of hepatocellular carcinoma by random amplified polymorphic DNA analysis and cloning sequencing of tumor differential DNA fragment

    Institute of Scientific and Technical Information of China (English)

    Zhi-Hong Xian; Wen-Ming Cong; Shu-Hui Zhang; Meng-Chao Wu

    2005-01-01

    AIM: To study the genetic alterations and their association with clinicopathological characteristics of hepatocellular carcinoma (HCC), and to find the tumor related DNA fragments.METHODS: DNA isolated from tumors and corresponding noncancerous liver tissues of 56 HCC patients was amplified by random amplified polymorphic DNA (RAPD)with 10 random 10-mer arbitrary primers. The RAPD bands showing obvious differences in tumor tissue DNA corresponding to that of normal tissue were separated,purified, cloned and sequenced. DNA sequences were analyzed and compared with GenBank data.RESULTS: A total of 56 cases of HCC were demonstrated to have genetic alterations, which were detected by at least one primer. The detestability of genetic alterations ranged from 20% to 70% in each case, and 17.9% to 50% in each primer. Serum HBV infection, tumor size,histological grade, tumor capsule, as well as tumor intrahepatic metastasis, might be correlated with genetic alterations on certain primers. A band with a higher intensity of 480 bp or so amplified fragments in tumor DNA relative to normal DNA could be seen in 27 of 56 tumor samples using primer 4. Sequence analysis of these fragments showed 91% homology with Homo sapiens double homeobox protein DUX10 gene.CONCLUSION: Genetic alterations are a frequent event in HCC, and tumor related DNA fragments have been found in this study, which may be associated with hepatocarcinogenesis. RAPD is an effective method for the identification and analysis of genetic alterations in HCC, and may provide new information for further evaluating the molecular mechanism of hepatocarcinogenesis.

  10. Cloning of C-Terminal of Opioid μ-Receptor and Construction of Its Expression Plasmid for Yeast Two Hybrid System

    Institute of Scientific and Technical Information of China (English)

    YANHui; GONGZe-hui

    2004-01-01

    Aim: To obtain the C-terminal DNA and construct the expression plasmid in yeast two-hybrid. Methods: About 177bp DNA fragment was amplified from the complete sequence of ( receptor by PCR. After being sequenced, the C-terminal fragment was ligased into EcoR I-BamH I site of pGBKT7 vector to form recombinants. The recombinant plasmid

  11. Purification of plasmid DNA from clarified and non-clarified Escherichia coli lysates by berenil pseudo-affinity chromatography.

    Science.gov (United States)

    Caramelo-Nunes, C; Gabriel, M F; Almeida, P; Marcos, J C; Tomaz, C T

    2012-09-01

    In this study, berenil was tested as a ligand, specifically to purify plasmids of different sizes pVAX1-LacZ (6.05 Kbp) and pCAMBIA-1303 (12.361 Kbp) from clarified Escherichia coli alkaline lysates. For this purpose, chromatographic experiments were performed using Sepharose derivatized with berenil. The results showed that both pDNA molecules are completely purified using lower amounts of salt in the eluent than those previously reported for other pseudo-affinity and hydrophobic interaction chromatography based processes. Total retention of all lysate components was achieved with 1.3M ammonium sulphate in the eluent buffer and pDNA elution was obtained by decreasing the salt concentration to 0.55 M. All impurities were eluted after decreasing the concentration to 0M. The recovery yield for pCAMBIA-1303 (45%) was lower than that obtained for pVAX1-LacZ (85%), however the larger pDNA showed a higher purity level. Purification of pVAX1-LacZ was also performed using non-clarified E. coli process streams, replacing the clarification step with a second chromatographic run on the berenil-Sepharose. Using the same binding and elution conditions as before, a pure plasmid sample was obtained with a 33% yield and with all host impurity levels in accordance with the requirements established by the regulatory agencies. These results suggest that this chromatographic method is a promising alternative to purify pDNA for therapeutic use. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Development of procedures for the identification of human papilloma virus DNA fragments in laser plume

    Science.gov (United States)

    Woellmer, Wolfgang; Meder, Tom; Jappe, Uta; Gross, Gerd; Riethdorf, Sabine; Riethdorf, Lutz; Kuhler-Obbarius, Christina; Loening, Thomas

    1996-01-01

    For the investigation of laser plume for the existence of HPV DNA fragments, which possibly occur during laser treatment of virus infected tissue, human papillomas and condylomas were treated in vitro with the CO2-laser. For the sampling of the laser plume a new method for the trapping of the material was developed by use of water-soluble gelatine filters. These samples were analyzed with the polymerase chain reaction (PCR) technique, which was optimized in regard of the gelatine filters and the specific primers. Positive PCR results for HPV DNA fragments up to the size of a complete oncogene were obtained and are discussed regarding infectiousity.

  13. Targeted Multifunctional Lipid ECO Plasmid DNA Nanoparticles as Efficient Non-viral Gene Therapy for Leber's Congenital Amaurosis.

    Science.gov (United States)

    Sun, Da; Sahu, Bhubanananda; Gao, Songqi; Schur, Rebecca M; Vaidya, Amita M; Maeda, Akiko; Palczewski, Krzysztof; Lu, Zheng-Rong

    2017-06-16

    Development of a gene delivery system with high efficiency and a good safety profile is essential for successful gene therapy. Here we developed a targeted non-viral delivery system using a multifunctional lipid ECO for treating Leber's congenital amaurosis type 2 (LCA2) and tested this in a mouse model. ECO formed stable nanoparticles with plasmid DNA (pDNA) at a low amine to phosphate (N/P) ratio and mediated high gene transfection efficiency in ARPE-19 cells because of their intrinsic properties of pH-sensitive amphiphilic endosomal escape and reductive cytosolic release (PERC). All-trans-retinylamine, which binds to interphotoreceptor retinoid-binding protein (IRBP), was incorporated into the nanoparticles via a polyethylene glycol (PEG) spacer for targeted delivery of pDNA into the retinal pigmented epithelium. The targeted ECO/pDNA nanoparticles provided high GFP expression in the RPE of 1-month-old Rpe65(-/-) mice after subretinal injection. Such mice also exhibited a significant increase in electroretinographic activity, and this therapeutic effect continued for at least 120 days. A safety study in wild-type BALB/c mice indicated no irreversible retinal damage following subretinal injection of these targeted nanoparticles. All-trans-retinylamine-modified ECO/pDNA nanoparticles provide a promising non-viral platform for safe and effective treatment of RPE-specific monogenic eye diseases such as LCA2. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  14. ASSOCIATION OF DIFFERENTIALLY EXPRESSED cDNA FRAGMENT OF FGG WITH HEPATOCELLULAR CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    范秉琳; 朱武凌; 邹国林; 段芳龄

    2002-01-01

    Objective: To identify a cDNA clone from the subtracted library of human hepatocellular carcinoma (HCC). Methods: Suppression subtractive hybridization was used to isolated a panel of genes that are differentially expressed in hepatocellular carcinoma as compared with cirrhotic liver. T/A cloning method was used to construct a subtracted cDNA library. DNA sequencing analysis and Northern blot analysis were also utilized. Results: The cloned cDNA is 787 nucleotides in length and contains an open reading frame of 230 amino acids, which is a cDNA fragment of reported human fibrinogen, gamma polypeptide (FGG). Northern analysis revealed that this gene was overexpressed in two hepatocellular carcinoma cell lines, SMMC-7721 and HepG2. Conclusion: Sequence identity proved the cDNA clone fragment of as FGG gene. Differential expression of the cDNA fragment in HCC suggested that FGG is related to HCC, indicating a new clue for developing a novel diagnostic and prognostic marker.

  15. Development of a Novel Reference Plasmid for Accurate Quantification of Genetically Modified Kefeng6 Rice DNA in Food and Feed Samples

    Directory of Open Access Journals (Sweden)

    Liang Li

    2013-01-01

    Full Text Available Reference plasmids are an essential tool for the quantification of genetically modified (GM events. Quantitative real-time PCR (qPCR is the most commonly used method to characterize and quantify reference plasmids. However, the precision of this method is often limited by calibration curves, and qPCR data can be affected by matrix differences between the standards and samples. Here, we describe a digital PCR (dPCR approach that can be used to accurately measure the novel reference plasmid pKefeng6 and quantify the unauthorized variety of GM rice Kefeng6, eliminating the issues associated with matrix effects in calibration curves. The pKefeng6 plasmid was used as a calibrant for the quantification of Kefeng6 rice by determining the copy numbers of event- (77 bp and taxon-specific (68 bp fragments, their ratios, and their concentrations. The plasmid was diluted to five different concentrations. The third sample (S3 was optimized for the quantification range of dPCR according to previous reports. The ratio between the two fragments was 1.005, which closely approximated the value certified by sequencing, and the concentration was found to be 792 copies/μL. This method was precise, with an RSD of ~3%. These findings demonstrate the advantages of using the dPCR method to characterize reference materials.

  16. The angiogenic response to PLL-g-PEG-mediated HIF-1α plasmid DNA delivery in healthy and diabetic rats.

    Science.gov (United States)

    Thiersch, Markus; Rimann, Markus; Panagiotopoulou, Vasiliki; Öztürk, Ece; Biedermann, Thomas; Textor, Marcus; Lühmann, Tessa C; Hall, Heike

    2013-05-01

    Impaired angiogenesis is a major clinical problem and affects wound healing especially in diabetic patients. Improving angiogenesis is a reasonable strategy to increase diabetes-impaired wound healing. Recently, our lab described a system of transient gene expression due to pegylated poly-l-lysine (PLL-g-PEG) polymer-mediated plasmid DNA delivery in vitro. Here we synthesized peptide-modified PLL-g-PEG polymers with two functionalities, characterized them in vitro and utilized them in vivo via a fibrin-based delivery matrix to induce dermal wound angiogenesis in diabetic rats. The two peptides were 1) a TG-peptide to covalently bind these nanocondensates to the fibrin matrix (TG-peptide) for a sustained release and 2) a polyR peptide to improve cellular uptake of these nanocondensates. In order to induce angiogenesis in vivo we condensed modified and non-modified polymers with plasmid DNA encoding a truncated form of the therapeutic candidate gene hypoxia-inducible transcription factor 1α (HIF-1α). HIF-1α is the primarily oxygen-dependent regulated subunit of the heterodimeric transcription factor HIF-1, which controls angiogenesis among other physiological pathways. The truncated form of HIF-1α lacks the oxygen-dependent degradation domain (ODD) and therefore escapes degradation under normoxic conditions. PLL-g-PEG polymer-mediated HIF-1α-ΔODD plasmid DNA delivery was found to lead to a transiently induced gene expression of angiogenesis-related genes Acta2 and Pecam1 as well as the HIF-1α target gene Vegf in vivo. Furthermore, HIF-1α gene delivery was shown to enhance the number endothelial cells and smooth muscle cells - precursors for mature blood vessels - during wound healing. We show that - depending on the selection of the therapeutic target gene - PLL-g-PEG nanocondensates are a promising alternative to viral DNA delivery approaches, which might pose a risk to health. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Effect of excessive cadmium chloride on the plasmids of E. coli HB 101 in vivo

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    After Escherichia coli HB101 with plasmid pWH58, pWH98, or pTBa5 were cultered respectively in amp LB broth which contained 50 mg/L CdCl2 constantly for 24 h, these plasmids were isolated from E. coli, and the effect of excessive CdCl2 on the E. coli HB101 and plasmid DNA was studied by surveying the growth of E. coli HB101 and plasmid, argarose gel electrophoresis and analysis of restriction fragment length polymorphism (RFLP) of plasmids, and plasmid transformation. The results showed that 50 mg/L CdCl2 treatment lagged the growth of E. coli HB101 for at least 4h, but after grown for 24h there were not significant differences in the growths of E. coli HB101s and the productions of plasmids between the treatment and control. These results implified that E. coli HB101 have induced adaptability to cadmium stress and excessive CdCl2 did not inhibit the replication and amp+ gene's expression of plasmid DNA in vivo of E. coli significantly. 50 mg/L CdCl2 treatment for 24 hours might cause the sequence's change of plasmid DNA, but could not lead to the random breakage of plasmid DNA strands. Moreover, after 50 mg/L of CdCl2 treatment in vivo the transformation activities of plasmid did not altered, implied excessive CdCl2 could not affect the superhelical structure of plasmid and also not break the loop of plasmid DNA evidently.

  18. 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.

    Science.gov (United States)

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Plasmid DNA Supercoiling and Gyrase Activity in Escherichia coli Wild-Type and rpoS Stationary-Phase Cells

    Science.gov (United States)

    Reyes-Domínguez, Yazmid; Contreras-Ferrat, Gabriel; Ramírez-Santos, Jesús; Membrillo-Hernández, Jorge; Gómez-Eichelmann, M. Carmen

    2003-01-01

    Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase rpoS cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that rpoS plays a role in the regulation of plasmid topology during the stationary phase. PMID:12533486

  20. DNA rearrangement has occurred in the carbazole-degradative plasmid pCAR1 and the chromosome of its unsuitable host, Pseudomonas fluorescens Pf0-1.

    Science.gov (United States)

    Shintani, Masaki; Matsumoto, Takashi; Yoshikawa, Hirofumi; Yamane, Hisakazu; Ohkuma, Moriya; Nojiri, Hideaki

    2011-12-01

    The carbazole-degradative plasmid pCAR1 carries the class II transposon Tn4676, which contains the car and ant genes, essential for conversion of carbazole into anthranilate, and anthranilate into catechol, respectively. In our previous study, DNA rearrangements in pCAR1 were frequently detected in the host Pseudomonas fluorescens Pf0-1 in the presence of carbazole, resulting in the improvement of host survivability. Several Pf0-1 mutants harbouring pCAR1 were isolated, and deletion of DNA in the plasmid ant gene was found. Here, we compared genome sequences of the parent strain Pf0-1L(pCAR1::rfp) and one of its mutants, 5EP83, to assess whether other DNA rearrangements occurred in either the plasmid or the host chromosome. We found transposition of Tn4676 into the 5EP83 chromosome. In addition, ISPre1 had transposed into the car gene intergenic region on the pCAR1-derivative plasmid of 5EP83, which inhibited car transcription. As a result of these transpositions, 5EP83 was able to metabolize carbazole due to the Tn4676 on its chromosome, although the car genes on its plasmid were non-functional. We also found that one copy of duplicate carAa genes had been deleted, and that ISPre4 had transposed into both the host chromosome and the plasmid. Our findings suggest that Pf0-1 harbouring pCAR1 is subjected to DNA rearrangements not only on the plasmid but also on its chromosome in the presence of carbazole.

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

    Science.gov (United States)

    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

  2. Statistical methods for detecting periodic fragments in DNA sequence data

    Directory of Open Access Journals (Sweden)

    Ying Hua

    2011-04-01

    Full Text Available Abstract Background Period 10 dinucleotides are structurally and functionally validated factors that influence the ability of DNA to form nucleosomes, histone core octamers. Robust identification of periodic signals in DNA sequences is therefore required to understand nucleosome organisation in genomes. While various techniques for identifying periodic components in genomic sequences have been proposed or adopted, the requirements for such techniques have not been considered in detail and confirmatory testing for a priori specified periods has not been developed. Results We compared the estimation accuracy and suitability for confirmatory testing of autocorrelation, discrete Fourier transform (DFT, integer period discrete Fourier transform (IPDFT and a previously proposed Hybrid measure. A number of different statistical significance procedures were evaluated but a blockwise bootstrap proved superior. When applied to synthetic data whose period-10 signal had been eroded, or for which the signal was approximately period-10, the Hybrid technique exhibited superior properties during exploratory period estimation. In contrast, confirmatory testing using the blockwise bootstrap procedure identified IPDFT as having the greatest statistical power. These properties were validated on yeast sequences defined from a ChIP-chip study where the Hybrid metric confirmed the expected dominance of period-10 in nucleosome associated DNA but IPDFT identified more significant occurrences of period-10. Application to the whole genomes of yeast and mouse identified ~ 21% and ~ 19% respectively of these genomes as spanned by period-10 nucleosome positioning sequences (NPS. Conclusions For estimating the dominant period, we find the Hybrid period estimation method empirically to be the most effective for both eroded and approximate periodicity. The blockwise bootstrap was found to be effective as a significance measure, performing particularly well in the problem of

  3. A Novel Sieving Medium for Separation of DNA Fragments- Poly(acrylamide-dimethylacrylamide)

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A short chain poly (acrylamide-dimethylacrylamide) was synthesized in water phase using isopropanol as a chain transfer agent. This copolymer can form a stable dynamic coating on the inner surface of the capillary, thereby suppressing the electroosmotic flow and DNA-capillary wall interaction. The high efficient separation of DNA fragments and SSCP analysis were obtained in bare capillaries using this copolymer as a sieving medium.

  4. Comparison of plasmid DNA versus PCR amplified gene of insert DNA for nucleofection in Kasumi-1 cells.

    Science.gov (United States)

    Wu, Kang; Zhao, Xu-Jie; Wong, Ka-Wing; Fan, Xiao-Yong

    2015-03-01

    Plasmid electroporation, or its optimized version nucleofection, is an important technique for gene transfection of cells in suspension. However, substantial cell death and/or low transfection efficiency are still common for some cell lines. By using enhanced green fluorescent protein (EGFP) as a reporter, we compared the use of PCR amplified EGFP (PaEGFP) and its parental plasmid (pEGFP-N2) for nucleofection in Kasumi-1 cells. We found that PaEGFP induced significantly lower cell death but had similar transfection efficiency compared to its parent plasmid (pEGFP-N2). Most importantly, contrary to the pEGFP-N2-nucleofected cells, the PaEGFP-nucleofected cells subsequently grew properly. Tests in other cell lines also implied that PaEGFP indeed induced consistently less cell death, but transfection efficiencies varied, being good in suspension cell lines but lower in adhesive cell lines. We suggest that direct transfection with PCR amplified genes can be a simple and useful approach for optimization of electropulse-based transfection not only of Kasumi-1 cells, but also may be useful for other cell lines that are difficult to transfect in suspension.

  5. Decreased sperm DNA fragmentation after surgical varicocelectomy is associated with increased pregnancy rate.

    Science.gov (United States)

    Smit, Marij; Romijn, Johannes C; Wildhagen, Mark F; Veldhoven, Joke L M; Weber, Robertus F A; Dohle, Gert R

    2013-01-01

    We prospectively evaluated changes in sperm chromatin structure in infertile patients before and after surgical repair of varicocele, and the impact on the pregnancy rate. Included in the study were 49 men with at least a 1-year history of infertility, a palpable varicocele and oligospermia. World Health Organization semen analysis and sperm DNA damage expressed as the DNA fragmentation index using the sperm chromatin structure assay were assessed preoperatively and postoperatively. Pregnancy (spontaneous and after assisted reproductive technique) was recorded 2 years after surgery. Mean sperm count, sperm concentration and sperm progressive motility improved significantly after varicocelectomy from 18.3 × 10(6) to 44.4 × 10(6), 4.8 × 10(6)/ml to 14.3 × 10(6)/ml and 16.7% to 26.6%, respectively (p DNA fragmentation index decreased significantly after surgery from 35.2% to 30.2% (p = 0.019). When the definition of greater than 50% improvement in sperm concentration after varicocelectomy was applied, 31 of 49 patients (63%) responded to varicocelectomy. After varicocelectomy 37% of the couples conceived spontaneously and 24% achieved pregnancy with assisted reproductive technique. The mean postoperative DNA fragmentation index was significantly higher in couples who did not conceive spontaneously or with assisted reproductive technique (p = 0.033). After varicocelectomy sperm parameters significantly improved and sperm DNA fragmentation was significantly decreased. Low DNA fragmentation index values are associated with a higher pregnancy rate (spontaneous and with assisted reproductive technique). We suggest that varicocelectomy should be considered in infertile men with palpable varicocele, abnormal semen analysis and no major female factors. Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  6. Plasmid DNA Vaccine Co-Immunisation Modulates Cellular and Humoral Immune Responses Induced by Intranasal Inoculation in Mice.

    Directory of Open Access Journals (Sweden)

    Deborah F L King

    Full Text Available An effective HIV vaccine will likely require induction of both mucosal and systemic cellular and humoral immune responses. We investigated whether intramuscular (IM delivery of electroporated plasmid DNA vaccine and simultaneous protein vaccinations by intranasal (IN and IM routes could be combined to induce mucosal and systemic cellular and humoral immune responses to a model HIV-1 CN54 gp140 antigen in mice.Co-immunisation of DNA with intranasal protein successfully elicited both serum and vaginal IgG and IgA responses, whereas DNA and IM protein co-delivery did not induce systemic or mucosal IgA responses. Cellular IFNγ responses were preserved in co-immunisation protocols compared to protein-only vaccination groups. The addition of DNA to IN protein vaccination reduced the strong Th2 bias observed with IN protein vaccination alone. Luminex analysis also revealed that co-immunisation with DNA and IN protein induced expression of cytokines that promote B-cell function, generation of TFH cells and CCR5 ligands that can reduce HIV infectivity.These data suggest that while IN inoculation alone elicits both cellular and humoral responses, co-administration with homologous DNA vaccination can tailor these towards a more balanced Th1/Th2 phenotype modulating the cellular cytokine profile while eliciting high-levels of antigen-specific antibody. This work provides insights on how to generate differential immune responses within the same vaccination visit, and supports co-immunisation with DNA and protein by a mucosal route as a potential delivery strategy for HIV vaccines.

  7. Fenton fragmentation for faster electrophoretic on chip purification of amplifiable genomic DNA.

    Science.gov (United States)

    Hakenberg, S; Hügle, M; Meyer, P; Behrmann, O; Dame, G; Urban, G A

    2015-05-15

    With a rapid and simple actuation protocol electrophoretic nucleic acid extraction is easy automatable, requires no moving parts, is easy to miniaturize and furthermore possesses a size dependent cut-off filter adjustable by the pore size of the hydrogel. However electrophoretic nucleic acid extraction from bacteria has so far been applied mainly for short RNA targets. One of the reasons is that electrophoretic processing of unfragmented genomic DNA strands is time-consuming, because of the length. Here DNA fragmentation would accelerate extraction and isolation. We introduce on-chip lysis and non-enzymatic DNA cleavage directly followed by a purifying step for receiving amplifiable DNA fragments from bacteria in less than 25 min. In contrast to restriction enzymes the Fenton reaction is known to cleave DNA without nucleotide specificity. The reaction mix contains iron(II) EDTA, sodium ascorbate, hydrogen peroxide and lysozyme. The degree of fragmentation can be adjusted by the concentration of reagents. The results enable electrophoretic extraction methods to unspecifically process long genomic DNA in a short time frame, e.g. for pathogen detection in a lab-on-a-chip format. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. A Monte Carlo study of the radiation quality dependence of DNA fragmentation spectra.

    Science.gov (United States)

    Alloni, D; Campa, A; Belli, M; Esposito, G; Facoetti, A; Friedland, W; Liotta, M; Mariotti, L; Paretzke, H G; Ottolenghi, A

    2010-03-01

    We simulated the irradiation of human fibroblasts with gamma rays, protons and helium, carbon and iron ions at a fixed dose of 5 Gy. The simulations were performed with the biophysical Monte Carlo code PARTRAC. From the output of the code, containing in particular the genomic positions of the radiation-induced DNA double-strand breaks (DSBs), we obtained the DNA fragmentation spectra. Very small fragments, in particular those related to "complex lesions" (few tens of base pairs), are probably very important for the late cellular consequences, but their detection is not possible with the common experimental techniques. We paid special attention to the differences among the various ions in the production of these very small fragments; in particular, we compared the fragmentation spectra for ions of the same specific energy and for ions of the same LET (linear energy transfer). As found previously for iron ions, we found that the RBE (relative biological effectiveness) for DSB production was considerably higher than 1 for all high-LET radiations considered. This is at variance with the results obtainable from experimental data, and it is due to the ability to count the contribution of small fragments. It should be noted that for a given LET this RBE decreases with increasing ion charge, due mainly to the increasing mean energy of secondary electrons. A precise quantification of the DNA initial damage can be of great importance for both radiation protection, particularly in open-space long-term manned missions, and hadrontherapy.

  9. Plasmid DNA Analysis of Pathogenic Escherichia coli in Musk Deer%麝致病性大肠杆菌的质粒DNA分析

    Institute of Scientific and Technical Information of China (English)

    罗燕; 程建国; 郑士华; 赵翠; 李蓓; 李敏

    2009-01-01

    [Objective] The pathogenic Escherichia coli in musk deer was classified at molecular level to provide basic materials for molecular epidemiology of pathogenic Escherichia coli in musk deer. [Method] Plasmids from 24 pathogenic Escherichia coli in musk deer were extracted by the Lysis Triton method, and then identified by single enzyme digestion with three endonucleases of Hind Ⅲ, EcoR Ⅰ and BamH Ⅰ. [Result] The yield rate of plasmids was 91.6%, and 24 pathogenic Escherichia coli in musk deer had the identical or similar plasmid profiles. [Conclusion] Plasmid DNA analysis offers scientific basis for molecular epidemiology of pathogenic Escherichia coli in musk deer in Sichuan Institute of Musk Deer Breeding.

  10. Restriction fragment length polymorphisms of the DNA of selected Naegleria and Acanthamoeba amebae.

    Science.gov (United States)

    McLaughlin, G L; Brandt, F H; Visvesvara, G S

    1988-09-01

    Fourteen strains of Naegleria fowleri, two strains of N. gruberi, and one strain each of N. australiensis, N. jadini, N. lovaniensis, Acanthamoeba sp., A. castellanii, A. polyphaga, and A. comandoni isolated from patients, soil, or water were characterized by restriction fragment length polymorphisms. Total cellular DNA (1 microgram) was digested with either HindIII, BglII, or EcoRI; separated on agarose gels; and stained with ethidium bromide. From 2 to 15 unusually prominent repetitive restriction fragment bands, totaling 15 to 50 kilobases in length and constituting probably more than 30% of the total DNA, were detected for all ameba strains. Each species displayed a characteristic pattern of repetitive restriction fragments. Digests of the four Acanthamoeba spp. displayed fewer, less intensely staining repetitive fragments than those of the Naegleria spp. All N. fowleri strains, whether isolated from the cerebrospinal fluid of patients from different parts of the world or from hot springs, had repetitive restriction fragment bands of similar total lengths (ca. 45 kilobases), and most repetitive bands displayed identical mobilities. However, polymorphic bands were useful in identifying particular isolates. Restriction fragment length polymorphism analysis generally was consistent with taxonomy based on studies of infectivity, morphology, isoenzyme patterns, and antibody reactivity and suggests that this technique may help classify amebae isolated from clinical specimens or from the environment.

  11. Direct and Auger Electron-Induced, Single- and Double-Strand Breaks on Plasmid DNA Caused by 99mTc-Labeled Pyrene Derivatives and the Effect of Bonding Distance

    Science.gov (United States)

    Reissig, Falco; Mamat, Constantin; Steinbach, Joerg; Pietzsch, Hans-Juergen; Freudenberg, Robert; Navarro-Retamal, Carlos; Caballero, Julio; Kotzerke, Joerg; Wunderlich, Gerd

    2016-01-01

    It is evident that 99mTc causes radical-mediated DNA damage due to Auger electrons, which were emitted simultaneously with the known γ-emission of 99mTc. We have synthesized a series of new 99mTc-labeled pyrene derivatives with varied distances between the pyrene moiety and the radionuclide. The pyrene motif is a common DNA intercalator and allowed us to test the influence of the radionuclide distance on damages of the DNA helix. In general, pUC 19 plasmid DNA enables the investigation of the unprotected interactions between the radiotracers and DNA that results in single-strand breaks (SSB) or double-strand breaks (DSB). The resulting DNA fragments were separated by gel electrophoresis and quantified by fluorescent staining. Direct DNA damage and radical-induced indirect DNA damage by radiolysis products of water were evaluated in the presence or absence of the radical scavenger DMSO. We demonstrated that Auger electrons directly induced both SSB and DSB in high efficiency when 99mTc was tightly bound to the plasmid DNA and this damage could not be completely prevented by DMSO, a free radical scavenger. For the first time, we were able to minimize this effect by increasing the carbon chain lengths between the pyrene moiety and the 99mTc nuclide. However, a critical distance between the 99mTc atom and the DNA helix could not be determined due to the significantly lowered DSB generation resulting from the interaction which is dependent on the type of the 99mTc binding motif. The effect of variable DNA damage caused by the different chain length between the pyrene residue and the Tc-core as well as the possible conformations of the applied Tc-complexes was supplemented with molecular dynamics (MD) calculations. The effectiveness of the DNA-binding 99mTc-labeled pyrene derivatives was demonstrated by comparison to non-DNA-binding 99mTcO4–, since nearly all DNA damage caused by 99mTcO4– was prevented by incubating with DMSO. PMID:27583677

  12. DermaVir: a plasmid DNA-based nanomedicine therapeutic vaccine for the treatment of HIV/AIDS.

    Science.gov (United States)

    Lori, Franco

    2011-10-01

    The HIV global pandemic continues to rage with over 33 million people living with the disease. Although multidrug therapy has improved the prognosis for those infected by the virus, it has not eradicated the infection. Immunological therapies, including therapeutic vaccines, are needed to supplement drug therapy in the search for a 'functional cure' for HIV. DermaVir (Genetic Immunity Kft, Budapest, Hungary and McLean, Virginia, USA), an experimental HIV/AIDS therapeutic vaccine, combines three key elements of rational therapeutic vaccine design: a single plasmid DNA (pDNA) immunogen expressing 15 HIV antigens, a synthetic pDNA nanomedicine formulation and a dendritic cell-targeting topical-vaccine administration. DermaVir's novel mechanism of action, natural transport by epidermal Langerhans cells to the lymph nodes to express the pDNA-encoded HIV antigens and induce precursor/memory T cells with high proliferation capacity, has been consistently demonstrated in mouse, rabbit, primate and human subjects. Safety, immunogenicity and preliminary efficacy of DermaVir have been clinically demonstrated in HIV-infected human subjects. The DermaVir technology platform for dendritic cell-based therapeutic vaccination might offer a new treatment paradigm for cancer and infectious diseases.

  13. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo.

    Science.gov (United States)

    Wegman, F; Bijenhof, A; Schuijff, L; Oner, F C; Dhert, W J A; Alblas, J

    2011-03-15

    Bone regeneration is one of the major focus points in the field of regenerative medicine. A well-known stimulus of bone formation is bone morphogenetic protein-2 (BMP-2), which has already been extensively used in clinical applications. We investigated the possibility of achieving osteogenic differentiation both in vitro and in vivo as a result of prolonged presence of BMP-2 using plasmid DNA-based gene therapy. By delivering BMP-2 cDNA in an alginate hydrogel, a versatile formulation is developed. High transfection efficiencies of up to 95% were obtained in both human multipotent stromal cells (MSCs) and MG-63 cells using naked DNA in vitro. Over a period of 5 weeks, an increasing amount of biologically active BMP-2 was released from the cells and remained present in the gel. In vivo, transfected cells were found after both two and six weeks implantation in naked mice, even in groups without seeded cells, thus indicating in vivo transfection of endogenous cells. The protein levels were effective in inducing osteogenic differentiation in vitro, as seen by elevated alkaline phosphatase (ALP) production and in vivo, as demonstrated by the production of collagen I and osteocalcin in a mineralised alginate matrix. We conclude that BMP-2 cDNA incorporated in alginate hydrogel appears to be a promising new strategy for minimal-invasive delivery of growth factors in bone regeneration.

  14. Novel PEI/Poly-γ-Gutamic Acid Nanoparticles for High Efficient siRNA and Plasmid DNA Co-Delivery

    Directory of Open Access Journals (Sweden)

    Shu-Fen Peng

    2017-01-01

    Full Text Available The efficient delivery of sufficient amounts of nucleic acids into target cells is critical for successful gene therapy and gene knockdown. The DNA/siRNA co-delivery system has been considered a promising approach for cancer therapy to simultaneously express and inhibit tumor suppressor genes and overexpressed oncogenes, respectively, triggering synergistic anti-cancer effects. Polyethylenimine (PEI has been identified as an efficient non-viral vector for transgene expression. In this study, we created a very high efficient DNA/siRNA co-delivery system by incorporating a negatively-charged poly-γ-glutamic acid (γ-PGA into PEI/nucleic acid complexes. Spherical nanoparticles with about 200 nm diameter were formed by mixing PEI/plasmid DNA/siRNA/γ-PGA (dual delivery nanoparticles; DDNPs with specific ratio (N/P/C ratio and the particles present positive surface charge under all manufacturing conditions. The gel retardation assay shows both nucleic acids were effectively condensed by PEI, even at low N/P ratios. The PEI-based DDNPs reveal excellent DNA/siRNA transfection efficiency in the human hepatoma cell line (Hep 3B by simultaneously providing high transgene expression efficiency and high siRNA silencing effect. The results indicated that DDNP can be an effective tool for gene therapy against hepatoma.

  15. Novel PEI/Poly-γ-Gutamic Acid Nanoparticles for High Efficient siRNA and Plasmid DNA Co-Delivery.

    Science.gov (United States)

    Peng, Shu-Fen; Hsu, Hung-Kun; Lin, Chun-Cheng; Cheng, Ya-Ming; Hsu, Kuang-Hsing

    2017-01-04

    The efficient delivery of sufficient amounts of nucleic acids into target cells is critical for successful gene therapy and gene knockdown. The DNA/siRNA co-delivery system has been considered a promising approach for cancer therapy to simultaneously express and inhibit tumor suppressor genes and overexpressed oncogenes, respectively, triggering synergistic anti-cancer effects. Polyethylenimine (PEI) has been identified as an efficient non-viral vector for transgene expression. In this study, we created a very high efficient DNA/siRNA co-delivery system by incorporating a negatively-charged poly-γ-glutamic acid (γ-PGA) into PEI/nucleic acid complexes. Spherical nanoparticles with about 200 nm diameter were formed by mixing PEI/plasmid DNA/siRNA/γ-PGA (dual delivery nanoparticles; DDNPs) with specific ratio (N/P/C ratio) and the particles present positive surface charge under all manufacturing conditions. The gel retardation assay shows both nucleic acids were effectively condensed by PEI, even at low N/P ratios. The PEI-based DDNPs reveal excellent DNA/siRNA transfection efficiency in the human hepatoma cell line (Hep 3B) by simultaneously providing high transgene expression efficiency and high siRNA silencing effect. The results indicated that DDNP can be an effective tool for gene therapy against hepatoma.

  16. Direct and precise length measurement of single, stretched DNA fragments by dynamic molecular combing and STED nanoscopy.

    Science.gov (United States)

    Kim, Namdoo; Kim, Hyung Jun; Kim, Younggyu; Min, Kyung Suk; Kim, Seong Keun

    2016-09-01

    A combination of DNA stretching method and super-resolution nanoscopy allows an accurate and precise measurement of the length of DNA fragments ranging widely in size from 117 to 23,130 bp. BstEII- and HindIII-treated λDNA fragments were stained with an intercalating dye and then linearly stretched on a coverslip by dynamic molecular combing. The image of individual DNA fragments was obtained by stimulated emission depletion nanoscopy. For DNA fragments longer than ∼1000 bp, the measured lengths of DNA fragments were consistently within ∼0.5 to 1.0 % of the reference values, raising the possibility of this method in a wide range of applications including facile detection for copy number variations and trinucleotide repeat disorder.

  17. Insertion mutagenesis of the yeast Candida famata (Debaryomyces hansenii) by random integration of linear DNA fragments.

    Science.gov (United States)

    Dmytruk, Kostyantyn V; Voronovsky, Andriy Y; Sibirny, Andriy A

    2006-09-01

    The feasibility of using random insertional mutagenesis to isolate mutants of the flavinogenic yeast Candida famata was explored. Mutagenesis was performed by transformation of the yeast with an integrative plasmid containing the Saccharomyces cerevisiae LEU2 gene as a selective marker. The addition of restriction enzyme together with the plasmid (restriction enzyme-mediated integration, REMI) increased the transformation frequency only slightly. Integration of the linearized plasmid occurred randomly in the C. famata genome. To investigate the potential of insertional mutagenesis, it was used for tagging genes involved in positive regulation of riboflavin synthesis in C. famata. Partial DNA sequencing of tagged genes showed that they were homologous to the S. cerevisiae genes RIB1, MET2, and SEF1. Intact orthologs of these genes isolated from Debaryomyces hansenii restored the wild phenotype of the corresponding mutants, i.e., the ability to overproduce riboflavin under iron limitation. The Staphylococcus aureus ble gene conferring resistance to phleomycin was used successfully in the study as a dominant selection marker for C. famata. The results obtained indicate that insertional mutagenesis is a powerful tool for tagging genes in C. famata.

  18. Correlation between aneuploidy, apoptotic markers and DNA fragmentation in spermatozoa from normozoospermic patients.

    Science.gov (United States)

    Vendrell, Xavier; Ferrer, Minerva; García-Mengual, Elena; Muñoz, Patricia; Triviño, Juan Carlos; Calatayud, Carmen; Rawe, Vanesa Y; Ruiz-Jorro, Miguel

    2014-04-01

    Genetic and biochemical sperm integrity is essential to ensure the reproductive competence. However, spermatogenesis involves physiological changes that could endanger sperm integrity. DNA protamination and apoptosis have been studied extensively. Furthermore, elevated rates of aneuploidy and DNA injury correlate with reproductive failures. Consequently, this study applied the conventional spermiogram method in combination with molecular tests to assess genetic integrity in ejaculate from normozoospermic patients with implantation failure by retrospectively analysing aneuploidy (chromosomes 18, X, Y), DNA fragmentation, externalization of phosphatidylserine and mitochondrial membrane potential status before and after magnetic activated cell sorting (MACS). Aneuploid, apoptotic and DNA-injured spermatozoa decreased significantly after MACS. A positive correlation was detected between reduction of aneuploidy and decreased DNA damage, but no correlation was determined with apoptotic markers. The interactions between apoptotic markers, DNA integrity and aneuploidy, and the effect of MACS on these parameters, remain unknown. In conclusion, use of MACS reduced aneuploidy, DNA fragmentation and apoptosis. A postulated mechanism relating aneuploidy and DNA injury is discussed; on the contrary, cell death markers could not be related. An 'apoptotic-like' route could explain this situation. Copyright © 2013 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  19. Assignment of ten DNA repair genes from Schizosaccharomyces pombe to chromosomal NotI restriction fragments

    NARCIS (Netherlands)

    B.C. Broughton; N.C. Barbet; J. Murray (Johanne); F.Z. Watts (Felicity); M.H.M. Koken (Marcel); A.R. Lehmann (Alan); A.M. Carr (Anthony)

    1991-01-01

    textabstractTen DNA repair (rad) genes from the fission yeast, Schizosaccharomyces pombe were mapped to the 17 NotI fragments of the three chromosomes. Nine of the genes map to chromosome I, but there is no evidence for significant clustering.

  20. A general method to modify BACs to generate large recombinant DNA fragments.

    Science.gov (United States)

    Shen, Wei; Huang, Yue; Tang, Yi; Liu, De-Pei; Liang, Chih-Chuan

    2005-11-01

    Bacterial artificial chromosome (BAC) has the capacity to clone DNA fragments in excess of 300 kb. It also has the considerable advantages of stable propagation and ease of purification. These features make BAC suitable in genetic research, such as library construction, transgenic mice production, and gene targeting constructs. Homologous recombination in Escherichia coli, a process named recombineering, has made the modification of BACs easy and reliable. We report here a modified recombineering method that can efficiently mediate the fusion of large DNA fragments from two or more different BACs. With the introduction of kanamycin-resistant gene and proposed rare-cutting restriction endonuclease (RCRE) sites into two BACs, a 82.6-kb DNA fragment containing the inverted human alpha-globin genes (theta, alpha1, alpha2, and zeta) from BAC191K2 and the locus control region (LCR) of human beta-globin gene locus (from the BAC186D7) was reconstructed. This approach for combining different BAC DNA fragments should facilitate many kinds of genomic experiments.

  1. Effect of sperm DNA fragmentation on assisted reproductive technology treatment category

    Institute of Scientific and Technical Information of China (English)

    Fei Qian-jin; Ni Wu-hua; Huang Xue-feng; Ye Bi-lu

    2012-01-01

    Objectives: To investigate the effect of sperm DNA fragmentation on outcomes of in vitro fertilization (IVF)and intracytoplasmic sperm injection (ICSI).Methods: A total of 242 cycles (154 IVF and 88 ICSI) from 235 couples were included.Sperm DNA fragmentation (SDF) and routine semen analysis were performed on the retrieval day.The rates of fertilization,embryo cleavage,good quality embryos,implantation and clinical pregnancy were measured.Results: Sperm DNA fragmentation index (DFI) in ICSI group was significantly higher than that in IVF group (P<0.01).The rates of fertilization,implantation and clinical pregnancy in ICSI were significantly higher than those in IVF with DFI≥24% (P<0.05).When DFI exceeded 24%,the OR for clinical pregnancy was 3.85 (95%CI 1.40-10.59) comparing ICSI with IVF,and the OR for clinical pregnancy increased to 4.61 (95% CI 1.09-19.57) after inclusion of sperm concentration,progressively motile sperm percentage and female age as covariates.Conclusions: High DNA fragmentation might affect the outcome of ICSI and IVF.When DFI exceeds 24%,ICSI should be chosen instead of IVF.

  2. Should sperm DNA fragmentation testing be included in the male infertility work-up?

    Science.gov (United States)

    Lewis, Sheena E M

    2015-08-01

    A response to the editorial 'Are we ready to incorporate sperm DNA fragmentation testing into our male infertility work-up? A plea for more robust studies' by Erma Drobnis and Martin Johnson. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  3. Molecular evidence of apoptotic pathway activation in semen samples with high DNA fragmentation.

    Science.gov (United States)

    Manente, Lucrezia; Pecoraro, Stefano; Picillo, Esther; Gargiulo, Umberto; Gargiulo, Paolo; De Luca, Antonio; Politano, Luisa

    2015-01-01

    Male infertility is diagnosed by semen parameters, such as concentration, motility and morphology; however, these are not sufficient for the prediction of male fertility capacity. In the clinical routine, several other sperm functions have been introduced, including the sperm DNA fragmentation test. The objective of the present study was to evaluate sperm chromatin integrity in semen samples. Sperm chromatin dispersion test (SCD) was used in ejaculates from men divided into five groups: normozoospermic, oligozoospermic, asthenozoospermic, oligoasthenozoospermic and cryptozoospermic. The data obtained showed that the SCD percentage appeared to be significantly associated with oligozoospermia diagnosis. We also evaluated total testosterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and inhibin B serum hormonal levels in all samples examined, in order to assess whether DNA fragmentation increase could correlate with abnormal hormonal values. Finally we selected certain samples with an increasing DNA fragmentation and analyzed the molecular activated apoptotic pathways. A significant relationship was found between caspase-3 activation and increased DNA fragmentation. Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  4. Subcloning and Sequencing of DNA Fragment Related to Salt Tolerance in Sinorhizobium fredii RT19%费氏中华根瘤菌与耐盐有关的DNA片段的亚克隆和测序

    Institute of Scientific and Technical Information of China (English)

    卞学琳; 葛世超; 杨苏声

    2000-01-01

    将费氏中华根瘤菌(Sinorhizobium fredii) RT19与耐盐有关的23kb DNA片段用BamH酶切成大小不同的长度,分别与质粒pML122连接,然后转化大肠杆菌(Escherichia coli) S17-1,筛选出3个转化子。以这些转化子为供体, RT19的盐敏感突变株RC3-3为受体,分别 进行二亲本杂交,筛选到接合子BR2,得到4.4kb与耐盐有关的DNA片段。根据其物理图谱,酶切成6个DNA片段,并分别连接到质粒pUC18进行测序。测序分析表明,该4.4kb DNA片段含有fixO、fixN基因和3个开放阅读框(ORF)。%A 23kb DNA fragment related to salt tolerance was obtained from the gene library of S. fredii strain RT19. In this study, BamH was selected to digest 23kb DNA fragment into different length of DNA fragments. The resulting fragments were ligated with plasmid pML122, then the recombinant plasmids were transformed to competent cells of E. coli S17-1 on selective medium and three transformants TR were obtained. Two-Parental mating experiments were carried out with these transformants as donor and salt sensitive S. fredii strain RC3-3 as recipient, and the transconjugant BR2 was selected on FY plates containing gentamycin and 0.4mol/L NaCl. Thus, a 4.4kb DNA fragment related to salt tolerance was obtained. Based on its physical map, six restriction fragments were subcloned into plasmid pUC18 for DNA sequencing. Subsequently, sequencing and analysis of 4.4kb DNA fragment showed that fixO, fixN genes and three ORFs were obtained.

  5. The One-Kilobase DNA Fragment Upstream of the ardC Actin Gene of Physarum polycephalum Is Both a Replicator and a Promoter

    Science.gov (United States)

    Pierron, Gérard; Pallotta, Dominick; Bénard, Marianne

    1999-01-01

    The 1-kb DNA fragment upstream of the ardC actin gene of Physarum polycephalum promotes the transcription of a reporter gene either in a transient-plasmid assay or as an integrated copy in an ectopic position, defining this region as the transcriptional promoter of the ardC gene (PardC). Since we mapped an origin of replication activated at the onset of S phase within this same fragment, we examined the pattern of replication of a cassette containing the PardC promoter and the hygromycin phosphotransferase gene, hph, integrated into two different chromosomal sites. In both cases, we show by two-dimensional agarose gel electrophoresis that an efficient, early activated origin coincides with the ectopic PardC fragment. One of the integration sites was a normally late-replicating region. The presence of the ectopic origin converted this late-replicating domain into an early-replicating domain in which replication forks propagate with kinetics indistinguishable from those of the native PardC replicon. This is the first demonstration that initiation sites for DNA replication in Physarum correspond to cis-acting replicator sequences. This work also confirms the close proximity of a replication origin and a promoter, with both functions being located within the 1-kb proximal region of the ardC actin gene. A more precise location of the replication origin with respect to the transcriptional promoter must await the development of a functional autonomously replicating sequence assay in Physarum. PMID:10207074

  6. Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein

    Science.gov (United States)

    Lipps, Georg; Stegert, Mario; Krauss, Gerhard

    2001-01-01

    There is still a lack of information on the specific characteristics of DNA-binding proteins from hyperthermophiles. Here we report on the product of the gene orf56 from plasmid pRN1 of the acidophilic and thermophilic archaeon Sulfolobus islandicus. orf56 has not been characterised yet but low sequence similarily to several eubacterial plasmid-encoded genes suggests that this 6.5 kDa protein is a sequence-specific DNA-binding protein. The DNA-binding properties of ORF56, expressed in Escherichia coli, have been investigated by EMSA experiments and by fluorescence anisotropy measurements. Recombinant ORF56 binds to double-stranded DNA, specifically to an inverted repeat located within the promoter of orf56. Binding to this site could down-regulate transcription of the orf56 gene and also of the overlapping orf904 gene, encoding the putative initiator protein of plasmid replication. By gel filtration and chemical crosslinking we have shown that ORF56 is a dimeric protein. Stoichiometric fluorescence anisotropy titrations further indicate that ORF56 binds as a tetramer to the inverted repeat of its target binding site. CD spectroscopy points to a significant increase in ordered secondary structure of ORF56 upon binding DNA. ORF56 binds without apparent cooperativity to its target DNA with a dissociation constant in the nanomolar range. Quantitative analysis of binding isotherms performed at various salt concentrations and at different temperatures indicates that approximately seven ions are released upon complex formation and that complex formation is accompanied by a change in heat capacity of –6.2 kJ/mol. Furthermore, recombinant ORF56 proved to be highly thermostable and is able to bind DNA up to 85°C. PMID:11160922

  7. Genetic analysis of an aphid endosymbiont DNA fragment homologous to the rnpA-rpmH-dnaA-dnaN-gyrB region of eubacteria.

    Science.gov (United States)

    Lai, C Y; Baumann, P

    1992-04-15

    Buchnera aphidicola is a Gram- eubacterium with a DNA G+C content of 28-30 mol%. This organism is an obligate intracellular symbiont of aphids. To determine its similarity to or difference from other eubacteria, a 4.9-kb DNA fragment from B. aphidicola containing the gene homologous to Escherichia coli dnaA (a gene involved in the initiation of chromosome replication) was cloned into E. coli and sequenced. The order of genes on this fragment, 60K-10K-rnpA-rpmH-dnaA-dnaN-gyrB, was similar to that found in other eubacteria. The sole difference was the absence of recF between dnaN and gyrB. The deduced amino acid sequence of these proteins resembled those of E. coli by a 41 to 83% identity. Except for E. coli, in all the eubacteria so far examined, dnaA is preceded by multiple 9-nucleotide repeats known as a DnaA boxes. No DnaA boxes were detected in the endosymbiont DNA. The possibility that this observation is a consequence of the low G+C content of this DNA fragment (14 mol% G+C) is unlikely since in Mycoplasma capricolum this fragment (19 mol% G+C) has eight DnaA boxes (Fujita et al., 1992). The presence of the sequence, GATC, recognized by the Dam methyl-transferase system, only within six regions coding for proteins suggests that methylation is not a factor in the regulation of the initiation of endosymbiont chromosome replication.

  8. Fragment-based discovery of DNA gyrase inhibitors targeting the ATPase subunit of GyrB.

    Science.gov (United States)

    Mesleh, Michael F; Cross, Jason B; Zhang, Jing; Kahmann, Jan; Andersen, Ole A; Barker, John; Cheng, Robert K; Felicetti, Brunella; Wood, Michael; Hadfield, Andrea T; Scheich, Christoph; Moy, Terence I; Yang, Qingyi; Shotwell, Joseph; Nguyen, Kien; Lippa, Blaise; Dolle, Roland; Ryan, M Dominic

    2016-02-15

    Inhibitors of the ATPase function of bacterial DNA gyrase, located in the GyrB subunit and its related ParE subunit in topoisomerase IV, have demonstrated antibacterial activity. In this study we describe an NMR fragment-based screening effort targeting Staphylococcus aureus GyrB that identified several attractive and novel starting points with good ligand efficiency. Fragment hits were further characterized using NMR binding studies against full-length S. aureus GyrB and Escherichia coli ParE. X-ray co-crystal structures of select fragment hits confirmed binding and suggested a path for medicinal chemistry optimization. The identification, characterization, and elaboration of one of these fragment series to a 0.265 μM inhibitor is described herein.

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

    CERN Document Server

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

    2011-01-01

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

  10. Conventional semen parameters and DNA fragmentation in relation to fertility status in a Greek population.

    Science.gov (United States)

    Evgeni, Evangelini; Lymberopoulos, George; Gazouli, Maria; Asimakopoulos, Byron

    2015-05-01

    Our study aimed to investigate the possible correlations between conventional seminal parameters and DNA fragmentation in specific groups of Greek men, selected in relation to their fertility history and to verify the validity of the recent WHO reference values for the basic semen analysis in this population. A total of 770 subject data were evaluated in three distinct groups: fertile men with children naturally conceived within one year of unprotected intercourse (n=78), subfertile men, having achieved pregnancies either naturally or by Assisted Reproduction Techniques, not resulting in live births (n=153) and infertile men, failing to produce either pregnancies or children (n=539). Semen volume, sperm concentration, total count, rapid and total progressive motility and morphology were evaluated following the World Health Organization (2010) methods. DNA fragmentation was assessed by the Sperm Chromatin Dispersion assay. The 5th percentile, as well as the 95% confidence intervals (CI) for each parameter, were calculated by the method of bootstrapping. Statistical correlations between the examined parameters were sought using the Spearman R test (pDNA fragmentation and sperm concentration, total count, progressive motility (rapid and total) and normal morphology in subfertile and infertile men (pDNA fragmentation in our group of fertile men. Concordance was established between the reference limits issued by the WHO 2010 for the basic semen parameters and semen quality of fertile men in the studied population. The variability of correlations established between DNA fragmentation and the conventional seminal profile in relation to fertility status indicates that they are independent attributes of semen quality, justifying the assessment of both during a comprehensive evaluation of male infertility. Moreover, the WHO 2010 reference limits were found adequately descriptive of seminal normality in Greek men. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  11. Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments

    Science.gov (United States)

    Dahl, Fredrik; Gullberg, Mats; Stenberg, Johan; Landegren, Ulf; Nilsson, Mats

    2005-01-01

    We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its respective target, forming individual circular complexes that are covalently closed by enzymatic ligation. Non-circularized fragments are removed by exonucleolysis, enriching for the selected fragments. The general sequence that is introduced into the circularized fragments allows them to be amplified in parallel using a universal primer pair. The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences. We demonstrate the specificity, reproducibility and flexibility of this process by performing a 96-plex amplification of an arbitrary set of specific DNA sequences, followed by hybridization to a cDNA microarray. Eighty-nine percent of the selectors generated PCR products that hybridized to the expected positions on the array, while little or no amplification artifacts were observed. PMID:15860768

  12. Chromosomal aneuploidies and DNA fragmentation of human spermatozoa from patients exposed to perfluorinated compounds.

    Science.gov (United States)

    Governini, L; Guerranti, C; De Leo, V; Boschi, L; Luddi, A; Gori, M; Orvieto, R; Piomboni, P

    2015-11-01

    This study investigated chromosomal aneuploidies and DNA damage in spermatozoa from male patients contaminated by perfluorinated compounds (PFCs) in whole blood and seminal plasma. Sperm aneuploidy and diploidy rate for chromosomes 18, X and Y were evaluated by FISH; sperm DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling technique coupled to flow cytometry. Our results indicated that PFC contamination was present in 58% of subjects included in the study. A significant increase in alterations of sperm parameters was observed in PFC-positive subjects compared to PFC-negative subjects. As regards the sperm aneuploidy, both disomy and diploidy rates resulted significantly increased in subjects positive for PFC contamination compared to PFC-negative samples. In addition, sperm DNA fragmentation index resulted significantly increased in PFC-contaminated subjects compared to PFC-non-contaminated subjects, with a significant increased level of dimmer DNA fragmentation index. Our results clearly indicate that PFC contamination may detrimentally affect spermatogenesis, disturbing both meiotic segregation and DNA integrity. We could therefore suggest cautions to reduce or eliminate any contact with these compounds because the long-term effects of PFC accumulation in the body are not predictable. © 2014 Blackwell Verlag GmbH.

  13. Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis

    NARCIS (Netherlands)

    Cissen, M.; Wely, M.V.; Scholten, I.; Mansell, S.; Bruin, J.P. de; Mol, B.W.; Braat, D.; Repping, S.; Hamer, G.

    2016-01-01

    Sperm DNA fragmentation has been associated with reduced fertilization rates, embryo quality, pregnancy rates and increased miscarriage rates. Various methods exist to test sperm DNA fragmentation such as the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion (SCD) test, the term

  14. Evaluation of sperm head shape at high magnification revealed correlation of sperm DNA fragmentation with aberrant head ellipticity and angularity.

    Science.gov (United States)

    Utsuno, Hiroki; Oka, Kenji; Yamamoto, Ayako; Shiozawa, Tanri

    2013-05-01

    To test for an association between DNA fragmentation and head shape at high magnification in fresh motile spermatozoa. Observational study. Academic tertiary care center. A total of 60 men in our assisted reproductive program. Quantifying sperm head shape using elliptic Fourier analysis, and detecting DNA fragmentation by use of a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Correlation between percentage of spermatozoa with abnormal head shape and percentage of DNA fragmentation. Elliptic Fourier analysis decomposed sperm head shapes into four quantitative parameters: ellipticity, anteroposterior (AP) symmetry, lateral symmetry, and angularity. The DNA fragmentation was statistically significantly correlated with abnormal angularity, and moderately with abnormal ellipticity but not with abnormal AP symmetry or lateral symmetry. Forward stepwise multiple logistic regression analysis revealed a statistically significantly higher percentage of DNA fragmentation in spermatozoa with abnormal ellipticity and abnormal angularity than in spermatozoa with normal-shaped head (6.1% and 5.4% vs. 2.8%). Spermatozoa with large nuclear vacuoles also correlated with sperm DNA fragmentation, and had a statistically significantly higher percentage of DNA fragmentation (4.7%). Among the morphologic features of the sperm head, abnormal ellipticity, angularity, and large nuclear vacuoles are associated with DNA fragmentation. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  15. Effects of reduced seminal enzymatic antioxidants on sperm DNA fragmentation and semen quality of Tunisian infertile men.

    Science.gov (United States)

    Atig, Fatma; Kerkeni, Abdelhamid; Saad, Ali; Ajina, Mounir

    2017-03-01

    To evaluate levels of sperm DNA fragmentation and enzymatic antioxidant status in seminal plasma of Tunisian fertile and infertile men in order to assess the effects of seminal oxidative stress on sperm DNA integrity and semen quality. Semen samples from 100 infertile patients (40 oligoasthenoteratozoospermics, 31 teratozoospermics and 29 asthenozoospermics) and 50 fertile men (controls) were analyzed for DNA fragmentation by TUNEL assay and biochemical parameters. Seminal antioxidant activities (Superoxide dismutase, Glutathione peroxidase and Catalase) and malondialdehyde concentrations were measured spectrophotometrically. Sperm DNA fragmentation and malondialdehyde levels in infertile groups were more elevated than controls. Nevertheless, the activities of the antioxidant enzymes were significantly lower in abnormal groups compared to normozoospermics. Sperm DNA fragmentation was closely and positively correlated to malondialdehyde levels (r = 0.37, P = 0.008); meanwhile, reduced seminal antioxidant profile was negatively associated to sperm DNA fragmentation. Interestingly, we noted also that sperm DNA fragmentation was negatively correlated to sperm motility (r = -0.54, P fragmentation can be due to the impaired seminal enzymatic antioxidant profile and increased Lipid peroxidation. Our results sustain that the evaluation of sperm DNA fragmentation and seminal oxidative biomarkers in infertile men is recommended as a consistent prognostic tool for male infertility assessment.

  16. Comparison of DNA Fragmentation Assay in Frozen-Thawed Cat Epididymal Sperm.

    Science.gov (United States)

    Kunkitti, P; Sjödahl, A; Bergqvist, A-S; Johannisson, A; Axnér, E

    2016-08-01

    DNA fragmentation of frozen-thawed feline epididymal sperm from corpus and cauda regions was evaluated by three different techniques. The DNA fragmentation index (DFI) was compared between techniques: the sperm chromatin structural assay (SCSA(®) ), acridine orange staining techniques (AOT) and the sperm chromatin dispersion (SCD). There were significant differences in DFI among the techniques (p < 0.05) with no correlations. Only DFI values obtained from SCD revealed a significantly higher DFI in corpus compared with cauda spermatozoa (p < 0.05). The discrepancy between techniques might be due to the sensitivity of each technique, differences in severity of DNA damaged that can be detected. The difference in DFI between epididymal regions from SCD technique might indicate different maturational stages of spermatozoa, with less chromatin condensation of spermatozoa in corpus compared with cauda epididymis. © 2016 Blackwell Verlag GmbH.

  17. Do Pilea Microphylla Improve Sperm DNA Fragmentation and Sperm Parameters in Varicocelized Rats?

    Science.gov (United States)

    Heidari, Reza; Alizadeh, Rafieh; Abbasi, Niloufar; Pasbakhsh, Parichehr; Hedayatpour, Azim; Farajpour, Mostafa; Khaleghi, Mohammad Reza; Abbasi, Mehdi; Dehpour, Ahmad Reza

    2015-01-01

    Varicocele is one of the most common causes of primary male infertility. Pilea microphylla (PM) is being used as folk medicine. This study was aimed to investigate the effects of PM in a rat model of varicocele. A total of 30 male Wistar rats were divided into control, sham, varicocele, accessory varicocele and PM-treated groups. After 10 weeks of varicocele induction, sperm parameters and chromatin (Aniline blue, acridine orange and toluidine blue) were evaluated, except for the treated and accessory groups that received 50 mg/kg PM orally daily for 10 weeks and then were sacrificed. Sperm parameters significantly decreased in varicocele groups (P DNA fragmentation and sperm parameters in varicocelized rats. Administration of PM led to significantly increased sperm parameters and AO staining (P DNA fragmentation in varicocelized rats. PM can reduce the damage to sperm DNA but not chromatin condensation.

  18. Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments.

    Science.gov (United States)

    Dabney, Jesse; Knapp, Michael; Glocke, Isabelle; Gansauge, Marie-Theres; Weihmann, Antje; Nickel, Birgit; Valdiosera, Cristina; García, Nuria; Pääbo, Svante; Arsuaga, Juan-Luis; Meyer, Matthias

    2013-09-24

    Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp.

  19. Types, Causes, Detection and Repair of DNA Fragmentation in Animal and Human Sperm Cells

    Directory of Open Access Journals (Sweden)

    Rosa Roy

    2012-10-01

    Full Text Available Concentration, motility and morphology are parameters commonly used to determine the fertilization potential of an ejaculate. These parameters give a general view on the quality of sperm but do not provide information about one of the most important components of the reproductive outcome: DNA. Either single or double DNA strand breaks can set the difference between fertile and infertile males. Sperm DNA fragmentation can be caused by intrinsic factors like abortive apoptosis, deficiencies in recombination, protamine imbalances or oxidative stress. Damage can also occur due to extrinsic factors such as storage temperatures, extenders, handling conditions, time after ejaculation, infections and reaction to medicines or post-testicular oxidative stress, among others. Two singular characteristics differentiate sperm from somatic cells: Protamination and absence of DNA repair. DNA repair in sperm is terminated as transcription and translation stops post-spermiogenesis, so these cells have no mechanism to repair the damage occurred during their transit through the epididymis and post-ejaculation. Oocytes and early embryos have been shown to repair sperm DNA damage, so the effect of sperm DNA fragmentation depends on the combined effects of sperm chromatin damage and the capacity of the oocyte to repair it. In this contribution we review some of these issues.

  20. Types, Causes, Detection and Repair of DNA Fragmentation in Animal and Human Sperm Cells

    Science.gov (United States)

    González-Marín, Clara; Gosálvez, Jaime; Roy, Rosa

    2012-01-01

    Concentration, motility and morphology are parameters commonly used to determine the fertilization potential of an ejaculate. These parameters give a general view on the quality of sperm but do not provide information about one of the most important components of the reproductive outcome: DNA. Either single or double DNA strand breaks can set the difference between fertile and infertile males. Sperm DNA fragmentation can be caused by intrinsic factors like abortive apoptosis, deficiencies in recombination, protamine imbalances or oxidative stress. Damage can also occur due to extrinsic factors such as storage temperatures, extenders, handling conditions, time after ejaculation, infections and reaction to medicines or post-testicular oxidative stress, among others. Two singular characteristics differentiate sperm from somatic cells: Protamination and absence of DNA repair. DNA repair in sperm is terminated as transcription and translation stops post-spermiogenesis, so these cells have no mechanism to repair the damage occurred during their transit through the epididymis and post-ejaculation. Oocytes and early embryos have been shown to repair sperm DNA damage, so the effect of sperm DNA fragmentation depends on the combined effects of sperm chromatin damage and the capacity of the oocyte to repair it. In this contribution we review some of these issues. PMID:23203048

  1. Mitochondrial permeability transition increases reactive oxygen species production and induces DNA fragmentation in human spermatozoa.

    Science.gov (United States)

    Treulen, Favián; Uribe, Pamela; Boguen, Rodrigo; Villegas, Juana V

    2015-04-01

    Does mitochondrial permeability transition (MPT) induced by calcium overload cause reactive oxygen species (ROS) production and DNA fragmentation in human spermatozoa? Studies conducted in vitro suggest that in human spermatozoa, MPT occurs in response to intracellular calcium increase and is associated with mitochondrial membrane potential (ΔΨm) dissipation, increased ROS production and DNA fragmentation. Oxidative stress is a major cause of defective sperm function in male infertility. By opening calcium-dependent pores in the inner mitochondrial membrane (IMM), MPT causes, among other things, increased ROS production and ΔΨm dissipation in somatic cells. MPT as a mechanism for generating oxidative stress and DNA fragmentation in human spermatozoa has not been studied. Human sperm were exposed to ionomycin for 1.5 h (n = 8) followed by analysis of sperm IMM permeability, ΔΨm, ROS production and DNA fragmentation. To evaluate the MPT in sperm cells, the calcein-AM and cobalt chloride method was used. The ΔΨm was evaluated by JC-1 staining, intracellular ROS production was evaluated with dihydroethidium and DNA fragmentation was evaluated by a modified TUNEL assay. Measurements were performed by fluorescence microscopy, confocal laser microscopy and flow cytometry. Decreased calcein fluorescence after treatment with ionomycin (P fragmentation. ROS production occurred prior to the decrease in ΔΨm. The study was carried out in vitro using motile sperm from healthy donors; tests on sperm from infertile patients were not carried out. We propose that the MPT, due to pores opening in sperm IMM, is an important mechanism of increased ROS and DNA fragmentation. Therefore, agents that modulate the opening of these pores might contribute to the prevention of damage by oxidative stress in human spermatozoa. This study was funded by grant DI12-0102 from the Universidad de La Frontera (J.V.V.) and a doctoral scholarship from CONICYT Chile (F.T.). The authors disclose

  2. Pregnancy prediction by free sperm DNA and sperm DNA fragmentation in semen specimens of IVF/ICSI-ET patients.

    Science.gov (United States)

    Bounartzi, Theofania; Dafopoulos, Konstantinos; Anifandis, George; Messini, Christina I; Koutsonikou, Chrysoula; Kouris, Spyros; Satra, Maria; Sotiriou, Sotirios; Vamvakopoulos, Nicholas; Messinis, Ioannis E

    2016-04-01

    The purpose of this study was to evaluate the predictive value of free sperm plasma DNA (f-spDNA) and sperm DNA fragmentation (SDF), in semen specimens from men undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) treatments. Fifty-five semen samples were evaluated during 55 consecutive IVF/ICSI-ET cycles. F-spDNA was determined by conventional quantitative real-time PCR-Sybr green detection approach, while evaluation of sperm DNA damage was performed using the sperm chromatin dispersion (SCD) assay. While f-spDNA only correlated with total sperm count, SDF correlated with many semen parameters (including sperm concentration, total sperm count and the per cent of non-progressive sperm). Neither SDF nor the proportion of sperm with small or no halos correlated with f-spDNA. Interestingly, smoking status correlated with f-spDNA but not with SDF. Although these two factors seem to interact for the prediction of pregnancy, receiver-operating characteristics (ROC) analysis revealed that SDF had a stronger predictive value (AUC = 0.7, p  0.05). SDF and f-spDNA may not be associated together but they interact at a significant level in order to exert their actions on pregnancy outcome. Among the two markers, SDF appears to have stronger and significantly predictive value for pregnancy success.

  3. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    F Wegman

    2011-03-01

    Full Text Available Bone regeneration is one of the major focus points in the field of regenerative medicine. A well-known stimulus of bone formation is bone morphogenetic protein-2 (BMP-2, which has already been extensively used in clinical applications. We investigated the possibility of achieving osteogenic differentiation both in vitro and in vivo as a result of prolonged presence of BMP-2 using plasmid DNA-based gene therapy. By delivering BMP-2 cDNA in an alginate hydrogel, a versatile formulation is developed. High transfection efficiencies of up to 95% were obtained in both human multipotent stromal cells (MSCs and MG-63 cells using naked DNA in vitro. Over a period of 5 weeks, an increasing amount of biologically active BMP-2 was released from the cells and remained present in the gel. In vivo, transfected cells were found after both two and six weeks implantation in naked mice, even in groups without seeded cells, thus indicating in vivo transfection of endogenous cells. The protein levels were effective in inducing osteogenic differentiation in vitro, as seen by elevated alkaline phosphatase (ALP production and in vivo, as demonstrated by the production of collagen I and osteocalcin in a mineralised alginate matrix.

  4. Effects of Sperm DNA Fragmentation on Semen Parameters and ICSI Outcome Determined by an Improved SCD Test,Halosperm

    Directory of Open Access Journals (Sweden)

    Asuman Demiroglu Zergeroğlu

    2010-01-01

    Full Text Available Background: Sperm DNA fragmentation is known as an important cause of male infertility.The influence of sperm DNA damage on reproductive potential has been subject of many studiesindicating various results and remaining the subject controversial. In this study, we investigateddifferences of the semen parameters and intracytoplasmic sperm injection (ICSI outcome accordingto sperm DNA fragmentation levels (DFLs of patients.Materials and Methods: The DFLs were determined by Halosperm, a new improved spermchromatin dispersion (SCD test. Patients were grouped as low DNA fragmentation group (LFG≤30% and high fragmentation group (HFG >30%.Results: Our analysis showed that semen parameters including concentration of untreated spermand motility of prepared semen were low in HGF, whereas other parameters were not different.Sperm DNA fragmentation levels decreased in both groups after semen preparation by densitygradient technique.Conclusion: No difference was detected on ICSI outcomes (fertilization, embryo development,embryo cleavage, embryo quality and pregnancy rates between two group.

  5. Increased receptor for advanced glycation end products in spermatozoa of diabetic men and its association with sperm nuclear DNA fragmentation.

    Science.gov (United States)

    Karimi, J; Goodarzi, M T; Tavilani, H; Khodadadi, I; Amiri, I

    2012-05-01

    Although the majority of patients with diabetes have disorders in sexual function, associations between diabetes mellitus and sperm function at the molecular level are largely unknown. As receptor for advanced glycation end products plays a key role in many diabetic complications, we hypothesised that it may be involved in sperm nuclear DNA fragmentation. RAGE levels were determined using ELISA and western blot analysis in sperm samples from 32 diabetic and 35 nondiabetic men. Sperm DNA fragmentation was assessed using TUNEL assay. Diabetic men had significantly higher mean levels of RAGE protein (P DNA fragmentation (P DNA fragmentation in diabetic men (r = 0.81, P DNA fragmentation in spermatozoa of diabetic men suggests a central role of RAGE in disturbances in sexual function of diabetic men. © 2011 Blackwell Verlag GmbH.

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

    Science.gov (United States)

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

    2016-05-01

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

  7. 亲和色谱纯化超螺旋质粒DNA的研究进展%RESEARCH PROGRESS OF AFFINITY CHROMATOGRAPHY IN PURIFICATION OF SUPERCOILED PLASMID DNA

    Institute of Scientific and Technical Information of China (English)

    白金山; 白姝

    2013-01-01

    非病毒载体质粒DNA已被广泛应用于基因治疗和DNA疫苗,目前迫切需要开发其大规模制备和分离纯化方法.亲和色谱是一种高分辨率、高选择性的分离技术,在蛋白质、抗体、核酸等生物大分子的分离纯化方面显示了良好的应用前景.本文综述了亲和色谱技术在超螺旋质粒DNA分离纯化中的研究进展,总结了各种亲和色谱方法分离超螺旋质粒DNA的机理和优缺点,并展望了亲和纯化技术在质粒DNA生产和制备中的应用前景.%Non-viral vector,plasmid DNA has been widely used in gene therapy and DNA vaccines.It is imperative to develop large-scale preparation and purification methods of plasmid DNA at present.As a separation technology of high resolution and high selectivity,affinity chromatography shows great application potential in terms of separation and purification of biological macromolecules such as proteins,antibodies,nucleic acids and so on.The domestic and foreign research progress of High Performance Liquid Chromatography (HPLC) technology,used in separation and purification of supercoiled plasmid DNA was reviewed in this paper.The advantages and disadvantages of various affinity chromatographic methods for separating supercoiled plasmid DNA were also summarized.At last,the affinity chromatography technology for preparation and purification of plasmid DNA was prospected.

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

    Science.gov (United States)

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

    1995-12-01

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

  9. Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA

    Directory of Open Access Journals (Sweden)

    Metsis Madis

    2008-06-01

    Full Text Available Abstract Background In a traditional electrophoresis mobility shift assay (EMSA a 32P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE in nondenaturing conditions. An extension of this method uses the large population of fragments derived from long genomic regions (approximately 600 kb for the identification of fragments containing protein binding regions. With this method, genomic DNA is fragmented by restriction enzymes, fragments are amplified by PCR, radiolabeled, incubated with nuclear proteins and the resulting DNA-protein complexes are separated by two-dimensional PAGE. Shifted DNA fragments containing protein binding sites are identified by using additional procedures, i. e. gel elution, PCR amplification, cloning and sequencing. Although the method allows simultaneous analysis of a large population of fragments, it is relatively laborious and can be used to detect only high affinity protein binding sites. Here we propose an alternative and straightforward strategy which is based on a combination of native and denaturing PAGE. This strategy allows the identification of DNA fragments containing low as well as high affinity protein binding regions, derived from genomic DNA ( Results We have combined an EMSA-based selection step with subsequent denaturing PAGE for the localization of protein binding regions in long (up to10 kb fragments of genomic DNA. Our strategy consists of the following steps: digestion of genomic DNA with a 4-cutter restriction enzyme (AluI, BsuRI, TruI, etc, separation of low and high molecular weight fractions of resultant DNA fragments, 32P-labeling with Klenow polymerase, traditional EMSA, gel elution and identification of the shifted bands (or smear by denaturing PAGE. The identification of DNA fragments containing protein binding sites is carried out by running the gel-eluted fragments alongside

  10. Linear induction of DNA double-strand breakage with X-ray dose, as determined from DNA fragment size distribution

    Energy Technology Data Exchange (ETDEWEB)

    Erixon, K.; Cedervall, B. [Karolinksa Institutet, Stockholm (Sweden)

    1995-05-01

    Pulsed-field gel electrophoresis has been applied to separate DNA from mouse L1210 cells exposed to X-ray doses of 1 to 50 Gy. Simultaneous separation of marker chromosomes in the range 0.1 to 12.6 Mbp allowed calculation of the size distribution of the radiation-induced fragments. The distribution was consistent with a random induction of double-strand breaks (DSBs). A theoretical relationship between the size distribution of such fragments and the average number of induced breaks was used to calculate the yield and dose response. The DNA distribution was determined by both radiolabeling and fluorescence staining. Two independent methods were use to evaluate the radiation-induced yield of DSBs, both assuming that all DNA is broken at random. In the first method we compared the theoretical and experimental fraction of DNA that is below a given size limit. By this method we estimated the yield to be 0.006-0.007 DSB/GY per million base pairs using the radiolabel and 0.004-0.008 DSB/Gy per million base pairs by fluorescence staining. The dose response was linear in both cases. In the second method we looked only at the size distribution in the resolving part of the gel and compared it to the theoretical distribution. By this method a value of approximately 0.012 DSB/Gy/Mb was found, using fluorescence as a measure of DNA distribution. In a normal diploid mammalian genome of size 60000 Mbp, this is equivalent to a yield of 25-50 DSBs/Gy or 70 DSBs/GY, respectively. The second approach, which looks only at the smaller fragments, may overestimate the yield, while the first approach suffers from uncertainties about the fraction of DNA irreversibly trapped in the well. The assay has the capacity to detect a dose of less than 1 Gy. 58 refs., 10 figs.

  11. Selection against spermatozoa with fragmented DNA after postovulatory mating depends on the type of damage

    Directory of Open Access Journals (Sweden)

    Pintado Belén

    2010-01-01

    Full Text Available Abstract Background Before ovulation, sperm-oviduct interaction mechanisms may act as checkpoint for the selection of fertilizing spermatozoa in mammals. Postovulatory mating does not allow the sperm to attach to the oviduct, and spermatozoa may only undergo some selection processes during the transport through the female reproductive tract and/or during the zona pellucida (ZP binding/penetration. Methods We have induced DNA damage in spermatozoa by two treatments, (a a scrotal heat treatment (42 degrees C, 30 min and (b irradiation with 137Cs gamma-rays (4 Gy, 1.25 Gy/min. The effects of the treatments were analyzed 21-25 days post heat stress or gamma-radiation. Postovulatory females mated either with treated or control males were sacrificed at Day 14 of pregnancy, and numbers of fetuses and resorptions were recorded. Results Both treatments decreased significantly implantation rates however, the proportion of fetuses/resorptions was only reduced in those females mated to males exposed to radiation, indicating a selection favoring fertilization of sperm with unfragmented DNA on the heat treatment group. To determine if DNA integrity is one of the keys of spermatozoa selection after postovulatory mating, we analyzed sperm DNA fragmentation by COMET assay in: a sperm recovered from mouse epididymides; b sperm recovered from three different regions of female uterine horns after mating; and c sperm attached to the ZP after in vitro fertilization (IVF. Similar results were found for control and both treatments, COMET values decreased significantly during the transit from the uterine section close to the uterotubal junction to the oviduct, and in the spermatozoa attached to ZP. However, fertilization by IVF and intracytoplasmatic sperm injection (ICSI showed that during sperm ZP-penetration, a stringent selection against fragmented-DNA sperm is carried out when the damage was induced by heat stress, but not when DNA fragmentation was induced by

  12. LDFF, the large molecular weight DNA fragmentation factor, is responsible for the large molecular weight DNA degradation during apoptosis in Xenopus egg extracts

    Institute of Scientific and Technical Information of China (English)

    Zhi Gang LU; Chuan Mao ZHANG; Zhong He ZHAI

    2004-01-01

    DNA degradation is a biochemical hallmark in apoptosis. It has been demonstrated in many cell types that there are two stages of DNA fragmentation during the apoptotic execution. In the early stage, chromatin DNA is cut into large molecular weight DNA fragments, although the responsible nuclease(s) has not been recognized. In the late stage, the chromatin DNA is cleaved further into short oligonucleosomal fragments by a well-characterized nuclease in apoptosis,the caspase-activated DNase (CAD/DFF40). In this study, we demonstrate that large molecular weight DNA fragmentation also occurs in Xenopus egg extracts in apoptosis. We show that the large molecular weight DNA fragmentation factor (LDFF) is not the Xenopus CAD homolog XCAD. LDFF is activated by caspase-3. The large molecular weight DNA fragmentation activity of LDFF is Mg2+-dependent and Ca2+-independent, can occur in both acidic and neutral pH conditions and can tolerate 45℃ treatment. These results indicate that LDFF in Xenopus egg extracts might be a new DNase (or DNases) responsible for the large DNA fragmentation.

  13. Chemoaffinity material for plasmid DNA analysis by high-performance liquid chromatography with condition-dependent switching between isoform and topoisomer selectivity.

    Science.gov (United States)

    Mahut, Marek; Gargano, Andrea; Schuchnigg, Hermann; Lindner, Wolfgang; Lämmerhofer, Michael

    2013-03-05

    Plasmid DNA may exist in three isoforms, the linear, open-circular (oc, "nicked"), and covalently closed circular (ccc, "supercoiled") form. We have recently reported on the chromatographic separation of supercoiled plasmid topoisomers on cinchona-alkaloid modified silica-based stationary phases. Herein, we present a selectivity switching mechanism to achieve separation of isoforms and/or supercoiled topoisomers using the very same chromatographic column and system. While salt gradient elution facilitates topoisomer separation, the supercoiled species are eluting as a single peak upon elution by a mixed pH and organic modifier gradient, still well separated from the other isoforms. We have found that a mobile phase pH value near the pI of the zwitterionic adsorbent surface leads to full recovery of all plasmid DNA isoforms, which is a major issue when using anion exchange-based resins. Furthermore, the observed elution pattern, oc < linear < ccc, is constant upon changes of mobile phase composition, gradient slope, and plasmid size. The remarkable isoform selectivity found on quinine-based selectors is explained by van't Hoff plots, revealing a different binding mechanism between the supercoiled plasmid on one hand and the oc and linear isoforms on the other hand.

  14. [Fingerprints identification of Gynostemma pentaphyllum by RAPD and cloning and analysis of its specific DNA fragment].

    Science.gov (United States)

    Jiang, Jun-fu; Li, Xiong-ying; Wu, Yao-sheng; Luo, Yu; Zhao, Rui-qiang; Lan, Xiu-wan

    2009-02-01

    To identify the resources of Gynostemma pentaphyllum and its spurious breed plant Cayratia japonica at level of DNA. Two random primers ( WGS001, WGS004) screened were applied to do random amplification with genomic DNA extracted from Gynostemma pentaphyllum and Cayratia japonica which were collected from different habitats. After amplificated with WGS004, one characteristic fragment about 500 bp which was common to all Gynostemma pentaphyllum samples studied but not to Cayratia japonica was cloned and sequenced. Then these sequences obtained were analyzed for identity and compared by Blastn program in GenBank. There were obvious different bands amplified by above two primers in their fingerprints of genomic DNA. On the basis of these different bands of DNA fingerprints, they could distinguish Gynostemma pentaphyllum and Cayratia japonica obviously. Sequence alignment of seven cloned bands showed that their identities ranged from 45.7% - 94.5%. There was no similar genome sequences searched in GenBank. This indicated that these seven DNA fragments had not been reported before and they should be new sequences. RAPD technique can be used for the accurate identification of Gynostemma pentaphyllum and its counterfeit goods Cayratia japonica. Besides, these specific DNA sequences for Gynostemmna pentaphyllum in this study are useful for the further research on identification of species and assisted selection breeding in Gynostemma pentaphyllum.

  15. Human Sperm DNA Fragmentation and its Correlation with Conventional Semen Parameters

    Science.gov (United States)

    Evgeni, Evangelini; Charalabopoulos, Konstantinos; Asimakopoulos, Byron

    2014-01-01

    Background The initial step in the diagnostic investigation of male infertility has been traditionally based on the conventional seminal profile. However, there are significant limitations regarding its ability to determine the underlying mechanisms that cause the disorder. Sperm DNA fragmentation has emerged as a potential causative factor of reproductive failure and its assessment has been suggested as a useful adjunct to the laboratory methodology of male infertility evaluation, especially before the application of assisted reproduction technology (ART). Methods A review of recent bibliography was carried out in PubMed by the use of relevant keywords, in order to evaluate the possible correlation between the conventional seminal parameters and sperm DNA fragmentation assessment as diagnostic tools in male infertility evaluation. Results A comprehensive diagnostic approach of male infertility should be based on a combination of diagnostic attributes, derived from the conventional semen analysis, as well as the investigation of genomic integrity testing. Conclusion Due to its strong correlation with several aspects of ART procedures and further consequences for the offspring, sperm DNA fragmentation is a parameter worth integrating in routine clinical practice. However, additional large scale studies focusing on specific subgroups of infertile men who may benefit from an efficient therapeutic management based on the optimization of sperm DNA integrity are needed. PMID:24696791

  16. A cost for high levels of sperm competition in rodents: increased sperm DNA fragmentation.

    Science.gov (United States)

    delBarco-Trillo, Javier; García-Álvarez, Olga; Soler, Ana Josefa; Tourmente, Maximiliano; Garde, José Julián; Roldan, Eduardo R S

    2016-03-16

    Sperm competition, a prevalent evolutionary process in which the spermatozoa of two or more males compete for the fertilization of the same ovum, leads to morphological and physiological adaptations, including increases in energetic metabolism that may serve to propel sperm faster but that may have negative effects on DNA integrity. Sperm DNA damage is associated with reduced rates of fertilization, embryo and fetal loss, offspring mortality, and mutations leading to genetic disease. We tested whether high levels of sperm competition affect sperm DNA integrity. We evaluated sperm DNA integrity in 18 species of rodents that differ in their levels of sperm competition using the sperm chromatin structure assay. DNA integrity was assessed upon sperm collection, in response to incubation under capacitating or non-capacitating conditions, and after exposure to physical and chemical stressors. Sperm DNA was very resistant to physical and chemical stressors, whereas incubation in non-capacitating and capacitating conditions resulted in only a small increase in sperm DNA damage. Importantly, levels of sperm competition were positively associated with sperm DNA fragmentation across rodent species. This is the first evidence showing that high levels of sperm competition lead to an important cost in the form of increased sperm DNA damage. © 2016 The Author(s).

  17. Influence of an extract of Juglans regia on the growth of Escherichia coli, on the electrophoretic profile of plasmid DNA and on the radiolabeling of blood constituents

    Energy Technology Data Exchange (ETDEWEB)

    Santos-Filho, Sebastiao David; Diniz, Claudia Leite; Carmo, Fernanda Santos do; Fonseca, Adenilson de Souza da; Bernardo-Filho, Mario [Universidade do Estado do Rio de Janeiro (UERJ), RJ (Brazil). Instituto de Biologia Roberto Alcantara Gomes. Dept. de Biofisica e Biometria]. E-mail: santos-filho@uer.br

    2008-12-15

    The aim of this work was to study the influence of a walnut (Juglans regia) extract on the growth of Escherichia coli (E. coli) AB1157, on the plasmid DNA topology and on the labeling of blood constituents with technetium-99m ({sup 99m}Tc). An E. coli AB1157 culture, in stationary phase, was incubated with walnut and the growth of the culture was evaluated by optical density at 600 nm for 7 hours. Plasmid DNA samples were incubated with SnCl{sub 2} in presence or absence of walnut for 40 minutes, 0.8% agarose gel electrophoresis was performed, the gel was stained and the plasmid topological forms were visualized. Blood samples from Wistar rats were incubated with walnut extract and an assay of labeling of blood constituents with technetium-99m ({sup 99m}Tc) was performed. Blood cells and plasma were separated. The radioactivity in each fraction was counted and percentage of incorporated radioactivity (%ATI) was determined. The results presented an inhibitory action of the growth of the E. coli AB1157 culture, no protective action of the walnut extract in plasmid DNA treated with SnCl{sub 2}. Moreover, walnut was also not capable to induce modifications in the DNA mobility in agarose gel but walnut was capable to decrease the distribution of {sup 99}'mTc on the blood cell compartment. In conclusion, our experimental data suggest that in the walnut extract has substances with an effect on the growth of E. coli culture, a potential action to increase the SnCl{sub 2} effect on plasmid DNA and also is capable to alter the distribution of {sup 99m}Tc on the blood cell compartment probably due to redoxi properties. (author)

  18. DNA large restriction fragment patterns of sporadic and epidemic nosocomial strains of Mycobacterium chelonae and Mycobacterium abscessus.

    OpenAIRE

    Wallace, R J; Zhang, Y; Brown, B.A.; Fraser, V; Mazurek, G H; S. Maloney

    1993-01-01

    Large restriction fragment (LRF) pattern analysis of genomic DNA using pulsed-field gel electrophoresis was performed on three reference strains, 32 sporadic isolates, and 92 nosocomial isolates from 12 epidemics of Mycobacterium chelonae and Mycobacterium abscessus. Only 17 of 30 (57%) unrelated strains of M. abscessus, compared with 10 of 11 (91%) of M. chelonae strains, gave satisfactory DNA extractions, with the remainder resulting in highly fragmented DNA. DraI, AsnI, XbaI, and SpeI gave...

  19. Effects of Sperm DNA Fragmentation on Semen Parameters and ICSI Outcome Determined by an Improved SCD Test,Halosperm

    OpenAIRE

    Asuman Demiroglu Zergeroğlu; Seda Yılmaz; Pelin Kutlu; Nuri Delikara; Kenan Sofuoglu

    2010-01-01

    Background: Sperm DNA fragmentation is known as an important cause of male infertility.The influence of sperm DNA damage on reproductive potential has been subject of many studiesindicating various results and remaining the subject controversial. In this study, we investigateddifferences of the semen parameters and intracytoplasmic sperm injection (ICSI) outcome accordingto sperm DNA fragmentation levels (DFLs) of patients.Materials and Methods: The DFLs were determined by Halosperm, a new im...

  20. Preparation of chitosan-plasmid DNA nanoparticles encoding interleukin-12 and their expression in CT-26 colon carcinoma cells.

    Science.gov (United States)

    Hallaj-Nezhadi, Somayeh; Valizadeh, Hadi; Dastmalchi, Siavoush; Baradaran, Behzad; Jalali, Mohammad Barzegar; Dobakhti, Faramarz; Lotfipour, Farzaneh

    2011-01-01

    Interleukin-12 (Il-12) as a cytokine has been proved to possess antitumor effects via stimulating the immune system. Non-viral gene delivery systems exhibit low toxicity and are easier to prepare compared to their viral counterparts. In this study, we aimed to prepare plasmid DNA loaded chitosan nanoparticles for expression of Il-12 and to evaluate their physicochemical characteristics, cytotoxicity and transfection efficiency in Murine CT-26 colon carcinoma cells. Nanoparticles were prepared using a complex coacervation process at different N/P ratios and characterized in terms of size, zeta potential, polydispersity index, morphology, encapsulation efficiency and polyplex formation. The cytotoxicities and transfection efficiencies of the prepared polyplexes were evaluated by MTT assay and ELISA (for hIL-12, p70), respectively. Size and zeta potential varied from 76.73 to 867.03 nm and between 5.68 and 16.77 mV, respectively. Strong attachment of the DNA to chitosan was observed after polyplex preparation. Encapsulation efficiencies were high (72.97-94.87%). The transfection efficiencies of the prepared complexes were obviously higher than those of naked pDNA when N/P ratios were between 16 and 60. Maximum level of phIL-12 expression was obtained at (N/P = 16) with mean particle size of 381.83±82.77 nm (polydispersity index=0.44) indicating the improved transfection of pUMVC3-hIL12 about 2.80 times compared to that of the naked pUMVC3-hIL12. Prepared polyplexes were nontoxic to CT-26 cells. Chitosan-DNA nanoparticles at N/P = 16 with minimal cytotoxicity, can be used as suitable candidate for Il-12 delivery. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

  1. An efficient algorithm for DNA fragment assembly in MapReduce.

    Science.gov (United States)

    Xu, Baomin; Gao, Jin; Li, Chunyan

    2012-09-28

    Fragment assembly is one of the most important problems of sequence assembly. Algorithms for DNA fragment assembly using de Bruijn graph have been widely used. These algorithms require a large amount of memory and running time to build the de Bruijn graph. Another drawback of the conventional de Bruijn approach is the loss of information. To overcome these shortcomings, this paper proposes a parallel strategy to construct de Bruijin graph. Its main characteristic is to avoid the division of de Bruijin graph. A novel fragment assembly algorithm based on our parallel strategy is implemented in the MapReduce framework. The experimental results show that the parallel strategy can effectively improve the computational efficiency and remove the memory limitations of the assembly algorithm based on Euler superpath. This paper provides a useful attempt to the assembly of large-scale genome sequence using Cloud Computing.

  2. Mobilization of Bacillus thuringiensis plasmid pTX14-3.

    Science.gov (United States)

    Andrup, L; Bendixen, H H; Jensen, G B

    1995-05-01

    The Bacillus thuringiensis subsp. israelensis (Bti) plasmid pTX14-3 has been reported to contain a gene, mob14-3, with considerable homology to genes encoding mobilization proteins from other gram-positive bacteria. We have used the aggregation-mediated conjugation system recently discovered in Bti to compare the mobilization kinetics of different derivatives of plasmid pTX14-3. Plasmid pTX14-3 has been found to replicate by the rolling-circle mechanism and to contain a locus suppressing the formation of high-molecular-weight DNA. We found that deleting a DNA fragment containing this locus increased the transfer frequency about twofold. The mobilization frequency of the plasmid containing the intact mob14-3 gene did not indicate a mobilization-enhancing activity of the encoded polypeptide. However, the presence of the mob14-3 gene seemed to increase the stability of the plasmid in exponential growth.

  3. Effect of serotonin on the expression of antigens and DNA levels in Yersinia pestis cells with different plasmid content

    Science.gov (United States)

    Klueva, Svetlana N.; Korsukov, Vladimir N.; Schukovskaya, Tatyana N.; Kravtsov, Alexander L.

    2004-08-01

    Using flow cytometry (FCM) the influence of exogenous serotonin on culture growth, DNA content and fluorescence intensity of cells binding FITC-labelled plague polyclonal immunoglobulins was studied in Yersinia pestis EV (pFra+, pCad+, pPst+), Yersinia pestis KM218 (pFra-, pCad-, pPst-), Yersinia pestis KM 216 (pFra-, pCad-, pPst+). The results have been obtained by FCM showed serotonin accelerated Yersinia pestis EV (pFra+, pCad+, pPst+), Yersinia pestis KM218 (pFra-, pCad-, pPst-) culture growth during cultivation in Hottinger broth pH 7.2 at 28°C at concentration of 10-5 M. The presence of 10-5 M serotonin in nutrient broth could modulate DNA content in 37°C growing population of plague microbe independently of their plasmid content. Serotonin have been an impact on the distribution pattern of the cells according to their phenotypical characteristics, which was reflected in the levels of population heterogeneity in the intensity of specific immunofluorescence determined by FMC.

  4. Three-dimensional imaging of <