WorldWideScience

Sample records for a-dna

  1. Ligand inducible assembly of a DNA tetrahedron.

    Science.gov (United States)

    Dohno, Chikara; Atsumi, Hiroshi; Nakatani, Kazuhiko

    2011-03-28

    Here we show that a small synthetic ligand can be used as a key building component for DNA nanofabrication. Using naphthyridinecarbamate dimer (NCD) as a molecular glue for DNA hybridization, we demonstrate NCD-triggered formation of a DNA tetrahedron.

  2. DNA encoding a DNA repair protein

    Science.gov (United States)

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-08-15

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  3. Stepwise oscillatory circuits of a DNA molecule

    OpenAIRE

    Xu, Kunming

    2009-01-01

    A DNA molecule is characterized by a stepwise oscillatory circuit where every base pair is a capacitor, every phosphate bridge is an inductance, and every deoxyribose is a charge router. The circuitry accounts for DNA conductivity through both short and long distances in good agreement with experimental evidence that has led to the identification of the so-called super-exchange and multiple-step hopping mechanisms. However, in contrast to the haphazard hopping and super-exchanging events, the...

  4. Methods to alter levels of a DNA repair protein

    Science.gov (United States)

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-10-17

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  5. Formal description of a DNA oriented computer language.

    OpenAIRE

    Schroeder, J L; Blattner, F R

    1982-01-01

    A computer language termed DNA has been devised to aid in the description of DNA sequence manipulations. This was an outgrowth of a DNA sequence editor which has been implemented for a microcomputer. A formal description of the language in the BNF formalism is presented.

  6. Theoretical description of biomolecular hydration - Application to A-DNA

    Energy Technology Data Exchange (ETDEWEB)

    Garcia, A.E.; Hummer, G. [Los Alamos National Laboratory, NM (United States); Soumpasis, D.M. [Max Planck Inst. for Biophysical Chemistry, Goettingen (Germany)

    1994-12-31

    The local density of water molecules around a biomolecule is constructed from calculated two- and three-points correlation functions of polar solvents in water using a Potential-of-Mean-Force (PMF) expansion. As a simple approximation, the hydration of all polar (including charged) groups in a biomolecule is represented by the hydration of water oxygen in bulk water, and the effect of non-polar groups on hydration are neglected, except for excluded volume effects. Pair and triplet correlation functions are calculated by molecular dynamics simulations. We present calculations of the structural hydration for ideal A-DNA molecules with sequences [d(CG){sub 5}]{sub 2} and [d(C{sub 5}G{sub 5})]{sub 2}. We find that this method can accurately reproduce the hydration patterns of A-DNA observed in neutron diffraction experiments on oriented DNA fibers.

  7. A DNA methylation fingerprint of 1628 human samples

    OpenAIRE

    Fernandez, A. F.; Assenov, Y.; Martin-Subero, J.I. (José Ignacio); Balint, B.; Siebert, R.; Taniguchi, H; Yamamoto, H.; M. Hidalgo; Tan, A.-C.; Galm, O; Ferrer, I.; Sanchez-Cespedes, M.; Villanueva, A; Carmona, J; Sanchez-Mut, J. V.

    2012-01-01

    Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns reve...

  8. A DNA Barcoding Approach to Characterize Pollen Collected by Honeybees

    OpenAIRE

    Andrea Galimberti; Fabrizio De Mattia; Ilaria Bruni; Daniela Scaccabarozzi; Anna Sandionigi; Michela Barbuto; Maurizio Casiraghi; Massimo Labra

    2014-01-01

    In the present study, we investigated DNA barcoding effectiveness to characterize honeybee pollen pellets, a food supplement largely used for human nutrition due to its therapeutic properties. We collected pollen pellets using modified beehives placed in three zones within an alpine protected area (Grigna Settentrionale Regional Park, Italy). A DNA barcoding reference database, including rbcL and trnH-psbA sequences from 693 plant species (104 sequenced in this study) was assembled. The datab...

  9. A DNA enzyme with N-glycosylase activity

    OpenAIRE

    Sheppard, Terry L.; Ordoukhanian, Phillip; Joyce, Gerald F.

    2000-01-01

    In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 106-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of...

  10. Developmental self-assembly of a DNA tetrahedron.

    Science.gov (United States)

    Sadowski, John P; Calvert, Colby R; Zhang, David Yu; Pierce, Niles A; Yin, Peng

    2014-04-22

    Kinetically controlled isothermal growth is fundamental to biological development, yet it remains challenging to rationally design molecular systems that self-assemble isothermally into complex geometries via prescribed assembly and disassembly pathways. By exploiting the programmable chemistry of base pairing, sophisticated spatial and temporal control have been demonstrated in DNA self-assembly, but largely as separate pursuits. By integrating temporal with spatial control, here we demonstrate the "developmental" self-assembly of a DNA tetrahedron, where a prescriptive molecular program orchestrates the kinetic pathways by which DNA molecules isothermally self-assemble into a well-defined three-dimensional wireframe geometry. In this reaction, nine DNA reactants initially coexist metastably, but upon catalysis by a DNA initiator molecule, navigate 24 individually characterizable intermediate states via prescribed assembly pathways, organized both in series and in parallel, to arrive at the tetrahedral final product. In contrast to previous work on dynamic DNA nanotechnology, this developmental program coordinates growth of ringed substructures into a three-dimensional wireframe superstructure, taking a step toward the goal of kinetically controlled isothermal growth of complex three-dimensional geometries.

  11. Reversibly switching the surface porosity of a DNA tetrahedron.

    Science.gov (United States)

    Zhang, Chuan; Tian, Cheng; Li, Xiang; Qian, Hang; Hao, Chenhui; Jiang, Wen; Mao, Chengde

    2012-07-25

    The ability to reversibly switch the surface porosity of nanocages would allow controllable matter transport in and out of the nanocages. This would be a desirable property for many technological applications, such as drug delivery. To achieve such capability, however, is challenging. Herein we report a strategy for reversibly changing the surface porosity of a self-assembled DNA nanocage (a DNA tetrahedron) that is based on DNA hydridization and strand displacement. The involved DNA nanostructures were thoroughly characterized by multiple techniques, including polyacrylamide gel electrophoresis, dynamic light scattering, atomic force microscopy, and cryogenic electron microscopy. This work may lead to the design and construction of stimuli-responsive nanocages that might find applications as smart materials.

  12. A DNA barcoding approach to characterize pollen collected by honeybees.

    Science.gov (United States)

    Galimberti, Andrea; De Mattia, Fabrizio; Bruni, Ilaria; Scaccabarozzi, Daniela; Sandionigi, Anna; Barbuto, Michela; Casiraghi, Maurizio; Labra, Massimo

    2014-01-01

    In the present study, we investigated DNA barcoding effectiveness to characterize honeybee pollen pellets, a food supplement largely used for human nutrition due to its therapeutic properties. We collected pollen pellets using modified beehives placed in three zones within an alpine protected area (Grigna Settentrionale Regional Park, Italy). A DNA barcoding reference database, including rbcL and trnH-psbA sequences from 693 plant species (104 sequenced in this study) was assembled. The database was used to identify pollen collected from the hives. Fifty-two plant species were identified at the molecular level. Results suggested rbcL alone could not distinguish among congeneric plants; however, psbA-trnH identified most of the pollen samples at the species level. Substantial variability in pollen composition was observed between the highest elevation locality (Alpe Moconodeno), characterized by arid grasslands and a rocky substrate, and the other two sites (Cornisella and Ortanella) at lower altitudes. Pollen from Ortanella and Cornisella showed the presence of typical deciduous forest species; however in samples collected at Ortanella, pollen of the invasive Lonicera japonica, and the ornamental Pelargonium x hortorum were observed. Our results indicated pollen composition was largely influenced by floristic local biodiversity, plant phenology, and the presence of alien flowering species. Therefore, pollen molecular characterization based on DNA barcoding might serve useful to beekeepers in obtaining honeybee products with specific nutritional or therapeutic characteristics desired by food market demands. PMID:25296114

  13. A DNA origami nanorobot controlled by nucleic acid hybridization

    KAUST Repository

    Torelli, Emanuela

    2014-03-20

    A prototype for a DNA origami nanorobot is designed, produced, and tested. The cylindrical nanorobot (diameter of 14 nm and length of 48 nm) with a switchable flap, is able to respond to an external stimulus and reacts by a physical switch from a disarmed to an armed configuration able to deliver a cellular compatible message. In the tested design the robot weapon is a nucleic acid fully contained in the inner of the tube and linked to a single point of the internal face of the flap. Upon actuation the nanorobot moves the flap extracting the nucleic acid that assembles into a hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme catalyzing a colorimetric reaction or chemiluminescence generation. The actuation switch is triggered by an external nucleic acid (target) that interacts with a complementary nucleic acid that is beard externally by the nanorobot (probe). Hybridization of probe and target produces a localized structural change that results in flap opening. The flap movement is studied on a two-dimensional prototype origami using Förster resonance energy transfer and is shown to be triggered by a variety of targets, including natural RNAs. The nanorobot has potential for in vivo biosensing and intelligent delivery of biological activators. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Arduino-based automation of a DNA extraction system.

    Science.gov (United States)

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile. PMID:26409535

  15. A DNA barcoding approach to characterize pollen collected by honeybees.

    Directory of Open Access Journals (Sweden)

    Andrea Galimberti

    Full Text Available In the present study, we investigated DNA barcoding effectiveness to characterize honeybee pollen pellets, a food supplement largely used for human nutrition due to its therapeutic properties. We collected pollen pellets using modified beehives placed in three zones within an alpine protected area (Grigna Settentrionale Regional Park, Italy. A DNA barcoding reference database, including rbcL and trnH-psbA sequences from 693 plant species (104 sequenced in this study was assembled. The database was used to identify pollen collected from the hives. Fifty-two plant species were identified at the molecular level. Results suggested rbcL alone could not distinguish among congeneric plants; however, psbA-trnH identified most of the pollen samples at the species level. Substantial variability in pollen composition was observed between the highest elevation locality (Alpe Moconodeno, characterized by arid grasslands and a rocky substrate, and the other two sites (Cornisella and Ortanella at lower altitudes. Pollen from Ortanella and Cornisella showed the presence of typical deciduous forest species; however in samples collected at Ortanella, pollen of the invasive Lonicera japonica, and the ornamental Pelargonium x hortorum were observed. Our results indicated pollen composition was largely influenced by floristic local biodiversity, plant phenology, and the presence of alien flowering species. Therefore, pollen molecular characterization based on DNA barcoding might serve useful to beekeepers in obtaining honeybee products with specific nutritional or therapeutic characteristics desired by food market demands.

  16. Arduino-based automation of a DNA extraction system.

    Science.gov (United States)

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile.

  17. A DNA origami nanorobot controlled by nucleic acid hybridization.

    Science.gov (United States)

    Torelli, Emanuela; Marini, Monica; Palmano, Sabrina; Piantanida, Luca; Polano, Cesare; Scarpellini, Alice; Lazzarino, Marco; Firrao, Giuseppe

    2014-07-23

    A prototype for a DNA origami nanorobot is designed, produced, and tested. The cylindrical nanorobot (diameter of 14 nm and length of 48 nm) with a switchable flap, is able to respond to an external stimulus and reacts by a physical switch from a disarmed to an armed configuration able to deliver a cellular compatible message. In the tested design the robot weapon is a nucleic acid fully contained in the inner of the tube and linked to a single point of the internal face of the flap. Upon actuation the nanorobot moves the flap extracting the nucleic acid that assembles into a hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme catalyzing a colorimetric reaction or chemiluminescence generation. The actuation switch is triggered by an external nucleic acid (target) that interacts with a complementary nucleic acid that is beard externally by the nanorobot (probe). Hybridization of probe and target produces a localized structural change that results in flap opening. The flap movement is studied on a two-dimensional prototype origami using Förster resonance energy transfer and is shown to be triggered by a variety of targets, including natural RNAs. The nanorobot has potential for in vivo biosensing and intelligent delivery of biological activators.

  18. Untangling taxonomy: a DNA barcode reference library for Canadian spiders.

    Science.gov (United States)

    Blagoev, Gergin A; deWaard, Jeremy R; Ratnasingham, Sujeevan; deWaard, Stephanie L; Lu, Liuqiong; Robertson, James; Telfer, Angela C; Hebert, Paul D N

    2016-01-01

    Approximately 1460 species of spiders have been reported from Canada, 3% of the global fauna. This study provides a DNA barcode reference library for 1018 of these species based upon the analysis of more than 30,000 specimens. The sequence results show a clear barcode gap in most cases with a mean intraspecific divergence of 0.78% vs. a minimum nearest-neighbour (NN) distance averaging 7.85%. The sequences were assigned to 1359 Barcode index numbers (BINs) with 1344 of these BINs composed of specimens belonging to a single currently recognized species. There was a perfect correspondence between BIN membership and a known species in 795 cases, while another 197 species were assigned to two or more BINs (556 in total). A few other species (26) were involved in BIN merges or in a combination of merges and splits. There was only a weak relationship between the number of specimens analysed for a species and its BIN count. However, three species were clear outliers with their specimens being placed in 11-22 BINs. Although all BIN splits need further study to clarify the taxonomic status of the entities involved, DNA barcodes discriminated 98% of the 1018 species. The present survey conservatively revealed 16 species new to science, 52 species new to Canada and major range extensions for 426 species. However, if most BIN splits detected in this study reflect cryptic taxa, the true species count for Canadian spiders could be 30-50% higher than currently recognized. PMID:26175299

  19. PPARGC1A DNA methylation in subcutaneous adipose tissue in low birth weight subjects

    DEFF Research Database (Denmark)

    Gillberg, Linn; Jacobsen, Stine; Rönn, Tina;

    2014-01-01

    -fat overfeeding increases PPARGC1A DNA methylation in muscle in a birth weight dependent manner. However, PPARGC1A DNA methylation in subcutaneous adipose tissue (SAT) in LBW subjects has not previously been investigated. Our objective was to determine PPARGC1A DNA methylation and mRNA expression in basal......OBJECTIVE: Increased DNA methylation of the metabolic regulator peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) has been reported in skeletal muscle from type 2 diabetes (T2D) subjects and from low birth weight (LBW) subjects with an increased risk of T2D. High...

  20. A DNA nanocapsule with aptamer-controlled open-closure function for targeted delivery

    DEFF Research Database (Denmark)

    Bentin, Thomas

    2012-01-01

    A DNA capsule fitted with aptamer controlled target sensing has been "woven" using a 7308-base single-stranded DNA "thread" and 196 staple oligonucleotides. The capsule enables logic-gated molecular cargo delivery to targeted cell surfaces.......A DNA capsule fitted with aptamer controlled target sensing has been "woven" using a 7308-base single-stranded DNA "thread" and 196 staple oligonucleotides. The capsule enables logic-gated molecular cargo delivery to targeted cell surfaces....

  1. Paleoparasitological report on Ascaris aDNA from an ancient East Asian sample

    Directory of Open Access Journals (Sweden)

    Chang Seok Oh

    2010-03-01

    Full Text Available In this study, Ascaris DNA was extracted and sequenced from a medieval archaeological sample in Korea. While Ascaris eggs were confirmed to be of human origin by archaeological evidence, it was not possible to pinpoint the exact species due to close genetic relationships among them. Despite this shortcoming, this is the first Ascaris ancient DNA (aDNA report from a medieval Asian country and thus will expand the scope of Ascaris aDNA research.

  2. A-DNA and B-DNA: Comparing Their Historical X-Ray Fiber Diffraction Images

    Science.gov (United States)

    Lucas, Amand A.

    2008-01-01

    A-DNA and B-DNA are two secondary molecular conformations (among other allomorphs) that double-stranded DNA drawn into a fiber can assume, depending on the relative water content and other chemical parameters of the fiber. They were the first two forms to be observed by X-ray fiber diffraction in the early 1950s, respectively by Wilkins and…

  3. Use PCR and a Single Hair To Produce a "DNA Fingerprint."

    Science.gov (United States)

    Campbell, A. Malcolm; And Others

    1997-01-01

    Presents a laboratory procedure that involves students extracting their own DNA from a single hair follicle, using the polymerase chain reaction (PCR) to amplify a polymorphic locus, performing electrophoresis on the PCR products on an agarose gel, and visualizing the alleles to generate a "DNA fingerprint." Discusses theoretical background,…

  4. Q-Bank Phytoplasma: A DNA Barcoding Tool for Phytoplasma Identification

    DEFF Research Database (Denmark)

    Contaldo, Nicoletta; Paltrinieri, Samanta; Makarova, Olga;

    2015-01-01

    DNA barcoding is an identification method based on comparison of a short DNA sequence with known sequences from a database. A DNA barcoding tool has been developed for phytoplasma identification. This phytoplasma DNA barcoding protocol based on the tuf gene has been shown to identify phytoplasmas...

  5. 78 FR 58514 - Availability of an Environmental Assessment for Field Testing of a DNA Immunostimulant

    Science.gov (United States)

    2013-09-24

    ... of a DNA Immunostimulant AGENCY: Animal and Plant Health Inspection Service, USDA. ACTION: Notice of... then to field test, an unlicensed DNA Immunostimulant recommended for reduction in morbidity and.... Product: DNA Immunostimulant. Possible Field Test Locations: Texas, Mississippi, and Georgia for...

  6. Activation of a DNA damage checkpoint response in a TAF1-defective cell line.

    Science.gov (United States)

    Buchmann, Ann M; Skaar, Jeffrey R; DeCaprio, James A

    2004-06-01

    Although the link between transcription and DNA repair is well established, defects in the core transcriptional complex itself have not been shown to elicit a DNA damage response. Here we show that a cell line with a temperature-sensitive defect in TBP-associated factor 1 (TAF1), a component of the TFIID general transcription complex, exhibits hallmarks of an ATR-mediated DNA damage response. Upon inactivation of TAF1, ATR rapidly localized to subnuclear foci and contributed to the phosphorylation of several downstream targets, including p53 and Chk1, resulting in cell cycle arrest. The increase in p53 expression and the G(1) phase arrest could be blocked by caffeine, an inhibitor of ATR. In addition, dominant negative forms of ATR but not ATM were able to override the arrest in G(1). These results suggest that a defect in TAF1 can elicit a DNA damage response. PMID:15169897

  7. A DNA glycosylase from human lymphoblasts that releases cis-thymine glycol from oxidized DNA

    International Nuclear Information System (INIS)

    An endonuclease (termed the UVX endonuclease) partially purified from cultured human leukemic lymphoblasts (CEM-CCRF line) was previously shown to act specifically on DNA irradiated by ionizing radiation, UV light, or treated with osmium tetroxide. This activity is tightly associated with an endonuclease specific for apurinic /apyrimidinic (AP) sites in DNA, suggesting that the initial attack on the radiation or oxidation induced lesions is by a DNA glycosylase that generates such AP sites. Thymine glycol has been shown to be a product common to γ-irradiated and oxidized DNA and in the present studies the authors have shown that the UVX endonuclease releases free cis-thymine glycol from osmium tetroxide treated DNA. HPLC analysis of the reaction products failed to detect any cis-thymidine glycol or nucleotide material indicating that the activity responsible is truly a DNA glycosylase. This enzyme thus resembles Endonuclease III of E. coli which contains both AP endonuclease and thymine glucol-DNA glycosylase activities

  8. Activation of a DNA Damage Checkpoint Response in a TAF1-Defective Cell Line

    OpenAIRE

    Buchmann, Ann M.; Skaar, Jeffrey R.; DeCaprio, James A.

    2004-01-01

    Although the link between transcription and DNA repair is well established, defects in the core transcriptional complex itself have not been shown to elicit a DNA damage response. Here we show that a cell line with a temperature-sensitive defect in TBP-associated factor 1 (TAF1), a component of the TFIID general transcription complex, exhibits hallmarks of an ATR-mediated DNA damage response. Upon inactivation of TAF1, ATR rapidly localized to subnuclear foci and contributed to the phosphoryl...

  9. Effects of Cytokine IL-18 Gene on Antibody Production Induced by Ag85A DNA Vaccine

    Institute of Scientific and Technical Information of China (English)

    CHENHai-wen; WANGZi-ming; FANXiong-lin; GANWei-min; SHITao; XUZhi-kai; LIYuan

    2004-01-01

    Objective: To investigate the effects of plasmid containing human IL-18 gene on the humoral immune response of mice immunized by Ag85A DNA vaccines of Mycobacterium tuberculosis H37 Rv strain. Methods: Human IL-18 cDNA was amplified from RNA of peripheral blood mononuclear cells(PBMCs)by RT-PCR and cloned into the pGEM-TEasy vector.After sequencing IL-18 gene was subcloned into the the sites of BamH I and EooR I digestion of pcDNA3.1. BALB/c mice were injected intramuscularly with eukaryotic expression plasmid pclL18, together with MTB pcAg85A DNA vaccines. The same immunization was repeated three times at intervals of two weeks. Mouse serawere collected at two weeks after the each injection. The titers of anti-Ag85A antibody were detected by ELISA. Results:IL-18 cDNA was amplified successfully from RNA of human PBMCs by RT-PCR and the result of sequencing was correct. The IL-18 gene was correctly inserted into the vector pcDNA3.1, which was confirmed with BamH I and EooR I digestion analysis. The positive plasmid was called pcIL18.After being immtmized with DNA vaccines,the titers of antibody obtained from mice being immtmized by pcAg85A combining with pclL18 were superior to mice inmunized by pcAg85A independently. Conc/us/on: Combination of IL-18 gene with MTB pcAg85A DNA vaccine could observably enhance the humoral immune responses to pcAg85A. It remains further investigated whether IL-18 gene plus MTB pcAg85A DNA vaccine could markedly induce the cellular mediated immune response to Ag85A or not.

  10. A DNA Structure-Based Bionic Wavelet Transform and Its Application to DNA Sequence Analysis

    OpenAIRE

    Fei Chen; Yuan-Ting Zhang

    2003-01-01

    DNA sequence analysis is of great significance for increasing our understanding of genomic functions. An important task facing us is the exploration of hidden structural information stored in the DNA sequence. This paper introduces a DNA structure-based adaptive wavelet transform (WT) – the bionic wavelet transform (BWT) – for DNA sequence analysis. The symbolic DNA sequence can be separated into four channels of indicator sequences. An adaptive symbol-to-number mapping, determined from the s...

  11. A DNA Crystal Designed to Contain Two Molecules per Asymmetric Unit

    Energy Technology Data Exchange (ETDEWEB)

    T Wang; R Sha; J Birktoft; J Zheng; C Mao; N Seeman

    2011-12-31

    We describe the self-assembly of a DNA crystal that contains two tensegrity triangle molecules per asymmetric unit. We have used X-ray crystallography to determine its crystal structure. In addition, we have demonstrated control over the colors of the crystals by attaching either Cy3 dye (pink) or Cy5 dye (blue-green) to the components of the crystal, yielding crystals of corresponding colors. Attaching the pair of dyes to the pair of molecules yields a purple crystal.

  12. A DNA-based system for selecting and displaying the combined result of two input variables

    DEFF Research Database (Denmark)

    Liu, Huajie; Wang, Jianbang; Song, S;

    2015-01-01

    demonstrate this capability in a DNA-based system that takes two input numbers, represented in DNA strands, and returns the result of their multiplication, writing this as a number in a display. Unlike a conventional calculator, this system operates by selecting the result from a library of solutions rather...... than through logic operations. The multiplicative example demonstrated here illustrates a much more general capability—to generate a unique output for any distinct pair of DNA inputs. The system thereby functions as a lookup table and could be a key component in future, more powerful data...

  13. Molecular and immunological characterization of a DNA-launched yellow fever virus 17D infectious clone

    OpenAIRE

    Jiang, Xiaohong; Dalebout, Tim J.; Lukashevich, Igor S.; Bredenbeek, Peter J.; Franco, David

    2015-01-01

    Yellow fever virus (YFV)-17D is an empirically developed, highly effective live-attenuated vaccine that has been administered to human beings for almost a century. YFV-17D has stood as a paradigm for a successful viral vaccine, and has been exploited as a potential virus vector for the development of recombinant vaccines against other diseases. In this study, a DNA-launched YFV-17D construct (pBeloBAC-FLYF) was explored as a new modality to the standard vaccine to combine the commendable feat...

  14. A DNA crystal designed to contain two molecules per asymmetric unit.

    Science.gov (United States)

    Wang, Tong; Sha, Ruojie; Birktoft, Jens; Zheng, Jianping; Mao, Chengde; Seeman, Nadrian C

    2010-11-10

    We describe the self-assembly of a DNA crystal that contains two tensegrity triangle molecules per asymmetric unit. We have used X-ray crystallography to determine its crystal structure. In addition, we have demonstrated control over the colors of the crystals by attaching either Cy3 dye (pink) or Cy5 dye (blue-green) to the components of the crystal, yielding crystals of corresponding colors. Attaching the pair of dyes to the pair of molecules yields a purple crystal. PMID:20958065

  15. ATP–stimulated DNA–mediated Redox Signaling by XPD, a DNA Repair and Transcription Helicase

    OpenAIRE

    Mui, Timothy P.; Fuss, Jill O.; Ishida, Justin P.; Tainer, John A.; Barton, Jacqueline K.

    2011-01-01

    Using DNA-modified electrodes, we show DNA-mediated signaling by XPD, a helicase that contains a [4Fe-4S] cluster and is critical for nucleotide excision repair and transcription. The DNA-mediated redox signal resembles that of base excision repair proteins, with a DNA-bound redox potential of ~80 mV versus NHE. Significantly, this signal increases with ATP hydrolysis. Moreover, the redox signal is substrate-dependent, reports on the DNA conformational changes associated with enzymatic functi...

  16. Protein–DNA charge transport: Redox activation of a DNA repair protein by guanine radical

    OpenAIRE

    Yavin, Eylon; Boal, Amie K.; Stemp, Eric D. A.; Boon, Elizabeth M; Livingston, Alison L.; O'Shea, Valerie L.; David, Sheila S.; Barton, Jacqueline K.

    2005-01-01

    DNA charge transport (CT) chemistry provides a route to carry out oxidative DNA damage from a distance in a reaction that is sensitive to DNA mismatches and lesions. Here, DNA-mediated CT also leads to oxidation of a DNA-bound base excision repair enzyme, MutY. DNA-bound Ru(III), generated through a flash/quench technique, is found to promote oxidation of the [4Fe-4S](2+) cluster of MutY to [4Fe-4S](3+) and its decomposition product [3Fe-4S](1+). Flash/quench experiments monitored by EPR spec...

  17. A DNA self-assembled monolayer for the specific attachment of unmodified double- or single-stranded DNA.

    OpenAIRE

    Bamdad, C

    1998-01-01

    A novel method for DNA surface immobilization and a paradigm for the attachment of unmodified DNA of any length or sequence are described herein. The development of a DNA self-assembled monolayer (DNA-SAM) that incorporates a DNA-thiol into a monolayer of inert alkane thiolates is reported. This DNA-SAM specifically hybridized complementary oligonucleotides while resisting the nonspecific adsorption of noncomplementary DNA and irrelevant proteins. Duplex DNA, having a single-stranded "capture...

  18. Decreased uv mutagenesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Prakash, L.; Hinkle, D.; Prakash, S.

    1978-01-01

    A DNA replication mutant of yeast, cdc8, was found to decrease uv-induced reversion of lys2-1, arg4-17, tryl and ural. This effect was observed with all three alleles of cdc8 tested. Survival curves obtained following uv irradiation in cdc8 rad double mutants show that cdc8 is epistatic to rad6, as well as to rad1; cdc8 rad51 double mutants seem to be more sensitive than the single mutants. Since uv-induced reversion in cdc8 rad1 and cdc8 rad51 double mutants is like that of the cdc8 single mutants, we conclude that CDC8 plays a direct role in error-prone repair. To test whether CDC8 codes for a DNA polymerase, we have purified both DNA polymerase I and DNA polymerase II from cdc8 and CDC+ cells. The purified DNA polymerases from cdc8 were no more heat labile than those from CDC+, suggesting that CDC8 is not a structural gene for either enzyme.

  19. A DNA immunoprecipitation assay used in quantitative detection of in vitro DNA-protein complex binding.

    Science.gov (United States)

    Kim, Min Young; Chae, Ji Hyung; Oh, Chang-Ho; Kim, Chul Geun

    2013-10-15

    To begin gene transcription, several transcription factors must bind to specific DNA sequences to form a complex via DNA-protein interactions. We established an in vitro method for specific and sensitive analyses of DNA-protein interactions based on a DNA immunoprecipitation (DIP) method. We verified the accuracy and efficiency of the DIP assay in quantitatively measuring DNA-protein binding using transcription factor CP2c as a model. With our DIP assay, we could detect specific interactions within a DNA-CP2c complex, with reproducible and quantitative binding values. In addition, we were able to effectively measure the changes in DNA-CP2c binding by the addition of a small molecule, FQI1 (factor quinolinone inhibitor 1), previously identified as a specific inhibitor of this binding. To identify a new regulator of DNA-CP2c binding, we analyzed several CP2c binding peptides and found that only one class of peptide severely inhibits DNA-CP2c binding. These data show that our DIP assay is very useful in quantitatively detecting the binding dynamics of DNA-protein complex. Because DNA-protein interaction is very dynamic in different cellular environments, our assay can be applied to the detection of active transcription factors, including promoter occupancy in normal and disease conditions. Moreover, it may be used to develop a targeted regulator of specific DNA-protein interaction.

  20. Scanning a DNA molecule for bound proteins using hybrid magnetic and optical tweezers.

    Directory of Open Access Journals (Sweden)

    Marijn T J van Loenhout

    Full Text Available The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ~17 nm spatial resolution. An offset of 33 ± 5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/[Formula: see text]. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.

  1. Enzymatic conjugation of multiple proteins on a DNA aptamer in a tail-specific manner.

    Science.gov (United States)

    Takahara, Mari; Hayashi, Kounosuke; Goto, Masahiro; Kamiya, Noriho

    2016-06-01

    Conjugation of single-strand DNA aptamers and enzymes has been of great significance in bioanalytical and biomedical applications because of the unlimited functions provided by DNA aptamer direction. Therefore, we developed efficient tailing of a DNA aptamer, with end-specific conjugation of multiple enzymes, through enzymatic catalysis. Terminal deoxynucleotidyl transferase (TdT) added multiple Z-Gln-Gly (Z-QG) moieties to the 3'-end of a DNA aptamer via the addition of Z-QG-modified deoxyuridine triphosphate (Z-QG-dUTP) and deoxynucleoside triphosphates (dNTPs). The resultant (Z-QG)m -(dN)l-aptamer, whose Z-QGs with dN spacers served as stickers for microbial transglutaminase (MTG), were crosslinked between the Z-QGs on the DNA and a substrate peptide sequence containing lysine (K), fused to a recombinant enzyme (i.e. bacterial alkaline phosphatase; BAP) by MTG. The incorporation efficiency of Z-QG moieties on the aptamer tail and the subsequent conjugation efficiency with multiple enzyme molecules were dramatically altered by the presence of dNTPs, revealing that a combination of Z-QG-dUTP/dTTP comprised the best labeling efficiency and corresponding properties during analytical performance. Thus, a novel optimized platform for designing (BAP)n -(dT)l-DNA aptamers was demonstrated for the first time in this article, offering unique opportunities for tailoring new types of covalent protein-nucleic acid conjugates in a controllable way. PMID:27119459

  2. Yeast redoxyendonuclease, a DNA repair enzyme similar to Escherichia coli endonuclease III

    Energy Technology Data Exchange (ETDEWEB)

    Gossett, J.; Lee, K.; Cunningham, R.P.; Doetsch, P.W.

    1988-04-05

    A DNA repair endonuclease (redoxyendonuclease) was isolated from bakers' yeast (Saccharomyces cerevisiae). The enzyme has been purified by a series of column chromatography steps and cleaves OsO/sub 4/-damaged, double-stranded DNA at sites of thymine glycol and heavily UV-irradiated DNA at sites of cytosine, thymine, and guanine photoproducts. The base specificity and mechanism of phosphodiester bond cleavage for the yeast redoxyendonuclease appear to be identical with those of Escherichia coli endonuclease III when thymine glycol containing, end-labeled DNA fragments of defined sequence are employed as substrates. Yeast redoxyendonuclease has an apparent molecular size of 38,000-42,000 daltons and is active in the absence of divalent metal cations. The identification of such an enzyme in yeast may be of value in the elucidation of the biochemical basis for radiation sensitivity in certain yeast mutants.

  3. Ultrasensitive detection of microRNA through rolling circle amplification on a DNA tetrahedron decorated electrode.

    Science.gov (United States)

    Miao, Peng; Wang, Bidou; Meng, Fanyu; Yin, Jian; Tang, Yuguo

    2015-03-18

    MicroRNAs are a class of evolutionally conserved, small noncoding RNAs involved in the regulation of gene expression and affect a variety of biological processes including cellular differentiation, immunological response, tumor development, and so on. Recently, microRNAs have been identified as promising disease biomarkers. In this work, we have fabricated a novel electrochemical method for ultrasensitive detection of microRNA. Generally, a DNA tetrahedron decorated gold electrode is employed as the recognition interface. Then, hybridizations between DNA tetrahedron, microRNA, and primer probe initiate rolling circle amplification (RCA) on the electrode surface. Silver nanoparticles attached to the RCA products provide significant electrochemical signals and a limit of detection as low as 50 aM is achieved. Moreover, homology microRNA family members with only one or two mismatches can be successfully distinguished. Therefore, this proposed method reveals great advancements toward improved disease diagnosis and prognosis.

  4. Superexchange interaction enhancement of the quantum transport in a DNA-type molecule

    Institute of Scientific and Technical Information of China (English)

    Wang Rui; Zhang Cun-Xi; Zhou Yun-Qing; Kong Ling-Min

    2011-01-01

    We use the transfer matrix method and the Green function technique to theoretically study the quantum tunnelling through a DNA-type molecule.Ferromagnetic electrodes are used to produce the spin-polarized transmission probability and therefore the spin current.The distance-dependent crossover comes from the topological variation from the onedimensional to the two-dimensional model transform as we switch on the interstrand coupling; a new base pair will present N - 1 extrachannels for the charge and spin as N being the total base pairs.This will restrain the decay of the transmission and improve the stability of the quantum transport.The spin and charge transfer through the DNA-type molecule is consistent with the quantum tunneling barrier.

  5. A DNA Structure-Based Bionic Wavelet Transform and Its Application to DNA Sequence Analysis

    Directory of Open Access Journals (Sweden)

    Fei Chen

    2003-01-01

    Full Text Available DNA sequence analysis is of great significance for increasing our understanding of genomic functions. An important task facing us is the exploration of hidden structural information stored in the DNA sequence. This paper introduces a DNA structure-based adaptive wavelet transform (WT – the bionic wavelet transform (BWT – for DNA sequence analysis. The symbolic DNA sequence can be separated into four channels of indicator sequences. An adaptive symbol-to-number mapping, determined from the structural feature of the DNA sequence, was introduced into WT. It can adjust the weight value of each channel to maximise the useful energy distribution of the whole BWT output. The performance of the proposed BWT was examined by analysing synthetic and real DNA sequences. Results show that BWT performs better than traditional WT in presenting greater energy distribution. This new BWT method should be useful for the detection of the latent structural features in future DNA sequence analysis.

  6. Voltammetric Detection of Damage to DNA by Arsenic Compounds at a DNA Biosensor

    Directory of Open Access Journals (Sweden)

    R. Wennrich

    2005-11-01

    Full Text Available DNA biosensor can serve as a powerfull tool for simple in vitro tests of chemicaltoxicity. In this paper, damage to DNA attached to the surface of screen-printed carbonelectrode by arsenic compounds in solution is described. Using the Co(III complex with1,10-phenanthroline, [Co(phen3]3+ , as an electrochemical DNA marker and the Ru(IIcomplex with bipyridyne, [Ru(bipy3]2+ , as a DNA oxidation catalyst, the portion of originaldsDNA which survives an incubation of the biosensor in the cleavage medium was evaluated.The model cleavage mixture was composed of an arsenic compound at 10-3 mol/Lconcentration corresponding to real contaminated water, 2x10-4 mol/L Fe(II or Cu(II ions asthe redox catalyst, and 1.5x10-2 mol/L hydrogen peroxide. DNA damage by arsenite,dimethylarsinic acid as the metabolic product of inorganic arsenic and widely used herbicide,as well as phenylarsonic acid and p-arsanilic acid as the representatives of feed additives wasfound in difference to arsenate.

  7. A fluorescent aptasensor based on a DNA pyramid nanostructure for ultrasensitive detection of ochratoxin A.

    Science.gov (United States)

    Nameghi, Morteza Alinezhad; Danesh, Noor Mohammad; Ramezani, Mohammad; Hassani, Faezeh Vahdati; Abnous, Khalil; Taghdisi, Seyed Mohammad

    2016-08-01

    Analytical techniques for detection of ochratoxin A (OTA) in food products and blood serum are of great significance. In this study, a fluorescent aptasensor was developed for sensitive and specific detection of OTA, based on a DNA pyramid nanostructure (DPN) and PicoGreen (PG) dye. The designed aptasensor inherits characteristics of DPN, such as high stability and capacity for PG loading. PG, as a fluorescent dye, could bind to double-stranded DNA (dsDNA). In the absence of OTA, the pyramid structure of DPN remains intact, leading to a very strong fluorescence emission. Because of higher affinity of aptamer for its target relative to its complementary strand, upon addition of target, the pyramid structure of DPN is disassembled, leading to a weak fluorescence emission. The presented aptasensor showed high specificity toward OTA with a limit of detection (LOD) as low as 0.135 nM. Besides, the designed sensing strategy was successfully utilized to recognize OTA in serum and grape juice with LODs of 0.184 and 0.149 nM, respectively.

  8. Regulation of MUTYH, a DNA Repair Enzyme, in Renal Proximal Tubular Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Jianping Lu

    2015-01-01

    Full Text Available MUTYH is a DNA repair enzyme that initiates a base excision repair (BER by recognizing and removing 8-Oxoguanine (8-oxoG and its paired adenine. We demonstrated that both TGF-β1 and H2O2 treatment led to an increased 8-oxoG in cultured human proximal tubule epithelial (HK-2 cells, while the former induced epithelial-mesenchymal transition and the latter caused cell apoptosis. Without stimulation, HK-2 cells showed MUTYH expression in mitochondria. TGF-β1 triggered a transient upregulation of mitochondrial MUTYH and induced the expression of nuclear isoforms, while H2O2 showed no role on MUTYH expression. Ureteral obstruction (UUO mice exhibited high 8-oxoG reactivity with tubulointerstitial lesions. After obstruction, the MUTYH expression was increased only in tubules at day 3 and decreased with obvious tubular atrophy at day 10. Particularly, MUTYH was primarily located in normal tubular cytoplasm with a dominant mitochondrial form. A few cells with nuclear MUTYH expression were observed in the fibrotic interstitium. We confirmed that increased MUTYH expression was upregulated and positively correlated with the severity of kidney fibrosis. Thus, renal fibrosis caused a cell-type-specific and time-dependent response of oxidative DNA repairs, even within the same tissues. It suggests that intervention of MUTYH might be effective for therapies.

  9. Prevention of Helicobacter pylori-induced gastric cancers in gerbils by a DNA demethylating agent.

    Science.gov (United States)

    Niwa, Tohru; Toyoda, Takeshi; Tsukamoto, Tetsuya; Mori, Akiko; Tatematsu, Masae; Ushijima, Toshikazu

    2013-04-01

    Suppression of aberrant DNA methylation is a novel approach to cancer prevention, but, so far, the efficacy of the strategy has not been evaluated in cancers associated with chronic inflammation. Gastric cancers induced by Helicobacter pylori infection are known to involve aberrant DNA methylation and associated with severe chronic inflammation in their early stages. Here, we aimed to clarify whether suppression of aberrant DNA methylation can prevent H. pylori-induced gastric cancers using a Mongolian gerbil model. Administration of a DNA demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), to gerbils (0.125 mg/kg for 50-55 weeks) decreased the incidence of gastric cancers induced by H. pylori infection and N-methyl-N-nitrosourea (MNU) treatment from 55.2% to 23.3% (P testicular atrophy. These results showed that 5-aza-dC treatment can prevent H. pylori-induced gastric cancers and suggested that removal of induced DNA methylation and/or suppression of DNA methylation induction can become a target for prevention of chronic inflammation-associated cancers. PMID:23559452

  10. Methods for transforming and expression screening of filamentous fungal cells with a DNA library

    Energy Technology Data Exchange (ETDEWEB)

    Teter, Sarah; Lamsa, Michael; Cherry, Joel; Ward, Connie

    2015-06-02

    The present invention relates to methods for expression screening of filamentous fungal transformants, comprising: (a) isolating single colony transformants of a DNA library introduced into E. coli; (b) preparing DNA from each of the single colony E. coli transformants; (c) introducing a sample of each of the DNA preparations of step (b) into separate suspensions of protoplasts of a filamentous fungus to obtain transformants thereof, wherein each transformant contains one or more copies of an individual polynucleotide from the DNA library; (d) growing the individual filamentous fungal transformants of step (c) on selective growth medium, thereby permitting growth of the filamentous fungal transformants, while suppressing growth of untransformed filamentous fungi; and (e) measuring activity or a property of each polypeptide encoded by the individual polynucleotides. The present invention also relates to isolated polynucleotides encoding polypeptides of interest obtained by such methods, to nucleic acid constructs, expression vectors, and recombinant host cells comprising the isolated polynucleotides, and to methods of producing the polypeptides encoded by the isolated polynucleotides.

  11. Inhibition of BACE1 Activity by a DNA Aptamer in an Alzheimer's Disease Cell Model.

    Directory of Open Access Journals (Sweden)

    Huiyu Liang

    Full Text Available An initial step in amyloid-β (Aβ production includes amyloid precursor protein (APP cleavage via β-Site amyloid precursor protein cleaving enzyme 1 (BACE1. Increased levels of brain Aβ have been implicated in the pathogenesis of Alzheimer's disease (AD. Thus, β-secretase represents a primary target for inhibitor drug development in AD. In this study, aptamers were obtained from combinatorial oligonucleotide libraries using a technology referred to as systematic evolution of ligands by exponential enrichment (SELEX. A purified human BACE1 extracellular domain was used as a target to conduct an in vitro selection process using SELEX. Two DNA aptamers were capable of binding to BACE1 with high affinity and good specificity, with Kd values in the nanomolar range. We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model. We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment. Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media. Furthermore, Western blot analysis indicated that sAPPβ expression significantly decreased in the A1 treated versus control groups. These findings support the preliminary feasibility of an aptamer evolved from a SELEX strategy to function as a potential BACE1 inhibitor. To our knowledge, this is the first study to acquire a DNA aptamer that exhibited binding specificity to BACE1 and inhibited its activity.

  12. Amperometric detection of gold by differential pulse voltammetry using a DNA biosensor

    Institute of Scientific and Technical Information of China (English)

    GAN Ning; WANG Zhiying; XU Weiming; PAN Jianguo

    2007-01-01

    A DNA biosensor with [Ru(DA-bpy)3]Cl2(DA-bpy:4,4'-diamino-2,2'-bipyridine) (RuL) as the electrochemical probe was prepared on pyrolytic graphite electrode (PGE) through the supramolecular interaction between RuL complex and DNA template. Cyclic voltammetry of RuL-DNA film showed a pair of stable and reversible peaks corresponding to the Ru(Ⅲ)/Ru(Ⅱ) redox potential of-0.165 V versus Ag|AgCl in pH 7.4 0.1 mol· L-1 Tris-HCl. The electron transfer was expected across the double-strand DNA by an "electron tunneling" mechanism. When the DNA biosensor was immerged in gold (Ⅲ) buffer solution, the current peak signal (Ⅰ) of the RuL-DNA supramolecular depressed and △Ⅰ was linear in the concentration range of Au ion from 1 × 10-7 to 2 × 10-5 mol·L-1 with a regression coefficient of 0.9879. The detection limit was 5 × 10-8 mol·L-1. The developed procedures were applied to the analysis of synthetic samples of real materials with good sensitivity and selectivity.

  13. HIF3A DNA Methylation Is Associated with Childhood Obesity and ALT.

    Directory of Open Access Journals (Sweden)

    Shuo Wang

    Full Text Available Gene polymorphisms associated so far with body mass index (BMI can explain only 1.18-1.45% of observed variation in BMI. Recent studies suggest that epigenetic modifications, especially DNA methylation, could contribute to explain part of the missing heritability, and two epigenetic genome-wide analysis studies (EWAS have reported that Hypoxia Inducible Factor 3 Alpha Subunit (HIF3A methylation was associated with BMI or BMI change. We therefore assessed whether the HIF3A methylation is associated with obesity and other obesity-related phenotypes in Chinese children. The subjects included 110 severe obese cases aged 7-17y and 110 normal-weight controls matched by age and gender for measurement of blood DNA methylation levels at the HIF3A gene locus using the Sequenom's MassARRAY system. We observed significantly higher methylation levels in obese children than in controls at positions 46801642 and 46801699 in HIF3A gene (P<0.05, and found positive associations between methylation and alanine aminotransferase (ALT levels adjusted by gender, age and BMI at the position 46801699 (r = 0.226, P = 0.007. These results suggest that HIF3A DNA methylation is associated with childhood obesity, and has a BMI-independent association with ALT. The results provide evidence for identifying epigenetic factors of elivated ALT and may be useful for risk assessment and personalized medicine of liver diseases such as non-alcoholic fatty liver disease (NAFLD.

  14. Immunoglobulin variable region hypermutation is associated with a DNA repair deficit

    International Nuclear Information System (INIS)

    The molecular mechanism of Ig variable region hypermutation is unknown, but has been hypothesized to involve an error-prone DNA repair process. In this study, the authors used a novel PCR-based assay to compare repair of UV-induced DNA damage in mantle zone versus germinal center B lymphocytes. They observed that DNA repair activity within rearranged VDJ loci was sluggish in germinal center B lymphocytes compared to repair activity monitored in mantle zone B lymphocytes. In contrast, DNA repair times within the germline VH5 gene family, the variable region JH endash CH intron, and the N-ras gene was rapid and similar in both germinal center and mantle zone B cells. These results reflect a DNA repair deficit which, as expected for hypermutation, is selective for rearranged Ig VDG in germinal center cells. To directly measure the fidelity of DNA repair, the repaired PCR-amplified gene segments were analyzed for sequence changes by restriction enzyme digestion. In experiments thus far, repair of germline VH5 was error-free in both germinal center and mantle zone B cells. However, while rearranged VH5 segments were also error-free in mantle zone cells, they were highly mutated in germinal center cells. These findings provide direct biochemical evidence for the role of a sequence- and stage-specific error-prone DNA repair pathway in Ig V gene hypermutation

  15. Average probability that a "cold hit" in a DNA database search results in an erroneous attribution.

    Science.gov (United States)

    Song, Yun S; Patil, Anand; Murphy, Erin E; Slatkin, Montgomery

    2009-01-01

    We consider a hypothetical series of cases in which the DNA profile of a crime-scene sample is found to match a known profile in a DNA database (i.e., a "cold hit"), resulting in the identification of a suspect based only on genetic evidence. We show that the average probability that there is another person in the population whose profile matches the crime-scene sample but who is not in the database is approximately 2(N - d)p(A), where N is the number of individuals in the population, d is the number of profiles in the database, and p(A) is the average match probability (AMP) for the population. The AMP is estimated by computing the average of the probabilities that two individuals in the population have the same profile. We show further that if a priori each individual in the population is equally likely to have left the crime-scene sample, then the average probability that the database search attributes the crime-scene sample to a wrong person is (N - d)p(A).

  16. The formation of A-DNA in NaDNA films is suppressed by netropsin.

    Science.gov (United States)

    Fritzsche, H; Brandes, R; Rupprecht, A; Song, Z; Weidlich, T; Kearns, D R

    1992-01-01

    Oriented films of NaDNA complexed with netropsin were studied with deuterium nuclear magnetic resonance (2H NMR), X-ray diffraction and ultraviolet (UV) linear dichroism to obtain information about the influence of netropsin on the structural arrangement of the DNA bases and on the B-A transition. The results of these studies clearly demonstrate a strong suppression of the formation of A-DNA at relative humidities (RHs) down to about 50%. The suppression was complete in the NaDNA-netropsin complex studied with 2H NMR which had a netropsin input ratio, r, of 0.22 drug/base pair. The sample used for UV linear dichroism had a similar input ratio while the X-ray diffraction samples had input ratios between 0.033 and 0.39 drug/base pair. Together, the results of these studies are in agreement with previous infrared (IR) linear dichroism studies of the conformation of the sugar-phosphate backbone in NaDNA-netropsin complexes, which showed that the B-A transition is suppressed for r-values down to approximately 0.1 drug/base pair (Fritzsche, H., Rupprecht, A. and Richter, M., Nucleic Acids Res. 12 (1984) 9165-9177). Images PMID:1313963

  17. A DNA-Inspired Encryption Methodology for Secure, Mobile Ad Hoc Networks

    Science.gov (United States)

    Shaw, Harry

    2012-01-01

    Users are pushing for greater physical mobility with their network and Internet access. Mobile ad hoc networks (MANET) can provide an efficient mobile network architecture, but security is a key concern. A figure summarizes differences in the state of network security for MANET and fixed networks. MANETs require the ability to distinguish trusted peers, and tolerate the ingress/egress of nodes on an unscheduled basis. Because the networks by their very nature are mobile and self-organizing, use of a Public Key Infra structure (PKI), X.509 certificates, RSA, and nonce ex changes becomes problematic if the ideal of MANET is to be achieved. Molecular biology models such as DNA evolution can provide a basis for a proprietary security architecture that achieves high degrees of diffusion and confusion, and resistance to cryptanalysis. A proprietary encryption mechanism was developed that uses the principles of DNA replication and steganography (hidden word cryptography) for confidentiality and authentication. The foundation of the approach includes organization of coded words and messages using base pairs organized into genes, an expandable genome consisting of DNA-based chromosome keys, and a DNA-based message encoding, replication, and evolution and fitness. In evolutionary computing, a fitness algorithm determines whether candidate solutions, in this case encrypted messages, are sufficiently encrypted to be transmitted. The technology provides a mechanism for confidential electronic traffic over a MANET without a PKI for authenticating users.

  18. A DNA sequence alignment algorithm using quality information and a fuzzy inference method

    Institute of Scientific and Technical Information of China (English)

    Kwangbaek Kim; Minhwan Kim; Youngwoon Woo

    2008-01-01

    DNA sequence alignment algorithms in computational molecular biology have been improved by diverse methods.In this paper.We propose a DNA sequence alignment that Uses quality information and a fuzzy inference method developed based on the characteristics of DNA fragments and a fuzzy logic system in order to improve conventional DNA sequence alignment methods that uses DNA sequence quality information.In conventional algorithms.DNA sequence alignment scores are calculated by the global sequence alignment algorithm proposed by Needleman-Wunsch,which is established by using quality information of each DNA fragment.However,there may be errors in the process of calculating DNA sequence alignment scores when the quality of DNA fragment tips is low.because only the overall DNA sequence quality information are used.In our proposed method.an exact DNA sequence alignment can be achieved in spite of the low quality of DNA fragment tips by improvement of conventional algorithms using quality information.Mapping score parameters used to calculate DNA sequence alignment scores are dynamically adjusted by the fuzzy logic system utilizing lengths of DNA fragments and frequencies of low quality DNA bases in the fragments.From the experiments by applying real genome data of National Center for Bioteclmology Information,we could see that the proposed method is more efficient than conventional algorithms.

  19. A DNA tetrahedron-based molecular beacon for tumor-related mRNA detection in living cells.

    Science.gov (United States)

    Xie, Nuli; Huang, Jin; Yang, Xiaohai; Yang, Yanjing; Quan, Ke; Wang, He; Ying, Le; Ou, Min; Wang, Kemin

    2016-02-01

    Due to its low cytotoxicity, high resistance to enzymatic degradation, and cellular permeability, a DNA tetrahedron-based molecular beacon (DTMB) is designed for tumor-related TK1 mRNA detection in living cells, where the target sequence can induce the tetrahedron from contraction to extension, resulting in fluorescence restoration. PMID:26729323

  20. A DNA tetrahedron-based molecular beacon for tumor-related mRNA detection in living cells.

    Science.gov (United States)

    Xie, Nuli; Huang, Jin; Yang, Xiaohai; Yang, Yanjing; Quan, Ke; Wang, He; Ying, Le; Ou, Min; Wang, Kemin

    2016-02-01

    Due to its low cytotoxicity, high resistance to enzymatic degradation, and cellular permeability, a DNA tetrahedron-based molecular beacon (DTMB) is designed for tumor-related TK1 mRNA detection in living cells, where the target sequence can induce the tetrahedron from contraction to extension, resulting in fluorescence restoration.

  1. Low Dose Iron Treatments Induce a DNA Damage Response in Human Endothelial Cells within Minutes.

    Directory of Open Access Journals (Sweden)

    Inês G Mollet

    Full Text Available Spontaneous reports from patients able to report vascular sequelae in real time, and recognition that serum non transferrin bound iron may reach or exceed 10μmol/L in the blood stream after iron tablets or infusions, led us to hypothesize that conventional iron treatments may provoke acute vascular injury. This prompted us to examine whether a phenotype could be observed in normal human endothelial cells treated with low dose iron.Confluent primary human endothelial cells (EC were treated with filter-sterilized iron (II citrate or fresh media for RNA sequencing and validation studies. RNA transcript profiles were evaluated using directional RNA sequencing with no pre-specification of target sequences. Alignments were counted for exons and junctions of the gene strand only, blinded to treatment types.Rapid changes in RNA transcript profiles were observed in endothelial cells treated with 10μmol/L iron (II citrate, compared to media-treated cells. Clustering for Gene Ontology (GO performed on all differentially expressed genes revealed significant differences in biological process terms between iron and media-treated EC, whereas 10 sets of an equivalent number of randomly selected genes from the respective EC gene datasets showed no significant differences in any GO terms. After 1 hour, differentially expressed genes clustered to vesicle mediated transport, protein catabolism, and cell cycle (Benjamini p = 0.0016, 0.0024 and 0.0032 respectively, and by 6 hours, to cellular response to DNA damage stimulus most significantly through DNA repair genes FANCG, BLM, and H2AFX. Comet assays demonstrated that 10μM iron treatment elicited DNA damage within 1 hour. This was accompanied by a brisk DNA damage response pulse, as ascertained by the development of DNA damage response (DDR foci, and p53 stabilization.These data suggest that low dose iron treatments are sufficient to modify the vascular endothelium, and induce a DNA damage response.

  2. Approaches to enzyme and substrate design of the murine Dnmt3a DNA methyltransferase.

    Science.gov (United States)

    Jurkowska, Renata Z; Siddique, Abu Nasar; Jurkowski, Tomasz P; Jeltsch, Albert

    2011-07-01

    Dnmt3a-C, the catalytic domain of the Dnmt3a DNA-(cytosine-C5)-methyltransferase, is active in an isolated form but, like the full-length Dnmt3a, shows only weak DNA methylation activity. To improve this activity by directed evolution, we set up a selection system in which Dnmt3a-C methylated its own expression plasmid in E. coli, and protected it from cleavage by methylation-sensitive restriction enzymes. However, despite screening about 400 clones that were selected in three rounds from a random mutagenesis library of 60 000 clones, we were not able to isolate a variant with improved activity, most likely because of a background of uncleaved plasmids and plasmids that had lost the restriction sites. To improve the catalytic activity of Dnmt3a-C by optimization of the sequence of the DNA substrate, we analyzed its flanking-sequence preference in detail by bisulfite DNA-methylation analysis and sequencing of individual clones. Based on the enrichment and depletion of certain bases in the positions flanking >1300 methylated CpG sites, we were able to define a sequence-preference profile for Dnmt3a-C from the -6 to the +6 position of the flanking sequence. This revealed preferences for T over a purine at position -2, A over G at -1, a pyrimidine at +1, and A and T over G at +3. We designed one "good" substrate optimized for methylation and one "bad" substrate designed not to be efficiently methylated, and showed that the optimized substrate is methylated >20 times more rapidly at its central CpG site. The optimized Dnmt3a-C substrate can be applied in enzymatic high-throughput assays with Dnmt3a-C (e.g., for inhibitor screening), because the increased activity provides an improved dynamic range and better signal/noise ratio.

  3. Methods for interpreting lists of affected genes obtained in a DNA microarray experiment

    Directory of Open Access Journals (Sweden)

    Hedegaard Jakob

    2009-07-01

    Full Text Available Abstract Background The aim of this paper was to describe and compare the methods used and the results obtained by the participants in a joint EADGENE (European Animal Disease Genomic Network of Excellence and SABRE (Cutting Edge Genomics for Sustainable Animal Breeding workshop focusing on post analysis of microarray data. The participating groups were provided with identical lists of microarray probes, including test statistics for three different contrasts, and the normalised log-ratios for each array, to be used as the starting point for interpreting the affected probes. The data originated from a microarray experiment conducted to study the host reactions in broilers occurring shortly after a secondary challenge with either a homologous or heterologous species of Eimeria. Results Several conceptually different analytical approaches, using both commercial and public available software, were applied by the participating groups. The following tools were used: Ingenuity Pathway Analysis, MAPPFinder, LIMMA, GOstats, GOEAST, GOTM, Globaltest, TopGO, ArrayUnlock, Pathway Studio, GIST and AnnotationDbi. The main focus of the approaches was to utilise the relation between probes/genes and their gene ontology and pathways to interpret the affected probes/genes. The lack of a well-annotated chicken genome did though limit the possibilities to fully explore the tools. The main results from these analyses showed that the biological interpretation is highly dependent on the statistical method used but that some common biological conclusions could be reached. Conclusion It is highly recommended to test different analytical methods on the same data set and compare the results to obtain a reliable biological interpretation of the affected genes in a DNA microarray experiment.

  4. Next-generation sequencing reveals recent horizontal transfer of a DNA transposon between divergent mosquitoes.

    Directory of Open Access Journals (Sweden)

    Yupu Diao

    Full Text Available Horizontal transfer of genetic material between complex organisms often involves transposable elements (TEs. For example, a DNA transposon mariner has been shown to undergo horizontal transfer between different orders of insects and between different phyla of animals. Here we report the discovery and characterization of an ITmD37D transposon, MJ1, in Anopheles sinensis. We show that some MJ1 elements in Aedes aegypti and An. sinensis contain intact open reading frames and share nearly 99% nucleotide identity over the entire transposon, which is unexpectedly high given that these two genera had diverged 145-200 million years ago. Chromosomal hybridization and TE-display showed that MJ1 copy number is low in An. sinensis. Among 24 mosquito species surveyed, MJ1 is only found in Ae. aegypti and the hyrcanus group of anopheline mosquitoes to which An. sinensis belongs. Phylogenetic analysis is consistent with horizontal transfer and provides the basis for inference of its timing and direction. Although report of horizontal transfer of DNA transposons between higher eukaryotes is accumulating, our analysis is one of a small number of cases in which horizontal transfer of nearly identical TEs among highly divergent species has been thoroughly investigated and strongly supported. Horizontal transfer involving mosquitoes is of particular interest because there are ongoing investigations of the possibility of spreading pathogen-resistant genes into mosquito populations to control malaria and other infectious diseases. The initial indication of horizontal transfer of MJ1 came from comparisons between a 0.4x coverage An. sinensis 454 sequence database and available TEs in mosquito genomes. Therefore we have shown that it is feasible to use low coverage sequencing to systematically uncover horizontal transfer events. Expanding such efforts across a wide range of species will generate novel insights into the relative frequency of horizontal transfer of

  5. Development of a DNA barcoding system for seagrasses: successful but not simple.

    Directory of Open Access Journals (Sweden)

    Christina Lucas

    Full Text Available Seagrasses, a unique group of submerged flowering plants, profoundly influence the physical, chemical and biological environments of coastal waters through their high primary productivity and nutrient recycling ability. They provide habitat for aquatic life, alter water flow, stabilize the ground and mitigate the impact of nutrient pollution. at the coast region. Although on a global scale seagrasses represent less than 0.1% of the angiosperm taxa, the taxonomical ambiguity in delineating seagrass species is high. Thus, the taxonomy of several genera is unsolved. While seagrasses are capable of performing both, sexual and asexual reproduction, vegetative reproduction is common and sexual progenies are always short lived and epimeral in nature. This makes species differentiation often difficult, especially for non-taxonomists since the flower as a distinct morphological trait is missing. Our goal is to develop a DNA barcoding system assisting also non-taxonomists to identify regional seagrass species. The results will be corroborated by publicly available sequence data. The main focus is on the 14 described seagrass species of India, supplemented with seagrasses from temperate regions. According to the recommendations of the Consortium for the Barcoding of Life (CBOL rbcL and matK were used in this study. After optimization of the DNA extraction method from preserved seagrass material, the respective sequences were amplified from all species analyzed. Tree- and character-based approaches demonstrate that the rbcL sequence fragment is capable of resolving up to family and genus level. Only matK sequences were reliable in resolving species and partially the ecotype level. Additionally, a plastidic gene spacer was included in the analysis to confirm the identification level. Although the analysis of these three loci solved several nodes, a few complexes remained unsolved, even when constructing a combined tree for all three loci. Our approaches

  6. Molecular and immunological characterization of a DNA-launched yellow fever virus 17D infectious clone.

    Science.gov (United States)

    Jiang, Xiaohong; Dalebout, Tim J; Lukashevich, Igor S; Bredenbeek, Peter J; Franco, David

    2015-04-01

    Yellow fever virus (YFV)-17D is an empirically developed, highly effective live-attenuated vaccine that has been administered to human beings for almost a century. YFV-17D has stood as a paradigm for a successful viral vaccine, and has been exploited as a potential virus vector for the development of recombinant vaccines against other diseases. In this study, a DNA-launched YFV-17D construct (pBeloBAC-FLYF) was explored as a new modality to the standard vaccine to combine the commendable features of both DNA vaccine and live-attenuated viral vaccine. The DNA-launched YFV-17D construct was characterized extensively both in cell culture and in mice. High titres of YFV-17D were generated upon transfection of the DNA into cells, whereas a mutant with deletion in the capsid-coding region (pBeloBAC-YF/ΔC) was restricted to a single round of infection, with no release of progeny virus. Homologous prime-boost immunization of AAD mice with both pBeloBAC-FLYF and pBeloBAC-YF/ΔC elicited specific dose-dependent cellular immune response against YFV-17D. Vaccination of A129 mice with pBeloBAC-FLYF resulted in the induction of YFV-specific neutralizing antibodies in all vaccinated subjects. These promising results underlined the potential of the DNA-launched YFV both as an alternative to standard YFV-17D vaccination and as a vaccine platform for the development of DNA-based recombinant YFV vaccines. PMID:25516543

  7. Shotgun metagenomics indicates novel family A DNA polymerases predominate within marine virioplankton.

    Science.gov (United States)

    Schmidt, Helen F; Sakowski, Eric G; Williamson, Shannon J; Polson, Shawn W; Wommack, K Eric

    2014-01-01

    Virioplankton have a significant role in marine ecosystems, yet we know little of the predominant biological characteristics of aquatic viruses that influence the flow of nutrients and energy through microbial communities. Family A DNA polymerases, critical to DNA replication and repair in prokaryotes, are found in many tailed bacteriophages. The essential role of DNA polymerase in viral replication makes it a useful target for connecting viral diversity with an important biological feature of viruses. Capturing the full diversity of this polymorphic gene by targeted approaches has been difficult; thus, full-length DNA polymerase genes were assembled out of virioplankton shotgun metagenomic sequence libraries (viromes). Within the viromes novel DNA polymerases were common and found in both double-stranded (ds) DNA and single-stranded (ss) DNA libraries. Finding DNA polymerase genes in ssDNA viral libraries was unexpected, as no such genes have been previously reported from ssDNA phage. Surprisingly, the most common virioplankton DNA polymerases were related to a siphovirus infecting an α-proteobacterial symbiont of a marine sponge and not the podoviral T7-like polymerases seen in many other studies. Amino acids predictive of catalytic efficiency and fidelity linked perfectly to the environmental clades, indicating that most DNA polymerase-carrying virioplankton utilize a lower efficiency, higher fidelity enzyme. Comparisons with previously reported, PCR-amplified DNA polymerase sequences indicated that the most common virioplankton metagenomic DNA polymerases formed a new group that included siphoviruses. These data indicate that slower-replicating, lytic or lysogenic phage populations rather than fast-replicating, highly lytic phages may predominate within the virioplankton. PMID:23985748

  8. Carbinolamine formation and dehydration in a DNA repair enzyme active site.

    Directory of Open Access Journals (Sweden)

    M L Dodson

    Full Text Available In order to suggest detailed mechanistic hypotheses for the formation and dehydration of a key carbinolamine intermediate in the T4 pyrimidine dimer glycosylase (T4PDG reaction, we have investigated these reactions using steered molecular dynamics with a coupled quantum mechanics-molecular mechanics potential (QM/MM. We carried out simulations of DNA abasic site carbinolamine formation with and without a water molecule restrained to remain within the active site quantum region. We recovered potentials of mean force (PMF from thirty replicate reaction trajectories using Jarzynski averaging. We demonstrated feasible pathways involving water, as well as those independent of water participation. The water-independent enzyme-catalyzed reaction had a bias-corrected Jarzynski-average barrier height of approximately (6.5 kcal mol(-1 (27.2 kJ mol(-1 for the carbinolamine formation reaction and 44.5 kcal mol(-1 (186 kJ mol(-1 for the reverse reaction at this level of representation. When the proton transfer was facilitated with an intrinsic quantum water, the barrier height was approximately 15 kcal mol(-1 (62.8 kJ mol(-1 in the forward (formation reaction and 19 kcal mol(-1 (79.5 kJ mol(-1 for the reverse. In addition, two modes of unsteered (free dynamics carbinolamine dehydration were observed: in one, the quantum water participated as an intermediate proton transfer species, and in the other, the active site protonated glutamate hydrogen was directly transferred to the carbinolamine oxygen. Water-independent unforced proton transfer from the protonated active site glutamate carboxyl to the unprotonated N-terminal amine was also observed. In summary, complex proton transfer events, some involving water intermediates, were studied in QM/MM simulations of T4PDG bound to a DNA abasic site. Imine carbinolamine formation was characterized using steered QM/MM molecular dynamics. Dehydration of the carbinolamine intermediate to form the final imine product

  9. Probing the recognition surface of a DNA triplex: binding studies with intercalator-neomycin conjugates.

    Science.gov (United States)

    Xue, Liang; Xi, Hongjuan; Kumar, Sunil; Gray, David; Davis, Erik; Hamilton, Paris; Skriba, Michael; Arya, Dev P

    2010-07-01

    Thermodynamic studies on the interactions between intercalator-neomycin conjugates and a DNA polynucleotide triplex [poly(dA).2poly(dT)] were conducted. To draw a complete picture of such interactions, naphthalene diimide-neomycin (3) and anthraquinone-neomycin (4) conjugates were synthesized and used together with two other analogues, previously synthesized pyrene-neomycin (1) and BQQ-neomycin (2) conjugates, in our investigations. A combination of experiments, including UV denaturation, circular dichroism (CD) titration, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC), revealed that all four conjugates (1-4) stabilized poly(dA).2poly(dT) much more than its parent compound, neomycin. UV melting experiments clearly showed that the temperature (T(m3-->2)) at which poly(dA).2poly(dT) dissociated into poly(dA).poly(dT) and poly(dT) increased dramatically (>12 degrees C) in the presence of intercalator-neomycin conjugates (1-4) even at a very low concentration (2 muM). In contrast to intercalator-neomycin conjugates, the increment of T(m3-->2) of poly(dA).2poly(dT) induced by neomycin was negligible under the same conditions. The binding preference of intercalator-neomycin conjugates (1-4) to poly(dA).2poly(dT) was also confirmed by competition dialysis and a fluorescent intercalator displacement assay. Circular dichroism titration studies revealed that compounds 1-4 had slightly larger binding site size ( approximately 7-7.5) with poly(dA).2poly(dT) as compared to neomycin ( approximately 6.5). The thermodynamic parameters of these intercalator-neomycin conjugates with poly(dA).2poly(dT) were derived from an integrated van't Hoff equation using the T(m3-->2) values, the binding site size numbers, and other parameters obtained from DSC and ITC. The binding affinity of all tested ligands with poly(dA).2poly(dT) increased in the following order: neomycin neomycin. The binding of compounds 1-4 with poly(dA).2poly(dT) was mostly enthalpy

  10. Quantification of Plasmodiophora brassicae Using a DNA-Based Soil Test Facilitates Sustainable Oilseed Rape Production.

    Science.gov (United States)

    Wallenhammar, Ann-Charlotte; Gunnarson, Albin; Hansson, Fredrik; Jonsson, Anders

    2016-01-01

    Outbreaks of clubroot disease caused by the soil-borne obligate parasite Plasmodiophora brassicae are common in oilseed rape (OSR) in Sweden. A DNA-based soil testing service that identifies fields where P. brassicae poses a significant risk of clubroot infection is now commercially available. It was applied here in field surveys to monitor the prevalence of P. brassicae DNA in field soils intended for winter OSR production and winter OSR field experiments. In 2013 in Scania, prior to planting, P. brassicae DNA was detected in 60% of 45 fields on 10 of 18 farms. In 2014, P. brassicae DNA was detected in 44% of 59 fields in 14 of 36 farms, in the main winter OSR producing region in southern Sweden. P. brassicae was present indicative of a risk for >10% yield loss with susceptible cultivars (>1300 DNA copies g soil(-1)) in 47% and 44% of fields in 2013 and 2014 respectively. Furthermore, P. brassicae DNA was indicative of sites at risk of complete crop failure if susceptible cultivars were grown (>50 000 copies g(-1) soil) in 14% and 8% of fields in 2013 and 2014, respectively. A survey of all fields at Lanna research station in western Sweden showed that P. brassicae was spread throughout the farm, as only three of the fields (20%) showed infection levels below the detection limit for P.brassicae DNA, while the level was >50,000 DNA copies g(-1) soil in 20% of the fields. Soil-borne spread is of critical importance and soil scraped off footwear showed levels of up to 682 million spores g(-1) soil. Soil testing is an important tool for determining the presence of P. brassicae and providing an indication of potential yield loss, e.g., in advisory work on planning for a sustainable OSR crop rotation. This soil test is gaining acceptance as a tool that increases the likelihood of success in precision agriculture and in applied research conducted in commercial oilseed fields and at research stations. The present application highlights the importance of prevention of

  11. Quantification of Plasmodiophora brassicae Using a DNA-Based Soil Test Facilitates Sustainable Oilseed Rape Production

    Directory of Open Access Journals (Sweden)

    Ann-Charlotte Wallenhammar

    2016-04-01

    Full Text Available Outbreaks of clubroot disease caused by the soil-borne obligate parasite Plasmodiophora brassicae are common in oilseed rape (OSR in Sweden. A DNA-based soil testing service that identifies fields where P. brassicae poses a significant risk of clubroot infection is now commercially available. It was applied here in field surveys to monitor the prevalence of P. brassicae DNA in field soils intended for winter OSR production and winter OSR field experiments. In 2013 in Scania, prior to planting, P. brassicae DNA was detected in 60% of 45 fields on 10 of 18 farms. In 2014, P. brassicae DNA was detected in 44% of 59 fields in 14 of 36 farms, in the main winter OSR producing region in southern Sweden. P. brassicae was present indicative of a risk for >10% yield loss with susceptible cultivars (>1300 DNA copies g soil−1 in 47% and 44% of fields in 2013 and 2014 respectively. Furthermore, P. brassicae DNA was indicative of sites at risk of complete crop failure if susceptible cultivars were grown (>50 000 copies g−1 soil in 14% and 8% of fields in 2013 and 2014, respectively. A survey of all fields at Lanna research station in western Sweden showed that P. brassicae was spread throughout the farm, as only three of the fields (20% showed infection levels below the detection limit for P.brassicae DNA, while the level was >50,000 DNA copies g−1 soil in 20% of the fields. Soil-borne spread is of critical importance and soil scraped off footwear showed levels of up to 682 million spores g−1 soil. Soil testing is an important tool for determining the presence of P. brassicae and providing an indication of potential yield loss, e.g., in advisory work on planning for a sustainable OSR crop rotation. This soil test is gaining acceptance as a tool that increases the likelihood of success in precision agriculture and in applied research conducted in commercial oilseed fields and at research stations. The present application highlights the importance of

  12. Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection.

    Science.gov (United States)

    Wu, Fengchi; Wu, Yuqiang; Niu, Zhongwei; Vollmer, Frank

    2016-07-29

    Mercury is an extremely toxic chemical pollutant of our environment. It has attracted the world's attention due to its high mobility and the ease with which it accumulates in organisms. Sensitive devices and methods specific for detecting mercury ions are, hence, in great need. Here, we have integrated a DNA strand displacement reaction with a whispering gallery mode (WGM) sensor for demonstrating the detection of Hg(2+) ions. Our approach relies on the displacement of a DNA hairpin structure, which forms after the binding of mercury ions to an aptamer DNA sequence. The strand displacement reaction of the DNA aptamer provides highly specific and quantitative means for determining the mercury ion concentration on a label-free WGM sensor platform. Our approach also shows the possibility for manipulating the kinetics of a strand displacement reaction with specific ionic species.

  13. Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection.

    Science.gov (United States)

    Wu, Fengchi; Wu, Yuqiang; Niu, Zhongwei; Vollmer, Frank

    2016-01-01

    Mercury is an extremely toxic chemical pollutant of our environment. It has attracted the world's attention due to its high mobility and the ease with which it accumulates in organisms. Sensitive devices and methods specific for detecting mercury ions are, hence, in great need. Here, we have integrated a DNA strand displacement reaction with a whispering gallery mode (WGM) sensor for demonstrating the detection of Hg(2+) ions. Our approach relies on the displacement of a DNA hairpin structure, which forms after the binding of mercury ions to an aptamer DNA sequence. The strand displacement reaction of the DNA aptamer provides highly specific and quantitative means for determining the mercury ion concentration on a label-free WGM sensor platform. Our approach also shows the possibility for manipulating the kinetics of a strand displacement reaction with specific ionic species. PMID:27483277

  14. Coupling a DNA-Based Machine with Glucometer Readouts for Amplified Detection of Telomerase Activity in Cancer Cells

    OpenAIRE

    Wenjing Wang; Shan Huang; Jingjing Li; Kai Rui; Jian-Rong Zhang; Jun-Jie Zhu

    2016-01-01

    The strong correlation between cancer and telomerase activity has inspired the development of new strategies to evaluate telomerase activity. Here, a personal glucose meter (PGM) system that uses DNA-based machine amplification to detect telomerase in cancer cells is reported. In this assay, telomerase elongation products are amplified in the form of another type of product by a DNA-based machine. This process can only be activated by the hybridization of the extended telomerase substrate (TS...

  15. Construction of a nrdA::luxCDABE Fusion and Its Use in Escherichia coli as a DNA Damage Biosensor

    Directory of Open Access Journals (Sweden)

    Man Bock Gu

    2008-02-01

    Full Text Available The promoter of nrdA gene which is related with DNA synthesis was used to construct a DNA damage sensitive biosensor. A recombinant bioluminescent E. coli strain, BBTNrdA, harboring a plasmid with the nrdA promoter fused to the luxCDABE operon, was successfully constructed. Its response to various chemicals including genotoxic chemicals substantiates it as a DNA damage biosensor. In characterization, three different classes of toxicants were used: DNA damaging chemicals, oxidative stress chemicals, and phenolics. BBTNrdA only responded strongly to DNA damaging chemicals, such as nalidixic acid (NDA, mitomycin C (MMC, 1-methyl-1-nitroso-N-methylguanidine (MNNG, and 4-nitroquinoline N-oxide (4-NQO. In contrast, there were no responses from the oxidative stress chemicals and phenolics, except from hydrogen peroxide (H2O2 which is known to cause DNA damage indirectly. Therefore, the results of the study demonstrate that BBTNrdA can be used as a DNA damage biosensor.

  16. Examining the effects of a DNA fingerprinting workshop on science teachers' professional development and student learning

    Science.gov (United States)

    Sonmez, Duygu

    behavior. The goal is to understand what factors affect teachers' decision making to implement the new knowledge and skills in their classrooms. For this purpose, the study focuses on the effects of a DNA fingerprinting workshop, which has been developed and is regularly offered by a large Midwestern university in the United States for secondary science teachers and their students through cooperation between the university and a large Midwestern public school district. The workshop focuses on the biotechnology applications of genetics---specifically, use of DNA fingerprinting technology in different areas of social life---while forensic science is emphasized. Results indicate that the teachers' motivation to attend the DNA Fingerprinting professional development workshop was mainly influenced by two variables: (1) the need to improve content knowledge and skills, and (2) requirements associated with current educational policies. Level of content knowledge was also found to be a factor contributing to teachers' motivation to implement the workshop. Concerns related to student maturity and classroom management were also identified as factors influencing teachers' implementation behavior. Evidence that the DNA Fingerprinting workshop can be successfully implemented by classroom teachers was obtained. The DNA fingerprinting workshop was found to be a successful model for packaging professional development experiences for content intensive areas.

  17. Correction of the DNA repair defect in xeroderma pigmentosum group E by injection of a DNA damage-binding protein.

    OpenAIRE

    Keeney, S.; Eker, André; Brody, T.; Vermeulen, Wim; Bootsma, Dirk; Hoeijmakers, Jan; Linn, S.(Florida International University, Miami, USA)

    1994-01-01

    textabstractCells from a subset of patients with the DNA-repair-defective disease xeroderma pigmentosum complementation group E (XP-E) are known to lack a DNA damage-binding (DDB) activity. Purified human DDB protein was injected into XP-E cells to test whether the DNA-repair defect in these cells is caused by a defect in DDB activity. Injected DDB protein stimulated DNA repair to normal levels in those strains that lack the DDB activity but did not stimulate repair in cells from other xerode...

  18. Infection with Plasmodium berghei Boosts Antibody Responses Primed by a DNA Vaccine Encoding Gametocyte Antigen Pbs48/45

    OpenAIRE

    Haddad, Diana; Maciel, Jorge; Kumar, Nirbhay

    2006-01-01

    An important consideration in the development of a malaria vaccine for individuals living in areas of endemicity is whether vaccine-elicited immune responses can be boosted by natural infection. To investigate this question, we used Plasmodium berghei ANKA blood-stage parasites for the infection of mice that were previously immunized with a DNA vaccine encoding the P. berghei sexual-stage antigen Pbs48/45. Intramuscular immunization in mice with one or two doses of DNA-Pbs48/45 or of empty DN...

  19. Protection against Vibrio alginolyticus in crimson snapper Lutjanus erythropterus immunized with a DNA vaccine containing the ompW gene.

    Science.gov (United States)

    Cai, Shuang-Hu; Lu, Yi-Shan; Jian, Ji-Chang; Wang, Bei; Huang, Yu-Cong; Tang, Ju-Fen; Ding, Yu; Wu, Zao-He

    2013-09-24

    The outer membrane proteins of Vibrio alginolyticus play an important role in the virulence of the bacterium and are potential candidates for vaccine development. In the present study, the ompW gene was cloned, expressed and purified. A DNA vaccine was constructed by inserting the ompW gene into a pcDNA plasmid. Crimson snapper Lutjanus erythropterus (Bloch) were injected intramuscularly with the recombinant plasmid pcDNA-ompW. The expression of the DNA vaccine was detected in gill, head kidney, heart, liver, spleen and injection site muscle of crimson snapper by RT-PCR 7 and 28 d post-vaccination. The ELISA results demonstrated that the DNA vaccine produced an observable antibody response in all sera of the vaccinated fish. In addition, crimson snapper immunized with the DNA vaccine showed a relative percentage survival (RPS) of 92.53%, indicating effective protection against V. alginolyticus infection.

  20. Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against alpha-Bungarotoxin

    DEFF Research Database (Denmark)

    Lauridsen, Lasse Holm; Shamaileh, Hadi A.; Edwards, Stacey L.;

    2012-01-01

    by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 mu M. Conclusion: We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low...... in one-step, technique is required for developing aptamers in limited time period. Principal Findings: Herein, we present a simple one-step selection of DNA aptamers against alpha-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed...... micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way...

  1. Nano-formulation of a photosensitizer using a DNA tetrahedron and its potential for in vivo photodynamic therapy.

    Science.gov (United States)

    Kim, Kyoung-Ran; Bang, Duhee; Ahn, Dae-Ro

    2016-04-01

    Photodynamic therapy (PDT) is a cytotoxic treatment using singlet oxygen produced by photosensitizers. Approved porphyrinoid PDT still suffers from a lack of robust production methods and low water solubility. Methylene blue (MB) is a good candidate for the PDT drug, because the dye is an effective photosensitizer, can be easily synthesized, and is already being used in other clinical fields. However, its poor cell/tissue penetration and low stability against the reducible biological conditions should be addressed by using a proper delivery vehicle. Here, we employed a DNA tetrahedron, a self-assembled nanostructure as the carrier for intracellular delivery of MB by taking advantage of the DNA binding property of the photosensitizer and demonstrated photo-induced cytotoxicity by the MB delivered by the DNA nanocarrier. We also evaluated the PDT potency of the MB-loaded DNA nanoconstruct in vivo tumor model to suppress tumor growth.

  2. Discovery of TNF inhibitors from a DNA-encoded chemical library based on diels-alder cycloaddition.

    Science.gov (United States)

    Buller, Fabian; Zhang, Yixin; Scheuermann, Jörg; Schäfer, Juliane; Bühlmann, Peter; Neri, Dario

    2009-10-30

    DNA-encoded chemical libraries are promising tools for the discovery of ligands toward protein targets of pharmaceutical relevance. DNA-encoded small molecules can be enriched in affinity-based selections and their unique DNA "barcode" allows the amplification and identification by high-throughput sequencing. We describe selection experiments using a DNA-encoded 4000-compound library generated by Diels-Alder cycloadditions. High-throughput sequencing enabled the identification and relative quantification of library members before and after selection. Sequence enrichment profiles corresponding to the "bar-coded" library members were validated by affinity measurements of single compounds. We were able to affinity mature trypsin inhibitors and identify a series of albumin binders for the conjugation of pharmaceuticals. Furthermore, we discovered a ligand for the antiapoptotic Bcl-xL protein and a class of tumor necrosis factor (TNF) binders that completely inhibited TNF-mediated killing of L-M fibroblasts in vitro.

  3. A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif

    Science.gov (United States)

    Reddy, A. S.; Reddy, V. S.; Golovkin, M.

    2000-01-01

    Calmodulin (CaM), a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that interacts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcription factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similarity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high sequence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.

  4. Non-covalent interactions between ATP and RecA DNA-repairing proteins: DFT and semiempirical calculations

    Science.gov (United States)

    Rodriguez, Jorge

    2015-03-01

    The role of Bacterial RecA in the structural maintenance of genomes and the genetic information they carry has been established. In particular, the RecA DNA-repairing protein from D. Radiodurans, a radiation-resistant bacteria, is crucial for the repair of double strand breaks (DSBs). We have performed semi-empirical free-energy calculations and QM/MM calculations to study their non-covalent interactions with ATP and ADP. Such studies provide insight into the mechanisms of ATP/ADP --> RecA energy transfer and, therefore, about specific functional uses of incoming energy for DNA repairing mechanisms. We present a detailed analysis of the non-covalent interactions which minimize the interaction Gibbs free energies leading to the most stable non-covalent binding sites. Van der Waal, hydrogen bonding and electrostatic interactions has been quantified which provides a detailed insight into the mechanisms of ATP-RecA interaction. Further, possible chemical interactions and functional roles of RecA proteins are explored based on the previously mentioned studies. Acknowledgements: Funded, in part, by DTRA award 106339 (JHR). Dr. Mark C. Palenik and Mrs. Lora Beard are gratefully acknowledged Supported in part by DTRA Award 106339.

  5. A DNA polymerase alpha accessory protein, Mcl1, is required for propagation of centromere structures in fission yeast.

    Directory of Open Access Journals (Sweden)

    Toyoaki Natsume

    Full Text Available Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A(Cnp1 kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase alpha (Pol alpha accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A(Cnp1 in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7(+, which encodes a catalytic subunit of Pol alpha. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha. These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication.

  6. Potential of the NBP method for the study of alkylation mechanisms: NBP as a DNA-model.

    Science.gov (United States)

    Gómez-Bombarelli, Rafael; González-Pérez, Marina; Calle, Emilio; Casado, Julio

    2012-06-18

    Alkylating agents are considered to be archetypal carcinogens. One suitable technique to evaluate the activity of alkylating compounds is the NBP assay. This method is based on the formation of a chromophore in the reaction between the alkylating agent and the nucleophile 4-(p-nitrobenzyl)pyridine (NBP), a trap for alkylating agents with nucleophilic characteristics similar to those of DNA bases. NBP is known to react with strong and weak alkylating agents, and much insight into such alkylation mechanisms in vivo can be gained from kinetic study of some alkylation reactions in vitro. Since 1925, the NBP assay has evolved from being a qualitative, analytical tool to becoming a useful physicochemical method that not only allows the rules of chemical reactivity that govern electrophilicity and nucleophilicity to be applied to the reaction of DNA with alkylating agents but also helps to understand some significant relationships between the structure of many alkylation substrates (including DNA) and their chemical and biological responses. Given that advances in this area have the potential to yield both fundamental and practical advances in chemistry, biology, predictive toxicology, and anticancer drug development, this review is designed to provide an overview of the evolution of the NBP method from its early inception until its recent kinetic-mechanistic approach, which allows the pros and cons of NBP as a DNA-model to be analyzed. The validity of NBP as a nucleophilicity model for DNA in general and the position of guanosine at N7 in particular are discussed. PMID:22480281

  7. Structure and Binding Energy of Double-Stranded A-DNA Mini-helices: Quantum-Chemical Study.

    Science.gov (United States)

    Zubatiuk, Tetiana; Kukuev, Maxim A; Korolyova, Alexandra S; Gorb, Leonid; Nyporko, Alexey; Hovorun, Dmytro; Leszczynski, Jerzy

    2015-10-01

    A-DNA is thought to play a significant biological role in gene expression due to its specific conformation and binding features. In this study, double-stranded mini-helices (dA:dT)3 and (dG:dC)3 in A-like DNA conformation were investigated. M06-2X/6-31G(d,p) method has been utilized to identify the optimal geometries and predict physicochemical parameters of these systems. The results show the ability of the corresponding mini-helices to preserve their A-like conformation under the influences of solvent, charge, and Na(+) counterions. Presented structural and energetic data offer evidence that two steps of GG/CC or AA/TT are already enough to turn the DNA helix to generate different forms by favoring specific values of roll and slide at a local level. Our calculations support the experimentally known fact that AA/TT steps prefer the B-form over the A-ones, whereas GG/CC steps may be found in either the B- or A-form. The stability of mini-helices at the level of total energy analysis, ΔEtotal((A–B)), is discussed. PMID:26356008

  8. A DNA polymerase alpha accessory protein, Mcl1, is required for propagation of centromere structures in fission yeast.

    Science.gov (United States)

    Natsume, Toyoaki; Tsutsui, Yasuhiro; Sutani, Takashi; Dunleavy, Elaine M; Pidoux, Alison L; Iwasaki, Hiroshi; Shirahige, Katsuhiko; Allshire, Robin C; Yamao, Fumiaki

    2008-01-01

    Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A(Cnp1) kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase alpha (Pol alpha) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7(+), which encodes a catalytic subunit of Pol alpha. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha. These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication. PMID:18493607

  9. The Species and Origin of Shark Fins in Taiwan's Fishing Ports, Markets, and Customs Detention: A DNA Barcoding Analysis.

    Science.gov (United States)

    Chuang, Po-Shun; Hung, Tzu-Chiao; Chang, Hung-An; Huang, Chien-Kang; Shiao, Jen-Chieh

    2016-01-01

    The increasing consumption of shark products, along with the shark's fishing vulnerabilities, has led to the decrease in certain shark populations. In this study we used a DNA barcoding method to identify the species of shark landings at fishing ports, shark fin products in retail stores, and shark fins detained by Taiwan customs. In total we identified 23, 24, and 14 species from 231 fishing landings, 316 fin products, and 113 detained shark fins, respectively. All the three sample sources were dominated by Prionace glauca, which accounted for more than 30% of the collected samples. Over 60% of the species identified in the fin products also appeared in the port landings, suggesting the domestic-dominance of shark fin products in Taiwan. However, international trade also contributes a certain proportion of the fin product markets, as four species identified from the shark fin products are not found in Taiwan's waters, and some domestic-available species were also found in the customs-detained sample. In addition to the species identification, we also found geographical differentiation in the cox1 gene of the common thresher sharks (Alopias vulpinus), the pelagic thresher shark (A. pelagicus), the smooth hammerhead shark (Sphyrna zygaena), and the scalloped hammerhead shark (S. lewini). This result might allow fishing authorities to more effectively trace the origins as well as enforce the management and conservation of these sharks.

  10. The Species and Origin of Shark Fins in Taiwan's Fishing Ports, Markets, and Customs Detention: A DNA Barcoding Analysis.

    Directory of Open Access Journals (Sweden)

    Po-Shun Chuang

    Full Text Available The increasing consumption of shark products, along with the shark's fishing vulnerabilities, has led to the decrease in certain shark populations. In this study we used a DNA barcoding method to identify the species of shark landings at fishing ports, shark fin products in retail stores, and shark fins detained by Taiwan customs. In total we identified 23, 24, and 14 species from 231 fishing landings, 316 fin products, and 113 detained shark fins, respectively. All the three sample sources were dominated by Prionace glauca, which accounted for more than 30% of the collected samples. Over 60% of the species identified in the fin products also appeared in the port landings, suggesting the domestic-dominance of shark fin products in Taiwan. However, international trade also contributes a certain proportion of the fin product markets, as four species identified from the shark fin products are not found in Taiwan's waters, and some domestic-available species were also found in the customs-detained sample. In addition to the species identification, we also found geographical differentiation in the cox1 gene of the common thresher sharks (Alopias vulpinus, the pelagic thresher shark (A. pelagicus, the smooth hammerhead shark (Sphyrna zygaena, and the scalloped hammerhead shark (S. lewini. This result might allow fishing authorities to more effectively trace the origins as well as enforce the management and conservation of these sharks.

  11. Building a DNA barcode reference library for the true butterflies (Lepidoptera of Peninsula Malaysia: what about the subspecies?

    Directory of Open Access Journals (Sweden)

    John-James Wilson

    Full Text Available The objective of this study was to build a DNA barcode reference library for the true butterflies of Peninsula Malaysia and assess the value of attaching subspecies names to DNA barcode records. A new DNA barcode library was constructed with butterflies from the Museum of Zoology, University of Malaya collection. The library was analysed in conjunction with publicly available DNA barcodes from other Asia-Pacific localities to test the ability of the DNA barcodes to discriminate species and subspecies. Analyses confirmed the capacity of the new DNA barcode reference library to distinguish the vast majority of species (92% and revealed that most subspecies possessed unique DNA barcodes (84%. In some cases conspecific subspecies exhibited genetic distances between their DNA barcodes that are typically seen between species, and these were often taxa that have previously been regarded as full species. Subspecies designations as shorthand for geographically and morphologically differentiated groups provide a useful heuristic for assessing how such groups correlate with clustering patterns of DNA barcodes, especially as the number of DNA barcodes per species in reference libraries increases. Our study demonstrates the value in attaching subspecies names to DNA barcode records as they can reveal a history of taxonomic concepts and expose important units of biodiversity.

  12. Building a DNA Barcode Reference Library for the True Butterflies (Lepidoptera) of Peninsula Malaysia: What about the Subspecies?

    Science.gov (United States)

    Wilson, John-James; Sing, Kong-Wah; Sofian-Azirun, Mohd

    2013-01-01

    The objective of this study was to build a DNA barcode reference library for the true butterflies of Peninsula Malaysia and assess the value of attaching subspecies names to DNA barcode records. A new DNA barcode library was constructed with butterflies from the Museum of Zoology, University of Malaya collection. The library was analysed in conjunction with publicly available DNA barcodes from other Asia-Pacific localities to test the ability of the DNA barcodes to discriminate species and subspecies. Analyses confirmed the capacity of the new DNA barcode reference library to distinguish the vast majority of species (92%) and revealed that most subspecies possessed unique DNA barcodes (84%). In some cases conspecific subspecies exhibited genetic distances between their DNA barcodes that are typically seen between species, and these were often taxa that have previously been regarded as full species. Subspecies designations as shorthand for geographically and morphologically differentiated groups provide a useful heuristic for assessing how such groups correlate with clustering patterns of DNA barcodes, especially as the number of DNA barcodes per species in reference libraries increases. Our study demonstrates the value in attaching subspecies names to DNA barcode records as they can reveal a history of taxonomic concepts and expose important units of biodiversity. PMID:24282514

  13. "Giant surfactants" created by the fast and efficient functionalization of a DNA tetrahedron with a temperature-responsive polymer.

    Science.gov (United States)

    Wilks, Thomas R; Bath, Jonathan; de Vries, Jan Willem; Raymond, Jeffery E; Herrmann, Andreas; Turberfield, Andrew J; O'Reilly, Rachel K

    2013-10-22

    Copper catalyzed azide-alkyne cycloaddition (CuAAC) was employed to synthesize DNA block copolymers (DBCs) with a range of polymer blocks including temperature-responsive poly(N-isoproylacrylamide) (poly(NIPAM)) and highly hydrophobic poly(styrene). Exceptionally high yields were achieved at low DNA concentrations, in organic solvents, and in the absence of any solid support. The DNA segment of the DBC remained capable of sequence-specific hybridization: it was used to assemble a precisely defined nanostructure, a DNA tetrahedron, with pendant poly(NIPAM) segments. In the presence of an excess of poly(NIPAM) homopolymer, the tetrahedron-poly(NIPAM) conjugate nucleated the formation of large, well-defined nanoparticles at 40 °C, a temperature at which the homopolymer precipitated from solution. These composite nanoparticles were observed by dynamic light scattering and cryoTEM, and their hybrid nature was confirmed by AFM imaging. As a result of the large effective surface area of the tetrahedron, only very low concentrations of the conjugate were required in order for this surfactant-like behavior to be observed.

  14. The Species and Origin of Shark Fins in Taiwan's Fishing Ports, Markets, and Customs Detention: A DNA Barcoding Analysis.

    Science.gov (United States)

    Chuang, Po-Shun; Hung, Tzu-Chiao; Chang, Hung-An; Huang, Chien-Kang; Shiao, Jen-Chieh

    2016-01-01

    The increasing consumption of shark products, along with the shark's fishing vulnerabilities, has led to the decrease in certain shark populations. In this study we used a DNA barcoding method to identify the species of shark landings at fishing ports, shark fin products in retail stores, and shark fins detained by Taiwan customs. In total we identified 23, 24, and 14 species from 231 fishing landings, 316 fin products, and 113 detained shark fins, respectively. All the three sample sources were dominated by Prionace glauca, which accounted for more than 30% of the collected samples. Over 60% of the species identified in the fin products also appeared in the port landings, suggesting the domestic-dominance of shark fin products in Taiwan. However, international trade also contributes a certain proportion of the fin product markets, as four species identified from the shark fin products are not found in Taiwan's waters, and some domestic-available species were also found in the customs-detained sample. In addition to the species identification, we also found geographical differentiation in the cox1 gene of the common thresher sharks (Alopias vulpinus), the pelagic thresher shark (A. pelagicus), the smooth hammerhead shark (Sphyrna zygaena), and the scalloped hammerhead shark (S. lewini). This result might allow fishing authorities to more effectively trace the origins as well as enforce the management and conservation of these sharks. PMID:26799827

  15. A DNA-PKcs mutation in a radiosensitive T-B- SCID patient inhibits Artemis activation and nonhomologous end-joining

    NARCIS (Netherlands)

    M. van der Burg (Mirjam); H. IJspeert (Hanna); N.S. Verkaik (Nicole); T. Turul (Tuba); W.W. Wiegant (Wouter); K. Morotomi-Yano (Keiko); P.O. Mari (Pierre-Olivier); I. Tezcan (Ilhan); D.J. Chen (David); M.Z. Zdzienicka (Malgorzata); J.J.M. van Dongen (Jacques); D.C. van Gent (Dik)

    2009-01-01

    textabstractRadiosensitive T-B- severe combined immunodeficiency (RS-SCID) is caused by defects in the nonhomologous end-joining (NHEJ) DNA repair pathway, which results in failure of functional V(D)J recombination. Here we have identified the first human RS-SCID patient to our knowledge with a DNA-

  16. Targeted delivery and pH-triggered release of a saporin toxin conjugated to transferrin via a DNA i-motif

    DEFF Research Database (Denmark)

    Liu, Tianqiang

    2016-01-01

    investigated a type of transferrin-saporin conjugate complex where saporin and transferrin, each conjugated to one DNA strand, are linked via hybridization to a common template. In this construct one of the strands can form a DNA i-motif at pH

  17. Rapid one-step selection method for generating nucleic acid aptamers: development of a DNA aptamer against α-bungarotoxin.

    Directory of Open Access Journals (Sweden)

    Lasse H Lauridsen

    Full Text Available BACKGROUND: Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF for the treatment of age related macular degeneration (AMD. Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period. PRINCIPAL FINDINGS: Herein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM. CONCLUSION: We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers.

  18. Mate-Pair Sequencing as a Powerful Clinical Tool for the Characterization of Cancers with a DNA Viral Etiology

    Directory of Open Access Journals (Sweden)

    Ge Gao

    2015-08-01

    Full Text Available DNA viruses are known to be associated with a variety of different cancers. Human papillomaviruses (HPV are a family of viruses and several of its sub-types are classified as high-risk HPVs as they are found to be associated with the development of a number of different cancers. Almost all cervical cancers appear to be driven by HPV infection and HPV is also found in most cancers of the anus and at least half the cancers of the vulva, penis and vagina, and increasingly found in one sub-type of head and neck cancers namely oropharyngeal squamous cell carcinoma. Our understanding of HPVs role in cancer development comes from extensive studies done on cervical cancer and it has just been assumed that HPV plays an identical role in the development of all other cancers arising in the presence of HPV sequences, although this has not been proven. Most invasive cervical cancers have the HPV genome integrated into one or more sites within the human genome. One powerful tool to examine all the sites of HPV integration in a cancer but that also provides a comprehensive view of genomic alterations in that cancer is the use of next generation sequencing of mate-pair libraries produced from the DNA isolated. We will describe how this powerful technology can provide important information about the genomic organization within an individual cancer genome, and how this has demonstrated that HPVs role in oropharyngeal squamous cell carcinoma is distinct from that in cervical cancer. We will also describe why the sequencing of mate-pair libraries could be a powerful clinical tool for the management of patients with a DNA viral etiology and how this could quickly transform the care of these patients.

  19. Mate-Pair Sequencing as a Powerful Clinical Tool for the Characterization of Cancers with a DNA Viral Etiology.

    Science.gov (United States)

    Gao, Ge; Smith, David I

    2015-08-01

    DNA viruses are known to be associated with a variety of different cancers. Human papillomaviruses (HPV) are a family of viruses and several of its sub-types are classified as high-risk HPVs as they are found to be associated with the development of a number of different cancers. Almost all cervical cancers appear to be driven by HPV infection and HPV is also found in most cancers of the anus and at least half the cancers of the vulva, penis and vagina, and increasingly found in one sub-type of head and neck cancers namely oropharyngeal squamous cell carcinoma. Our understanding of HPVs role in cancer development comes from extensive studies done on cervical cancer and it has just been assumed that HPV plays an identical role in the development of all other cancers arising in the presence of HPV sequences, although this has not been proven. Most invasive cervical cancers have the HPV genome integrated into one or more sites within the human genome. One powerful tool to examine all the sites of HPV integration in a cancer but that also provides a comprehensive view of genomic alterations in that cancer is the use of next generation sequencing of mate-pair libraries produced from the DNA isolated. We will describe how this powerful technology can provide important information about the genomic organization within an individual cancer genome, and how this has demonstrated that HPVs role in oropharyngeal squamous cell carcinoma is distinct from that in cervical cancer. We will also describe why the sequencing of mate-pair libraries could be a powerful clinical tool for the management of patients with a DNA viral etiology and how this could quickly transform the care of these patients. PMID:26262638

  20. Effects of Regular Treadmill Exercise on a DNA Oxidative-Damage Marker and Total Antioxidant Capacity in Rat Hippocampal Tissue

    Science.gov (United States)

    Mahjoub, Soleiman; Ghadi, Arezoo; Pourbagher, Roghayeh; Hajian-Tilaki, Karimollah

    2016-01-01

    Background and Purpose Regular exercise can result in changes in the levels of oxidative stress in the hippocampus; however, little attention has been paid to physical-activity-induced neuronal protection to exposure to lead compounds. This study investigated the effects of regular treadmill exercise on a DNA oxidative-damage marker [8-hydroxy-2'-deoxyguanosine (8-OHdG)] and the total antioxidant capacity (TAC) of hippocampal tissue in lead-acetate exposed rats. Methods This study investigated the effects of 8 weeks of regular treadmill exercise on 8-OHdG and the TAC of hippocampal tissue in lead-acetate-exposed rats. Wistar rats were randomly divided into four groups: baseline, sham (control), lead, and exercise+lead. The exercise program involved running on a treadmill with increasing intensity five times a week for 8 weeks. Animals in the lead and exercise+lead groups received lead acetate at 20 mg/kg body weight intraperitoneally three times weekly for 8 weeks. Animals in the sham group received solvent (ethyl oleate) at 30 mg/kg body weight three times weekly for 8 weeks. TAC and 8-OHdG were measured by spectrophotometric and ELISA techniques, respectively. Data were analyzed by ANOVA and Tukey's post-hoc test with a significance cutoff of p≤0.05. Results The level of 8-OHdG and the TAC were significantly higher and lower, respectively, in the lead group than in the baseline and sham groups (p<0.01). However, the 8-OHdG level and TAC value in hippocampal tissue were significantly decreased and increased, respectively, in the exercise+lead group relative to the lead group (p<0.05). Conclusions The TAC of hippocampal tissue may be directly associated with neural protection mechanisms of exercise following lead acetate injection, and the beneficial effects of regular exercise in preventing hippocampal neuronal damage could be due to decreased hippocampal oxidative stress such as reflected by a lower 8-OHdG level and increased TAC.

  1. Boundary conditions for free A-DNA in solution and the relation of local to global DNA structures at reduced water activity.

    Science.gov (United States)

    Porschke, Dietmar

    2016-07-01

    Because of repeated claims that A-DNA cannot exist without aggregation or condensation, the state of DNA restriction fragments with 84-859 bp has been analyzed in aqueous solutions upon reduction of the water activity. Rotational diffusion times τ (d) measured by electric dichroism at different water activities with a wide variation of viscosities are normalized to values τ (c) at the viscosity of water, which indicate DNA structures at a high sensitivity. For short helices (chain lengths [Formula: see text] ≤ persistence length p), cooperative formation of A-DNA is reflected by the expected reduction of the hydrodynamic length; the transition to the A-form is without aggregation or condensation upon addition of ethanol at monovalent salt ≤1 mM. The aggregation boundary, indicated by a strong increase of τ (c), is shifted to higher monovalent salt (≥4 mM) when ethanol is replaced by trifluoroethanol. The BA transition is not indicated anymore by a cooperative change of τ (c) for [Formula: see text] » p; τ (c) values for these long chains decrease upon reduction of the water activity continuously over the full range, including the BA transition interval. This suggests a non-cooperative BC transition, which induces DNA curvature. The resulting wide distribution of global structures hides changes of local length during the BA transition. Free A-DNA without aggregation/condensation is found at low-salt concentrations where aggregation is inhibited and/or very slow. In an intermediate range of solvent conditions, where the A-form starts to aggregate, a time window remains that can be used for analysis of free A-DNA in a quasi-equilibrium state. PMID:26872482

  2. The Species and Origin of Shark Fins in Taiwan’s Fishing Ports, Markets, and Customs Detention: A DNA Barcoding Analysis

    OpenAIRE

    Po-Shun Chuang; Tzu-Chiao Hung; Hung-An Chang; Chien-Kang Huang; Jen-Chieh Shiao

    2016-01-01

    The increasing consumption of shark products, along with the shark's fishing vulnerabilities, has led to the decrease in certain shark populations. In this study we used a DNA barcoding method to identify the species of shark landings at fishing ports, shark fin products in retail stores, and shark fins detained by Taiwan customs. In total we identified 23, 24, and 14 species from 231 fishing landings, 316 fin products, and 113 detained shark fins, respectively. All the three sample sources w...

  3. Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models

    Institute of Scientific and Technical Information of China (English)

    Yi-Ping Li; Hye Na Kang; Lorne A Babiuk; Qiang Liu

    2006-01-01

    AIM: To characterize the immunogenicity of a hepatitis C virus (HCV) E2 DNA vaccine alone or with a protein vaccine boost in murine and porcine animal models.METHODS: A DNA vaccine expressing a secreted form of HCV E2 protein was constructed and used to vaccinate mice and piglets with or without boosting with a recombinant E2 protein vaccine formulated with CpG ODN and 10% Emulsigen. The immunogenicity of HCV E2 vaccines was analyzed by ELISA for antibody responses, MTT assay for lymphocyte proliferation,ELISPOT for the number of interferon-γ secreting cells,and cytotoxic T lymphocyte assays.RESULTS: Intradermal injection of E2 DNA vaccine induced strong Th1-like immune responses in mice. In piglets, E2 DNA vaccine elicited moderate and more balanced immune responses. A DNA vaccine prime and protein boost vaccination strategy induced significantly higher E2-specific antibody levels and shifted the immune response towards Th2-like ones in piglets.CONCLUSION: A DNA vaccine expressing a secreted form of HCV E2 protein elicited E2-specific immune responses in mice and piglets. Recombinant E2 protein vaccination following DNA immunization significantly increased the antibody response in piglets. These HCV E2 vaccines may represent promising hepatitis C vaccine candidates for further investigations.

  4. DVC1 (C1orf124) is a DNA damage-targeting p97 adaptor that promotes ubiquitin-dependent responses to replication blocks

    DEFF Research Database (Denmark)

    Mosbech, Anna; Gibbs-Seymour, Ian; Kagias, Konstantinos;

    2012-01-01

    Ubiquitin-mediated processes orchestrate critical DNA-damage signaling and repair pathways. We identify human DVC1 (C1orf124; Spartan) as a cell cycle-regulated anaphase-promoting complex (APC) substrate that accumulates at stalled replication forks. DVC1 recruitment to sites of replication stress...... synthesis (TLS) DNA polymerase η (Pol η) from monoubiquitylated PCNA. DVC1 knockdown enhances UV light-induced mutagenesis, and depletion of human DVC1 or the Caenorhabditis elegans ortholog DVC-1 causes hypersensitivity to replication stress-inducing agents. Our findings establish DVC1 as a DNA damage...

  5. The stability region of the Streptomyces lividans plasmid pIJ101 encodes a DNA-binding protein recognizing a highly conserved short palindromic sequence motif

    OpenAIRE

    Thoma, Lina; Sepulveda, Edgardo; Latus, Annette; Muth, Günther

    2014-01-01

    Conjugation is a driving force in the evolution and shaping of bacterial genomes. In antibiotic producing streptomycetes even small plasmids replicating via the rolling-circle mechanism are conjugative. Although they encode only genes involved in replication and transfer, the molecular function of most plasmid encoded proteins is unknown. In this work we show that the conjugative plasmid pIJ101 encodes an overlooked protein, SpdA2. We show that SpdA2 is a DNA binding protein which specificall...

  6. Delivery of antigenic candidates by a DNA/MVA heterologous approach elicits effector CD8+T cell mediated immunity against Trypanosoma cruzi

    OpenAIRE

    Gupta, Shivali; Garg, Nisha Jain

    2012-01-01

    In this study, we have characterized the immune mechanisms elicited by antigenic candidates, TcG2 and TcG4, delivered by a DNA-prime/MVA-boost approach, and evaluated the host responses to T. cruzi infection in C57BL/6 mice. Immunization of mice with antigenic candidates elicited antigen-specific, high-avidity, trypanolytic antibody response (IgG2b>IgG1) and CD8+T cells that exhibited type-1 cytolytic effector (CD8+CD107a+IFN-γ+Perforin+) phenotype. The extent of TcG2-dependent type 1 B and T...

  7. Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models

    DEFF Research Database (Denmark)

    Li, Yiping; Kang, H.N.; Babiuk, L.A.;

    2006-01-01

    AIM: To characterize the immunogenicity of a hepatitis C virus (HCV) E2 DNA vaccine alone or with a protein vaccine boost in murine and porcine animal models. METHODS: A DNA vaccine expressing a secreted form of HCV E2 protein was constructed and used to vaccinate mice and piglets with or without...... boosting with a recombinant E2 protein vaccine formulated with CpG ODN and 10% Emulsigen. The immunogenicity of HCV E2 vaccines was analyzed by ELISA for antibody responses, MTT assay for lymphocyte proliferation, ELISPOT for the number of interferon-gamma secreting cells, and cytotoxic T lymphocyte assays...

  8. Adjuvanting a DNA vaccine with a TLR9 ligand plus Flt3 ligand results in enhanced cellular immunity against the simian immunodeficiency virus.

    Science.gov (United States)

    Kwissa, Marcin; Amara, Rama R; Robinson, Harriet L; Moss, Bernard; Alkan, Sefik; Jabbar, Abdul; Villinger, Francois; Pulendran, Bali

    2007-10-29

    DNA vaccines offer promising strategies for immunization against infections. However, their clinical use requires improvements in immunogenicity. We explored the efficacy of Toll-like receptor (TLR) ligands (TLR-Ls) on augmenting the immunogenicity of a DNA prime-modified vaccinia virus Ankara (MVA) boost vaccine against SIV. Rhesus macaques were injected with Fms-like tyrosine kinase 3 (Flt3)-ligand (FL) to expand dendritic cells (DCs) and were primed with a DNA vaccine encoding immunodeficiency virus antigens mixed with ligands for TLR9 or TLR7/8. Subsequently, the animals were boosted with DNA and twice with recombinant MVA expressing the same antigens. TLR9-L (CpG DNA) mediated activation of DCs in vivo and enhanced the magnitude of antigen-specific CD8(+) interferon (IFN) gamma(+) T cells and polyfunctional CD8(+) T cells producing IFN-gamma, tumor necrosis factor alpha, and interleukin 2. Although this trial was designed primarily as an immunogenicity study, we challenged the animals with pathogenic SIVmac(251) and observed a reduction in peak viremia and cumulative viral loads in the TLR9-L plus FL-adjuvanted group relative to the unvaccinated group; however, the study design precluded comparisons between the adjuvanted groups and the group vaccinated with DNA/MVA alone. Viral loads were inversely correlated with the magnitude and quality of the immune response. Thus, the immunogenicity of DNA vaccines can be augmented with TLR9-L plus FL. PMID:17954572

  9. Methods to assess the nucleocytoplasmic shuttling of the HPV E1 helicase and its effects on cellular proliferation and induction of a DNA damage response.

    Science.gov (United States)

    Lehoux, Michaël; Fradet-Turcotte, Amélie; Archambault, Jacques

    2015-01-01

    Replication of the human papillomavirus (HPV) double-stranded DNA genome in the nucleus of infected cells relies on the viral proteins E1 and E2 in conjunction with the host DNA replication machinery. This process is tightly linked to the replication of cellular DNA, in part through the cyclin-dependent phosphorylation of E1, which inhibits its export out of the nucleus to promote its accumulation in this compartment during S-phase. It has been recently shown that accumulation of E1 in the nucleus, while a prerequisite for viral DNA replication, leads to the inhibition of cellular proliferation and the activation of a DNA damage response (DDR). Here we describe methods to monitor the subcellular localization of E1 and to assess the deleterious effects of its nuclear accumulation on cellular proliferation, cell cycle progression and the induction of a DDR, using a combination of colony formation assays, immunofluorescence microcopy, and flow cytometry approaches. PMID:25348298

  10. THAP5 is a DNA-binding transcriptional repressor that is regulated in melanoma cells during DNA damage-induced cell death

    International Nuclear Information System (INIS)

    Research highlights: → THAP5 is a DNA-binding protein and a transcriptional repressor. → THAP5 is induced in melanoma cells upon exposure to UV or treatment with cisplatin. → THAP5 induction correlates with the degree of apoptosis in melanoma cell population. → THAP5 is a pro-apoptotic protein involved in melanoma cell death. -- Abstract: THAP5 was originally isolated as a specific interactor and substrate of the mitochondrial pro-apoptotic Omi/HtrA2 protease. It is a human zinc finger protein characterized by a restricted pattern of expression and the lack of orthologs in mouse and rat. The biological function of THAP5 is unknown but our previous studies suggest it could regulate G2/M transition in kidney cells and could be involved in human cardiomyocyte cell death associated with coronary artery disease (CAD). In this report, we expanded our studies on the properties and function of THAP5 in human melanoma cells. THAP5 was expressed in primary human melanocytes as well as in all melanoma cell lines that were tested. THAP5 protein level was significantly induced by UV irradiation or cisplatin treatment, conditions known to cause DNA damage. The induction of THAP5 correlated with a significant increase in apoptotic cell death. In addition, we show that THAP5 is a nuclear protein that could recognize and bind a specific DNA motif. THAP5 could also repress the transcription of a reporter gene in a heterologous system. Our work suggests that THAP5 is a DNA-binding protein and a transcriptional repressor. Furthermore, THAP5 has a pro-apoptotic function and it was induced in melanoma cells under conditions that promoted cell death.

  11. THAP5 is a DNA-binding transcriptional repressor that is regulated in melanoma cells during DNA damage-induced cell death

    Energy Technology Data Exchange (ETDEWEB)

    Balakrishnan, Meenakshi P.; Cilenti, Lucia; Ambivero, Camilla [Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL (United States); Goto, Yamafumi [Department of Dermatology, Shinshu University School of Medicine, Matsumoto (Japan); Takata, Minoru [Department of Dermatology, Okayama University Graduate School of Medical Dentistry and Pharmaceutical Sciences, Okayama (Japan); Turkson, James; Li, Xiaoman Shawn [Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL (United States); Zervos, Antonis S., E-mail: azervos@mail.ucf.edu [Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL (United States)

    2011-01-07

    Research highlights: {yields} THAP5 is a DNA-binding protein and a transcriptional repressor. {yields} THAP5 is induced in melanoma cells upon exposure to UV or treatment with cisplatin. {yields} THAP5 induction correlates with the degree of apoptosis in melanoma cell population. {yields} THAP5 is a pro-apoptotic protein involved in melanoma cell death. -- Abstract: THAP5 was originally isolated as a specific interactor and substrate of the mitochondrial pro-apoptotic Omi/HtrA2 protease. It is a human zinc finger protein characterized by a restricted pattern of expression and the lack of orthologs in mouse and rat. The biological function of THAP5 is unknown but our previous studies suggest it could regulate G2/M transition in kidney cells and could be involved in human cardiomyocyte cell death associated with coronary artery disease (CAD). In this report, we expanded our studies on the properties and function of THAP5 in human melanoma cells. THAP5 was expressed in primary human melanocytes as well as in all melanoma cell lines that were tested. THAP5 protein level was significantly induced by UV irradiation or cisplatin treatment, conditions known to cause DNA damage. The induction of THAP5 correlated with a significant increase in apoptotic cell death. In addition, we show that THAP5 is a nuclear protein that could recognize and bind a specific DNA motif. THAP5 could also repress the transcription of a reporter gene in a heterologous system. Our work suggests that THAP5 is a DNA-binding protein and a transcriptional repressor. Furthermore, THAP5 has a pro-apoptotic function and it was induced in melanoma cells under conditions that promoted cell death.

  12. Electrochemical detection of avian influenza virus H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode

    International Nuclear Information System (INIS)

    Highlights: → A sensitive electrochemical biosensor for the detection of gene sequence was developed. → The biosensor was assembled by MWNT, polypyrrole nanowires and gold nanoparticles. → The hybrid nanomaterials could provide a porous structure with good properties. → The biosensor has highly selectivity and sensitivity. → The design strategy is expected to have extensive applications in other biosensors - Abstract: A sensitive electrochemical method for the detection of avian influenza virus (AIV) H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode was developed. To enhance the selectivity and sensitivity, the modified electrode was assembled with multi-wall carbon nanotubes (MWNT), polypyrrole nanowires (PPNWs) and gold nanoparticles (GNPs). This electrode offered a porous structure with a large effective surface area, highly electrocatalytic activities and electronic conductivity. Therefore, the amount of DNA aptamer immobilized onto the electrode was increased while the accessibility of the detection target was maintained. The biosensor is based on the hybridization and preferred orientation of a DNA aptamer immobilized onto a modified electrode surface with its target (H5N1 specific sequence) present in solution. It is selective for the H5N1 specific sequence, and the signal of the indicator was approximately linear to log(concentration) of the H5N1 specific sequence from 5.0 x 10-12 to 1.0 x 10-9 M (R = 0.9863) with a detection limit of 4.3 x 10-13 M. These studies showed that the new hybrid nanomaterial (MWNT/PPNWs/GNPs) and the DNA aptamer could be used to fabricate an electrochemical biosensor for gene sequence detection. Furthermore, this design strategy is expected to have extensive applications in other biosensors.

  13. Electrochemical detection of avian influenza virus H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode

    Energy Technology Data Exchange (ETDEWEB)

    Liu Xianggang [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China); Cheng Ziqiang, E-mail: czqsd@126.com [College of Animal Science and Technology, Shandong Agricultural University, Taian 271018, Shandong (China); Fan Hai [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China); Ai Shiyun, E-mail: ashy@sdau.edu.cn [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China); Han Ruixia [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China)

    2011-07-15

    Highlights: > A sensitive electrochemical biosensor for the detection of gene sequence was developed. > The biosensor was assembled by MWNT, polypyrrole nanowires and gold nanoparticles. > The hybrid nanomaterials could provide a porous structure with good properties. > The biosensor has highly selectivity and sensitivity. > The design strategy is expected to have extensive applications in other biosensors - Abstract: A sensitive electrochemical method for the detection of avian influenza virus (AIV) H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode was developed. To enhance the selectivity and sensitivity, the modified electrode was assembled with multi-wall carbon nanotubes (MWNT), polypyrrole nanowires (PPNWs) and gold nanoparticles (GNPs). This electrode offered a porous structure with a large effective surface area, highly electrocatalytic activities and electronic conductivity. Therefore, the amount of DNA aptamer immobilized onto the electrode was increased while the accessibility of the detection target was maintained. The biosensor is based on the hybridization and preferred orientation of a DNA aptamer immobilized onto a modified electrode surface with its target (H5N1 specific sequence) present in solution. It is selective for the H5N1 specific sequence, and the signal of the indicator was approximately linear to log(concentration) of the H5N1 specific sequence from 5.0 x 10{sup -12} to 1.0 x 10{sup -9} M (R = 0.9863) with a detection limit of 4.3 x 10{sup -13} M. These studies showed that the new hybrid nanomaterial (MWNT/PPNWs/GNPs) and the DNA aptamer could be used to fabricate an electrochemical biosensor for gene sequence detection. Furthermore, this design strategy is expected to have extensive applications in other biosensors.

  14. Modulation of immune response to Toxoplasma gondii in sheep by immunization with a DNA vaccine encoding ROP1 antigen as a fusion protein with ovine CD154.

    Science.gov (United States)

    Hiszczyńska-Sawicka, Elżbieta; Li, Hong; Xu, Janet Boyu; Holec-Gąsior, Lucyna; Kur, Józef; Sedcole, Richard; Bickerstaffe, Roy; Stankiewicz, Mirosław

    2011-12-29

    CD154 is a cell surface molecule expressed by activated T cells. CD40 and CD154 interaction is critically important in regulating humoral and cell-mediated immune responses. In this study we have investigated whether a DNA vaccine encoding rhoptry protein 1 (ROP1) of Toxoplasma gondii, and encoding ovine CD154 induces an enhanced ROP1-specific immune response in sheep. Two groups of twelve animals received two intramuscular injections, of a DNA plasmid encoding T. gondii ROP1 antigen (group 1) or an ROP1 antigen fused to ovine CD154 (group 2). There were two control groups of sheep. One was injected with an empty vector (group 3) and the other received no injections at all (group 4). The injection of the plasmid containing ROP1 (group 1) at weeks 0 and 4 induced a significant IgG2 response at week 2 which was amplified at week 4 after the booster injection and persisted to week 8 compared to the control animals in groups 3 and 4. For IgG1, significant differences from the control animals were only observed from week 5 onwards. The fusion of CD154 and ROP1 elicited significant IgG1 and IgG2 responses from week 1 which were amplified from weeks 5 to 8 compared to the control animals in groups 3 and 4. The IgG1 response was significantly higher in group 2 animals receiving pROP1-CD154 compared to group 1 receiving pROP1 only. There was no significant difference in IgG2 responses between groups 1 and 2. Significant differences in IFN-γ levels were only observed in treatment group 1 at week 2 and treatment group 2 at weeks 1 and 2 compared to the control animals. The results demonstrated that an intramuscular injection of pROP1-CD154 gene to sheep significantly enhanced their immune response and induced a mixed Th1/Th2 response while the intramuscular injection of pROP1 only induced a Th1-specific immune response.

  15. DVC1 (C1orf124) is a DNA damage-targeting p97 adaptor that promotes ubiquitin-dependent responses to replication blocks.

    Science.gov (United States)

    Mosbech, Anna; Gibbs-Seymour, Ian; Kagias, Konstantinos; Thorslund, Tina; Beli, Petra; Povlsen, Lou; Nielsen, Sofie Vincents; Smedegaard, Stine; Sedgwick, Garry; Lukas, Claudia; Hartmann-Petersen, Rasmus; Lukas, Jiri; Choudhary, Chunaram; Pocock, Roger; Bekker-Jensen, Simon; Mailand, Niels

    2012-11-01

    Ubiquitin-mediated processes orchestrate critical DNA-damage signaling and repair pathways. We identify human DVC1 (C1orf124; Spartan) as a cell cycle-regulated anaphase-promoting complex (APC) substrate that accumulates at stalled replication forks. DVC1 recruitment to sites of replication stress requires its ubiquitin-binding UBZ domain and PCNA-binding PIP box motif but is independent of RAD18-mediated PCNA monoubiquitylation. Via a conserved SHP box, DVC1 recruits the ubiquitin-selective chaperone p97 to blocked replication forks, which may facilitate p97-dependent removal of translesion synthesis (TLS) DNA polymerase η (Pol η) from monoubiquitylated PCNA. DVC1 knockdown enhances UV light-induced mutagenesis, and depletion of human DVC1 or the Caenorhabditis elegans ortholog DVC-1 causes hypersensitivity to replication stress-inducing agents. Our findings establish DVC1 as a DNA damage-targeting p97 adaptor that protects cells from deleterious consequences of replication blocks and suggest an important role of p97 in ubiquitin-dependent regulation of TLS. PMID:23042605

  16. Pulsed Field Gel Electrophoresis (PFGE: a DNA finger printing technique to study the genetic diversity of blood disease bacterium of banana

    Directory of Open Access Journals (Sweden)

    HADIWIYONO

    2011-01-01

    Full Text Available Hadiwiyono, Widada J, Subandiyah S, Fegan F (2011 Pulsed Field Gel Electrophoresis (PFGE: a DNA finger printing technique to study the genetic diversity of blood disease bacterium of banana. Biodiversitas 12: 12-16. Blood disease bacterium (BDB is the most important pathogen of bananas in Indonesia. In some field, the disease incidence reaches over 80%. Epidemiologically, the disease is similar to moko disease in South America and bugtok disease in the Philippines caused by Ralstonia solanacearum race 2. However, BDB is different in phenotype and genotype from the two diseases. Previously BDB was limited in South Sulawesi since 1920s – 1980s and recently was reported in 27 of 30 provinces in Indonesia. Pulsed-Field Gel Electrophoresis (PFGE is a genomic DNA fingerprinting method, which employs rare cutting restriction endonucleases to digest genome prior to electrophoresis using specialized condition to separate of large DNA fragments. The results showed that PFGE analysis was a discriminative tool to study the genetic diversity of BDB. Based on the PFGE analysis, BDB isolates obtained from different localities in Yogyakarta and Central Java were quit diverse.

  17. A DNA-based method for identification of krill species and its application to analysing the diet of marine vertebrate predators.

    Science.gov (United States)

    Jarman, S N; Gales, N J; Tierney, M; Gill, P C; Elliott, N G

    2002-12-01

    Accurate identification of species that are consumed by vertebrate predators is necessary for understanding marine food webs. Morphological methods for identifying prey components after consumption often fail to make accurate identifications of invertebrates because prey morphology becomes damaged during capture, ingestion and digestion. Another disadvantage of morphological methods for prey identification is that they often involve sampling procedures that are disruptive for the predator, such as stomach flushing or lethal collection. We have developed a DNA-based method for identifying species of krill (Crustacea: Malacostraca), an enormously abundant group of invertebrates that are directly consumed by many groups of marine vertebrates. The DNA-based approach allows identification of krill species present in samples of vertebrate stomach contents, vomit, and, more importantly, faeces. Utilizing samples of faeces from vertebrate predators minimizes the impact of dietary studies on the subject animals. We demonstrate our method first on samples of Adelie penguin (Pygoscelis adeliae) stomach contents, where DNA-based species identification can be confirmed by prey morphology. We then apply the method to faeces of Adelie penguins and to faeces of the endangered pygmy blue whale (Balaenoptera musculus brevicauda). In each of these cases, krill species consumed by the predators could be identified from their DNA present in faeces or stomach contents. PMID:12453250

  18. THAP5 is a DNA binding transcriptional repressor that is regulated in melanoma cells during DNA damage-induced cell death

    Science.gov (United States)

    Balakrishnan, Meenakshi P.; Cilenti, Lucia; Ambivero, Camilla; Goto, Yamafumi; Takata, Minoru; Turkson, James; Li, Xiaoman Shawn; Zervos, Antonis S.

    2011-01-01

    THAP5 was originally isolated as a specific interactor and substrate of the mitochondrial pro-apoptotic Omi/HtrA2 protease. It is a human zinc finger protein characterized by a restricted pattern of expression and the lack of orthologs in mouse and rat. The biological function of THAP5 is unknown but our previous studies suggest it could regulate G2/M transition in kidney cells and could be involved in human cardiomyocyte cell death associated with coronary artery disease (CAD). In this report, we expanded our studies on the properties and function of THAP5 in human melanoma cells. THAP5 was expressed in primary human melanocytes as well as in all melanoma cell lines that were tested. THAP5 protein level was significantly induced by UV irradiation or cisplatin treatment, conditions known to cause DNA damage. The induction of THAP5 correlated with a significant increase in apoptotic cell death. In addition, we show that THAP5 is a nuclear protein that could recognize and bind a specific DNA motif. THAP5 could also repress the transcription of a reporter gene in a heterologous system. Our work suggests that THAP5 is a DNA binding protein and a transcriptional repressor. Furthermore, THAP5 has a pro-apoptotic function and it was induced in melanoma cells under conditions that promoted cell death. PMID:21110952

  19. Gene Gun Bombardment with DNA-Coated Golden Particles Enhanced the Protective Effect of a DNA Vaccine Based on Thioredoxin Glutathione Reductase of Schistosoma japonicum

    Directory of Open Access Journals (Sweden)

    Yan Cao

    2013-01-01

    Full Text Available Schistosomiasis, caused by infection with Schistosoma species, remains an important parasitic zoonosis. Thioredoxin glutathione reductase of Schistosoma japonicum (SjTGR plays an important role in the development of the parasite and for its survival. Here we present a recombinant plasmid DNA vaccine, pVAX1/SjTGR, to estimate its protection against S. japonicum in BALB/c mice. The DNA vaccine administrated by particle bombardment induced higher protection than by intramuscular injection. All animals vaccinated with pVAX1/SjTGR developed significant specific anti-SjTGR antibodies than control groups. Moreover, animals immunized by gene gun exhibited a splenocyte proliferative response, with an increase in IFN-γ and IL-4. The recombinant plasmid administrated by gene gun achieved a medium protective efficacy of 27.83–38.83% ( of worm reduction and 40.38–44.51% ( of liver egg count reduction. It suggests that different modes of administering a DNA vaccine can influence the protective efficacy induced by the vaccine. Interestingly, from the enzymatic activity results, we found that worms obtained from pVAX1/SjTGR-vaccinated animals expressed lower enzymatic activity than the control group and the antibodies weakened the enzymatic activity of SjTGR in vitro, too. It implies that the high-level antibodies may contribute to the protective effects.

  20. The SET-domain protein SUVR5 mediates H3K9me2 deposition and silencing at stimulus response genes in a DNA methylation-independent manner.

    Directory of Open Access Journals (Sweden)

    Elena Caro

    Full Text Available In eukaryotic cells, environmental and developmental signals alter chromatin structure and modulate gene expression. Heterochromatin constitutes the transcriptionally inactive state of the genome and in plants and mammals is generally characterized by DNA methylation and histone modifications such as histone H3 lysine 9 (H3K9 methylation. In Arabidopsis thaliana, DNA methylation and H3K9 methylation are usually colocated and set up a mutually self-reinforcing and stable state. Here, in contrast, we found that SUVR5, a plant Su(var3-9 homolog with a SET histone methyltransferase domain, mediates H3K9me2 deposition and regulates gene expression in a DNA methylation-independent manner. SUVR5 binds DNA through its zinc fingers and represses the expression of a subset of stimulus response genes. This represents a novel mechanism for plants to regulate their chromatin and transcriptional state, which may allow for the adaptability and modulation necessary to rapidly respond to extracellular cues.

  1. A quantum dot-aptamer beacon using a DNA intercalating dye as the FRET reporter: application to label-free thrombin detection.

    Science.gov (United States)

    Chi, Chun-Wei; Lao, Yeh-Hsing; Li, Yi-Shan; Chen, Lin-Chi

    2011-03-15

    A new quantum dot (QD)-aptamer (apt) beacon that acts by folding-induced dissociation of a DNA intercalating dye, BOBO-3(B), is demonstrated with label-free thrombin detection. The beacon, denoted as QD-apt:B, is constructed by (1) coupling of a single-stranded thrombin aptamer to Qdot 565 via EDC/Sulfo-NHS chemistry and (2) staining the duplex regions of the aptamer on QD with excess BOBO-3 before thrombin binding. When mixing a thrombin sample with QD-apt:B, BOBO-3 is competed away from the beacon due to target-induced aptamer folding, which then causes a decrease in QD fluorescence resonance energy transfer (FRET)-mediated BOBO-3 emission and achieves thrombin quantitation. In this work, the effects of Mg(2+), coupling time, and aptamer type on the beacon's performances are investigated and discussed thoroughly with various methods, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and two-color differential gel electrophoresis. Using the best aptamer beacon (HTQ37), we attain highly specific and wide-range detection (from nM to μM) of thrombin in buffer, and the beacon can sense nM-range thrombin in 15% diluted serum. Compared to the reported QD aptamer assays, our method is advantageous from the aspect of using a simple sensory unit design without losing the detection sensitivity. Therefore, we consider the QD-apt:B beacon a potential alternative to immuno-reagents and an effective tool to study nucleic acid folding on QD as well. PMID:21306887

  2. Aspects of T Cell-Mediated Immunity Induced in Mice by a DNA Vaccine Based on the Dengue-NS1 Antigen after Challenge by the Intracerebral Route.

    Science.gov (United States)

    Oliveira, Edson R A; Gonçalves, Antônio J S; Costa, Simone M; Azevedo, Adriana S; Mantuano-Barradas, Marcio; Nogueira, Ana Cristina M A; Alves, Ada M B

    2016-01-01

    Dengue disease has emerged as a major public health issue across tropical and subtropical countries. Infections caused by dengue virus (DENV) can evolve to life-threatening forms, resulting in about 20,000 deaths every year worldwide. Several animal models have been described concerning pre-clinical stages in vaccine development against dengue, each of them presenting limitations and advantages. Among these models, a traditional approach is the inoculation of a mouse-brain adapted DENV variant in immunocompetent animals by the intracerebral (i.c.) route. Despite the historical usage and relevance of this model for vaccine testing, little is known about the mechanisms by which the protection is developed upon vaccination. To cover this topic, a DNA vaccine based on the DENV non-structural protein 1 (pcTPANS1) was considered and investigations were focused on the induced T cell-mediated immunity against i.c.-DENV infection. Immunophenotyping assays by flow cytometry revealed that immunization with pcTPANS1 promotes a sustained T cell activation in spleen of i.c.-infected mice. Moreover, we found that the downregulation of CD45RB on T cells, as an indicator of cell activation, correlated with absence of morbidity upon virus challenge. Adoptive transfer procedures supported by CFSE-labeled cell tracking showed that NS1-specific T cells induced by vaccination, proliferate and migrate to peripheral organs of infected mice, such as the liver. Additionally, in late stages of infection (from the 7th day onwards), vaccinated mice also presented reduced levels of circulating IFN-γ and IL-12p70 in comparison to non-vaccinated animals. In conclusion, this work presented new aspects about the T cell-mediated immunity concerning DNA vaccination with pcTPANS1 and the i.c. infection model. These insights can be explored in further studies of anti-dengue vaccine efficacy. PMID:27631083

  3. Modulatory effect of CpG oligodeoxynucleotide on a DNA vaccine against nervous necrosis virus in orange-spotted grouper (Epinephelus coioides).

    Science.gov (United States)

    Chen, Shiang-Peng; Peng, Ran-Hong; Chiou, Pinwen P

    2015-08-01

    We report the development of a DNA vaccine pcMGNNV2 against nervous necrosis virus (NNV), a leading cause of mass mortality in grouper larvae. In addition, the modulatory effect of CpG oligodeoxynucleotide (ODN), a Toll-like receptor 9 agonist, on the DNA vaccine was evaluated. The DNA vaccine alone elicited the production of NNV-specific antibodies, indicating that the vaccine was capable of triggering adaptive humoral response. Furthermore, significant induction of TLR9, Mx and IL-1β was observed in the spleen on day 7 post-vaccination, supporting that the vaccine could trigger TLR9 signaling. The incorporation of CpG ODN at high dose did not significantly affect the level of NNV-specific antibodies, but was able to moderately enhance the expression of Mx and IL-1β on day 7, indicating its ability in modulating innate response. After challenge with NNV, the vaccine alone enhanced the survival rate in infected larvae at both 1 and 2 weeks post-vaccination. The combination of CpG ODN further increased the survival rate at week 1 but not week 2. Interestingly, at week 2 the ODN appeared to induce a Th1-like response, as indicated by upregulation of T-bet (a Th1 marker) and downregulation of GATA-3 (a Th2 marker). Thus, the results suggest that the boosted Th1 response by CpG ODN does not augment the protection efficacy of pcMGNNV2 vaccine. To our best knowledge, this is the first report of a successful DNA vaccine against NNV in grouper.

  4. Vector optimization and needle-free intradermal application of a broadly protective polyvalent influenza A DNA vaccine for pigs and humans.

    Science.gov (United States)

    Borggren, Marie; Nielsen, Jens; Bragstad, Karoline; Karlsson, Ingrid; Krog, Jesper S; Williams, James A; Fomsgaard, Anders

    2015-01-01

    The threat posed by the 2009 pandemic H1N1 virus emphasized the need for new influenza A virus vaccines inducing a broad cross-protective immune response for use in both humans and pigs. An effective and broad influenza vaccine for pigs would greatly benefit the pork industry and contribute to public health by diminishing the risk of emerging highly pathogenic reassortants. Current inactivated protein vaccines against swine influenza produce only short-lived immunity and have no efficacy against heterologous strains. DNA vaccines are a potential alternative with advantages such as the induction of cellular and humoral immunity, inherent safety and rapid production time. We have previously developed a DNA vaccine encoding selected influenza proteins of pandemic origin and demonstrated broad protective immune responses in ferrets and pigs. In this study, we evaluated our DNA vaccine expressed by next-generation vectors. These new vectors can improve gene expression, but they are also efficiently produced on large scales and comply with regulatory guidelines by avoiding antibiotic resistance genes. In addition, a new needle-free delivery of the vaccine, convenient for mass vaccinations, was compared with intradermal needle injection followed by electroporation. We report that when our DNA vaccine is expressed by the new vectors and delivered to the skin with the needle-free device in the rabbit model, it can elicit an antibody response with the same titers as a conventional vector with intradermal electroporation. The needle-free delivery is already in use for traditional protein vaccines in pigs but should be considered as a practical alternative for the mass administration of broadly protective influenza DNA vaccines. PMID:25746201

  5. Aspects of T Cell-Mediated Immunity Induced in Mice by a DNA Vaccine Based on the Dengue-NS1 Antigen after Challenge by the Intracerebral Route

    Science.gov (United States)

    Oliveira, Edson R. A.; Gonçalves, Antônio J. S.; Costa, Simone M.; Azevedo, Adriana S.; Mantuano-Barradas, Marcio; Nogueira, Ana Cristina M. A.

    2016-01-01

    Dengue disease has emerged as a major public health issue across tropical and subtropical countries. Infections caused by dengue virus (DENV) can evolve to life-threatening forms, resulting in about 20,000 deaths every year worldwide. Several animal models have been described concerning pre-clinical stages in vaccine development against dengue, each of them presenting limitations and advantages. Among these models, a traditional approach is the inoculation of a mouse-brain adapted DENV variant in immunocompetent animals by the intracerebral (i.c.) route. Despite the historical usage and relevance of this model for vaccine testing, little is known about the mechanisms by which the protection is developed upon vaccination. To cover this topic, a DNA vaccine based on the DENV non-structural protein 1 (pcTPANS1) was considered and investigations were focused on the induced T cell-mediated immunity against i.c.-DENV infection. Immunophenotyping assays by flow cytometry revealed that immunization with pcTPANS1 promotes a sustained T cell activation in spleen of i.c.-infected mice. Moreover, we found that the downregulation of CD45RB on T cells, as an indicator of cell activation, correlated with absence of morbidity upon virus challenge. Adoptive transfer procedures supported by CFSE-labeled cell tracking showed that NS1-specific T cells induced by vaccination, proliferate and migrate to peripheral organs of infected mice, such as the liver. Additionally, in late stages of infection (from the 7th day onwards), vaccinated mice also presented reduced levels of circulating IFN-γ and IL-12p70 in comparison to non-vaccinated animals. In conclusion, this work presented new aspects about the T cell-mediated immunity concerning DNA vaccination with pcTPANS1 and the i.c. infection model. These insights can be explored in further studies of anti-dengue vaccine efficacy. PMID:27631083

  6. Comparative analysis and molecular characterization of a gene BANF1 encoded a DNA-binding protein during mitosis from the Giant Panda and Black Bear.

    Science.gov (United States)

    Zeng, Yichun; Hou, Yi-Ling; Ding, Xiang; Hou, Wan-Ru; Li, Jian

    2014-01-01

    Barrier to autointegration factor 1 (BANF1) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mitosis. The cDNA and the genomic sequence of BANF1 were cloned from the Giant Panda (Ailuropoda melanoleuca) and Black Bear (Ursus thibetanus mupinensis) using RT-PCR technology and Touchdown-PCR, respectively. The cDNA of the BANF1 cloned from Giant Panda and Black Bear is 297 bp in size, containing an open reading frame of 270 bp encoding 89 amino acids. The length of the genomic sequence from Giant Panda is 521 bp, from Black Bear is 536 bp, which were found both to possess 2 exons. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to some mammalian species studied. Topology prediction showed there is one Protein kinase C phosphorylation site, one Casein kinase II phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Giant Panda, and there is one Protein kinase C phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Black Bear. The BANF1 gene can be readily expressed in E. coli. Results showed that the protein BANF1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 14 kD polypeptide that formed inclusion bodies. The expression products obtained could be used to purify the proteins and study their function further. PMID:25009988

  7. A quantum dot-aptamer beacon using a DNA intercalating dye as the FRET reporter: application to label-free thrombin detection.

    Science.gov (United States)

    Chi, Chun-Wei; Lao, Yeh-Hsing; Li, Yi-Shan; Chen, Lin-Chi

    2011-03-15

    A new quantum dot (QD)-aptamer (apt) beacon that acts by folding-induced dissociation of a DNA intercalating dye, BOBO-3(B), is demonstrated with label-free thrombin detection. The beacon, denoted as QD-apt:B, is constructed by (1) coupling of a single-stranded thrombin aptamer to Qdot 565 via EDC/Sulfo-NHS chemistry and (2) staining the duplex regions of the aptamer on QD with excess BOBO-3 before thrombin binding. When mixing a thrombin sample with QD-apt:B, BOBO-3 is competed away from the beacon due to target-induced aptamer folding, which then causes a decrease in QD fluorescence resonance energy transfer (FRET)-mediated BOBO-3 emission and achieves thrombin quantitation. In this work, the effects of Mg(2+), coupling time, and aptamer type on the beacon's performances are investigated and discussed thoroughly with various methods, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and two-color differential gel electrophoresis. Using the best aptamer beacon (HTQ37), we attain highly specific and wide-range detection (from nM to μM) of thrombin in buffer, and the beacon can sense nM-range thrombin in 15% diluted serum. Compared to the reported QD aptamer assays, our method is advantageous from the aspect of using a simple sensory unit design without losing the detection sensitivity. Therefore, we consider the QD-apt:B beacon a potential alternative to immuno-reagents and an effective tool to study nucleic acid folding on QD as well.

  8. Direct Electrochemistry and Electrocatalysis of Horseradish Peroxidase Immobilized in a DNA/Chitosan-Fe3O4 Magnetic Nanoparticle Bio-Complex Film

    Directory of Open Access Journals (Sweden)

    Tingting Gu

    2014-02-01

    Full Text Available A DNA/chitosan-Fe3O4 magnetic nanoparticle bio-complex film was constructed for the immobilization of horseradish peroxidase (HRP on a glassy carbon electrode. HRP was simply mixed with DNA, chitosan and Fe3O4 nanoparticles, and then applied to the electrode surface to form an enzyme-incorporated polyion complex film. Scanning electron microscopy (SEM was used to study the surface features of DNA/chitosan/Fe3O4/HRP layer. The results of electrochemical impedance spectroscopy (EIS show that Fe3O4 and enzyme were successfully immobilized on the electrode surface by the DNA/chitosan bio-polyion complex membrane. Direct electron transfer (DET and bioelectrocatalysis of HRP in the DNA/chitosan/Fe3O4 film were investigated by cyclic voltammetry (CV and constant potential amperometry. The HRP-immobilized electrode was found to undergo DET and exhibited a fast electron transfer rate constant of 3.7 s−1. The CV results showed that the modified electrode gave rise to well-defined peaks in phosphate buffer, corresponding to the electrochemical redox reaction between HRP(Fe(III and HRP(Fe(II. The obtained electrode also displayed an electrocatalytic reduction behavior towards H2O2. The resulting DNA/chitosan/Fe3O4/HRP/glassy carbon electrode (GCE shows a high sensitivity (20.8 A·cm−2·M−1 toward H2O2. A linear response to H2O2 measurement was obtained over the range from 2 µM to 100 µM (R2 = 0.99 and an amperometric detection limit of 1 µM (S/N = 3. The apparent Michaelis-Menten constant of HRP immobilized on the electrode was 0.28 mM. Furthermore, the electrode exhibits both good operational stability and storage stability.

  9. The C-terminal domain of the bacterial SSB protein acts as a DNA maintenance hub at active chromosome replication forks.

    Directory of Open Access Journals (Sweden)

    Audrey Costes

    Full Text Available We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSB(Cter as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSB(Cter interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSB(Cter deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSB(Cter acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome.

  10. The C-terminal domain of the bacterial SSB protein acts as a DNA maintenance hub at active chromosome replication forks.

    Science.gov (United States)

    Costes, Audrey; Lecointe, François; McGovern, Stephen; Quevillon-Cheruel, Sophie; Polard, Patrice

    2010-01-01

    We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSB(Cter)) as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSB(Cter) interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSB(Cter) deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSB(Cter) acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome. PMID:21170359

  11. Improved Immunogenicity of a Vaccination Regimen Combining a DNA Vaccine Encoding Brucella melitensis Outer Membrane Protein 31 (Omp31) and Recombinant Omp31 Boosting▿

    Science.gov (United States)

    Cassataro, Juliana; Velikovsky, Carlos A.; Bruno, Laura; Estein, Silvia M.; de la Barrera, Silvia; Bowden, Raúl; Fossati, Carlos A.; Giambartolomei, Guillermo H.

    2007-01-01

    In the present study, we report an attempt to improve the immunogenicity of the Omp31 antigen by a DNA prime-protein boost immunization regimen. We immunized BALB/c mice with an Omp31 DNA vaccine (pCIOmp31) followed by boosting with recombinant Omp31 (rOmp31) in incomplete Freund's adjuvant and characterized the resulting immune responses and the protective efficacy against Brucella ovis and B. melitensis infection. Immunoglobulin G1 (IgG1) and IgG2a titers were higher in sera from pCIOmp31/rOmp31-immunized mice than in sera from mice immunized with pCIOmp31 or rOmp31 alone. Splenocytes from pCIOmp31/rOmp31-immunized mice produced significantly higher levels of gamma interferon than did those from mice given rOmp31 alone. In contrast, interleukin 2 (IL-2) production levels were comparable between the two groups of immunized mice. Cells from all immunized mice produced undetectable levels of IL-4. Notably, rOmp31 stimulated IL-10 production in the pCIOmp31/rOmp31-immunized group but not in the pCIOmp31- or rOmp31-immunized group. Although the prime-boost regimen induced specific cytotoxic responses, these responses could not reach the levels achieved by the pCIOmp31 immunization. In conclusion, pCIOmp31 priming followed by rOmp31 boosting led to moderately improved protection against a challenge with B. ovis or B. melitensis. PMID:17428946

  12. A DNA vaccine against extracellular domains 1-3 of flk-1 and its immune preventive and therapeutic effects against H22 tumor cell in vivo

    Institute of Scientific and Technical Information of China (English)

    Fan Lü; Zhao-Yin Qin; Wen-Bin Yang; Yin-Xin Qi; Yi-Min Li

    2004-01-01

    AIM: To construct a DNA vaccine against extracellular domains 1-3 of fetal liver kinase-1 (flk-1), and to investigate its preventive and therapeutic effect against H22 cellin vivo.METHODS: Flk-1 DNA vaccine was produced by cloning extracellular domains 1-3 of flk-1 and by inserting the cloned gene into pcDNA3.1 (+). Fifteen mice were divided into 3 groups and inoculated by vaccine, plasmid and saline respectively to detect specific T lymphocyte response. Thirty Mice were equally divided into preventive group and therapeutic group. Preventive group was further divided into V, P, and S subgroups, namely immunized by vaccine,pcDNA3.1 (+) and saline, respectively, and attacked by H22 cell. Therapeutical group was divided into 3 subgroups of V, P and S, and attacked by H22, then treated with vaccine, pcDNA3.1 (+) and saline, respectively. The tumor size, tumor weight, mice survival time and tumor latency period were compared within these groups. Furthermore,intratumoral microvessel density (MVD) was assessed by immunohistochemistry.RESULTS: DNA vaccine pcDNA3.1 (+) flk-1-domains 1-3 was successfully constructed and could raise specific CTL activity. In the preventive group and therapeutic group,tumor latency period and survival time were significantly longer in vaccine subgroup than that in P and S subgroups (P<0.05); the tumor size, weight and MVD were significantly less in vaccine subgroup than that in P and S subgroups (P<0.05). The survival time of therapeutic vaccine subgroup was significantly shorter than that of preventive vaccine subgroup (P<0.05); the tumor size, and MVD of therapeutic vaccine subgroup were significantly greater than that of preventive vaccine subgroup (P<0.05).CONCLUSION: DNA vaccine against flk-1 domains 1-3 can stimulate potent specific CTL activity; and has distinctive prophylactic effect on tumor H22; and also can inhibit the tumor growthin vivo. This vaccine may be used as an adjuvant therapy because it is less effective on

  13. Cooperation between CD4+ T Cells and Humoral Immunity Is Critical for Protection against Dengue Using a DNA Vaccine Based on the NS1 Antigen.

    Directory of Open Access Journals (Sweden)

    Antônio J S Gonçalves

    2015-12-01

    Full Text Available Dengue virus (DENV is spread through most tropical and subtropical areas of the world and represents a serious public health problem. At present, the control of dengue disease is mainly hampered by the absence of antivirals or a vaccine, which results in an estimated half worldwide population at risk of infection. The immune response against DENV is not yet fully understood and a better knowledge of it is now recognized as one of the main challenge for vaccine development. In previous studies, we reported that a DNA vaccine containing the signal peptide sequence from the human tissue plasminogen activator (t-PA fused to the DENV2 NS1 gene (pcTPANS1 induced protection against dengue in mice. In the present work, we aimed to elucidate the contribution of cellular and humoral responses elicited by this vaccine candidate for protective immunity. We observed that pcTPANS1 exerts a robust protection against dengue, inducing considerable levels of anti-NS1 antibodies and T cell responses. Passive immunization with anti-NS1 antibodies conferred partial protection in mice infected with low virus load (4 LD50, which was abrogated with the increase of viral dose (40 LD50. The pcTPANS1 also induced activation of CD4+ and CD8+ T cells. We detected production of IFN-γ and a cytotoxic activity by CD8+ T lymphocytes induced by this vaccine, although its contribution in the protection was not so evident when compared to CD4+ cells. Depletion of CD4+ cells in immunized mice completely abolished protection. Furthermore, transfer experiments revealed that animals receiving CD4+ T cells combined with anti-NS1 antiserum, both obtained from vaccinated mice, survived virus infection with survival rates not significantly different from pcTPANS1-immunized animals. Taken together, results showed that the protective immune response induced by the expression of NS1 antigen mediated by the pcTPANS1 requires a cooperation between CD4+ T cells and the humoral immunity.

  14. É possível uma vacina gênica auxiliar no controle da tuberculose? Could a DNA vaccine be useful in the control of tuberculosis?

    Directory of Open Access Journals (Sweden)

    José Maciel Rodrigues Júnior

    2004-08-01

    vaccines currently under pre-clinical and clinical development may prove to be important tools in combating infectious diseases, such as tuberculosis, for which no safe and effective form of prevention has yet been developed. In recent years, several studies have aimed to develop a DNA vaccine encoding mycobacterial proteins such as antigen 85 (Ag85 and the 65-kDa mycobacterial heat shock protein (hsp65. The latter is protective against virulent infection with Mycobacterium tuberculosis (including multidrug-resistant strains. The hsp65 DNA vaccine, currently under clinical evaluation in Brazil for cancer therapy, is able to induce the secretion of Th1 cytokines, such as gamma-interferon, associated with disease control. Furthermore, this vaccine stimulates cytotoxic CD8 and CD4 T-cell clones that can be characterized as memory cells, which are responsible for effective and long-lasting immunity against tuberculosis. When used as a therapeutic agent in inoculated mice, the hsp65 DNA vaccine promotes changes in the immunity profile, triggering the secretion of Th1 cytokines and establishing a favorable environment for the elimination of bacilli. The results also demonstrate that the route of administration, as well as the formulation in which the vaccine is administered, fundamentally influence the pattern and duration of the immune response induced. Taking all currently available data into account, we can conclude that a DNA vaccine against tuberculosis could contribute significantly to the control of the disease.

  15. Use of S-[2,3-Bispalmitoyiloxy-(2R)-Propyl]-R-Cysteinyl-Amido-Monomethoxy Polyethylene Glycol as an Adjuvant Improved Protective Immunity Associated with a DNA Vaccine Encoding Cu,Zn Superoxide Dismutase of Brucella abortus in Mice

    OpenAIRE

    Retamal-Díaz, Angello; Riquelme-Neira, Roberto; Sáez, Darwin; Rivera, Alejandra; Fernández, Pablo; Cabrera, Alex; Guzmán, Carlos A.; Oñate, Angel

    2014-01-01

    This study was conducted to evaluate the immunogenicity and protective efficacy of a DNA vaccine encoding Brucella abortus Cu,Zn superoxide dismutase (SOD) using the Toll-like receptor 2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol (BPPcysMPEG) as an adjuvant. Intranasal coadministration of BPPcysMPEG with a plasmid carrying the SOD-encoding gene (pcDNA-SOD) into BALB/c mice elicited antigen-specific humoral and cellular immune responses. Hu...

  16. 一种含不同流感病毒血凝素抗原表位的核酸疫苗%A DNA Vaccine Containing Different Influenza Hemagglutinin Epitopes

    Institute of Scientific and Technical Information of China (English)

    彭波; 常海艳; 张风华; 李小曼; 陈则; 方芳

    2011-01-01

    流感病毒表面抗原血凝素( hemagglutinin,HA)是流感核酸疫苗重要的靶抗原,针对HA的保护性中和抗体主要由HA上的五个抗原表位诱导产生.在本文中,我们构建了一种以新甲型H1N1流感病毒HA1为骨架的含2个A/PR/8( H1N1)流感病毒HA抗原表位和3个新甲型H1N1流感病毒HA抗原表位的核酸疫苗,并在BALB/c小鼠致死量病毒攻击模型中检验其免疫保护效果.结果显示,两次电击免疫后,该核酸疫苗对新甲型H1N1流感病毒和A/PR/8流感病毒的攻击都能提供完全的保护,达到了和含相应病毒株全长HA的核酸疫苗相同的效果.因此我们认为,含流感病毒血凝素不同抗原表位的核酸疫苗可以作为一种新型的具有交叉保护效果的流感疫苗.%Hemagglutinin (HA) , the surface antigen of influenza virus, is a very important target for influenza DNA vaccines. On the surface of the protein, there are five main neutralizing epitopes that induce protective antibodies against influenza. In this paper, a DNA vaccine was constructed based on the HA1 from the new influenza A (H1N1) virus, which contains two epitopes of the A/PR/8 (H1N1) influenza virus HA. Female BALB/c mice were immunized twice by electroporation before challenged with a lethal dose of new influenza A ( H1N1) virus or A/PR/8 influenza virus. The result showed that the DNA vaccine could provide mice a complete cross-protection against these two influenza viruses. The protective effects were comparable to those provided by DNA vaccines containing an intact HA gene. Thus, a DNA vaccine that contains epitopes of different HA genes from influenza viruses of the same subtype could be a new influenza vaccine to offer cross-protection against influenza antigenic drift.

  17. A DNA tweezer-actuated enzyme nanoreactor.

    Science.gov (United States)

    Liu, Minghui; Fu, Jinglin; Hejesen, Christian; Yang, Yuhe; Woodbury, Neal W; Gothelf, Kurt; Liu, Yan; Yan, Hao

    2013-01-01

    The functions of regulatory enzymes are essential to modulating cellular pathways. Here we report a tweezer-like DNA nanodevice to actuate the activity of an enzyme/cofactor pair. A dehydrogenase and NAD(+) cofactor are attached to different arms of the DNA tweezer structure and actuation of enzymatic function is achieved by switching the tweezers between open and closed states. The enzyme/cofactor pair is spatially separated in the open state with inhibited enzyme function, whereas in the closed state, enzyme is activated by the close proximity of the two molecules. The conformational state of the DNA tweezer is controlled by the addition of specific oligonucleotides that serve as the thermodynamic driver (fuel) to trigger the change. Using this approach, several cycles of externally controlled enzyme inhibition and activation are successfully demonstrated. This principle of responsive enzyme nanodevices may be used to regulate other types of enzymes and to introduce feedback or feed-forward control loops.

  18. Modelling of a DNA packaging motor

    Institute of Scientific and Technical Information of China (English)

    Qian Jun; Xie Ping; Xue Xiao-Guang; Wang Peng-Ye

    2009-01-01

    During the assembly of many viruses, a powerful molecular motor packages the genome into a preassembled capsid. The Bacillus subtilis phage φ29 is an excellent model system to investigate the DNA packaging mechanism because of its highly efficient in vitro DNA packaging activity and the development of a single-molecule packaging assay. Here we make use of structural and biochemical experimental data to build a physical model of DNA packaging by the φ29 DNA packaging motor. Based on the model, various dynamic behaviours such as the packaging rate, pause frequency and slip frequency under different ATP concentrations, ADP concentrations, external loads as well as capsid fillings are studied by using Monte Carlo simulation. Good agreement is obtained between the simulated and available experimental results. Moreover, we make testable predictions that should guide future experiments related to motor function.

  19. A DNA recombinant database management system.

    OpenAIRE

    Tolstoshev, C M; Jeltsch, J M; Fritz, R.; Oudet, P

    1983-01-01

    A set of computer programs is described which constitutes a clone database management system. Maintenance of the database and the stocks of material is designed to be under the control of one person or group of people, who may insert, delete or modify data entries, and who may interrogate the database as to which stocks are in need of checking. The system is organised in such a way that information is freely and speedily available to all users. Database entries may be accessed by name or key ...

  20. A DNA based model for addition computation

    Institute of Scientific and Technical Information of China (English)

    GAO Lin; YANG Xiao; LIU Wenbin; XU Jin

    2004-01-01

    Much effort has been made to solve computing problems by using DNA-an organic simulating method, which in some cases is preferable to the current electronic computer. However, No one at present has proposed an effective and applicable method to solve addition problem with molecular algorithm due to the difficulty in solving the carry problem which can be easily solved by hardware of an electronic computer. In this article, we solved this problem by employing two kinds of DNA strings, one is called result and operation string while the other is named carrier. The result and operation string contains some carry information by its own and denotes the ultimate result while the carrier is just for carrying use. The significance of this algorithm is the original code, the fairly easy steps to follow and the feasibility under current molecular biological technology.

  1. A DNA barcode for land plants

    OpenAIRE

    Hollingsworth, Peter M.; Forrest, Laura L.; Spouge, John L.; Hajibabaei, Mehrdad; Ratnasingham, Sujeevan; van der Bank,Michelle; Chase, Mark W.; Cowan, Robyn S; Erickson, David L.; Fazekas, Aron J.; Graham, Sean W.; James, Karen E.; Kim, Ki-Joong; Kress, W. John; Schneider, Harald

    2009-01-01

    DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer). Based on assessments of recoverability, sequence quali...

  2. A DNA barcode for land plants.

    Science.gov (United States)

    2009-08-01

    DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF-atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK-psbI spacer, and trnH-psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants. PMID:19666622

  3. A DNA barcode for land plants

    Science.gov (United States)

    Hollingsworth, Peter M.; Forrest, Laura L.; Spouge, John L.; Hajibabaei, Mehrdad; Ratnasingham, Sujeevan; van der Bank, Michelle; Chase, Mark W.; Cowan, Robyn S.; Erickson, David L.; Fazekas, Aron J.; Graham, Sean W.; James, Karen E.; Kim, Ki-Joong; Kress, W. John; Schneider, Harald; van AlphenStahl, Jonathan; Barrett, Spencer C.H.; van den Berg, Cassio; Bogarin, Diego; Burgess, Kevin S.; Cameron, Kenneth M.; Carine, Mark; Chacón, Juliana; Clark, Alexandra; Clarkson, James J.; Conrad, Ferozah; Devey, Dion S.; Ford, Caroline S.; Hedderson, Terry A.J.; Hollingsworth, Michelle L.; Husband, Brian C.; Kelly, Laura J.; Kesanakurti, Prasad R.; Kim, Jung Sung; Kim, Young-Dong; Lahaye, Renaud; Lee, Hae-Lim; Long, David G.; Madriñán, Santiago; Maurin, Olivier; Meusnier, Isabelle; Newmaster, Steven G.; Park, Chong-Wook; Percy, Diana M.; Petersen, Gitte; Richardson, James E.; Salazar, Gerardo A.; Savolainen, Vincent; Seberg, Ole; Wilkinson, Michael J.; Yi, Dong-Keun; Little, Damon P.

    2009-01-01

    DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants. PMID:19666622

  4. Molecular electronics : A DNA that conducts

    OpenAIRE

    Scheer, Elke

    2014-01-01

    Experiments with conducting atomic force microscopy provide a clear demonstration of long-range charge transport in G-quadruplex DNA molecules, and allow a hopping transport model to be developed that could also be applied to other conductive polymers.

  5. Molecular electronics: A DNA that conducts

    Science.gov (United States)

    Scheer, Elke

    2014-12-01

    Experiments with conducting atomic force microscopy provide a clear demonstration of long-range charge transport in G-quadruplex DNA molecules, and allow a hopping transport model to be developed that could also be applied to other conductive polymers.

  6. The nuclear retention of transcription factor FOXO3a correlates with a DNA damage response and increased glutamine synthetase expression by astrocytes suggesting a neuroprotective role in the ageing brain.

    Science.gov (United States)

    Fluteau, Adeline; Ince, Paul G; Minett, Thais; Matthews, Fiona E; Brayne, Carol; Garwood, Claire J; Ratcliffe, Laura E; Morgan, Sarah; Heath, Paul R; Shaw, Pamela J; Wharton, Stephen B; Simpson, Julie E

    2015-11-16

    The accumulation of reactive oxygen species leading to oxidative damage and cell death plays an important role in a number of neurodegenerative disorders. FOXO3a, the main isoform of FOXO transcription factors, mediates the cellular response to oxidative stress by regulating the expression of genes involved in DNA repair and glutamine metabolism, including glutamine synthetase (GS). Immunohistochemical investigation of the population-based neuropathology cohort of the Medical Research Council's Cognitive Function and Ageing Study (MRC CFAS) demonstrates that nuclear retention of FOXO3a significantly correlates with a DNA damage response and with GS expression by astrocytes. Furthermore, we show that GS expression correlates with increasing Alzheimer-type pathology in this ageing cohort. Our findings suggest that in response to oxidative stress, the nuclear retention of FOXO3a in astrocytes upregulates expression of GS as a neuroprotective mechanism. However, the activity of GS may be compromised by increasing levels of oxidative stress in the ageing brain resulting in dysfunctional enzyme activity, neuronal excitotoxic damage and cognitive impairment.

  7. A DNA-binding protein factor in K562 nuclear extract interacts with positive control region (PCR) in the 5'-flanking sequence of human β-globin gene

    Institute of Scientific and Technical Information of China (English)

    HUYULONG; YADICHEN; TONGSUN; RUOLANQIAN

    1993-01-01

    It has been known that there are at least three regulatory regions (NCR1. NCR2 and PCR) in the 5'-flanking sequence (from -610 bp to +1 bp) of human β-glohin geneand that the function of PCR is unique to the human erythroleukemia (Ksfi2) ceils. Here we have detected a DNA-binding protein factor (termed NFEa) in K562 ceils. which can bind specifically to the PCR of human β-globin gene. The sequence of the binding site is 5'ACTGATG3' (between -222 bp and -216 bp). The NFEa is erythroidspecific and perhaps specific for K562 cells. It seemed that this factor differed from the erythroid-specific transcriptional factor (NFE-1) ,nsing competition assay. The presence of the NFEa further supported that the funciton of the cis-acting element PCR was specitic for K562 cells. and helps us to understand the mechauism of the regulation of the expression of lmman β-globin gene in the human K562 cells.

  8. Synthesis and antitumor activity evaluation of a novel combi-nitrosourea prodrug: Designed to release a DNA cross-linking agent and an inhibitor of O(6)-alkylguanine-DNA alkyltransferase.

    Science.gov (United States)

    Sun, Guohui; Zhang, Na; Zhao, Lijiao; Fan, Tengjiao; Zhang, Shufen; Zhong, Rugang

    2016-05-01

    The drug resistance of CENUs induced by O(6)-alkylguanine-DNA alkyltransferase (AGT), which repairs the O(6)-alkylated guanine and subsequently inhibits the formation of dG-dC cross-links, hinders the application of CENU chemotherapies. Therefore, the discovery of CENU analogs with AGT inhibiting activity is a promising approach leading to novel CENU chemotherapies with high therapeutic index. In this study, a new combi-nitrosourea prodrug 3-(3-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)-1-(2-chloroethyl)-1-nitrosourea (6), designed to release a DNA cross-linking agent and an inhibitor of AGT, was synthesized and evaluated for its antitumor activity and ability to induce DNA interstrand cross-links (ICLs). The results indicated that 6 exhibited higher cytotoxicity against mer(+) glioma cells compared with ACNU, BCNU, and their respective combinations with O(6)-benzylguanine (O(6)-BG). Quantifications of dG-dC cross-links induced by 6 were performed using HPLC-ESI-MS/MS. Higher levels of dG-dC cross-link were observed in 6-treated human glioma SF763 cells (mer(+)), whereas lower levels of dG-dC cross-link were observed in 6-treated calf thymus DNA, when compared with the groups treated with BCNU and ACNU. The results suggested that the superiority of 6 might result from the AGT inhibitory moiety, which specifically functions in cells with AGT activity. Molecular docking studies indicated that five hydrogen bonds were formed between the O(6)-BG analogs released from 6 and the five residues in the active pocket of AGT, which provided a reasonable explanation for the higher AGT-inhibitory activity of 6 than O(6)-BG. PMID:27041398

  9. 3D-DART: a DNA structure modelling server

    NARCIS (Netherlands)

    van Dijk, M.; Bonvin, A.M.J.J.

    2009-01-01

    There is a growing interest in structural studies of DNA by both experimental and computational approaches. Often, 3D-structural models of DNA are required, for instance, to serve as templates for homology modeling, as starting structures for macro-molecular docking or as scaffold for NMR structure

  10. Electrophoretic Capture of a DNA Chain into a Nanopore

    CERN Document Server

    Rowghanian, Payam

    2013-01-01

    Based on our formulation of the DNA electrophoresis near a pore [P. Rowghanian and A. Y. Grosberg, Phys. Rev. E 87, 042723 (2013)], we address the electrophoretic DNA capture into a nanopore as a steady-state process of particle absorption to a sink placed on top of an energy barrier. Reproducing the previously observed diffusion-limited and barrier-limited regimes as two different limits of the particle absorption process and matching the data, our model suggests a slower growth of the capture rate with the DNA length for very large DNA molecules than the previous model, motivating more experiments beyond the current range of electric field and DNA length. At moderately weak electric fields, our model predicts a different effect, stating that the DNA length dependence of the capture rate first disappears as the field is reduced and eventually reverses to a decreasing trend with $N$.

  11. A DNA vaccine against yellow fever virus: development and evaluation.

    Directory of Open Access Journals (Sweden)

    Milton Maciel

    2015-04-01

    Full Text Available Attenuated yellow fever (YF virus 17D/17DD vaccines are the only available protection from YF infection, which remains a significant source of morbidity and mortality in the tropical areas of the world. The attenuated YF virus vaccine, which is used worldwide, generates both long-lasting neutralizing antibodies and strong T-cell responses. However, on rare occasions, this vaccine has toxic side effects that can be fatal. This study presents the design of two non-viral DNA-based antigen formulations and the characterization of their expression and immunological properties. The two antigen formulations consist of DNA encoding the full-length envelope protein (p/YFE or the full-length envelope protein fused to the lysosomal-associated membrane protein signal, LAMP-1 (pL/YFE, aimed at diverting antigen processing/presentation through the major histocompatibility complex II precursor compartments. The immune responses triggered by these formulations were evaluated in H2b and H2d backgrounds, corresponding to the C57Bl/6 and BALB/c mice strains, respectively. Both DNA constructs were able to induce very strong T-cell responses of similar magnitude against almost all epitopes that are also generated by the YF 17DD vaccine. The pL/YFE formulation performed best overall. In addition to the T-cell response, it was also able to stimulate high titers of anti-YF neutralizing antibodies comparable to the levels elicited by the 17DD vaccine. More importantly, the pL/YFE vaccine conferred 100% protection against the YF virus in intracerebrally challenged mice. These results indicate that pL/YFE DNA is an excellent vaccine candidate and should be considered for further developmental studies.

  12. Telomerase RNA is more than a DNA template.

    Science.gov (United States)

    Webb, Christopher J; Zakian, Virginia A

    2016-08-01

    The addition of telomeric DNA to chromosome ends is an essential cellular activity that compensates for the loss of genomic DNA that is due to the inability of the conventional DNA replication apparatus to duplicate the entire chromosome. The telomerase reverse transcriptase and its associated RNA bind to the very end of the telomere via a sequence in the RNA and specific protein-protein interactions. Telomerase RNA also provides the template for addition of new telomeric repeats by the reverse-transcriptase protein subunit. In addition to the template, there are 3 other conserved regions in telomerase RNA that are essential for normal telomerase activity. Here we briefly review the conserved core regions of telomerase RNA and then focus on a recent study in fission yeast that determined the function of another conserved region in telomerase RNA called the Stem Terminus Element (STE). (1) The STE is distant from the templating core of telomerase in both the linear and RNA secondary structure, but, nonetheless, affects the fidelity of telomere sequence addition and, in turn, the ability of telomere binding proteins to bind and protect chromosome ends. We will discuss possible mechanisms of STE action and the suitability of the STE as an anti-cancer target. PMID:27245259

  13. Electrophoresis of a DNA Coil Near a Nanopore

    CERN Document Server

    Rowghanian, Payam

    2013-01-01

    Motivated by DNA electrophoresis near a nanopore, we consider the flow field around an "elongated jet", a long thin source which injects momentum into a liquid. This solution qualitatively describes the electro-osmotic flow around a long rigid polymer, where due to electrohydrodynamic coupling, the solvent receives momentum from the electric field. Based on the qualitative behavior of the elongated jet solution, we develop a coarse-grained scheme which reproduces the known theoretical results regarding the electrophoretic behavior of a long rigid polymer and a polymer coil in a uniform field, which we then exploit to analyze the electrophoresis of a polymer coil in the non-uniform field near a nanopore.

  14. Development of a DNA sensor using molecular logic gate

    CERN Document Server

    Bhattacharjee, D; Chakraborty, S; Hussain, Syed Arshad

    2014-01-01

    This communication reports the increase in fluorescence resonance energy transfer (FRET) efficiency between two laser dyes in presence of Deoxyribonucleic acid (DNA). Two types of molecular logic gates have been designed where DNA acts as input signal and fluorescence intensity of different bands are taken as output signal. Use of these logic gates as DNA sensor has been demonstrated

  15. A DNA barcoding approach in the study of tardigrades

    Directory of Open Access Journals (Sweden)

    Michele Cesari

    2013-05-01

    Full Text Available DNA barcoding is a technique proposed by Hebert and co-workers in 2003 for discriminating species through analysis of a single gene barcode locus. It aims to obtain a better taxonomic resolution than that achieved through morphological studies, and to avoid the decline in taxonomic knowledge. Today DNA barcoding is a global enterprise, and the implementation of the idea has seen a rapid rise (more than 1900 papers published to date on different organisms. Nonetheless, controversy still arises regarding barcoding and taxonomy. It is important to note that DNA barcoding does not focus on building a tree-of-life or on doing DNA taxonomy, even though sometimes it has been used for these purposes. DNA barcoding rather focuses on producing a universal molecular identification key based on strong taxonomic knowledge that should be included in the barcode reference library. In the phylum Tardigrada, DNA barcoding represents a recent approach to species identification and to help in solving taxonomic problems, especially considering the diminutive size of these animals and the paucity of morphological characters useful for taxonomy. In the framework of the MoDNA Project (Morphology and DNA, carried out by our research group in collaboration with several colleagues, we are combining the study of a fragment of the mitochondrial cytochrome c oxidase subunit I gene (cox1 with morphological data, in a wide sense (cuticular structures, chromosomes, data on sex ratio and reproduction, to form an integrative taxonomy approach for tardigrade species identification. We believe that without verified reference sequences from voucher specimens that have been authenticated by qualified taxonomists, there is no reliable library for newly generated sequences with which to be compared. Methods and protocols for standardized results are focused on obtaining tight correspondence between tardigrade morphology (and egg shell morphology, when useful, possibly both light and scanning electron microscopy images, and molecular sequence. This approach is particularly useful in describing new species, and important when applied on material collected in species type localities. Results using this approach are presented, primarily focusing on a number of species from the so-called Macrobiotus hufelandi group.

  16. Conformation dependent electronic transport in a DNA double-helix

    International Nuclear Information System (INIS)

    We present a tight-binding study of conformation dependent electronic transport properties of DNA double-helix including its helical symmetry. We have studied the changes in the localization properties of DNA as we alter the number of stacked bases within every pitch of the double-helix keeping fixed the total number of nitrogen bases within the DNA molecule. We take three DNA sequences, two of them are periodic and one is random and observe that in all the cases localization length increases as we increase the radius of DNA double-helix i.e., number of nucleobases within a pitch. We have also investigated the effect of backbone energetic on the I-V response of the system and found that in presence of helical symmetry, depending on the interplay of conformal variation and disorder, DNA can be found in either metallic, semiconducting and insulating phases, as observed experimentally

  17. Divergent Requirement for a DNA Repair Enzyme during Enterovirus Infections

    Directory of Open Access Journals (Sweden)

    Sonia Maciejewski

    2015-12-01

    Full Text Available Viruses of the Enterovirus genus of picornaviruses, including poliovirus, coxsackievirus B3 (CVB3, and human rhinovirus, commandeer the functions of host cell proteins to aid in the replication of their small viral genomic RNAs during infection. One of these host proteins is a cellular DNA repair enzyme known as 5′ tyrosyl-DNA phosphodiesterase 2 (TDP2. TDP2 was previously demonstrated to mediate the cleavage of a unique covalent linkage between a viral protein (VPg and the 5′ end of picornavirus RNAs. Although VPg is absent from actively translating poliovirus mRNAs, the removal of VPg is not required for the in vitro translation and replication of the RNA. However, TDP2 appears to be excluded from replication and encapsidation sites during peak times of poliovirus infection of HeLa cells, suggesting a role for TDP2 during the viral replication cycle. Using a mouse embryonic fibroblast cell line lacking TDP2, we found that TDP2 is differentially required among enteroviruses. Our single-cycle viral growth analysis shows that CVB3 replication has a greater dependency on TDP2 than does poliovirus or human rhinovirus replication. During infection, CVB3 protein accumulation is undetectable (by Western blot analysis in the absence of TDP2, whereas poliovirus protein accumulation is reduced but still detectable. Using an infectious CVB3 RNA with a reporter, CVB3 RNA could still be replicated in the absence of TDP2 following transfection, albeit at reduced levels. Overall, these results indicate that TDP2 potentiates viral replication during enterovirus infections of cultured cells, making TDP2 a potential target for antiviral development for picornavirus infections.

  18. A DNA mini-barcode for land plants.

    Science.gov (United States)

    Little, Damon P

    2014-05-01

    Small portions of the barcode region - mini-barcodes - may be used in place of full-length barcodes to overcome DNA degradation for samples with poor DNA preservation. 591,491,286 rbcL mini-barcode primer combinations were electronically evaluated for PCR universality, and two novel highly universal sets of priming sites were identified. Novel and published rbcL mini-barcode primers were evaluated for PCR amplification [determined with a validated electronic simulation (n = 2765) and empirically (n = 188)], Sanger sequence quality [determined empirically (n = 188)], and taxonomic discrimination [determined empirically (n = 30,472)]. PCR amplification for all mini-barcodes, as estimated by validated electronic simulation, was successful for 90.2-99.8% of species. Overall Sanger sequence quality for mini-barcodes was very low - the best mini-barcode tested produced sequences of adequate quality (B20 ≥ 0.5) for 74.5% of samples. The majority of mini-barcodes provide correct identifications of families in excess of 70.1% of the time. Discriminatory power noticeably decreased at lower taxonomic levels. At the species level, the discriminatory power of the best mini-barcode was less than 38.2%. For samples believed to contain DNA from only one species, an investigator should attempt to sequence, in decreasing order of utility and probability of success, mini-barcodes F (rbcL1/rbcLB), D (F52/R193) and K (F517/R604). For samples believed to contain DNA from more than one species, an investigator should amplify and sequence mini-barcode D (F52/R193). PMID:24286499

  19. Detection of Toxoplasma gondii with a DNA molecular beacon probe

    Science.gov (United States)

    Zhou, Cun; Xu, Shichao; Yang, Juan; Zhang, Jimei; Dai, Zhao; Zheng, Guo; Sun, Bo; Sun, Shuqing; Feng, Teilin; Zi, Yan; Liang, Chu; Luo, Hao

    2009-07-01

    Toxoplasma gondii is a kind of microscopic parasite that may infect humans, and there are increasing concerns on the early detection of latent Toxoplasma gondii infection in recent years. This research highlights a new type of molecular beacon (MB) fluorescent probe for Toxoplasma DNA testing. We combined high-efficiency fluorescent inorganic core-shell quantum dots-CdTe/ZnS (as fluorescent energy donor) and BHQ-2 (energy acceptor) to the single-strand DNA of Toxoplasma gondii, and a molecular beacon sensing system based on fluorescence resonance energy transfer (FRET) was achieved. Core-shell quantum dots CdTe/ZnS was firstly prepared in aqueous solution, and the influencing factor of its fluorescent properties, including CdTe/Na2S/Zn(CH3COO)2 (v/v), dependence of reaction time, temperature, and pH, is investigated systematically. The synthesized quantum dots and molecular beacon were characterized by transmission electron microscopy (TEM), ultraviolet-visible spectrophotometer (UV-vis), fluorescent spectrophotometer (FS), respectively. The TEM results showed that CdTe/ZnS core-shell quantum dots is ~11nm in size, and the quantum dots is water-soluble well. The sensing ability of target DNA of assembled MB was investigated, and results showed that the target Toxoplasma gonddi DNA can be successfully detected by measuring the change of fluorescence intensity. The results showed that the current sensing probe will be a useful and convenient tool in Toxoplasma gondii early detection.

  20. The path for metal complexes to a DNA target.

    Science.gov (United States)

    Komor, Alexis C; Barton, Jacqueline K

    2013-05-01

    The discovery of cisplatin as a therapeutic agent stimulated a new era in the application of transition metal complexes for therapeutic design. Here we describe recent results on a variety of transition metal complexes targeted to DNA to illustrate many of the issues involved in new therapeutic design. We describe first structural studies of complexes bound covalently and non-covalently to DNA to identify potential lesions within the cell. We then review the biological fates of these complexes, illustrating the key elements in obtaining potent activity, the importance of uptake and subcellular localization of the complexes, as well as the techniques used to delineate these characteristics. Genomic DNA provides a challenging but valuable target for new transition metal-based therapeutics.

  1. Scaffolded DNA Origami of a DNA Tetrahedron Molecular Container

    DEFF Research Database (Denmark)

    Ke, Yongang; Sharma, Jaswinder; Liu, Minghui;

    2009-01-01

    We describe a strategy of scaffolded DNA origami to design and construct 3D molecular cages of tetrahedron geometry with inside volume closed by triangular faces. Each edge of the triangular face is ∼54 nm in dimension. The estimated total external volume and the internal cavity of the triangular...... pyramid are about 1.8 × 10-23 and 1.5 × 10-23 m3, respectively. Correct formation of the tetrahedron DNA cage was verified by gel electrophoresis, atomic force microscopy, transmission electron microscopy, and dynamic light scattering techniques....

  2. A DNA vaccine against yellow fever virus: development and evaluation.

    Science.gov (United States)

    Maciel, Milton; Cruz, Fábia da Silva Pereira; Cordeiro, Marli Tenório; da Motta, Márcia Archer; Cassemiro, Klécia Marília Soares de Melo; Maia, Rita de Cássia Carvalho; de Figueiredo, Regina Célia Bressan Queiroz; Galler, Ricardo; Freire, Marcos da Silva; August, Joseph Thomas; Marques, Ernesto T A; Dhalia, Rafael

    2015-04-01

    Attenuated yellow fever (YF) virus 17D/17DD vaccines are the only available protection from YF infection, which remains a significant source of morbidity and mortality in the tropical areas of the world. The attenuated YF virus vaccine, which is used worldwide, generates both long-lasting neutralizing antibodies and strong T-cell responses. However, on rare occasions, this vaccine has toxic side effects that can be fatal. This study presents the design of two non-viral DNA-based antigen formulations and the characterization of their expression and immunological properties. The two antigen formulations consist of DNA encoding the full-length envelope protein (p/YFE) or the full-length envelope protein fused to the lysosomal-associated membrane protein signal, LAMP-1 (pL/YFE), aimed at diverting antigen processing/presentation through the major histocompatibility complex II precursor compartments. The immune responses triggered by these formulations were evaluated in H2b and H2d backgrounds, corresponding to the C57Bl/6 and BALB/c mice strains, respectively. Both DNA constructs were able to induce very strong T-cell responses of similar magnitude against almost all epitopes that are also generated by the YF 17DD vaccine. The pL/YFE formulation performed best overall. In addition to the T-cell response, it was also able to stimulate high titers of anti-YF neutralizing antibodies comparable to the levels elicited by the 17DD vaccine. More importantly, the pL/YFE vaccine conferred 100% protection against the YF virus in intracerebrally challenged mice. These results indicate that pL/YFE DNA is an excellent vaccine candidate and should be considered for further developmental studies. PMID:25875109

  3. 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.

  4. Molecular dynamics simulation of peeling a DNA molecule on substrate

    Institute of Scientific and Technical Information of China (English)

    Xinghua Shi; Yong Kong; Yapu Zhao; Huajian Gao

    2005-01-01

    Molecular dynamics (MD) simulations are performed to study adhesion and peeling of a short fragment of single strand DNA (ssDNA) molecule from a graphite surface. The critical peel-off force is found to depend on both the peeling angle and the elasticity of ssDNA. For the short ssDNA strand under investigation, we show that the simulation results can be explained by a continuum model of an adhesive elastic band on substrate. The analysis suggests that it is often the peak value, rather than the mean value, of adhesion energy which determines the peeling of a nanoscale material.

  5. Dissolving Hydroxyolite: A DNA Molecule into Its Hydroxyapatite Mold.

    Science.gov (United States)

    Bertran, Oscar; Revilla-López, Guillermo; Casanovas, Jordi; Del Valle, Luis J; Turon, Pau; Puiggalí, Jordi; Alemán, Carlos

    2016-05-01

    In spite of the clinical importance of hydroxyapatite (HAp), the mechanism that controls its dissolution in acidic environments remains unclear. Knowledge of such a process is highly desirable to provide better understanding of different pathologies, as for example osteoporosis, and of the HAp potential as vehicle for gene delivery to replace damaged DNA. In this work, the mechanism of dissolution in acid conditions of HAp nanoparticles encapsulating double-stranded DNA has been investigated at the atomistic level using computer simulations. For this purpose, four consecutive (multi-step) molecular dynamics simulations, involving different temperatures and proton transfer processes, have been carried out. Results are consistent with a polynuclear decalcification mechanism in which proton transfer processes, from the surface to the internal regions of the particle, play a crucial role. In addition, the DNA remains protected by the mineral mold and transferred proton from both temperature and chemicals. These results, which indicate that biomineralization imparts very effective protection to DNA, also have important implications in other biomedical fields, as for example in the design of artificial bones or in the fight against osteoporosis by promoting the fixation of Ca(2+) ions.

  6. Ag85A DNA疫苗加强免疫显著提高卡介苗初免小鼠的抗结核T细胞免疫应答%Ag85A DNA vaccination boosting enhances BCG primed-mice anti-tuberculosis T cell responses

    Institute of Scientific and Technical Information of China (English)

    康涵; 范小勇; 袁琴; 吴福明; 沈芳

    2013-01-01

    Objective To construct DNA vaccine expressing Mycobacterium tuberculosis(Mtb) immunodominant antigen Ag85A and analyze its anti-tuberculosis T cell responses in BCG primed-mice after DNA vaccination boosting.Methods The coding gene of Ag85A mature fragment was amplified by PCR with H37Rv genomic DNA as template,and then cloned into the eukaryotic expression vector pVAX1 to construct Ag85A DNA vaccine.After purification,Ag85A DNA vaccine was injected intramuscularly twice in BCG primed-mice with BCG vaccination and DNA vaccination alone as control.Eight weeks post-vaccination,spleen lymphocytes were separated and were then used to analyze Mtb antigen specific effector T cell response and polyfuntional IFN-γ/TNF-α/IL-2 secreting CD4+ T cell frequencies and intensities,and CD8+T cell responses by IFN-γ ELISPOT assay and intracellular staining,respectively.Results Compared to BCG vaccinated-and DNA vaccinated-mice,Ag85A DNA boosting not only enhanced significantly BCG primed-mice IFN-γ+TNF-α+IL-2+,IFN-γ+ IL-2+,TNF-α+IL-2+ and IL-2+ CD4+ T cell frequencies and IL-2 secretion,but also improved significantly IFN-γ-secreting and IL-2-secreting CD8+ T cell frequencies.Condusion Ag85A DNA vaccine was constructed successfully and was demonstrated to enhance significantly BCG primed-mice Mtb antigen specific CD4+ and CD8+ T cell responses when boosting,which is beneficial to improve BCG immunogenicity and its waning immune protection against Mtb.%目的 构建表达结核分枝杆菌(Mycobacterium tuberculosis,Mtb)免疫优势抗原Ag85A的DNA疫苗,分析其加强免疫后提高卡介苗(BCG)初免小鼠的抗结核T细胞免疫应答.方法 以Mtb毒株H37Rv基因组DNA为模板,PCR扩增Ag85A抗原编码的结构基因并克隆至真核表达载体pVAX1中构建其DNA疫苗;接着,将纯化后的该DNA疫苗加强免疫BCG初免小鼠2针,以BCG和DNA单独免疫小鼠为对照,免疫8周后无菌分离脾淋巴细胞,分别应用IFN-γ ELISPOT和多

  7. The use of cycleave PCR for the differentiation of the rejuvenating herb species Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea), and Mucuna macrocarpa (Black Kwao Khruea), and the simultaneous detection of multiple DNA targets in a DNA admixture.

    Science.gov (United States)

    Wiriyakarun, Suchaya; Zhu, Shu; Komatsu, Katsuko; Sukrong, Suchada

    2014-01-01

    Kwao Khruea, the tuberous roots of Pueraria candollei Graham ex Benth. (White Kwao Khruea), Butea superba Roxb. (Red Kwao Khruea), and Mucuna macrocarpa Wall. (Black Kwao Khruea), are used as rejuvenating herbs in traditional medicine in many tropical countries. Although Kwao Khruea has attracted strong interest because of its rejuvenation properties, each species is used for specific purposes and effects. P. candollei shows estrogenic effects in females. In contrast, B. superba and M. macrocarpa show androgenic effects in males. The potential misidentification of dried tuberous roots of various Kwao Khruea species might cause problems in the drug market, especially when they are reduced into powders. A cycleave PCR, which is based on the sequence of chloroplast matK gene, was developed to differentiate P. candollei, B. superba, and M. macrocarpa. The results showed that cycleave PCR is able to identify specific Kwao Khruea species. A multiplex cycleave PCR was optimized for the simultaneous detection of two different DNA targets in a DNA admixture. The specificity of this technique was confirmed by its ability to distinguish M. macrocarpa from five related Mucuna species. Cycleave PCR can be a specific, sensitive, and rapid method for the identification of medicinal plants and crude plant samples. PMID:24660477

  8. The design and synthesis of novel heterodinuclear complexes combining a DNA-cleaving agent and a DNA-targeting moiety

    NARCIS (Netherlands)

    Hoog, Paul de

    2008-01-01

    Cancer is a leading cause of death worldwide. Nowadays, the treatment of cancer by chemotherapy can consist of a combination of antitumor drugs. Nevertheless, chemotherapy is accompanied by serious side effects and intrinsic and acquired resistance to the drugs. This thesis describes the design and

  9. Immunogenic and protective effects of a DNA vaccine containing flagellin flaC gene against Vibrio alginolyticus in red snapper(Lutjanus sanguineus)%溶藻弧菌鞭毛蛋白flaC基因DNA疫苗对红笛鲷的免疫保护

    Institute of Scientific and Technical Information of China (English)

    梁海鹰; 陈永新; 简纪常; 吴灶和

    2013-01-01

    In order to study the immunogenic and protective effects of DNA vaccine, plasmid DNA encoding flagellin flaC gene ( designated as pcDNA-flaC ) was used as a DNA vaccine to immunize red snapper (Lutjanus sanguineus). The distribution, expression and immunoprotection of the DNA vaccine were analyzed in tissues of the red snapper by PCR, RT-PCR and challenge test. PCR results indicated that pcDNA-flaC was distributed in liver, spleen, kidney, gill and injection site muscle at 7 - 28 days after vaccination. RT-PCR results indicated that the flaC gene was expressed in all above tissues of vaccinated fish at 7 -28 days after vaccination. These results demonstrated that the DNA vaccine was distributed and flaC gene was expressed in various tissues of vaccinated fish. Red snapper immunized with DNA vaccine showed higher serum antibody levels at 7 - 28 days after vaccination, compared to fish vaccinated with the control eukaryotic expression vector pcDNA3. 1 and PBS. In addition,fish immunized with DNA vaccine developed a protective response to live V. alginolyticus challenge 28 days post-inoculation, as demonstrated by increased survival of vaccinated fish over the control fish. This study indicates that pcDNA-flaC is an effective vaccine candidate against V. alginolyticus infection.%为研究溶藻弧菌鞭毛蛋白flaC基因DNA疫苗对红笛鲷的免疫保护作用,实验构建了重组真核表达质粒pcDNA-flaC并将该质粒肌肉注射红笛鲷,采用PCR、RT-PCR、ELISA和攻毒试验等方法检测了该真核表达质粒在红笛鲷组织内的分布、表达和对红笛鲷的免疫保护.PCR结果显示,免疫接种7和28 d,注射点周围肌肉、鳃、肾脏、肝脏和脾脏都存在质粒分布;RT-PCR结果显示,免疫接种后第7天、14天和28天,红笛鲷不同组织内均有目的基因表达.ELISA结果表明,鱼血清内产生了抗FlaC蛋白的抗体,表明DNA疫苗免疫后鱼体表达了目的蛋白,并诱导产生了相应抗体.攻毒实验

  10. Effects of Gold Nanoparticles on Quantum Dot Electrochemiluminescence Obtained Using a DNA Electrochemiluminescence Sensor%基于DNA电化学发光传感器研究金纳米颗粒对量子点的电化学发光影响

    Institute of Scientific and Technical Information of China (English)

    鲁理平; 李娇; 武静; 康天放; 程水源

    2015-01-01

    Gold nanoparticles (AuNPs) have a high extinction coefficient and a strong surface plasmon resonance, the latter of which is influenced by the size of AuNPs and the surrounding environment. In this article, a DNA electrochemiluminescence (ECL) sensor was fabricated based on the distance-dependence of semiconductor nanocrystals' ECL signal to AuNPs. AuNPs were first deposited on the surface of glassy carbon electrode (GCE) by cyclic voltammetry (CV). The mercaptopropionic acid-capped CdS quantum dots (QDs) used in this study can covalently bind with amino-terminated double-stranded DNA (dsDNA), via the―CO―NH bond to obtain a QDs-dsDNA compound. The QDs-dsDNA compounds were assembled on the surface of AuNPs via an Au―S bond, using the other distal of dsDNA that is labeled with thiol, to create the CdS QDs-DNA/AuNPs/GCE ECL sensor. Experimental conditions, such as the QDs-dsDNA density on the surface of electrode and the deposition method of AuNPs, were then optimized. The surface properties of different modified electrodes were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), and electrochemical impedance spectroscopy (EIS). The effect of AuNPs on the ECL intensity of CdS QDs was investigated by control ing the DNA which lies between the AuNPs and the CdS QDs. The ECL signal was affected significantly by the length and type of DNA strands. The sensor was used to detect DNA damage from environmental pol utants and exhibited a highly sensitive response.%纳米金颗粒具有高的消光系数和良好的表面等离子体共振特性,其等离子体共振特性受纳米金颗粒的尺寸和周围环境等因素的影响。本文基于半导体纳米晶电化学发光信号对金纳米颗粒的距离依赖性制备了DNA电化学发光传感器。首先利用循环伏安法(CV)在玻碳电极(GCE)表面原位沉积金纳米颗粒(AuNPs),巯基丙酸包裹的CdS量子点(QDs)与氨基修饰的双链DNA (dsDNA)通过酰胺键

  11. The use of Listeria monocytogenes as a DNA delivery vector for cancer gene therapy.

    LENUS (Irish Health Repository)

    Tangney, Mark

    2012-01-31

    Listeria monocytogenes is an intracellular pathogen that lyses the phagosomal vacuole of infected cells, proliferates in the host cell cytoplasm and can actively enter adjacent cells. The pathogen is therefore well suited to exploitation as a vector for the delivery of DNA to target cells as the lifecycle favors cellular targeting with vector amplification and the potential for cell-to-cell spread. We have recently demonstrated DNA transfer by L. monocytogenes in growing tumors in murine models. Our approach exploited an ampicillin sensitive stain of L. monocytogenes which can be lysed through systemic administration of ampicillin to facilitate release of plasmid DNA for expression by infected mammalian cells. Here, we discuss the implications of this technology and the potential for future improvements of the system.

  12. Bacterial pathogen gene regulation: a DNA-structure-centred view of a protein-dominated domain.

    Science.gov (United States)

    Dorman, Charles J; Colgan, Aoife; Dorman, Matthew J

    2016-07-01

    The mechanisms used by bacterial pathogens to regulate the expression of their genes, especially their virulence genes, have been the subject of intense investigation for several decades. Whole genome sequencing projects, together with more targeted studies, have identified hundreds of DNA-binding proteins that contribute to the patterns of gene expression observed during infection as well as providing important insights into the nature of the gene products whose expression is being controlled by these proteins. Themes that have emerged include the importance of horizontal gene transfer to the evolution of pathogens, the need to impose regulatory discipline upon these imported genes and the important roles played by factors normally associated with the organization of genome architecture as regulatory principles in the control of virulence gene expression. Among these architectural elements is the structure of DNA itself, its variable nature at a topological rather than just at a base-sequence level and its ability to play an active (as well as a passive) part in the gene regulation process. PMID:27252403

  13. Force-Induced Rupture of a DNA Duplex: From Fundamentals to Force Sensors.

    Science.gov (United States)

    Mosayebi, Majid; Louis, Ard A; Doye, Jonathan P K; Ouldridge, Thomas E

    2015-12-22

    The rupture of double-stranded DNA under stress is a key process in biophysics and nanotechnology. In this article, we consider the shear-induced rupture of short DNA duplexes, a system that has been given new importance by recently designed force sensors and nanotechnological devices. We argue that rupture must be understood as an activated process, where the duplex state is metastable and the strands will separate in a finite time that depends on the duplex length and the force applied. Thus, the critical shearing force required to rupture a duplex depends strongly on the time scale of observation. We use simple models of DNA to show that this approach naturally captures the observed dependence of the force required to rupture a duplex within a given time on duplex length. In particular, this critical force is zero for the shortest duplexes, before rising sharply and then plateauing in the long length limit. The prevailing approach, based on identifying when the presence of each additional base pair within the duplex is thermodynamically unfavorable rather than allowing for metastability, does not predict a time-scale-dependent critical force and does not naturally incorporate a critical force of zero for the shortest duplexes. We demonstrate that our findings have important consequences for the behavior of a new force-sensing nanodevice, which operates in a mixed mode that interpolates between shearing and unzipping. At a fixed time scale and duplex length, the critical force exhibits a sigmoidal dependence on the fraction of the duplex that is subject to shearing.

  14. Complementation of a DNA repair defect in xeroderma pigmentosum cells by transfer of human chromosome 9

    International Nuclear Information System (INIS)

    Complementation of the repair defect in xeroderma pigmentosum cells of complementation group A was achieved by the transfer of human chromosome 9. A set of mouse-human hybrid cell lines, each containing a single Ecogpt-marked human chromosome, was used as a source of donor chromosomes. Chromosome transfer to XPTG-1 cells, a hypoxanthine/guanine phosphoribosyltransferase-deficient mutant of simian virus 40-transformed complementation group A cells, was achieved by microcell fusion and selection for Ecogpt. Chromosome-transfer clones of XPTG-1 cells, each containing a different human donor chromosome, were analyzed for complementation of sensitivity to UV irradiation. Among all the clones, increased levels of resistance to UV was observed only in clones containing chromosome 9. Since our recipient cell line XPTG-1 is hypoxanthine/guanine phosphoribosyltransferase deficient, cultivation of Ecogpt+ clones in medium containing 6-thioguanine permits selection of cells for loss of the marker and, by inference, transferred chromosome 9. Clones isolated for growth in 6-thioguanine, which have lost the Ecogpt-marked chromosome, exhibited a UV-sensitive phenotype, confirming the presence of the repair gene(s) for complementation group A on chromosome 9

  15. Characterisation of a DNA sequence element that directs Dictyostelium stalk cell-specific gene expression.

    Science.gov (United States)

    Ceccarelli, A; Zhukovskaya, N; Kawata, T; Bozzaro, S; Williams, J

    2000-12-01

    The ecmB gene of Dictyostelium is expressed at culmination both in the prestalk cells that enter the stalk tube and in ancillary stalk cell structures such as the basal disc. Stalk tube-specific expression is regulated by sequence elements within the cap-site proximal part of the promoter, the stalk tube (ST) promoter region. Dd-STATa, a member of the STAT transcription factor family, binds to elements present in the ST promoter-region and represses transcription prior to entry into the stalk tube. We have characterised an activatory DNA sequence element, that lies distal to the repressor elements and that is both necessary and sufficient for expression within the stalk tube. We have mapped this activator to a 28 nucleotide region (the 28-mer) within which we have identified a GA-containing sequence element that is required for efficient gene transcription. The Dd-STATa protein binds to the 28-mer in an in vitro binding assay, and binding is dependent upon the GA-containing sequence. However, the ecmB gene is expressed in a Dd-STATa null mutant, therefore Dd-STATa cannot be responsible for activating the 28-mer in vivo. Instead, we identified a distinct 28-mer binding activity in nuclear extracts from the Dd-STATa null mutant, the activity of this GA binding activity being largely masked in wild type extracts by the high affinity binding of the Dd-STATa protein. We suggest, that in addition to the long range repression exerted by binding to the two known repressor sites, Dd-STATa inhibits transcription by direct competition with this putative activator for binding to the GA sequence.

  16. Novel dnaG mutation in a dnaP mutant of Escherichia coli.

    OpenAIRE

    Murakami, Y.; Nagata, T; Schwarz, W.; Wada, C.; Yura, T

    1985-01-01

    Reexamination of the dnaP18 mutant strain of Escherichia coli revealed that the mutation responsible for the arrest of DNA replication and cell growth at high temperatures resides in the dnaG gene rather than in the dnaP locus as previously thought; this mutation has been designated dnaG2903.

  17. Design of a DNA chip for detection of unknown genetically modified organisms (GMOs)

    OpenAIRE

    2003-01-01

    Knowing the extent and nature of genetically modified (GM) ingredients in food products has become increasingly important for food exporters, importers, retailers and consumers. In this thesis a model for detecting unknown genetically modified organisms (GMOs) by utilization of a high-density synthetic oligonucleotide array (DNA chip) is presented. Biological and combinatorial reduction rules are applied on a set of probes containing all possible sequences of a uniform length n, ...

  18. A DNA extraction protocol for improved DNA yield from individual mosquitoes [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Catelyn C. Nieman

    2015-11-01

    Full Text Available Typical DNA extraction protocols from commercially available kits provide an adequate amount of DNA from a single individual mosquito sufficient for PCR-based assays. However, next-generation sequencing applications and high-throughput SNP genotyping assays exposed the limitation of DNA quantity one usually gets from a single individual mosquito. Whole genome amplification could alleviate the issue but it also creates bias in genome representation. While trying to find alternative DNA extraction protocols for improved DNA yield, we found that a combination of the tissue lysis protocol from Life Technologies and the DNA extraction protocol from Qiagen yielded a higher DNA amount than the protocol using the Qiagen or Life Technologies kit only. We have not rigorously tested all the possible combinations of extraction protocols; we also only tested this on mosquito samples. Therefore, our finding should be noted as a suggestion for improving people’s own DNA extraction protocols and not as an advertisement of a commercially available product.

  19. Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays.

    Science.gov (United States)

    Kojima, Takaaki; Mizoguchi, Takuro; Ota, Eri; Hata, Jumpei; Homma, Keisuke; Zhu, Bo; Hitomi, Kiyotaka; Nakano, Hideo

    2016-02-01

    A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities.

  20. A DNA damage-induced, SOS-independent checkpoint regulates cell division in Caulobacter crescentus.

    Science.gov (United States)

    Modell, Joshua W; Kambara, Tracy K; Perchuk, Barrett S; Laub, Michael T

    2014-10-01

    Cells must coordinate DNA replication with cell division, especially during episodes of DNA damage. The paradigm for cell division control following DNA damage in bacteria involves the SOS response where cleavage of the transcriptional repressor LexA induces a division inhibitor. However, in Caulobacter crescentus, cells lacking the primary SOS-regulated inhibitor, sidA, can often still delay division post-damage. Here we identify didA, a second cell division inhibitor that is induced by DNA damage, but in an SOS-independent manner. Together, DidA and SidA inhibit division, such that cells lacking both inhibitors divide prematurely following DNA damage, with lethal consequences. We show that DidA does not disrupt assembly of the division machinery and instead binds the essential division protein FtsN to block cytokinesis. Intriguingly, mutations in FtsW and FtsI, which drive the synthesis of septal cell wall material, can suppress the activity of both SidA and DidA, likely by causing the FtsW/I/N complex to hyperactively initiate cell division. Finally, we identify a transcription factor, DriD, that drives the SOS-independent transcription of didA following DNA damage. PMID:25350732

  1. Design of a DNA panel for genomic studies in Russian cattle breeds

    Science.gov (United States)

    A panel of 96 DNA samples (Russian Cattle Genomic Diversity Panel 1.0 or RCGDP 1.0) characterizing the breadth of genetic diversity in popular Russian cattle breeds was designed. The panel contains from four to eight animals from each of 11 dairy and six dairy-meat and meat breeds. The main criterio...

  2. Quantification of Fewer than Ten Copies of a DNA Biomarker without Amplification or Labeling.

    Science.gov (United States)

    Lee, Yoonhee; Kim, Youngkyu; Lee, Donggyu; Roy, Dhruvajyoti; Park, Joon Won

    2016-06-01

    Polymerase chain reaction (PCR) is a highly sensitive diagnosis technique for detection of nucleic acids and for monitoring residual disease; however, PCR can be unreliable for samples containing very few target molecules. Here, we describe a quantification method, using force-distance (FD) curve based atomic force microscopy (AFM) to detect a target DNA bound to small (1.4-1.9 μm diameter) probe DNA spots, allowing mapping of entire spots to nanometer resolution. Using a synthetic BCR-ABL fusion gene sequence target, we examined samples containing between one and 10 target copies. A high degree of correlation (r(2) = 0.994) between numbers of target copies and detected probe clusters was observed, and the approach could detect the BCR-ABL biomarker when only a single copy was present, although multiple screens were required. Our results clearly demonstrate that FD curve-based imaging is suitable for quantitative analysis of fewer than 10 copies of DNA biomarkers without amplification, modification, or labeling. PMID:27175474

  3. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

    Directory of Open Access Journals (Sweden)

    Regina Stoltenburg

    Full Text Available A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

  4. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

    Science.gov (United States)

    Díaz-Badillo, Alvaro; de Lourdes Muñoz, María; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G.; Martínez-Muñoz, Jorge P.; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano

    2014-01-01

    Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples. PMID:24776933

  5. Priming of microglia in a DNA-repair deficient model of accelerated aging

    NARCIS (Netherlands)

    Raj, Divya D. A.; Jaarsma, Dick; Holtman, Inge R.; Olah, Marta; Ferreira, Filipa M.; Schaafsma, Wandert; Brouwer, Nieske; Meijer, Michel M.; de Waard, Monique C.; van der Pluijm, Ingrid; Brandt, Renata; Kreft, Karim L.; Laman, Jon D.; de Haan, Gerald; Biber, Knut P. H.; Hoeijmakers, Jan H. J.; Eggen, Bart J. L.; Boddeke, Hendrikus W. G. M.

    2014-01-01

    Aging is associated with reduced function, degenerative changes, and increased neuroinflammation of the central nervous system (CNS). Increasing evidence suggests that changes in microglia cells contribute to the age-related deterioration of the CNS. The most prominent age-related change of microgli

  6. A DNA damage-induced, SOS-independent checkpoint regulates cell division in Caulobacter crescentus.

    Directory of Open Access Journals (Sweden)

    Joshua W Modell

    2014-10-01

    Full Text Available Cells must coordinate DNA replication with cell division, especially during episodes of DNA damage. The paradigm for cell division control following DNA damage in bacteria involves the SOS response where cleavage of the transcriptional repressor LexA induces a division inhibitor. However, in Caulobacter crescentus, cells lacking the primary SOS-regulated inhibitor, sidA, can often still delay division post-damage. Here we identify didA, a second cell division inhibitor that is induced by DNA damage, but in an SOS-independent manner. Together, DidA and SidA inhibit division, such that cells lacking both inhibitors divide prematurely following DNA damage, with lethal consequences. We show that DidA does not disrupt assembly of the division machinery and instead binds the essential division protein FtsN to block cytokinesis. Intriguingly, mutations in FtsW and FtsI, which drive the synthesis of septal cell wall material, can suppress the activity of both SidA and DidA, likely by causing the FtsW/I/N complex to hyperactively initiate cell division. Finally, we identify a transcription factor, DriD, that drives the SOS-independent transcription of didA following DNA damage.

  7. A DNA damage-induced, SOS-independent checkpoint regulates cell division in Caulobacter crescentus.

    Science.gov (United States)

    Modell, Joshua W; Kambara, Tracy K; Perchuk, Barrett S; Laub, Michael T

    2014-10-01

    Cells must coordinate DNA replication with cell division, especially during episodes of DNA damage. The paradigm for cell division control following DNA damage in bacteria involves the SOS response where cleavage of the transcriptional repressor LexA induces a division inhibitor. However, in Caulobacter crescentus, cells lacking the primary SOS-regulated inhibitor, sidA, can often still delay division post-damage. Here we identify didA, a second cell division inhibitor that is induced by DNA damage, but in an SOS-independent manner. Together, DidA and SidA inhibit division, such that cells lacking both inhibitors divide prematurely following DNA damage, with lethal consequences. We show that DidA does not disrupt assembly of the division machinery and instead binds the essential division protein FtsN to block cytokinesis. Intriguingly, mutations in FtsW and FtsI, which drive the synthesis of septal cell wall material, can suppress the activity of both SidA and DidA, likely by causing the FtsW/I/N complex to hyperactively initiate cell division. Finally, we identify a transcription factor, DriD, that drives the SOS-independent transcription of didA following DNA damage.

  8. Evaluation of a DNA Vaccine for Immunocontraceptive Potential Against Zona Pellucida Glycoproteins in Cattle

    Directory of Open Access Journals (Sweden)

    C. A. Foley

    2007-01-01

    Full Text Available Holstein cows were administered zona pellucida (ZP DNA vaccine and used to determine the potential of recombinant rabbit ZP glycoproteins (rZP as immunocontraceptive antigens. Zona pellucida proteins were purified and quantified. Cows were assigned to one of four treatment groups in which plasmids encoding rabbit ZP proteins were administered, i.d., using a gene gun (ZP55, n=2; ZP75, n=2; Hep55, n=2; and Control, n=3. Blood samples were taken before initial vaccination, once weekly for 5 wk and at 148 wk post-immunization. An ELISA was developed to assess anti-ZP titer levels in cow serum and ovarian function in cows was monitored using trans-rectal ultrasonography. Four of the six cows in ZP treatment groups developed antibody titer levels with similar linear responses over time. These cows also experienced reduced ovarian function as indicated by decreases in follicular and luteal activity. Estrous activity was observed in all cows and decreased in ZP treatment cows in comparison to Controls. Further research is needed to determine the relationship between ZP immunocontraception and ovarian function. Still, this study provides a basis for future researchers to use in developing a contraceptive vaccine for cattle.

  9. The antibiotic microcin B17 is a DNA gyrase poison: characterisation of the mode of inhibition.

    Science.gov (United States)

    Heddle, J G; Blance, S J; Zamble, D B; Hollfelder, F; Miller, D A; Wentzell, L M; Walsh, C T; Maxwell, A

    2001-04-13

    Microcin B17 is a 3.1-kDa bactericidal peptide; the putative target of this antibiotic is DNA gyrase. Microcin B17 has no detectable effect on gyrase-catalysed DNA supercoiling or relaxation activities in vitro and is unable to stabilise DNA cleavage in the absence of nucleotides. However, in the presence of ATP, or the non-hydrolysable analogue 5'-adenylyl beta,gamma-imidodiphosphate, microcin B17 stabilises a gyrase-dependent DNA cleavage complex in a manner reminiscent of quinolones, Ca(2+), or the bacterial toxin CcdB. The pattern of DNA cleavage produced by gyrase in the presence of microcin B17 is different from that produced by quinolones and more closely resembles Ca(2+)-mediated cleavage. Several gyrase mutants, including well-known quinolone-resistant mutants, are cross resistant to microcin-induced DNA cleavage. We suggest that microcin exerts its effects through a mechanism that has similarities to those of both the bacterial toxin CcdB and the quinolone antibacterial agents.

  10. Application of graphene–pyrenebutyric acid nanocomposite as probe oligonucleotide immobilization platform in a DNA biosensor

    International Nuclear Information System (INIS)

    A stable and uniform organic–inorganic nanocomposite that consists of graphene (GR) and pyrenebutyric acid (PBA) was obtained by ultrasonication, which was characterized by scanning electron microscopy (SEM) and UV–vis absorption spectra. The dispersion was dropped onto a gold electrode surface to obtain GR–PBA modified electrode (GR–PBA/Au). Electrochemical behaviors of the modified electrode were characterized by cyclic voltammetry and electrochemical impedance spectroscopy using [Fe(CN)6]3−/4− as the electroactive probe. A novel DNA biosensor was constructed based on the covalent coupling of amino modified oligonucleotides with the carboxylic group on PBA. By using methylene blue (MB) as a redox-active hybridization indicator, the biosensor was applied to electrochemically detect the complementary sequence, and the results suggested that the peak currents of MB showed a good linear relationship with the logarithm values of target DNA concentrations in the range from 1.0 × 10−15 to 5.0 × 10−12 M with a detection limit of 3.8 × 10−16 M. The selectivity experiment also showed that the biosensor can well distinguish the target DNA from the non-complementary sequences. - Highlights: • A nanocomposite containing graphene and pyrenebutyric acid was prepared. • The nanocomposite was applied as a function platform for DNA immobilization platform. • The developed biosensor shows excellent selectivity and sensitivity for target DNA detection

  11. Ultrafast dynamics of solvation and charge transfer in a DNA-based biomaterial.

    Science.gov (United States)

    Choudhury, Susobhan; Batabyal, Subrata; Mondol, Tanumoy; Sao, Dilip; Lemmens, Peter; Pal, Samir Kumar

    2014-05-01

    Charge migration along DNA molecules is a key factor for DNA-based devices in optoelectronics and biotechnology. The association of a significant amount of water molecules in DNA-based materials for the intactness of the DNA structure and their dynamic role in the charge-transfer (CT) dynamics is less documented in contemporary literature. In the present study, we have used a genomic DNA-cetyltrimethyl ammonium chloride (CTMA) complex, a technological important biomaterial, and Hoechest 33258 (H258), a well-known DNA minor groove binder, as fluorogenic probe for the dynamic solvation studies. The CT dynamics of CdSe/ZnS quantum dots (QDs; 5.2 nm) embedded in the as-prepared and swollen biomaterial have also been studied and correlated with that of the timescale of solvation. We have extended our studies on the temperature-dependent CT dynamics of QDs in a nanoenvironment of an anionic, sodium bis(2-ethylhexyl)sulfosuccinate reverse micelle (AOT RMs), whereby the number of water molecules and their dynamics can be tuned in a controlled manner. A direct correlation of the dynamics of solvation and that of the CT in the nanoenvironments clearly suggests that the hydration barrier within the Arrhenius framework essentially dictates the charge-transfer dynamics.

  12. Early development and characterization of a DNA-based radiation dosimeter

    Science.gov (United States)

    Avarmaa, Kirsten A.

    It is the priority of first responders to minimize damage to persons and infrastructure in the case of a nuclear emergency due to an accident or deliberate terrorist attack -- if this emergency includes a radioactive hazard, first responders require a simple-to-use, accurate and complete dosimeter for radiation protection purposes in order to minimize the health risk to these individuals and the general population at large. This work consists of the early evaluation of the design and performance of a biologically relevant dosimeter which uses DNA material that can respond to the radiation of any particle type. The construct consists of fluorescently tagged strands of DNA. The signalling components of this dosimeter are also investigated for their sensitivity to radiation damage and light exposure. The dual-labelled dosimeter that is evaluated in this work gave a measurable response to gamma radiation at dose levels of 10 Gy for the given detector design and experimental setup. Further testing outside of this work confirmed this finding and indicated a working range of 100 mGy to 10 Gy using a custom-built fluorimeter as part of a larger CRTI initiative. Characterization of the chromatic components of the dosimeter showed that photobleaching is not expected to have an effect on dosimeter performance, but that radiation can damage the non-DNA signalling components at higher dose levels, although this damage is minimal at lower doses over the expected operating ranges. This work therefore describes the early steps in the quantification of the behaviour of the DNA dosimeter as a potential biologically-based device to measure radiation dose.

  13. Attenuated Shigella as a DNA Delivery Vehicle for DNA-Mediated Immunization

    Science.gov (United States)

    Sizemore, Donata R.; Branstrom, Arthur A.; Sadoff, Jerald C.

    1995-10-01

    Direct inoculation of DNA, in the form of purified bacterial plasmids that are unable to replicate in mammalian cells but are able to direct cell synthesis of foreign proteins, is being explored as an approach to vaccine development. Here, a highly attenuated Shigella vector invaded mammalian cells and delivered such plasmids into the cytoplasm of cells, and subsequent production of functional foreign protein was measured. Because this Shigella vector was designed to deliver DNA to colonic mucosa, the method is a potential basis for oral and other mucosal DNA immunization and gene therapy strategies.

  14. Placental pseudo-malignancy from a DNA methylation perspective: unanswered questions and future directions

    Directory of Open Access Journals (Sweden)

    Boris eNovakovic

    2013-12-01

    Full Text Available The growing fetus is dependent on adequate placental function for delivery of essential nutrients and oxygen, and for waste removal. The placenta also plays an important protective role; shielding the developing baby from the maternal immune system and adverse environmental exposures. Fundamental to these processes is correct invasion of the decidua and remodelling of maternal vasculature, each of which show remarkable parallels to tumourogenesis, with the obvious exception that the former is usually a tightly controlled process. It is not surprising that these physiological similarities are mirrored in gene expression and epigenetic parallels, many not found in any other aspect of human development. In this perspective, we summarise known DNA methylation similarities between placenta and human tumours, and discuss the implications and knowledge gaps associated with these findings. We also speculate on the potential origin of common DNA methylation features in these two disparate aspects of human physiology.

  15. Development of a robust, versatile, and scalable inoculum train for the production of a DNA vaccine.

    Science.gov (United States)

    Okonkowski, J; Kizer-Bentley, L; Listner, K; Robinson, D; Chartrain, M

    2005-01-01

    For many microbial fermentation processes, the inoculum train can have a substantial impact on process performance in terms of productivity, profitability, and process control. In general, it is understood that a well-characterized and flexible inoculum train is essential for future scale-up and implementation of the process in a pilot plant or manufacturing setting. A fermentation process utilizing E. coli DH5 for the production of plasmid DNA carrying the HIV gag gene for use as a vaccine is currently under development in our laboratory. As part of the development effort, we evaluated inoculum train schemes that incorporate one, two, or three stages. In addition, we investigated the effect of inoculum viable-cell concentrations, either thawed or actively growing, over a wide range (from 2.5 x 10(4) to 1.0 x 10(8) viable cells/mL or approximately 0.001% to 4% of final working volume). The various inoculum trains were evaluated in terms of final plasmid yield, process time, reproducibility, robustness, and feasibility at large scale. The results of these studies show that final plasmid yield remained in the desired range, despite the number of stages or inoculation viable-cell concentrations comprising the inoculum train. On the basis of these observations and because it established a large database, the first part of these investigations supports an exceptional flexibility in the design of scalable inoculum trains for this DNA vaccine process. This work also highlighted that a slightly higher level of process reproducibility, as measured by the time for the culture to reach mid-exponential growth, was observed when using actively growing versus frozen cells. It also demonstrated the existence of a viable-cell concentration threshold for the one-stage process, since we observed that inoculation of the production stage with very low amounts of viable cells from a frozen source could lead to increased process sensitivity to external factors such as variation in the quality of the raw materials used in the medium formulation. However, our analysis indicates that, despite this slight disadvantage, a one-stage inoculum train was a viable option in many situations, especially if the inoculation viable-cell concentration was kept above 4.8 x 10(6) viable cells/mL. Because it leads to a reduction in process steps and eliminates some capital investments (i.e., inoculum fermenter), when feasible a one-stage process configuration will positively impact process economics. PMID:16080681

  16. TALEored Epigenetics: A DNA-Binding Scaffold for Programmable Epigenome Editing and Analysis.

    Science.gov (United States)

    Kubik, Grzegorz; Summerer, Daniel

    2016-06-01

    Epigenetic modification of the cytosine 5-position is an important regulator of gene expression with essential roles in genome stability, development, and disease. In addition to 5-methylcytosine (mC), the oxidized mC derivatives 5-hydroxymethyl-, 5-formyl-, and 5-carboxylcytosine (hmC, fC, and caC) have recently been discovered. These are intermediates of an active demethylation pathway but might also represent new epigenetic marks with individual biological roles. This increase in chemical complexity of DNA-encoded information has created a pressing need for new approaches that allow reading and editing of this information. Transcription-activator-like effectors (TALEs) are DNA-binding domains with programmable sequence selectivity that enable the direct reading of epigenetic cytosine modifications but can also guide enzymatic editing domains to genomic loci of choice. Here, we review recent advances in employing TALEs for these applications. PMID:26972580

  17. Genetic suppression of a dnaG mutation in Escherichia coli.

    OpenAIRE

    Katayama, T; Murakami, Y.; Wada, C.; Ohmori, H; Yura, T; Nagata, T

    1989-01-01

    Escherichia coli strains with a temperature-sensitive mutation, dnaG2903, in the primase-encoding gene spontaneously reverted to the temperature-insensitive phenotype at a high frequency. Many of the reversions were caused by extragenic sdg suppressors. About 100 independently isolated sdg suppressors were analyzed. They fall into two classes. The sdgA mutations were genetically mapped very close to and upstream of the dnaG gene and were found to be cis dominant. DNA sequencing of two of them...

  18. A DNA-based semantic fusion model for remote sensing data.

    Science.gov (United States)

    Sun, Heng; Weng, Jian; Yu, Guangchuang; Massawe, Richard H

    2013-01-01

    Semantic technology plays a key role in various domains, from conversation understanding to algorithm analysis. As the most efficient semantic tool, ontology can represent, process and manage the widespread knowledge. Nowadays, many researchers use ontology to collect and organize data's semantic information in order to maximize research productivity. In this paper, we firstly describe our work on the development of a remote sensing data ontology, with a primary focus on semantic fusion-driven research for big data. Our ontology is made up of 1,264 concepts and 2,030 semantic relationships. However, the growth of big data is straining the capacities of current semantic fusion and reasoning practices. Considering the massive parallelism of DNA strands, we propose a novel DNA-based semantic fusion model. In this model, a parallel strategy is developed to encode the semantic information in DNA for a large volume of remote sensing data. The semantic information is read in a parallel and bit-wise manner and an individual bit is converted to a base. By doing so, a considerable amount of conversion time can be saved, i.e., the cluster-based multi-processes program can reduce the conversion time from 81,536 seconds to 4,937 seconds for 4.34 GB source data files. Moreover, the size of result file recording DNA sequences is 54.51 GB for parallel C program compared with 57.89 GB for sequential Perl. This shows that our parallel method can also reduce the DNA synthesis cost. In addition, data types are encoded in our model, which is a basis for building type system in our future DNA computer. Finally, we describe theoretically an algorithm for DNA-based semantic fusion. This algorithm enables the process of integration of the knowledge from disparate remote sensing data sources into a consistent, accurate, and complete representation. This process depends solely on ligation reaction and screening operations instead of the ontology.

  19. A DNA-based semantic fusion model for remote sensing data.

    Directory of Open Access Journals (Sweden)

    Heng Sun

    Full Text Available Semantic technology plays a key role in various domains, from conversation understanding to algorithm analysis. As the most efficient semantic tool, ontology can represent, process and manage the widespread knowledge. Nowadays, many researchers use ontology to collect and organize data's semantic information in order to maximize research productivity. In this paper, we firstly describe our work on the development of a remote sensing data ontology, with a primary focus on semantic fusion-driven research for big data. Our ontology is made up of 1,264 concepts and 2,030 semantic relationships. However, the growth of big data is straining the capacities of current semantic fusion and reasoning practices. Considering the massive parallelism of DNA strands, we propose a novel DNA-based semantic fusion model. In this model, a parallel strategy is developed to encode the semantic information in DNA for a large volume of remote sensing data. The semantic information is read in a parallel and bit-wise manner and an individual bit is converted to a base. By doing so, a considerable amount of conversion time can be saved, i.e., the cluster-based multi-processes program can reduce the conversion time from 81,536 seconds to 4,937 seconds for 4.34 GB source data files. Moreover, the size of result file recording DNA sequences is 54.51 GB for parallel C program compared with 57.89 GB for sequential Perl. This shows that our parallel method can also reduce the DNA synthesis cost. In addition, data types are encoded in our model, which is a basis for building type system in our future DNA computer. Finally, we describe theoretically an algorithm for DNA-based semantic fusion. This algorithm enables the process of integration of the knowledge from disparate remote sensing data sources into a consistent, accurate, and complete representation. This process depends solely on ligation reaction and screening operations instead of the ontology.

  20. A universal design for a DNA probe providing ratiometric fluorescence detection by generation of silver nanoclusters.

    Science.gov (United States)

    Del Bonis-O'Donnell, Jackson Travis; Vong, Daniel; Pennathur, Sumita; Fygenson, Deborah Kuchnir

    2016-08-14

    DNA-stabilized silver nanoclusters (AgNCs), the fluorescence emission of which can rival that of typical organic fluorophores, have made possible a new class of label-free molecular beacons for the detection of single-stranded DNA. Like fluorophore-quencher molecular beacons (FQ-MBs) AgNC-based molecular beacons (AgNC-MBs) are based on a single-stranded DNA that undergoes a conformational change upon binding a target sequence. The new conformation exposes a stretch of single-stranded DNA capable of hosting a fluorescent AgNC upon reduction in the presence of Ag(+) ions. The utility of AgNC-MBs has been limited, however, because changing the target binding sequence unpredictably alters cluster fluorescence. Here we show that the original AgNC-MB design depends on bases in the target-binding (loop) domain to stabilize its AgNC. We then rationally alter the design to overcome this limitation. By separating and lengthening the AgNC-stabilizing domain, we create an AgNC-hairpin probe with consistent performance for arbitrary target sequence. This new design supports ratiometric fluorescence measurements of DNA target concentration, thereby providing a more sensitive, responsive and stable signal compared to turn-on AgNC probes. Using the new design, we demonstrate AgNC-MBs with nanomolar sensitivity and singe-nucleotide specificity, expanding the breadth of applicability of these cost-effective probes for biomolecular detection. PMID:27406901

  1. Constructing Bio-molecular Databases on a DNA-based Computer

    CERN Document Server

    Chang, Weng-Long; Ho,; Guo, Minyi

    2007-01-01

    Codd [Codd 1970] wrote the first paper in which the model of a relational database was proposed. Adleman [Adleman 1994] wrote the first paper in which DNA strands in a test tube were used to solve an instance of the Hamiltonian path problem. From [Adleman 1994], it is obviously indicated that for storing information in molecules of DNA allows for an information density of approximately 1 bit per cubic nm (nanometer) and a dramatic improvement over existing storage media such as video tape which store information at a density of approximately 1 bit per 1012 cubic nanometers. This paper demonstrates that biological operations can be applied to construct bio-molecular databases where data records in relational tables are encoded as DNA strands. In order to achieve the goal, DNA algorithms are proposed to perform eight operations of relational algebra (calculus) on bio-molecular relational databases, which include Cartesian product, union, set difference, selection, projection, intersection, join and division. Fu...

  2. The Benzyl Moiety in a Quinoxaline-Based Scaffold Acts as a DNA Intercalation Switch.

    Science.gov (United States)

    Mahata, Tridib; Kanungo, Ajay; Ganguly, Sudakshina; Modugula, Eswar Kalyan; Choudhury, Susobhan; Pal, Samir Kumar; Basu, Gautam; Dutta, Sanjay

    2016-06-27

    Quinoxaline antibiotics intercalate dsDNA and exhibit antitumor properties. However, they are difficult to synthesize and their structural complexity impedes a clear mechanistic understanding of DNA binding. Therefore design and synthesis of minimal-intercalators, using only part of the antibiotic scaffold so as to retain the key DNA-binding property, is extremely important. Reported is a unique example of a monomeric quinoxaline derivative of a 6-nitroquinoxaline-2,3-diamine scaffold which binds dsDNA by two different modes. While benzyl derivatives bound DNA in a sequential fashion, with intercalation as the second event, nonbenzyl derivatives showed only the first binding event. The benzyl intercalation switch provides important insights about molecular architecture which control specific DNA binding modes and would be useful in designing functionally important monomeric quinoxaline DNA binders and benchmarking molecular simulations.

  3. Determining plant-leaf miner-parasitoid interactions: a DNA barcoding approach.

    Directory of Open Access Journals (Sweden)

    Stéphane A P Derocles

    Full Text Available A major challenge in network ecology is to describe the full-range of species interactions in a community to create highly-resolved food-webs. We developed a molecular approach based on DNA full barcoding and mini-barcoding to describe difficult to observe plant-leaf miner-parasitoid interactions, consisting of animals commonly regarded as agricultural pests and their natural enemies. We tested the ability of universal primers to amplify the remaining DNA inside leaf miner mines after the emergence of the insect. We compared the results of a morphological identification of adult specimens; b identification based on the shape of the mines; c the COI Mini-barcode (130 bp and d the COI full barcode (658 bp fragments to accurately identify the leaf-miner species. We used the molecular approach to build and analyse a tri-partite ecological network of plant-leaf miner-parasitoid interactions. We were able to detect the DNA of leaf-mining insects within their feeding mines on a range of host plants using mini-barcoding primers: 6% for the leaves collected empty and 33% success after we observed the emergence of the leaf miner. We suggest that the low amplification success of leaf mines collected empty was mainly due to the time since the adult emerged and discuss methodological improvements. Nevertheless our approach provided new species-interaction data for the ecological network. We found that the 130 bp fragment is variable enough to identify all the species included in this study. Both COI fragments reveal that some leaf miner species could be composed of cryptic species. The network built using the molecular approach was more accurate in describing tri-partite interactions compared with traditional approaches based on morphological criteria.

  4. Molecular adjuvant IL-33 enhances the potency of a DNA vaccine in a lethal challenge model

    Science.gov (United States)

    Villarreal, Daniel O.; Svoronos, Nikos; Wise, Megan C.; Shedlock, Devon J.; Morrow, Matthew P.; Garcia, Jose-Conejo; Weiner, David B.

    2015-01-01

    Identifying new molecular adjuvants that elicit effective vaccine-induced CD8+ T cell immunity may be critical for the elimination of many challenging diseases including Tuberculosis, HIV and cancer. Here, we report that co-administration of molecular adjuvant IL-33 during vaccination enhanced the magnitude and function of antigen (Ag)-specific CD8+ T cells against a model Ag, LCMV NP target protein. These enhanced responses were characterized by higher frequencies of Ag-specific, polyfunctional CD8+ T cells exhibiting cytotoxic characteristics. Importantly, these cells were capable of robust expansion upon Ag-specific restimulation in vivo and conferred remarkable protection against a high dose lethal LCMV challenge. In addition, we demonstrate the ability of IL-33 to amplifying the frequency of Ag-specific KLRG1+ effector CD8+ T cells. These data show that IL-33 is a promising immunoadjuvant at improving T cell immunity in a vaccine setting and suggest further development and understanding of this molecular adjuvant for strategies against many obstinate infectious diseases and cancer. PMID:25887087

  5. A contamination assessment of the CI carbonaceous meteorite Orgueil using a DNA-directed approach

    Science.gov (United States)

    Aerts, J. W.; Elsaesser, A.; RöLing, W. F. M.; Ehrenfreund, P.

    2016-05-01

    The Orgueil meteorite has become one of the most well-studied carbonaceous meteorites, after it fell in France 150 yr ago. Extraterrestrial organic compounds such as amino acids and nucleobases in the parts per billion ranges were identified in Orgueil samples with supporting isotopic analyses. However, speculations of terrestrial contamination such as organic inclusions in the form of microbes and seeds accompanied the analyses of the Orgueil meteorite ever since its fall. By using molecular analysis, we performed DNA extractions and spiking experiments combined with 16S and 18S rRNA gene targeted PCR amplification to quantify the level of terrestrial biocontamination. Our results indicate that terrestrial contamination with DNA was insignificant in the investigated meteorite fraction. We also remeasured and confirmed concentrations of amino acids found in previous studies and conclude that their rather high concentrations and distribution cannot be explained by terrestrial contamination with microorganisms alone. These results represent the first analysis using DNA-directed tools in the analysis of the Orgueil meteorite to determine trace levels of biomarkers.

  6. UVI31+ is a DNA endonuclease that dynamically localizes to chloroplast pyrenoids in C. reinhardtii.

    Directory of Open Access Journals (Sweden)

    Manish Shukla

    Full Text Available UVI31+ is an evolutionarily conserved BolA family protein. In this study we examine the presence, localization and possible functions of this protein in the context of a unicellular alga, Chlamydomonas reinhardtii. UVI31+ in C. reinhardtii exhibits DNA endonuclease activity and is induced upon UV stress. Further, UVI31+ that normally localizes to the cell wall and pyrenoid regions gets redistributed into punctate foci within the whole chloroplast, away from the pyrenoid, upon UV stress. The observed induction upon UV-stress as well as the endonuclease activity suggests plausible role of this protein in DNA repair. We have also observed that UV31+ is induced in C. reinhardtii grown in dark conditions, whereby the protein localization is enhanced in the pyrenoid. Biomolecular interaction between the purified pyrenoids and UVI31+ studied by NMR demonstrates the involvement of the disordered loop domain of the protein in its interaction.

  7. Methods for interpreting lists of affected genes obstained in a DNA microarray experiment

    NARCIS (Netherlands)

    Hedegaard, J.; Arce, A.M.G.; Bicciato, S.; Bonnet, A.; Buitenhuis, B.; Collado, M.C.; Conley, L.N.; San Cristobal, M.; Ferrari, F.; Garrido, J.J.; Groenen, M.A.M.; Hornshoj, H.; Hulsegge, B.; Jiang, L.; Jimenez-Marin, A.; Kommadath, A.; Lagarrigue, S.; Leunissen, J.A.M.; Liaubet, L.; Neerincx, P.; Nie, H.; Poel, van der W.H.M.; Prickett, D.; Ramirez-Boo, M.; Rebel, J.M.J.; Robert-Granie, C.; Skarman, A.; Smits, M.A.; Sorensen, P.; Tosser-klopp, G.; Watson, M.

    2009-01-01

    Background - The aim of this paper was to describe and compare the methods used and the results obtained by the participants in a joint EADGENE (European Animal Disease Genomic Network of Excellence) and SABRE (Cutting Edge Genomics for Sustainable Animal Breeding) workshop focusing on post analysis

  8. A DNA fingerprint probe from Mycosphaerella graminicola identifies an active transposable element

    NARCIS (Netherlands)

    Goodwin, S.B.; Cavaletto, J.R.; Waalwijk, C.; Kema, G.H.J.

    2001-01-01

    DNA fingerprinting has been used extensively to characterize populations of Mycosphaerella graminicola, the Septoria tritici blotch pathogen of wheat. The highly polymorphic DNA fingerprints of Mycosphaerella graminicola were assumed to reflect the action of transposable elements. However, there was

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

    Institute of Scientific and Technical Information of China (English)

    Yan-xia Bai; Qing-yong Ma; Guang-xiao Yang

    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) apoptotic peptide was amplified by using self-complementation polymerase chain reaction (PCR) method and cloned into the pGEM-T Easy vector. The constructed plasmid was confirmed by endonuclease analysis and sequencing. Results The insertion of objective DNA fragment was confirmed by plasmid DNA enzyme spectrum analysis, p53 (N37) gene was successfully synthesized chemically in vitro. The sequencing result of positive clone was completely identical to the human p53(N37) sequence in GenBank using BLAST software (http://www. ncbi. him. nih. gov/cgi-bin /BLASTn). Conclusion The cloning of DNA fragment encoding p53(N37) apoptotic peptide was constructed by using DNA synthesis and pGEM-T Easy cloning methods. With the constructed plasmid, we could further investigate the function of N37 peptide.

  10. Growth control switch by a DNA-damage-inducible toxin-antitoxin system in Caulobacter crescentus.

    Science.gov (United States)

    Kirkpatrick, Clare L; Martins, Daniel; Redder, Peter; Frandi, Antonio; Mignolet, Johann; Chapalay, Julien Bortoli; Chambon, Marc; Turcatti, Gerardo; Viollier, Patrick H

    2016-01-01

    Bacterial toxin-antitoxin systems (TASs) are thought to respond to various stresses, often inducing growth-arrested (persistent) sub-populations of cells whose housekeeping functions are inhibited. Many such TASs induce this effect through the translation-dependent RNA cleavage (RNase) activity of their toxins, which are held in check by their cognate antitoxins in the absence of stress. However, it is not always clear whether specific mRNA targets of orthologous RNase toxins are responsible for their phenotypic effect, which has made it difficult to accurately place the multitude of TASs within cellular and adaptive regulatory networks. Here, we show that the TAS HigBA of Caulobacter crescentus can promote and inhibit bacterial growth dependent on the dosage of HigB, a toxin regulated by the DNA damage (SOS) repressor LexA in addition to its antitoxin HigA, and the target selectivity of HigB's mRNA cleavage activity. HigB reduced the expression of an efflux pump that is toxic to a polarity control mutant, cripples the growth of cells lacking LexA, and targets the cell cycle circuitry. Thus, TASs can have outcome switching activity in bacterial adaptive (stress) and systemic (cell cycle) networks. PMID:27572440

  11. Screening of differentially expressed genes associated with human glioblastoma and functional analysis using a DNA microarray.

    Science.gov (United States)

    Wang, Lina; Wei, Bo; Hu, Guozhang; Wang, Le; Bi, Miaomiao; Sun, Zhigang; Jin, Ying

    2015-08-01

    Glioblastoma multiforme (GBM) is the most malignant type of human glioma, and has a poor prognosis. Screening differentially expressed genes (DEGs) in brain tumor samples and normal brain samples is of importance for identifying GBM and to design specific-targeting drugs. The transcriptional profile of GSE30563, containing three genechips of brain tumor samples and three genechips of normal brain samples, was downloaded from Gene Expression Omnibus to identify the DEGs. The differences in the expression of the DEGs in the two different samples were compared through hierarchical biclustering. The co-expression coefficient of the DEGs was calculated using the information from COXPRESdb, the network of the DEGs was constructed and functional enrichment and pathway analysis were performed. Finally, the transcription factors of important DEGs were predicted. A total of 1,006 DEGs, including 368 upregulated and 638 downregulated DEGs, were identified. A close correlation was demonstrated between six important genes, associated with immune response, HLA-DQB1, HLA-DRB1, HLA-DPA1, HLA-B, HLA-DMA and HLA-DRA, and the immune response. Allograft rejection was selected as the most significant pathway. A total of 17 transcription factors, including nuclear factor (NF)-κB and NF-κB1, and their binding sites containing these six DEGs, were also identified. The DEGs, including major histocompatibility complex (MHC) class II, DQβ1, MHC class II, DRβ1, MHC class IB, MHC class II, DMα, MHC class II, DPα1, MHC class II, DRα, may provide novel targets for the diagnosis and treatment of GBM. The transcription factors of these six genes and their binding sites may also provide evidence and direction for identifying target-specific drugs. PMID:25901754

  12. Diagnostic and prognostic utility of a DNA hypermethylated gene signature in prostate cancer.

    Directory of Open Access Journals (Sweden)

    Liang Kee Goh

    Full Text Available We aimed to identify a prostate cancer DNA hypermethylation microarray signature (denoted as PHYMA that differentiates prostate cancer from benign prostate hyperplasia (BPH, high from low-grade and lethal from non-lethal cancers. This is a non-randomized retrospective study in 111 local Asian men (87 prostate cancers and 24 BPH treated from 1995 to 2009 in our institution. Archival prostate epithelia were laser-capture microdissected and genomic DNA extracted and bisulfite-converted. Samples were profiled using Illumina GoldenGate Methylation microarray, with raw data processed by GenomeStudio. A classification model was generated using support vector machine, consisting of a 55-probe DNA methylation signature of 46 genes. The model was independently validated on an internal testing dataset which yielded cancer detection sensitivity and specificity of 95.3% and 100% respectively, with overall accuracy of 96.4%. Second validation on another independent western cohort yielded 89.8% sensitivity and 66.7% specificity, with overall accuracy of 88.7%. A PHYMA score was developed for each sample based on the state of methylation in the PHYMA signature. Increasing PHYMA score was significantly associated with higher Gleason score and Gleason primary grade. Men with higher PHYMA scores have poorer survival on univariate (p = 0.0038, HR = 3.89 and multivariate analyses when controlled for (i clinical stage (p = 0.055, HR = 2.57, and (ii clinical stage and Gleason score (p = 0.043, HR = 2.61. We further performed bisulfite genomic sequencing on 2 relatively unknown genes to demonstrate robustness of the assay results. PHYMA is thus a signature with high sensitivity and specificity for discriminating tumors from BPH, and has a potential role in early detection and in predicting survival.

  13. Facile preparation of a DNA sensor for rapid herpes virus detection

    Energy Technology Data Exchange (ETDEWEB)

    Tam, Phuong Dinh, E-mail: tampd-hast@mail.hut.edu.vn [Hanoi Advanced School of Science and Technology, Hanoi University of Technology (Viet Nam); Tuan, Mai Anh, E-mail: tuanma-itims@mail.hut.edu.vn [International Training Institute for Materials Science, Hanoi University of Technology (Viet Nam); Huy, Tran Quang [National Institute of Hygiene and Epidemiology (NIHE), 01 Yersin, Hai Ba Trung District, Hanoi (Viet Nam); Le, Anh-Tuan [Hanoi Advanced School of Science and Technology, Hanoi University of Technology (Viet Nam); Hieu, Nguyen Van, E-mail: hieu@itims.edu.vn [International Training Institute for Materials Science, Hanoi University of Technology (Viet Nam)

    2010-10-12

    In this paper, a simple DNA sensor platform was developed for rapid herpes virus detection in real samples. The deoxyribonucleic acid (DNA) sequences of the herpes simplex virus (DNA probe) were directly immobilized on the surface of interdigitated electrodes by electrochemical polymerization along with pyrrole monomers. The potential was scanned from - 0.7 to + 0.6 V, and the scanning rate was 100 mV/s. Fourier transform infrared spectroscopy was employed to verify specific DNA sequence binding and the conducting polymer. The morphology of the conducting polymer doped with DNA strands was characterized using a field emission scanning electron microscope. As-obtained DNA sensor was used to detect the herpes virus DNA in the real samples. The results show that the current DNA sensors detected the lowest DNA concentration of 2 nM. This sensitivity appears to be better than that of the DNA sensors prepared by immobilization of the DNA probe on the 3-aminopropyl-triethoxy-silance (APTS) membrane.

  14. Development of a DNA-based microarray for the detection of zoonotic pathogens in rodent species.

    Science.gov (United States)

    Giles, Timothy; Yon, Lisa; Hannant, Duncan; Barrow, Paul; Abu-Median, Abu-Bakr

    2015-12-01

    The demand for diagnostic tools that allow simultaneous screening of samples for multiple pathogens is increasing because they overcome the limitations of other methods, which can only screen for a single or a few pathogens at a time. Microarrays offer the advantages of being capable to test a large number of samples simultaneously, screening for multiple pathogen types per sample and having comparable sensitivity to existing methods such as PCR. Array design is often considered the most important process in any microarray experiment and can be the deciding factor in the success of a study. There are currently no microarrays for simultaneous detection of rodent-borne pathogens. The aim of this report is to explicate the design, development and evaluation of a microarray platform for use as a screening tool that combines ease of use and rapid identification of a number of rodent-borne pathogens of zoonotic importance. Nucleic acid was amplified by multiplex biotinylation PCR prior to hybridisation onto microarrays. The array sensitivity was comparable to standard PCR, though less sensitive than real-time PCR. The array presented here is a prototype microarray identification system for zoonotic pathogens that can infect rodent species. PMID:26188129

  15. PARP-1 modulation of mTOR signaling in response to a DNA alkylating agent.

    Directory of Open Access Journals (Sweden)

    Chantal Ethier

    Full Text Available Poly(ADP-ribose polymerase-1 (PARP-1 is widely involved in cell death responses. Depending on the degree of injury and on cell type, PARP activation may lead to autophagy, apoptosis or necrosis. In HEK293 cells exposed to the alkylating agent N-methyl-N'-nitro-N'-nitrosoguanine (MNNG, we show that PARP-1 activation triggers a necrotic cell death response. The massive poly(ADP-ribose (PAR synthesis following PARP-1 activation leads to the modulation of mTORC1 pathway. Shortly after MNNG exposure, NAD⁺ and ATP levels decrease, while AMP levels drastically increase. We characterized at the molecular level the consequences of these altered nucleotide levels. First, AMP-activated protein kinase (AMPK is activated and the mTORC1 pathway is inhibited by the phosphorylation of Raptor, in an attempt to preserve cellular energy. Phosphorylation of the mTORC1 target S6 is decreased as well as the phosphorylation of the mTORC2 component Rictor on Thr1135. Finally, Akt phosphorylation on Ser473 is lost and then, cell death by necrosis occurs. Inhibition of PARP-1 with the potent PARP inhibitor AG14361 prevents all of these events. Moreover, the antioxidant N-acetyl-L-cysteine (NAC can also abrogate all the signaling events caused by MNNG exposure suggesting that reactive oxygen species (ROS production is involved in PARP-1 activation and modulation of mTOR signaling. In this study, we show that PARP-1 activation and PAR synthesis affect the energetic status of cells, inhibit the mTORC1 signaling pathway and possibly modulate the mTORC2 complex affecting cell fate. These results provide new evidence that cell death by necrosis is orchestrated by the balance between several signaling pathways, and that PARP-1 and PAR take part in these events.

  16. Optimization of a DNA Nicking Assay to Evaluate Oenocarpus bataua and Camellia sinensis Antioxidant Capacity

    Directory of Open Access Journals (Sweden)

    Louis-Jérôme Leba

    2014-10-01

    Full Text Available This study was aimed at assessing the DNA damage protective activity of different types of extracts (aqueous, methanolic and acetonic using an in vitro DNA nicking assay. Several parameters were optimized using the pUC18 plasmid, especially FeSO4, EDTA, solvent concentrations and incubation time. Special attention has been paid to removing the protective and damaging effect of the solvent and FeSO4 respectively, as well as to identifying the relevant positive and negative controls. For each solvent, the optimal conditions were determined: (i for aqueous extracts, 0.33 mM of FeSO4 and 0.62 mM of EDTA were incubated for 20 min at 37 °C; (ii for acetone extracts, 1.16% solvent were incubated for 15 min at 37 °C with 1.3 mM of FeSO4 and 2.5 mM of EDTA and (iii for methanol extracts, 0.16% solvent, were incubated for 1.5 h at 37 °C with 0.33 mM of FeSO4 and 0.62 mM of EDTA. Using the optimized conditions, the DNA damage protective activity of aqueous, methanolic and acetonic extracts of an Amazonian palm berry (Oenocarpus bataua and green tea (Camellia sinensis was assessed. Aqueous and acetonic Oenocarpus bataua extracts were protective against DNA damage, whereas aqueous, methanolic and acetonic extracts of Camellia sinensis extracts induced DNA damage.

  17. Methods for interpreting lists of affected genes obtained in a DNA microarray experiment

    DEFF Research Database (Denmark)

    Hedegaard, Jakob; Arce, Christina; Bicciato, Silvio;

    2009-01-01

    The aim of this paper was to describe and compare the methods used and the results obtained by the participants in a joint EADGENE (European Animal Disease Genomic Network of Excellence) and SABRE (Cutting Edge Genomics for Sustainable Animal Breeding) workshop focusing on post analysis of microa...

  18. Plasmonic coupling and long-range transfer of an excitation along a DNA nanowire.

    Science.gov (United States)

    Toppari, J Jussi; Wirth, Janina; Garwe, Frank; Stranik, Ondrej; Csaki, Andrea; Bergmann, Joachim; Paa, Wolfgang; Fritzsche, Wolfgang

    2013-02-26

    We demonstrate an excitation transfer along a fluorescently labeled dsDNA nanowire over a length of several micrometers. Launching of the excitation is done by exciting a localized surface plasmon mode of a 40 nm silver nanoparticle by 800 nm femtosecond laser pulses via two-photon absorption. The plasmonic mode is subsequently coupled or transformed to excitation in the nanowire in contact with the particle and propagated along it, inducing bleaching of the dyes on its way. In situ as well as ex situ fluorescence microscopy is utilized to observe the phenomenon. In addition, transfer of the excitation along the nanowire to another nanoparticle over a separation of 5.7 μm was clearly observed. The nature of the excitation coupling and transfer could not be fully resolved here, but injection of an electron into the DNA from the excited nanoparticle and subsequent coupled transfer of charge (Dexter) and delocalized exciton (Frenkel) is the most probable mechanism. However, a direct plasmonic or optical coupling and energy transfer along the nanowire cannot be totally ruled out either. By further studies the observed phenomenon could be utilized in novel molecular systems, providing a long-needed communication method between molecular devices. PMID:23305550

  19. Arabidopsis thaliana GYRB3 Does Not Encode a DNA Gyrase Subunit

    Science.gov (United States)

    Evans-Roberts, Katherine M.; Breuer, Christian; Wall, Melisa K.; Sugimoto-Shirasu, Keiko; Maxwell, Anthony

    2010-01-01

    Background DNA topoisomerases are enzymes that control the topology of DNA in all cells. DNA gyrase is unique among the topoisomerases in that it is the only enzyme that can actively supercoil DNA using the free energy of ATP hydrolysis. Until recently gyrase was thought to be unique to bacteria, but has now been discovered in plants. The genome of the model plant, Arabidopsis thaliana, is predicted to encode four gyrase subunits: AtGyrA, AtGyrB1, AtGyrB2 and AtGyrB3. Methodology/Principal Findings We found, contrary to previous data, that AtGyrB3 is not essential to the survival of A. thaliana. Bioinformatic analysis suggests AtGyrB3 is considerably shorter than other gyrase B subunits, lacking part of the ATPase domain and other key motifs found in all type II topoisomerases; but it does contain a putative DNA-binding domain. Partially purified AtGyrB3 cannot bind E. coli GyrA or support supercoiling. AtGyrB3 cannot complement an E. coli gyrB temperature-sensitive strain, whereas AtGyrB2 can. Yeast two-hybrid analysis suggests that AtGyrB3 cannot bind to AtGyrA or form a dimer. Conclusions/Significance These data strongly suggest that AtGyrB3 is not a gyrase subunit but has another unknown function. One possibility is that it is a nuclear protein with a role in meiosis in pollen. PMID:20360860

  20. Arabidopsis thaliana GYRB3 Does Not Encode a DNA Gyrase Subunit

    OpenAIRE

    Evans-Roberts, Katherine M.; Christian Breuer; Wall, Melisa K.; Keiko Sugimoto-Shirasu; Anthony Maxwell

    2010-01-01

    Background DNA topoisomerases are enzymes that control the topology of DNA in all cells. DNA gyrase is unique among the topoisomerases in that it is the only enzyme that can actively supercoil DNA using the free energy of ATP hydrolysis. Until recently gyrase was thought to be unique to bacteria, but has now been discovered in plants. The genome of the model plant, Arabidopsis thaliana, is predicted to encode four gyrase subunits: AtGyrA, AtGyrB1, AtGyrB2 and AtGyrB3. Methodology/Principal Fi...

  1. MoS2 nanocrystals confined in a DNA matrix exhibiting energy transfer.

    Science.gov (United States)

    Goswami, Nirmal; Giri, Anupam; Pal, Samir Kumar

    2013-09-10

    We report the wet chemical synthesis of MoS2 nanocrystals (NCs), a transition-metal dichalcogenide, using DNA as a host matrix. As evidenced from transmission electron microscopy (TEM), the NCs are highly crystalline, with an average diameter of ~5 nm. Ultraviolet-visible (UV-vis) absorption studies along with band gap calculations confirm that NCs are in quantum confinement. A prominent red shift of the optical absorption bands has been observed upon formation of the thin film using hexadecyltrimethylammonium chloride (CTAC), i.e., in the case of MoS2@DNA-CTAC. In the thin film, strong electron-phonon coupling arises because of the resonance effect, which is reflected from the emergence of intense first-, second-, and third-order Raman peaks, whenever excited with the 488 nm line. We have established that our as-synthesized MoS2 NCs quench the fluorescence of a well-known DNA minor groove binding probe, Hoechst 33258. Unprecedented fluorescence quenching (94%) of donor (Hoechst 33258) emission and efficient energy transfer (89%) between Hoechst 33258 and MoS2 NCs (acceptor) are obtained. The donor-acceptor distance of these conjugates has been described by a Förster resonance energy transfer (FRET)-based model. Furthermore, employing a statistical method, we have estimated the probability of the distance distribution between the donor and acceptor. We believe that the study described herein may enable substantial advances in fields of optoelectronics, photovoltaics, catalysis, and many others.

  2. Long-Range Interactions and Wave Patterns in a DNA Model

    International Nuclear Information System (INIS)

    We propose a spin-like model of DNA nonlinear dynamics with long-range interactions between adjacent base pairs. We show that the model equation is a modified sine-Gordon equation. We perform the linear stability analysis of a plane wave, which predicts high amplitude and extended oscillating waves for high values of the long-range parameter. This is confirmed numerically and biological implications of the obtained patterns are suggested. (author)

  3. A DNA extraction protocol for improved DNA yield from individual mosquitoes [version 1; referees: 2 approved

    OpenAIRE

    Nieman, Catelyn C.; Youki Yamasaki; Travis C. Collier; Yoosook Lee

    2015-01-01

    Typical DNA extraction protocols from commercially available kits provide an adequate amount of DNA from a single individual mosquito sufficient for PCR-based assays. However, next-generation sequencing applications and high-throughput SNP genotyping assays exposed the limitation of DNA quantity one usually gets from a single individual mosquito. Whole genome amplification could alleviate the issue but it also creates bias in genome representation. While trying to find alternative DNA extract...

  4. The role of cytosine methylation on charge transport through a DNA strand

    Science.gov (United States)

    Qi, Jianqing; Govind, Niranjan; Anantram, M. P.

    2015-09-01

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Büttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit.

  5. Algebraic Statistics of Poincaré Recurrences in a DNA Molecule.

    Science.gov (United States)

    Mazur, Alexey K; Shepelyansky, D L

    2015-10-30

    The statistics of Poincaré recurrences is studied for the base-pair breathing dynamics of an all-atom DNA molecule in a realistic aqueous environment with thousands of degrees of freedom. It is found that at least over five decades in time the decay of recurrences is described by an algebraic law with the Poincaré exponent close to β=1.2. This value is directly related to the correlation decay exponent ν=β-1, which is close to ν≈0.15 observed in the time resolved Stokes shift experiments. By applying the virial theorem we analyze the chaotic dynamics in polynomial potentials and demonstrate analytically that an exponent β=1.2 is obtained assuming the dominance of dipole-dipole interactions in the relevant DNA dynamics. Molecular dynamics simulations also reveal the presence of strong low frequency noise with the exponent η=1.6. We trace parallels with the chaotic dynamics of symplectic maps with a few degrees of freedom characterized by the Poincaré exponent β~1.5. PMID:26565502

  6. [Identification and diagnosis of Taylorella equigenitalis by a DNA amplification method (PCR)].

    Science.gov (United States)

    Miserez, R; Frey, J; Krawinkler, M; Nicolet, J

    1996-01-01

    A polymerase chain reaction (PCR) for identification of Taylorella equigenitalis was developed. The oligonucleotide primers are based on the DNA sequence of the rrs gene of T. equigenitalis, encoding for the 16S ribosomal RNA. Analysis of 21 strains of T. equigenitalis from England, USA and Switzerland showed an amplification product of 410 bp with identical Sau3A restriction profile. The sensitivity of the PCR-Assay was estimated to detect 50 to 500 bacteria of T. equigenitalis in a mixture with frequently found contaminants. Further analysis of culture from 60 genital swabs, taken in the course of the control of the contagious equine metritis in horses and donkeys, of experimental assays as well as of two positive cases from the diagnostic showed that this PCR-assay can be used to identify and to detect strains of T. equigenitalis. In addition, preliminary results indicate that the method is also applicable for direct in vitro establishment of the presence of T. equigenitalis in clinical samples.

  7. Model of a DNA-protein complex of the architectural monomeric protein MC1 from Euryarchaea.

    Directory of Open Access Journals (Sweden)

    Françoise Paquet

    Full Text Available In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1 from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.

  8. Construction of a DNA library from chromosome 4 of rice (Oryza sativa) by microdissection

    Institute of Scientific and Technical Information of China (English)

    MAOYINGWEI; SIYUANLIANG; 等

    1998-01-01

    A simple method to create a chromosome-specific DNA librqary of rice,including microdissection,amplification,charterization and cloning,is described.Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR).The PCR products were labeled as probes with DIG-11-dUTP using the random priming method.Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4.A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed.Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences.The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.

  9. Identification of mealybug pest species (Hemiptera: Pseudococcidae) in Egypt and France, using a DNA barcoding approach.

    Science.gov (United States)

    Abd-Rabou, S; Shalaby, H; Germain, J-F; Ris, N; Kreiter, P; Malausa, T

    2012-10-01

    Pseudococcidae (mealybugs) is a large taxonomic group, including a number of agronomic pests. Taxonomic identification of mealybug species is a recurrent problem and represents a major barrier to the establishment of adequate pest management strategies. We combined molecular analysis of three DNA markers (28S-D2, cytochrome oxidase I and internal transcribed spacer 2) with morphological examination, for the identification of 176 specimens collected from 40 mealybug populations infesting various crops and ornamental plants in Egypt and France. This combination of DNA and morphological analyses led to the identification of 17 species: seven in Egypt (Planococcus citri (Risso), Planococcus ficus (Signoret), Maconellicoccus hirsutus (Green), Ferrisia virgata (Cockerell), Phenacoccus solenopsis Tinsley, Phenacoccus parvus Morrison and Saccharicoccus sacchari (Cockerell)) and 11 in France (Planococcus citri, Pseudococcus viburni Signoret, Pseudococcus longispinus (Targioni-Tozzetti), Pseudococcus comstocki (Kuwana), Rhizoecus amorphophalli Betrem, Trionymus bambusae (Green), Balanococcus diminutus (Leonardi), Phenacoccus madeirensis Green, Planococcus vovae (Nasonov), Dysmicoccus brevipes (Cockerell) and Phenacoccus aceris Signoret), Pl. citri being found in both countries. We also found genetic variation between populations considered to belong to the same species, justifying further investigation of the possible occurrence of complexes of cryptic taxa.

  10. A DNA-based registry for all animal species: the barcode index number (BIN system.

    Directory of Open Access Journals (Sweden)

    Sujeevan Ratnasingham

    Full Text Available Because many animal species are undescribed, and because the identification of known species is often difficult, interim taxonomic nomenclature has often been used in biodiversity analysis. By assigning individuals to presumptive species, called operational taxonomic units (OTUs, these systems speed investigations into the patterning of biodiversity and enable studies that would otherwise be impossible. Although OTUs have conventionally been separated through their morphological divergence, DNA-based delineations are not only feasible, but have important advantages. OTU designation can be automated, data can be readily archived, and results can be easily compared among investigations. This study exploits these attributes to develop a persistent, species-level taxonomic registry for the animal kingdom based on the analysis of patterns of nucleotide variation in the barcode region of the cytochrome c oxidase I (COI gene. It begins by examining the correspondence between groups of specimens identified to a species through prior taxonomic work and those inferred from the analysis of COI sequence variation using one new (RESL and four established (ABGD, CROP, GMYC, jMOTU algorithms. It subsequently describes the implementation, and structural attributes of the Barcode Index Number (BIN system. Aside from a pragmatic role in biodiversity assessments, BINs will aid revisionary taxonomy by flagging possible cases of synonymy, and by collating geographical information, descriptive metadata, and images for specimens that are likely to belong to the same species, even if it is undescribed. More than 274,000 BIN web pages are now available, creating a biodiversity resource that is positioned for rapid growth.

  11. Chaos-based image encryption using a hybrid genetic algorithm and a DNA sequence

    Science.gov (United States)

    Enayatifar, Rasul; Abdullah, Abdul Hanan; Isnin, Ismail Fauzi

    2014-05-01

    The paper studies a recently developed evolutionary-based image encryption algorithm. A novel image encryption algorithm based on a hybrid model of deoxyribonucleic acid (DNA) masking, a genetic algorithm (GA) and a logistic map is proposed. This study uses DNA and logistic map functions to create the number of initial DNA masks and applies GA to determine the best mask for encryption. The significant advantage of this approach is improving the quality of DNA masks to obtain the best mask that is compatible with plain images. The experimental results and computer simulations both confirm that the proposed scheme not only demonstrates excellent encryption but also resists various typical attacks.

  12. Thermal and mechanical denaturation properties of a DNA model with three sites per nucleotide

    CERN Document Server

    Florescu, Ana-Maria; 10.1063/1.3626870

    2011-01-01

    In this paper, we show that the coarse grain model for DNA, which has been proposed recently by Knotts, Rathore, Schwartz and de Pablo (J. Chem. Phys. 126, 084901 (2007)), can be adapted to describe the thermal and mechanical denaturation of long DNA sequences by adjusting slightly the base pairing contribution. The adjusted model leads to (i) critical temperatures for long homogeneous sequences that are in good agreement with both experimental ones and those obtained from statistical models, (ii) a realistic step-like denaturation behaviour for long inhomogeneous sequences, and (iii) critical forces at ambient temperature of the order of 10 pN, close to measured values. The adjusted model furthermore supports the conclusion that the thermal denaturation of long homogeneous sequences corresponds to a first-order phase transition and yields a critical exponent for the critical force equal to sigma=0.70. This model is both geometrically and energetically realistic, in the sense that the helical structure and th...

  13. Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries

    Institute of Scientific and Technical Information of China (English)

    Ya-ping YANG; Yu-hong LI; Ai-hua ZHANG; Lan BI; Ming-wen FAN

    2009-01-01

    Aim: To prepare a clinical-grade anti-caries DNA vaccine pGJA-P/VAX and explore its immune effect and protective efficacy against a cariogenic bacterial challenge.Methods: A large-scale industrial production process was developed under Good Manufacturing Practices (GMP) by combining and optimizing common unit operations such as alkaline lysis, precipitation, endotoxin removal and column chromatography. Quality controls of the purified bulk and final lyophilized vaccine were conducted according to authoritative guidelines. Mice and gnotobiotic rats were intranasally immunized with clinical-grade pGJA-P/VAX with chitosan. Antibody levels of serum IgG and salivary SlgA were assessed by an enzyme-linked immunosorbent assay (ELISA), and caries activity was evaluated by the Keyes method. pGJA-P/VAX and pVAX1 prepared by a laboratory-scale commercial kit were used as controls.Results: The production process proved to be scalable and reproducible. Impurities including host protein, residual RNA, genomic DNA and endotoxin in the purified plasmid were all under the limits of set specifications. Intranasal vaccination with clinical-grade pGJA-P/ VAX induced higher serum IgG and salivary SlgA in both mice and gnotobiotic rats. While in the experimental caries model, the enamel (E), dentinal slight (Ds), and dentinal moderate (Dm) caries lesions were reduced by 21.1%, 33.0%, and 40.9%, respectively. Conclusion: The production process under GMP was efficient in preparing clinical-grade pGJA-P/VAX with high purity and intended effectiveness, thus facilitating future clinical trials for the anti-caries DNA vaccine.

  14. Remyelination Induced by a DNA Aptamer in a Mouse Model of Multiple Sclerosis

    OpenAIRE

    Branislav Nastasijevic; Wright, Brent R.; John Smestad; Warrington, Arthur E.; Moses Rodriguez; L James Maher

    2012-01-01

    Multiple sclerosis (MS) is a debilitating inflammatory disease of the central nervous system (CNS) characterized by local destruction of the insulating myelin surrounding neuronal axons. With more than 200 million MS patients worldwide, the absence of treatments that prevent progression or induce repair poses a major challenge. Anti-inflammatory therapies have met with limited success only in preventing relapses. Previous screening of human serum samples revealed natural IgM antibodies that b...

  15. Remyelination induced by a DNA aptamer in a mouse model of multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Branislav Nastasijevic

    Full Text Available Multiple sclerosis (MS is a debilitating inflammatory disease of the central nervous system (CNS characterized by local destruction of the insulating myelin surrounding neuronal axons. With more than 200 million MS patients worldwide, the absence of treatments that prevent progression or induce repair poses a major challenge. Anti-inflammatory therapies have met with limited success only in preventing relapses. Previous screening of human serum samples revealed natural IgM antibodies that bind oligodendrocytes and promote both cell signaling and remyelination of CNS lesions in an MS model involving chronic infection of susceptible mice by Theiler's encephalomyelitis virus and in the lysolecithin model of focal demyelination. This intriguing result raises the possibility that molecules with binding specificity for oligodendrocytes or myelin components may promote therapeutic remyelination in MS. Because of the size and complexity of IgM antibodies, it is of interest to identify smaller myelin-specific molecules with the ability to promote remyelination in vivo. Here we show that a 40-nucleotide single-stranded DNA aptamer selected for affinity to murine myelin shows this property. This aptamer binds multiple myelin components in vitro. Peritoneal injection of this aptamer results in distribution to CNS tissues and promotes remyelination of CNS lesions in mice infected by Theiler's virus. Interestingly, the selected DNA aptamer contains guanosine-rich sequences predicted to induce folding involving guanosine quartet structures. Relative to monoclonal antibodies, DNA aptamers are small, stable, and non-immunogenic, suggesting new possibilities for MS treatment.

  16. Algebraic Statistics of Poincaré Recurrences in a DNA Molecule.

    Science.gov (United States)

    Mazur, Alexey K; Shepelyansky, D L

    2015-10-30

    The statistics of Poincaré recurrences is studied for the base-pair breathing dynamics of an all-atom DNA molecule in a realistic aqueous environment with thousands of degrees of freedom. It is found that at least over five decades in time the decay of recurrences is described by an algebraic law with the Poincaré exponent close to β=1.2. This value is directly related to the correlation decay exponent ν=β-1, which is close to ν≈0.15 observed in the time resolved Stokes shift experiments. By applying the virial theorem we analyze the chaotic dynamics in polynomial potentials and demonstrate analytically that an exponent β=1.2 is obtained assuming the dominance of dipole-dipole interactions in the relevant DNA dynamics. Molecular dynamics simulations also reveal the presence of strong low frequency noise with the exponent η=1.6. We trace parallels with the chaotic dynamics of symplectic maps with a few degrees of freedom characterized by the Poincaré exponent β~1.5.

  17. Human Replication Protein A Melts a DNA Triple Helix Structure in a Potent and Specific Manner†

    OpenAIRE

    Wu, Yuliang; Rawtani, Nina; Thazhathveetil, Arun Kalliat; Kenny, Mark K.; Seidman, Michael M.; Brosh, Robert M.

    2008-01-01

    Alternate DNA structures other than double-stranded B-form DNA can potentially impede cellular processes such as transcription and replication. The DNA triplex helix and G4 tetraplex structures that form by Hoogsteen hydrogen bonding are two examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human replication protein A (RPA), a single-stranded DNA binding protein that is implicated in all facets of DNA metabolism, t...

  18. A DNA biosensor based on resonance light scattering using unmodified gold bipyramids

    International Nuclear Information System (INIS)

    We report on a novel biosensor for determining sequence-specific DNA. It is based on resonance light scattering (RLS) caused by the aggregation of gold bipyramids. These display localized surface plasmon resonance and can be used as a bioprobe. The absorption spectra and the transmission electron micrographs provide visual evidence of the aggregation of the gold bipyramids in the presence of DNA. The RLS intensity of the gold bipyramids increases with the concentration of the target DNA. The method was successfully applied to the determination of a 30-mer single-stranded oligonucleotide and works over the 0.1-10 nM concentration range. (author)

  19. A DNA biosensor based on the electrocatalytic oxidation of amine by a threading intercalator

    Energy Technology Data Exchange (ETDEWEB)

    Gao Zhiqiang [Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669 (Singapore)], E-mail: zqgao@ibn.a-star.edu.sg; Tansil, Natalia [Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669 (Singapore)

    2009-03-16

    An electrochemical biosensor for the detection of DNA based a peptide nucleic acid (PNA) capture probe (CP) modified indium tin oxide electrode (ITO) is described in this report. After hybridization, a threading intercalator, N,N'-bis[(3-propyl)-imidazole]-1,4,5,8-naphthalene diimide (PIND) imidazole complexed with Ru(bpy){sub 2}Cl (PIND-Ru, bpy = 2,2'-bipyridine), was introduced to the biosensor. PIND-Ru selectively intercalated to double-stranded DNA (ds-DNA) and became immobilized on the biosensor surface. Voltammetric tests showed highly stable and reversible electrochemical oxidation/reduction processes and the peak currents can directly be utilized for DNA quantification. When the tests were conducted in an amine-containing medium, Tris-HCl buffer for example, a remarkable improvement in the voltammetric response and noticeable enhancements of voltammetric and amperometric sensitivities were observed due to the electrocatalytic activity of the [Ru(bpy){sub 2}Cl] redox moieties. Electrocatalytic current was observed when as little as 3.0 attomoles of DNA was present in the sample solution.

  20. Thermal and mechanical properties of a DNA model with solvation barrier

    CERN Document Server

    Tapia-Rojo, Rafael; Falo, Fernando

    2010-01-01

    We study the thermal and mechanical behavior of DNA denaturation in the frame of the mesoscopic Peyrard- Bishop-Dauxois model with the inclusion of solvent interaction. By analyzing the melting transition of a homogeneous A-T sequence, we are able to set suitable values of the parameters of the model and study the formation and stability of bubbles in the system. Then, we focus on the case of the P5 promoter sequence and use the Principal Component Analysis of the trajectories to extract the main information on the dynamical behavior of the system. We find that this analysis method gives an excellent agreement with previous biological results.

  1. MoS2 nanocrystals confined in a DNA matrix exhibiting energy transfer.

    Science.gov (United States)

    Goswami, Nirmal; Giri, Anupam; Pal, Samir Kumar

    2013-09-10

    We report the wet chemical synthesis of MoS2 nanocrystals (NCs), a transition-metal dichalcogenide, using DNA as a host matrix. As evidenced from transmission electron microscopy (TEM), the NCs are highly crystalline, with an average diameter of ~5 nm. Ultraviolet-visible (UV-vis) absorption studies along with band gap calculations confirm that NCs are in quantum confinement. A prominent red shift of the optical absorption bands has been observed upon formation of the thin film using hexadecyltrimethylammonium chloride (CTAC), i.e., in the case of MoS2@DNA-CTAC. In the thin film, strong electron-phonon coupling arises because of the resonance effect, which is reflected from the emergence of intense first-, second-, and third-order Raman peaks, whenever excited with the 488 nm line. We have established that our as-synthesized MoS2 NCs quench the fluorescence of a well-known DNA minor groove binding probe, Hoechst 33258. Unprecedented fluorescence quenching (94%) of donor (Hoechst 33258) emission and efficient energy transfer (89%) between Hoechst 33258 and MoS2 NCs (acceptor) are obtained. The donor-acceptor distance of these conjugates has been described by a Förster resonance energy transfer (FRET)-based model. Furthermore, employing a statistical method, we have estimated the probability of the distance distribution between the donor and acceptor. We believe that the study described herein may enable substantial advances in fields of optoelectronics, photovoltaics, catalysis, and many others. PMID:23931064

  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. Immunogenicity and protective efficacy of Vibrio harveyi pcFlaA DNA vaccine in Epinephelus awoara

    Institute of Scientific and Technical Information of China (English)

    QIN Yingxue; SU Yongquan; WANG Shifeng; YAN Qingpi

    2009-01-01

    The FlaA gene from Vibrio harveyi, with a short nucleotide sequence encoding the Flag marker, was cloned into the eukaryotic expression vector pcDNA3.1(+) (designated as pcFlaA). Ninety grouper (Epinephelus awoara) were separated into three equal size groups. An experimental group was immunized with pcFlaA, Control I group was immunized with the vector pcDNA3.1(+), and Control II group was immunized with PBS. The expression of pcFlaA mRNA and protein was examined using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. We also evaluated the immunogenicity and protective efficacy of pcFlaA against V. harveyi by measuring the lymphocyte proliferation response and serum levels of specific antibody and conducting a bacterial challenge test. We successfully transfected the fish muscle with pcFlaA. The pcFlaA mRNA and protein was expressed in the muscle cells for up to one month following injection. The proliferation response of lymphocytes in fish immunized with pcFlaA was significantly higher than in control group II. Furthermore, the immunized fish generated specific antibody. The vaccination also resulted in significantly higher survival during the bacterial challenge test.

  4. Screening and functional pathway analysis of genes associated with pediatric allergic asthma using a DNA microarray

    OpenAIRE

    Lu, Li-Qun; Liao, Wei

    2015-01-01

    The present study aimed to identify differentially expressed genes (DEGs) associated with pediatric allergic asthma, and to analyze the functional pathways of the selected target genes, in order to explore the pathogenesis of the disease. The GSE18965 gene expression profile was downloaded from the Gene Expression Omnibus database and was preprocessed. This gene expression profile consisted of seven normal samples and nine samples from patients with pediatric allergic asthma. The DEGs between...

  5. Arabidopsis thaliana GYRB3 does not encode a DNA gyrase subunit.

    Directory of Open Access Journals (Sweden)

    Katherine M Evans-Roberts

    Full Text Available DNA topoisomerases are enzymes that control the topology of DNA in all cells. DNA gyrase is unique among the topoisomerases in that it is the only enzyme that can actively supercoil DNA using the free energy of ATP hydrolysis. Until recently gyrase was thought to be unique to bacteria, but has now been discovered in plants. The genome of the model plant, Arabidopsis thaliana, is predicted to encode four gyrase subunits: AtGyrA, AtGyrB1, AtGyrB2 and AtGyrB3.We found, contrary to previous data, that AtGyrB3 is not essential to the survival of A. thaliana. Bioinformatic analysis suggests AtGyrB3 is considerably shorter than other gyrase B subunits, lacking part of the ATPase domain and other key motifs found in all type II topoisomerases; but it does contain a putative DNA-binding domain. Partially purified AtGyrB3 cannot bind E. coli GyrA or support supercoiling. AtGyrB3 cannot complement an E. coli gyrB temperature-sensitive strain, whereas AtGyrB2 can. Yeast two-hybrid analysis suggests that AtGyrB3 cannot bind to AtGyrA or form a dimer.These data strongly suggest that AtGyrB3 is not a gyrase subunit but has another unknown function. One possibility is that it is a nuclear protein with a role in meiosis in pollen.

  6. A universal design for a DNA probe providing ratiometric fluorescence detection by generation of silver nanoclusters

    Science.gov (United States)

    Del Bonis-O'Donnell, Jackson Travis; Vong, Daniel; Pennathur, Sumita; Fygenson, Deborah Kuchnir

    2016-07-01

    DNA-stabilized silver nanoclusters (AgNCs), the fluorescence emission of which can rival that of typical organic fluorophores, have made possible a new class of label-free molecular beacons for the detection of single-stranded DNA. Like fluorophore-quencher molecular beacons (FQ-MBs) AgNC-based molecular beacons (AgNC-MBs) are based on a single-stranded DNA that undergoes a conformational change upon binding a target sequence. The new conformation exposes a stretch of single-stranded DNA capable of hosting a fluorescent AgNC upon reduction in the presence of Ag+ ions. The utility of AgNC-MBs has been limited, however, because changing the target binding sequence unpredictably alters cluster fluorescence. Here we show that the original AgNC-MB design depends on bases in the target-binding (loop) domain to stabilize its AgNC. We then rationally alter the design to overcome this limitation. By separating and lengthening the AgNC-stabilizing domain, we create an AgNC-hairpin probe with consistent performance for arbitrary target sequence. This new design supports ratiometric fluorescence measurements of DNA target concentration, thereby providing a more sensitive, responsive and stable signal compared to turn-on AgNC probes. Using the new design, we demonstrate AgNC-MBs with nanomolar sensitivity and singe-nucleotide specificity, expanding the breadth of applicability of these cost-effective probes for biomolecular detection.DNA-stabilized silver nanoclusters (AgNCs), the fluorescence emission of which can rival that of typical organic fluorophores, have made possible a new class of label-free molecular beacons for the detection of single-stranded DNA. Like fluorophore-quencher molecular beacons (FQ-MBs) AgNC-based molecular beacons (AgNC-MBs) are based on a single-stranded DNA that undergoes a conformational change upon binding a target sequence. The new conformation exposes a stretch of single-stranded DNA capable of hosting a fluorescent AgNC upon reduction in the presence of Ag+ ions. The utility of AgNC-MBs has been limited, however, because changing the target binding sequence unpredictably alters cluster fluorescence. Here we show that the original AgNC-MB design depends on bases in the target-binding (loop) domain to stabilize its AgNC. We then rationally alter the design to overcome this limitation. By separating and lengthening the AgNC-stabilizing domain, we create an AgNC-hairpin probe with consistent performance for arbitrary target sequence. This new design supports ratiometric fluorescence measurements of DNA target concentration, thereby providing a more sensitive, responsive and stable signal compared to turn-on AgNC probes. Using the new design, we demonstrate AgNC-MBs with nanomolar sensitivity and singe-nucleotide specificity, expanding the breadth of applicability of these cost-effective probes for biomolecular detection. Electronic supplementary information (ESI) available: Tables containing DNA sequences of probes and targets; fluorescence emission spectra of AgNC12-MB probes containing a 4-dT spacer between domains; comparison of ratiometric fluorescence emission of AgNC22-MB probes using UV excitation and visible excitation; emission intensities of the green and red AgNCs generated by AgNC22-MBs with and without target and excited using peak visible excitation. See DOI: 10.1039/c6nr03827a

  7. Active destabilization of base pairs by a DNA glycosylase wedge initiates damage recognition

    Science.gov (United States)

    Kuznetsov, Nikita A.; Bergonzo, Christina; Campbell, Arthur J.; Li, Haoquan; Mechetin, Grigory V.; de los Santos, Carlos; Grollman, Arthur P.; Fedorova, Olga S.; Zharkov, Dmitry O.; Simmerling, Carlos

    2015-01-01

    Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal guanine. We combined molecular dynamics simulation and stopped-flow kinetics with fluorescence detection to track the events in the recognition of oxoG by Fpg and its mutants with a key phenylalanine residue, which intercalates next to the damaged base, changed to either alanine (F110A) or fluorescent reporter tryptophan (F110W). Guanine was sampled by Fpg, as evident from the F110W stopped-flow traces, but less extensively than oxoG. The wedgeless F110A enzyme could bend DNA but failed to proceed further in oxoG recognition. Modeling of the base eversion with energy decomposition suggested that the wedge destabilizes the intrahelical base primarily through buckling both surrounding base pairs. Replacement of oxoG with abasic (AP) site rescued the activity, and calculations suggested that wedge insertion is not required for AP site destabilization and eversion. Our results suggest that Fpg, and possibly other DNA glycosylases, convert part of the binding energy into active destabilization of their substrates, using the energy differences between normal and damaged bases for fast substrate discrimination. PMID:25520195

  8. Breather trapping and breather transmission in a DNA model with an interface

    DEFF Research Database (Denmark)

    Alvarez, A.; Romero, F.R.; Archilla, J.F.R.;

    2006-01-01

    in a region around the interface collecting vibrational energy. For an energy larger than the critical value, the breather is transmitted and continues travelling along the double strand with lower velocity. Reflection phenomena never occur. The same study has been carried out when a single dipole is oriented...... of the Peyrard-Bishop model is augmented with a term that includes the dipole-dipole coupling between base pairs. Numerical simulations show the existence of two dynamical regimes. If the translational kinetic energy of a moving breather launched towards the interface is below a critical value, it is trapped...

  9. Study on Electrochemical Insulin Sensing Utilizing a DNA Aptamer-Immobilized Gold Electrode

    Directory of Open Access Journals (Sweden)

    Izumi Kubo

    2015-07-01

    Full Text Available We investigated an insulin-sensing method by utilizing an insulin-binding aptamer IGA3, which forms an anti-parallel G-quadruplex with folded single strands. Spectroscopic observation indicates that some anti-parallel G-quadruplex bind hemin and show peroxidase activity. In this study, the peroxidase activity of IGA3 with hemin was confirmed by spectrophotometric measurements, i.e., the activity was three-times higher than hemin itself. IGA3 was then immobilized onto a gold electrode to determine its electrochemical activity. The peroxidase activity of the immobilized IGA3-hemin complex was determined by cyclic voltammetry, and a cathodic peak current of the electrode showed a dependence on the concentration of H2O2. The cathodic peak current of the IGA3-hemin complex decreased by binding it to insulin, and this decrease depended on the concentration of insulin.

  10. A DNA minor groove electronegative potential genome map based on photo-chemical probing

    DEFF Research Database (Denmark)

    Lindemose, Søren; Nielsen, Peter Eigil; Hansen, Morten;

    2011-01-01

    of any given sequence. We have validated this model on a series of protein-DNA binding sites known to involve minor groove electrostatic recognition as well as on stable nucleosome core complexes. The algorithm allows for the first time a full minor groove electrostatic description at the nucleotide...

  11. A DNA microarray for the authentication of toxic traditional Chinese medicinal plants.

    Science.gov (United States)

    Carles, Maria; Cheung, Matthew Kin; Moganti, Shanti; Dong, Tina T; Tsim, Karl W; Ip, Nancy Y; Sucher, Nikolaus J

    2005-06-01

    A silicon-based DNA microarray was designed and fabricated for the identification of toxic traditional Chinese medicinal plants. Species-specific oligonucleotide probes were derived from the 5S ribosomal RNA gene of Aconitum carmichaeli, A. kusnezoffi, Alocasia macrorrhiza, Croton tiglium, Datura inoxia, D. metel, D. tatula, Dysosma pleiantha, Dy. versipellis, Euphorbia kansui, Hyoscyamus niger, Pinellia cordata, P. pedatisecta, P. ternata, Rhododendron molle, Strychnos nux-vomica, Typhonium divaricatum and T. giganteum and the leucine transfer RNA gene of Aconitum pendulum and Stellera chamaejasme. The probes were immobilized via dithiol linkage on a silicon chip. Genomic target sequences were amplified and fluorescently labeled by asymmetric polymerase chain reaction. Multiple toxic plant species were identified by parallel genotyping. Chip-based authentication of medicinal plants may be useful as inexpensive and rapid tool for quality control and safety monitoring of herbal pharmaceuticals and neutraceuticals. PMID:15971136

  12. Identification of the vascular plants of Churchill, Manitoba, using a DNA barcode library

    Directory of Open Access Journals (Sweden)

    Kuzmina Maria L

    2012-11-01

    Full Text Available Abstract Background Because arctic plant communities are highly vulnerable to climate change, shifts in their composition require rapid, accurate identifications, often for specimens that lack diagnostic floral characters. The present study examines the role that DNA barcoding can play in aiding floristic evaluations in the arctic by testing the effectiveness of the core plant barcode regions (rbcL, matK and a supplemental ribosomal DNA (ITS2 marker for a well-studied flora near Churchill, Manitoba. Results This investigation examined 900 specimens representing 312 of the 354 species of vascular plants known from Churchill. Sequencing success was high for rbcL: 95% for fresh specimens and 85% for herbarium samples (mean age 20 years. ITS2 worked equally well for the fresh and herbarium material (89% and 88%. However, sequencing success was lower for matK, despite two rounds of PCR amplification, which reflected less effective primer binding and sensitivity to the DNA degradation (76% of fresh, 45% of herbaria samples. A species was considered as taxonomically resolved if its members showed at least one diagnostic difference from any other taxon in the study and formed a monophyletic clade. The highest species resolution (69% was obtained by combining information from all three genes. The joint sequence information for rbcL and matK distinguished 54% of 286 species, while rbcL and ITS2 distinguished 63% of 285 species. Discrimination of species within Salix, which constituted 8% of the flora, was particularly problematic. Despite incomplete resolution, the barcode results revealed 22 misidentified herbarium specimens, and enabled the identification of field specimens which were otherwise too immature to identify. Although seven cases of ITS2 paralogy were noted in the families Cyperaceae, Juncaceae and Juncaginaceae, this intergenic spacer played an important role in resolving congeneric plant species at Churchill. Conclusions Our results provided fast and cost-effective solution to create a comprehensive, effective DNA barcode reference library for a local flora.

  13. Solving the SAT problem using a DNA computing algorithm based on ligase chain reaction.

    Science.gov (United States)

    Wang, Xiaolong; Bao, Zhenmin; Hu, Jingjie; Wang, Shi; Zhan, Aibin

    2008-01-01

    A new DNA computing algorithm based on a ligase chain reaction is demonstrated to solve an SAT problem. The proposed DNA algorithm can solve an n-variable m-clause SAT problem in m steps and the computation time required is O (3m+n). Instead of generating the full-solution DNA library, we start with an empty test tube and then generate solutions that partially satisfy the SAT formula. These partial solutions are then extended step by step by the ligation of new variables using Taq DNA ligase. Correct strands are amplified and false strands are pruned by a ligase chain reaction (LCR) as soon as they fail to satisfy the conditions. If we score and sort the clauses, we can use this algorithm to markedly reduce the number of DNA strands required throughout the computing process. In a computer simulation, the maximum number of DNA strands required was 2(0.48n) when n=50, and the exponent ratio varied inversely with the number of variables n and the clause/variable ratio m/n. This algorithm is highly space-efficient and error-tolerant compared to conventional brute-force searching, and thus can be scaled-up to solve large and hard SAT problems. PMID:17904730

  14. The Role of Cytosine Methylation on Charge Transport through a DNA Strand

    Energy Technology Data Exchange (ETDEWEB)

    Qi, Jianqing [Univ. of Washington, Seattle, WA (United States); Govind, Niranjan [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Anantram, M. P. [Univ. of Washington, Seattle, WA (United States)

    2015-09-04

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modifi-cation remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Buttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. Specifically, we compare the results generated with the widely used B3LYP exchange-correlation (XC) functional and CAM-B3LYP based tuned range-separated hybrid density functional. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that with both functionals, the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and interstrand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital (HOMO) level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with both functionals. We also study the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit. Our results suggest that the effect of the two different functionals is to alter the on-site energies of the DNA bases at the HOMO level, while the transport properties don't depend much on the two functionals.

  15. The role of cytosine methylation on charge transport through a DNA strand

    Energy Technology Data Exchange (ETDEWEB)

    Qi, Jianqing, E-mail: jqqi@uw.edu; Anantram, M. P., E-mail: anantmp@uw.edu [Department of Electrical Engineering, University of Washington, Seattle, Washington 98195-2500 (United States); Govind, Niranjan, E-mail: niri.govind@pnnl.gov [William R. Wiley Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352 (United States)

    2015-09-07

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Büttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit.

  16. Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins

    Science.gov (United States)

    Gaspar, Miguel; Shenk, Thomas

    2006-02-01

    The DNA damage checkpoint pathway responds to DNA damage and induces a cell cycle arrest to allow time for DNA repair. Several viruses are known to activate or modulate this cellular response. Here we show that the ataxia-telangiectasia mutated checkpoint pathway, which responds to double-strand breaks in DNA, is activated in response to human cytomegalovirus DNA replication. However, this activation does not propagate through the pathway; it is blocked at the level of the effector kinase, checkpoint kinase 2 (Chk2). Late after infection, several checkpoint proteins, including ataxia-telangiectasia mutated and Chk2, are mislocalized to a cytoplasmic virus assembly zone, where they are colocalized with virion structural proteins. This colocalization was confirmed by immunoprecipitation of virion proteins with an antibody that recognizes Chk2. Virus replication was resistant to ionizing radiation, which causes double-strand breaks in DNA. We propose that human CMV DNA replication activates the checkpoint response to DNA double-strand breaks, and the virus responds by altering the localization of checkpoint proteins to the cytoplasm and thereby inhibiting the signaling pathway. ionizing radiation | ataxia-telangiectasia mutated pathway

  17. A High Phosphorus Diet Affects Lipid Metabolism in Rat Liver: A DNA Microarray Analysis.

    Directory of Open Access Journals (Sweden)

    Sunwoo Chun

    Full Text Available A high phosphorus (HP diet causes disorders of renal function, bone metabolism, and vascular function. We previously demonstrated that DNA microarray analysis is an appropriate method to comprehensively evaluate the effects of a HP diet on kidney dysfunction such as calcification, fibrillization, and inflammation. We reported that type IIb sodium-dependent phosphate transporter is significantly up-regulated in this context. In the present study, we performed DNA microarray analysis to investigate the effects of a HP diet on the liver, which plays a pivotal role in energy metabolism. DNA microarray analysis was performed with total RNA isolated from the livers of rats fed a control diet (containing 0.3% phosphorus or a HP diet (containing 1.2% phosphorus. Gene Ontology analysis of differentially expressed genes (DEGs revealed that the HP diet induced down-regulation of genes involved in hepatic amino acid catabolism and lipogenesis, while genes related to fatty acid β-oxidation process were up-regulated. Although genes related to fatty acid biosynthesis were down-regulated in HP diet-fed rats, genes important for the elongation and desaturation reactions of omega-3 and -6 fatty acids were up-regulated. Concentrations of hepatic arachidonic acid and eicosapentaenoic acid were increased in HP diet-fed rats. These essential fatty acids activate peroxisome proliferator-activated receptor alpha (PPARα, a transcription factor for fatty acid β-oxidation. Evaluation of the upstream regulators of DEGs using Ingenuity Pathway Analysis indicated that PPARα was activated in the livers of HP diet-fed rats. Furthermore, the serum concentration of fibroblast growth factor 21, a hormone secreted from the liver that promotes fatty acid utilization in adipose tissue as a PPARα target gene, was higher (p = 0.054 in HP diet-fed rats than in control diet-fed rats. These data suggest that a HP diet enhances energy expenditure through the utilization of free fatty acids released via lipolysis of white adipose tissue.

  18. The Protective Mechanisms Induced by a DNA Vaccine in Fish Depend on Temperature

    DEFF Research Database (Denmark)

    Lorenzen, Ellen; Einer-Jensen, Katja; Rasmussen, Jesper Skou;

    2011-01-01

    In veterinary vaccinology, DNA-vaccines encoding the viral glycoproteins of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV) have proved highly efficient in fish under experimental conditions. In the early phase following vaccination, innate cross-protective...... mechanisms are dominating but the protection becomes highly specific within 3–4 weeks at 12–15 C. Temperature is known as an important external parameter affecting the immune response in fish and the present study aimed at characterizing temperature effects on the immune response to a VHS DNA vaccine....... Rainbow trout fingerlings acclimated at 5, 10 or 15 C, were given an intramuscular injection of 1 lg purified plasmid DNA and challenged with virulent VHSV 9 or 36–40 days later. The vaccine protected the fish well at all three temperatures, however the non-specific mechanisms lasted for a longer period...

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

    OpenAIRE

    Houtteman, S W; Elder, R T

    1993-01-01

    Yeast autonomously replicating sequence (ARS) plasmids exhibit an unusual segregation pattern during mitosis. While the nucleus divides equally into mother and daughter cells, all copies of the ARS plasmid will often remain in the mother cell. A screen was designed to isolate mutations that suppress this segregation bias. A plasmid with a weak ARS (wARS) that displayed an extremely high segregation bias was constructed. When cells were grown under selection for the wARS plasmid, the resulting...

  20. Molecular Cloning and Analysis of a DNA Repetitive Element from the Mouse Genome

    Science.gov (United States)

    Geisinger, Adriana; Cossio, Gabriela; Wettstein, Rodolfo

    2006-01-01

    We report the development of a 3-week laboratory activity for an undergraduate molecular biology course. This activity introduces students to the practice of basic molecular techniques such as restriction enzyme digestion, agarose gel electrophoresis, cloning, plasmid DNA purification, Southern blotting, and sequencing. Students learn how to carry…

  1. Algebraic Statistics of Poincaré Recurrences in a DNA Molecule

    Science.gov (United States)

    Mazur, Alexey K.; Shepelyansky, D. L.

    2015-10-01

    The statistics of Poincaré recurrences is studied for the base-pair breathing dynamics of an all-atom DNA molecule in a realistic aqueous environment with thousands of degrees of freedom. It is found that at least over five decades in time the decay of recurrences is described by an algebraic law with the Poincaré exponent close to β =1.2 . This value is directly related to the correlation decay exponent ν =β -1 , which is close to ν ≈0.15 observed in the time resolved Stokes shift experiments. By applying the virial theorem we analyze the chaotic dynamics in polynomial potentials and demonstrate analytically that an exponent β =1.2 is obtained assuming the dominance of dipole-dipole interactions in the relevant DNA dynamics. Molecular dynamics simulations also reveal the presence of strong low frequency noise with the exponent η =1.6 . We trace parallels with the chaotic dynamics of symplectic maps with a few degrees of freedom characterized by the Poincaré exponent β ˜1.5 .

  2. A DNA-Mediated Homogeneous Binding Assay for Proteins and Small Molecules

    DEFF Research Database (Denmark)

    Zhang, Zhao; Hejesen, Christian; Kjelstrup, Michael Brøndum;

    2014-01-01

    Optical detection of molecular targets typically requires immobilization, separation, or chemical or enzymatic processing. An important exception is aptamers that allow optical detection in solution based on conformational changes. This method, however, requires the laborious selection of aptamers...... with high target specificity and affinity, and the ability to undergo the required conformational changes. Here we report on an alternative generic scheme for detecting small molecules and proteins in solution based on a shift in the equilibrium of DNA-based strand displacement competition reaction....... The shift occurs upon binding of a protein, for example, an antibody to its target. We demonstrate nanomolar detection of small molecules such as biotin, digoxigenin, vitamin D, and folate, in buffer and in plasma. The method is flexible, and we also show nanomolar detection of the respective antibodies...

  3. A DNA barcode library for North American Ephemeroptera: progress and prospects.

    Directory of Open Access Journals (Sweden)

    Jeffrey M Webb

    Full Text Available DNA barcoding of aquatic macroinvertebrates holds much promise as a tool for taxonomic research and for providing the reliable identifications needed for water quality assessment programs. A prerequisite for identification using barcodes is a reliable reference library. We gathered 4165 sequences from the barcode region of the mitochondrial cytochrome c oxidase subunit I gene representing 264 nominal and 90 provisional species of mayflies (Insecta: Ephemeroptera from Canada, Mexico, and the United States. No species shared barcode sequences and all can be identified with barcodes with the possible exception of some Caenis. Minimum interspecific distances ranged from 0.3-24.7% (mean: 12.5%, while the average intraspecific divergence was 1.97%. The latter value was inflated by the presence of very high divergences in some taxa. In fact, nearly 20% of the species included two or three haplotype clusters showing greater than 5.0% sequence divergence and some values are as high as 26.7%. Many of the species with high divergences are polyphyletic and likely represent species complexes. Indeed, many of these polyphyletic species have numerous synonyms and individuals in some barcode clusters show morphological attributes characteristic of the synonymized species. In light of our findings, it is imperative that type or topotype specimens be sequenced to correctly associate barcode clusters with morphological species concepts and to determine the status of currently synonymized species.

  4. Colloidal Au-enhanced surface plasmon resonance imaging: application in a DNA hybridization process

    Science.gov (United States)

    Manera, M. G.; Spadavecchia, J.; Taurino, A.; Rella, R.

    2010-03-01

    The detection of the DNA hybridization mechanism using monodispersed gold nanoparticles as labels is an interesting alternative to increase the sensitivity of the SPR imaging technique. DNA-modified Au nanoparticles (DNA-Au NPs) containing single-stranded (ss) portions of DNA were prepared by monitoring their monolayer formation by UV-vis spectroscopy. The hybridization process between specific thio-oligonucleotides immobilized on the DNA-Au NPs and the corresponding complementary strands is reported and compared with the traditional hybridization process on properly self-assembled thin gold films deposited on glass substrates. A remarkable signal amplification is observed, following the incorporation of colloidal Au into a SPR biosensing experiment, resulting in an increased SPR response to DNA-DNA interactions. In particular Fusarium thiolated DNA (5'HS poly(T)15ATC CCT CAA AAA CTG CCG CT-3) and trichothecenes complementary DNA (5'-AGC GGC AGT TTT TGA GGG AT-3') sequences have been explored due to their possible application to agro-industry for the control of food quality.

  5. Identification of a DNA binding protein that recognizes the nonamer recombinational signal sequence of immunoglobulin genes.

    Science.gov (United States)

    Halligan, B D; Desiderio, S V

    1987-10-01

    Extracts of nuclei from B- and T-lymphoid cells contain a protein that binds specifically to the conserved nonamer DNA sequence within the recombinational signals of immunoglobulin genes. Complexes with DNA fragments from four kappa light-chain joining (J) segments have the same electrophoretic mobility. Nonamer-containing DNA fragments from heavy-chain and light-chain genes compete for binding. Within the 5'-flanking DNA of the J kappa 4 gene segment, the binding site has been localized to a 27-base-pair interval spanning the nonamer region. The binding activity is recovered as a single peak after ion-exchange chromatography. The site of binding of the protein and its presence in nuclei of lymphoid cells suggest that it may function in the assembly of immunoglobulin genes.

  6. The 75-kilodalton cytoplasmic Chlamydia trachomatis L2 polypeptide is a DnaK-like protein

    DEFF Research Database (Denmark)

    Birkelund, Svend; Lundemose, AG; Christiansen, Gunna

    1990-01-01

    The gene coding for the 75-kilodalton cytoplasmic Chlamydia trachomatis L2 polypeptide has been cloned in Escherichia coli, and the nucleotide sequence has been determined. The cloned DNA fragment contained the coding region as well as the putative promoter. The deduced amino acid sequence of the 1...

  7. Evidence of a genetic instability induced by the incorporation of a DNA precursor marked with tritium

    International Nuclear Information System (INIS)

    The authors report a molecular geno-toxicology investigation which allowed molecular events induced par intracellular incorporation of tritium to be studied, and the genetic instability resulting from a chronic exposure even at low dose to be analysed. For this purpose, they developed cell models (hamster tumorous cells and human fibroblasts) in which they know how to incorporate given quantities of marked nucleotides in the DNA. They show that the incorporation of tritium, even with doses which are said to be non toxic, causes a prolonged exposure of the cell to a genotoxic stress, and maybe a genetic instability due to a too great number of recombination events

  8. Colour patterns do not diagnose species: quantitative evaluation of a DNA barcoded cryptic bumblebee complex.

    Directory of Open Access Journals (Sweden)

    James C Carolan

    Full Text Available Cryptic diversity within bumblebees (Bombus has the potential to undermine crucial conservation efforts designed to reverse the observed decline in many bumblebee species worldwide. Central to such efforts is the ability to correctly recognise and diagnose species. The B. lucorum complex (Bombus lucorum, B. cryptarum and B. magnus comprises one of the most abundant and important group of wild plant and crop pollinators in northern Europe. Although the workers of these species are notoriously difficult to diagnose morphologically, it has been claimed that queens are readily diagnosable from morphological characters. Here we assess the value of colour-pattern characters in species identification of DNA-barcoded queens from the B. lucorum complex. Three distinct molecular operational taxonomic units were identified each representing one species. However, no uniquely diagnostic colour-pattern character state was found for any of these three molecular units and most colour-pattern characters showed continuous variation among the units. All characters previously deemed to be unique and diagnostic for one species were displayed by specimens molecularly identified as a different species. These results presented here raise questions on the reliability of species determinations in previous studies and highlights the benefits of implementing DNA barcoding prior to ecological, taxonomic and conservation studies of these important key pollinators.

  9. A DNA Barcode Library for Korean Chironomidae (Insecta: Diptera) and Indexes for Defining Barcode Gap

    OpenAIRE

    Kim, Sungmin; Song, Kyo-Hong; Ree, Han-Il; Kim, Won

    2011-01-01

    Non-biting midges (Diptera: Chironomidae) are a diverse population that commonly causes respiratory allergies in humans. Chironomid larvae can be used to indicate freshwater pollution, but accurate identification on the basis of morphological characteristics is difficult. In this study, we constructed a mitochondrial cytochrome c oxidase subunit I (COI)-based DNA barcode library for Korean chironomids. This library consists of 211 specimens from 49 species, including adults and unidentified l...

  10. Identification of the vascular plants of Churchill, Manitoba, using a DNA barcode library

    Science.gov (United States)

    2012-01-01

    Background Because arctic plant communities are highly vulnerable to climate change, shifts in their composition require rapid, accurate identifications, often for specimens that lack diagnostic floral characters. The present study examines the role that DNA barcoding can play in aiding floristic evaluations in the arctic by testing the effectiveness of the core plant barcode regions (rbcL, matK) and a supplemental ribosomal DNA (ITS2) marker for a well-studied flora near Churchill, Manitoba. Results This investigation examined 900 specimens representing 312 of the 354 species of vascular plants known from Churchill. Sequencing success was high for rbcL: 95% for fresh specimens and 85% for herbarium samples (mean age 20 years). ITS2 worked equally well for the fresh and herbarium material (89% and 88%). However, sequencing success was lower for matK, despite two rounds of PCR amplification, which reflected less effective primer binding and sensitivity to the DNA degradation (76% of fresh, 45% of herbaria samples). A species was considered as taxonomically resolved if its members showed at least one diagnostic difference from any other taxon in the study and formed a monophyletic clade. The highest species resolution (69%) was obtained by combining information from all three genes. The joint sequence information for rbcL and matK distinguished 54% of 286 species, while rbcL and ITS2 distinguished 63% of 285 species. Discrimination of species within Salix, which constituted 8% of the flora, was particularly problematic. Despite incomplete resolution, the barcode results revealed 22 misidentified herbarium specimens, and enabled the identification of field specimens which were otherwise too immature to identify. Although seven cases of ITS2 paralogy were noted in the families Cyperaceae, Juncaceae and Juncaginaceae, this intergenic spacer played an important role in resolving congeneric plant species at Churchill. Conclusions Our results provided fast and cost-effective solution to create a comprehensive, effective DNA barcode reference library for a local flora. PMID:23190419

  11. A DNA 'barcode blitz': rapid digitization and sequencing of a natural history collection.

    Directory of Open Access Journals (Sweden)

    Paul D N Hebert

    Full Text Available DNA barcoding protocols require the linkage of each sequence record to a voucher specimen that has, whenever possible, been authoritatively identified. Natural history collections would seem an ideal resource for barcode library construction, but they have never seen large-scale analysis because of concerns linked to DNA degradation. The present study examines the strength of this barrier, carrying out a comprehensive analysis of moth and butterfly (Lepidoptera species in the Australian National Insect Collection. Protocols were developed that enabled tissue samples, specimen data, and images to be assembled rapidly. Using these methods, a five-person team processed 41,650 specimens representing 12,699 species in 14 weeks. Subsequent molecular analysis took about six months, reflecting the need for multiple rounds of PCR as sequence recovery was impacted by age, body size, and collection protocols. Despite these variables and the fact that specimens averaged 30.4 years old, barcode records were obtained from 86% of the species. In fact, one or more barcode compliant sequences (>487 bp were recovered from virtually all species represented by five or more individuals, even when the youngest was 50 years old. By assembling specimen images, distributional data, and DNA barcode sequences on a web-accessible informatics platform, this study has greatly advanced accessibility to information on thousands of species. Moreover, much of the specimen data became publically accessible within days of its acquisition, while most sequence results saw release within three months. As such, this study reveals the speed with which DNA barcode workflows can mobilize biodiversity data, often providing the first web-accessible information for a species. These results further suggest that existing collections can enable the rapid development of a comprehensive DNA barcode library for the most diverse compartment of terrestrial biodiversity - insects.

  12. Starting a DNA barcode reference library for shallow water polychaetes from the southern European Atlantic coast.

    Science.gov (United States)

    Lobo, Jorge; Teixeira, Marcos A L; Borges, Luisa M S; Ferreira, Maria S G; Hollatz, Claudia; Gomes, Pedro T; Sousa, Ronaldo; Ravara, Ascensão; Costa, Maria H; Costa, Filipe O

    2016-01-01

    Annelid polychaetes have been seldom the focus of dedicated DNA barcoding studies, despite their ecological relevance and often dominance, particularly in soft-bottom estuarine and coastal marine ecosystems. Here, we report the first assessment of the performance of DNA barcodes in the discrimination of shallow water polychaete species from the southern European Atlantic coast, focusing on specimens collected in estuaries and coastal ecosystems of Portugal. We analysed cytochrome oxidase I DNA barcodes (COI-5P) from 164 specimens, which were assigned to 51 morphospecies. To our data set from Portugal, we added available published sequences selected from the same species, genus or family, to inspect for taxonomic congruence among studies and collection location. The final data set comprised 290 specimens and 79 morphospecies, which generated 99 Barcode Index Numbers (BINs) within Barcode of Life Data Systems (BOLD). Among these, 22 BINs were singletons, 47 other BINs were concordant, confirming the initial identification based on morphological characters, and 30 were discordant, most of which consisted on multiple BINs found for the same morphospecies. Some of the most prominent cases in the latter category include Hediste diversicolor (O.F. Müller, 1776) (7), Eulalia viridis (Linnaeus, 1767) (2) and Owenia fusiformis (delle Chiaje, 1844) (5), all of them reported from Portugal and frequently used in ecological studies as environmental quality indicators. Our results for these species showed discordance between molecular lineages and morphospecies, or added additional relatively divergent lineages. The potential inaccuracies in environmental assessments, where underpinning polychaete species diversity is poorly resolved or clarified, demand additional and extensive investigation of the DNA barcode diversity in this group, in parallel with alpha taxonomy efforts. PMID:26129849

  13. A DNA ‘Barcode Blitz’: Rapid Digitization and Sequencing of a Natural History Collection

    Science.gov (United States)

    Hebert, Paul D. N.; deWaard, Jeremy R.; Zakharov, Evgeny V.; Prosser, Sean W. J.; Sones, Jayme E.; McKeown, Jaclyn T. A.; Mantle, Beth; La Salle, John

    2013-01-01

    DNA barcoding protocols require the linkage of each sequence record to a voucher specimen that has, whenever possible, been authoritatively identified. Natural history collections would seem an ideal resource for barcode library construction, but they have never seen large-scale analysis because of concerns linked to DNA degradation. The present study examines the strength of this barrier, carrying out a comprehensive analysis of moth and butterfly (Lepidoptera) species in the Australian National Insect Collection. Protocols were developed that enabled tissue samples, specimen data, and images to be assembled rapidly. Using these methods, a five-person team processed 41,650 specimens representing 12,699 species in 14 weeks. Subsequent molecular analysis took about six months, reflecting the need for multiple rounds of PCR as sequence recovery was impacted by age, body size, and collection protocols. Despite these variables and the fact that specimens averaged 30.4 years old, barcode records were obtained from 86% of the species. In fact, one or more barcode compliant sequences (>487 bp) were recovered from virtually all species represented by five or more individuals, even when the youngest was 50 years old. By assembling specimen images, distributional data, and DNA barcode sequences on a web-accessible informatics platform, this study has greatly advanced accessibility to information on thousands of species. Moreover, much of the specimen data became publically accessible within days of its acquisition, while most sequence results saw release within three months. As such, this study reveals the speed with which DNA barcode workflows can mobilize biodiversity data, often providing the first web-accessible information for a species. These results further suggest that existing collections can enable the rapid development of a comprehensive DNA barcode library for the most diverse compartment of terrestrial biodiversity – insects. PMID:23874660

  14. Identification of the vascular plants of Churchill, Manitoba, using a DNA barcode library

    OpenAIRE

    Kuzmina Maria L; Johnson Karen L; Barron Hannah R; Hebert Paul DN

    2012-01-01

    Abstract Background Because arctic plant communities are highly vulnerable to climate change, shifts in their composition require rapid, accurate identifications, often for specimens that lack diagnostic floral characters. The present study examines the role that DNA barcoding can play in aiding floristic evaluations in the arctic by testing the effectiveness of the core plant barcode regions (rbcL, matK) and a supplemental ribosomal DNA (ITS2) marker for a well-studied flora near Churchill, ...

  15. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

    Directory of Open Access Journals (Sweden)

    Alvaro Díaz-Badillo

    2014-04-01

    Full Text Available Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.

  16. Single Molecule Atomic Force Microscopy Studies of Photosensitized Singlet Oxygen Behavior on a DNA Origami Template

    DEFF Research Database (Denmark)

    Helmig, Sarah Wendelboe; Rotaru, Alexandru; Arian, Dumitru;

    2010-01-01

    DNA origami, the folding of a long single-stranded DNA sequence (scaffold strand) by hundreds of short synthetic oligonucleotides (staple strands) into parallel aligned helices, is a highly efficient method to form advanced self-assembled DNA-architectures. Since molecules and various materials can...... a single photosensitizer molecule conjugated to a selected DNA origami staple strand on an origami structure. We demonstrate a distance-dependent oxidation of organic moieties incorporated in specific positions on DNA origami by singlet oxygen produced from a single photosensitizer located at the...

  17. A DNA test for Indiana/Swiss hereditary amyloidosis (FAP II).

    OpenAIRE

    Wallace, M. R.; Conneally, P M; Benson, M D

    1988-01-01

    Autosomal dominant amyloidosis of the Indiana/Swiss type is a late-onset disorder characterized by carpal tunnel syndrome, progressive peripheral neuropathy, vitreous deposits, and cardiomyopathy. This disorder was originally described in a large Indiana family of Swiss descent and is also known as familial amyloidotic polyneuropathy (FAP) type II. In the Indiana family, the genetic basis of the disease is a variant of plasma prealbumin (transthyretin), which has a serine-for-isoleucine subst...

  18. Bio-recognitive photonics of a DNA-guided organic semiconductor.

    Science.gov (United States)

    Back, Seung Hyuk; Park, Jin Hyuk; Cui, Chunzhi; Ahn, Dong June

    2016-01-01

    Incorporation of duplex DNA with higher molecular weights has attracted attention for a new opportunity towards a better organic light-emitting diode (OLED) capability. However, biological recognition by OLED materials is yet to be addressed. In this study, specific oligomeric DNA-DNA recognition is successfully achieved by tri (8-hydroxyquinoline) aluminium (Alq3), an organic semiconductor. Alq3 rods crystallized with guidance from single-strand DNA molecules show, strikingly, a unique distribution of the DNA molecules with a shape of an 'inverted' hourglass. The crystal's luminescent intensity is enhanced by 1.6-fold upon recognition of the perfect-matched target DNA sequence, but not in the case of a single-base mismatched one. The DNA-DNA recognition forming double-helix structure is identified to occur only in the rod's outer periphery. This study opens up new opportunities of Alq3, one of the most widely used OLED materials, enabling biological recognition.

  19. On some surprising statistical properties of a DNA fingerprinting technique called AFLP

    NARCIS (Netherlands)

    Gort, G.

    2010-01-01

    AFLP is a widely used DNA fingerprinting technique, resulting in band absence - presence profiles, like a bar code. Bands represent DNA fragments, sampled from the genome of an individual plant or other organism. The DNA fragments travel through a lane of an electrophoretic gel or microcapillary sys

  20. Design tools for a DNA-guided self-assembling carbon nanotube technology

    Science.gov (United States)

    Dwyer, C.; Johri, V.; Cheung, M.; Patwardhan, J.; Lebeck, A.; Sorin, D.

    2004-09-01

    The shift in technology away from silicon complementary metal-oxide semiconductors (CMOS) to novel nanoscale technologies requires new design tools. In this paper, we explore one particular nanotechnology: carbon nanotube transistors that are self-assembled into circuits by using DNA. We develop design tools and demonstrate how to use them to develop circuitry based on this nanotechnology.

  1. Embryonic stem cells or induced pluripotent stem cells? A DNA integrity perspective.

    Science.gov (United States)

    Bai, Qiang; Desprat, Romain; Klein, Bernard; Lemaître, Jean-Marc; De Vos, John

    2013-04-01

    Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are two types of pluripotent stem cells that hold great promise for biomedical research and medical applications. iPSCs were initially favorably compared to ESCs. This view was first based on ethical arguments (the generation of iPSCs does not require the destruction of an embryo) and on immunological reasons (it is easier to derive patient HLA-matched iPSCs than ESCs). However, several reports suggest that iPSCs might be characterized by higher occurrence of epigenetic and genetic aberrations than ESCs as a consequence of the reprogramming process. We focus here on the DNA integrity of pluripotent stem cells and examine the three main sources of genomic abnormalities in iPSCs: (1) genomic variety of the parental cells, (2) cell reprogramming, and (3) in vitro cell culture. Recent reports claim that it is possible to generate mouse or human iPSC lines with a mutation level similar to that of the parental cells, suggesting that "genome-friendly" reprogramming techniques can be developed. The issue of iPSC DNA integrity clearly highlights the crucial need of guidelines to define the acceptable level of genomic integrity of pluripotent stem cells for biomedical applications. We discuss here the main issues that such guidelines should address.

  2. Preferential DNA Cleavage under Anaerobic Conditions by a DNA Binding Ruthenium Dimer

    OpenAIRE

    Janaratne, Thamara K.; Ongeri, Fiona; Yadav, Abishek; MacDonnell, Frederick M.

    2007-01-01

    In the absence of O2, the cationic complex, [(phen)2Ru(tatpp)Ru(phen)2]4+ (P4+), undergoes in situ reduction by glutathione (GSH) to form a species that induces DNA cleavage. Exposure to air strongly attenuates the cleavage activity, even in the presence of a large excess of reducing agent (e.g., 40 equiv GSH per P4+) suggesting the complex may be useful in targeting cells with a low oxygen microenvironment (hypoxia) for destruction via DNA cleavage. The active species is identified as the do...

  3. Clitocybe nuda Activates Dendritic Cells and Acts as a DNA Vaccine Adjuvant

    Directory of Open Access Journals (Sweden)

    Mei-Hsing Chen

    2013-01-01

    Full Text Available This work represents the first evaluation of the effects of water extract of C. nuda (WE-CN, an edible mushroom, on murine bone marrow-derived dendritic cells (BMDCs and the potential pathway through which the effects are mediated. Our experimental results show that WE-CN could induce phenotypic maturation of DCs, as shown by the increased expression of MHC and costimulatory molecules. In addition, it also induced the proinflammatory cytokines expression on DCs and enhanced both the proliferation and IFN-γ secretion of allogenic T cells. Therefore, since WE-CN did not induce maturation of DCs generated from mice with mutated TLR-4 or TLR-2, suggesting that TLR4 and TLR2 might function as membrane receptors for WE-CN. Moreover, the mechanism of action of WE-CN may be mediated by increased phosphorylation of ERK, p38, and JNK mitogen-activated protein kinase (MAPK and increased NF-κB p65 activity, which are important signaling molecules downstream of TLR-4 and TLR-2. Finally, coimmunization of mice with WE-CN and a HER-2/neu DNA vaccine induced a HER-2/neu-specific Th1 response that resulted in significant inhibition of HER-2/neu overexpressing mouse bladder tumor (MBT-2 growth. These data suggest that WE-CN induces DC maturation through TLR-4 and/or TLR-2 and that WE-CN can be used as an adjuvant in cancer vaccine immunotherapy.

  4. Evidence for a DNA-based mechanism of intron-mediated enhancement

    Directory of Open Access Journals (Sweden)

    Alan B. Rose

    2011-12-01

    Full Text Available Many introns significantly increase gene expression through a process termed Intron-Mediated Enhancement (IME. Introns exist in the transcribed DNA and the nascent RNA, and could affect expression from either location. To determine which is more relevant to IME, hybrid introns were constructed that contain sequences from stimulating Arabidopsis thaliana introns either in their normal orientation or as the reverse complement. Both ends of each intron are from the non-stimulatory COR15a intron in their normal orientation to allow splicing. The inversions create major alterations to the sequence of the transcribed RNA with relatively minor changes to the DNA structure. Introns containing portions of either the UBQ10 or ATPK1 intron increased expression to a similar degree regardless of orientation. Also, computational predictions of IME improve when both intron strands are considered. These findings are more consistent with models of IME that act at the level of DNA rather than RNA.

  5. Discovery of small-molecule interleukin-2 inhibitors from a DNA-encoded chemical library.

    Science.gov (United States)

    Leimbacher, Markus; Zhang, Yixin; Mannocci, Luca; Stravs, Michael; Geppert, Tim; Scheuermann, Jörg; Schneider, Gisbert; Neri, Dario

    2012-06-18

    Libraries of chemical compounds individually coupled to encoding DNA tags (DNA-encoded chemical libraries) hold promise to facilitate exceptionally efficient ligand discovery. We constructed a high-quality DNA-encoded chemical library comprising 30,000 drug-like compounds; this was screened in 170 different affinity capture experiments. High-throughput sequencing allowed the evaluation of 120 million DNA codes for a systematic analysis of selection strategies and statistically robust identification of binding molecules. Selections performed against the tumor-associated antigen carbonic anhydrase IX (CA IX) and the pro-inflammatory cytokine interleukin-2 (IL-2) yielded potent inhibitors with exquisite target specificity. The binding mode of the revealed pharmacophore against IL-2 was confirmed by molecular docking. Our findings suggest that DNA-encoded chemical libraries allow the facile identification of drug-like ligands principally to any protein of choice, including molecules capable of disrupting high-affinity protein-protein interactions.

  6. A DNA 'barcode blitz': rapid digitization and sequencing of a natural history collection.

    Science.gov (United States)

    Hebert, Paul D N; Dewaard, Jeremy R; Zakharov, Evgeny V; Prosser, Sean W J; Sones, Jayme E; McKeown, Jaclyn T A; Mantle, Beth; La Salle, John

    2013-01-01

    DNA barcoding protocols require the linkage of each sequence record to a voucher specimen that has, whenever possible, been authoritatively identified. Natural history collections would seem an ideal resource for barcode library construction, but they have never seen large-scale analysis because of concerns linked to DNA degradation. The present study examines the strength of this barrier, carrying out a comprehensive analysis of moth and butterfly (Lepidoptera) species in the Australian National Insect Collection. Protocols were developed that enabled tissue samples, specimen data, and images to be assembled rapidly. Using these methods, a five-person team processed 41,650 specimens representing 12,699 species in 14 weeks. Subsequent molecular analysis took about six months, reflecting the need for multiple rounds of PCR as sequence recovery was impacted by age, body size, and collection protocols. Despite these variables and the fact that specimens averaged 30.4 years old, barcode records were obtained from 86% of the species. In fact, one or more barcode compliant sequences (>487 bp) were recovered from virtually all species represented by five or more individuals, even when the youngest was 50 years old. By assembling specimen images, distributional data, and DNA barcode sequences on a web-accessible informatics platform, this study has greatly advanced accessibility to information on thousands of species. Moreover, much of the specimen data became publically accessible within days of its acquisition, while most sequence results saw release within three months. As such, this study reveals the speed with which DNA barcode workflows can mobilize biodiversity data, often providing the first web-accessible information for a species. These results further suggest that existing collections can enable the rapid development of a comprehensive DNA barcode library for the most diverse compartment of terrestrial biodiversity - insects.

  7. Self-assembly of two-dimensional binary quasicrystals: A possible route to a DNA quasicrystal

    CERN Document Server

    Reinhardt, Aleks; Romano, Flavio; Doye, Jonathan P K

    2016-01-01

    We use Monte Carlo simulations and free-energy techniques to show that binary solutions of penta- and hexavalent two-dimensional patchy particles can form thermodynamically stable quasicrystals even at very narrow patch widths, provided their patch interactions are chosen in an appropriate way. Such patchy particles can be thought of as a coarse-grained representation of DNA multi-arm `star' motifs, which can be chosen to bond with one another very specifically by tuning the DNA sequences of the protruding arms. We explore several possible design strategies and conclude that DNA star tiles that are designed to interact with one another in a specific but not overly constrained way could potentially be used to construct soft quasicrystals in experiment. We verify that such star tiles can form stable dodecagonal motifs using oxDNA, a realistic coarse-grained model of DNA.

  8. Ag85A DNA Vaccine Delivery by Nanoparticles: Influence of the Formulation Characteristics on Immune Responses.

    Science.gov (United States)

    Poecheim, Johanna; Barnier-Quer, Christophe; Collin, Nicolas; Borchard, Gerrit

    2016-01-01

    The influence of DNA vaccine formulations on immune responses in combination with adjuvants was investigated with the aim to increase cell-mediated immunity against plasmid DNA (pDNA) encoding Mycobacterium tuberculosis antigen 85A. Different ratios of pDNA with cationic trimethyl chitosan (TMC) nanoparticles were characterized for their morphology and physicochemical characteristics (size, zeta potential, loading efficiency and pDNA release profile) applied in vitro for cellular uptake studies and in vivo, to determine the dose-dependent effects of pDNA on immune responses. A selected pDNA/TMC nanoparticle formulation was optimized by the incorporation of muramyl dipeptide (MDP) as an immunostimulatory agent. Cellular uptake investigations in vitro showed saturation to a maximum level upon the increase in the pDNA/TMC nanoparticle ratio, correlating with increasing Th1-related antibody responses up to a definite pDNA dose applied. Moreover, TMC nanoparticles induced clear polarization towards a Th1 response, indicated by IgG2c/IgG1 ratios above unity and enhanced numbers of antigen-specific IFN-γ producing T-cells in the spleen. Remarkably, the incorporation of MDP in TMC nanoparticles provoked a significant additional increase in T-cell-mediated responses induced by pDNA. In conclusion, pDNA-loaded TMC nanoparticles are capable of provoking strong Th1-type cellular and humoral immune responses, with the potential to be further optimized by the incorporation of MDP. PMID:27626449

  9. Facile preparation of a DNA sensor for rapid herpes virus detection

    International Nuclear Information System (INIS)

    In this paper, a simple DNA sensor platform was developed for rapid herpes virus detection in real samples. The deoxyribonucleic acid (DNA) sequences of the herpes simplex virus (DNA probe) were directly immobilized on the surface of interdigitated electrodes by electrochemical polymerization along with pyrrole monomers. The potential was scanned from - 0.7 to + 0.6 V, and the scanning rate was 100 mV/s. Fourier transform infrared spectroscopy was employed to verify specific DNA sequence binding and the conducting polymer. The morphology of the conducting polymer doped with DNA strands was characterized using a field emission scanning electron microscope. As-obtained DNA sensor was used to detect the herpes virus DNA in the real samples. The results show that the current DNA sensors detected the lowest DNA concentration of 2 nM. This sensitivity appears to be better than that of the DNA sensors prepared by immobilization of the DNA probe on the 3-aminopropyl-triethoxy-silance (APTS) membrane.

  10. The Replication Focus Targeting Sequence (RFTS) Domain Is a DNA-competitive Inhibitor of Dnmt1

    Energy Technology Data Exchange (ETDEWEB)

    Syeda, Farisa; Fagan, Rebecca L.; Wean, Matthew; Avvakumov, George V.; Walker, John R.; Xue, Sheng; Dhe-Paganon, Sirano; Brenner, Charles (Iowa); (Toronto)

    2015-11-30

    Dnmt1 (DNA methyltransferase 1) is the principal enzyme responsible for maintenance of cytosine methylation at CpG dinucleotides in the mammalian genome. The N-terminal replication focus targeting sequence (RFTS) domain of Dnmt1 has been implicated in subcellular localization, protein association, and catalytic function. However, progress in understanding its function has been limited by the lack of assays for and a structure of this domain. Here, we show that the naked DNA- and polynucleosome-binding activities of Dnmt1 are inhibited by the RFTS domain, which functions by virtue of binding the catalytic domain to the exclusion of DNA. Kinetic analysis with a fluorogenic DNA substrate established the RFTS domain as a 600-fold inhibitor of Dnmt1 enzymatic activity. The crystal structure of the RFTS domain reveals a novel fold and supports a mechanism in which an RFTS-targeted Dnmt1-binding protein, such as Uhrf1, may activate Dnmt1 for DNA binding.

  11. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis

    Energy Technology Data Exchange (ETDEWEB)

    Santiago-Felipe, S.; Tortajada-Genaro, L.A.; Puchades, R.; Maquieira, A., E-mail: amaquieira@qim.upv.es

    2014-02-06

    Graphical abstract: -- Highlights: •Recombinase polymerase amplification is a powerful DNA method operating at 40 °C. •The combination RPA–ELISA gives excellent performances for high-throughput analysis. •Screening of food safety threats has been done using standard laboratory equipment. •Allergens, GMOs, bacteria, and fungi have been successfully determined. -- Abstract: Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR–ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA–ELISA combination is proposed for amplification at a low, constant temperature (40 °C) in a short time (40 min), for the hybridisation of labelled products to specific 5′-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA–ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA–ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings.

  12. A DNA vaccine encoding p39 and sp41 of Brucella melitensis induces protective immunity in BALB/c mice

    Directory of Open Access Journals (Sweden)

    A Al-Mariri

    2014-01-01

    Full Text Available Brucella species are facultative intracellular gram-negative bacteria that can multiply within phagocytic and non-phagocytic cells of humans or animals as end hosts. B. melitensis causes abortion in pregnant animals and undulant fever in humans. A 41 kDa surface protein (sp41 is associated with bacterial adherence and invasion of HeLa cells. The role of this protein a is important for the interaction with host cells. Previously, the putative periplasmic binding protein p39 had been described as T-cell immunodominant Brucella antigens. Both vectors (pCIp39 and pCIsp41 induced antigen-specific serum immunoglobulin as well as a T-cell-proliferative response and a strong gamma interferon production upon re-stimulation with either the specific antigens or Brucella extract. The level of protection was significant in pCIp39 and pCIsp41 treated mice but it was lower than the required level.

  13. The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element

    Science.gov (United States)

    Fukunaga, Junichi; Nomura, Yusuke; Tanaka, Yoichiro; Amano, Ryo; Tanaka, Taku; Nakamura, Yoshikazu; Kawai, Gota; Sakamoto, Taiichi; Kozu, Tomoko

    2013-01-01

    AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5′-NNCCAC-3′ and 5′-GCGMGN′N′-3′ (M:A or C; N and N′ form Watson–Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt domain. Mutational analyses of the Runt domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt domain in vitro. These results demonstrated that the Runt domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences. PMID:23709277

  14. High-Resolution Profiling of Drosophila Replication Start Sites Reveals a DNA Shape and Chromatin Signature of Metazoan Origins

    Directory of Open Access Journals (Sweden)

    Federico Comoglio

    2015-05-01

    Full Text Available At every cell cycle, faithful inheritance of metazoan genomes requires the concerted activation of thousands of DNA replication origins. However, the genetic and chromatin features defining metazoan replication start sites remain largely unknown. Here, we delineate the origin repertoire of the Drosophila genome at high resolution. We address the role of origin-proximal G-quadruplexes and suggest that they transiently stall replication forks in vivo. We dissect the chromatin configuration of replication origins and identify a rich spatial organization of chromatin features at initiation sites. DNA shape and chromatin configurations, not strict sequence motifs, mark and predict origins in higher eukaryotes. We further examine the link between transcription and origin firing and reveal that modulation of origin activity across cell types is intimately linked to cell-type-specific transcriptional programs. Our study unravels conserved origin features and provides unique insights into the relationship among DNA topology, chromatin, transcription, and replication initiation across metazoa.

  15. [Serologic response to a DNA recombinant vaccine against hepatitis B in natives of the Peruvian Amazonian jungle].

    Science.gov (United States)

    Colichón, A; Vildósola, H; Sjogren, M; Cantella, R; Rojas, C

    1990-01-01

    Large areas of the Amazon basin in Brazil, Colombia, Ecuador, and in the nonoriental region of the peruvian jungle have been found to be hyperendemic to Hepatitis B with high prevalence of asymptomatic carriers (11 to 25%) and, in more selected areas, Hepatitis Delta has been also reported. In the present report, we have studied 108 volunteers from six different Jivaroes communities living in a hyperendemic Hepatitis B area. They received 2 doses of DNA recombinant yeast derivated HBV vaccine. All the selected persons were HBsAb negatives, but many (80%) had antibodies to HBc. Following immunization schedule, 80% responded with the formation of HBsAb; a better seroconversion was achieved in those negatives to anticore IgG compared with those having HBcAb. We obtained 90% of seroconversion in spite of the fact that our vaccination schedule was prolonged up to 10 months from the one recommended by the manufacturer. The vaccination schedule 0,4, 14 months, and the schedule 0,4 months, had 76 and 29% of seroconversion, respectively. We want to point out three observations: 1) It is quite possible that many of the Anti-core positives, that did not respond to vaccination were carriers of HBsAg undetectable by the conventional EIA test carried out; 2) The seroconversion rate in these natives was low (up to six months after the vaccination schedule); and 3) Many of the HBcAb were false positives and many of them were recently infected. We conclude: A) It is highly important to assess the anti-HBs hyperendemic areas before attempting vaccinations; B) All persons negative to anti-HBs should be vaccinated in spite to anticore antibodies; C) Areas with difficult access could be vaccinated even until 10 months without affecting good results, and D) DNA recombinant vaccine (ENGERIX B) was well tolerated. No side effects were observed.

  16. A DNA-binding-site landscape and regulatory network analysis for NAC transcription factors in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Lindemose, Søren; Jensen, Michael Krogh; de Velde, Jan Van;

    2014-01-01

    Target gene identification for transcription factors is a prerequisite for the systems wide understanding of organismal behaviour. NAM-ATAF1/2-CUC2 (NAC) transcription factors are amongst the largest transcription factor families in plants, yet limited data exist from unbiased approaches to resolve...... regulatory networks of 12 NAC transcription factors. Our data offer specific single-base resolution fingerprints for most TFs studied and indicate that NAC DNA-binding specificities might be predicted from their DNA-binding domain's sequence. The developed methodology, including the application...... with the workflow associated with functional modules offer a strong resource to unravel the regulatory potential of NAC genes and that this workflow could be used to study other families of transcription factors....

  17. TET1 is a DNA-binding protein that modulates DNA methylation and gene transcription via hydroxylation of 5-methylcytosine

    Institute of Scientific and Technical Information of China (English)

    Haikuo Zhang; Xin Zhang; Erin Clark; Michelle Mulcahey; Stephen Huang; Yujiang Geno Shi

    2010-01-01

    @@ Dear Editor, DNA methylation, which often occurs at the 5-carbon position of cytosine (5mC) located in CpG dinucleotide, is a key epigenetic hallmark and serves as a major epigenetic mechanism for establishing X-inactivation, paren tal imprinting and silencing retrotransposable elements during early embryogenesis in mammals.

  18. den V gene of bacteriophage T4 determines a DNA glycosylase specific for pyrimidine dimers in DNA.

    OpenAIRE

    Seawell, P C; Smith, C A; Ganesan, A K

    1980-01-01

    Endonuclease V of bacteriophage T4 has been described as an enzyme, coded for by the denV gene, that incises UV-irradiated DNA. It has recently been proposed that incision of irradiated DNA by this enzyme and the analogous "correndonucleases" I and II of Micrococcus luteus requires the sequential action of a pyrimidine dimer-specific DNA glycosylase and an apyrimidinic/apurinic endonuclease. In support of this two-step mechanism, we found that our preparations of T4 endonuclease V contained a...

  19. Yeast Rad5 Protein Required for Postreplication Repair Has a DNA Helicase Activity Specific for Replication Fork Regression

    OpenAIRE

    Blastyák, András; Pintér, Lajos; Unk, Ildiko; Prakash, Louise; Prakash, Satya; Haracska, Lajos

    2007-01-01

    Summary Lesions in the template DNA strand block the progression of the replication fork. In the yeast Saccharomyces cerevisiae, replication through DNA lesions is mediated by different Rad6-Rad18-dependent means, which include translesion synthesis and a Rad5-dependent postreplicational repair pathway that repairs the discontinuities that form in the DNA synthesized from damaged templates. Although translesion synthesis is well characterized, little is known about the mechanisms that modulat...

  20. Immunogenicity of a DNA-launched replicon-based canine parvovirus DNA vaccine expressing VP2 antigen in dogs.

    Science.gov (United States)

    Dahiya, Shyam S; Saini, Mohini; Kumar, Pankaj; Gupta, Praveen K

    2012-10-01

    A replicon-based DNA vaccine encoding VP2 gene of canine parvovirus (CPV) was developed by cloning CPV-VP2 gene into a replicon-based DNA vaccine vector (pAlpha). The characteristics of a replicon-based DNA vaccine like, self-amplification of transcripts and induction of apoptosis were analyzed in transfected mammalian cells. When the pAlpha-CPV-VP2 was injected intradermal as DNA-launched replicon-based DNA vaccine in dogs, it induced CPV-specific humoral and cell mediated immune responses. The virus neutralization antibody and lymphocyte proliferative responses were higher than conventional CPV DNA vaccine and commercial CPV vaccine. These results indicated that DNA-launched replicon-based CPV DNA vaccine was effective in inducing both CPV-specific humoral and cellular immune responses and can be considered as effective alternative to conventional CPV DNA vaccine and commercial CPV vaccine.

  1. A DNA sequence directed mutual transcription regulation of HSF1 and NFIX involves novel heat sensitive protein interactions.

    Directory of Open Access Journals (Sweden)

    Umashankar Singh

    Full Text Available BACKGROUND: Though the Nuclear factor 1 family member NFIX has been strongly implicated in PDGFB-induced glioblastoma, its molecular mechanisms of action remain unknown. HSF1, a heat shock-related transcription factor is also a powerful modifier of carcinogenesis by several factors, including PDGFB. How HSF1 transcription is controlled has remained largely elusive. METHODOLOGY/PRINCIPAL FINDINGS: By combining microarray expression profiling and a yeast-two-hybrid screen, we identified that NFIX and its interactions with CGGBP1 and HMGN1 regulate expression of HSF1. We found that CGGBP1 organizes a bifunctional transcriptional complex at small CGG repeats in the HSF1 promoter. Under chronic heat shock, NFIX uses CGGBP1 and HMGN1 to get recruited to this promoter and in turn affects their binding to DNA. Results show that the interactions of NFIX with CGGBP1 and HMGN1 in the soluble fraction are heat shock sensitive due to preferential localization of CGGBP1 to heterochromatin after heat shock. HSF1 in turn was found to bind to the NFIX promoter and repress its expression in a heat shock sensitive manner. CONCLUSIONS/SIGNIFICANCE: NFIX and HSF1 exert a mutual transcriptional repressive effect on each other which requires CGG repeat in HSF1 promoter and HSF1 binding site in NFIX promoter. We unravel a unique mechanism of heat shock sensitive DNA sequence-directed reciprocal transcriptional regulation between NFIX and HSF1. Our findings provide new insights into mechanisms of transcription regulation under stress.

  2. A DNA damage checkpoint in Caulobacter crescentus inhibits cell division through a direct interaction with FtsW.

    Science.gov (United States)

    Modell, Joshua W; Hopkins, Alexander C; Laub, Michael T

    2011-06-15

    Following DNA damage, cells typically delay cell cycle progression and inhibit cell division until their chromosomes have been repaired. The bacterial checkpoint systems responsible for these DNA damage responses are incompletely understood. Here, we show that Caulobacter crescentus responds to DNA damage by coordinately inducing an SOS regulon and inhibiting the master regulator CtrA. Included in the SOS regulon is sidA (SOS-induced inhibitor of cell division A), a membrane protein of only 29 amino acids that helps to delay cell division following DNA damage, but is dispensable in undamaged cells. SidA is sufficient, when overproduced, to block cell division. However, unlike many other regulators of bacterial cell division, SidA does not directly disrupt the assembly or stability of the cytokinetic ring protein FtsZ, nor does it affect the recruitment of other components of the cell division machinery. Instead, we provide evidence that SidA inhibits division by binding directly to FtsW to prevent the final constriction of the cytokinetic ring.

  3. Explorative study to identify novel candidate genes related to oxaliplatin efficacy and toxicity using a DNA repair array.

    NARCIS (Netherlands)

    Kweekel, D.M.; Antonini, N.F.; Nortier, J.W.; Punt, C.J.A.; Gelderblom, H.; Guchelaar, H.J.

    2009-01-01

    PURPOSE: To identify new polymorphisms (single nucleotide polymorphisms, SNPs) in DNA repair pathways that are associated with efficacy and toxicity in patients receiving oxaliplatin and capecitabine for advanced colorectal cancer (ACC). METHODS: We studied progression-free survival (PFS) in 91 ACC

  4. A DNA barcode library for ground beetles (Insecta, Coleoptera, Carabidae) of Germany: The genus Bembidion Latreille, 1802 and allied taxa.

    Science.gov (United States)

    Raupach, Michael J; Hannig, Karsten; Morinière, Jérome; Hendrich, Lars

    2016-01-01

    As molecular identification method, DNA barcoding based on partial cytochrome c oxidase subunit 1 (COI) sequences has been proven to be a useful tool for species determination in many insect taxa including ground beetles. In this study we tested the effectiveness of DNA barcodes to discriminate species of the ground beetle genus Bembidion and some closely related taxa of Germany. DNA barcodes were obtained from 819 individuals and 78 species, including sequences from previous studies as well as more than 300 new generated DNA barcodes. We found a 1:1 correspondence between BIN and traditionally recognized species for 69 species (89%). Low interspecific distances with maximum pairwise K2P values below 2.2% were found for three species pairs, including two species pairs with haplotype sharing (Bembidion atrocaeruleum/Bembidion varicolor and Bembidion guttula/Bembidion mannerheimii). In contrast to this, deep intraspecific sequence divergences with distinct lineages were revealed for two species (Bembidion geniculatum/Ocys harpaloides). Our study emphasizes the use of DNA barcodes for the identification of the analyzed ground beetles species and represents an important step in building-up a comprehensive barcode library for the Carabidae in Germany and Central Europe as well. PMID:27408547

  5. Inhibition of aflatoxin metabolism and growth of Aspergillus flavus in liquid culture by a DNA methylation inhibitor.

    Science.gov (United States)

    Yang, Kunlong; Zhuang, Zhenhong; Zhang, Feng; Song, Fengqin; Zhong, Hong; Ran, Fanlei; Yu, Song; Xu, Gaopo; Lan, Faxiu; Wang, Shihua

    2015-01-01

    Aflatoxins (AFs) are a group of highly oxygenated polyketidese-derived toxins mainly produced by Aspergillus flavus and A. parasiticus, whose biosynthesis mechanisms are extremely sophisticated. Methylation is known as the major form of epigenetic regulation, which is correlated with gene expression. As the DNA methylation inhibitor 5-azacytidine (5-AC) blocks AF production, we studied AFB1 metabolism and morphological changes of A. flavus by treatment with 5-AC in liquid culture. The results show that 5-AC caused a decrease in AF production and concurrent changes in morphology. In addition, we isolated a non-aflatoxigenic mutant of A. flavus, showing a significant reduction in pigment production, after 5-AC treatment. This mutant showed significant reduction in the expression of genes in the AF biosynthesis pathway, and conidia formation. Furthermore, as AF biosynthesis and oxidative stress are intimately related events, we assessed the viability of A. flavus to oxidative stress after treatment with 5-AC, which showed that the mutant was more sensitive to the strong oxidant hydrogen peroxide. We found that the non-aflatoxigenic mutant showed a decrease in reactive oxygen species (ROS) and metabolites indicative of oxidative stress, which may be caused by the disruption of the defence system against excessive ROS formation after 5-AC treatment. These data indicate that 5-AC, as an inactivator of DNA methyltransferase, plays a very important role in AFB1 metabolism and the development of A. flavus, which might provide an effective strategy to pre- or post-harvest control of AFs. PMID:25312249

  6. Examining the relationship between hemolymph phenoloxidase and resistance to a DNA virus, Plodia interpunctella granulosis virus (PiGV).

    Science.gov (United States)

    Saejeng, A; Tidbury, H; Siva-Jothy, M T; Boots, M

    2010-09-01

    We have a detailed understanding of invertebrate immune responses to bacteria and fungal pathogens, but we know less about how insects respond to virus challenge. Phenoloxidase (PO) functions as an important immune response against many parasites and pathogens and is routinely used as a measure of immune competance. We examine the role of haemolymph PO activity in Plodia interpuncetella's response to its natural granulosis virus (PiGV). Larvae were challenged with virus by both oral inoculation of occluded virus (the natural infection route) and direct intrahaemocoelic injection of budded virus. Haemolymph was collected at time points post-viral challenge using a novel method that allows the volume of haemolymph to be quanitified. The haemolmyph was collected without killing the larvae so that haemolymph samples from individuals that developed viral disease could be distinguished from samples collected from those that fought off infection. The level of haemolymph PO activity in resistant larvae did not differ from control larvae. Therefore we have no evidence that PO is involved in resistance to virus in the haemocoel whether larvae are challenged naturally by oral innoculation or directly by intraheamocoelic injection. Phenoloxidase may therefore not be a relevant metric of immunocompetence for viral infection.

  7. A DNA replicon system for rapid high-level production of virus-like particles in plants

    OpenAIRE

    Huang, Zhong; Chen, Qiang; Hjelm, Brooke; Arntzen, Charles; Mason, Hugh

    2009-01-01

    Recombinant virus-like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low level antigen accumulation and long time frame to produce transgenic plants are the two major roadblocks in the practical development of plant-based VLP production. In this paper, we describe the optimization of geminivirus-derived DNA replicon vectors for rapi...

  8. An Assessment of Whole Blood and Fractions by Nested PCR as a DNA Source for Diagnosing Canine Ehrlichiosis and Anaplasmosis

    Directory of Open Access Journals (Sweden)

    Tereza Emmanuelle de Farias Rotondano

    2012-01-01

    Full Text Available Ehrlichiosis and anaplasmosis are tick-borne diseases. Ehrlichia canis and Anaplasma platys infect mainly white cells and platelets, respectively. The main DNA source for PCR is peripheral blood, but the potential of blood cell fractions has not been extensively investigated. This study aims at assessment of whole blood (WB and blood fractions potential in nested PCR (nPCR to diagnose canine ehrlichiosis and anaplasmosis. The 16S rRNA gene was amplified in 71.4, 17.8, 31.57, and 30% of the WB, granulocyte (G, mononuclear cells (M, and buffy coat (BC samples. Compared to the WB, the sensitivity of the PCR was 42.86% for the M, and BC fractions, 21.43% for the G, and 33.33% for the blood clot (C. There was fair agreement between the WB and M, BC and C, and slight with the G. Fair agreement occurred between the nPCR and morulae in the blood smear. One animal was coinfected with A. platys and E. canis. This study provided the first evidence of A. platys infection in dogs in Paraíba, Brazil, and demonstrated that WB is a better DNA source than blood fractions to detect Ehrlichia and Anaplasma by nPCR, probably because of the plasma bacterial concentration following host cell lysis.

  9. Transcriptional stimulation via SC site of Bombyx sericin-1 gene through an interaction with a DNA binding protein SGF-3.

    OpenAIRE

    Matsuno, K.; Takiya, S; Hui, C C; Suzuki, T.; Fukuta, M.; Ueno, K.; Suzuki, Y

    1990-01-01

    Three protein binding sites have been identified in the upstream region of the sericin-1 gene. Two of them, SA and SC sites, have been known as putative cis-acting elements. Using synthetic oligonucleotides of these binding sites, it was found that silk gland factor-1 (SGF-1) binds to the SA site, and silk gland factor-3 (SGF-3) binds to the SC site but not to a mutated SC site, SCM. Tissue distribution of the two factors was different. SGF-3 is present abundantly in the middle silk gland (MS...

  10. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis.

    Science.gov (United States)

    Santiago-Felipe, S; Tortajada-Genaro, L A; Puchades, R; Maquieira, A

    2014-02-01

    Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR-ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA-ELISA combination is proposed for amplification at a low, constant temperature (40°C) in a short time (40 min), for the hybridisation of labelled products to specific 5'-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA-ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA-ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings.

  11. Characterization of a DNA Adenine Methyltransferase Gene of Borrelia hermsii and Its Dispensability for Murine Infection and Persistence

    Science.gov (United States)

    James, Allison E.; Rogovskyy, Artem S.; Crowley, Michael A.; Bankhead, Troy

    2016-01-01

    DNA methyltransferases have been implicated in the regulation of virulence genes in a number of pathogens. Relapsing fever Borrelia species harbor a conserved, putative DNA methyltransferase gene on their chromosome, while no such ortholog can be found in the annotated genome of the Lyme disease agent, Borrelia burgdorferi. In the relapsing fever species Borrelia hermsii, the locus bh0463A encodes this putative DNA adenine methyltransferase (dam). To verify the function of the BH0463A protein product as a Dam, the gene was cloned into a Dam-deficient strain of Escherichia coli. Restriction fragment analysis subsequently demonstrated that complementation of this E. coli mutant with bh0463A restored adenine methylation, verifying bh0463A as a Dam. The requirement of bh0463A for B. hermsii viability, infectivity, and persistence was then investigated by genetically disrupting the gene. The dam- mutant was capable of infecting immunocompetent mice, and the mean level of spirochetemia in immunocompetent mice was not significantly different from wild type B. hermsii. Collectively, the data indicate that dam is dispensable for B. hermsii viability, infectivity, and persistence. PMID:27195796

  12. A DNA Fingerprinting Simulation Laboratory for Biology Students: Hands-on Experimentation To Solve a Mock Forensic Problem.

    Science.gov (United States)

    Palladino, Michael A.; Cosentino, Emily

    2001-01-01

    Presents an alternative approach to DNA fingerprinting. Demonstrates how undergraduate students can be involved in many aspects of this type of experiment and how DNA fingerprinting experiments can be incorporated into the laboratory curriculum of courses for majors and nonmajors. (NB)

  13. Detection of respiratory syncytial virus by reverse transcription-PCR and hybridization with a DNA enzyme immunoassay.

    OpenAIRE

    Freymuth, F.; Eugene, G; Vabret, A.; Petitjean, J.; Gennetay, E; Brouard, J; Duhamel, J F; Guillois, B

    1995-01-01

    Nasal aspirates from 238 infants hospitalized with acute respiratory infections during the winter of 1994 and 1995 were tested for respiratory syncytial virus (RSV) by immunofluorescence assay (IFA) and the viral isolation technique (VIT) and by two PCR and hybridization methods: reverse transcription PCR 1 (RT-PCR1), which amplifies the RNAs of all RSV strains, and RT-PCR-2, which allows subgroup classification of RSV. RT-PCR-1 and RT-PCR-2 detected viral sequences in 56.7% (135 of 238) and ...

  14. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis

    International Nuclear Information System (INIS)

    Graphical abstract: -- Highlights: •Recombinase polymerase amplification is a powerful DNA method operating at 40 °C. •The combination RPA–ELISA gives excellent performances for high-throughput analysis. •Screening of food safety threats has been done using standard laboratory equipment. •Allergens, GMOs, bacteria, and fungi have been successfully determined. -- Abstract: Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR–ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA–ELISA combination is proposed for amplification at a low, constant temperature (40 °C) in a short time (40 min), for the hybridisation of labelled products to specific 5′-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA–ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA–ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings

  15. Development of a DNA Microarray-Based Assay for the Detection of Sugar Beet Root Rot Pathogens.

    Science.gov (United States)

    Liebe, Sebastian; Christ, Daniela S; Ehricht, Ralf; Varrelmann, Mark

    2016-01-01

    Sugar beet root rot diseases that occur during the cropping season or in storage are accompanied by high yield losses and a severe reduction of processing quality. The vast diversity of microorganism species involved in rot development requires molecular tools allowing simultaneous identification of many different targets. Therefore, a new microarray technology (ArrayTube) was applied in this study to improve diagnosis of sugar beet root rot diseases. Based on three marker genes (internal transcribed spacer, translation elongation factor 1 alpha, and 16S ribosomal DNA), 42 well-performing probes enabled the identification of prevalent field pathogens (e.g., Aphanomyces cochlioides), storage pathogens (e.g., Botrytis cinerea), and ubiquitous spoilage fungi (e.g., Penicillium expansum). All probes were proven for specificity with pure cultures from 73 microorganism species as well as for in planta detection of their target species using inoculated sugar beet tissue. Microarray-based identification of root rot pathogens in diseased field beets was successfully confirmed by classical detection methods. The high discriminatory potential was proven by Fusarium species differentiation based on a single nucleotide polymorphism. The results demonstrate that the ArrayTube constitute an innovative tool allowing a rapid and reliable detection of plant pathogens particularly when multiple microorganism species are present. PMID:26524545

  16. West Nile virus seroconversion in penguins after vaccination with a killed virus vaccine or a DNA vaccine.

    Science.gov (United States)

    Davis, Michelle R; Langan, Jennifer N; Johnson, Yvette J; Ritchie, Branson W; Van Bonn, William

    2008-12-01

    To investigate the serologic response of penguins to West Nile virus (WNV) vaccines, four species of exclusively indoor-housed penguins, negative for WNV by serology, were evaluated: Humboldt (Spheniscus humboldti), Magellanic (Spheniscus magellanicus), Gentoo (Pygoscelis papua), and Rockhopper (Eudyptes chrysoscome) penguins. Birds were inoculated with either a killed virus vaccine or a plasmid-mediated DNA WNV vaccine, and postinoculation serology was evaluated. Both vaccines induced seroconversion in all four species, and no adverse reactions were noted. Postvaccination serology results varied across species and vaccine types. However, in all four species, the killed virus vaccine resulted in a greater seroconversion rate than the DNA vaccine and in a significantly shorter time period. Additionally, the duration of the seropositive titer was significantly longer in those birds vaccinated with the killed virus vaccine compared with those vaccinated with the DNA vaccine. A subset of unvaccinated penguins serving as negative controls remained negative throughout the duration of the study despite the presence of WNV in the geographic locations of the study, suggesting that indoor housing may minimize exposure to the virus and may be an additional means of preventing WNV infection in penguins. PMID:19110700

  17. A DNA based method to detect the grapevine root-rotting fungus Roesleria subterranea in soil and root samples

    OpenAIRE

    Neuhauser, Sigrid; Huber, Lars; Kirchmair, Martin

    2009-01-01

    Roesleria subterranea causes root rot in grapevine and fruit trees. The fungus has long been underestimated as a weak parasite, but during the last years it has been reported to cause severe damages in German vineyards. Direct, observation-based detection of the parasite is time consuming and destructive, as large parts of the rootstocks have to be uprooted and screened for the tiny, stipitate, hypogeous ascomata of R. subterranea. To facilitate rapid detection in vineyards, protocols to extr...

  18. A DNA-based nanomechanical device used to characterize the distortion of DNA by Apo-SoxR protein.

    Science.gov (United States)

    Liu, Chunhua; Kim, Eunsuk; Demple, Bruce; Seeman, Nadrian C

    2012-02-01

    DNA-based nanomechanical devices can be used to characterize the action of DNA-distorting proteins. Here, we have constructed a device wherein two DNA triple-crossover (TX) molecules are connected by a shaft, similar to a previous device that measured the binding free energy of integration host factor. In our case, the binding site on the shaft contains the sequence recognized by SoxR protein, the apo form of which is a transcriptional activator. Another active form is oxidized [2Fe-2S] SoxR formed during redox sensing, and previous data suggest that activated Fe-SoxR distorts its binding site by localized DNA untwisting by an amount that corresponds to ~2 bp. A pair of dyes report the fluorescence resonance energy transfer (FRET) signal between the two TX domains, reflecting changes in the shape of the device upon binding of the protein. The TX domains are used to amplify the signal expected from a relatively small distortion of the DNA binding site. From FRET analysis of apo-SoxR binding, the effect of apo-SoxR on the original TX device is similar to the effect of shortening the TX device by 2 bp. We estimate that the binding free energy of apo-SoxR on the DNA target site is 3.2-6.1 kcal/mol. PMID:22257222

  19. A DNA target-enrichment approach to detect mutations, copy number changes and immunoglobulin translocations in multiple myeloma.

    Science.gov (United States)

    Bolli, N; Li, Y; Sathiaseelan, V; Raine, K; Jones, D; Ganly, P; Cocito, F; Bignell, G; Chapman, M A; Sperling, A S; Anderson, K C; Avet-Loiseau, H; Minvielle, S; Campbell, P J; Munshi, N C

    2016-01-01

    Genomic lesions are not investigated during routine diagnostic workup for multiple myeloma (MM). Cytogenetic studies are performed to assess prognosis but with limited impact on therapeutic decisions. Recently, several recurrently mutated genes have been described, but their clinical value remains to be defined. Therefore, clinical-grade strategies to investigate the genomic landscape of myeloma samples are needed to integrate new and old prognostic markers. We developed a target-enrichment strategy followed by next-generation sequencing (NGS) to streamline simultaneous analysis of gene mutations, copy number changes and immunoglobulin heavy chain (IGH) translocations in MM in a high-throughput manner, and validated it in a panel of cell lines. We identified 548 likely oncogenic mutations in 182 genes. By integrating published data sets of NGS in MM, we retrieved a list of genes with significant relevance to myeloma and found that the mutational spectrum of primary samples and MM cell lines is partially overlapping. Gains and losses of chromosomes, chromosomal segments and gene loci were identified with accuracy comparable to conventional arrays, allowing identification of lesions with known prognostic significance. Furthermore, we identified IGH translocations with high positive and negative predictive value. Our approach could allow the identification of novel biomarkers with clinical relevance in myeloma. PMID:27588520

  20. Ion-dependent conformational switching by a DNA aptamer that induces remyelination in a mouse model of multiple sclerosis

    OpenAIRE

    Smestad, John; Maher, L. James

    2012-01-01

    We recently reported that a guanosine-rich 40-mer DNA aptamer (LJM-3064) mediates remyelination in the Theiler’s murine encephalomyelitis virus mouse model of multiple sclerosis. Here, we characterize the G-quadruplex forms of this aptamer in vitro, and demonstrate using circular dichroism spectroscopy that LJM-3064 undergoes a monovalent ion-dependent conformational switch. In the presence of sodium ions and no potassium ions, LJM-3064 adopts an antiparallel-stranded G-quadruplex structure. ...

  1. Identification of a DNA transformation gene required for com101A+ expression and supertransformer phenotype in Haemophilus influenzae.

    OpenAIRE

    Zulty, J J; Barcak, G J

    1995-01-01

    DNA sequencing, RNA mapping, and protein expression experiments revealed the presence of a gene, tfoX+, encoding a 24.9-kDa polypeptide, that is transcribed divergently from a common promoter region with the Haemophilus influenzae rec-1+ gene. H. influenzae strains mutant for tfoX failed to bind transforming DNA and were transformation deficient. Primer extension experiments utilizing in vivo total RNA from precompetent and competent H. influenzae cells demonstrated that transcription of tfoX...

  2. Super-specific DNA methylation by a DNA methyltransferase coupled with a triple helix-forming oligonucleotide

    OpenAIRE

    Maluszynska-Hoffman, Maria

    2008-01-01

    In this work, an engineered variant of the DNA cytosine-C5 methyltransferase (MTase) M.SssI has been conjugated with a triple helix-forming oligonucleotide (TFO) and the properties of the enzyme-TFO conjugate were evaluated using a variety of biochemical and biophysical methods. M.SssI is the only known prokaryotic DNA MTase which shares the 5’-CG-3’ recognition sequence (CpG) with mammalian DNA MTases. It has been rendered a super-specific MTase by conjugation with a TFO designed to bind a t...

  3. Thermodynamic characterization of the stability and the melting behavior of a DNA triplex: A spectroscopic and calorimetric study

    OpenAIRE

    Plum, G E; Park, Y. W.; Singleton, S F; Dervan, P B; Breslauer, K J

    1990-01-01

    We report a complete thermodynamic characterization of the stability and the melting behavior of an oligomeric DNA triplex. The triplex chosen for study forms by way of major-groove Hoogsteen association of an all-pyrimidine 15-mer single strand (termed y15) with a Watson-Crick 21-mer duplex composed of one purine-rich strand (termed u21) and one pyrimidine-rich strand (termed y21). We find that the near-UV CD spectrum of the triplex can be duplicated by the addition of the B-like CD spectrum...

  4. An in silico study of the protonated DNA triplex: In vivo stability of C +GC in a DNA triple helix

    Science.gov (United States)

    Jung, Jiwon; Lee, Sang Hak

    2010-07-01

    Protonated DNA base pairs, particularly the Cytosine-Guanine-Cytosine (CGC) triple pairs, were investigated its relative energetics by theoretical calculations. The energetic stabilities of C +GC and CG +C are almost the same in the gas phase, although the C +G pair is more stable than CG + by ca. 1150 cm -1 (3.34 kcal/mol). It is to be noted, however, that only C +GC was found in vivo, which seems to indicate different chemical environments for the triple pairs in vivo.

  5. The effect of a DNA repair gene on cellular invasiveness: XRCC3 over-expression in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Veronica L Martinez-Marignac

    Full Text Available Over-expression of DNA repair genes has been associated with resistance to radiation and DNA-damage induced by chemotherapeutic agents such as cisplatin. More recently, based on the analysis of genome expression profiling, it was proposed that over-expression of DNA repair genes enhances the invasive behaviour of tumour cells. In this study we present experimental evidence utilizing functional assays to test this hypothesis. We assessed the effect of the DNA repair proteins known as X-ray complementing protein 3 (XRCC3 and RAD51, to the invasive behavior of the MCF-7 luminal epithelial-like and BT20 basal-like triple negative human breast cancer cell lines. We report that stable or transient over-expression of XRCC3 but not RAD51 increased invasiveness in both cell lines in vitro. Moreover, XRCC3 over-expressing MCF-7 cells also showed a higher tumorigenesis in vivo and this phenotype was associated with increased activity of the metalloproteinase MMP-9 and the expression of known modulators of cell-cell adhesion and metastasis such as CD44, ID-1, DDR1 and TFF1. Our results suggest that in addition to its' role in facilitating repair of DNA damage, XRCC3 affects invasiveness of breast cancer cell lines and the expression of genes associated with cell adhesion and invasion.

  6. GEOPHYSICS, ASTRONOMY AND ASTROPHYSICS: Second-harmonic generation as a DNA malignancy indicator of prostate glandular epithelial cells

    Science.gov (United States)

    Zhuang, Zheng-Fei; Liu, Han-Ping; Guo, Zhou-Yi; Zhuo, Shuang-Mu; Yu, Bi-Ying; Deng, Xiao-Yuan

    2010-04-01

    This paper first demonstrates second-harmonic generation (SHG) in the intact cell nucleus, which acts as an optical indicator of DNA malignancy in prostate glandular epithelial cells. Within a scanning region of 2.7 μm×2.7 μm in cell nuclei, SHG signals produced from benign prostatic hyperplasia (BPH) and prostate carcinoma (PC) tissues (mouse model C57BL/6) have been investigated. Statistical analyses (t test) of a total of 405 measurements (204 nuclei from BPH and 201 nuclei from PC) show that SHG signals from BPH and PC have a distinct difference (p prostate glandular epithelial cells based upon induced biochemical and/or biophysical modifications in DNA.

  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. Endohedral confinement of a DNA dodecamer onto pristine carbon nanotubes and the stability of the canonical B form

    CERN Document Server

    Cruz, Fernando J A L; Mota, José P B

    2016-01-01

    Although carbon nanotubes are potential candidates for DNA encapsulation and subsequent delivery of biological payloads to living cells, the thermodynamical spontaneity of DNA encapsulation under physiological conditions is still a matter of debate. Using enhanced sampling techniques, we show for the first time that, given a sufficiently large carbon nanotube, the confinement of a double-stranded DNA segment, 5'-D(*CP*GP*CP*GP*AP*AP*TP*TP*CP*GP*CP*G)-3', is thermodynamically favourable under physiological environments (134 mM, 310 K, 1 bar), leading to DNA-nanotube hybrids with lower free energy than the unconfined biomolecule. A diameter threshold of 3 nm is established below which encapsulation is inhibited. The confined DNA segment maintains its translational mobility and exhibits the main geometrical features of the canonical B form. To accommodate itself within the nanopore, the DNA end-to-end length increases from 3.85 nm up to approximately 4.1 nm, due to a 0.3 nm elastic expansion of the strand termin...

  9. Epigenetic inactivation of the CpG demethylase TET1 as a DNA methylation feedback loop in human cancers

    Science.gov (United States)

    Li, Lili; Li, Chen; Mao, Haitao; Du, Zhenfang; Chan, Wai Yee; Murray, Paul; Luo, Bing; Chan, Anthony TC; Mok, Tony SK; Chan, Francis KL; Ambinder, Richard F; Tao, Qian

    2016-01-01

    Promoter CpG methylation is a fundamental regulatory process of gene expression. TET proteins are active CpG demethylases converting 5-methylcytosine to 5-hydroxymethylcytosine, with loss of 5 hmC as an epigenetic hallmark of cancers, indicating critical roles of TET proteins in epigenetic tumorigenesis. Through analysis of tumor methylomes, we discovered TET1 as a methylated target, and further confirmed its frequent downregulation/methylation in cell lines and primary tumors of multiple carcinomas and lymphomas, including nasopharyngeal, esophageal, gastric, colorectal, renal, breast and cervical carcinomas, as well as non-Hodgkin, Hodgkin and nasal natural killer/T-cell lymphomas, although all three TET family genes are ubiquitously expressed in normal tissues. Ectopic expression of TET1 catalytic domain suppressed colony formation and induced apoptosis of tumor cells of multiple tissue types, supporting its role as a broad bona fide tumor suppressor. Furthermore, TET1 catalytic domain possessed demethylase activity in cancer cells, being able to inhibit the CpG methylation of tumor suppressor gene (TSG) promoters and reactivate their expression, such as SLIT2, ZNF382 and HOXA9. As only infrequent mutations of TET1 have been reported, compared to TET2, epigenetic silencing therefore appears to be the dominant mechanism for TET1 inactivation in cancers, which also forms a feedback loop of CpG methylation during tumorigenesis. PMID:27225590

  10. Cdk2 silencing via a DNA/PCL electrospun scaffold suppresses proliferation and increases death of breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Clément Achille

    Full Text Available RNA interference (RNAi is a promising approach for cancer treatment. Site specific and controlled delivery of RNAi could be beneficial to the patient, while at the same time reducing undesirable off-target side effects. We utilized electrospinning to generate a biodegradable scaffold capable of incorporating and delivering a bioactive plasmid encoding for short hairpin (sh RNA against the cell cycle specific protein, Cdk2. Three electrospun scaffolds were constructed, one using polycaprolactone (PCL alone (Control and PCL with plasmid DNA encoding for either Cdk2 (Cdk2i and EGFP (EGFPi, also served as a control shRNA. Scaffold fiber diameters ranged from 1 to 20 µm (DNA containing and 0.2-3 µm (Control. While the electrospun fibers remained intact for more than two weeks in physiological buffer, degradation was visible during the third week of incubation. Approximately 20-60 ng/ml (~2.5% cumulative release of intact and bioactive plasmid DNA was released over 21 days. Further, Cdk2 mRNA expression in cells plated on the Cdk2i scaffold was decreased by ~51% and 30%, in comparison with that of cells plated on Control or EGFPi scaffold, respectively. This decrease in Cdk2 mRNA by the Cdk2i scaffold translated to a ~40% decrease in the proliferation of the breast cancer cell line, MCF-7, as well as the presence of increased number of dead cells. Taken together, these results represent the first successful demonstration of the delivery of bioactive RNAi-based plasmid DNA from an electrospun polymer scaffold, specifically, in disrupting cell cycle regulation and suppressing proliferation of cancer cells.

  11. Cisplatin adducts on a GGG sequence within a DNA duplex studied by NMR spectroscopy and molecular dynamics simulations.

    Science.gov (United States)

    Téletchéa, Stéphane; Skauge, Tormod; Sletten, Einar; Kozelka, Jirí

    2009-11-16

    The antitumor drug cisplatin(cis-[PtCl2(NH3)2]) reacts with cellular DNA to form GG intrastrand adducts between adjacent guanines as predominant lesions. GGG sites have been shown to be hotspots of platination. To study the structural perturbation induced by binding of cisplatin to two adjacent guanines of a GGG trinucleotide,we examined here the decanucleotide duplex d[(G1C2C3G*4 G*5 G6T7-C8G9C10).d(G11C12G13A14C15C16C17G18-G19C20)] (dsCG*G*G) intrastrand cross-linked at the G* guanines by cis-{Pt(NH3)2}2+ using NMR spectroscopy and molecular dynamics (MD) simulations.The NMR spectra of dsCG*G*G were found to be similar to those of previously characterized DNA duplexes cross-linked by cisplatin at apyG*G*X site (py=pyrimidine; X=C,T, A). This similarity of NMR spectra indicates that the base at the 3'-side of the G*G*-Pt cross-link does not affect the structure to a large extent. An unprecedented reversible isomerization between the duplex dsCG*G*G (bearing a G*4 G*5 -Pt chelate) and duplex dsGG*G*T (bearing a G*5 G*6 -Pt chelate)was observed, which yielded a 40:60 equilibrium between the two intrastrand GG-Pt cross-links. No formation of interstrand cross-links was observed.NMR spectroscopic data of dsCG*G*G indicated that the deoxyribose of the 5'-G* adopts an N-type conformation, and the cytidines C3, C15,and C16 have average phase angles intermediate between S and N. The NMR spectroscopic chemical shifts of dsGG*G*T showed some fundamental differences to those of pyG*G*-platinum adducts but were in agreement with the NMR spectra reported previously for the DNA duplexes crosslinked at an AG*G*C sequence by cisplatin or oxaliplatin. The presence of apurine instead of a pyrimidine at the 5'-side of the G*G* cross-link seems therefore to affect the structure of the XG* step significantly. PMID:19813235

  12. A DNA vaccine directed against a rainbow trout rhabdovirus induces early protection against a nodavirus challenge in turbot

    DEFF Research Database (Denmark)

    Sommerset, I.; Lorenzen, Ellen; Lorenzen, Niels;

    2003-01-01

    encoding the capsid protein of AHNV revealed no protective properties against the nodavirus challenge. Histological examination of muscle tissue sections from the vaccine injection site showed that the DNA vaccine against VHSV triggered a pronounced inflammatory response in turbot similar to what has...

  13. A DNA vaccine against chikungunya virus is protective in mice and induces neutralizing antibodies in mice and nonhuman primates.

    Directory of Open Access Journals (Sweden)

    Karthik Mallilankaraman

    Full Text Available Chikungunya virus (CHIKV is an emerging mosquito-borne alphavirus indigenous to tropical Africa and Asia. Acute illness is characterized by fever, arthralgias, conjunctivitis, rash, and sometimes arthritis. Relatively little is known about the antigenic targets for immunity, and no licensed vaccines or therapeutics are currently available for the pathogen. While the Aedes aegypti mosquito is its primary vector, recent evidence suggests that other carriers can transmit CHIKV thus raising concerns about its spread outside of natural endemic areas to new countries including the U.S. and Europe. Considering the potential for pandemic spread, understanding the development of immunity is paramount to the development of effective counter measures against CHIKV. In this study, we isolated a new CHIKV virus from an acutely infected human patient and developed a defined viral challenge stock in mice that allowed us to study viral pathogenesis and develop a viral neutralization assay. We then constructed a synthetic DNA vaccine delivered by in vivo electroporation (EP that expresses a component of the CHIKV envelope glycoprotein and used this model to evaluate its efficacy. Vaccination induced robust antigen-specific cellular and humoral immune responses, which individually were capable of providing protection against CHIKV challenge in mice. Furthermore, vaccine studies in rhesus macaques demonstrated induction of nAb responses, which mimicked those induced in convalescent human patient sera. These data suggest a protective role for nAb against CHIKV disease and support further study of envelope-based CHIKV DNA vaccines.

  14. Development of a DNA microarray to detect antimicrobial resistance genes identified in the national center for biotechnology information database

    Science.gov (United States)

    High density genotyping techniques are needed for investigating antimicrobial resistance especially in the case of multi-drug resistant (MDR) isolates. To achieve this all antimicrobial resistance genes in the NCBI Genbank database were identified by key word searches of sequence annotations and the...

  15. Identification and characterization of a DNA primase activity present in herpes simplex virus type 1-infected HeLa cells

    International Nuclear Information System (INIS)

    A novel DNA primase activity has been identified in HeLa cells infected with herpes simplex virus type 1 (HSV-1). Such an activity has not been detected in mock-infected cells. The primase activity coeluted with a portion of HSV-1 DNA polymerase from single-stranded DNA agarose columns loaded with high-salt extracts derived from infected cells. This DNA primase activity could be distinguished from host HeLa cell DNA primase by several criteria. First, the pH optimum of the HSV primase was relatively broad and peaked at 8.2 to 8.7 pH units. Second, freshly isolated HSV DNA primase was less salt sensitive than the HeLa primase. Third, antibodies raised against individual peptides of the calf thymus DNA polymerase:primase complex cross-reacted with the HeLa primase but did not react with the HSV DNA primase. Fourth, freshly prepared HSV DNA primase appeared to be associated with the HSV polymerase, but after storage at 4 degree C for several weeks, the DNA primase separated from the viral DNA polymerase. This free DNA primase had an apparent molecular size of approximately 40 kilodaltons, whereas free HeLa DNA primase had an apparent molecular size of approximately 110 kilodaltons. On the basis of these data, the authors believe that the novel DNA primase activity in HSV-infected cells may be virus coded and that this enzyme represents a new and important function involved in the replication of HSV DNA

  16. Rapid detection and identification of viral and bacterial fish pathogens using a DNA array‐based multiplex assay

    DEFF Research Database (Denmark)

    Lievens, B.; Frans, I.; Heusdens, C.;

    2011-01-01

    Fish diseases can be caused by a variety of diverse organisms, including bacteria, fungi, viruses and protozoa, and pose a universal threat to the ornamental fish industry and aquaculture. The lack of rapid, accurate and reliable means by which fish pathogens can be detected and identified has been...... for sensitive pathogen detection and identification in complex samples such as infected tissue is demonstrated in this study....

  17. The incorporation of radiolabelled sulphur from captan into protein and its impact on a DNA binding study.

    Science.gov (United States)

    Provan, W M; Eyton-Jones, H; Lappin, G; Pritchard, D; Moore, R B; Green, T

    1995-05-19

    Repeated administration of high doses of captan is known to produce tumours specifically in the duodenum of mice. Captan is not carcinogenic in the rat. In this study, DNA purified from the liver, stomach, duodenum and jejenum of mice dosed with 35S radiolabelled captan was found to contain radioactivity equivalent to Covalent Binding Indices in the range 38-91; that from the bone marrow had a CBI of 2.8. The distribution of radioactivity between the various tissues did not reflect the target organ specificity of captan. Attempts to further purify the DNA samples using caesium chloride gradients resulted in partial separation of the radioactivity from the DNA suggesting that covalent binding to the DNA may not have occurred. A study of the chemical breakdown of captan showed that captan is unstable, producing a variety of potentially reactive species containing sulphur. Evidence was further obtained to show that the sulphur of captan is incorporated into endogenous amino acids and protein. Hepatic DNA from mice dosed with 35S radiolabelled N-acetylcysteine, and two thiazolidine derivatives which are analogous to known metabolites of captan, was radiolabelled to a similar extent to that from captan treated mice. Furthermore, the DNA from each of these treatments had similar properties on caesium chloride gradients. It was concluded that the radioactivity associated with DNA in the captan DNA binding study was present in the low levels of protein which are always associated with purified DNA samples.

  18. [Development of a DNA biochip for detection of known mtDNA mutations associated with MELAS and MERRF syndromes.].

    Science.gov (United States)

    Chen, Gang; Li, Wei; DU, Wei-Dong; Cao, Hui-Min; Tang, Hua-Yang; Tang, Xian-Fa; Sun, Zhong-Wu; Zhao, Hui; Jin, Qing-Hui; Zhao, Jian-Long; Zhang, Xue-Jun

    2008-10-01

    We developed an oligonucleotide biochip for synchronous multiplex detection of 31 known mitochondrial DNA mutations associated with MELAS (Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) and MERRF (Myoclonic epilepsy with ragged red fibers). Allele-specific oligonucleotide probes were covalently immobilized on aldehyde modified glass slides, and then hybridized with Cy5-labled DNA fragments amplified from sample DNAs by a multiplex asymmetric PCR (MAP) method. Five patients with MELAS, 5 patients with MERRF and 20 healthy controls were investigated using the oligonucleotide biochip. The results showed that all the cases with MELAS had an A3243G mutation in the MT-TL1 gene. In the MERRF group, 4 cases were found to be an A8344G mutation and 1 case was a T8356C mutation, and both mutations were in the MT-TK gene. In the healthy controls, none of the 31 related mutations was found. The results of the DNA biochip were consistent with those by DNA sequencing. Clearly, the DNA biochip combined with MAP method would become a valuable tool in multiplex detecting of the point mutations in mtDNA leading to MELAS and/or MERRF syndrome. Moreover, this biochip format could be modified to extend to the screening scope of SNPs for any other human mitochondrial diseases.

  19. Mate-Pair Sequencing as a Powerful Clinical Tool for the Characterization of Cancers with a DNA Viral Etiology

    OpenAIRE

    2015-01-01

    DNA viruses are known to be associated with a variety of different cancers. Human papillomaviruses (HPV) are a family of viruses and several of its sub-types are classified as high-risk HPVs as they are found to be associated with the development of a number of different cancers. Almost all cervical cancers appear to be driven by HPV infection and HPV is also found in most cancers of the anus and at least half the cancers of the vulva, penis and vagina, and increasingly found in one sub-type ...

  20. The extension of a DNA double helix by an additional Watson-Crick base pair on the same backbone

    DEFF Research Database (Denmark)

    Kumar, P.; Sharma, P. K.; Madsen, Charlotte S.;

    2013-01-01

    Additional base pair: The DNA duplex can be extended with an additional Watson-Crick base pair on the same backbone by the use of double-headed nucleotides. These also work as compressed dinucleotides and form two base pairs with cognate nucleobases on the opposite strand....

  1. A DNA barcode library of the beetle reference collection (Insecta: Coleoptera in the National Science Museum, Korea

    Directory of Open Access Journals (Sweden)

    Sang Woo Jung

    2016-06-01

    Full Text Available Coleoptera is a group of insects that are most diverse among insect resources. Although used as indicator species and applied in developing new drugs, it is difficult to identify them quickly. Since the development of a method using mitochondrial DNA information for identification, studies have been conducted in Korea to swiftly and accurately identify species. The National Science Museum of Korea (NSMK has been collecting and morphologically identifying domestic reference insects since 2013, and building a database of DNA barcodes with digital images. The NSMK completed construction of a database of digital images and DNA barcodes of 60 beetle species in the Korean National Research Information System. A total of 179 specimens and 60 species were used for the analysis, and the averages of intraspecific and interspecific variations were 0.70±0.45% and 26.34±6.01%, respectively, with variation rates ranging from 0% to 1.45% and 9.83% to 56.23%, respectively.

  2. The phytoplankton chip - development and assessment of a DNA microarray as a reliable tool for monitoring of phytoplankton

    OpenAIRE

    Gescher, Christine

    2007-01-01

    One microarray, the Phytoplankton Chip was developed . Phytoplankton field samples were taken at the island of Helgoland in the North Sea from 2004 to 2006 at regular intervals. For the phytoplankton community, only the > 20 mikrometer size fraction is identified on a daily basis. For picoplanktonic groups, light microscopy can not differentiate taxa or species. The phyto- and especially picoplanktonic dynamics were successfully analyzed with the Phytoplankton Chip in these three annual cycle...

  3. A biomolecule-compatible visible-light-induced azide reduction from a DNA-encoded reaction-discovery system.

    Science.gov (United States)

    Chen, Yiyun; Kamlet, Adam S; Steinman, Jonathan B; Liu, David R

    2011-02-01

    Using a system that accelerates the serendipitous discovery of new reactions by evaluating hundreds of DNA-encoded substrate combinations in a single experiment, we explored a broad range of reaction conditions for new bond-forming reactions. We discovered reactivity that led to a biomolecule-compatible, Ru(II)-catalysed azide-reduction reaction induced by visible light. In contrast to current azide-reduction methods, this reaction is highly chemoselective and is compatible with alcohols, phenols, acids, alkenes, alkynes, aldehydes, alkyl halides, alkyl mesylates and disulfides. The remarkable functional group compatibility and mild conditions of the reaction enabled the azide reduction of nucleic acid and oligosaccharide substrates, with no detectable occurrence of side reactions. The reaction was also performed in the presence of a protein enzyme without the loss of enzymatic activity, in contrast to two commonly used azide-reduction methods. The visible-light dependence of this reaction provides a means of photouncaging functional groups, such as amines and carboxylates, on biological macromolecules without using ultraviolet irradiation.

  4. A DNA barcode library for ground beetles (Insecta, Coleoptera, Carabidae) of Germany: The genus Bembidion Latreille, 1802 and allied taxa.

    Science.gov (United States)

    Raupach, Michael J; Hannig, Karsten; Morinière, Jérome; Hendrich, Lars

    2016-01-01

    As molecular identification method, DNA barcoding based on partial cytochrome c oxidase subunit 1 (COI) sequences has been proven to be a useful tool for species determination in many insect taxa including ground beetles. In this study we tested the effectiveness of DNA barcodes to discriminate species of the ground beetle genus Bembidion and some closely related taxa of Germany. DNA barcodes were obtained from 819 individuals and 78 species, including sequences from previous studies as well as more than 300 new generated DNA barcodes. We found a 1:1 correspondence between BIN and traditionally recognized species for 69 species (89%). Low interspecific distances with maximum pairwise K2P values below 2.2% were found for three species pairs, including two species pairs with haplotype sharing (Bembidion atrocaeruleum/Bembidion varicolor and Bembidion guttula/Bembidion mannerheimii). In contrast to this, deep intraspecific sequence divergences with distinct lineages were revealed for two species (Bembidion geniculatum/Ocys harpaloides). Our study emphasizes the use of DNA barcodes for the identification of the analyzed ground beetles species and represents an important step in building-up a comprehensive barcode library for the Carabidae in Germany and Central Europe as well.

  5. Spontaneous germline excision of Tol1, a DNA-based transposable element naturally occurring in the medaka fish genome.

    Science.gov (United States)

    Watanabe, Kohei; Koga, Hajime; Nakamura, Kodai; Fujita, Akiko; Hattori, Akimasa; Matsuda, Masaru; Koga, Akihiko

    2014-04-01

    DNA-based transposable elements are ubiquitous constituents of eukaryotic genomes. Vertebrates are, however, exceptional in that most of their DNA-based elements appear to be inactivated. The Tol1 element of the medaka fish, Oryzias latipes, is one of the few elements for which copies containing an undamaged gene have been found. Spontaneous transposition of this element in somatic cells has previously been demonstrated, but there is only indirect evidence for its germline transposition. Here, we show direct evidence of spontaneous excision in the germline. Tyrosinase is the key enzyme in melanin biosynthesis. In an albino laboratory strain of medaka fish, which is homozygous for a mutant tyrosinase gene in which a Tol1 copy is inserted, we identified de novo reversion mutations related to melanin pigmentation. The gamete-based reversion rate was as high as 0.4%. The revertant fish carried the tyrosinase gene from which the Tol1 copy had been excised. We previously reported the germline transposition of Tol2, another DNA-based element that is thought to be a recent invader of the medaka fish genome. Tol1 is an ancient resident of the genome. Our results indicate that even an old element can contribute to genetic variation in the host genome as a natural mutator.

  6. Molecular Identification of Atlantic Bluefin Tuna (Thunnus thynnus, Scombridae Larvae and Development of a DNA Character-Based Identification Key for Mediterranean Scombrids.

    Directory of Open Access Journals (Sweden)

    Gregory Neils Puncher

    Full Text Available The Atlantic bluefin tuna, Thunnus thynnus, is a commercially important species that has been severely over-exploited in the recent past. Although the eastern Atlantic and Mediterranean stock is now showing signs of recovery, its current status remains very uncertain and as a consequence their recovery is dependent upon severe management informed by rigorous scientific research. Monitoring of early life history stages can inform decision makers about the health of the species based upon recruitment and survival rates. Misidentification of fish larvae and eggs can lead to inaccurate estimates of stock biomass and productivity which can trigger demands for increased quotas and unsound management conclusions. Herein we used a molecular approach employing mitochondrial and nuclear genes (CO1 and ITS1, respectively to identify larvae (n = 188 collected from three spawning areas in the Mediterranean Sea by different institutions working with a regional fisheries management organization. Several techniques were used to analyze the genetic sequences (sequence alignments using search algorithms, neighbour joining trees, and a genetic character-based identification key and an extensive comparison of the results is presented. During this process various inaccuracies in related publications and online databases were uncovered. Our results reveal important differences in the accuracy of the taxonomic identifications carried out by different ichthyoplanktologists following morphology-based methods. While less than half of larvae provided were bluefin tuna, other dominant taxa were bullet tuna (Auxis rochei, albacore (Thunnus alalunga and little tunny (Euthynnus alletteratus. We advocate an expansion of expertise for a new generation of morphology-based taxonomists, increased dialogue between morphology-based and molecular taxonomists and increased scrutiny of public sequence databases.

  7. Suppression of thermosensitive initiation of DNA replication in a dnaR mutant of Escherichia coli by a rifampin resistance mutation in the rpoB gene.

    OpenAIRE

    Sakakibara, Y

    1995-01-01

    The thermosensitivity of the Escherichia coli dnaR130 mutant in initiation of DNA replication was suppressed by a spontaneous rifampin resistance mutation in rpoB, the gene for the beta subunit of RNA polymerase. Among the dnaR-suppressing rpoB alleles obtained was rpoB22, which was able to suppress the thermosensitivity of the dnaA46 or dnaA167 mutant, but not that of the dnaA5 mutant, in initiation of replication. Some dnaA-suppressing rpoB alleles obtained from rifampin-resistant derivativ...

  8. A mechanism for DNA-PK activation requiring unique contributions from each strand of a DNA terminus and implications for microhomology-mediated nonhomologous DNA end joining

    OpenAIRE

    Pawelczak, Katherine S; Turchi, John J.

    2008-01-01

    DNA-dependent protein kinase (DNA-PK) is an essential component of the nonhomologous end joining pathway (NHEJ), responsible for the repair of DNA double-strand breaks. Ku binds a DSB and recruits the catalytic subunit, DNA-PKcs, where it is activated once the kinase is bound to the DSB. The precise mechanism by which DNA activates DNA-PK remains unknown. We have investigated the effect of DNA structure on DNA-PK activation and results demonstrate that in Ku-dependent DNA-PKcs reactions, DNA-...

  9. Cloning of cDNA and expression analysis of a DnaJ-like gene under heavy metal stress in bean

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A clone of PvSR6 encoding a new member of the DnaJ-like protein family was isolated from a mer curic-chloride-treated bean ( Phaseolus vulgaris L. )cDNA library by differential screening using cDNAs derived from treated and untreated plants. The predicted protein contains the highly conserved J domain only, which is present in all DnaJ-like proteins and is considered to play a critical role in DnaJ protein-protein interactions. PvSR6 gene is constitu tively expressed in roots but weakly expressed in stems and leaf tissue. Northern blot analysis revealed the transcripts of PvSR6 were at low levels in unstressed bean leaves, but the genes expression was strongly stimulated by heavy metals. These suggest that the PvSR6 might play an important role in resistance to the damage caused by heavy metals.

  10. Construction of a DNA library representing 15q11-13 by subtraction of two flow sorted marker chromosome-specific libraries

    Energy Technology Data Exchange (ETDEWEB)

    Blennow, E.; Werelius, B.; Nordenskjoeld, M. [Karolinska Hospital, Stockholm (Sweden)] [and others

    1994-09-01

    Constitutional extra {open_quotes}marker chromosomes{close_quotes} are found in {approx}0.5/1000 of newborns. Of these, 50% are inverted duplications of the pericentromeric region of chromosome 15, including two variants; (1) inv dup(15)(pter{yields}q11:q11{yields}pter) and (2) inv dup(15) (pter{yields}q12-13::q12-13{yields}pter). Variant (1) is found in phenotypically normal individuals, whereas variant (2) will produce a typical clinical picture including mental retardation, autism, hyperactivity and discrete dysmorphic features. Fluorescence in situ hybridization (FISH) using single copy probes from the Prader-Willi region confirms these observations as well as chromosome painting using a flow-sorted marker chromosome-specific library from a variant (1) marker, hybridized to the chromosomes of a patient with a variant (2) marker chromosome. Followingly, a flow-sorted biotinylated variant (1) library was subtracted from a non-labeled variant (2) library using magnetic beads and subsequent amplification by degenerate oligonucleotide-primed PCR (DOP-PCR). The successful result was demonstrated by using the amplified material for chromosome painting on chromosome slides from variant (1) and variant (2) patients. We have constructed a library from 15q11-13. This region contains genes producing a specific abnormal phenotype when found in a tri- or tetrasomic state. The region also contains the genes responsible for the Prader-Willi and Angelman syndromes when the paternal/maternal copy is missing, respectively. It is therefore a region where parental imprinting plays an important role. The isolated library may be used to isolate single copy clones which will allow further investigations of this region.

  11. Selection and characterization of a DNA aptamer that can discriminate between cJun/cJun and cJun/cFos.

    Directory of Open Access Journals (Sweden)

    Ryan D Walters

    Full Text Available The AP-1 family of transcriptional activators plays pivotal roles in regulating a wide range of biological processes from the immune response to tumorigenesis. Determining the roles of specific AP-1 dimers in cells, however, has remained challenging because common molecular biology techniques are unable to distinguish between the role of, for example, cJun/cJun homodimers versus cJun/cFos heterodimers. Here we used SELEX (systematic evolution of ligands by exponential enrichment to identify and characterize DNA aptamers that are >100-fold more specific for binding cJun/cJun compared to cJun/cFos, setting the foundation to investigate the biological functions of different AP-1 dimer compositions.

  12. Miscoding properties of 1,N{sup 6}-ethanoadenine, a DNA adduct derived from reaction with antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea

    Energy Technology Data Exchange (ETDEWEB)

    Hang, Bo; Guliaev, Anton B.; Chenna, Ahmed; Singer, B.

    2003-03-05

    1,N{sup 6}-Ethanoadenine (EA) is an exocyclic adduct formed from DNA reaction with the antitumor agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). To understand the role of this adduct in the mechanism of mutagenicity or carcinogenicity by BCNU, an oligonucleotide with a site-specific EA was synthesized using phosphoramidite chemistry. We now report the in vitro miscoding properties of EA in translesion DNA synthesis catalyzed by mammalian DNA polymerases (pols) {alpha}, {beta}, {eta} and {iota}. These data were also compared with those obtained for the structurally related exocyclic adduct, 1,N{sup 6}-ethenoadenine ({var_epsilon}A). Using a primer extension assay, both pols {alpha} and {beta} were primarily blocked by EA or {var_epsilon}A with very minor extension. Pol {eta} a member of the Y family of polymerases, was capable of catalyzing a significant amount of bypass across both adducts. Pol {eta} incorporated all four nucleotides opposite EA and {var_epsilon}A, but with differential preferences and mainly in an error-prone manner. Human pol {iota}, a paralog of human pol {eta}, was blocked by both adducts with a very small amount of synthesis past {var_epsilon}A. It incorporated C and, to a much lesser extent, T, opposite either adduct. In addition, the presence of an A adduct, e.g. {var_epsilon}A, could affect the specificity of pol {iota} toward the template T immediately 3 feet to the adduct. In conclusion, the four polymerases assayed on templates containing an EA or {var_epsilon}A showed differential bypass capacity and nucleotide incorporation specificity, with the two adducts not completely identical in influencing these properties. Although there was a measurable extent of error-free nucleotide incorporation, all these polymerases primarily misincorporated opposite EA, indicating that the adduct, similar to {var_epsilon}A, is a miscoding lesion.

  13. Studies of monocytopoiesis in patients with malignant disease and after imunostimulation with BCG, using 3H-thymidine as a DNA-label

    International Nuclear Information System (INIS)

    Monocytopoiesis and blood monocytes were investigated in patients with Hodgkin's disease, non-Hodgkin lymphomas, mycosis fungoides, breast cancer or melanoma. The investigation was carried out before surgery and just before each application of BCG. Monocyte production was increased in untreated patients. Postoperative prophylactic BCG-vaccination gave rise to increased proliferation activity. However monocyte production returned to normal between the 4th and 6th month of BCG immunotherapy. These results indicate that monocytopoiesis is stimulated by human tumors. BCG immunostimulation is able to increase proliferation activity during the first month of treatment only. (orig.)

  14. Synthesis of a DNA-targeting nickel (II) complex with testosterone thiosemicarbazone which exhibits selective cytotoxicity towards human prostate cancer cells (LNCaP).

    Science.gov (United States)

    Heng, Mok Piew; Sinniah, Saravana Kumar; Teoh, Wuen Yew; Sim, Kae Shin; Ng, Seik Weng; Cheah, Yoke Kqueen; Tan, Kong Wai

    2015-01-01

    Testosterone thiosemicarbazone, L and its nickel (II) complex 1 were synthesized and characterized by using FTIR, CHN, (1)H NMR, and X-ray crystallography. X-ray diffraction study confirmed the formation of L from condensation of testosterone and thiosemicarbazide. Mononuclear complex 1 is coordinated to two Schiff base ligands via two imine nitrogens and two tautomeric thiol sulfurs. The cytotoxicity of both compounds was investigated via MTT assay with cisplatin as positive reference standard. L is more potent towards androgen-dependent LNCaP (prostate) and HCT 116 (colon). On the other hand, complex 1, which is in a distorted square planar environment with L acting as a bidentate NS-donor ligand, is capable of inhibiting the growth of all the cancer cell lines tested, including PC-3 (prostate). It is noteworthy that both compounds are less toxic towards human colon cell CCD-18Co. The intrinsic DNA binding constant (Kb) of both compounds were evaluated via UV-Vis spectrophotometry. Both compounds showed Kb values which are comparable to the reported Kb value of typical classical intercalator such as ethidium bromide. The binding constant of the complex is almost double compared with ligand L. Both compounds were unable to inhibit the action topoisomerase I, which is the common target in cancer treatment (especially colon cancer). This suggest a topoisomerase I independent-cell death mechanism. PMID:26057090

  15. Failure to detect a DNA repair-related defect in the transfection of ataxia-telangiectasia cells by enzymatically restricted plasmid

    International Nuclear Information System (INIS)

    Two SV40-transformed human fibroblast cell lines were transfected with plasmids in which double-strand breaks had been introduced by restriction enzymes, within or near the selected gene. Restriction of pSV2gpt with KpnI reduced the frequency of transfection more in the ionizing radiation-sensitive ataxia-telangiectasia line AT5BIVA than in the resistant line MRC5V1. When the related plasmid pSV2neo was restricted with SmaI, the reduction in transfection was less in the ataxia-telangiectasia than in the normal cells. The apparent defect in transfection of AT5BIVA by pSV2gpt appeared to be a result of the unusual sensitivity of the repair-deficient recipient to the selective agent. Loss of potential transfectants is exacerbated when transient gene expression is reduced by restriction of the plasmid. It is suggested that a reduction in yield of transfectants with restricted plasmid in ataxia-telangiectasia cells cannot readily be used as evidence of a defect in DNA repair. The results are also relevant to standard transfection experiments; they emphasize the importance of optimizing selection when transient expression may be reduced, to ensure that potential transfectants are not killed by the selection regime. (author)

  16. A DNA vaccine encoding mutated HPV58 mE6E7-Fc-GPI fusion antigen and GM-CSF and B7.1

    Directory of Open Access Journals (Sweden)

    Wang H

    2015-10-01

    Full Text Available He Wang,1 Jiyun Yu,2 Li Li1 1Department of Gynecologic Oncology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning, Guangxi, 2Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing, People’s Republic of China Background: Persistent infection with high-risk human papillomavirus (HPV is a predominant cause of cervical cancer, and HPV58 is the third most common virus detected in the patients with cervical cancer in Asia. E6 and E7 are the viral oncogenes which are constitutively expressed in HPV-associated tumor cells and can be used as target antigens for related immunotherapy. In this study, we modified the HPV58 E6 and E7 oncogenes to eliminate their oncogenic potential and constructed a recombinant DNA vaccine that coexpresses the sig-HPV58 mE6E7-Fc-GPI fusion antigen in addition to granulocyte-macrophage colony-stimulating factor (GM-CSF and B7.1 as molecular adjuvants (PVAX1-HPV58 mE6E7FcGB for the treatment of HPV58 (+ cancer. Methods: PVAX1-HPV58 mE6E7FcGB recombinant DNA vaccine was constructed to express a fusion protein containing a signal peptide, a modified HPV58 mE6E7 gene, and human IgG Fc and glycosylphosphatidylinositol (GPI-anchoring sequences using the modified DNA vaccine vector PVAX1-IRES-GM/B7.1 that coexpresses GM-CSF, and B7.1. C57BL/6 mice were challenged by HPV58 E6E7-expressing B16-HPV58 E6E7 cells, followed by immunization by PVAX1-HPV58 mE6E7FcGB vaccine on days 7, 14, 21 after tumor challenge. The cellular immune responses in immunized mice were assessed by measuring IFN-γ production in splenocytes upon stimulation by HPV58 E6E7-GST protein and the lysis of B16-HPV58 E6E7 target cells by splenocytes after restimulation with HPV58 E6E7-GST protein. The antitumor efficacy was evaluated by monitoring the growth of the tumor. Results: PVAX1-HPV58 mE6E7FcGB elicited varying levels of IFN-lsgdB58onn T-cell immune responses and lysis of target cell in mice in response to the recombinant antigen HPV58 E6E7-GST. Furthermore, the vaccine also induced antitumor responses in the HPV58 (+ B16-HPV58 E6E7 tumor challenge model as evidenced by delayed tumor development. Conclusion: The recombinant DNA vaccine PVAX1-HPV58 mE6E7FcGB efficiently generates cellular immunity and antitumor efficacy in immunized mice. These data provide a basis for the further study of this recombinant vaccine as a potential candidate vaccine. Keywords: human papillomavirus type 58, E6 gene, E7 gene, DNA vaccine, immunogenicity

  17. Interplay of nonlinearity and geometry in a DNA-related, Klein-Gordon model with long-range dipole-dipole interaction

    DEFF Research Database (Denmark)

    Archilla, J. F.R.; Christiansen, Peter Leth; Gaididei, Yuri Borisovich

    2002-01-01

    Most of the studies on mathematical models of DNA are limited to next neighbor interaction. However, the coupling between base pairs is thought to be caused by dipole interaction, and, when the DNA strand is bent, the distances between base pairs become shorter, therefore the interactions...... with distant base pairs have to be taken into account. In this, paper we analyze the existence and stability of breathers, i.e., localized oscillations in a simple model of bent DNA with long-range dipole interaction. Breathers have been suggested as precursors of the denaturation bubble....

  18. Development and applications of a DNA labeling method with magnetic nanoparticles to study the role of horizontal gene transfer events between bacteria in soil pollutant bioremediation processes.

    Science.gov (United States)

    Pivetal, J; Frénéa-Robin, M; Haddour, N; Vézy, C; Zanini, L F; Ciuta, G; Dempsey, N M; Dumas-Bouchiat, F; Reyne, G; Bégin-Colin, S; Felder-Flesh, D; Ghobril, C; Pourroy, G; Simonet, P

    2015-12-01

    Horizontal gene transfers are critical mechanisms of bacterial evolution and adaptation that are involved to a significant level in the degradation of toxic molecules such as xenobiotic pesticides. However, understanding how these mechanisms are regulated in situ and how they could be used by man to increase the degradation potential of soil microbes is compromised by conceptual and technical limitations. This includes the physical and chemical complexity and heterogeneity in such environments leading to an extreme bacterial taxonomical diversity and a strong redundancy of genes and functions. In addition, more than 99 % of soil bacteria fail to develop colonies in vitro, and even new DNA-based investigation methods (metagenomics) are not specific and sensitive enough to consider lysis recalcitrant bacteria and those belonging to the rare biosphere. The objective of the ANR funded project “Emergent” was to develop a new culture independent approach to monitor gene transfer among soil bacteria by labeling plasmid DNA with magnetic nanoparticles in order to specifically capture and isolate recombinant cells using magnetic microfluidic devices. We showed the feasibility of the approach by using electrotransformation to transform a suspension of Escherichia coli cells with biotin-functionalized plasmid DNA molecules linked to streptavidin-coated superparamagnetic nanoparticles. Our results have demonstrated that magnetically labeled cells could be specifically retained on micromagnets integrated in a microfluidic channel and that an efficient selective separation can be achieved with the microfluidic device. Altogether, the project offers a promising alternative to traditional culture-based approaches for deciphering the extent of horizontal gene transfer events mediated by electro or natural genetic transformation mechanisms in complex environments such as soil. PMID:26498963

  19. A DNA-dependent stress response involving DNA-PK occurs in hypoxic cells and contributes to cellular adaptation to hypoxia.

    Science.gov (United States)

    Bouquet, Fanny; Ousset, Marielle; Biard, Denis; Fallone, Frédérique; Dauvillier, Stéphanie; Frit, Philippe; Salles, Bernard; Muller, Catherine

    2011-06-01

    DNA-dependent protein kinase (DNA-PK) is involved in DNA double-strand break (DSB) signalling and repair. We report that DNA-PK is activated by mild hypoxia conditions (0.1-1% O₂) as shown by (1) its autophosphorylation on Ser2056, and (2) its mobilisation from a soluble nucleoplasmic compartment to a less extractable nuclear fraction. The recruitment of DNA-PK was not followed by activation and recruitment of the XRCC4-DNA-ligase-IV complex, suggesting that DSBs are not responsible for activation of DNA-PK. To unravel the mechanism of DNA-PK activation, we show that exposure of cells to trichostatin A, a histone deacetylase inhibitor, leads to DNA-PK autophosphorylation and relocalisation to DNA. Histone acetylation (mainly H3K14) is increased in hypoxic cells and treatment with anacardic acid, an inhibitor of histone acetyl transferase, prevented both histone modifications and DNA-PK activation in hypoxic conditions. Importantly, in using either silenced DNA-PK cells or cells exposed to a specific DNA-PK inhibitor (NU7026), we demonstrated that hypoxic DNA-PK activation positively regulates the key transcription factor HIF-1 and one subsequent target gene, GLUT1. Our results show that hypoxia initiates chromatin modification and consequently DNA-PK activation, which positively regulate cellular oxygen-sensing and oxygen-signalling pathways. PMID:21576354

  20. Search for genes essential for pneumococcal transformation : The RadA DNA repair protein plays a role in genomic recombination of donor DNA

    NARCIS (Netherlands)

    Burghout, Peter; Bootsma, Hester J.; Kloosterman, Tomas G.; Bijlsma, Jetta J. E.; de Jongh, Christa E.; Kuipers, Oscar P.; Hermans, Peter W. M.

    2007-01-01

    We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner transposon mutant libraries in S. pneumoniae strain R6 were challenged by transfor

  1. Development and evaluation of a DNA microarray assay for the simultaneous detection of nine harmful algal species in ship ballast and seaport waters

    Science.gov (United States)

    Chen, Xianfeng; Zhou, Qianjin; Duan, Weijun; Zhou, Chengxu; Duan, Lijun; Zhang, Huili; Sun, Aili; Yan, Xiaojun; Chen, Jiong

    2016-01-01

    Rapid, high-throughput and reliable methods are urgently required to accurately detect and monitor harmful algae, which are responsible for algal blooms, such as red and green tides. In this study, we successfully developed a multiplex PCR-based DNA microarray method capable of detecting nine harmful algal species simultaneously, namely Alexandrium tamarense, Gyrodinium instriatum, Heterosigma akashiwo, Karenia mikimotoi, Prorocentrum donghaiense, Prorocentrum minimum, Ulva compressa, Ulva ohnoi and Ulva prolifera. This method achieved a limit of detection (LOD) of 0.5 ng of genomic DNA (orders of magnitude of the deci-nanogram range) in the tested algae cultures. Altogether, 230 field samples from ship ballast waters and seaport waters were used to evaluate the DNA microarray. The clinical sensitivity and specificity of the DNA microarray assay in detecting field samples were 96.4% and 90.9%, respectively, relative to conventional morphological methods. This indicated that this high-throughput, automatic, and specific method is well suited for the detection of algae in water samples.

  2. The Effects of a DNA Virus Infection on the Reproductive Potential of Female Tsetse Flies, Glossina morsitans centralis and Glossina morsitans morsitans (Diptera: Glossinidae

    Directory of Open Access Journals (Sweden)

    Sang Rosemary C

    1998-01-01

    Full Text Available Reproductive anomalies associated with the tsetse DNA virus infection in the female tsetse hosts, Glossina morsitans centralis Machado and Glossina morsitans morsitans Westwood, inoculated with the virus during the 3rd instar larval stage were studied and the data compared to those obtained from the control females injected with sterile physiological saline. Virus infected flies had significantly longer first and second pregnancy cycles (P<0.0001 and produced pupae that were of significantly less weight in milligrams (P<0.0001 compared to controls. Transmission of the virus to progeny was not absolute and only 21% of G. m. centralis and 48% of G. m. morsitans first progeny flies from infected females developed salivary gland hypertrophy as a result of transmission from mother to progeny. The virus infected females produced significantly fewere pupae compared to the controls during the experimental period (P<0.00001.

  3. Design and performance testing of a DNA extraction assay for sensitive and reliable quantification of acetic acid bacteria directly in red wine using real time PCR

    Directory of Open Access Journals (Sweden)

    Cédric eLONGIN

    2016-06-01

    Full Text Available Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence there is a real need for a rapid, specific, sensitive and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR. Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP at 1% (v/v during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 mL to 10 mL. Thus the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage.

  4. Molecular Identification of Atlantic Bluefin Tuna (Thunnus thynnus, Scombridae) Larvae and Development of a DNA Character-Based Identification Key for Mediterranean Scombrids.

    Science.gov (United States)

    Puncher, Gregory Neils; Arrizabalaga, Haritz; Alemany, Francisco; Cariani, Alessia; Oray, Isik K; Karakulak, F Saadet; Basilone, Gualtiero; Cuttitta, Angela; Mazzola, Salvatore; Tinti, Fausto

    2015-01-01

    The Atlantic bluefin tuna, Thunnus thynnus, is a commercially important species that has been severely over-exploited in the recent past. Although the eastern Atlantic and Mediterranean stock is now showing signs of recovery, its current status remains very uncertain and as a consequence their recovery is dependent upon severe management informed by rigorous scientific research. Monitoring of early life history stages can inform decision makers about the health of the species based upon recruitment and survival rates. Misidentification of fish larvae and eggs can lead to inaccurate estimates of stock biomass and productivity which can trigger demands for increased quotas and unsound management conclusions. Herein we used a molecular approach employing mitochondrial and nuclear genes (CO1 and ITS1, respectively) to identify larvae (n = 188) collected from three spawning areas in the Mediterranean Sea by different institutions working with a regional fisheries management organization. Several techniques were used to analyze the genetic sequences (sequence alignments using search algorithms, neighbour joining trees, and a genetic character-based identification key) and an extensive comparison of the results is presented. During this process various inaccuracies in related publications and online databases were uncovered. Our results reveal important differences in the accuracy of the taxonomic identifications carried out by different ichthyoplanktologists following morphology-based methods. While less than half of larvae provided were bluefin tuna, other dominant taxa were bullet tuna (Auxis rochei), albacore (Thunnus alalunga) and little tunny (Euthynnus alletteratus). We advocate an expansion of expertise for a new generation of morphology-based taxonomists, increased dialogue between morphology-based and molecular taxonomists and increased scrutiny of public sequence databases.

  5. Overall structure and sugar dynamics of a DNA dodecamer from homo- and heteronuclear dipolar couplings and 31P chemical shift anisotropy

    International Nuclear Information System (INIS)

    The solution structure of d(CGCGAATTCGCG)2 has been determined on the basis of an exceptionally large set of residual dipolar couplings. In addition to the heteronuclear 13C-1H and 15N-1H and qualitative homonuclear 1H-1H dipolar couplings, previously measured in bicelle medium, more than 300 quantitative 1H-1H and 22 31P-1H dipolar restraints were obtained in liquid crystalline Pf1 medium, and 22 31P chemical shift anisotropy restraints. High quality DNA structures can be obtained solely on the basis of these new restraints, and these structures are in close agreement with those calculated previously on the basis of 13C-1H and 15N-1H dipolar couplings. In the newly calculated structures, 31P-1H dipolar and 3JsubH3'Psub couplings and 31P CSA data restrain the phosphodiester backbone torsion angles. The final structure represents a quite regular B-form helix with a modest bending of ∼10 deg., which is essentially independent of whether or not electrostatic terms are used in the calculation. Combined, the number of homo- and heteronuclear dipolar couplings significantly exceeds the number of degrees of freedom in the system. Results indicate that the dipolar coupling data cannot be fit by a single structure, but are compatible with the presence of rapid equilibria between C2'-endo and C3'-endo deoxyribose puckers (sugar switching). The C2'-H2'/H2'' dipolar couplings in B-form DNA are particularly sensitive to sugar pucker and yield the largest discrepancies when fit to a single structure. To resolve these discrepancies, we suggest a simplified dipolar coupling analysis that yields N/S equilibria for the ribose sugar puckers, which are in good agreement with previous analyses of NMR JHH couplings, with a population of the minor C3'-endo form higher for pyrimidines than for purines

  6. A HIV-Tat/C4-binding protein chimera encoded by a DNA vaccine is highly immunogenic and contains acute EcoHIV infection in mice.

    Science.gov (United States)

    Tomusange, Khamis; Wijesundara, Danushka; Gummow, Jason; Garrod, Tamsin; Li, Yanrui; Gray, Lachlan; Churchill, Melissa; Grubor-Bauk, Branka; Gowans, Eric J

    2016-01-01

    DNA vaccines are cost-effective to manufacture on a global scale and Tat-based DNA vaccines have yielded protective outcomes in preclinical and clinical models of human immunodeficiency virus (HIV), highlighting the potential of such vaccines. However, Tat-based DNA vaccines have been poorly immunogenic, and despite the administration of multiple doses and/or the addition of adjuvants, these vaccines are not in general use. In this study, we improved Tat immunogenicity by fusing it with the oligomerisation domain of a chimeric C4-binding protein (C4b-p), termed IMX313, resulting in Tat heptamerisation and linked Tat to the leader sequence of tissue plasminogen activator (TPA) to ensure that the bulk of heptamerised Tat is secreted. Mice vaccinated with secreted Tat fused to IMX313 (pVAX-sTat-IMX313) developed higher titres of Tat-specific serum IgG, mucosal sIgA and cell-mediated immune (CMI) responses, and showed superior control of EcoHIV infection, a surrogate murine HIV challenge model, compared with animals vaccinated with other test vaccines. Given the crucial contribution of Tat to HIV-1 pathogenesis and the precedent of Tat-based DNA vaccines in conferring some level of protection in animal models, we believe that the virologic control demonstrated with this novel multimerised Tat vaccine highlights the promise of this vaccine candidate for humans. PMID:27358023

  7. MicroRNA expression in rainbow trout (Oncorhynchus mykiss) vaccinated with a DNA vaccine encoding the glycoprotein gene of Viral hemorrhagic septicemia virus

    DEFF Research Database (Denmark)

    Bela-Ong, Dennis; Schyth, Brian Dall; Lorenzen, Niels

    Viral hemorrhagic septicemia caused by a fish rhabdovirus, Viral hemorrhagic septicemia virus (VHSV), results in significant mortality in farmed rainbow trout (Oncorhynchus mykiss Walbaum). Although the disease had been eradicated in Denmark, wildlife marine reservoir of VHSV poses a threat parti...

  8. A SILAC-based screen for Methyl-CpG binding proteins identifies RBP-J as a DNA methylation and sequence-specific binding protein.

    Directory of Open Access Journals (Sweden)

    Stefanie J J Bartels

    Full Text Available BACKGROUND: DNA methylation is an epigenetic modification that plays a crucial role in a variety of biological processes. Methylated DNA is specifically bound by Methyl-CpG Binding Proteins (MBPs. Three different types of MBPs have been identified so far: the Methyl-CpG Binding Domain (MBD family proteins, three BTB/POZ-Zn-finger proteins, and UHRF1. Most of the known MBPs have been identified via homology with the MBD and Zn-finger domains as present in MeCP2 and Kaiso, respectively. It is conceivable that other proteins are capable of recognizing methylated DNA. METHODOLOGY/PRINCIPAL FINDINGS: For the purpose of identifying novel 'readers' we set up a methyl-CpG pull-down assay combined with stable-isotope labeling by amino acids in cell culture (SILAC. In a methyl-CpG pull-down with U937 nuclear extracts, we recovered several known MBPs and almost all subunits of the MBD2/NuRD complex as methylation specific binders, providing proof-of-principle. Interestingly, RBP-J, the transcription factor downstream of Notch receptors, also bound the DNA in a methylation dependent manner. Follow-up pull-downs and electrophoretic mobility shift assays (EMSAs showed that RBP-J binds methylated DNA in the context of a mutated RBP-J consensus motif. CONCLUSIONS/SIGNIFICANCE: The here described SILAC/methyl-CpG pull-down constitutes a new approach to identify potential novel DNAme readers and will advance unraveling of the complete methyl-DNA interactome.

  9. Study of a DNA Duplex by Nuclear Magnetic Resonance and Molecular Dynamics Simulations. Validation of Pulsed Dipolar Electron Paramagnetic Resonance Distance Measurements Using Triarylmethyl-Based Spin Labels.

    Science.gov (United States)

    Lomzov, Alexander A; Sviridov, Eugeniy A; Shernuykov, Andrey V; Shevelev, Georgiy Yu; Pyshnyi, Dmitrii V; Bagryanskaya, Elena G

    2016-06-16

    Pulse dipole-dipole electron paramagnetic resonance (EPR) spectroscopy (double electron-electron resonance [DEER] or pulse electron-electron double resonance [PELDOR] and double quantum coherence [DQC]) allows for measurement of distances in biomolecules and can be used at low temperatures in a frozen solution. Recently, the possibility of distance measurement in a nucleic acid at a physiological temperature using pulse EPR was demonstrated. In these experiments, triarylmethyl (TAM) radicals with long memory time of the electron spin served as a spin label. In addition, the duplex was immobilized on modified silica gel particles (Nucleosil DMA); this approach enables measurement of interspin distances close to 4.5 nm. Nevertheless, the possible influence of TAM on the structure of a biopolymer under study and validity of the data obtained by DQC are debated. In this paper, a combination of molecular dynamics (MD) and nuclear magnetic resonance (NMR) methods was used for verification of interspin distances measured by the X-band DQC method. NMR is widely used for structural analysis of biomolecules under natural conditions (room temperature and an aqueous solution). The ultraviolet (UV) melting method and thermal series (1)H NMR in the range 5-95 °C revealed the presence of only the DNA duplex in solution at oligonucleotide concentrations 1 μM to 1.1 mM at temperatures below 40 °C. The duplex structures and conformation flexibility of native and TAM-labeled DNA complexes obtained by MD simulation were the same as the structure obtained by NMR refinement. Thus, we showed that distance measurements at physiological temperatures by the X-band DQC method allow researchers to obtain valid structural information on an unperturbed DNA duplex using terminal TAM spin labels.

  10. Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule

    OpenAIRE

    van der Gun, Bernardina T. F.; Maluszynska-Hoffman, Maria; Kiss, Antal; Arendzen, Alice J.; Ruiters, Marcel H.J.; McLaughlin, Pamela M. J.; Weinhold, Elmar; Rots, Marianne G.

    2010-01-01

    The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that has been identified as a marker of cancer-initiating cells. EpCAM is highly expressed on most carcinomas, and transient silencing of EpCAM expression leads to reduced oncogenic potential. To silence the EpCAM gene in a persistent manner via targeted DNA methylation, a low activity mutant (C141S) of the CpG-specific DNA methyltransferase M.SssI was coupled to a triple-helix-forming oligonucleotide (TFO−C141S) specifi...

  11. Click Addition of a DNA Thread to the N-Termini of Peptides for Their Translocation through Solid-State Nanopores.

    Science.gov (United States)

    Biswas, Sudipta; Song, Weisi; Borges, Chad; Lindsay, Stuart; Zhang, Peiming

    2015-10-27

    Foremost among the challenges facing single molecule sequencing of proteins by nanopores is the lack of a universal method for driving proteins or peptides into nanopores. In contrast to nucleic acids, the backbones of which are uniformly negatively charged nucleotides, proteins carry positive, negative and neutral side chains that are randomly distributed. Recombinant proteins carrying a negatively charged oligonucleotide or polypeptide at the C-termini can be translocated through a α-hemolysin (α-HL) nanopore, but the required genetic engineering limits the generality of these approaches. In this present study, we have developed a chemical approach for addition of a charged oligomer to peptides so that they can be translocated through nanopores. As an example, an oligonucleotide PolyT20 was tethered to peptides through first selectively functionalizing their N-termini with azide followed by a click reaction. The data show that the peptide-PolyT20 conjugates translocated through nanopores, whereas the unmodified peptides did not. Surprisingly, the conjugates with their peptides tethered at the 5'-end of PolyT20 passed the nanopores more rapidly than the PolyT20 alone. The PolyT20 also yielded a wider distribution of blockade currents. The same broad distribution was found for a conjugate with its peptide tethered at the 3'-end of PolyT20, suggesting that the larger blockades (and longer translocation times) are associated with events in which the 5'-end of the PolyT20 enters the pore first.

  12. Cloning of a DNA-binding protein that interacts with the ethylene-responsive enhancer element of the carnation GST1 gene.

    Science.gov (United States)

    Maxson, J M; Woodson, W R

    1996-07-01

    Ethylene transcriptionally activates a glutathione S-transferase gene (GST1) at the onset of the senescence program in carnation (Dianthus caryophyllus L.) flower petals. A 126 bp region of the GST1 promoter sequence has been identified as an ethylene-responsive enhancer element (ERE). In this paper, we demonstrate the ability of nuclear proteins from senescing petals to recognize a 22 bp sequence within the ERE (ERE oligonucleotide). Mutation of the ERE oligonucleotide sequence significantly alters the strength of this nuclear protein-DNA association. The wild-type ERE oligonucleotide sequence was used to isolate a cDNA clone encoding a sequence-specific DNA binding protein. Nucleotide sequencing and deduced amino acid sequence analysis of this cDNA predicted a 32 kDa protein which we have designated carnation ethylene-responsive element-binding protein-1 (CEBP-1). The mRNA expression pattern of CEBP-1 suggests that it is not transcriptionally regulated by ethylene. The amino acid sequence homology of CEBP-1 with other plant nucleic acid binding proteins indicates a conserved nucleic acid binding domain. Within this domain are two highly conserved RNA-binding motifs, RNP-1 and RNP-2. An acidic region and a putative nuclear localization signal are also identified.

  13. Effect of initial ion positions on the interactions of monovalent and divalent ions with a DNA duplex as revealed with atomistic molecular dynamics simulations.

    Science.gov (United States)

    Robbins, Timothy J; Wang, Yongmei

    2013-01-01

    Monovalent (Na(+)) and divalent (Mg(2+)) ion distributions around the Dickerson-Drew dodecamer were studied by atomistic molecular dynamics (MD) simulations with AMBER molecular modeling software. Different initial placements of ions were tried and the resulting effects on the ion distributions around DNA were investigated. For monovalent ions, results were found to be nearly independent of initial cation coordinates. However, Mg(2+) ions demonstrated a strong initial coordinate dependent behavior. While some divalent ions initially placed near the DNA formed essentially permanent direct coordination complexes with electronegative DNA atoms, Mg(2+) ions initially placed further away from the duplex formed a full, nonexchanging, octahedral first solvation shell. These fully solvated cations were still capable of binding with DNA with events lasting up to 20 ns, and in comparison were bound much longer than Na(+) ions. Force field parameters were also investigated with modest and little differences arising from ion (ions94 and ions08) and nucleic acid description (ff99, ff99bsc0, and ff10), respectively. Based on known Mg(2+) ion solvation structure, we conclude that in most cases Mg(2+) ions retain their first solvation shell, making only solvent-mediated contacts with DNA duplex. The proper way to simulate Mg(2+) ions around DNA duplex, therefore, should begin with ions placed in the bulk water.

  14. Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins.

    Science.gov (United States)

    Colin, Didier J; Hain, Karolina O; Allan, Lindsey A; Clarke, Paul R

    2015-03-01

    Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.

  15. A DNA vaccine encoding a chimeric allergen derived from major group 1 allergens of dust mite can be used for specific immunotherapy

    OpenAIRE

    Sun, Tong; Yin, Kang; Wu, Lu-Yi; Jin, Wen-Jie; Li, Yang; Sheng, Bin; Jiang, Yu-Xin

    2014-01-01

    Immunization with DNA-based constructs has been shown to be against the antigen and the response is skewed in such a way as to ameliorate the symptoms of allergic disease. This approach is particularly useful in the treatment of allergic inflammatory diseases, such as asthma. The major group 1 allergen from house dust mites is one of the triggers of allergic asthma. This study explores whether a chimeric gene R8, derived from the major group 1 allergen of house dust mite species (Dermatophago...

  16. Development of PCR primers and a DNA macroarray for the simultaneous detection of major Staphylococcus species using groESL gene.

    Science.gov (United States)

    Chiang, Yu-Cheng; Lu, Hsi-Chi; Li, Sheng-Chih; Chang, Yu-Hsin; Chen, Hsin-Yen; Lin, Chia-Wei; Tsen, Hau-Yang

    2012-03-01

    Staphylococcus spp., including S. aureus, S. intermedius, S. hyicus, S. epidermidis, S. saprophyticus, S. haemolyticus, S. xylosus, and S. carnosus, are major bacterial species associated with food poisoning, and human and veterinary clinics. Traditional methods for the identification of these staphylococci are time-consuming, laborious, or inaccurate. Therefore, rapid and accurate diagnostic methods are needed. In this study, we designed the DNA probes and polymerase chain reaction (PCR) primers for the detection of the aforementioned Staphylococcus species. These primers were proved to be specific for the detection of their corresponding target strains. Furthermore, by using a consensus primer pair, we were able to co-amplify the intergenic region of groES-groEL for these staphylococci. Followed by a chromogenic macroarray system with the specific probes on the plastic chips, these staphylococci in milk products or clinical samples could be simultaneously detected. When the system was used for the inspection of milk or urine samples containing N × 10⁰ target cells per milliliter of the sample, all these staphylococcal species could be identified after an 8-h pre-enrichment step. This system also allowed the adequate diagnosis of bacteremia, since N × 10⁰ target cells per milliliter of the blood samples could be detected after a 12-h pre-enrichment. Compared to the multiplex PCR method, this approach has the additional advantage that it allowed the discrimination of more bacterial strains-even some bacterial strains that may generate PCR products with the same molecular sizes.

  17. The proteasome inhibitor MG-132 sensitizes PC-3 prostate cancer cells to ionizing radiation by a DNA-PK-independent mechanism

    Directory of Open Access Journals (Sweden)

    McBride William H

    2005-07-01

    Full Text Available Abstract Background By modulating the expression levels of specific signal transduction molecules, the 26S proteasome plays a central role in determining cell cycle progression or arrest and cell survival or death in response to stress stimuli, including ionizing radiation. Inhibition of proteasome function by specific drugs results in cell cycle arrest, apoptosis and radiosensitization of many cancer cell lines. This study investigates whether there is also a concomitant increase in cellular radiosensitivity if proteasome inhibition occurs only transiently before radiation. Further, since proteasome inhibition has been shown to activate caspase-3, which is involved in apoptosis, and caspase-3 can cleave DNA-PKcs, which is involved in DNA-double strand repair, the hypothesis was tested that caspase-3 activation was essential for both apoptosis and radiosensitization following proteasome inhibition. Methods Prostate carcinoma PC-3 cells were treated with the reversible proteasome inhibitor MG-132. Cell cycle distribution, apoptosis, caspase-3 activity, DNA-PKcs protein levels and DNA-PK activity were monitored. Radiosensitivity was assessed using a clonogenic assay. Results Inhibition of proteasome function caused cell cycle arrest and apoptosis but this did not involve early activation of caspase-3. Short-time inhibition of proteasome function also caused radiosensitization but this did not involve a decrease in DNA-PKcs protein levels or DNA-PK activity. Conclusion We conclude that caspase-dependent cleavage of DNA-PKcs during apoptosis does not contribute to the radiosensitizing effects of MG-132.

  18. Electrospray ionisation mass spectrometry of non-convalent complexes of a DNA-binding protein, TUS with its DNA recognition sequence, TER B

    International Nuclear Information System (INIS)

    Full text: Electrospray ionization mass spectrometry (ESI MS) is a powerful tool for analysis of biological macromolecules. More recently, the technique has been applied to characterization of non covalent complexes. The application of ESI MS to the study of these biochemical complexes presents a number of technical difficulties. For example traditional buffer components are not volatile and are therefore not compatible with this technique. Observations of macromolecular complexes in the gas phase require careful scrutiny. Are they representative of the solution structure of the complex or are they the result of non-specific gas phase associations of molecules? In order to examine some of these questions, we have studied the complex of Tus protein (35,652 Da), with its double-stranded 21 mer DNA recognition sequence, TerB. Binding of Tus to termination sequences (Ter) on the E coll chromosome halts DNA replication. The Tus Ter B complex is well-characterized, a crystal structure is available, and the binding characteristics of a range of protein mutants and variant DNA sequences have been examined using several techniques. We have determined conditions for detecting a Tus Ter B complex by ESI MS. Under these conditions, only a negligible amount of a Tus random DNA complex was observed. We have also been able to distinguish binding between wild-type and mutant Tus to Ter B, and between Ter B and variant DNA sequences to Tus

  19. SDA, a DNA aptamer inhibiting E- and P-selectin mediated adhesion of cancer and leukemia cells, the first and pivotal step in transendothelial migration during metastasis formation.

    Directory of Open Access Journals (Sweden)

    Rassa Faryammanesh

    Full Text Available Endothelial (E- and platelet (P- selectin mediated adhesion of tumor cells to vascular endothelium is a pivotal step of hematogenous metastasis formation. Recent studies have demonstrated that selectin deficiency significantly reduces metastasis formation in vivo. We selected an E- and P-Selectin specific DNA Aptamer (SDA via SELEX (Systematic Evolution of Ligands by EXponential enrichment with a K(d value of approximately 100 nM and the capability of inhibiting the interaction between selectin and its ligands. Employing human colorectal cancer (HT29 and leukemia (EOL-1 cell lines we could demonstrate an anti-adhesive effect for SDA in vitro. Under physiological shear stress conditions in a laminar flow adhesion assay, SDA inhibited dynamic tumor cell adhesion to immobilized E- or P-selectin. The stability of SDA for more than two hours allowed its application in cell-cell adhesion assays in cell culture medium. When adhesion of HT29 cells to TNFα-stimulated E-selectin presenting human pulmonary microvascular endothelial cells was analyzed, inhibition via SDA could be demonstrated as well. In conclusion, SDA is a potential new therapeutic agent that antagonizes selectin-mediated adhesion during metastasis formation in human malignancies.

  20. Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule

    NARCIS (Netherlands)

    van der Gun, Bernardina T. F.; Maluszynska-Hoffman, Maria; Kiss, Antal; Arendzen, Alice J.; Ruiters, Marcel H. J.; McLaughlin, Pamela M. J.; Weinhold, Elmar; Rots, Marianne G.

    2010-01-01

    The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that has been identified as a marker of cancer-initiating cells. EpCAM is highly expressed on most carcinomas, and transient silencing of EpCAM expression leads to reduced oncogenic potential. To silence (he EpCAM gene in a per

  1. Embryonic neural inducing factor churchill is not a DNA-binding zinc finger protein: solution structure reveals a solvent-exposed beta-sheet and zinc binuclear cluster.

    Science.gov (United States)

    Lee, Brian M; Buck-Koehntop, Bethany A; Martinez-Yamout, Maria A; Dyson, H Jane; Wright, Peter E

    2007-08-31

    Churchill is a zinc-containing protein that is involved in neural induction during embryogenesis. At the time of its discovery, it was thought on the basis of sequence alignment to contain two zinc fingers of the C4 type. Further, binding of an N-terminal GST-Churchill fusion protein to a particular DNA sequence was demonstrated by immunoprecipitation selection assay, suggesting that Churchill may function as a transcriptional regulator by sequence-specific DNA binding. We show by NMR solution structure determination that, far from containing canonical C4 zinc fingers, the protein contains three bound zinc ions in novel coordination sites, including an unusual binuclear zinc cluster. The secondary structure of Churchill is also unusual, consisting of a highly solvent-exposed single-layer beta-sheet. Hydrogen-deuterium exchange and backbone relaxation measurements reveal that Churchill is unusually dynamic on a number of time scales, with the exception of regions surrounding the zinc coordinating sites, which serve to stabilize the otherwise unstructured N terminus and the single-layer beta-sheet. No binding of Churchill to the previously identified DNA sequence could be detected, and extensive searches using DNA sequence selection techniques could find no other DNA sequence that was bound by Churchill. Since the N-terminal amino acids of Churchill form part of the zinc-binding motif, the addition of a fusion protein at the N terminus causes loss of zinc and unfolding of Churchill. This observation most likely explains the published DNA-binding results, which would arise due to non-specific interaction of the unfolded protein in the immunoprecipitation selection assay. Since Churchill does not appear to bind DNA, we suggest that it may function in embryogenesis as a protein-interaction factor.

  2. A HIV-Tat/C4-binding protein chimera encoded by a DNA vaccine is highly immunogenic and contains acute EcoHIV infection in mice

    Science.gov (United States)

    Tomusange, Khamis; Wijesundara, Danushka; Gummow, Jason; Garrod, Tamsin; Li, Yanrui; Gray, Lachlan; Churchill, Melissa; Grubor-Bauk, Branka; Gowans, Eric J.

    2016-01-01

    DNA vaccines are cost-effective to manufacture on a global scale and Tat-based DNA vaccines have yielded protective outcomes in preclinical and clinical models of human immunodeficiency virus (HIV), highlighting the potential of such vaccines. However, Tat-based DNA vaccines have been poorly immunogenic, and despite the administration of multiple doses and/or the addition of adjuvants, these vaccines are not in general use. In this study, we improved Tat immunogenicity by fusing it with the oligomerisation domain of a chimeric C4-binding protein (C4b-p), termed IMX313, resulting in Tat heptamerisation and linked Tat to the leader sequence of tissue plasminogen activator (TPA) to ensure that the bulk of heptamerised Tat is secreted. Mice vaccinated with secreted Tat fused to IMX313 (pVAX-sTat-IMX313) developed higher titres of Tat-specific serum IgG, mucosal sIgA and cell-mediated immune (CMI) responses, and showed superior control of EcoHIV infection, a surrogate murine HIV challenge model, compared with animals vaccinated with other test vaccines. Given the crucial contribution of Tat to HIV-1 pathogenesis and the precedent of Tat-based DNA vaccines in conferring some level of protection in animal models, we believe that the virologic control demonstrated with this novel multimerised Tat vaccine highlights the promise of this vaccine candidate for humans. PMID:27358023

  3. Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays

    Directory of Open Access Journals (Sweden)

    Kelly J. Henrickson

    2009-10-01

    Full Text Available Assays to simultaneously detect multiple potential agents of bioterrorism are limited. Two multiplex PCR and RT-PCR enzyme hybridization assays (mPCR-EHA, mRT-PCR-EHA were developed to simultaneously detect many of the CDC category “A” bioterrorism agents. The “Bio T” DNA assay was developed to detect: Variola major (VM, Bacillus anthracis (BA, Yersinia pestis (YP, Francisella tularensis (FT and Varicella zoster virus (VZV. The “Bio T” RNA assay (mRT-PCR-EHA was developed to detect: Ebola virus (Ebola, Lassa fever virus (Lassa, Rift Valley fever (RVF, Hantavirus Sin Nombre species (HSN and dengue virus (serotypes 1-4. Sensitivity and specificity of the 2 assays were tested by using genomic DNA, recombinant plasmid positive controls, RNA transcripts controls, surrogate (spiked clinical samples and common respiratory pathogens. The analytical sensitivity (limit of detection (LOD of the DNA asssay for genomic DNA was 1×100~1×102 copies/mL for BA, FT and YP. The LOD for VZV whole organism was 1×10-2 TCID50/mL. The LOD for recombinant controls ranged from 1×102~1×103copies/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1×104~1×106 copies/mL without extraction and 1×105~1×106 copies/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ~1×10-4 dilution for dengue 1 and 2, 1×104 LD50/mL and 1×102 LD50/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ~1×103 copies/mL (1.5 input copies/reaction for Ebola, RVF, Lassa and HSN. No cross-reactivity of primers and probes used in both assays was detected with common respiratory pathogens or between targeted analytes. Clinical sensitivity was estimated using 264 surrogate clinical samples tested with the BioT DNA assay and 549 samples tested with the BioT RNA assay. The clinical specificity is 99.6% and 99.8% for BioT DNA assay and BioT RNA assay, respectively. The surrogate sensitivities of these two assays were 100% (95%CI 83-100 for FT, BA (pX02, YP, VM, VZV, dengue 2,3,4 and 95% (95%CI 75-100 for BA (pX01 and dengue 1 using spiked clinical specimens. The specificity of both BioT multiplex assays on spiked specimens was 100% (95% CI 99-100. Compared to other available assays (culture, serology, PCR, etc. both the BioT DNA mPCR-EHA and BioT RNA mRT-PCR-EHA are rapid, sensitive and specific assays for detecting many category “A” Bioterrorism agents using a standard thermocycler.

  4. Exonuclease I-aided homogeneous electrochemical strategy for organophosphorus pesticide detection based on enzyme inhibition integrated with a DNA conformational switch.

    Science.gov (United States)

    Wang, Xiuzhong; Dong, Shanshan; Hou, Ting; Liu, Lei; Liu, Xiaojuan; Li, Feng

    2016-03-01

    A novel enzyme inhibition-based homogeneous electrochemical biosensing strategy was designed for an organophosphorus pesticide assay based on exploiting the resistance of a mercury ion-mediated helper probe (HP) toward nuclease-catalyzed digestion and the remarkable diffusivity difference between HPs and the mononucleotides toward a negatively charged indium tin oxide (ITO) electrode. In particular, the mercury ion-mediated T-Hg(2+)-T base pairs facilitate the HP labeled with methylene blue (MB) to fold into a hairpin structure, preventing its digestion by exonuclease I, and thus resulting in a low electrochemical response because of the large electrostatic repulsion between the negatively charged ITO electrode and the HPs. The competitive binding by a thiol group (-SH), produced in the hydrolysis reaction of acetylthiocholine (ACh) chloride with acetylcholinesterase (AChE), removes mercury ions from the base pairs, causing a nuclease-catalyzed digestion, and the subsequent electrochemical response increase due to the weak electrostatic repulsion between the product-mononucleotides and the ITO electrode. Mercury ion-mediated HPs were first designed for pesticide detection and diazinon was chosen as the model target. Under the optimal experimental conditions, the approach exhibited high sensitivity for diazinon detection with a detection limit of 0.25 μg L(-1). The satisfactory results in the determination of diazinon in real samples demonstrate that the method possesses great potential for detecting organophosphorus pesticides. This new approach is expected to promote the exploitation of mercury-mediated base pair-based homogenous electrochemical biosensors in biochemical studies and in the food safety field.

  5. Unbiased mutagenesis of MHV68 LANA reveals a DNA-binding domain required for LANA function in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Clinton R Paden

    2012-09-01

    Full Text Available The Latency-Associated Nuclear Antigen (LANA, encoded by ORF73, is a conserved gene among the γ2-herpesviruses (rhadinoviruses. The Kaposi's Sarcoma-Associated Herpesvirus (KSHV LANA is consistently expressed in KSHV-associated malignancies. In the case of the rodent γ2-herpesvirus, murine gammaherpesvirus 68 (MHV68, the LANA homolog (mLANA is required for efficient virus replication, reactivation from latency and immortalization of murine fetal liver-derived B cells. To gain insights into mLANA function(s, knowing that KSHV LANA binds DNA and can modulate transcription of a variety of promoters, we sought out and identified a mLANA-responsive promoter which maps to the terminal repeat (TR of MHV68. Notably, mLANA strongly repressed activity from this promoter. We extended these analyses to demonstrate direct, sequence-specific binding of recombinant mLANA to TR DNA by DNase I footprinting. To assess whether the DNA-binding and/or transcription modulating function is important in the known mLANA phenotypes, we generated an unbiased library of mLANA point mutants using error-prone PCR, and screened a large panel of mutants for repression of the mLANA-responsive promoter to identify loss of function mutants. Notably, among the mutant mLANA proteins recovered, many of the mutations are in a predicted EBNA-1-like DNA-binding domain. Consistent with this prediction, those tested displayed loss of DNA binding activity. We engineered six of these mLANA mutants into the MHV68 genome and tested the resulting mutant viruses for: (i replication fitness; (ii efficiency of latency establishment; and (iii reactivation from latency. Interestingly, each of these mLANA-mutant viruses exhibited phenotypes similar to the mLANA-null mutant virus, indicating that DNA-binding is critical for mLANA function.

  6. Leading-edge forensic DNA analyses and the necessity of including crime scene investigators, police officers and technicians in a DNA elimination database.

    Science.gov (United States)

    Lapointe, Martine; Rogic, Anita; Bourgoin, Sarah; Jolicoeur, Christine; Séguin, Diane

    2015-11-01

    In recent years, sophisticated technology has significantly increased the sensitivity and analytical power of genetic analyses so that very little starting material may now produce viable genetic profiles. This sensitivity however, has also increased the risk of detecting unknown genetic profiles assumed to be that of the perpetrator, yet originate from extraneous sources such as from crime scene workers. These contaminants may mislead investigations, keeping criminal cases active and unresolved for long spans of time. Voluntary submission of DNA samples from crime scene workers is fairly low, therefore we have created a promotional method for our staff elimination database that has resulted in a significant increase in voluntary samples since 2011. Our database enforces privacy safeguards and allows for optional anonymity to all staff members. We also offer information sessions at various police precincts to advise crime scene workers of the importance and success of our staff elimination database. This study, a pioneer in its field, has obtained 327 voluntary submissions from crime scene workers to date, of which 46 individual profiles (14%) have been matched to 58 criminal cases. By implementing our methods and respect for individual privacy, forensic laboratories everywhere may see similar growth and success in explaining unidentified genetic profiles in stagnate criminal cases.

  7. A DNA fragment from Xq21 replaces a deleted region containing the entire FVIII gene in a severe hemophilia A patient

    Energy Technology Data Exchange (ETDEWEB)

    Murru, S.; Casula, L.; Moi, P. [Insituto di Clinica e Biologia dell` Eta Evolutiva, Cagliari (Italy)] [and others

    1994-09-15

    In this paper the authors report the molecular characterization of a large deletion that removes the entire Factor VIII gene in a severe hemophilia A patient. Accurate DNA analysis of the breakpoint region revealed that a large DNA fragment replaced the 300-kb one, which was removed by the deletion. Pulsed-field gel electrophoresis analysis revealed that the size of the inserted fragment is about 550 kb. In situ hybridization demonstrated that part of the inserted region normally maps to Xq21 and to the tip of the short arm of the Y chromosome (Yp). In this patient this locus is present both in Xq21 and in Xq28, in addition to the Yp, being thus duplicated in the X chromosome. Sequence analysis of the 3` breakpoint suggested that an illegitimate recombination is probably the cause of this complex rearrangement. 52 refs., 7 figs.

  8. Chemo/Photoacoustic Dual Therapy with mRNA-Triggered DOX Release and Photoinduced Shockwave Based on a DNA-Gold Nanoplatform.

    Science.gov (United States)

    Zang, Yundong; Wei, Yanchun; Shi, Yujiao; Chen, Qun; Xing, Da

    2016-02-10

    A multifunctional nanoparticle based on gold nanorod (GNR), utilizing mRNA triggered chemo-drug release and near-infrared photoacoustic effect, is developed for a combined chemo-photoacoustic therapy. The constructed nanoparticle (GNR-DNA/FA:DOX) comprises three functional components: (i) GNR as the drug delivery platform and photoacoustic effect enhancer; (ii) toehold-possessed DNA dressed on the GNR to load doxorubicin (DOX) to implement a tumor cell specific chemotherapy; and (iii) folate acid (FA) modified on GNR to guide the nanoparticle to target tumor cells. The results show that, upon an effective and specific delivery of the nanoparticles to the tumor cells with overexpressed folate receptors, the cytotoxic DOX loaded on the GNR-DNA nanoplatform can be released through DNA displacement reaction in melanoma-associated antigen gene mRNA expressed cells. With 808 nm pulse laser irradiation, the photoacoustic effect of the GNR leads to a direct physical damage to the cells. The combined treatment of the two modalities can effectively destroy tumor cells and eradicate the tumors with two distinctively different and supplementing mechanisms. With the nanoparticle, photoacoustic imaging is successfully performed in situ to monitor the drug distribution and tumor morphology for therapeutical guidance. With further in-depth investigation, the proposed nanoparticle may provide an effective and safe alternative cancer treatment modality.

  9. Orally administered lactoperoxidase increases expression of the FK506 binding protein 5 gene in epithelial cells of the small intestine of mice: a DNA microarray study.

    Science.gov (United States)

    Wakabayashi, Hiroyuki; Miyauchi, Hirofumi; Shin, Kouichirou; Yamauchi, Koji; Matsumoto, Ichiro; Abe, Keiko; Takase, Mitsunori

    2007-09-01

    Lactoperoxidase (LPO) is a component of milk and other external secretions. To study the influence of ingested LPO on the digestive tract, we performed DNA microarray analysis of the small intestine of mice administered LPO. LPO administration upregulated 78 genes, including genes involved in metabolism, immunity, apoptosis, and the cell cycle, and downregulated nine genes, including immunity-related genes. The most upregulated gene was FK506 binding protein 5 (FKBP5), a glucocorticoid regulating immunophilin. The upregulation of this gene was confirmed by quantitative RT-PCR in other samples. In situ hybridization revealed that expression of the FKBP5 gene in the crypt epithelial cells of the small intestine was enhanced by LPO. These results suggest that ingested LPO modulates gene expression in the small intestine and especially increases FKBP5 gene expression in the epithelial cells of the intestine.

  10. Building-Up of a DNA Barcode Library for True Bugs (Insecta: Hemiptera: Heteroptera) of Germany Reveals Taxonomic Uncertainties and Surprises

    Science.gov (United States)

    Raupach, Michael J.; Hendrich, Lars; Küchler, Stefan M.; Deister, Fabian; Morinière, Jérome; Gossner, Martin M.

    2014-01-01

    During the last few years, DNA barcoding has become an efficient method for the identification of species. In the case of insects, most published DNA barcoding studies focus on species of the Ephemeroptera, Trichoptera, Hymenoptera and especially Lepidoptera. In this study we test the efficiency of DNA barcoding for true bugs (Hemiptera: Heteroptera), an ecological and economical highly important as well as morphologically diverse insect taxon. As part of our study we analyzed DNA barcodes for 1742 specimens of 457 species, comprising 39 families of the Heteroptera. We found low nucleotide distances with a minimum pairwise K2P distance 2.2% were detected for 16 traditionally recognized and valid species. With a successful identification rate of 91.5% (418 species) our study emphasizes the use of DNA barcodes for the identification of true bugs and represents an important step in building-up a comprehensive barcode library for true bugs in Germany and Central Europe as well. Our study also highlights the urgent necessity of taxonomic revisions for various taxa of the Heteroptera, with a special focus on various species of the Miridae. In this context we found evidence for on-going hybridization events within various taxonomically challenging genera (e.g. Nabis Latreille, 1802 (Nabidae), Lygus Hahn, 1833 (Miridae), Phytocoris Fallén, 1814 (Miridae)) as well as the putative existence of cryptic species (e.g. Aneurus avenius (Duffour, 1833) (Aradidae) or Orius niger (Wolff, 1811) (Anthocoridae)). PMID:25203616

  11. A theoretical analysis of codon adaptation index of the Boophilus microplus bm86 gene directed to the optimization of a DNA vaccine.

    Science.gov (United States)

    Ruiz, Lina María; Armengol, Gemma; Habeych, Edwin; Orduz, Sergio

    2006-04-21

    DNA vaccines utilize host cell molecules for gene transcription and translation to proteins, and the interspecific difference of codon usage is one of the major obstacles for effective induction of specific and strong immune response. In an attempt to improve codon usage effects of DNA vaccine on protein expression, a quantitative study was conducted to clarify the relationship of codon usage in the tick gene bm86 and its potential expression in bovine cells. The calculated relative synonymous codon usage (RSCU) and codon adaptation index (CAI) values of bm86 from Boophilus microplus and a set of 14 highly expressed genes from Bos taurus indicated that some codons utilized frequently in bm86 are rarely used in B. taurus genes and vice versa. The different translational efficiencies obtained suggested that after DNA vaccination using the wild bm86 gene, the protein Bm86 would be expressed in bovines, but it would not be the optimum sequence. However, using the codon-optimized bm86 gene to bovines, whose sequence was theoretically designed, would probably improve the level of the immune response generated against ticks. PMID:16171828

  12. Label-free ultrasensitive detection of telomerase activity via multiple telomeric hemin/G-quadruplex triggered polyaniline deposition and a DNA tetrahedron-structure regulated signal.

    Science.gov (United States)

    Liu, Yuanjian; Wei, Min; Liu, Xu; Wei, Wei; Zhao, Hongyu; Zhang, Yuanjian; Liu, Songqin

    2016-01-31

    Label-free detection of telomerase activity was done by using telomeric hemin/G-quadruplex triggered polyaniline deposition, not only on themselves but also on the DNA tetrahedron-structure (DTS). DTS size has a great impact on telomerase accessibility, reactivity and detection sensitivity. The method has been used to evaluate bladder cancer development.

  13. Toward a DNA taxonomy of Alpine Rhithrogena (Ephemeroptera: Heptageniidae using a mixed Yule-coalescent analysis of mitochondrial and nuclear DNA.

    Directory of Open Access Journals (Sweden)

    Laurent Vuataz

    Full Text Available Aquatic larvae of many Rhithrogena mayflies (Ephemeroptera inhabit sensitive Alpine environments. A number of species are on the IUCN Red List and many recognized species have restricted distributions and are of conservation interest. Despite their ecological and conservation importance, ambiguous morphological differences among closely related species suggest that the current taxonomy may not accurately reflect the evolutionary diversity of the group. Here we examined the species status of nearly 50% of European Rhithrogena diversity using a widespread sampling scheme of Alpine species that included 22 type localities, general mixed Yule-coalescent (GMYC model analysis of one standard mtDNA marker and one newly developed nDNA marker, and morphological identification where possible. Using sequences from 533 individuals from 144 sampling localities, we observed significant clustering of the mitochondrial (cox1 marker into 31 GMYC species. Twenty-one of these could be identified based on the presence of topotypes (expertly identified specimens from the species' type locality or unambiguous morphology. These results strongly suggest the presence of both cryptic diversity and taxonomic oversplitting in Rhithrogena. Significant clustering was not detected with protein-coding nuclear PEPCK, although nine GMYC species were congruent with well supported terminal clusters of nDNA. Lack of greater congruence in the two data sets may be the result of incomplete sorting of ancestral polymorphism. Bayesian phylogenetic analyses of both gene regions recovered four of the six recognized Rhithrogena species groups in our samples as monophyletic. Future development of more nuclear markers would facilitate multi-locus analysis of unresolved, closely related species pairs. The DNA taxonomy developed here lays the groundwork for a future revision of the important but cryptic Rhithrogena genus in Europe.

  14. Toward a DNA taxonomy of Alpine Rhithrogena (Ephemeroptera: Heptageniidae) using a mixed Yule-coalescent analysis of mitochondrial and nuclear DNA.

    Science.gov (United States)

    Vuataz, Laurent; Sartori, Michel; Wagner, André; Monaghan, Michael T

    2011-01-01

    Aquatic larvae of many Rhithrogena mayflies (Ephemeroptera) inhabit sensitive Alpine environments. A number of species are on the IUCN Red List and many recognized species have restricted distributions and are of conservation interest. Despite their ecological and conservation importance, ambiguous morphological differences among closely related species suggest that the current taxonomy may not accurately reflect the evolutionary diversity of the group. Here we examined the species status of nearly 50% of European Rhithrogena diversity using a widespread sampling scheme of Alpine species that included 22 type localities, general mixed Yule-coalescent (GMYC) model analysis of one standard mtDNA marker and one newly developed nDNA marker, and morphological identification where possible. Using sequences from 533 individuals from 144 sampling localities, we observed significant clustering of the mitochondrial (cox1) marker into 31 GMYC species. Twenty-one of these could be identified based on the presence of topotypes (expertly identified specimens from the species' type locality) or unambiguous morphology. These results strongly suggest the presence of both cryptic diversity and taxonomic oversplitting in Rhithrogena. Significant clustering was not detected with protein-coding nuclear PEPCK, although nine GMYC species were congruent with well supported terminal clusters of nDNA. Lack of greater congruence in the two data sets may be the result of incomplete sorting of ancestral polymorphism. Bayesian phylogenetic analyses of both gene regions recovered four of the six recognized Rhithrogena species groups in our samples as monophyletic. Future development of more nuclear markers would facilitate multi-locus analysis of unresolved, closely related species pairs. The DNA taxonomy developed here lays the groundwork for a future revision of the important but cryptic Rhithrogena genus in Europe.

  15. Studies of monocytopoiesis in patients with malignant disease and after imunostimulation with BCG, using /sup 3/H-thymidine as a DNA-label

    Energy Technology Data Exchange (ETDEWEB)

    Schmitt, E.; Meuret, G.; Waldermann, F.; Hoffmann, G.

    1982-04-01

    Monocytopoiesis and blood monocytes were investigated in patients with Hodgkin's disease, non-Hodgkin lymphomas, mycosis fungoides, breast cancer or melanoma. The investigation was carried out before surgery and just before each application of BCG. Monocyte production was increased in untreated patients. Postoperative prophylactic BCG-vaccination gave rise to increased proliferation activity. However monocyte production returned to normal between the 4th and 6th month of BCG immunotherapy. These results indicate that monocytopoiesis is stimulated by human tumors. BCG immunostimulation is able to increase proliferation activity during the first month of treatment only.

  16. Vector optimization and needle-free intradermal application of a broadly protective polyvalent influenza A DNA vaccine for pigs and humans

    DEFF Research Database (Denmark)

    Borggren, Marie; Nielsen, Jens; Bragstad, Karoline;

    2015-01-01

    , was compared with intradermal needle injection followed by electroporation. We report that when our DNA vaccine is expressed by the new vectors and delivered to the skin with the needle-free device in the rabbit model, it can elicit an antibody response with the same titers as a conventional vector...... with intradermal electroporation. The needle-free delivery is already in use for traditional protein vaccines in pigs but should be considered as a practical alternative for the mass administration of broadly protective influenza DNA vaccines....

  17. A polyvalent influenza A DNA vaccine induces heterologous immunity and protects pigs against pandemic A(H1N1)pdm09 virus infection

    DEFF Research Database (Denmark)

    Bragstad, Karoline; Vinner, Lasse; Hansen, Mette Sif;

    2013-01-01

    vaccine components.We found that pigs challenged with a virus homologous to the HA and NA DNA vaccine components were well protected from infection. In addition, heterologous challenge virus was cleared rapidly compared to the unvaccinated control pigs. Immunisation by electroporation induced HI...

  18. A DNA element recognised by the molybdenum-responsive transcription factor ModE is conserved in Proteobacteria, green sulphur bacteria and Archaea

    Directory of Open Access Journals (Sweden)

    Pau Richard N

    2003-12-01

    Full Text Available Abstract Background The transition metal molybdenum is essential for life. Escherichia coli imports this metal into the cell in the form of molybdate ions, which are taken up via an ABC transport system. In E. coli and other Proteobacteria molybdenum metabolism and homeostasis are regulated by the molybdate-responsive transcription factor ModE. Results Orthologues of ModE are widespread amongst diverse prokaryotes, but not ubiquitous. We identified probable ModE-binding sites upstream of genes implicated in molybdenum metabolism in green sulphur bacteria and methanogenic Archaea as well as in Proteobacteria. We also present evidence of horizontal transfer of nitrogen fixation genes between green sulphur bacteria and methanogenic Archaea. Conclusions Whereas most of the archaeal helix-turn-helix-containing transcription factors belong to families that are Archaea-specific, ModE is unusual in that it is found in both Archaea and Bacteria. Moreover, its cognate upstream DNA recognition sequence is also conserved between Archaea and Bacteria, despite the fundamental differences in their core transcription machinery. ModE is the third example of a transcriptional regulator with a binding signal that is conserved in Bacteria and Archaea.

  19. Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR

    Science.gov (United States)

    Longin, Cédric; Guilloux-Benatier, Michèle; Alexandre, Hervé

    2016-01-01

    Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage. PMID:27313572

  20. Archaeal DNA Polymerase-B as a DNA Template Guardian: Links between Polymerases and Base/Alternative Excision Repair Enzymes in Handling the Deaminated Bases Uracil and Hypoxanthine

    Directory of Open Access Journals (Sweden)

    Javier Abellón-Ruiz

    2016-01-01

    Full Text Available In Archaea repair of uracil and hypoxanthine, which arise by deamination of cytosine and adenine, respectively, is initiated by three enzymes: Uracil-DNA-glycosylase (UDG, which recognises uracil; Endonuclease V (EndoV, which recognises hypoxanthine; and Endonuclease Q (EndoQ, (which recognises both uracil and hypoxanthine. Two archaeal DNA polymerases, Pol-B and Pol-D, are inhibited by deaminated bases in template strands, a feature unique to this domain. Thus the three repair enzymes and the two polymerases show overlapping specificity for uracil and hypoxanthine. Here it is demonstrated that binding of Pol-D to primer-templates containing deaminated bases inhibits the activity of UDG, EndoV, and EndoQ. Similarly Pol-B almost completely turns off EndoQ, extending earlier work that demonstrated that Pol-B reduces catalysis by UDG and EndoV. Pol-B was observed to be a more potent inhibitor of the enzymes compared to Pol-D. Although Pol-D is directly inhibited by template strand uracil, the presence of Pol-B further suppresses any residual activity of Pol-D, to near-zero levels. The results are compatible with Pol-D acting as the replicative polymerase and Pol-B functioning primarily as a guardian preventing deaminated base-induced DNA mutations.

  1. A Novel DNA Binding Mechanism for maf Basic Region-Leucine Zipper Factors Inferred from a MafA-DNA Complex Structure and Binding Specificities

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Xun; Guanga, Gerald P; Wan, Cheng; Rose, Robert B [Z; (W Elec.); (NCSU)

    2012-11-13

    MafA is a proto-oncoprotein and is critical for insulin gene expression in pancreatic β-cells. Maf proteins belong to the AP1 superfamily of basic region-leucine zipper (bZIP) transcription factors. Residues in the basic helix and an ancillary N-terminal domain, the Extended Homology Region (EHR), endow maf proteins with unique DNA binding properties: binding a 13 bp consensus site consisting of a core AP1 site (TGACTCA) flanked by TGC sequences and binding DNA stably as monomers. To further characterize maf DNA binding, we determined the structure of a MafA–DNA complex. MafA forms base-specific hydrogen bonds with the flanking G–5C–4 and central C0/G0 bases, but not with the core-TGA bases. However, in vitro binding studies utilizing a pulse–chase electrophoretic mobility shift assay protocol revealed that mutating either the core-TGA or flanking-TGC bases dramatically increases the binding off rate. Comparing the known maf structures, we propose that DNA binding specificity results from positioning the basic helix through unique phosphate contacts. The EHR does not contact DNA directly but stabilizes DNA binding by contacting the basic helix. Collectively, these results suggest a novel multistep DNA binding process involving a conformational change from contacting the core-TGA to contacting the flanking-TGC bases.

  2. Characterization of a DNA sequence family in the Prader-Willi/Angelman syndrome chromosome region in 15q11-q13

    Energy Technology Data Exchange (ETDEWEB)

    Dittrich, B.; Knoblauch, H.; Buiting, K.; Horsthemke, B. (Universitaetsklinikum Essen (Germany))

    1993-04-01

    IR4-3R (D15S11) is an anonymous DNA sequence from human chromosome 15. Using YAC cloning and restriction enzyme analysis, the authors have found that IR4-3R detects five related DNA sequences, which are spread over 700 kb within the Prader-Willi/Angelman syndrome chromosome region in 15q11-q 13. The RsaI and StyI polymorphisms, which were described previously, are associated with the most proximal copy of IR4-3R and are in strong linkage disequilibrium. IR4-3R represents the third DNA sequence family that has been identified in 15q11-q13. 14 refs., 2 figs., 1 tab.

  3. Induction of Immune Response in BALB/c Mice with a DNA Vaccine Encoding Bacterioferritin or P39 of Brucella spp.

    OpenAIRE

    Al-Mariri, Ayman; Tibor, Anne; Mertens, Pascal; De Bolle, Xavier; Michel, Patrick; Godfroid, Jacques; Walravens, Karl; Letesson, Jean-Jacques

    2001-01-01

    In this study, we evaluated the ability of DNA vaccines encoding the bacterioferritin (BFR) or P39 proteins of Brucella spp. to induce cellular and humoral immune responses and to protect BALB/c mice against a challenge with B. abortus 544. We constructed eukaryotic expression vectors called pCIBFR and pCIP39, encoding BFR or P39 antigens, respectively, and we verified that these proteins were produced after transfection of COS-7 cells. PCIBFR or pCIP39 was injected intramuscularly three time...

  4. Genome-wide profiling identifies a DNA methylation signature that associates with TET2 mutations in diffuse large B-cell lymphoma

    DEFF Research Database (Denmark)

    Asmar, Fazila; Punj, Vasu; Christensen, Jesper Aagaard;

    2013-01-01

    The discovery that the Ten-Eleven Translocation (TET) hydroxylases cause DNA demethylation has fundamentally changed the notion of how DNA methylation is regulated. Clonal analysis of the hematopoetic stem cell compartment suggests that TET2 mutations can be early events in hematologic cancers...

  5. A DNA Vaccine Coding for the Brucella Outer Membrane Protein 31 Confers Protection against B. melitensis and B. ovis Infection by Eliciting a Specific Cytotoxic Response

    Science.gov (United States)

    Cassataro, Juliana; Velikovsky, Carlos A.; de la Barrera, Silvia; Estein, Silvia M.; Bruno, Laura; Bowden, Raúl; Pasquevich, Karina A.; Fossati, Carlos A.; Giambartolomei, Guillermo H.

    2005-01-01

    The development of an effective subunit vaccine against brucellosis is a research area of intense interest. The outer membrane proteins (Omps) of Brucella spp. have been extensively characterized as potential immunogenic and protective antigens. This study was conducted to evaluate the immunogenicity and protective efficacy of the B. melitensis Omp31 gene cloned in the pCI plasmid (pCIOmp31). Immunization of BALB/c mice with pCIOmp31 conferred protection against B. ovis and B. melitensis infection. Mice vaccinated with pCIOmp31 developed a very weak humoral response, and in vitro stimulation of their splenocytes with recombinant Omp31 did not induced the secretion of gamma interferon. Splenocytes from Omp31-vaccinated animals induced a specific cytotoxic-T-lymphocyte activity, which leads to the in vitro lysis of Brucella-infected macrophages. pCIOmp31 immunization elicited mainly CD8+ T cells, which mediate cytotoxicity via perforins, but also CD4+ T cells, which mediate lysis via the Fas-FasL pathway. In vivo depletion of T-cell subsets showed that the pCIOmp31-induced protection against Brucella infection is mediated predominantly by CD8+ T cells, although CD4+T cells also contribute. Our results demonstrate that the Omp31 DNA vaccine induces cytotoxic responses that have the potential to contribute to protection against Brucella infection. The protective response could be related to the induction of CD8+ T cells that eliminate Brucella-infected cells via the perforin pathway. PMID:16177328

  6. Tlys, a newly identified Sulfolobus spindle-shaped virus 1 transcript expressed in the lysogenic state, encodes a DNA-binding protein interacting at the promoters of the early genes

    DEFF Research Database (Denmark)

    Fusco, Salvatore; She, Qunxin; Bartolucci, Simonetta;

    2013-01-01

    sites of F55 are included within a tandem-repeated sequence overlapping the transcription start sites and/or the B recognition element of the pertinent genes. The strongest binding was observed with the promoters of T5 and T6, and an apparent cooperativity in binding was observed with the Tind promoter......While studying the gene expression of the Sulfolobus spindle-shaped virus 1 (SSV1) in Sulfolobus solfataricus lysogenic cells, a novel viral transcript (Tlys) was identified. Transcriptional analysis revealed that Tlys is expressed only in the absence of UV irradiation and is downregulated during...... the growth of the lysogenic host. The correponding gene f55 lies between two transcriptional units (T6 and Tind) that are upregulated upon UV irradiation. The open reading frame f55 encodes a 6.3-kDa protein which shows sequence identity with negative regulators that fold into the ribbon-helix-helix DNA...

  7. Cloning and sequence analysis of novel DNA polymerases from thermophilic Geobacillus species isolated from hot springs in Turkey: characterization of a DNA polymerase I from Geobacillus kaue strain NB.

    Science.gov (United States)

    Çağlayan, Melike; Bilgin, Neş'e

    2011-11-01

    The complete coding sequences of the polA genes from seven thermophilic Geobacillus species, isolated from hot springs of Gönen and Hisaralan in Turkey, were cloned and sequenced. The polA genes of these Geobacillus species contain a long open reading frame of 2,637 bp encoding DNA polymerase I with a calculated molecular mass of 99 kDa. Amino acid sequences of these Geobacillus DNA polymerases are closely related. The multiple sequence alignments show all include the conserved amino acids in the polymerase and 5'-3' exonuclease domains, but the catalytic residues varied in 3'-5' exonuclease domain of these Geobacillus DNA polymerases. One of them, DNA polymerase I from Geobacillus kaue strain NB (Gkaue polI) is purified to homogeneity and biochemically characterized in vitro. The optimum temperature for enzymatic activity of Gkaue polI is 70 °C at pH 7.5-8.5 in the presence of 8 mM Mg(2+) and 80-100 mM of monovalent ions. The addition of polyamines stimulates the polymerization activity of the enzyme. Three-dimensional structure of Gkaue polI predicted using homology modeling confirmed the conservation of all the functionally important regions in the polymerase active site.

  8. A DNA Study of the Neolithic Human Bones from the Jiangjialiang Site in Yangyuan County,Hebei%河北阳原县姜家梁遗址新石器时代人骨DNA的研究

    Institute of Scientific and Technical Information of China (English)

    吉林大学考古DNA实验室

    2001-01-01

    Mitochondrial DNA has been successfully extracted from 10 specimens from tour tombs at Jiangjialiang. Hypervariable region I (HVRI) in the mitochondrial DNA has been amplified and sequenced. Altogether 10 distinct haplotypes with 15 polymorphic sites have been obtained. A phylogenitic tree of the 10 sequences has been constructed by means of the maximum likelihood method. Four clusters,corresponding to the four tombs, have been presented in the tree. The fact that mitochondrial DNA is inherited exclusively in the female line and the features of graves in matriarchal society suggest that the Jiangjialiang population should not be taken to be a matriarchal community.

  9. Reduced repair capacity of a DNA clustered damage site comprised of 8-oxo-7,8-dihydro-2′-deoxyguanosine and 2-deoxyribonolactone results in an increased mutagenic potential of these lesions

    Energy Technology Data Exchange (ETDEWEB)

    Cunniffe, Siobhan [CRUK-MRC Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ (United Kingdom); O’Neill, Peter, E-mail: peter.oneill@oncology.ox.ac.uk [CRUK-MRC Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ (United Kingdom); Greenberg, Marc M. [Johns Hopkins University, Department of Chemistry, 3400 N. Charles St. , Baltimore, MD 21218 (United States); Lomax, Martine E. [CRUK-MRC Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ (United Kingdom)

    2014-04-15

    Highlights: • A dL lesion is not repaired as effectively as an AP site. • The repair of a cluster with dL and 8-oxodGuo lesions is compromised. • Delayed repair of the cluster leads to an increase in mutation frequency. - Abstract: A signature of ionizing radiation is the induction of DNA clustered damaged sites. Non-double strand break (DSB) clustered damage has been shown to compromise the base excision repair pathway, extending the lifetimes of the lesions within the cluster, compared to isolated lesions. This increases the likelihood the lesions persist to replication and thus increasing the mutagenic potential of the lesions within the cluster. Lesions formed by ionizing radiation include 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 2-deoxyribonolactone (dL). dL poses an additional challenge to the cell as it is not repaired by the short-patch base excision repair pathway. Here we show recalcitrant dL repair is reflected in mutations observed when DNA containing it and a proximal 8-oxodGuo is replicated in Escherichia coli. 8-oxodGuo in close proximity to dL on the opposing DNA strand results in an enhanced frequency of mutation of the lesions within the cluster and a 20 base sequence flanking the clustered damage site in an E. coli based plasmid assay. In vitro repair of a dL lesion is reduced when compared to the repair of an abasic (AP) site and a tetrahydrofuran (THF), and this is due mainly to a reduction in the activity of polymerase β, leading to retarded FEN1 and ligase 1 activities. This study has given insights in to the biological effects of clusters containing dL.

  10. A DNA nanomachine based on rolling circle amplification-bridged two-stage exonuclease III-assisted recycling strategy for label-free multi-amplified biosensing of nucleic acid.

    Science.gov (United States)

    Xue, Qingwang; Lv, Yanqin; Cui, Hui; Gu, Xiaohong; Zhang, Shuqiu; Liu, Jifeng

    2015-01-26

    An autonomous DNA nanomachine based on rolling circle amplification (RCA)-bridged two-stage exonuclease III (Exo III)-induced recycling amplification (Exo III-RCA-Exo III) was developed for label-free and highly sensitive homogeneous multi-amplified detection of DNA combined with sensitive fluorescence detection technique. According to the configuration, the analysis of DNA is accomplished by recognizing the target to a unlabeled molecular beacon (UMB) that integrates target-binding and signal transducer within one multifunctional design, followed by the target-binding of UMB in duplex DNA removed stepwise by Exo III accompanied by the releasing of target DNA for the successive hybridization and cleavage process and autonomous generation of the primer that initiate RCA process with a rational designed padlock DNA. The RCA products containing thousands of repeated catalytic sequences catalytically hybridize with a hairpin reporter probe that includes a "caged" inactive G-quadruplex sequence (HGP) and were then detected by Exo III-assisted recycling amplification, liberating the active G-quadruplex and generating remarkable ZnPPIX/G-quadruplex fluorescence signals with the help of zinc(II)-protoporphyrin IX (ZnPPIX). The proposed strategy showed a wide dynamic range over 7 orders of magnitude with a low limit of detection of 0.51 aM. In addition, this designed protocol can discriminate mismatched DNA from perfectly matched target DNA, and holds a great potential for early diagnosis in gene-related diseases.

  11. Correlation of mRNA Profiles, miRNA Profiles, and Functional Immune Response in Rainbow Trout (Oncorrhynkus Mykiss) Infected With Viral Hemorrhagic Septicemia Virus (VHSV) and in Fish Vaccinated With a DNA Vaccine Against VHSV

    DEFF Research Database (Denmark)

    Bela-Ong, Dennis; Schyth, Brian Dall; Jørgensen, Hanne;

    2011-01-01

    Micro ribonucleic acids (miRNAs) are a diverse class of small (18-22 nucleotides) endogenous RNAs that potently mediate post-transcriptional silencing of a wide range of genes and are emerging as critical regulators of cellular processes. They are transcribed and processed from larger precursors......RNAs, miRNAs could be involved in controlling the expression of fish immune response genes. This project aims to analyze mRNA and miRNA expression in organs of vaccinated and non-vaccinated rainbow trout (Oncorhynchus mykiss) families showing differential mortality in previous infection trials...... encoding the VHSV glycoprotein gene. We will link mRNA and miRNA profiles with phenotypic, genotypic, and immunological data, which will provide an integrated view of the mechanisms of resistance and the strong protective immune responses provided by vaccination. This information is important in designing...

  12. Induction of Broad and Potent Anti-Human Immunodeficiency Virus Immune Responses in Rhesus Macaques by Priming with a DNA Vaccine and Boosting with Protein-Adsorbed Polylactide Coglycolide Microparticles

    OpenAIRE

    Otten, Gillis; Schaefer, Mary; Greer, Catherine; Calderon-Cacia, Maria; Coit, Doris; Kazzaz, Jina; Medina-Selby, Angelica; Selby, Mark; Singh, Manmohan; Ugozzoli, Mildred; zur Megede, Jan; Barnett, Susan W.; O'Hagan, Derek; Donnelly, John; Ulmer, Jeffrey

    2003-01-01

    Several vaccine technologies were evaluated for their abilities to induce anti-human immunodeficiency virus Gag immune responses in rhesus macaques. While no vaccine alone was able to induce broad and strong immune responses, these were achieved by priming with Gag DNA and boosting with Gag protein adsorbed to polylactide coglycolide microparticles. This regimen elicited strong antibodies, helper T cells, and cytotoxic T lymphocytes and thus holds promise as an effective vaccination scheme.

  13. STUDY OF A DNA SEQUENCE FROM BRINE SHRIMP ARTEMIA CONTAINING A NOVEL DM DOMAIN%卤虫中编码DM结构域的DNA序列的克隆和初步研究(简报)

    Institute of Scientific and Technical Information of China (English)

    曾辉; 宋文芹; 陈瑞阳

    2004-01-01

    性别决定的分子机制复杂多样,但是处于动物性别决定的基因调控网络底部的一些调控基因具有相当高的保守性。doublesex(dsx)基因和male abnomal-3(mab-3)基因分别是果蝇(Drosophila melanogaster)和线虫(Caenorhabditis elegans)性别决定调控途径末端的重要基因,对这两个基因序列的比较导致了DM结构域的发现,它是已知在性别发育过程中最为保守的DNA结合结构域。目前,已

  14. Structure and stability of a DNA triple helix in solution: NMR studies on d(T) sub 6 ter dot d(T) sub 6 and its complex with a minor groove binding drug

    Energy Technology Data Exchange (ETDEWEB)

    Umemoto, Kimiko; Sarma, Mukti H.; Gupta, Goutam; Luo, Jia; Sarma, Ramaswamy H. (State Univ. of New York, Albany (USA))

    1990-05-23

    The possibility of both Watson-Crick and Hoogsteen A{center dot}T pairs can result in a triple helical structure for d(T){sub 6}{center dot}d(A){sub 6}{center dot}d(T){sub 6} in solution. In the triple helix the Watson-Crick paired T strand can run antiparallel, while the Hoogsteen paired T strand can run parallel to the A strand. On the basis of 1D/2D NMR studies, we have characterized the structural properties of the triple helix in terms of (a) nature of H-bonding, (b) chain conformations and relative chain orientations, (c) location of triplets T{center dot}A{center dot}T with respect to the helix axis, and (d) effects of NaCl and MgCl{sub 2}. In addition, we experimentally demonstrate that a minor groove specific drug Dst2 (a distamycin analogue) can bind to the triple helix. We show that the nature of thermal transition is altered by Dst2 binding; i.e., the host triple helix shows triple {yields} coil (monophasic) transition in the absence of Dst2, while in its presence the helix shows a triplex {yields} duplex {yields} coil (biphasic) transition.

  15. Comparison of Immune Responses against FMD by a DNA Vaccine Encoding the FMDV/O/IRN/2007 VP1 Gene and the Conventional Inactivated Vaccine in an Animal Model

    Institute of Scientific and Technical Information of China (English)

    Farahnaz Motamedi Sedeh; Hoorieh Soleimanjahi; AmirReza Jalilian; Homayoon Mahravani

    2012-01-01

    Foot-and-mouth disease virus (FMDV) is highly contagious and responsible for huge outbreaks among cloven hoofed animals.The aim of the present study is to evaluate a plasmid DNA immunization system that expresses the FMDV/O/IRN/2007 VP1 gene and compare it with the conventional inactivated vaccine in an animal model.The VP1 gene was sub-cloned into the unique Kpn I and BamH I cloning sites of the pcDNA3.1+ and pEGFP-N1 vectors to construct the VP1 gene cassettes.The transfected BHKT7 cells with sub-cloned pEGFP-N1-VP1 vector expressed GFP-VP1 fusion protein and displayed more green fluorescence spots than the transfected BHKT7 cells with pEGFP-N1 vector,which solely expressed the GFP protein.Six mice groups were respectively immunized by the sub-cloned pcDNA3.1+-VP1 gene cassette as the DNA vaccine,DNA vaccine and PCMV-SPORT-GMCSF vector (as molecular adjuvant) together,conventional vaccine,PBS (as negative control),pcDNA3.1+ vector (as control group) and PCMV-SPORT vector that contained the GMCSF gene (as control group).Significant neutralizing antibody responses were induced in the mice which were immunized using plasmid vectors expressing the VP1 and GMCSF genes together,the DNA vaccine alone and the conventional inactivated vaccine (P<0.05).Co-administration of DNA vaccine and GMCSF gene improved neutralizing antibody response in comparison with administration of the DNA vaccine alone,but this response was the most for the conventional vaccine group.However,induction of humeral immunity response in the conventional vaccine group was more protective than for the DNA vaccine,but T-cell proliferation and IFN-γ concentration were the most in DNA vaccine with the GMCSF gene.Therefore the group that was vaccinated by DNA vaccine with the GMCSF gene,showed protective neutralizing antibody response and the most Th1 cellular immunity.

  16. Binding sequences for RdgB, a DNA damage-responsive transcriptional activator, and temperature-dependent expression of bacteriocin and pectin lyase genes in Pectobacterium carotovorum subsp. carotovorum.

    Science.gov (United States)

    Yamada, Kazuteru; Kaneko, Jun; Kamio, Yoshiyuki; Itoh, Yoshifumi

    2008-10-01

    Pectobacterium carotovorum subsp. carotovorum strain Er simultaneously produces the phage tail-like bacteriocin carotovoricin (Ctv) and pectin lyase (Pnl) in response to DNA-damaging agents. The regulatory protein RdgB of the Mor/C family of proteins activates transcription of pnl through binding to the promoter. However, the optimal temperature for the synthesis of Ctv (23 degrees C) differs from that for synthesis of Pnl (30 degrees C), raising the question of whether RdgB directly activates ctv transcription. Here we report that RdgB directly regulates Ctv synthesis. Gel mobility shift assays demonstrated RdgB binding to the P(0), P(1), and P(2) promoters of the ctv operons, and DNase I footprinting determined RdgB-binding sequences (RdgB boxes) on these and on the pnl promoters. The RdgB box of the pnl promoter included a perfect 7-bp inverted repeat with high binding affinity to the regulator (K(d) [dissociation constant] = 150 nM). In contrast, RdgB boxes of the ctv promoters contained an imperfect inverted repeat with two or three mismatches that consequently reduced binding affinity (K(d) = 250 to 350 nM). Transcription of the rdgB and ctv genes was about doubled at 23 degrees C compared with that at 30 degrees C. In contrast, the amount of pnl transcription tripled at 30 degrees C. Thus, the inverse synthesis of Ctv and Pnl as a function of temperature is apparently controlled at the transcriptional level, and reduced rdgB expression at 30 degrees C obviously affected transcription from the ctv promoters with low-affinity RdgB boxes. Pathogenicity toward potato tubers was reduced in an rdgB knockout mutant, suggesting that the RdgAB system contributes to the pathogenicity of this bacterium, probably by activating pnl expression.

  17. Prime-Boost Immunization Using a DNA Vaccine Delivered by Attenuated Salmonella enterica Serovar Typhimurium and a Killed Vaccine Completely Protects Chickens from H5N1 Highly Pathogenic Avian Influenza Virus▿

    OpenAIRE

    Pan, Zhiming; Zhang, Xiaoming; Geng, Shizhong; Fang, Qiang; You, Meng; Zhang, Lei; Jiao, Xinan; Liu, Xiufan

    2010-01-01

    H5N1 highly pathogenic avian influenza virus (HPAIV) has posed a great threat not only for the poultry industry but also for human health. However, an effective vaccine to provide a full spectrum of protection is lacking in the poultry field. In the current study, a novel prime-boost vaccination strategy against H5N1 HPAIV was developed: chickens were first orally immunized with a hemagglutinin (HA) DNA vaccine delivered by attenuated Salmonella enterica serovar Typhimurium, and boosting with...

  18. RecFOR proteins target RecA protein to a DNA gap with either DNA or RNA at the 5' terminus: implication for repair of stalled replication forks.

    Science.gov (United States)

    Morimatsu, Katsumi; Wu, Yun; Kowalczykowski, Stephen C

    2012-10-12

    The repair of single-stranded gaps in duplex DNA by homologous recombination requires the proteins of the RecF pathway. The assembly of RecA protein onto gapped DNA (gDNA) that is complexed with the single-stranded DNA-binding protein is accelerated by the RecF, RecO, and RecR (RecFOR) proteins. Here, we show the RecFOR proteins specifically target RecA protein to gDNA even in the presence of a thousand-fold excess of single-stranded DNA (ssDNA). The binding constant of RecF protein, in the presence of the RecOR proteins, to the junction of ssDNA and dsDNA within a gap is 1-2 nm, suggesting that a few RecF molecules in the cell are sufficient to recognize gDNA. We also found that the nucleation of a RecA filament on gDNA in the presence of the RecFOR proteins occurs at a faster rate than filament elongation, resulting in a RecA nucleoprotein filament on ssDNA for 1000-2000 nucleotides downstream (5' → 3') of the junction with duplex DNA. Thus, RecA loading by RecFOR is localized to a region close to a junction. RecFOR proteins also recognize RNA at the 5'-end of an RNA-DNA junction within an ssDNA gap, which is compatible with their role in the repair of lagging strand gaps at stalled replication forks. PMID:22902627

  19. High in vitro activity of the novel spiropyrimidinetrione AZD0914, a DNA gyrase inhibitor, against multidrug-resistant Neisseria gonorrhoeae isolates suggests a new effective option for oral treatment of gonorrhea.

    Science.gov (United States)

    Jacobsson, Susanne; Golparian, Daniel; Alm, Richard A; Huband, Michael; Mueller, John; Jensen, Jorgen Skov; Ohnishi, Makoto; Unemo, Magnus

    2014-09-01

    We evaluated the activity of the novel spiropyrimidinetrione AZD0914 (DNA gyrase inhibitor) against clinical gonococcal isolates and international reference strains (n=250), including strains with diverse multidrug resistance and extensive drug resistance. The AZD0914 MICs were substantially lower than those of most other currently or previously recommended antimicrobials. AZD0914 should be further evaluated, including in vitro selection, in vivo emergence and mechanisms of resistance, pharmacokinetics/pharmacodynamics in humans, optimal dosing, and performance, in appropriate randomized and controlled clinical trials. PMID:24982070

  20. Reduced repair capacity of a DNA clustered damage site comprised of 8-oxo-7,8-dihydro-2′-deoxyguanosine and 2-deoxyribonolactone results in an increased mutagenic potential of these lesions

    International Nuclear Information System (INIS)

    Highlights: • A dL lesion is not repaired as effectively as an AP site. • The repair of a cluster with dL and 8-oxodGuo lesions is compromised. • Delayed repair of the cluster leads to an increase in mutation frequency. - Abstract: A signature of ionizing radiation is the induction of DNA clustered damaged sites. Non-double strand break (DSB) clustered damage has been shown to compromise the base excision repair pathway, extending the lifetimes of the lesions within the cluster, compared to isolated lesions. This increases the likelihood the lesions persist to replication and thus increasing the mutagenic potential of the lesions within the cluster. Lesions formed by ionizing radiation include 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 2-deoxyribonolactone (dL). dL poses an additional challenge to the cell as it is not repaired by the short-patch base excision repair pathway. Here we show recalcitrant dL repair is reflected in mutations observed when DNA containing it and a proximal 8-oxodGuo is replicated in Escherichia coli. 8-oxodGuo in close proximity to dL on the opposing DNA strand results in an enhanced frequency of mutation of the lesions within the cluster and a 20 base sequence flanking the clustered damage site in an E. coli based plasmid assay. In vitro repair of a dL lesion is reduced when compared to the repair of an abasic (AP) site and a tetrahydrofuran (THF), and this is due mainly to a reduction in the activity of polymerase β, leading to retarded FEN1 and ligase 1 activities. This study has given insights in to the biological effects of clusters containing dL

  1. Tlys, a Newly Identified Sulfolobus Spindle-Shaped Virus 1 Transcript Expressed in the Lysogenic State, Encodes a DNA-Binding Protein Interacting at the Promoters of the Early Genes

    OpenAIRE

    Fusco, Salvatore; She, Qunxin; Bartolucci, Simonetta; Contursi, Patrizia

    2013-01-01

    While studying the gene expression of the Sulfolobus spindle-shaped virus 1 (SSV1) in Sulfolobus solfataricus lysogenic cells, a novel viral transcript (Tlys) was identified. Transcriptional analysis revealed that Tlys is expressed only in the absence of UV irradiation and is downregulated during the growth of the lysogenic host. The correponding gene f55 lies between two transcriptional units (T6 and Tind) that are upregulated upon UV irradiation. The open reading frame f55 encodes a 6.3-kDa...

  2. T(lys), a newly identified Sulfolobus spindle-shaped virus 1 transcript expressed in the lysogenic state, encodes a DNA-binding protein interacting at the promoters of the early genes.

    Science.gov (United States)

    Fusco, Salvatore; She, Qunxin; Bartolucci, Simonetta; Contursi, Patrizia

    2013-05-01

    While studying the gene expression of the Sulfolobus spindle-shaped virus 1 (SSV1) in Sulfolobus solfataricus lysogenic cells, a novel viral transcript (T(lys)) was identified. Transcriptional analysis revealed that T(lys) is expressed only in the absence of UV irradiation and is downregulated during the growth of the lysogenic host. The correponding gene f55 lies between two transcriptional units (T6 and T(ind)) that are upregulated upon UV irradiation. The open reading frame f55 encodes a 6.3-kDa protein which shows sequence identity with negative regulators that fold into the ribbon-helix-helix DNA-binding motif. DNA-binding assays demonstrated that the recombinant F55, purified from Escherichia coli, is indeed a putative transcription factor able to recognize site specifically target sequences in the promoters of the early induced T5, T6, and T(ind) transcripts, as well as of its own promoter. Binding sites of F55 are included within a tandem-repeated sequence overlapping the transcription start sites and/or the B recognition element of the pertinent genes. The strongest binding was observed with the promoters of T5 and T6, and an apparent cooperativity in binding was observed with the T(ind) promoter. Taking together the transcriptional analysis data and the biochemical evidences, we surmise that the protein F55 is involved in the regulation of the lysogenic state of SSV1. PMID:23514883

  3. An Improved Quantitative Real-Time PCR Assay for the Enumeration of Heterosigma akashiwo (Raphidophyceae) Cysts Using a DNA Debris Removal Method and a Cyst-Based Standard Curve.

    Science.gov (United States)

    Kim, Joo-Hwan; Kim, Jin Ho; Wang, Pengbin; Park, Bum Soo; Han, Myung-Soo

    2016-01-01

    The identification and quantification of Heterosigma akashiwo cysts in sediments by light microscopy can be difficult due to the small size and morphology of the cysts, which are often indistinguishable from those of other types of algae. Quantitative real-time PCR (qPCR) based assays represent a potentially efficient method for quantifying the abundance of H. akashiwo cysts, although standard curves must be based on cyst DNA rather than on vegetative cell DNA due to differences in gene copy number and DNA extraction yield between these two cell types. Furthermore, qPCR on sediment samples can be complicated by the presence of extracellular DNA debris. To solve these problems, we constructed a cyst-based standard curve and developed a simple method for removing DNA debris from sediment samples. This cyst-based standard curve was compared with a standard curve based on vegetative cells, as vegetative cells may have twice the gene copy number of cysts. To remove DNA debris from the sediment, we developed a simple method involving dilution with distilled water and heating at 75°C. A total of 18 sediment samples were used to evaluate this method. Cyst abundance determined using the qPCR assay without DNA debris removal yielded results up to 51-fold greater than with direct counting. By contrast, a highly significant correlation was observed between cyst abundance determined by direct counting and the qPCR assay in conjunction with DNA debris removal (r2 = 0.72, slope = 1.07, p cysts in sediment samples.

  4. Marked dependence on carrier-ligand bulk but not on carrier-ligand chirality of the duplex versus single-strand forms of a DNA oligonucleotide with a series of G-Pt(II)-G intrastrand cross-links modeling cisplatin-DNA adducts.

    Science.gov (United States)

    Beljanski, Vladimir; Villanueva, Julie M; Doetsch, Paul W; Natile, Giovanni; Marzilli, Luigi G

    2005-11-16

    The N7-Pt-N7 adjacent G,G intrastrand DNA cross-link responsible for cisplatin anticancer activity is dynamic, promotes local "melting" in long DNA, and converts many oligomer duplexes to single strands. For 5'-d(A1T2G3G4G5T6A7C8C9C10A11T12)-3' (G3), treatment of the (G3)2 duplex with five pairs of [LPt(H2O)2]2+ enantiomers (L = an asymmetric diamine) formed mixtures of LPt-G3 products (1 Pt per strand) cross-linked at G3,G4 or at G4,G5 in all cases. L chirality exerted little influence. For primary diamines L with bulk on chelate ring carbons (e.g., 1,2-diaminocyclohexane), the duplex was converted completely into single strands (G3,G4 coils and G4,G5 hairpins), exactly mirroring results for cisplatin, which lacks bulk. In sharp contrast, for secondary diamines L with bulk on chelate ring nitrogens (e.g., 2,2'-bipiperidine, Bip), unexpectedly stable duplexes having two platinated strands (even a unique G3,G4/G4,G5 heteroduplex) were formed. After enzymatic digestion of BipPt-G3 duplexes, the conformation of the relatively nondynamic G,G units was shown to be head-to-head (HH) by HPLC/mass spectrometric characterization. Because the HH conformation dominates at the G,G lesion in duplex DNA and in the BipPt-G3 duplexes, the stabilization of the duplex form only when the L nitrogen adducts possess bulk suggests that H-bonding interactions of the Pt-NH groups with the flanking DNA lead to local melting and to destabilization of oligomer duplexes. The marked dependence of adduct properties on L bulk and the minimal dependence on L chirality underscore the need for future exploration of the roles of the L periphery in affecting anticancer activity. PMID:16277526

  5. A Polydiacetylene Vesicle-based Colorimetric Detection of Campylobacter jejuni Using a DNA Aptamer as Recognition Element%聚丁二炔纳米囊泡生物传感器比色检测空肠弯曲菌的初步研究

    Institute of Scientific and Technical Information of China (English)

    董建涛; 欧阳后先; 徐柯; 吴文鹤; 吕建新

    2012-01-01

    A polydiacetylene vesicle-based biosensor for rapid colorimetric detection of Campylobacter jejuni was developed in this work. Aptamers coupled to the surface of polydiacetylene nanovesicle by chemical reaction in order to achieve vesicle functionalization. Molecular recognition between Campylobacter jejuni and aptamers on the surface of the vesicle lead to absorption spectrum transition of polydiacetylene by which quantitative detection of Campylobacter jejuni could be realized. This method can detect Campylobacter jejuni in 30 mins with high specificity and simple operation.%本实验构建一种可以快速比色检测空肠弯曲菌的聚丁二炔纳米囊泡生物传感器.适配体通过化学反应耦合到聚丁二炔纳米囊泡表面,完成对囊泡的功能化.适配体与空肠弯曲菌特异识别,引起纳米囊泡溶液吸收光谱的改变,通过比色响应值衡量检测体系中有无空肠弯曲菌.该方法特异性好,与PCR对比实验相符;操作简便,检测时间短,仅需30 min.

  6. Evidence of a genetic instability induced by the incorporation of a DNA precursor marked with tritium; Mise en evidence d'une instabilite genetique induite par l'incorporation d'un precurseur de L'ADN marque au tritium

    Energy Technology Data Exchange (ETDEWEB)

    Saintigny, Y.; Laurent, D.; Lahayel, J.B. [CEA Fontenay-aux-Roses, IRCM-LRTS, U967 - CEA/INSERM/Universites Paris 7 and Paris-11, 92 (France); Roche, St.; Meynard, D.; Lopez, B.S. [CEA Fontenay-aux-Roses, LMR - UMR 217 - CEA/CNRS, Institut de Radiobiologie Cellulaire et moleculaire, Direction des Sciences du Vivant, 92 (France)

    2009-07-01

    The authors report a molecular geno-toxicology investigation which allowed molecular events induced par intracellular incorporation of tritium to be studied, and the genetic instability resulting from a chronic exposure even at low dose to be analysed. For this purpose, they developed cell models (hamster tumorous cells and human fibroblasts) in which they know how to incorporate given quantities of marked nucleotides in the DNA. They show that the incorporation of tritium, even with doses which are said to be non toxic, causes a prolonged exposure of the cell to a genotoxic stress, and maybe a genetic instability due to a too great number of recombination events

  7. MELAS 和 MERRF 综合征相关 mtDNA 突变位点检测集成芯片的建立%Development of a DNA biochip for detection of known mtDNA mutations associated with MELAS and MERRF Syndromes

    Institute of Scientific and Technical Information of China (English)

    陈刚; 赵建龙; 张学军; 李伟; 杜卫东; 曹慧敏; 汤华阳; 唐先发; 孙中武; 赵辉; 金庆辉

    2008-01-01

    文中建立了一种新型的寡核苷酸芯片,用于线粒体脑肌病伴高乳酸血症和卒中样发作(Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes,MELAS)和肌阵挛性癫痫伴发不规整红纤维(Myoclonic epilepsy with ragged red fibers,MERRF)线粒体DNA所有已知突变位点的集成检测.将31对allele位点特异性的寡核苷酸探针包被在醛基修饰的载玻片表面,以多重不对称PCR方法制备Cy5荧光标记靶基因.利用此芯片对5例MELAS患者,5例MERRF患者及20例健康对照进行筛查,结果发现,MELAS患者均为MT-T1基因A3243G突变;在MERRF患者组,MT-TK基因A8344G突变4例,T8356C突变1例;健康对照组均未发现31种相关mtDNA突变.芯片检测与DNA测序结果完全一致.结果表明,这种寡核苷酸芯片可以对MELAS和MERRF综合征已知突变位点进行同步快速检测,具有较高的灵敏度和特异性.这一模式的基因芯片经过适当改装后也可用于其他人类线粒体疾病的基因诊断.

  8. Immunological competence of a DNA vaccine containing human papillomavirus type 16 E2 combined with IL-12%HPV16 E2与IL-12联合基因疫苗的免疫效果初步研究

    Institute of Scientific and Technical Information of China (English)

    唐双阳; 李乐; 余敏君; 粟盛梅; 蔡恒玲; 周良; 万艳平

    2012-01-01

    目的 将HPV16 E2与IL-12真核表达载体联合免疫小鼠,检测小鼠产生的特异性体液免疫和细胞免疫应答水平,为HPV16诱发的宫颈癌治疗性核酸疫苗的研制奠定实验基础. 方法 40只BALB/c小鼠随机分成PBS组、pcD-NA3.1(+)空质粒组、pcDNA3.1(+)/IL-12组、pcDNA3.1(+)/HPV16 E2组以及pcDNA3.1(+)/HPV16 E2+pcD-NA3.1(+)/IL-12联合组,每组8只,共免疫4次,每2周1次.于免疫前1d及第8周采血,分离血清,-20℃保存;颈椎脱臼处死小鼠,无菌取脾脏,制备脾细胞悬液.ELISA法测定小鼠血清HPV16 E2 IgG抗体水平及脾细胞培养上清IFN-γ、IL-4水平;MTT比色法检测脾淋巴细胞的增殖. 结果 免疫8周后,血清IgG抗体A450值E2组和联合组与PBS组和空质粒组比较差异有统计学意义(P<0.05);但在每个时间点,联合组与E2组比较差异无统计学意义(P>0.05).IL-12组、E2组、联合组脾淋巴细胞培养上清中的IFN γ和IL-4含量与PBS组和空质粒组比较差异有统计学意义(P<0.05);联合组与E2组比较IFN-γ含量差异有统计学意义(P<0.05),两组的IL-4含量差异无统计学意义(P>0.05).IL-12组、E2组、联合组脾淋巴细胞SI值与PBS组和空质粒组比较差异有统计学意义(P<0.05);联合组与E2组比较差异有统计学意义(P<0.05). 结论 HPV16 E2单基因疫苗及其与IL-12联合基因疫苗能刺激小鼠产生特异性抗体,脾淋巴细胞培养上清IFN-γ含量升高,且联合基因疫苗优于E2单基因疫苗.%Objective In order to provide an experimental basis for studying remedial vaccines to treat infection with human papillomavirus type 16 (HPV16), the immunological competence of BALB/c mice was analyzed after they were inoculated with the eukaryotic expression vectors HPV16 E2 and IL-12. Methods A total of 40 BALB/c mice was randomly divided into 5 groups, the PBS Group, pcDNA3. 1 ( + ) Group, pcDNA3. 1( + )/IL-12 Group, pcDNA3. 1( + )/ HPV16 E2 Group(the single DNA vaccine group), and the pcDNA3. 1( + )/HPV16 E2 + pcDNA3. 1( + )/IL-12 Group (the combined DNA vaccine group). The mice were injected every four times every two weeks. The sera of mice were collected and stored at — 20 ℃ the day before every immunization or the day before sacrifice. Spleen cells were cultured from the spleens of the mice. ELISA was used to measure the serum-specific IgG antibody and IFN-γ or IL-4 levels in the supernatant from cultured spleen cells. The proliferative response of the spleen cells was measured with an MTT assay. Results The A45O value for IgG in the final immunized sera from the combined DNA vaccine group and the single DNA vaccine group was significantly higher than that from the pcDNA3. 1( + ) Group and PBS Group (P0. 05) in the A450 value for the combined DNA vaccine group and the single DNA vaccine group at any of the vaccination times. IFN-γ and IL-4 levels in the supernatant of he spleen lymphocytes from the pcDNA3. 1( + )/IL-12 Group, the combined DNA vaccine group, and the single DNA vaccine group were significantly higher than those from the pcDNA3. 1( + ) Group and PBS Group (P0. 05). The stimulation index (SI) of the spleen cells from the pcDNA3. 1( + )/IL-12 Group, the combined DNA vaccine group, and the single DNA vaccine group was higher than that from the pcDNA3. 1( + ) Group and PBS Group (P0. 05). Conclusion The HPV16 E2 vaccine may stimulate the level of specific antibodies and increase the level of IFN-γ. A combination DNA vaccine may have a more potent immune effect.

  9. 嗜肺军团菌15种血清型基因芯片检测方法的建立%Development of a DNA Microarray for Detection and Identification of 15 Distinct O Antigen Forms of Legionella Pneumophila

    Institute of Scientific and Technical Information of China (English)

    吴冬雪; 王乃福; 黄晨; 高旗利; 关淳

    2014-01-01

    To establish a rapid, accurate detection method for 15 distinct O antigen forms of Legionella pneumophila using DNA microarray combined with multiplex PCR. The special genes of these 15 distinct O antigen forms of L. Pneumophila were as target genes for multiplex PCR respectively, then primers and captured oligonucleotide probes were hybridizes with DNA microarray, which contained specific probes of Legionella pneumophila. Scanner was used to determinant the types of bacterium. The DNA microarray assay can detect 15 distinct O antigen forms of L. Pneumophila specially. The sensitivity of the DNA microarray was 10 ng. The detection method of 15 distinct O antigen forms of Legionella pneumophila by DNA microarray was established. The detection method has better specificity, sensitivity and repeatability.%建立一种多重PCR技术结合基因芯片检测方法,实现嗜肺军团菌15种血清型的快速、准确检测。根据嗜肺军团菌15种血清型的O抗原特异性基因设计并筛选合适的引物和探针,进行多重PCR扩增,制备寡核苷酸芯片。将多重PCR扩增产物与带有特异探针的芯片杂交。用扫描仪扫描,判定嗜肺军团菌的血清型。该基因芯片可特异性的检测嗜肺军团菌的15种血清型,具有良好的特异性,芯片纯菌DNA检测灵敏度为10 ng。所建立的嗜肺军团菌15种血清型基因芯片检测方法特异性好,灵敏度高,具有较好的实用性。

  10. Comparative study of ancient DNA extraction methods for archaeological plant remains

    OpenAIRE

    Moore, Jason Nicholas

    2011-01-01

    Despite the potential for plant ancient DNA (aDNA) to address important archaeological questions, there are significantly fewer studies of plant aDNA compared to human and animal aDNA, partially due to a lack of research on DNA extraction methods for ancient plant remains. The current study uses heat to degrade modern corn, pea, and squash seeds to simulate degraded DNA associated with archaeological macro-botanical remains. I then compare DNA recovery efficiencies of three common DNA extract...

  11. High-throughput sequencing of ancient plant and mammal DNA preserved in herbivore middens

    DEFF Research Database (Denmark)

    Murray, D.C.; Pearson, S.G.; Fullagar, R.;

    2012-01-01

    of information regarding past environments and the nature of ecological fluctuations within arid zones. The application of ancient DNA (aDNA) techniques to hot, arid zone middens remains unexplored. This paper attempts to retrieve and characterise aDNA from four Southern Hemisphere fossil middens; three located...

  12. Base excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase.

    NARCIS (Netherlands)

    B.P. Engelward (Bevin); G. Weeda (Geert); M.D. Wyatt; J.L.M. Broekhof (Jose'); J. de Wit (Jan); I. Donker (Ingrid); J.M. Allan (James); B. Gold (Bert); J.H.J. Hoeijmakers (Jan); L.D. Samson (Leona)

    1997-01-01

    textabstract3-methyladenine (3MeA) DNA glycosylases remove 3MeAs from alkylated DNA to initiate the base excision repair pathway. Here we report the generation of mice deficient in the 3MeA DNA glycosylase encoded by the Aag (Mpg) gene. Alkyladenine DNA glycosylase turns out to be the major DNA glyc

  13. Microarrays/DNA Chips for the Detection of Waterborne Pathogens.

    Science.gov (United States)

    Vale, Filipa F

    2016-01-01

    DNA microarrays are useful for the simultaneous detection of microorganisms in water samples. Specific probes targeting waterborne pathogens are selected with bioinformatics tools, synthesized and spotted onto a DNA array. Here, the construction of a DNA chip for waterborne pathogen detection is described, including the processes of probe in silico selection, synthesis, validation, and data analysis. PMID:27460375

  14. Effect of X-ray irradiation on ancient DNA in sub-fossil bones – Guidelines for safe X-ray imaging

    Science.gov (United States)

    Immel, Alexander; Le Cabec, Adeline; Bonazzi, Marion; Herbig, Alexander; Temming, Heiko; Schuenemann, Verena J.; Bos, Kirsten I.; Langbein, Frauke; Harvati, Katerina; Bridault, Anne; Pion, Gilbert; Julien, Marie-Anne; Krotova, Oleksandra; Conard, Nicholas J.; Münzel, Susanne C.; Drucker, Dorothée G.; Viola, Bence; Hublin, Jean-Jacques; Tafforeau, Paul; Krause, Johannes

    2016-01-01

    Sub-fossilised remains may still contain highly degraded ancient DNA (aDNA) useful for palaeogenetic investigations. Whether X-ray computed [micro-] tomography ([μ]CT) imaging of these fossils may further damage aDNA remains debated. Although the effect of X-ray on DNA in living organisms is well documented, its impact on aDNA molecules is unexplored. Here we investigate the effects of synchrotron X-ray irradiation on aDNA from Pleistocene bones. A clear correlation appears between decreasing aDNA quantities and accumulating X-ray dose-levels above 2000 Gray (Gy). We further find that strong X-ray irradiation reduces the amount of nucleotide misincorporations at the aDNA molecule ends. No representative effect can be detected for doses below 200 Gy. Dosimetry shows that conventional μCT usually does not reach the risky dose level, while classical synchrotron imaging can degrade aDNA significantly. Optimised synchrotron protocols and simple rules introduced here are sufficient to ensure that fossils can be scanned without impairing future aDNA studies. PMID:27615365

  15. Repair and genetic consequences of DNA double strand breaks during animal development

    NARCIS (Netherlands)

    Lemmens, Bennie Benjamin Lodewijk Gerardus

    2014-01-01

    The genetic code of life is stored in DNA molecules that consist of two parallel strands of coupled nucleotides that form a DNA double helix. One of the most deleterious forms of DNA damage is a DNA double-strand break (DSB) in which both strands of the helix are broken. When not repaired adequately

  16. Novel high-resolution characterization of ancient DNA reveals C > U-type base modification events as the sole cause of post mortem miscoding lesions

    Science.gov (United States)

    Brotherton, Paul; Endicott, Phillip; Sanchez, Juan J.; Beaumont, Mark; Barnett, Ross; Austin, Jeremy; Cooper, Alan

    2007-01-01

    Ancient DNA (aDNA) research has long depended on the power of PCR to amplify trace amounts of surviving genetic material from preserved specimens. While PCR permits specific loci to be targeted and amplified, in many ways it can be intrinsically unsuited to damaged and degraded aDNA templates. PCR amplification of aDNA can produce highly-skewed distributions with significant contributions from miscoding lesion damage and non-authentic sequence artefacts. As traditional PCR-based approaches have been unable to fully resolve the molecular nature of aDNA damage over many years, we have developed a novel single primer extension (SPEX)-based approach to generate more accurate sequence information. SPEX targets selected template strands at defined loci and can generate a quantifiable redundancy of coverage; providing new insights into the molecular nature of aDNA damage and fragmentation. SPEX sequence data reveals inherent limitations in both traditional and metagenomic PCR-based approaches to aDNA, which can make current damage analyses and correct genotyping of ancient specimens problematic. In contrast to previous aDNA studies, SPEX provides strong quantitative evidence that C > U-type base modifications are the sole cause of authentic endogenous damage-derived miscoding lesions. This new approach could allow ancient specimens to be genotyped with unprecedented accuracy. PMID:17715147

  17. Threading Immobilized DNA Molecules through a Solid-State Nanopore at >100 μs per Base Rate

    OpenAIRE

    Hyun, Changbae; Kaur, Harpreet; Rollings, Ryan; Xiao, Min; Li, Jiali

    2013-01-01

    In pursuit of developing solid-state nanopore based DNA sequencing technology, we have designed and constructed an apparatus that can place a DNA tethered probe tip near a solid-state nanopore, control the DNA moving speed, and measure the ionic current change when a DNA is captured and released from a nanopore. The probe tip’s position is sensed and controlled by a tuning fork based feedback force sensor and a nanopositioning system. Using this newly constructed apparatus, a DNA strand movin...

  18. Ancestral Polymorphisms and Sex-Biased Migration Shaped the Demographic History of Brown Bears and Polar Bears

    OpenAIRE

    Shigeki Nakagome; Shuhei Mano; Masami Hasegawa

    2013-01-01

    Recent studies have reported discordant gene trees in the evolution of brown bears and polar bears. Genealogical histories are different among independent nuclear loci and between biparentally inherited autosomal DNA (aDNA) and matrilineal mitochondrial DNA (mtDNA). Based on multi-locus genomic sequences from aDNA and mtDNA, we inferred the population demography of brown and polar bears and found that brown bears have 6 times (aDNA) or more than 14 times (mtDNA) larger population sizes than p...

  19. More on contamination: the use of asymmetric molecular behavior to identify authentic ancient human DNA

    DEFF Research Database (Denmark)

    Malmström, Helena; Svensson, Emma M; Gilbert, M Thomas P;

    2007-01-01

    the reliability of one of the proposed criteria, that of appropriate molecular behavior. Using real-time polymerase chain reaction (PCR) and pyrosequencing, we have quantified the relative levels of authentic aDNA and contaminant human DNA sequences recovered from archaeological dog and cattle remains. In doing....... Furthermore, we find that there is a substantial increase in the relative proportions of authentic DNA to contaminant DNA as the PCR target fragment size is decreased. We therefore conclude that the degradation pattern in aDNA provides a quantifiable difference between authentic aDNA and modern contamination...

  20. Evaluation of smoking genotoxicity in Turkish young adults

    Directory of Open Access Journals (Sweden)

    Ayse G Zamani

    2011-01-01

    Conclusions: Our data showed that cigarette smoke is a DNA damage causitive agent on exfoliative buccal mucosa and urothelial cells and peripheric blood lymphocytes of young smokers, but it has most destructive effect on urothelial cells.

  1. Inability of ‘Whole Genome Amplification’ to Improve Success Rates for the Biomolecular Detection of Tuberculosis in Archaeological Samples

    Science.gov (United States)

    Forst, Jannine; Brown, Terence A.

    2016-01-01

    We assessed the ability of whole genome amplification (WGA) to improve the efficiency of downstream polymerase chain reactions (PCRs) directed at ancient DNA (aDNA) of members of the Mycobacterium tuberculosis complex (MTBC). Using extracts from a variety of bones and a tooth from human skeletons with or without lesions indicative of tuberculosis, from multiple time periods, we obtained inconsistent results. We conclude that WGA does not provide any advantage in studies of MTBC aDNA. The sporadic nature of our results are probably due to the fact that WGA is itself a PCR-based procedure which, although designed to deal with fragmented DNA, might be inefficient with the low concentration of templates in an aDNA extract. As such, WGA is subject to similar, if not the same, restrictions as PCR when applied to aDNA. PMID:27654468

  2. ATM modulates the loading of recombination proteins onto a chromosomal translocation breakpoint hotspot

    NARCIS (Netherlands)

    J. Sun (Jiying); Y. Oma (Yukako); M. Harata (Masahiko); K. Kono (Kazuteru); H. Shima (Hiroki); A. Kinomura (Aiko); T. Ikura (Tsuyoshi); H. Suzuki (Hidekazu); S. Mizutani (Shuki); R. Kanaar (Roland); S. Tashiro (Satoshi)

    2010-01-01

    textabstractChromosome translocations induced by DNA damaging agents, such as ionizing radiation and certain chemotherapies, alter genetic information resulting in malignant transformation. Abrogation or loss of the ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, incre

  3. Detection of Multiple Waterborne Pathogens Using Microsequencing Arrays

    Science.gov (United States)

    Aims: A microarray was developed to simultaneously detect Cryptosporidium parvum, Cryptosporidium hominis, Enterococcus faecium, Bacillus anthracis and Francisella tularensis in water. Methods and Results: A DNA microarray was designed to contain probes that specifically dete...

  4. A reversible metal ion fueled DNA three-way junction molecular device for ``turn-on and -off'' fluorescence detection of mercury ions (II) and biothiols respectively with high selectivity and sensitivity

    Science.gov (United States)

    Ma, Long; Wu, Guanrong; Li, Yufeng; Qin, Ping; Meng, Lingpei; Liu, Haiyan; Li, Yuyin; Diao, Aipo

    2015-10-01

    We constructed a reversible molecular device in the nanoscale based on a DNA three-way junction (3WJ) fueled by Hg2+ binding and sequestration. It is highly responsive to external stimuli, which brings about optically detectable global structural changes. Such a DNA device can serve as a novel ``turn-on and -off'' fluorescent sensor for Hg2+ and biothiol detection with high selectivity and sensitivity.We constructed a reversible molecular device in the nanoscale based on a DNA three-way junction (3WJ) fueled by Hg2+ binding and sequestration. It is highly responsive to external stimuli, which brings about optically detectable global structural changes. Such a DNA device can serve as a novel ``turn-on and -off'' fluorescent sensor for Hg2+ and biothiol detection with high selectivity and sensitivity. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr04688b

  5. Cell encoding recombinant human erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    Beck, A.K.; Withy, R.M.; Zabrecky, J.R.; Masiello, N.C.

    1990-09-04

    This patent describes a C127 cell transformed with a recombinant DNA vector. It comprises: a DNA sequence encoding human erythropoietin, the transformed cell being capable of producing N-linked and O-linked glycosylated human erythropoietin.

  6. Sequence Classification: 894068 [

    Lifescience Database Archive (English)

    Full Text Available ity control; catalyzes polyadenylation of unmodified tRNAs, and snoRNA and rRNA precursors; disputed role as a DNA polymerase; Pap2p || http://www.ncbi.nlm.nih.gov/protein/6324457 ...

  7. Evaluation of cellular responses for a chimeric HBsAg-HCV core DNA vaccine in BALB/c mice

    Directory of Open Access Journals (Sweden)

    Maryam Yazdanian

    2015-01-01

    Conclusion: Fusion of HBsAg to HCVcp in the context of a DNA vaccine modality could augment Th1-oriented cellular and CTL responses toward a protective epitope, comparable to that of HCVcp (subunit HCV vaccine immunization.

  8. Peptide nucleic acid: a new artificial biomacromolecular with great potential applications in molecular biology and biomedicine

    Institute of Scientific and Technical Information of China (English)

    WANG Jin-ke; LU Zu-hong; HE Nong-yue

    2001-01-01

    @@ Peptide nucleic acid (PNA) is a DNA mimic that was originally developed by Peter E Nielsen in 1991 as a reagent for sequence-specific recognition of double stranded (ds) DNA by a conventional triple helix type principle.

  9. The RecQ DNA helicases: Jacks-of-all-trades or master-tradesmen?

    Institute of Scientific and Technical Information of China (English)

    Neil Hunter

    2008-01-01

    @@ Homologous recombination occurs when a damaged chromosome uses an intact homologous chromosome as a template for its repair. The main steps of recombination are most readily illustrated for the repair of a DNA double-strand-break (DSB).

  10. Method of inactivation of an end product of energy metabolism in Zymomonas mobilis

    Science.gov (United States)

    Zhang, Min; Chou, Yat-Chen

    2008-05-20

    The present invention briefly provides a method of site-specific insertion in Zymomonas, comprising, providing a Zymomonas gene fragment, interrupting a DNA sequence the fragment, and transforming the Zymomonas through homologous recombination with the interrupted fragment.

  11. Optical dating of perennially frozen deposits associated with preserved ancient plant and animal DNA in north-central Siberia

    DEFF Research Database (Denmark)

    Arnold, L.J.; Roberts, R.G.; Macphee, R.D.E.;

    2008-01-01

    We present chronological constraints on a suite of permanently frozen fluvial deposits which contain ancient DNA (aDNA) from the Taimyr Peninsula of north-central Siberia. The luminescence phenomenology of these samples is first discussed, focusing on the optically stimulated luminescence (OSL) d...... of providing a reliable chronometric framework for sedimentary aDNA records in permafrost environments. (C) 2007 Elsevier Ltd. All rights reserved Udgivelsesdato: 2008...

  12. Analysis of ancient DNA from coprolites: a perspective with random amplified polymorphic DNA-polymerase chain reaction approach

    OpenAIRE

    Iñiguez Alena M; Araújo Adauto; Ferreira Luiz Fernando; Vicente Ana Carolina P

    2003-01-01

    The aim of this work was to determine approaches that would improve the quality of ancient DNA (aDNA) present in coprolites to enhance the possibility of success in retrieving specific sequence targets. We worked with coprolites from South American archaeological sites in Brazil and Chile dating up to 7,000 years ago. Using established protocols for aDNA extraction we obtained samples showing high degradation as usually happens with this kind of material. The reconstructive polymerization pre...

  13. DNA Microarray Assessment of Putative Borrelia burgdorferi Lipoprotein Genes

    OpenAIRE

    Liang, Fang Ting; Nelson, F. Kenneth; Fikrig, Erol

    2002-01-01

    A DNA microarray containing fragments of 137 Borrelia burgdorferi B31 putative lipoprotein genes was used to examine Lyme disease spirochetes. DNA from B. burgdorferi sensu stricto B31, 297, and N40; Borrelia garinii IP90; and Borrelia afzelii P/Gau was fluorescently labeled and hybridized to the microarray, demonstrating the degree to which the individual putative lipoprotein genes were conserved among the genospecies. These data show that a DNA microarray can globally examine the genes enco...

  14. Immune response to a hepatitis B DNA vaccine in Aotus monkeys: a comparison of vaccine formulation, route, and method of administration.

    OpenAIRE

    Gramzinski, R. A.; Millan, C. L.; Obaldia, N.; Hoffman, S L; Davis, H L

    1998-01-01

    BACKGROUND: Attempts to optimize DNA vaccines in mice include using different routes of administration and different formulations. It may be more relevant to human use to carry such studies out in nonhuman primates. Here we compare different approaches to delivery of a DNA vaccine against the hepatitis B virus (HBV) in Aotus monkeys. MATERIALS AND METHODS: Thirty-two adult Aotus l. lemurinus monkeys divided into 8 groups of four were immunized with 400 microg of a DNA vaccine which encoded he...

  15. Alternative lengthening of telomeres in normal mammalian somatic cells

    OpenAIRE

    Neumann, Axel A.; Watson, Catherine M.; Noble, Jane R.; Hilda A Pickett; Tam, Patrick P.L.; Reddel, Roger R

    2013-01-01

    Alternative lengthening of telomeres (ALT), a mechanism involving the replication of new telomeric DNA from a DNA template, is used by some cancer cells to lengthen their telomeres. Reddel and colleagues now show that ALT activity exists in normal somatic tissues as well. A telomere with a DNA tag is found to be intertelomerically copied in normal somatic cells but not germline cells, providing important implications for understanding telomere maintenance and its evolution.

  16. ycf1, the most promising plastid DNA barcode of land plants

    OpenAIRE

    Wenpan Dong; Chao Xu; Changhao Li; Jiahui Sun; Yunjuan Zuo; Shuo Shi; Tao Cheng; Junjie Guo; Shiliang Zhou

    2015-01-01

    A DNA barcode is a DNA fragment used to identify species. For land plants, DNA fragments of plastid genome could be the primary consideration. Unfortunately, most of the plastid candidate barcodes lack species-level resolution. The identification of DNA barcodes of high resolution at species level is critical to the success of DNA barcoding in plants. We searched the available plastid genomes for the most variable regions and tested the best candidates using both a large number of tree specie...

  17. Population genetic studies in Northeastern Atlantic minke whales

    OpenAIRE

    Berube, M.; Skaug, Hans Julius; Andersen, Liselotte W.; Haug, Tore; Øien, Nils

    2007-01-01

    Minke whales are the most abundance species of baleen whales in the North Atlantic. As part of current management of minke whales in Norwegian and adjacent waters, a DNA-register have been established. The register ensures that samples are taken of each animal caught under the Norwegian catch quota, and that a DNA-profile is established and stored in a database from each individual whale. Previous studies have indicated that genetic population sub-structure exists within the North Atlantic, b...

  18. Conformational Transitions and Stop-and-Go Nanopore Transport of Single Stranded DNA on Charged Graphene

    OpenAIRE

    Shankla, Manish; Aksimentiev, Aleksei

    2014-01-01

    Control over interactions with biomolecules holds the key to applications of graphene in biotechnology. One such application is nanopore sequencing, where a DNA molecule is electrophoretically driven through a graphene nanopore. Here, we investigate how interactions of single-stranded DNA and a graphene membrane can be controlled by electrically biasing the membrane. The results of our molecular dynamics simulations suggest that electric charge on graphene can force a DNA homopolymer to adopt...

  19. Toward high-resolution population genomics using archaeological samples

    Science.gov (United States)

    Morozova, Irina; Flegontov, Pavel; Mikheyev, Alexander S.; Bruskin, Sergey; Asgharian, Hosseinali; Ponomarenko, Petr; Klyuchnikov, Vladimir; ArunKumar, GaneshPrasad; Prokhortchouk, Egor; Gankin, Yuriy; Rogaev, Evgeny; Nikolsky, Yuri; Baranova, Ancha; Elhaik, Eran; Tatarinova, Tatiana V.

    2016-01-01

    The term ‘ancient DNA’ (aDNA) is coming of age, with over 1,200 hits in the PubMed database, beginning in the early 1980s with the studies of ‘molecular paleontology’. Rooted in cloning and limited sequencing of DNA from ancient remains during the pre-PCR era, the field has made incredible progress since the introduction of PCR and next-generation sequencing. Over the last decade, aDNA analysis ushered in a new era in genomics and became the method of choice for reconstructing the history of organisms, their biogeography, and migration routes, with applications in evolutionary biology, population genetics, archaeogenetics, paleo-epidemiology, and many other areas. This change was brought by development of new strategies for coping with the challenges in studying aDNA due to damage and fragmentation, scarce samples, significant historical gaps, and limited applicability of population genetics methods. In this review, we describe the state-of-the-art achievements in aDNA studies, with particular focus on human evolution and demographic history. We present the current experimental and theoretical procedures for handling and analysing highly degraded aDNA. We also review the challenges in the rapidly growing field of ancient epigenomics. Advancement of aDNA tools and methods signifies a new era in population genetics and evolutionary medicine research. PMID:27436340

  20. Toward high-resolution population genomics using archaeological samples.

    Science.gov (United States)

    Morozova, Irina; Flegontov, Pavel; Mikheyev, Alexander S; Bruskin, Sergey; Asgharian, Hosseinali; Ponomarenko, Petr; Klyuchnikov, Vladimir; ArunKumar, GaneshPrasad; Prokhortchouk, Egor; Gankin, Yuriy; Rogaev, Evgeny; Nikolsky, Yuri; Baranova, Ancha; Elhaik, Eran; Tatarinova, Tatiana V

    2016-08-01

    The term 'ancient DNA' (aDNA) is coming of age, with over 1,200 hits in the PubMed database, beginning in the early 1980s with the studies of 'molecular paleontology'. Rooted in cloning and limited sequencing of DNA from ancient remains during the pre-PCR era, the field has made incredible progress since the introduction of PCR and next-generation sequencing. Over the last decade, aDNA analysis ushered in a new era in genomics and became the method of choice for reconstructing the history of organisms, their biogeography, and migration routes, with applications in evolutionary biology, population genetics, archaeogenetics, paleo-epidemiology, and many other areas. This change was brought by development of new strategies for coping with the challenges in studying aDNA due to damage and fragmentation, scarce samples, significant historical gaps, and limited applicability of population genetics methods. In this review, we describe the state-of-the-art achievements in aDNA studies, with particular focus on human evolution and demographic history. We present the current experimental and theoretical procedures for handling and analysing highly degraded aDNA. We also review the challenges in the rapidly growing field of ancient epigenomics. Advancement of aDNA tools and methods signifies a new era in population genetics and evolutionary medicine research. PMID:27436340

  1. The first attested extraction of ancient DNA in legumes (Fabaceae

    Directory of Open Access Journals (Sweden)

    Aleksandar M. Mikić

    2015-11-01

    Full Text Available Ancient DNA (aDNA is any DNA extracted from ancient specimens, important for diverse evolutionary researches. The major obstacles in aDNA studies are mutations, contamination and fragmentation. Its studies may be crucial for crop history if integrated with human aDNA research and historical linguistics, both general and relating to agriculture. Legumes (Fabaceae are one of the richest end economically most important plant families, not only from Neolithic onwards, since they were used as food by Neanderthals and Paleolithic modern man. The idea of extracting and analysing legume aDNA was considered beneficial for both basic science and applied research, with an emphasis on genetic resources and plant breeding. The first reported successful and attested extraction of the legume aDNA was done from the sample of charred seeds of pea (Pisum sativum and bitter vetch (Vicia ervilia from Hissar, southeast Serbia, dated to 1,350 - 1,000 Before Christ. A modified version of cetyltrimethylammonium bromide (CTAB method and the commercial kit for DNA extraction QIAGEN DNAesy yielded several ng μl-1 of aDNA of both species and, after the whole genome amplification and with a fragment of nuclear ribosomal DNA gene 26S rDNA, resulted in the detection of the aDNA among the PCR products. A comparative analysis of four informative chloroplast DNA regions (trnSG, trnK, matK and rbcL among the modern wild and cultivated pea taxa demonstrated not only that the extracted aDNA was genuine, on the basis of mutation rate, but also that the ancient Hissar pea was most likely an early domesticated crop, related to the modern wild pea of a neighbouring region. It is anticipated that this premier extraction of legume aDNA may provide taxonomists with the answers to diverse questions, such as leaf development in legumes, as well as with novel data on the single steps in domesticating legume crops worldwide.

  2. The First Attested Extraction of Ancient DNA in Legumes (Fabaceae).

    Science.gov (United States)

    Mikić, Aleksandar M

    2015-01-01

    Ancient DNA (aDNA) is any DNA extracted from ancient specimens, important for diverse evolutionary researches. The major obstacles in aDNA studies are mutations, contamination and fragmentation. Its studies may be crucial for crop history if integrated with human aDNA research and historical linguistics, both general and relating to agriculture. Legumes (Fabaceae) are one of the richest end economically most important plant families, not only from Neolithic onwards, since they were used as food by Neanderthals and Paleolithic modern man. The idea of extracting and analyzing legume aDNA was considered beneficial for both basic science and applied research, with an emphasis on genetic resources and plant breeding. The first reported successful and attested extraction of the legume aDNA was done from the sample of charred seeds of pea (Pisum sativum) and bitter vetch (Vicia ervilia) from Hissar, southeast Serbia, dated to 1,350-1,000 Before Christ. A modified version of cetyltrimethylammonium bromide (CTAB) method and the commercial kit for DNA extraction QIAGEN DNAesy yielded several ng μl(-1) of aDNA of both species and, after the whole genome amplification and with a fragment of nuclear ribosomal DNA gene 26S rDNA, resulted in the detection of the aDNA among the PCR products. A comparative analysis of four informative chloroplast DNA regions (trnSG, trnK, matK, and rbcL) among the modern wild and cultivated pea taxa demonstrated not only that the extracted aDNA was genuine, on the basis of mutation rate, but also that the ancient Hissar pea was most likely an early domesticated crop, related to the modern wild pea of a neighboring region. It is anticipated that this premier extraction of legume aDNA may provide taxonomists with the answers to diverse questions, such as leaf development in legumes, as well as with novel data on the single steps in domesticating legume crops worldwide. PMID:26635833

  3. Optimization of DNA recovery and amplification from non-carbonized archaeobotanical remains.

    Directory of Open Access Journals (Sweden)

    Nathan Wales

    Full Text Available Ancient DNA (aDNA recovered from archaeobotanical remains can provide key insights into many prominent archaeological research questions, including processes of domestication, past subsistence strategies, and human interactions with the environment. However, it is often difficult to isolate aDNA from ancient plant materials, and furthermore, such DNA extracts frequently contain inhibitory substances that preclude successful PCR amplification. In the age of high-throughput sequencing, this problem is even more significant because each additional endogenous aDNA molecule improves analytical resolution. Therefore, in this paper, we compare a variety of DNA extraction techniques on primarily desiccated archaeobotanical remains and identify which method consistently yields the greatest amount of purified DNA. In addition, we test five DNA polymerases to determine how well they replicate DNA extracted from non-charred ancient plant remains. Based upon the criteria of resistance to enzymatic inhibition, behavior in quantitative real-time PCR, replication fidelity, and compatibility with aDNA damage, we conclude these polymerases have nuanced properties, requiring researchers to make educated decisions as to which one to use for a given task. The experimental findings should prove useful to the aDNA and archaeological communities by guiding future research methodologies and ensuring precious archaeobotanical remains are studied in optimal ways, and may thereby yield important new perspectives on the interactions between humans and past plant communities.

  4. Ancestral polymorphisms and sex-biased migration shaped the demographic history of brown bears and polar bears.

    Directory of Open Access Journals (Sweden)

    Shigeki Nakagome

    Full Text Available Recent studies have reported discordant gene trees in the evolution of brown bears and polar bears. Genealogical histories are different among independent nuclear loci and between biparentally inherited autosomal DNA (aDNA and matrilineal mitochondrial DNA (mtDNA. Based on multi-locus genomic sequences from aDNA and mtDNA, we inferred the population demography of brown and polar bears and found that brown bears have 6 times (aDNA or more than 14 times (mtDNA larger population sizes than polar bears and that polar bear lineage is derived from within brown bear diversity. In brown bears, the effective population size ratio of mtDNA to aDNA was at least 0.62, which deviated from the expected value of 0.25, suggesting matriarchal population due to female philopatry and male-biased migration. These results emphasize that ancestral polymorphisms and sex-biased migration may have contributed to conflicting branching patterns in brown and polar bears across aDNA genes and mtDNA.

  5. Damage and repair of ancient DNA

    DEFF Research Database (Denmark)

    Mitchell, David; Willerslev, Eske; Hansen, Anders

    2005-01-01

    , and extensive degradation. In the course of this review, we will discuss the current aDNA literature describing the importance of aDNA studies as they relate to important biological questions and the difficulties associated with extracting useful information from highly degraded and damaged substrates derived......Under certain conditions small amounts of DNA can survive for long periods of time and can be used as polymerase chain reaction (PCR) substrates for the study of phylogenetic relationships and population genetics of extinct plants and animals, including hominids. Because of extensive DNA...... degradation, these studies are limited to species that lived within the past 10(4)-10(5) years (Late Pleistocene), although DNA sequences from 10(6) years have been reported. Ancient DNA (aDNA) has been used to study phylogenetic relationships of protists, fungi, algae, plants, and higher eukaryotes...

  6. Analysis of ancient DNA from coprolites: a perspective with random amplified polymorphic DNA-polymerase chain reaction approach

    Directory of Open Access Journals (Sweden)

    Iñiguez Alena M

    2003-01-01

    Full Text Available The aim of this work was to determine approaches that would improve the quality of ancient DNA (aDNA present in coprolites to enhance the possibility of success in retrieving specific sequence targets. We worked with coprolites from South American archaeological sites in Brazil and Chile dating up to 7,000 years ago. Using established protocols for aDNA extraction we obtained samples showing high degradation as usually happens with this kind of material. The reconstructive polymerization pretreatment was essential to overcome the DNA degradation and the serial dilutions helped with to prevent polymerase chain reaction (PCR inhibitors. Moreover, the random amplified polymorphic DNA-PCR has been shown to be a reliable technique for further experiments to recover specific aDNA sequences.

  7. Interactions of DNA replication factors in vivo as detected by introduction of suppressor alleles of dnaA into other temperature-sensitive dna mutants.

    OpenAIRE

    Blinkowa, A; J. R. Walker

    1983-01-01

    Suppressor mutations located within dnaA can suppress the temperature sensitivity of a dnaZ polymerization mutant, indicating in vivo interaction of the products of these genes. The suppressor allele of dnaA [designated dnaA(SUZ, Cs)] could not be introduced, even at the permissive temperature, by transduction into temperature-sensitive (Ts) dnaC or dnaG recipients; it was transduced into dnaB(Ts) and dnaE(Ts) strains but at very low frequency. Recipient cells which were dnaA+ dnaE(Ts) were k...

  8. Deoxyribonucleic acid initiation mutation dnaB252 is suppressed by elevated dnaC+ gene dosage.

    OpenAIRE

    Sclafani, R A; Wechsler, J A

    1981-01-01

    The Escherichia coli dnaB252 allele is the only dnaB mutation which confers a deoxyribonucleic acid initiation-defective phenotype on the cell. The presence of a multicopy hybrid plasmid containing the dnaC+ gene in a dnaB252 strain completely suppressed the temperature-sensitive phenotype. It is suggested that at high temperature the dnaB252 protein has a lowered affinity for dnaC protein, and that the formation of a dnaB-dnaC complex is mandatory for initiation.

  9. Custom-Designed MLPA Using Multiple Short Synthetic Probes Application to Methylation Analysis of Five Promoter CpG Islands in Tumor and Urine Specimens from Patients with Bladder Cancer

    DEFF Research Database (Denmark)

    Serizawa, R.R.; Ralfkiaer, U.; Dahl, C.;

    2010-01-01

    Ligation of two oligonucleotide probes hybridized adjacently to a DNA template has been widely used for detection of genome alterations. The multiplex ligation-dependent probe amplification (MLPA) technique allows simultaneous screening of multiple target sequences in a single reaction by using......-stranded bacteriophage vector to introduce a sequence of defined length between the primer binding site and the specific target sequence. Here we demonstrate that differences in amplicon length can be achieved by using multiple short synthetic probes for each target sequence. When joined by a DNA ligase, these probes...

  10. Custom-designed MLPA using multiple short synthetic probes

    DEFF Research Database (Denmark)

    Serizawa, R.R.; Ralfkiaer, U.; Dahl, C.;

    2010-01-01

    Ligation of two oligonucleotide probes hybridized adjacently to a DNA template has been widely used for detection of genome alterations. The multiplex ligation-dependent probe amplification (MLPA) technique allows simultaneous screening of multiple target sequences in a single reaction by using......-stranded bacteriophage vector to introduce a sequence of defined length between the primer binding site and the specific target sequence. Here we demonstrate that differences in amplicon length can be achieved by using multiple short synthetic probes for each target sequence. When joined by a DNA ligase, these probes...

  11. Life after the synthetic cell

    DEFF Research Database (Denmark)

    Rasmussen, Steen

    2010-01-01

    Nature asked eight synthetic-biology experts about the implications for science and society of the “synthetic cell” made by the J. Craig Venter Institute (JCVI). The institute's team assembled, modified and implanted a synthesized genome into a DNA-free bacterial shell to make a self-replicating ......Nature asked eight synthetic-biology experts about the implications for science and society of the “synthetic cell” made by the J. Craig Venter Institute (JCVI). The institute's team assembled, modified and implanted a synthesized genome into a DNA-free bacterial shell to make a self...

  12. DNA repair: keeping it together

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2004-01-01

    A protein scaffold has been identified that holds a chromosome together in the event of a DNA double-strand break. This scaffold is dependent on Rad52 and the Rad50-Mre11-Xrs2 complex and withstands the pulling forces of the mitotic spindle during DNA damage checkpoint arrest.......A protein scaffold has been identified that holds a chromosome together in the event of a DNA double-strand break. This scaffold is dependent on Rad52 and the Rad50-Mre11-Xrs2 complex and withstands the pulling forces of the mitotic spindle during DNA damage checkpoint arrest....

  13. Nutlin-3-induced redistribution of chromatin-bound IFI16 in human hepatocellular carcinoma cells in vitro is associated with p53 activation

    OpenAIRE

    Shi, Xin-li; Yang, Jing; Mao, Nan; Wu, Jing-hua; Ren, Lai-feng; Yang, Yuan; Yin, Xiao-Lin; Wei, Lin; Li, Ming-Yuan; Wang, Bao-ning

    2014-01-01

    Aim: Interferon-γ inducible protein 16 (IFI16), a DNA sensor for DNA double-strand break (DSB), is expressed in most human hepatocellular carcinoma cell (HCC) lines. In this study we investigated the re-localization of chromatin-bound IFI16 by Nutlin-3, a DNA damage agent, in HCC cells in vitro, and the potential mechanisms. Methods: Human HCC SMMC-7721 (wild-type TP53), Huh-7 (mutant TP53), Hep3B (null TP53) and normal fetal liver L02 cell lines were examined. DSB damage in HCC cells was det...

  14. The DNA damage response at eroded telomeres and tethering to the nuclear pore complex

    DEFF Research Database (Denmark)

    Khadaroo, Basheer; Teixeira, M Teresa; Luciano, Pierre;

    2009-01-01

    to induce the recruitment of checkpoint and recombination proteins. Notably, a DNA damage response at eroded telomeres starts many generations before senescence and is characterized by the recruitment of Cdc13 (cell division cycle 13), replication protein A, DNA damage checkpoint proteins and the DNA repair...... protein Rad52 into a single focus. Moreover, we show that eroded telomeres, although remaining at the nuclear periphery, move to the nuclear pore complex. Our results link the DNA damage response at eroded telomeres to changes in subnuclear localization and suggest the existence of collapsed replication...... forks at eroded telomeres....

  15. Assembly-associated structural changes of bacteriophage T7 capsids. Detection by use of a protein-specific probe.

    OpenAIRE

    Khan, S. A.; Griess, G A; Serwer, P

    1992-01-01

    To detect changes in capsid structure that occur when a preassembled bacteriophage T7 capsid both packages and cleaves to mature-size longer (concatameric) DNA, the kinetics and thermodynamics are determined here for the binding of the protein-specific probe, 1,1'-bi(4-anilino)naphthalene-5,5'-di-sulfonic acid (bis-ANS), to bacteriophage T7, a T7 DNA deletion (8.4%) mutant, and a DNA-free T7 capsid (metrizamide low density capsid II) known to be a DNA packaging intermediate that has a permeab...

  16. Modulation of UvrD Helicase Activity by Covalent DNA-Protein Cross-links*

    OpenAIRE

    Kumari, Anuradha; Minko, Irina G.; Smith, Rebecca L.; Lloyd, R. Stephen; McCullough, Amanda K.

    2010-01-01

    UvrD (DNA helicase II) has been implicated in DNA replication, DNA recombination, nucleotide excision repair, and methyl-directed mismatch repair. The enzymatic function of UvrD is to translocate along a DNA strand in a 3′ to 5′ direction and unwind duplex DNA utilizing a DNA-dependent ATPase activity. In addition, UvrD interacts with many other proteins involved in the above processes and is hypothesized to facilitate protein turnover, thus promoting further DNA processing. Although UvrD int...

  17. Structural and Mechanistic Insight into DNA Unwinding by Deinococcus radiodurans UvrD

    OpenAIRE

    Meike Stelter; Samira Acajjaoui; Sean McSweeney; Joanna Timmins

    2013-01-01

    International audience DNA helicases are responsible for unwinding the duplex DNA, a key step in many biological processes. UvrD is a DNA helicase involved in several DNA repair pathways. We report here crystal structures of Deinococcus radiodurans UvrD (drUvrD) in complex with DNA in different nucleotide-free and bound states. These structures provide us with three distinct snapshots of drUvrD in action and for the first time trap a DNA helicase undergoing a large-scale spiral movement ar...

  18. Mutational analysis of Mycobacterium UvrD1 identifies functional groups required for ATP hydrolysis, DNA unwinding, and chemomechanical coupling

    OpenAIRE

    Sinha, Krishna Murari; Glickman, Michael S.; Shuman, Stewart

    2009-01-01

    Mycobacterial UvrD1 is a DNA-dependent ATPase and a Ku-dependent 3’ to 5’ DNA helicase. The UvrD1 motor domain resembles that of the prototypal superfamily I helicases UvrD and PcrA. Here we performed a mutational analysis of UvrD1 guided by the crystal structure of a DNA-bound E. coli UvrD-ADP-MgF3 transition state mimetic. Alanine scanning and conservative substitutions identified five amino acids essential for both ATP hydrolysis and duplex unwinding, including those implicated in phosphoh...

  19. Quantitative analysis of the ion-dependent folding stability of DNA triplexes

    OpenAIRE

    Chen, Gengsheng; Chen, Shi-Jie (Gary)

    2011-01-01

    A DNA triplex is formed through binding of a third strand to the major groove of a duplex. Due to the high charge density of a DNA triplex, metal ions are critical for its stability. We recently developed the tightly bound ion (TBI) model for ion-nucleic acids interactions. The model accounts for the potential correlation and fluctuations of the ion distribution. We now apply the TBI model to analyze the ion-dependence of the thermodynamic stability for DNA triplexes. We focus on two experime...

  20. The formation of adjacent triplex-duplex domainsin DNA.

    OpenAIRE

    Nam, K. H.; Abhiraman, S; Wartell, R M

    1999-01-01

    The ability of single-stranded DNA oligomers to form adjacent triplex and duplex domains with two DNA structural motifs was examined. Helix-coil transition curves and a gel mobility shift assay were used to characterize the interaction of single-stranded oligomers 12-20 nt in length with a DNA hairpin and with a DNA duplex that has a dangling end. The 12 nt on the 5'-ends of the oligomers could form a triplex structure with the 12 bp stem of the hairpin or the duplex portion of the DNA with a...

  1. Crenarchaeota in Lake Michigan sediment.

    OpenAIRE

    MacGregor, B.J.; Moser, D. P.; Alm, E W; Nealson, K. H.; Stahl, D. A.

    1997-01-01

    RNA from Lake Michigan sediment was hybridized with a DNA probe for archaeal 16S rRNA. There was a peak of archaeal rRNA abundance in the oxic zone and another immediately below it. Six contributing species were identified by PCR amplification of extracted DNA with primers specific for archaeal rDNA: two related to Methanosarcina acetivorans and four related to marine crenarchaeotal sequences. rRNA quantification using a DNA probe specific for this crenarchaeotal assemblage showed it is most ...

  2. A highly conserved repeated chromosomal sequence in the radioresistant bacterium Deinococcus radiodurans SARK.

    Science.gov (United States)

    Lennon, E; Gutman, P D; Yao, H L; Minton, K W

    1991-03-01

    A DNA fragment containing a portion of a DNA damage-inducible gene from Deinococcus radiodurans SARK hybridized to numerous fragments of SARK genomic DNA because of a highly conserved repetitive chromosomal element. The element is of variable length, ranging from 150 to 192 bp, depending on the absence or presence of one or two 21-bp sequences located internally. A putative translational start site of the damage-inducible gene is within the reiterated element. The element contains dyad symmetries that suggest modes of transcriptional and/or translational control.

  3. Purification, reconstitution, and I kappa B association of the c-Rel-p65 (RelA) complex, a strong activator of transcription.

    OpenAIRE

    Hansen, S K; Baeuerle, P A; Blasi, F.

    1994-01-01

    HeLa cells contain a DNA-binding activity which associates with a kappa B-like DNA element, termed Rel-related protein-binding element (RRBE), localized upstream of the human urokinase promoter. We have purified this activity from the HeLa cell cytosol and have shown that it represents a performed heteromeric complex between p65 (RelA) and c-Rel. Coexpression of c-Rel and p65 (RelA) by in vitro translation formed a DNA-binding complex indistinguishable from purified cellular c-Rel-p65 (RelA) ...

  4. DNA-mediated charge transport for DNA repair

    OpenAIRE

    Boon, Elizabeth M; Livingston, Alison L.; Chmiel, Nikolas H.; David, Sheila S.; Barton, Jacqueline K.

    2003-01-01

    MutY, like many DNA base excision repair enzymes, contains a [4Fe4S](2+) cluster of undetermined function. Electrochemical studies of MutY bound to a DNA-modified gold electrode demonstrate that the [4Fe4S] cluster of MutY can be accessed in a DNA-mediated redox reaction. Although not detectable without DNA, the redox potential of DNA-bound MutY is approximate to275 mV versus NHE, which is characteristic of HiPiP iron proteins. Binding to DNA is thus associated with a change in [4Fe4S](3+/2+)...

  5. Recharacterization of ancient DNA miscoding lesions: insights in the era of sequencing-by-synthesis

    DEFF Research Database (Denmark)

    Gilbert, M Thomas P; Binladen, Jonas; Miller, Webb;

    2007-01-01

    Although ancient DNA (aDNA) miscoding lesions have been studied since the earliest days of the field, their nature remains a source of debate. A variety of conflicting hypotheses exist about which miscoding lesions constitute true aDNA damage as opposed to PCR polymerase amplification error...... strand of origin of observed damage events. With the advent of emulsion-based clonal amplification (emPCR) and the sequencing-by-synthesis technology this has changed. In this paper we demonstrate how data produced on the Roche GS20 genome sequencer can determine miscoding lesion strands of origin...

  6. Protective antiviral immune responses to pseudorabies virus induced by DNA vaccination using dimethyldioctadecylammonium bromide as an adjuvant

    NARCIS (Netherlands)

    Rooij, van E.M.A.; Glansbeek, H.L.; Hilgers, L.A.T.; Lintelo, te E.G.; Visser, de Y.E.; Boersma, W.J.A.; Haagmans, B.L.; Bianchi, A.T.J.

    2002-01-01

    To enhance the efficacy of a DNA vaccine against pseudorabies virus (PRV), we evaluated the adjuvant properties of plasmids coding for gamma interferon or interleukin-12, of CpG immunostimulatory motifs, and of the conventional adjuvants dimethyldioctadecylammonium bromide in water (DDA) and sulfoli

  7. Elevated ERCC-1 Gene Expression in blood cells associated with exposure to arsenic from drinking water in Inner Mongolia

    Science.gov (United States)

    Background: Chronic arsenic exposure has been associated with human cancers. The objective of this study was to investigate arsenic effects on a DNA nucleotide excision repair gene, ERCC1, expression in human blood cells. Material and Methods: Water and toe nail samples were coll...

  8. Connexin43 confers Temozolomide resistance in human glioma cells by modulating the mitochondrial apoptosis pathway

    NARCIS (Netherlands)

    Gielen, P.R.; Aftab, Q.; Ma, N.; Chen, V.C.; Hong, X.; Lozinsky, S.; Naus, C.C.; Sin, W.C.

    2013-01-01

    Glioblastoma multiforme (GBM) is the most aggressive astrocytoma, and therapeutic options are generally limited to surgical resection, radiotherapy, and Temozolomide (TMZ) chemotherapy. TMZ is a DNA alkylating agent that causes DNA damage and induces cell death. Unfortunately, glioma cells often dev

  9. Inhibition of methyleugenol bioactivation by the herb-based constituent nevadensin and prediction of possible in vivo consequences using physiologically based kinetic modeling.

    NARCIS (Netherlands)

    Al-Subeihi, A.A.; Alhusainy, W.; Paini, A.; Punt, A.; Vervoort, J.; Bladeren, van P.J.; Rietjens, I.M.C.M.

    2013-01-01

    Methyleugenol (ME) occurs naturally in a variety of spices, herbs, including basil, and their essential oils. ME induces hepatomas in rodent bioassays following its conversion to a DNA reactive metabolite. In the present study, the basil constituent nevadensin was shown to be able to inhibit SULT-me

  10. Fossil avian eggshell preserves ancient DNA

    DEFF Research Database (Denmark)

    Oskam, Charlotte L; Haile, James; McLay, Emma;

    2010-01-01

    Owing to exceptional biomolecule preservation, fossil avian eggshell has been used extensively in geochronology and palaeodietary studies. Here, we show, to our knowledge, for the first time that fossil eggshell is a previously unrecognized source of ancient DNA (aDNA). We describe the successful...

  11. Functions and Dynamics of DNA Repair Proteins in Mitosis and Meiosis

    NARCIS (Netherlands)

    E.J. Uringa

    2005-01-01

    textabstractMy PhD project encompassed studies on the functions of several different proteins, all involved in DNA repair, in somatic and germ-line cells. Hr6b and Rad18Sc are involved in a DNA repair mechanism called ‘Replicative Damage Bypass’ (RDB), and function as ubiquitin conjugating enzym

  12. Bleomycin resistance : a new dominant selectable marker for plant cell transformation

    NARCIS (Netherlands)

    Hille, Jacques; Verheggen, Frank; Roelvink, Peter; Franssen, Henk; Kammen, Ab van; Zabel, Pim

    1986-01-01

    Plant cells are sensitive to the antibiotic bleomycin, a DNA damaging glycopeptide. A bleomycin resistance determinant, located on transposon Tn5 and functional in bacteria, has been cloned in a plant expression vector and introduced into Nicotiana plumbaginifolia using Agrobacterium tumefaciens. Th

  13. Charcot-Marie-Tooth disease type 1A

    DEFF Research Database (Denmark)

    Hertz, Jens Michael; Børglum, A D; Brandt, C A;

    1994-01-01

    Charcot-Marie-Tooth disease type 1A (CMT1A) is an autosomal dominant peripheral neuropathy associated with a DNA duplication on chromosome 17p11.2-p12 in the majority of cases. Most of the sporadic cases are due to a de novo duplication. We have screened for this duplication in 11 Danish patients...

  14. DNA Fingerprint Analysis of Three Short Tandem Repeat (STR) Loci for Biochemistry and Forensic Science Laboratory Courses

    Science.gov (United States)

    McNamara-Schroeder, Kathleen; Olonan, Cheryl; Chu, Simon; Montoya, Maria C.; Alviri, Mahta; Ginty, Shannon; Love, John J.

    2006-01-01

    We have devised and implemented a DNA fingerprinting module for an upper division undergraduate laboratory based on the amplification and analysis of three of the 13 short tandem repeat loci that are required by the Federal Bureau of Investigation Combined DNA Index System (FBI CODIS) data base. Students first collect human epithelial (cheek)…

  15. Polymerase-directed synthesis of C5-ethynyl locked nucleic acids

    DEFF Research Database (Denmark)

    Veedu, Rakesh N; Burri, Harsha Vardhan Reddy; Kumar, Pawan;

    2010-01-01

    -U and C5-ethynyl LNA-U nucleotides into oligonucleotides. Phusion High Fidelity and KOD DNA polymerases efficiently incorporated LNA-U and C5-ethynyl LNA-U nucleotides into a DNA strand and T7 RNA polymerase successfully accepted the LNA-U nucleoside 5'-triphosphate as substrate for RNA transcripts....

  16. Temperature influences the expression profiling of immune response genes in rainbow trout following DNA vaccination and VHS virus infection

    DEFF Research Database (Denmark)

    Einer-Jensen, Katja; Gautier, Laurent; Rasmussen, Jesper Skou;

    A DNA vaccine encoding the glycoprotein (G) genes of the salmonid rhabdovirus viral haemorrhagic septicaemia virus (VHSV) has proven highly efficient against the disease caused by this virus in rainbow trout (Oncorhynchus mykiss). Several studies have demonstrated that this vaccine induces both a...

  17. Bacterial mitosis: partitioning protein ParA oscillates in spiral-shaped structures and positions plasmids at mid-cell

    DEFF Research Database (Denmark)

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

    2004-01-01

    The par2 locus of Escherichia coli plasmid pB171 encodes oscillating ATPase ParA, DNA binding protein ParB and two cis-acting DNA regions to which ParB binds (parC1 and parC2). Three independent techniques were used to investigate the subcellular localization of plasmids carrying par2. In cells w...

  18. Enhancement of DNA vaccine potency through linkage of antigen to filamentous bacteriophage coat protein III domain I

    DEFF Research Database (Denmark)

    Cuesta, Àngel M; Suárez, Eduardo; Larsen, Martin;

    2006-01-01

    immune pathways by adding immune-activating genes to the tumour antigen sequence. In this work, we converted a model non-immunogenic antigen into a vaccine by fusing it to domain I of the filamentous bacteriophage coat protein III gene. Vaccination with a DNA construct encoding the domain I fusion...

  19. A global heuristically search algorithm for DNA encoding

    Institute of Scientific and Technical Information of China (English)

    Zhang Kai; Pan Linqiang; Xu Jin

    2007-01-01

    A new efficient algorithm is developed to design DNA words with equal length for DNA computing. The algorithm uses a global heuristic optimizing search approach and converts constraints to a carry number to accelerate the convergence, which can generate a DNA words set satisfying some thermodynamic and combinatorial constraints. Based on the algorithm, a software for DNA words design is developed.

  20. A TaqMan-based real-time PCR assay for porcine parvovirus 4 detection and quantification in reproductive tissues of sows

    Science.gov (United States)

    Porcine parvovirus 4 (PPV4) is a DNA virus, and a member of the Parvoviridae family within the Bocavirus genera. It was recently detected in swine, but its epidemiology and pathology remain unclear. A TaqMan-based real-time polymerase chain reaction (qPCR) assay targeting a conserved region of the O...