Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

Energy Technology Data Exchange (ETDEWEB)

Chattopadhyay, Saket; Ely, Abdullah; Bloom, Kristie; Weinberg, Marc S. [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa); Arbuthnot, Patrick, E-mail: Patrick.Arbuthnot@wits.ac.za [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa)

2009-11-20

RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

International Nuclear Information System (INIS)

Chattopadhyay, Saket; Ely, Abdullah; Bloom, Kristie; Weinberg, Marc S.; Arbuthnot, Patrick

2009-01-01

RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

Sub-wavelength plasmonic readout for direct linear analysis of optically tagged DNA

Science.gov (United States)

Varsanik, Jonathan; Teynor, William; LeBlanc, John; Clark, Heather; Krogmeier, Jeffrey; Yang, Tian; Crozier, Kenneth; Bernstein, Jonathan

2010-02-01

This work describes the development and fabrication of a novel nanofluidic flow-through sensing chip that utilizes a plasmonic resonator to excite fluorescent tags with sub-wavelength resolution. We cover the design of the microfluidic chip and simulation of the plasmonic resonator using Finite Difference Time Domain (FDTD) software. The fabrication methods are presented, with testing procedures and preliminary results. This research is aimed at improving the resolution limits of the Direct Linear Analysis (DLA) technique developed by US Genomics [1]. In DLA, intercalating dyes which tag a specific 8 base-pair sequence are inserted in a DNA sample. This sample is pumped though a nano-fluidic channel, where it is stretched into a linear geometry and interrogated with light which excites the fluorescent tags. The resulting sequence of optical pulses produces a characteristic "fingerprint" of the sample which uniquely identifies any sample of DNA. Plasmonic confinement of light to a 100 nm wide metallic nano-stripe enables resolution of a higher tag density compared to free space optics. Prototype devices have been fabricated and are being tested with fluorophore solutions and tagged DNA. Preliminary results show evanescent coupling to the plasmonic resonator is occurring with 0.1 micron resolution, however light scattering limits the S/N of the detector. Two methods to reduce scattered light are presented: index matching and curved waveguides.

Synthesis and antimalarial activity of new chloroquine analogues carrying a multifunctional linear side chain

OpenAIRE

Iwaniuk, Daniel P.; Whetmore, Eric D.; Rosa, Nicholas; Ekoue-Kovi, Kekeli; Alumasa, John; de Dios, Angel C.; Roepe, Paul D.; Wolf, Christian

2009-01-01

We report the synthesis and in vitro antimalarial activity of several new 4-amino-and 4-alkoxy-7-chloroquinolines carrying a linear dibasic side chain. Many of these chloroquine analogues have submicromolar antimalarial activity versus HB3 (chloroquine sensitive) and Dd2 (chloroquine resistant strain of P. falciparum) and low resistance indices were obtained in most cases. Importantly, compounds 11–15 and 24 proved to be more potent against Dd2 than chloroquine. Branching of the side chain st...

Synthesis and antimalarial activity of new chloroquine analogues carrying a multifunctional linear side chain.

Science.gov (United States)

Iwaniuk, Daniel P; Whetmore, Eric D; Rosa, Nicholas; Ekoue-Kovi, Kekeli; Alumasa, John; de Dios, Angel C; Roepe, Paul D; Wolf, Christian

2009-09-15

We report the synthesis and in vitro antimalarial activity of several new 4-amino- and 4-alkoxy-7-chloroquinolines carrying a linear dibasic side chain. Many of these chloroquine analogues have submicromolar antimalarial activity versus HB3 (chloroquine sensitive) and Dd2 (chloroquine resistant strain of Plasmodium falciparum) and low resistance indices were obtained in most cases. Importantly, compounds 11-15 and 24 proved to be more potent against Dd2 than chloroquine. Branching of the side chain structure proved detrimental to the activity against the CQR strain.

Electrogenerated chemiluminescence detection for deoxyribonucleic acid hybridization based on gold nanoparticles carrying multiple probes

International Nuclear Information System (INIS)

Wang Hui; Zhang Chengxiao; Li Yan; Qi Honglan

2006-01-01

A novel sensitive electrogenerated chemiluminescence (ECL) method for the detection deoxyribonucleic acid (DNA) hybridization based on gold nanoparticles carrying multiple probes was developed. Ruthenium bis(2,2'-bipyridine)(2,2'-bipyridine-4,4'-dicarboxylic acid)-N-hydroxysuccinimide ester (Ru(bpy) 2 (dcbpy)NHS) was used as a ECL label and gold nanoparticle as a carrier. Probe single strand DNA (ss-DNA) was self-assembled at the 3'-terminal with a thiol group to the surface of gold nanoparticle and covalently labeled at the 5'-terminal of a phosphate group with Ru(bpy) 2 (dcbpy)NHS and the resulting conjugate (Ru(bpy) 2 (dcbpy)NHS)-ss-DNA-Au, was taken as a ECL probe. When target analyte ss-DNA was immobilized on a gold electrode by self-assembled monolayer technique and then hybridized with the ECL probe to form a double-stranded DNA (ds-DNA), a strong ECL response was electrochemically generated. The ECL intensity was linearly related to the concentration of the complementary sequence (target ss-DNA) in the range from 1.0 x 10 -11 to 1.0 x 10 -8 mol L -1 , and the linear regression equation was S = 57301 + 4579.6 lg C (unit of C is mol L -1 ). A detection limit of 5.0 x 10 -12 mol L -1 for target ss-DNA was achieved. The ECL signal generated from many reporters of ECL probe prepared is greatly amplified, compared to the convention scheme which is based on one reporter per hybridization event

The Infection Efficiency and Replication Ability of Circularized HBV DNA Optimized the Linear HBV DNA in Vitro and in Vivo

Science.gov (United States)

Li, Xiaosong; Zhu, Junke; Lai, Guoqi; Yan, Lei; Hu, Jieli; Chen, Juan; Tang, Ni; Huang, Ailong

2015-01-01

Studies on molecular mechanisms of the persist infection of hepatitis B virus have been hampered by a lack of a robust animal model. We successfully established a simple, versatile, and reproducible HBV persist infection model in vitro and in vivo with the circularized HBV DNA. The cells and mice were transfected or injected with circularized HBV DNA and pAAV/HBV1.2, respectively. At the indicated time, the cells, supernatants, serum samples, and liver tissues were collected for virological and serological detection. Both in vitro and in vivo, the circularized HBV DNA and pAAV/HBV1.2 could replicate and transcribe efficiently, but the infection effect of the former was superior to the latter (p HBV genome DNA into the mice robustly supported HBV infection and approximately 80% of HBV infected mice established persistent infection for at least 10 weeks. This study demonstrated that the infection efficiency and replication ability of the circularized structure of HBV DNA overmatched that of the expression plasmid containing the linear structure of HBV DNA in vitro and in vivo. Meanwhile, this research results could provide useful tools and methodology for further study of pathogenic mechanisms and potential antiviral treatments of human chronic HBV infection in vitro and in vivo. PMID:25751726

Infrared A radiation promotes survival of human melanocytes carrying ultraviolet radiation-induced DNA damage.

Science.gov (United States)

Kimeswenger, Susanne; Schwarz, Agatha; Födinger, Dagmar; Müller, Susanne; Pehamberger, Hubert; Schwarz, Thomas; Jantschitsch, Christian

2016-06-01

The link between solar radiation and melanoma is still elusive. Although infrared radiation (IR) accounts for over 50% of terrestrial solar energy, its influence on human skin is not well explored. There is increasing evidence that IR influences the expression patterns of several molecules independently of heat. A previous in vivo study revealed that pretreatment with IR might promote the development of UVR-induced non-epithelial skin cancer and possibly of melanoma in mice. To expand on this, the aim of the present study was to evaluate the impact of IR on UVR-induced apoptosis and DNA repair in normal human epidermal melanocytes. The balance between these two effects is a key factor of malignant transformation. Human melanocytes were exposed to physiologic doses of IR and UVR. Compared to cells irradiated with UVR only, simultaneous exposure to IR significantly reduced the apoptotic rate. However, IR did not influence the repair of UVR-induced DNA damage. IR partly reversed the pro-apoptotic effects of UVR via modification of the expression and activity of proteins mainly of the extrinsic apoptotic pathway. In conclusion, IR enhances the survival of melanocytes carrying UVR-induced DNA damage and thereby might contribute to melanomagenesis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Physical manipulation of single-molecule DNA using microbead and its application to analysis of DNA-protein interaction

International Nuclear Information System (INIS)

Kurita, Hirofumi; Yasuda, Hachiro; Takashima, Kazunori; Katsura, Shinji; Mizuno, Akira

2009-01-01

We carried out an individual DNA manipulation using an optical trapping for a microbead. This manipulation system is based on a fluorescent microscopy equipped with an IR laser. Both ends of linear DNA molecule were labeled with a biotin and a thiol group, respectively. Then the biotinylated end was attached to a microbead, and the other was immobilized on a thiol-linkable glass surface. We controlled the form of an individual DNA molecule by moving the focal point of IR laser, which trapped the microbead. In addition, we applied single-molecule approach to analyze DNA hydrolysis. We also used microchannel for single-molecule observation of DNA hydrolysis. The shortening of DNA in length caused by enzymatic hydrolysis was observed in real-time. The single-molecule DNA manipulation should contribute to elucidate detailed mechanisms of DNA-protein interactions

A study of the linear free energy model for DNA structures using the generalized Hamiltonian formalism

Energy Technology Data Exchange (ETDEWEB)

Yavari, M., E-mail: yavari@iaukashan.ac.ir [Islamic Azad University, Kashan Branch (Iran, Islamic Republic of)

2016-06-15

We generalize the results of Nesterenko [13, 14] and Gogilidze and Surovtsev [15] for DNA structures. Using the generalized Hamiltonian formalism, we investigate solutions of the equilibrium shape equations for the linear free energy model.

Construction of expression vectors carrying mouse peroxisomal ...

African Journals Online (AJOL)

PRECIOUS

2009-11-16

Nov 16, 2009 ... The aim of this study was to construct expression vectors carrying mouse peroxisomal protein gene. (PEP-cDNA) in prokaryotic and mammalian expression vectors in chimeric cDNA types, encompassing. GST and FLAG with PEP-cDNA. PEP-cDNA was sub-cloned in pGEX6p2 prokaryotic expression ...

A linear concatenation strategy to construct 5'-enriched amplified cDNA libraries using multiple displacement amplification.

Science.gov (United States)

Gadkar, Vijay J; Filion, Martin

2013-06-01

In various experimental systems, limiting available amounts of RNA may prevent a researcher from performing large-scale analyses of gene transcripts. One way to circumvent this is to 'pre-amplify' the starting RNA/cDNA, so that sufficient amounts are available for any downstream analysis. In the present study, we report the development of a novel protocol for constructing amplified cDNA libraries using the Phi29 DNA polymerase based multiple displacement amplification (MDA) system. Using as little as 200 ng of total RNA, we developed a linear concatenation strategy to make the single-stranded cDNA template amenable for MDA. The concatenation, made possible by the template switching property of the reverse transcriptase enzyme, resulted in the amplified cDNA library with intact 5' ends. MDA generated micrograms of template, allowing large-scale polymerase chain reaction analyses or other large-scale downstream applications. As the amplified cDNA library contains intact 5' ends, it is also compatible with 5' RACE analyses of specific gene transcripts. Empirical validation of this protocol is demonstrated on a highly characterized (tomato) and an uncharacterized (corn gromwell) experimental system.

From stripe to slab confinement for DNA linearization in nanochannels

Science.gov (United States)

Cifra, Peter; Benkova, Zuzana; Namer, Pavol

We investigate suggested advantageous analysis in the linearization experiments with macromolecules confined in a stripe-like channel using Monte Carlo simulations. The enhanced chain extension in a stripe that is due to significant excluded volume interactions between monomers in two dimensions weakens on transition to experimentally feasible slit-like channel. Based on the chain extension-confinement strength dependence and the structure factor behavior for the chain in stripe we infer the excluded volume regime typical for two-dimensional systems. On transition to the slab geometry, the advantageous chain extension decreases and the Gaussian regime is observed for not very long semiflexible chains. The evidence for pseudo-ideality in confined chains is based on indicators such as the extension curves, variation of the extension with the persistence length or the structure factor. The slab behavior is observed when the stripe (originally of monomer thickness) reaches the thickness larger than cca 10nm in the third dimension. This maximum height of the slab to retain the advantage of the stripe is very low and this have implication for DNA linearization experiments. The presented analysis, however, has a broader relevance for confined polymers. Support from Slovak R&D Agency (SRDA-0451-11) is acknowledged.

Flexible bent rod model with a saturating induced dipole moment to study the electric linear dichroism of DNA fragments

Directory of Open Access Journals (Sweden)

Jorge A. Bertolotto

2016-06-01

Full Text Available In the present work we make a theoretical study of the steady state electric linear dichroism of DNA fragments in aqueous solution. The here developed theoretical approach considers a flexible bent rod model with a saturating induced dipole moment. The electric polarizability tensor of bent DNA fragments is calculated considering a phenomenological model which theoretical and experimental backgroung is presented here. The model has into account the electric polarizability longitudinal and transversal to the macroion. Molecular flexibility is described using an elastic potential. We consider DNA fragments originally bent with bending fluctuations around an average bending angle. The induced dipole moment is supposed constant once the electric field strength grows up at critical value. To calculate the reduced electric linear dichroism we determine the optical factor considering the basis of the bent DNA perpendicular to the molecular axis. The orientational distribution function has into account the anisotropic electric properties and the molecule flexibility. We applied the present theoretical background to fit electric dichroism experimental data of DNA fragments reported in the bibliography in a wide range of molecular weight and electric field. From these fits, values of DNA physical properties are estimated. We compare and discuss the results here obtained with the theoretical and experimental data presented by other authors. The original contributions of this work are: the inclusion of the transversal electric polarizability saturating with the electric field, the description of the electric properties with an electric polarizability tensor dependant on the bending angle and the use of an arc model originally bent.

Flexible bent rod model with a saturating induced dipole moment to study the electric linear dichroism of DNA fragments

Science.gov (United States)

Bertolotto, Jorge A.; Umazano, Juan P.

2016-06-01

In the present work we make a theoretical study of the steady state electric linear dichroism of DNA fragments in aqueous solution. The here developed theoretical approach considers a flexible bent rod model with a saturating induced dipole moment. The electric polarizability tensor of bent DNA fragments is calculated considering a phenomenological model which theoretical and experimental backgroung is presented here. The model has into account the electric polarizability longitudinal and transversal to the macroion. Molecular flexibility is described using an elastic potential. We consider DNA fragments originally bent with bending fluctuations around an average bending angle. The induced dipole moment is supposed constant once the electric field strength grows up at critical value. To calculate the reduced electric linear dichroism we determine the optical factor considering the basis of the bent DNA perpendicular to the molecular axis. The orientational distribution function has into account the anisotropic electric properties and the molecule flexibility. We applied the present theoretical background to fit electric dichroism experimental data of DNA fragments reported in the bibliography in a wide range of molecular weight and electric field. From these fits, values of DNA physical properties are estimated. We compare and discuss the results here obtained with the theoretical and experimental data presented by other authors. The original contributions of this work are: the inclusion of the transversal electric polarizability saturating with the electric field, the description of the electric properties with an electric polarizability tensor dependant on the bending angle and the use of an arc model originally bent.

Complete DNA sequence of the linear mitochondrial genome of the pathogenic yeast Candida parapsilosis

Czech Academy of Sciences Publication Activity Database

Nosek, J.; Novotná, Marcela; Hlavaticová, Z.; Ussery, D. W.; Fajkus, Jiří; Tomáška, L.

2004-01-01

Roč. 272, č. 2 (2004), s. 173-180 ISSN 1617-4615 Grant - others:Howard Hughes Medical Institute(US) 55000327; VEGA MŠ SR(SK) 1/9153/02; VEGA MŠ SR(SK) 1/0006/03; APVT(SK) 20-003902; Fogarty International NIH(US) 1-R03-TW05654-01 Institutional research plan: CEZ:AV0Z5004920 Keywords : Candida parapsilosis * linear mitochondrial DNA * telomeric circles (t-circles) Subject RIV: BO - Biophysics Impact factor: 2.371, year: 2004

Deformation of the free surface of a conducting fluid in the magnetic field of current-carrying linear conductors

International Nuclear Information System (INIS)

Zubarev, N.M.; Zubareva, O.V.

2017-01-01

The magnetic shaping problem is studied for the situation where a cylindrical column of a perfectly conducting fluid is deformed by the magnetic field of a system of linear current-carrying conductors. Equilibrium is achieved due to the balance of capillary and magnetic pressures. Two two-parametric families of exact solutions of the problem are obtained with the help of conformal mapping technique. In accordance with them, the column essentially deforms in the cross section up to its disintegration.

Deformation of the free surface of a conducting fluid in the magnetic field of current-carrying linear conductors

Science.gov (United States)

Zubarev, N. M.; Zubareva, O. V.

2017-06-01

The magnetic shaping problem is studied for the situation where a cylindrical column of a perfectly conducting fluid is deformed by the magnetic field of a system of linear current-carrying conductors. Equilibrium is achieved due to the balance of capillary and magnetic pressures. Two two-parametric families of exact solutions of the problem are obtained with the help of conformal mapping technique. In accordance with them, the column essentially deforms in the cross section up to its disintegration.

The effect of co-administration of DNA carrying chicken interferon-gamma gene on protection of chickens against infectious bursal disease by DNA-mediated vaccination.

Science.gov (United States)

Hsieh, Ming Kun; Wu, Ching Ching; Lin, Tsang Long

2006-11-17

The purpose of the present study was to determine whether DNA vaccination by co-administration of DNA coding for chicken interferon-gamma (IFN-gamma) gene and DNA encoding for the VP243 gene of IBDV could enhance immune response and protection efficacy of chickens against challenge by IBDV. Plasmids carrying VP243 gene of IBDV strain variant E (VE) (P/VP243/E) and chicken IFN-gamma gene (P/cIFN-gamma) were constructed, respectively. One-day-old chickens were intramuscularly injected with P/VP243/E, or P/cIFN-gamma, or both once, twice, or three times into the thigh muscle of one leg or the thigh muscles of two separate legs at weekly intervals. Chickens were orally challenged with IBDV strain VE at 3 weeks of age and observed for 10 days. Chickens receiving two plasmids in the same site two times had significantly higher (Pprotection and those receiving two plasmids in the same sites did not have any protection against IBD. The enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) titers to IBDV of chickens in the groups with three doses of P/VP243/E were significantly higher (Pprotected by DNA vaccination did not have detectable IBDV antigen in the bursae as determined by immunofluorescent antibody assay (IFA). The results indicated that co-administration of plasmid encoding chicken IFN-gamma gene with plasmid encoding a large segment gene of the IBDV did not enhance immune response and protection against challenge by IBDV.

Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents

Science.gov (United States)

Klapacz, Joanna; Pottenger, Lynn H.; Engelward, Bevin P.; Heinen, Christopher D.; Johnson, George E.; Clewell, Rebecca A.; Carmichael, Paul L.; Adeleye, Yeyejide; Andersen, Melvin E.

2016-01-01

From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations. PMID:27036068

Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents.

Science.gov (United States)

Klapacz, Joanna; Pottenger, Lynn H; Engelward, Bevin P; Heinen, Christopher D; Johnson, George E; Clewell, Rebecca A; Carmichael, Paul L; Adeleye, Yeyejide; Andersen, Melvin E

2016-01-01

From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance of a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations. Copyright © 2015 Elsevier B.V. All rights reserved.

A polymer, random walk model for the size-distribution of large DNA fragments after high linear energy transfer radiation

Science.gov (United States)

Ponomarev, A. L.; Brenner, D.; Hlatky, L. R.; Sachs, R. K.

2000-01-01

DNA double-strand breaks (DSBs) produced by densely ionizing radiation are not located randomly in the genome: recent data indicate DSB clustering along chromosomes. Stochastic DSB clustering at large scales, from > 100 Mbp down to simulations and analytic equations. A random-walk, coarse-grained polymer model for chromatin is combined with a simple track structure model in Monte Carlo software called DNAbreak and is applied to data on alpha-particle irradiation of V-79 cells. The chromatin model neglects molecular details but systematically incorporates an increase in average spatial separation between two DNA loci as the number of base-pairs between the loci increases. Fragment-size distributions obtained using DNAbreak match data on large fragments about as well as distributions previously obtained with a less mechanistic approach. Dose-response relations, linear at small doses of high linear energy transfer (LET) radiation, are obtained. They are found to be non-linear when the dose becomes so large that there is a significant probability of overlapping or close juxtaposition, along one chromosome, for different DSB clusters from different tracks. The non-linearity is more evident for large fragments than for small. The DNAbreak results furnish an example of the RLC (randomly located clusters) analytic formalism, which generalizes the broken-stick fragment-size distribution of the random-breakage model that is often applied to low-LET data.

Extended DNA Tile Actuators

DEFF Research Database (Denmark)

Kristiansen, Martin; Kryger, Mille; Zhang, Zhao

2012-01-01

A dynamic linear DNA tile actuator is expanded to three new structures of higher complexity. The original DNA actuator was constructed from a central roller strand which hybridizes with two piston strands by forming two half-crossover junctions. A linear expansion of the actuator is obtained...

Investigations on the influence of radiation with variable linear energy transfer (LET) on the DNA-content and DNA-repair-mechanisms in Vicia faba

International Nuclear Information System (INIS)

Eckl, P.

1981-01-01

This study was initiated to investigate, whether there are any radiation-induced changes in DNA-content and if these changes can be repaired. Seeds of Vicia faba L. were grown in glass culture vessels. After 10 to 20 days the seedings were irradiated using a 1 C1 60 Co gammasource (90mrad/h and 33 rad/h) and a 5 mCi 252 Cf neutronsource (90 mrad/h). Both, neutron and gamma irradiation cause a reduction in nuclear DNA-content even after low doses (1 to 10 rad). The extent of depression is only depending on linear energy transfer. Parallel to the induced minimum in DNA-content, but shifted to higher doses, also the mitotic activity reaches a minimum. Whereas neutron irradiation results in a total stop after doses of 8 rad, gamma-irradiation only induces a depression of 80 %. Whith higher doses the mitotic activity increases again. The neutron-induced changes in DNA-content seem to be restored within 90 minutes after irradiation. No continuous increase could be found after low gamma-doses. Gamma-irradiation with higher dose rates ( 60 Co, 33 rad/h) causes a general decrease over the dose-range studied (100 to 1600 rad). Following doses of 100 rad the mitotic activity increases significantly. With higher doses the decrease is exponential. A dose-dependent mitotic delay could also be observed. As described by many authors, unscheduled DNA-synthesis (UDS) could not be detected in nuclei of Vicia faba. This indicates that an other system, perhaps acting in situ - at the damaged place - is responsible for the repair of radiation-induced thymine-damages. (Author)

DNA Open states and DNA hydratation; Estados abiertos del ADN e hidratacion del ADN

Energy Technology Data Exchange (ETDEWEB)

Lema-Larre, B de [Universidad Central de Venezuela (UCV), Facultad de Medicina, Caracas (Venezuela); Martin-Landrove, M [Universidad Central de Venezuela (UCV), Facultad de Ciencias, Centro de Resonancia Magnetica Caracas (Venezuela)

1995-07-01

It is a very well-known fact that an protonic exchange exists among natural DNA filaments and synthetic polynucleotides with the solvent (1--2). The existence of DNA open states, that is to say states for which the interior of the DNA molecule is exposed to the external environment, it has been demonstrated by means of proton-deuterium exchange (3). This work has carried out experiments measuring the dispersion of the traverse relaxation rate (4), as a pulsation rate function in a Carr-Purcell-Meiboom-Gill (CPMG) pulses sequence rate, to determine changes in the moist layer of the DNA molecule. The experiments were carried out under different experimental conditions in order to vary the probability that open states occurs, such as temperature or the exposure to electromagnetic fields. Some theoretical models were supposed to adjust the experimental results including those related to DNA non linear dynamic. [Spanish] Es un hecho bien conocido que existe un intercambio protonico entre filamentos naturales de ADN y polinucleotidos sinteticos con el solvente (1--2). La existencia de estados abiertos en el ADN, es decir estados para los cuales el interior de la molecula del ADN es expuesto al ambiente exterior, ha sido demostrado mediante experimentos de intercambio proton-deuterio (3). En el presente trabajo hemos realizado experimentos midiendo la dispersion de la tasa de relajacion transversal (4), como una funcion de la tasa de pulsacion en una secuencia de pulsos de Carr-Purcell-Meiboom-Gill (CPMG), para determinar cambios en la capa de hidratacion de la molecula de ADN. Los experimentos fueron realizados bajo diferentes condiciones experimentales para asi variar la probabilidad de que ocurran estados abiertos, tales como la temperatura o la exposicion a campos electromagneticos. Algunos modelos teoricos fueron supuestos para ajustar los resultados experimentales incluyendo aquellos relacionados con dinamica no lineal del ADN. (autor)

Dynamic constitutional frameworks for DNA biomimetic recognition.

Science.gov (United States)

Catana, Romina; Barboiu, Mihail; Moleavin, Ioana; Clima, Lilia; Rotaru, Alexandru; Ursu, Elena-Laura; Pinteala, Mariana

2015-02-07

Linear and cross-linked dynamic constitutional frameworks generated from reversibly interacting linear PEG/core constituents and cationic sites shed light on the dominant coiling versus linear DNA binding behaviours, closer to the histone DNA binding wrapping mechanism.

The Pedersen current carried by electrons: a non-linear response of the ionosphere to magnetospheric forcing

Directory of Open Access Journals (Sweden)

S. C. Buchert

2008-09-01

Full Text Available Observations by the EISCAT Svalbard radar show that electron temperatures T_e in the cusp electrojet reach up to about 4000 K. The heat is tapped and converted from plasma convection in the near Earth space by a Pedersen current that is carried by electrons due to the presence of irregularities and their demagnetising effect. The heat is transfered to the neutral gas by collisions. In order to enhance T_e to such high temperatures the maximally possible dissipation at 50% demagnetisation must nearly be reached. The effective Pedersen conductances are found to be enhanced by up to 60% compared to classical values. Conductivities and conductances respond significantly to variations of the electric field strength E, and "Ohm's law" for the ionosphere becomes non-linear for large E.

Highly Effective Non-Viral Antitumor Gene Therapy System Comprised of Biocompatible Small Plasmid Complex Particles Consisting of pDNA, Anionic Polysaccharide, and Fully Deprotected Linear Polyethylenimine

Directory of Open Access Journals (Sweden)

Yoshiyuki Koyama

2015-07-01

Full Text Available We have reported that ternary complexes of plasmid DNA with conventional linear polyethylenimine (l-PEI and certain polyanions were very stably dispersed, and, with no cryoprotectant, they could be freeze-dried and re-hydrated without the loss of transfection ability. These properties enabled the preparation of a concentrated suspension of very small pDNA complex, by preparing the complexes at highly diluted conditions, followed by condensation via lyophilization-and-rehydration procedure. Recently, a high potency linear polyethylenimine having no residual protective groups, i.e., Polyethylenimine “Max” (PEI “Max”, is available, which has been reported to induce much higher gene expression than conventional l-PEI. We tried to prepare the small DNA/PEI “Max”/polyanion complexes by a similar freeze-drying method. Small complex particles could be obtained without apparent aggregation, but transfection activity of the rehydrated complexes was severely reduced. Complex-preparation conditions were investigated in details to achieve the freeze-dried DNA/PEI “Max”/polyanion small ternary complexes with high transfection efficiency. DNA/PEI “Max”/polyanion complexes containing cytokine-coding plasmids were then prepared, and their anti-tumor therapeutic efficacy was examined in tumor-bearing mice.

Nuclear DNA but not mtDNA controls tumor phenotypes in mouse cells

International Nuclear Information System (INIS)

Akimoto, Miho; Niikura, Mamoru; Ichikawa, Masami; Yonekawa, Hiromichi; Nakada, Kazuto; Honma, Yoshio; Hayashi, Jun-Ichi

2005-01-01

Recent studies showed high frequencies of homoplasmic mtDNA mutations in various human tumor types, suggesting that the mutated mtDNA haplotypes somehow contribute to expression of tumor phenotypes. We directly addressed this issue by isolating mouse mtDNA-less (ρ 0 ) cells for complete mtDNA replacement between normal cells and their carcinogen-induced transformants, and examined the effect of the mtDNA replacement on expression of tumorigenicity, a phenotype forming tumors in nude mice. The results showed that genome chimera cells carrying nuclear DNA from tumor cells and mtDNA from normal cells expressed tumorigenicity, whereas those carrying nuclear DNA from normal cells and mtDNA from tumor cells did not. These observations provided direct evidence that nuclear DNA, but not mtDNA, is responsible for carcinogen-induced malignant transformation, although it remains possible that mtDNA mutations and resultant respiration defects may influence the degree of malignancy, such as invasive or metastatic properties

Development of DNA-based radiopharmaceuticals carrying Auger-electron emitters for anti-gene radiotherapy

International Nuclear Information System (INIS)

Panyutin, I.G.; Winters, T.A.; Feinendegen, L.E.; Neumann, R.D.

2000-01-01

Targeting of radiation damage to specific DNA sequences is the essence of antigene radiotherapy. This technique also provides a tool to study molecular mechanisms of DNA repair on a defined, single radio damaged site. It was achieved such sequence-specific radio damage by combining the highly localized DNA damage produced by the decay of Auger-electron-emitters such as 125 I with the sequence-specific action of triplex-forming oligonucleotides (TFO). TFO complementary to polypurine-polypyrimidine regions of human genes were synthesized and labeled with 125 I-dCTP by the primer extension method. 125 I-TFO were delivered into cells with several delivery systems. In addition, human enzymes capable of supporting DNA single-strand-break repair were isolated and assessed for their role in the repair of this lesion. Also, the mutagenicity and repairability of 125 I-TFO-induced double strand breaks (DSB) were assessed by repair of plasmid possessing a site-specific DSB lesion. Using plasmids containing target polypurine-polypyrimidine tracts, it was obtained the fine structure of sequence-specific DNA breaks produced by decay of 125 I with single-nucleotide resolution. It was showed that the designed 125 I-TFO in nanomolar concentrations could bind to and introduce double-strand breaks into the target sequences in situ, i.e., within isolated nuclei and intact digitonin-permeabilized cells. It was also showed 125 I-TFO-induced DSB to be highly mutagenic lesions resulting in a mutation frequency of nearly 80%, with deletions comprising the majority of mutations. The results obtained demonstrate the ability of 125 I-TFO to target specific sequences in their natural environment - within eukaryotic nucleus. Repair of 125 I-TFO-induced DNA damage should typically result in mutagenic gene inactivation

Plasmid DNA linearization in the antibacterial action of a new fluorescent Ag nanoparticle-paracetamol dimer composite

Science.gov (United States)

Sahoo, Amaresh Kumar; Sk, Md Palashuddin; Ghosh, Siddhartha Sankar; Chattopadhyay, Arun

2011-10-01

Herein, we report the generation of a composite comprised of p-hydroxyacetanilide dimer and Ag nanoparticles (NPs) by reaction of AgNO3 and p-hydroxyacetanilide. The formation of the composite was established by UV-vis, FTIR and NMR spectroscopy, transmission electron microscopy and X-ray diffraction along with substantiation by mass spectrometry. Interestingly, the composite exhibited an emission spectrum with a peak at 435 nm when excited by light of wavelength 320 nm. The composite showed superior antimicrobial activity with respect to its individual components against a wide range of Gram positive and Gram negative bacteria at relatively low concentrations of Ag NPs and at which there was no apparent cytotoxicity against mammalian cells. Our results suggest that the composite strongly interacted with the bacterial cell walls leading to cell bursting. Interestingly, enhancement in the reactive oxygen species (ROS) generation in bacteria was observed in the presence of the composite. It is proposed that the ROS generation led to oxidation of the dimer to N-acetyl-p-benzoquinone imine (NAPQI). The generated NAPQI acted as a DNA gyrase inhibitor causing cell death following linearization of DNA.Herein, we report the generation of a composite comprised of p-hydroxyacetanilide dimer and Ag nanoparticles (NPs) by reaction of AgNO3 and p-hydroxyacetanilide. The formation of the composite was established by UV-vis, FTIR and NMR spectroscopy, transmission electron microscopy and X-ray diffraction along with substantiation by mass spectrometry. Interestingly, the composite exhibited an emission spectrum with a peak at 435 nm when excited by light of wavelength 320 nm. The composite showed superior antimicrobial activity with respect to its individual components against a wide range of Gram positive and Gram negative bacteria at relatively low concentrations of Ag NPs and at which there was no apparent cytotoxicity against mammalian cells. Our results suggest that the

Distinct kinetics of human DNA ligases I, IIIalpha, IIIbeta, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair

Energy Technology Data Exchange (ETDEWEB)

Chen, Xi; Ballin, Jeff D.; Della-Maria, Julie; Tsai, Miaw-Sheue; White, Elizabeth J.; Tomkinson, Alan E.; Wilson, Gerald M.

2009-05-11

The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIII{beta} and the hLigIII{alpha}/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/XRCC4 molecules.

Modeling DNA

Science.gov (United States)

Robertson, Carol

2016-01-01

Deoxyribonucleic acid (DNA) is life's most amazing molecule. It carries the genetic instructions that almost every organism needs to develop and reproduce. In the human genome alone, there are some three billion DNA base pairs. The most difficult part of teaching DNA structure, however, may be getting students to visualize something as small as a…

Comprehensive preimplantation genetic screening and sperm deoxyribonucleic acid fragmentation from three males carrying balanced chromosome rearrangements.

Science.gov (United States)

Ramos, Laia; Daina, Gemma; Del Rey, Javier; Ribas-Maynou, Jordi; Fernández-Encinas, Alba; Martinez-Passarell, Olga; Boada, Montserrat; Benet, Jordi; Navarro, Joaquima

2015-09-01

To assess whether preimplantation genetic screening can successfully identify cytogenetically normal embryos in couples carrying balanced chromosome rearrangements in addition to increased sperm DNA fragmentation. Comprehensive preimplantation genetic screening was performed on three couples carrying chromosome rearrangements. Sperm DNA fragmentation was assessed for each patient. Academic center. One couple with the male partner carrying a chromosome 2 pericentric inversion and two couples with the male partners carrying a Robertsonian translocation (13:14 and 14:21, respectively). A single blastomere from each of the 18 cleavage-stage embryos obtained was analysed by metaphase comparative genomic hybridization. Single- and double-strand sperm DNA fragmentation was determined by the alkaline and neutral Comet assays. Single- and double-strand sperm DNA fragmentation values and incidence of chromosome imbalances in the blastomeres were analyzed. The obtained values of single-strand sperm DNA fragmentation were between 47% and 59%, and the double-strand sperm DNA fragmentation values were between 43% and 54%. No euploid embryos were observed in the couple showing the highest single-strand sperm DNA fragmentation. However, euploid embryos were observed in the other two couples: embryo transfer was performed, and pregnancy was achieved by the couple showing the lowest sperm DNA fragmentation values. Preimplantation genetic screening enables the detection of euploid embryos in couples affected by balanced chromosome rearrangements and increased sperm DNA fragmentation. Even though sperm DNA fragmentation may potentially have clinical consequences on fertility, comprehensive preimplantation genetic screening allows for the identification and transfer of euploid embryos. Copyright © 2015. Published by Elsevier Inc.

DNA is structured as a linear "jigsaw puzzle" in the genomes of Arabidopsis, rice, and budding yeast.

Science.gov (United States)

Liu, Yun-Hua; Zhang, Meiping; Wu, Chengcang; Huang, James J; Zhang, Hong-Bin

2014-01-01

Knowledge of how a genome is structured and organized from its constituent elements is crucial to understanding its biology and evolution. Here, we report the genome structuring and organization pattern as revealed by systems analysis of the sequences of three model species, Arabidopsis, rice and yeast, at the whole-genome and chromosome levels. We found that all fundamental function elements (FFE) constituting the genomes, including genes (GEN), DNA transposable elements (DTE), retrotransposable elements (RTE), simple sequence repeats (SSR), and (or) low complexity repeats (LCR), are structured in a nonrandom and correlative manner, thus leading to a hypothesis that the DNA of the species is structured as a linear "jigsaw puzzle". Furthermore, we showed that different FFE differ in their importance in the formation and evolution of the DNA jigsaw puzzle structure between species. DTE and RTE play more important roles than GEN, LCR, and SSR in Arabidopsis, whereas GEN and RTE play more important roles than LCR, SSR, and DTE in rice. The genes having multiple recognized functions play more important roles than those having single functions. These results provide useful knowledge necessary for better understanding genome biology and evolution of the species and for effective molecular breeding of rice.

Eukaryotic DNA Replication Fork.

Science.gov (United States)

Burgers, Peter M J; Kunkel, Thomas A

2017-06-20

This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

Reaction of misonidazole with DNA radicals and its effect on the template activity of DNA

International Nuclear Information System (INIS)

Endoh, Daiji; Kuwabara, Mikinori; Sato, Fumiaki; Yoshii, Giichi.

1985-01-01

After calf thymus DNA was gamma-irradiated in the solid state in vacuo and subsequently dissolved in aqueous solution containing misonidazole (3 mM) under hypoxic condition, the frequency of single-strand breaks and alkali-labile sites in DNA and the amount of misonidazole bound to DNA were measured. The presence of misonidazole converted the precursor radicals, which otherwise results in single-strand breaks, to alkali-labile sites, and the amount of alkali-labile sites increased linearly with increasing radiation dose. The amount of misonidazole bound to DNA also increased linearly with increasing radiation dose. The biological meaning of the changes in the frequency of single-strand breaks and alkali-labile sites by the reaction of misonidazole with DNA radicals and of binding misonidazole with DNA was examined using a model system to measure the template activity of DNA for RNA synthesis in vitro. The conversion of DNA radicals to alkali-labile sites protected the radiation-induced decrease in the template activity of DNA, while the adduct formation of misonidazole had no effect on it. (author)

DnaB gene product-independence of DNA polymerase III-directed repair synthesis in Escherichia coli K-12

International Nuclear Information System (INIS)

Billen, D.; Hellermann, G.R.

1977-01-01

An investigation has been carried out into the role of dnaB gene product in X-ray-induced repair synthesis carried out by DNA polymerase III in toluene-treated Escherichia coli K-12. A polAl polBlOO dnaB mutant deficient in both DNA polymerase I and II activities was used, and it was shown that the level of X-ray-induced, ATP-dependent, non-conservative DNA synthesis was, unlike semi-conservative DNA synthesis, unaffected by a temperature shift from 30 0 to 42 0 C. The dnaB gene product was not therefore necessary for DNA polymerase III-directed repair synthesis, which occurred in the absence of replicative synthesis. (U.K.)

Construction of expression vectors carrying mouse peroxisomal ...

African Journals Online (AJOL)

The aim of this study was to construct expression vectors carrying mouse peroxisomal protein gene (PEP-cDNA) in prokaryotic and mammalian expression vectors in ... pGEX6p2-PEP and pUcD3-FLAG-PEP constructed vectors were transformed into the one shot TOP10 and JM105 bacterial competent cells, respectively.

DNA maintenance in plastids and mitochondria of plants

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Delene J Oldenburg

2015-10-01

Full Text Available The DNA molecules in plastids and mitochondria of plants have been studied for over 40 years. Here, we review the data on the circular or linear form, replication, repair, and persistence of the organellar DNA (orgDNA in plants. The bacterial origin of orgDNA appears to have profoundly influenced ideas about the properties of chromosomal DNA molecules in these organelles to the point of dismissing data inconsistent with ideas from the 1970s. When found at all, circular genome-sized molecules comprise a few percent of orgDNA. In cells active in orgDNA replication, most orgDNA is found as linear and branched-linear forms larger than the size of the genome, likely a consequence of a virus-like DNA replication mechanism. In contrast to the stable chromosomal DNA molecules in bacteria and the plant nucleus, the molecular integrity of orgDNA declines during leaf development at a rate that varies among plant species. This decline is attributed to degradation of damaged-but-not-repaired molecules, with a proposed repair cost-saving benefit most evident in grasses. All orgDNA maintenance activities are proposed to occur on the nucleoid tethered to organellar membranes by developmentally-regulated proteins.

Evolution of linear chromosomes and multipartite genomes in yeast mitochondria

Science.gov (United States)

Valach, Matus; Farkas, Zoltan; Fricova, Dominika; Kovac, Jakub; Brejova, Brona; Vinar, Tomas; Pfeiffer, Ilona; Kucsera, Judit; Tomaska, Lubomir; Lang, B. Franz; Nosek, Jozef

2011-01-01

Mitochondrial genome diversity in closely related species provides an excellent platform for investigation of chromosome architecture and its evolution by means of comparative genomics. In this study, we determined the complete mitochondrial DNA sequences of eight Candida species and analyzed their molecular architectures. Our survey revealed a puzzling variability of genome architecture, including circular- and linear-mapping and multipartite linear forms. We propose that the arrangement of large inverted repeats identified in these genomes plays a crucial role in alterations of their molecular architectures. In specific arrangements, the inverted repeats appear to function as resolution elements, allowing genome conversion among different topologies, eventually leading to genome fragmentation into multiple linear DNA molecules. We suggest that molecular transactions generating linear mitochondrial DNA molecules with defined telomeric structures may parallel the evolutionary emergence of linear chromosomes and multipartite genomes in general and may provide clues for the origin of telomeres and pathways implicated in their maintenance. PMID:21266473

DNA damage, homology-directed repair, and DNA methylation.

Directory of Open Access Journals (Sweden)

Concetta Cuozzo

2007-07-01

Full Text Available To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP genes (DR-GFP. A total of 2%-4% of the cells generated a functional GFP by homology-directed repair (HR and gene conversion. However, approximately 50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2'-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

Self-assembled DNA Structures for Nanoconstruction

Science.gov (United States)

Yan, Hao; Yin, Peng; Park, Sung Ha; Li, Hanying; Feng, Liping; Guan, Xiaoju; Liu, Dage; Reif, John H.; LaBean, Thomas H.

2004-09-01

In recent years, a number of research groups have begun developing nanofabrication methods based on DNA self-assembly. Here we review our recent experimental progress to utilize novel DNA nanostructures for self-assembly as well as for templates in the fabrication of functional nano-patterned materials. We have prototyped a new DNA nanostructure known as a cross structure. This nanostructure has a 4-fold symmetry which promotes its self-assembly into tetragonal 2D lattices. We have utilized the tetragonal 2D lattices as templates for highly conductive metallic nanowires and periodic 2D protein nano-arrays. We have constructed and characterized a DNA nanotube, a new self-assembling superstructure composed of DNA tiles. We have also demonstrated an aperiodic DNA lattice composed of DNA tiles assembled around a long scaffold strand; the system translates information encoded in the scaffold strand into a specific and reprogrammable barcode pattern. We have achieved metallic nanoparticle linear arrays templated on self-assembled 1D DNA arrays. We have designed and demonstrated a 2-state DNA lattice, which displays expand/contract motion switched by DNA nanoactuators. We have also achieved an autonomous DNA motor executing unidirectional motion along a linear DNA track.

Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing.

Science.gov (United States)

Hu, Jiazhi; Meyers, Robin M; Dong, Junchao; Panchakshari, Rohit A; Alt, Frederick W; Frock, Richard L

2016-05-01

Unbiased, high-throughput assays for detecting and quantifying DNA double-stranded breaks (DSBs) across the genome in mammalian cells will facilitate basic studies of the mechanisms that generate and repair endogenous DSBs. They will also enable more applied studies, such as those to evaluate the on- and off-target activities of engineered nucleases. Here we describe a linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) method for the detection of genome-wide 'prey' DSBs via their translocation in cultured mammalian cells to a fixed 'bait' DSB. Bait-prey junctions are cloned directly from isolated genomic DNA using LAM-PCR and unidirectionally ligated to bridge adapters; subsequent PCR steps amplify the single-stranded DNA junction library in preparation for Illumina Miseq paired-end sequencing. A custom bioinformatics pipeline identifies prey sequences that contribute to junctions and maps them across the genome. LAM-HTGTS differs from related approaches because it detects a wide range of broken end structures with nucleotide-level resolution. Familiarity with nucleic acid methods and next-generation sequencing analysis is necessary for library generation and data interpretation. LAM-HTGTS assays are sensitive, reproducible, relatively inexpensive, scalable and straightforward to implement with a turnaround time of <1 week.

Nucleotide sequence analysis of regions of adenovirus 5 DNA containing the origins of DNA replication

International Nuclear Information System (INIS)

Steenbergh, P.H.

1979-01-01

The purpose of the investigations described is the determination of nucleotide sequences at the molecular ends of the linear adenovirus type 5 DNA. Knowledge of the primary structure at the termini of this DNA molecule is of particular interest in the study of the mechanism of replication of adenovirus DNA. The initiation- and termination sites of adenovirus DNA replication are located at the ends of the DNA molecule. (Auth.)

Noncoding DNA in lipofection of HeLa cells-a few insights.

Science.gov (United States)

Symens, Nathalie; Rejman, Joanna; Lucas, Bart; Demeester, Joseph; De Smedt, Stefaan C; Remaut, Katrien

2013-03-04

In cationic carrier-mediated gene delivery, the disproportional relationship between the quantity of delivered DNA and the amount of encoded protein produced is a well-known phenomenon. The numerous intracellular barriers which need to be overcome by pDNA to reach the nucleoplasm play a major role in it. In contrast to what one would expect, a partial replacement of coding pDNA by noncoding DNA does not lead to a decrease in transfection efficiency. The mechanism underlying this observation is still unclear. Therefore, we investigated which constituents of the transfection process might contribute to this phenomenon. Our data reveal that the topology of the noncoding plasmid DNA plays a major role. Noncoding pDNA can be used only in a supercoiled form to replace coding pDNA in Lipofectamine lipoplexes, without a loss in transfection levels. When noncoding pDNA is linearized or partly digested, it diminishes the transfection potential of coding pDNA, as does noncoding salmon DNA. The difference in transfection efficiencies could not be attributed to diverse physicochemical characteristics of the Lipofectamine lipoplexes containing different types of noncoding DNA or to the extent of their internalization. At the level of endosomal release, however, nucleic acid release from the endosomal compartment proceeds faster when lipoplexes contain noncoding salmon DNA. Since the half-life of pDNA in the cytosol hardly exceeds 90 min, it is conceivable that prolonged release of coding pDNA from complexes carrying supercoiled noncoding pDNA may explain its positive effect on transfection, while this depot effect does not exist when noncoding salmon DNA is used.

Exploration of methods to localize DNA sequences missing from c-locus deletions

International Nuclear Information System (INIS)

Albritton, L.M.; Russell, L.B.; Montgomery, C.S.

1987-01-01

The authors have earlier characterized a large number of radiation-induced mutations at the c locus (on Chromosome 7) through genetic analysis, including extensive complementation tests. Based on this work, they have postulated that many of these mutations are deletions of various lengths, overlapping at c (the marker used in the mutation-rate experiments that generated the mutants). It was possible to apportion these deletions among 13 complementation groups and to fit them to a linear map of 8 functional units. Collectively, the deletions extend from a point between tp and c to one between sh-1 and Hbb, i.e., a genetic distance of from 6 to 10 cM, corresponding to at least 10 4 Kb of DNA. This year, the authors completed a pilot study designed to explore methods for finding DNA sequences that map to the region covered by the various c-deletions. The general plan was to probe DNA with clones derived from Chromosome-7-enriched libraries or with sequences known (or suspected) to reside in Chromosome 7. Three methods were explored for deriving the c-region-deficient DNA: (a) from mouse-hamster somatic-cell hydrids retaining a deleted mouse Chromosome 7, but no homologue; (b) from F 1 hybrids of M. musculus domesticus (carrying a c-locus deletion) by M. spretus; and (c) from F 1 hybrids of M. domesticus stocks carrying complementing deletions

Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation

Energy Technology Data Exchange (ETDEWEB)

Banks, G.R.; Taylor, S.Y. (National Institute for Medical Research, London (England))

1988-12-01

The Ustilago maydis PYR3 gene encoding dihydroorotase activity was cloned by direct complementation of Escherichia coli pyrC mutations. PYR3 transformants of E. coli pyrC mutants expressed homologous transcripts of a variety of sizes and regained dihydroorotase activity. PYR3 also complemented Saccharomyces cerevisiae ura4 mutations, and again multiple transcripts were expressed in transformants, and enzyme activity was regained. A 1.25-kilobase poly(rA)+ PYR3 transcript was detected in U. maydis itself. Linear DNA carrying the PYR3 gene transformed a U. maydis pyr3-1 pyrimidine auxotroph to prototrophy. Hybridization analysis revealed that three different types of transformants could be generated, depending on the structure of the transforming DNA used. The first type involved exchange of chromosomal mutant gene sequences with the cloned wild-type plasmid sequences. A second type had integrated linear transforming DNA at the chromosomal PYR3 locus, probably via a single crossover event. The third type had integrated transforming DNA sequences at multiple sites in the U. maydis genome. In the last two types, tandemly reiterated copies of the transforming DNA were found to have been integrated. All three types had lost the sensitivity of the parental pyr3-1 mutant to UV irradiation. They had also regained dihydroorotase activity, although its level did not correlate with the PYR3 gene copy number.

Transfer of stem cells carrying engineered chromosomes with XY clone laser system.

Science.gov (United States)

Sinko, Ildiko; Katona, Robert L

2011-01-01

Current transgenic technologies for gene transfer into the germline of mammals cause a random integration of exogenous naked DNA into the host genome that can generate undesirable position effects as well as insertional mutations. The vectors used to generate transgenic animals are limited by the amount of foreign DNA they can carry. Mammalian artificial chromosomes have large DNA-carrying capacity and ability to replicate in parallel with, but without integration into, the host genome. Hence they are attractive vectors for transgenesis, cellular protein production, and gene therapy applications as well. ES cells mediated chromosome transfer by conventional blastocyst injection has a limitation in unpredictable germline transmission. The demonstrated protocol of laser-assisted microinjection of artificial chromosome containing ES cells into eight-cell mouse embryos protocol described here can solve the problem for faster production of germline transchromosomic mice.

Electron microscopic visualization of the RecA protein-mediated pairing and branch migration phases of DNA strand exchange

DEFF Research Database (Denmark)

Register, JC; Christiansen, Gunna; Griffith, J

1987-01-01

examined by electron microscopy: supertwisted double-stranded (ds) DNA and linear single-stranded (ss) DNA, linear dsDNA and circular ssDNA, and linear dsDNA and colinear ssDNA. Several major observations were: (i) with RecA protein bound to the DNA, plectonemic joints were ultrastructurally...

Conserved linear dynamics of single-molecule Brownian motion

KAUST Repository

Serag, Maged F.

2017-06-06

Macromolecular diffusion in homogeneous fluid at length scales greater than the size of the molecule is regarded as a random process. The mean-squared displacement (MSD) of molecules in this regime increases linearly with time. Here we show that non-random motion of DNA molecules in this regime that is undetectable by the MSD analysis can be quantified by characterizing the molecular motion relative to a latticed frame of reference. Our lattice occupancy analysis reveals unexpected sub-modes of motion of DNA that deviate from expected random motion in the linear, diffusive regime. We demonstrate that a subtle interplay between these sub-modes causes the overall diffusive motion of DNA to appear to conform to the linear regime. Our results show that apparently random motion of macromolecules could be governed by non-random dynamics that are detectable only by their relative motion. Our analytical approach should advance broad understanding of diffusion processes of fundamental relevance.

Conserved linear dynamics of single-molecule Brownian motion

Science.gov (United States)

Serag, Maged F.; Habuchi, Satoshi

2017-06-01

Macromolecular diffusion in homogeneous fluid at length scales greater than the size of the molecule is regarded as a random process. The mean-squared displacement (MSD) of molecules in this regime increases linearly with time. Here we show that non-random motion of DNA molecules in this regime that is undetectable by the MSD analysis can be quantified by characterizing the molecular motion relative to a latticed frame of reference. Our lattice occupancy analysis reveals unexpected sub-modes of motion of DNA that deviate from expected random motion in the linear, diffusive regime. We demonstrate that a subtle interplay between these sub-modes causes the overall diffusive motion of DNA to appear to conform to the linear regime. Our results show that apparently random motion of macromolecules could be governed by non-random dynamics that are detectable only by their relative motion. Our analytical approach should advance broad understanding of diffusion processes of fundamental relevance.

Conserved linear dynamics of single-molecule Brownian motion

KAUST Repository

Serag, Maged F.; Habuchi, Satoshi

2017-01-01

Macromolecular diffusion in homogeneous fluid at length scales greater than the size of the molecule is regarded as a random process. The mean-squared displacement (MSD) of molecules in this regime increases linearly with time. Here we show that non-random motion of DNA molecules in this regime that is undetectable by the MSD analysis can be quantified by characterizing the molecular motion relative to a latticed frame of reference. Our lattice occupancy analysis reveals unexpected sub-modes of motion of DNA that deviate from expected random motion in the linear, diffusive regime. We demonstrate that a subtle interplay between these sub-modes causes the overall diffusive motion of DNA to appear to conform to the linear regime. Our results show that apparently random motion of macromolecules could be governed by non-random dynamics that are detectable only by their relative motion. Our analytical approach should advance broad understanding of diffusion processes of fundamental relevance.

Synthesis and optical properties of pyrrolidinyl peptide nucleic acid carrying a clicked Nile red label

Directory of Open Access Journals (Sweden)

Nattawut Yotapan

2014-09-01

Full Text Available DNA or its analogues with an environment-sensitive fluorescent label are potentially useful as a probe for studying the structure and dynamics of nucleic acids. In this work, pyrrolidinyl peptide nucleic acid (acpcPNA was labeled at its backbone with Nile red, a solvatochromic benzophenoxazine dye, by means of click chemistry. The optical properties of the Nile red-labeled acpcPNA were investigated by UV–vis and fluorescence spectroscopy in the absence and in the presence of DNA. In contrast to the usual quenching observed in Nile red-labeled DNA, the hybridization with DNA resulted in blue shifting and an enhanced fluorescence regardless of the neighboring bases. More pronounced blue shifts and fluorescence enhancements were observed when the DNA target carried a base insertion in close proximity to the Nile red label. The results indicate that the Nile red label is located in a more hydrophobic environment in acpcPNA–DNA duplexes than in the single-stranded acpcPNA. The different fluorescence properties of the acpcPNA hybrids of complementary DNA and DNA carrying a base insertion are suggestive of different interactions between the Nile red label and the duplexes.

Blood extracellular DNA after irradiation

International Nuclear Information System (INIS)

Vladimirov, V.G.; Tishchenko, L.I.; Surkova, E.A.; Vasil'eva, I.N.

1993-01-01

It has been shown that blood extracellular DNA of irradiated rats largely consists of the low-molecular DNA and its oligomers. Molecular masses of oligomers are multiple to molecular mass of monomer fragment with nucleosome size. The low-molecular DNA has linear form. The average content of GC-pairs in low-molecular DNA is higher than in total rat's DNA (48.5% against 41.5%). The low-molecular DNA is a part of complex containing RNA, acidic proteins and lipids. It is assumed that the formation of low-molecular DNA is a result of Ca/Mg - dependent nuclear endonuclease action

Elimination Voltammetry with Linear Scan as a New Detection Method for DNA Sensors

Directory of Open Access Journals (Sweden)

Rene Kizek

2005-11-01

Full Text Available The paper describes successful coupling of adsorptive transfer stripping (AdTS andelimination voltammetry with linear scan (EVLS for the resolution of reduction signals of cytosine (Cand adenine (A residues in hetero-oligodeoxynucleotides (ODNs. Short ODNs (9-mers and 20-merswere adsorbed from a small volume on a hanging mercury drop electrode (HMDE. After washing ofthe ODN-modified electrode by water and its transferring to an electrochemical cell, voltammetric curves were measured. The AdTS EVLS was able to determine of C/A ratio of ODNs through theelimination function conserving the diffusion current component and eliminating kinetic and chargingcurrent components. This function, which provides the elimination signal in a peak-counterpeak form,increased the current sensitivity for A and C resolution, and for the recognition of bases sequences inODN chains. Optimal conditions of elimination experiments such as pH, time of adsorption, and scanrate were found. The combination of EVLS with AdTS procedure can be considered as a newdetection method in a DNA sensor.

Efficacy of a DNA Vaccine Carrying Eimeria maxima Gam56 Antigen Gene against Coccidiosis in Chickens

Science.gov (United States)

Xu, Jinjun; Zhang, Yan

2013-01-01

To control coccidiosis without using prophylactic medications, a DNA vaccine targeting the gametophyte antigen Gam56 from Eimeria maxima in chickens was constructed, and the immunogenicity and protective effects were evaluated. The ORF of Gam56 gene was cloned into an eukaryotic expression vector pcDNA3.1(zeo)+. Expression of Gam56 protein in COS-7 cells transfected with recombinant plasmid pcDNA-Gam56 was confirmed by indirect immunofluorescence assay. The DNA vaccine was injected intramuscularly to yellow feathered broilers of 1-week old at 3 dosages (25, 50, and 100 µg/chick). Injection was repeated once 1 week later. One week after the second injection, birds were challenged orally with 5×104 sporulated oocysts of E. maxima, then weighed and killed at day 8 post challenge. Blood samples were collected and examined for specific peripheral blood lymphocyte proliferation activity and serum antibody levels. Compared with control groups, the administration of pcDNA-Gam56 vaccine markedly increased the lymphocyte proliferation activity (P<0.05) at day 7 and 14 after the first immunization. The level of lymphocyte proliferation started to decrease on day 21 after the first immunization. A similar trend was seen in specific antibody levels. Among the 3 pcDNA-Gam56 immunized groups, the median dosage group displayed the highest lymphocyte proliferation and antibody levels (P<0.05). The median dosage group had the greatest relative body weight gain (89.7%), and the greatest oocyst shedding reduction (53.7%). These results indicate that median dosage of DNA vaccine had good immunogenicity and immune protection effects, and may be used in field applications for coccidiosis control. PMID:23710081

Radiation damage of DNA. Model for direct ionization of DNA

International Nuclear Information System (INIS)

Kobayashi, Kazuo; Tagawa, Seiichi

2004-01-01

Current aspects of radiation damage of DNA, particularly induced by the direct effect of radiation, and author's method of pulse radiolysis are described in relation to behavior of ions formed by radiation and active principles to induce the strand break. In irradiation of DNA solution in water, the direct effect of radiation is derived from ionization of DNA itself and indirect one, from the reaction between DNA and radicals generated from water molecules and the former direct one has been scarcely investigated due to difficulty of experimental approach. Radicals generated in sugar moiety of DNA are shown important in the strand break by recent studies on crystalline DNA irradiated by X-ray, DNA solution by electron and photon beams, hydrated DNA by γ-ray and by high linear energy transfer (LET) ion. Author's pulse radiolysis studies have revealed behaviors of guanine and adenine radical cations in dynamics of DNA oxidation. Since reactions described are the model, the experimental approach is thought necessary for elucidation of the actually occurring DNA damage in living cells. (N.I.)

Programmable motion of DNA origami mechanisms.

Science.gov (United States)

Marras, Alexander E; Zhou, Lifeng; Su, Hai-Jun; Castro, Carlos E

2015-01-20

DNA origami enables the precise fabrication of nanoscale geometries. We demonstrate an approach to engineer complex and reversible motion of nanoscale DNA origami machine elements. We first design, fabricate, and characterize the mechanical behavior of flexible DNA origami rotational and linear joints that integrate stiff double-stranded DNA components and flexible single-stranded DNA components to constrain motion along a single degree of freedom and demonstrate the ability to tune the flexibility and range of motion. Multiple joints with simple 1D motion were then integrated into higher order mechanisms. One mechanism is a crank-slider that couples rotational and linear motion, and the other is a Bennett linkage that moves between a compacted bundle and an expanded frame configuration with a constrained 3D motion path. Finally, we demonstrate distributed actuation of the linkage using DNA input strands to achieve reversible conformational changes of the entire structure on ∼ minute timescales. Our results demonstrate programmable motion of 2D and 3D DNA origami mechanisms constructed following a macroscopic machine design approach.

Programmable motion of DNA origami mechanisms

Science.gov (United States)

Marras, Alexander E.; Zhou, Lifeng; Su, Hai-Jun; Castro, Carlos E.

2015-01-01

DNA origami enables the precise fabrication of nanoscale geometries. We demonstrate an approach to engineer complex and reversible motion of nanoscale DNA origami machine elements. We first design, fabricate, and characterize the mechanical behavior of flexible DNA origami rotational and linear joints that integrate stiff double-stranded DNA components and flexible single-stranded DNA components to constrain motion along a single degree of freedom and demonstrate the ability to tune the flexibility and range of motion. Multiple joints with simple 1D motion were then integrated into higher order mechanisms. One mechanism is a crank–slider that couples rotational and linear motion, and the other is a Bennett linkage that moves between a compacted bundle and an expanded frame configuration with a constrained 3D motion path. Finally, we demonstrate distributed actuation of the linkage using DNA input strands to achieve reversible conformational changes of the entire structure on ∼minute timescales. Our results demonstrate programmable motion of 2D and 3D DNA origami mechanisms constructed following a macroscopic machine design approach. PMID:25561550

Mutagenic DNA repair in Escherichia coli: Pt. 17

International Nuclear Information System (INIS)

Sharif, F.; Bridges, B.A.

1990-01-01

In contrast to the dnaE485 mutation, which is nearly 'dead-stop', dnaE1026 allows DNA synthesis for some time at restrictive temperatures. When bacteria carrying the dnaE486 or dnaE1026 temperature-sensitive mutations were incubated at restrictive temperature after exposure to UV light they showed little or no fixation of mutations as determined by loss of photoreversibility and the mutation frequency fell progressively. These results confirm a role for DnaE protein in UV mutagenesis. A derivative of dnaE1026 carrying the umuC122 allele which blocks normal UV mutagenesis showed induction of mutations when photoreversing light was given to UV-irradiated bacteria after a period of incubation at either permissive or restrictive temperature. The defective DnaE1026 protein is therefore able to carry out the misincorporations postulated to be the first step in the mutagenic process. Its inability to give rise to mutations in umu + bacteria may therefore be attributed to its inability to participate in a later step. In contrast, UV-irradiated dnaE486 umuC122 bacteria did not show mutagenesis when incubated at restrictive temperature before photoreversal, suggesting that the altered DnaE486 protein was not able to carry out the postulate misincorporation step at 43 0 C. DNA polymerase III α-subunit therefore appears to be required for both the misincorporation and bypass steps in the two-step model for UV mutagenesis. (Author)

The effect of volume exclusion on the formation of DNA minicircle networks: implications to kinetoplast DNA

International Nuclear Information System (INIS)

Diao, Y; Hinson, K; Sun, Y; Arsuaga, J

2015-01-01

Kinetoplast DNA (kDNA) is the mitochondrial of DNA of disease causing organisms such as Trypanosoma Brucei (T. Brucei) and Trypanosoma Cruzi (T. Cruzi). In most organisms, KDNA is made of thousands of small circular DNA molecules that are highly condensed and topologically linked forming a gigantic planar network. In our previous work we have developed mathematical and computational models to test the confinement hypothesis, that is that the formation of kDNA minicircle networks is a product of the high DNA condensation achieved in the mitochondrion of these organisms. In these studies we studied three parameters that characterize the growth of the network topology upon confinement: the critical percolation density, the mean saturation density and the mean valence (i.e. the number of mini circles topologically linked to any chosen minicircle). Experimental results on insect-infecting organisms showed that the mean valence is equal to three, forming a structure similar to those found in medieval chain-mails. These same studies hypothesized that this value of the mean valence was driven by the DNA excluded volume. Here we extend our previous work on kDNA by characterizing the effects of DNA excluded volume on the three descriptive parameters. Using computer simulations of polymer swelling we found that (1) in agreement with previous studies the linking probability of two minicircles does not decrease linearly with the distance between the two minicircles, (2) the mean valence grows linearly with the density of minicircles and decreases with the thickness of the excluded volume, (3) the critical percolation and mean saturation densities grow linearly with the thickness of the excluded volume. Our results therefore suggest that the swelling of the DNA molecule, due to electrostatic interactions, has relatively mild implications on the overall topology of the network. Our results also validate our topological descriptors since they appear to reflect the changes in the

The effect of volume exclusion on the formation of DNA minicircle networks: implications to kinetoplast DNA

Science.gov (United States)

Diao, Y.; Hinson, K.; Sun, Y.; Arsuaga, J.

2015-10-01

Kinetoplast DNA (kDNA) is the mitochondrial of DNA of disease causing organisms such as Trypanosoma Brucei (T. Brucei) and Trypanosoma Cruzi (T. Cruzi). In most organisms, KDNA is made of thousands of small circular DNA molecules that are highly condensed and topologically linked forming a gigantic planar network. In our previous work we have developed mathematical and computational models to test the confinement hypothesis, that is that the formation of kDNA minicircle networks is a product of the high DNA condensation achieved in the mitochondrion of these organisms. In these studies we studied three parameters that characterize the growth of the network topology upon confinement: the critical percolation density, the mean saturation density and the mean valence (i.e. the number of mini circles topologically linked to any chosen minicircle). Experimental results on insect-infecting organisms showed that the mean valence is equal to three, forming a structure similar to those found in medieval chain-mails. These same studies hypothesized that this value of the mean valence was driven by the DNA excluded volume. Here we extend our previous work on kDNA by characterizing the effects of DNA excluded volume on the three descriptive parameters. Using computer simulations of polymer swelling we found that (1) in agreement with previous studies the linking probability of two minicircles does not decrease linearly with the distance between the two minicircles, (2) the mean valence grows linearly with the density of minicircles and decreases with the thickness of the excluded volume, (3) the critical percolation and mean saturation densities grow linearly with the thickness of the excluded volume. Our results therefore suggest that the swelling of the DNA molecule, due to electrostatic interactions, has relatively mild implications on the overall topology of the network. Our results also validate our topological descriptors since they appear to reflect the changes in the

Studies on the mechanism of replication of adenovirus DNA. III. Electron microscopy of replicating DNA

NARCIS (Netherlands)

Ellens, D.J.; Sussenbach, J.S.; Jansz, H.S.

1974-01-01

Replicating Ad5 DNA was isolated from nuclei of infected KB cells and studied by electron microscopy. Branched as well as unbranched linear intermediates were observed containing extended regions of single-stranded DNA. The relationship between the branched and unbranched structures was studied

Insights into the quality of DnaA boxes and their cooperativity

DEFF Research Database (Denmark)

Hansen, Flemming G.; Christensen, Bjarke Bak; Nielsen, Christina Bang

2006-01-01

Plasmids carrying the mioC promoter region with its two DnaA boxes are as efficient in titration of DnaA protein as plasmids carrying a replicationinactivated oriC region with its five DnaA boxes. The two DnaA boxes upstream of the mioC promoter were mutated in various ways to study the cooperati......Plasmids carrying the mioC promoter region with its two DnaA boxes are as efficient in titration of DnaA protein as plasmids carrying a replicationinactivated oriC region with its five DnaA boxes. The two DnaA boxes upstream of the mioC promoter were mutated in various ways to study...... the cooperativity between the DnaA boxes, and to study in vivo the in vitrodefined 9mer DnaA box consensus sequence TTA/TTNCACA). The quality and cooperativity of the DnaA oxes were determined in two complementary ways: as titration of DnaA protein leading to derepression of the dnaA promoter, and as repression...... of the mioC promoter caused by the DnaA protein binding to the DnaA boxes. Titration of DnaA protein correlated with repression of the mioC promoter. The level of titration and repression with the normal promoter-proximal box (TTTTCCACA) depends strongly on the presence and the quality of a DnaA box...

Quantized, piecewise linear filter network

DEFF Research Database (Denmark)

Sørensen, John Aasted

1993-01-01

A quantization based piecewise linear filter network is defined. A method for the training of this network based on local approximation in the input space is devised. The training is carried out by repeatedly alternating between vector quantization of the training set into quantization classes...... and equalization of the quantization classes linear filter mean square training errors. The equalization of the mean square training errors is carried out by adapting the boundaries between neighbor quantization classes such that the differences in mean square training errors are reduced...

Phosphate vibrations as reporters of DNA hydration

Science.gov (United States)

Corcelli, Steven

The asymmetric phosphate stretch vibrational frequency is extraordinarily sensitive to its local solvent environment. Using density functional theory calculations on the model compound dimethyl phosphate, the asymmetric phosphate stretch vibrational frequency was found to shift linearly with the magnitude of an electric field along the symmetry axis of the PO2 moiety (i.e. the asymmetric phosphate stretch is an excellent linear vibrational Stark effect probe). With this linear relationship established, asymmetric phosphate stretch vibrational frequencies were computed during the course of a molecular dynamics simulation of fully hydrated DNA. Moreover, contributions to shifts in the frequencies from subpopulations of water molecules (e.g. backbone, minor groove, major groove, etc.) were calculated to reveal how phosphate vibrations report the onset of DNA hydration in experiments that vary the relative humidity of non-condensing (dry) DNA samples.

DNA adducts-chemical addons

Directory of Open Access Journals (Sweden)

T R Rajalakshmi

2015-01-01

Full Text Available DNA adduct is a piece of DNA covalently bond to a chemical (safrole, benzopyrenediol epoxide, acetaldehyde. This process could be the start of a cancerous cell. When a chemical binds to DNA, it gets damaged resulting in abnormal replication. This could be the start of a mutation and without proper DNA repair, this can lead to cancer. It is this chemical that binds with the DNA is our prime area of concern. Instead of performing the whole body analysis for diagnosing cancer, this test could be carried out for early detection of cancer. When scanning tunneling microscope is used, the DNA results can be obtained earlier. DNA adducts in scientific experiments are used as biomarkers.

PFGE analysis of DNA double-strand breaks and DNA repair process in human osteosarcoma cells irradiated by X-ray

International Nuclear Information System (INIS)

Cao Jianping; Majima, H.; Yamaguchi, C.

2000-01-01

Objective: To study the induction of DNA double-strand breaks (DSBs) in human osteosarcoma cells irradiated by X-ray, the DNA DSBs repair process and the tumour cell radiosensitivity. Methods: Two cell lines of human osteosarcoma, Rho0 and 143. B were used. Initial DNA damage of DSBs by X-ray irradiation was measured using clamped homogeneous electrical field (CHEF) electrophoresis. Results: X-ray-induced DNA DSBs of human osteosarcoma cells after CHEF-electrophoresis increased linearly with the irradiation dose between 0 and 50 Gy. The repair of DNA DSBs in human osteosarcoma cells increased with the post-irradiation incubation time. In contrast to 14.3B cell line at the same dose point, much more DNA DSBs were induced in Rho0 cell line after X-ray irradiation. Conclusion: CHEF pulsed-field gel electrophoresis (PEGE) is a sensitive method for the determination of radiation-induced DNA DSBs in high molecular weight DNA of human osteosarcoma cells. Radiation-induced DNA DSBs of osteosarcoma increase with the dose in a linear manner. After incubation, both Rho0 cell line and 143. B cell line can repair the DNA DSBs. Between two cell lines of human osteosarcoma, Rho0 and 143.B, Rho0 cell line is more sensitive to ionizing radiation than 143.B line

The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli.

Science.gov (United States)

Burgers, P M; Kornberg, A; Sakakibara, Y

1981-09-01

An Escherichia coli mutant, dnaN59, stops DNA synthesis promptly upon a shift to a high temperature; the wild-type dnaN gene carried in a transducing phage encodes a polypeptide of about 41,000 daltons [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553; Yuasa, S. & Sakakibara, Y. (1980) Mol. Gen. Genet. 180, 267-273]. We now find that the product of dnaN gene is the beta subunit of DNA polymerase III holoenzyme, the principal DNA synthetic multipolypeptide complex in E. coli. The conclusion is based on the following observations: (i) Extracts from dnaN59 cells were defective in phage phi X174 and G4 DNA synthesis after the mutant cells had been exposed to the increased temperature. (ii) The enzymatic defect was overcome by addition of purified beta subunit but not by other subunits of DNA polymerase III holoenzyme or by other replication proteins required for phi X174 DNA synthesis. (iii) Partially purified beta subunit from the dnaN mutant, unlike that from the wild type, was inactive in reconstituting the holoenzyme when mixed with the other purified subunits. (iv) Increased dosage of the dnaN gene provided by a plasmid carrying the gene raised cellular levels of the beta subunit 5- to 6-fold.

Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

Directory of Open Access Journals (Sweden)

Nynne Sharma

2013-01-01

Full Text Available DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system.

Linear sweep polarographic determination of nucleic acids using acridine orange as a bioprobe

Directory of Open Access Journals (Sweden)

WEI SUN

2007-11-01

Full Text Available The interaction of acridine orange (AO with double-stranded (ds DNA in aqueous solution was investigated by linear sweep polarography (LSP on a dropping mercury working electrode (DME. In pH 2.5 Britton–Robinson (B–R buffer solution, AO had a sensitive linear sweep polarographic reductive peak at –0.89 V (vs. SCE, which could be greatly inhibited by the addition of dsDNA, with a positive shift of the peak potential. Based on the decrease of the reductive peak current, a new quantitative electrochemical determination method for dsDNA was developed with a linear range of 2.0−20.0 mg l-1 and the linear regression equation: ΔIp” (nA = 111.90 C (mg l-1+125.32 (n = 9, γ = 0.997. The influences of commonly co-existing substances, such as metal ions, amino acid, etc., on the determination were also investigated. The method is sensitive, rapid and simple with good selectivity. The new proposed method was further applied to the detection of RNA and three synthetic samples containing dsDNA with satisfactory results. The binding number and the equilibrium constant between dsDNA and AO were calculated by an electrochemical method.

Specificity of DNA import into isolated mitochondria from plants and mammals

Directory of Open Access Journals (Sweden)

Koulintchenko M. V.

2014-01-01

Full Text Available Aim. Investigation of different features of DNA import into plant and human mitochondria, for a better understanding of mitochondrial genetics and generation of biotechnological tools. Methods. DNA up-take experiments with isolated plant mitochondria, using as substrates various sequences associated or not with the specific terminal inverted repeats (TIRs present at each end of the plant mitochondrial linear plasmids. Results. It was established that the DNA import efficiency has a non-linear dependence on DNA size. It was shown that import into plant mitochondria of DNA molecules of «medium» sizes, i. e. between 4 and 7 kb, barely has any sequence specificity: neither TIRs from the 11.6 kb Brassica plasmid, nor TIRs from the Zea mays S-plasmids influenced DNA import into Solanum tuberosum mitochondria. Conclusions. The data obtained support the hypothesis about species-specific import mechanism operating under the mitochondrial linear plasmids transfer into plant mitochondria.

Psoralens cleave pBR322 DNA under ultraviolet radiation

International Nuclear Information System (INIS)

Kagan, J.; Xinsheng Chen; Wang, T.P.

1992-01-01

Supercoiled (SC) pBR322 was used to probe the recent claim that 5-geranoxylpsoralen (5-GOP) did not photoreact with DNA. Contrary to expectations, 5-GOP was found to damage DNA in the presence of UV-A through two competing pathways; (a) single strand breaks, identified by the conversion of supercoiled into open circular and linear DNA, and (b) cross-linking, revealed by the fluence-dependent decrease in the extent of denaturation of the double stranded supercoiled DNA to single stranded circular DNA. In addition, a fluence-dependent modification reduced the ability of the restriction enzyme EcoR I to linearize the photosensitized DNA, and alkali-labile lesions were generated. Psoralen, 5-methoxypsoralen, and 8-methoxypsoralen, which are well-known to undergo cycloaddition to DNA, had a more pronounced effect on supercoiled DNA. Single strand breaks occurred more readily than with 5-GOP, and the surviving SC form remaining had reduced electrophoretic mobility in agarose gels. In all cases, the DNA damage was more prominent when oxygen was absent. (author)

DNA polymerase zeta cooperates with polymerases kappa and iota in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients.

Science.gov (United States)

Ziv, Omer; Geacintov, Nicholas; Nakajima, Satoshi; Yasui, Akira; Livneh, Zvi

2009-07-14

Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase eta, the POLH gene product. A deficiency in DNA polymerase eta due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase zeta cooperates with DNA polymerases kappa and iota to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases zeta and kappa, but not iota, protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load.

DNA polymerase ζ cooperates with polymerases κ and ι in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients

Science.gov (United States)

Ziv, Omer; Geacintov, Nicholas; Nakajima, Satoshi; Yasui, Akira; Livneh, Zvi

2009-01-01

Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase η, the POLH gene product. A deficiency in DNA polymerase η due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase ζ cooperates with DNA polymerases κ and ι to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases ζ and κ, but not ι, protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load. PMID:19564618

Radicals of DNA and DNA nucleotides generated by ionising radiation

International Nuclear Information System (INIS)

Przybytniak, G.

2004-01-01

A first stage of cell processes leading to DNA damage of initiated by radical reactions. In a model system such transformations were generated by ionising radiation which involves production of electron loss and electron gain centers of the substrate and radical formation. Using cryogenic ESR spectroscopy it was found that the DNA nucleotides, which convert to radical anions upon electron capture undergo the separation of unpaired spin and charge due to protonation. Circular and linear dichroism studies enabled to conclude that iron ions(III) induce strong changes in the DNA helical structure indicating their coordination with nitrogen bases. The repair of DNA radicals produced via radiolytic oxidation, i.e. the guanine radical cation and the allyl type radical of thymine, is possible at elevated temperatures due to the involvement of sulphydryl groups. The influence of the thiol charge is then limited

A DNA biosensor based on gold nanoparticle decorated on carboxylated multi-walled carbon nanotubes for gender determination of Arowana fish.

Science.gov (United States)

Saeedfar, Kasra; Heng, Lee Yook; Chiang, Chew Poh

2017-12-01

Multi-wall carbon nanotubes (MWCNTs) were modified to design a new DNA biosensor. Functionalized MWCNTs were equipped with gold nanoparticles (GNPs) (~15nm) (GNP-MWCNTCOOH) to construct DNA biosensors based on carbon-paste screen-printed (SPE) electrodes. GNP attachment onto functionalized MWCNTs was carried out by microwave irradiation and was confirmed by spectroscopic studies and surface analysis. DNA biosensors based on differential pulse voltammetry (DPV) were constructed by immobilizing thiolated single-stranded DNA probes onto GNP-MWCNTCOOH. Ruthenium (III) chloride hexaammoniate [Ru(NH 3 ) 6 ,2Cl - ] (RuHex) was used as hybridization redox indicator. RuHex and MWCNT interaction was low in compared to other organic redox hybridization indicators. The linear response range for DNA determination was 1×10 -21 to 1×10 -9 M with a lower detection limit of 1.55×10 -21 M. Thus, the attachment of GNPs onto functionalized MWCNTs yielded sensitive DNA biosensor with low detection limit and stability more than 30days. Constructed electrode was used to determine gender of arowana fish. Copyright © 2017 Elsevier B.V. All rights reserved.

DNA probe for strain typing of Cryptococcus neoformans.

OpenAIRE

Varma, A; Kwon-Chung, K J

1992-01-01

A 7-kb linear plasmid, harbored by a URA5 transformant, hybridized to all the chromosomes of Cryptococcus neoformans separated by contour-clamped homogeneous electric field electrophoresis. Its linear maintenance was determined to have been facilitated by the presence of telomere-like sequences at its free ends. Hybridization of this plasmid to AccI-digested genomic DNAs of 26 C. neoformans strains generated 21 unique DNA fingerprints. The DNA fingerprints of isolates within the same serotype...

DNA confinement in nanochannels: physics and biological applications

DEFF Research Database (Denmark)

Reisner, Walter; Pedersen, Jonas Nyvold; Austin, Robert H

2012-01-01

in nanochannels, creating a linear unscrolling of the genome along the channel for analysis. We will first review the fundamental physics of DNA nanochannel confinement—including the effect of varying ionic strength—and then discuss recent applications of these systems to genomic mapping. Apart from the intense...... direct assessment of the genome in its native state). In this review, we will discuss how the information contained in genomic-length single DNA molecules can be accessed via physical confinement in nanochannels. Due to self-avoidance interactions, DNA molecules will stretch out when confined...... biological interest in extracting linear sequence information from elongated DNA molecules, from a physics view these systems are fascinating as they enable probing of single-molecule conformation in environments with dimensions that intersect key physical length-scales in the 1 nm to 100μm range. (Some...

Structure of human DNA polymerase iota and the mechanism of DNA synthesis.

Science.gov (United States)

Makarova, A V; Kulbachinskiy, A V

2012-06-01

Cellular DNA polymerases belong to several families and carry out different functions. Highly accurate replicative DNA polymerases play the major role in cell genome replication. A number of new specialized DNA polymerases were discovered at the turn of XX-XXI centuries and have been intensively studied during the last decade. Due to the special structure of the active site, these enzymes efficiently perform synthesis on damaged DNA but are characterized by low fidelity. Human DNA polymerase iota (Pol ι) belongs to the Y-family of specialized DNA polymerases and is one of the most error-prone enzymes involved in DNA synthesis. In contrast to other DNA polymerases, Pol ι is able to use noncanonical Hoogsteen interactions for nucleotide base pairing. This allows it to incorporate nucleotides opposite various lesions in the DNA template that impair Watson-Crick interactions. Based on the data of X-ray structural analysis of Pol ι in complexes with various DNA templates and dNTP substrates, we consider the structural peculiarities of the Pol ι active site and discuss possible mechanisms that ensure the unique behavior of the enzyme on damaged and undamaged DNA.

Physical association of pyrimidine dimer DNA glycosylase and apurinic/apyrimidinic DNA endonuclease essential for repair of ultraviolet-damaged DNA

International Nuclear Information System (INIS)

Nakabeppu, Y.; Sekiguchi, M.

1981-01-01

T4 endonuclease, which is involved in repair of uv-damaged DNA, has been purified to apparent physical homogeneity. Incubation of uv-irradiated poly(dA).poly(dT) with the purified enzyme preparations resulted in production of alkali-labile apyrimidinic sites, followed by formation of nicks in the polymer. By performing a limited reaction with T4 endonuclease V at pH 8.5, irradiated polymer was converted to an intermediate form that carried a large number of alkali-labile sites but only a few nicks. The intermediate was used as substrate for the assay of apurinic/apyrimidinic DNA endonuclease activity. The two activities, a pyrimidine dimer DNA glycosylase and an apurinic/apyrimidinic DNA endonuclease, were copurified and found in enzyme preparations that contained only a 16,000-dalton polypeptide. These results strongly suggested that a DNA glycosylase specific for pyrimidine dimers and an apurinic/apyrimidinic DNA endonuclease reside in a single polypeptide chain coded by the denV gene of bacteriophage T4

Genomic analysis of Bacillus subtilis lytic bacteriophage ϕNIT1 capable of obstructing natto fermentation carrying genes for the capsule-lytic soluble enzymes poly-γ-glutamate hydrolase and levanase.

Science.gov (United States)

Ozaki, Tatsuro; Abe, Naoki; Kimura, Keitarou; Suzuki, Atsuto; Kaneko, Jun

2017-01-01

Bacillus subtilis strains including the fermented soybean (natto) starter produce capsular polymers consisting of poly-γ-glutamate and levan. Capsular polymers may protect the cells from phage infection. However, bacteriophage ϕNIT1 carries a γ-PGA hydrolase gene (pghP) that help it to counteract the host cell's protection strategy. ϕNIT had a linear double stranded DNA genome of 155,631-bp with a terminal redundancy of 5,103-bp, containing a gene encoding an active levan hydrolase. These capsule-lytic enzyme genes were located in the possible foreign gene cluster regions between central core and terminal redundant regions, and were expressed at the late phase of the phage lytic cycle. All tested natto origin Spounavirinae phages carried both genes for capsule degrading enzymes similar to ϕNIT1. A comparative genomic analysis revealed the diversity among ϕNIT1 and Bacillus phages carrying pghP-like and levan-hydrolase genes, and provides novel understanding on the acquisition mechanism of these enzymatic genes.

DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair

Directory of Open Access Journals (Sweden)

Elisa Mentegari

2016-08-01

Full Text Available DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell’s genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair.

Science.gov (United States)

Mentegari, Elisa; Kissova, Miroslava; Bavagnoli, Laura; Maga, Giovanni; Crespan, Emmanuele

2016-08-31

DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell's genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

Enzymatic properties of the bacteriophage phi X174 A protein on superhelical phi X174 DNA: a model for the termination of the rolling circle DNA replication

NARCIS (Netherlands)

van der Ende, A.; Langeveld, S. A.; Teertstra, R.; van Arkel, G. A.; Weisbeek, P. J.

1981-01-01

Incubation of phi X174 replication form I DNA with the A* protein of phi X174 in the presence of MN2+ results in the formation of three different types of DNA molecules: open circular form DNA (RFII), linear form DNA (RFIII) and the relaxed covalently closed form DNA (RFIV). The RFII and RFIII DNAs

Cellular Uptake of Tile-Assembled DNA Nanotubes.

Science.gov (United States)

Kocabey, Samet; Meinl, Hanna; MacPherson, Iain S; Cassinelli, Valentina; Manetto, Antonio; Rothenfusser, Simon; Liedl, Tim; Lichtenegger, Felix S

2014-12-30

DNA-based nanostructures have received great attention as molecular vehicles for cellular delivery of biomolecules and cancer drugs. Here, we report on the cellular uptake of tubule-like DNA tile-assembled nanostructures 27 nm in length and 8 nm in diameter that carry siRNA molecules, folic acid and fluorescent dyes. In our observations, the DNA structures are delivered to the endosome and do not reach the cytosol of the GFP -expressing HeLa cells that were used in the experiments. Consistent with this observation, no elevated silencing of the GFP gene could be detected. Furthermore, the presence of up to six molecules of folic acid on the carrier surface did not alter the uptake behavior and gene silencing. We further observed several challenges that have to be considered when performing in vitro and in vivo experiments with DNA structures: (i) DNA tile tubes consisting of 42 nt-long oligonucleotides and carrying single- or double-stranded extensions degrade within one hour in cell medium at 37 °C, while the same tubes without extensions are stable for up to eight hours. The degradation is caused mainly by the low concentration of divalent ions in the media. The lifetime in cell medium can be increased drastically by employing DNA tiles that are 84 nt long. (ii) Dyes may get cleaved from the oligonucleotides and then accumulate inside the cell close to the mitochondria, which can lead to misinterpretation of data generated by flow cytometry and fluorescence microscopy. (iii) Single-stranded DNA carrying fluorescent dyes are internalized at similar levels as the DNA tile-assembled tubes used here.

Linear ubiquitin chain induces apoptosis and inhibits tumor growth.

Science.gov (United States)

Qin, Zhoushuai; Jiang, Wandong; Wang, Guifen; Sun, Ying; Xiao, Wei

2018-01-01

Ubiquitination of proliferating cell nuclear antigen (PCNA) plays an important role in DNA damage response. Ectopic expression of PCNA fused at either terminus with ubiquitin (Ub) lacking two C-terminal glycine residues induces translesion DNA synthesis which resembles synthesis mediated by PCNA monoubiquitination. PCNA fused with Ub containing the C-terminal Gly residues at the C-terminus can be further polyubiquitinated in a Gly-dependent manner, which inhibits cell proliferation and induces ATR-dependent replication checkpoint. In this study, we surprisingly found that PCNA fused to a head-to-tail linear Ub chain induces apoptosis in a Ub chain length-dependent manner. Further investigation revealed that the apoptotic effect is actually induced by the linear Ub chain independently from PCNA, as the Ub chain fused to GFP or an epitope tag still efficiently induces apoptosis. It is revealed that the artificial linear Ub chain differs from endogenously encoded linear Ub chains in that its Ubs contain a Ub-G76S substitution, making the Ub chain resistant to cleavage by deubiquitination enzymes. We demonstrated in this study that ectopic expression of the artificial Ub chain alone in cultured human cancer cells is sufficient to inhibit tumor growth in a xenograft mouse model, making the linear Ub chain a putative anti-cancer agent.

DNA damage induced by radionuclide internal irradiation

International Nuclear Information System (INIS)

Cui Fengmei; Zhao Jingyong; Hong Chengjiao; Lao Qinhua; Wang Liuyi; Yang Shuqin

2004-01-01

Objective: To study the DNA damage of peripheral blood mononuclear cell (PBMC) in rats exposed to radionuclide internal irradiation. Methods: The radionuclides were injected into the rats and single cell get electrophoresis (SCGE) was performed to detect the length of DNA migration in the rat PBMC. Results: DNA migration in the rat PBMC increased with accumulative dose or dose-rate. It showed good relationship of dose vs. response and of dose-rate vs. response, both relationship could be described as linear models. Conclusion: Radionuclide internal irradiation could cause DNA damage in rat PBMC. (authors)

An extended sequence specificity for UV-induced DNA damage.

Science.gov (United States)

Chung, Long H; Murray, Vincent

2018-01-01

The sequence specificity of UV-induced DNA damage was determined with a higher precision and accuracy than previously reported. UV light induces two major damage adducts: cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs). Employing capillary electrophoresis with laser-induced fluorescence and taking advantages of the distinct properties of the CPDs and 6-4PPs, we studied the sequence specificity of UV-induced DNA damage in a purified DNA sequence using two approaches: end-labelling and a polymerase stop/linear amplification assay. A mitochondrial DNA sequence that contained a random nucleotide composition was employed as the target DNA sequence. With previous methodology, the UV sequence specificity was determined at a dinucleotide or trinucleotide level; however, in this paper, we have extended the UV sequence specificity to a hexanucleotide level. With the end-labelling technique (for 6-4PPs), the consensus sequence was found to be 5'-GCTC*AC (where C* is the breakage site); while with the linear amplification procedure, it was 5'-TCTT*AC. With end-labelling, the dinucleotide frequency of occurrence was highest for 5'-TC*, 5'-TT* and 5'-CC*; whereas it was 5'-TT* for linear amplification. The influence of neighbouring nucleotides on the degree of UV-induced DNA damage was also examined. The core sequences consisted of pyrimidine nucleotides 5'-CTC* and 5'-CTT* while an A at position "1" and C at position "2" enhanced UV-induced DNA damage. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Microwave Radiometer Linearity Measured by Simple Means

DEFF Research Database (Denmark)

Skou, Niels

2002-01-01

Modern spaceborne radiometer systems feature an almost perfect on-board calibration, hence the primary calibration task to be carried out before launch is a check of radiometer linearity. This paper describes two ways of measuring linearity of microwave radiometers only requiring relatively simple...

Intracellular responsive dual delivery by endosomolytic polyplexes carrying DNA anchored porous silicon nanoparticles.

Science.gov (United States)

Shahbazi, Mohammad-Ali; Almeida, Patrick Vingadas; Correia, Alexandra; Herranz-Blanco, Barbara; Shrestha, Neha; Mäkilä, Ermei; Salonen, Jarno; Hirvonen, Jouni; Santos, Hélder A

2017-03-10

Bioresponsive cytosolic nanobased multidelivery has been emerging as an enormously challenging novel concept due to the intrinsic protective barriers of the cells and hardly controllable performances of nanomaterials. Here, we present a new paradigm to advance nano-in-nano integration technology amenable to create multifunctional nanovehicles showing considerable promise to overcome restrictions of intracellular delivery, solve impediments of endosomal localization and aid effectual tracking of nanoparticles. A redox responsive intercalator chemistry comprised of cystine and 9-aminoacridine is designed as a cross-linker to cap carboxylated porous silicon nanoparticles with DNA. These intelligent nanocarriers are then encapsulated within novel one-pot electrostatically complexed nano-networks made of a zwitterionic amino acid (cysteine), an anionic bioadhesive polymer (poly(methyl vinyl ether-alt-maleic acid)) and a cationic endosomolytic polymer (polyethyleneimine). This combined nanocomposite is successfully tested for the co-delivery of hydrophobic (sorafenib) or hydrophilic (calcein) molecules loaded within the porous core, and an imaging agent covalently integrated into the polyplex shell by click chemistry. High loading capacity, low cyto- and hemo-toxicity, glutathione responsive on-command drug release, and superior cytosolic delivery are shown as achievable key features of the proposed formulation. Overall, formulating drug molecules, DNA and imaging agents, without any interference, in a physico-chemically optimized carrier may open a path towards broad applicability of these cost-effective multivalent nanocomposites for treating different diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Performance verification and comparison of TianLong automatic hypersensitive hepatitis B virus DNA quantification system with Roche CAP/CTM system.

Science.gov (United States)

Li, Ming; Chen, Lin; Liu, Li-Ming; Li, Yong-Li; Li, Bo-An; Li, Bo; Mao, Yuan-Li; Xia, Li-Fang; Wang, Tong; Liu, Ya-Nan; Li, Zheng; Guo, Tong-Sheng

2017-10-07

To investigate and compare the analytical and clinical performance of TianLong automatic hypersensitive hepatitis B virus (HBV) DNA quantification system and Roche CAP/CTM system. Two hundred blood samples for HBV DNA testing, HBV-DNA negative samples and high-titer HBV-DNA mixture samples were collected and prepared. National standard materials for serum HBV and a worldwide HBV DNA panel were employed for performance verification. The analytical performance, such as limit of detection, limit of quantification, accuracy, precision, reproducibility, linearity, genotype coverage and cross-contamination, was determined using the TianLong automatic hypersensitive HBV DNA quantification system (TL system). Correlation and Bland-Altman plot analyses were carried out to compare the clinical performance of the TL system assay and the CAP/CTM system. The detection limit of the TL system was 10 IU/mL, and its limit of quantification was 30 IU/mL. The differences between the expected and tested concentrations of the national standards were less than ± 0.4 Log 10 IU/mL, which showed high accuracy of the system. Results of the precision, reproducibility and linearity tests showed that the multiple test coefficient of variation (CV) of the same sample was less than 5% for 10 2 -10 6 IU/mL; and for 30-10 8 IU/mL, the linear correlation coefficient r 2 = 0.99. The TL system detected HBV DNA (A-H) genotypes and there was no cross-contamination during the "checkerboard" test. When compared with the CAP/CTM assay, the two assays showed 100% consistency in both negative and positive sample results (15 negative samples and 185 positive samples). No statistical differences between the two assays in the HBV DNA quantification values were observed ( P > 0.05). Correlation analysis indicated a significant correlation between the two assays, r 2 = 0.9774. The Bland-Altman plot analysis showed that 98.9% of the positive data were within the 95% acceptable range, and the maximum difference

Protection of free-radical induced DNA strand breaks in vitro by flavonoids

International Nuclear Information System (INIS)

Fisher, L.; Anderson, R.F.

1998-01-01

Full text: We have used both plasmid and cosmid test systems to assay the effect of antioxidant flavonoids (AO) on DNA strand breakage in supercoiled closed circular DNA (DNA SC ) following the formation oxidative radical damage on DNA (DNA OXID + . ) in aqueous solution. Single strand breaks in DNA SC result in the formation of the relaxed circular form (DNA RC ) and double strand breaks give linear DNA (DNA L ). Dose response curves were constructed for the log of the loss of [DNA S C] against dose (0-600 Gy). The D 37 (dose for 37% unchanged DNA SC ) values determined in the presence of increasing amounts of flavonoids were compared as ratios to the D 37 control value to give dose modification factor (DMF). Irradiations were carried out under 'constant scavenging' conditions to separate out the effect of direct radical scavenging from the possible electron transfer reaction. Control irradiation experiments, were performed in aerated TRIS buffer, concentration 10 mM, which has a scavenging capacity, k s (defined as the summation of the rate constants for the reaction of OH radicals with all species in solution, multiplied by their concentrations) of 1.5 x 10 7 s -1 . The concentration of TRIS was reduced upon addition of AO to maintain k s at this level. Data will be presented for examples from all four major types of flavonoids (flavonols, isoflavones, flavones and flavon-3-ols) showing DMF values plateau at near 2.0 even at low concentrations (ca. 20 μM) of the flavonoids. Increased DNA strand breaks following post irradiation incubation with endo III protein was unaffected by having the flavonoids present at the time of irradiation. This result suggests that the protection afforded by the flavonoids is unlikely to be in repairing radical damage on pyrimidine bases that are precursors of DNA strand breaks. Overall these studies provide evidence for an additional mechanism of antioxidant activity

Carry

DEFF Research Database (Denmark)

Koijen, Ralph S.J.; Moskowitz, Tobias J.; Heje Pedersen, Lasse

that include global equities, global bonds, currencies, commodities, US Treasuries, credit, and equity index options. This predictability underlies the strong returns to "carry trades" that go long high-carry and short low-carry securities, applied almost exclusively to currencies, but shown here...

Influence of molecular weight of DNA on the determination of anti-DNA antibodies in systemic lupus erythematosus (SLE) sera by radioimmunoassay

Energy Technology Data Exchange (ETDEWEB)

Geisert, M; Heicke, B; Metzmann, E; Zahn, R K

1975-04-01

Using a radioimmunoassay (RIA) based on the Farr technique with radioactively labeled /sup 3/H-DNA for quantitative measurements of anti-DNA antibodies in sera of patients with systemic lupus erythematosus (SLE), the influence of molecular weight of DNA (ranging from 0.1 x 10/sup 6/ to 22.0 x 10/sup 6/ daltons) on binding and precipitation in this system has been investigated. Comparing our results with mathematical models it follows that one antibody molecule is fixed on the average to a statistical DNA segment of 2 x 10/sup 6/ to 4 x 10/sup 6/ daltons. Furthermore binding capacity of the DNA was found to be independent of the molecular weight, as demonstrated in a double label experiment using /sup 14/C and /sup 3/H-labeled DNA of different size. However, the amount of radioactivity precipitated was found to depend on the molecular weight of the labeled DNA following a non-linear function. It was calculated that a minimal ratio of fixed antibody molecules per a certain size of DNA was necessary for precipitation. The mathematical treatment of the observed non-linear precipitation dependence will be discussed using various statistical models. The results indicate that the quantitative measurements of anti-DNA antibodies with the Farr technique e.g., for diagnosis and control of SLE in clinical immunology is highly dependent on the molecular weight of the labeled DNA used in the assay system and reliable results are only obtained with DNA of a sufficiently high molecular weight. (auth)

A Theoretical and Experimental Study of DNA Self-assembly

Science.gov (United States)

Chandran, Harish

The control of matter and phenomena at the nanoscale is fast becoming one of the most important challenges of the 21st century with wide-ranging applications from energy and health care to computing and material science. Conventional top-down approaches to nanotechnology, having served us well for long, are reaching their inherent limitations. Meanwhile, bottom-up methods such as self-assembly are emerging as viable alternatives for nanoscale fabrication and manipulation. A particularly successful bottom up technique is DNA self-assembly where a set of carefully designed DNA strands form a nanoscale object as a consequence of specific, local interactions among the different components, without external direction. The final product of the self-assembly process might be a static nanostructure or a dynamic nanodevice that performs a specific function. Over the past two decades, DNA self-assembly has produced stunning nanoscale objects such as 2D and 3D lattices, polyhedra and addressable arbitrary shaped substrates, and a myriad of nanoscale devices such as molecular tweezers, computational circuits, biosensors and molecular assembly lines. In this dissertation we study multiple problems in the theory, simulations and experiments of DNA self-assembly. We extend the Turing-universal mathematical framework of self-assembly known as the Tile Assembly Model by incorporating randomization during the assembly process. This allows us to reduce the tile complexity of linear assemblies. We develop multiple techniques to build linear assemblies of expected length N using far fewer tile types than previously possible. We abstract the fundamental properties of DNA and develop a biochemical system, which we call meta-DNA, based entirely on strands of DNA as the only component molecule. We further develop various enzyme-free protocols to manipulate meta-DNA systems and provide strand level details along with abstract notations for these mechanisms. We simulate DNA circuits by

DNA barcode goes two-dimensions: DNA QR code web server.

Science.gov (United States)

Liu, Chang; Shi, Linchun; Xu, Xiaolan; Li, Huan; Xing, Hang; Liang, Dong; Jiang, Kun; Pang, Xiaohui; Song, Jingyuan; Chen, Shilin

2012-01-01

The DNA barcoding technology uses a standard region of DNA sequence for species identification and discovery. At present, "DNA barcode" actually refers to DNA sequences, which are not amenable to information storage, recognition, and retrieval. Our aim is to identify the best symbology that can represent DNA barcode sequences in practical applications. A comprehensive set of sequences for five DNA barcode markers ITS2, rbcL, matK, psbA-trnH, and CO1 was used as the test data. Fifty-three different types of one-dimensional and ten two-dimensional barcode symbologies were compared based on different criteria, such as coding capacity, compression efficiency, and error detection ability. The quick response (QR) code was found to have the largest coding capacity and relatively high compression ratio. To facilitate the further usage of QR code-based DNA barcodes, a web server was developed and is accessible at http://qrfordna.dnsalias.org. The web server allows users to retrieve the QR code for a species of interests, convert a DNA sequence to and from a QR code, and perform species identification based on local and global sequence similarities. In summary, the first comprehensive evaluation of various barcode symbologies has been carried out. The QR code has been found to be the most appropriate symbology for DNA barcode sequences. A web server has also been constructed to allow biologists to utilize QR codes in practical DNA barcoding applications.

DNA barcode goes two-dimensions: DNA QR code web server.

Directory of Open Access Journals (Sweden)

Chang Liu

Full Text Available The DNA barcoding technology uses a standard region of DNA sequence for species identification and discovery. At present, "DNA barcode" actually refers to DNA sequences, which are not amenable to information storage, recognition, and retrieval. Our aim is to identify the best symbology that can represent DNA barcode sequences in practical applications. A comprehensive set of sequences for five DNA barcode markers ITS2, rbcL, matK, psbA-trnH, and CO1 was used as the test data. Fifty-three different types of one-dimensional and ten two-dimensional barcode symbologies were compared based on different criteria, such as coding capacity, compression efficiency, and error detection ability. The quick response (QR code was found to have the largest coding capacity and relatively high compression ratio. To facilitate the further usage of QR code-based DNA barcodes, a web server was developed and is accessible at http://qrfordna.dnsalias.org. The web server allows users to retrieve the QR code for a species of interests, convert a DNA sequence to and from a QR code, and perform species identification based on local and global sequence similarities. In summary, the first comprehensive evaluation of various barcode symbologies has been carried out. The QR code has been found to be the most appropriate symbology for DNA barcode sequences. A web server has also been constructed to allow biologists to utilize QR codes in practical DNA barcoding applications.

LEDGF/p75 Deficiency Increases Deletions at the HIV-1 cDNA Ends.

Science.gov (United States)

Bueno, Murilo T D; Reyes, Daniel; Llano, Manuel

2017-09-15

Processing of unintegrated linear HIV-1 cDNA by the host DNA repair system results in its degradation and/or circularization. As a consequence, deficient viral cDNA integration generally leads to an increase in the levels of HIV-1 cDNA circles containing one or two long terminal repeats (LTRs). Intriguingly, impaired HIV-1 integration in LEDGF/p75-deficient cells does not result in a correspondent increase in viral cDNA circles. We postulate that increased degradation of unintegrated linear viral cDNA in cells lacking the lens epithelium-derived growth factor (LEDGF/p75) account for this inconsistency. To evaluate this hypothesis, we characterized the nucleotide sequence spanning 2-LTR junctions isolated from LEDGF/p75-deficient and control cells. LEDGF/p75 deficiency resulted in a significant increase in the frequency of 2-LTRs harboring large deletions. Of note, these deletions were dependent on the 3' processing activity of integrase and were not originated by aberrant reverse transcription. Our findings suggest a novel role of LEDGF/p75 in protecting the unintegrated 3' processed linear HIV-1 cDNA from exonucleolytic degradation.

Synchronization of DNA array replication kinetics

Science.gov (United States)

Manturov, Alexey O.; Grigoryev, Anton V.

2016-04-01

In the present work we discuss the features of the DNA replication kinetics at the case of multiplicity of simultaneously elongated DNA fragments. The interaction between replicated DNA fragments is carried out by free protons that appears at the every nucleotide attachment at the free end of elongated DNA fragment. So there is feedback between free protons concentration and DNA-polymerase activity that appears as elongation rate dependence. We develop the numerical model based on a cellular automaton, which can simulate the elongation stage (growth of DNA strands) for DNA elongation process with conditions pointed above and we study the possibility of the DNA polymerases movement synchronization. The results obtained numerically can be useful for DNA polymerase movement detection and visualization of the elongation process in the case of massive DNA replication, eg, under PCR condition or for DNA "sequencing by synthesis" sequencing devices evaluation.

Expansion during PCR of short single-stranded DNA fragments carrying nonselfcomplementary dinucleotide or trinucleotide repeats

Czech Academy of Sciences Publication Activity Database

Reichová, Naďa; Kypr, Jaroslav

2003-01-01

Roč. 30, č. 3 (2003), s. 155-163 ISSN 0301-4851 R&D Projects: GA ČR GA301/01/0590 Institutional research plan: CEZ:AV0Z5004920 Keywords : DNA * PCR * expansion Subject RIV: BO - Biophysics Impact factor: 0.565, year: 2003

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

Directory of Open Access Journals (Sweden)

Trina Ekawati Tallei

2015-10-01

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

DNA damage induced by radiation plasmodial mixed + gamma thermal neutrons in the presence and absence of free radical scavenger

International Nuclear Information System (INIS)

Rodriguez Gual, Maritza; Mas Milian, Felix; Gouveia, Andreia; Deppman, Airton

2010-01-01

In this work is quantified the damage in DNA plasmid induced by mixed radiation (thermal neutron and gamma rays) for first time. For the study was used the pBs KS+ plasmid of 2961 bp in aqueous solution of the 88 ng/μL with 0, 2 and 20 mmol/L of glycerol which acts as a free radicals scavenger. This plasmid changes its form of supercoiled to circular when a simple strand break is produced, and passes to a linear form when a double strand break is produced in the chain. Quantifying the fractions that exist in each of these forms is possible to estimate the effect of radiation on DNA. The irradiations were carried out in the radial channel 3 at IEA-R1 research reactor of the Instituto de Pesquisas Energeticas y Nucleares in Sao Paulo, Brazil. DNA forms were separated by agarose gel electrophoresis. For quantification the program GelAnalis was used. The values of the fractions of DNA in various forms were plotted as a function of dose and fitted to exponential and linear functions to obtaining the probabilities of simple and double strand breaks normalized by dose and molecular mass. The results showed the protective action of free radical scavenger against damage induced for radiation which corroborates the previous results found with other ionizing radiations. Yields of SSB and DSB will be of interest for the validation of the different models that attempt to reproduce the experimental results

On load carrying capacity of frames taking into account finite displacements

International Nuclear Information System (INIS)

Borkoaski, A.; Saran, M.

1981-01-01

An approximate method that takes into account the influence of finite displacements upon the load carrying capacity of planar unbraced frames is described. It is the iterative procedure where Quadratic Programming is applied for evaluation of subsequent configurations of the frame, whereas Linear Programming serves to find ultimate load factors for each configuration. Numerical tests show that the procedure gives practically acceptable results being at the same time much cheaper than the exact geometrically and physically non-linear incremental analysis. (orig.)

The essential DNA polymerases δ and ε are involved in repair of UV-damaged DNA in the yeast Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Halas, A.; Policinska, Z.; Baranowska, H.; Jachymczyk, W.J.

1999-01-01

We have studied the ability of yeast DNA polymerases to carry out repair of lesions caused by UV irradiation in Saccharomyces cerevisiae. By the analysis of postirradiation relative molecular mass changes in cellular DNA of different DNA polymerases mutant strains, it was established that mutations in DNA polymerases δ and ε showed accumulation of single-strand breaks indicating defective repair. Mutations in other DNA polymerase genes exhibited no defects in DNA repair. Thus, the data obtained suggest that DNA polymerases δ and ε are both necessary for DNA replication and for repair of lesions caused by UV irradiation. The results are discussed in the light of current concepts concerning the specificity of DNA polymerases in DNA repair. (author)

An enzyme-catalyzed multistep DNA refolding mechanism in hairpin telomere formation.

Directory of Open Access Journals (Sweden)

Ke Shi

Full Text Available Hairpin telomeres of bacterial linear chromosomes are generated by a DNA cutting-rejoining enzyme protelomerase. Protelomerase resolves a concatenated dimer of chromosomes as the last step of chromosome replication, converting a palindromic DNA sequence at the junctions between chromosomes into covalently closed hairpins. The mechanism by which protelomerase transforms a duplex DNA substrate into the hairpin telomeres remains largely unknown. We report here a series of crystal structures of the protelomerase TelA bound to DNA that represent distinct stages along the reaction pathway. The structures suggest that TelA converts a linear duplex substrate into hairpin turns via a transient strand-refolding intermediate that involves DNA-base flipping and wobble base-pairs. The extremely compact di-nucleotide hairpin structure of the product is fully stabilized by TelA prior to strand ligation, which drives the reaction to completion. The enzyme-catalyzed, multistep strand refolding is a novel mechanism in DNA rearrangement reactions.

Accurate quantification of supercoiled DNA by digital PCR

Science.gov (United States)

Dong, Lianhua; Yoo, Hee-Bong; Wang, Jing; Park, Sang-Ryoul

2016-01-01

Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry. PMID:27063649

Tranformasi Fragmen Dna Kromosom Xanthomonas Campestris ke dalam Escherichia Coli

Directory of Open Access Journals (Sweden)

Wibowo Mangunwardoyo

2002-04-01

Full Text Available Research on DNA transformation of Xanthomonas campestris into Escherichia coli DH5αα using plasmid vector Escherichia coli (pUC19. was carried out. DNA chromosome was isolated using CTAB method, alkali lysis method was used to isolate DNA plasmid. Both of DNA plasmid and chromosome were digested using restriction enzyme EcoRI. Competent cell was prepared with CaCl2 and heat shock method for transformation procedure. The result revealed transformation obtain 5 white colonies, with transformation frequency was 1,22 x 10-8 colony/competent cell. Electrophoresis analysis showed the DNA fragment (insert in range 0.5 – 7,5 kb. Further research should be carried out to prepare the genomic library to obtain better result of transformant.

Spontaneous mutation by mutagenic repair of spontaneous lesions in DNA

International Nuclear Information System (INIS)

Hastings, P.J.; Quah, S.-K.; Borstel, R.C. von

1976-01-01

It is stated that strains of yeast carrying mutations in many of the steps in pathways repairing radiation-induced damage to DNA have enhanced spontaneous mutation rates. Most strains isolated because they have enhanced spontaneous mutation carry mutations in DNA repair systems. This suggests that much spontaneous mutation arises by mutagenic repair of spontaneous lesions. (author)

Chemical and biological consequences of the radioactive decay of iodine-125 in plasmid DNA

International Nuclear Information System (INIS)

Linz, U.

1983-09-01

The consequences of the decay of iodine-125 incorporated into DNA were studied on a molecular basis. Doubly ( 14 C and 125 I) labelled 5-iodo-2'-deoxycytidine 5'-triphosphate (IdCTP) was synthesized and incorporated enzymatically into the SalI-cutting site of the plasmid pBR 322. Part of the radioiodinated DNA was treated with T4-DNA ligase in order to restore the circular structure of the native plasmid molecule. After 4 months of storage under various conditions the stable end products were analyzed by radio GC, radio HPLC and electron microscopy. The experiments were not only carried out with doubly-labelled DNA but also with solutions of 14 C-labelled DNA containing Na 125 I as internal radiation source. The results clearly indicate that radiolysis alone causes only minor damage. Transmutation of the covalently bound iodine, on the other hand, leads to complete destruction of the labelled nucleotide, giving rise to 14 CO 2 and 14 CO as main products. The production of 14 CO 2 which originates from both the base as well as the sugar component shows a strong solvent effect. The electron microscopy analysis of the DNA reveals that the local effects are always connected with at least one double strand break directly at the site of decay. In addition, one finds DNA double strand breaks in areas which are hundreds of base pairs apart from that site. Under certain circumstances most of the DNA molecules exhibit up to 10 breaks. A comparison between ligase-treated and untreated DNA shows that the configuration of the DNA and the position of the labelled nucleotide play in important role in the extent of the overall damage. It could be demonstrated that there is a linear correlation between gaseous fragmentation products and the number of double strand breaks. (orig./MG) [de

Construction of Infectious cDNA Clone of a Chrysanthemum stunt viroid Korean Isolate

Directory of Open Access Journals (Sweden)

Ju-Yeon Yoon

2014-03-01

Full Text Available Chrysanthemum stunt viroid (CSVd, a noncoding infectious RNA molecule, causes seriously economic losses of chrysanthemum for 3 or 4 years after its first infection. Monomeric cDNA clones of CSVd isolate SK1 (CSVd-SK1 were constructed in the plasmids pGEM-T easy vector and pUC19 vector. Linear positive-sense transcripts synthesized in vitro from the full-length monomeric cDNA clones of CSVd-SK1 could infect systemically tomato seedlings and chrysanthemum plants, suggesting that the linear CSVd RNA transcribed from the cDNA clones could be replicated as efficiently as circular CSVd in host species. However, direct inoculation of plasmid cDNA clones containing full-length monomeric cDNA of CSVd-SK1 failed to infect tomato and chrysanthemum and linear negative-sense transcripts from the plasmid DNAs were not infectious in the two plant species. The cDNA sequences of progeny viroid in systemically infected tomato and chrysanthemum showed a few substitutions at a specific nucleotide position, but there were no deletions and insertions in the sequences of the CSVd progeny from tomato and chrysanthemum plants.

Protective role of OH scavengers and DNA/chromatin organization in the induction of DNA breaks: mechanistic models and Monte Carlo simulations

International Nuclear Information System (INIS)

Ballarini, F.; Rossetti, M.; Scannicchio, D.; Jacob, P.; Molinelli, S.; Ottolenghi, A.; Volata, A.

2003-01-01

Radiation-induced DNA damage can be modulated by various factors, including the environment scavenging capacity (SC) and the DNA organization within the cell nucleus (chromatin compactness, DNA-binding proteins etc.). In this context the induction of ssb and dsb by photons and light ions of different energies impinging on different DNA structures (e.g. linear DNA, SV40 'minichromosomes' and cellular DNA) at different OH-radical SC values was modelled with the Monte Carlo PARTRAC code. Presently PARTRAC can transport electrons, photons, protons and alpha particles in liquid water with an 'event-by-event' approach, and can simulate the DNA content of mammalian cells with an 'atom-by-atom' description, from nucleotide pairs to chromatin fibre loops and chromosome territories. Energy depositions in the sugar-phosphate were considered as potential (direct) ssb. The production, diffusion and reaction of chemical species were explicitly simulated; reactions of OH radicals with the sugar-phosphate were assumed to lead to 'indirect' ssb with probability 65%. Two ssb on opposite strands within 10 bp were considered as a dsb. Yields of ssb and dsb/Gy/Dalton were calculated for different DNA structures as a function of the OH mean life time. By Zyuzikov, N.; Michael, B.D. (Gray Cancer Institute, (GB)); Wu, L. (Ch Zyuzdirect damage yields. In general, also depending on radiation quality, linear DNA was found to be more susceptible to strand breakage than SV40 minichromosomes, which in turn showed higher damage yields with respect to cellular DNA. The very good agreement found with available experimental data provided a validation of the model and allowed us to quantify separately the protective effect of OH scavengers and DNA/chromatin organization. Comparisons with data on nucleoids (DNA unfolded and depleted of histones) suggested that the experimental procedures used to obtain such targets might lower the environment SC, due to the loss of cellular scavenging compounds

Spectral analysis of linear relations and degenerate operator semigroups

International Nuclear Information System (INIS)

Baskakov, A G; Chernyshov, K I

2002-01-01

Several problems of the spectral theory of linear relations in Banach spaces are considered. Linear differential inclusions in a Banach space are studied. The construction of the phase space and solutions is carried out with the help of the spectral theory of linear relations, ergodic theorems, and degenerate operator semigroups

Adenovirus 36 DNA in human adipose tissue.

Science.gov (United States)

Ponterio, E; Cangemi, R; Mariani, S; Casella, G; De Cesare, A; Trovato, F M; Garozzo, A; Gnessi, L

2015-12-01

Recent studies have suggested a possible correlation between obesity and adenovirus 36 (Adv36) infection in humans. As information on adenoviral DNA presence in human adipose tissue are limited, we evaluated the presence of Adv36 DNA in adipose tissue of 21 adult overweight or obese patients. Total DNA was extracted from adipose tissue biopsies. Virus detection was performed using PCR protocols with primers against specific Adv36 fiber protein and the viral oncogenic E4orf1 protein nucleotide sequences. Sequences were aligned with the NCBI database and phylogenetic analyses were carried out with MEGA6 software. Adv36 DNA was found in four samples (19%). This study indicates that some individuals carry Adv36 in the visceral adipose tissue. Further studies are needed to determine the specific effect of Adv36 infection on adipocytes, the prevalence of Adv36 infection and its relationship with obesity in the perspective of developing a vaccine that could potentially prevent or mitigate infection.

Site-specific integration of CAR gene into Jurkat T cells with a linear close-ended AAV-based DNA vector for CAR-T engineering.

Science.gov (United States)

Zhang, Yun; Liu, Xiaomei; Zhang, Jinju; Zhang, Chun

2016-09-01

To develop a site-specific integration strategy for CAR-T engineering by using a non-viral vector dependent on adeno-associated viral (AAV) genome, which tends to be integrated into AAVS1 site with the help of its Rep proteins. AAV-dependent vectors were produced in Sf9 cells. Structural analyses revealed the vector as covalently close-ended, linear duplex molecules, which was termed "CELiD" DNA. A plasmid CMV-Rep was constructed to express the integrases Rep78 and Rep68. Jurkat cells were co-electroporated with "CELiD" DNA and plasmid CMV-Rep in order to specifically integrate CAR gene into AAVS1 site. We examined 71 stably transfected Jurkat clones by nested PCR, sequencing and southern blotting, of which 30 clones bore CAR gene within AAVS1 site. The site-specific integration efficiency was nearly 42.2 %. The AAV-dependent vector preferentially integrated CAR into AAVS1 site, which could be further used in human T cell modification and enhance the security of CAR-T therapy.

A virus of hyperthermophilic archaea with a unique architecture among DNA viruses.

Science.gov (United States)

Rensen, Elena Ilka; Mochizuki, Tomohiro; Quemin, Emmanuelle; Schouten, Stefan; Krupovic, Mart; Prangishvili, David

2016-03-01

Viruses package their genetic material in diverse ways. Most known strategies include encapsulation of nucleic acids into spherical or filamentous virions with icosahedral or helical symmetry, respectively. Filamentous viruses with dsDNA genomes are currently associated exclusively with Archaea. Here, we describe a filamentous hyperthermophilic archaeal virus, Pyrobaculum filamentous virus 1 (PFV1), with a type of virion organization not previously observed in DNA viruses. The PFV1 virion, 400 ± 20 × 32 ± 3 nm, contains an envelope and an inner core consisting of two structural units: a rod-shaped helical nucleocapsid formed of two 14-kDa major virion proteins and a nucleocapsid-encompassing protein sheath composed of a single major virion protein of 18 kDa. The virion organization of PFV1 is superficially similar to that of negative-sense RNA viruses of the family Filoviridae, including Ebola virus and Marburg virus. The linear dsDNA of PFV1 carries 17,714 bp, including 60-bp-long terminal inverted repeats, and contains 39 predicted ORFs, most of which do not show similarities to sequences in public databases. PFV1 is a lytic virus that completely disrupts the host cell membrane at the end of the infection cycle.

DNA-binding proteins essential for protein-primed bacteriophage ø29 DNA replication

Directory of Open Access Journals (Sweden)

Margarita Salas

2016-08-01

Full Text Available Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5’ ends of the DNA. This protein, called terminal protein (TP, is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3’-5’ exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding

Correlation of iris biometrics and DNA

DEFF Research Database (Denmark)

Harder, Stine; Clemmensen, Line Katrine Harder; Dahl, Anders Bjorholm

2013-01-01

The presented work concerns prediction of complex human phenotypes from genotypes. We were interested in correlating iris color and texture with DNA. Our data consist of 212 eye images along with DNA: 32 single-nucleotide polymorphisms (SNPs). We used two types of biometrics to describe the eye...... images: One for iris color and one for iris texture. Both biometrics were high dimensional and a sparse principle component analysis (SPCA) reduced the dimensions and resulted in a representation of data with good interpretability. The correlations between the sparse principal components (SPCs......) and the 32 SNPs were found using a canonical correlation analysis (CCA). The result was a single significant canonical correlation (CC) for both biometrics. Each CC comprised two correlated canonical variables, consisting of a linear combination of SPCs and a linear combination of SNPs, respectively...

[Association of etheno-DNA adduct and DNA methylation level among workers exposed to diesel engine exhaust].

Science.gov (United States)

Shen, M L; He, Z N; Zhang, X; Duan, H W; Niu, Y; Bin, P; Ye, M; Meng, T; Dai, Y F; Yu, S F; Chen, W; Zheng, Y X

2017-06-06

Objective: To investigate the association between etheno-DNA adduct and the promoter of DNA methylation levels of cyclin dependent kinase inhibitor 2A (P16), Ras association domain family 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in workers with occupational exposure to diesel engine exhaust (DEE). Methods: We recruited 124 diesel engine testing workers as DEE exposure group and 112 water pump operator in the same area as control group in Henan province in 2012 using cluster sampling. The demographic data were obtained by questionnaire survey; urine after work and venous blood samples were collected from each subject. The urinary etheno-DNA adducts were detected using UPLC-MS/MS, including 1,N6-etheno-2'-deoxyadenosine (εdA) and 3,N4-etheno-2'-deoxycytidine(εdC). The DNA methylation levels of P16, RASSF1A, and MGMT were evaluated using bisulfite-pyrosequencing assay. The percentage of methylation was expressed as the 5-methylcytosine (5mC) over the sum of cytosines (%5mC). Spearman correlation and multiple linear regression were applied to analyze the association between etheno-DNA adducts and DNA methylation of P16, RASSF1A, and MGMT. Results: The median ( P (25)- P (75)) of urinary εdA level was 230.00 (98.04-470.91) pmol/g creatinine in DEE exposure group, and 102.10 (49.95-194.48) creatinine in control group. The level of εdA was higher in DEE exposure group than control group ( P 0.05) . Multiple linear regression confirmed the negative correlation between εdA and DNA methylation levels of P16, RASSF1A, and MGMT in non-smoking group (β (95 %CI ) was -0.068 (-0.132--0.003), -0.082 (-0.159--0.004) and -0.048 (-0.090--0.007), P values were 0.039, 0.039 and 0.024, respectively). Moreover, εdC was negative associated with DNA methylation level of MGMT in non-smoking group (β (95 %CI ) was -0.094 (-0.179--0.008), P= 0.032). Conclusion: DEE exposure could induce the increased of εdA and decreased of DNA methylation levels of P16, RASSF1A

Mitochondrial DNA mutations in mutator mice confer respiration defects and B-cell lymphoma development.

Directory of Open Access Journals (Sweden)

Takayuki Mito

Full Text Available Mitochondrial DNA (mtDNA mutator mice are proposed to express premature aging phenotypes including kyphosis and hair loss (alopecia due to their carrying a nuclear-encoded mtDNA polymerase with a defective proofreading function, which causes accelerated accumulation of random mutations in mtDNA, resulting in expression of respiration defects. On the contrary, transmitochondrial mito-miceΔ carrying mtDNA with a large-scale deletion mutation (ΔmtDNA also express respiration defects, but not express premature aging phenotypes. Here, we resolved this discrepancy by generating mtDNA mutator mice sharing the same C57BL/6J (B6J nuclear background with that of mito-miceΔ. Expression patterns of premature aging phenotypes are very close, when we compared between homozygous mtDNA mutator mice carrying a B6J nuclear background and selected mito-miceΔ only carrying predominant amounts of ΔmtDNA, in their expression of significant respiration defects, kyphosis, and a short lifespan, but not the alopecia. Therefore, the apparent discrepancy in the presence and absence of premature aging phenotypes in mtDNA mutator mice and mito-miceΔ, respectively, is partly the result of differences in the nuclear background of mtDNA mutator mice and of the broad range of ΔmtDNA proportions of mito-miceΔ used in previous studies. We also provided direct evidence that mtDNA abnormalities in homozygous mtDNA mutator mice are responsible for respiration defects by demonstrating the co-transfer of mtDNA and respiration defects from mtDNA mutator mice into mtDNA-less (ρ(0 mouse cells. Moreover, heterozygous mtDNA mutator mice had a normal lifespan, but frequently developed B-cell lymphoma, suggesting that the mtDNA abnormalities in heterozygous mutator mice are not sufficient to induce a short lifespan and aging phenotypes, but are able to contribute to the B-cell lymphoma development during their prolonged lifespan.

Specificity of binding to four-way junctions in DNA by bacteriophage T7 endonuclease I.

OpenAIRE

Parsons, C A; West, S C

1990-01-01

T7 endonuclease I binds specifically to four-way junctions in duplex DNA and promotes their resolution into linear duplexes. Under conditions in which the nuclease activity is blocked by the absence of divalent cations, the enzyme forms a distinct protein-DNA complex with the junction, as detected by gel retardation and filter binding assays. The formation of this complex is structure-specific and contrasts with the short-lived binding complexes formed on linear duplex DNA. The binding comple...

Induction and Persistence of Large γH2AX Foci by High Linear Energy Transfer Radiation in DNA-Dependent protein kinase–Deficient Cells

Energy Technology Data Exchange (ETDEWEB)

Bracalente, Candelaria; Ibañez, Irene L. [Departamento de Micro y Nanotecnología, Comisión Nacional de Energía Atómica, San Martín, Buenos Aires (Argentina); Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires (Argentina); Molinari, Beatriz [Departamento de Radiobiología, Comisión Nacional de Energía Atómica, San Martín, Buenos Aires (Argentina); Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires (Argentina); Palmieri, Mónica [Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires (Argentina); Kreiner, Andrés [Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires (Argentina); Gerencia de Investigación y Aplicaciones, Comisión Nacional de Energía Atómica, San Martín, Buenos Aires (Argentina); Escuela de Ciencia y Tecnología, Universidad Nacional de San Martín, San Martín, Buenos Aires (Argentina); Valda, Alejandro [Escuela de Ciencia y Tecnología, Universidad Nacional de San Martín, San Martín, Buenos Aires (Argentina); and others

2013-11-15

Purpose: To evaluate the cell response to DNA double-strand breaks induced by low and high linear energy transfer (LET) radiations when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an essential protein of the nonhomologous end-joining repair pathway, lacks kinase activity. Methods and Materials: CHO10B2, a Chinese hamster ovary cell line, and its derived radiosensitive mutant cell line, irs-20, lacking DNA-PKcs activity, were evaluated after 0 to 3 Gy of γ-rays, plateau and Bragg peak protons, and lithium beams by clonogenic assay, and as a measurement of double-strand breaks, phosphorylated H2AX (γH2AX) foci number and size were quantified by immunocytofluorescence. Results: Irs-20 exhibited greater radiosensitivity and a higher amount of γH2AX foci than CHO10B2 at 6 hours after irradiation for all types of radiations. Remarkably, CHO10B2 and irs-20 maintained their difference in radiosensitivity after high-LET radiation. Six hours after low-LET radiations, irs-20 did not reach basal levels of γH2AX at high doses, whereas CHO10B2 recovered basal levels for all doses. After high-LET radiation, only CHO10B2 exhibited a reduction in γH2AX foci, but it never reached basal levels. Persistent foci in irs-20 confirmed a repair deficiency. Interestingly, after 30 minutes of high-LET radiation both cell lines exhibited large foci (size >0.9 μm{sup 2}) related to the damage nature, whereas at 6 hours irs-20 showed a higher amount of large foci than CHO10B2, with a 7-fold increase at 3 Gy, that could also be associated to radiosensitivity. Conclusions: We demonstrated, for the first time, an association between deficient DNA-PKcs activity and not only high levels of H2AX phosphorylation but also persistence and size increase of γH2AX foci after high-LET irradiation.

Induction and Persistence of Large γH2AX Foci by High Linear Energy Transfer Radiation in DNA-Dependent protein kinase–Deficient Cells

International Nuclear Information System (INIS)

Bracalente, Candelaria; Ibañez, Irene L.; Molinari, Beatriz; Palmieri, Mónica; Kreiner, Andrés; Valda, Alejandro

2013-01-01

Purpose: To evaluate the cell response to DNA double-strand breaks induced by low and high linear energy transfer (LET) radiations when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an essential protein of the nonhomologous end-joining repair pathway, lacks kinase activity. Methods and Materials: CHO10B2, a Chinese hamster ovary cell line, and its derived radiosensitive mutant cell line, irs-20, lacking DNA-PKcs activity, were evaluated after 0 to 3 Gy of γ-rays, plateau and Bragg peak protons, and lithium beams by clonogenic assay, and as a measurement of double-strand breaks, phosphorylated H2AX (γH2AX) foci number and size were quantified by immunocytofluorescence. Results: Irs-20 exhibited greater radiosensitivity and a higher amount of γH2AX foci than CHO10B2 at 6 hours after irradiation for all types of radiations. Remarkably, CHO10B2 and irs-20 maintained their difference in radiosensitivity after high-LET radiation. Six hours after low-LET radiations, irs-20 did not reach basal levels of γH2AX at high doses, whereas CHO10B2 recovered basal levels for all doses. After high-LET radiation, only CHO10B2 exhibited a reduction in γH2AX foci, but it never reached basal levels. Persistent foci in irs-20 confirmed a repair deficiency. Interestingly, after 30 minutes of high-LET radiation both cell lines exhibited large foci (size >0.9 μm 2 ) related to the damage nature, whereas at 6 hours irs-20 showed a higher amount of large foci than CHO10B2, with a 7-fold increase at 3 Gy, that could also be associated to radiosensitivity. Conclusions: We demonstrated, for the first time, an association between deficient DNA-PKcs activity and not only high levels of H2AX phosphorylation but also persistence and size increase of γH2AX foci after high-LET irradiation

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

International Nuclear Information System (INIS)

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

1994-01-01

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

A device for extraction, manipulation and stretching of DNA from single human chromosomes

DEFF Research Database (Denmark)

Rasmussen, Kristian Hagsted; Marie, Rodolphe; Moresco, Jacob Lange

2011-01-01

by time-lapse imaging; pressure-driven flow was then used to shunt the chromosomal DNA package into a nanoslit. A long linear DNA strand (>1.3 Mbp) was seen to stretch out from the DNA package and along the length of the nanoslit. Delivery of DNA in its native metaphase chromosome package as well...

Iterated function systems for DNA replication

Science.gov (United States)

Gaspard, Pierre

2017-10-01

The kinetic equations of DNA replication are shown to be exactly solved in terms of iterated function systems, running along the template sequence and giving the statistical properties of the copy sequences, as well as the kinetic and thermodynamic properties of the replication process. With this method, different effects due to sequence heterogeneity can be studied, in particular, a transition between linear and sublinear growths in time of the copies, and a transition between continuous and fractal distributions of the local velocities of the DNA polymerase along the template. The method is applied to the human mitochondrial DNA polymerase γ without and with exonuclease proofreading.

Protocol for quantitative tracing of surface water with synthetic DNA

Science.gov (United States)

Foppen, J. W.; Bogaard, T. A.

2012-04-01

Based on experiments we carried out in 2010 with various synthetic single stranded DNA markers with a size of 80 nucleotides (ssDNA; Foppen et al., 2011), we concluded that ssDNA can be used to carry out spatially distributed multi-tracer experiments in the environment. Main advantages are in principle unlimited amount of tracers, environmental friendly and tracer recovery at very high dilution rates (detection limit is very low). However, when ssDNA was injected in headwater streams, we found that at selected downstream locations, the total mass recovery was less than 100%. The exact reason for low mass recovery was unknown. In order to start identifying the cause of the loss of mass in these surface waters, and to increase our knowledge of the behaviour of synthetic ssDNA in the environment, we examined the effect of laboratory and field protocols working with artificial DNA by performing numerous batch experiments. Then, we carried out several field tests in different headwater streams in the Netherlands and in Luxembourg. The laboratory experiments consisted of a batch of water in a vessel with in the order of 10^10 ssDNA molecules injected into the batch. The total duration of each experiment was 10 hour, and, at regular time intervals, 100 µl samples were collected in a 1.5 ml Eppendorf vial for qPCR analyses. The waters we used ranged from milliQ water to river water with an Electrical Conductivity of around 400 μS/cm. The batch experiments were performed in different vessel types: polyethylene bottles, polypropylene copolymer bottles , and glass bottles. In addition, two filter types were tested: 1 µm pore size glass fibre filters and 0.2 µm pore size cellulose acetate filters. Lastly, stream bed sediment was added to the batch experiments to quantify interaction of the DNA with sediment. For each field experiment around 10^15 ssDNA molecules were injected, and water samples were collected 100 - 600 m downstream of the point of injection. Additionally

Modifying and adapting a plant-based DNA extraction protocol for ...

African Journals Online (AJOL)

... a 100 apparently healthy individuals residing in Calabar. The modified DNA procedure yielded good quality genomic DNA which was used in carrying out allele specific polymerase chain reaction which also yielded good quality amplicons. This method is simple and suitable for the extraction of DNA from human red cell.

Topological and metric properties of linear and circular DNA chains in nano-slits and nano-channels

Science.gov (United States)

Orlandini, Enzo; Micheletti, Cristian

2014-03-01

Motivated by recent advancements in single DNA molecule experiments, based on nanofluidic devices, we investigate numerically the metric and topological properties of a modelof open and circular DNA chains confined inside nano-slits and nano-channles. The results reveal an interesting characterization of the metric crossover behaviour in terms of the abundance, type and length of occuring knots. In particular we find that the knotting probability is nonmonotonic for increasing confinement and can be largely enhanced or suppressed, compared to the bulk case, by simply varying the slit or channel trasversal dimension. The observed knot population consists of knots that are far simpler than for DNA chains in spherical (i.e. cavities or capsids) confinement. These results suggest that nanoslits and nanochannels can be properly designed to produce open DNA chains hosting simple knots or to sieve DNA rings according to their knotted state. Finally we discuss the implications that the presence of knots may have on the dynamical properties of confined DNA chains such as chain elongation, injection/ejection processes and entanglement relaxation. We acknowledge financial support from the Italian ministry of education, grant PRIN 2010HXAW77.

High Performance Systolic Array Core Architecture Design for DNA Sequencer

Directory of Open Access Journals (Sweden)

Saiful Nurdin Dayana

2018-01-01

Full Text Available This paper presents a high performance systolic array (SA core architecture design for Deoxyribonucleic Acid (DNA sequencer. The core implements the affine gap penalty score Smith-Waterman (SW algorithm. This time-consuming local alignment algorithm guarantees optimal alignment between DNA sequences, but it requires quadratic computation time when performed on standard desktop computers. The use of linear SA decreases the time complexity from quadratic to linear. In addition, with the exponential growth of DNA databases, the SA architecture is used to overcome the timing issue. In this work, the SW algorithm has been captured using Verilog Hardware Description Language (HDL and simulated using Xilinx ISIM simulator. The proposed design has been implemented in Xilinx Virtex -6 Field Programmable Gate Array (FPGA and improved in the core area by 90% reduction.

Linear dose response curves in fungi and tradescantia

International Nuclear Information System (INIS)

Unrau, P.

1999-07-01

Tradescantia Clone 02 data suggests that linear non-threshold dose responses are expected to the lowest doses and dose rates of low linear energy transfer (LET) radiation. This is likely to be true for other living organisms even though Clone 02 is radiation sensitive. It is concluded that Clone 02 is partially defective in the RAD 6 pathway for the repair of DNA interstrand cross-links (ISCL) and other loss of coding damage (LCD), based on its cross sensitivities to EMS and ionizing radiation. Tradescantia Clone 02 data showing linear non-threshold induction of somatic genetic events in part reflects the repair deficiency of this Clone. More DNA damage is repaired by recombinational mechanisms in Clone 02 than would occur in a wild-type strain. Two important classes of DNA lesions are induced by ionizing radiation in DNA - double strand breaks (DSB) which are repaired by recombination mechanisms, and loss of coding information damage (LCD), which is repaired by error prone mechanisms but may also be a substrate for recombinational repair. Based on data from yeast, there are two different repair pathways which deal with these differing lesions with different somatic genetic consequences. From yeast, yield cross sections can be derived and applied to DNA damage and repair in Tradescantia. For Clone 02, per lesion, more visible genetic events are scored than in wild-type strains. In a radiation-derived sub-clone, Clone 0106, which is more variable than Clone 02, even more events occur per lesion. This derivative clone, plus breeding experiments, indicate that Clone 02 is heterozygous, or a 'carrier' for a mutant version of a gene in the Tradescantia RAD 6 repair pathway. Clone 02 is, therefore, much like a Fanconi's anemia carrier in a human population, while the Clone 0106 derivative is much like a homozygous Fanconi's anemia patient, with respect to its response to ionizing radiation damage. Two anomalies in its dose response curves for 'pink' loss of

Linear dose response curves in fungi and tradescantia

Energy Technology Data Exchange (ETDEWEB)

Unrau, P. [Atomic Energy of Canada Ltd., Chalk River, Ontario (Canada)

1999-07-15

Tradescantia Clone 02 data suggests that linear non-threshold dose responses are expected to the lowest doses and dose rates of low linear energy transfer (LET) radiation. This is likely to be true for other living organisms even though Clone 02 is radiation sensitive. It is concluded that Clone 02 is partially defective in the RAD 6 pathway for the repair of DNA interstrand cross-links (ISCL) and other loss of coding damage (LCD), based on its cross sensitivities to EMS and ionizing radiation. Tradescantia Clone 02 data showing linear non-threshold induction of somatic genetic events in part reflects the repair deficiency of this Clone. More DNA damage is repaired by recombinational mechanisms in Clone 02 than would occur in a wild-type strain. Two important classes of DNA lesions are induced by ionizing radiation in DNA - double strand breaks (DSB) which are repaired by recombination mechanisms, and loss of coding information damage (LCD), which is repaired by error prone mechanisms but may also be a substrate for recombinational repair. Based on data from yeast, there are two different repair pathways which deal with these differing lesions with different somatic genetic consequences. From yeast, yield cross sections can be derived and applied to DNA damage and repair in Tradescantia. For Clone 02, per lesion, more visible genetic events are scored than in wild-type strains. In a radiation-derived sub-clone, Clone 0106, which is more variable than Clone 02, even more events occur per lesion. This derivative clone, plus breeding experiments, indicate that Clone 02 is heterozygous, or a 'carrier' for a mutant version of a gene in the Tradescantia RAD 6 repair pathway. Clone 02 is, therefore, much like a Fanconi's anemia carrier in a human population, while the Clone 0106 derivative is much like a homozygous Fanconi's anemia patient, with respect to its response to ionizing radiation damage. Two anomalies in its dose response curves for &apos

Single Molecule Screening of Disease DNA Without Amplification

Energy Technology Data Exchange (ETDEWEB)

Lee, Ji-Young [Iowa State Univ., Ames, IA (United States)

2006-01-01

The potential of single molecule detection as an analysis tool in biological and medical fields is well recognized today. This fast evolving technique will provide fundamental sensitivity to pick up individual pathogen molecules, and therefore contribute to a more accurate diagnosis and a better chance for a complete cure. Many studies are being carried out to successfully apply this technique in real screening fields. In this dissertation, several attempts are shown that have been made to test and refine the application of the single molecule technique as a clinical screening method. A basic applicability was tested with a 100% target content sample, using electrophoretic mobility and multiple colors as identification tools. Both electrophoretic and spectral information of individual molecule were collected within a second, while the molecule travels along the flow in a capillary. Insertion of a transmission grating made the recording of the whole spectrum of a dye-stained molecule possible without adding complicated instrumental components. Collecting two kinds of information simultaneously and combining them allowed more thorough identification, up to 98.8% accuracy. Probing mRNA molecules with fluorescently labeled cDNA via hybridization was also carried out. The spectral differences among target, probe, and hybrid were interpreted in terms of dispersion distances after transmission grating, and used for the identification of each molecule. The probes were designed to have the least background when they are free, but have strong fluorescence after hybridization via fluorescence resonance energy transfer. The mRNA-cDNA hybrids were further imaged in whole blood, plasma, and saliva, to test how far a crude preparation can be tolerated. Imaging was possible with up to 50% of clear bio-matrix contents, suggesting a simple lysis and dilution would be sufficient for imaging for some cells. Real pathogen DNA of human papillomavirus (HPV) type-I6 in human genomic DNA

JET VELOCITY OF LINEAR SHAPED CHARGES

Directory of Open Access Journals (Sweden)

Vječislav Bohanek

2012-12-01

Full Text Available Shaped explosive charges with one dimension significantly larger than the other are called linear shaped charges. Linear shaped charges are used in various industries and are applied within specific technologies for metal cutting, such as demolition of steel structures, separating spent rocket fuel tanks, demining, cutting holes in the barriers for fire service, etc. According to existing theories and models efficiency of linear shaped charges depends on the kinetic energy of the jet which is proportional to square of jet velocity. The original method for measuring velocity of linear shaped charge jet is applied in the aforementioned research. Measurements were carried out for two different linear materials, and the results are graphically presented, analysed and compared. Measurement results show a discrepancy in the measured velocity of the jet for different materials with the same ratio between linear and explosive mass (M/C per unit of surface, which is not described by presented models (the paper is published in Croatian.

Binding branched and linear DNA structures: From isolated clusters to fully bonded gels

Science.gov (United States)

Fernandez-Castanon, J.; Bomboi, F.; Sciortino, F.

2018-01-01

The proper design of DNA sequences allows for the formation of well-defined supramolecular units with controlled interactions via a consecution of self-assembling processes. Here, we benefit from the controlled DNA self-assembly to experimentally realize particles with well-defined valence, namely, tetravalent nanostars (A) and bivalent chains (B). We specifically focus on the case in which A particles can only bind to B particles, via appropriately designed sticky-end sequences. Hence AA and BB bonds are not allowed. Such a binary mixture system reproduces with DNA-based particles the physics of poly-functional condensation, with an exquisite control over the bonding process, tuned by the ratio, r, between B and A units and by the temperature, T. We report dynamic light scattering experiments in a window of Ts ranging from 10 °C to 55 °C and an interval of r around the percolation transition to quantify the decay of the density correlation for the different cases. At low T, when all possible bonds are formed, the system behaves as a fully bonded network, as a percolating gel, and as a cluster fluid depending on the selected r.

Consumerism and the Sister Carrie's American Dream%Consumerism and the Sister Carrie''s American Dream

Institute of Scientific and Technical Information of China (English)

卢亚丽

2017-01-01

From the aspect of consumerism to this text analyze Sister Carrie's"American dream"destruction. The author wholly and deeply analyzes the embodiment of consumerism in Dreiser's Sister Carrie and Dreiser's outlook and values under the effect of consumerism. To prove that the reason for destruction of Carrie's American dream is consumerism.

Comparison of the cobas Human Papillomavirus (HPV) Test with the Hybrid Capture 2 and Linear Array HPV DNA Tests

Science.gov (United States)

Sadorra, Mark; LaMere, Brandon J.; Kail, Randi; Aldrich, Carrie; Kinney, Walter; Fetterman, Barbara; Lorey, Thomas; Schiffman, Mark; Castle, Philip E.

2012-01-01

The cobas human papillomavirus (HPV) test (cobas) was recently approved by the U.S. Food and Drug Administration (FDA) and identifies HPV16 and HPV18 separately as well as detecting a pool of 11 HR-HPV genotypes (HPV31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -68) and also HPV66. We compared cobas, Linear Array (LA), and Hybrid Capture 2 (HC2) assays for detection of carcinogenic HPV DNA, and cobas and LA for detection of HPV16 and HPV18 DNA, among the first 1,852 women enrolled in the HPV Persistence and Progression Cohort (PaP Cohort) study. Specimens were tested by all 3 assays 1 year after an HC2-positive result. In 1,824 specimens with cobas results, cobas had an 85.9% agreement with HC2 and 91.0% agreement with LA for carcinogenic HPV detection. When results between cobas and HC2 disagreed, cobas tended to call more women HPV positive (P < 0.01). Categorizing cobas and LA results hierarchically according to cancer risk (HPV16, HPV18, other carcinogenic HPV genotypes, or carcinogen negative), there was a 90% agreement for all categories of HPV (n = 1,824). We found good agreement between the two U.S. FDA-approved HPV tests, with discrepancies between the two assays due to specific characteristics of the individual assays. Additional studies are needed to compare HC2 and cobas for detecting and predicting CIN3 to understand the clinical implications of the discrepant test results between the two tests. PMID:22075592

Cellular radiosensitivity and DNA damage in primary human fibroblasts

International Nuclear Information System (INIS)

Wurm, R.; Burnet, N.G.; Duggal, N.

1994-01-01

To evaluate the relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts to decide whether the initial or residual DNA damage levels are more predictive of normal tissue cellular radiosensitivity. Five primary human nonsyndromic and two primary ataxia telangiectasia fibroblast strains grown in monolayer were studied. Cell survival was assessed by clonogenic assay. Irradiation was given at high dose rate (HDR) 1-2 Gy/min. DNA damage was measured in stationary phase cells and expressed as fraction released from the well by pulsed-field gel electrophoresis (PFGE). For initial damage, cells were embedded in agarose and irradiated at HDR on ice. Residual DNA damage was measured in monolayer by allowing a 4-h repair period after HDR irradiation. Following HDR irradiation, cell survival varied between SF 2 0.025 to 0.23. Measurement of initial DNA damage demonstrated linear induction up to 30 Gy, with small differences in the slope of the dose-response curve between strains. No correlation between cell survival and initial damage was found. Residual damage increased linearly up to 80 Gy with a variation in slope by a factor of 3.2. Cell survival correlated with the slope of the dose-response curves for residual damage of the different strains (p = 0.003). The relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts of differing radiosensitivity is closest with the amount of DNA damage remaining after repair. If assays of DNA damage are to be used as predictors of normal tissue response to radiation, residual DNA damage provides the most likely correlation with cell survival. 52 refs., 5 figs., 2 tabs

Infinite sets of conservation laws for linear and non-linear field equations

International Nuclear Information System (INIS)

Niederle, J.

1984-01-01

The work was motivated by a desire to understand group theoretically the existence of an infinite set of conservation laws for non-interacting fields and to carry over these conservation laws to the case of interacting fields. The relation between an infinite set of conservation laws of a linear field equation and the enveloping algebra of its space-time symmetry group was established. It is shown that in the case of the Korteweg-de Vries (KdV) equation to each symmetry of the corresponding linear equation delta sub(o)uxxx=u sub() determined by an element of the enveloping algebra of the space translation algebra, there corresponds a symmetry of the full KdV equation

Flavisolibacter carri sp. nov., isolated from an automotive air-conditioning system.

Science.gov (United States)

Lee, Hyosun; Kim, Dong-Uk; Lee, Suyeon; Kim, Song-Gun; Park, A-Young; Ahn, Jae-Hyung; Ka, Jong-Ok

2018-04-18

A Gram-stain negative, aerobic, non-motile, rod-shaped and yellow bacterium, designated TX0651 T , was isolated from an automotive air-conditioning system. Phylogenetically, the strain groups with the members of the genus Flavisolibacter and exhibits high 16S rRNA gene sequence similarities with Flavisolibacter ginsenosidimutans Gsoil 636 T (97.4%), Flavisolibacter ginsengiterrae Gsoil 492 T (96.3%) and Flavisolibacter ginsengisoli Gsoil 643 T (96.2%). DNA-DNA relatedness between TX0651 T and F. ginsenosidimutans KCTC 22818 T and F. ginsengiterrae KCTC 12656 T were determined to be less than 40%. The low levels of DNA-DNA relatedness identifies the strain TX0651 T as a novel species in the genus Flavisolibacter. The major cellular fatty acids were identified as iso-C 15:0 , summed feature 3 (C 16:1 ω7c and/or C 16:1 ω6c), iso-C 15:1 G and iso-C 17:0 3-OH. The predominant respiratory quinone was identified as MK-7. The polar lipids were found to be comprised of phosphatidylethanolamine, unidentified amino-glycophospholipids, an unidentified aminophospholipid, an unidentified amino lipid and unidentified lipids. The DNA G+C content of the strain was determined to be 31.2 mol%. On the basis of the phenotypic, genotypic and chemotaxonomic characteristics, strain TX0651 T should be classified in a novel species in the genus Flavisolibacter, for which the name Flavisolibacter carri sp. nov. (= KACC 19014 T  = KCTC 52836 T  = NBRC 111784 T ) is proposed.

Radiolabeled Cu-ATSM as a novel indicator of overreduced intracellular state due to mitochondrial dysfunction: studies with mitochondrial DNA-less ρ0 cells and cybrids carrying MELAS mitochondrial DNA mutation

International Nuclear Information System (INIS)

Yoshii, Yukie; Yoneda, Makoto; Ikawa, Masamichi; Furukawa, Takako; Kiyono, Yasushi; Mori, Tetsuya; Yoshii, Hiroshi; Oyama, Nobuyuki; Okazawa, Hidehiko; Saga, Tsuneo; Fujibayashi, Yasuhisa

2012-01-01

Objectives: Radiolabeled Cu-diacetyl-bis (N 4 -methylthiosemicarbazone) ( ⁎ Cu-ATSM), including 60/62/64 Cu-ATSM, is a potential imaging agent of hypoxic tumors for positron emission tomography (PET). We have reported that ⁎ Cu-ATSM is trapped in tumor cells under intracellular overreduced states, e.g., hypoxia. Here we evaluated ⁎ Cu-ATSM as an indicator of intracellular overreduced states in mitochondrial disorders using cell lines with mitochondrial dysfunction. Methods: Mitochondrial DNA-less ρ 0 206 cells; the parental 143B human osteosarcoma cells; the cybrids carrying mutated mitochondria from a patient of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) (2SD); and that carrying wild-type one (2SA) were used. Cells were treated under normoxia or hypoxia, and 64 Cu-ATSM uptake was examined to compare it with levels of biological reductant NADH and NADPH. Results: ρ 0 206 cells showed higher 64 Cu-ATSM uptake than control 143B cells under normoxia, whereas 64 Cu-ATSM uptake was not significantly increased under hypoxia in ρ 0 206 cells. Additionally, 64 Cu-ATSM uptake showed correlate change to the NADH and NADPH levels, but not oxygenic conditions. 2SD cells showed increased 64 Cu-ATSM uptake under normoxia as compared with the control 2SA, and 64 Cu-ATSM uptake followed NADH and NADPH levels, but not oxygenic conditions. Conclusions: 64 Cu-ATSM accumulated in cells with overreduced states due to mitochondrial dysfunction, even under normoxia. We recently reported that 62 Cu-ATSM-PET can visualize stroke-like episodes maintaining oxygen supply in MELAS patients. Taken together, our data indicate that ⁎ Cu-ATSM uptake reflects overreduced intracellular states, despite oxygenic conditions; thus, ⁎ Cu-ATSM would be a promising marker of intracellular overreduced states for disorders with mitochondrial dysfunction, such as MELAS, Parkinson's disease and Alzheimer's disease.

Direct detection of chicken genomic DNA for gender determination by thymine-DNA glycosylase.

Science.gov (United States)

Porat, N; Bogdanov, K; Danielli, A; Arie, A; Samina, I; Hadani, A

2011-02-01

1. Birds, especially nestlings, are generally difficult to sex by morphology and early detection of chick gender in ovo in the hatchery would facilitate removal of unwanted chicks and diminish welfare objections regarding culling after hatch. 2. We describe a method to determine chicken gender without the need for PCR via use of Thymine-DNA Glycosylase (TDG). TDG restores thymine (T)/guanine (G) mismatches to cytosine (C)/G. We show here, that like DNA Polymerase, TDG can recognise, bind and function on a primer hybridised to chicken genomic DNA. 3. The primer contained a T to mismatch a G in a chicken genomic template and the T/G was cleaved with high fidelity by TDG. Thus, the chicken genomic DNA can be identified without PCR amplification via direct and linear detection. Sensitivity was increased using gender specific sequences from the chicken genome. 4. Currently, these are laboratory results, but we anticipate that further development will allow this method to be used in non-laboratory settings, where PCR cannot be employed.

Intricate and Cell Type-Specific Populations of Endogenous Circular DNA (eccDNA) in Caenorhabditis elegans and Homo sapiens.

Science.gov (United States)

Shoura, Massa J; Gabdank, Idan; Hansen, Loren; Merker, Jason; Gotlib, Jason; Levene, Stephen D; Fire, Andrew Z

2017-10-05

Investigations aimed at defining the 3D configuration of eukaryotic chromosomes have consistently encountered an endogenous population of chromosome-derived circular genomic DNA, referred to as extrachromosomal circular DNA (eccDNA). While the production, distribution, and activities of eccDNAs remain understudied, eccDNA formation from specific regions of the linear genome has profound consequences on the regulatory and coding capabilities for these regions. Here, we define eccDNA distributions in Caenorhabditis elegans and in three human cell types, utilizing a set of DNA topology-dependent approaches for enrichment and characterization. The use of parallel biophysical, enzymatic, and informatic approaches provides a comprehensive profiling of eccDNA robust to isolation and analysis methodology. Results in human and nematode systems provide quantitative analysis of the eccDNA loci at both unique and repetitive regions. Our studies converge on and support a consistent picture, in which endogenous genomic DNA circles are present in normal physiological states, and in which the circles come from both coding and noncoding genomic regions. Prominent among the coding regions generating DNA circles are several genes known to produce a diversity of protein isoforms, with mucin proteins and titin as specific examples. Copyright © 2017 Shoura et al.

DNA clasping by mycobacterial HU: the C-terminal region of HupB mediates increased specificity of DNA binding.

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Sandeep Kumar

Full Text Available BACKGROUND: HU a small, basic, histone like protein is a major component of the bacterial nucleoid. E. coli has two subunits of HU coded by hupA and hupB genes whereas Mycobacterium tuberculosis (Mtb has only one subunit of HU coded by ORF Rv2986c (hupB gene. One noticeable feature regarding Mtb HupB, based on sequence alignment of HU orthologs from different bacteria, was that HupB(Mtb bears at its C-terminal end, a highly basic extension and this prompted an examination of its role in Mtb HupB function. METHODOLOGY/PRINCIPAL FINDINGS: With this objective two clones of Mtb HupB were generated; one expressing full length HupB protein (HupB(Mtb and another which expresses only the N terminal region (first 95 amino acid of hupB (HupB(MtbN. Gel retardation assays revealed that HupB(MtbN is almost like E. coli HU (heat stable nucleoid protein in terms of its DNA binding, with a binding constant (K(d for linear dsDNA greater than 1000 nM, a value comparable to that obtained for the HUalphaalpha and HUalphabeta forms. However CTR (C-terminal Region of HupB(Mtb imparts greater specificity in DNA binding. HupB(Mtb protein binds more strongly to supercoiled plasmid DNA than to linear DNA, also this binding is very stable as it provides DNase I protection even up to 5 minutes. Similar results were obtained when the abilities of both proteins to mediate protection against DNA strand cleavage by hydroxyl radicals generated by the Fenton's reaction, were compared. It was also observed that both the proteins have DNA binding preference for A:T rich DNA which may occur at the regulatory regions of ORFs and the oriC region of Mtb. CONCLUSIONS/SIGNIFICANCE: These data thus point that HupB(Mtb may participate in chromosome organization in-vivo, it may also play a passive, possibly an architectural role.

Effect of temperature on DNA double helix: An insight from ...

Indian Academy of Sciences (India)

2012-06-25

Jun 25, 2012 ... We have carried out detailed MD simulations of DNA double ..... Wireframe representation of B-DNA crystal structures with PDB identifier (a) 1EHV, ..... nucleic acid structures, PhD thesis, University of Calcutta, Kolkata.

Thermal denaturation of A-DNA

International Nuclear Information System (INIS)

Valle-Orero, J; Wildes, A R; Theodorakopoulos, N; Cuesta-López, S; Peyrard, M; Garden, J-L; Danilkin, S

2014-01-01

The DNA molecule can take various conformational forms. Investigations focus mainly on the so-called ‘B-form’, schematically drawn in the famous paper by Watson and Crick [1]. This is the usual form of DNA in a biological environment and is the only form that is stable in an aqueous environment. Other forms, however, can teach us much about DNA. They have the same nucleotide base pairs for ‘building blocks’ as B-DNA, but with different relative positions, and studying these forms gives insight into the interactions between elements under conditions far from equilibrium in the B-form. Studying the thermal denaturation is particularly interesting because it provides a direct probe of those interactions which control the growth of the fluctuations when the ‘melting’ temperature is approached. Here we report such a study on the ‘A-form’ using calorimetry and neutron scattering. We show that it can be carried further than a similar study on B-DNA, requiring the improvement of thermodynamic models for DNA. (paper)

Comparison of Quantifiler(®) Trio and InnoQuant™ human DNA quantification kits for detection of DNA degradation in developed and aged fingerprints.

Science.gov (United States)

Goecker, Zachary C; Swiontek, Stephen E; Lakhtakia, Akhlesh; Roy, Reena

2016-06-01

The development techniques employed to visualize fingerprints collected from crime scenes as well as post-development ageing may result in the degradation of the DNA present in low quantities in such evidence samples. Amplification of the DNA samples with short tandem repeat (STR) amplification kits may result in partial DNA profiles. A comparative study of two commercially available quantification kits, Quantifiler(®) Trio and InnoQuant™, was performed on latent fingerprint samples that were either (i) developed using one of three different techniques and then aged in ambient conditions or (ii) undeveloped and then aged in ambient conditions. The three fingerprint development techniques used were: cyanoacrylate fuming, dusting with black powder, and the columnar-thin-film (CTF) technique. In order to determine the differences between the expected quantities and actual quantities of DNA, manually degraded samples generated by controlled exposure of DNA standards to ultraviolet radiation were also analyzed. A total of 144 fingerprint and 42 manually degraded DNA samples were processed in this study. The results indicate that the InnoQuant™ kit is capable of producing higher degradation ratios compared to the Quantifiler(®) Trio kit. This was an expected result since the degradation ratio is a relative value specific for a kit based on the length and extent of amplification of the two amplicons that vary from one kit to the other. Additionally, samples with lower concentrations of DNA yielded non-linear relationships of degradation ratio with the duration of aging, whereas samples with higher concentrations of DNA yielded quasi-linear relationships. None of the three development techniques produced a noticeably different degradation pattern when compared to undeveloped fingerprints, and therefore do not impede downstream DNA analysis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Minisequencing mitochondrial DNA pathogenic mutations

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Carracedo Ángel

2008-04-01

Full Text Available Abstract Background There are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA diseases. In order to overcome technical problems related to the analysis of complete mtDNA genomes, a variety of different techniques have been proposed that allow the screening of coding region pathogenic mutations. Methods We here propose a minisequencing assay for the analysis of mtDNA mutations. In a single reaction, we interrogate a total of 25 pathogenic mutations distributed all around the whole mtDNA genome in a sample of patients suspected for mtDNA disease. Results We have detected 11 causal homoplasmic mutations in patients suspected for Leber disease, which were further confirmed by standard automatic sequencing. Mutations m.11778G>A and m.14484T>C occur at higher frequency than expected by change in the Galician (northwest Spain patients carrying haplogroup J lineages (Fisher's Exact test, P-value Conclusion We here developed a minisequencing genotyping method for the screening of the most common pathogenic mtDNA mutations which is simple, fast, and low-cost. The technique is robust and reproducible and can easily be implemented in standard clinical laboratories.

Precise Sequential DNA Ligation on A Solid Substrate: Solid-Based Rapid Sequential Ligation of Multiple DNA Molecules

Science.gov (United States)

Takita, Eiji; Kohda, Katsunori; Tomatsu, Hajime; Hanano, Shigeru; Moriya, Kanami; Hosouchi, Tsutomu; Sakurai, Nozomu; Suzuki, Hideyuki; Shinmyo, Atsuhiko; Shibata, Daisuke

2013-01-01

Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation. PMID:23897972

DNA degradation in minicells of Escherichia coli K-12. Pt. 2. Effect of recA1 and recB21 mutations on DNA degradation in minicells and detection of exonuclease V activity

Energy Technology Data Exchange (ETDEWEB)

Khachatourians, G G [Saskatchewan Univ., Saskatoon (Canada). Dept. of Microbiology; Oak Ridge National Lab., Tenn. (USA). Biology Div.); Paterson, M C [Tennessee Univ., Oak Ridge (USA). Graduate School of Biomedical Sciences; Rijksuniversiteit Leiden (Netherlands). Lab. voor Stralengenetica); Sheehy, R J [Tennessee Univ., Oak Ridge (USA). Graduate School of Biomedical Sciences; Dorp, B Van [Rijksuniversiteit Leiden (Netherlands). Lab. voor Stralengenetica; Worthy, T E [Tennessee Univ., Knoxville (USA). Inst. of Radiation Biology

1975-06-01

The properties of minicell producing mutants of Escherichia coli deficient in genetic recombination were examined. Experiments were designed to test recombinant formation in conjugal crosses, survival following UV-irradiation in cells, and the state of DNA metabolism in minicells. The REC-phenotypes are unaffected by min/sup +///sup -/ genotypes in whole cells. In contrast to minicells produced by rec/sup +/ parental cells, minicells from a recB21 strain have limited capacity to degrade linear, Hfr transferred DNA. The lack of a functional recA gene product, presumably involved in inhibiting the recBC nuclease action(s), permits unrestricted Hfr DNA breakdown in minicells produced by a recA1 strain. This results in an increase in TGA soluble products and in the formation of small DNA molecules that sediment near the top of an alkaline sucrose gradient. Unlike the linear DNA, circular duplex DNA from plasmids R64-11 or lambdadv, segregated into the minicells, is resistant to breakdown. By using in vitro criteria, and (/sup 32/P)-labelled linear DNA from bacteriophage T/sub 7/ for substrate, we found that the ATP-dependent exonuclease of the recBC complex (exo V) is present in rec/sup +/ and recA/sup -/ minicells, and is lacking in the recB21 mutant. In fact, the absence of a functional exo V in recBC/sup -/ minicells results in isolation of larger than average Hfr DNA from minicells. We suggest that recombination (REC) enzymes segregate into the polar minicells at the time of minicell biogenesis. This system should be useful for studies on DNA metabolism and functions of the recBC and recA gene products.

Analysis of T-DNA/Host-Plant DNA Junction Sequences in Single-Copy Transgenic Barley Lines

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Joanne G. Bartlett

2014-01-01

Full Text Available Sequencing across the junction between an integrated transfer DNA (T-DNA and a host plant genome provides two important pieces of information. The junctions themselves provide information regarding the proportion of T-DNA which has integrated into the host plant genome, whilst the transgene flanking sequences can be used to study the local genetic environment of the integrated transgene. In addition, this information is important in the safety assessment of GM crops and essential for GM traceability. In this study, a detailed analysis was carried out on the right-border T-DNA junction sequences of single-copy independent transgenic barley lines. T-DNA truncations at the right-border were found to be relatively common and affected 33.3% of the lines. In addition, 14.3% of lines had rearranged construct sequence after the right border break-point. An in depth analysis of the host-plant flanking sequences revealed that a significant proportion of the T-DNAs integrated into or close to known repetitive elements. However, this integration into repetitive DNA did not have a negative effect on transgene expression.

Controlling Function and Structure with DNA

DEFF Research Database (Denmark)

Tørring, Thomas

2011-01-01

and ideas are presented. The second research topic concerns our contributions to the field of DNA origami. This includes investigations of single molecule reactions on a DNA origami platform. The reaction between an amine and an activated ester, as well as the Huisgen-Meldal-Sharpless reaction were...... investigated on a two dimensional DNA origami platform. This was done by incorporating functional groups on the surface of the origami, and reacting these with biotin analogues carrying the complementary functional groups. Successful reactions could then be observed using atomic force microscopy after addition...... of the protein streptavidin. While the implementation of chemical functionalities on origami can be achieved during automated DNA synthesis, this is laborious and costly. In a separate research project we aimed at improving the accessibility by applying an enzymatic labelling method. We demonstrated that the DNA...

Ancient mtDNA genetic variants modulate mtDNA transcription and replication.

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Sarit Suissa

2009-05-01

Full Text Available Although the functional consequences of mitochondrial DNA (mtDNA genetic backgrounds (haplotypes, haplogroups have been demonstrated by both disease association studies and cell culture experiments, it is not clear which of the mutations within the haplogroup carry functional implications and which are "evolutionary silent hitchhikers". We set forth to study the functionality of haplogroup-defining mutations within the mtDNA transcription/replication regulatory region by in vitro transcription, hypothesizing that haplogroup-defining mutations occurring within regulatory motifs of mtDNA could affect these processes. We thus screened >2500 complete human mtDNAs representing all major populations worldwide for natural variation in experimentally established protein binding sites and regulatory regions comprising a total of 241 bp in each mtDNA. Our screen revealed 77/241 sites showing point mutations that could be divided into non-fixed (57/77, 74% and haplogroup/sub-haplogroup-defining changes (i.e., population fixed changes, 20/77, 26%. The variant defining Caucasian haplogroup J (C295T increased the binding of TFAM (Electro Mobility Shift Assay and the capacity of in vitro L-strand transcription, especially of a shorter transcript that maps immediately upstream of conserved sequence block 1 (CSB1, a region associated with RNA priming of mtDNA replication. Consistent with this finding, cybrids (i.e., cells sharing the same nuclear genetic background but differing in their mtDNA backgrounds harboring haplogroup J mtDNA had a >2 fold increase in mtDNA copy number, as compared to cybrids containing haplogroup H, with no apparent differences in steady state levels of mtDNA-encoded transcripts. Hence, a haplogroup J regulatory region mutation affects mtDNA replication or stability, which may partially account for the phenotypic impact of this haplogroup. Our analysis thus demonstrates, for the first time, the functional impact of particular mtDNA

Hair Dye–DNA Interaction: Plausible Cause of Mutation

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Swati Maiti

2015-09-01

Full Text Available Hair dye is one of the most popular cosmetic products which are used more widely and frequently to improve an individual’s appearance. Although the genotoxic effects of dye ingredients are widely reported, hair dye in its usable form is not reported extensively. In this contribution, we report the possible mode of interaction of hair dye with DNA which leads to genotoxicity. The effect of dye DNA interaction was studied on the most popular and globally used hair dye with Calf Thymus DNA and plasmid DNA. This interaction of dye DNA was studied by spectroscopic analyses and gel electrophoresis. The result had shown positive interaction of dye with DNA. Gel electrophoresis study confirms the binding of dye with DNA which results in linearization and fragmentation of the plasmid DNA. Dye–DNA interaction causes fragmentation and oxidation of DNA in absence of any catalyst, implies high toxicity of commercial hair dyes. Thus, it can be deduced from the present studies that hair dye in its usable form may lead to its penetration through skin affecting genomic DNA possesses genotoxic property and can be treated as one of the most common mutagen.

Giant linear plasmids in Streptomyces: a treasure trove of antibiotic biosynthetic clusters.

Science.gov (United States)

Kinashi, Haruyasu

2011-01-01

Many giant linear plasmids have been isolated from Streptomyces by using pulsed-field gel electrophoresis and some of them were found to carry an antibiotic biosynthetic cluster(s); SCP1 carries biosynthetic genes for methylenomycin, pSLA2-L for lankacidin and lankamycin, and pKSL for lasalocid and echinomycin. Accumulated data suggest that giant linear plasmids have played critical roles in genome evolution and horizontal transfer of secondary metabolism. In this review, I summarize typical examples of giant linear plasmids whose involvement in antibiotic production has been studied in some detail, emphasizing their finding processes and interaction with the host chromosomes. A hypothesis on horizontal transfer of secondary metabolism involving giant linear plasmids is proposed at the end.

Helicobacter pylori ATCC 43629/NCTC 11639 Outer Membrane Vesicles (OMVs) from Biofilm and Planktonic Phase Associated with Extracellular DNA (eDNA)

Science.gov (United States)

Grande, Rossella; Di Marcantonio, Maria C.; Robuffo, Iole; Pompilio, Arianna; Celia, Christian; Di Marzio, Luisa; Paolino, Donatella; Codagnone, Marilina; Muraro, Raffaella; Stoodley, Paul; Hall-Stoodley, Luanne; Mincione, Gabriella

2015-01-01

Helicobacter pylori persistence is associated with its capacity to develop biofilms as a response to changing environmental conditions and stress. Extracellular DNA (eDNA) is a component of H. pylori biofilm matrix but the lack of DNase I activity supports the hypothesis that eDNA might be protected by other extracellular polymeric substances (EPS) and/or Outer Membrane Vesicles (OMVs), which bleb from the bacteria surface during growth. The aim of the present study was to both identify the eDNA presence on OMVs segregated from H. pylori ATCC 43629/NCTC 11639 biofilm (bOMVs) and its planktonic phase (pOMVs) and to characterize the physical-chemical properties of the OMVs. The presence of eDNA in bOMVs and pOMVs was initially carried out using DNase I-gold complex labeling and Transmission Electron Microscope analysis (TEM). bOMVs and pOMVs were further isolated and physical-chemical characterization carried out using dynamic light scattering (DLS) analysis. eDNA associated with OMVs was detected and quantified using a PicoGreen spectrophotometer assay, while its extraction was performed with a DNA Kit. TEM images showed that eDNA was mainly associated with the OMV membrane surfaces; while PicoGreen staining showed a four-fold increase of dsDNA in bOMVs compared with pOMVs. The eDNA extracted from OMVs was visualized using gel electrophoresis. DLS analysis indicated that both planktonic and biofilm H. pylori phenotypes generated vesicles, with a broad distribution of sizes on the nanometer scale. The DLS aggregation assay suggested that eDNA may play a role in the aggregation of OMVs, in the biofilm phenotype. Moreover, the eDNA associated with vesicle membrane may impede DNase I activity on H. pylori biofilms. These results suggest that OMVs derived from the H. pylori biofilm phenotype may play a structural role by preventing eDNA degradation by nucleases, providing a bridging function between eDNA strands on OMV surfaces and promoting aggregation. PMID:26733944

Studies on sildenafil citrate (Viagra) interaction with DNA using electrochemical DNA biosensor.

Science.gov (United States)

Rauf, Sakandar; Nawaz, Haq; Akhtar, Kalsoom; Ghauri, Muhammad A; Khalid, Ahmad M

2007-05-15

The interaction of sildenafil citrate (Viagra) with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of sildenafil citrate was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current was used as an indicator for the interaction in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 2.01+/-0.05 x 10(5) and 1.97+/-0.01 x 10(5)M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak current was observed within the range of 1-40 microM sildenafil citrate with slope=-2.74 x 10(-4)s/microM, r=0.989 and slope=-2.78 x 10(-3)microA/microM, r=0.995 by using constant current potentiometry and differential pulse voltammetry, respectively. Additionally, binding constant values for sildenafil citrate-DNA interaction were determined for the pH range of 4-8 and in biological fluids (serum and urine) at pH 5. The influence of sodium and calcium ions was also studied to elucidate the mechanism of sildenafil citrate-DNA interaction under different solution conditions. The present study may prove to be helpful in extending our understanding of the anticancer activity of sildenafil citrate from cellular to DNA level.

X-ray induced degradation of DNA in Aspergillus nidulans cells comparative analysis of UV- and X-ray induced DNA degradation

International Nuclear Information System (INIS)

Zinchenko, V.V.; Babykin, M.M.

1980-01-01

Irradiating cells of Aspergillus nidulans of the wild type in the logarythmical growth phase with X-rays leads to a certain retention in DNA synthesis. This period is characterized by an insignificant fermentative DNA degradation connected with a process of its repair. There is no direct dependence between the radiation dose and the level of DNA degradation. The investigation of X-ray induced DNA degradation in a number of UVS-mutants permits to show the existence of two branches of DNA degradation - dependent and independent of the exogenic energy source. The dependence of DNA degradation on albumen synthesis prior to irradiation and after it, is demonstrated. It is supposed that the level of X-ray induced DNA degradation is determined by two albumen systems, one of which initiates degradation and the other terminates it. The comparative analysis of UV and X-ray induced DNA degradation is carried out

Sensitive detection of mercury and copper ions by fluorescent DNA/Ag nanoclusters in guanine-rich DNA hybridization.

Science.gov (United States)

Peng, Jun; Ling, Jian; Zhang, Xiu-Qing; Bai, Hui-Ping; Zheng, Liyan; Cao, Qiu-E; Ding, Zhong-Tao

2015-02-25

In this work, we designed a new fluorescent oligonucleotides-stabilized silver nanoclusters (DNA/AgNCs) probe for sensitive detection of mercury and copper ions. This probe contains two tailored DNA sequence. One is a signal probe contains a cytosine-rich sequence template for AgNCs synthesis and link sequence at both ends. The other is a guanine-rich sequence for signal enhancement and link sequence complementary to the link sequence of the signal probe. After hybridization, the fluorescence of hybridized double-strand DNA/AgNCs is 200-fold enhanced based on the fluorescence enhancement effect of DNA/AgNCs in proximity of guanine-rich DNA sequence. The double-strand DNA/AgNCs probe is brighter and stable than that of single-strand DNA/AgNCs, and more importantly, can be used as novel fluorescent probes for detecting mercury and copper ions. Mercury and copper ions in the range of 6.0-160.0 and 6-240 nM, can be linearly detected with the detection limits of 2.1 and 3.4 nM, respectively. Our results indicated that the analytical parameters of the method for mercury and copper ions detection are much better than which using a single-strand DNA/AgNCs. Copyright © 2014 Elsevier B.V. All rights reserved.

Involvement of specialized DNA polymerases Pol II, Pol IV and DnaE2 in DNA replication in the absence of Pol I in Pseudomonas putida

International Nuclear Information System (INIS)

Sidorenko, Julia; Jatsenko, Tatjana; Saumaa, Signe; Teras, Riho; Tark-Dame, Mariliis; Horak, Rita; Kivisaar, Maia

2011-01-01

The majority of bacteria possess a different set of specialized DNA polymerases than those identified in the most common model organism Escherichia coli. Here, we have studied the ability of specialized DNA polymerases to substitute Pol I in DNA replication in Pseudomonas putida. Our results revealed that P. putida Pol I-deficient cells have severe growth defects in LB medium, which is accompanied by filamentous cell morphology. However, growth of Pol I-deficient bacteria on solid rich medium can be restored by reduction of reactive oxygen species in cells. Also, mutants with improved growth emerge rapidly. Similarly to the initial Pol I-deficient P. putida, its adapted derivatives express a moderate mutator phenotype, which indicates that DNA replication carried out in the absence of Pol I is erroneous both in the original Pol I-deficient bacteria and the adapted derivatives. Analysis of the spectra of spontaneous Rif r mutations in P. putida strains lacking different DNA polymerases revealed that the presence of specialized DNA polymerases Pol II and Pol IV influences the frequency of certain base substitutions in Pol I-proficient and Pol I-deficient backgrounds in opposite ways. Involvement of another specialized DNA polymerase DnaE2 in DNA replication in Pol I-deficient bacteria is stimulated by UV irradiation of bacteria, implying that DnaE2-provided translesion synthesis partially substitutes the absence of Pol I in cells containing heavily damaged DNA.

Specific interaction of the nonstructural protein NS1 of minute virus of mice (MVM) with [ACCA](2) motifs in the centre of the right-end MVM DNA palindrome induces hairpin-primed viral DNA replication.

Science.gov (United States)

Willwand, Kurt; Moroianu, Adela; Hörlein, Rita; Stremmel, Wolfgang; Rommelaere, Jean

2002-07-01

The linear single-stranded DNA genome of minute virus of mice (MVM) is replicated via a double-stranded replicative form (RF) intermediate DNA. Amplification of viral RF DNA requires the structural transition of the right-end palindrome from a linear duplex into a double-hairpin structure, which serves for the repriming of unidirectional DNA synthesis. This conformational transition was found previously to be induced by the MVM nonstructural protein NS1. Elimination of the cognate NS1-binding sites, [ACCA](2), from the central region of the right-end palindrome next to the axis of symmetry was shown to markedly reduce the efficiency of hairpin-primed DNA replication, as measured in a reconstituted in vitro replication system. Thus, [ACCA](2) sequence motifs are essential as NS1-binding elements in the context of the structural transition of the right-end MVM palindrome.

Diffusive limits for linear transport equations

International Nuclear Information System (INIS)

Pomraning, G.C.

1992-01-01

The authors show that the Hibert and Chapman-Enskog asymptotic treatments that reduce the nonlinear Boltzmann equation to the Euler and Navier-Stokes fluid equations have analogs in linear transport theory. In this linear setting, these fluid limits are described by diffusion equations, involving familiar and less familiar diffusion coefficients. Because of the linearity extant, one can carry out explicitly the initial and boundary layer analyses required to obtain asymptotically consistent initial and boundary conditions for the diffusion equations. In particular, the effects of boundary curvature and boundary condition variation along the surface can be included in the boundary layer analysis. A brief review of heuristic (nonasymptotic) diffusion description derivations is also included in our discussion

My journey to DNA repair.

Science.gov (United States)

Lindahl, Tomas

2013-02-01

I completed my medical studies at the Karolinska Institute in Stockholm but have always been devoted to basic research. My longstanding interest is to understand fundamental DNA repair mechanisms in the fields of cancer therapy, inherited human genetic disorders and ancient DNA. I initially measured DNA decay, including rates of base loss and cytosine deamination. I have discovered several important DNA repair proteins and determined their mechanisms of action. The discovery of uracil-DNA glycosylase defined a new category of repair enzymes with each specialized for different types of DNA damage. The base excision repair pathway was first reconstituted with human proteins in my group. Cell-free analysis for mammalian nucleotide excision repair of DNA was also developed in my laboratory. I found multiple distinct DNA ligases in mammalian cells, and led the first genetic and biochemical work on DNA ligases I, III and IV. I discovered the mammalian exonucleases DNase III (TREX1) and IV (FEN1). Interestingly, expression of TREX1 was altered in some human autoimmune diseases. I also showed that the mutagenic DNA adduct O(6)-methylguanine (O(6)mG) is repaired without removing the guanine from DNA, identifying a surprising mechanism by which the methyl group is transferred to a residue in the repair protein itself. A further novel process of DNA repair discovered by my research group is the action of AlkB as an iron-dependent enzyme carrying out oxidative demethylation. Copyright © 2013. Production and hosting by Elsevier Ltd.

Expression of protein-coding genes embedded in ribosomal DNA

DEFF Research Database (Denmark)

Johansen, Steinar D; Haugen, Peik; Nielsen, Henrik

2007-01-01

Ribosomal DNA (rDNA) is a specialised chromosomal location that is dedicated to high-level transcription of ribosomal RNA genes. Interestingly, rDNAs are frequently interrupted by parasitic elements, some of which carry protein genes. These are non-LTR retrotransposons and group II introns that e...... in the nucleolus....

Clustered DNA damage induced by proton and heavy ion irradiation

International Nuclear Information System (INIS)

Davidkova, M.; Pachnerova Brabcova, K; Stepan, V.; Vysin, L.; Sihver, L.; Incerti, S.

2014-01-01

Ionizing radiation induces in DNA strand breaks, damaged bases and modified sugars, which accumulate with increasing density of ionizations in charged particle tracks. Compared to isolated DNA damage sites, the biological toxicity of damage clusters can be for living cells more severe. We investigated the clustered DNA damage induced by protons (30 MeV) and high LET radiation (C 290 MeV/u and Fe 500 MeV/u) in pBR322 plasmid DNA. To distinguish between direct and indirect pathways of radiation damage, the plasmid was irradiated in pure water or in aqueous solution of one of the three scavengers (coumarin-3-carboxylic acid, dimethylsulfoxide, and glycylglycine). The goal of the contribution is the analysis of determined types of DNA damage in dependence on radiation quality and related contribution of direct and indirect radiation effects. The yield of double strand breaks (DSB) induced in the DNA plasmid-scavenger system by heavy ion radiation was found to decrease with increasing scavenging capacity due to reaction with hydroxyl radical, linearly with high correlation coefficients. The yield of non-DSB clusters was found to occur twice as much as the DSB. Their decrease with increasing scavenging capacity had lower linear correlation coefficients. This indicates that the yield of non-DSB clusters depends on more factors, which are likely connected to the chemical properties of individual scavengers. (authors)

Aligned deposition and electrical measurements on single DNA molecules

International Nuclear Information System (INIS)

Eidelshtein, Gennady; Kotlyar, Alexander; Hashemi, Mohtadin; Gurevich, Leonid

2015-01-01

A reliable method of deposition of aligned individual dsDNA molecules on mica, silicon, and micro/nanofabricated circuits is presented. Complexes of biotinylated double stranded poly(dG)–poly(dC) DNA with avidin were prepared and deposited on mica and silicon surfaces in the absence of Mg 2+ ions. Due to its positive charge, the avidin attached to one end of the DNA anchors the complex to negatively charged substrates. Subsequent drying with a directional gas flow yields DNA molecules perfectly aligned on the surface. In the avidin–DNA complex only the avidin moiety is strongly and irreversibly bound to the surface, while the DNA counterpart interacts with the substrates much more weakly and can be lifted from the surface and realigned in any direction. Using this technique, avidin–DNA complexes were deposited across platinum electrodes on a silicon substrate. Electrical measurements on the deposited DNA molecules revealed linear IV-characteristics and exponential dependence on relative humidity. (paper)

Polymer multilayer tattooing for enhanced DNA vaccination

Science.gov (United States)

Demuth, Peter C.; Min, Younjin; Huang, Bonnie; Kramer, Joshua A.; Miller, Andrew D.; Barouch, Dan H.; Hammond, Paula T.; Irvine, Darrell J.

2013-04-01

DNA vaccines have many potential benefits but have failed to generate robust immune responses in humans. Recently, methods such as in vivo electroporation have demonstrated improved performance, but an optimal strategy for safe, reproducible, and pain-free DNA vaccination remains elusive. Here we report an approach for rapid implantation of vaccine-loaded polymer films carrying DNA, immune-stimulatory RNA, and biodegradable polycations into the immune-cell-rich epidermis, using microneedles coated with releasable polyelectrolyte multilayers. Films transferred into the skin following brief microneedle application promoted local transfection and controlled the persistence of DNA and adjuvants in the skin from days to weeks, with kinetics determined by the film composition. These ‘multilayer tattoo’ DNA vaccines induced immune responses against a model HIV antigen comparable to electroporation in mice, enhanced memory T-cell generation, and elicited 140-fold higher gene expression in non-human primate skin than intradermal DNA injection, indicating the potential of this strategy for enhancing DNA vaccination.

Polymer multilayer tattooing for enhanced DNA vaccination

Science.gov (United States)

DeMuth, Peter C.; Min, Younjin; Huang, Bonnie; Kramer, Joshua A.; Miller, Andrew D.; Barouch, Dan H.; Hammond, Paula T.; Irvine, Darrell J.

2014-01-01

DNA vaccines have many potential benefits but have failed to generate robust immune responses in humans. Recently, methods such as in vivo electroporation have demonstrated improved performance, but an optimal strategy for safe, reproducible, and pain-free DNA vaccination remains elusive. Here we report an approach for rapid implantation of vaccine-loaded polymer films carrying DNA, immune-stimulatory RNA, and biodegradable polycations into the immune-cell-rich epidermis, using microneedles coated with releasable polyelectrolyte multilayers. Films transferred into the skin following brief microneedle application promoted local transfection and controlled the persistence of DNA and adjuvants in the skin from days to weeks, with kinetics determined by the film composition. These “multilayer tattoo” DNA vaccines induced immune responses against a model HIV antigen comparable to electroporation in mice, enhanced memory T-cell generation, and elicited 140-fold higher gene expression in non-human primate skin than intradermal DNA injection, indicating the potential of this strategy for enhancing DNA vaccination. PMID:23353628

Multifunctional DNA Nanomaterials for Biomedical Applications

Directory of Open Access Journals (Sweden)

Dick Yan Tam

2015-01-01

Full Text Available The rapidly emerging DNA nanotechnology began with pioneer Seeman’s hypothesis that DNA not only can carry genetic information but also can be used as molecular organizer to create well-designed and controllable nanomaterials for applications in materials science, nanotechnology, and biology. DNA-based self-assembly represents a versatile system for nanoscale construction due to the well-characterized conformation of DNA and its predictability in the formation of base pairs. The structural features of nucleic acids form the basis of constructing a wide variety of DNA nanoarchitectures with well-defined shapes and sizes, in addition to controllable permeability and flexibility. More importantly, self-assembled DNA nanostructures can be easily functionalized to construct artificial functional systems with nanometer scale precision for multipurposes. Apparently scientists envision artificial DNA-based nanostructures as tool for drug loading and in vivo targeted delivery because of their abilities in selective encapsulation and stimuli-triggered release of cargo. Herein, we summarize the strategies of creating multidimensional self-assembled DNA nanoarchitectures and review studies investigating their stability, toxicity, delivery efficiency, loading, and control release of cargos in addition to their site-specific targeting and delivery of drug or cargo molecules to cellular systems.

Efficient condensation of DNA into environmentally responsive polyplexes produced from block catiomers carrying amine or diamine groups

Czech Academy of Sciences Publication Activity Database

Albuquerque, L. J. C.; Annes, K.; Milazzotto, M. P.; Mattei, B.; Riske, K. A.; Jäger, Eliezer; Pánek, Jiří; Štěpánek, Petr; Kapusta, Peter; Muraro, P. I. R.; De Freitas, A. G. O.; Schmidt, V.; Giacomelli, C.; Bonvent, J.-J.; Giacomelli, F. C.

2016-01-01

Roč. 32, č. 2 (2016), s. 577-586 ISSN 0743-7463 R&D Projects: GA MŠk(CZ) LH14292 Institutional support: RVO:61389013 ; RVO:61388955 Keywords : polyplexes * DNA condensation * fluorescence Subject RIV: CC - Organic Chemistry; CF - Physical ; Theoretical Chemistry (UFCH-W) Impact factor: 3.833, year: 2016

The influence of bromodeoxyuridine on the induction and repair of DNA double-strand breaks in glioblastoma cells

International Nuclear Information System (INIS)

Nusser, N.N.; Bartkowiak, D.; Roettinger, E.M.

2002-01-01

Aims: To examine the dose response of DNA damage and its modification by the radiosensitizer, 5-bromo-2'-deoxyuridine (BrdU). The sensitizing mechanism is analyzed with regard to its influence on the induction and repair of DNA double-strand breaks (DSBs). Material and Methods: Cells from three different human glioblastoma lines, A7, LH and U87MG, were X-irradiated with and without exposure to BrdU. DNA fragments were separated by field-inversion gel electrophoresis (FIGE) and quantified by fluorometry immediately and 24 h after irradiation. Results: In all cell lines, the dose response followed a linear-quadratic rather than a purely linear function. BrdU-treated cells exhibited a significantly higher amount of mobile DNA. In repair experiments with and without BrdU, the amount of mobile DNA fell close to control values within 24 h. Conclusions: The linear-quadratic model appropriately describes the X-ray induced fragmentation of DNA. BrdU sensitizing acts predominantly by increasing DNA fragility, and not by impairing damage repair. The amount of DSBs persistent after 24 h of repair is minimal, even after highly cytotoxic doses. However, it appears to depend on the extent of initial damage, causing sensitized cells to retain more DSBs than unsensitized cells. (orig.)

DNA nanoparticles with core-shell morphology.

Science.gov (United States)

Chandran, Preethi L; Dimitriadis, Emilios K; Lisziewicz, Julianna; Speransky, Vlad; Horkay, Ferenc

2014-10-14

Mannobiose-modified polyethylenimines (PEI) are used in gene therapy to generate nanoparticles of DNA that can be targeted to the antigen-presenting cells of the immune system. We report that the sugar modification alters the DNA organization within the nanoparticles from homogenous to shell-like packing. The depth-dependent packing of DNA within the nanoparticles was probed using AFM nano-indentation. Unmodified PEI-DNA nanoparticles display linear elastic properties and depth-independent mechanics, characteristic of homogenous materials. Mannobiose-modified nanoparticles, however, showed distinct force regimes that were dependent on indentation depth, with 'buckling'-like response that is reproducible and not due to particle failure. By comparison with theoretical studies of spherical shell mechanics, the structure of mannobiosylated particles was deduced to be a thin shell with wall thickness in the order of few nanometers, and a fluid-filled core. The shell-core structure is also consistent with observations of nanoparticle denting in altered solution conditions, with measurements of nanoparticle water content from AFM images, and with images of DNA distribution in Transmission Electron Microscopy.

Depletion-induced instability in protein-DNA mixtures: Influence of protein charge and size

NARCIS (Netherlands)

Vries, de R.J.

2006-01-01

While there is abundant experimental and theoretical work on polymer-induced DNA condensation, it is still unclear whether globular proteins can condense linear DNA or not. We develop a simple analytical approximation for the depletion attraction between rodlike segments of semiflexible

Design and Assembly of DNA Nano-Objects and 2D DNA Origami Arrays

Science.gov (United States)

Liu, Wenyan

DNA, which plays a central role in biology as the carrier of genetic information, is also an excellent candidate for structural nanotechnology. Researches have proven that a variety of complicated DNA assemblies, such as objects, 2D & 3D crystals, and nanomechanical devices, can be fabricated through the combination of robust branched DNA motifs and sticky ends. This dissertation focuses on the design and construction of DNA nano--objects and 2D DNA origami arrays. In this dissertation, we first describe the formation of a triangular species that has four strands per edge, held together by PX interactions. We demonstrate by nondenaturing gel electrophoresis and by atomic force microscopy (AFM) that we can combine a partial triangle with other strands to form a robust four--stranded molecule. By combining them with a novel three--domain molecule, we also demonstrate by AFM that these triangles can be self--assembled into a linear array. Second, we demonstrate our attempts to design and self--assemble 2D DNA origami arrays using several different strategies. Specifically, we introduce the self--assembly of 2D DNA origami lattices using a symmetric cross--like design. This design strategy resulted in a well--ordered woven latticework array with edge dimensions of 2--3 mum. This size is likely to be large enough to connect bottom-up methods of patterning with top--down approaches. Third, we illustrate the design and construction of DNA nano--objects for exploring the substrate preferences of topoisomerase (topo) II. We designed and fabricated four double rhombus--like DNA molecules, each of which contains a different conformation of crossover in the middle, as possible substrates to establish the structural preferences for topo II. We characterized the formation of each substrate molecule by gel electrophoresis. Finally, we study the effect of M13 DNA knotting on the formation of the DNA origami tiles. We demonstrate by atomic force microscopy (AFM) that knotted M13

Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

Directory of Open Access Journals (Sweden)

Caroline M Li

Full Text Available We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE. In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40 origin of DNA replication and the viral large tumor antigen (T-antigen protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA, DNA topoisomerase I (topo I, DNA polymerase δ (Pol δ, DNA polymerase ɛ (Pol ɛ, replication protein A (RPA and replication factor C (RFC. Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

Science.gov (United States)

Li, Caroline M; Miao, Yunan; Lingeman, Robert G; Hickey, Robert J; Malkas, Linda H

2016-01-01

We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

Artificial Intelligence, DNA Mimicry, and Human Health.

Science.gov (United States)

Stefano, George B; Kream, Richard M

2017-08-14

The molecular evolution of genomic DNA across diverse plant and animal phyla involved dynamic registrations of sequence modifications to maintain existential homeostasis to increasingly complex patterns of environmental stressors. As an essential corollary, driver effects of positive evolutionary pressure are hypothesized to effect concerted modifications of genomic DNA sequences to meet expanded platforms of regulatory controls for successful implementation of advanced physiological requirements. It is also clearly apparent that preservation of updated registries of advantageous modifications of genomic DNA sequences requires coordinate expansion of convergent cellular proofreading/error correction mechanisms that are encoded by reciprocally modified genomic DNA. Computational expansion of operationally defined DNA memory extends to coordinate modification of coding and previously under-emphasized noncoding regions that now appear to represent essential reservoirs of untapped genetic information amenable to evolutionary driven recruitment into the realm of biologically active domains. Additionally, expansion of DNA memory potential via chemical modification and activation of noncoding sequences is targeted to vertical augmentation and integration of an expanded cadre of transcriptional and epigenetic regulatory factors affecting linear coding of protein amino acid sequences within open reading frames.

Identification of novel linear megaplasmids carrying a ß-lactamase gene in neurotoxigenic Clostridium butyricum type E strains.

Directory of Open Access Journals (Sweden)

Giovanna Franciosa

Full Text Available Since the first isolation of type E botulinum toxin-producing Clostridium butyricum from two infant botulism cases in Italy in 1984, this peculiar microorganism has been implicated in different forms of botulism worldwide. By applying particular pulsed-field gel electrophoresis run conditions, we were able to show for the first time that ten neurotoxigenic C. butyricum type E strains originated from Italy and China have linear megaplasmids in their genomes. At least four different megaplasmid sizes were identified among the ten neurotoxigenic C. butyricum type E strains. Each isolate displayed a single sized megaplasmid that was shown to possess a linear structure by ATP-dependent exonuclease digestion. Some of the neurotoxigenic C. butyricum type E strains possessed additional smaller circular plasmids. In order to investigate the genetic content of the newly identified megaplasmids, selected gene probes were designed and used in Southern hybridization experiments. Our results revealed that the type E botulinum neurotoxin gene was chromosome-located in all neurotoxigenic C. butyricum type E strains. Similar results were obtained with the 16S rRNA, the tetracycline tet(P and the lincomycin resistance protein lmrB gene probes. A specific mobA gene probe only hybridized to the smaller plasmids of the Italian C. butyricum type E strains. Of note, a ß-lactamase gene probe hybridized to the megaplasmids of eight neurotoxigenic C. butyricum type E strains, of which seven from clinical sources and the remaining one from a food implicated in foodborne botulism, whereas this ß-lactam antibiotic resistance gene was absent form the megaplasmids of the two soil strains examined. The widespread occurrence among C. butyricum type E strains associated to human disease of linear megaplasmids harboring an antibiotic resistance gene strongly suggests that the megaplasmids could have played an important role in the emergence of C. butyricum type E as a human

No evidence of Neandertal mtDNA contribution to early modern humans.

Directory of Open Access Journals (Sweden)

David Serre

2004-03-01

Full Text Available The retrieval of mitochondrial DNA (mtDNA sequences from four Neandertal fossils from Germany, Russia, and Croatia has demonstrated that these individuals carried closely related mtDNAs that are not found among current humans. However, these results do not definitively resolve the question of a possible Neandertal contribution to the gene pool of modern humans since such a contribution might have been erased by genetic drift or by the continuous influx of modern human DNA into the Neandertal gene pool. A further concern is that if some Neandertals carried mtDNA sequences similar to contemporaneous humans, such sequences may be erroneously regarded as modern contaminations when retrieved from fossils. Here we address these issues by the analysis of 24 Neandertal and 40 early modern human remains. The biomolecular preservation of four Neandertals and of five early modern humans was good enough to suggest the preservation of DNA. All four Neandertals yielded mtDNA sequences similar to those previously determined from Neandertal individuals, whereas none of the five early modern humans contained such mtDNA sequences. In combination with current mtDNA data, this excludes any large genetic contribution by Neandertals to early modern humans, but does not rule out the possibility of a smaller contribution.

Enhancement of DNA-transfection frequency by X-rays

Energy Technology Data Exchange (ETDEWEB)

Iwamoto, Ryota; Fushimi, Kazuo; Hiraki, Yoshio; Namba, Masayoshi [Okayama University Medical School (Japan). Institute of Cellular and Molecular Biology

1997-02-01

This study was conducted to evaluate the frequency of DNA transfection into human cells following X-ray irradiation. We transfected plasmid DNA (pSV2neo) into human cells, HeLa and PA-1, by either calcium phosphate precipitation or the lipofection method immediately after irradiating the cells with various doses of X-rays. The transfection frequency was evaluated by counting the number of G418-resistant colonies. When circular plasmid DNA was used, irradiation up to a dose of 2 Gy dose-dependently increased the transfection frequency, which reached a maximum of 5 to 10-fold that of the control unirradiated cells. When linear plasmid DNA was used, the transfection frequency was 2 times higher than that of circular DNA. All five of the clones that were randomly chosen expressed the transfected neo gene. In addition, the pSV2neo gene was randomly integrated into the genomic DNA of each clone. These findings indicate that X-ray treatment can facilitate foreign DNA transfer into human cells and that radiation-induced DNA breaks may promote the insertion of foreign DNA into host DNA. The enhancement of DNA transfection with X-rays may be instrumental in practicing gene therapy. (author)

Enhancement of DNA-transfection frequency by X-rays

International Nuclear Information System (INIS)

Iwamoto, Ryota; Fushimi, Kazuo; Hiraki, Yoshio; Namba, Masayoshi

1997-01-01

This study was conducted to evaluate the frequency of DNA transfection into human cells following X-ray irradiation. We transfected plasmid DNA (pSV2neo) into human cells, HeLa and PA-1, by either calcium phosphate precipitation or the lipofection method immediately after irradiating the cells with various doses of X-rays. The transfection frequency was evaluated by counting the number of G418-resistant colonies. When circular plasmid DNA was used, irradiation up to a dose of 2 Gy dose-dependently increased the transfection frequency, which reached a maximum of 5 to 10-fold that of the control unirradiated cells. When linear plasmid DNA was used, the transfection frequency was 2 times higher than that of circular DNA. All five of the clones that were randomly chosen expressed the transfected neo gene. In addition, the pSV2neo gene was randomly integrated into the genomic DNA of each clone. These findings indicate that X-ray treatment can facilitate foreign DNA transfer into human cells and that radiation-induced DNA breaks may promote the insertion of foreign DNA into host DNA. The enhancement of DNA transfection with X-rays may be instrumental in practicing gene therapy. (author)

Effects of fluorescence excitation geometry on the accuracy of DNA fragment sizing by flow cytometry

Energy Technology Data Exchange (ETDEWEB)

Werner, James H. [Division of Bioscience, Los Alamos National Laboratory, Mail Stop M888, Los Alamos, New Mexico 87545-0001 (United States); Larson, Erica J. [Division of Bioscience, Los Alamos National Laboratory, Mail Stop M888, Los Alamos, New Mexico 87545-0001 (United States); Goodwin, Peter M. [Division of Bioscience, Los Alamos National Laboratory, Mail Stop M888, Los Alamos, New Mexico 87545-0001 (United States); Ambrose, W. Patrick [Division of Bioscience, Los Alamos National Laboratory, Mail Stop M888, Los Alamos, New Mexico 87545-0001 (United States); Keller, Richard A. [Division of Bioscience, Los Alamos National Laboratory, Mail Stop M888, Los Alamos, New Mexico 87545-0001 (United States)

2000-06-01

We report on various excitation geometries used in ultrasensitive flow cytometry that yield a linear relation between the fluorescence intensity measured from individual strained DNA fragments and the lengths of the fragments (in base pairs). This linearity holds for DNA samples that exhibit a wide range of conformations. The variety of DNA conformations leads to a distribution of dipole moment orientations for the dye molecules intercalated into the DNA. It is consequently important to use an excitation geometry such that all dye molecules are detected with similar efficiency. To estimate the conformation and the extent of elongation of the strained fragments in the flow, fluorescence polarization anisotropy and autocorrelation measurements were performed. Significant extension was observed for DNA fragments under the flow conditions frequently used for DNA fragment sizing. Classical calculations of the fluorescence emission collected over a finite solid angle are in agreement with the experimental measurements and have confirmed the relative insensitivity to DNA conformation of an orthogonal excitation geometry. Furthermore, the calculations suggested a modified excitation geometry that has increased our sizing resolution. (c) 2000 Optical Society of America.

Effects of fluorescence excitation geometry on the accuracy of DNA fragment sizing by flow cytometry

International Nuclear Information System (INIS)

Werner, James H.; Larson, Erica J.; Goodwin, Peter M.; Ambrose, W. Patrick; Keller, Richard A.

2000-01-01

We report on various excitation geometries used in ultrasensitive flow cytometry that yield a linear relation between the fluorescence intensity measured from individual strained DNA fragments and the lengths of the fragments (in base pairs). This linearity holds for DNA samples that exhibit a wide range of conformations. The variety of DNA conformations leads to a distribution of dipole moment orientations for the dye molecules intercalated into the DNA. It is consequently important to use an excitation geometry such that all dye molecules are detected with similar efficiency. To estimate the conformation and the extent of elongation of the strained fragments in the flow, fluorescence polarization anisotropy and autocorrelation measurements were performed. Significant extension was observed for DNA fragments under the flow conditions frequently used for DNA fragment sizing. Classical calculations of the fluorescence emission collected over a finite solid angle are in agreement with the experimental measurements and have confirmed the relative insensitivity to DNA conformation of an orthogonal excitation geometry. Furthermore, the calculations suggested a modified excitation geometry that has increased our sizing resolution. (c) 2000 Optical Society of America

The Variation Analysis of DNA Methylation in Wheat Carrying Gametocidal Chromosome 3C from Aegilops triuncialis.

Science.gov (United States)

Wang, Dan; Zhao, Jieyu; Bai, Yan; Ao, You; Guo, Changhong

2017-08-10

Gametocidal (Gc) chromosomes can ensure their preferential transmission by killing the gametes without themselves through causing chromosome breakage and therefore have been exploited as an effective tool for genetic breeding. However, to date very little is known about the molecular mechanism of Gc action. In this study, we used methylation-sensitive amplified polymorphism (MSAP) technique to assess the extent and pattern of cytosine methylation alterations at the whole genome level between two lines of wheat Gc addition line and their common wheat parent. The results indicated that the overall levels of cytosine methylation of two studied Gc addition lines (CS-3C and CS-3C3C, 48.68% and 48.65%, respectively) were significantly increased when compared to common wheat CS (41.31%) and no matter fully methylated or hemimethylated rates enhanced in Gc addition lines. A set of 30 isolated fragments that showed different DNA methylation or demethylation patterns between the three lines were sequenced and the results indicated that 8 fragments showed significant homology to known sequences, of which three were homologous to MITE transposon (Miniature inverted-repeat transposable elements), LTR-retrotransposon WIS-1p and retrotransposon Gypsy , respectively. Overall, our results showed that DNA methylation could play a role in the Gc action.

The Variation Analysis of DNA Methylation in Wheat Carrying Gametocidal Chromosome 3C from Aegilops triuncialis

Directory of Open Access Journals (Sweden)

Dan Wang

2017-08-01

Full Text Available Gametocidal (Gc chromosomes can ensure their preferential transmission by killing the gametes without themselves through causing chromosome breakage and therefore have been exploited as an effective tool for genetic breeding. However, to date very little is known about the molecular mechanism of Gc action. In this study, we used methylation-sensitive amplified polymorphism (MSAP technique to assess the extent and pattern of cytosine methylation alterations at the whole genome level between two lines of wheat Gc addition line and their common wheat parent. The results indicated that the overall levels of cytosine methylation of two studied Gc addition lines (CS–3C and CS–3C3C, 48.68% and 48.65%, respectively were significantly increased when compared to common wheat CS (41.31% and no matter fully methylated or hemimethylated rates enhanced in Gc addition lines. A set of 30 isolated fragments that showed different DNA methylation or demethylation patterns between the three lines were sequenced and the results indicated that 8 fragments showed significant homology to known sequences, of which three were homologous to MITE transposon (Miniature inverted–repeat transposable elements, LTR-retrotransposon WIS-1p and retrotransposon Gypsy, respectively. Overall, our results showed that DNA methylation could play a role in the Gc action.

RBE comparison between rapid electrons of 20 MeV and 45 MeV with survival rate, DNA synthesis, DNA reparation and nucleoid sedimentation

International Nuclear Information System (INIS)

Alth, G.; Weniger, P.; Turtzer, K.; Klein, W.; Kocsis, F.; Krankenhaus der Stadt Wien-Lainz; Oesterreichisches Forschungszentrum Seibersdorf G.m.b.H. Inst. fuer Biologie)

1982-01-01

In order to find out possible differences of the biologic efficacy of rapid electrons of different energies, the authors examined the influence of rapid electrons of 20 MeV and 45 MeV upon the survival rate of Hela cells S3, their cell growth, DNA synthesis, DNA reparation, and sedimentation of nucleoids. The results show a difference only for the nucleoid sedimentation, i.e. there are more fractured DNA cords after 45 MeV irradiation. No significant differences could be demonstrated for the parameters of the survival curve, DNA synthesis and DNA reparation. Four double tests were carried out corresponding to the indicated types of examination. (orig.) [de

Hierarchically assembled DNA origami tubules with reconfigurable chirality

International Nuclear Information System (INIS)

Chen, Haorong; Cha, Tae-Gon; Pan, Jing; Choi, Jong Hyun

2013-01-01

The dynamic reconfiguration of a hierarchically assembled tubular structure is demonstrated using the DNA origami technique. Short cylindrical DNA origami monomers are synthesized and linked into elongated tubules, which can then be disassembled via toehold-mediated strand displacement. The disassembled subunits are subsequently linked into tubules of a different chirality. The reconfiguration is performed with the subunits carrying dumbbell hairpin DNA oligonucleotides or gold nanoparticles (AuNPs). The reconfiguration of higher order origami structures presented here is useful for constructing dynamic nanostructures that exceed the size limit of single DNA origami and may facilitate the study of molecular or particle interactions by tuning their relative distance and organization. (paper)

Nuclear DNA contents of Echinchloa crus-galli and its Gaussian relationships with environments

Science.gov (United States)

Li, Dan-Dan; Lu, Yong-Liang; Guo, Shui-Liang; Yin, Li-Ping; Zhou, Ping; Lou, Yu-Xia

2017-02-01

Previous studies on plant nuclear DNA content variation and its relationships with environmental gradients produced conflicting results. We speculated that the relationships between nuclear DNA content of a widely-distributed species and its environmental gradients might be non-linear if it was sampled in a large geographical gradient. Echinochloa crus-galli (L.) P. Beauv. is a worldwide species, but without documents on its intraspecific variation of nuclear DNA content. Our objectives are: 1) to detect intraspecific variation scope of E. crus-galli in its nuclear DNA content, and 2) to testify whether nuclear DNA content of the species changes with environmental gradients following Gaussian models if its populations were sampled in a large geographical gradient. We collected seeds of 36 Chinese populations of E. crus-galli across a wide geographical gradient, and sowed them in a homogeneous field to get their offspring to determine their nuclear DNA content. We analyzed the relationships of nuclear DNA content of these populations with latitude, longitude, and nineteen bioclimatic variables by using Gaussian and linear models. (1) Nuclear DNA content varied from 2.113 to 2.410 pg among 36 Chinese populations of E. crus-galli, with a mean value of 2.256 pg. (2) Gaussian correlations of nuclear DNA content (y) with geographical gradients were detected, with latitude (x) following y = 2.2923*e -(x - 24.9360)2/2*63.79452 (r = 0.546, P correlations of its Nuclear DNA content with geographical and most bioclimatic gradients.

A newly identified DNA ligase of Saccharomyces cerevisiae involved in RAD52-independent repair of DNA double-strand breaks

Science.gov (United States)

Schär, Primo; Herrmann, Gernot; Daly, Graham; Lindahl, Tomas

1997-01-01

Eukaryotic DNA ligases are ATP-dependent DNA strand-joining enzymes that participate in DNA replication, repair, and recombination. Whereas mammalian cells contain several different DNA ligases, encoded by at least three distinct genes, only one DNA ligase has been detected previously in either budding yeast or fission yeast. Here, we describe a newly identified nonessential Saccharomyces cerevisiae gene that encodes a DNA ligase distinct from the CDC9 gene product. This DNA ligase shares significant amino acid sequence homology with human DNA ligase IV; accordingly, we designate the yeast gene LIG4. Recombinant LIG4 protein forms a covalent enzyme-AMP complex and can join a DNA single-strand break in a DNA/RNA hybrid duplex, the preferred substrate in vitro. Disruption of the LIG4 gene causes only marginally increased cellular sensitivity to several DNA damaging agents, and does not further sensitize cdc9 or rad52 mutant cells. In contrast, lig4 mutant cells have a 1000-fold reduced capacity for correct recircularization of linearized plasmids by illegitimate end-joining after transformation. Moreover, homozygous lig4 mutant diploids sporulate less efficiently than isogenic wild-type cells, and show retarded progression through meiotic prophase I. Spore viability is normal, but lig4 mutants appear to produce a higher proportion of tetrads with only three viable spores. The mutant phenotypes are consistent with functions of LIG4 in an illegitimate DNA end-joining pathway and ensuring efficient meiosis. PMID:9271115

DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Budd, M.E.; Wittrup, K.D.; Bailey, J.E.; Campbell, J.L.

1989-01-01

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I

DNA supercoiling enhances cooperativity and efficiency of an epigenetic switch

DEFF Research Database (Denmark)

Norregaard, Kamilla; Andersson, Magnus; Sneppen, Kim

2013-01-01

Bacteriophage λ stably maintains its dormant prophage state but efficiently enters lytic development in response to DNA damage. The mediator of these processes is the λ repressor protein, CI, and its interactions with λ operator DNA. This λ switch is a model on the basis of which epigenetic switch...... with relaxed DNA, the presence of supercoils greatly enhances juxtaposition probability. Also, the efficiency and cooperativity of the λ switch is significantly increased in the supercoiled system compared with a linear assay, increasing the Hill coefficient....

Carry

DEFF Research Database (Denmark)

Koijen, Ralph S.J.; Moskowitz, Tobias; Pedersen, Lasse Heje

2018-01-01

-sectionally and in time series for a host of different asset classes, including global equities, global bonds, commodities, US Treasuries, credit, and options. Carry is not explained by known predictors of returns from these asset classes, and it captures many of these predictors, providing a unifying framework...... for return predictability. We reject a generalized version of Uncovered Interest Parity and the Expectations Hypothesis in favor of models with varying risk premia, in which carry strategies are commonly exposed to global recession, liquidity, and volatility risks, though none fully explains carry’s premium....

First report of linear megaplasmids in the genus Micrococcus.

Science.gov (United States)

Dib, Julian R; Wagenknecht, Martin; Hill, Russell T; Farías, María E; Meinhardt, Friedhelm

2010-01-01

High-altitude wetlands (above 4200m) in the northwest of Argentina are considered pristine and extreme environments. Micrococcus sp. A1, H5, and V7, isolated from such environments, were shown to contain linear megaplasmids, designated pLMA1, pLMH5, and pLMV7, respectively. As known from linear plasmids of other actinomycetes, all three plasmids were resistant to lambda exonuclease treatment, which is consistent with having terminal proteins covalently attached to their 5' DNA ends. Electrophoretic mobility, Southern analysis, and restriction endonuclease patterns revealed pLMA1 and pLMH5 being indistinguishable plasmids, even though they were found in different strains isolated from two distant wetlands - Laguna Azul and Laguna Huaca Huasi. Analysis of 16S rDNA sequences of Micrococcus sp. A1, H5, and V7 suggested a close relationship to Micrococcus luteus. Typing of isolates was performed using fingerprint patterns generated by BOX-PCR. Plasmid-deficient strains, generated from Micrococcus sp. A1, showed a significantly decreased resistance level for erythromycin. Copyright 2009 Elsevier Inc. All rights reserved.

Sphingomonas carri sp. nov., isolated from a car air-conditioning system.

Science.gov (United States)

Lee, Hyosun; Kim, Dong-Uk; Lee, Suyeon; Yun, Jungpyo; Park, Sooyeon; Yoon, Jung-Hoon; Park, So Yoon; Ka, Jong-Ok

2017-10-01

A Gram-stain-negative, yellow-pigmented bacterial strain, designated PR0302 T , was isolated from a car evaporator core collected in Korea. The cells were strictly aerobic, non-spore-forming and rod-shaped. The strain grew at 15-37 °C (optimum, 25 °C), at pH 6.0-8.0 (optimum, 7.0) and in the presence of 0-1 % (w/v) NaCl. Phylogenetically, the strain was closely related to members of the genus Sphingomonas(97.04-91.22 % 16S rRNA gene sequence similarities) and showed the highest sequence similarity of 97.04 % to Sphingomonas kyeonggiensis THG-DT81 T . It contained C16 : 0, summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and C14 : 0 2-OH as the predominant fatty acids and Q-10 as the major ubiquinone. The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and sphingoglycolipid. The major polyamine was sym-homospermidine. The serine palmitoyl transferase gene (spt) was detected and sphingolipid synthesis was confirmed. The mean DNA G+C content of the strain was 67.8±0.5 mol%. DNA-DNA relatedness between strain PR0302 T and closely related type strains of Sphingomonas species was less than 30 %. The low levels of DNA-DNA relatedness identified strain PR0302 T as a member of a novel species in the genus Sphingomonas. Based on phenotypic, genotypic and chemotaxonomic data, strain PR0302 T represents a novel species in the genus Sphingomonas, for which the name Sphingomonas carri sp. nov. is proposed. The type strain is PR0302 T (=KACC 18487 T =NBRC 111532 T ).

DNA repair mechanism in radioresistant bacteria

International Nuclear Information System (INIS)

Kitayama, Shigeru

1992-01-01

Many radiation resistant bacteria have been isolated from various sources which are not in high background field. Since Deinococcus radiodurans had been isolated first in 1956, studies on the mechanism for radioresistance were carried out mostly using this bacterium. DNA in this bacterium isn't protected against injury induced by not only ionizing radiation but also ultraviolet light. Therefore, DNA damages induced by various treatments are efficiently and accurately repaired in this cells. Damages in base and/or sugar in DNA are removed by endonucleases which, if not all, are synthesized during postirradiation incubation. Following the endonucleolytic cleavage the strand scissions in DNA are seemed to be rejoined by a process common for the repair of strand scissions induced by such as ionizing radiations. Induce protein(s) is also involved in this rejoining process of strand scissions. DNA repair genes were classified into three phenotypic groups. (1)Genes which are responsible for the endonucleolytic activities. (2) Genes involved in the rejoining of DNA strand scissions. (3) Genes which participate in genetic recombination and repair. Three genes belong to (1) and (2) were cloned onto approximately 1 kbp DNA fragments which base sequences have been determined. (author)

DNA repair mechanism in radioresistant bacteria

International Nuclear Information System (INIS)

Kitayama, Shigeru

1992-01-01

Many radiation resistant bacteria have been isolated from various sources which are not in high background field. Since Deinococcus radiodurans had been isolated first in 1956, the studies on the mechanism of radioresistance were mostly carried out using this bacterium. DNA in this bacterium isn't protected against injury induced by not only ionizing radiation but also ultraviolet light. Therefore, DNA damages induced by various treatments are efficiently and accurately repaired in this cells. Damages in base and/or sugar in DNA are removed by endonucleases which, if not all, are synthesized during postirradiation incubation. Following the endonucleolytic cleavage the strand scissions in DNA are seemed to be rejoined by a process common for the repair of strand scissions induced by such as ionizing radiations. Induce protein(s) is also involved in this rejoining process of strand scissions. DNA repair genes were classified into three phenotypic groups. (1) Genes which are responsible for the endonucleolytic activities. (2) Genes involved in the rejoining of DNA strand scissions. (3) Genes which participate in genetic recombination and repair. Three genes belong to (1) and (2) were cloned onto approximately 1 kbp DNA fragments which base sequences have been determined. (author)

Comparison of equivalent linear and non linear methods on ground response analysis: case study at West Bangka site

International Nuclear Information System (INIS)

Eko Rudi Iswanto; Eric Yee

2016-01-01

Within the framework of identifying NPP sites, site surveys are performed in West Bangka (WB), Bangka-Belitung Island Province. Ground response analysis of a potential site has been carried out using peak strain profiles and peak ground acceleration. The objective of this research is to compare Equivalent Linear (EQL) and Non Linear (NL) methods of ground response analysis on the selected NPP site (West Bangka) using Deep Soil software. Equivalent linear method is widely used because requires soil data in simple way and short time of computational process. On the other hand, non linear method is capable of representing the actual soil behaviour by considering non linear soil parameter. The results showed that EQL method has similar trends to NL method. At surface layer, the acceleration values for EQL and NL methods are resulted as 0.425 g and 0.375 g respectively. NL method is more reliable in capturing higher frequencies of spectral acceleration compared to EQL method. (author)

Recovery of DNA from agarose gel by trap method | Xia | African ...

African Journals Online (AJOL)

Recovery of DNA from agarose gel electrophoresis is a basic operation during molecular cloning. Circular or linear DNA fragments which vary from 1.5 to 6.5 kb and correspond to 1 kb marker can be recovered from 0.8 to 1.0% agarose gel smoothly with a simple and rapid trap method. The recovery efficiency could be ...

Accumulation of pathogenic ΔmtDNA induced deafness but not diabetic phenotypes in mito-mice

International Nuclear Information System (INIS)

Nakada, Kazuto; Sato, Akitsugu; Sone, Hideyuki; Kasahara, Atsuko; Ikeda, Katsuhisa; Kagawa, Yasuo; Yonekawa, Hiromichi; Hayashi, Jun-Ichi

2004-01-01

Mito-mice carrying various proportions of deletion mutant mtDNA (ΔmtDNA) were generated by introduction of the ΔmtDNA from cultured cells into fertilized eggs of C57BL/6J (B6) strain mice. Great advantages of mito-mice are that they share exactly the same nuclear-genome background, and that their genetic variations are restricted to proportions of pathogenic ΔmtDNA. Since accumulation of ΔmtDNA to more than 75% induced respiration defects, the disease phenotypes observed exclusively in mito-mice carrying more than 75% ΔmtDNA should be due to accumulated ΔmtDNA. In this study, we focused on the expressions of hearing loss and diabetic phenotypes, since these common age-associated abnormalities have sometimes been reported to be inherited maternally and to be associated with pathogenic mutant mtDNAs. The results showed that accumulation of exogenously introduced ΔmtDNA was responsible for hearing loss, but not for expression of diabetic phenotypes in mito-mice

The sequence specificity of UV-induced DNA damage in a systematically altered DNA sequence.

Science.gov (United States)

Khoe, Clairine V; Chung, Long H; Murray, Vincent

2018-06-01

The sequence specificity of UV-induced DNA damage was investigated in a specifically designed DNA plasmid using two procedures: end-labelling and linear amplification. Absorption of UV photons by DNA leads to dimerisation of pyrimidine bases and produces two major photoproducts, cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs). A previous study had determined that two hexanucleotide sequences, 5'-GCTC*AC and 5'-TATT*AA, were high intensity UV-induced DNA damage sites. The UV clone plasmid was constructed by systematically altering each nucleotide of these two hexanucleotide sequences. One of the main goals of this study was to determine the influence of single nucleotide alterations on the intensity of UV-induced DNA damage. The sequence 5'-GCTC*AC was designed to examine the sequence specificity of 6-4PPs and the highest intensity 6-4PP damage sites were found at 5'-GTTC*CC nucleotides. The sequence 5'-TATT*AA was devised to investigate the sequence specificity of CPDs and the highest intensity CPD damage sites were found at 5'-TTTT*CG nucleotides. It was proposed that the tetranucleotide DNA sequence, 5'-YTC*Y (where Y is T or C), was the consensus sequence for the highest intensity UV-induced 6-4PP adduct sites; while it was 5'-YTT*C for the highest intensity UV-induced CPD damage sites. These consensus tetranucleotides are composed entirely of consecutive pyrimidines and must have a DNA conformation that is highly productive for the absorption of UV photons. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

Cloning of the human androgen receptor cDNA

International Nuclear Information System (INIS)

Govindan, M.V.; Burelle, M.; Cantin, C.; Kabrie, C.; Labrie, F.; Lachance, Y.; Leblanc, G.; Lefebvre, C.; Patel, P.; Simard, J.

1988-01-01

The authors discuss how in order to define the functional domains of the human androgen receptor, complementary DNA (cDNA) clones encoding the human androgen receptor (hAR) have been isolated from a human testis λgtll cDNA library using synthetic oligonnucleotide probes, homologous to segments of the human glucocorticoid, estradiol and progesterone receptors. The cDNA clones corresponding to the human glucocorticoid, estradiol and progesterone receptors were eliminated after cross-hybridization with their respective cDNA probes and/or after restriction mapping of the cDNA clones. The remaining cDNA clones were classified into different groups after analysis by restriction digestion and cross-hybridization. Two of the largest cDNA clones from each group were inserted into an expression vector in both orientations. The linearized plasmids were used as templates in in vitro transcription with T7 RNA polymerase. Subsequent in vitro translation of the purified transcripts in rabbit reticulocyte lysate followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) permitted the characterization of the encoded polyeptides. The expressed proteins larger than 30,000 Da were analyzed for their ability to bind tritium-labelled dihydrotestosterone ([ 3 H] DHT) with high affinity and specificity

DNA methylation patterns in cord blood DNA and body size in childhood.

Directory of Open Access Journals (Sweden)

Caroline L Relton

Full Text Available Epigenetic markings acquired in early life may have phenotypic consequences later in development through their role in transcriptional regulation with relevance to the developmental origins of diseases including obesity. The goal of this study was to investigate whether DNA methylation levels at birth are associated with body size later in childhood.A study design involving two birth cohorts was used to conduct transcription profiling followed by DNA methylation analysis in peripheral blood. Gene expression analysis was undertaken in 24 individuals whose biological samples and clinical data were collected at a mean ± standard deviation (SD age of 12.35 (0.95 years, the upper and lower tertiles of body mass index (BMI were compared with a mean (SD BMI difference of 9.86 (2.37 kg/m(2. This generated a panel of differentially expressed genes for DNA methylation analysis which was then undertaken in cord blood DNA in 178 individuals with body composition data prospectively collected at a mean (SD age of 9.83 (0.23 years. Twenty-nine differentially expressed genes (>1.2-fold and p<10(-4 were analysed to determine DNA methylation levels at 1-3 sites per gene. Five genes were unmethylated and DNA methylation in the remaining 24 genes was analysed using linear regression with bootstrapping. Methylation in 9 of the 24 (37.5% genes studied was associated with at least one index of body composition (BMI, fat mass, lean mass, height at age 9 years, although only one of these associations remained after correction for multiple testing (ALPL with height, p(Corrected = 0.017.DNA methylation patterns in cord blood show some association with altered gene expression, body size and composition in childhood. The observed relationship is correlative and despite suggestion of a mechanistic epigenetic link between in utero life and later phenotype, further investigation is required to establish causality.

Electrochemical DNA probe for Hg(2+) detection based on a triple-helix DNA and Multistage Signal Amplification Strategy.

Science.gov (United States)

Wang, Huan; Zhang, Yihe; Ma, Hongmin; Ren, Xiang; Wang, Yaoguang; Zhang, Yong; Wei, Qin

2016-12-15

In this work, an ultrasensitive electrochemical sensor was developed for detection of Hg(2+). Gold nanoparticles decorated bovine serum albumin reduction of graphene oxide (AuNP-BSA-rGO) were used as subsurface material for the immobilization of triple-helix DNA. The triple-helix DNA containing a thiol labelled single-stranded DNA (sDNA) and a thymine-rich DNA (T-rich DNA), which could be unwinded in the present of Hg(2+) to form more stable thymine-Hg(2+)-thymine (T-Hg(2+)-T) complex. T-Hg(2+)-T complex was then removed and the sDNA was left on the electrode. At this time, gold nanoparticle carrying thiol labelled cytosine-rich complementary DNA (cDNA-AuNP) could bind with the free sDNA. Meanwhile, the other free cDNA on AuNP could bind with each other in the present of Ag(+) to form the stable cytosine-Ag(+)-cytosine (C-Ag(+)-C) complex and circle amplification. Plenty of C-Ag(+)-C could form silver nanoclusters by electrochemical reduction and the striping signal of Ag could be measured for purpose of the final electrochemical detection of Hg(2+). This sensor could detect Hg(2+) over a wide concentration range from 0.1 to 130nM with a detection limit of 0.03nM. Copyright © 2016 Elsevier B.V. All rights reserved.

Enhancement of DNA polymerase activity in potato tuber slices

International Nuclear Information System (INIS)

Watanabe, Akira; Imaseki, Hidemasa

1977-01-01

DNA polymerase was extracted from potato (Soleum tuberosum L.) tuber discs and the temporal correlation of its activity change to DNA synthesis in vivo was examined during aging of the discs. Most of the DNA polymerase was recovered as a bound form in the 18,000 x g precipitate. Reaction with the bound-form enzyme was dependent on the presence of four deoxynucleoside triphosphates, Mg 2+ , and a template. ''Activated'' DNA and heat-denatured DNA, but not native DNA, were utilized as templates. The polymerase activity was sensitive to SH reagents. Fresh discs, which do not synthesize DNA in vivo, contained a significant amount of DNA polymerase and its activity increased linearly with time until 48 hr after slicing and became four times that of fresh discs after 72 hr, whereas the activity of DNA synthesis in vivo increased with time and decreased after reaching a maximum at 30 hr. Cycloheximide inhibited the enhancement of polymerase activity. DNA polymerase from aged and fresh discs had identical requirements for deoxynucleotides and a template in their reactions, sensitivity to SH reagent, and affinity to thymidine triphosphate. (auth.)

Purification of supercoiled DNA of plasmid Col E1 by RPC-5 chromatography

Energy Technology Data Exchange (ETDEWEB)

Best, A.N.; Allison, D.P.; Novelli, G.D.

1981-07-01

Col E1 DNA can be purified to a high degree by RPC-5 chromatography of a partially purified cell lysate with a very shallow linear NaC1 gradient at pH 7.8. Electron micrographs demonstrated that the purest fractions were composed of 93% supercoiled (form I) DNA and 7% open circular (form II) DNA. The actual chromatography can be accomplished in 13 to 14 h and is designed for the production of several milligrams of plasmid DNA.

Amplification of DNA mixtures--Missing data approach

DEFF Research Database (Denmark)

Tvedebrink, Torben; Eriksen, Poul Svante; Mogensen, Helle Smidt

2008-01-01

This paper presents a model for the interpretation of results of STR typing of DNA mixtures based on a multivariate normal distribution of peak areas. From previous analyses of controlled experiments with mixed DNA samples, we exploit the linear relationship between peak heights and peak areas...... DNA samples, it is only possible to observe the cumulative peak heights and areas. Complying with this latent structure, we use the EM-algorithm to impute the missing variables based on a compound symmetry model. That is the measurements are subject to intra- and inter-loci correlations not depending...... on the actual alleles of the DNA profiles. Due to factorization of the likelihood, properties of the normal distribution and use of auxiliary variables, an ordinary implementation of the EM-algorithm solves the missing data problem. We estimate the parameters in the model based on a training data set. In order...

Ten helical twist angles of B-DNA

Energy Technology Data Exchange (ETDEWEB)

Kabsch, W; Sander, C; Trifonov, E N

1982-01-01

On the assumption that the twist angles between adjacent base-pairs in the DNA molecule are additive a linear system of 40 equations was derived from experimental measurements of the total twist angles for different pieces of DNA of known sequences. This system of equations is found to be statistically consistent providing a solution for all ten possible twist angles of B-DNA by a least squares fitting procedure. Four of the calculated twist angles were not known before. The other six twist angles calculated are very close to the experimentally measured ones. The data used were obtained by the electrophoretic band-shift method, crystallography and nuclease digestion of DNA adsorbed to mica or Ca-phosphate surface. The validity of the principle of additivity of the twist angles implies that the angle between any particular two base-pairs is a function of only these base-pairs, independent of nearest neighbors.

Repair of O6-methylguanine adducts in human telomeric G-quadruplex DNA by O6-alkylguanine-DNA alkyltransferase

Science.gov (United States)

Hellman, Lance M.; Spear, Tyler J.; Koontz, Colton J.; Melikishvili, Manana; Fried, Michael G.

2014-01-01

O6-alkylguanine-DNA alkyltransferase (AGT) is a single-cycle DNA repair enzyme that removes pro-mutagenic O6-alkylguanine adducts from DNA. Its functions with short single-stranded and duplex substrates have been characterized, but its ability to act on other DNA structures remains poorly understood. Here, we examine the functions of this enzyme on O6-methylguanine (6mG) adducts in the four-stranded structure of the human telomeric G-quadruplex. On a folded 22-nt G-quadruplex substrate, binding saturated at 2 AGT:DNA, significantly less than the ∼5 AGT:DNA found with linear single-stranded DNAs of similar length, and less than the value found with the telomere sequence under conditions that inhibit quadruplex formation (4 AGT:DNA). Despite these differences, AGT repaired 6mG adducts located within folded G-quadruplexes, at rates that were comparable to those found for a duplex DNA substrate under analogous conditions. Repair was kinetically biphasic with the amplitudes of rapid and slow phases dependent on the position of the adduct within the G-quadruplex: in general, adducts located in the top or bottom tetrads of a quadruplex stack exhibited more rapid-phase repair than did adducts located in the inner tetrad. This distinction may reflect differences in the conformational dynamics of 6mG residues in G-quadruplex DNAs. PMID:25080506

Construction of recombinant DNA clone for bovine viral diarrhea virus

International Nuclear Information System (INIS)

Yeo, S.G.; Cho, H.J.; Masri, S.A.

1992-01-01

Molecular cloning was carried out on the Danish strain of bovine viral diarrhea virus (BVDV) to construct strategy for the diagnostic tools and effective vaccine of BVD afterwards. A recombinant DNA clone (No. 29) was established successfully from cDNA for viral RNA tailed with adenine homopolymer at 3 -end. 32 P-labeled DNA probes of 300~1, 800bp fragments, originating from the clone 29, directed specific DNA-RNA hybridization results with BVDV RNA. Recombinant DNA of the clone 29 was about 5,200bp representing 41.6% of the full length of Danish strain's RNA, and restriction sites were recognized for EooR I, Sst I, Hind III and Pst I restriction enzymes in the DNA fragment

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.

Method for assessing damage to mitochondrial DNA caused by radiation and epichlorohydrin

International Nuclear Information System (INIS)

Singh, G.; Hauswirth, W.W.; Ross, W.E.; Neims, A.H.

1985-01-01

This paper describes a rapid and reliable method for quantification of damage to mitochondrial DNA (mtDNA), especially strand breaks. The degree of damage to mtDNA is assessed by the proportion of physical forms (i.e., supercoiled versus open-circular and linear forms) upon agarose gel electrophoresis, blotting, and visualization by hybridization with [ 32 P]mtDNA probes. The use of a radiolabeled probe is a crucial step in the procedure because it provides both a means to quantify by radioautography and to obtain the mtDNA specificity required to eliminate misinterpretation due to nuclear DNA contamination. To demonstrate the utility of this technique, X-irradiation and epichlorohydrin are shown to damage both isolated mtDNA and mtDNA in whole cells in a dose-dependent fashion

Development of a defined-sequence DNA system for use in DNA misrepair studies

International Nuclear Information System (INIS)

Sutton, S.; Tobias, C.A.

1984-01-01

The authors have developed a system that allows them to study cellular DNA repair processes at the molecular level. In particular, the authors are using this system to examine the consequences of a misrepair of radiation-induced DNA damage, as a function of dose. The cells being used are specially engineered haploid yeast cells. Maintained in the cells, at one copy per cell, is a cen plasmid, a plasmid that behaves like a functional chromosome. This plasmid carries a small defined sequence of DNA from the E. coli lac z gene. It is this lac z region (called the alpha region) that serves as the target for radiation damage. Two copies of the complimentary portion of the lac z gene are integrated into the yeast genome. Irradiated cells are screened for possible mutation in the alpha region by testing the cells' ability to hydrolyze xgal, a lactose substrate. The DNA of interest is then extracted from the cells, sequenced, and the sequence is compared to that of the control. Unlike the usual defined-sequence DNA systems, theirs is an in vivo system. A disadvantage is the relatively high background mutation rate. Results achieved with this system, as well as future applications, are discussed

Only male matrilineal relatives with Leber's hereditary optic neuropathy in a large Chinese family carrying the mitochondrial DNA G11778A mutation

International Nuclear Information System (INIS)

Qu Jia; Li Ronghua; Tong Yi; Hu Yongwu; Zhou Xiangtian; Qian Yaping; Lu Fan; Guan Minxin

2005-01-01

We report here the characterization of a five-generation large Chinese family with Leber's hereditary optic neuropathy (LHON). Very strikingly, six affected individuals of 38 matrilineal relatives (17 females/21 males) are exclusively males in this Chinese family. These matrilineal relatives in this family exhibited late-onset/progressive visual impairment with a wide range of severity, ranging from blindness to normal vision. The age of onset in visual impairment varies from 17 to 30 years. Sequence analysis of the complete mitochondrial genome in this pedigree revealed the presence of the G11778A mutation in ND4 gene and 29 other variants. This mitochondrial genome belongs to the Southern Chinese haplogroup B5b. We showed that the G11778A mutation is present at near homoplasmy in matrilineal relatives of this Chinese family but not in 164 Chinese controls. Incomplete penetrance of LHON in this family indicates the involvement of modulatory factors in the phenotypic expression of visual dysfunction associated with the G11778A mutation. However, none of other mtDNA variants are evolutionarily conserved and implicated to have significantly functional consequence. Thus, nuclear modifier gene(s) or environmental factor(s) seem to account for the penetrance and phenotypic variability of LHON in this Chinese family carrying the G11778A mutation

Asynchronous Magnetic Bead Rotation (AMBR Microviscometer for Label-Free DNA Analysis

Directory of Open Access Journals (Sweden)

Yunzi Li

2014-03-01

Full Text Available We have developed a label-free viscosity-based DNA detection system, using paramagnetic beads as an asynchronous magnetic bead rotation (AMBR microviscometer. We have demonstrated experimentally that the bead rotation period is linearly proportional to the viscosity of a DNA solution surrounding the paramagnetic bead, as expected theoretically. Simple optical measurement of asynchronous microbead motion determines solution viscosity precisely in microscale volumes, thus allowing an estimate of DNA concentration or average fragment length. The response of the AMBR microviscometer yields reproducible measurement of DNA solutions, enzymatic digestion reactions, and PCR systems at template concentrations across a 5000-fold range. The results demonstrate the feasibility of viscosity-based DNA detection using AMBR in microscale aqueous volumes.

Detection of circular telomeric DNA without 2D gel electrophoresis.

Science.gov (United States)

Dlaska, Margit; Anderl, Conrad; Eisterer, Wolfgang; Bechter, Oliver E

2008-09-01

The end of linear chromosomes forms a lasso-like structure called the t-loop. Such t-loops resemble a DNA recombination intermediate, where the single-stranded 3' overhang is arrested in a stretch of duplex DNA. Presumably, such a t-loop can also be deleted via a recombination process. This would result in the occurrence of circular extrachromosomal telomeric DNA (t-circles), which are known to be abundantly present in immortal cells engaging the recombination-based alternative lengthening of telomeres pathway (ALT pathway). Little is known about the basic mechanism of telomeric recombination in these cells and what ultimately causes the generation of such t-circles. Current standard procedures for detecting these molecules involve 2D gel electrophoresis or electron microscopy. However, both methods are labor intense and sophisticated to perform. Here, we present a simpler, faster, and equally sensitive method for detecting t-circles. Our approach is a telomere restriction fragment assay that involves the enzymatic preservation of circular DNA with Klenow enzyme followed by Bal31 degradation of the remaining linear DNA molecules. We show that with this approach t-circles can be detected in ALT cell lines, whereas no t-circles are present in telomerase-positive cell lines. We consider our approach a valid method in which t-circle generation is the experimental readout.

DNA damage assessment by visualization and quantification of DNA damage response

International Nuclear Information System (INIS)

Matsuda, Shun; Matsuda, Tomonari; Ikura, Tsuyoshi

2017-01-01

DNA damage response (DDR) carries out signal transduction for DNA repair, activation of cell cycle checkpoint, and apoptosis to maintain genome integrity, in response to DNA damage. Many proteins and their post-translational modifications participate in the process. Especially, S139-phosphorylated histone H2AX (γH2AX), which is formed by DNA double-strand breaks (DSBs), is an important factor to bring and retain other DDR proteins to DSB sites, Thus, γH2AX is used as a good indicator of DSBs in clinical study and pharmacology for efficacy evaluation of chemotherapy and radiotherapy, detection of precancerous regions, and others. In regulatory science, γH2AX is also a useful biomarker of genotoxicity of chemicals, since a wide range of genotoxic chemicals induce γH2AX. However, conventional detection methods of γH2AX absolutely require anti-γH2AX antibody whose staining is burdensome and time-consuming, and some of these methods are not so superior in quantitativity. In this review, we introduce two new methods to overcome these limitations, involving an easy-to-use genotoxicity assay using DDR-visualizing cells and an absolute quantification method of γH2AX using liquid chromatography-tandem mass spectrometry (LC/MS/MS). (author)

Patterning nanocrystals using DNA

Energy Technology Data Exchange (ETDEWEB)

Williams, Shara Carol [Univ. of California, Berkeley, CA (United States)

2003-01-01

One of the goals of nanotechnology is to enable programmed self-assembly of patterns made of various materials with nanometer-sized control. This dissertation describes the results of experiments templating arrangements of gold and semiconductor nanocrystals using 2'-deoxyribonucleic acid (DNA). Previously, simple DNA-templated linear arrangements of two and three nanocrystals structures have been made.[1] Here, we have sought to assemble larger and more complex nanostructures. Gold-DNA conjugates with 50 to 100 bases self-assembled into planned arrangements using strands of DNA containing complementary base sequences. We used two methods to increase the complexity of the arrangements: using branched synthetic doublers within the DNA covalent backbone to create discrete nanocrystal groupings, and incorporating the nanocrystals into a previously developed DNA lattice structure [2][3] that self-assembles from tiles made of DNA double-crossover molecules to create ordered nanoparticle arrays. In the first project, the introduction of a covalently-branched synthetic doubler reagent into the backbone of DNA strands created a branched DNA ''trimer.'' This DNA trimer templated various structures that contained groupings of three and four gold nanoparticles, giving promising, but inconclusive transmission electron microscopy (TEM) results. Due to the presence of a variety of possible structures in the reaction mixtures, and due to the difficulty of isolating the desired structures, the TEM and gel electrophoresis results for larger structures having four particles, and for structures containing both 5 and 10 nm gold nanoparticles were inconclusive. Better results may come from using optical detection methods, or from improved sample preparation. In the second project, we worked toward making two-dimensional ordered arrays of nanocrystals. We replicated and improved upon previous results for making DNA lattices, increasing the size of the lattices

Development and application of compact and on-chip electron linear accelerators for dynamic tracking cancer therapy and DNA damage/repair analysis

Science.gov (United States)

Uesaka, M.; Demachi, K.; Fujiwara, T.; Dobashi, K.; Fujisawa, H.; Chhatkuli, R. B.; Tsuda, A.; Tanaka, S.; Matsumura, Y.; Otsuki, S.; Kusano, J.; Yamamoto, M.; Nakamura, N.; Tanabe, E.; Koyama, K.; Yoshida, M.; Fujimori, R.; Yasui, A.

2015-06-01

We are developing compact electron linear accelerators (hereafter linac) with high RF (Radio Frequency) frequency (9.3 GHz, wavelength 32.3 mm) of X-band and applying to medicine and non-destructive testing. Especially, potable 950 keV and 3.95 MeV linac X-ray sources have been developed for on-site transmission testing at several industrial plants and civil infrastructures including bridges. 6 MeV linac have been made for pinpoint X-ray dynamic tracking cancer therapy. The length of the accelerating tube is ∼600 mm. The electron beam size at the X-ray target is less than 1 mm and X-ray spot size at the cancer is less than 3 mm. Several hardware and software are under construction for dynamic tracking therapy for moving lung cancer. Moreover, as an ultimate compact linac, we are designing and manufacturing a laser dielectric linac of ∼1 MeV with Yr fiber laser (283 THz, wavelength 1.06 pm). Since the wavelength is 1.06 μm, the length of one accelerating strcture is tens pm and the electron beam size is in sub-micro meter. Since the sizes of cell and nuclear are about 10 and 1 μm, respectively, we plan to use this “On-chip” linac for radiation-induced DNA damage/repair analysis. We are thinking a system where DNA in a nucleus of cell is hit by ∼1 μm electron or X-ray beam and observe its repair by proteins and enzymes in live cells in-situ.

Hepatitis B virus DNA quantification with the three-in-one (3io) method allows accurate single-step differentiation of total HBV DNA and cccDNA in biopsy-size liver samples.

Science.gov (United States)

Taranta, Andrzej; Tien Sy, Bui; Zacher, Behrend Johan; Rogalska-Taranta, Magdalena; Manns, Michael Peter; Bock, Claus Thomas; Wursthorn, Karsten

2014-08-01

Hepatitis B virus (HBV) replicates via reverse transcription converting its partially double stranded genome into the covalently closed circular DNA (cccDNA). The long-lasting cccDNA serves as a replication intermediate in the nuclei of hepatocytes. It is an excellent, though evasive, parameter for monitoring the course of liver disease and treatment efficiency. To develop and test a new approach for HBV DNA quantification in serum and small-size liver samples. The p3io plasmid contains an HBV fragment and human β-actin gene (hACTB) as a standard. Respective TaqMan probes were labeled with different fluorescent dyes. A triplex real-time PCR for simultaneous quantification of total HBV DNA, cccDNA and hACTB could be established. Three-in-one method allows simultaneous analysis of 3 targets with a lower limit of quantification of 48 copies per 20 μl PCR reaction and a wide range of linearity (R(2)>0.99, pDNA samples from HBV infected patients. Total HBV DNA and cccDNA could be quantified in 32 and 22 of 33 FFPE preserved liver specimens, respectively. Total HBV DNA concentrations quantified by the 3io method remained comparable with Cobas TaqMan HBV Test v2.0. The three-in-one protocol allows the single step quantification of viral DNA in samples from different sources. Therefore lower sample input, faster data acquisition, a lowered error and significantly lower costs are the advantages of the method. Copyright © 2014 Elsevier B.V. All rights reserved.

Highly sensitive DNA sensors based on cerium oxide nanorods

Science.gov (United States)

Nguyet, Nguyen Thi; Hai Yen, Le Thi; Van Thu, Vu; lan, Hoang; Trung, Tran; Vuong, Pham Hung; Tam, Phuong Dinh

2018-04-01

In this work, a CeO2 nanorod (NR)-based electrochemical DNA sensor was developed to identify Salmonella that causes food-borne infections. CeO2 NRs were synthesized without templates via a simple and unexpensive hydrothermal approach at 170 °C for 12 h by using CeO(NO3)3·6H2O as a Ce source. The DNA probe was immobilized onto the CeO2 NR-modified electrode through covalent attachment. The characteristics of the hybridized DNA were analyzed through electrochemical impedance spectroscopy (EIS) with [Fe(CN)6]3-/4- as a redox probe. Experimental results showed that electron transfer resistance (Ret) increased after the DNA probe was attached to the electrode surface and increased further after the DNA probe hybridized with its complementary sequence. A linear response of Ret to the target DNA concentration was found from 0.01 μM to 2 μM. The detection limit and sensitivity of the DNA sensor were 0.01 μM and 3362.1 Ω μM-1 cm-2, respectively. Various parameters, such as pH value, ionic strength, DNA probe concentration, and hybridization time, influencing DNA sensor responses were also investigated.

The interaction of taurine-salicylaldehyde Schiff base copper(II) complex with DNA and the determination of DNA using the complex as a fluorescence probe

Science.gov (United States)

Zhang, Xiaoyan; Wang, Yong; Zhang, Qianru; Yang, Zhousheng

2010-09-01

The interaction of taurine-salicylaldehyde Schiff base copper(II) (Cu(TSSB) 22+) complex with DNA was explored by using UV-vis, fluorescence spectrophotometry, and voltammetry. In pH 7.4 Tris-HCl buffer solution, the binding constant of the Cu(TSSB) 22+ complex interaction with DNA was 3.49 × 10 4 L mol -1. Moreover, due to the fluorescence enhancing of Cu(TSSB) 22+ complex in the presence of DNA, a method for determination of DNA with Cu(TSSB) 22+ complex as a fluorescence probe was developed. The fluorescence spectra indicated that the maximum excitation and emission wavelength were 389 nm and 512 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range of 0.03-9.03 μg mL -1 for calf thymus DNA (CT-DNA), 0.10-36 μg mL -1 for yeast DNA and 0.01-10.01 μg mL -1 for salmon DNA (SM-DNA), respectively. The corresponding detection limits are 7 ng mL -1 for CT-DNA, 3 ng mL -1 for yeast DNA and 3 ng mL -1 for SM-DNA. Using this method, DNA in synthetic samples was determined with satisfactory results.

Improved chaos-based video steganography using DNA alphabets

Directory of Open Access Journals (Sweden)

Nirmalya Kar

2018-03-01

Full Text Available DNA based steganography plays a vital role in the field of privacy and secure communication. Here, we propose a DNA properties-based mechanism to send data hidden inside a video file. Initially, the video file is converted into image frames. Random frames are then selected and data is hidden in these at random locations by using the Least Significant Bit substitution method. We analyze the proposed architecture in terms of peak signal-to-noise ratio as well as mean squared error measured between the original and steganographic files averaged over all video frames. The results show minimal degradation of the steganographic video file. Keywords: Chaotic map, DNA, Linear congruential generator, Video steganography, Least significant bit

Quantitation of the repair of gamma-radiation-induced double-strand DNA breaks in human fibroblasts

International Nuclear Information System (INIS)

Woods, W.G.

1981-01-01

The quantitation and repair of double-strand DNA breaks in human fibroblasts has been determined using a method involving the nondenaturing elution of DNA from a filter. DNA from cells from two human fibroblast lines exposed to γ-radiation from 0 to 10000 rad showed increasing retention on a filter with decreasing radiation dose, and the data suggest a linear relationship between double-strand breaks induced and radiation dose. The ability of normal human fibroblasts to repair double-strand breaks with various doses of radiation was demonstrated, with a tsub(1/2) of 10 min for repair of 5000 rad exposure and 39 min for repair of 10000 rad damage. The kinetics of the DNA rejoining were not linear and suggest that, as in the repair of single-strand breaks, both an initial fast and a later slow mechanism may be involved. (Auth.)

Flexibility of the genetic code with respect to DNA structure

DEFF Research Database (Denmark)

Baisnée, P. F.; Baldi, Pierre; Brunak, Søren

2001-01-01

Motivation. The primary function of DNA is to carry genetic information through the genetic code. DNA, however, contains a variety of other signals related, for instance, to reading frame, codon bias, pairwise codon bias, splice sites and transcription regulation, nucleosome positioning and DNA...... structure. Here we study the relationship between the genetic code and DNA structure and address two questions. First, to which degree does the degeneracy of the genetic code and the acceptable amino acid substitution patterns allow for the superimposition of DNA structural signals to protein coding...... sequences? Second, is the origin or evolution of the genetic code likely to have been constrained by DNA structure? Results. We develop an index for code flexibility with respect to DNA structure. Using five different di- or tri-nucleotide models of sequence-dependent DNA structure, we show...

Study on DNA damages induced by UV radiation

International Nuclear Information System (INIS)

Doan Hong Van; Dinh Ba Tuan; Tran Tuan Anh; Nguyen Thuy Ngan; Ta Bich Thuan; Vo Thi Thuong Lan; Tran Minh Quynh; Nguyen Thi Thom

2015-01-01

DNA damages in Escherichia coli (E. coli) exposed to UV radiation have been investigated. After 30 min of exposure to UV radiation of 5 mJ/cm"2, the growth of E. coli in LB broth medium was about only 10% in compared with non-irradiated one. This results suggested that the UV radiation caused the damages for E. coli genome resulted in reduction in its growth and survival, and those lesions can be somewhat recovered. For both solutions of plasmid DNAs and E. coli cells containing plasmid DNA, this dose also caused the breakage on single and double strands of DNA, shifted the morphology of DNA plasmid from supercoiled to circular and linear forms. The formation of pyrimidine dimers upon UV radiation significantly reduced when the DNA was irradiated in the presence of Ganoderma lucidum extract. Thus, studies on UV-induced DNA damage at molecular level are very essential to determine the UV radiation doses corresponding to the DNA damages, especially for creation and selection of useful radiation-induced mutants, as well as elucidation the protective effects of the specific compounds against UV light. (author)

Ribosomal DNA-binding proteins in the nucleolus of Physarum polycephalum

International Nuclear Information System (INIS)

Graham-Lorence, S.E.

1987-01-01

In Physarum polycephalum, the nucleoli are extra chromosomal structures containing 200 to 400 copies of a linear 60 kilobase palindromic rDNA molecule. These rDNA molecules are organized into minichromosomes which apparently are held within a nucleolar protein matrix. To obtained evidence for attachment of the rDNA to such a matrix, both intact and lithium diiodosalicylate/NaCl-extracted nucleoli were digested for various lengths of time with micrococcal nuclease, so that portions of the rDNA molecules not attached within the nucleolar structure would be released. Nucleolar DNA-binding proteins were determined by blotting electrophoretically separated proteins from SDS-polyacrylamide gels onto nitrocellulose paper and probing them with radiolabeled DNA. In addition to the histones and lexosome proteins, eight DNA-binding proteins were identified having molecular weights of 25, 38, 47, 53, 55, 67, and 70 kD, with the 47, 53, 67, and 70 kD proteins requiring Ca 2+ for binding

Studies of DNA repair in saccharomyces cerevisiae. I. Characterization of a new allele of RAD6. II. Investigation of events in the first cell cycle after DNA damage

International Nuclear Information System (INIS)

Douthwright-Fasse, J.A.

1979-01-01

Studies in two independent, but related, areas of DNA repair have been carried out in Saccharomyces cerevisiae; characterization of a new allele in the RAD6 gene which suggests that the gene is multifunctional, and utilization of photoreactivation as a probe of events occurring during the first cell cycle after DNA damage. Strains carrying the new allele, designated rad6-4, are as sensitive to uv and ionizing radiation as those carrying rad6-1 or rad6-3 but, unlike them, are capable of induced mutagenesis and sporulation. Although rad6-4 may well be a missense mutation, the evidence shows that it is unlikely that this phenotype is due to leakiness. Instead, the data suggest that the RAD6 gene is multifunctional. One function is necessary to recover from DNA damage in an error-free manner, and the other is concerned with mutagenic processes and sporulation. Rad6-1 and rad6-3 strains are deficient in both of these functions, while rad6-4 strains are deficient only in the error-free function. The loss of photoreversibility (LOP) of ultraviolet induced mutations to arginine independence in an excision defective strain carrying arg4-17 examines the events occurring in the first cell cycle after DNA damage. LOP is dependent upon de novo protein synthesis. LOP begins immediately after UV irradiation, before semiconservative DNA synthesis takes place, and is complete after four hours in growth medium.There is no evidence indicating whether the normal function of the protein is involved in excision repair, or in one of the two repair processes believed to be inducible; induced mutagenesis or recombinational repair

Checking the foundation: recent radiobiology and the linear no-threshold theory.

Science.gov (United States)

Ulsh, Brant A

2010-12-01

The linear no-threshold (LNT) theory has been adopted as the foundation of radiation protection standards and risk estimation for several decades. The "microdosimetric argument" has been offered in support of the LNT theory. This argument postulates that energy is deposited in critical cellular targets by radiation in a linear fashion across all doses down to zero, and that this in turn implies a linear relationship between dose and biological effect across all doses. This paper examines whether the microdosimetric argument holds at the lowest levels of biological organization following low dose, low dose-rate exposures to ionizing radiation. The assumptions of the microdosimetric argument are evaluated in light of recent radiobiological studies on radiation damage in biological molecules and cellular and tissue level responses to radiation damage. There is strong evidence that radiation initially deposits energy in biological molecules (e.g., DNA) in a linear fashion, and that this energy deposition results in various forms of prompt DNA damage that may be produced in a pattern that is distinct from endogenous (e.g., oxidative) damage. However, a large and rapidly growing body of radiobiological evidence indicates that cell and tissue level responses to this damage, particularly at low doses and/or dose-rates, are nonlinear and may exhibit thresholds. To the extent that responses observed at lower levels of biological organization in vitro are predictive of carcinogenesis observed in vivo, this evidence directly contradicts the assumptions upon which the microdosimetric argument is based.

Interfacing of DNA with carbon nanotubes for nanodevice applications

International Nuclear Information System (INIS)

Rastogi, Richa; Dhindsa, Navneet; Suri, C. Raman; Pant, B.D.; Tripathi, S.K.; Kaur, Inderpreet; Bharadwaj, Lalit M.

2012-01-01

In nanotechnology, carbon nanotubes are evolving as ‘hot spot’ due to their applications as most sensitive biosensors. Thus, study of effect of biomolecular interaction is prerequisite for their electrical application in biosensors and bioelectronics. Here, we have explored this effect on electrical properties of carbon nanotubes with DNA as a model biomolecule. A stable conjugate of carbon nanotubes with DNA is formed via covalent methodology employing quantum dot as fluoropore and characterized with various spectroscopic, fluoroscopic and microscopic techniques. CNT–DNA adduct showed decreased transconductance (from 614.46 μS to 1.34 μS) and shift of threshold voltage (from −0.85 V to 2.5 V) due to change in Schottky barriers at metal–nanotube contact. In addition, decrease in hole mobility (from 4.46 × 10 6 to 9.72 × 10 3 cm 2 V −1 s −1 ) and increase in ON-linear resistance (from 74 kΩ to 0.44 MΩ) conclude large change in device parameters. On the one hand, this substantial change in device parameters after interfacing with biomolecules supports application of carbon nanotubes in the field of biosensors while on the other hand, the same can limit their use in future power electronic devices where stability in device parameters is essential. -- Graphical abstract: Carbon nanotubes are interfaced with DNA via covalent interactions and characterized with spectroscopic, fluoroscopic and microscopic techniques. Electrical characterization of this stable SWNT–DNA conjugate shows decreased transconductance and shift of threshold voltage towards positive gate voltages. On the one hand, this substantial change in device parameters after interfacing with biomolecules supports application of carbon nanotubes in the field of biosensors while on the other hand, the same can limit their use in future power electronic devices where stability in device parameters is essential. Highlights: ► Effect of biomolecular (DNA) interaction on electrical

Interstrand DNA crosslinking by 4,5',8-trimethylpsoralen plus monochromatic ultraviolet light

International Nuclear Information System (INIS)

Cohen, L.F.; Ewig, R.A.G.; Kohn, K.W.; Glaubiger, D.

1980-01-01

DNA crosslinking by 4,5',8-trimethylpsoralen plus monochromatic ultraviolet light of wavelength 365 nm was studied in mouse L1210 leukemia cells. DNA breaks and crosslinking were evaluated by alkaline elution of DNA from poly(vinyl chloride) filters. Trimethylpsoralen plus 365 nm light produced DNA crosslinks but not breaks. The kinetics of crosslinging were linear with respect to concentration and second-order with respect to light exposure time. The latter finding supports the proposed two photon mechanism for the formation of diadducts. In contrast to DNA crosslinking agents such as nitrogen mustard, nitrosoureas and platinums, trimethylpsoralen crosslinks were resistant to proteolytic digestion. Thus, trimethylpsoralen plus 365 nm light produced interstrand crosslinks, as proposed for a bifunctional agent binding to bases on opposite DNA strands. (Auth.)

Mitochondrial DNA replication: a PrimPol perspective

Science.gov (United States)

Bailey, Laura J.

2017-01-01

PrimPol, (primase–polymerase), the most recently identified eukaryotic polymerase, has roles in both nuclear and mitochondrial DNA maintenance. PrimPol is capable of acting as a DNA polymerase, with the ability to extend primers and also bypass a variety of oxidative and photolesions. In addition, PrimPol also functions as a primase, catalysing the preferential formation of DNA primers in a zinc finger-dependent manner. Although PrimPol's catalytic activities have been uncovered in vitro, we still know little about how and why it is targeted to the mitochondrion and what its key roles are in the maintenance of this multicopy DNA molecule. Unlike nuclear DNA, the mammalian mitochondrial genome is circular and the organelle has many unique proteins essential for its maintenance, presenting a differing environment within which PrimPol must function. Here, we discuss what is currently known about the mechanisms of DNA replication in the mitochondrion, the proteins that carry out these processes and how PrimPol is likely to be involved in assisting this vital cellular process. PMID:28408491

TEMPORAL MODELING OF DNA DEGRADATION IN BONE REMAINS

Directory of Open Access Journals (Sweden)

Andrei Stefan

2012-06-01

Full Text Available The aim of this study is to follow the changes that occur, in time, at DNA level and to establish an efficient and reliable protocol for ancestral DNA extraction from bones found in archaeological sites. To test whether the protocol is efficient and capable of yielding good quality DNA, extraction was first performed on fresh bones. The material consists of fresh pig (Sus scrofa and cow (Bos taurus bones that were grounded by using a drill operating at low speed. The bone powder was then incubated in lysis buffer in the presence of proteinase K. DNA isolation and purification were done by using the phenol:chloroform protocol and DNA was precipitated with absolute ethanol stored at -20oC. The extractions were carried out once every month for a total of four extractions

[DNA complexes, formed on aqueous phase surfaces: new planar polymeric and composite nanostructures].

Science.gov (United States)

Antipina, M N; Gaĭnutdinov, R V; Rakhnianskaia, A A; Sergeev-Cherenkov, A N; Tolstikhina, A L; Iurova, T V; Kislov, V V; Khomutov, G B

2003-01-01

The formation of DNA complexes with Langmuir monolayers of the cationic lipid octadecylamine (ODA) and the new amphiphilic polycation poly-4-vinylpyridine with 16% of cetylpyridinium groups (PVP-16) on the surface of an aqueous solution of native DNA of low ionic strength was studied. Topographic images of Langmuir-Blodgett films of DNA/ODA and DNA/PVP-16 complexes applied to micaceous substrates were investigated by the method of atomic force microscopy. It was found that films of the amphiphilic polycation have an ordered planar polycrystalline structure. The morphology of planar DNA complexes with the amphiphilic cation substantially depended on the incubation time and the phase state of the monolayer on the surface of the aqueous DNA solution. Complex structures and individual DNA molecules were observed on the surface of the amphiphilic monolayer. Along with quasi-linear individual bound DNA molecules, characteristic extended net-like structures and quasi-circular toroidal condensed conformations of planar DNA complexes were detected. Mono- and multilayer films of DNA/PVP-16 complexes were used as templates and nanoreactors for the synthesis of inorganic nanostructures via the binding of metal cations from the solution and subsequent generation of the inorganic phase. As a result, ultrathin polymeric composite films with integrated DNA building blocks and quasi-linear arrays of inorganic semiconductor (CdS) and iron oxide nanoparticles and nanowires were obtained. The nanostructures obtained were characterized by scanning probe microscopy and transmission electron microscopy techniques. The methods developed are promising for investigating the mechanisms of structural organization and transformation in DNA and polyelectrolyte complexes at the gas-liquid interface and for the design of new extremely thin highly ordered planar polymeric and composite materials, films, and coatings with controlled ultrastructure for applications in nanoelectronics and

Effect of multipurpose solutions against Acinetobacter carrying QAC genes.

Science.gov (United States)

Boost, Maureen V; Chan, Jessica; Shi, Guang-sen; Cho, Pauline

2014-03-01

Acinetobacter has low virulence but causes infections in subjects with reduced immunity. It has been reported in ocular infections including those of patients using contact lenses. Treatment is difficult because Acinetobacter is frequently multidrug resistant. Antibiotic-resistant strains frequently also harbor genes for antiseptic resistance (quaternary ammonium compound [QAC]) genes. Because Acinetobacter is part of the normal flora, it may contaminate contact lens and accessories. This study aims to investigate carriage rates of QAC genes in household and clinical isolates of Acinetobacter and to determine the effectiveness of two multipurpose solutions (MPSs) for soft lenses against organisms carrying QAC genes. DNA was extracted from 11 bathroom isolates and 15 clinical isolates and amplified by polymerase chain reaction to determine the presence of qacEΔ1. Gene-positive and gene-negative control strains were used to challenge the two MPSs, and minimum inhibitory concentrations (MICs) of these organisms to benzalkonium chloride and chlorhexidine gluconate were determined. More than 90% of isolates carried qacEΔ1. The MICs of clinical isolates were higher than those of isolates of bathrooms. Both MPSs were able to produce a 3-log reduction in the numbers of all isolates. Although most isolates carried qacEΔ1 and elevated MICs to benzalkonium chloride and chlorhexidine gluconate were observed, all were susceptible to both MPSs tested. However, if there were to be poor compliance with care procedures, it is probable that such organisms could survive in the presence of diluted or expired solutions.

Non-invasive prenatal detection of achondroplasia using circulating fetal DNA in maternal plasma.

Science.gov (United States)

Lim, Ji Hyae; Kim, Mee Jin; Kim, Shin Young; Kim, Hye Ok; Song, Mee Jin; Kim, Min Hyoung; Park, So Yeon; Yang, Jae Hyug; Ryu, Hyun Mee

2011-02-01

To perform a reliable non-invasive detection of the fetal achondroplasia using maternal plasma. We developed a quantitative fluorescent-polymerase chain reaction (QF-PCR) method suitable for detection of the FGFR3 mutation (G1138A) causing achondroplasia. This method was applied in a non-invasive detection of the fetal achondroplasia using circulating fetal-DNA (cf-DNA) in maternal plasma. Maternal plasmas were obtained at 27 weeks of gestational age from women carrying an achondroplasia fetus or a normal fetus. Two percent or less achondroplasia DNA was reliably detected by QF-PCR. In a woman carrying a normal fetus, analysis of cf-DNA showed only one peak of the wild-type G allele. In a woman expected an achondroplasia fetus, analysis of cf-DNA showed the two peaks of wild-type G allele and mutant-type A allele and accurately detected the fetal achondroplasia. The non-invasive method using maternal plasma and QF-PCR may be useful for diagnosis of the fetal achondroplasia.

Fractals in DNA sequence analysis

Institute of Scientific and Technical Information of China (English)

Yu Zu-Guo(喻祖国); Vo Anh; Gong Zhi-Min(龚志民); Long Shun-Chao(龙顺潮)

2002-01-01

Fractal methods have been successfully used to study many problems in physics, mathematics, engineering, finance,and even in biology. There has been an increasing interest in unravelling the mysteries of DNA; for example, how can we distinguish coding and noncoding sequences, and the problems of classification and evolution relationship of organisms are key problems in bioinformatics. Although much research has been carried out by taking into consideration the long-range correlations in DNA sequences, and the global fractal dimension has been used in these works by other people, the models and methods are somewhat rough and the results are not satisfactory. In recent years, our group has introduced a time series model (statistical point of view) and a visual representation (geometrical point of view)to DNA sequence analysis. We have also used fractal dimension, correlation dimension, the Hurst exponent and the dimension spectrum (multifractal analysis) to discuss problems in this field. In this paper, we introduce these fractal models and methods and the results of DNA sequence analysis.

Photoconductivity in DNA-Porphyrin Complexes

Science.gov (United States)

Myint, Peco; Oxford, Emma; Nyazenga, Collence; Smith, Walter; Qi, Zhengqing; Johnson, A. T.

2015-03-01

We have measured the photoconductivity of λ - DNA that is modified by intercalating a porphyrin compound, meso-tetrakis(N-methyl-4-pyridiniumyl)porphyrin (TMPyP), into its base stacks. Intercalation was verified by a red shift and hypochromism of the Soret absorption peak. The DNA/porphyrin strands were then deposited onto oxidized silicon substrates which had been patterned with interdigitated electrodes, and blown dry. Electrical measurements were carried out under nitrogen, using illumination from a 445 nm laser; this wavelength falls within the absorption peak of the DNA/porphyrin complexes. When initially measured under dry nitrogen, the complexes show no photoconductivity or dark conductivity. However, at relative humidities of 30% and above, we do observe dark conductivity, and also photoconductivity that grows with time. Photoconductivity gets larger at higher relative humidity. Remarkably, when the humidity is lowered again, some photoconductivity is now observed, indicating a change that persists for more than 24 hours. It may be that the humidity alters the structure of the DNA, perhaps allowing for better alignment of the bases. This work was supported by NSF Grant BMAT-1306170.

Local hyperspectral data multisharpening based on linear/linear-quadratic nonnegative matrix factorization by integrating lidar data

Science.gov (United States)

Benhalouche, Fatima Zohra; Karoui, Moussa Sofiane; Deville, Yannick; Ouamri, Abdelaziz

2015-10-01

In this paper, a new Spectral-Unmixing-based approach, using Nonnegative Matrix Factorization (NMF), is proposed to locally multi-sharpen hyperspectral data by integrating a Digital Surface Model (DSM) obtained from LIDAR data. In this new approach, the nature of the local mixing model is detected by using the local variance of the object elevations. The hyper/multispectral images are explored using small zones. In each zone, the variance of the object elevations is calculated from the DSM data in this zone. This variance is compared to a threshold value and the adequate linear/linearquadratic spectral unmixing technique is used in the considered zone to independently unmix hyperspectral and multispectral data, using an adequate linear/linear-quadratic NMF-based approach. The obtained spectral and spatial information thus respectively extracted from the hyper/multispectral images are then recombined in the considered zone, according to the selected mixing model. Experiments based on synthetic hyper/multispectral data are carried out to evaluate the performance of the proposed multi-sharpening approach and literature linear/linear-quadratic approaches used on the whole hyper/multispectral data. In these experiments, real DSM data are used to generate synthetic data containing linear and linear-quadratic mixed pixel zones. The DSM data are also used for locally detecting the nature of the mixing model in the proposed approach. Globally, the proposed approach yields good spatial and spectral fidelities for the multi-sharpened data and significantly outperforms the used literature methods.

Detection of Avian Influenza Virus by Fluorescent DNA Barcode-based Immunoassay with Sensitivity Comparable to PCR

DEFF Research Database (Denmark)

Cao, Cuong; Dhumpa, Raghuram; Bang, Dang Duong

2010-01-01

involves the sandwiching of the target AIV between magnetic immunoprobes and barcode-carrying immunoprobes. Because each barcode-carrying immunoprobe is functionalized with a multitude of fluorophore-DNA barcode strands, many DNA barcodes are released for each positive binding event resulting......In this paper, a coupling of fluorophore-DNA barcode and bead-based immunoassay for detecting avian influenza virus (AIV) with PCR-like sensitivity is reported. The assay is based on the use of sandwich immunoassay and fluorophore-tagged oligonucleotides as representative barcodes. The detection...

Varicella-zoster virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription.

Science.gov (United States)

Khalil, Mohamed I; Sommer, Marvin H; Hay, John; Ruyechan, William T; Arvin, Ann M

2015-07-01

The VZV genome has two origins of DNA replication (oriS), each of which consists of an AT-rich sequence and three origin binding protein (OBP) sites called Box A, C and B. In these experiments, the mutation in the core sequence CGC of the Box A and C not only inhibited DNA replication but also inhibited both ORF62 and ORF63 expression in reporter gene assays. In contrast the Box B mutation did not influence DNA replication or flanking gene transcription. These results suggest that efficient DNA replication enhances ORF62 and ORF63 transcription. Recombinant viruses carrying these mutations in both sites and one with a deletion of the whole oriS were constructed. Surprisingly, the recombinant virus lacking both copies of oriS retained the capacity to replicate in melanoma and HELF cells suggesting that VZV has another origin of DNA replication. Copyright © 2015 Elsevier Inc. All rights reserved.

A novel technique using DNA denaturation to detect multiply induced single-strand breaks in a hydrated plasmid DNA molecule by X-ray and 4He2+ ion irradiation

International Nuclear Information System (INIS)

Yokoya, A.; Shikazono, N.; Fujii, K.; Noguchi, M.; Urushibara, A.

2011-01-01

To detect multiple single-strand breaks (SSBs) produced in plasmid DNA molecules by direct energy deposition from radiation tracks, we have developed a novel technique using DNA denaturation by which irradiated DNA is analysed as single-strand DNA (SS-DNA). The multiple SSBs that arise in both strands of DNA, but do not induce a double-strand break, are quantified as loss of SS-DNA using agarose gel electrophoresis. We have applied this method to X-ray and 4 He 2+ ion-irradiated samples of fully hydrated pUC18 plasmid DNA. The fractions of both SS-DNA and closed circular DNA (CC-DNA) exponentially decrease with the increasing dose of X rays and 4 He 2+ ions. The efficiency of the loss of SS-DNA was half that of CC-DNA for both types of irradiation, indicating that one of two strands in DNA is not broken when one SSB is produced in CC-DNA by irradiation. Contrary to our initial expectation, these results indicate that SSBs are not multiply induced even by high linear energy transfer radiation distributed in both strands. (authors)

Reversible Modulation of DNA-Based Hydrogel Shapes by Internal Stress Interactions.

Science.gov (United States)

Hu, Yuwei; Kahn, Jason S; Guo, Weiwei; Huang, Fujian; Fadeev, Michael; Harries, Daniel; Willner, Itamar

2016-12-14

We present the assembly of asymmetric two-layer hybrid DNA-based hydrogels revealing stimuli-triggered reversibly modulated shape transitions. Asymmetric, linear hydrogels that include layer-selective switchable stimuli-responsive elements that control the hydrogel stiffness are designed. Trigger-induced stress in one of the layers results in the bending of the linear hybrid structure, thereby minimizing the elastic free energy of the systems. The removal of the stress by a counter-trigger restores the original linear bilayer hydrogel. The stiffness of the DNA hydrogel layers is controlled by thermal, pH (i-motif), K + ion/crown ether (G-quadruplexes), chemical (pH-doped polyaniline), or biocatalytic (glucose oxidase/urease) triggers. A theoretical model relating the experimental bending radius of curvatures of the hydrogels with the Young's moduli and geometrical parameters of the hydrogels is provided. Promising applications of shape-regulated stimuli-responsive asymmetric hydrogels include their use as valves, actuators, sensors, and drug delivery devices.

An electrochemical study of neutral red-DNA interaction

International Nuclear Information System (INIS)

Heli, H.; Bathaie, S.Z.; Mousavi, M.F.

2005-01-01

Electrochemical methods were used to investigate the interaction of neutral red (NR) with double-stranded calf thymus DNA, in solution as well as using a DNA-modified glassy carbon (GC-DNA) electrode. The results were compared with those obtained from bare glassy carbon (GC) electrode. The formal potential of NR was more positive when GC-DNA electrode was used although the rate of heterogeneous electron transfer is as high as that of using GC electrode. GC-DNA electrode enables preconcentration of NR for chosen times on the electrode surface, despite the fact that the mass transfer effects in the thin DNA layer adsorbed on the surface was still observed using cyclic voltammetry and electrochemical impedance spectroscopy techniques. Parameters, such as the diffusion coefficient of NR, binding site size in base pairs and the ratio of the binding constants for the oxidized and reduced forms of the bound species were obtained. A binding isotherm for NR at GC-DNA electrode was obtained from coulometric titrations and gave an affinity constant equal to 2.76 x 10 4 L mol -1 . From the studies of the interaction in solution, the diffusion coefficient of free and DNA-bound NR, binding constant and binding site size of the DNA-NR complex was also obtained simultaneously by non-linear fitting analysis of voltammetric data

Probe DNA-Cisplatin Interaction with Solid-State Nanopores

Science.gov (United States)

Zhou, Zhi; Hu, Ying; Li, Wei; Xu, Zhi; Wang, Pengye; Bai, Xuedong; Shan, Xinyan; Lu, Xinghua; Nanopore Collaboration

2014-03-01

Understanding the mechanism of DNA-cisplatin interaction is essential for clinical application and novel drug design. As an emerging single-molecule technology, solid-state nanopore has been employed in biomolecule detection and probing DNA-molecule interactions. Herein, we reported a real-time monitoring of DNA-cisplatin interaction by employing solid-state SiN nanopores. The DNA-cisplatin interacting process is clearly classified into three stages by measuring the capture rate of DNA-cisplatin adducts. In the first stage, the negative charged DNA molecules were partially discharged due to the bonding of positive charged cisplatin and forming of mono-adducts. In the second stage, forming of DNA-cisplatin di-adducts with the adjacent bases results in DNA bending and softening. The capture rate increases since the softened bi-adducts experience a lower barrier to thread into the nanopores. In the third stage, complex structures, such as micro-loop, are formed and the DNA-cisplatin adducts are aggregated. The capture rate decreases to zero as the aggregated adduct grows to the size of the pore. The characteristic time of this stage was found to be linear with the diameter of the nanopore and this dynamic process can be described with a second-order reaction model. We are grateful to Laboratory of Microfabrication, Dr. Y. Yao, and Prof. R.C. Yu (Institute of Physics, Chinese Academy of Sciences) for technical assistance.

Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation

Science.gov (United States)

Sutherland, B. M.; Bennett, P. V.; Sidorkina, O.; Laval, J.; Lowenstein, D. I. (Principal Investigator)

2000-01-01

Clustered DNA damages-two or more closely spaced damages (strand breaks, abasic sites, or oxidized bases) on opposing strands-are suspects as critical lesions producing lethal and mutagenic effects of ionizing radiation. However, as a result of the lack of methods for measuring damage clusters induced by ionizing radiation in genomic DNA, neither the frequencies of their production by physiological doses of radiation, nor their repairability, nor their biological effects are known. On the basis of methods that we developed for quantitating damages in large DNAs, we have devised and validated a way of measuring ionizing radiation-induced clustered lesions in genomic DNA, including DNA from human cells. DNA is treated with an endonuclease that induces a single-strand cleavage at an oxidized base or abasic site. If there are two closely spaced damages on opposing strands, such cleavage will reduce the size of the DNA on a nondenaturing gel. We show that ionizing radiation does induce clustered DNA damages containing abasic sites, oxidized purines, or oxidized pyrimidines. Further, the frequency of each of these cluster classes is comparable to that of frank double-strand breaks; among all complex damages induced by ionizing radiation, double-strand breaks are only about 20%, with other clustered damage constituting some 80%. We also show that even low doses (0.1-1 Gy) of high linear energy transfer ionizing radiation induce clustered damages in human cells.

Synthesis of DNA block copolymers with extended nucleic acid segments by enzymatic ligation : cut and paste large hybrid architectures

NARCIS (Netherlands)

Ayaz, Meryem S.; Kwak, Minseok; Alemdaroglu, Fikri E.; Wang, Jie; Berger, Ruediger; Herrmann, Andreas; Berger, Rüdiger

2011-01-01

Ultra-high molecular weight DNA/polymer hybrid materials were prepared employing molecular biology techniques. Nucleic acid restriction and ligation enzymes were used to generate linear DNA di- and triblock copolymers that contain up to thousands of base pairs in the DNA segments.

DNA double strand break repair is enhanced by P53 following induction by DNA damage and is dependent on the C-terminal domain of P53

International Nuclear Information System (INIS)

Wei Tang; Powell, Simon N.

1996-01-01

Purpose: The tumor suppressor gene p53 can mediate cell cycle arrest or apoptosis in response to DNA damage. Accumulating evidence suggests that it may also directly or indirectly influence the DNA repair machinery. In the present study, we investigated whether p53, induced by DNA damage, could enhance the rejoining of double-strand DNA breaks. Materials and Methods: DNA double-strand breaks (dsb) were made by restriction enzyme digestion of a plasmid, between a promoter and a 'reporter' gene: luciferase (LUC) or chloramphenicol acetyl-transferase (CAT). Linear or circular plasmid DNA (LUC or CAT) was co-transfected with circular β-Gal plasmid (to normalize for uptake) into mouse embryonic fibroblasts genetically matched to be (+/+) or (-/-) for p53. Their ability to rejoin linearized plasmid was measured by the luciferase or CAT activity detected in rescued plasmids. The activity detected in cells transfected with linear plasmid was scored relative to the activity detected in cells transfected with circular plasmid. Results: Ionizing radiation (IR, 2 Gy) enhanced the dsb repair activity in wild type p53 cells; however, p53 null cells lose this effect, indicating that the enhancement of dsb repair was p53-dependent. REF cells with dominant-negative mutant p53 showed a similar induction compared with the parental REF cells with wild-type p53. This ala-143 mutant p53 prevents cell cycle arrest and transactivation of p21 WAF1/cip1) following IR, indicating that the p53-dependent enhancement of DNA repair is distinct from transactivation. Immortalized murine embryonic fibroblasts, 10(1)VasK1 cells, which express p53 cDNA encoding a temperature-sensitive mutant in the DNA sequence specific binding domain (ala135 to val135) with an alternatively spliced C-terminal domain (ASp53: amino-acids 360-381) and, 10(1)Val5 cells, which express the normal spliced p53 (NSp53) with the same temperature-sensitive mutant were compared. It was found that 10(1)VasK1 cells showed no DNA

Ultrasensitive Electrochemical Detection of Clostridium perfringens DNA Based Morphology-Dependent DNA Adsorption Properties of CeO2 Nanorods in Dairy Products

Directory of Open Access Journals (Sweden)

Xingcan Qian

2018-06-01

Full Text Available Foodborne pathogens such as Clostridium perfringens can cause diverse illnesses and seriously threaten to human health, yet far less attention has been given to detecting these pathogenic bacteria. Herein, two morphologies of nanoceria were synthesized via adjusting the concentration of NaOH, and CeO2 nanorod has been utilized as sensing material to achieve sensitive and selective detection of C. perfringens DNA sequence due to its strong adsorption ability towards DNA compared to nanoparticle. The DNA probe was tightly immobilized on CeO2/chitosan modified electrode surface via metal coordination, and the DNA surface density was 2.51 × 10−10 mol/cm2. Under optimal experimental conditions, the electrochemical impedance biosensor displays favorable selectivity toward target DNA in comparison with base-mismatched and non-complementary DNA. The dynamic linear range of the proposed biosensor for detecting oligonucleotide sequence of Clostridium perfringens was from 1.0 × 10−14 to 1.0 × 10−7 mol/L. The detection limit was 7.06 × 10−15 mol/L. In comparison, differential pulse voltammetry (DPV method quantified the target DNA with a detection limit of 1.95 × 10−15 mol/L. Moreover, the DNA biosensor could detect C. perfringens extracted DNA in dairy products and provided a potential application in food quality control.

Carrying Capacity

DEFF Research Database (Denmark)

Schroll, Henning; Andersen, Jan; Kjærgård, Bente

2012-01-01

A spatial planning act was introduced inIndonesia 1992 and renewed in 2008. It emphasised the planning role of decentralised authorities. The spatial planning act covers both spatial and environmental issues. It defines the concept of carrying capacity and includes definitions of supportive....../cities. Four different sectors (water, food production, waste, and forests) were selected as core areas for decentralised spatial planning. Indicators for SCC and ACC were identified and assessed with regard to relevance and quantifiability. For each of the indicators selected, a legal threshold or guiding...... was introduced inIndonesia 1992 and renewed in 2008. It emphasised the planning role of decentralised authorities. The spatial planning act covers both spatial and environmental issues. It defines the concept of carrying capacity and includes definitions of supportive carrying capacity (SCC) and assimilative...

Multimodal Image Alignment via Linear Mapping between Feature Modalities.

Science.gov (United States)

Jiang, Yanyun; Zheng, Yuanjie; Hou, Sujuan; Chang, Yuchou; Gee, James

2017-01-01

We propose a novel landmark matching based method for aligning multimodal images, which is accomplished uniquely by resolving a linear mapping between different feature modalities. This linear mapping results in a new measurement on similarity of images captured from different modalities. In addition, our method simultaneously solves this linear mapping and the landmark correspondences by minimizing a convex quadratic function. Our method can estimate complex image relationship between different modalities and nonlinear nonrigid spatial transformations even in the presence of heavy noise, as shown in our experiments carried out by using a variety of image modalities.

A geometrical model for DNA organization in bacteria.

Directory of Open Access Journals (Sweden)

Mathias Buenemann

Full Text Available Recent experimental studies have revealed that bacteria, such as C. crescentus, show a remarkable spatial ordering of their chromosome. A strong linear correlation has been found between the position of genes on the chromosomal map and their spatial position in the cellular volume. We show that this correlation can be explained by a purely geometrical model. Namely, self-avoidance of DNA, specific positioning of one or few DNA loci (such as origin or terminus together with the action of DNA compaction proteins (that organize the chromosome into topological domains are sufficient to get a linear arrangement of the chromosome along the cell axis. We develop a Monte-Carlo method that allows us to test our model numerically and to analyze the dependence of the spatial ordering on various physiologically relevant parameters. We show that the proposed geometrical ordering mechanism is robust and universal (i.e. does not depend on specific bacterial details. The geometrical mechanism should work in all bacteria that have compacted chromosomes with spatially fixed regions. We use our model to make specific and experimentally testable predictions about the spatial arrangement of the chromosome in mutants of C. crescentus and the growth-stage dependent ordering in E. coli.

CSA: An efficient algorithm to improve circular DNA multiple alignment

Directory of Open Access Journals (Sweden)

Pereira Luísa

2009-07-01

Full Text Available Abstract Background The comparison of homologous sequences from different species is an essential approach to reconstruct the evolutionary history of species and of the genes they harbour in their genomes. Several complete mitochondrial and nuclear genomes are now available, increasing the importance of using multiple sequence alignment algorithms in comparative genomics. MtDNA has long been used in phylogenetic analysis and errors in the alignments can lead to errors in the interpretation of evolutionary information. Although a large number of multiple sequence alignment algorithms have been proposed to date, they all deal with linear DNA and cannot handle directly circular DNA. Researchers interested in aligning circular DNA sequences must first rotate them to the "right" place using an essentially manual process, before they can use multiple sequence alignment tools. Results In this paper we propose an efficient algorithm that identifies the most interesting region to cut circular genomes in order to improve phylogenetic analysis when using standard multiple sequence alignment algorithms. This algorithm identifies the largest chain of non-repeated longest subsequences common to a set of circular mitochondrial DNA sequences. All the sequences are then rotated and made linear for multiple alignment purposes. To evaluate the effectiveness of this new tool, three different sets of mitochondrial DNA sequences were considered. Other tests considering randomly rotated sequences were also performed. The software package Arlequin was used to evaluate the standard genetic measures of the alignments obtained with and without the use of the CSA algorithm with two well known multiple alignment algorithms, the CLUSTALW and the MAVID tools, and also the visualization tool SinicView. Conclusion The results show that a circularization and rotation pre-processing step significantly improves the efficiency of public available multiple sequence alignment

DNA synthesis in vitro in human fibroblast preparations

Energy Technology Data Exchange (ETDEWEB)

Kaufmann, W.K.

1983-01-01

When confluent cultures of human fibroblasts were ultraviolet irradiated and either permeabilized or lysed, three types of DNA synthesis were subsequently observed during incubation in vitro: (A) a low level of DNA replication, which ceased after 15-30 min incubation at 37/sup 0/C; (B) radiation-dependent reparative gap-filling, which also ceased after 15 min at 37/sup 0/C; and (C) radiation-independent DNA synthesis, which was not semiconservative and proceeded at a linear rate for 1 hr at 37/sup 0/C. Normal and xeroderma pigmentosum fibroblasts displayed different rates of radiation-dependent reparative gap-filling after lysis but similar rates of radiation-independent DNA synthesis. The rates of DNA replication and radiation-independent DNA synthesis were less in the permeable cell system than in the lysed cell system, whereas radiation-dependent reparative gap-filling was the same in both. Preparations of permeable and lysed cells activated radiation-dependent reparative gap-filling at about 15% of the rate estimated for intact cells. No radiation-dependent DNA strand breaks, as assayed by alkaline elution, were observed in the lysed cell preparation. Some radiation-dependent breaks were observed in the permeable cell preparation, but radiation-dependent DNA breakage was less than that seen in intact cells. This inability to incise DNA at damaged sites could account for the low rate of activation of reparative gap-filling in vitro. DNA strand breaks were produced in fibroblast preparations nonspecifically during lysis or permeabilization and incubation in vitro, and this breakage of DNA probably was responsible for the radiation-independent DNA synthesis.

Antigen-antibody reactions of UV-irradiated phage DNA

International Nuclear Information System (INIS)

Fink, A.

1976-01-01

The observation of others could be confirmed that UV-irradiated DNA is a better immunogen than unirradiated DNA. The author's immune sera contained a high amount of antibodies with a specific action against photoproducts in the DNA. The thymine dimer was identified as relevant photoproduct and thus as antigenic determinant. In comparison, the amount of unspecific antibodies reacting with denaturated DNA was low and varied between sera. Thymin-dimer antibodies showed a high specificity without cross-reaction with other pyrimidine dimers such as anti CC and anti CT; they belong to the class of IgG molecules. UV-irradiated dinucleotide dTpT is sufficient to induce the formation of antibodies reacting with the cis-syn thymine dimers in UV-irradiated DNA. Antibody binding is proportional to the UV doses applied to the DNA. When using completely denaturated DNA, there is a linear increase changing into a plateau at higher doses. The extent of antigen-antibody binding is strongly dependent on the degree of denaturation of the DNA. With increasing denaturation, the antibody binding of the DNA increases. The antigen-antibody reaction can thus be used to estimate the degree of denaturation of the DNA. There were no signs of an influence of the degree of denaturation of the DNA on the quantum yield of thymine dimers. The different amounts of antibodies is therefore due to the masking of thymine dimers in native DNA. When irradiating intact phage particles, there was no sign of an influence of the phages' protein covers on the antibody binding capacity of DNA compared with DNA irradiated in vitro. (orig.) [de

Thermoluminescent dosimetry applied to radiotherapy carried out around the Therac-20-Saturne linear accelerator

International Nuclear Information System (INIS)

Bennab, K.

1984-10-01

In the first part of this work, the physical and technical aspects necessary to a radiotherapy treatment planning elaboration are presented. These include the apparatus description as well as the measurements of the dosimetric quantities relative to the delivered beams. Since in-vivo measurements are very useful to optimise the treatment conditions, the second part deals with thermoluminescent dosimeters, their characteristics and their setting out such as they be used with maximum efficiency. In-vivo measurements carried out enabled us to estimate the delivered dose to a given tumor with a precision down to +-4%. The registered differences between the measured dose and the prescribed one made possible an appropriate corrective action. Because of the difficulties encountered with conventional dosimetric systems when used in narrow beam measurements, we have developed a new method making use of TL dosimeters. The results obtained will allow treatments of small volume tumors with the significant advantage of saving healthy surrounding tissues from irradiation

U1 snDNA clusters in grasshoppers: chromosomal dynamics and genomic organization

Science.gov (United States)

Anjos, A; Ruiz-Ruano, F J; Camacho, J P M; Loreto, V; Cabrero, J; de Souza, M J; Cabral-de-Mello, D C

2015-01-01

The spliceosome, constituted by a protein set associated with small nuclear RNA (snRNA), is responsible for mRNA maturation through intron removal. Among snRNA genes, U1 is generally a conserved repetitive sequence. To unveil the chromosomal/genomic dynamics of this multigene family in grasshoppers, we mapped U1 genes by fluorescence in situ hybridization in 70 species belonging to the families Proscopiidae, Pyrgomorphidae, Ommexechidae, Romaleidae and Acrididae. Evident clusters were observed in all species, indicating that, at least, some U1 repeats are tandemly arrayed. High conservation was observed in the first four families, with most species carrying a single U1 cluster, frequently located in the third or fourth longest autosome. By contrast, extensive variation was observed among Acrididae, from a single chromosome pair carrying U1 to all chromosome pairs carrying it, with occasional occurrence of two or more clusters in the same chromosome. DNA sequence analysis in Eyprepocnemis plorans (species carrying U1 clusters on seven different chromosome pairs) and Locusta migratoria (carrying U1 in a single chromosome pair) supported the coexistence of functional and pseudogenic lineages. One of these pseudogenic lineages was truncated in the same nucleotide position in both species, suggesting that it was present in a common ancestor to both species. At least in E. plorans, this U1 snDNA pseudogenic lineage was associated with 5S rDNA and short interspersed elements (SINE)-like mobile elements. Given that we conclude in grasshoppers that the U1 snDNA had evolved under the birth-and-death model and that its intragenomic spread might be related with mobile elements. PMID:25248465

Development of non-linear vibration analysis code for CANDU fuelling machine

International Nuclear Information System (INIS)

Murakami, Hajime; Hirai, Takeshi; Horikoshi, Kiyomi; Mizukoshi, Kaoru; Takenaka, Yasuo; Suzuki, Norio.

1988-01-01

This paper describes the development of a non-linear, dynamic analysis code for the CANDU 600 fuelling machine (F-M), which includes a number of non-linearities such as gap with or without Coulomb friction, special multi-linear spring connections, etc. The capabilities and features of the code and the mathematical treatment for the non-linearities are explained. The modeling and numerical methodology for the non-linearities employed in the code are verified experimentally. Finally, the simulation analyses for the full-scale F-M vibration testing are carried out, and the applicability of the code to such multi-degree of freedom systems as F-M is demonstrated. (author)

Feasibility of using microbeads with holographic barcodes to track DNA specimens in the clinical molecular laboratory

Directory of Open Access Journals (Sweden)

Jason D. Merker

2013-07-01

Full Text Available We demonstrate the feasibility of using glass microbeads with a holographic barcode identifier to track DNA specimens in the molecular pathology laboratory. These beads can be added to peripheral blood specimens and are carried through automated DNA extraction protocols that use magnetic glass particles. We found that an adequate number of microbeads are consistently carried over during genomic DNA extraction to allow specimen identification, that the beads do not interfere with the performance of several different molecular assays, and that the beads and genomic DNA remain stable when stored together under regular storage conditions in the molecular pathology laboratory. The beads function as an internal, easily readable specimen barcode. This approach may be useful for identifying DNA specimens and reducing errors associated with molecular laboratory testing.

On the distinction of the mechanisms of DNA cleavage by restriction enzymes—The I-, II-, and III-type molecular motors

Science.gov (United States)

Pikin, S. A.

2008-09-01

A comparative physical description is given for the functioning of various restriction enzymes and for their processes of DNA cleavage. The previously proposed model system of kinetic equations is applied to the I-and III-type enzymes, which use ATP molecules as an energy source, while the II-type enzymes work thanks to catalytic reactions with participation of an electric field. All the enzymes achieved bending and twisting DNA, providing for either the linear motion of the II-type enzyme along the DNA chain or the DNA translocation by the I-and III-type enzymes due to moving chiral kinks. A comparative estimation of the considered linear and angular velocities is performed. The role of stalling forces for enzyme-DNA complexes, which induce the observed cutting of the DNA either inside the enzyme (II) or in some “weak” places outside enzymes I and III, which results in the supercoiling of the DNA, is shown. The role of ionic screening for the described processes is discussed.

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

NARCIS (Netherlands)

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

We have investigated the damage of synthetic plasmid pBR322 DNA in dilute aqueous solutions induced by fast carbon ions. The relative contribution of indirect damage and direct damage to the DNA itself is expected to vary with linear energy transfer along the ion track, with the direct damage

Cross-linking and relaxation of supercoiled DNA by psoralen and light

International Nuclear Information System (INIS)

Yoakum, G.H.; Cole, R.S.

1978-01-01

Photoreaction of 4,5',8-trimethylpsoralen with superhelical ColE1 and ColE1amp DNA was studied. Changes in mobilities in agarose gels, formation of interstrand cross-links, and DNA strand breaks were determined. Psoralen and light treatment removed negative superhelical turns, and extensive treatments failed to produce positive superhelical turns in covalently closed plasmid DNA. The rate of relaxation of superhelical turns by psoralen photobinding appeared to be directly proportional to the number of superhelical turns remaining. A unique reaction mechanism is presented to explain these results. By this interpretation the initial rate of psoralen photobinding to superhelical DNA was estimated to be 3 times that for linear DNA, and the ratio of cross-linking to monofunctional adducts appears to be dependent on the superhelical conformation of the DNA. The estimated ratio of psoralen molecules bound to DNA strand breaks was 1.7 . 10 4 :1, and 70% of this breakage is caused by the light alone. (Auth.)

On the identification techniques for ionizing radiation structure breaks in the DNA molecule

International Nuclear Information System (INIS)

Kamluk, A.N.; Shirko, A.V.; Zhavarankau, I.S.

2012-01-01

In this paper, we propose a theoretical method for evaluation of the number and locations of single-strand breaks in DNA using a change in the passage of a longitudinal wave along the double helix. A linear chain of n interacting particles connected by a pair of springs is taken as a model of the DNA molecule. (authors)

Chromosomal Aberrations in DNA Repair Defective Cell Lines: Comparisons of Dose Rate and Radiation Quality

Science.gov (United States)

George, K. A.; Hada, M.; Patel, Z.; Huff, J.; Pluth, J. M.; Cucinotta, F. A.

2009-01-01

Chromosome aberration yields were assessed in DNA double-strand break repair (DSB) deficient cells after acute doses of gamma-rays or high-LET iron nuclei, or low dose-rate (0.018 Gy/hr) gamma-rays. We studied several cell lines including fibroblasts deficient in ATM (product of the gene that is mutated in ataxia telangiectasia patients) or NBS (product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase, DNA-PK activity. Chromosomes were analyzed using the fluorescence in-situ hybridization (FISH) chromosome painting method in cells at the first division post-irradiation and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma radiation induced higher yields of both simple and complex exchanges in the DSB repair defective cells than in the normal cells. The quadratic dose-response terms for both chromosome exchange types were significantly higher for the ATM and NBS defective lines than for normal fibroblasts. However, the linear dose-response term was significantly higher only for simple exchanges in the NBS cells. Large increases in the quadratic dose response terms indicate the important roles of ATM and NBS in chromatin modifications that facilitate correct DSB repair and minimize aberration formation. Differences in the response of AT and NBS deficient cells at lower doses suggests important questions about the applicability of observations of radiation sensitivity at high dose to low dose exposures. For all iron nuclei irradiated cells, regression models preferred purely linear and quadratic dose responses for simple and complex exchanges, respectively. All the DNA repair defective cell lines had lower Relative biological effectiveness (RBE) values than normal cells, the lowest being for the DNA-PK-deficient cells, which was near unity. To further

Viral evasion of DNA-stimulated innate immune responses.

Science.gov (United States)

Christensen, Maria H; Paludan, Søren R

2017-01-01

Cellular sensing of virus-derived nucleic acids is essential for early defenses against virus infections. In recent years, the discovery of DNA sensing proteins, including cyclic GMP-AMP synthase (cGAS) and gamma-interferon-inducible protein (IFI16), has led to understanding of how cells evoke strong innate immune responses against incoming pathogens carrying DNA genomes. The signaling stimulated by DNA sensors depends on the adaptor protein STING (stimulator of interferon genes), to enable expression of antiviral proteins, including type I interferon. To facilitate efficient infections, viruses have evolved a wide range of evasion strategies, targeting host DNA sensors, adaptor proteins and transcription factors. In this review, the current literature on virus-induced activation of the STING pathway is presented and we discuss recently identified viral evasion mechanisms targeting different steps in this antiviral pathway.

LINEAR2007, Linear-Linear Interpolation of ENDF Format Cross-Sections

International Nuclear Information System (INIS)

2007-01-01

1 - Description of program or function: LINEAR converts evaluated cross sections in the ENDF/B format into a tabular form that is subject to linear-linear interpolation in energy and cross section. The code also thins tables of cross sections already in that form. Codes used subsequently need thus to consider only linear-linear data. IAEA1311/15: This version include the updates up to January 30, 2007. Changes in ENDF/B-VII Format and procedures, as well as the evaluations themselves, make it impossible for versions of the ENDF/B pre-processing codes earlier than PREPRO 2007 (2007 Version) to accurately process current ENDF/B-VII evaluations. The present code can handle all existing ENDF/B-VI evaluations through release 8, which will be the last release of ENDF/B-VI. Modifications from previous versions: - Linear VERS. 2007-1 (JAN. 2007): checked against all ENDF/B-VII; increased page size from 60,000 to 600,000 points 2 - Method of solution: Each section of data is considered separately. Each section of File 3, 23, and 27 data consists of a table of cross section versus energy with any of five interpolation laws. LINEAR will replace each section with a new table of energy versus cross section data in which the interpolation law is always linear in energy and cross section. The histogram (constant cross section between two energies) interpolation law is converted to linear-linear by substituting two points for each initial point. The linear-linear is not altered. For the log-linear, linear-log and log- log laws, the cross section data are converted to linear by an interval halving algorithm. Each interval is divided in half until the value at the middle of the interval can be approximated by linear-linear interpolation to within a given accuracy. The LINEAR program uses a multipoint fractional error thinning algorithm to minimize the size of each cross section table

Cellular responses of Saccharomyces cerevisiae to DNA damage

International Nuclear Information System (INIS)

Ciesla, Z.; Sledziewska-Gojska, E.; Nowicka, A.; Mieczkowski, P.; Fikus, M.U.; Koprowski, P.

1998-01-01

Full text. Several experimental strategies have been used to study responses of S. cerevisiae cells to DNA damage. One approach was based on the isolation of novel genes, the expression of which is induced by lesions in DNA. One of these genes, DIN7, was cloned and partially characterized previously. The product of DIN7 belongs to a large family of proteins involved in DNA repair and mutagenesis. This family includes Rad2, Rad27 and ExoI proteins of S. cerevisiae and their respective human homologues, all of which are endowed with DNA nuclease activity. To study cellular function of Din7 we constructed the pPK3 plasmid carrying DIN7 fused to the GAL1 promoter. Effects of DIN7 overproduction on the phenotypes of wild-type cells and of rad27 and exoI mutants were examined. Overproduction of Din7 does not seem to affect the proficiency of wild-type S. cerevisiae cells in recombination and mutagenesis. Also, overexpression of DIN7 does not suppress the deficiency of the EXOI gene product, the closest homologue of Din7, both in recombination and in controlling the fidelity of DNA replication. Unexpectedly, we found that elevated levels of Din7 result in a very high frequency of mitochondrial rho - mutants. A high frequency of production of rho - mutants wa s also observed in strains defective in the functioning of the Dun1 protein kinase involved in signal transmission in cells exposed to DNA damaging agents. Interestingly, deficiency of Dun1 results also in a significant derepression of the DIN7 gene. Experiments are under way to distinguish whether a high cellular level of Din7 specifically decreases stability of mitochondrial DNA or affects stability of chromosomal DNA as well. Analysis of previously constructed S. cerevisiae strains carrying random geno mic fusions with reporter lacZ gene, allowed us to identify the reading frame YBR173c, on chromosome II as a novel damage inducible gene - DIN8. We have shown that DIN8-lacZ fusion is induced in yeast cells treated

Visualization of complex DNA damage along accelerated ions tracks

Science.gov (United States)

Kulikova, Elena; Boreyko, Alla; Bulanova, Tatiana; Ježková, Lucie; Zadneprianetc, Mariia; Smirnova, Elena

2018-04-01

The most deleterious DNA lesions induced by ionizing radiation are clustered DNA double-strand breaks (DSB). Clustered or complex DNA damage is a combination of a few simple lesions (single-strand breaks, base damage etc.) within one or two DNA helix turns. It is known that yield of complex DNA lesions increases with increasing linear energy transfer (LET) of radiation. For investigation of the induction and repair of complex DNA lesions, human fibroblasts were irradiated with high-LET 15N ions (LET = 183.3 keV/μm, E = 13MeV/n) and low-LET 60Co γ-rays (LET ≈ 0.3 keV/μm) radiation. DNA DSBs (γH2AX and 53BP1) and base damage (OGG1) markers were visualized by immunofluorecence staining and high-resolution microscopy. The obtained results showed slower repair kinetics of induced DSBs in cells irradiated with accelerated ions compared to 60Co γ-rays, indicating induction of more complex DNA damage. Confirming previous assumptions, detailed 3D analysis of γH2AX/53BP1 foci in 15N ions tracks revealed more complicated structure of the foci in contrast to γ-rays. It was shown that proteins 53BP1 and OGG1 involved in repair of DNA DSBs and modified bases, respectively, were colocalized in tracks of 15N ions and thus represented clustered DNA DSBs.

Retroviral DNA Integration Directed by HIV Integration Protein in Vitro

Science.gov (United States)

Bushman, Frederic D.; Fujiwara, Tamio; Craigie, Robert

1990-09-01

Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a λ DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.

DNA functionalization by dynamic chemistry

Directory of Open Access Journals (Sweden)

Zeynep Kanlidere

2016-10-01

Full Text Available Dynamic combinatorial chemistry (DCC is an attractive method to efficiently generate libraries of molecules from simpler building blocks by reversible reactions under thermodynamic control. Here we focus on the chemical modification of DNA oligonucleotides with acyclic diol linkers and demonstrate their potential for the deoxyribonucleic acid functionalization and generation of libraries of reversibly interconverting building blocks. The syntheses of phosphoramidite building blocks derived from D-threoninol are presented in two variants with protected amino or thiol groups. The threoninol building blocks were successfully incorporated via automated solid-phase synthesis into 13mer oligonucleotides. The amino group containing phosphoramidite was used together with complementary single-strand DNA templates that influenced the Watson–Crick base-pairing equilibrium in the mixture with a set of aldehyde modified nucleobases. A significant fraction of all possible base-pair mismatches was obtained, whereas, the highest selectivity (over 80% was found for the guanine aldehyde templated by the complementary cytosine containing DNA. The elevated occurrence of mismatches can be explained by increased backbone plasticity derived from the linear threoninol building block as a cyclic deoxyribose analogue.

DNA replication after mutagenic treatment in Hordeum vulgare.

Science.gov (United States)

Kwasniewska, Jolanta; Kus, Arita; Swoboda, Monika; Braszewska-Zalewska, Agnieszka

2016-12-01

The temporal and spatial properties of DNA replication in plants related to DNA damage and mutagenesis is poorly understood. Experiments were carried out to explore the relationships between DNA replication, chromatin structure and DNA damage in nuclei from barley root tips. We quantitavely analysed the topological organisation of replication foci using pulse EdU labelling during the S phase and its relationship with the DNA damage induced by mutagenic treatment with maleic hydrazide (MH), nitroso-N-methyl-urea (MNU) and gamma ray. Treatment with mutagens did not change the characteristic S-phase patterns in the nuclei; however, the frequencies of the S-phase-labelled cells after treatment differed from those observed in the control cells. The analyses of DNA replication in barley nuclei were extended to the micronuclei induced by mutagens. Replication in the chromatin of the micronuclei was rare. The results of simultanous TUNEL reaction to identify cells with DNA strand breaks and the labelling of the S-phase cells with EdU revealed the possibility of DNA replication occurring in damaged nuclei. For the first time, the intensity of EdU fluorescence to study the rate of DNA replication was analysed. Copyright © 2016 Elsevier B.V. All rights reserved.

Structural Transitions in Supercoiled Stretched DNA

Science.gov (United States)

v, Croquette

1998-03-01

Using magnetic micromanipulation techniques [Strick 96]( uc(T.R.) Strick, J.-F. Allemand, D. Bensimon, A. Bensimon) and uc(V.) Croquette, "The elasticity of a single supercoiled DNA molecule", Science, 271, 1835 (1996)., we have studied the mechanical properties (force versus extension) of single DNA molecules under a wide range of torsional stresses (supercoiling). We show that unwinding the DNA double helix leads to a phase separation between regular B-DNA and denaturation bubbles. The fraction of denatured molecule increases linearly with the degree of unwinding, beginning at a value of 1% unwinding. We have confirmed this denatured state by hybridization of homologous single-stranded DNA probes and by a chemical attack of the exposed bases. Surprisingly, when we overwind the molecule, the elasticity curves we obtain may also be interpreted by the coexistence of two phases, B-DNA and a new phase which we note P-DNA. The fraction of this new phase increases smoothly with overwinding, beginning at 3 % and continuing up to 300 %. Our results indicate that this new phase is four times more twisted that the standard B-DNA and is 1.75 times longer. Although the structure of this phase is not yet known, such a high twisting can only be attained if the sugar-phosphate backbones of the two strands are twisted closely while the bases are expelled outside of the molecule's core, in a structure reminiscent of the one proposed by Pauling. Indeed we have shown that this new phase is sensitive to chemical attack whereas the B-DNA is not. This new phase begins to appear on a molecule overwound by 3 % and stretched by a force of 5 pN, conditions typically encountered in vivo during gene transcription. This new phase may thus play a biological role (for more details).

A New Linearization Technique Using Multi-sinh Doublet

Directory of Open Access Journals (Sweden)

CEHAN, V.

2009-06-01

Full Text Available In this paper a new linearization technique using multi-sinh doublet, implemented with a second generation current conveyor is presented. This new linearization technique is compared with the one based on multi-tanh doublets with linearization series connected diodes on the branches. The comparative study of the two linearization techniques is carried out using both dynamic range analysis, expressed by linearity error and the THD value calculation of output current, and the noise behavior of the two analyzed doublets. For the multi-sinh linearization technique proposed in the paper a method which assures the increase of the dynamic range, keeping the transconductance value constant is presented. This is done by using two design parameters: the number of series connected diodes N, which specifies the desired linear operating range and the k emitters areas ratio of the input stage transistors, which establishes the transconductance value. In the paper is also shown that if the transconductances of the two analyzed doublets are identical, and for the same values of N and k parameters, respectively, the current consumption of the multi-sinh doublet is always smaller than for the multi-tanh doublet.

What Information is Stored in DNA: Does it Contain Digital Error Correcting Codes?

Science.gov (United States)

Liebovitch, Larry

1998-03-01

The longest term correlations in living systems are the information stored in DNA which reflects the evolutionary history of an organism. The 4 bases (A,T,G,C) encode sequences of amino acids as well as locations of binding sites for proteins that regulate DNA. The fidelity of this important information is maintained by ANALOG error check mechanisms. When a single strand of DNA is replicated the complementary base is inserted in the new strand. Sometimes the wrong base is inserted that sticks out disrupting the phosphate backbone. The new base is not yet methylated, so repair enzymes, that slide along the DNA, can tear out the wrong base and replace it with the right one. The bases in DNA form a sequence of 4 different symbols and so the information is encoded in a DIGITAL form. All the digital codes in our society (ISBN book numbers, UPC product codes, bank account numbers, airline ticket numbers) use error checking code, where some digits are functions of other digits to maintain the fidelity of transmitted informaiton. Does DNA also utitlize a DIGITAL error chekcing code to maintain the fidelity of its information and increase the accuracy of replication? That is, are some bases in DNA functions of other bases upstream or downstream? This raises the interesting mathematical problem: How does one determine whether some symbols in a sequence of symbols are a function of other symbols. It also bears on the issue of determining algorithmic complexity: What is the function that generates the shortest algorithm for reproducing the symbol sequence. The error checking codes most used in our technology are linear block codes. We developed an efficient method to test for the presence of such codes in DNA. We coded the 4 bases as (0,1,2,3) and used Gaussian elimination, modified for modulus 4, to test if some bases are linear combinations of other bases. We used this method to analyze the base sequence in the genes from the lac operon and cytochrome C. We did not find

Self-DNA inhibitory effects: Underlying mechanisms and ecological implications.

Science.gov (United States)

Cartenì, Fabrizio; Bonanomi, Giuliano; Giannino, Francesco; Incerti, Guido; Vincenot, Christian Ernest; Chiusano, Maria Luisa; Mazzoleni, Stefano

2016-01-01

DNA is usually known as the molecule that carries the instructions necessary for cell functioning and genetic inheritance. A recent discovery reported a new functional role for extracellular DNA. After fragmentation, either by natural or artificial decomposition, small DNA molecules (between ∼50 and ∼2000 bp) exert a species specific inhibitory effect on individuals of the same species. Evidence shows that such effect occurs for a wide range of organisms, suggesting a general biological process. In this paper we explore the possible molecular mechanisms behind those findings and discuss the ecological implications, specifically those related to plant species coexistence.

Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.

Directory of Open Access Journals (Sweden)

Jason D Thompson

Full Text Available Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.

Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.

Science.gov (United States)

Thompson, Jason D; Shibahara, Gosuke; Rajan, Sweta; Pel, Joel; Marziali, Andre

2012-01-01

Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.

Investigation of DNA double strand breaks induced by α particle and 7Li ions

International Nuclear Information System (INIS)

Kong Fuquan; Cai Minghui; Zhao Kui; Guo Jiyu; Ni Meinan; Sui Li; Yang Mingjian; Zhan Yong

2006-01-01

α particles and Lithium ions were produced by 241 Am radiation source and HI-13 tandem accelerator at China Institute of Atomic Energy (CIAE) respectively to simulate ionizing radiation in Boron Neutron Capture Therapy (BNCT) process. Plasmid DNA in aqueous solution was irradiated and the DNA fragments were imaged by AFM. The image software ImageJ was used to measure the length of DNA fragments. The length distribution and conformation changes of DNA fragments were assessed. Our results showed that the mean length of DNA fragments as well as the fraction of linear and open circle DNA molecules decreased by dose. At higher dose, Lithium ions induced more pronounced relative biological effects than α particles. (author)

Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament.

KAUST Repository

Fornander, Louise H

2013-12-03

The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction.

Evaluation of assays for quantification of DNA in canine plasma as an indirect marker of NETosis.

Science.gov (United States)

Smith, Stephanie A; Lawson, Corinne M; McMichael, Maureen A; Jung, Katrina; O'Brien, Mauria; Achiel, Ron

2017-06-01

Neutrophil extracellular traps (NET), consisting of a filamentous DNA/chromatin-histone scaffold originating from neutrophils are part of the innate immune response, may be released under a variety of inflammatory conditions and are associated with an increased risk for thrombosis. The purpose of this study was to evaluate a SYTOX green fluorescence assay and a histone-DNA complex (hisDNA) ELISA for quantification of NET-related DNA in canine plasma. The influence of variations in blood sample handling on assay results was tested. Accuracy of the SYTOX green fluorescence and the hisDNA ELISA was evaluated with dilutional linearity using serial dilutions. Interference was assessed by addition of purified bilirubin or hemoglobin. Precision was determined by calculating the intra- and inter-assay CV. Preanalytic sample handling did not influence DNA measurements by either assay. Citrate and EDTA plasma samples were equivalent. For the DNA fluorescence assay, dilutional linearity was poor due to autofluorescence, which was corrected by addition of canine plasma to the diluent. The presence of bilirubin and hemoglobin also increased autofluorescence, and resulted in falsely low concentrations of DNA. On the hisDNA ELISA, pigmentemia had no effect. Both assays as modified in this study are suitable for measuring DNA in canine EDTA or citrate plasma. However, performance of the fluorescence assay was impacted by pigmentemia, and it was less sensitive than the ELISA in detecting the presence of nucleosome material in the plasma. © 2017 American Society for Veterinary Clinical Pathology.

Characterization of polymer, DNA-based, and silk thin film resistivities and of DNA-based films prepared for enhanced electrical conductivity

Science.gov (United States)

Yaney, Perry P.; Ouchen, Fahima; Grote, James G.

2009-08-01

DC resistivity studies were carried out on biopolymer films of DNA-CTMA and silk fibroin, and on selected traditional polymer films, including PMMA and APC. Films of DNA-CTMA versus molecular weight and with conductive dopants PCBM, BAYTRON P and ammonium tetrachloroplatinate are reported. The films were spin coated on glass slides configured for measurements of volume dc resistance. The measurements used the alternating polarity method to record the applied voltage-dependent current independent of charging and background currents. The Arrhenius equation plus a constant was fitted to the conductivity versus temperature data of the polymers and the non-doped DNA-based biopolymers with activation energies ranging from 0.8 to 1.4 eV.

Electron paramagnetic relaxation studies of free radicals in. gamma. -irradiated DNA

Energy Technology Data Exchange (ETDEWEB)

Kuwabara, M; Yoshi, G [Hokkaido Univ., Sapporo (Japan)

1980-01-01

Using the continuous microwave power saturation method the T/sub 1/ spin-lattice relaxation time and T/sub 2/ spin-spin relaxation time for DNA radicals (measured at 297/sup 0/K) are reported. Identical experiments carried out on thymidine-5'-monophosphate sodium salt (TMP) and deoxycytidine-5'-monophosphate sodium salt (dCMP) are also reported. Irradiated DNA produces TMP radicals on the base moiety and dCMP radicals on the sugar moiety. Comparing the relaxation times of DNA with those of TMP and dCMP provided a reliable analysis of the nature of DNA radicals.

Design of Linear - and Minimum-phase FIR-equalizers

DEFF Research Database (Denmark)

Bysted, Tommy Kristensen; Jensen, K.J.; Gaunholt, Hans

1996-01-01

an error function which is quadratic in the filtercoefficients. The advantage of the quadratic function is the ability to find the optimal coefficients solving a system of linear equations without iterations.The transformation to a minimum-phase equalizer is carried out by homomorphic deconvolution...

Precision polymers and 3D DNA nanostructures: emergent assemblies from new parameter space.

Science.gov (United States)

Serpell, Christopher J; Edwardson, Thomas G W; Chidchob, Pongphak; Carneiro, Karina M M; Sleiman, Hanadi F

2014-11-05

Polymer self-assembly and DNA nanotechnology have both proved to be powerful nanoscale techniques. To date, most attempts to merge the fields have been limited to placing linear DNA segments within a polydisperse block copolymer. Here we show that, by using hydrophobic polymers of a precisely predetermined length conjugated to DNA strands, and addressable 3D DNA prisms, we are able to effect the formation of unprecedented monodisperse quantized superstructures. The structure and properties of larger micelles-of-prisms were probed in depth, revealing their ability to participate in controlled release of their constituent nanostructures, and template light-harvesting energy transfer cascades, mediated through both the addressability of DNA and the controlled aggregation of the polymers.

Interfacing of DNA with carbon nanotubes for nanodevice applications

Energy Technology Data Exchange (ETDEWEB)

Rastogi, Richa, E-mail: richa.bend@gmail.com [Biomolecular Electronics and Nanotechnology Division (BEND), Central Scientific Instruments Organisation (CSIO), Sector-30C, Chandigarh 160030 (India); Centre of Advanced Studies in Physics, Punjab University, Sector-14, Chandigarh 160014 (India); Dhindsa, Navneet [Biomolecular Electronics and Nanotechnology Division (BEND), Central Scientific Instruments Organisation (CSIO), Sector-30C, Chandigarh 160030 (India); Suri, C. Raman [Biosensor Division, Institute of Microbial Technology (IMTECH), Sector-39, Chandigarh 160039 (India); Pant, B.D. [Central Electronics Engineering Research Institute, Pilani, Rajasthan (India); Tripathi, S.K. [Centre of Advanced Studies in Physics, Punjab University, Sector-14, Chandigarh 160014 (India); Kaur, Inderpreet; Bharadwaj, Lalit M. [Biomolecular Electronics and Nanotechnology Division (BEND), Central Scientific Instruments Organisation (CSIO), Sector-30C, Chandigarh 160030 (India)

2012-08-15

In nanotechnology, carbon nanotubes are evolving as 'hot spot' due to their applications as most sensitive biosensors. Thus, study of effect of biomolecular interaction is prerequisite for their electrical application in biosensors and bioelectronics. Here, we have explored this effect on electrical properties of carbon nanotubes with DNA as a model biomolecule. A stable conjugate of carbon nanotubes with DNA is formed via covalent methodology employing quantum dot as fluoropore and characterized with various spectroscopic, fluoroscopic and microscopic techniques. CNT-DNA adduct showed decreased transconductance (from 614.46 {mu}S to 1.34 {mu}S) and shift of threshold voltage (from -0.85 V to 2.5 V) due to change in Schottky barriers at metal-nanotube contact. In addition, decrease in hole mobility (from 4.46 Multiplication-Sign 10{sup 6} to 9.72 Multiplication-Sign 10{sup 3} cm{sup 2} V{sup -1} s{sup -1}) and increase in ON-linear resistance (from 74 k Ohm-Sign to 0.44 M Ohm-Sign ) conclude large change in device parameters. On the one hand, this substantial change in device parameters after interfacing with biomolecules supports application of carbon nanotubes in the field of biosensors while on the other hand, the same can limit their use in future power electronic devices where stability in device parameters is essential. -- Graphical abstract: Carbon nanotubes are interfaced with DNA via covalent interactions and characterized with spectroscopic, fluoroscopic and microscopic techniques. Electrical characterization of this stable SWNT-DNA conjugate shows decreased transconductance and shift of threshold voltage towards positive gate voltages. On the one hand, this substantial change in device parameters after interfacing with biomolecules supports application of carbon nanotubes in the field of biosensors while on the other hand, the same can limit their use in future power electronic devices where stability in device parameters is essential

Assessment of Hepatitis B Virus DNA Stability in Serum by the Chiron Quantiplex Branched-DNA Assay

Science.gov (United States)

Krajden, Mel; Comanor, Lorraine; Rifkin, Oretta; Grigoriew, Anna; Minor, James M.; Kapke, Gordon F.

1998-01-01

Quantification of hepatitis B virus (HBV) DNA in serum is used to establish eligibility for treatment and to monitor therapeutic response. With the trend toward centralized testing, defining the conditions that preserve sample integrity is of paramount importance. We therefore evaluated the stability of HBV DNA in 26 previously frozen (PF) and 5 fresh, never previously frozen serum specimens. PF specimens, covering a 3-log10 HBV DNA dynamic range, were thawed and stored at −70, 4, 23, 37, and 45°C (±1.5°C) for 0, 24, 72, and 120 h (±2 h) and were refrozen at −70°C prior to testing. Five fresh specimens were split into two groups. Both group FG1 and group FG2 specimens were handled as described above; however, group FG1 specimens were subsequently maintained at 4°C and were never frozen prior to testing. Linear regression analysis of PF specimens demonstrated no significant HBV DNA degradation at ≤4°C over 5 days; however, HBV DNA levels decreased by 1.8, 3.4, and 20% per day at 23, 37, and 45°C, respectively. Three independent statistical methods confirmed that the probability of specimen failure, defined as a loss of 20% or more of HBV DNA and/or coagulation of serum, was lowest at ≤4°C and increased with temperature. Because only 10 to 20% of individual patient specimens demonstrated losses of HBV DNA of ≥20% at 23 or 37°C, sufficient numbers of serum specimens must be evaluated to determine overall statistical trends. In conclusion, HBV DNA integrity in separated serum specimens is preserved for at least 5 days when the specimens are stored at −70 or 4°C. PMID:9466745

DNA damage response and role of shelterin complex in human peripheral blood mononuclear cells exposed to gamma radiation

International Nuclear Information System (INIS)

Saini, Divyalakshmi; Das, Birajalaxmi

2013-01-01

Telomeres are the DNA protein structures that cap the ends of linear DNA. It consists of short repetitive DNA sequences (TTAGGG)n and specialized telomere binding proteins. There are six telomeric proteins (TRF1, TRF2, TIN2, TERF2, PTOP and POT1) called as shelterin complex/telosome which maintains telomere integrity. The function of this 'telosome' is to protect the natural ends of the chromosomes from being recognized as artificial DNA breaks, thereby preventing chromosome end-to-end fusions. DNA Damage Response (DDR) induced by radiation and its interaction with telomeric protein complex is poorly understood in human PBMCs at G 0 stage. Alterations in either telomeric DNA or telomere binding proteins can impair the function of the telosome, which may lead to senescence or apoptosis. Ionizing radiation which induces a plethora of DNA lesions in human cell may also alter the expression of telomere associated proteins. In the present study, we have made an attempt to study the DNA damage response of telomere proteins in human peripheral blood mononuclear cells exposed to gamma radiation. Venous blood samples were collected from eight random healthy volunteers and PBMCs were separated. Dose response as well as time point kinetics study was carried out at transcription as well as protein level. PBMCs were irradiated at various doses between 10 cGy to 2.0 Gy at a dose rate of 1.0 Gy/min. Total RNA was isolated for gene expression analysis at 0 hour and 4 hours respectively. cDNA was prepared and transcriptional pattern as studied using real time q-PCR where Taqman probes were used. Time point kinetics of transcriptional pattern of TRF1, TRF2, TIN2, TERF2, PTOP and POT1 was carried out at 0 min, 15 min, 30 min, 60 min, and 120 min for two different doses (1.0 Gy and 2.0 Gy). Dose response and time point kinetics of TRF2 was studied at similar doses using confocal microscopy. Our results revealed that at 2.0 Gy there was a two fold increase at the level of transcription

Repair of DNA DSB in higher eukaryotes

International Nuclear Information System (INIS)

Wang, H.; Perrault, A.R.; Takeda, Y.; Iliakis, G.

2003-01-01

Cells of higher eukaryotes process within minutes double strand breaks (DSBs) in their genome using a NHEJ apparatus that engages DNA-PKcs, Ku, DNA ligase IV, XRCC4, and other as of yet unidentified factors. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DNA DSBs using an alternative pathway operating with slower kinetics. This alternative pathway is active in mutants deficient in genes of the RAD52 epistasis group. We proposed, therefore, that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK- dependent (D-NHEJ) pathway, rather than homology directed repair of DSBs. We studied the role of Ku and DNA-PKcs in the coordination of these pathways using as a model end joining of restriction endonuclease linearized plasmid DNA in whole cell extracts. Efficient error-free endjoining observed in such in-vitro reactions is strongly inhibited by anti-Ku antibodies. The inhibition requires DNA-PKcs, despite that fact that Ku efficiently binds DNA ends in the presence of antibodies, or in the absence of DNA-PKcs. Strong inhibition of DNA endjoining is also mediated by wortmannin, an inhibitor of DNA-PKcs, in the presence but not in the absence of Ku, and this inhibition can be rescued by pre-incubating the reaction with double stranded oligonucleotides. The results are compatible with a role of Ku in directing endjoining to a DNA-PK dependent pathway, mediated by efficient end binding and productive interactions with DNA-PKcs. On the other hand, efficient end joining is observed in extracts of cells lacking DNA-PKcs, as well as in Ku-depleted extracts sugggesting the operation of alternative pathways. Extracts depleted of Ku and DNA-PKcs rejoin blunt ends, as well as homologous ends with 3' or 5' protruding single strands with similar efficiency, but addition of Ku suppresses joining of blunt ends and homologous ends with 3' overhangs. We propose that the

Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes.

Science.gov (United States)

Kachhap, Sangita; Singh, Balvinder

2015-01-01

In most of homeodomain-DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1-DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein-DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein-DNA complexes. The order of stability of protein-DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein-DNA complexes. Among specific protein-DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.

A Low Glycaemic Index Diet in Pregnancy Induces DNA Methylation Variation in Blood of Newborns: Results from the ROLO Randomised Controlled Trial

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Aisling A. Geraghty

2018-04-01

Full Text Available The epigenetic profile of the developing fetus is sensitive to environmental influence. Maternal diet has been shown to influence DNA methylation patterns in offspring, but research in humans is limited. We investigated the impact of a low glycaemic index dietary intervention during pregnancy on offspring DNA methylation patterns using a genome-wide methylation approach. Sixty neonates were selected from the ROLO (Randomised cOntrol trial of LOw glycaemic index diet to prevent macrosomia study: 30 neonates from the low glycaemic index intervention arm and 30 from the control, whose mothers received no specific dietary advice. DNA methylation was investigated in 771,484 CpG sites in free DNA from cord blood serum. Principal component analysis and linear regression were carried out comparing the intervention and control groups. Gene clustering and pathway analysis were also explored. Widespread variation was identified in the newborns exposed to the dietary intervention, accounting for 11% of the total level of DNA methylation variation within the dataset. No association was found with maternal early-pregnancy body mass index (BMI, infant sex, or birthweight. Pathway analysis identified common influences of the intervention on gene clusters plausibly linked to pathways targeted by the intervention, including cardiac and immune functioning. Analysis in 60 additional samples from the ROLO study failed to replicate the original findings. Using a modest-sized discovery sample, we identified preliminary evidence of differential methylation in progeny of mothers exposed to a dietary intervention during pregnancy.

"Isogaba Maware": quality control of genome DNA by checkpoints.

Science.gov (United States)

Kitazono, A; Matsumoto, T

1998-05-01

Checkpoints maintain the interdependency of cell cycle events by permitting the onset of an event only after the completion of the preceding event. The DNA replication checkpoint induces a cell cycle arrest until the completion of the DNA replication. Similarly, the DNA damage checkpoint arrests cell cycle progression if DNA repair is incomplete. A number of genes that play a role in the two checkpoints have been identified through genetic studies in yeasts, and their homologues have been found in fly, mouse, and human. They form signaling cascades activated by a DNA replication block or DNA damage and subsequently generate the negative constraints on cell cycle regulators. The failure of these signaling cascades results in producing offspring that carry mutations or that lack a portion of the genome. In humans, defects in the checkpoints are often associated with cancer-prone diseases. Focusing mainly on the studies in budding and fission yeasts, we summarize the recent progress.

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

International Nuclear Information System (INIS)

Adelstein, S.J.; Kassis, A.I.

1996-01-01

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

2-Aminopurine hairpin probes for the detection of ultraviolet-induced DNA damage

International Nuclear Information System (INIS)

El-Yazbi, Amira F.; Loppnow, Glen R.

2012-01-01

Highlights: ► Molecular beacon with 2AP bases detects DNA damage in a simple mix-and-read assay. ► Molecular beacons with 2AP bases detect damage at a 17.2 nM limit of detection. ► The 2AP molecular beacon is linear over a 0–3.5 μM concentration range for damage. - Abstract: Nucleic acid exposure to radiation and chemical insults leads to damage and disease. Thus, detection and understanding DNA damage is important for elucidating molecular mechanisms of disease. However, current methods of DNA damage detection are either time-consuming, destroy the sample, or are too specific to be used for generic detection of damage. In this paper, we develop a fluorescence sensor of 2-aminopurine (2AP), a fluorescent analogue of adenine, incorporated in the loop of a hairpin probe for the quantification of ultraviolet (UV) C-induced nucleic acid damage. Our results show that the selectivity of the 2AP hairpin probe to UV-induced nucleic acid damage is comparable to molecular beacon (MB) probes of DNA damage. The calibration curve for the 2AP hairpin probe shows good linearity (R 2 = 0.98) with a limit of detection of 17.2 nM. This probe is a simple, fast and economic fluorescence sensor for the quantification of UV-induced damage in DNA.

Linearization Method and Linear Complexity

Science.gov (United States)

Tanaka, Hidema

We focus on the relationship between the linearization method and linear complexity and show that the linearization method is another effective technique for calculating linear complexity. We analyze its effectiveness by comparing with the logic circuit method. We compare the relevant conditions and necessary computational cost with those of the Berlekamp-Massey algorithm and the Games-Chan algorithm. The significant property of a linearization method is that it needs no output sequence from a pseudo-random number generator (PRNG) because it calculates linear complexity using the algebraic expression of its algorithm. When a PRNG has n [bit] stages (registers or internal states), the necessary computational cost is smaller than O(2n). On the other hand, the Berlekamp-Massey algorithm needs O(N2) where N(≅2n) denotes period. Since existing methods calculate using the output sequence, an initial value of PRNG influences a resultant value of linear complexity. Therefore, a linear complexity is generally given as an estimate value. On the other hand, a linearization method calculates from an algorithm of PRNG, it can determine the lower bound of linear complexity.

Performance Characteristics of Different Anti-Double-Stranded DNA Antibody Assays in the Monitoring of Systemic Lupus Erythematosus

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Michael Mahler

2017-01-01

Full Text Available Objective. We sought to evaluate different anti-double-stranded DNA assays for their performance characteristics in monitoring disease activity fluctuations in systemic lupus erythematosus (SLE. Methods. 36 active SLE patients were followed monthly. At each study visit (total n=371, blood was collected and disease activity was scored using the SELENA-SLEDAI (excluding anti-dsDNA or complement components and by a physician’s global assessment (PGA. Four anti-dsDNA tests were compared. Linear mixed-effects models with random intercept and fixed slopes were used to evaluate the relationship between the longitudinal fluctuations of disease activity and anti-dsDNA titers. Results. At enrollment, positivity for QUANTA Lite and high-avidity anti-dsDNA assay was both 64% and significantly lower than anti-dsDNA positivity by QUANTA Flash (83% and CLIFT (96%. Linear mixed-effects modeling indicated that the change in clinical SELENA-SLEDAI scores was associated with the titers of all anti-dsDNA with QUANTA Flash yielding the highest marginal R2 (0.15; p<0.01. QUANTA Flash was the only anti-dsDNA assay significantly associated with the change in PGA (marginal R2=0.05; p<0.01. Conclusion. These data indicate that anti-dsDNA antibodies determined by QUANTA Flash have a value in monitoring SLE disease activity.

Yields of clustered DNA damage induced by charged-particle radiations of similar kinetic energy per nucleon: LET dependence in different DNA microenvironments

International Nuclear Information System (INIS)

Keszenman, D.J.; Sutherland, B.M.

2010-01-01

To determine the linear energy transfer (LET) dependence of the biological effects of densely ionizing radiation in relation to changes in the ionization density along the track, we measured the yields and spectrum of clustered DNA damages induced by charged particles of different atomic number but similar kinetic energy per nucleon in different DNA microenvironments. Yeast DNA embedded in agarose in solutions of different free radical scavenging capacity was irradiated with 1 GeV protons, 1 GeV/nucleon oxygen ions, 980 MeV/nucleon titanium ions or 968 MeV/nucleon iron ions. The frequencies of double-strand breaks (DSBs), abasic sites and oxypurine clusters were quantified. The total DNA damage yields per absorbed dose induced in non-radioquenching solution decreased with LET, with minor variations in radioquenching conditions being detected. However, the total damage yields per particle fluence increased with LET in both conditions, indicating a higher efficiency per particle to induce clustered DNA damages. The yields of DSBs and non-DSB clusters as well as the damage spectra varied with LET and DNA milieu, suggesting the involvement of more than one mechanism in the formation of the different types of clustered damages.

DNA damage induced by the direct effect of He ion particles

International Nuclear Information System (INIS)

Urushibara, A.; Shikazono, N.; Watanabe, R.; Fujii, K.; O'Neill, P.; Yokoya, A.

2006-01-01

We present here evidence showing that the yields of DNA lesions induced by He 2+ ions strongly depend on Linear energy transfer (LET). In this study, hydrated plasmid DNA was irradiated with He 2+ ions with LET values of 19, 63 and 95 keVμm -1 . The yields of prompt single-strand breaks (SSBs) are very similar at the varying LET values, whereas the yields of prompt double-strand breaks (DSBs) increase with increasing LET. Further, base lesions were revealed as additional strand breaks by post-irradiation treatment of the DNA with endonuclease III (Nth) and formamido-pyrimidine-DNA glycosylase (Fpg). The reduction in the yield of these enzymatically induced SSBs and DSBs becomes significant as the LET increases. These results suggest that the clustering of DNA lesions becomes more probable in regions of high LET. (authors)

SiPM as miniaturised optical biosensor for DNA-microarray applications

Directory of Open Access Journals (Sweden)

M.F. Santangelo

2015-12-01

Full Text Available A miniaturized optical biosensor for low-level fluorescence emitted by DNA strands labelled with CY5 is showed. Aim of this work is to demonstrate that a Si-based photodetector, having a low noise and a high sensitivity, can replace traditional detection systems in DNA-microarray applications. The photodetector used is a photomultiplier (SiPM, with 25 pixels. It exhibits a higher sensitivity than commercial optical readers and we experimentally found a detection limit for spotted dried samples of ∼1 nM. We measured the fluorescence signal in different operating conditions (angle of analysis, fluorophores concentrations, solution volumes and support. Once fixed the angle of analysis, for samples spotted on Al-TEOS slide dried, the system is proportional to the concentration of the analyte in the sample and is linear in the range 1 nM–1 μM. For solutions, the range of linearity ranges from 100 fM to 10 nM. The system potentialities and the device low costs suggest it as basic component for the design and fabrication of a cheap, easy and portable optical system. Keywords: Optical Biosensor, SiPM, DNA microarray, Fluorophore detection

Robust estimation for partially linear models with large-dimensional covariates.

Science.gov (United States)

Zhu, LiPing; Li, RunZe; Cui, HengJian

2013-10-01

We are concerned with robust estimation procedures to estimate the parameters in partially linear models with large-dimensional covariates. To enhance the interpretability, we suggest implementing a noncon-cave regularization method in the robust estimation procedure to select important covariates from the linear component. We establish the consistency for both the linear and the nonlinear components when the covariate dimension diverges at the rate of [Formula: see text], where n is the sample size. We show that the robust estimate of linear component performs asymptotically as well as its oracle counterpart which assumes the baseline function and the unimportant covariates were known a priori. With a consistent estimator of the linear component, we estimate the nonparametric component by a robust local linear regression. It is proved that the robust estimate of nonlinear component performs asymptotically as well as if the linear component were known in advance. Comprehensive simulation studies are carried out and an application is presented to examine the finite-sample performance of the proposed procedures.

DNA dynamics play a role as a basal transcription factor in the positioning and regulation of gene transcription initiation

OpenAIRE

Alexandrov, Boian S.; Gelev, Vladimir; Yoo, Sang Wook; Alexandrov, Ludmil B.; Fukuyo, Yayoi; Bishop, Alan R.; Rasmussen, Kim ?.; Usheva, Anny

2009-01-01

We assess the role of DNA breathing dynamics as a determinant of promoter strength and transcription start site (TSS) location. We compare DNA Langevin dynamic profiles of representative gene promoters, calculated with the extended non-linear PBD model of DNA with experimental data on transcription factor binding and transcriptional activity. Our results demonstrate that DNA dynamic activity at the TSS can be suppressed by mutations that do not affect basal transcription factor binding–DNA co...

Comparison of impedimetric detection of DNA hybridization on the various biosensors based on modified glassy carbon electrodes with PANHS and nanomaterials of RGO and MWCNTs.

Science.gov (United States)

Benvidi, Ali; Tezerjani, Marzieh Dehghan; Jahanbani, Shahriar; Mazloum Ardakani, Mohammad; Moshtaghioun, Seyed Mohammad

2016-01-15

In this research, we have developed lable free DNA biosensors based on modified glassy carbon electrodes (GCE) with reduced graphene oxide (RGO) and carbon nanotubes (MWCNTs) for detection of DNA sequences. This paper compares the detection of BRCA1 5382insC mutation using independent glassy carbon electrodes (GCE) modified with RGO and MWCNTs. A probe (BRCA1 5382insC mutation detection (ssDNA)) was then immobilized on the modified electrodes for a specific time. The immobilization of the probe and its hybridization with the target DNA (Complementary DNA) were performed under optimum conditions using different electrochemical techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The proposed biosensors were used for determination of complementary DNA sequences. The non-modified DNA biosensor (1-pyrenebutyric acid-N- hydroxysuccinimide ester (PANHS)/GCE), revealed a linear relationship between ∆Rct and logarithm of the complementary target DNA concentration ranging from 1.0×10(-16)molL(-1) to 1.0×10(-10)mol L(-1) with a correlation coefficient of 0.992, for DNA biosensors modified with multi-wall carbon nanotubes (MWCNTs) and reduced graphene oxide (RGO) wider linear range and lower detection limit were obtained. For ssDNA/PANHS/MWCNTs/GCE a linear range 1.0×10(-17)mol L(-1)-1.0×10(-10)mol L(-1) with a correlation coefficient of 0.993 and for ssDNA/PANHS/RGO/GCE a linear range from 1.0×10(-18)mol L(-1) to 1.0×10(-10)mol L(-1) with a correlation coefficient of 0.985 were obtained. In addition, the mentioned biosensors were satisfactorily applied for discriminating of complementary sequences from noncomplementary sequences, so the mentioned biosensors can be used for the detection of BRCA1-associated breast cancer. Copyright © 2015. Published by Elsevier B.V.

Viral evasion of DNA-stimulated innate immune responses

Science.gov (United States)

Christensen, Maria H; Paludan, Søren R

2017-01-01

Cellular sensing of virus-derived nucleic acids is essential for early defenses against virus infections. In recent years, the discovery of DNA sensing proteins, including cyclic GMP–AMP synthase (cGAS) and gamma-interferon-inducible protein (IFI16), has led to understanding of how cells evoke strong innate immune responses against incoming pathogens carrying DNA genomes. The signaling stimulated by DNA sensors depends on the adaptor protein STING (stimulator of interferon genes), to enable expression of antiviral proteins, including type I interferon. To facilitate efficient infections, viruses have evolved a wide range of evasion strategies, targeting host DNA sensors, adaptor proteins and transcription factors. In this review, the current literature on virus-induced activation of the STING pathway is presented and we discuss recently identified viral evasion mechanisms targeting different steps in this antiviral pathway. PMID:26972769

A novel fluorescent DNA sensor for ultrasensitive detection of Helicobacter pylori.

Science.gov (United States)

Liu, Ziping; Su, Xingguang

2017-01-15

In this work, a novel fluorescent DNA sensor for ultrasensitive detection of Helicobacter pylori (H. pylori) DNA was developed. This strategy took advantage of DNA hybridization between single-stranded DNA (ssDNA, which had been designed as an aptamer specific for H. pylori DNA) and the complementary target H. pylori DNA, and the feature that ssDNA bound to graphene oxide (GO) with significantly higher affinity than double-stranded DNA (dsDNA). ssDNA were firstly covalent conjugated with CuInS 2 quantum dots (QDs) by reaction between the carboxy group of QDs and amino group modified ssDNA, forming ssDNA-QDs genosensor. In the absence of the complementary target H. pylori DNA, GO could adsorb ssDNA-QDs DNA sensor and efficiently quench the fluorescence of ssDNA-QDs. While the complementary target H. pylori DNA was introduced, the ssDNA-QDs preferentially bound with the H. pylori DNA. The formation of dsDNA would alter the conformation of ssDNA and disturb the interaction between ssDNA and GO. Thus, the dsDNA-QDs/GO system exhibited a stronger fluorescence emission than that of the ssDNA-QDs/GO system. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I 0 and the concentration of H. pylori DNA in the range of 1.25-875pmolL -1 with a detection limit of 0.46pmolL -1 . The proposed method was applied to the determination of H. pylori DNA sequence in milk samples with satisfactory results. Copyright © 2016 Elsevier B.V. All rights reserved.

DNA origami-based nanoribbons: assembly, length distribution, and twist

Energy Technology Data Exchange (ETDEWEB)

Jungmann, Ralf; Scheible, Max; Kuzyk, Anton; Pardatscher, Guenther; Simmel, Friedrich C [Lehrstuhl fuer Bioelektronik, Physik-Department and ZNN/WSI, Technische Universitaet Muenchen, Am Coulombwall 4a, 85748 Garching (Germany); Castro, Carlos E, E-mail: simmel@ph.tum.de [Labor fuer Biomolekulare Nanotechnologie, Physik-Department and ZNN/WSI, Technische Universitaet Muenchen, Am Coulombwall 4a, 85748 Garching (Germany)

2011-07-08

A variety of polymerization methods for the assembly of elongated nanoribbons from rectangular DNA origami structures are investigated. The most efficient method utilizes single-stranded DNA oligonucleotides to bridge an intermolecular scaffold seam between origami monomers. This approach allows the fabrication of origami ribbons with lengths of several micrometers, which can be used for long-range ordered arrangement of proteins. It is quantitatively shown that the length distribution of origami ribbons obtained with this technique follows the theoretical prediction for a simple linear polymerization reaction. The design of flat single layer origami structures with constant crossover spacing inevitably results in local underwinding of the DNA helix, which leads to a global twist of the origami structures that also translates to the nanoribbons.

DNA origami-based nanoribbons: assembly, length distribution, and twist

International Nuclear Information System (INIS)

Jungmann, Ralf; Scheible, Max; Kuzyk, Anton; Pardatscher, Guenther; Simmel, Friedrich C; Castro, Carlos E

2011-01-01

A variety of polymerization methods for the assembly of elongated nanoribbons from rectangular DNA origami structures are investigated. The most efficient method utilizes single-stranded DNA oligonucleotides to bridge an intermolecular scaffold seam between origami monomers. This approach allows the fabrication of origami ribbons with lengths of several micrometers, which can be used for long-range ordered arrangement of proteins. It is quantitatively shown that the length distribution of origami ribbons obtained with this technique follows the theoretical prediction for a simple linear polymerization reaction. The design of flat single layer origami structures with constant crossover spacing inevitably results in local underwinding of the DNA helix, which leads to a global twist of the origami structures that also translates to the nanoribbons.

Quantification of cellular uptake of DNA nanostructures by qPCR.

Science.gov (United States)

Okholm, Anders Hauge; Nielsen, Jesper Sejrup; Vinther, Mathias; Sørensen, Rasmus Schøler; Schaffert, David; Kjems, Jørgen

2014-05-15

DNA nanostructures facilitating drug delivery are likely soon to be realized. In the past few decades programmed self-assembly of DNA building blocks have successfully been employed to construct sophisticated nanoscale objects. By conjugating functionalities to DNA, other molecules such as peptides, proteins and polymers can be precisely positioned on DNA nanostructures. This exceptional ability to produce modular nanoscale devices with tunable and controlled behavior has initiated an interest in employing DNA nanostructures for drug delivery. However, to obtain this the relationship between cellular interactions and structural and functional features of the DNA delivery device must be thoroughly investigated. Here, we present a rapid and robust method for the precise quantification of the component materials of DNA origami structures capable of entering cells in vitro. The quantification is performed by quantitative polymerase chain reaction, allowing a linear dynamic range of detection of five orders of magnitude. We demonstrate the use of this method for high-throughput screening, which could prove efficient to identify key features of DNA nanostructures enabling cell penetration. The method described here is suitable for quantification of in vitro uptake studies but should easily be extended to quantify DNA nanostructures in blood or tissue samples. Copyright © 2014 Elsevier Inc. All rights reserved.

Radiation metagenesis and inhibition of DNA synthesis

International Nuclear Information System (INIS)

Dubinina, L.G.; Sergievskaya, S.P.; Kurashova, Z.I.; Dubinin, N.P.

1983-01-01

The study of modification of radiation mutagenesis and inhibition of the DNA synthesis by means of 1-β-D arabinofuranosylcytosine (ara-C) is carried out. It is shown that ara-C-acting on chromosomes in the G 1 phase and G 2 phase does not cause mutations in the C capillaris cells. The modification by means of ara-C radiation effect in the G 1 phase and G 2 phase correlates with duration and time of administering ara-C before and after irradiation. A new form of ara-C DNA synthesis inhibitor interaction with mutation processes has been found out. Protective effect of the DNA synthesis inhibitor (ara-C) from mutageneous radiation effect is stressed. Sensibilization of the radiation mutagenesis during cell treafment by the DNA synthesis inhibitor (ara-C) is shown. It is pointed out that emergence of sensibilization or protective effect, i. e. antimutagenesis phenomenon depends on conditions under which the synthesis inhibitor acted in G 1 and G 2 phases

Core seismic behaviour: linear and non-linear models

International Nuclear Information System (INIS)

Bernard, M.; Van Dorsselaere, M.; Gauvain, M.; Jenapierre-Gantenbein, M.

1981-08-01

The usual methodology for the core seismic behaviour analysis leads to a double complementary approach: to define a core model to be included in the reactor-block seismic response analysis, simple enough but representative of basic movements (diagrid or slab), to define a finer core model, with basic data issued from the first model. This paper presents the history of the different models of both kinds. The inert mass model (IMM) yielded a first rough diagrid movement. The direct linear model (DLM), without shocks and with sodium as an added mass, let to two different ones: DLM 1 with independent movements of the fuel and radial blanket subassemblies, and DLM 2 with a core combined movement. The non-linear (NLM) ''CORALIE'' uses the same basic modelization (Finite Element Beams) but accounts for shocks. It studies the response of a diameter on flats and takes into account the fluid coupling and the wrapper tube flexibility at the pad level. Damping consists of one modal part of 2% and one part due to shocks. Finally, ''CORALIE'' yields the time-history of the displacements and efforts on the supports, but damping (probably greater than 2%) and fluid-structures interaction are still to be precised. The validation experiments were performed on a RAPSODIE core mock-up on scale 1, in similitude of 1/3 as to SPX 1. The equivalent linear model (ELM) was developed for the SPX 1 reactor-block response analysis and a specified seismic level (SB or SM). It is composed of several oscillators fixed to the diagrid and yields the same maximum displacements and efforts than the NLM. The SPX 1 core seismic analysis with a diagrid input spectrum which corresponds to a 0,1 g group acceleration, has been carried out with these models: some aspects of these calculations are presented here

Electrostatic assembly of CTAB-capped silver nanoparticles along predefined λ-DNA template

International Nuclear Information System (INIS)

Wei Gang; Wang Li; Zhou Hualan; Liu Zhiguo; Song Yonghai; Li Zhuang

2005-01-01

Cetyltrimethylammonium bromide (CTAB)-capped positively-charged silver nanoparticles synthesized in water-ethanol system was electrostatic assembled on predefined aligned λ-DNA template. Silver nanowire can be obtained by changing the reaction time and the particles concentration. In our work, the length of the silver nanowire obtained is about 10 μm, and the dimension of the wires is about 20 nm. AFM data reveal that the assembly of CTAB-capped silver nanoparticles on DNA is ordered, but there is space between two particles absorbed on the DNA template. X-ray photoelectron spectroscopy (XPS) was applied to characterize the linear silver clusters, which provides an additional proof that the silver particles were assembled onto DNA template with fine order

Molecular Precision at Micrometer Length Scales: Hierarchical Assembly of DNA-Protein Nanostructures.

Science.gov (United States)

Schiffels, Daniel; Szalai, Veronika A; Liddle, J Alexander

2017-07-25

Robust self-assembly across length scales is a ubiquitous feature of biological systems but remains challenging for synthetic structures. Taking a cue from biology-where disparate molecules work together to produce large, functional assemblies-we demonstrate how to engineer microscale structures with nanoscale features: Our self-assembly approach begins by using DNA polymerase to controllably create double-stranded DNA (dsDNA) sections on a single-stranded template. The single-stranded DNA (ssDNA) sections are then folded into a mechanically flexible skeleton by the origami method. This process simultaneously shapes the structure at the nanoscale and directs the large-scale geometry. The DNA skeleton guides the assembly of RecA protein filaments, which provides rigidity at the micrometer scale. We use our modular design strategy to assemble tetrahedral, rectangular, and linear shapes of defined dimensions. This method enables the robust construction of complex assemblies, greatly extending the range of DNA-based self-assembly methods.

Microanalysis of DNA by stripping transfer voltammetry

Czech Academy of Sciences Publication Activity Database

Jelen, František; Kouřilová, Alena; Pečinka, Petr; Paleček, Emil

2004-01-01

Roč. 63, 1-2 (2004), s. 249-252 ISSN 1567-5394 R&D Projects: GA ČR GA203/02/0422; GA AV ČR IAA1163201; GA AV ČR IBS5004107 Institutional research plan: CEZ:AV0Z5004920 Keywords : DNA determination * determination of purine base s * linear sweep voltammetry Subject RIV: BO - Biophysics Impact factor: 2.261, year: 2004

Sensitive Leptospira DNA detection using tapered optical fiber sensor.

Science.gov (United States)

Zainuddin, Nurul H; Chee, Hui Y; Ahmad, Muhammad Z; Mahdi, Mohd A; Abu Bakar, Muhammad H; Yaacob, Mohd H

2018-03-23

This paper presents the development of tapered optical fiber sensor to detect a specific Leptospira bacteria DNA. The bacteria causes Leptospirosis, a deadly disease but with common early flu-like symptoms. Optical single mode fiber (SMF) of 125 μm diameter is tapered to produce 12 μm waist diameter and 15 cm length. The novel DNA-based optical fiber sensor is functionalized by incubating the tapered region with sodium hydroxide (NaOH), (3-Aminopropyl) triethoxysilane and glutaraldehyde. Probe DNA is immobilized onto the tapered region and subsequently hybridized by its complementary DNA (cDNA). The transmission spectra of the DNA-based optical fiber sensor are measured in the 1500 to 1600 nm wavelength range. It is discovered that the shift of the wavelength in the SMF sensor is linearly proportional with the increase in the cDNA concentrations from 0.1 to 1.0 nM. The sensitivity of the sensor toward DNA is measured to be 1.2862 nm/nM and able to detect as low as 0.1 fM. The sensor indicates high specificity when only minimal shift is detected for non-cDNA testing. The developed sensor is able to distinguish between actual DNA of Leptospira serovars (Canicola and Copenhageni) against Clostridium difficile (control sample) at very low (femtomolar) target concentrations. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-speed DNA-based rolling motors powered by RNase H

Science.gov (United States)

Yehl, Kevin; Mugler, Andrew; Vivek, Skanda; Liu, Yang; Zhang, Yun; Fan, Mengzhen; Weeks, Eric R.

2016-01-01

DNA-based machines that walk by converting chemical energy into controlled motion could be of use in applications such as next generation sensors, drug delivery platforms, and biological computing. Despite their exquisite programmability, DNA-based walkers are, however, challenging to work with due to their low fidelity and slow rates (~1 nm/min). Here, we report DNA-based machines that roll rather than walk, and consequently have a maximum speed and processivity that is three-orders of magnitude greater than conventional DNA motors. The motors are made from DNA-coated spherical particles that hybridise to a surface modified with complementary RNA; motion is achieved through the addition of RNase H, which selectively hydrolyses hybridised RNA. Spherical motors move in a self-avoiding manner, whereas anisotropic particles, such as dimerised particles or rod-shaped particles travel linearly without a track or external force. Finally, we demonstrate detection of single nucleotide polymorphism by measuring particle displacement using a smartphone camera. PMID:26619152

Use of real-time PCR to evaluate two DNA extraction methods from food

Directory of Open Access Journals (Sweden)

Maria Regina Branquinho

2012-03-01

Full Text Available The DNA extraction is a critical step in Genetically Modified Organisms analysis based on real-time PCR. In this study, the CTAB and DNeasy methods provided good quality and quantity of DNA from the texturized soy protein, infant formula, and soy milk samples. Concerning the Certified Reference Material consisting of 5% Roundup Ready® soybean, neither method yielded DNA of good quality. However, the dilution test applied in the CTAB extracts showed no interference of inhibitory substances. The PCR efficiencies of lectin target amplification were not statistically different, and the coefficients of correlation (R² demonstrated high degree of correlation between the copy numbers and the threshold cycle (Ct values. ANOVA showed suitable adjustment of the regression and absence of significant linear deviations. The efficiencies of the p35S amplification were not statistically different, and all R² values using DNeasy extracts were above 0.98 with no significant linear deviations. Two out of three R² values using CTAB extracts were lower than 0.98, corresponding to lower degree of correlation, and the lack-of-fit test showed significant linear deviation in one run. The comparative analysis of the Ct values for the p35S and lectin targets demonstrated no statistical significant differences between the analytical curves of each target.

Enhanced dielectric-wall linear accelerator

Science.gov (United States)

Sampayan, Stephen E.; Caporaso, George J.; Kirbie, Hugh C.

1998-01-01

A dielectric-wall linear accelerator is enhanced by a high-voltage, fast e-time switch that includes a pair of electrodes between which are laminated alternating layers of isolated conductors and insulators. A high voltage is placed between the electrodes sufficient to stress the voltage breakdown of the insulator on command. A light trigger, such as a laser, is focused along at least one line along the edge surface of the laminated alternating layers of isolated conductors and insulators extending between the electrodes. The laser is energized to initiate a surface breakdown by a fluence of photons, thus causing the electrical switch to close very promptly. Such insulators and lasers are incorporated in a dielectric wall linear accelerator with Blumlein modules, and phasing is controlled by adjusting the length of fiber optic cables that carry the laser light to the insulator surface.

DNA repair decline during mouse spermiogenesis results in the accumulation of heritable DNA damage

Energy Technology Data Exchange (ETDEWEB)

Marchetti, Francesco; Marchetti, Francesco; Wryobek, Andrew J

2008-02-21

The post-meiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the three weeks of spermiogenesis affected the accumulation of DEB-induced heritable damage in early spermatids (21-15 days before fertilization, dbf), late spermatids (14-8 dbf) and sperm (7- 1 dbf). Analysis of chromosomalaberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than two weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of post-meioitic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e, smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.

DNA Repair Decline During Mouse Spermiogenesis Results in the Accumulation of Heritable DNA Damage

Energy Technology Data Exchange (ETDEWEB)

Marchetti, Francesco; Marchetti, Francesco; Wyrobek, Andrew J.

2007-12-01

The post-meiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the three weeks of spermiogenesis affected the accumulation of DEB-induced heritable damage in early spermatids (21-15 days before fertilization, dbf), late spermatids (14-8 dbf) and sperm (7-1 dbf). Analysis of chromosomal aberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than two weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of post-meioitic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e, smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.

DNA repair efficiency in germ cells and early mouse embryos and consequences for radiation-induced transgenerational genomic damage

Energy Technology Data Exchange (ETDEWEB)

Marchetti, Francesco; Wyrobek, Andrew J.

2009-01-18

Exposure to ionizing radiation and other environmental agents can affect the genomic integrity of germ cells and induce adverse health effects in the progeny. Efficient DNA repair during gametogenesis and the early embryonic cycles after fertilization is critical for preventing transmission of DNA damage to the progeny and relies on maternal factors stored in the egg before fertilization. The ability of the maternal repair machinery to repair DNA damage in both parental genomes in the fertilizing egg is especially crucial for the fertilizing male genome that has not experienced a DNA repair-competent cellular environment for several weeks prior to fertilization. During the DNA repair-deficient period of spermatogenesis, DNA lesions may accumulate in sperm and be carried into the egg where, if not properly repaired, could result in the formation of heritable chromosomal aberrations or mutations and associated birth defects. Studies with female mice deficient in specific DNA repair genes have shown that: (i) cell cycle checkpoints are activated in the fertilized egg by DNA damage carried by the sperm; and (ii) the maternal genotype plays a major role in determining the efficiency of repairing genomic lesions in the fertilizing sperm and directly affect the risk for abnormal reproductive outcomes. There is also growing evidence that implicates DNA damage carried by the fertilizing gamete as a mediator of postfertilization processes that contribute to genomic instability in subsequent generations. Transgenerational genomic instability most likely involves epigenetic mechanisms or error-prone DNA repair processes in the early embryo. Maternal and embryonic DNA repair processes during the early phases of mammalian embryonic development can have far reaching consequences for the genomic integrity and health of subsequent generations.

LET-effects in DNA

International Nuclear Information System (INIS)

Kraft, G.; Taucher-Scholz, G.; Heilmann, J.

1995-01-01

The use of heavy particles in radiobiological experiments provides a fundamental tool to study the influence of different ionization densities and to prove the physical basis of models and theories. The knowledge of the interaction of high LET radiation (LET: linear energy transfer [keV/microm]) with biological matter is of great importance for the application of neutrons, protons and heavier ions in radiotherapy. It is also essential in radioprotection to estimate the risk in case of exposure to high LET radiation. In this contribution, an introductory view on the physical properties of ions is given and the cellular response to high LET radiation is summarized. Then the measurements of strand break induction of DNA in solution and in intracellular DNA are reported and compared to cell survival. The possibility of changes in the quality of the lesions is discussed and finally the present status of model calculations in comparison to the experiments is given

Expression, purification, and DNA-binding activity of the Herbaspirillum seropedicae RecX protein.

Science.gov (United States)

Galvão, Carolina W; Pedrosa, Fábio O; Souza, Emanuel M; Yates, M Geoffrey; Chubatsu, Leda S; Steffens, Maria Berenice R

2004-06-01

The Herbaspirillum seropedicae RecX protein participates in the SOS response: a process in which the RecA protein plays a central role. The RecX protein of the H. seropedicae, fused to a His-tag sequence (RecX His-tagged), was over-expressed in Escherichia coli and purified by metal-affinity chromatography to yield a highly purified and active protein. DNA band-shift assays showed that the RecX His-tagged protein bound to both circular and linear double-stranded DNA and also to circular single-stranded DNA. The apparent affinity of RecX for DNA decreased in the presence of Mg(2+) ions. The ability of RecX to bind DNA may be relevant to its function in the SOS response.

Linearized models for a new magnetic control in MAST

International Nuclear Information System (INIS)

Artaserse, G.; Maviglia, F.; Albanese, R.; McArdle, G.J.; Pangione, L.

2013-01-01

Highlights: ► We applied linearized models for a new magnetic control on MAST tokamak. ► A suite of procedures, conceived to be machine independent, have been used. ► We carried out model-based simulations, taking into account eddy currents effects. ► Comparison with the EFIT flux maps and the experimental magnetic signals are shown. ► A current driven model for the dynamic simulations of the experimental data have been performed. -- Abstract: The aim of this work is to provide reliable linearized models for the design and assessment of a new magnetic control system for MAST (Mega Ampère Spherical Tokamak) using rtEFIT, which can easily be exported to MAST Upgrade. Linearized models for magnetic control have been obtained using the 2D axisymmetric finite element code CREATE L. MAST linearized models include equivalent 2D axisymmetric schematization of poloidal field (PF) coils, vacuum vessel, and other conducting structures. A plasmaless and a double null configuration have been chosen as benchmark cases for the comparison with experimental data and EFIT reconstructions. Good agreement has been found with the EFIT flux map and the experimental signals coming from magnetic probes with only few mismatches probably due to broken sensors. A suite of procedures (equipped with a user friendly interface to be run even remotely) to provide linearized models for magnetic control is now available on the MAST linux machines. A new current driven model has been used to obtain a state space model having the PF coil currents as inputs. Dynamic simulations of experimental data have been carried out using linearized models, including modelling of the effects of the passive structures, showing a fair agreement. The modelling activity has been useful also to reproduce accurately the interaction between plasma current and radial position control loops

Linearized models for a new magnetic control in MAST

Energy Technology Data Exchange (ETDEWEB)

Artaserse, G., E-mail: giovanni.artaserse@enea.it [Associazione Euratom-ENEA sulla Fusione, Via Enrico Fermi 45, I-00044 Frascati (RM) (Italy); Maviglia, F.; Albanese, R. [Associazione Euratom-ENEA-CREATE sulla Fusione, Via Claudio 21, I-80125 Napoli (Italy); McArdle, G.J.; Pangione, L. [EURATOM/CCFE Fusion Association, Culham Science Centre, Abingdon, Oxon, OX14 3DB (United Kingdom)

2013-10-15

Highlights: ► We applied linearized models for a new magnetic control on MAST tokamak. ► A suite of procedures, conceived to be machine independent, have been used. ► We carried out model-based simulations, taking into account eddy currents effects. ► Comparison with the EFIT flux maps and the experimental magnetic signals are shown. ► A current driven model for the dynamic simulations of the experimental data have been performed. -- Abstract: The aim of this work is to provide reliable linearized models for the design and assessment of a new magnetic control system for MAST (Mega Ampère Spherical Tokamak) using rtEFIT, which can easily be exported to MAST Upgrade. Linearized models for magnetic control have been obtained using the 2D axisymmetric finite element code CREATE L. MAST linearized models include equivalent 2D axisymmetric schematization of poloidal field (PF) coils, vacuum vessel, and other conducting structures. A plasmaless and a double null configuration have been chosen as benchmark cases for the comparison with experimental data and EFIT reconstructions. Good agreement has been found with the EFIT flux map and the experimental signals coming from magnetic probes with only few mismatches probably due to broken sensors. A suite of procedures (equipped with a user friendly interface to be run even remotely) to provide linearized models for magnetic control is now available on the MAST linux machines. A new current driven model has been used to obtain a state space model having the PF coil currents as inputs. Dynamic simulations of experimental data have been carried out using linearized models, including modelling of the effects of the passive structures, showing a fair agreement. The modelling activity has been useful also to reproduce accurately the interaction between plasma current and radial position control loops.

Studies of DNA repair in Saccharomyces cerevisiae. I. Characterization of a new allele of RAD6. II. Investigation of events in the first cell cycle after DNA damage

International Nuclear Information System (INIS)

Dolthwright-Fasse, J.A.

1980-01-01

Studies in two independent, but related, areas of DNA repair have been carried out in the eucaryotic yeast, Saccharomyces cerevisiae. The first is the characterization of a new allele in the RAD6 gene suggesting that the gene is multifunctional. The second is the utilization of photoreactivation as a probe of events occurring during the first cell cycle after DNA damage. Strains carrying the new allele, designated rad6-4, of the RAD6 locus are about as sensitive to uv and ionizing radiation as those carrying rad6-1 or rad6-3. Although rad6-4 may well be a missense mutation, the data suggest that the RAD6 gene is multifunctional. One function is necessary to recover from DNA damage in an error-free manner, and the other is concerned with mutagenic processes and sporulation. The loss of photoreversibility (LOP) of ultraviolet induced mutations to arginine independence in an excision defective strain carrying arg4-17 examines the events occurring in the first cell cycle. The post uv protein synthesis causes pyrimidine dimmers to become inaccessible to the photoreactivating enzyme in some unknown manner. There is no evidence indicating whether the normal function of the protein is involved in excision repair, or in one of the two repair processes believed to be inducible; induced mutagenesis or recombinational repair

Molecular beacon based biosensor for the sequence-specific detection of DNA using DNA-capped gold nanoparticles-streptavidin conjugates for signal amplification

International Nuclear Information System (INIS)

Fang, Xian; Jiang, Wei; Han, Xiaowei; Zhang, Yuzhong

2013-01-01

We describe a highly sensitive and selective molecular beacon-based electrochemical impedance biosensor for the sequence-specific detection of DNA. DNA-capped conjugates between gold nanoparticles (Au-NPs) and streptavidin are used for signal amplification. The molecular beacon was labeled with a thiol at its 5′ end and with biotin at its 3′ end, and then immobilized on the surface of a bare gold electrode through the formation of Au-S bonds. Initially, the molecular beacon is present in the “closed” state, and this shields the biotin from being approached by streptavidin due to steric hindrance. In the presence of the target DNA, the target DNA molecules hybridize with the loop and cause a conformational change that moves the biotin away from the surface of the electrode. The biotin thereby becomes accessible for the reporter (the DNA-streptavidin capped Au-NPs), and this results in a distinct increase in electron transfer resistance. Under optimal conditions, the increase in resistance is linearly related to the logarithm of the concentration of complementary target DNA in the range from 1.0 fM to 0.1 μM, with a detection limit of 0.35 fM (at an S/N of 3). This biosensor exhibits good selectivity, and acceptable stability and reproducibility. (author)

Cell sensitivity to irradiation and DNA repair processes. II

International Nuclear Information System (INIS)

Kozubek, S.; Krasavin, E.A.

1984-01-01

A new model of DNA single-strand break (SSB) and double-strand break (DSB) induction by radiations of different linear energy transfer (LET) has been developed. Utilizing quadratic dependence of the dose that delta-electrons depart in the track of heavy particles the fraction of heavy particle energy deposited in the target of DNA dimensions has been calculated. SSBs arise from energy depositions in one strand of DNA, direct DSBs arise from two SSBs on opposite strands of DNA in the track of one particle. It is concluded that DSB's induced by γ-radiation are mostly of enzymatic origin, meanwhile DSB's induced by high-LET radiation are direct DSB's. The dependence of radiosensitivity D 0 -1 on LET (L) for isogenic mutants of E. coli with different sensitivity to γ-radiation has been determined on the bases of the model and considering microscopic energy fluctuations. The shape of D 0 -1 (L) function is formed both by physical characteristics of radiation and by the ability of cells to repair some types of DNA damage. The model provides a basis for further investigation. (author)

Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles.

Directory of Open Access Journals (Sweden)

Stefanie Fischer

Full Text Available The biological relevance of extracellular vesicles (EV in intercellular communication has been well established. Thus far, proteins and RNA were described as main cargo. Here, we show that EV released from human bone marrow derived mesenchymal stromal cells (BM-hMSC also carry high-molecular DNA in addition. Extensive EV characterization revealed this DNA mainly associated with the outer EV membrane and to a smaller degree also inside the EV. Our EV purification protocol secured that DNA is not derived from apoptotic or necrotic cells. To analyze the relevance of EV-associated DNA we lentivirally transduced Arabidopsis thaliana-DNA (A.t.-DNA as indicator into BM-hMSC and generated EV. Using quantitative polymerase chain reaction (qPCR techniques we detected high copy numbers of A.t.-DNA in EV. In recipient hMSC incubated with tagged EV for two weeks we identified A.t.-DNA transferred to recipient cells. Investigation of recipient cell DNA using quantitative PCR and verification of PCR-products by sequencing suggested stable integration of A.t.-DNA. In conclusion, for the first time our proof-of-principle experiments point to horizontal DNA transfer into recipient cells via EV. Based on our results we assume that eukaryotic cells are able to exchange genetic information in form of DNA extending the known cargo of EV by genomic DNA. This mechanism might be of relevance in cancer but also during cell evolution and development.

Linear and Nonlinear Analysis of Brain Dynamics in Children with Cerebral Palsy

Science.gov (United States)

Sajedi, Firoozeh; Ahmadlou, Mehran; Vameghi, Roshanak; Gharib, Masoud; Hemmati, Sahel

2013-01-01

This study was carried out to determine linear and nonlinear changes of brain dynamics and their relationships with the motor dysfunctions in CP children. For this purpose power of EEG frequency bands (as a linear analysis) and EEG fractality (as a nonlinear analysis) were computed in eyes-closed resting state and statistically compared between 26…

Is DNA a worm-like chain in Couette flow? In search of persistence length, a critical review.

Science.gov (United States)

Rittman, Martyn; Gilroy, Emma; Koohya, Hashem; Rodger, Alison; Richards, Adair

2009-01-01

Persistence length is the foremost measure of DNA flexibility. Its origins lie in polymer theory which was adapted for DNA following the determination of BDNA structure in 1953. There is no single definition of persistence length used, and the links between published definitions are based on assumptions which may, or may not be, clearly stated. DNA flexibility is affected by local ionic strength, solvent environment, bound ligands and intrinsic sequence-dependent flexibility. This article is a review of persistence length providing a mathematical treatment of the relationships between four definitions of persistence length, including: correlation, Kuhn length, bending, and curvature. Persistence length has been measured using various microscopy, force extension and solution methods such as linear dichroism and transient electric birefringence. For each experimental method a model of DNA is required to interpret the data. The importance of understanding the underlying models, along with the assumptions required by each definition to determine a value of persistence length, is highlighted for linear dichroism data, where it transpires that no model is currently available for long DNA or medium to high shear rate experiments.

A laboratory information management system for DNA barcoding workflows.

Science.gov (United States)

Vu, Thuy Duong; Eberhardt, Ursula; Szöke, Szániszló; Groenewald, Marizeth; Robert, Vincent

2012-07-01

This paper presents a laboratory information management system for DNA sequences (LIMS) created and based on the needs of a DNA barcoding project at the CBS-KNAW Fungal Biodiversity Centre (Utrecht, the Netherlands). DNA barcoding is a global initiative for species identification through simple DNA sequence markers. We aim at generating barcode data for all strains (or specimens) included in the collection (currently ca. 80 k). The LIMS has been developed to better manage large amounts of sequence data and to keep track of the whole experimental procedure. The system has allowed us to classify strains more efficiently as the quality of sequence data has improved, and as a result, up-to-date taxonomic names have been given to strains and more accurate correlation analyses have been carried out.

DNA Assembly in 3D Printed Fluidics (Open Access, Publisher’s Version)

Science.gov (United States)

2015-12-30

millifluidics to mix linear double stranded DNA with enzymes to assemble plasmids via Golden Gate biochemistry . The assembly reac- tions occurred at...used, a constitutively expressed yellow fluorescent protein (YFP) reporter, and a plasmid backbone containing an ampicillin selection marker ( AMP ) and...proof-of- principle that 3D printed devices can adequately mix the Golden Gate enzymes and DNA and that the Golden Gate biochemistry proceeds

StpA and Hha stimulate pausing by RNA polymerase by promoting DNA-DNA bridging of H-NS filaments.

Science.gov (United States)

Boudreau, Beth A; Hron, Daniel R; Qin, Liang; van der Valk, Ramon A; Kotlajich, Matthew V; Dame, Remus T; Landick, Robert

2018-06-20

In enterobacteria, AT-rich horizontally acquired genes, including virulence genes, are silenced through the actions of at least three nucleoid-associated proteins (NAPs): H-NS, StpA and Hha. These proteins form gene-silencing nucleoprotein filaments through direct DNA binding by H-NS and StpA homodimers or heterodimers. Both linear and bridged filaments, in which NAPs bind one or two DNA segments, respectively, have been observed. Hha can interact with H-NS or StpA filaments, but itself lacks a DNA-binding domain. Filaments composed of H-NS alone can inhibit transcription initiation and, in the bridged conformation, slow elongating RNA polymerase (RNAP) by promoting backtracking at pause sites. How the other NAPs modulate these effects of H-NS is unknown, despite evidence that they help regulate subsets of silenced genes in vivo (e.g. in pathogenicity islands). Here we report that Hha and StpA greatly enhance H-NS-stimulated pausing by RNAP at 20°C. StpA:H-NS or StpA-only filaments also stimulate pausing at 37°C, a temperature at which Hha:H-NS or H-NS-only filaments have much less effect. In addition, we report that both Hha and StpA greatly stimulate DNA-DNA bridging by H-NS filaments. Together, these observations indicate that Hha and StpA can affect H-NS-mediated gene regulation by stimulating bridging of H-NS/DNA filaments.

Studies on the arctiin and its interaction with DNA by spectral methods

International Nuclear Information System (INIS)

Sun Yantao; Zhang Hanqi; Bi Shuyun; Zhou Xiaofu; Wang Liang; Yan Yongsheng

2011-01-01

The emission spectra of arctiin were determined under various experimental conditions. In addition, a fluorescence method was developed to obtain the binding constants and sites of the interaction between arctiin and DNA. A competitive binding experiment and melting temperature mensuration were carried out to investigate the binding mechanism of arctiin and DNA. The experimental results showed that the interaction between arctiin and DNA belongs to a groove binding mode. - Highlights: → Determined the emission spectra of arctiin by fluorescence spectrometry. → Obtain the binding constants and sites of interaction between arctiin and DNA. → Calculate the binding parameters according an improved calculation method.

Fluorescence bio-barcode DNA assay based on gold and magnetic nanoparticles for detection of Exotoxin A gene sequence.

Science.gov (United States)

Amini, Bahram; Kamali, Mehdi; Salouti, Mojtaba; Yaghmaei, Parichehreh

2017-06-15

Bio-barcode DNA based on gold nanoparticle (bDNA-GNPs) as a new generation of biosensor based detection tools, holds promise for biological science studies. They are of enormous importance in the emergence of rapid and sensitive procedures for detecting toxins of microorganisms. Exotoxin A (ETA) is the most toxic virulence factor of Pseudomonas aeruginosa. ETA has ADP-ribosylation activity and decisively affects the protein synthesis of the host cells. In the present study, we developed a fluorescence bio-barcode technology to trace P. aeruginosa ETA. The GNPs were coated with the first target-specific DNA probe 1 (1pDNA) and bio-barcode DNA, which acted as a signal reporter. The magnetic nanoparticles (MNPs) were coated with the second target-specific DNA probe 2 (2pDNA) that was able to recognize the other end of the target DNA. After binding the nanoparticles with the target DNA, the following sandwich structure was formed: MNP 2pDNA/tDNA/1pDNA-GNP-bDNA. After isolating the sandwiches by a magnetic field, the DNAs of the probes which have been hybridized to their complementary DNA, GNPs and MNPs, via the hydrogen, electrostatic and covalently bonds, were released from the sandwiches after dissolving in dithiothreitol solution (DTT 0.8M). This bio-barcode DNA with known DNA sequence was then detected by fluorescence spectrophotometry. The findings showed that the new method has the advantages of fast, high sensitivity (the detection limit was 1.2ng/ml), good selectivity, and wide linear range of 5-200ng/ml. The regression analysis also showed that there was a good linear relationship (∆F=0.57 [target DNA]+21.31, R 2 =0.9984) between the fluorescent intensity and the target DNA concentration in the samples. Copyright © 2016. Published by Elsevier B.V.

Linearly Polarized Gluons and the Higgs Transverse Momentum Distribution

NARCIS (Netherlands)

Boer, Daniel; den Dunnen, Wilco J.; Pisano, Cristian; Schlegel, Marc; Vogelsang, Werner

2012-01-01

We study how gluons carrying linear polarization inside an unpolarized hadron contribute to the transverse momentum distribution of Higgs bosons produced in hadronic collisions. They modify the distribution produced by unpolarized gluons in a characteristic way that could be used to determine

Molecular verification on male sterile mutant after injected exogenous λDNA into wheat

International Nuclear Information System (INIS)

Yang Jingcheng; Yu Yuanjie; Liu Fengzhen; Qi Yanfang; Shen Fafu

2000-01-01

A cytoplasmic male sterile mutant and then a stable CMS line named D-type sterile line were obtained after injected exogenous λDNA into wheat line 814527, and line 814527 could be its maintainer line. By using λDNA labelled with 32 P as probe, unlabelled λDNA as positive check, dot blotting of nuclear DNA and chloroplast DNA of receptor 814527, D-type sterile line and its hybrid F 1 with Lumai 14 were carried out. Positive dots appeared in nuclear DNA and chloroplast DNA of D-type sterile line and its hybrid F 1 , but did not appear in the receptor. It showed that fragments of exogenous λDNA existed in nuclear genome and chloroplast genome of D-type sterile line, and could be inherited stably. All these results, on a molecular level, proved the reliability of exogenous DNA injection

Properties of the chromatin assembled on DNA injected into Xenopus oocytes and eggs

International Nuclear Information System (INIS)

Gargiulo, G.; Wasserman, W.; Worcel, A.

1983-01-01

The onset of DNA synthesis occurs between 10 and 30 minutes after activation of the egg and thus the transition from nuclease-sensitive to nuclease-resistant supercoils may take place on the newly replicated DNA. To test this possibility, the nonradioactive circular 5-kb DNA carrying the Drosophila histone gene repeat and [α -32 P]dCTP were coinjected into fertilized eggs. Such protocol labels both the injected, replicated heterologous DNA and the replicated endogenous, maternal Xenopus DNA. The labeled, presumably replicated, supercoiled DNA is resistant to micrococcal nuclease as expected. The endogenous, high-molecular-weight Xenopus DNA is degraded to 180-bp nucleosomal DNA. Thus, the nuclease resistance is not a general property of chromatin during the cleavage stage of the Xenopus embryo but is a peculiar feature of the injected DNA. 42 references, 5 figures

DNA damage and repair process in earthworm after in-vivo and in vitro exposure to soils irrigated by wastewaters

International Nuclear Information System (INIS)

Qiao Min; Chen Ying; Wang Chunxia; Wang Zijian; Zhu Yongguan

2007-01-01

In this study, DNA damage to earthworms (Eisenia fetida) after in vivo exposure to contaminated soils was measured by detecting DNA strand breakages (DSBs) and causality was analyzed through fractionation based bioassays. A non-linear dose-response relationship existed between DNA damage and total soil PAHs levels. DNA damage, measured with the comet assay, and its repair process, were observed. To identify the chemical causality, an in vitro comet assay using coelomocytes was subsequently performed on the fractionated organic extracts from soils. The results showed that the PAHs in the soils were responsible for the exerting genotoxic effects on earthworms. When normalized to benzo(a)pyrene toxic equivalent (TEQ BaP ), the saturation dose in the dose-response curve was about 10 ng TEQ BaP g -1 soil (dw). - A non-linear dose-response relationship exists between earthworm DNA damage, measured with comet assay, and total PAHs levels in soils irrigated by wastewaters

DNA damage and repair process in earthworm after in-vivo and in vitro exposure to soils irrigated by wastewaters

Energy Technology Data Exchange (ETDEWEB)

Qiao Min [State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Chen Ying [State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Wang Chunxia [State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Wang Zijian [State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China)]. E-mail: wangzj@rcees.ac.cn; Zhu Yongguan [State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China)

2007-07-15

In this study, DNA damage to earthworms (Eisenia fetida) after in vivo exposure to contaminated soils was measured by detecting DNA strand breakages (DSBs) and causality was analyzed through fractionation based bioassays. A non-linear dose-response relationship existed between DNA damage and total soil PAHs levels. DNA damage, measured with the comet assay, and its repair process, were observed. To identify the chemical causality, an in vitro comet assay using coelomocytes was subsequently performed on the fractionated organic extracts from soils. The results showed that the PAHs in the soils were responsible for the exerting genotoxic effects on earthworms. When normalized to benzo(a)pyrene toxic equivalent (TEQ{sub BaP}), the saturation dose in the dose-response curve was about 10 ng TEQ{sub BaP} g{sup -1} soil (dw). - A non-linear dose-response relationship exists between earthworm DNA damage, measured with comet assay, and total PAHs levels in soils irrigated by wastewaters.

A family of quantization based piecewise linear filter networks

DEFF Research Database (Denmark)

Sørensen, John Aasted

1992-01-01

A family of quantization-based piecewise linear filter networks is proposed. For stationary signals, a filter network from this family is a generalization of the classical Wiener filter with an input signal and a desired response. The construction of the filter network is based on quantization...... of the input signal x(n) into quantization classes. With each quantization class is associated a linear filter. The filtering at time n is carried out by the filter belonging to the actual quantization class of x(n ) and the filters belonging to the neighbor quantization classes of x(n) (regularization......). This construction leads to a three-layer filter network. The first layer consists of the quantization class filters for the input signal. The second layer carries out the regularization between neighbor quantization classes, and the third layer constitutes a decision of quantization class from where the resulting...

Salmonella Typhimurium ST213 is associated with two types of IncA/C plasmids carrying multiple resistance determinants.

Science.gov (United States)

Wiesner, Magdalena; Calva, Edmundo; Fernández-Mora, Marcos; Cevallos, Miguel A; Campos, Freddy; Zaidi, Mussaret B; Silva, Claudia

2011-01-11

Salmonella Typhimurium ST213 was first detected in the Mexican Typhimurium population in 2001. It is associated with a multi-drug resistance phenotype and a plasmid-borne blaCMY-2 gene conferring resistance to extended-spectrum cephalosporins. The objective of the current study was to examine the association between the ST213 genotype and blaCMY-2 plasmids. The blaCMY-2 gene was carried by an IncA/C plasmid. ST213 strains lacking the blaCMY-2 gene carried a different IncA/C plasmid. PCR analysis of seven DNA regions distributed throughout the plasmids showed that these IncA/C plasmids were related, but the presence and absence of DNA stretches produced two divergent types I and II. A class 1 integron (dfrA12, orfF and aadA2) was detected in most of the type I plasmids. Type I contained all the plasmids carrying the blaCMY-2 gene and a subset of plasmids lacking blaCMY-2. Type II included all of the remaining blaCMY-2-negative plasmids. A sequence comparison of the seven DNA regions showed that both types were closely related to IncA/C plasmids found in Escherichia, Salmonella, Yersinia, Photobacterium, Vibrio and Aeromonas. Analysis of our Typhimurium strains showed that the region containing the blaCMY-2 gene is inserted between traA and traC as a single copy, like in the E. coli plasmid pAR060302. The floR allele was identical to that of Newport pSN254, suggesting a mosaic pattern of ancestry with plasmids from other Salmonella serovars and E. coli. Only one of the tested strains was able to conjugate the IncA/C plasmid at very low frequencies (10-7 to 10-9). The lack of conjugation ability of our IncA/C plasmids agrees with the clonal dissemination trend suggested by the chromosomal backgrounds and plasmid pattern associations. The ecological success of the newly emerging Typhimurium ST213 genotype in Mexico may be related to the carriage of IncA/C plasmids. We conclude that types I and II of IncA/C plasmids originated from a common ancestor and that the

Universal Linear Precoding for NBI-Proof Widely Linear Equalization in MC Systems

Directory of Open Access Journals (Sweden)

Donatella Darsena

2007-09-01

Full Text Available In multicarrier (MC systems, transmitter redundancy, which is introduced by means of finite-impulse response (FIR linear precoders, allows for perfect or zero-forcing (ZF equalization of FIR channels (in the absence of noise. Recently, it has been shown that the noncircular or improper nature of some symbol constellations offers an intrinsic source of redundancy, which can be exploited to design efficient FIR widely-linear (WL receiving structures for MC systems operating in the presence of narrowband interference (NBI. With regard to both cyclic-prefixed and zero-padded transmission techniques, it is shown in this paper that, with appropriately designed precoders, it is possible to synthesize in both cases WL-ZF universal equalizers, which guarantee perfect symbol recovery for any FIR channel. Furthermore, it is theoretically shown that the intrinsic redundancy of the improper symbol sequence also enables WL-ZF equalization, based on the minimum mean output-energy criterion, with improved NBI suppression capabilities. Finally, results of numerical simulations are presented, which assess the merits of the proposed precoding designs and validate the theoretical analysis carried out.

One-electron oxidation of DNA: mechanism and consequences.

Science.gov (United States)

Schuster, Gary B

2009-01-01

All living organisms store the information necessary to maintain life in their DNA. Any process that damages DNA and causes loss or corruption of that information threatens the viability of the organism. One-electron oxidation is such a process. Loss of an electron from DNA generates a radical cation that is located primarily on its nucleobases. The radical cation migrates reversibly through duplex DNA by hopping until it is eventually trapped in an irreversible chemical reaction. The particular sequence of nucleobases in a DNA oligomer determines both the efficiency of hopping and the specific location and nature of the damaging chemical reaction. In its normal aqueous solutions, DNA is a polyanion because of the negative charge carried by its phosphate groups. Counter ions (typically Na(+)) to the phosphate groups play an important role in facilitating both the migration of the radical cation and in its eventual reaction with H(2)O. Irreversible reaction of a radical cation with H(2)O in duplex DNA occurs preferentially at the most reactive site. In normal DNA that is comprised of the four common DNA nucleobases, reaction occurs most commonly at a guanine and results in its conversion primarily to 8-oxo-7,8-dihydroguanine (8-OxoG). Both electronic and steric effects control the outcome of this process. If the DNA oligomer does not contain a suitable guanine, then reaction of the radical cation occurs at the thymine of a TT step primarily by a tandem process. The general outcomes revealed in the one-electron oxidation of DNA oligomers in solution appear to be generally valid also for more complex DNA structures and for the cellular DNA of living organisms.

E11 and the non-linear dual graviton

Science.gov (United States)

Tumanov, Alexander G.; West, Peter

2018-04-01

The non-linear dual graviton equation of motion as well as the duality relation between the gravity and dual gravity fields are found in E theory by carrying out E11 variations of previously found equations of motion. As a result the equations of motion in E theory have now been found at the full non-linear level up to, and including, level three, which contains the dual graviton field. When truncated to contain fields at levels three and less, and the spacetime is restricted to be the familiar eleven dimensional space time, the equations are equivalent to those of eleven dimensional supergravity.

Spatiotemporal kinetics of γ-H2AX protein on charged particles induced DNA damage

Energy Technology Data Exchange (ETDEWEB)

Niu, H., E-mail: hniu@mx.nthu.edu.tw [Nuclear Science and Technology Development Center, National Tsing Hua University, Hsinchu, Taiwan (China); Chang, H.C. [Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan (China); Cho, I.C. [Institute for Radiological Research, Chang Gung University and Chang Gung Memorial Hospital, Taoyuan, Taiwan (China); Chen, C.H. [Nuclear Science and Technology Development Center, National Tsing Hua University, Hsinchu, Taiwan (China); Liu, C.S. [Cancer Center of Taipei Veterans General Hospital, Taipei, Taiwan (China); Chou, W.T. [Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan (China)

2014-08-15

Highlights: • Charged particles can induce more complex DNA damages, and these complex damages have higher ability to cause the cell death or cell carcinogenesis. • In this study, we used γ-H2AX protein to investigate the spatiotemporal kinetics of DNA double strand breaks in particle irradiated HeLa cells. • The HeLa cells were irradiated by 400 keV alpha-particles in four different dosages. • The result shows that a good linear relationship can be observed between foci number and radiation dose. • The data shows that the dissolution rate of γ-H2AX foci agree with the two components DNA repairing model, and it was decreasing as the radiation dose increased. • These results suggest that charged particles can induce more complex DNA damages and causing the retardation of DNA repair. - Abstract: In several researches, it has been demonstrated that charged particles can induce more complex DNA damages. These complex damages have higher ability to cause the cell death or cell carcinogenesis. For this reason, clarifying the DNA repair mechanism after charged particle irradiation plays an important role in the development of charged particle therapy and space exploration. Unfortunately, the detail spatiotemporal kinetic of DNA damage repair is still unclear. In this study, we used γ-H2AX protein to investigate the spatiotemporal kinetics of DNA double strand breaks in alpha-particle irradiated HeLa cells. The result shows that the intensity of γ-H2AX foci increased gradually, and reached to its maximum at 30 min after irradiation. A good linear relationship can be observed between foci intensity and radiation dose. After 30 min, the γ-H2AX foci intensity was decreased with time passed, but remained a large portion (∼50%) at 48 h passed. The data show that the dissolution rate of γ-H2AX foci agreed with two components DNA repairing model. These results suggest that charged particles can induce more complex DNA damages and causing the retardation of DNA

Unidirectional variable stiffness hydraulic actuator for load-carrying knee exoskeleton

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Jun Zhu

2017-01-01

Full Text Available This article presents the design and experimental testing of a unidirectional variable stiffness hydraulic actuator for load-carrying knee exoskeleton. The proposed actuator is designed for mimicking the high-efficiency passive behavior of biological knee and providing actively assistance in locomotion. The adjustable passive compliance of exoskeletal knee is achieved through a variable ratio lever mechanism with linear elastic element. A compact customized electrohydraulic system is also designed to accommodate application demands. Preliminary experimental results show the prototype has good performances in terms of stiffness regulation and joint torque control. The actuator is also implemented in an exoskeleton knee joint, resulting in anticipant human-like passive compliance behavior.

Linearizing control of continuous anaerobic fermentation processes

Energy Technology Data Exchange (ETDEWEB)

Babary, J.P. [Centre National d`Etudes Spatiales (CNES), 31 - Toulouse (France). Laboratoire d`Analyse et d`Architecture des Systemes; Simeonov, I. [Institute of Microbiology, Bulgarian Academy of Sciences (Bulgaria); Ljubenova, V. [Institute of Control and System Research, BAS (Country unknown/Code not available); Dochain, D. [Universite Catholique de Louvain (UCL), Louvain-la-Neuve (Belgium)

1997-09-01

Biotechnological processes (BTP) involve living organisms. In the anaerobic fermentation (biogas production process) the organic matter is mineralized by microorganisms into biogas (methane and carbon dioxide) in the absence of oxygen. The biogas is an additional energy source. Generally this process is carried out as a continuous BTP. It has been widely used in life process and has been confirmed as a promising method of solving some energy and ecological problems in the agriculture and industry. Because of the very restrictive on-line information the control of this process in continuous mode is often reduced to control of the biogas production rate or the concentration of the polluting organic matter (de-pollution control) at a desired value in the presence of some perturbations. Investigations show that classical linear controllers have good performances only in the linear zone of the strongly non-linear input-output characteristics. More sophisticated robust and with variable structure (VSC) controllers are studied. Due to the strongly non-linear dynamics of the process the performances of the closed loop system may be degrading in this case. The aim of this paper is to investigate different linearizing algorithms for control of a continuous non-linear methane fermentation process using the dilution rate as a control action and taking into account some practical implementation aspects. (authors) 8 refs.

Perturbed soliton excitations in inhomogeneous DNA

International Nuclear Information System (INIS)

Daniel, M.; Vasumathi, V.

2005-05-01

We study nonlinear dynamics of inhomogeneous DNA double helical chain under dynamic plane-base rotator model by considering angular rotation of bases in a plane normal to the helical axis. The DNA dynamics in this case is found to be governed by a perturbed sine-Gordon equation when taking into account the interstrand hydrogen bonding energy and intrastrand inhomogeneous stacking energy and making an analogy with the Heisenberg model of the Hamiltonian for an inhomogeneous anisotropic spin ladder with ferromagnetic legs and antiferromagentic rung coupling. In the homogeneous limit the dynamics is governed by the kink-antikink soliton of the sine-Gordon equation which represents the formation of open state configuration in DNA double helix. The effect of inhomogeneity in stacking energy in the form of localized and periodic variations on the formation of open states in DNA is studied under perturbation. The perturbed soliton is obtained using a multiple scale soliton perturbation theory by solving the associated linear eigen value problem and constructing the complete set of eigen functions. The inhomogeneity in stacking energy is found to modulate the width and speed of the soliton depending on the nature of inhomogeneity. Also it introduces fluctuations in the form of train of pulses or periodic oscillation in the open state configuration (author)

An atomistic geometrical model of the B-DNA configuration for DNA-radiation interaction simulations

Science.gov (United States)

Bernal, M. A.; Sikansi, D.; Cavalcante, F.; Incerti, S.; Champion, C.; Ivanchenko, V.; Francis, Z.

2013-12-01

In this paper, an atomistic geometrical model for the B-DNA configuration is explained. This model accounts for five organization levels of the DNA, up to the 30 nm chromatin fiber. However, fragments of this fiber can be used to construct the whole genome. The algorithm developed in this work is capable to determine which is the closest atom with respect to an arbitrary point in space. It can be used in any application in which a DNA geometrical model is needed, for instance, in investigations related to the effects of ionizing radiations on the human genetic material. Successful consistency checks were carried out to test the proposed model. Catalogue identifier: AEPZ_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEPZ_v1_0.html Program obtainable from: CPC Program Library, Queen’s University, Belfast, N. Ireland Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 1245 No. of bytes in distributed program, including test data, etc.: 6574 Distribution format: tar.gz Programming language: FORTRAN. Computer: Any. Operating system: Multi-platform. RAM: 2 Gb Classification: 3. Nature of problem: The Monte Carlo method is used to simulate the interaction of ionizing radiation with the human genetic material in order to determine DNA damage yields per unit absorbed dose. To accomplish this task, an algorithm to determine if a given energy deposition lies within a given target is needed. This target can be an atom or any other structure of the genetic material. Solution method: This is a stand-alone subroutine describing an atomic-resolution geometrical model of the B-DNA configuration. It is able to determine the closest atom to an arbitrary point in space. This model accounts for five organization levels of the human genetic material, from the nucleotide pair up to the 30 nm chromatin fiber. This subroutine carries out a series of coordinate transformations

Enzyme-linked immunosorbent assays for Z-DNA.

Science.gov (United States)

Thomas, M J; Strobl, J S

1988-10-01

Dot blot and transblot enzyme-linked immunosorbent assays (e.l.i.s.a.) are described which provide sensitive non-radioactive methods for screening Z-DNA-specific antisera and for detecting Z-DNA in polydeoxyribonucleotides and supercoiled plasmids. In the alkaline phosphatase dot blot e.l.i.s.a., Z-DNA, Br-poly(dG-dC).poly(dG-dC), or B-DNA, poly(dG-dC).poly(dG-dC), poly(dA-dT).poly(dA-dT), Br-poly(dI-dC).poly(dI-dC), or salmon sperm DNA were spotted onto nitrocellulose discs and baked. The e.l.i.s.a. was conducted in 48-well culture dishes at 37 degrees C using a rabbit polyclonal antiserum developed against Br-poly(dG-dC).poly(dG-dC), an alkaline phosphatase-conjugated second antibody, and p-nitrophenol as the substrate. Under conditions where antibody concentrations were not limiting, alkaline phosphatase activity was linear for 2 h. Dot blot e.l.i.s.a. conditions are described which allow quantification of Z-DNA [Br-poly(dG-dC).poly(dG-dC)] within the range 5-250 ng. Dot blot and transblot horseradish peroxidase e.l.i.s.a. are described that detect Z-DNA within supercoiled plasmid DNAs immobilized on diazophenylthioether (DPT) paper. In the transblot e.l.i.s.a., plasmid pUC8 derivatives containing 16, 24, or 32 residues of Z-DNA were electrophoresed in agarose gels and electrophoretically transferred to DPT paper. Z-DNA-antibody complexes were detected by the horseradish peroxidase-catalysed conversion of 4-chloro-1-naphthol to a coloured product that was covalently bound to the DPT paper. Z-DNA antibody reactivity was specific for supercoiled Z-DNA containing plasmids after removal of the antibodies cross-reactive with B-DNA by absorption onto native DNA-cellulose. The transblot e.l.i.s.a. was sensitive enough to detect 16 base pairs of alternating G-C residues in 100 ng of pUC8 DNA.

Isolation of a sex-linked DNA sequence in cranes.

Science.gov (United States)

Duan, W; Fuerst, P A

2001-01-01

A female-specific DNA fragment (CSL-W; crane sex-linked DNA on W chromosome) was cloned from female whooping cranes (Grus americana). From the nucleotide sequence of CSL-W, a set of polymerase chain reaction (PCR) primers was identified which amplify a 227-230 bp female-specific fragment from all existing crane species and some other noncrane species. A duplicated versions of the DNA segment, which is found to have a larger size (231-235 bp) than CSL-W in both sexes, was also identified, and was designated CSL-NW (crane sex-linked DNA on non-W chromosome). The nucleotide similarity between the sequences of CSL-W and CSL-NW from whooping cranes was 86.3%. The CSL primers do not amplify any sequence from mammalian DNA, limiting the potential for contamination from human sources. Using the CSL primers in combination with a quick DNA extraction method allows the noninvasive identification of crane gender in less than 10 h. A test of the methodology was carried out on fully developed body feathers from 18 captive cranes and resulted in 100% successful identification.

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

International Nuclear Information System (INIS)

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

2009-01-01

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

A comprehensive characterization of mitochondrial DNA mutations in glioblastoma multiforme.

Science.gov (United States)

Vidone, Michele; Clima, Rosanna; Santorsola, Mariangela; Calabrese, Claudia; Girolimetti, Giulia; Kurelac, Ivana; Amato, Laura Benedetta; Iommarini, Luisa; Trevisan, Elisa; Leone, Marco; Soffietti, Riccardo; Morra, Isabella; Faccani, Giuliano; Attimonelli, Marcella; Porcelli, Anna Maria; Gasparre, Giuseppe

2015-06-01

Glioblastoma multiforme (GBM) is the most malignant brain cancer in adults, with a poor prognosis, whose molecular stratification still represents a challenge in pathology and clinics. On the other hand, mitochondrial DNA (mtDNA) mutations have been found in most tumors as modifiers of the bioenergetics state, albeit in GBM a characterization of the mtDNA status is lacking to date. Here, a characterization of the burden of mtDNA mutations in GBM samples was performed. First, investigation of tumor-specific vs. non tumor-specific mutations was carried out with the MToolBox bioinformatics pipeline by analyzing 45 matched tumor/blood samples, from whole genome or whole exome sequencing datasets obtained from The Cancer Genome Atlas (TCGA) consortium. Additionally, the entire mtDNA sequence was obtained in a dataset of 104 fresh-frozen GBM samples. Mitochondrial mutations with potential pathogenic interest were prioritized based on heteroplasmic fraction, nucleotide variability, and in silico prediction of pathogenicity. A preliminary biochemical analysis of the activity of mitochondrial respiratory complexes was also performed on fresh-frozen GBM samples. Although a high number of mutations was detected, we report that the large majority of them does not pass the prioritization filters. Therefore, a relatively limited burden of pathogenic mutations is indeed carried by GBM, which did not appear to determine a general impairment of the respiratory chain. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genotoxic thresholds, DNA repair, and susceptibility in human populations

International Nuclear Information System (INIS)

Jenkins, Gareth J.S.; Zair, Zoulikha; Johnson, George E.; Doak, Shareen H.

2010-01-01

It has been long assumed that DNA damage is induced in a linear manner with respect to the dose of a direct acting genotoxin. Thus, it is implied that direct acting genotoxic agents induce DNA damage at even the lowest of concentrations and that no 'safe' dose range exists. The linear (non-threshold) paradigm has led to the one-hit model being developed. This 'one hit' scenario can be interpreted such that a single DNA damaging event in a cell has the capability to induce a single point mutation in that cell which could (if positioned in a key growth controlling gene) lead to increased proliferation, leading ultimately to the formation of a tumour. There are many groups (including our own) who, for a decade or more, have argued, that low dose exposures to direct acting genotoxins may be tolerated by cells through homeostatic mechanisms such as DNA repair. This argument stems from the existence of evolutionary adaptive mechanisms that allow organisms to adapt to low levels of exogenous sources of genotoxins. We have been particularly interested in the genotoxic effects of known mutagens at low dose exposures in human cells and have identified for the first time, in vitro genotoxic thresholds for several mutagenic alkylating agents (Doak et al., 2007). Our working hypothesis is that DNA repair is primarily responsible for these thresholded effects at low doses by removing low levels of DNA damage but becoming saturated at higher doses. We are currently assessing the roles of base excision repair (BER) and methylguanine-DNA methyltransferase (MGMT) for roles in the identified thresholds (Doak et al., 2008). This research area is currently important as it assesses whether 'safe' exposure levels to mutagenic chemicals can exist and allows risk assessment using appropriate safety factors to define such exposure levels. Given human variation, the mechanistic basis for genotoxic thresholds (e.g. DNA repair) has to be well defined in order that susceptible individuals are

A Review of Circulating Tumor DNA in Hepatobiliary Malignancies

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Kabir Mody

2018-06-01

Full Text Available Circulating tumor DNA (ctDNA is released into circulation (blood specifically from tumor cells undergoing metabolic secretion, apoptosis, or necrosis, carries tumor-specific genetic or epigenetic alterations. Technologies enabling clinical evaluation of ctDNA continue to advance rapidly and allow for the assessment of patient-specific tumoral genetic and epigenetic alterations. This holds great potential for earlier detection of disease, serial monitoring of tumor heterogeneity, identification of therapeutic targets, and evaluation of treatment response and mechanisms of resistance. Hepatobiliary malignancies are often diagnosed late, recur commonly, yield limited available tumor on biopsy, and harbor several genomic alterations with potential therapeutic impacts. Patients suffering from or at risk for these diseases thus stand to benefit immensely from this technology. Herein, we review the limited literature pertaining to the potential for ctDNA technologies in such patients. Patients with these cancers stand to benefit greatly from the application of ctDNA technologies, and concerted efforts at further investigation of such are ongoing and greatly needed.

Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome

International Nuclear Information System (INIS)

Dai Heqiao; Liu Jianying; Malkas, Linda H.; Catalano, Jennifer; Alagharu, Srilakshmi; Hickey, Robert J.

2009-01-01

Hexavalent chromium Cr(VI) is known to be a carcinogenic metal ion, with a complicated mechanism of action. It can be found within our environment in soil and water contaminated by manufacturing processes. Cr(VI) ion is readily taken up by cells, and is recognized to be both genotoxic and cytotoxic; following its reduction to the stable trivalent form of the ion, chromium(Cr(III)), within cells. This form of the ion is known to impede the activity of cellular DNA polymerase and polymerase-mediated DNA replication. Here, we report the effects of chromium on the activity and fidelity of the DNA replication process mediated by the human cell DNA synthesome. The DNA synthesome is a functional multiprotein complex that is fully competent to carry-out each phase of the DNA replication process. The IC 50 of Cr(III) toward the activity of DNA synthesome-associated DNA polymerases α, δ and ε is 15, 45 and 125 μM, respectively. Cr(III) inhibits synthesome-mediated DNA synthesis (IC 50 = 88 μM), and significantly reduces the fidelity of synthesome-mediated DNA replication. The mutation frequency induced by the different concentrations of Cr(III) ion used in our assays ranges from 2-13 fold higher than that which occurs spontaneously, and the types of mutations include single nucleotide substitutions, insertions, and deletions. Single nucleotide substitutions are the predominant type of mutation, and they occur primarily at GC base-pairs. Cr(III) ion produces a lower number of transition and a higher number of transversion mutations than occur spontaneously. Unlike Cr(III), Cr(VI) ion has little effect on the in vitro DNA synthetic activity and fidelity of the DNA synthesome, but does significantly inhibit DNA synthesis in intact cells. Cell growth and proliferation is also arrested by increasing concentrations of Cr(VI) ion. Our studies provide evidence indicating that the chromium ion induced decrease in the fidelity and activity of synthesome mediated DNA replication

Virulence properties and random amplification of polymorphic DNA ...

African Journals Online (AJOL)

Genotypic and phenotypic characterization as well as studies on the virulence factors of Candida albicans isolates obtained from oral cavity of patients was carried out using random amplified polymorphic DNA (RAPD) fingerprinting and epithelial cells adherence assay, respectively. RAPD patterns revealed the presence of ...

Proximity hybridization-regulated catalytic DNA hairpin assembly for electrochemical immunoassay based on in situ DNA template-synthesized Pd nanoparticles

International Nuclear Information System (INIS)

Zhou, Fuyi; Yao, Yao; Luo, Jianjun; Zhang, Xing; Zhang, Yu; Yin, Dengyang; Gao, Fenglei; Wang, Po

2017-01-01

Novel hybridization proximity-regulated catalytic DNA hairpin assembly strategy has been proposed for electrochemical immunoassay based on in situ DNA template-synthesized Pd nanoparticles as signal label. The DNA template-synthesized Pd nanoparticles were characterized with atomic force microscopic and X-ray photoelectron spectroscopy. The highly efficient electrocatalysis by DNA template synthesized Pd nanoparticles for NaBH 4 oxidation produced an intense detection signal. The label-free electrochemical method achieved the detection of carcinoembryonic antigen (CEA) with a linear range from 10 −15 to 10 −11  g mL −1 and a detection limit of 0.43 × 10 −15  g mL −1 . Through introducing a supersandwich reaction to increase the DNA length, the electrochemical signal was further amplified, leading to a detection limit of 0.52 × 10 −16  g mL −1 . And it rendered satisfactory analytical performance for the determination of CEA in serum samples. Furthermore, it exhibited good reproducibility and stability; meanwhile, it also showed excellent specificity due to the specific recognition of antigen by antibody. Therefore, the DNA template synthesized Pd nanoparticles based signal amplification approach has great potential in clinical applications and is also suitable for quantification of biomarkers at ultralow level. - Graphical abstract: A novel label-free and enzyme-free electrochemical immunoassay based on proximity hybridization-regulated catalytic DNA hairpin assemblies for recycling of the CEA. - Highlights: • A novel enzyme-free electrochemical immunosensor was developed for detection of CEA. • The signal amplification was based on catalytic DNA hairpin assembly and DNA-template-synthesized Pd nanoparticles. • The biosensor could detect CEA down to 0.52 × 10 −16  g mL −1 level with a dynamic range spanning 5 orders of magnitude.

Estimation of strength in different extra Watson-Crick hydrogen bonds in DNA double helices through quantum chemical studies.

Science.gov (United States)

Bandyopadhyay, D; Bhattacharyya, D

2006-10-15

It was shown earlier, from database analysis, model building studies, and molecular dynamics simulations that formation of cross-strand bifurcated or Extra Watson-Crick hydrogen (EWC) bonds between successive base pairs may lead to extra rigidity to DNA double helices of certain sequences. The strengths of these hydrogen bonds are debatable, however, as they do not have standard linear geometry criterion. We have therefore carried out detailed ab initio quantum chemical studies using RHF/6-31G(2d,2p) and B3LYP/6-31G(2p,2d) basis sets to determine strengths of several bent hydrogen bonds with different donor and acceptors. Interaction energy calculations, corrected for the basis set superposition errors, suggest that N-H...O type bent EWC hydrogen bonds are possible along same strands or across the strands between successive base pairs, leading to significant stability (ca. 4-9 kcal/mol). The N-H...N and C-H...O type interactions, however, are not so stabilizing. Hence, consideration of EWC N-H...O H-bonds can lead to a better understanding of DNA sequence directed structural features. Copyright (c) 2006 Wiley Periodicals, Inc.

Properties of Confined Star-Branched and Linear Chains. A Monte Carlo Simulation Study

International Nuclear Information System (INIS)

Romiszowski, P.; Sikorski, A.

2004-01-01

A model of linear and star-branched polymer chains confined between two parallel and impenetrable surfaces was built. The polymer chains were restricted to a simple cubic lattice. Two macromolecular architectures of the chain: linear and star branched (consisted of f = 3 branches of equal length) were studied. The excluded volume was the only potential introduced into the model (the athermal system). Monte Carlo simulations were carried out using a sampling algorithm based on chain's local changes of conformation. The simulations were carried out at different confinement conditions: from light to high chain's compression. The scaling of chain's size with the chain length was studied and discussed. The influence of the confinement and the macromolecular architecture on the shape of a chain was studied. The differences in the shape of linear and star-branched chains were pointed out. (author)

Investigation of DNA strand breaks induced by 7Li and 12C ions

International Nuclear Information System (INIS)

Sui Li; Zhao Kui; Ni Meinan; Guo Jiyu; Luo Hongbing; Mei Junping; Lu Xiuqin; Zhou Ping

2004-01-01

Deoxyribonucleic acid (DNA) is an important biomacromolecule. It is a carrier of genetic information and a critical target for radiobiological effects. Numerous lesions have been identified in irradiated DNA. DNA double strand breaks (DSBs) are considered as the most important initial damage of all biological effects induced by ionizing radiation. In this experiment, DNA DSBs induced by heavy ions in the early period were investigated with atomic force microscopy (AFM). Choosing 7 Li and 12 C heavy ions with different LET values delivered by HI-13 tandem accelerator respectively, purified plasmid DNA samples in aqueous solution were irradiated at different doses. AFM was used for nanometer-level-structure analysis of DNA damage induced by these two kinds of heavy ions. Measurement of the DNA fragment lengths was accomplished with the Scion Image analyzed soft-ware. Change laws of three forms of DNA, supercoils, open circular and linear form as dose increased were obtained. Distributed function of DNA fragment length was also obtained, and fitted with Tsallis entropy statistical theory. (author)

Modes of Escherichia coli Dps Interaction with DNA as Revealed by Atomic Force Microscopy.

Directory of Open Access Journals (Sweden)

Vladislav V Melekhov

Full Text Available Multifunctional protein Dps plays an important role in iron assimilation and a crucial role in bacterial genome packaging. Its monomers form dodecameric spherical particles accumulating ~400 molecules of oxidized iron ions within the protein cavity and applying a flexible N-terminal ends of each subunit for interaction with DNA. Deposition of iron is a well-studied process by which cells remove toxic Fe2+ ions from the genetic material and store them in an easily accessible form. However, the mode of interaction with linear DNA remained mysterious and binary complexes with Dps have not been characterized so far. It is widely believed that Dps binds DNA without any sequence or structural preferences but several lines of evidence have demonstrated its ability to differentiate gene expression, which assumes certain specificity. Here we show that Dps has a different affinity for the two DNA fragments taken from the dps gene regulatory region. We found by atomic force microscopy that Dps predominantly occupies thermodynamically unstable ends of linear double-stranded DNA fragments and has high affinity to the central part of the branched DNA molecule self-assembled from three single-stranded oligonucleotides. It was proposed that Dps prefers binding to those regions in DNA that provide more contact pads for the triad of its DNA-binding bundle associated with one vertex of the protein globule. To our knowledge, this is the first study revealed the nucleoid protein with an affinity to branched DNA typical for genomic regions with direct and inverted repeats. As a ubiquitous feature of bacterial and eukaryotic genomes, such structural elements should be of particular care, but the protein system evolutionarily adapted for this function is not yet known, and we suggest Dps as a putative component of this system.

DNA repair by MGMT, but not AAG, causes a threshold in alkylation-induced colorectal carcinogenesis.

Science.gov (United States)

Fahrer, Jörg; Frisch, Janina; Nagel, Georg; Kraus, Alexander; Dörsam, Bastian; Thomas, Adam D; Reißig, Sonja; Waisman, Ari; Kaina, Bernd

2015-10-01

Epidemiological studies indicate that N-nitroso compounds (NOC) are causally linked to colorectal cancer (CRC). NOC induce DNA alkylations, including O (6)-methylguanine (O (6)-MeG) and N-methylated purines, which are repaired by O (6)-MeG-DNA methyltransferase (MGMT) and N-alkyladenine-DNA glycosylase (AAG)-initiated base excision repair, respectively. In view of recent evidence of nonlinear mutagenicity for NOC-like compounds, the question arises as to the existence of threshold doses in CRC formation. Here, we set out to determine the impact of DNA repair on the dose-response of alkylation-induced CRC. DNA repair proficient (WT) and deficient (Mgmt (-/-), Aag (-/-) and Mgmt (-/-)/Aag (-/-)) mice were treated with azoxymethane (AOM) and dextran sodium sulfate to trigger CRC. Tumors were quantified by non-invasive mini-endoscopy. A non-linear increase in CRC formation was observed in WT and Aag (-/-) mice. In contrast, a linear dose-dependent increase in tumor frequency was found in Mgmt (-/-) and Mgmt (-/-)/Aag (-/-) mice. The data were corroborated by hockey stick modeling, yielding similar carcinogenic thresholds for WT and Aag (-/-) and no threshold for MGMT lacking mice. O (6)-MeG levels and depletion of MGMT correlated well with the observed dose-response in CRC formation. AOM induced dose-dependently DNA double-strand breaks in colon crypts including Lgr5-positive colon stem cells, which coincided with ATR-Chk1-p53 signaling. Intriguingly, Mgmt (-/-) mice displayed significantly enhanced levels of γ-H2AX, suggesting the usefulness of γ-H2AX as an early genotoxicity marker in the colorectum. This study demonstrates for the first time a non-linear dose-response for alkylation-induced colorectal carcinogenesis and reveals DNA repair by MGMT, but not AAG, as a key node in determining a carcinogenic threshold. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Optimization Research of Generation Investment Based on Linear Programming Model

Science.gov (United States)

Wu, Juan; Ge, Xueqian

Linear programming is an important branch of operational research and it is a mathematical method to assist the people to carry out scientific management. GAMS is an advanced simulation and optimization modeling language and it will combine a large number of complex mathematical programming, such as linear programming LP, nonlinear programming NLP, MIP and other mixed-integer programming with the system simulation. In this paper, based on the linear programming model, the optimized investment decision-making of generation is simulated and analyzed. At last, the optimal installed capacity of power plants and the final total cost are got, which provides the rational decision-making basis for optimized investments.

Inhibition of in vitro SV40 DNA replication by ultraviolet light

International Nuclear Information System (INIS)

Gough, G.; Wood, R.W.

1989-01-01

Ultraviolet light-induced DNA damage was found to inhibit SV40 origin-dependent DNA synthesis carried out by soluble humancell extracts. Replication of SV40-based plasmids was reduced to approx. 35% of that in unirradiated controls after irradiation with 50-100 J/m 2 germicidal ultraviolet light, where an average of 3-6 pyrimidine dimer photoproducts were formed per plasmid circle. Inhibition of the DNA helicase activity of T antigen (required for initiation of replication in the in vitro system) was also investigated, and was only significant after much higher fluences, 1000-5000 J/m 2 . The data indicate that DNA damage by ultraviolet light inhibits DNA synthesis in cell-free extracts principally by affecting components of the replication complex other than the DNA helicase activity of T antigen. The soluble system could be used to biochemically investigate the possible bypass or tolerance of DNA damage during replication (author). 21 refs.; 2 figs

Collective spin by linearization of the Schrodinger equation for nuclear collective motion

International Nuclear Information System (INIS)

Greiner, M.; Scheid, W.; Herrmann, R.

1988-01-01

The free Schrodinger equation for multipole degrees of freedom is linearized so that energy and momentum operators appear only in first order. As an example, the authors demonstrate the linearization procedure for quadrupole degrees of freedom. The wave function solving this equation carries a spin. The authors derive the operator of the collective spin and its eigen values depending on multipolarity

Targeting DNA Methyltranferases in Urological Tumors

Directory of Open Access Journals (Sweden)

Ângela Marques-Magalhães

2018-04-01

Full Text Available Urological cancers are a heterogeneous group of malignancies accounting for a considerable proportion of cancer-related morbidity and mortality worldwide. Aberrant epigenetic traits, especially altered DNA methylation patterns constitute a hallmark of these tumors. Nonetheless, these alterations are reversible, and several efforts have been carried out to design and test several epigenetic compounds that might reprogram tumor cell phenotype back to a normal state. Indeed, several DNMT inhibitors are currently under evaluation for therapeutic efficacy in clinical trials. This review highlights the critical role of DNA methylation in urological cancers and summarizes the available data on pre-clinical assays and clinical trials with DNMT inhibitors in bladder, kidney, prostate, and testicular germ cell cancers.

New environmental metabarcodes for analysing soil DNA

DEFF Research Database (Denmark)

Epp, Laura S.; Boessenkool, Sanne; Bellemain, Eva P.

2012-01-01

was systematically evaluated by (i) in silico PCRs using all standard sequences in the EMBL public database as templates, (ii) in vitro PCRs of DNA extracts from surface soil samples from a site in Varanger, northern Norway and (iii) in vitro PCRs of DNA extracts from permanently frozen sediment samples of late......Metabarcoding approaches use total and typically degraded DNA from environmental samples to analyse biotic assemblages and can potentially be carried out for any kinds of organisms in an ecosystem. These analyses rely on specific markers, here called metabarcodes, which should be optimized...... for taxonomic resolution, minimal bias in amplification of the target organism group and short sequence length. Using bioinformatic tools, we developed metabarcodes for several groups of organisms: fungi, bryophytes, enchytraeids, beetles and birds. The ability of these metabarcodes to amplify the target groups...

A bio-hybrid DNA rotor-stator nanoengine that moves along predefined tracks.

Science.gov (United States)

Valero, Julián; Pal, Nibedita; Dhakal, Soma; Walter, Nils G; Famulok, Michael

2018-06-01

Biological motors are highly complex protein assemblies that generate linear or rotary motion, powered by chemical energy. Synthetic motors based on DNA nanostructures, bio-hybrid designs or synthetic organic chemistry have been assembled. However, unidirectionally rotating biomimetic wheel motors with rotor-stator units that consume chemical energy are elusive. Here, we report a bio-hybrid nanoengine consisting of a catalytic stator that unidirectionally rotates an interlocked DNA wheel, powered by NTP hydrolysis. The engine consists of an engineered T7 RNA polymerase (T7RNAP-ZIF) attached to a dsDNA nanoring that is catenated to a rigid rotating dsDNA wheel. The wheel motor produces long, repetitive RNA transcripts that remain attached to the engine and are used to guide its movement along predefined ssDNA tracks arranged on a DNA nanotube. The simplicity of the design renders this walking nanoengine adaptable to other biological nanoarchitectures, facilitating the construction of complex bio-hybrid structures that achieve NTP-driven locomotion.

Oral DNA Vaccine in Chickens

Directory of Open Access Journals (Sweden)

Seyed Davoud Jazayeri

2012-01-01

Full Text Available Attenuated Salmonella has been used as a carrier for DNA vaccine. However, in vitro and in vivo studies on the bacteria following transfection of plasmid DNA were poorly studied. In this paper, eukaryotic expression plasmids encoding avian influenza virus (AIV subtype H5N1 genes, pcDNA3.1/HA, NA, and NP, were transfected into an attenuated Salmonella enteric typhimurium SV4089. In vitro stability of the transfected plasmids into Salmonella were over 90% after 100 generations. The attenuated Salmonella were able to invade MCF-7 (1.2% and MCF-10A (0.5% human breast cancer cells. Newly hatched specific-pathogen-free (SPF chicks were inoculated once by oral gavage with 109 colony-forming unit (CFU of the attenuated Salmonella. No abnormal clinical signs or deaths were recorded after inoculation. Viable bacteria were detected 3 days after inoculation by plating from spleen, liver, and cecum. Fluorescent in situ hybridization (FISH and polymerase chain reaction (PCR were carried out for confirmation. Salmonella was not detected in blood cultures although serum antibody immune responses to Salmonella O antiserum group D1 factor 1, 9, and 12 antigens were observed in all the inoculated chickens after 7 days up to 35 days. Our results showed that live attenuated S. typhimurium SV4089 harboring pcDNA3.1/HA, NA, and NP may provide a unique alternative as a carrier for DNA oral vaccine in chickens.

Integrating DNA barcode data and taxonomic practice: determination, discovery, and description.

Science.gov (United States)

Goldstein, Paul Z; DeSalle, Rob

2011-02-01

DNA barcodes, like traditional sources of taxonomic information, are potentially powerful heuristics in the identification of described species but require mindful analytical interpretation. The role of DNA barcoding in generating hypotheses of new taxa in need of formal taxonomic treatment is discussed, and it is emphasized that the recursive process of character evaluation is both necessary and best served by understanding the empirical mechanics of the discovery process. These undertakings carry enormous ramifications not only for the translation of DNA sequence data into taxonomic information but also for our comprehension of the magnitude of species diversity and its disappearance. This paper examines the potential strengths and pitfalls of integrating DNA sequence data, specifically in the form of DNA barcodes as they are currently generated and analyzed, with taxonomic practice.

Optimization of Neutral Comet Assay for studying DNA double-strand breaks in pea and wheat

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Ivelina Nikolova

2013-01-01

Full Text Available This study describes an adaptation of the Comet assay under neutral conditions for mono- and dicotyledonous plants pea (Pisum sativum L. and wheat (Triticum aestivum L.. Modifications concern lysis and electrophoresis steps, respectively. Electrophoresis was carried out varying the intensity of the electric field. A linear relationship between the percentages of DNA in the tail from control background with alteration of intensity was found. Trypan blue dye exclusion test was used in order to determine the intactness of nuclear membrane of the isolated nuclei from both plant model systems. Assessment was conducted on non-irradiated and irradiated nuclei on a monolayer with three doses of UVC. It was found that the share of intact nuclei (trypan blue negative ones is about 95% in controls. Gradual dose-related increase of damaged nuclei was observed in both species, reaching statistical significance only at the higher dose applied.

DNAzyme Feedback Amplification: Relaying Molecular Recognition to Exponential DNA Amplification.

Science.gov (United States)

Liu, Meng; Yin, Qingxin; McConnell, Erin M; Chang, Yangyang; Brennan, John D; Li, Yingfu

2018-03-26

Technologies capable of linking DNA amplification to molecular recognition are very desirable for ultrasensitive biosensing applications. We have developed a simple but powerful isothermal DNA amplification method, termed DNAzyme feedback amplification (DFA), that is capable of relaying molecular recognition to exponential DNA amplification. The method incorporates both an RNA-cleaving DNAzyme (RCD) and rolling circle amplification (RCA) carried out by a special DNA polymerase using a circular DNA template. DFA begins with a stimulus-dependent RCA reaction, producing tandemly linked RCDs in long-chain DNA products. These RCDs cleave an RNA-containing DNA sequence to form additional primers that hybridize to the circular DNA molecule, giving rise to DNA assemblies that act as the new inputs for RCA. The RCA reaction and the cleavage event keep on feeding each other autonomously, resulting in exponential growth of repetitive DNA sequences that can be easily detected. This method can be used for the detection of both nucleic acid based targets and non-nucleic acid analytes. In this article, we discuss the conceptual framework of the feedback amplification approach, the essential features of this method as well as remaining challenges and possible solutions. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Slope Safety Factor Calculations With Non-Linear Yield Criterion Using Finite Elements

DEFF Research Database (Denmark)

Clausen, Johan; Damkilde, Lars

2006-01-01

The factor of safety for a slope is calculated with the finite element method using a non-linear yield criterion of the Hoek-Brown type. The parameters of the Hoek-Brown criterion are found from triaxial test data. Parameters of the linear Mohr-Coulomb criterion are calibrated to the same triaxial...... are carried out at much higher stress levels than present in a slope failure, this leads to the conclusion that the use of the non-linear criterion leads to a safer slope design...

Enzyme-enhanced fluorescence detection of DNA on etched optical fibers.

Science.gov (United States)

Niu, Shu-yan; Li, Quan-yi; Ren, Rui; Zhang, Shu-sheng

2009-05-15

A novel DNA biosensor based on enzyme-enhanced fluorescence detection on etched optical fibers was developed. The hybridization complex of DNA probe and biotinylated target was formed on the etched optical fiber, and was then bound with streptavidin labeled horseradish peroxidase (streptavidin-HRP). The target DNA was quantified through the fluorescent detection of bi-p,p'-4-hydroxyphenylacetic acid (DBDA) generated from the substrate 4-hydroxyphenylacetic acid (p-HPA) under the catalysis of HRP, with a detection limit of 1 pM and a linear range from 1.69 pM to 169 pM. It is facile to regenerate this sensor through surface treatment with concentrated urea solution. It was discovered that the sensor can retain 70% of its original activity after three detection-regeneration cycles.

Are mini DNA-barcodes sufficiently informative to resolve species ...

Indian Academy of Sciences (India)

... between mini-barcode and the full- length DNA barcode was carried out in Microsoft Excel. (http://www.office.microsoft.com). ..... Received 15 June 2013, in final revised form 5 April 2014; accepted 3 June 2014. Unedited version published ...

Space Radiation Effects on Human Cells: Modeling DNA Breakage, DNA Damage Foci Distribution, Chromosomal Aberrations and Tissue Effects

Science.gov (United States)

Ponomarev, A. L.; Huff, J. L.; Cucinotta, F. A.

2011-01-01

Future long-tem space travel will face challenges from radiation concerns as the space environment poses health risk to humans in space from radiations with high biological efficiency and adverse post-flight long-term effects. Solar particles events may dramatically affect the crew performance, while Galactic Cosmic Rays will induce a chronic exposure to high-linear-energy-transfer (LET) particles. These types of radiation, not present on the ground level, can increase the probability of a fatal cancer later in astronaut life. No feasible shielding is possible from radiation in space, especially for the heavy ion component, as suggested solutions will require a dramatic increase in the mass of the mission. Our research group focuses on fundamental research and strategic analysis leading to better shielding design and to better understanding of the biological mechanisms of radiation damage. We present our recent effort to model DNA damage and tissue damage using computational models based on the physics of heavy ion radiation, DNA structure and DNA damage and repair in human cells. Our particular area of expertise include the clustered DNA damage from high-LET radiation, the visualization of DSBs (DNA double strand breaks) via DNA damage foci, image analysis and the statistics of the foci for different experimental situations, chromosomal aberration formation through DSB misrepair, the kinetics of DSB repair leading to a model-derived spectrum of chromosomal aberrations, and, finally, the simulation of human tissue and the pattern of apoptotic cell damage. This compendium of theoretical and experimental data sheds light on the complex nature of radiation interacting with human DNA, cells and tissues, which can lead to mutagenesis and carcinogenesis later in human life after the space mission.

Relative controllability and null controllability of linear delay systems ...

African Journals Online (AJOL)

Necessary and sufficient conditions are established for the relative, absolute controllability and null controllability of the generalized linear delay system and its discrete prototype. The paper presents illuminating examples on previous controllability results by Manitius and Olbrot [7] and carries over the results of Onwuatu [8] ...

Experimental setup and first measurement of DNA damage induced along and around an antiproton beam

International Nuclear Information System (INIS)

Kavanagh, J.N.; Currell, F.J.; Prise, K.M.; Schettino, G.; Currell, F.J.; Timson, D.J.; Holzscheiter, M.H.; Bassler, N.; Herrmann, R.

2010-01-01

Radiotherapy employs ionizing radiation to induce lethal DNA lesions in cancer cells while minimizing damage to healthy tissues. Due to their pattern of energy deposition, better therapeutic outcomes can, in theory, be achieved with ions compared to photons. Antiprotons have been proposed to offer a further enhancement due to their annihilation at the end of the path. The work presented here aimed to establish and validate an experimental procedure for the quantification of plasmid and genomic DNA damage resulting from antiproton exposure. Immunocytochemistry was used to assess DNA damage in directly and indirectly exposed human fibroblasts irradiated in both plateau and Bragg peak regions of a 126 MeV antiproton beam at CERN. Cells were stained post irradiation with an anti-γ-H2AX antibody. Quantification of the γ-H2AX foci-dose relationship is consistent with a linear increase in the Bragg peak region. A qualitative analysis of the foci detected in the Bragg peak and plateau region indicates significant differences highlighting the different severity of DNA lesions produced along the particle path. Irradiation of desalted plasmid DNA with 5 Gy antiprotons at the Bragg peak resulted in a significant portion of linear plasmid in the resultant solution. (authors)

Detection of influenza A virus using carbon nanotubes field effect transistor based DNA sensor

Science.gov (United States)

Tran, Thi Luyen; Nguyen, Thi Thuy; Huyen Tran, Thi Thu; Chu, Van Tuan; Thinh Tran, Quang; Tuan Mai, Anh

2017-09-01

The carbon nanotubes field effect transistor (CNTFET) based DNA sensor was developed, in this paper, for detection of influenza A virus DNA. Number of factors that influence the output signal and analytical results were investigated. The initial probe DNA, decides the available DNA strands on CNTs, was 10 μM. The hybridization time for defined single helix was 120 min. The hybridization temperature was set at 30 °C to get a net change in drain current of the DNA sensor without altering properties of any biological compounds. The response time of the DNA sensor was less than one minute with a high reproducibility. In addition, the DNA sensor has a wide linear detection range from 1 pM to 10 nM, and a very low detection limit of 1 pM. Finally, after 7-month storage in 7.4 pH buffer, the output signal of DNA sensor recovered 97%.

A Selective G-Quadruplex DNA-Stabilizing Ligand Based on a Cyclic Naphthalene Diimide Derivative

Directory of Open Access Journals (Sweden)