Multiple regulatory systems coordinate DNA replication with cell growth in Bacillus subtilis.

Science.gov (United States)

Murray, Heath; Koh, Alan

2014-10-01

In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s) that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes.

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

Some Effective Tight-Binding Models for Electrons in DNA Conduction: A Review

International Nuclear Information System (INIS)

Yamada, H.; Iguchi, K.

2010-01-01

Quantum transport for DNA conduction has been widely studied with interest in application as a candidate in making nanowires as well as interest in the scientific mechanism. In this paper, we review recent works concerning the electronic states and the conduction/transfer in DNA polymers. We have mainly investigated the energy-band structure and the correlation effects of localization property in the two- and three-chain systems (ladder model) with long-range correlation as a simple model for electronic property in a double strand of DNA by using the tight-bindingmodel. In addition, we investigated the localization properties of electronic states in several actual DNA sequences such as bacteriophages of Escherichia coli, human-chromosome 22, compared with those of the artificial disordered sequences with correlation. The charge-transfer properties for poly(dA)-poly(dT) and poly(dG)-poly(dC) DNA polymers are also presented in terms of localization lengths within the frameworks of the polaron models due to the coupling between the charge carriers and the lattice vibrations of the double strand of DNA

Multiple regulatory systems coordinate DNA replication with cell growth in Bacillus subtilis.

Directory of Open Access Journals (Sweden)

Heath Murray

2014-10-01

Full Text Available In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes.

Multiple Regulatory Systems Coordinate DNA Replication with Cell Growth in Bacillus subtilis

Science.gov (United States)

Murray, Heath; Koh, Alan

2014-01-01

In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s) that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes. PMID:25340815

Introducing improved structural properties and salt dependence into a coarse-grained model of DNA

Energy Technology Data Exchange (ETDEWEB)

Snodin, Benedict E. K., E-mail: benedict.snodin@chem.ox.ac.uk; Mosayebi, Majid; Schreck, John S.; Romano, Flavio; Doye, Jonathan P. K., E-mail: jonathan.doye@chem.ox.ac.uk [Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ (United Kingdom); Randisi, Ferdinando [Life Sciences Interface Doctoral Training Center, South Parks Road, Oxford OX1 3QU (United Kingdom); Rudolf Peierls Centre for Theoretical Physics, 1 Keble Road, Oxford OX1 3NP (United Kingdom); Šulc, Petr [Center for Studies in Physics and Biology, The Rockefeller University, 1230 York Avenue, New York, New York 10065 (United States); Ouldridge, Thomas E. [Department of Mathematics, Imperial College, 180 Queen’s Gate, London SW7 2AZ (United Kingdom); Tsukanov, Roman; Nir, Eyal [Department of Chemistry and the Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer Sheva (Israel); Louis, Ard A. [Rudolf Peierls Centre for Theoretical Physics, 1 Keble Road, Oxford OX1 3NP (United Kingdom)

2015-06-21

We introduce an extended version of oxDNA, a coarse-grained model of deoxyribonucleic acid (DNA) designed to capture the thermodynamic, structural, and mechanical properties of single- and double-stranded DNA. By including explicit major and minor grooves and by slightly modifying the coaxial stacking and backbone-backbone interactions, we improve the ability of the model to treat large (kilobase-pair) structures, such as DNA origami, which are sensitive to these geometric features. Further, we extend the model, which was previously parameterised to just one salt concentration ([Na{sup +}] = 0.5M), so that it can be used for a range of salt concentrations including those corresponding to physiological conditions. Finally, we use new experimental data to parameterise the oxDNA potential so that consecutive adenine bases stack with a different strength to consecutive thymine bases, a feature which allows a more accurate treatment of systems where the flexibility of single-stranded regions is important. We illustrate the new possibilities opened up by the updated model, oxDNA2, by presenting results from simulations of the structure of large DNA objects and by using the model to investigate some salt-dependent properties of DNA.

Introducing improved structural properties and salt dependence into a coarse-grained model of DNA

International Nuclear Information System (INIS)

Snodin, Benedict E. K.; Mosayebi, Majid; Schreck, John S.; Romano, Flavio; Doye, Jonathan P. K.; Randisi, Ferdinando; Šulc, Petr; Ouldridge, Thomas E.; Tsukanov, Roman; Nir, Eyal; Louis, Ard A.

2015-01-01

We introduce an extended version of oxDNA, a coarse-grained model of deoxyribonucleic acid (DNA) designed to capture the thermodynamic, structural, and mechanical properties of single- and double-stranded DNA. By including explicit major and minor grooves and by slightly modifying the coaxial stacking and backbone-backbone interactions, we improve the ability of the model to treat large (kilobase-pair) structures, such as DNA origami, which are sensitive to these geometric features. Further, we extend the model, which was previously parameterised to just one salt concentration ([Na + ] = 0.5M), so that it can be used for a range of salt concentrations including those corresponding to physiological conditions. Finally, we use new experimental data to parameterise the oxDNA potential so that consecutive adenine bases stack with a different strength to consecutive thymine bases, a feature which allows a more accurate treatment of systems where the flexibility of single-stranded regions is important. We illustrate the new possibilities opened up by the updated model, oxDNA2, by presenting results from simulations of the structure of large DNA objects and by using the model to investigate some salt-dependent properties of DNA

Bacterial DNA Sequence Compression Models Using Artificial Neural Networks

Directory of Open Access Journals (Sweden)

Armando J. Pinho

2013-08-01

Full Text Available It is widely accepted that the advances in DNA sequencing techniques have contributed to an unprecedented growth of genomic data. This fact has increased the interest in DNA compression, not only from the information theory and biology points of view, but also from a practical perspective, since such sequences require storage resources. Several compression methods exist, and particularly, those using finite-context models (FCMs have received increasing attention, as they have been proven to effectively compress DNA sequences with low bits-per-base, as well as low encoding/decoding time-per-base. However, the amount of run-time memory required to store high-order finite-context models may become impractical, since a context-order as low as 16 requires a maximum of 17.2 x 109 memory entries. This paper presents a method to reduce such a memory requirement by using a novel application of artificial neural networks (ANN to build such probabilistic models in a compact way and shows how to use them to estimate the probabilities. Such a system was implemented, and its performance compared against state-of-the art compressors, such as XM-DNA (expert model and FCM-Mx (mixture of finite-context models , as well as with general-purpose compressors. Using a combination of order-10 FCM and ANN, similar encoding results to those of FCM, up to order-16, are obtained using only 17 megabytes of memory, whereas the latter, even employing hash-tables, uses several hundreds of megabytes.

Multiscale modelling of DNA mechanics

Czech Academy of Sciences Publication Activity Database

Dršata, Tomáš; Lankaš, Filip

2015-01-01

Roč. 27, č. 32 (2015), 323102/1-323102/12 ISSN 0953-8984 R&D Projects: GA ČR(CZ) GA14-21893S Institutional support: RVO:61388963 Keywords : DNA elasticity * DNA coarse-grained models * molecular dynamics simulations Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.209, year: 2015

Modeling spin selectivity in charge transfer across the DNA/Gold interface

Energy Technology Data Exchange (ETDEWEB)

Behnia, S., E-mail: s.behnia@sci.uut.ac.ir [Department of Physics, Urmia University of Technology, Urmia (Iran, Islamic Republic of); Fathizadeh, S. [Department of Physics, Urmia University of Technology, Urmia (Iran, Islamic Republic of); Akhshani, A. [Department of Physics, Urmia Branch, Islamic Azad University, Urmia (Iran, Islamic Republic of)

2016-09-30

Highlights: • DNA in spintronics is applied. Nearly pure spin current is observed in the system. • A combined spin-polaronic PBH model is proposed for spin transfer in DNA molecule. • Spin Hall effect in DNA due to spin–orbit coupling is verified. • The temperature dependence of Hall conductivity is appeared. • Regions of parameters were determined that polarization of spin current is maximum. - Abstract: Experimental results show that the photoelectrons emitted from the gold substrate due to laser radiation, passe through DNA nanowires with spin-polarized nature. This study proposes the use of chiral DNA molecule in spintronics and information processing. To investigate the spin transfer in DNA molecules, we established a theoretical model based on a combined spin-polaronic Peyrard–Bishop–Holstein model. Accordingly, a nearly pure spin current is appeared. The simultaneous effects of the incident radiation and external magnetic field create characteristic islands corresponding to the pure spin currents, which can be predicted and detected using the multifractal dimensions spectrum. We can verify the spin Hall effect on DNA oligomers through spin–orbit coupling. As such, we can proceed to our significant purpose, which is to create a nearly pure spin current for information transfer and determine the regions of parameter values from which the maximal polarization in spin current emerges.

A universal DNA-based protein detection system.

Science.gov (United States)

Tran, Thua N N; Cui, Jinhui; Hartman, Mark R; Peng, Songming; Funabashi, Hisakage; Duan, Faping; Yang, Dayong; March, John C; Lis, John T; Cui, Haixin; Luo, Dan

2013-09-25

Protein immune detection requires secondary antibodies which must be carefully selected in order to avoid interspecies cross-reactivity, and is therefore restricted by the limited availability of primary/secondary antibody pairs. Here we present a versatile DNA-based protein detection system using a universal adapter to interface between IgG antibodies and DNA-modified reporter molecules. As a demonstration of this capability, we successfully used DNA nano-barcodes, quantum dots, and horseradish peroxidase enzyme to detect multiple proteins using our DNA-based labeling system. Our system not only eliminates secondary antibodies but also serves as a novel method platform for protein detection with modularity, high capacity, and multiplexed capability.

Intermolecular G-quadruplex structure-based fluorescent DNA detection system.

Science.gov (United States)

Zhou, Hui; Wu, Zai-Sheng; Shen, Guo-Li; Yu, Ru-Qin

2013-03-15

Adopting multi-donors to pair with one acceptor could improve the performance of fluorogenic detection probes. However, common dyes (e.g., fluorescein) in close proximity to each other would self-quench the fluorescence, and the fluorescence is difficult to restore. In this contribution, we constructed a novel "multi-donors-to-one acceptor" fluorescent DNA detection system by means of the intermolecular G-quadruplex (IGQ) structure-based fluorescence signal enhancement combined with the hairpin oligonucleotide. The novel IGQ-hairpin system was characterized using the p53 gene as the model target DNA. The proposed system showed an improved assay performance due to the introduction of IGQ-structure into fluorescent signaling probes, which could inhibit the background fluorescence and increase fluorescence restoration amplitude of fluoresceins upon target DNA hybridization. The proof-of-concept scheme is expected to provide new insight into the potential of G-quadruplex structure and promote the application of fluorescent oligonucleotide probes in fundamental research, diagnosis, and treatment of genetic diseases. Copyright © 2012 Elsevier B.V. All rights reserved.

Understanding DNA Under Oxidative Stress and Sensitization: The Role of Molecular Modeling

Directory of Open Access Journals (Sweden)

Antonio eMonari

2015-07-01

Full Text Available DNA is constantly exposed to damaging threats coming from oxidative stress, i.e. from the presence of free radicals and reactive oxygen species. Sensitization from exogenous and endogenous compounds that strongly enhance the frequency of light-induced lesions also plays an important role. The experimental determination of DNA lesions, though a difficult subject, is somehow well established and allows to elucidate even extremely rare DNA lesions. In parallel, molecular modeling has become fundamental to clearly understand the fine mechanisms related to DNA defects induction. Indeed, it offers an unprecedented possibility to get access to an atomistic or even electronic resolution. Ab initio molecular dynamics may also describe the time-evolution of the molecular system and its reactivity. Yet the modeling of DNA (photo-reactions does necessitate elaborate multi-scale methodologies to tackle a damage induction reactivity that takes place in a complex environment. The double-stranded DNA environment is first characterized by a very high flexibility, that dynamical effects are to be taken into account, but also a strongly inhomogeneous electrostatic embedding. Additionally, one aims at capturing more subtle effects, such as the sequence selectivity which is of critical important for DNA damage. The structure and dynamics of the DNA/sensitizers complexes, as well as the photo-induced electron- and energy-transfer phenomena taking place upon sensitization, should be carefully modeled. Finally the factors inducing different repair ratios for different lesions should also be rationalized.In this review we will critically analyze the different computational strategies used to model DNA lesions. A clear picture of the complex interplay between reactivity and structural factors will be sketched. The use of proper multi-scale modeling leads to the in-depth comprehension of DNA lesions mechanism and also to the rational design of new chemo-therapeutic agents.

A model capturing novel strand symmetries in bacterial DNA

International Nuclear Information System (INIS)

Sobottka, Marcelo; Hart, Andrew G.

2011-01-01

Highlights: → We propose a simple stochastic model to construct primitive DNA sequences. → The model provide an explanation for Chargaff's second parity rule in primitive DNA sequences. → The model is also used to predict a novel type of strand symmetry in primitive DNA sequences. → We extend the results for bacterial DNA sequences and compare distributional properties intrinsic to the model to statistical estimates from 1049 bacterial genomes. → We find out statistical evidences that the novel type of strand symmetry holds for bacterial DNA sequences. -- Abstract: Chargaff's second parity rule for short oligonucleotides states that the frequency of any short nucleotide sequence on a strand is approximately equal to the frequency of its reverse complement on the same strand. Recent studies have shown that, with the exception of organellar DNA, this parity rule generally holds for double-stranded DNA genomes and fails to hold for single-stranded genomes. While Chargaff's first parity rule is fully explained by the Watson-Crick pairing in the DNA double helix, a definitive explanation for the second parity rule has not yet been determined. In this work, we propose a model based on a hidden Markov process for approximating the distributional structure of primitive DNA sequences. Then, we use the model to provide another possible theoretical explanation for Chargaff's second parity rule, and to predict novel distributional aspects of bacterial DNA sequences.

Modeling inhomogeneous DNA replication kinetics.

Directory of Open Access Journals (Sweden)

Michel G Gauthier

Full Text Available In eukaryotic organisms, DNA replication is initiated at a series of chromosomal locations called origins, where replication forks are assembled proceeding bidirectionally to replicate the genome. The distribution and firing rate of these origins, in conjunction with the velocity at which forks progress, dictate the program of the replication process. Previous attempts at modeling DNA replication in eukaryotes have focused on cases where the firing rate and the velocity of replication forks are homogeneous, or uniform, across the genome. However, it is now known that there are large variations in origin activity along the genome and variations in fork velocities can also take place. Here, we generalize previous approaches to modeling replication, to allow for arbitrary spatial variation of initiation rates and fork velocities. We derive rate equations for left- and right-moving forks and for replication probability over time that can be solved numerically to obtain the mean-field replication program. This method accurately reproduces the results of DNA replication simulation. We also successfully adapted our approach to the inverse problem of fitting measurements of DNA replication performed on single DNA molecules. Since such measurements are performed on specified portion of the genome, the examined DNA molecules may be replicated by forks that originate either within the studied molecule or outside of it. This problem was solved by using an effective flux of incoming replication forks at the model boundaries to represent the origin activity outside the studied region. Using this approach, we show that reliable inferences can be made about the replication of specific portions of the genome even if the amount of data that can be obtained from single-molecule experiments is generally limited.

Computational Approach for Studying Optical Properties of DNA Systems in Solution

DEFF Research Database (Denmark)

Nørby, Morten Steen; Svendsen, Casper Steinmann; Olsen, Jógvan Magnus Haugaard

2016-01-01

In this paper we present a study of the methodological aspects regarding calculations of optical properties for DNA systems in solution. Our computational approach will be built upon a fully polarizable QM/MM/Continuum model within a damped linear response theory framework. In this approach...... the environment is given a highly advanced description in terms of the electrostatic potential through the polarizable embedding model. Furthermore, bulk solvent effects are included in an efficient manner through a conductor-like screening model. With the aim of reducing the computational cost we develop a set...... of averaged partial charges and distributed isotropic dipole-dipole polarizabilities for DNA suitable for describing the classical region in ground-state and excited-state calculations. Calculations of the UV-spectrum of the 2-aminopurine optical probe embedded in a DNA double helical structure are presented...

DNA damage, metabolism and aging in pro-inflammatory T cells: Rheumatoid arthritis as a model system.

Science.gov (United States)

Li, Yinyin; Goronzy, Jörg J; Weyand, Cornelia M

2018-05-01

The aging process is the major driver of morbidity and mortality, steeply increasing the risk to succumb to cancer, cardiovascular disease, infection and neurodegeneration. Inflammation is a common denominator in age-related pathologies, identifying the immune system as a gatekeeper in aging overall. Among immune cells, T cells are long-lived and exposed to intense replication pressure, making them sensitive to aging-related abnormalities. In successful T cell aging, numbers of naïve cells, repertoire diversity and activation thresholds are preserved as long as possible; in maladaptive T cell aging, protective T cell functions decline and pro-inflammatory effector cells are enriched. Here, we review in the model system of rheumatoid arthritis (RA) how maladaptive T cell aging renders the host susceptible to chronic, tissue-damaging inflammation. In T cells from RA patients, known to be about 20years pre-aged, three interconnected functional domains are altered: DNA damage repair, metabolic activity generating energy and biosynthetic precursor molecules, and shaping of plasma membranes to promote T cell motility. In each of these domains, key molecules and pathways have now been identified, including the glycolytic enzymes PFKFB3 and G6PD; the DNA repair molecules ATM, DNA-PKcs and MRE11A; and the podosome marker protein TKS5. Some of these molecules may help in defining targetable pathways to slow the T cell aging process. Copyright © 2017 Elsevier Inc. All rights reserved.

Context dependent DNA evolutionary models

DEFF Research Database (Denmark)

Jensen, Jens Ledet

This paper is about stochastic models for the evolution of DNA. For a set of aligned DNA sequences, connected in a phylogenetic tree, the models should be able to explain - in probabilistic terms - the differences seen in the sequences. From the estimates of the parameters in the model one can...... start to make biologically interpretations and conclusions concerning the evolutionary forces at work. In parallel with the increase in computing power, models have become more complex. Starting with Markov processes on a space with 4 states, and extended to Markov processes with 64 states, we are today...... studying models on spaces with 4n (or 64n) number of states with n well above one hundred, say. For such models it is no longer possible to calculate the transition probability analytically, and often Markov chain Monte Carlo is used in connection with likelihood analysis. This is also the approach taken...

A passive physical model for DnaK chaperoning

Science.gov (United States)

Uhl, Lionel; Dumont, Audrey; Dukan, Sam

2018-03-01

Almost all living organisms use protein chaperones with a view to preventing proteins from misfolding or aggregation either spontaneously or during cellular stress. This work uses a reaction-diffusion stochastic model to describe the dynamic localization of the Hsp70 chaperone DnaK in Escherichia coli cells during transient proteotoxic collapse characterized by the accumulation of insoluble proteins. In the model, misfolded (‘abnormal’) proteins are produced during alcoholic stress and have the propensity to aggregate with a polymerization-like kinetics. When aggregates diffuse more slowly they grow larger. According to Michaelis-Menten-type kinetics, DnaK has the propensity to bind with misfolded proteins or aggregates in order to catalyse refolding. To match experimental fluorescence microscopy data showing clusters of DnaK-GFP localized in multiple foci, the model includes spatial zones with local reduced diffusion rates to generate spontaneous assemblies of DnaK called ‘foci’. Numerical simulations of our model succeed in reproducing the kinetics of DnaK localization experimentally observed. DnaK starts from foci, moves to large aggregates during acute stress, resolves those aggregates during recovery and finally returns to its initial punctate localization pattern. Finally, we compare real biological events with hypothetical repartitions of the protein aggregates or DnaK. We then notice that DnaK action is more efficient on protein aggregates than on protein homogeneously distributed.

Predicting Variation of DNA Shape Preferences in Protein-DNA Interaction in Cancer Cells with a New Biophysical Model.

Science.gov (United States)

Batmanov, Kirill; Wang, Junbai

2017-09-18

DNA shape readout is an important mechanism of transcription factor target site recognition, in addition to the sequence readout. Several machine learning-based models of transcription factor-DNA interactions, considering DNA shape features, have been developed in recent years. Here, we present a new biophysical model of protein-DNA interactions by integrating the DNA shape properties. It is based on the neighbor dinucleotide dependency model BayesPI2, where new parameters are restricted to a subspace spanned by the dinucleotide form of DNA shape features. This allows a biophysical interpretation of the new parameters as a position-dependent preference towards specific DNA shape features. Using the new model, we explore the variation of DNA shape preferences in several transcription factors across various cancer cell lines and cellular conditions. The results reveal that there are DNA shape variations at FOXA1 (Forkhead Box Protein A1) binding sites in steroid-treated MCF7 cells. The new biophysical model is useful for elucidating the finer details of transcription factor-DNA interaction, as well as for predicting cancer mutation effects in the future.

Molecular models for DNA damaged by photoreaction

International Nuclear Information System (INIS)

Pearlman, D.A.; Holbrook, S.R.; Pirkle, D.H.; Kim, S.H.

1985-01-01

Structural models of a DNA molecule containing a radiation-induced psoralen cross-link and of a DNA containing a thymine photodimer were constructed by applying energy-minimization techniques and model-building procedures to data from x-ray crystallographic studies. The helical axes of the models show substantial kinking and unwinding at the sites of the damage, which may have long-range as well as local effects arising from the concomitant changes in the supercoiling and overall structure of the DNA. The damaged areas may also serve as recognition sites for repair enzymes. These results should help in understanding the biologic effects of radiation-induced damage on cells

DNA Profiling of Convicted Offender Samples for the Combined DNA Index System

Science.gov (United States)

Millard, Julie T

2011-01-01

The cornerstone of forensic chemistry is that a perpetrator inevitably leaves trace evidence at a crime scene. One important type of evidence is DNA, which has been instrumental in both the implication and exoneration of thousands of suspects in a wide range of crimes. The Combined DNA Index System (CODIS), a network of DNA databases, provides…

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

Probing the role of interfacial waters in protein-DNA recognition using a hybrid implicit/explicit solvation model

Science.gov (United States)

Li, Shen; Bradley, Philip

2013-01-01

When proteins bind to their DNA target sites, ordered water molecules are often present at the protein-DNA interface bridging protein and DNA through hydrogen bonds. What is the role of these ordered interfacial waters? Are they important determinants of the specificity of DNA sequence recognition, or do they act in binding in a primarily non-specific manner, by improving packing of the interface, shielding unfavorable electrostatic interactions, and solvating unsatisfied polar groups that are inaccessible to bulk solvent? When modeling details of structure and binding preferences, can fully implicit solvent models be fruitfully applied to protein-DNA interfaces, or must the individualistic properties of these interfacial waters be accounted for? To address these questions, we have developed a hybrid implicit/explicit solvation model that specifically accounts for the locations and orientations of small numbers of DNA-bound water molecules while treating the majority of the solvent implicitly. Comparing the performance of this model to its fully implicit counterpart, we find that explicit treatment of interfacial waters results in a modest but significant improvement in protein sidechain placement and DNA sequence recovery. Base-by-base comparison of the performance of the two models highlights DNA sequence positions whose recognition may be dependent on interfacial water. Our study offers large-scale statistical evidence for the role of ordered water for protein DNA recognition, together with detailed examination of several well-characterized systems. In addition, our approach provides a template for modeling explicit water molecules at interfaces that should be extensible to other systems. PMID:23444044

Intrinsic Dynamics Analysis of a DNA Octahedron by Elastic Network Model

Directory of Open Access Journals (Sweden)

Guang Hu

2017-01-01

Full Text Available DNA is a fundamental component of living systems where it plays a crucial role at both functional and structural level. The programmable properties of DNA make it an interesting building block for the construction of nanostructures. However, molecular mechanisms for the arrangement of these well-defined DNA assemblies are not fully understood. In this paper, the intrinsic dynamics of a DNA octahedron has been investigated by using two types of Elastic Network Models (ENMs. The application of ENMs to DNA nanocages include the analysis of the intrinsic flexibilities of DNA double-helices and hinge sites through the calculation of the square fluctuations, as well as the intrinsic collective dynamics in terms of cross-collective map calculation coupled with global motions analysis. The dynamics profiles derived from ENMs have then been evaluated and compared with previous classical molecular dynamics simulation trajectories. The results presented here revealed that ENMs can provide useful insights into the intrinsic dynamics of large DNA nanocages and represent a useful tool in the field of structural DNA nanotechnology.

A novel Listeria monocytogenes-based DNA delivery system for cancer gene therapy.

LENUS (Irish Health Repository)

van Pijkeren, Jan Peter

2012-01-31

Bacteria-mediated transfer of plasmid DNA to mammalian cells (bactofection) has been shown to have significant potential as an approach to express heterologous proteins in various cell types. This is achieved through entry of the entire bacterium into cells, followed by release of plasmid DNA. In a murine model, we show that Listeria monocytogenes can invade and spread in tumors, and establish the use of Listeria to deliver genes to tumors in vivo. A novel approach to vector lysis and release of plasmid DNA through antibiotic administration was developed. Ampicillin administration facilitated both plasmid transfer and safety control of vector. To further improve on the gene delivery system, we selected a Listeria monocytogenes derivative that is more sensitive to ampicillin, and less pathogenic than the wild-type strain. Incorporation of a eukaryotic-transcribed lysin cassette in the plasmid further increased bacterial lysis. Successful gene delivery of firefly luciferase to growing tumors in murine models and to patient breast tumor samples ex vivo was achieved. The model described encompasses a three-phase treatment regimen, involving (1) intratumoral administration of vector followed by a period of vector spread, (2) systemic ampicillin administration to induce vector lysis and plasmid transfer, and (3) systemic administration of combined moxifloxacin and ampicillin to eliminate systemic vector. For the first time, our results reveal the potential of Listeria monocytogenes for in vivo gene delivery.

Chronic hepatitis B infection and HBV DNA-containing capsids: Modeling and analysis

Science.gov (United States)

Manna, Kalyan; Chakrabarty, Siddhartha P.

2015-05-01

We analyze the dynamics of chronic HBV infection taking into account both uninfected and infected hepatocytes along with the intracellular HBV DNA-containing capsids and the virions. While previous HBV models have included either the uninfected hepatocytes or the intracellular HBV DNA-containing capsids, our model accounts for both these two populations. We prove the conditions for local and global stability of both the uninfected and infected steady states in terms of the basic reproduction number. Further, we incorporate a time lag in the model to encompass the intracellular delay in the production of the infected hepatocytes and find that this delay does not affect the overall dynamics of the system. The results for the model and the delay model are finally numerically illustrated.

Repair of uv damaged DNA in systemic lupus erythematosus. [Mice

Energy Technology Data Exchange (ETDEWEB)

Beighlie, D J; Teplitz, R L

1975-06-01

The NZB NZW hybrid mouse is an animal model of human systemic lupus erythematosus (SLE). Two breeding schemes were devised using NZB, NZW, B/W, and CBA mice, which permit definitive decisions regarding genetic and/or viral origin of the disease. It is proposed that at least two factors must be involved: a genetic abnormality producing hyper-responsiveness to nucleic acid antigens, and a DNA repair defect which results in liberation of DNA and RNA when cells are lethally injured. Evidence is presented for a DNA repair deficit in human SLE lymphocytes following in vitro irradiation with ultraviolet (uv) light. Lymphocytes from adult New Zealand and control mice were found to lack normal amounts of endonuclease necessary for repairing uv damage.

A Paper Model of DNA Structure and Replication.

Science.gov (United States)

Sigismondi, Linda A.

1989-01-01

A paper model which is designed to give students a hands-on experience during lecture and blackboard instruction on DNA structure is provided. A list of materials, paper patterns, and procedures for using the models to teach DNA structure and replication are given. (CW)

Early models of DNA damage formation

International Nuclear Information System (INIS)

Śmiałek, Małgorzata A

2012-01-01

Quantification of DNA damage, induced by various types of incident radiation as well as chemical agents, has been the subject of many theoretical and experimental studies, supporting the development of modern cancer therapy. The primary observations showed that many factors can lead to damage of DNA molecules. It became clear that the development of experimental techniques for exploring this phenomenon is required. Another problem was simultaneously dealt with, anticipating on how the damage is distributed within the double helix of the DNA molecule and how the single strand break formation and accumulation can influence the lethal double strand break formation. In this work the most important probabilistic models for DNA strand breakage and damage propagation are summarized and compared.

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…

3D-DART: a DNA structure modelling server

NARCIS (Netherlands)

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

2009-01-01

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

Moving beyond Watson-Crick models of coarse grained DNA dynamics.

Science.gov (United States)

Linak, Margaret C; Tourdot, Richard; Dorfman, Kevin D

2011-11-28

DNA produces a wide range of structures in addition to the canonical B-form of double-stranded DNA. Some of these structures are stabilized by Hoogsteen bonds. We developed an experimentally parameterized, coarse-grained model that incorporates such bonds. The model reproduces many of the microscopic features of double-stranded DNA and captures the experimental melting curves for a number of short DNA hairpins, even when the open state forms complicated secondary structures. We demonstrate the utility of the model by simulating the folding of a thrombin aptamer, which contains G-quartets, and strand invasion during triplex formation. Our results highlight the importance of including Hoogsteen bonding in coarse-grained models of DNA.

Assessing environmental DNA detection in controlled lentic systems.

Science.gov (United States)

Moyer, Gregory R; Díaz-Ferguson, Edgardo; Hill, Jeffrey E; Shea, Colin

2014-01-01

Little consideration has been given to environmental DNA (eDNA) sampling strategies for rare species. The certainty of species detection relies on understanding false positive and false negative error rates. We used artificial ponds together with logistic regression models to assess the detection of African jewelfish eDNA at varying fish densities (0, 0.32, 1.75, and 5.25 fish/m3). Our objectives were to determine the most effective water stratum for eDNA detection, estimate true and false positive eDNA detection rates, and assess the number of water samples necessary to minimize the risk of false negatives. There were 28 eDNA detections in 324, 1-L, water samples collected from four experimental ponds. The best-approximating model indicated that the per-L-sample probability of eDNA detection was 4.86 times more likely for every 2.53 fish/m3 (1 SD) increase in fish density and 1.67 times less likely for every 1.02 C (1 SD) increase in water temperature. The best section of the water column to detect eDNA was the surface and to a lesser extent the bottom. Although no false positives were detected, the estimated likely number of false positives in samples from ponds that contained fish averaged 3.62. At high densities of African jewelfish, 3-5 L of water provided a >95% probability for the presence/absence of its eDNA. Conversely, at moderate and low densities, the number of water samples necessary to achieve a >95% probability of eDNA detection approximated 42-73 and >100 L, respectively. Potential biases associated with incomplete detection of eDNA could be alleviated via formal estimation of eDNA detection probabilities under an occupancy modeling framework; alternatively, the filtration of hundreds of liters of water may be required to achieve a high (e.g., 95%) level of certainty that African jewelfish eDNA will be detected at low densities (i.e., <0.32 fish/m3 or 1.75 g/m3).

Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA

NARCIS (Netherlands)

Etson, Candice M.; Hamdan, Samir M.; Richardson, Charles C.; Oijen, Antoine M. van; Richardson, Charles C.

2010-01-01

The DNA polymerases involved in DNA replication achieve high processivity of nucleotide incorporation by forming a complex with processivity factors. A model system for replicative DNA polymerases, the bacteriophage T7 DNA polymerase (gp5), encoded by gene 5, forms a tight, 1:1 complex with

DNA sequence modeling based on context trees

NARCIS (Netherlands)

Kusters, C.J.; Ignatenko, T.; Roland, J.; Horlin, F.

2015-01-01

Genomic sequences contain instructions for protein and cell production. Therefore understanding and identification of biologically and functionally meaningful patterns in DNA sequences is of paramount importance. Modeling of DNA sequences in its turn can help to better understand and identify such

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

Directory of Open Access Journals (Sweden)

Heng Sun

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

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

Science.gov (United States)

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

2013-01-01

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

DNA-Enabled Integrated Molecular Systems for Computation and Sensing

Science.gov (United States)

2014-05-21

Computational devices can be chemically conjugated to different strands of DNA that are then self-assembled according to strict Watson − Crick binding rules... DNA -Enabled Integrated Molecular Systems for Computation and Sensing Craig LaBoda,† Heather Duschl,† and Chris L. Dwyer*,†,‡ †Department of...guided folding of DNA , inspired by nature, allows designs to manipulate molecular-scale processes unlike any other material system. Thus, DNA can be

Force-extension behavior of DNA in the presence of DNA-bending nucleoid associated proteins

Science.gov (United States)

Dahlke, K.; Sing, C. E.

2018-02-01

Interactions between nucleoid associated proteins (NAPs) and DNA affect DNA polymer conformation, leading to phenomena such as concentration dependent force-extension behavior. These effects, in turn, also impact the local binding behavior of the protein, such as high forces causing proteins to unbind, or proteins binding favorably to locally bent DNA. We develop a coarse-grained NAP-DNA simulation model that incorporates both force- and concentration-dependent behaviors, in order to study the interplay between NAP binding and DNA conformation. This model system includes multi-state protein binding and unbinding, motivated by prior work, but is now dependent on the local structure of the DNA, which is related to external forces acting on the DNA strand. We observe the expected qualitative binding behavior, where more proteins are bound at lower forces than at higher forces. Our model also includes NAP-induced DNA bending, which affects DNA elasticity. We see semi-quantitative matching of our simulated force-extension behavior to the reported experimental data. By using a coarse-grained simulation, we are also able to look at non-equilibrium behaviors, such as dynamic extension of a DNA strand. We stretch a DNA strand at different rates and at different NAP concentrations to observe how the time scales of the system (such as pulling time and unbinding time) work in concert. When these time scales are similar, we observe measurable rate-dependent changes in the system, which include the number of proteins bound and the force required to extend the DNA molecule. This suggests that the relative time scales of different dynamic processes play an important role in the behavior of NAP-DNA systems.

DNA-Based Enzyme Reactors and Systems

Directory of Open Access Journals (Sweden)

Veikko Linko

2016-07-01

Full Text Available During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications.

Heavy ion-induced lesions in DNA: A theoretical model for the initial induction of DNA strand breaks and chromatin breaks

International Nuclear Information System (INIS)

Schmidt, J.B.

1993-01-01

A theoretical model has been developed and used to calculate yields and spatial distributions of DNA strand breaks resulting from the interactions of heavy ions with chromatin in aqueous systems. The three dimensional spatial distribution of ionizing events has been modeled for charged particles as a function of charge and velocity. Chromatin has been modeled as a 30 nm diameter solenoid of nucleosomal DNA. The Monte Carlo methods used by Chatterjee et al. have been applied to DNA in a chromatin conformation. Refinements to their methods include: a combined treatment of primary and low energy (<2 keV) secondary electron interactions, an improved low energy delta ray model, and the combined simulation of direct energy deposition on the DNA and attack by diffusing hydroxyl radicals. Individual particle tracks are treated independently, which is assumed to be applicable to low fluence irradiations in which multiple particle effects are negligible. Single strand break cross section open-quotes hooksclose quotes seen in experiments at very high LET appear to be due to the collapsing radial extent of the track, as predicted in the open-quotes deep sieveclose quotes hypothesis proposed by Tobias et al. Spatial distributions of lesions produced by particles have been found to depend on chromatin structure. In the future, heavy ions may be used as a tool to probe the organization of DNA in chromatin. A Neyman A-binomial variation of the open-quotes cluster modelclose quotes for the distribution of chromatin breaks per irradiated cell has been theoretically tested. The model includes a treatment of the chromatin fragment detection technique's resolution, which places a limitation on the minimum size of fragments which can be detected. The model appears to fit some of the experimental data reasonably well. However, further experimental and theoretical refinements are desirable

Modelling DNA origami self-assembly at the domain level

Energy Technology Data Exchange (ETDEWEB)

Dannenberg, Frits; Kwiatkowska, Marta [Department of Computer Science, University of Oxford, Wolfson Building, Parks Road, Oxford OX1 3QD (United Kingdom); Dunn, Katherine E. [Department of Physics, University of Oxford, Clarendon Laboratory, Parks Road, Oxford OX1 3PU (United Kingdom); Department of Electronics, University of York, York YO10 5DD (United Kingdom); Bath, Jonathan; Turberfield, Andrew J. [Department of Physics, University of Oxford, Clarendon Laboratory, Parks Road, Oxford OX1 3PU (United Kingdom); Ouldridge, Thomas E. [Department of Physics, University of Oxford, Rudolf Peierls Centre for Theoretical Physics, 1 Keble Road, Oxford OX1 3NP (United Kingdom); Department of Mathematics, Imperial College, 180 Queen’s Gate, London SW7 2AZ (United Kingdom)

2015-10-28

We present a modelling framework, and basic model parameterization, for the study of DNA origami folding at the level of DNA domains. Our approach is explicitly kinetic and does not assume a specific folding pathway. The binding of each staple is associated with a free-energy change that depends on staple sequence, the possibility of coaxial stacking with neighbouring domains, and the entropic cost of constraining the scaffold by inserting staple crossovers. A rigorous thermodynamic model is difficult to implement as a result of the complex, multiply connected geometry of the scaffold: we present a solution to this problem for planar origami. Coaxial stacking of helices and entropic terms, particularly when loop closure exponents are taken to be larger than those for ideal chains, introduce interactions between staples. These cooperative interactions lead to the prediction of sharp assembly transitions with notable hysteresis that are consistent with experimental observations. We show that the model reproduces the experimentally observed consequences of reducing staple concentration, accelerated cooling, and absent staples. We also present a simpler methodology that gives consistent results and can be used to study a wider range of systems including non-planar origami.

Modelling DNA origami self-assembly at the domain level

International Nuclear Information System (INIS)

Dannenberg, Frits; Kwiatkowska, Marta; Dunn, Katherine E.; Bath, Jonathan; Turberfield, Andrew J.; Ouldridge, Thomas E.

2015-01-01

We present a modelling framework, and basic model parameterization, for the study of DNA origami folding at the level of DNA domains. Our approach is explicitly kinetic and does not assume a specific folding pathway. The binding of each staple is associated with a free-energy change that depends on staple sequence, the possibility of coaxial stacking with neighbouring domains, and the entropic cost of constraining the scaffold by inserting staple crossovers. A rigorous thermodynamic model is difficult to implement as a result of the complex, multiply connected geometry of the scaffold: we present a solution to this problem for planar origami. Coaxial stacking of helices and entropic terms, particularly when loop closure exponents are taken to be larger than those for ideal chains, introduce interactions between staples. These cooperative interactions lead to the prediction of sharp assembly transitions with notable hysteresis that are consistent with experimental observations. We show that the model reproduces the experimentally observed consequences of reducing staple concentration, accelerated cooling, and absent staples. We also present a simpler methodology that gives consistent results and can be used to study a wider range of systems including non-planar origami

Modelling DNA origami self-assembly at the domain level

Science.gov (United States)

Dannenberg, Frits; Dunn, Katherine E.; Bath, Jonathan; Kwiatkowska, Marta; Turberfield, Andrew J.; Ouldridge, Thomas E.

2015-10-01

We present a modelling framework, and basic model parameterization, for the study of DNA origami folding at the level of DNA domains. Our approach is explicitly kinetic and does not assume a specific folding pathway. The binding of each staple is associated with a free-energy change that depends on staple sequence, the possibility of coaxial stacking with neighbouring domains, and the entropic cost of constraining the scaffold by inserting staple crossovers. A rigorous thermodynamic model is difficult to implement as a result of the complex, multiply connected geometry of the scaffold: we present a solution to this problem for planar origami. Coaxial stacking of helices and entropic terms, particularly when loop closure exponents are taken to be larger than those for ideal chains, introduce interactions between staples. These cooperative interactions lead to the prediction of sharp assembly transitions with notable hysteresis that are consistent with experimental observations. We show that the model reproduces the experimentally observed consequences of reducing staple concentration, accelerated cooling, and absent staples. We also present a simpler methodology that gives consistent results and can be used to study a wider range of systems including non-planar origami.

A laboratory information management system for DNA barcoding workflows

NARCIS (Netherlands)

Vu, D.; Eberhardt, U.; Szöke, S.; Groenewald, M.; Robert, V.

2012-01-01

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

Statistical-mechanical lattice models for protein-DNA binding in chromatin

International Nuclear Information System (INIS)

Teif, Vladimir B; Rippe, Karsten

2010-01-01

Statistical-mechanical lattice models for protein-DNA binding are well established as a method to describe complex ligand binding equilibria measured in vitro with purified DNA and protein components. Recently, a new field of applications has opened up for this approach since it has become possible to experimentally quantify genome-wide protein occupancies in relation to the DNA sequence. In particular, the organization of the eukaryotic genome by histone proteins into a nucleoprotein complex termed chromatin has been recognized as a key parameter that controls the access of transcription factors to the DNA sequence. New approaches have to be developed to derive statistical-mechanical lattice descriptions of chromatin-associated protein-DNA interactions. Here, we present the theoretical framework for lattice models of histone-DNA interactions in chromatin and investigate the (competitive) DNA binding of other chromosomal proteins and transcription factors. The results have a number of applications for quantitative models for the regulation of gene expression.

DNA hosted and aligned in aqueous interstitia of a lamellar liquid crystal – a membrane–biomacromolecule interaction model system

KAUST Repository

Carlsson, Nils; Jonsson, Fabian; Wilhelmsson, L. Marcus; Nordé n, Bengt; Å kerman, Bjö rn

2013-01-01

We report that DNA molecules can be intercalated and macroscopically oriented in the aqueous interstitia of a lyotropic lamellar liquid crystal. Using UV-vis linear dichroism and fluorescence spectroscopy we show that double-stranded oligonucleotides (25 base pairs) in the water-octanoate-decanol system remain base-paired in the B conformation and are confined in two dimensions, with the helix axis preferentially parallel to the lipid bilayer surfaces but free to rotate within this plane. The degree of helix confinement and the corresponding 2-D orientation can be improved by decreasing the thickness of the water interstitia via the fraction of water in the ternary mixture. Not surprisingly, the corresponding single-stranded oligonucleotides are not aligned, with their persistence length being short in comparison to the lamellar interstitium thickness. We propose this as a model system for studying interactions of DNA-ligand complexes near a lipid bilayer membrane which we demonstrate by using dye probes that are either covalently attached to one end of the oligonucleotide or reversibly bound by intercalation between the base pairs. Three cationic dyes, all strongly bound by intercalation to DNA when free in solution, are found to not bind to DNA but to prefer the membrane surface. The covalently attached Cy5 also binds to the bilayer while Cy3 tends to end-stack to the oligonucleotide duplex. The orientation of Cy5 parallel to the membrane indicates that electrostatic surface binding predominates over insertion into the hydrophobic interior of the membrane. Anionic and zwitterionic dyes (FAM and ROX) are found to remain randomly oriented in the water between the lipid bilayer surfaces. © The Royal Society of Chemistry.

Modeling photoionization of aqueous DNA and its components.

Science.gov (United States)

Pluhařová, Eva; Slavíček, Petr; Jungwirth, Pavel

2015-05-19

Radiation damage to DNA is usually considered in terms of UVA and UVB radiation. These ultraviolet rays, which are part of the solar spectrum, can indeed cause chemical lesions in DNA, triggered by photoexcitation particularly in the UVB range. Damage can, however, be also caused by higher energy radiation, which can ionize directly the DNA or its immediate surroundings, leading to indirect damage. Thanks to absorption in the atmosphere, the intensity of such ionizing radiation is negligible in the solar spectrum at the surface of Earth. Nevertheless, such an ionizing scenario can become dangerously plausible for astronauts or flight personnel, as well as for persons present at nuclear power plant accidents. On the beneficial side, ionizing radiation is employed as means for destroying the DNA of cancer cells during radiation therapy. Quantitative information about ionization of DNA and its components is important not only for DNA radiation damage, but also for understanding redox properties of DNA in redox sensing or labeling, as well as charge migration along the double helix in nanoelectronics applications. Until recently, the vast majority of experimental and computational data on DNA ionization was pertinent to its components in the gas phase, which is far from its native aqueous environment. The situation has, however, changed for the better due to the advent of photoelectron spectroscopy in liquid microjets and its most recent application to photoionization of aqueous nucleosides, nucleotides, and larger DNA fragments. Here, we present a consistent and efficient computational methodology, which allows to accurately evaluate ionization energies and model photoelectron spectra of aqueous DNA and its individual components. After careful benchmarking, the method based on density functional theory and its time-dependent variant with properly chosen hybrid functionals and polarizable continuum solvent model provides ionization energies with accuracy of 0.2-0.3 e

The HIrisPlex-S system for eye, hair and skin colour prediction from DNA: Introduction and forensic developmental validation.

Science.gov (United States)

Chaitanya, Lakshmi; Breslin, Krystal; Zuñiga, Sofia; Wirken, Laura; Pośpiech, Ewelina; Kukla-Bartoszek, Magdalena; Sijen, Titia; Knijff, Peter de; Liu, Fan; Branicki, Wojciech; Kayser, Manfred; Walsh, Susan

2018-07-01

Forensic DNA Phenotyping (FDP), i.e. the prediction of human externally visible traits from DNA, has become a fast growing subfield within forensic genetics due to the intelligence information it can provide from DNA traces. FDP outcomes can help focus police investigations in search of unknown perpetrators, who are generally unidentifiable with standard DNA profiling. Therefore, we previously developed and forensically validated the IrisPlex DNA test system for eye colour prediction and the HIrisPlex system for combined eye and hair colour prediction from DNA traces. Here we introduce and forensically validate the HIrisPlex-S DNA test system (S for skin) for the simultaneous prediction of eye, hair, and skin colour from trace DNA. This FDP system consists of two SNaPshot-based multiplex assays targeting a total of 41 SNPs via a novel multiplex assay for 17 skin colour predictive SNPs and the previous HIrisPlex assay for 24 eye and hair colour predictive SNPs, 19 of which also contribute to skin colour prediction. The HIrisPlex-S system further comprises three statistical prediction models, the previously developed IrisPlex model for eye colour prediction based on 6 SNPs, the previous HIrisPlex model for hair colour prediction based on 22 SNPs, and the recently introduced HIrisPlex-S model for skin colour prediction based on 36 SNPs. In the forensic developmental validation testing, the novel 17-plex assay performed in full agreement with the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, as previously shown for the 24-plex assay. Sensitivity testing of the 17-plex assay revealed complete SNP profiles from as little as 63 pg of input DNA, equalling the previously demonstrated sensitivity threshold of the 24-plex HIrisPlex assay. Testing of simulated forensic casework samples such as blood, semen, saliva stains, of inhibited DNA samples, of low quantity touch (trace) DNA samples, and of artificially degraded DNA samples as well as

DNA in the Criminal Justice System: The DNA Success Story in Perspective.

Science.gov (United States)

Mapes, Anna A; Kloosterman, Ate D; de Poot, Christianne J

2015-07-01

Current figures on the efficiency of DNA as an investigative tool in criminal investigations only tell part of the story. To get the DNA success story in the right perspective, we examined all forensic reports from serious (N = 116) and high-volume crime cases (N = 2791) over the year 2011 from one police region in the Netherlands. These data show that 38% of analyzed serious crime traces (N = 384) and 17% of analyzed high-volume crime traces (N = 386) did not result in a DNA profile. Turnaround times (from crime scene to DNA report) were 66 days for traces from serious crimes and 44 days for traces from high-volume crimes. Suspects were truly identified through a match with the Offender DNA database of the Netherlands in 3% of the serious crime cases and in 1% of the high-volume crime cases. These data are important for both the forensic laboratory and the professionals in the criminal justice system to further optimize forensic DNA testing as an investigative tool. © 2015 American Academy of Forensic Sciences.

Seasonal variability in the persistence of dissolved environmental DNA (eDNA in a marine system: The role of microbial nutrient limitation.

Directory of Open Access Journals (Sweden)

Ian Salter

Full Text Available Environmental DNA (eDNA can be defined as the DNA pool recovered from an environmental sample that includes both extracellular and intracellular DNA. There has been a significant increase in the number of recent studies that have demonstrated the possibility to detect macroorganisms using eDNA. Despite the enormous potential of eDNA to serve as a biomonitoring and conservation tool in aquatic systems, there remain some important limitations concerning its application. One significant factor is the variable persistence of eDNA over natural environmental gradients, which imposes a critical constraint on the temporal and spatial scales of species detection. In the present study, a radiotracer bioassay approach was used to quantify the kinetic parameters of dissolved eDNA (d-eDNA, a component of extracellular DNA, over an annual cycle in the coastal Northwest Mediterranean. Significant seasonal variability in the biological uptake and turnover of d-eDNA was observed, the latter ranging from several hours to over one month. Maximum uptake rates of d-eDNA occurred in summer during a period of intense phosphate limitation (turnover <5 hrs. Corresponding increases in bacterial production and uptake of adenosine triphosphate (ATP demonstrated the microbial utilization of d-eDNA as an organic phosphorus substrate. Higher temperatures during summer may amplify this effect through a general enhancement of microbial metabolism. A partial least squares regression (PLSR model was able to reproduce the seasonal cycle in d-eDNA persistence and explained 60% of the variance in the observations. Rapid phosphate turnover and low concentrations of bioavailable phosphate, both indicative of phosphate limitation, were the most important parameters in the model. Abiotic factors such as pH, salinity and oxygen exerted minimal influence. The present study demonstrates significant seasonal variability in the persistence of d-eDNA in a natural marine environment that can

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.

Comparison of methods of DNA extraction for real-time PCR in a model of pleural tuberculosis.

Science.gov (United States)

Santos, Ana; Cremades, Rosa; Rodríguez, Juan Carlos; García-Pachón, Eduardo; Ruiz, Montserrat; Royo, Gloria

2010-01-01

Molecular methods have been reported to have different sensitivities in the diagnosis of pleural tuberculosis and this may in part be caused by the use of different methods of DNA extraction. Our study compares nine DNA extraction systems in an experimental model of pleural tuberculosis. An inoculum of Mycobacterium tuberculosis was added to 23 pleural liquid samples with different characteristics. DNA was subsequently extracted using nine different methods (seven manual and two automatic) for analysis with real-time PCR. Only two methods were able to detect the presence of M. tuberculosis DNA in all the samples: extraction using columns (Qiagen) and automated extraction with the TNAI system (Roche). The automatic method is more expensive, but requires less time. Almost all the false negatives were because of the difficulty involved in extracting M. tuberculosis DNA, as in general, all the methods studied are capable of eliminating inhibitory substances that block the amplification reaction. The method of M. tuberculosis DNA extraction used affects the results of the diagnosis of pleural tuberculosis by molecular methods. DNA extraction systems that have been shown to be effective in pleural liquid should be used.

Modeling spatiotemporal dynamics of DNA methylation

DEFF Research Database (Denmark)

Lövkvist, Cecilia Elisabet

into how epigenetic marks are distributed in the human genome. In the first part of the thesis, we investigate DNA methylation and maintenance of methylation patterns throughout cell division. We argue that collaborative models, those where the methylation of CpG sites depends on the methylation status...... into the game more explicitly in another type of model that speaks out the duality of the two aspects. Using statistical analysis of experimental data, this thesis further explores a link between DNA methylation and nucleosome occupancy. By comparing the patterns on promoters to regions with similar Cp...... division. The patterns of epigentic marks depend on enzymes that ensure their maintenance and introduction. Using theoretical models, this thesis proposes new mechanisms for how enzymes operate to maintain patterns of epigenetic marks. Through analysis of experimental data this work gives new insight...

21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

DNA Repair Systems

Indian Academy of Sciences (India)

DNA molecule which makes it ideal for storage and propagation of genetic information. ... of these errors are broadly referred to as DNA repair. DNA can ... changes occur in the human genome per day. ..... nails, frequent physical and mental.

DNA repair systems as targets of cadmium toxicity

International Nuclear Information System (INIS)

Giaginis, Constantinos; Gatzidou, Elisavet; Theocharis, Stamatios

2006-01-01

Cadmium (Cd) is a heavy metal and a potent carcinogen implicated in tumor development through occupational and environmental exposure. Recent evidence suggests that proteins participating in the DNA repair systems, especially in excision and mismatch repair, are sensitive targets of Cd toxicity. Cd by interfering and inhibiting these DNA repair processes might contribute to increased risk for tumor formation in humans. In the present review, the information available on the interference of Cd with DNA repair systems and their inhibition is summarized. These actions could possibly explain the indirect contribution of Cd to mutagenic effects and/or carcinogenicity

Establishment of Cre-mediated HBV recombinant cccDNA (rcccDNA) cell line for cccDNA biology and antiviral screening assays.

Science.gov (United States)

Wu, Min; Li, Jin; Yue, Lei; Bai, Lu; Li, Yaming; Chen, Jieliang; Zhang, Xiaonan; Yuan, Zhenghong

2018-04-01

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), existing in hepatocyte nuclei as a stable minichromosome, plays a central role in the life cycle of the virus and permits the persistence of infection. Despite being essential for HBV infection, little is known about the molecular mechanisms of cccDNA formation, regulation and degradation, and there is no therapeutic agents directly targeting cccDNA, fore mostly due to the lack of robust, reliable and quantifiable HBV cccDNA models. In this study, combined the Cre/loxP and sleeping beauty transposons system, we established HepG2-derived cell lines integrated with 2-60 copies of monomeric HBV genome flanked by loxP sites (HepG2-HBV/loxP). After Cre expression via adenoviral transduction, 3.3-kb recombinant cccDNA (rcccDNA) bearing a chimeric intron can be produced in the nuclei of these HepG2-HBV/loxP cells. The rcccDNA could be accurately quantified by quantitative PCR using specific primers and cccDNA pool generated in this model could be easily detected by Southern blotting using the digoxigenin probe system. We demonstrated that the rcccDNA was epigenetically organized as the natural minichromosome and served as the template supporting pgRNA transcription and viral replication. As the expression of HBV S antigen (HBsAg) is dependent on the newly generated cccDNA, HBsAg is the surrogate marker of cccDNA. Additionally, the efficacies of 3 classes of anti-HBV agents were evaluated in HepG2-HBV/loxP cells and antiviral activities with different mechanisms were confirmed. These data collectively suggested that HepG2-HBV/loxP cell system will be powerful platform for studying cccDNA related biological mechanisms and developing novel cccDNA targeting drugs. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

BOLDMirror: a global mirror system of DNA barcode data.

Science.gov (United States)

Liu, D; Liu, L; Guo, G; Wang, W; Sun, Q; Parani, M; Ma, J

2013-11-01

DNA barcoding is a novel concept for taxonomic identification using short, specific genetic markers and has been applied to study a large number of eukaryotes. The huge amount of data output generated by DNA barcoding requires well-organized information systems. Besides the Barcode of Life Data system (BOLD) established in Canada, the mirror system is also important for the international barcode of life project (iBOL). For this purpose, we developed the BOLDMirror, a global mirror system of DNA barcode data. It is open-sourced and can run on the LAMP (Linux + Apache + MySQL + PHP) environment. BOLDMirror has data synchronization, data representation and statistics modules, and also provides spaces to store user operation history. BOLDMirror can be accessed at http://www.boldmirror.net and several countries have used it to setup their site of DNA barcoding. © 2012 John Wiley & Sons Ltd.

DNA repair genes

International Nuclear Information System (INIS)

Morimyo, Mitsuoki

1995-01-01

Fission yeast S. pombe is assumed to be a good model for cloning of human DNA repair genes, because human gene is normally expressed in S. pombe and has a very similar protein sequence to yeast protein. We have tried to elucidate the DNA repair mechanisms of S. pombe as a model system for those of mammals. (J.P.N.)

Discrete persistent-chain model for protein binding on DNA.

Science.gov (United States)

Lam, Pui-Man; Zhen, Yi

2011-04-01

We describe and solve a discrete persistent-chain model of protein binding on DNA, involving an extra σ(i) at a site i of the DNA. This variable takes the value 1 or 0, depending on whether or not the site is occupied by a protein. In addition, if the site is occupied by a protein, there is an extra energy cost ɛ. For a small force, we obtain analytic expressions for the force-extension curve and the fraction of bound protein on the DNA. For higher forces, the model can be solved numerically to obtain force-extension curves and the average fraction of bound proteins as a function of applied force. Our model can be used to analyze experimental force-extension curves of protein binding on DNA, and hence deduce the number of bound proteins in the case of nonspecific binding. ©2011 American Physical Society

Torsion of DNA modeled as a heterogeneous fluctuating rod

Science.gov (United States)

Argudo, David; Purohit, Prashant K.

2014-01-01

We discuss the statistical mechanics of a heterogeneous elastic rod with bending, twisting and stretching. Our model goes beyond earlier works where only homogeneous rods were considered in the limit of high forces and long lengths. Our methods allow us to consider shorter fluctuating rods for which boundary conditions can play an important role. We use our theory to study structural transitions in torsionally constrained DNA where there is coexistence of states with different effective properties. In particular, we examine whether a newly discovered left-handed DNA conformation called L-DNA is a mixture of two known states. We also use our model to investigate the mechanical effects of the binding of small molecules to DNA. For both these applications we make experimentally falsifiable predictions.

Influence of oxygen on the repair of direct radiation damage to DNA by thiols in model systems

International Nuclear Information System (INIS)

Becker, D.; Summerfield, S.; Gillich, S.; Sevilla, M.D.

1994-01-01

Here the reactions of thiols with DNA primary radical intermediates formed after γ-irradiation of frozen (77K) anoxic and oxic solutions of DNA/thiol mixtures are investigated. Through analysis of the experimental composite spectra at each annealing temperature, the relative concentrations of individual radicals present are estimated and reaction sequences inferred. In all samples the primary DNA radical anions and cations (DNA · + and DNA · - ) are suggested to be the predominant radicals at low temperatures. In anoxic samples, TH · (5,6-dihydrothym-5-yl radical), RSSR · - and, in glutathione samples, · GSH [γ-glu-NHC(CH 2 SH) CO-gly] radicals are observed as the temperature is increased. The presence of oxygen efficiently suppresses the formation of RSSR · - and · GSH; instead, in oxic samples, O 2 · - , DNAOO · , RSOO · and RSO · are observed at higher temperatures. The photolytic conversion of RSOO · to RSO 2 · is used to verify the presence of RSOO · in γ-irradiated DNA/thiol systems and confirm that the computer analysis employed yields reasonable estimates of the relative DNAOO · and RSOO · concentrations. (Author)

Cell-free DNA in a three-dimensional spheroid cell culture model

DEFF Research Database (Denmark)

Aucamp, Janine; Calitz, Carlemi; Bronkhorst, Abel J.

2017-01-01

Background Investigating the biological functions of cell-free DNA (cfDNA) is limited by the interference of vast numbers of putative sources and causes of DNA release into circulation. Utilization of three-dimensional (3D) spheroid cell cultures, models with characteristics closer to the in vivo...... cultures can serve as effective, simplified in vivo-simulating “closed-circuit” models since putative sources of cfDNA are limited to only the targeted cells. In addition, cfDNA can also serve as an alternative or auxiliary marker for tracking spheroid growth, development and culture stability. Biological...... significance 3D cell cultures can be used to translate “closed-circuit” in vitro model research into data that is relevant for in vivo studies and clinical applications. In turn, the utilization of cfDNA during 3D culture research can optimize sample collection without affecting the stability of the growth...

[DNA Extraction from Old Bones by AutoMate Express™ System].

Science.gov (United States)

Li, B; Lü, Z

2017-08-01

To establish a method for extracting DNA from old bones by AutoMate Express™ system. Bones were grinded into powder by freeze-mill. After extraction by AutoMate Express™, DNA were amplified and genotyped by Identifiler®Plus and MinFiler™ kits. DNA were extracted from 10 old bone samples, which kept in different environments with the postmortem interval from 10 to 20 years, in 3 hours by AutoMate Express™ system. Complete STR typing results were obtained from 8 samples. AutoMate Express™ system can quickly and efficiently extract DNA from old bones, which can be applied in forensic practice. Copyright© by the Editorial Department of Journal of Forensic Medicine

Distribution of free radical products among the bases of x-irradiated DNA model systems: an ESR study

International Nuclear Information System (INIS)

Spalletta, R.A.

1984-01-01

Exposure of solid state DNA to ionizing radiation results in an ESR spectrum that has been attributed to a nonstoichiometric distribution of free radicals among the bases. At low temperatures radical cations appear to be stabilized on the purines while radical anions are stabilized on the pyrimidines. This distribution could arise from at least two different mechanisms. The first, charge transfer, involves the transfer of electrons and/or holes between stacked bases. In the second, saturation asymmetry, the free radical distribution arises from differences in the dose saturation characteristics of individual bases. The present study addresses the relative importance of charge transfer versus saturation asymmetry in the production of these population differences. Radicals formed by dissolving irradiated polycrystalline pyrimidines in aqueous solutions containing NtB or PBN spin traps were analyzed using ESR. The relative importance of the two free radical production and distribution mechanisms was assessed using DNA model systems. Saturation asymmetry plays a significant role in determining the free radical population while charge transfer was unambiguously observed in only one, the complex of dAMP and TMP. The results demonstrate that any quantitative analysis of charge transfer must take saturation asymmetry into account

Charge transfer through DNA/DNA duplexes and DNA/RNA hybrids: complex theoretical and experimental studies.

Science.gov (United States)

Kratochvílová, Irena; Vala, Martin; Weiter, Martin; Špérová, Miroslava; Schneider, Bohdan; Páv, Ondřej; Šebera, Jakub; Rosenberg, Ivan; Sychrovský, Vladimír

2013-01-01

Oligonucleotides conduct electric charge via various mechanisms and their characterization and understanding is a very important and complicated task. In this work, experimental (temperature dependent steady state fluorescence spectroscopy, time-resolved fluorescence spectroscopy) and theoretical (Density Functional Theory) approaches were combined to study charge transfer processes in short DNA/DNA and RNA/DNA duplexes with virtually equivalent sequences. The experimental results were consistent with the theoretical model - the delocalized nature of HOMO orbitals and holes, base stacking, electronic coupling and conformational flexibility formed the conditions for more effective short distance charge transfer processes in RNA/DNA hybrids. RNA/DNA and DNA/DNA charge transfer properties were strongly connected with temperature affected structural changes of molecular systems - charge transfer could be used as a probe of even tiny changes of molecular structures and settings. © 2013. Published by Elsevier B.V. All rights reserved.

LAMMPS Framework for Dynamic Bonding and an Application Modeling DNA

DEFF Research Database (Denmark)

Svaneborg, Carsten

2012-01-01

and bond types. When breaking bonds, all angular and dihedral interactions involving broken bonds are removed. The framework allows chemical reactions to be modeled, and use it to simulate a simplistic, coarse-grained DNA model. The resulting DNA dynamics illustrates the power of the present framework....

A comprehensive complex systems approach to the study and analysis of mammalian cell cycle control system in the presence of DNA damage stress.

Science.gov (United States)

Abroudi, Ali; Samarasinghe, Sandhya; Kulasiri, Don

2017-09-21

Not many models of mammalian cell cycle system exist due to its complexity. Some models are too complex and hard to understand, while some others are too simple and not comprehensive enough. Moreover, some essential aspects, such as the response of G1-S and G2-M checkpoints to DNA damage as well as the growth factor signalling, have not been investigated from a systems point of view in current mammalian cell cycle models. To address these issues, we bring a holistic perspective to cell cycle by mathematically modelling it as a complex system consisting of important sub-systems that interact with each other. This retains the functionality of the system and provides a clearer interpretation to the processes within it while reducing the complexity in comprehending these processes. To achieve this, we first update a published ODE mathematical model of cell cycle with current knowledge. Then the part of the mathematical model relevant to each sub-system is shown separately in conjunction with a diagram of the sub-system as part of this representation. The model sub-systems are Growth Factor, DNA damage, G1-S, and G2-M checkpoint signalling. To further simplify the model and better explore the function of sub-systems, they are further divided into modules. Here we also add important new modules of: chk-related rapid cell cycle arrest, p53 modules expanded to seamlessly integrate with the rapid arrest module, Tyrosine phosphatase modules that activate Cyc_Cdk complexes and play a crucial role in rapid and delay arrest at both G1-S and G2-M, Tyrosine Kinase module that is important for inactivating nuclear transport of CycB_cdk1 through Wee1 to resist M phase entry, Plk1-Related module that is crucial in activating Tyrosine phosphatases and inactivating Tyrosine kinase, and APC-Related module to show steps in CycB degradation. This multi-level systems approach incorporating all known aspects of cell cycle allowed us to (i) study, through dynamic simulation of an ODE model

Deficiency of double-strand DNA break repair does not impair Mycobacterium tuberculosis virulence in multiple animal models of infection.

Science.gov (United States)

Heaton, Brook E; Barkan, Daniel; Bongiorno, Paola; Karakousis, Petros C; Glickman, Michael S

2014-08-01

Mycobacterium tuberculosis persistence within its human host requires mechanisms to resist the effector molecules of host immunity, which exert their bactericidal effects through damaging pathogen proteins, membranes, and DNA. Substantial evidence indicates that bacterial pathogens, including M. tuberculosis, require DNA repair systems to repair the DNA damage inflicted by the host during infection, but the role of double-strand DNA break (DSB) repair systems is unclear. Double-strand DNA breaks are the most cytotoxic form of DNA damage and must be repaired for chromosome replication to proceed. M. tuberculosis elaborates three genetically distinct DSB repair systems: homologous recombination (HR), nonhomologous end joining (NHEJ), and single-strand annealing (SSA). NHEJ, which repairs DSBs in quiescent cells, may be particularly relevant to M. tuberculosis latency. However, very little information is available about the phenotype of DSB repair-deficient M. tuberculosis in animal models of infection. Here we tested M. tuberculosis strains lacking NHEJ (a Δku ΔligD strain), HR (a ΔrecA strain), or both (a ΔrecA Δku strain) in C57BL/6J mice, C3HeB/FeJ mice, guinea pigs, and a mouse hollow-fiber model of infection. We found no difference in bacterial load, histopathology, or host mortality between wild-type and DSB repair mutant strains in any model of infection. These results suggest that the animal models tested do not inflict DSBs on the mycobacterial chromosome, that other repair pathways can compensate for the loss of NHEJ and HR, or that DSB repair is not required for M. tuberculosis pathogenesis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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.

Induction of a systemic lupus erythematosus-like disease in mice by a common human anti-DNA idiotype

International Nuclear Information System (INIS)

Mendlovic, S.; Brocke, S.; Meshorer, A.; Mozes, E.; Shoenfeld, Y.; Bakimer, R.; Ben-Bassat, M.

1988-01-01

Systemic lupus erythematosus (SLE) is considered to be the quintessential autoimmune disease. It has not been possible to induce SLE in animal models by DNA immunization or by challenge with anti-DNA antibodies. The authors report a murine model of SLE-like disease induced by immunization of C3H.SW female mice with a common human monoclonal anti-DNA idiotype (16/6 idiotype). Following a booster injection with the 16/6 idiotype, high levels of murine anti-16/6 and anti-anti-16/6 antibodies (associated with anti-DNA activity) were detected in the sera of the immunized mice. Elevated titers of autoantibodies reacting with DNA, poly(I), poly(dT), ribonucleoprotein, autoantigens [Sm, SS-A (Ro), and SS-B (La)], and cardiolipin were noted. The serological findings were associated with increased erythrocyte sedimentation rate, leukopenia, proteinuria, immune complex deposition in the glomerular mesangium, and sclerosis of the glomeruli. The immune complexes in the kidneys were shown to contain the 16/6 idiotype. This experimental SLE-like model may be used to elucidate the mechanisms underlying SLE

DNA Self-Assembly and Computation Studied with a Coarse-grained Dynamic Bonded Model

DEFF Research Database (Denmark)

Svaneborg, Carsten; Fellermann, Harold; Rasmussen, Steen

2012-01-01

We utilize a coarse-grained directional dynamic bonding DNA model [C. Svaneborg, Comp. Phys. Comm. (In Press DOI:10.1016/j.cpc.2012.03.005)] to study DNA self-assembly and DNA computation. In our DNA model, a single nucleotide is represented by a single interaction site, and complementary sites can...

Ecological niche modelling and nDNA sequencing support a new, morphologically cryptic beetle species unveiled by DNA barcoding.

Science.gov (United States)

Hawlitschek, Oliver; Porch, Nick; Hendrich, Lars; Balke, Michael

2011-02-09

DNA sequencing techniques used to estimate biodiversity, such as DNA barcoding, may reveal cryptic species. However, disagreements between barcoding and morphological data have already led to controversy. Species delimitation should therefore not be based on mtDNA alone. Here, we explore the use of nDNA and bioclimatic modelling in a new species of aquatic beetle revealed by mtDNA sequence data. The aquatic beetle fauna of Australia is characterised by high degrees of endemism, including local radiations such as the genus Antiporus. Antiporus femoralis was previously considered to exist in two disjunct, but morphologically indistinguishable populations in south-western and south-eastern Australia. We constructed a phylogeny of Antiporus and detected a deep split between these populations. Diagnostic characters from the highly variable nuclear protein encoding arginine kinase gene confirmed the presence of two isolated populations. We then used ecological niche modelling to examine the climatic niche characteristics of the two populations. All results support the status of the two populations as distinct species. We describe the south-western species as Antiporus occidentalis sp.n. In addition to nDNA sequence data and extended use of mitochondrial sequences, ecological niche modelling has great potential for delineating morphologically cryptic species.

Present status of DNA repair mechanisms in uv irradiated yeast taken as a model eukaryotic system

International Nuclear Information System (INIS)

Moustacchi, E.; Waters, R.; Heude, M.; Chanet, R.

1975-01-01

The repair mechanisms of altered DNA are generally less well understood for eukaryotes than they are for prokaryotes and bacteriophages. For mammalian cell lines cultured in vitro the specific labelling of DNA has allowed the biochemical analysis of some of the steps of the repair processes whereas the determination of their genetic controls is, with a few exceptions, obviously difficult. On the other hand, with fungi and more specifically with yeast taken as a model unicellular eukaryotic system, the genetic approach has been extensively explored: radiosensitive mutants are readily detected and genetically analyzed, double and multiple mutants can be constructed and from their responses to irradiation the number of repair pathways involved can be suggested. The lack of thymidine kinase in these organisms has hampered for a certain time the biochemical analysis of repair. However, the recent isolation of yeast strains capable of taking up and incorporating thymidine 5'-monophosphate into their DNA opens new possibilities for the future. In spite of this difficulty, attempts to measure the induction and removal of uv-induced pyrimidine dimers were performed by several groups during the last three years. The two main repair pathways described for E. coli, i.e., the excision-resynthesis and post-replicative recombinational repair pathways, do exist in yeast. The existence of the former pathway is supported not only by indirect evidence but also by biochemical analysis. The rad 1 and rad 2 mutants for instance have been shown to be blocked in the excision of uv-induced pyrimidine dimers. Other loci are epistatic to rad 1 and rad 2 (rad 3 , rad 4 ) and are likely to act on this excision pathway. The genetic control of the mitochondrial response to a uv treatment involves nuclear genes and mitochondrial determinants

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.

Effects of microbial DNA on human DNA profiles generated using the PowerPlex® 16 HS system.

Science.gov (United States)

Dembinski, Gina M; Picard, Christine J

2017-11-01

Most crime scenes are not sterile and therefore may be contaminated with environmental DNA, especially if a decomposing body is found. Collecting biological evidence from this individual will yield DNA samples mixed with microbial DNA. This also becomes important if postmortem swabs are collected from sexually assaulted victims. Although genotyping kits undergo validation tests, including bacterial screens, they do not account for the diverse microbial load during decomposition. We investigated the effect of spiking human DNA samples with known concentrations of DNA from 17 microbe species associated with decomposition on DNA profiles produced using the Promega PowerPlex ® HS system. Two species, Bacillus subtilis and Mycobacterium smegmatis, produced an extraneous allele at the TPOX locus. When repeated with the PowerPlex ® Fusion kit, the extra allele no longer amplified with these two species. This experiment demonstrates that caution should be exhibited if microbial load is high and the PowerPlex ® 16HS system is used. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Chaos game representation (CGR)-walk model for DNA sequences

International Nuclear Information System (INIS)

Jie, Gao; Zhen-Yuan, Xu

2009-01-01

Chaos game representation (CGR) is an iterative mapping technique that processes sequences of units, such as nucleotides in a DNA sequence or amino acids in a protein, in order to determine the coordinates of their positions in a continuous space. This distribution of positions has two features: one is unique, and the other is source sequence that can be recovered from the coordinates so that the distance between positions may serve as a measure of similarity between the corresponding sequences. A CGR-walk model is proposed based on CGR coordinates for the DNA sequences. The CGR coordinates are converted into a time series, and a long-memory ARFIMA (p, d, q) model, where ARFIMA stands for autoregressive fractionally integrated moving average, is introduced into the DNA sequence analysis. This model is applied to simulating real CGR-walk sequence data of ten genomic sequences. Remarkably long-range correlations are uncovered in the data, and the results from these models are reasonably fitted with those from the ARFIMA (p, d, q) model. (cross-disciplinary physics and related areas of science and technology)

A new triple system DNA-Nanosilver-Berberine for cancer therapy

Science.gov (United States)

Grebinyk, Anna; Yashchuk, Valeriy; Bashmakova, Nataliya; Gryn, Dmytro; Hagemann, Tobias; Naumenko, Antonina; Kutsevol, Nataliya; Dandekar, Thomas; Frohme, Marcus

2018-03-01

The isoquinoline quaternary alkaloid Berberine possesses a variety of pharmacological properties that suggests its promising application for an anticancer delivery system design utilizing its ability to intercalate DNA. In the current work, we have investigated the effects of Berberine on the human T cell leukemia cell line in vitro. Fluorescent microscopy of leukemic cells revealed Berberine nuclear localization. The results showed that Berberine inhibited leukemic cell growth in a time- and dose-dependent manner, that was associated with reactive oxygen species production intensification and caspase 3/7 activity increase with followed apoptosis induction. Berberine was used as a toxic and phototoxic agent for triple system synthesis along with DNA as a carrier and nanosilver as a plasmonic accelerator of Berberine electronic transitions and high energy emission absorbent centers. The proposed method allows to obtain the complex of DNA with Berberine molecules and silver nanoparticles. The optical properties of free components as well as their various combinations, including the final triple system DNA-Nanosilver-Berberine, were investigated. Obtained results support the possibility to use the triple system DNA-Nanosilver-Berberine as an alternative therapeutic agent for cancer treatment.

Predictors of hepatitis B cure using gene therapy to deliver DNA cleavage enzymes: a mathematical modeling approach.

Directory of Open Access Journals (Sweden)

Joshua T Schiffer

Full Text Available Most chronic viral infections are managed with small molecule therapies that inhibit replication but are not curative because non-replicating viral forms can persist despite decades of suppressive treatment. There are therefore numerous strategies in development to eradicate all non-replicating viruses from the body. We are currently engineering DNA cleavage enzymes that specifically target hepatitis B virus covalently closed circular DNA (HBV cccDNA, the episomal form of the virus that persists despite potent antiviral therapies. DNA cleavage enzymes, including homing endonucleases or meganucleases, zinc-finger nucleases (ZFNs, TAL effector nucleases (TALENs, and CRISPR-associated system 9 (Cas9 proteins, can disrupt specific regions of viral DNA. Because DNA repair is error prone, the virus can be neutralized after repeated cleavage events when a target sequence becomes mutated. DNA cleavage enzymes will be delivered as genes within viral vectors that enter hepatocytes. Here we develop mathematical models that describe the delivery and intracellular activity of DNA cleavage enzymes. Model simulations predict that high vector to target cell ratio, limited removal of delivery vectors by humoral immunity, and avid binding between enzyme and its DNA target will promote the highest level of cccDNA disruption. Development of de novo resistance to cleavage enzymes may occur if DNA cleavage and error prone repair does not render the viral episome replication incompetent: our model predicts that concurrent delivery of multiple enzymes which target different vital cccDNA regions, or sequential delivery of different enzymes, are both potentially useful strategies for avoiding multi-enzyme resistance. The underlying dynamics of cccDNA persistence are unlikely to impact the probability of cure provided that antiviral therapy is given concurrently during eradication trials. We conclude by describing experiments that can be used to validate the model, which

DNA computing models

CERN Document Server

Ignatova, Zoya; Zimmermann, Karl-Heinz

2008-01-01

In this excellent text, the reader is given a comprehensive introduction to the field of DNA computing. The book emphasizes computational methods to tackle central problems of DNA computing, such as controlling living cells, building patterns, and generating nanomachines.

Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders.

Science.gov (United States)

Lorenz, Carmen; Lesimple, Pierre; Bukowiecki, Raul; Zink, Annika; Inak, Gizem; Mlody, Barbara; Singh, Manvendra; Semtner, Marcus; Mah, Nancy; Auré, Karine; Leong, Megan; Zabiegalov, Oleksandr; Lyras, Ekaterini-Maria; Pfiffer, Vanessa; Fauler, Beatrix; Eichhorst, Jenny; Wiesner, Burkhard; Huebner, Norbert; Priller, Josef; Mielke, Thorsten; Meierhofer, David; Izsvák, Zsuzsanna; Meier, Jochen C; Bouillaud, Frédéric; Adjaye, James; Schuelke, Markus; Wanker, Erich E; Lombès, Anne; Prigione, Alessandro

2017-05-04

Mitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Modeling of these defects has been difficult because of the challenges associated with engineering mtDNA. We show here that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) retain the parental mtDNA profile and exhibit a metabolic switch toward oxidative phosphorylation. NPCs derived in this way from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) showed defective ATP production and abnormally high mitochondrial membrane potential (MMP), plus altered calcium homeostasis, which represents a potential cause of neural impairment. High-content screening of FDA-approved drugs using the MMP phenotype highlighted avanafil, which we found was able to partially rescue the calcium defect in patient NPCs and differentiated neurons. Overall, our results show that iPSC-derived NPCs provide an effective model for drug screening to target mtDNA disorders that affect the nervous system. Copyright © 2016 Elsevier Inc. All rights reserved.

Mesoscopic modeling of DNA denaturation rates: Sequence dependence and experimental comparison

Energy Technology Data Exchange (ETDEWEB)

Dahlen, Oda, E-mail: oda.dahlen@ntnu.no; Erp, Titus S. van, E-mail: titus.van.erp@ntnu.no [Department of Chemistry, Norwegian University of Science and Technology (NTNU), Høgskoleringen 5, Realfagbygget D3-117 7491 Trondheim (Norway)

2015-06-21

Using rare event simulation techniques, we calculated DNA denaturation rate constants for a range of sequences and temperatures for the Peyrard-Bishop-Dauxois (PBD) model with two different parameter sets. We studied a larger variety of sequences compared to previous studies that only consider DNA homopolymers and DNA sequences containing an equal amount of weak AT- and strong GC-base pairs. Our results show that, contrary to previous findings, an even distribution of the strong GC-base pairs does not always result in the fastest possible denaturation. In addition, we applied an adaptation of the PBD model to study hairpin denaturation for which experimental data are available. This is the first quantitative study in which dynamical results from the mesoscopic PBD model have been compared with experiments. Our results show that present parameterized models, although giving good results regarding thermodynamic properties, overestimate denaturation rates by orders of magnitude. We believe that our dynamical approach is, therefore, an important tool for verifying DNA models and for developing next generation models that have higher predictive power than present ones.

High Efficiency Hydrodynamic DNA Fragmentation in a Bubbling System.

Science.gov (United States)

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

2017-01-18

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

DNA methylation regulates transcriptional homeostasis of algal endosymbiosis in the coral model Aiptasia

KAUST Repository

Li, Yong

2017-11-03

The symbiotic relationship between cnidarians and dinoflagellates is the cornerstone of coral reef ecosystems. Although research is focusing on the molecular mechanisms underlying this symbiosis, the role of epigenetic mechanisms, which have been implicated in transcriptional regulation and acclimation to environmental change, is unknown. To assess the role of DNA methylation in the cnidarian-dinoflagellate symbiosis, we analyzed genome-wide CpG methylation, histone associations, and transcriptomic states of symbiotic and aposymbiotic anemones in the model system Aiptasia. We find methylated genes are marked by histone H3K36me3 and show significant reduction of spurious transcription and transcriptional noise, revealing a role of DNA methylation in the maintenance of transcriptional homeostasis. Changes in DNA methylation and expression show enrichment for symbiosis-related processes such as immunity, apoptosis, phagocytosis recognition and phagosome formation, and unveil intricate interactions between the underlying pathways. Our results demonstrate that DNA methylation provides an epigenetic mechanism of transcriptional homeostasis during symbiosis.

DNA methylation regulates transcriptional homeostasis of algal endosymbiosis in the coral model Aiptasia

KAUST Repository

Li, Yong; Liew, Yi Jin; Cui, Guoxin; Cziesielski, Maha J; Zahran, Noura Ibrahim Omar; Michell, Craig T; Voolstra, Christian R.; Aranda, Manuel

2017-01-01

The symbiotic relationship between cnidarians and dinoflagellates is the cornerstone of coral reef ecosystems. Although research is focusing on the molecular mechanisms underlying this symbiosis, the role of epigenetic mechanisms, which have been implicated in transcriptional regulation and acclimation to environmental change, is unknown. To assess the role of DNA methylation in the cnidarian-dinoflagellate symbiosis, we analyzed genome-wide CpG methylation, histone associations, and transcriptomic states of symbiotic and aposymbiotic anemones in the model system Aiptasia. We find methylated genes are marked by histone H3K36me3 and show significant reduction of spurious transcription and transcriptional noise, revealing a role of DNA methylation in the maintenance of transcriptional homeostasis. Changes in DNA methylation and expression show enrichment for symbiosis-related processes such as immunity, apoptosis, phagocytosis recognition and phagosome formation, and unveil intricate interactions between the underlying pathways. Our results demonstrate that DNA methylation provides an epigenetic mechanism of transcriptional homeostasis during symbiosis.

Interaction of bacteriophage T4 and T7 single-stranded DNA-binding proteins with DNA

International Nuclear Information System (INIS)

Shokri, Leila; Williams, Mark C; Rouzina, Ioulia

2009-01-01

Bacteriophages T4 and T7 are well-studied model replication systems, which have allowed researchers to determine the roles of many proteins central to DNA replication, recombination and repair. Here we summarize and discuss the results from two recently developed single-molecule methods to determine the salt-dependent DNA-binding kinetics and thermodynamics of the single-stranded DNA (ssDNA)-binding proteins (SSBs) from these systems. We use these methods to characterize both the equilibrium double-stranded DNA (dsDNA) and ssDNA binding of the SSBs T4 gene 32 protein (gp32) and T7 gene 2.5 protein (gp2.5). Despite the overall two-orders-of-magnitude weaker binding of gp2.5 to both forms of DNA, we find that both proteins exhibit four-orders-of-magnitude preferential binding to ssDNA relative to dsDNA. This strong preferential ssDNA binding as well as the weak dsDNA binding is essential for the ability of both proteins to search dsDNA in one dimension to find available ssDNA-binding sites at the replication fork

Charge transfer in pi-stacked systems including DNA

International Nuclear Information System (INIS)

Siebbeles, L.D.A.

2003-01-01

Charge migration in DNA is a subject of intense current study motivated by long-range detection of DNA damage and the potential application of DNA as a molecular wire in nanoscale electronic devices. A key structural element, which makes DNA a medium for long-range charge transfer, is the array of stacked base pairs in the interior of the double helix. The overlapping pi-orbitals of the nucleobases provide a pathway for motion of charge carriers generated on the stack. This 'pi-pathway' resembles the columnarly stacked macrocyclic cores in discotic materials such as triphenylenes. The structure of these pi-stacked systems is highly disordered with dynamic fluctuations occurring on picosecond to nanosecond time scales. Theoretical calculations, concerning the effects of structural disorder and nucleobase sequence in DNA, on the dynamics of charge carriers are presented. Electronic couplings and localization energies of charge carriers were calculated using density functional theory (DFT). Results for columnarly stacked triphenylenes and DNA nucleobases are compared. The results are used to provide insight into the factors that control the mobility of charge carriers. Further, experimental results on the site-selective oxidation of guanine nucleobases in DNA (hot spots for DNA damage) are analyzed on basis of the theoretical results

Botanical DNA evidence in criminal cases: Knotgrass (Polygonum aviculare L.) as a model species

NARCIS (Netherlands)

Koopman, W.J.M.; Kuiper, I.; Klein Geltink, D.J.A.; Sabatino, G.J.H.; Smulders, M.J.M.

2012-01-01

The possibilities and strategies for using DNA characteristics to link a botanical sample to a specific source plant or location vary with its breeding system. For inbreeding species, which often form small patches of identical genotypes, knotgrass (Polygonum aviculare L.) is a suitable model

Twist-writhe partitioning in a coarse-grained DNA minicircle model

Science.gov (United States)

Sayar, Mehmet; Avşaroǧlu, Barış; Kabakçıoǧlu, Alkan

2010-04-01

Here we present a systematic study of supercoil formation in DNA minicircles under varying linking number by using molecular-dynamics simulations of a two-bead coarse-grained model. Our model is designed with the purpose of simulating long chains without sacrificing the characteristic structural properties of the DNA molecule, such as its helicity, backbone directionality, and the presence of major and minor grooves. The model parameters are extracted directly from full-atomistic simulations of DNA oligomers via Boltzmann inversion; therefore, our results can be interpreted as an extrapolation of those simulations to presently inaccessible chain lengths and simulation times. Using this model, we measure the twist/writhe partitioning in DNA minicircles, in particular its dependence on the chain length and excess linking number. We observe an asymmetric supercoiling transition consistent with experiments. Our results suggest that the fraction of the linking number absorbed as twist and writhe is nontrivially dependent on chain length and excess linking number. Beyond the supercoiling transition, chains of the order of one persistence length carry equal amounts of twist and writhe. For longer chains, an increasing fraction of the linking number is absorbed by the writhe.

Integration of Principles of Systems Biology and Radiation Biology: Toward Development of in silico Models to Optimize IUdR-Mediated Radiosensitization of DNA Mismatch Repair Deficient (Damage Tolerant) Human Cancers

International Nuclear Information System (INIS)

Kinsella, Timothy J.; Gurkan-Cavusoglu, Evren; Du, Weinan; Loparo, Kenneth A.

2011-01-01

Over the last 7 years, we have focused our experimental and computational research efforts on improving our understanding of the biochemical, molecular, and cellular processing of iododeoxyuridine (IUdR) and ionizing radiation (IR) induced DNA base damage by DNA mismatch repair (MMR). These coordinated research efforts, sponsored by the National Cancer Institute Integrative Cancer Biology Program (ICBP), brought together system scientists with expertise in engineering, mathematics, and complex systems theory and translational cancer researchers with expertise in radiation biology. Our overall goal was to begin to develop computational models of IUdR- and/or IR-induced base damage processing by MMR that may provide new clinical strategies to optimize IUdR-mediated radiosensitization in MMR deficient (MMR − ) “damage tolerant” human cancers. Using multiple scales of experimental testing, ranging from purified protein systems to in vitro (cellular) and to in vivo (human tumor xenografts in athymic mice) models, we have begun to integrate and interpolate these experimental data with hybrid stochastic biochemical models of MMR damage processing and probabilistic cell cycle regulation models through a systems biology approach. In this article, we highlight the results and current status of our integration of radiation biology approaches and computational modeling to enhance IUdR-mediated radiosensitization in MMR − damage tolerant cancers.

Cascade of chromosomal rearrangements caused by a heterogeneous T-DNA integration supports the double-stranded break repair model for T-DNA integration.

Science.gov (United States)

Hu, Yufei; Chen, Zhiyu; Zhuang, Chuxiong; Huang, Jilei

2017-06-01

Transferred DNA (T-DNA) from Agrobacterium tumefaciens can be integrated into the plant genome. The double-stranded break repair (DSBR) pathway is a major model for T-DNA integration. From this model, we expect that two ends of a T-DNA molecule would invade into a single DNA double-stranded break (DSB) or independent DSBs in the plant genome. We call the later phenomenon a heterogeneous T-DNA integration, which has never been observed. In this work, we demonstrated it in an Arabidopsis T-DNA insertion mutant seb19. To resolve the chromosomal structural changes caused by T-DNA integration at both the nucleotide and chromosome levels, we performed inverse PCR, genome resequencing, fluorescence in situ hybridization and linkage analysis. We found, in seb19, a single T-DNA connected two different chromosomal loci and caused complex chromosomal rearrangements. The specific break-junction pattern in seb19 is consistent with the result of heterogeneous T-DNA integration but not of recombination between two T-DNA insertions. We demonstrated that, in seb19, heterogeneous T-DNA integration evoked a cascade of incorrect repair of seven DSBs on chromosomes 4 and 5, and then produced translocation, inversion, duplication and deletion. Heterogeneous T-DNA integration supports the DSBR model and suggests that two ends of a T-DNA molecule could be integrated into the plant genome independently. Our results also show a new origin of chromosomal abnormalities. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System.

Directory of Open Access Journals (Sweden)

Tina Y Liu

Full Text Available CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus Type III-A Csm complex (TthCsm with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (5' tag of the crRNA and the 3' flanking region of the ssRNA. Mutation of the histidine-aspartate (HD nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryo-electron microscopy and x-ray crystallography.

RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System.

Science.gov (United States)

Liu, Tina Y; Iavarone, Anthony T; Doudna, Jennifer A

2017-01-01

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA) to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus) Type III-A Csm complex (TthCsm) with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA) by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (5' tag) of the crRNA and the 3' flanking region of the ssRNA. Mutation of the histidine-aspartate (HD) nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA) during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryo-electron microscopy and x-ray crystallography.

New applications of CRISPR/Cas9 system on mutant DNA detection.

Science.gov (United States)

Jia, Chenqiang; Huai, Cong; Ding, Jiaqi; Hu, Lingna; Su, Bo; Chen, Hongyan; Lu, Daru

2018-01-30

The detection of mutant DNA is critical for precision medicine, but low-frequency DNA mutation is very hard to be determined. CRISPR/Cas9 is a robust tool for in vivo gene editing, and shows the potential for precise in vitro DNA cleavage. Here we developed a DNA mutation detection system based on CRISPR/Cas9 that can detect gene mutation efficiently even in a low-frequency condition. The system of CRISPR/Cas9 cleavage in vitro showed a high accuracy similar to traditional T7 endonuclease I (T7E1) assay in estimating mutant DNA proportion in the condition of normal frequency. The technology was further used for low-frequency mutant DNA detection of EGFR and HBB somatic mutations. To the end, Cas9 was employed to cleave the wild-type (WT) DNA and to enrich the mutant DNA. Using amplified fragment length polymorphism analysis (AFLPA) and Sanger sequencing, we assessed the sensitivity of CRISPR/Cas9 cleavage-based PCR, in which mutations at 1%-10% could be enriched and detected. When combined with blocker PCR, its sensitivity reached up to 0.1%. Our results suggested that this new application of CRISPR/Cas9 system is a robust and potential method for heterogeneous specimens in the clinical diagnosis and treatment management. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Directory of Open Access Journals (Sweden)

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.

Quantifying DNA melting transitions using single-molecule force spectroscopy

International Nuclear Information System (INIS)

Calderon, Christopher P; Chen, W-H; Harris, Nolan C; Kiang, C-H; Lin, K-J

2009-01-01

We stretched a DNA molecule using an atomic force microscope (AFM) and quantified the mechanical properties associated with B and S forms of double-stranded DNA (dsDNA), molten DNA, and single-stranded DNA. We also fit overdamped diffusion models to the AFM time series and used these models to extract additional kinetic information about the system. Our analysis provides additional evidence supporting the view that S-DNA is a stable intermediate encountered during dsDNA melting by mechanical force. In addition, we demonstrated that the estimated diffusion models can detect dynamical signatures of conformational degrees of freedom not directly observed in experiments.

Quantifying DNA melting transitions using single-molecule force spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Calderon, Christopher P [Department of Computational and Applied Mathematics, Rice University, Houston, TX (United States); Chen, W-H; Harris, Nolan C; Kiang, C-H [Department of Physics and Astronomy, Rice University, Houston, TX (United States); Lin, K-J [Department of Chemistry, National Chung Hsing University, Taichung, Taiwan (China)], E-mail: chkiang@rice.edu

2009-01-21

We stretched a DNA molecule using an atomic force microscope (AFM) and quantified the mechanical properties associated with B and S forms of double-stranded DNA (dsDNA), molten DNA, and single-stranded DNA. We also fit overdamped diffusion models to the AFM time series and used these models to extract additional kinetic information about the system. Our analysis provides additional evidence supporting the view that S-DNA is a stable intermediate encountered during dsDNA melting by mechanical force. In addition, we demonstrated that the estimated diffusion models can detect dynamical signatures of conformational degrees of freedom not directly observed in experiments.

DNA Repair Systems

Indian Academy of Sciences (India)

Thanks to the pioneering research work of Lindahl, Sancar, Modrich and their colleagues, we now have an holistic awareness of how DNA damage occurs and how the damage is rectified in bacteria as well as in higher organisms including human beings. A comprehensive understanding of DNA repair has proven crucial ...

Quantification of Cooperativity in Heterodimer-DNA Binding Improves the Accuracy of Binding Specificity Models*

Science.gov (United States)

Isakova, Alina; Berset, Yves; Hatzimanikatis, Vassily; Deplancke, Bart

2016-01-01

Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ):retinoid X receptor α (RXRα) heterodimer. Using the high throughput MITOMI (mechanically induced trapping of molecular interactions) platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer-DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers. PMID:26912662

Quantification of Cooperativity in Heterodimer-DNA Binding Improves the Accuracy of Binding Specificity Models.

Science.gov (United States)

Isakova, Alina; Berset, Yves; Hatzimanikatis, Vassily; Deplancke, Bart

2016-05-06

Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ):retinoid X receptor α (RXRα) heterodimer. Using the high throughput MITOMI (mechanically induced trapping of molecular interactions) platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer-DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A Comparison Study for DNA Motif Modeling on Protein Binding Microarray

KAUST Repository

Wong, Ka-Chun; Li, Yue; Peng, Chengbin; Wong, Hau-San

2015-01-01

Transcription Factor Binding Sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, Protein Binding Microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k=810). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build motif models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement using di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

A Comparison Study for DNA Motif Modeling on Protein Binding Microarray

KAUST Repository

Wong, Ka-Chun

2015-06-11

Transcription Factor Binding Sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, Protein Binding Microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k=810). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build motif models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement using di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

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.

Evaluation of DNA bending models in their capacity to predict electrophoretic migration anomalies of satellite DNA sequences.

Science.gov (United States)

Matyášek, Roman; Fulneček, Jaroslav; Kovařík, Aleš

2013-09-01

DNA containing a sequence that generates a local curvature exhibits a pronounced retardation in electrophoretic mobility. Various theoretical models have been proposed to explain relationship between DNA structural features and migration anomaly. Here, we studied the capacity of 15 static wedge-bending models to predict electrophoretic behavior of 69 satellite monomers derived from four divergent families. All monomers exhibited retarded mobility in PAGE corresponding to retardation factors ranging 1.02-1.54. The curvature varied both within and across the groups and correlated with the number, position, and lengths of A-tracts. Two dinucleotide models provided strong correlation between gel mobility and curvature prediction; two trinucleotide models were satisfactory while remaining dinucleotide models provided intermediate results with reliable prediction for subsets of sequences only. In some cases, similarly shaped molecules exhibited relatively large differences in mobility and vice versa. Generally less accurate predictions were obtained in groups containing less homogeneous sequences possessing distinct structural features. In conclusion, relatively universal theoretical models were identified suitable for the analysis of natural sequences known to harbor relatively moderate curvature. These models could be potentially applied to genome wide studies. However, in silico predictions should be viewed in context of experimental measurement of intrinsic DNA curvature. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Model of biological quantum logic in DNA.

Science.gov (United States)

Mihelic, F Matthew

2013-08-02

The DNA molecule has properties that allow it to act as a quantum logic processor. It has been demonstrated that there is coherent conduction of electrons longitudinally along the DNA molecule through pi stacking interactions of the aromatic nucleotide bases, and it has also been demonstrated that electrons moving longitudinally along the DNA molecule are subject to a very efficient electron spin filtering effect as the helicity of the DNA molecule interacts with the spin of the electron. This means that, in DNA, electrons are coherently conducted along a very efficient spin filter. Coherent electron spin is held in a logically and thermodynamically reversible chiral symmetry between the C2-endo and C3-endo enantiomers of the deoxyribose moiety in each nucleotide, which enables each nucleotide to function as a quantum gate. The symmetry break that provides for quantum decision in the system is determined by the spin direction of an electron that has an orbital angular momentum that is sufficient to overcome the energy barrier of the double well potential separating the C2-endo and C3-endo enantiomers, and that enantiomeric energy barrier is appropriate to the Landauer limit of the energy necessary to randomize one bit of information.

DNA accumulation on ventilation system filters in university buildings in Singapore.

Science.gov (United States)

Luhung, Irvan; Wu, Yan; Xu, Siyu; Yamamoto, Naomichi; Chang, Victor Wei-Chung; Nazaroff, William W

2017-01-01

Biological particles deposit on air handling system filters as they process air. This study reports and interprets abundance and diversity information regarding biomass accumulation on ordinarily used filters acquired from several locations in a university environment. DNA-based analysis was applied both to quantify (via DNA fluorometry and qPCR) and to characterize (via high-throughput sequencing) the microbial material on filters, which mainly processed recirculated indoor air. Results were interpreted in relation to building occupancy and ventilation system operational parameters. Based on accumulated biomass, average DNA concentrations per AHU filter surface area across nine indoor locations after twelve weeks of filter use were in the respective ranges 1.1 to 41 ng per cm2 for total DNA, 0.02 to 3.3 ng per cm2 for bacterial DNA and 0.2 to 2.0 ng DNA per cm2 for fungal DNA. The most abundant genera detected on the AHU filter samples were Clostridium, Streptophyta, Bacillus, Acinetobacter and Ktedonobacter for bacteria and Aspergillus, Cladosporium, Nigrospora, Rigidoporus and Lentinus for fungi. Conditional indoor airborne DNA concentrations (median (range)) were estimated to be 13 (2.6-107) pg/m3 for total DNA, 0.4 (0.05-8.4) pg/m3 for bacterial DNA and 2.3 (1.0-5.1) pg/m3 for fungal DNA. Conditional airborne concentrations and the relative abundances of selected groups of genera correlate well with occupancy level. Bacterial DNA was found to be more responsive than fungal DNA to differences in occupancy level and indoor environmental conditions.

A model for the mechanism of strand passage by DNA gyrase

DEFF Research Database (Denmark)

Kampranis, S C; Bates, A D; Maxwell, A

1999-01-01

this mechanism by probing the topology of the bound DNA segment at distinct steps of the catalytic cycle. We propose a model in which gyrase captures a contiguous DNA segment with high probability, irrespective of the superhelical density of the DNA substrate, setting up an equilibrium of the transported segment......The mechanism of type II DNA topoisomerases involves the formation of an enzyme-operated gate in one double-stranded DNA segment and the passage of another segment through this gate. DNA gyrase is the only type II topoisomerase able to introduce negative supercoils into DNA, a feature that requires...... the enzyme to dictate the directionality of strand passage. Although it is known that this is a consequence of the characteristic wrapping of DNA by gyrase, the detailed mechanism by which the transported DNA segment is captured and directed through the DNA gate is largely unknown. We have addressed...

Design and construction of a DNA origami drug delivery system based on MPT64 antibody aptamer for tuberculosis treatment.

Science.gov (United States)

Ranjbar, Reza; Hafezi-Moghadam, Mohammad Sadegh

2016-02-01

With all of the developments on infectious diseases, tuberculosis (TB) remains a cause of death among people. One of the most promising assembly techniques in nano-technology is "scaffolded DNA origami" to design and construct a nano-scale drug delivery system. Because of the global health problems of tuberculosis, the development of potent new anti-tuberculosis drug delivery system without cross-resistance with known anti-mycobacterial agents is urgently needed. The aim of this study was to design a nano-scale drug delivery system for TB treatment using the DNA origami method. In this study, we presented an experimental research on a DNA drug delivery system for treating Tuberculosis. TEM images were visualized with an FEI Tecnai T12 BioTWIN at 120 kV. The model was designed by caDNAno software and computational prediction of the 3D solution shape and its flexibility was calculated with a CanDo server. Synthesizing the product was imaged using transmission electron microscopy after negative-staining by uranyl formate. We constructed a multilayer 3D DNA nanostructure system by designing square lattice geometry with the scaffolded-DNA-origami method. With changes in the lock and key sequences, we recommend that this system be used for other infectious diseases to target the pathogenic bacteria.

A Rewritable, Random-Access DNA-Based Storage System.

Science.gov (United States)

Yazdi, S M Hossein Tabatabaei; Yuan, Yongbo; Ma, Jian; Zhao, Huimin; Milenkovic, Olgica

2015-09-18

We describe the first DNA-based storage architecture that enables random access to data blocks and rewriting of information stored at arbitrary locations within the blocks. The newly developed architecture overcomes drawbacks of existing read-only methods that require decoding the whole file in order to read one data fragment. Our system is based on new constrained coding techniques and accompanying DNA editing methods that ensure data reliability, specificity and sensitivity of access, and at the same time provide exceptionally high data storage capacity. As a proof of concept, we encoded parts of the Wikipedia pages of six universities in the USA, and selected and edited parts of the text written in DNA corresponding to three of these schools. The results suggest that DNA is a versatile media suitable for both ultrahigh density archival and rewritable storage applications.

Assessment of evidence for nanosized titanium dioxide-generated DNA strand breaks and oxidatively damaged DNA in cells and animal models

DEFF Research Database (Denmark)

Møller, Peter; Jensen, Ditte Marie; Wils, Regitze Sølling

2017-01-01

Nanosized titanium dioxide (TiO2) has been investigated in numerous studies on genotoxicity, including comet assay endpoints and oxidatively damaged DNA in cell cultures and animal models. The results have been surprisingly mixed, which might be attributed to physico-chemical differences...... culture studies also demonstrate increased levels of oxidatively damaged DNA after exposure to TiO2. There are relatively few studies on animal models where DNA strand breaks and oxidatively damaged DNA have been tested with reliable methods. Collectively, this review shows that exposure to nanosized TiO2...... of the tested TiO2. In the present review, we assess the role of certain methodological issues and publication bias. The analysis shows that studies on DNA strand breaks without proper assay controls or very low intra-group variation tend to show statistically significant effects. Levels of oxidatively damaged...

DNA-Inspired Online Behavioral Modeling and Its Application to Spambot Detection

DEFF Research Database (Denmark)

Cresci, Stefano; Di Pietro, Roberto; Petrocchi, Marinella

2016-01-01

A novel, simple, and effective approach to modeling online user behavior extracts and analyzes digital DNA sequences from user online actions and uses Twitter as a benchmark to test the proposal. Specifically, the model obtains an incisive and compact DNA-inspired characterization of user actions...... methodology is platform and technology agnostic, paving the way for diverse behavioral characterization tasks....

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.

Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks.

Science.gov (United States)

Uematsu, Naoya; Weterings, Eric; Yano, Ken-ichi; Morotomi-Yano, Keiko; Jakob, Burkhard; Taucher-Scholz, Gisela; Mari, Pierre-Olivier; van Gent, Dik C; Chen, Benjamin P C; Chen, David J

2007-04-23

The DNA-dependent protein kinase catalytic subunit (DNA-PK(CS)) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PK(CS) recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PK(CS) accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PK(CS) influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PK(CS) at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PK(CS) influence the stability of its binding to DNA ends. We suggest a model in which DNA-PK(CS) phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PK(CS) with the DNA ends.

Optimization of DNA Sensor Model Based Nanostructured Graphene Using Particle Swarm Optimization Technique

Directory of Open Access Journals (Sweden)

Hediyeh Karimi

2013-01-01

Full Text Available It has been predicted that the nanomaterials of graphene will be among the candidate materials for postsilicon electronics due to their astonishing properties such as high carrier mobility, thermal conductivity, and biocompatibility. Graphene is a semimetal zero gap nanomaterial with demonstrated ability to be employed as an excellent candidate for DNA sensing. Graphene-based DNA sensors have been used to detect the DNA adsorption to examine a DNA concentration in an analyte solution. In particular, there is an essential need for developing the cost-effective DNA sensors holding the fact that it is suitable for the diagnosis of genetic or pathogenic diseases. In this paper, particle swarm optimization technique is employed to optimize the analytical model of a graphene-based DNA sensor which is used for electrical detection of DNA molecules. The results are reported for 5 different concentrations, covering a range from 0.01 nM to 500 nM. The comparison of the optimized model with the experimental data shows an accuracy of more than 95% which verifies that the optimized model is reliable for being used in any application of the graphene-based DNA sensor.

Administration of the optimized β-Lapachone-poloxamer-cyclodextrin ternary system induces apoptosis, DNA damage and reduces tumor growth in a human breast adenocarcinoma xenograft mouse model.

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Seoane, Samuel; Díaz-Rodríguez, Patricia; Sendon-Lago, Juan; Gallego, Rosalia; Pérez-Fernández, Román; Landin, Mariana

2013-08-01

β-Lapachone (β-Lap) is a 1,2-orthonaphthoquinone that selectively induces cell death in human cancer cells through NAD(P)H:quinone oxidoreductase-1 (NQO1). NQO1 is overexpressed in a variety of tumors, as compared to normal adjacent tissue. However, the low solubility and non-specific distribution of β-Lap limit its suitability for clinical assays. We formulated β-Lap in an optimal random methylated-β-cyclodextrin/poloxamer 407 mixture (i.e., β-Lap ternary system) and, using human breast adenocarcinoma MCF-7 cells and immunodeficient mice, performed in vitro and in vivo evaluation of its anti-tumor effects on proliferation, cell cycle, apoptosis, DNA damage, and tumor growth. This ternary system is fluid at room temperature, gels over 29 °C, and provides a significant amount of drug, thus facilitating intratumoral delivery, in situ gelation, and the formation of a depot for time-release. Administration of β-Lap ternary system to MCF-7 cells induces an increase in apoptosis and DNA damage, while producing no changes in cell cycle. Moreover, in a mouse xenograft tumor model, intratumoral injection of the system significantly reduces tumor volume, while increasing apoptosis and DNA damage without visible toxicity to liver or kidney. These anti-tumoral effects and lack of visible toxicity make this system a promising new therapeutic agent for breast cancer treatment. Copyright © 2013 Elsevier B.V. All rights reserved.

The validation of forensic DNA extraction systems to utilize soil contaminated biological evidence.

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Kasu, Mohaimin; Shires, Karen

2015-07-01

The production of full DNA profiles from biological evidence found in soil has a high failure rate due largely to the inhibitory substance humic acid (HA). Abundant in various natural soils, HA co-extracts with DNA during extraction and inhibits DNA profiling by binding to the molecular components of the genotyping assay. To successfully utilize traces of soil contaminated evidence, such as that found at many murder and rape crime scenes in South Africa, a reliable HA removal extraction system would often be selected based on previous validation studies. However, for many standard forensic DNA extraction systems, peer-reviewed publications detailing the efficacy on soil evidence is either lacking or is incomplete. Consequently, these sample types are often not collected or fail to yield suitable DNA material due to the use of unsuitable methodology. The aim of this study was to validate the common forensic DNA collection and extraction systems used in South Africa, namely DNA IQ, FTA elute and Nucleosave for processing blood and saliva contaminated with HA. A forensic appropriate volume of biological evidence was spiked with HA (0, 0.5, 1.5 and 2.5 mg/ml) and processed through each extraction protocol for the evaluation of HA removal using QPCR and STR-genotyping. The DNA IQ magnetic bead system effectively removed HA from highly contaminated blood and saliva, and generated consistently acceptable STR profiles from both artificially spiked samples and crude soil samples. This system is highly recommended for use on soil-contaminated evidence over the cellulose card-based systems currently being preferentially used for DNA sample collection. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Robustness of Component Models in Energy System Simulators

DEFF Research Database (Denmark)

Elmegaard, Brian

2003-01-01

During the development of the component-based energy system simulator DNA (Dynamic Network Analysis), several obstacles to easy use of the program have been observed. Some of these have to do with the nature of the program being based on a modelling language, not a graphical user interface (GUI......). Others have to do with the interaction between models of the nature of the substances in an energy system (e.g., fuels, air, flue gas), models of the components in a system (e.g., heat exchangers, turbines, pumps), and the solver for the system of equations. This paper proposes that the interaction...

DNA Nanocarriers for Systemic Administration: Characterization and In Vivo Bioimaging in Healthy Mice

Directory of Open Access Journals (Sweden)

Stephanie David

2013-01-01

Full Text Available We hereby present different DNA nanocarriers consisting of new multimodular systems (MMS, containing the cationic lipid dioleylaminesuccinylparomomycin (DNA MMS DOSP, or bis (guanidinium-tren-cholesterol (DNA MMS BGTC, and DNA lipid nanocapsules (DNA LNCs. Active targeting of the asialoglycoprotein receptor (ASGP-R using galactose as a ligand for DNA MMS (GAL DNA MMS and passive targeting using a polyethylene glycol coating for DNA LNCs (PEG DNA LNCs should improve the properties of these DNA nanocarriers. All systems were characterized via physicochemical methods and the DNA payload of DNA LNCs was quantified for the first time. Afterwards, their biodistribution in healthy mice was analyzed after encapsulation of a fluorescent dye via in vivo biofluorescence imaging (BFI, revealing various distribution profiles depending on the cationic lipid used and their surface characteristics. Furthermore, the two vectors with the best prolonged circulation profile were administered twice in healthy mice revealing that the new DNA MMS DOSP vectors showed no toxicity and the same distribution profile for both injections, contrary to PEG DNA LNCs which showed a rapid clearance after the second injection, certainly due to the accelerated blood clearance phenomenon.

Fluctuations in the DNA double helix

Science.gov (United States)

Peyrard, M.; López, S. C.; Angelov, D.

2007-08-01

DNA is not the static entity suggested by the famous double helix structure. It shows large fluctuational openings, in which the bases, which contain the genetic code, are temporarily open. Therefore it is an interesting system to study the effect of nonlinearity on the physical properties of a system. A simple model for DNA, at a mesoscopic scale, can be investigated by computer simulation, in the same spirit as the original work of Fermi, Pasta and Ulam. These calculations raise fundamental questions in statistical physics because they show a temporary breaking of equipartition of energy, regions with large amplitude fluctuations being able to coexist with regions where the fluctuations are very small, even when the model is studied in the canonical ensemble. This phenomenon can be related to nonlinear excitations in the model. The ability of the model to describe the actual properties of DNA is discussed by comparing theoretical and experimental results for the probability that base pairs open an a given temperature in specific DNA sequences. These studies give us indications on the proper description of the effect of the sequence in the mesoscopic model.

Cell-based DNA demethylation detection system for screening of epigenetic drugs in 2D, 3D, and xenograft models

Czech Academy of Sciences Publication Activity Database

Agrawal, K.; Das, V.; Otmar, Miroslav; Krečmerová, Marcela; Džubák, P.; Hajdúch, M.

91A, č. 2 (2017), s. 133-143 ISSN 1552-4922 R&D Projects: GA MZd(CZ) NV15-31984A; GA MŠk(CZ) LO1304; GA MŠk(CZ) LM2015064; GA TA ČR(CZ) TE01020028 Institutional support: RVO:61388963 Keywords : DNA methylation * DNA methylation inhibitors * demethylation detection system * epigenetic drugs * high content screening Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 3.222, year: 2016

Investigations on the interactions of diclofenac sodium with HSA and ctDNA using molecular modeling and multispectroscopic methods

Science.gov (United States)

Cui, Yanrui; Hao, Erjun; Hui, Guangquan; Guo, Wei; Cui, Fengling

2013-06-01

A tentative study on interaction of diclofenac sodium (DF-Na) with human serum albumin (HSA) and calf thymus DNA (ctDNA) was conducted by using multi-spectroscopic and molecular modeling techniques under simulative physiological conditions. The results of spectroscopic measurements suggested that the quenching mechanisms were static quenching. Three-dimensional fluorescence spectroscopy clearly demonstrated the occurrence of conformational changes of HSA with addition of DF-Na. In addition, competitive studies with ethidium bromide (EB) have shown that DF-Na can bind to ctDNA relatively strong via groove binding. Based on the values of thermodynamic parameters and the results of molecular modeling, it was confirmed that hydrophobic forces and hydrogen bond were the mainly binding forces in DF-Na-HSA and DF-Na-DNA systems. The binding distance between DF-Na and HSA was also determined using the theory of the Förster energy transference.

Sequence-based prediction of protein-binding sites in DNA: comparative study of two SVM models.

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Park, Byungkyu; Im, Jinyong; Tuvshinjargal, Narankhuu; Lee, Wook; Han, Kyungsook

2014-11-01

As many structures of protein-DNA complexes have been known in the past years, several computational methods have been developed to predict DNA-binding sites in proteins. However, its inverse problem (i.e., predicting protein-binding sites in DNA) has received much less attention. One of the reasons is that the differences between the interaction propensities of nucleotides are much smaller than those between amino acids. Another reason is that DNA exhibits less diverse sequence patterns than protein. Therefore, predicting protein-binding DNA nucleotides is much harder than predicting DNA-binding amino acids. We computed the interaction propensity (IP) of nucleotide triplets with amino acids using an extensive dataset of protein-DNA complexes, and developed two support vector machine (SVM) models that predict protein-binding nucleotides from sequence data alone. One SVM model predicts protein-binding nucleotides using DNA sequence data alone, and the other SVM model predicts protein-binding nucleotides using both DNA and protein sequences. In a 10-fold cross-validation with 1519 DNA sequences, the SVM model that uses DNA sequence data only predicted protein-binding nucleotides with an accuracy of 67.0%, an F-measure of 67.1%, and a Matthews correlation coefficient (MCC) of 0.340. With an independent dataset of 181 DNAs that were not used in training, it achieved an accuracy of 66.2%, an F-measure 66.3% and a MCC of 0.324. Another SVM model that uses both DNA and protein sequences achieved an accuracy of 69.6%, an F-measure of 69.6%, and a MCC of 0.383 in a 10-fold cross-validation with 1519 DNA sequences and 859 protein sequences. With an independent dataset of 181 DNAs and 143 proteins, it showed an accuracy of 67.3%, an F-measure of 66.5% and a MCC of 0.329. Both in cross-validation and independent testing, the second SVM model that used both DNA and protein sequence data showed better performance than the first model that used DNA sequence data. To the best of

A DNA Mini-Barcoding System for Authentication of Processed Fish Products.

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Shokralla, Shadi; Hellberg, Rosalee S; Handy, Sara M; King, Ian; Hajibabaei, Mehrdad

2015-10-30

Species substitution is a form of seafood fraud for the purpose of economic gain. DNA barcoding utilizes species-specific DNA sequence information for specimen identification. Previous work has established the usability of short DNA sequences-mini-barcodes-for identification of specimens harboring degraded DNA. This study aims at establishing a DNA mini-barcoding system for all fish species commonly used in processed fish products in North America. Six mini-barcode primer pairs targeting short (127-314 bp) fragments of the cytochrome c oxidase I (CO1) DNA barcode region were developed by examining over 8,000 DNA barcodes from species in the U.S. Food and Drug Administration (FDA) Seafood List. The mini-barcode primer pairs were then tested against 44 processed fish products representing a range of species and product types. Of the 44 products, 41 (93.2%) could be identified at the species or genus level. The greatest mini-barcoding success rate found with an individual primer pair was 88.6% compared to 20.5% success rate achieved by the full-length DNA barcode primers. Overall, this study presents a mini-barcoding system that can be used to identify a wide range of fish species in commercial products and may be utilized in high throughput DNA sequencing for authentication of heavily processed fish products.

Exciplex fluorescence emission from simple organic intramolecular constructs in non-polar and highly polar media as model systems for DNA-assembled exciplex detectors.

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Bichenkova, Elena V; Sardarian, Ali R; Wilton, Amanda N; Bonnet, Pascal; Bryce, Richard A; Douglas, Kenneth T

2006-01-21

Organic intramolecular exciplexes, N-(4-dimethylaminobenzyl)-N-(1-pyrenemethyl)amine (1) and N'-4-dimethylaminonaphthyl-N-(1-pyrenemethyl)amine (2), were used as model systems to reveal major factors affecting their exciplex fluorescence, and thus lay the basis for developing emissive target-assembled exciplexes for DNA-mounted systems in solution. These models with an aromatic pyrenyl hydrocarbon moiety as an electron acceptor appropriately connected to an aromatic dimethylamino electron donor component (N,N-dimethylaminophenyl or N,N-dimethylaminonaphthyl) showed strong intramolecular exciplex emission in both non-polar and highly polar solvents. The effect of dielectric constant on the maximum wavelength for exciplex emission was studied, and emission was observed for 1 and 2 over the full range of solvent from non-polar hydrocarbons up to N-methylformamide with a dielectric constant of 182. Quantum yields were determined for these intramolecular exciplexes in a range of solvents relative to that for Hoechst 33,258. Conformational analysis of 1 was performed both computationally and via qualitative 2D NMR using (1)H-NOESY experiments. The results obtained indicated the contribution of pre-folded conformation(s) to the ground state of 1 conducive to exciplex emission. This research provides the initial background for design of self-assembled, DNA-mounted exciplexes and underpins further development of exciplex-based hybridisation bioassays.

Computer-assisted design for scaling up systems based on DNA reaction networks.

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Aubert, Nathanaël; Mosca, Clément; Fujii, Teruo; Hagiya, Masami; Rondelez, Yannick

2014-04-06

In the past few years, there have been many exciting advances in the field of molecular programming, reaching a point where implementation of non-trivial systems, such as neural networks or switchable bistable networks, is a reality. Such systems require nonlinearity, be it through signal amplification, digitalization or the generation of autonomous dynamics such as oscillations. The biochemistry of DNA systems provides such mechanisms, but assembling them in a constructive manner is still a difficult and sometimes counterintuitive process. Moreover, realistic prediction of the actual evolution of concentrations over time requires a number of side reactions, such as leaks, cross-talks or competitive interactions, to be taken into account. In this case, the design of a system targeting a given function takes much trial and error before the correct architecture can be found. To speed up this process, we have created DNA Artificial Circuits Computer-Assisted Design (DACCAD), a computer-assisted design software that supports the construction of systems for the DNA toolbox. DACCAD is ultimately aimed to design actual in vitro implementations, which is made possible by building on the experimental knowledge available on the DNA toolbox. We illustrate its effectiveness by designing various systems, from Montagne et al.'s Oligator or Padirac et al.'s bistable system to new and complex networks, including a two-bit counter or a frequency divider as well as an example of very large system encoding the game Mastermind. In the process, we highlight a variety of behaviours, such as enzymatic saturation and load effect, which would be hard to handle or even predict with a simpler model. We also show that those mechanisms, while generally seen as detrimental, can be used in a positive way, as functional part of a design. Additionally, the number of parameters included in these simulations can be large, especially in the case of complex systems. For this reason, we included the

All-Atom Polarizable Force Field for DNA Based on the Classical Drude Oscillator Model

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Savelyev, Alexey; MacKerell, Alexander D.

2014-01-01

Presented is a first generation atomistic force field for DNA in which electronic polarization is modeled based on the classical Drude oscillator formalism. The DNA model is based on parameters for small molecules representative of nucleic acids, including alkanes, ethers, dimethylphosphate, and the nucleic acid bases and empirical adjustment of key dihedral parameters associated with the phosphodiester backbone, glycosidic linkages and sugar moiety of DNA. Our optimization strategy is based on achieving a compromise between satisfying the properties of the underlying model compounds in the gas phase targeting QM data and reproducing a number of experimental properties of DNA duplexes in the condensed phase. The resulting Drude force field yields stable DNA duplexes on the 100 ns time scale and satisfactorily reproduces (1) the equilibrium between A and B forms of DNA and (2) transitions between the BI and BII sub-states of B form DNA. Consistency with the gas phase QM data for the model compounds is significantly better for the Drude model as compared to the CHARMM36 additive force field, which is suggested to be due to the improved response of the model to changes in the environment associated with the explicit inclusion of polarizability. Analysis of dipole moments associated with the nucleic acid bases shows the Drude model to have significantly larger values than those present in CHARMM36, with the dipoles of individual bases undergoing significant variations during the MD simulations. Additionally, the dipole moment of water was observed to be perturbed in the grooves of DNA. PMID:24752978

A sequence-dependent rigid-base model of DNA

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Gonzalez, O.; Petkevičiutė, D.; Maddocks, J. H.

2013-02-01

A novel hierarchy of coarse-grain, sequence-dependent, rigid-base models of B-form DNA in solution is introduced. The hierarchy depends on both the assumed range of energetic couplings, and the extent of sequence dependence of the model parameters. A significant feature of the models is that they exhibit the phenomenon of frustration: each base cannot simultaneously minimize the energy of all of its interactions. As a consequence, an arbitrary DNA oligomer has an intrinsic or pre-existing stress, with the level of this frustration dependent on the particular sequence of the oligomer. Attention is focussed on the particular model in the hierarchy that has nearest-neighbor interactions and dimer sequence dependence of the model parameters. For a Gaussian version of this model, a complete coarse-grain parameter set is estimated. The parameterized model allows, for an oligomer of arbitrary length and sequence, a simple and explicit construction of an approximation to the configuration-space equilibrium probability density function for the oligomer in solution. The training set leading to the coarse-grain parameter set is itself extracted from a recent and extensive database of a large number of independent, atomic-resolution molecular dynamics (MD) simulations of short DNA oligomers immersed in explicit solvent. The Kullback-Leibler divergence between probability density functions is used to make several quantitative assessments of our nearest-neighbor, dimer-dependent model, which is compared against others in the hierarchy to assess various assumptions pertaining both to the locality of the energetic couplings and to the level of sequence dependence of its parameters. It is also compared directly against all-atom MD simulation to assess its predictive capabilities. The results show that the nearest-neighbor, dimer-dependent model can successfully resolve sequence effects both within and between oligomers. For example, due to the presence of frustration, the model can

A sequence-dependent rigid-base model of DNA.

Science.gov (United States)

Gonzalez, O; Petkevičiūtė, D; Maddocks, J H

2013-02-07

A novel hierarchy of coarse-grain, sequence-dependent, rigid-base models of B-form DNA in solution is introduced. The hierarchy depends on both the assumed range of energetic couplings, and the extent of sequence dependence of the model parameters. A significant feature of the models is that they exhibit the phenomenon of frustration: each base cannot simultaneously minimize the energy of all of its interactions. As a consequence, an arbitrary DNA oligomer has an intrinsic or pre-existing stress, with the level of this frustration dependent on the particular sequence of the oligomer. Attention is focussed on the particular model in the hierarchy that has nearest-neighbor interactions and dimer sequence dependence of the model parameters. For a Gaussian version of this model, a complete coarse-grain parameter set is estimated. The parameterized model allows, for an oligomer of arbitrary length and sequence, a simple and explicit construction of an approximation to the configuration-space equilibrium probability density function for the oligomer in solution. The training set leading to the coarse-grain parameter set is itself extracted from a recent and extensive database of a large number of independent, atomic-resolution molecular dynamics (MD) simulations of short DNA oligomers immersed in explicit solvent. The Kullback-Leibler divergence between probability density functions is used to make several quantitative assessments of our nearest-neighbor, dimer-dependent model, which is compared against others in the hierarchy to assess various assumptions pertaining both to the locality of the energetic couplings and to the level of sequence dependence of its parameters. It is also compared directly against all-atom MD simulation to assess its predictive capabilities. The results show that the nearest-neighbor, dimer-dependent model can successfully resolve sequence effects both within and between oligomers. For example, due to the presence of frustration, the model can

Targeting DNA repair systems in antitubercular drug development.

Science.gov (United States)

Minias, Alina; Brzostek, Anna; Dziadek, Jaroslaw

2018-01-28

Infections with Mycobacterium tuberculosis, the causative agent of tuberculosis, are difficult to treat using currently available chemotherapeutics. Clinicians agree on the urgent need for novel drugs to treat tuberculosis. In this mini review, we summarize data that prompts the consideration of DNA repair-associated proteins as targets for the development of new antitubercular compounds. We discuss data, including gene expression data, that highlight the importance of DNA repair genes during the pathogenic cycle as well as after exposure to antimicrobials currently in use. Specifically, we report experiments on determining the essentiality of DNA repair-related genes. We report the availability of protein crystal structures and summarize discovered protein inhibitors. Further, we describe phenotypes of available gene mutants of M. tuberculosis and model organisms Mycobacterium bovis and Mycobacterium smegmatis. We summarize experiments regarding the role of DNA repair-related proteins in pathogenesis and virulence performed both in vitro and in vivo during the infection of macrophages and animals. We detail the role of DNA repair genes in acquiring mutations, which influence the rate of drug resistance acquisition. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

DNA Damage Repair System in Plants: A Worldwide Research Update.

Science.gov (United States)

Gimenez, Estela; Manzano-Agugliaro, Francisco

2017-10-30

Living organisms are usually exposed to various DNA damaging agents so the mechanisms to detect and repair diverse DNA lesions have developed in all organisms with the result of maintaining genome integrity. Defects in DNA repair machinery contribute to cancer, certain diseases, and aging. Therefore, conserving the genomic sequence in organisms is key for the perpetuation of life. The machinery of DNA damage repair (DDR) in prokaryotes and eukaryotes is similar. Plants also share mechanisms for DNA repair with animals, although they differ in other important details. Plants have, surprisingly, been less investigated than other living organisms in this context, despite the fact that numerous lethal mutations in animals are viable in plants. In this manuscript, a worldwide bibliometric analysis of DDR systems and DDR research in plants was made. A comparison between both subjects was accomplished. The bibliometric analyses prove that the first study about DDR systems in plants (1987) was published thirteen years later than that for other living organisms (1975). Despite the increase in the number of papers about DDR mechanisms in plants in recent decades, nowadays the number of articles published each year about DDR systems in plants only represents 10% of the total number of articles about DDR. The DDR research field was done by 74 countries while the number of countries involved in the DDR & Plant field is 44. This indicates the great influence that DDR research in the plant field currently has, worldwide. As expected, the percentage of studies published about DDR systems in plants has increased in the subject area of agricultural and biological sciences and has diminished in medicine with respect to DDR studies in other living organisms. In short, bibliometric results highlight the current interest in DDR research in plants among DDR studies and can open new perspectives in the research field of DNA damage repair.

Localization of radiolabeled anti-DNA monoclonal antibodies in murine systemic lupus erythematosus (SLE)

International Nuclear Information System (INIS)

Wahl, R.; Hahn, B.; Ebling, F.

1984-01-01

The diagnosis of SLE can be extremely difficult. This multi-system disease is characterized by the deposition of DNA-anti-DNA antibody (Ab) complexes in many tissues, producing glomerulonephritis and systemic vasculitis. This study evaluates an IGG monoclonal (Mo) Ab directe3d against DNA (MrSSl) for potential radioimmunodiagnosis of SLE. Six 15 wk. old F-1 female hybrids of NZB+NZW mice (an animal SLE model that develops vasculitis and nephritis) were injected with 50 μCl of I-131 MrSSl and 15 μCl of I-125 isotype-matched control mouse myeloma (LPC-1) (non-reactive with DNA). Imaging and tissue distribution were studied. Two animals were also imaged using I-131 LPC Ab. Images at 2 and 9 days showed no clear differences in scan patterns using MrSSl or LPC-1 Ab. Tissue distribution studies at six days, however, showed a significantly higher accumulation of MrSSl in the kidneys vs. control Ab (2.7% vs. 1.8% of injected dose) (p < .04). Similarly, higher levels of MrSS were also seen in the spleen, liver and lungs (p < .03). Blood levels tended to be higher with the specific antibody as well. These differences were not apparent at 3 days post injection. The increased concentration of MrSSl present at 9 days in several organs may be secondary to MrSSl binding to DNA containing immune complexes present in diseased tissues. Blocked clearance by immune complexes or DNA, or differences in electrical charges of the antibodies could be contributing to the higher MrSSl levels seen. Images did not suggest deiodination as responsible. Further studies are necessary to determine if the amount of MrSSl retained by diseased animals is indicative of SLE disease activity

Quantum entanglement and quantum information in biological systems (DNA)

Science.gov (United States)

Hubač, Ivan; Švec, Miloslav; Wilson, Stephen

2017-12-01

Recent studies of DNA show that the hydrogen bonds between given base pairs can be treated as diabatic systems with spin-orbit coupling. For solid state systems strong diabaticity and spin-orbit coupling the possibility of forming Majorana fermions has been discussed. We analyze the hydrogen bonds in the base pairs in DNA from this perspective. Our analysis is based on a quasiparticle supersymmetric transformation which couples electronic and vibrational motion and includes normal coordinates and the corresponding momenta. We define qubits formed by Majorana fermions in the hydrogen bonds and also discuss the entangled states in base pairs. Quantum information and quantum entropy are introduced. In addition to the well-known classical information connected with the DNA base pairs, we also consider quantum information and show that the classical and quantum information are closely connected.

Vibronic dephasing model for coherent-to-incoherent crossover in DNA

Science.gov (United States)

Karasch, Patrick; Ryndyk, Dmitry A.; Frauenheim, Thomas

2018-05-01

In this paper, we investigate the interplay between coherent and incoherent charge transport in cytosine-guanine (GC-) rich DNA molecules. Our objective is to introduce a physically grounded approach to dephasing in large molecules and to understand the length-dependent charge transport characteristics, and especially the crossover from the coherent tunneling to incoherent hopping regime at different temperatures. Therefore, we apply the vibronic dephasing model and compare the results to the Büttiker probe model which is commonly used to describe decoherence effects in charge transport. Using the full ladder model and simplified one-dimensional model of DNA, we consider molecular junctions with alternating and stacked GC sequences and compare our results to recent experimental measurements.

DNA nanocarriers for systemic administration: characterization and in vivo bioimaging in healthy mice.

Science.gov (United States)

David, Stephanie; Passirani, Catherine; Carmoy, Nathalie; Morille, Marie; Mevel, Mathieu; Chatin, Benoit; Benoit, Jean-Pierre; Montier, Tristan; Pitard, Bruno

2013-01-08

We hereby present different DNA nanocarriers consisting of new multimodular systems (MMS), containing the cationic lipid dioleylaminesuccinylparomomycin (DNA MMS DOSP), or bis (guanidinium)-tren-cholesterol (DNA MMS BGTC), and DNA lipid nanocapsules (DNA LNCs). Active targeting of the asialoglycoprotein receptor (ASGP-R) using galactose as a ligand for DNA MMS (GAL DNA MMS) and passive targeting using a polyethylene glycol coating for DNA LNCs (PEG DNA LNCs) should improve the properties of these DNA nanocarriers. All systems were characterized via physicochemical methods and the DNA payload of DNA LNCs was quantified for the first time. Afterwards, their biodistribution in healthy mice was analyzed after encapsulation of a fluorescent dye via in vivo biofluorescence imaging (BFI), revealing various distribution profiles depending on the cationic lipid used and their surface characteristics. Furthermore, the two vectors with the best prolonged circulation profile were administered twice in healthy mice revealing that the new DNA MMS DOSP vectors showed no toxicity and the same distribution profile for both injections, contrary to PEG DNA LNCs which showed a rapid clearance after the second injection, certainly due to the accelerated blood clearance phenomenon.Molecular Therapy - Nucleic Acids (2013) 2, e64; doi:10.1038/mtna.2012.56; published online 8 January 2013.

Chip-Oriented Fluorimeter Design and Detection System Development for DNA Quantification in Nano-Liter Volumes

Directory of Open Access Journals (Sweden)

Da-Sheng Lee

2009-12-01

Full Text Available The chip-based polymerase chain reaction (PCR system has been developed in recent years to achieve DNA quantification. Using a microstructure and miniature chip, the volume consumption for a PCR can be reduced to a nano-liter. With high speed cycling and a low reaction volume, the time consumption of one PCR cycle performed on a chip can be reduced. However, most of the presented prototypes employ commercial fluorimeters which are not optimized for fluorescence detection of such a small quantity sample. This limits the performance of DNA quantification, especially low experiment reproducibility. This study discusses the concept of a chip-oriented fluorimeter design. Using the analytical model, the current study analyzes the sensitivity and dynamic range of the fluorimeter to fit the requirements for detecting fluorescence in nano-liter volumes. Through the optimized processes, a real-time PCR on a chip system with only one nano-liter volume test sample is as sensitive as the commercial real-time PCR machine using the sample with twenty micro-liter volumes. The signal to noise (S/N ratio of a chip system for DNA quantification with hepatitis B virus (HBV plasmid samples is 3 dB higher. DNA quantification by the miniature chip shows higher reproducibility compared to the commercial machine with respect to samples of initial concentrations from 103 to 105 copies per reaction.

Diffusion-controlled reactions modeling in Geant4-DNA

Energy Technology Data Exchange (ETDEWEB)

Karamitros, M., E-mail: matkara@gmail.com [CNRS, IN2P3, CENBG, UMR 5797, F-33170 Gradignan (France); CNRS, INCIA, UMR 5287, F-33400 Talence (France); Luan, S. [University of New Mexico, Department of Computer Science, Albuquerque, NM (United States); Bernal, M.A. [Instituto de Física Gleb Wataghin, Universidade Estadual de Campinas, SP (Brazil); Allison, J. [Geant4 Associates International Ltd (United Kingdom); Baldacchino, G. [CEA Saclay, IRAMIS, LIDYL, Radiation Physical Chemistry Group, F-91191 Gif sur Yvette Cedex (France); CNRS, UMR3299, SIS2M, F-91191 Gif sur Yvette Cedex (France); Davidkova, M. [Nuclear Physics Institute of the ASCR, Prague (Czech Republic); Francis, Z. [Saint Joseph University, Faculty of Sciences, Department of Physics, Mkalles, Beirut (Lebanon); Friedland, W. [Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Radiation Protection, Ingolstädter Landstr. 1, 85764 Neuherberg (Germany); Ivantchenko, V. [Ecoanalytica, 119899 Moscow (Russian Federation); Geant4 Associates International Ltd (United Kingdom); Ivantchenko, A. [Geant4 Associates International Ltd (United Kingdom); Mantero, A. [SwHaRD s.r.l., via Buccari 9, 16153 Genova (Italy); Nieminem, P.; Santin, G. [ESA-ESTEC, 2200 AG Noordwijk (Netherlands); Tran, H.N. [Division of Nuclear Physics and Faculty of Applied Sciences, Ton Duc Thang University, Tan Phong Ward, District 7, Ho Chi Minh City (Viet Nam); Stepan, V. [CNRS, IN2P3, CENBG, UMR 5797, F-33170 Gradignan (France); Nuclear Physics Institute of the ASCR, Prague (Czech Republic); Incerti, S., E-mail: incerti@cenbg.in2p3.fr [CNRS, IN2P3, CENBG, UMR 5797, F-33170 Gradignan (France)

2014-10-01

Context Under irradiation, a biological system undergoes a cascade of chemical reactions that can lead to an alteration of its normal operation. There are different types of radiation and many competing reactions. As a result the kinetics of chemical species is extremely complex. The simulation becomes then a powerful tool which, by describing the basic principles of chemical reactions, can reveal the dynamics of the macroscopic system. To understand the dynamics of biological systems under radiation, since the 80s there have been on-going efforts carried out by several research groups to establish a mechanistic model that consists in describing all the physical, chemical and biological phenomena following the irradiation of single cells. This approach is generally divided into a succession of stages that follow each other in time: (1) the physical stage, where the ionizing particles interact directly with the biological material; (2) the physico-chemical stage, where the targeted molecules release their energy by dissociating, creating new chemical species; (3) the chemical stage, where the new chemical species interact with each other or with the biomolecules; (4) the biological stage, where the repairing mechanisms of the cell come into play. This article focuses on the modeling of the chemical stage. Method This article presents a general method of speeding-up chemical reaction simulations in fluids based on the Smoluchowski equation and Monte-Carlo methods, where all molecules are explicitly simulated and the solvent is treated as a continuum. The model describes diffusion-controlled reactions. This method has been implemented in Geant4-DNA. The keys to the new algorithm include: (1) the combination of a method to compute time steps dynamically with a Brownian bridge process to account for chemical reactions, which avoids costly fixed time step simulations; (2) a k–d tree data structure for quickly locating, for a given molecule, its closest reactants. The

Failure to detect circulating DNA-anti-DNA complexes by four radioimmunological methods in patients with systemic lupus erythematosus

International Nuclear Information System (INIS)

Izui, S.; Lambert, P.H.; Miescher, P.A.

1977-01-01

The presence of DNA-anti-DNA complexes in sera from patients with systemic lupus erythematosus (SLE) was investigated by two new radioimmunoassays (RIA) developed for this purpose and by measuring the CLq and DNA binding activity of serum before and after treatment with DNAse. Two direct RIA developed in this study were based on the reactivity of [ 3 H]actinomycin D ([ 3 H]ACT-D) or solid-phase methylated bovine serum albumin (mBSA) with DNA-anti-DNA complexes. DNA-anti-DNA complexes prepared in vitro could be efficiently detected at various antigen-antibody ratios by these two RIA. Increased levels of circulating immune complexes as indicated by the CLq binding test were found in 52% of SLE sera. However, the frequency of specific DNA-anti-DNA complexes detected in SLE sera was very low. Only 6% of sera exhibited an increased value deviating by more than three s.d. from the normal mean when tested with the [ 3 H]ACT-D binding RIA or the solid-phase mBSA RIA. On the other hand, there was no significant difference in the serum CLq or DNA binding activity after treatment with DNAse. These results suggest that DNA-anti-DNA complexes do not occur frequently in circulating blood and represent only a very small portion of the immune complexes detected in serum from patients with SLE. (author)

Biophysics of DNA-Protein Interactions From Single Molecules to Biological Systems

CERN Document Server

Williams, Mark C

2011-01-01

This book presents a concise overview of current research on the biophysics of DNA-protein interactions. A wide range of new and classical methods are presented by authors investigating physical mechanisms by which proteins interact with DNA. For example, several chapters address the mechanisms by which proteins search for and recognize specific binding sites on DNA, a process critical for cellular function. Single molecule methods such as force spectroscopy as well as fluorescence imaging and tracking are described in these chapters as well as other parts of the book that address the dynamics of protein-DNA interactions. Other important topics include the mechanisms by which proteins engage DNA sequences and/or alter DNA structure. These simple but important model interactions are then placed in the broader biological context with discussion of larger protein-DNA complexes . Topics include replication forks, recombination complexes, DNA repair interactions, and ultimately, methods to understand the chromatin...

Modification of DNA radiolysis by DNA-binding proteins: Structural aspects

International Nuclear Information System (INIS)

Davidkova, M.; Stisova, V.; Goffinont, S.; Gillard, N.; Castaing, B.; Spotheim-Maurizot, M.

2006-01-01

Formation of specific complexes between proteins and their cognate DNA modulates the yields and the location of radiation damage on both partners of the complex. The radiolysis of DNA-protein complexes is studied for: (1) the Escherichia coli lactose operator-repressor complex, (2) the complex between DNA bearing an analogue of an abasic site and the repair protein Fpg of Lactococcus lactis. Experimental patterns of DNA damages are presented and compared to predicted damage distribution obtained using an improved version of the stochastic model RADACK. The same method is used for predicting the location of damages on the proteins. At doses lower than a threshold that depends on the system, proteins protect their specific binding site on DNA while at high doses, the studied complexes are disrupted mainly through protein damage. The loss of binding ability is the functional consequence of the amino-acids modification by OH . radicals. Many of the most probably damaged amino acids are essential for the DNA-protein interaction and within a complex are protected by DNA. (authors)

Systems Biology of Saccharomyces cerevisiae Physiology and its DNA Damage Response

DEFF Research Database (Denmark)

Fazio, Alessandro

The yeast Saccharomyces cerevisiae is a model organism in biology, being widely used in fundamental research, the first eukaryotic organism to be fully sequenced and the platform for the development of many genomics techniques. Therefore, it is not surprising that S. cerevisiae has also been widely...... used in the field of systems biology during the last decade. This thesis investigates S. cerevisiae growth physiology and DNA damage response by using a systems biology approach. Elucidation of the relationship between growth rate and gene expression is important to understand the mechanisms regulating...... set of growth dependent genes by using a multi-factorial experimental design. Moreover, new insights into the metabolic response and transcriptional regulation of these genes have been provided by using systems biology tools (Chapter 3). One of the prerequisite of systems biology should...

Systems and methods for modeling and analyzing networks

Science.gov (United States)

Hill, Colin C; Church, Bruce W; McDonagh, Paul D; Khalil, Iya G; Neyarapally, Thomas A; Pitluk, Zachary W

2013-10-29

The systems and methods described herein utilize a probabilistic modeling framework for reverse engineering an ensemble of causal models, from data and then forward simulating the ensemble of models to analyze and predict the behavior of the network. In certain embodiments, the systems and methods described herein include data-driven techniques for developing causal models for biological networks. Causal network models include computational representations of the causal relationships between independent variables such as a compound of interest and dependent variables such as measured DNA alterations, changes in mRNA, protein, and metabolites to phenotypic readouts of efficacy and toxicity.

Systems Biology Model of Interactions Between Tissue Growth Factors and DNA Damage Pathways: Low Dose Response and Cross-Talk in TGFbeta and ATM Signaling

Energy Technology Data Exchange (ETDEWEB)

O' Neill, Peter [University of Oxford; Anderson, Jennifer [University of Oxford

2014-10-02

The etiology of radiation carcinogenesis has been described in terms of aberrant changes that span several levels of biological organization. Growth factors regulate many important cellular and tissue functions including apoptosis, differentiation and proliferation. A variety of genetic and epigenetic changes of growth factors have been shown to contribute to cancer initiation and progression. It is known that cellular and tissue damage to ionizing radiation is in part initiated by the production of reactive oxygen species, which can activate cytokine signaling, and the DNA damage response pathways, most notably the ATM signaling pathway. Recently the transforming growth factor β (TGFβ) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation. The relevance of this interaction with the ATM pathway is not known although p53 becomes phosphorylated and DNA damage responses are involved. However, growth factor interactions with DNA damage responses have not been elucidated particularly at low doses and further characterization of their relationship to cancer processes is warranted. Our goal will be to use a systems biology approach to mathematically and experimentally describe the low dose responses and cross-talk between the ATM and TGFβ pathways initiated by low and high LET radiation. We will characterize ATM and TGFβ signaling in epithelial and fibroblast cells using 2D models and ultimately extending to 3D organotypic cell culture models to begin to elucidate possible differences that may occur for different cell types and/or inter-cellular communication. We will investigate the roles of the Smad and Activating transcription factor 2 (ATF2) proteins as the potential major contributors to cross- talk between the TGFβ and ATM pathways, and links to cell cycle control and/or the DNA damage response, and potential differences in their responses at low and high doses. We have developed various experimental

Systems Biology Model of Interactions between Tissue Growth Factors and DNA Damage Pathways: Low Dose Response and Cross-Talk in TGFβ and ATM Signaling

International Nuclear Information System (INIS)

Cucinotta, Francis A

2016-01-01

The etiology of radiation carcinogenesis has been described in terms of aberrant changes that span several levels of biological organization. Growth factors regulate many important cellular and tissue functions including apoptosis, differentiation and proliferation. A variety of genetic and epigenetic changes of growth factors have been shown to contribute to cancer initiation and progression. It is known that cellular and tissue damage to ionizing radiation is in part initiated by the production of reactive oxygen species, which can activate cytokine signaling, and the DNA damage response pathways, most notably the ATM signaling pathway. Recently, the transforming growth factor β (TGFβ) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation. The relevance of this interaction with the ATM pathway is not known although p53 becomes phosphorylated and DNA damage responses are involved. However, growth factor interactions with DNA damage responses have not been elucidated particularly at low doses, and further characterization of their relationship to cancer processes is warranted. Our goal will be to use a systems biology approach to mathematically and experimentally describe the low-dose responses and cross-talk between the ATM and TGFβ pathways initiated by low- and high-LET radiation. We will characterize ATM and TGFβ signaling in epithelial and fibroblast cells using 2D models and ultimately extending to 3D organotypic cell culture models to begin to elucidate possible differences that may occur for different cell types and/or inter-cellular communication. We will investigate the roles of the Smad and Activating transcription factor 2 (ATF2) proteins as the potential major contributors to crosstalk between the TGFβ and ATM pathways, and links to cell cycle control and/or the DNA damage response, and potential differences in their responses at low and high doses. We have developed various experimental

Systems Biology Model of Interactions between Tissue Growth Factors and DNA Damage Pathways: Low Dose Response and Cross-Talk in TGFβ and ATM Signaling

Energy Technology Data Exchange (ETDEWEB)

Cucinotta, Francis A [Univ. of Nevada, Las Vegas, NV (United States)

2016-09-01

The etiology of radiation carcinogenesis has been described in terms of aberrant changes that span several levels of biological organization. Growth factors regulate many important cellular and tissue functions including apoptosis, differentiation and proliferation. A variety of genetic and epigenetic changes of growth factors have been shown to contribute to cancer initiation and progression. It is known that cellular and tissue damage to ionizing radiation is in part initiated by the production of reactive oxygen species, which can activate cytokine signaling, and the DNA damage response pathways, most notably the ATM signaling pathway. Recently, the transforming growth factor β (TGFβ) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation. The relevance of this interaction with the ATM pathway is not known although p53 becomes phosphorylated and DNA damage responses are involved. However, growth factor interactions with DNA damage responses have not been elucidated particularly at low doses, and further characterization of their relationship to cancer processes is warranted. Our goal will be to use a systems biology approach to mathematically and experimentally describe the low-dose responses and cross-talk between the ATM and TGFβ pathways initiated by low- and high-LET radiation. We will characterize ATM and TGFβ signaling in epithelial and fibroblast cells using 2D models and ultimately extending to 3D organotypic cell culture models to begin to elucidate possible differences that may occur for different cell types and/or inter-cellular communication. We will investigate the roles of the Smad and Activating transcription factor 2 (ATF2) proteins as the potential major contributors to crosstalk between the TGFβ and ATM pathways, and links to cell cycle control and/or the DNA damage response, and potential differences in their responses at low and high doses. We have developed various experimental

DNA repair protocols

DEFF Research Database (Denmark)

Bjergbæk, Lotte

In its 3rd edition, this Methods in Molecular Biology(TM) book covers the eukaryotic response to genomic insult including advanced protocols and standard techniques in the field of DNA repair. Offers expert guidance for DNA repair, recombination, and replication. Current knowledge of the mechanisms...... that regulate DNA repair has grown significantly over the past years with technology advances such as RNA interference, advanced proteomics and microscopy as well as high throughput screens. The third edition of DNA Repair Protocols covers various aspects of the eukaryotic response to genomic insult including...... recent advanced protocols as well as standard techniques used in the field of DNA repair. Both mammalian and non-mammalian model organisms are covered in the book, and many of the techniques can be applied with only minor modifications to other systems than the one described. Written in the highly...

Modeling genetic imprinting effects of DNA sequences with multilocus polymorphism data

Directory of Open Access Journals (Sweden)

Staud Roland

2009-08-01

Full Text Available Abstract Single nucleotide polymorphisms (SNPs represent the most widespread type of DNA sequence variation in the human genome and they have recently emerged as valuable genetic markers for revealing the genetic architecture of complex traits in terms of nucleotide combination and sequence. Here, we extend an algorithmic model for the haplotype analysis of SNPs to estimate the effects of genetic imprinting expressed at the DNA sequence level. The model provides a general procedure for identifying the number and types of optimal DNA sequence variants that are expressed differently due to their parental origin. The model is used to analyze a genetic data set collected from a pain genetics project. We find that DNA haplotype GAC from three SNPs, OPRKG36T (with two alleles G and T, OPRKA843G (with alleles A and G, and OPRKC846T (with alleles C and T, at the kappa-opioid receptor, triggers a significant effect on pain sensitivity, but with expression significantly depending on the parent from which it is inherited (p = 0.008. With a tremendous advance in SNP identification and automated screening, the model founded on haplotype discovery and statistical inference may provide a useful tool for genetic analysis of any quantitative trait with complex inheritance.

Development and validation of open-source software for DNA mixture interpretation based on a quantitative continuous model.

Science.gov (United States)

Manabe, Sho; Morimoto, Chie; Hamano, Yuya; Fujimoto, Shuntaro; Tamaki, Keiji

2017-01-01

In criminal investigations, forensic scientists need to evaluate DNA mixtures. The estimation of the number of contributors and evaluation of the contribution of a person of interest (POI) from these samples are challenging. In this study, we developed a new open-source software "Kongoh" for interpreting DNA mixture based on a quantitative continuous model. The model uses quantitative information of peak heights in the DNA profile and considers the effect of artifacts and allelic drop-out. By using this software, the likelihoods of 1-4 persons' contributions are calculated, and the most optimal number of contributors is automatically determined; this differs from other open-source software. Therefore, we can eliminate the need to manually determine the number of contributors before the analysis. Kongoh also considers allele- or locus-specific effects of biological parameters based on the experimental data. We then validated Kongoh by calculating the likelihood ratio (LR) of a POI's contribution in true contributors and non-contributors by using 2-4 person mixtures analyzed through a 15 short tandem repeat typing system. Most LR values obtained from Kongoh during true-contributor testing strongly supported the POI's contribution even for small amounts or degraded DNA samples. Kongoh correctly rejected a false hypothesis in the non-contributor testing, generated reproducible LR values, and demonstrated higher accuracy of the estimated number of contributors than another software based on the quantitative continuous model. Therefore, Kongoh is useful in accurately interpreting DNA evidence like mixtures and small amounts or degraded DNA samples.

Development and validation of open-source software for DNA mixture interpretation based on a quantitative continuous model.

Directory of Open Access Journals (Sweden)

Sho Manabe

Full Text Available In criminal investigations, forensic scientists need to evaluate DNA mixtures. The estimation of the number of contributors and evaluation of the contribution of a person of interest (POI from these samples are challenging. In this study, we developed a new open-source software "Kongoh" for interpreting DNA mixture based on a quantitative continuous model. The model uses quantitative information of peak heights in the DNA profile and considers the effect of artifacts and allelic drop-out. By using this software, the likelihoods of 1-4 persons' contributions are calculated, and the most optimal number of contributors is automatically determined; this differs from other open-source software. Therefore, we can eliminate the need to manually determine the number of contributors before the analysis. Kongoh also considers allele- or locus-specific effects of biological parameters based on the experimental data. We then validated Kongoh by calculating the likelihood ratio (LR of a POI's contribution in true contributors and non-contributors by using 2-4 person mixtures analyzed through a 15 short tandem repeat typing system. Most LR values obtained from Kongoh during true-contributor testing strongly supported the POI's contribution even for small amounts or degraded DNA samples. Kongoh correctly rejected a false hypothesis in the non-contributor testing, generated reproducible LR values, and demonstrated higher accuracy of the estimated number of contributors than another software based on the quantitative continuous model. Therefore, Kongoh is useful in accurately interpreting DNA evidence like mixtures and small amounts or degraded DNA samples.

Alginate and DNA Gels Are Suitable Delivery Systems for Diabetic Wound Healing.

Science.gov (United States)

Tellechea, Ana; Silva, Eduardo A; Min, Jianghong; Leal, Ermelindo C; Auster, Michael E; Pradhan-Nabzdyk, Leena; Shih, William; Mooney, David J; Veves, Aristidis

2015-06-01

Diabetic foot ulcers (DFU) represent a severe health problem and an unmet clinical challenge. In this study, we tested the efficacy of novel biomaterials in improving wound healing in mouse models of diabetes mellitus (DM). The biomaterials are composed of alginate- and deoxyribonucleic acid (DNA)-based gels that allow incorporation of effector cells, such as outgrowth endothelial cells (OEC), and provide sustained release of bioactive factors, such as neuropeptides and growth factors, which have been previously validated in experimental models of DM wound healing or hind limb ischemia. We tested these biomaterials in mice and demonstrate that they are biocompatible and can be injected into the wound margins without major adverse effects. In addition, we show that the combination of OEC and the neuropeptide Substance P has a better healing outcome than the delivery of OEC alone, while subtherapeutic doses of vascular endothelial growth factor (VEGF) are required for the transplanted cells to exert their beneficial effects in wound healing. In summary, alginate and DNA scaffolds could serve as potential delivery systems for the next-generation DFU therapies. © The Author(s) 2015.

Failure to detect circulating DNA-anti-DNA complexes by four radioimmunological methods in patients with systemic lupus erythematosus

Energy Technology Data Exchange (ETDEWEB)

Izui, S; Lambert, P H; Miescher, P A [Hopital Cantonal Geneve (Switzerland)

1977-12-01

The presence of The DNA-anti-DNA complexes in sera from patients with systemic lupus erythematosus (SLE) was investigated by two new radioimmunoassays (RIA) developed for this purpose and by measuring the CLq and DNA binding activity of serum before and after treatment with DNAse. Two direct RIA developed in this study were based on the reactivity of (/sup 3/H)actinomycin D ((/sup 3/H)ACT-D) or solid-phase methylated bovine serum albumin (mBSA) with DNA-anti-DNA complexes. DNA-anti-DNA complexes prepared in vitro could be efficiently detected at various antigen-antibody ratios by these two RIA. Increased levels of circulating immune complexes as indicated by the CLq binding test were found in 52% of the SLE sera. However, the frequency of specific DNA-anti-DNA complexes detected in the SLE sera was very low. Only 6% of the sera exhibited an increased value deviating by more than three s.d. from the normal mean when tested with the (/sup 3/H)ACT-D binding RIA or the solid-phase mBSA RIA. On the other hand, there was no significant difference in the serum CLq or DNA binding activity after treatment with DNAse. These results suggest that DNA-anti-DNA complexes do not occur frequently in circulating blood and represent only a very small portion of the immune complexes detected in serum from patients with SLE.

Flow-injection chemiluminescence assay for ultra-trace determination of DNA using rhodamine B-Ce(IV)-DNA ternary system in sulfuric acid media

International Nuclear Information System (INIS)

Ma Yongjun; Zhou Min; Jin Xiaoyong; Zhang Ziyu; Teng Xiulan; Chen Hui

2004-01-01

A novel flow-injection chemiluminescence method for the determination of DNA at ultra-trace level has been established. In 0.8 M sulfuric acid media, the chemiluminescence of the rhodamine B-cerium (IV) or Ce(IV) system is enhanced by DNA, activated previously by imidazole-HCl buffer solution (pH 7.0). The enhanced intensity of chemiluminescence is in proportion to log DNA concentration 1.0x10 -8 to 0.1 μg ml -1 for herring sperm DNA and 2.0x10 -6 to 0.2 μg ml -1 for calf thymus DNA with 3σ detection limits of 8.3x10 -9 μg ml -1 for herring sperm DNA and 3.5x10 -7 μg ml -1 for calf thymus DNA, respectively. The relative standard deviation for 1.0x10 -4 μg ml -1 herring sperm DNA was 0.99% and 2.0x10 -3 μg ml -1 for calf thymus DNA was 1.1% (n=11). Using the optimized system, DNA contents in six synthetic samples has been determined with recoveries of 99.5-109.0%. The possible mechanism has also been studied in this paper

Flow-injection chemiluminescence assay for ultra-trace determination of DNA using rhodamine B-Ce(IV)-DNA ternary system in sulfuric acid media

Energy Technology Data Exchange (ETDEWEB)

Ma Yongjun; Zhou Min; Jin Xiaoyong; Zhang Ziyu; Teng Xiulan; Chen Hui

2004-01-09

A novel flow-injection chemiluminescence method for the determination of DNA at ultra-trace level has been established. In 0.8 M sulfuric acid media, the chemiluminescence of the rhodamine B-cerium (IV) or Ce(IV) system is enhanced by DNA, activated previously by imidazole-HCl buffer solution (pH 7.0). The enhanced intensity of chemiluminescence is in proportion to log DNA concentration 1.0x10{sup -8} to 0.1 {mu}g ml{sup -1} for herring sperm DNA and 2.0x10{sup -6} to 0.2 {mu}g ml{sup -1} for calf thymus DNA with 3{sigma} detection limits of 8.3x10{sup -9} {mu}g ml{sup -1} for herring sperm DNA and 3.5x10{sup -7} {mu}g ml{sup -1} for calf thymus DNA, respectively. The relative standard deviation for 1.0x10{sup -4} {mu}g ml{sup -1} herring sperm DNA was 0.99% and 2.0x10{sup -3} {mu}g ml{sup -1} for calf thymus DNA was 1.1% (n=11). Using the optimized system, DNA contents in six synthetic samples has been determined with recoveries of 99.5-109.0%. The possible mechanism has also been studied in this paper.

DNA-assisted swarm control in a biomolecular motor system.

Science.gov (United States)

Keya, Jakia Jannat; Suzuki, Ryuhei; Kabir, Arif Md Rashedul; Inoue, Daisuke; Asanuma, Hiroyuki; Sada, Kazuki; Hess, Henry; Kuzuya, Akinori; Kakugo, Akira

2018-01-31

In nature, swarming behavior has evolved repeatedly among motile organisms because it confers a variety of beneficial emergent properties. These include improved information gathering, protection from predators, and resource utilization. Some organisms, e.g., locusts, switch between solitary and swarm behavior in response to external stimuli. Aspects of swarming behavior have been demonstrated for motile supramolecular systems composed of biomolecular motors and cytoskeletal filaments, where cross-linkers induce large scale organization. The capabilities of such supramolecular systems may be further extended if the swarming behavior can be programmed and controlled. Here, we demonstrate that the swarming of DNA-functionalized microtubules (MTs) propelled by surface-adhered kinesin motors can be programmed and reversibly regulated by DNA signals. Emergent swarm behavior, such as translational and circular motion, can be selected by tuning the MT stiffness. Photoresponsive DNA containing azobenzene groups enables switching between solitary and swarm behavior in response to stimulation with visible or ultraviolet light.

Modeling DNA structure and processes through animation and kinesthetic visualizations

Science.gov (United States)

Hager, Christine

There have been many studies regarding the effectiveness of visual aids that go beyond that of static illustrations. Many of these have been concentrated on the effectiveness of visual aids such as animations and models or even non-traditional visual aid activities like role-playing activities. This study focuses on the effectiveness of three different types of visual aids: models, animation, and a role-playing activity. Students used a modeling kit made of Styrofoam balls and toothpicks to construct nucleotides and then bond nucleotides together to form DNA. Next, students created their own animation to depict the processes of DNA replication, transcription, and translation. Finally, students worked in teams to build proteins while acting out the process of translation. Students were given a pre- and post-test that measured their knowledge and comprehension of the four topics mentioned above. Results show that there was a significant gain in the post-test scores when compared to the pre-test scores. This indicates that the incorporated visual aids were effective methods for teaching DNA structure and processes.

A Penalty Method to Model Particle Interactions in DNA-laden Flows

International Nuclear Information System (INIS)

Trebotich, D; Miller, G H; Bybee, M D

2006-01-01

We present a hybrid fluid-particle algorithm to simulate flow and transport of DNA-laden fluids in microdevices. Relevant length scales in microfluidic systems range from characteristic channel sizes of millimeters to micron scale geometric variation (e.g., post arrays) to 10 nanometers for the length of a single rod in a bead-rod polymer representation of a biological material such as DNA. The method is based on a previous fluid-particle algorithm in which long molecules are represented as a chain of connected rods, but in which the physically unrealistic behavior of rod crossing occurred. We have extended this algorithm to include screened Coulombic forces between particles by implementing a Debye-Hueckel potential acting between rods. In the method an unsteady incompressible Newtonian fluid is discretized with a second-order finite difference method in the interior of the Cartesian grid domain; an embedded boundary volume-of-fluid formulation is used near boundaries. The bead-rod polymer model is fully coupled to the solvent through body forces representing hydrodynamic drag and stochastic thermal fluctuations. While intrapolymer interactions are modeled by a soft potential, polymer-structure interactions are treated as perfectly elastic collisions. We demonstrate this method on flow and transport of a polymer through a post array microchannel in 2D where the polymer incorporates more realistic physical parameters of DNA, and compare to previous simulations where rods are allowed to cross. We also show that the method is capable of simulating 3D flow in a packed bed micro-column

A Penalty Method to Model Particle Interactions in DNA-laden Flows

Energy Technology Data Exchange (ETDEWEB)

Trebotich, D; Miller, G H; Bybee, M D

2006-10-06

We present a hybrid fluid-particle algorithm to simulate flow and transport of DNA-laden fluids in microdevices. Relevant length scales in microfluidic systems range from characteristic channel sizes of millimeters to micron scale geometric variation (e.g., post arrays) to 10 nanometers for the length of a single rod in a bead-rod polymer representation of a biological material such as DNA. The method is based on a previous fluid-particle algorithm in which long molecules are represented as a chain of connected rods, but in which the physically unrealistic behavior of rod crossing occurred. We have extended this algorithm to include screened Coulombic forces between particles by implementing a Debye-Hueckel potential acting between rods. In the method an unsteady incompressible Newtonian fluid is discretized with a second-order finite difference method in the interior of the Cartesian grid domain; an embedded boundary volume-of-fluid formulation is used near boundaries. The bead-rod polymer model is fully coupled to the solvent through body forces representing hydrodynamic drag and stochastic thermal fluctuations. While intrapolymer interactions are modeled by a soft potential, polymer-structure interactions are treated as perfectly elastic collisions. We demonstrate this method on flow and transport of a polymer through a post array microchannel in 2D where the polymer incorporates more realistic physical parameters of DNA, and compare to previous simulations where rods are allowed to cross. We also show that the method is capable of simulating 3D flow in a packed bed micro-column.

Cell cycle regulation of DNA polymerase beta in rotenone-based Parkinson's disease models.

Directory of Open Access Journals (Sweden)

Hongcai Wang

Full Text Available In Parkinson's disease (PD, neuronal cells undergo mitotic catastrophe and endoreduplication prior to cell death; however, the regulatory mechanisms remain to be defined. In this study, we investigated cell cycle regulation of DNA polymerase β (poly β in rotenone-based dopaminergic cellular and animal models. Incubation with a low concentration (0.25 µM of rotenone for 1.5 to 7 days resulted in a flattened cell body and decreased DNA replication during S phase, whereas a high concentration (2 µM of rotenone exposure resulted in enlarged, multi-nucleated cells and converted the mitotic cycle into endoreduplication. Consistently, DNA poly β, which is mainly involved in DNA repair synthesis, was upregulated to a high level following exposure to 2 µM rotenone. The abrogation of DNA poly β by siRNA transfection or dideoxycytidine (DDC treatment attenuated the rotenone-induced endoreduplication. The cell cycle was reactivated in cyclin D-expressing dopaminergic neurons from the substantia nigra (SN of rats following stereotactic (ST infusion of rotenone. Increased DNA poly β expression was observed in the substantia nigra pars compacta (SNc and the substantia nigra pars reticulate (SNr of rotenone-treated rats. Collectively, in the in vitro model of rotenone-induced mitotic catastrophe, the overexpression of DNA poly β promotes endoreduplication; in the in vivo model, the upregulation of DNA poly β and cell cycle reentry were also observed in the adult rat substantia nigra. Therefore, the cell cycle regulation of DNA poly β may be involved in the pathological processes of PD, which results in the induction of endoreduplication.

Diversity of the DNA Replication System in the Archaea Domain

Directory of Open Access Journals (Sweden)

Felipe Sarmiento

2014-01-01

Full Text Available The precise and timely duplication of the genome is essential for cellular life. It is achieved by DNA replication, a complex process that is conserved among the three domains of life. Even though the cellular structure of archaea closely resembles that of bacteria, the information processing machinery of archaea is evolutionarily more closely related to the eukaryotic system, especially for the proteins involved in the DNA replication process. While the general DNA replication mechanism is conserved among the different domains of life, modifications in functionality and in some of the specialized replication proteins are observed. Indeed, Archaea possess specific features unique to this domain. Moreover, even though the general pattern of the replicative system is the same in all archaea, a great deal of variation exists between specific groups.

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

A cell-based model system links chromothripsis with hyperploidy

DEFF Research Database (Denmark)

Mardin, Balca R; Drainas, Alexandros P; Waszak, Sebastian M

2015-01-01

A remarkable observation emerging from recent cancer genome analyses is the identification of chromothripsis as a one-off genomic catastrophe, resulting in massive somatic DNA structural rearrangements (SRs). Largely due to lack of suitable model systems, the mechanistic basis of chromothripsis h...... in hyperploid cells. Analysis of primary medulloblastoma cancer genomes verified the link between hyperploidy and chromothripsis in vivo. CAST provides the foundation for mechanistic dissection of complex DNA rearrangement processes....

Structure of DNA toroids and electrostatic attraction of DNA duplexes

International Nuclear Information System (INIS)

Cherstvy, A G

2005-01-01

DNA-DNA electrostatic attraction is considered as the driving force for the formation of DNA toroids in the presence of DNA condensing cations. This attraction comes from the DNA helical charge distribution and favours hexagonal toroidal cross-sections. The latter is in agreement with recent cryo-electron microscopy studies on DNA condensed with cobalt hexammine. We treat the DNA-DNA interactions within the modern theory of electrostatic interaction between helical macromolecules. The size and thickness of the toroids is calculated within a simple model; other models of stability of DNA toroids are discussed and compared

Pitfalls of establishing DNA barcoding systems in protists: the cryptophyceae as a test case.

Science.gov (United States)

Hoef-Emden, Kerstin

2012-01-01

A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5'-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene). In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC), have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed.

Pitfalls of establishing DNA barcoding systems in protists: the cryptophyceae as a test case.

Directory of Open Access Journals (Sweden)

Kerstin Hoef-Emden

Full Text Available A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5'-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene. In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC, have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed.

Pitfalls of Establishing DNA Barcoding Systems in Protists: The Cryptophyceae as a Test Case

Science.gov (United States)

Hoef-Emden, Kerstin

2012-01-01

A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5′-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene). In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC), have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed. PMID:22970104

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

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

Science.gov (United States)

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

2014-10-01

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

Making sense of the noise: The effect of hydrology on silver carp eDNA detection in the Chicago area waterway system.

Science.gov (United States)

Song, Jeffery W; Small, Mitchell J; Casman, Elizabeth A

2017-12-15

Environmental DNA (eDNA) sampling is an emerging tool for monitoring the spread of aquatic invasive species. One confounding factor when interpreting eDNA sampling evidence is that eDNA can be present in the water in the absence of living target organisms, originating from excreta, dead tissue, boats, or sewage effluent, etc. In the Chicago Area Waterway System (CAWS), electric fish dispersal barriers were built to prevent non-native Asian carp species from invading Lake Michigan, and yet Asian carp eDNA has been detected above the barriers sporadically since 2009. In this paper the influence of stream flow characteristics in the CAWS on the probability of invasive Asian carp eDNA detection in the CAWS from 2009 to 2012 was examined. In the CAWS, the direction of stream flow is mostly away from Lake Michigan, though there are infrequent reversals in flow direction towards Lake Michigan during dry spells. We find that the flow reversal volume into the Lake has a statistically significant positive relationship with eDNA detection probability, while other covariates, like gage height, precipitation, season, water temperature, dissolved oxygen concentration, pH and chlorophyll concentration do not. This suggests that stream flow direction is highly influential on eDNA detection in the CAWS and should be considered when interpreting eDNA evidence. We also find that the beta-binomial regression model provides a stronger fit for eDNA detection probability compared to a binomial regression model. This paper provides a statistical modeling framework for interpreting eDNA sampling evidence and for evaluating covariates influencing eDNA detection. Copyright © 2017 Elsevier B.V. All rights reserved.

A statistical model for investigating binding probabilities of DNA nucleotide sequences using microarrays.

Science.gov (United States)

Lee, Mei-Ling Ting; Bulyk, Martha L; Whitmore, G A; Church, George M

2002-12-01

There is considerable scientific interest in knowing the probability that a site-specific transcription factor will bind to a given DNA sequence. Microarray methods provide an effective means for assessing the binding affinities of a large number of DNA sequences as demonstrated by Bulyk et al. (2001, Proceedings of the National Academy of Sciences, USA 98, 7158-7163) in their study of the DNA-binding specificities of Zif268 zinc fingers using microarray technology. In a follow-up investigation, Bulyk, Johnson, and Church (2002, Nucleic Acid Research 30, 1255-1261) studied the interdependence of nucleotides on the binding affinities of transcription proteins. Our article is motivated by this pair of studies. We present a general statistical methodology for analyzing microarray intensity measurements reflecting DNA-protein interactions. The log probability of a protein binding to a DNA sequence on an array is modeled using a linear ANOVA model. This model is convenient because it employs familiar statistical concepts and procedures and also because it is effective for investigating the probability structure of the binding mechanism.

Concentrations of environmental DNA (eDNA) reflect spawning salmon abundance at fine spatial and temporal scales

Science.gov (United States)

Tillotson, Michael D.; Kelly, Ryan P.; Duda, Jeff; Hoy, Marshal S.; Kralj, James; Quinn, Thomas P.

2018-01-01

Developing fast, cost-effective assessments of wild animal abundance is an important goal for many researchers, and environmental DNA (eDNA) holds much promise for this purpose. However, the quantitative relationship between species abundance and the amount of DNA present in the environment is likely to vary substantially among taxa and with ecological context. Here, we report a strong quantitative relationship between eDNA concentration and the abundance of spawning sockeye salmon in a small stream in Alaska, USA, where we took temporally- and spatially-replicated samples during the spawning period. This high-resolution dataset suggests that (1) eDNA concentrations vary significantly day-to-day, and likely within hours, in the context of the dynamic biological event of a salmon spawning season; (2) eDNA, as detected by species-specific quantitative PCR probes, seems to be conserved over short distances (tens of meters) in running water, but degrade quickly over larger scales (ca. 1.5 km); and (3) factors other than the mere presence of live, individual fish — such as location within the stream, live/dead ratio, and water temperature — can affect the eDNA-biomass correlation in space or time. A multivariate model incorporating both biotic and abiotic variables accounted for over 75% of the eDNA variance observed, suggesting that where a system is well-characterized, it may be possible to predict species' abundance from eDNA surveys, although we underscore that species- and system-specific variables are likely to limit the generality of any given quantitative model. Nevertheless, these findings provide an important step toward quantitative applications of eDNA in conservation and management.

Watson-Crick Base Pair Radical Cation as a Model for Oxidative Damage in DNA.

Science.gov (United States)

Feketeová, Linda; Chan, Bun; Khairallah, George N; Steinmetz, Vincent; Maitre, Philippe; Radom, Leo; O'Hair, Richard A J

2017-07-06

The deleterious cellular effects of ionizing radiation are well-known, but the mechanisms causing DNA damage are poorly understood. The accepted molecular events involve initial oxidation and deprotonation at guanine sites, triggering hydrogen atom abstraction reactions from the sugar moieties, causing DNA strand breaks. Probing the chemistry of the initially formed radical cation has been challenging. Here, we generate, spectroscopically characterize, and examine the reactivity of the Watson-Crick nucleobase pair radical cation in the gas phase. We observe rich chemistry, including proton transfer between the bases and propagation of the radical site in deoxyguanosine from the base to the sugar, thus rupturing the sugar. This first example of a gas-phase model system providing molecular-level details on the chemistry of an ionized DNA base pair paves the way toward a more complete understanding of molecular processes induced by radiation. It also highlights the role of radical propagation in chemistry, biology, and nanotechnology.

Repair of model compounds of photoinduced lesions in DNA. Electrochemical approaches

International Nuclear Information System (INIS)

Boussicault, F.

2006-09-01

The goal of this work is to better understand the repair mechanism of photoinduced lesions in DNA (cyclobutane dimers and pyrimidine (6-4) pyrimidone adducts) by photolyase redox enzymes, using tools and concepts of molecular electrochemistry. Thanks to the study of model compounds of cyclobutane lesions by cyclic voltametry, we have been able to mimic the key step of the enzymatic repair (dissociative electron transfer) and to monitor the repair of model compounds by Escherichia coli DNA photolyase. From these results, we have discussed the repair mechanism, especially the stepwise or concerted character of the process. Repair mechanism of (6-4) adducts is not known now, but a possible pathway implies an electron transfer coupled to the cleavage of two bonds in the closed form of the lesions (oxetanes). Voltammetric study of reduction and oxidation of model oxetanes and their repair by E. coli DNA photolyase gave some experimental evidence confirming the proposed mechanism and allowing a better understanding of it. (author)

EDNA-An expert software system for comparison and evaluation of DNA profiles in forensic casework

DEFF Research Database (Denmark)

Haldemann, B.; Dornseifer, S.; Heylen, T.

2015-01-01

eDNA is an expert software system for DNA profile comparison, match interpretation and automated report generation in forensic DNA casework. Process automation and intelligent graphical representation maximise reliability of DNA evidence, while facilitating and accelerating the work of DNA experts....

Double-Strand DNA Break Repair in Mycobacteria.

Science.gov (United States)

Glickman, Michael S

2014-10-01

Discontinuity of both strands of the chromosome is a lethal event in all living organisms because it compromises chromosome replication. As such, a diversity of DNA repair systems has evolved to repair double-strand DNA breaks (DSBs). In part, this diversity of DSB repair systems has evolved to repair breaks that arise in diverse physiologic circumstances or sequence contexts, including cellular states of nonreplication or breaks that arise between repeats. Mycobacteria elaborate a set of three genetically distinct DNA repair pathways: homologous recombination, nonhomologous end joining, and single-strand annealing. As such, mycobacterial DSB repair diverges substantially from the standard model of prokaryotic DSB repair and represents an attractive new model system. In addition, the presence in mycobacteria of a DSB repair system that can repair DSBs in nonreplicating cells (nonhomologous end joining) or when DSBs arise between repeats (single-strand annealing) has clear potential relevance to Mycobacterium tuberculosis pathogenesis, although the exact role of these systems in M. tuberculosis pathogenesis is still being elucidated. In this article we will review the genetics of mycobacterial DSB repair systems, focusing on recent insights.

A Hard Constraint Algorithm to Model Particle Interactions in DNA-laden Flows

Energy Technology Data Exchange (ETDEWEB)

Trebotich, D; Miller, G H; Bybee, M D

2006-08-01

We present a new method for particle interactions in polymer models of DNA. The DNA is represented by a bead-rod polymer model and is fully-coupled to the fluid. The main objective in this work is to implement short-range forces to properly model polymer-polymer and polymer-surface interactions, specifically, rod-rod and rod-surface uncrossing. Our new method is based on a rigid constraint algorithm whereby rods elastically bounce off one another to prevent crossing, similar to our previous algorithm used to model polymer-surface interactions. We compare this model to a classical (smooth) potential which acts as a repulsive force between rods, and rods and surfaces.

Detection of DNA via the fluorescence quenching of Mn-doped ZnSe D-dots/doxorubicin/DNA ternary complexes system.

Science.gov (United States)

Gao, Xue; Niu, Lu; Su, Xingguang

2012-01-01

This manuscript reports a method for the detection of double-stranded DNA, based on Mn:ZnSe d-dots and intercalating agent doxorubicin (DOX). DOX can quench the photoluminescence (PL) of Mn:ZnSe d-dots through photoinduced electron transfer process, after binding with Mn:ZnSe d-dots. The addition of DNA can result in the formation of the Mn:ZnSe d-dots-DOX-DNA ternary complexes, the fluorescence of the Mn:ZnSe d-dots-DOX complexes would be further quenched by the addition of DNA, thus allowing the detection of DNA. The formation mechanism of the Mn:ZnSe d-dots-DOX-DNA ternary complexes was studied in detail in this paper. Under optimal conditions, the quenched fluorescence intensity of Mn:ZnSe d-dots-DOX system are perfectly described by Stern-Volmer equation with the concentration of hsDNA ranging from 0.006 μg mL(-1) to 6.4 μg mL(-1). The detection limit (S/N = 3) for hsDNA is 0.5 ng mL(-1). The proposed method was successfully applied to the detection of DNA in synthetic samples and the results were satisfactory.

A model treating the DNA double-strand break repair inhibition by damage clustering

International Nuclear Information System (INIS)

Rosemann, M.; Abel, H.; Regel, K.

1992-01-01

A microdosimetric model for the interpretation of radiation induced irreparable DNA double-strand breaks was applied to the biological endpoint of chromosomal aberrations. The model explains irreparable DNA double-strand breaks in terms of break clustering in DNA subunits. The model predicts quite good chromosomal aberrations in gamma- and X-ray irradiated V79 cells and human lymphocytes. In the case of α-particle irradiation the presumption had to be made, that only the cells with indirect events in the nucleus (due to delta-electrons) reach the metaphase and are analysed. With the help of this model we are able to explain the peculiar effectiveness of ultrasoft C-X-rays in human lymphocytes. In addition, an interpretation of experiments with accelerated and spatially correlated particles is given. (author)

Compatibility of DNA IQ™, QIAamp® DNA Investigator, and QIAsymphony® DNA Investigator® with various fingerprint treatments.

Science.gov (United States)

Lin, Sze-Wah; Ip, Stephen C Y; Lam, Tze-Tsun; Tan, Tung-Fai; Yeung, Wai-Lung; Tam, Wai-Ming

2017-03-01

Latent fingerprint and touch DNA are the two most important contact evidence for individualization in forensic science which provide complementary information that can lead to direct and unequivocal identification of the culprit. In order to retrieve useful information from both fingerprints and DNA, which are usually mingled together, one strategy is to perform fingerprint examination prior to DNA analysis since common DNA sampling technique such as swabbing could disturb or even destroy fingerprint details. Here, we describe the compatibility of three automatic DNA extraction systems, namely, DNA IQ™, QIAamp ® DNA Investigator, and QIAsymphony ® DNA Investigator ® , with respective to the effects of various fingerprint detection techniques. Our results demonstrate that Super Glue fingerprint treatment followed by DNA IQ™ extraction shows better effectiveness in DNA profiling. Aluminum powder dusting offers the least interference to the three DNA extraction systems above. Magnetic powder dusting, on the other hand, strongly impedes DNA recovery. Physical Developer is the most intrusive, which yields profiles with poor quality, including lower peak heights, poor peak height ratios, and poor intra-color balance. In terms of the choice of extraction method, DNA IQ™ system is recommended for sampling after fingerprint treatments, but not the two DNA Investigator systems.

Structural aspects of DNA in its replication and repair

International Nuclear Information System (INIS)

Mitra, S.; Pal, B.C.; Foote, R.S.; Bates, R.C.; Bhattacharyya, A.; Snow, E.T.; Wobbe, C.R.; Morse, C.C.; Snyder, C.E.

1984-01-01

The research objective of this laboratory is to investigate the structure of DNA, the mechanism of DNA replication and its regulation, and the mechanism and role of repair of the altered DNA in the expression of heritable changes. This research has two broad aims, namely investigation of (a) the regulation of DNA replication in mammals, using parvovirus DNA as a model system and (b) the role of DNA repair in mutagenesis and carcinogenesis induced by simple alkylating mutagens

Using Environmental DNA to Improve Species Distribution Models for Freshwater Invaders

Directory of Open Access Journals (Sweden)

Teja P. Muha

2017-12-01

Full Text Available Species Distribution Models (SDMs have been reported as a useful tool for the risk assessment and modeling of the pathways of dispersal of freshwater invasive alien species (IAS. Environmental DNA (eDNA is a novel tool that can help detect IAS at their early stage of introduction and additionally improve the data available for a more efficient management. SDMs rely on presence and absence of the species in the study area to infer the predictors affecting species distributions. Presence is verified once a species is detected, but confirmation of absence can be problematic because this depends both on the detectability of the species and the sampling strategy. eDNA is a technique that presents higher detectability and accuracy in comparison to conventional sampling techniques, and can effectively differentiate between presence or absence of specific species or entire communities by using a barcoding or metabarcoding approach. However, a number of potential bias can be introduced during (i sampling, (ii amplification, (iii sequencing, or (iv through the usage of bioinformatics pipelines. Therefore, it is important to report and conduct the field and laboratory procedures in a consistent way, by (i introducing eDNA independent observations, (ii amplifying and sequencing control samples, (iii achieving quality sequence reads by appropriate clean-up steps, (iv controlling primer amplification preferences, (v introducing PCR-free sequence capturing, (vi estimating primer detection capabilities through controlled experiments and/or (vii post-hoc introduction of “site occupancy-detection models.” With eDNA methodology becoming increasingly routine, its use is strongly recommended to retrieve species distributional data for SDMs.

Development and production of Lab-on-Chip systems for DNA mapping

DEFF Research Database (Denmark)

Østergaard, Peter Friis

as low as 1:200. The developed polymer systems are tested by conducting two different experiments on DNA. Since such experiments are highly sensitive, efforts have been taken in order to lower the autofluorescence of the devices, resulting in a decrease of the background signal to roughly half...... several nanochannels can be placed parallel to each other, a large number of DNA molecules can be investigated. In the second experiment, mapping is performed on human DNA in nanoslit devices. A fluorescent profile is created by heating the sample up to a temperature, where the DNA is partially denatured....... The fluorescent dye will diffuse away from the denatured regions, and by analysing these black areas, the DNA molecule can be identified and potential mutations can be found. In the nanoslits, the DNA is stretched out via a shear flow, resulting in a stretching of more than 95% of the contour length meaning...

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

Colorimetric DNA detection of transgenic plants using gold nanoparticles functionalized with L-shaped DNA probes

Science.gov (United States)

Nourisaeid, Elham; Mousavi, Amir; Arpanaei, Ayyoob

2016-01-01

In this study, a DNA colorimetric detection system based on gold nanoparticles functionalized with L-shaped DNA probes was prepared and evaluated. We investigated the hybridization efficiency of the L-shaped probes and studied the effect of nanoparticle size and the L-shaped DNA probe length on the performance of the as-prepared system. Probes were attached to the surface of gold nanoparticles using an adenine sequence. An optimal sequence of 35S rRNA gene promoter from the cauliflower mosaic virus, which is frequently used in the development of transgenic plants, and the two complementary ends of this gene were employed as model target strands and probe molecules, respectively. The spectrophotometric properties of the as-prepared systems indicated that the large NPs show better changes in the absorption spectrum and consequently present a better performance. The results of this study revealed that the probe/Au-NPs prepared using a vertical spacer containing 5 thymine oligonucleotides exhibited a stronger spectrophotometric response in comparison to that of larger probes. These results in general indicate the suitable performance of the L-shaped DNA probe-functionalized Au-NPs, and in particular emphasize the important role of the gold nanoparticle size and length of the DNA probes in enhancing the performance of such a system.

The Helicobacter pylori HpyAXII restriction–modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components

Science.gov (United States)

Humbert, Olivier; Salama, Nina R.

2008-01-01

The naturally competent organism Helicobacter pylori encodes a large number of restriction–modification (R–M) systems that consist of a restriction endonuclease and a DNA methyltransferase. R–M systems are not only believed to limit DNA exchange among bacteria but may also have other cellular functions. We report a previously uncharacterized H. pylori type II R–M system, M.HpyAXII/R.HpyAXII. We show that this system targets GTAC sites, which are rare in the H. pylori chromosome but numerous in ribosomal RNA genes. As predicted, this type II R–M system showed attributes of a selfish element. Deletion of the methyltransferase M.HpyAXII is lethal when associated with an active endonuclease R.HpyAXII unless compensated by adaptive mutation or gene amplification. R.HpyAXII effectively restricted both unmethylated plasmid and chromosomal DNA during natural transformation and was predicted to belong to the novel ‘half pipe’ structural family of endonucleases. Analysis of a panel of clinical isolates revealed that R.HpyAXII was functional in a small number of H. pylori strains (18.9%, n = 37), whereas the activity of M.HpyAXII was highly conserved (92%, n = 50), suggesting that GTAC methylation confers a selective advantage to H. pylori. However, M.HpyAXII activity did not enhance H. pylori fitness during stomach colonization of a mouse infection model. PMID:18978016

PH- and salt-dependent molecular combing of DNA: experiments and phenomenological model

International Nuclear Information System (INIS)

Benke, Annegret; Pompe, Wolfgang; Mertig, Michael

2011-01-01

λ-DNA as well as plasmids can be successfully deposited by molecular combing on hydrophobic surfaces, for pH values ranging from 4 to 10. On polydimethylsiloxane (PDMS) substrates, the deposited DNA molecules are overstretched by about 60-100%. There is a significant influence of sodium ions (NaCl) on the surface density of the deposited DNA, with a maximum near to 100 mM NaCl for a DNA solution (28 ng μl -1 ) at pH 8. The combing process can be described by a micromechanical model including: (i) the adsorption of free moving coiled DNA at the substrate; (ii) the stretching of the coiled DNA by the preceding meniscus; (iii) the relaxation of the deposited DNA to the final length. The sticky ends of λ-DNA cause an adhesion force in the range of about 400 pN which allows a stable overstretching of the DNA by the preceding meniscus. The exposing of hidden hydrophobic bonds of the overstretched DNA leads to a stable deposition on the hydrophobic substrate. The pH-dependent density of deposited DNA as well as the observed influence of sodium ions can be explained by their screening of the negatively charged DNA backbone and sticky ends, respectively. The final DNA length can be derived from a balance of the stored elastic energy of the overstretched molecules and the energy of adhesion.

Essential and non-essential DNA replication genes in the model halophilic Archaeon, Halobacterium sp. NRC-1

Directory of Open Access Journals (Sweden)

DasSarma Shiladitya

2007-06-01

Full Text Available Abstract Background Information transfer systems in Archaea, including many components of the DNA replication machinery, are similar to those found in eukaryotes. Functional assignments of archaeal DNA replication genes have been primarily based upon sequence homology and biochemical studies of replisome components, but few genetic studies have been conducted thus far. We have developed a tractable genetic system for knockout analysis of genes in the model halophilic archaeon, Halobacterium sp. NRC-1, and used it to determine which DNA replication genes are essential. Results Using a directed in-frame gene knockout method in Halobacterium sp. NRC-1, we examined nineteen genes predicted to be involved in DNA replication. Preliminary bioinformatic analysis of the large haloarchaeal Orc/Cdc6 family, related to eukaryotic Orc1 and Cdc6, showed five distinct clades of Orc/Cdc6 proteins conserved in all sequenced haloarchaea. Of ten orc/cdc6 genes in Halobacterium sp. NRC-1, only two were found to be essential, orc10, on the large chromosome, and orc2, on the minichromosome, pNRC200. Of the three replicative-type DNA polymerase genes, two were essential: the chromosomally encoded B family, polB1, and the chromosomally encoded euryarchaeal-specific D family, polD1/D2 (formerly called polA1/polA2 in the Halobacterium sp. NRC-1 genome sequence. The pNRC200-encoded B family polymerase, polB2, was non-essential. Accessory genes for DNA replication initiation and elongation factors, including the putative replicative helicase, mcm, the eukaryotic-type DNA primase, pri1/pri2, the DNA polymerase sliding clamp, pcn, and the flap endonuclease, rad2, were all essential. Targeted genes were classified as non-essential if knockouts were obtained and essential based on statistical analysis and/or by demonstrating the inability to isolate chromosomal knockouts except in the presence of a complementing plasmid copy of the gene. Conclusion The results showed that ten

UV induced DNA-protein cross links in vitro and in vivo

International Nuclear Information System (INIS)

Kornhauser, A.

1976-01-01

The review was not intended to cover all the past year's literature in this field; only selective material published in 1974 and 1975 has been surveyed. Covalent linkage of DNA and RNA to proteins induced by UV is considered, but DNA-membrade attachment, amino acids covalently bound to DNA as functions of growth conditions and protein non-covalently bound to DNA involved in cell regulation are excluded. Studies of DNA-protein cross-links upon UV irradiation in chemical model systems, bacteria and tissue culture systems, and an in vivo mammalian system are all surveyed. (U.K.)

Fundamental study of the radiation monitoring system based on evaluation of DNA lesions

International Nuclear Information System (INIS)

Shimizu, K.; Matuo, Y.; Izumi, Y.; Ikeda, T.

2011-01-01

The biological dosemeter that measures biological responses to ionising radiation is useful for radiation protection. This paper presents the development and characterisation of a gamma ray irradiation dosimetry system based on real-time PCR (polymerase chain reaction) methodology. Real-time PCR is used to amplify and simultaneously quantify a targeted DNA molecule. If there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment. The essential point of this assay is that DNA lesions caused by ionising radiation block DNA synthesis by DNA polymerase, resulting in a decrease in the amplification of a damaged DNA template compared with that of non-damaged DNA templates. (authors)

Exploring the Feasibility of a DNA Computer: Design of an ALU Using Sticker-Based DNA Model.

Science.gov (United States)

Sarkar, Mayukh; Ghosal, Prasun; Mohanty, Saraju P

2017-09-01

Since its inception, DNA computing has advanced to offer an extremely powerful, energy-efficient emerging technology for solving hard computational problems with its inherent massive parallelism and extremely high data density. This would be much more powerful and general purpose when combined with other existing well-known algorithmic solutions that exist for conventional computing architectures using a suitable ALU. Thus, a specifically designed DNA Arithmetic and Logic Unit (ALU) that can address operations suitable for both domains can mitigate the gap between these two. An ALU must be able to perform all possible logic operations, including NOT, OR, AND, XOR, NOR, NAND, and XNOR; compare, shift etc., integer and floating point arithmetic operations (addition, subtraction, multiplication, and division). In this paper, design of an ALU has been proposed using sticker-based DNA model with experimental feasibility analysis. Novelties of this paper may be in manifold. First, the integer arithmetic operations performed here are 2s complement arithmetic, and the floating point operations follow the IEEE 754 floating point format, resembling closely to a conventional ALU. Also, the output of each operation can be reused for any next operation. So any algorithm or program logic that users can think of can be implemented directly on the DNA computer without any modification. Second, once the basic operations of sticker model can be automated, the implementations proposed in this paper become highly suitable to design a fully automated ALU. Third, proposed approaches are easy to implement. Finally, these approaches can work on sufficiently large binary numbers.

DNA repair in B. subtilis: an inducible dimer-specific W-reactivation system

International Nuclear Information System (INIS)

Fields, P.I.; Yasbin, R.E.

1982-01-01

The W-reactivation system of Bacillus subtilis can repair pyrimidine dimers in bacteriophage DNA. This inducible repair system can be activated by treatment of the bacteria with uv, alkylating agents, cross-linking agents and gamma irradiation. However, bacteriophage treated with agents other than those that cause pyrimidine dimers to be produced was not repaired by this unique form of W-reactivation. In contrast, the W-reactivation system of Escherichia coli can repair a variety of damages placed in the bacteriophage DNA

Guardians of the mycobacterial genome: A review on DNA repair systems in Mycobacterium tuberculosis.

Science.gov (United States)

Singh, Amandeep

2017-12-01

The genomic integrity of Mycobacterium tuberculosis is continuously threatened by the harsh survival conditions inside host macrophages, due to immune and antibiotic stresses. Faithful genome maintenance and repair must be accomplished under stress for the bacillus to survive in the host, necessitating a robust DNA repair system. The importance of DNA repair systems in pathogenesis is well established. Previous examination of the M. tuberculosis genome revealed homologues of almost all the major DNA repair systems, i.e. nucleotide excision repair (NER), base excision repair (BER), homologous recombination (HR) and non-homologous end joining (NHEJ). However, recent developments in the field have pointed to the presence of novel proteins and pathways in mycobacteria. Homologues of archeal mismatch repair proteins were recently reported in mycobacteria, a pathway previously thought to be absent. RecBCD, the major nuclease-helicase enzymes involved in HR in E. coli, were implicated in the single-strand annealing (SSA) pathway. Novel roles of archeo-eukaryotic primase (AEP) polymerases, previously thought to be exclusive to NHEJ, have been reported in BER. Many new proteins with a probable role in DNA repair have also been discovered. It is now realized that the DNA repair systems in M. tuberculosis are highly evolved and have redundant backup mechanisms to mend the damage. This review is an attempt to summarize our current understanding of the DNA repair systems in M. tuberculosis.

Model studies of radiation induced oxidation and reduction processes in DNA

International Nuclear Information System (INIS)

Hole, E.O.

1992-01-01

The papers presented in this thesis represent the major part of a systematic study of primary and secondary radiation induced damages in DNA. The magnetic resonance techniques EPR, ENDOR and FSE have been the experimental methods used. The study of radical formation in isolated DNA components under different environmental conditions demonstrates certain characteristics of the DNA components which are important in the study of DNA. It has been clearly demonstrated that the electrostatic environment, in particular the hydrogen bond pattern, is a vital factor for the secondary reaction scheme. Even radicals which are found in all related systems seem to be formed by different reaction pathways, depending upon the specific matrix. 92 refs., 2 figs., 6 tabs

Direct atomic force microscopy observation of DNA tile crystal growth at the single-molecule level.

Science.gov (United States)

Evans, Constantine G; Hariadi, Rizal F; Winfree, Erik

2012-06-27

While the theoretical implications of models of DNA tile self-assembly have been extensively researched and such models have been used to design DNA tile systems for use in experiments, there has been little research testing the fundamental assumptions of those models. In this paper, we use direct observation of individual tile attachments and detachments of two DNA tile systems on a mica surface imaged with an atomic force microscope (AFM) to compile statistics of tile attachments and detachments. We show that these statistics fit the widely used kinetic Tile Assembly Model and demonstrate AFM movies as a viable technique for directly investigating DNA tile systems during growth rather than after assembly.

Clusters of DNA induced by ionizing radiation: formation of short DNA fragments. I. Theoretical modeling

Science.gov (United States)

Holley, W. R.; Chatterjee, A.

1996-01-01

We have developed a general theoretical model for the interaction of ionizing radiation with chromatin. Chromatin is modeled as a 30-nm-diameter solenoidal fiber comprised of 20 turns of nucleosomes, 6 nucleosomes per turn. Charged-particle tracks are modeled by partitioning the energy deposition between primary track core, resulting from glancing collisions with 100 eV or less per event, and delta rays due to knock-on collisions involving energy transfers >100 eV. A Monte Carlo simulation incorporates damages due to the following molecular mechanisms: (1) ionization of water molecules leading to the formation of OH, H, eaq, etc.; (2) OH attack on sugar molecules leading to strand breaks: (3) OH attack on bases; (4) direct ionization of the sugar molecules leading to strand breaks; (5) direct ionization of the bases. Our calculations predict significant clustering of damage both locally, over regions up to 40 bp and over regions extending to several kilobase pairs. A characteristic feature of the regional damage predicted by our model is the production of short fragments of DNA associated with multiple nearby strand breaks. The shapes of the spectra of DNA fragment lengths depend on the symmetries or approximate symmetries of the chromatin structure. Such fragments have subsequently been detected experimentally and are reported in an accompanying paper (B. Rydberg, Radiat, Res. 145, 200-209, 1996) after exposure to both high- and low-LET radiation. The overall measured yields agree well quantitatively with the theoretical predictions. Our theoretical results predict the existence of a strong peak at about 85 bp, which represents the revolution period about the nucleosome. Other peaks at multiples of about 1,000 bp correspond to the periodicity of the particular solenoid model of chromatin used in these calculations. Theoretical results in combination with experimental data on fragmentation spectra may help determine the consensus or average structure of the

The radiation chemistry of the purine bases within DNA and related model compounds

International Nuclear Information System (INIS)

Cadet, J.; Berger, M.; Shaw, A.

1986-01-01

Both the direct and indirect effects of ionizing radiations are believed to contribute to the chemical changes induced in cellular DNA. Relevant information on the possible degradation pathways has been provided by studies using DNA model compounds, the major proportion of which have focused on pyrimidine components and sugar derivatives. With the development of powerful analytical tools such as high performance liquid chromatography and soft ionization mass spectrometry techniques, progress has recently been made in the elucidation of the nature of the radiation-induced chemical modifications of purine bases in DNA and related nucleosides and nucleotides. This short review details recent aspects of the radiation-induced degradation of adenine and guanine bases in DNA and its model compounds as the result of both direct and indirect effects. 11 refs., 2 figs., 1 tab

FBIS: A regional DNA barcode archival & analysis system for Indian fishes

Science.gov (United States)

Nagpure, Naresh Sahebrao; Rashid, Iliyas; Pathak, Ajey Kumar; Singh, Mahender; Singh, Shri Prakash; Sarkar, Uttam Kumar

2012-01-01

DNA barcode is a new tool for taxon recognition and classification of biological organisms based on sequence of a fragment of mitochondrial gene, cytochrome c oxidase I (COI). In view of the growing importance of the fish DNA barcoding for species identification, molecular taxonomy and fish diversity conservation, we developed a Fish Barcode Information System (FBIS) for Indian fishes, which will serve as a regional DNA barcode archival and analysis system. The database presently contains 2334 sequence records of COI gene for 472 aquatic species belonging to 39 orders and 136 families, collected from available published data sources. Additionally, it contains information on phenotype, distribution and IUCN Red List status of fishes. The web version of FBIS was designed using MySQL, Perl and PHP under Linux operating platform to (a) store and manage the acquisition (b) analyze and explore DNA barcode records (c) identify species and estimate genetic divergence. FBIS has also been integrated with appropriate tools for retrieving and viewing information about the database statistics and taxonomy. It is expected that FBIS would be useful as a potent information system in fish molecular taxonomy, phylogeny and genomics. Availability The database is available for free at http://mail.nbfgr.res.in/fbis/ PMID:22715304

Computational design and multiscale modeling of a nanoactuator using DNA actuation

International Nuclear Information System (INIS)

Hamdi, Mustapha

2009-01-01

Developments in the field of nano-biodevices coupling nanostructures and biological components are of great interest in medical nanorobotics. As the fundamentals of bio/non-bio interaction processes are still poorly understood in the design of these devices, design tools and multiscale dynamics modeling approaches are necessary at the fabrication pre-project stage. This paper proposes a new concept of optimized carbon nanotube based servomotor design for drug delivery and biomolecular transport applications. The design of an encapsulated DNA-multi-walled carbon nanotube actuator is prototyped using multiscale modeling. The system is parametrized by using a quantum level approach and characterized by using a molecular dynamics simulation. Based on the analysis of the simulation results, a servo nanoactuator using ionic current feedback is simulated and analyzed for application as a drug delivery carrier.

Real-time PCR Machine System Modeling and a Systematic Approach for the Robust Design of a Real-time PCR-on-a-Chip System

Directory of Open Access Journals (Sweden)

Da-Sheng Lee

2010-01-01

Full Text Available Chip-based DNA quantification systems are widespread, and used in many point-of-care applications. However, instruments for such applications may not be maintained or calibrated regularly. Since machine reliability is a key issue for normal operation, this study presents a system model of the real-time Polymerase Chain Reaction (PCR machine to analyze the instrument design through numerical experiments. Based on model analysis, a systematic approach was developed to lower the variation of DNA quantification and achieve a robust design for a real-time PCR-on-a-chip system. Accelerated lift testing was adopted to evaluate the reliability of the chip prototype. According to the life test plan, this proposed real-time PCR-on-a-chip system was simulated to work continuously for over three years with similar reproducibility in DNA quantification. This not only shows the robustness of the lab-on-a-chip system, but also verifies the effectiveness of our systematic method for achieving a robust design.

Looping and clustering model for the organization of protein-DNA complexes on the bacterial genome

Science.gov (United States)

Walter, Jean-Charles; Walliser, Nils-Ole; David, Gabriel; Dorignac, Jérôme; Geniet, Frédéric; Palmeri, John; Parmeggiani, Andrea; Wingreen, Ned S.; Broedersz, Chase P.

2018-03-01

The bacterial genome is organized by a variety of associated proteins inside a structure called the nucleoid. These proteins can form complexes on DNA that play a central role in various biological processes, including chromosome segregation. A prominent example is the large ParB-DNA complex, which forms an essential component of the segregation machinery in many bacteria. ChIP-Seq experiments show that ParB proteins localize around centromere-like parS sites on the DNA to which ParB binds specifically, and spreads from there over large sections of the chromosome. Recent theoretical and experimental studies suggest that DNA-bound ParB proteins can interact with each other to condense into a coherent 3D complex on the DNA. However, the structural organization of this protein-DNA complex remains unclear, and a predictive quantitative theory for the distribution of ParB proteins on DNA is lacking. Here, we propose the looping and clustering model, which employs a statistical physics approach to describe protein-DNA complexes. The looping and clustering model accounts for the extrusion of DNA loops from a cluster of interacting DNA-bound proteins that is organized around a single high-affinity binding site. Conceptually, the structure of the protein-DNA complex is determined by a competition between attractive protein interactions and loop closure entropy of this protein-DNA cluster on the one hand, and the positional entropy for placing loops within the cluster on the other. Indeed, we show that the protein interaction strength determines the ‘tightness’ of the loopy protein-DNA complex. Thus, our model provides a theoretical framework for quantitatively computing the binding profiles of ParB-like proteins around a cognate (parS) binding site.

Development of a Model for the Teaching of Mitochondrial DNA

Directory of Open Access Journals (Sweden)

A.P.S. Souza

2010-05-01

Full Text Available The Cellular Biology and Molecular Biology are fields of Science that use very abstract concepts, because they look into microscopic and molecular aspects of the nature. The process of teaching/learning of those disciplines requires didactic material, as an alternative approach for the students, to increase the chances of understanding these issues and to become an important tool in the synthesis of this knowledge. One of the methods that can be employed is the didactic models based on multimedia, because they allow an easy and fun interaction with these subjects. On this work was created a new educational model that represents the human mitochondrial DNA molecule, mtDNA, in its circular form, using the softwares Excel 2007 and PowerPoint 2007. The model was constructed in hypertext format, which allowed a quick and interactive access to the information contained in the genes found in the L and the H strands of mtDNA, and its function in the mitochondrial processes, like themechanism of energy production that occurs inside of the mitochondria by the coupling of electron transfer and ATP synthesis or still others uses like forensic identification.

Hepatitis B virus DNA integration occurs early in the viral life cycle in an in vitro infection model via NTCP-dependent uptake of enveloped virus particles.

Science.gov (United States)

Tu, Thomas; Budzinska, Magdalena A; Vondran, Florian W R; Shackel, Nicholas A; Urban, Stephan

2018-02-07

Chronic infection by the Hepatitis B Virus (HBV) is the major contributor to liver disease worldwide. Though HBV replicates via a nuclear episomal DNA (cccDNA), integration of HBV DNA into the host cell genome is regularly observed in the liver of infected patients. While reported as a pro-oncogenic alteration, the mechanism(s) and timing of HBV DNA integration are not well-understood, chiefly due to the lack of in vitro infection models that have detectable integration events. Here, we have established an in vitro system in which integration can be reliably detected following HBV infection. We measured HBV DNA integration using inverse nested PCR in primary human hepatocytes, HepaRG-NTCP, HepG2-NTCP, and Huh7-NTCP cells after HBV infection. Integration was detected in all cell types at a rate of >1 per 10000 cells, with the most consistent detection in Huh7-NTCP cells. Integration rate remained stable between 3 and 9 days post-infection. HBV DNA integration was efficiently blocked by treatment with 200nM of the HBV entry inhibitor Myrcludex B, but not with 10μM Tenofovir, 100U Interferon alpha, or 1μM of the capsid assembly inhibitor GLS4. This suggests integration of HBV DNA occurs immediately after infection of hepatocytes and is likely independent of de novo HBV replication in this model. Site analysis revealed that HBV DNA integrations were distributed over the entire human genome. Further, integrated HBV DNA sequences were consistent with double-stranded linear HBV DNA being the major precursor. Thus, we have established an in vitro system to interrogate the mechanisms of HBV DNA integration. Importance Hepatitis B Virus (HBV) is a common blood-borne pathogen and, following a chronic infection, can cause liver cancer and liver cirrhosis. Integration of HBV DNA into the host genome occurs in all known members of the hepadnaviridae family, despite this form not being necessary for viral replication. HBV DNA integration has been reported to drive liver cancer

Using DNA nanotechnology to produce a drug delivery system

International Nuclear Information System (INIS)

La, Thi Huyen; Nguyen, Thi Thu Thuy; Pham, Van Phuc; Nguyen, Thi Minh Huyen; Le, Quang Huan

2013-01-01

Drug delivery to cancer cells in chemotherapy is one of the most advanced research topics. The effectiveness of the current cancer treatment drugs is limited because they are not capable of distinguishing between cancer cells and normal cells so that they kill not only cancer cells but also normal ones. To overcome this disadvantage by profiting from the differences in physical and chemical properties between cancer and normal cells, nanoparticles (NPs) delivering a drug are designed in a specific manner such that they can distinguish the cancer cells from the normal ones and are targeted only to the cancer cells. Currently, there are various drug delivery systems with many advantages, but sharing some common disadvantages such as difficulty with controlling the size, low encapsulation capacity and low stability. With the development and success of DNA nanotechnology, DNA strands are used to create effective drug delivery NPs with precisely controlled size and structure, safety and high stability. This article presents our study on drug encapsulation in DNA nanostructure which loaded docetaxel and curcumin in a desire to create a new and effective drug delivery system with high biological compatibility. (paper)

Specific binding of a dihydropyrimidinone derivative with DNA: Spectroscopic, calorimetric and modeling investigations

International Nuclear Information System (INIS)

Wang Gongke; Yan Changling; Wang Dongchao; Li Dan; Lu Yan

2012-01-01

One of the dihydropyrimidinone derivative 5-(ethoxycarbonyl)-6-methyl-4-(4-methoxyphenyl) -3,4-dihydropyrimidin-2(1H)-one (EMMD) was synthesized, and its binding properties with calf-thymus DNA (ctDNA) were investigated using spectroscopic, viscometric, isothermal titration calorimetric (ITC) and molecular modeling techniques. Fluorescence spectra suggested that the fluorescence enhancement of the binding interaction of EMMD to ctDNA was a static process with ground state complex formation. The binding constant determined with spectroscopic titration and ITC was found to be in the same order of 10 4 M −1 . According to the results of the viscosity analysis, fluorescence competitive binding experiment, fluorescence quenching studies, absorption spectral and ITC investigations, it can be concluded that EMMD is intercalative binding to ctDNA. Furthermore, the results of molecular modeling confirmed those obtained from spectroscopic, viscosimetric and ITC investigations. Additionally, ITC studies also indicated that the binding interaction is predominantly enthalpy driven. - Highlights: ► Medically important dihydropyrimidinones derivative EMMD is synthesized. ► EMMD is intercalative binding into ctDNA helix. ► Hydrogen bonding may play an essential role in the binding of EMCD with ctDNA. ► This binding interaction is predominantly enthalpy driven.

A novel color image encryption scheme using fractional-order hyperchaotic system and DNA sequence operations

International Nuclear Information System (INIS)

Zhang Li-Min; Sun Ke-Hui; Liu Wen-Hao; He Shao-Bo

2017-01-01

In this paper, Adomian decomposition method (ADM) with high accuracy and fast convergence is introduced to solve the fractional-order piecewise-linear (PWL) hyperchaotic system. Based on the obtained hyperchaotic sequences, a novel color image encryption algorithm is proposed by employing a hybrid model of bidirectional circular permutation and DNA masking. In this scheme, the pixel positions of image are scrambled by circular permutation, and the pixel values are substituted by DNA sequence operations. In the DNA sequence operations, addition and substraction operations are performed according to traditional addition and subtraction in the binary, and two rounds of addition rules are used to encrypt the pixel values. The simulation results and security analysis show that the hyperchaotic map is suitable for image encryption, and the proposed encryption algorithm has good encryption effect and strong key sensitivity. It can resist brute-force attack, statistical attack, differential attack, known-plaintext, and chosen-plaintext attacks. (paper)

Human DNA quantification and sample quality assessment: Developmental validation of the PowerQuant(®) system.

Science.gov (United States)

Ewing, Margaret M; Thompson, Jonelle M; McLaren, Robert S; Purpero, Vincent M; Thomas, Kelli J; Dobrowski, Patricia A; DeGroot, Gretchen A; Romsos, Erica L; Storts, Douglas R

2016-07-01

Quantification of the total amount of human DNA isolated from a forensic evidence item is crucial for DNA normalization prior to short tandem repeat (STR) DNA analysis and a federal quality assurance standard requirement. Previous commercial quantification methods determine the total human DNA and total human male DNA concentrations, but provide limited information about the condition of the DNA sample. The PowerQuant(®) System includes targets for quantification of total human and total human male DNA as well as targets for evaluating whether the human DNA is degraded and/or PCR inhibitors are present in the sample. A developmental validation of the PowerQuant(®) System was completed, following SWGDAM Validation Guidelines, to evaluate the assay's specificity, sensitivity, precision and accuracy, as well as the ability to detect degraded DNA or PCR inhibitors. In addition to the total human DNA and total human male DNA concentrations in a sample, data from the degradation target and internal PCR control (IPC) provide a forensic DNA analyst meaningful information about the quality of the isolated human DNA and the presence of PCR inhibitors in the sample that can be used to determine the most effective workflow and assist downstream interpretation. Copyright © 2016 The Author(s). Published by Elsevier Ireland Ltd.. All rights reserved.

Design of a Clinical Information Management System to Support DNA Analysis Laboratory Operation

Science.gov (United States)

Dubay, Christopher J.; Zimmerman, David; Popovich, Bradley

1995-01-01

The LabDirector system has been developed at the Oregon Health Sciences University to support the operation of our clinical DNA analysis laboratory. Through an iterative design process which has spanned two years, we have produced a system that is both highly tailored to a clinical genetics production laboratory and flexible in its implementation, to support the rapid growth and change of protocols and methodologies in use in the field. The administrative aspects of the system are integrated with an enterprise schedule management system. The laboratory side of the system is driven by a protocol modeling and execution system. The close integration between these two aspects of the clinical laboratory facilitates smooth operations, and allows management to accurately measure costs and performance. The entire application has been designed and documented to provide utility to a wide range of clinical laboratory environments.

Are dinucleoside monophosphates relevant models for the study of DNA intrastrand cross-link lesions? The example of g[8-5m]T.

Science.gov (United States)

Garrec, Julian; Dumont, Elise

2014-07-21

Oxidatively generated tandem lesions such as G[8-5m]T pose a potent threat to genome integrity. Direct experimental studies of the kinetics and thermodynamics of a specific lesion within DNA are very challenging, mostly due to the variety of products that can be formed in oxidative conditions. Dinucleoside monophosphates (DM) involving only the reactive nucleobases in water represent appealing alternative models on which most physical chemistry and structural techniques can be applied. However, it is not yet clear how relevant these models are. Here, we present QM/MM MD simulations of the cyclization step involved in the formation of G[8-5m]T from the guanine-thymine (GpT) DM in water, with the aim of comparing our results to our previous investigation of the same reaction in DNA ( Garrec , J. , Patel , C. , Rothlisberger , U. , and Dumont , E. ( 2012 ) J. Am. Chem. Soc. 134 , 2111 - 2119 ). We show that, despite the different levels of preorganization of the two systems, the corresponding reactions share many energetic and structural characteristics. The main difference lies in the angle between the G and T bases, which is slightly higher in the transition state (TS) and product of the reaction in water than in the reaction in DNA. This effect is due to the Watson-Crick H-bonds, which are absent in the {GpT+water} system and restrain the relative positioning of the reactive nucleobases in DNA. However, since the lesion is accommodated easily in the DNA macromolecule, the induced energetic penalty is relatively small. The high similarity between the two reactions strongly supports the use of GpT in water as a model of the corresponding reaction in DNA.

A test of the transcription model for biased inheritance of yeast mitochondrial DNA.

Science.gov (United States)

Lorimer, H E; Brewer, B J; Fangman, W L

1995-09-01

Two strand-specific origins of replication appear to be required for mammalian mitochondrial DNA (mtDNA) replication. Structural equivalents of these origins are found in the rep sequences of Saccharomyces cerevisiae mtDNA. These striking similarities have contributed to a universal model for the initiation of mtDNA replication in which a primer is created by cleavage of an origin region transcript. Consistent with this model are the properties of deletion mutants of yeast mtDNA ([rho-]) with a high density of reps (HS [rho-]). These mutant mtDNAs are preferentially inherited by the progeny resulting from the mating of HS [rho-] cells with cells containing wild-type mtDNA ([rho+]). This bias is presumed to result from a replication advantage conferred on HS [rho-] mtDNA by the high density of rep sequences acting as origins. To test whether transcription is indeed required for the preferential inheritance of HS [rho-] mtDNA, we deleted the nuclear gene (RPO41) for the mitochondrial RNA polymerase, reducing transcripts by at least 1000-fold. Since [rho-] genomes, but not [rho+] genomes, are stable when RPO41 is deleted, we examined matings between HS [rho-] and neutral [rho-] cells. Neutral [rho-] mtDNAs lack rep sequences and are not preferentially inherited in [rho-] x [rho+] crosses. In HS [rho-] x neutral [rho-] matings, the HS [rho-] mtDNA was preferentially inherited whether both parents were wild type or both were deleted for RPO41. Thus, transcription from the rep promoter does not appear to be necessary for biased inheritance. Our results, and analysis of the literature, suggest that priming by transcription is not a universal mechanism for mtDNA replication initiation.

Low-energy electron dose-point kernel simulations using new physics models implemented in Geant4-DNA

Energy Technology Data Exchange (ETDEWEB)

Bordes, Julien, E-mail: julien.bordes@inserm.fr [CRCT, UMR 1037 INSERM, Université Paul Sabatier, F-31037 Toulouse (France); UMR 1037, CRCT, Université Toulouse III-Paul Sabatier, F-31037 (France); Incerti, Sébastien, E-mail: incerti@cenbg.in2p3.fr [Université de Bordeaux, CENBG, UMR 5797, F-33170 Gradignan (France); CNRS, IN2P3, CENBG, UMR 5797, F-33170 Gradignan (France); Lampe, Nathanael, E-mail: nathanael.lampe@gmail.com [Université de Bordeaux, CENBG, UMR 5797, F-33170 Gradignan (France); CNRS, IN2P3, CENBG, UMR 5797, F-33170 Gradignan (France); Bardiès, Manuel, E-mail: manuel.bardies@inserm.fr [CRCT, UMR 1037 INSERM, Université Paul Sabatier, F-31037 Toulouse (France); UMR 1037, CRCT, Université Toulouse III-Paul Sabatier, F-31037 (France); Bordage, Marie-Claude, E-mail: marie-claude.bordage@inserm.fr [CRCT, UMR 1037 INSERM, Université Paul Sabatier, F-31037 Toulouse (France); UMR 1037, CRCT, Université Toulouse III-Paul Sabatier, F-31037 (France)

2017-05-01

When low-energy electrons, such as Auger electrons, interact with liquid water, they induce highly localized ionizing energy depositions over ranges comparable to cell diameters. Monte Carlo track structure (MCTS) codes are suitable tools for performing dosimetry at this level. One of the main MCTS codes, Geant4-DNA, is equipped with only two sets of cross section models for low-energy electron interactions in liquid water (“option 2” and its improved version, “option 4”). To provide Geant4-DNA users with new alternative physics models, a set of cross sections, extracted from CPA100 MCTS code, have been added to Geant4-DNA. This new version is hereafter referred to as “Geant4-DNA-CPA100”. In this study, “Geant4-DNA-CPA100” was used to calculate low-energy electron dose-point kernels (DPKs) between 1 keV and 200 keV. Such kernels represent the radial energy deposited by an isotropic point source, a parameter that is useful for dosimetry calculations in nuclear medicine. In order to assess the influence of different physics models on DPK calculations, DPKs were calculated using the existing Geant4-DNA models (“option 2” and “option 4”), newly integrated CPA100 models, and the PENELOPE Monte Carlo code used in step-by-step mode for monoenergetic electrons. Additionally, a comparison was performed of two sets of DPKs that were simulated with “Geant4-DNA-CPA100” – the first set using Geant4′s default settings, and the second using CPA100′s original code default settings. A maximum difference of 9.4% was found between the Geant4-DNA-CPA100 and PENELOPE DPKs. Between the two Geant4-DNA existing models, slight differences, between 1 keV and 10 keV were observed. It was highlighted that the DPKs simulated with the two Geant4-DNA’s existing models were always broader than those generated with “Geant4-DNA-CPA100”. The discrepancies observed between the DPKs generated using Geant4-DNA’s existing models and “Geant4-DNA-CPA100” were

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)

Genetic coding and united-hypercomplex systems in the models of algebraic biology.

Science.gov (United States)

Petoukhov, Sergey V

2017-08-01

Structured alphabets of DNA and RNA in their matrix form of representations are connected with Walsh functions and a new type of systems of multidimensional numbers. This type generalizes systems of complex numbers and hypercomplex numbers, which serve as the basis of mathematical natural sciences and many technologies. The new systems of multi-dimensional numbers have interesting mathematical properties and are called in a general case as "systems of united-hypercomplex numbers" (or briefly "U-hypercomplex numbers"). They can be widely used in models of multi-parametrical systems in the field of algebraic biology, artificial life, devices of biological inspired artificial intelligence, etc. In particular, an application of U-hypercomplex numbers reveals hidden properties of genetic alphabets under cyclic permutations in their doublets and triplets. A special attention is devoted to the author's hypothesis about a multi-linguistic in DNA-sequences in a relation with an ensemble of U-numerical sub-alphabets. Genetic multi-linguistic is considered as an important factor to provide noise-immunity properties of the multi-channel genetic coding. Our results attest to the conformity of the algebraic properties of the U-numerical systems with phenomenological properties of the DNA-alphabets and with the complementary device of the double DNA-helix. It seems that in the modeling field of algebraic biology the genetic-informational organization of living bodies can be considered as a set of united-hypercomplex numbers in some association with the famous slogan of Pythagoras "the numbers rule the world". Copyright © 2017 Elsevier B.V. All rights reserved.

Is DNA a nonlinear dynamical system where solitary conformational ...

Indian Academy of Sciences (India)

Unknown

DNA is considered as a nonlinear dynamical system in which solitary conformational waves can be excited. The ... nonlinear differential equations and their soliton-like solu- .... structure and dynamics can be added till the most accurate.

CRISPR-Cas systems exploit viral DNA injection to establish and maintain adaptive immunity.

Science.gov (United States)

Modell, Joshua W; Jiang, Wenyan; Marraffini, Luciano A

2017-04-06

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide protection against viral and plasmid infection by capturing short DNA sequences from these invaders and integrating them into the CRISPR locus of the prokaryotic host. These sequences, known as spacers, are transcribed into short CRISPR RNA guides that specify the cleavage site of Cas nucleases in the genome of the invader. It is not known when spacer sequences are acquired during viral infection. Here, to investigate this, we tracked spacer acquisition in Staphylococcus aureus cells harbouring a type II CRISPR-Cas9 system after infection with the staphylococcal bacteriophage ϕ12. We found that new spacers were acquired immediately after infection preferentially from the cos site, the viral free DNA end that is first injected into the cell. Analysis of spacer acquisition after infection with mutant phages demonstrated that most spacers are acquired during DNA injection, but not during other stages of the viral cycle that produce free DNA ends, such as DNA replication or packaging. Finally, we showed that spacers acquired from early-injected genomic regions, which direct Cas9 cleavage of the viral DNA immediately after infection, provide better immunity than spacers acquired from late-injected regions. Our results reveal that CRISPR-Cas systems exploit the phage life cycle to generate a pattern of spacer acquisition that ensures a successful CRISPR immune response.

A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation—and Beyond?

Directory of Open Access Journals (Sweden)

Sabrina Schreiner

2017-05-01

Full Text Available Chronic hepatitis B virus (HBV infection puts more than 250 million people at a greatly increased risk to develop end-stage liver disease. Like all hepadnaviruses, HBV replicates via protein-primed reverse transcription of a pregenomic (pg RNA, yielding an unusually structured, viral polymerase-linked relaxed-circular (RC DNA as genome in infectious particles. Upon infection, RC-DNA is converted into nuclear covalently closed circular (ccc DNA. Associating with cellular proteins into an episomal minichromosome, cccDNA acts as template for new viral RNAs, ensuring formation of progeny virions. Hence, cccDNA represents the viral persistence reservoir that is not directly targeted by current anti-HBV therapeutics. Eliminating cccDNA will thus be at the heart of a cure for chronic hepatitis B. The low production of HBV cccDNA in most experimental models and the associated problems in reliable cccDNA quantitation have long hampered a deeper understanding of cccDNA molecular biology. Recent advancements including cccDNA-dependent cell culture systems have begun to identify select host DNA repair enzymes that HBV usurps for RC-DNA to cccDNA conversion. While this list is bound to grow, it may represent just one facet of a broader interaction with the cellular DNA damage response (DDR, a network of pathways that sense and repair aberrant DNA structures and in the process profoundly affect the cell cycle, up to inducing cell death if repair fails. Given the divergent interactions between other viruses and the DDR it will be intriguing to see how HBV copes with this multipronged host system.

A feature-based approach to modeling protein-DNA interactions.

Directory of Open Access Journals (Sweden)

Eilon Sharon

Full Text Available Transcription factor (TF binding to its DNA target site is a fundamental regulatory interaction. The most common model used to represent TF binding specificities is a position specific scoring matrix (PSSM, which assumes independence between binding positions. However, in many cases, this simplifying assumption does not hold. Here, we present feature motif models (FMMs, a novel probabilistic method for modeling TF-DNA interactions, based on log-linear models. Our approach uses sequence features to represent TF binding specificities, where each feature may span multiple positions. We develop the mathematical formulation of our model and devise an algorithm for learning its structural features from binding site data. We also developed a discriminative motif finder, which discovers de novo FMMs that are enriched in target sets of sequences compared to background sets. We evaluate our approach on synthetic data and on the widely used TF chromatin immunoprecipitation (ChIP dataset of Harbison et al. We then apply our algorithm to high-throughput TF ChIP data from mouse and human, reveal sequence features that are present in the binding specificities of mouse and human TFs, and show that FMMs explain TF binding significantly better than PSSMs. Our FMM learning and motif finder software are available at http://genie.weizmann.ac.il/.

Mechanisms for radiation damage in DNA

International Nuclear Information System (INIS)

Sevilla, M.D.

1987-01-01

Several mechanisms are proposed for radiation damage to DNA and its constituents, and a series of experiments utilizing electron spin resonance spectrometry have been used to test the proposed mechanisms. In the past we have concentrated chiefly on investigating irradiated systems of DNA constituents. In this year's effort we have concentrated on radiation effects on DNA itself. In addition studies of radiation effects on lipids and model compounds have been performed which shed light on the only other proposed site for cell kill, the membrane

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.

Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB

Science.gov (United States)

Munir, Ahsan; Waseem, Hassan; Williams, Maggie R.; Stedtfeld, Robert D.; Gulari, Erdogan; Tiedje, James M.; Hashsham, Syed A.

2017-01-01

Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics) to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs). Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R2 = 0.8131). PMID:28555058

Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB

Directory of Open Access Journals (Sweden)

Ahsan Munir

2017-05-01

Full Text Available Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs. Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R2 = 0.8131.

Validation of a sensitive DNA walking strategy to characterise unauthorised GMOs using model food matrices mimicking common rice products.

Science.gov (United States)

Fraiture, Marie-Alice; Herman, Philippe; Taverniers, Isabel; De Loose, Marc; Van Nieuwerburgh, Filip; Deforce, Dieter; Roosens, Nancy H

2015-04-15

To identify unauthorised GMOs in food and feed matrices, an integrated approach has recently been developed targeting pCAMBIA family vectors, highly present in transgenic plants. Their presence is first assessed by qPCR screening and is subsequently confirmed by characterising the transgene flanking regions, using DNA walking. Here, the DNA walking performance has been thoroughly tested for the first time, regarding the targeted DNA quality and quantity. Several assays, on model food matrices mimicking common rice products, have allowed to determine the limit of detection as well as the potential effects of food mixture and processing. This detection system allows the identification of transgenic insertions as low as 10 HGEs and was not affected by the presence of untargeted DNA. Moreover, despite the clear impact of food processing on DNA quality, this method was able to cope with degraded DNA. Given its specificity, sensitivity, reliability, applicability and practicability, the proposed approach is a key detection tool, easily implementable in enforcement laboratories. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

A DNA Computing Model for the Graph Vertex Coloring Problem Based on a Probe Graph

Directory of Open Access Journals (Sweden)

Jin Xu

2018-02-01

Full Text Available The biggest bottleneck in DNA computing is exponential explosion, in which the DNA molecules used as data in information processing grow exponentially with an increase of problem size. To overcome this bottleneck and improve the processing speed, we propose a DNA computing model to solve the graph vertex coloring problem. The main points of the model are as follows: ① The exponential explosion problem is solved by dividing subgraphs, reducing the vertex colors without losing the solutions, and ordering the vertices in subgraphs; and ② the bio-operation times are reduced considerably by a designed parallel polymerase chain reaction (PCR technology that dramatically improves the processing speed. In this article, a 3-colorable graph with 61 vertices is used to illustrate the capability of the DNA computing model. The experiment showed that not only are all the solutions of the graph found, but also more than 99% of false solutions are deleted when the initial solution space is constructed. The powerful computational capability of the model was based on specific reactions among the large number of nanoscale oligonucleotide strands. All these tiny strands are operated by DNA self-assembly and parallel PCR. After thousands of accurate PCR operations, the solutions were found by recognizing, splicing, and assembling. We also prove that the searching capability of this model is up to O(359. By means of an exhaustive search, it would take more than 896 000 years for an electronic computer (5 × 1014 s−1 to achieve this enormous task. This searching capability is the largest among both the electronic and non-electronic computers that have been developed since the DNA computing model was proposed by Adleman’s research group in 2002 (with a searching capability of O(220. Keywords: DNA computing, Graph vertex coloring problem, Polymerase chain reaction

Direct current hopping conductance along DNA chain

Institute of Scientific and Technical Information of China (English)

Ma Song-Shan; Xu Hui; Liu Xiao-Liang; Li Ming-Jun

2007-01-01

This paper proposes a model of direct current(DC) electron hopping transport in DNA,in which DNA is considered as a binary one-dimensional disordered system.To quantitatively study the DC conductivity in DNA,it numerically calculates the DC conductivity of DNA chains with difierent parameter values.The result shows that the DC conductivity of DNA chain increases with the increase of temperature.And the conductivity of DNA chain is depended on the probability P.which represents the degree of compositional disorder in a DNA sequence to some extent.For P＜0.5,the conductivity of DNA chain decreases with the increase of P,while for P≥0.5,the conductivity increases with the increase of p.The DC conductivity in DNA chain also varies with the change of the electric field,it presents non-Ohm's law conductivity characteristics.

Toward metrological traceability for DNA fragment ratios in GM quantification. 3. Suitability of DNA calibrants studied with a MON 810 corn model.

Science.gov (United States)

Charels, Diana; Broeders, Sylvia; Corbisier, Philippe; Trapmann, Stefanie; Schimmel, Heinz; Emons, Hendrik

2007-05-02

The quantification of GMOs by real-time PCR relies on an external calibrant. In this paper the suitability of two DNA calibrants, genomic DNA from plant leaves and plasmidic DNA, was investigated. The PCR efficiencies, the correlation coefficients of the calibration curves, and the ratios between PCR efficiencies of transgenic and endogenous sequences were compared for both calibrants using 59 data sets produced by 43 laboratories. There were no significant differences between plasmidic and genomic DNA except for the PCR efficiencies of the calibration curves for the transgene of the construct-specific real-time PCR method. In the GM system investigated, PCR efficiencies of plasmidic calibrants were slightly closer to the PCR efficiencies observed for the unknowns than those of the genomic DNA calibrant. Therefore, plasmidic DNA was the more suitable calibrant for the PCR measurements on genomic DNA extracted from MON 810 seeds. It is shown that plasmidic DNA is an appropriate choice for the calibration of measurements of MON 810 corn with respect to the DNA copy number ratio.

An integrated multiple capillary array electrophoresis system for high-throughput DNA sequencing

Energy Technology Data Exchange (ETDEWEB)

Lu, X.

1998-03-27

A capillary array electrophoresis system was chosen to perform DNA sequencing because of several advantages such as rapid heat dissipation, multiplexing capabilities, gel matrix filling simplicity, and the mature nature of the associated manufacturing technologies. There are two major concerns for the multiple capillary systems. One concern is inter-capillary cross-talk, and the other concern is excitation and detection efficiency. Cross-talk is eliminated through proper optical coupling, good focusing and immersing capillary array into index matching fluid. A side-entry excitation scheme with orthogonal detection was established for large capillary array. Two 100 capillary array formats were used for DNA sequencing. One format is cylindrical capillary with 150 {micro}m o.d., 75 {micro}m i.d and the other format is square capillary with 300 {micro}m out edge and 75 {micro}m inner edge. This project is focused on the development of excitation and detection of DNA as well as performing DNA sequencing. The DNA injection schemes are discussed for the cases of single and bundled capillaries. An individual sampling device was designed. The base-calling was performed for a capillary from the capillary array with the accuracy of 98%.

PIMS sequencing extension: a laboratory information management system for DNA sequencing facilities

Directory of Open Access Journals (Sweden)

Baldwin Stephen A

2011-03-01

Full Text Available Abstract Background Facilities that provide a service for DNA sequencing typically support large numbers of users and experiment types. The cost of services is often reduced by the use of liquid handling robots but the efficiency of such facilities is hampered because the software for such robots does not usually integrate well with the systems that run the sequencing machines. Accordingly, there is a need for software systems capable of integrating different robotic systems and managing sample information for DNA sequencing services. In this paper, we describe an extension to the Protein Information Management System (PIMS that is designed for DNA sequencing facilities. The new version of PIMS has a user-friendly web interface and integrates all aspects of the sequencing process, including sample submission, handling and tracking, together with capture and management of the data. Results The PIMS sequencing extension has been in production since July 2009 at the University of Leeds DNA Sequencing Facility. It has completely replaced manual data handling and simplified the tasks of data management and user communication. Samples from 45 groups have been processed with an average throughput of 10000 samples per month. The current version of the PIMS sequencing extension works with Applied Biosystems 3130XL 96-well plate sequencer and MWG 4204 or Aviso Theonyx liquid handling robots, but is readily adaptable for use with other combinations of robots. Conclusions PIMS has been extended to provide a user-friendly and integrated data management solution for DNA sequencing facilities that is accessed through a normal web browser and allows simultaneous access by multiple users as well as facility managers. The system integrates sequencing and liquid handling robots, manages the data flow, and provides remote access to the sequencing results. The software is freely available, for academic users, from http://www.pims-lims.org/.

PIMS sequencing extension: a laboratory information management system for DNA sequencing facilities.

Science.gov (United States)

Troshin, Peter V; Postis, Vincent Lg; Ashworth, Denise; Baldwin, Stephen A; McPherson, Michael J; Barton, Geoffrey J

2011-03-07

Facilities that provide a service for DNA sequencing typically support large numbers of users and experiment types. The cost of services is often reduced by the use of liquid handling robots but the efficiency of such facilities is hampered because the software for such robots does not usually integrate well with the systems that run the sequencing machines. Accordingly, there is a need for software systems capable of integrating different robotic systems and managing sample information for DNA sequencing services. In this paper, we describe an extension to the Protein Information Management System (PIMS) that is designed for DNA sequencing facilities. The new version of PIMS has a user-friendly web interface and integrates all aspects of the sequencing process, including sample submission, handling and tracking, together with capture and management of the data. The PIMS sequencing extension has been in production since July 2009 at the University of Leeds DNA Sequencing Facility. It has completely replaced manual data handling and simplified the tasks of data management and user communication. Samples from 45 groups have been processed with an average throughput of 10000 samples per month. The current version of the PIMS sequencing extension works with Applied Biosystems 3130XL 96-well plate sequencer and MWG 4204 or Aviso Theonyx liquid handling robots, but is readily adaptable for use with other combinations of robots. PIMS has been extended to provide a user-friendly and integrated data management solution for DNA sequencing facilities that is accessed through a normal web browser and allows simultaneous access by multiple users as well as facility managers. The system integrates sequencing and liquid handling robots, manages the data flow, and provides remote access to the sequencing results. The software is freely available, for academic users, from http://www.pims-lims.org/.

Nanoparticle-mediated rhodopsin cDNA but not intron-containing DNA delivery causes transgene silencing in a rhodopsin knockout model.

Science.gov (United States)

Zheng, Min; Mitra, Rajendra N; Filonov, Nazar A; Han, Zongchao

2016-03-01

Previously, we compared the efficacy of nanoparticle (NP)-mediated intron-containing rhodopsin (sgRho) vs. intronless cDNA in ameliorating retinal disease phenotypes in a rhodopsin knockout (RKO) mouse model of retinitis pigmentosa. We showed that NP-mediated sgRho delivery achieved long-term expression and phenotypic improvement in RKO mice, but not NP housing cDNA. However, the protein level of the NP-sgRho construct was only 5-10% of wild-type at 8 mo postinjection. To have a better understanding of the reduced levels of long-term expression of the vectors, in the present study, we evaluated the epigenetic changes of subretinal delivering NP-cDNA vs. NP-sgRho in the RKO mouse eyes. Following the administration, DNA methylation and histone status of specific regions (bacteria plasmid backbone, promoter, rhodopsin gene, and scaffold/matrix attachment region) of the vectors were evaluated at various time points. We documented that epigenetic transgene silencing occurred in vector-mediated gene transfer, which were caused by the plasmid backbone and the cDNA of the transgene, but not the intron-containing transgene. No toxicity or inflammation was found in the treated eyes. Our results suggest that cDNA of the rhodopsin transgene and bacteria backbone interfered with the host defense mechanism of DNA methylation-mediated transgene silencing through heterochromatin-associated modifications. © FASEB.

Protecting DNA from errors and damage: an overview of DNA repair mechanisms in plants compared to mammals.

Science.gov (United States)

Spampinato, Claudia P

2017-05-01

The genome integrity of all organisms is constantly threatened by replication errors and DNA damage arising from endogenous and exogenous sources. Such base pair anomalies must be accurately repaired to prevent mutagenesis and/or lethality. Thus, it is not surprising that cells have evolved multiple and partially overlapping DNA repair pathways to correct specific types of DNA errors and lesions. Great progress in unraveling these repair mechanisms at the molecular level has been made by several talented researchers, among them Tomas Lindahl, Aziz Sancar, and Paul Modrich, all three Nobel laureates in Chemistry for 2015. Much of this knowledge comes from studies performed in bacteria, yeast, and mammals and has impacted research in plant systems. Two plant features should be mentioned. Plants differ from higher eukaryotes in that they lack a reserve germline and cannot avoid environmental stresses. Therefore, plants have evolved different strategies to sustain genome fidelity through generations and continuous exposure to genotoxic stresses. These strategies include the presence of unique or multiple paralogous genes with partially overlapping DNA repair activities. Yet, in spite (or because) of these differences, plants, especially Arabidopsis thaliana, can be used as a model organism for functional studies. Some advantages of this model system are worth mentioning: short life cycle, availability of both homozygous and heterozygous lines for many genes, plant transformation techniques, tissue culture methods and reporter systems for gene expression and function studies. Here, I provide a current understanding of DNA repair genes in plants, with a special focus on A. thaliana. It is expected that this review will be a valuable resource for future functional studies in the DNA repair field, both in plants and animals.

A methodology to annotate systems biology markup language models with the synthetic biology open language.

Science.gov (United States)

Roehner, Nicholas; Myers, Chris J

2014-02-21

Recently, we have begun to witness the potential of synthetic biology, noted here in the form of bacteria and yeast that have been genetically engineered to produce biofuels, manufacture drug precursors, and even invade tumor cells. The success of these projects, however, has often failed in translation and application to new projects, a problem exacerbated by a lack of engineering standards that combine descriptions of the structure and function of DNA. To address this need, this paper describes a methodology to connect the systems biology markup language (SBML) to the synthetic biology open language (SBOL), existing standards that describe biochemical models and DNA components, respectively. Our methodology involves first annotating SBML model elements such as species and reactions with SBOL DNA components. A graph is then constructed from the model, with vertices corresponding to elements within the model and edges corresponding to the cause-and-effect relationships between these elements. Lastly, the graph is traversed to assemble the annotating DNA components into a composite DNA component, which is used to annotate the model itself and can be referenced by other composite models and DNA components. In this way, our methodology can be used to build up a hierarchical library of models annotated with DNA components. Such a library is a useful input to any future genetic technology mapping algorithm that would automate the process of composing DNA components to satisfy a behavioral specification. Our methodology for SBML-to-SBOL annotation is implemented in the latest version of our genetic design automation (GDA) software tool, iBioSim.

Electronic transport in methylated fragments of DNA

Energy Technology Data Exchange (ETDEWEB)

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

2015-11-16

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

Electronic transport in methylated fragments of DNA

International Nuclear Information System (INIS)

Almeida, M. L. de; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L.; Albuquerque, E. L.; Freire, V. N.; Caetano, E. W. S.; Moura, F. A. B. F. de; Lyra, M. L.

2015-01-01

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

Erythropoietin overrides the triggering effect of DNA platination products in a mouse model of Cisplatin-induced neuropathy

Directory of Open Access Journals (Sweden)

Egensperger Rupert

2009-07-01

Full Text Available Abstract Background Cisplatin mediates its antineoplastic activity by formation of distinct DNA intrastrand cross links. The clinical efficacy and desirable dose escalations of cisplatin are restricted by the accumulation of DNA lesions in dorsal root ganglion (DRG cells leading to sensory polyneuropathy (PNP. We investigated in a mouse model by which mechanism recombinant erythropoietin (rhEPO protects the peripheral nervous system from structural and functional damage caused by cisplatin treatment with special emphasis on DNA damage burden. Results A cumulative dose of 16 mg cisplatin/kg resulted in clear electrophysiological signs of neuropathy, which were significantly attenuated by concomitant erythropoietin (cisplatin 32,48 m/s ± 1,68 m/s; cisplatin + rhEPO 49,66 m/s ± 1,26 m/s; control 55,01 m/s ± 1,88 m/s; p Conclusion The protective effect of recombinant erythropoietin is not mediated by reducing the burden of DNA platination in the target cells, but it is likely to be due to a higher resistance of the target cells to the adverse effect of DNA damage. The increased frequency of intact mitochondria might also contribute to this protective role.

Social Fingerprinting: detection of spambot groups through DNA-inspired behavioral modeling

DEFF Research Database (Denmark)

Cresci, Stefano; Di Pietro, Roberto; Petrocchi, Marinella

2017-01-01

Spambot detection in online social networks is a long-lasting challenge involving the study and design of detection techniques capable of efficiently identifying ever-evolving spammers. Recently, a new wave of social spambots has emerged, with advanced human-like characteristics that allow them...... to go undetected even by current state-of-the-art algorithms. In this paper, we show that efficient spambots detection can be achieved via an in-depth analysis of their collective behaviors exploiting the digital DNA technique for modeling the behaviors of social network users. Inspired by its...... biological counterpart, in the digital DNA representation the behavioral lifetime of a digital account is encoded in a sequence of characters. Then, we define a similarity measure for such digital DNA sequences. We build upon digital DNA and the similarity between groups of users to characterize both genuine...

Impact of DNA mismatch repair system alterations on human fertility and related treatments.

Science.gov (United States)

Hu, Min-hao; Liu, Shu-yuan; Wang, Ning; Wu, Yan; Jin, Fan

2016-01-01

DNA mismatch repair (MMR) is one of the biological pathways, which plays a critical role in DNA homeostasis, primarily by repairing base-pair mismatches and insertion/deletion loops that occur during DNA replication. MMR also takes part in other metabolic pathways and regulates cell cycle arrest. Defects in MMR are associated with genomic instability, predisposition to certain types of cancers and resistance to certain therapeutic drugs. Moreover, genetic and epigenetic alterations in the MMR system demonstrate a significant relationship with human fertility and related treatments, which helps us to understand the etiology and susceptibility of human infertility. Alterations in the MMR system may also influence the health of offspring conceived by assisted reproductive technology in humans. However, further studies are needed to explore the specific mechanisms by which the MMR system may affect human infertility. This review addresses the physiological mechanisms of the MMR system and associations between alterations of the MMR system and human fertility and related treatments, and potential effects on the next generation.

Direct Atomic Force Microscopy Observation of DNA Tile Crystal Growth at the Single-Molecule Level

OpenAIRE

Evans, Constantine G.; Hariadi, Rizal F.; Winfree, Erik

2012-01-01

While the theoretical implications of models of DNA tile self-assembly have been extensively researched and such models have been used to design DNA tile systems for use in experiments, there has been little research testing the fundamental assumptions of those models. In this paper, we use direct observation of individual tile attachments and detachments of two DNA tile systems on a mica surface imaged with an atomic force microscope (AFM) to compile statistics of tile attachments and detach...

Using DNA nanotechnology to produce a drug delivery system

Science.gov (United States)

Huyen La, Thi; Thu Thuy Nguyen, Thi; Phuc Pham, Van; Huyen Nguyen, Thi Minh; Huan Le, Quang

2013-03-01

Drug delivery to cancer cells in chemotherapy is one of the most advanced research topics. The effectiveness of the current cancer treatment drugs is limited because they are not capable of distinguishing between cancer cells and normal cells so that they kill not only cancer cells but also normal ones. To overcome this disadvantage by profiting from the differences in physical and chemical properties between cancer and normal cells, nanoparticles (NPs) delivering a drug are designed in a specific manner such that they can distinguish the cancer cells from the normal ones and are targeted only to the cancer cells. Currently, there are various drug delivery systems with many advantages, but sharing some common disadvantages such as difficulty with controlling the size, low encapsulation capacity and low stability. With the development and success of DNA nanotechnology, DNA strands are used to create effective drug delivery NPs with precisely controlled size and structure, safety and high stability. This article presents our study on drug encapsulation in DNA nanostructure which loaded docetaxel and curcumin in a desire to create a new and effective drug delivery system with high biological compatibility. Invited talk at the 6th International Workshop on Advanced Materials Science and Nanotechnology, 30 October-2 November, 2012, Ha Long, Vietnam.

DNA vaccination for cervical cancer: Strategic optimisation of RALA mediated gene delivery from a biodegradable microneedle system.

Science.gov (United States)

Cole, Grace; Ali, Ahlam A; McCrudden, Cian M; McBride, John W; McCaffrey, Joanne; Robson, Tracy; Kett, Vicky L; Dunne, Nicholas J; Donnelly, Ryan F; McCarthy, Helen O

2018-03-03

Dissolvable microneedles can be employed to deliver DNA to antigen presenting cells within the skin. However, this technology faces two main challenges: the poor transfection efficacy of pDNA following release from the microneedle matrix, and the limited loading capacity of the micron-scale devices. Two-tier delivery systems combining microneedle platforms and DNA delivery vectors have increased efficacy but the challenge of increasing the loading capacity remains. This study utilised lyophilisation to increase the loading of RALA/pDNA nanoparticles within dissolvable PVA microneedles. As a result, delivery was significantly enhanced in vivo into an appropriate range for DNA vaccination (∼50 μg per array). Furthermore, modifying the manufacturing process was not detrimental to the microneedle mechanical properties or cargo functionality. It was demonstrated that arrays retained mechanical and functional stability over short term storage, and were able to elicit gene expression in vitro and in vivo. Finally, treatment with this novel formulation significantly retarded the growth of established tumours, and proved superior to standard intramuscular injection in a preclinical model of cervical cancer. Copyright © 2018. Published by Elsevier B.V.

Endurance exercise rescues progeroid aging and induces systemic mitochondrial rejuvenation in mtDNA mutator mice

Science.gov (United States)

Safdar, Adeel; Bourgeois, Jacqueline M.; Ogborn, Daniel I.; Little, Jonathan P.; Hettinga, Bart P.; Akhtar, Mahmood; Thompson, James E.; Melov, Simon; Mocellin, Nicholas J.; Kujoth, Gregory C.; Prolla, Tomas A.; Tarnopolsky, Mark A.

2011-01-01

A causal role for mitochondrial DNA (mtDNA) mutagenesis in mammalian aging is supported by recent studies demonstrating that the mtDNA mutator mouse, harboring a defect in the proofreading-exonuclease activity of mitochondrial polymerase gamma, exhibits accelerated aging phenotypes characteristic of human aging, systemic mitochondrial dysfunction, multisystem pathology, and reduced lifespan. Epidemiologic studies in humans have demonstrated that endurance training reduces the risk of chronic diseases and extends life expectancy. Whether endurance exercise can attenuate the cumulative systemic decline observed in aging remains elusive. Here we show that 5 mo of endurance exercise induced systemic mitochondrial biogenesis, prevented mtDNA depletion and mutations, increased mitochondrial oxidative capacity and respiratory chain assembly, restored mitochondrial morphology, and blunted pathological levels of apoptosis in multiple tissues of mtDNA mutator mice. These adaptations conferred complete phenotypic protection, reduced multisystem pathology, and prevented premature mortality in these mice. The systemic mitochondrial rejuvenation through endurance exercise promises to be an effective therapeutic approach to mitigating mitochondrial dysfunction in aging and related comorbidities. PMID:21368114

Track structure based modelling of light ion radiation effects on nuclear and mitochondrial DNA

Science.gov (United States)

Schmitt, Elke; Ottolenghi, Andrea; Dingfelder, Michael; Friedland, Werner; Kundrat, Pavel; Baiocco, Giorgio

2016-07-01

Space radiation risk assessment is of great importance for manned spaceflights in order to estimate risks and to develop counter-measures to reduce them. Biophysical simulations with PARTRAC can help greatly to improve the understanding of initial biological response to ionizing radiation. Results from modelling radiation quality dependent DNA damage and repair mechanisms up to chromosomal aberrations (e.g. dicentrics) can be used to predict radiation effects depending on the kind of mixed radiation field exposure. Especially dicentric yields can serve as a biomarker for an increased risk due to radiation and hence as an indicator for the effectiveness of the used shielding. PARTRAC [1] is a multi-scale biophysical research MC code for track structure based initial DNA damage and damage response modelling. It integrates physics, radiochemistry, detailed nuclear DNA structure and molecular biology of DNA repair by NHEJ-pathway to assess radiation effects on cellular level [2]. Ongoing experiments with quasi-homogeneously distributed compared to sub-micrometre focused bunches of protons, lithium and carbon ions allow a separation of effects due to DNA damage complexity on nanometre scale from damage clustering on (sub-) micrometre scale [3, 4]. These data provide an unprecedented benchmark for the DNA damage response model in PARTRAC and help understand the mechanisms leading to cell killing and chromosomal aberrations (e.g. dicentrics) induction. A large part of space radiation is due to a mixed ion field of high energy protons and few heavier ions that can be only partly absorbed by the shielding. Radiation damage induced by low-energy ions significantly contributes to the high relative biological efficiency (RBE) of ion beams around Bragg peak regions. For slow light ions the physical cross section data basis in PARTRAC has been extended to investigate radiation quality effects in the Bragg peak region [5]. The resulting range and LET values agree with ICRU data

A model for the induction of DNA damages and their evolution into cell clonogenic inactivation

International Nuclear Information System (INIS)

Yamaguchi, Hiroshi; Ohara, Hiroshi; Waker, A.J.

2006-01-01

The dependence of the initial production of DNA damages on radiation quality was examined by using a proposed new model on the basis of target theory. For the estimation of DNA damage-production by different radiation qualities, five possible modes of radiation action, including both direct and indirect effects, were assumed inside a target the molecular structure of which was defined to consist of 10 base-pairs of DNA surrounded by water molecules. The induction of DNA damage was modeled on the basis of comparisons between the primary ionization mean free path and the distance between pairs of ionized atoms, such distance being characteristic on the mode of radiation action. The OH radicals per average energy to produce an ion pair on the nanosecond time scale was estimated and used for indirect action. Assuming a relation between estimated yields of DNA damages and experimental inactivation cross sections for AT-cells, the present model enabled the quantitative reproduction of experimental results for AT-cell killing under aerobic or hypoxic conditions. The results suggest a higher order organization of DNA in a way that there will be at least two types of water environment, one filling half the space surrounding DNA with a depth of 3.7-4.3 nm and the other filling all space with a depth 4.6-4.9 nm. (author)

DNA damage response and DNA repair – dog as a model?

International Nuclear Information System (INIS)

Grosse, Nicole; Loon, Barbara van; Rohrer Bley, Carla

2014-01-01

Companion animals like dogs frequently develop tumors with age and similarly to human malignancies, display interpatient tumoral heterogeneity. Tumors are frequently characterized with regard to their mutation spectra, changes in gene expression or protein levels. Among others, these changes affect proteins involved in the DNA damage response (DDR), which served as a basis for the development of numerous clinically relevant cancer therapies. Even though the effects of different DNA damaging agents, as well as DDR kinetics, have been well characterized in mammalian cells in vitro, very little is so far known about the kinetics of DDR in tumor and normal tissues in vivo. Due to (i) the similarities between human and canine genomes, (ii) the course of spontaneous tumor development, as well as (iii) common exposure to environmental agents, canine tumors are potentially an excellent model to study DDR in vivo. This is further supported by the fact that dogs show approximately the same rate of tumor development with age as humans. Though similarities between human and dog osteosarcoma, as well as mammary tumors have been well established, only few studies using canine tumor samples addressed the importance of affected DDR pathways in tumor progression, thus leaving many questions unanswered. Studies in humans showed that misregulated DDR pathways play an important role during tumor development, as well as in treatment response. Since dogs are proposed to be a good tumor model in many aspects of cancer research, we herein critically investigate the current knowledge of canine DDR and discuss (i) its future potential for studies on the in vivo level, as well as (ii) its possible translation to veterinary and human medicine

From nonspecific DNA-protein encounter complexes to the prediction of DNA-protein interactions.

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Mu Gao

2009-03-01

Full Text Available DNA-protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA-protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA-protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA-protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA-protein interaction modes exhibit some similarity to specific DNA-protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Calpha deviation from native is up to 5 A from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA-protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein.

Genomic survey, gene expression analysis and structural modeling suggest diverse roles of DNA methyltransferases in legumes.

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Rohini Garg

Full Text Available DNA methylation plays a crucial role in development through inheritable gene silencing. Plants possess three types of DNA methyltransferases (MTases, namely Methyltransferase (MET, Chromomethylase (CMT and Domains Rearranged Methyltransferase (DRM, which maintain methylation at CG, CHG and CHH sites. DNA MTases have not been studied in legumes so far. Here, we report the identification and analysis of putative DNA MTases in five legumes, including chickpea, soybean, pigeonpea, Medicago and Lotus. MTases in legumes could be classified in known MET, CMT, DRM and DNA nucleotide methyltransferases (DNMT2 subfamilies based on their domain organization. First three MTases represent DNA MTases, whereas DNMT2 represents a transfer RNA (tRNA MTase. Structural comparison of all the MTases in plants with known MTases in mammalian and plant systems have been reported to assign structural features in context of biological functions of these proteins. The structure analysis clearly specified regions crucial for protein-protein interactions and regions important for nucleosome binding in various domains of CMT and MET proteins. In addition, structural model of DRM suggested that circular permutation of motifs does not have any effect on overall structure of DNA methyltransferase domain. These results provide valuable insights into role of various domains in molecular recognition and should facilitate mechanistic understanding of their function in mediating specific methylation patterns. Further, the comprehensive gene expression analyses of MTases in legumes provided evidence of their role in various developmental processes throughout the plant life cycle and response to various abiotic stresses. Overall, our study will be very helpful in establishing the specific functions of DNA MTases in legumes.

DNA Mismatch Repair System: Repercussions in Cellular Homeostasis and Relationship with Aging

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Juan Cristóbal Conde-Pérezprina

2012-01-01

Full Text Available The mechanisms that concern DNA repair have been studied in the last years due to their consequences in cellular homeostasis. The diverse and damaging stimuli that affect DNA integrity, such as changes in the genetic sequence and modifications in gene expression, can disrupt the steady state of the cell and have serious repercussions to pathways that regulate apoptosis, senescence, and cancer. These altered pathways not only modify cellular and organism longevity, but quality of life (“health-span”. The DNA mismatch repair system (MMR is highly conserved between species; its role is paramount in the preservation of DNA integrity, placing it as a necessary focal point in the study of pathways that prolong lifespan, aging, and disease. Here, we review different insights concerning the malfunction or absence of the DNA-MMR and its impact on cellular homeostasis. In particular, we will focus on DNA-MMR mechanisms regulated by known repair proteins MSH2, MSH6, PMS2, and MHL1, among others.

A programming language for composable DNA circuits.

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Phillips, Andrew; Cardelli, Luca

2009-08-06

Recently, a range of information-processing circuits have been implemented in DNA by using strand displacement as their main computational mechanism. Examples include digital logic circuits and catalytic signal amplification circuits that function as efficient molecular detectors. As new paradigms for DNA computation emerge, the development of corresponding languages and tools for these paradigms will help to facilitate the design of DNA circuits and their automatic compilation to nucleotide sequences. We present a programming language for designing and simulating DNA circuits in which strand displacement is the main computational mechanism. The language includes basic elements of sequence domains, toeholds and branch migration, and assumes that strands do not possess any secondary structure. The language is used to model and simulate a variety of circuits, including an entropy-driven catalytic gate, a simple gate motif for synthesizing large-scale circuits and a scheme for implementing an arbitrary system of chemical reactions. The language is a first step towards the design of modelling and simulation tools for DNA strand displacement, which complements the emergence of novel implementation strategies for DNA computing.

Development of an Automated Microfluidic System for DNA Collection, Amplification, and Detection of Pathogens

Energy Technology Data Exchange (ETDEWEB)

Hagan, Bethany S.; Bruckner-Lea, Cynthia J.

2002-12-01

This project was focused on developing and testing automated routines for a microfluidic Pathogen Detection System. The basic pathogen detection routine has three primary components; cell concentration, DNA amplification, and detection. In cell concentration, magnetic beads are held in a flow cell by an electromagnet. Sample liquid is passed through the flow cell and bacterial cells attach to the beads. These beads are then released into a small volume of fluid and delivered to the peltier device for cell lysis and DNA amplification. The cells are lysed during initial heating in the peltier device, and the released DNA is amplified using polymerase chain reaction (PCR) or strand displacement amplification (SDA). Once amplified, the DNA is then delivered to a laser induced fluorescence detection unit in which the sample is detected. These three components create a flexible platform that can be used for pathogen detection in liquid and sediment samples. Future developments of the system will include on-line DNA detection during DNA amplification and improved capture and release methods for the magnetic beads during cell concentration.

Integration of the Reconfigurable Self-Healing eDNA Architecture in an Embedded System

Science.gov (United States)

Boesen, Michael Reibel; Keymeulen, Didier; Madsen, Jan; Lu, Thomas; Chao, Tien-Hsin

2011-01-01

In this work we describe the first real world case study for the self-healing eDNA (electronic DNA) architecture by implementing the control and data processing of a Fourier Transform Spectrometer (FTS) on an eDNA prototype. For this purpose the eDNA prototype has been ported from a Xilinx Virtex 5 FPGA to an embedded system consisting of a PowerPC and a Xilinx Virtex 5 FPGA. The FTS instrument features a novel liquid crystal waveguide, which consequently eliminates all moving parts from the instrument. The addition of the eDNA architecture to do the control and data processing has resulted in a highly fault-tolerant FTS instrument. The case study has shown that the early stage prototype of the autonomous self-healing eDNA architecture is expensive in terms of execution time.

Application of a random walk model to geographic distributions of animal mitochondrial DNA variation.

Science.gov (United States)

Neigel, J E; Avise, J C

1993-12-01

In rapidly evolving molecules, such as animal mitochondrial DNA, mutations that delineate specific lineages may not be dispersed at sufficient rates to attain an equilibrium between genetic drift and gene flow. Here we predict conditions that lead to nonequilibrium geographic distributions of mtDNA lineages, test the robustness of these predictions and examine mtDNA data sets for consistency with our model. Under a simple isolation by distance model, the variance of an mtDNA lineage's geographic distribution is expected be proportional to its age. Simulation results indicated that this relationship is fairly robust. Analysis of mtDNA data from natural populations revealed three qualitative distributional patterns: (1) significant departure of lineage structure from equilibrium geographic distributions, a pattern exhibited in three rodent species with limited dispersal; (2) nonsignificant departure from equilibrium expectations, exhibited by two avian and two marine fish species with potentials for relatively long-distance dispersal; and (3) a progression from nonequilibrium distributions for younger lineages to equilibrium distributions for older lineages, a condition displayed by one surveyed avian species. These results demonstrate the advantages of considering mutation and genealogy in the interpretation of mtDNA geographic variation.

Sequential action of ATPase, ATP, ADP, Pi and dsDNA in procapsid-free system to enlighten mechanism in viral dsDNA packaging.

Science.gov (United States)

Schwartz, Chad; Fang, Huaming; Huang, Lisa; Guo, Peixuan

2012-03-01

Many cells and double-stranded DNA (dsDNA) viruses contain an AAA(+) ATPase that assembles into oligomers, often hexamers, with a central channel. The dsDNA packaging motor of bacteriophage phi29 also contains an ATPase to translocate dsDNA through a dodecameric channel. The motor ATPase has been investigated substantially in the context of the entire procapsid. Here, we report the sequential action between the ATPase and additional motor components. It is suggested that the contact of ATPase to ATP resulted in its conformational change to a higher binding affinity toward dsDNA. It was found that ATP hydrolysis led to the departure of dsDNA from the ATPase/dsDNA complex, an action that is speculated to push dsDNA to pass the connector channel. Our results suggest that dsDNA packaging goes through a combined effort of both the gp16 ATPase for pushing and the channel as a one-way valve to control the dsDNA translocation direction. Many packaging models have previously been proposed, and the packaging mechanism has been contingent upon the number of nucleotides packaged per ATP relative to the 10.5 bp per helical turn for B-type dsDNA. Both 2 and 2.5 bp per ATP have been used to argue for four, five or six discrete steps of dsDNA translocation. Combination of the two distinct roles of gp16 and connector renews the perception of previous dsDNA packaging energy calculations and provides insight into the discrepancy between 2 and 2.5 bp per ATP.

Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

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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.

Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains

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Sangalli, Juliano Rodrigues; Rodrigues, Thiago Bittencourt; Smith, Lawrence Charles; Meirelles, Flávio Vieira; Chiaratti, Marcos Roberto

2015-01-01

Mouse models are widely employed to study mitochondrial inheritance, which have implications to several human diseases caused by mutations in the mitochondrial genome (mtDNA). These mouse models take advantage of polymorphisms between the mtDNA of the NZB/BINJ and the mtDNA of common inbred laboratory (i.e., C57BL/6) strains to generate mice with two mtDNA haplotypes (heteroplasmy). Based on PCR followed by restriction fragment length polymorphism (PCR-RFLP), these studies determine the level of heteroplasmy across generations and in different cell types aiming to understand the mechanisms underlying mitochondrial inheritance. However, PCR-RFLP is a time-consuming method of low sensitivity and accuracy that dependents on the use of restriction enzyme digestions. A more robust method to measure heteroplasmy has been provided by the use of real-time quantitative PCR (qPCR) based on allelic refractory mutation detection system (ARMS-qPCR). Herein, we report an ARMS-qPCR assay for quantification of heteroplasmy using heteroplasmic mice with mtDNA of NZB/BINJ and C57BL/6 origin. Heteroplasmy and mtDNA copy number were estimated in germline and somatic tissues, providing evidence of the reliability of the approach. Furthermore, it enabled single-step quantification of heteroplasmy, with sensitivity to detect as low as 0.1% of either NZB/BINJ or C57BL/6 mtDNA. These findings are relevant as the ARMS-qPCR assay reported here is fully compatible with similar heteroplasmic mouse models used to study mitochondrial inheritance in mammals. PMID:26274500

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

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Françoise Paquet

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

The role of DNA restriction-modification systems in the biology of Bacillus anthracis

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Ramakrishnan eSitaraman

2016-01-01

Full Text Available Restriction-modification (R-M systems are widespread among prokaryotes and, depending on their type, may be viewed as selfish genetic elements that persist as toxin-antitoxin modules or as cellular defense systems against phage infection. Studies in the last decade have made it amply clear that these two options do not exhaust the list of possible biological roles for R-M systems. Their presence in a cell may also have a bearing on other processes such as horizontal gene transfer and gene regulation. From genome sequencing and experimental data, we know that Bacillus anthracis encodes at least three methylation-dependent (typeIV restriction endonucleases, and an orphan DNA methyltransferase. In this article, we first present an outline of our current knowledge of R-M systems in Bacillus anthracis. Based on available DNA sequence data, and on our current understanding of the functions of similar genes in other systems, we conclude with hypotheses on the possible roles of the three restriction endonucleases and the orphan DNA methyltransferase.

Efficacy and Safety Profile of Tricyclo-DNA Antisense Oligonucleotides in Duchenne Muscular Dystrophy Mouse Model

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Karima Relizani

2017-09-01

Full Text Available Antisense oligonucleotides (AONs hold promise for therapeutic splice-switching correction in many genetic diseases. However, despite advances in AON chemistry and design, systemic use of AONs is limited due to poor tissue uptake and sufficient therapeutic efficacy is still difficult to achieve. A novel class of AONs made of tricyclo-DNA (tcDNA is considered very promising for the treatment of Duchenne muscular dystrophy (DMD, a neuromuscular disease typically caused by frameshifting deletions or nonsense mutations in the gene-encoding dystrophin and characterized by progressive muscle weakness, cardiomyopathy, and respiratory failure in addition to cognitive impairment. Herein, we report the efficacy and toxicology profile of a 13-mer tcDNA in mdx mice. We show that systemic delivery of 13-mer tcDNA allows restoration of dystrophin in skeletal muscles and to a lower extent in the brain, leading to muscle function improvement and correction of behavioral features linked to the emotional/cognitive deficiency. More importantly, tcDNA treatment was generally limited to minimal glomerular changes and few cell necroses in proximal tubules, with only slight variation in serum and urinary kidney toxicity biomarker levels. These results demonstrate an encouraging safety profile for tcDNA, albeit typical of phosphorothiate AONs, and confirm its therapeutic potential for the systemic treatment of DMD patients. Keywords: antisense oligonucleotides, Duchenne muscular dystrophy, preclinical, splice switching, tcDNA-AONs

MiniX-STR multiplex system population study in Japan and application to degraded DNA analysis.

Science.gov (United States)

Asamura, H; Sakai, H; Kobayashi, K; Ota, M; Fukushima, H

2006-05-01

We sought to evaluate a more effective system for analyzing X-chromosomal short tandem repeats (X-STRs) in highly degraded DNA. To generate smaller amplicon lengths, we designed new polymerase chain reaction (PCR) primers for DXS7423, DXS6789, DXS101, GATA31E08, DXS8378, DXS7133, DXS7424, and GATA165B12 at X-linked short tandem repeat (STR) loci, devising two miniX-multiplex PCR systems. Among 333 Japanese individuals, these X-linked loci were detected in amplification products ranging in length from 76 to 169 bp, and statistical analyses of the eight loci indicated a high usefulness for the Japanese forensic practice. Results of tests on highly degraded DNA indicated the miniX-STR multiplex strategies to be an effective system for analyzing degraded DNA. We conclude that analysis by the current miniX-STR multiplex systems offers high effectiveness for personal identification from degraded DNA samples.

MIDAS: A Modular DNA Assembly System for Synthetic Biology.

Science.gov (United States)

van Dolleweerd, Craig J; Kessans, Sarah A; Van de Bittner, Kyle C; Bustamante, Leyla Y; Bundela, Rudranuj; Scott, Barry; Nicholson, Matthew J; Parker, Emily J

2018-04-20

A modular and hierarchical DNA assembly platform for synthetic biology based on Golden Gate (Type IIS restriction enzyme) cloning is described. This enabling technology, termed MIDAS (for Modular Idempotent DNA Assembly System), can be used to precisely assemble multiple DNA fragments in a single reaction using a standardized assembly design. It can be used to build genes from libraries of sequence-verified, reusable parts and to assemble multiple genes in a single vector, with full user control over gene order and orientation, as well as control of the direction of growth (polarity) of the multigene assembly, a feature that allows genes to be nested between other genes or genetic elements. We describe the detailed design and use of MIDAS, exemplified by the reconstruction, in the filamentous fungus Penicillium paxilli, of the metabolic pathway for production of paspaline and paxilline, key intermediates in the biosynthesis of a range of indole diterpenes-a class of secondary metabolites produced by several species of filamentous fungi. MIDAS was used to efficiently assemble a 25.2 kb plasmid from 21 different modules (seven genes, each composed of three basic parts). By using a parts library-based system for construction of complex assemblies, and a unique set of vectors, MIDAS can provide a flexible route to assembling tailored combinations of genes and other genetic elements, thereby supporting synthetic biology applications in a wide range of expression hosts.

Fixing the model for transcription: the DNA moves, not the polymerase.

Science.gov (United States)

Papantonis, Argyris; Cook, Peter R

2011-01-01

The traditional model for transcription sees active polymerases tracking along their templates. An alternative (controversial) model has active enzymes immobilized in "factories." Recent evidence supports the idea that the DNA moves, not the polymerase, and points to alternative explanations of how regulatory motifs like enhancers and silencers work.

Effects of benzo[a]pyrene-DNA adducts on a reconstituted replication system

International Nuclear Information System (INIS)

Brown, W.C.; Romano, L.J.

1991-01-01

The authors have used a partially reconstituted replication system consisting of T7 DNA polymerase and T7 gene 4 protein to examine the effect of benzo[a]pyrene (B[a]P) adducts on DNA synthesis and gene 4 protein activities. The gene 4 protein is required for T7 DNA replication because of its ability to act as both a primase and helicase. They show here that total synthesis decreases as the level of adducts per molecule of DNA increases, suggesting that the B[a]P adducts are blocking an aspect of the replication process. By challenging synthesis on oligonucleotide-primed B[a]P-modified DNA with unmodified DNA, they present evidence that the T7 DNA polymerase freely dissociates after encountering an adduct. Prior studies have shown that the gene 4 protein alone does not dissociate from the template during translocation upon encountering an adduct. However, when gene 4 protein primed DNA synthesis is challenged, they observe an increase in synthesis but to a lesser extent than observed on oligonucleotide-primed synthesis. Finally, they have examined DNA synthesis on duplex templates and show the B[a]P adducts inhibit synthesis by the T7 DNA polymerase and gene 4 protein to the same extent regardless of whether the adducts are positioned in the leading or lagging strand, while synthesis by the polymerase alone is inhibited only when the adducts are in the template strand

Feasibility of using DNA-immobilized nanocellulose-based immunoadsorbent for systemic lupus erythematosus plasmapheresis.

Science.gov (United States)

Xu, Changgang; Carlsson, Daniel O; Mihranyan, Albert

2016-07-01

The goal of this project was to study the feasibility of using a DNA-immobilized nanocellulose-based immunoadsorbent for possible application in medical apheresis such as systemic lupus erythematosus (SLE) treatment. Calf thymus DNA was bound to high surface area nanocellulose membrane at varying concentrations using UV-irradiation. The DNA-immobilized samples were characterized with scanning electron microscopy, atomic force microscopy, and phosphorus elemental analysis. The anti-ds-DNA IgG binding was tested in vitro using ELISA. The produced sample showed high affinity in vitro to bind anti-ds-DNA-antibodies from mice, as much as 80% of added IgG was bound by the membrane. Furthermore, the binding efficiency was quantitatively dependent on the amount of immobilized DNA onto nanocellulose membrane. The described nanocellulose membranes are interesting immunoadsorbents for continued clinical studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Information management in DNA replication modeled by directional, stochastic chains with memory

Science.gov (United States)

Arias-Gonzalez, J. Ricardo

2016-11-01

Stochastic chains represent a key variety of phenomena in many branches of science within the context of information theory and thermodynamics. They are typically approached by a sequence of independent events or by a memoryless Markov process. Stochastic chains are of special significance to molecular biology, where genes are conveyed by linear polymers made up of molecular subunits and transferred from DNA to proteins by specialized molecular motors in the presence of errors. Here, we demonstrate that when memory is introduced, the statistics of the chain depends on the mechanism by which objects or symbols are assembled, even in the slow dynamics limit wherein friction can be neglected. To analyze these systems, we introduce a sequence-dependent partition function, investigate its properties, and compare it to the standard normalization defined by the statistical physics of ensembles. We then apply this theory to characterize the enzyme-mediated information transfer involved in DNA replication under the real, non-equilibrium conditions, reproducing measured error rates and explaining the typical 100-fold increase in fidelity that is experimentally found when proofreading and edition take place. Our model further predicts that approximately 1 kT has to be consumed to elevate fidelity in one order of magnitude. We anticipate that our results are necessary to interpret configurational order and information management in many molecular systems within biophysics, materials science, communication, and engineering.

Formation of malonic dialdehyde and other 2-thiobarbituric-acid-active products in γ-radiolysis of DNA and DNA model substances in aqueous solution

International Nuclear Information System (INIS)

Langfinger, K.D.

1984-01-01

During radiation-induced DNA strand break, a product was observed which reacts positively with 2-thiobarbituric acid (TBA) to malonic dialdehyde (MDA) but is not a free MDA. The paper therefore discusses the formation of products during γ irradiation of DNA and DNA model substances which react positively with TBA to MDA. This reaction is highly sensitive but has low specificity, so that further analytical techniques were used for characterisation. These were: kinematic studies on chromophore formation using TBA, UV spectroscopy, and chromatography. The investigations comprised 1. Irradiation of sugars and polyalcohols. 2. Irradiation of nucleosides and nucleotides. 3. Irradiation of DNA. (orig./PW) [de

Bubble generation in a twisted and bent DNA-like model

DEFF Research Database (Denmark)

Larsen, Peter Ulrik Vingaard; Christiansen, Peter Leth; Bang, Ole

2004-01-01

The DNA molecule is modeled by a parabola embedded chain with long-range interactions between twisted base pair dipoles. A mechanism for bubble generation is presented and investigated in two different configurations. Using random normally distributed initial conditions to simulate thermal...

DNA-mediated gene transfer into human diploid fibroblasts derived from normal and ataxia-telangiectasia donors: parameters for DNA transfer and properties of DNA transformants

International Nuclear Information System (INIS)

Debenham, P.G.; Webb, M.B.T.; Masson, W.K.; Cox, R.

1984-01-01

An investigation was made of the feasibility of DNA-mediated gene transfer into human diploid fibroblasts derived from patients with the radiation sensitive syndrome ataxia-telangiectasia (A-T) and from a normal donor. Although they are markedly different in their growth characteristics, both normal and A-T strains give similar frequencies for DNA transfer in a model system using the recombinant plasmid pSV2-gpt. pSV2-gpt DNA transformants arise with a frequency between 10 -5 and 10 -4 per viable cell. Analysis of such transformants, although possible, is severely handicapped by the limited clonal life span of diploid human cells. Despite these problems it may be concluded that diploid human fibroblasts are competent recipients for DNA-mediated gene transfer and the putative repair deficiency of A-T does not markedly effect the efficiency of this process. (author)

Three job stress models/concepts and oxidative DNA damage in a sample of workers in Japan.

Science.gov (United States)

Inoue, Akiomi; Kawakami, Norito; Ishizaki, Masao; Tabata, Masaji; Tsuchiya, Masao; Akiyama, Miki; Kitazume, Akiko; Kuroda, Mitsuyo; Shimazu, Akihito

2009-04-01

Three job stress models/concepts (the job demands-control [DC] model, the effort-reward imbalance [ERI] model, and organizational justice) have been linked to coronary heart disease (CHD) at work. In recent years, oxidative DNA damage has been identified as a new risk factor for CHD. However, evidence for the association between these job stressors and oxidative DNA damage is limited. The present cross-sectional study investigated the association between these job stress models/concepts and oxidative DNA damage as a possible mediator of the adverse health effects of job stress. A total of 166 male and 51 female workers of a manufacturing factory in Japan were surveyed using a mailed questionnaire regarding job stressors and demographic, occupational, and lifestyle variables. Urinary concentrations of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage, were also measured. In male subjects, the urinary concentrations of 8-OHdG were significantly higher among the group with lower interactional justice, one of the two components of organizational justice; however, no association was observed with the DC model or the ERI model. In female subjects, high job demands/control ratio was significantly and positively associated with the urinary concentrations of 8-OHdG. Interactional justice among male workers and the DC model-based strain among female workers may be associated with increased urinary concentrations of 8-OHdG which possibly reflects oxidative DNA damage.

DNA repair systems and the pathogenesis of Mycobacterium tuberculosis: varying activities at different stages of infection.

Science.gov (United States)

Gorna, Alina E; Bowater, Richard P; Dziadek, Jaroslaw

2010-05-25

Mycobacteria, including most of all MTB (Mycobacterium tuberculosis), cause pathogenic infections in humans and, during the infectious process, are exposed to a range of environmental insults, including the host's immune response. From the moment MTB is exhaled by infected individuals, through an active and latent phase in the body of the new host, until the time they reach the reactivation stage, MTB is exposed to many types of DNA-damaging agents. Like all cellular organisms, MTB has efficient DNA repair systems, and these are believed to play essential roles in mycobacterial pathogenesis. As different stages of infection have great variation in the conditions in which mycobacteria reside, it is possible that different repair systems are essential for progression to specific phases of infection. MTB possesses homologues of DNA repair systems that are found widely in other species of bacteria, such as nucleotide excision repair, base excision repair and repair by homologous recombination. MTB also possesses a system for non-homologous end-joining of DNA breaks, which appears to be widespread in prokaryotes, although its presence is sporadic within different species within a genus. However, MTB does not possess homologues of the typical mismatch repair system that is found in most bacteria. Recent studies have demonstrated that DNA repair genes are expressed differentially at each stage of infection. In the present review, we focus on different DNA repair systems from mycobacteria and identify questions that remain in our understanding of how these systems have an impact upon the infection processes of these important pathogens.

Development of a recombinant DNA assay system for the detection of genetic change in astronauts' cells

International Nuclear Information System (INIS)

Atchley, S.V.; Chen, D.J.C.; Strniste, G.F.; Walters, R.A.; Moyzis, R.K.

1984-01-01

We are developing a new recombinant DNA system for the detection and measurement of genetic change in humans caused by exposure to low level ionizing radiation. A unique feature of the method is the use of cloned repetitive DNA probes to assay human DNA for structural changes during or after irradiation. Repetitive sequences exist in different families. Collectively they constitute over 25% of the DNA in a human cell. Repeat families have between 10 and 500,000 members. We have constructed repetitive DNA sequence libraries using recombinant DNA techniques. From these libraries we have isolated and characterized individual repeats comprising 75 to 90% of the mass of human repetitive DNA. Repeats used in our assay system exist in tandem arrays in the genome. Perturbation of these sequences in a cell, followed by detection with a repeat probe, produces a new, multimeric ''ladder'' pattern on an autoradiogram. The repeat probe used in our initial study is complementary to 1% of human DNA. Therefore, the sensitivity of this method is several orders of magnitude better than existing assays. Preliminary evidence from human skin cells exposed to acute, low-dose x-ray treatments indicates that DNA is affected at a dose as low as 5R. The radiation doses used in this system are well within the range of doses received by astronauts during spaceflight missions. Due to its small material requirements, this technique could easily be adapted for use in space. 16 refs., 1 fig

Differential Genetic Associations for Systemic Lupus Erythematosus Based on Anti–dsDNA Autoantibody Production

Science.gov (United States)

Chung, Sharon A.; Taylor, Kimberly E.; Graham, Robert R.; Nititham, Joanne; Lee, Annette T.; Ortmann, Ward A.; Jacob, Chaim O.; Alarcón-Riquelme, Marta E.; Tsao, Betty P.; Harley, John B.; Gaffney, Patrick M.; Moser, Kathy L.; Petri, Michelle; Demirci, F. Yesim; Kamboh, M. Ilyas; Manzi, Susan; Gregersen, Peter K.; Langefeld, Carl D.; Behrens, Timothy W.; Criswell, Lindsey A.

2011-01-01

Systemic lupus erythematosus (SLE) is a clinically heterogeneous, systemic autoimmune disease characterized by autoantibody formation. Previously published genome-wide association studies (GWAS) have investigated SLE as a single phenotype. Therefore, we conducted a GWAS to identify genetic factors associated with anti–dsDNA autoantibody production, a SLE–related autoantibody with diagnostic and clinical importance. Using two independent datasets, over 400,000 single nucleotide polymorphisms (SNPs) were studied in a total of 1,717 SLE cases and 4,813 healthy controls. Anti–dsDNA autoantibody positive (anti–dsDNA +, n = 811) and anti–dsDNA autoantibody negative (anti–dsDNA –, n = 906) SLE cases were compared to healthy controls and to each other to identify SNPs associated specifically with these SLE subtypes. SNPs in the previously identified SLE susceptibility loci STAT4, IRF5, ITGAM, and the major histocompatibility complex were strongly associated with anti–dsDNA + SLE. Far fewer and weaker associations were observed for anti–dsDNA – SLE. For example, rs7574865 in STAT4 had an OR for anti–dsDNA + SLE of 1.77 (95% CI 1.57–1.99, p = 2.0E-20) compared to an OR for anti–dsDNA – SLE of 1.26 (95% CI 1.12–1.41, p = 2.4E-04), with pheterogeneity<0.0005. SNPs in the SLE susceptibility loci BANK1, KIAA1542, and UBE2L3 showed evidence of association with anti–dsDNA + SLE and were not associated with anti–dsDNA – SLE. In conclusion, we identified differential genetic associations with SLE based on anti–dsDNA autoantibody production. Many previously identified SLE susceptibility loci may confer disease risk through their role in autoantibody production and be more accurately described as autoantibody propensity loci. Lack of strong SNP associations may suggest that other types of genetic variation or non-genetic factors such as environmental exposures have a greater impact on susceptibility to anti–dsDNA – SLE. PMID

Differential genetic associations for systemic lupus erythematosus based on anti-dsDNA autoantibody production.

Directory of Open Access Journals (Sweden)

Sharon A Chung

2011-03-01

Full Text Available Systemic lupus erythematosus (SLE is a clinically heterogeneous, systemic autoimmune disease characterized by autoantibody formation. Previously published genome-wide association studies (GWAS have investigated SLE as a single phenotype. Therefore, we conducted a GWAS to identify genetic factors associated with anti-dsDNA autoantibody production, a SLE-related autoantibody with diagnostic and clinical importance. Using two independent datasets, over 400,000 single nucleotide polymorphisms (SNPs were studied in a total of 1,717 SLE cases and 4,813 healthy controls. Anti-dsDNA autoantibody positive (anti-dsDNA +, n = 811 and anti-dsDNA autoantibody negative (anti-dsDNA -, n = 906 SLE cases were compared to healthy controls and to each other to identify SNPs associated specifically with these SLE subtypes. SNPs in the previously identified SLE susceptibility loci STAT4, IRF5, ITGAM, and the major histocompatibility complex were strongly associated with anti-dsDNA + SLE. Far fewer and weaker associations were observed for anti-dsDNA - SLE. For example, rs7574865 in STAT4 had an OR for anti-dsDNA + SLE of 1.77 (95% CI 1.57-1.99, p = 2.0E-20 compared to an OR for anti-dsDNA - SLE of 1.26 (95% CI 1.12-1.41, p = 2.4E-04, with p(heterogeneity<0.0005. SNPs in the SLE susceptibility loci BANK1, KIAA1542, and UBE2L3 showed evidence of association with anti-dsDNA + SLE and were not associated with anti-dsDNA - SLE. In conclusion, we identified differential genetic associations with SLE based on anti-dsDNA autoantibody production. Many previously identified SLE susceptibility loci may confer disease risk through their role in autoantibody production and be more accurately described as autoantibody propensity loci. Lack of strong SNP associations may suggest that other types of genetic variation or non-genetic factors such as environmental exposures have a greater impact on susceptibility to anti-dsDNA - SLE.

Influence of mobile DNA-protein-DNA bridges on DNA configurations: Coarse-grained Monte-Carlo simulations

NARCIS (Netherlands)

Vries, de R.

2011-01-01

A large literature exists on modeling the influence of sequence-specific DNA-binding proteins on the shape of the DNA double helix in terms of one or a few fixed constraints. This approach is inadequate for the many proteins that bind DNA sequence independently, and that are present in very large

A Phenotype-Driven Approach to Generate Mouse Models with Pathogenic mtDNA Mutations Causing Mitochondrial Disease

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Johanna H.K. Kauppila

2016-09-01

Full Text Available Mutations of mtDNA are an important cause of human disease, but few animal models exist. Because mammalian mitochondria cannot be transfected, the development of mice with pathogenic mtDNA mutations has been challenging, and the main strategy has therefore been to introduce mutations found in cell lines into mouse embryos. Here, we describe a phenotype-driven strategy that is based on detecting clonal expansion of pathogenic mtDNA mutations in colonic crypts of founder mice derived from heterozygous mtDNA mutator mice. As proof of concept, we report the generation of a mouse line transmitting a heteroplasmic pathogenic mutation in the alanine tRNA gene of mtDNA displaying typical characteristics of classic mitochondrial disease. In summary, we describe a straightforward and technically simple strategy based on mouse breeding and histology to generate animal models of mtDNA-mutation disease, which will be of great importance for studies of disease pathophysiology and preclinical treatment trials.

DnaA protein DNA-binding domain binds to Hda protein to promote inter-AAA+ domain interaction involved in regulatory inactivation of DnaA.

Science.gov (United States)

Keyamura, Kenji; Katayama, Tsutomu

2011-08-19

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.

DnaA Protein DNA-binding Domain Binds to Hda Protein to Promote Inter-AAA+ Domain Interaction Involved in Regulatory Inactivation of DnaA*

Science.gov (United States)

Keyamura, Kenji; Katayama, Tsutomu

2011-01-01

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis. PMID:21708944

Deficiency of Double-Strand DNA Break Repair Does Not Impair Mycobacterium tuberculosis Virulence in Multiple Animal Models of Infection

OpenAIRE

Heaton, Brook E.; Barkan, Daniel; Bongiorno, Paola; Karakousis, Petros C.; Glickman, Michael S.

2014-01-01

Mycobacterium tuberculosis persistence within its human host requires mechanisms to resist the effector molecules of host immunity, which exert their bactericidal effects through damaging pathogen proteins, membranes, and DNA. Substantial evidence indicates that bacterial pathogens, including M. tuberculosis, require DNA repair systems to repair the DNA damage inflicted by the host during infection, but the role of double-strand DNA break (DSB) repair systems is unclear. Double-strand DNA bre...

DNA-repair and mutations in immuncompetent cells from patients with rheumatic diseases and corresponding animal models

International Nuclear Information System (INIS)

Altmann, H.

1977-01-01

Unscheduled DNA synthesis was investigated in lymphocytes of patients with different inflammatory rheumatic diseases. After γ-irradiation H 3 -thymidin incorporation in DNA and DNA rejoining was reduced. After UV-irradiation the first step (90 min) of unscheduled DNA synthesis was above the controls. Some animal models for human diseases showed the same trend. An infectious ethiology was discussed for some of these diseases. (author)

Water versus DNA A new deal for proton transport modeling in biological matter

International Nuclear Information System (INIS)

Champion, C; Quinto, M A; Monti, J M; Galassi, M E; Fojón, O A; Hanssen, J; Rivarola, R D; Week, P F

2015-01-01

Water vapor is a common surrogate of DNA for modeling the proton-induced ionizing processes in living tissue exposed to radiations. The present study aims at scrutinizing the validity of this approximation and then revealing new insights into proton-induced energy transfers by a comparative analysis between water and realistic biological medium. In this context, self-consistent quantum mechanical modeling of the ionization and electron capture processes is reported within the continuum distorted wave-eikonal initial state framework for both isolated water molecules and DNA components impacted by proton beams. (paper)

Graphite-MicroMégas, a tool for DNA modeling

OpenAIRE

Hornus , Samuel; Larivière , Damien

2011-01-01

National audience; MicroMégas is the current state of an ongoing effort to develop tools for modeling biological assembly of molecules. We here present its DNA modeling part. MicroMégas is implemented as a plug-in to Graphite, which is a research plat- form for computer graphics, 3D modeling and numerical geometry that is developed by members of the ALICE team of INRIA.; Nous décrivons l'outils MicroMégas et les techniques qu'il met en jeu pour la modélisation d'assemblage de molécule, en par...

Biophysics of DNA

CERN Document Server

Vologodskii, Alexander

2015-01-01

Surveying the last sixty years of research, this book describes the physical properties of DNA in the context of its biological functioning. It is designed to enable both students and researchers of molecular biology, biochemistry and physics to better understand the biophysics of DNA, addressing key questions and facilitating further research. The chapters integrate theoretical and experimental approaches, emphasising throughout the importance of a quantitative knowledge of physical properties in building and analysing models of DNA functioning. For example, the book shows how the relationship between DNA mechanical properties and the sequence specificity of DNA-protein binding can be analyzed quantitatively by using our current knowledge of the physical and structural properties of DNA. Theoretical models and experimental methods in the field are critically considered to enable the reader to engage effectively with the current scientific literature on the physical properties of DNA.

A performance study on three qPCR quantification kits and their compatibilities with the 6-dye DNA profiling systems.

Science.gov (United States)

Lin, Sze-Wah; Li, Christina; Ip, Stephen C Y

2018-03-01

DNA quantification plays an integral role in forensic DNA profiling. Not only does it estimate the total amount of amplifiable human autosomal and male DNA to ensure optimal amplification of target DNA for subsequent analysis, but also assesses the extraction efficiency and purity of the DNA extract. Latest DNA quantification systems even offer an estimate for the degree of DNA degradation in a sample. Here, we report the performance of three new generation qPCR kits, namely Investigator ® Quantiplex HYres Kit from QIAGEN, Quantifiler ® Trio DNA Quantification Kit from Applied Biosystems™, and PowerQuant ® System from Promega, and their compatibilities with three 6-dye DNA profiling systems. Our results have demonstrated that all three kits generate standard curves with satisfactory consistency and reproducibility, and are capable of screening out traces of male DNA in the presence of 30-fold excess of female DNA. They also exhibit a higher tolerance to PCR inhibition than Quantifiler ® Human DNA Quantification Kit from Applied Biosystems™ in autosomal DNA quantification. PowerQuant ® , as compared to Quantiplex HYres and Quantifiler ® Trio, shows a better precision for both autosomal and male DNA quantifications. Quantifiler ® Trio and PowerQuant ® in contrast to Quantiplex HYres offer better correlations with lower discrepancies between autosomal and male DNA quantification, and their additional degradation index features provide a detection platform for inhibited and/or degraded DNA template. Regarding the compatibility between these quantification and profiling systems: (1) both Quantifiler ® Trio and PowerQuant ® work well with GlobalFiler and Fusion 6C, allowing a fairly accurate prediction of their DNA typing results based on the quantification values; (2) Quantiplex HYres offers a fairly reliable IPC system for detecting any potential inhibitions on Investigator 24plex, whereas Quantifiler ® Trio and PowerQuant ® suit better for Global

Application of high-resolution DNA melting for genotyping in lepidopteran non-model species: Ostrinia furnacalis (Crambidae.

Directory of Open Access Journals (Sweden)

FengBo Li

Full Text Available Development of an ideal marker system facilitates a better understanding of the genetic diversity in lepidopteran non-model organisms, which have abundant species, but relatively limited genomic resources. Single nucleotide polymorphisms (SNPs discovered within single-copy genes have proved to be desired markers, but SNP genotyping by current techniques remain laborious and expensive. High resolution melting (HRM curve analysis represents a simple, rapid and inexpensive genotyping method that is primarily confined to clinical and diagnostic studies. In this study, we evaluated the potential of HRM analysis for SNP genotyping in the lepidopteran non-model species Ostrinia furnacalis (Crambidae. Small amplicon and unlabeled probe assays were developed for the SNPs, which were identified in 30 females of O. furnacalis from 3 different populations by our direct sequencing. Both assays were then applied to genotype 90 unknown female DNA by prior mixing with known wild-type DNA. The genotyping results were compared with those that were obtained using bi-directional sequencing analysis. Our results demonstrated the efficiency and reliability of the HRM assays. HRM has the potential to provide simple, cost-effective genotyping assays and facilitates genotyping studies in any non-model lepidopteran species of interest.

Effects of radiation on DNA

International Nuclear Information System (INIS)

Braddock, M.

1985-07-01

The hydroxyl radical (OH radical) is the most damaging radical produced by the effect of ionizing radiation in water. The rate of reaction of the OH radical with purified, native and isodisperse DNA has been determined as compared with calf thymus DNA. This has been achieved by direct observation of the rate of formation of the DNA-OH radical adduct, and by competition with SCN - . Results obtained from direct observation are consistent with calculations which have been performed using the encounter frequency model of Braams and Ebert. However, results obtained for OH radical with DNA derived from competition plots suggest a rate constant somewhat lower than that obtained from direct observation. The relative merits of both techniques are discussed. In order to study the effect of energy deposited directly in the DNA, dry films of purified plasmid DNA have been irradiated in a system where the indirect effects of radical interaction have been minimized. The present results indicate that with different molecular lengths of plasmid DNA, non-random breakage may occur, and that additional damage may be brought about at sites of previously existing damage. Differences in the sensitivity of plasmid DNA molecules of varying lengths to radiation induced double strand breaks have been demonstrated. (author)

Dynamic In Vivo Profiling of DNA Damage and Repair after Radiotherapy Using Canine Patients as a Model

Directory of Open Access Journals (Sweden)

Nadine Schulz

2017-06-01

Full Text Available Time resolved data of DNA damage and repair after radiotherapy elucidates the relation between damage, repair, and cell survival. While well characterized in vitro, little is known about the time-course of DNA damage response in tumors sampled from individual patients. Kinetics of DNA damage after radiotherapy was assessed in eight dogs using repeated in vivo samples of tumor and co-irradiated normal tissue analyzed with comet assay and phosphorylated H2AX (γH2AX immunohistochemistry. In vivo results were then compared (in silico with a dynamic mathematical model for DNA damage formation and repair. Maximum %DNA in tail was observed at 15–60 min after irradiation, with a rapid decrease. Time-courses of γH2AX-foci paralleled these findings with a small time delay and were not influenced by covariates. The evolutionary parameter search based on %DNA in tail revealed a good fit of the DNA repair model to in vivo data for pooled sarcoma time-courses, but fits for individual sarcoma time-courses suffer from the heterogeneous nature of the in vivo data. It was possible to follow dynamics of comet tail intensity and γH2AX-foci during a course of radiation using a minimally invasive approach. DNA repair can be quantitatively investigated as time-courses of individual patients by integrating this resulting data into a dynamic mathematical model.

Purification of functionalized DNA origami nanostructures.

Science.gov (United States)

Shaw, Alan; Benson, Erik; Högberg, Björn

2015-05-26

The high programmability of DNA origami has provided tools for precise manipulation of matter at the nanoscale. This manipulation of matter opens up the possibility to arrange functional elements for a diverse range of applications that utilize the nanometer precision provided by these structures. However, the realization of functionalized DNA origami still suffers from imperfect production methods, in particular in the purification step, where excess material is separated from the desired functionalized DNA origami. In this article we demonstrate and optimize two purification methods that have not previously been applied to DNA origami. In addition, we provide a systematic study comparing the purification efficacy of these and five other commonly used purification methods. Three types of functionalized DNA origami were used as model systems in this study. DNA origami was patterned with either small molecules, antibodies, or larger proteins. With the results of our work we aim to provide a guideline in quality fabrication of various types of functionalized DNA origami and to provide a route for scalable production of these promising tools.

The DnaA N-terminal domain interacts with Hda to facilitate replicase clamp-mediated inactivation of DnaA.

Science.gov (United States)

Su'etsugu, Masayuki; Harada, Yuji; Keyamura, Kenji; Matsunaga, Chika; Kasho, Kazutoshi; Abe, Yoshito; Ueda, Tadashi; Katayama, Tsutomu

2013-12-01

DnaA activity for replication initiation of the Escherichia coli chromosome is negatively regulated by feedback from the DNA-loaded form of the replicase clamp. In this process, called RIDA (regulatory inactivation of DnaA), ATP-bound DnaA transiently assembles into a complex consisting of Hda and the DNA-clamp, which promotes inter-AAA+ domain association between Hda and DnaA and stimulates hydrolysis of DnaA-bound ATP, producing inactive ADP-DnaA. Using a truncated DnaA mutant, we previously demonstrated that the DnaA N-terminal domain is involved in RIDA. However, the precise role of the N-terminal domain in RIDA has remained largely unclear. Here, we used an in vitro reconstituted system to demonstrate that the Asn-44 residue in the N-terminal domain of DnaA is crucial for RIDA but not for replication initiation. Moreover, an assay termed PDAX (pull-down after cross-linking) revealed an unstable interaction between a DnaA-N44A mutant and Hda. In vivo, this mutant exhibited an increase in the cellular level of ATP-bound DnaA. These results establish a model in which interaction between DnaA Asn-44 and Hda stabilizes the association between the AAA+ domains of DnaA and Hda to facilitate DnaA-ATP hydrolysis during RIDA. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

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

Supercoil Formation During DNA Melting

Science.gov (United States)

Sayar, Mehmet; Avsaroglu, Baris; Kabakcioglu, Alkan

2009-03-01

Supercoil formation plays a key role in determining the structure-function relationship in DNA. Biological and technological processes, such as protein synthesis, polymerase chain reaction, and microarrays relys on separation of the two strands in DNA, which is coupled to the unwinding of the supercoiled structure. This problem has been studied theoretically via Peyrard-Bishop and Poland-Scheraga type models, which include a simple representation of the DNA structural properties. In recent years, computational models, which provide a more realtistic representaion of DNA molecule, have been used to study the melting behavior of short DNA chains. Here, we will present a new coarse-grained model of DNA which is capable of simulating sufficiently long DNA chains for studying the supercoil formation during melting, without sacrificing the local structural properties. Our coarse-grained model successfully reproduces the local geometry of the DNA molecule, such as the 3'-5' directionality, major-minor groove structure, and the helical pitch. We will present our initial results on the dynamics of supercoiling during DNA melting.

Physical principles for DNA tile self-assembly.

Science.gov (United States)

Evans, Constantine G; Winfree, Erik

2017-06-19

DNA tiles provide a promising technique for assembling structures with nanoscale resolution through self-assembly by basic interactions rather than top-down assembly of individual structures. Tile systems can be programmed to grow based on logical rules, allowing for a small number of tile types to assemble large, complex assemblies that can retain nanoscale resolution. Such algorithmic systems can even assemble different structures using the same tiles, based on inputs that seed the growth. While programming and theoretical analysis of tile self-assembly often makes use of abstract logical models of growth, experimentally implemented systems are governed by nanoscale physical processes that can lead to very different behavior, more accurately modeled by taking into account the thermodynamics and kinetics of tile attachment and detachment in solution. This review discusses the relationships between more abstract and more physically realistic tile assembly models. A central concern is how consideration of model differences enables the design of tile systems that robustly exhibit the desired abstract behavior in realistic physical models and in experimental implementations. Conversely, we identify situations where self-assembly in abstract models can not be well-approximated by physically realistic models, putting constraints on physical relevance of the abstract models. To facilitate the discussion, we introduce a unified model of tile self-assembly that clarifies the relationships between several well-studied models in the literature. Throughout, we highlight open questions regarding the physical principles for DNA tile self-assembly.

Modeling early physical and chemical events for DNA damage induced by photons and tritium beta particles

International Nuclear Information System (INIS)

Moiseenko, V.; Waker, A.J.; Prestwich, W.V.

1998-02-01

A method has been developed to model production of single-strand breaks (SSB) and double-strand breaks (DSB) in Deoxyribo Nucleic Acid (DNA) by ionizing radiations. Modeling is carried out by Monte Carlo means and includes consideration of direct energy depositions in DNA molecules, production of chemical species following water radiolysis, diffusion of chemical species, and their interactions with each other and DNA. Computer-generated electron tracks in liquid water are used to model energy deposition and to derive the initial localization of chemical species. Atomistic representation of the DNA with a first hydration shell is used to derive direct energy depositions in DNA molecules and the resulting consequences, and to derive coordinates of reactive sites for modeling of the chemical stage of radiation damage. Diffusion of chemical species is followed in time, and the reactions of species with each other and DNA are considered to occur in an encounter-controlled manner. Time of diffusion follow-up is restricted to 10 -12 - 10 -9 s, which yields a diffusion length of hydroxyl radicals comparable to that in the cellular environment. DNA SSB are assumed to result from any direct energy depositions in the sugar/phosphate moiety, ionizations in water molecules bound to sugar/phosphate and hydroxyl attacks on deoxyribose. DSB are assumed to result from two SSB on opposite strands separated by 10 or fewer base pairs. Photon radiations in the energy range 70 keV-1 MeV and tritium beta particles are considered. It is shown that for naked DNA in B-form (the configuration thought to be most biologically relevant) the effectiveness of tritium for SSB and DSB production is, within statistical uncertainties, comparable to photon radiation with energies in the range 70 keV-1 MeV, although a tendency for increased DSB production has been observed for 70 keV photons that represent orthovoltage X-rays and for tritium beta particles. It is predicted that hydroxyl radicals react

Repair of Clustered Damage and DNA Polymerase Iota.

Science.gov (United States)

Belousova, E A; Lavrik, O I

2015-08-01

Multiple DNA lesions occurring within one or two turns of the DNA helix known as clustered damage are a source of double-stranded DNA breaks, which represent a serious threat to the cells. Repair of clustered lesions is accomplished in several steps. If a clustered lesion contains oxidized bases, an individual DNA lesion is repaired by the base excision repair (BER) mechanism involving a specialized DNA polymerase after excising DNA damage. Here, we investigated DNA synthesis catalyzed by DNA polymerase iota using damaged DNA templates. Two types of DNA substrates were used as model DNAs: partial DNA duplexes containing breaks of different length, and DNA duplexes containing 5-formyluracil (5-foU) and uracil as a precursor of apurinic/apyrimidinic sites (AP) in opposite DNA strands. For the first time, we showed that DNA polymerase iota is able to catalyze DNA synthesis using partial DNA duplexes having breaks of different length as substrates. In addition, we found that DNA polymerase iota could catalyze DNA synthesis during repair of clustered damage via the BER system by using both undamaged and 5-foU-containing templates. We found that hPCNA (human proliferating cell nuclear antigen) increased efficacy of DNA synthesis catalyzed by DNA polymerase iota.

Model for how type I restriction enzymes select cleavage sites in DNA

International Nuclear Information System (INIS)

Studier, F.W.; Bandyopadhyay, P.K.

1988-01-01

Under appropriate conditions, digestion of phage T7 DNA by the type I restriction enzyme EcoK produces an orderly progression of discrete DNA fragments. All details of the fragmentation pattern can be explained on the basis of the known properties of type I enzymes, together with two further assumptions: (i) in the ATP-stimulated translocation reaction, the enzyme bound at the recognition sequence translocates DNA toward itself from both directions simultaneously; and (ii) when translocation causes neighboring enzymes to meet, they cut the DNA between them. The kinetics of digestion at 37 degree C indicates that the rate of translocation of DNA from each side of a bound enzyme is about 200 base pairs per second, and the cuts are completed within 15-25 sec of the time neighboring enzymes meet. The resulting DNA fragments each contain a single recognition site with an enzyme (or subunit) remaining bound to it. At high enzyme concentrations, such fragments can bu further degraded, apparently by cooperation between the specifically bound and excess enzymes. This model is consistent with a substantial body of previous work on the nuclease activity of EcoB and EcoK, and it explains in a simple way how cleavage sites are selected

Perturbations in DNA structure upon interaction with porphyrins revealed by chemical probes, DNA footprinting and molecular modelling.

Science.gov (United States)

Ford, K G; Neidle, S

1995-06-01

The interactions of several porphyrins with a 74 base-pair DNA sequence have been examined by footprinting and chemical protection methods. Tetra-(4-N-methyl-(pyridyl)) porphyrin (TMPy), two of its metal complexes and tetra-(4-trimethylanilinium) porphyrin (TMAP) bind to closely similar AT-rich sequences. The three TMPy ligands produce modest changes in DNA structure and base accessibility on binding, in contrast to the large-scale conformational changes observed with TMAP. Molecular modelling studies have been performed on TMPy and TMAP bound in the AT-rich minor groove of an oligonucleotide. These have shown that significant structural change is needed to accommodate the bulky trimethyl substituent groups of TMAP, in contrast to the facile minor groove fit of TMPy.

Improved recovery of DNA from polyacrylamide gels after in situ DNA footprinting

NARCIS (Netherlands)

van Keulen, G; Meijer, WG

Methods used to date for the isolation of DNA from polyacrylamide gels are elution based, time-consuming and with low yield in DNA. This paper describes an improved system employing polyacrylamide gels made of a meltable matrix. The new system was successfully applied to in situ DNA footprinting

Exact method for numerically analyzing a model of local denaturation in superhelically stressed DNA

International Nuclear Information System (INIS)

Fye, R.M.; Benham, C.J.

1999-01-01

Local denaturation, the separation at specific sites of the two strands comprising the DNA double helix, is one of the most fundamental processes in biology, required to allow the base sequence to be read both in DNA transcription and in replication. In living organisms this process can be mediated by enzymes which regulate the amount of superhelical stress imposed on the DNA. We present a numerically exact technique for analyzing a model of denaturation in superhelically stressed DNA. This approach is capable of predicting the locations and extents of transition in circular superhelical DNA molecules of kilobase lengths and specified base pair sequences. It can also be used for closed loops of DNA which are typically found in vivo to be kilobases long. The analytic method consists of an integration over the DNA twist degrees of freedom followed by the introduction of auxiliary variables to decouple the remaining degrees of freedom, which allows the use of the transfer matrix method. The algorithm implementing our technique requires O(N 2 ) operations and O(N) memory to analyze a DNA domain containing N base pairs. However, to analyze kilobase length DNA molecules it must be implemented in high precision floating point arithmetic. An accelerated algorithm is constructed by imposing an upper bound M on the number of base pairs that can simultaneously denature in a state. This accelerated algorithm requires O(MN) operations, and has an analytically bounded error. Sample calculations show that it achieves high accuracy (greater than 15 decimal digits) with relatively small values of M (M<0.05N) for kilobase length molecules under physiologically relevant conditions. Calculations are performed on the superhelical pBR322 DNA sequence to test the accuracy of the method. With no free parameters in the model, the locations and extents of local denaturation predicted by this analysis are in quantitatively precise agreement with in vitro experimental measurements

DNA sequence+shape kernel enables alignment-free modeling of transcription factor binding.

Science.gov (United States)

Ma, Wenxiu; Yang, Lin; Rohs, Remo; Noble, William Stafford

2017-10-01

Transcription factors (TFs) bind to specific DNA sequence motifs. Several lines of evidence suggest that TF-DNA binding is mediated in part by properties of the local DNA shape: the width of the minor groove, the relative orientations of adjacent base pairs, etc. Several methods have been developed to jointly account for DNA sequence and shape properties in predicting TF binding affinity. However, a limitation of these methods is that they typically require a training set of aligned TF binding sites. We describe a sequence + shape kernel that leverages DNA sequence and shape information to better understand protein-DNA binding preference and affinity. This kernel extends an existing class of k-mer based sequence kernels, based on the recently described di-mismatch kernel. Using three in vitro benchmark datasets, derived from universal protein binding microarrays (uPBMs), genomic context PBMs (gcPBMs) and SELEX-seq data, we demonstrate that incorporating DNA shape information improves our ability to predict protein-DNA binding affinity. In particular, we observe that (i) the k-spectrum + shape model performs better than the classical k-spectrum kernel, particularly for small k values; (ii) the di-mismatch kernel performs better than the k-mer kernel, for larger k; and (iii) the di-mismatch + shape kernel performs better than the di-mismatch kernel for intermediate k values. The software is available at https://bitbucket.org/wenxiu/sequence-shape.git. rohs@usc.edu or william-noble@uw.edu. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Radiation damage to DNA in DNA-protein complexes.

Science.gov (United States)

Spotheim-Maurizot, M; Davídková, M

2011-06-03

The most aggressive product of water radiolysis, the hydroxyl (OH) radical, is responsible for the indirect effect of ionizing radiations on DNA in solution and aerobic conditions. According to radiolytic footprinting experiments, the resulting strand breaks and base modifications are inhomogeneously distributed along the DNA molecule irradiated free or bound to ligands (polyamines, thiols, proteins). A Monte-Carlo based model of simulation of the reaction of OH radicals with the macromolecules, called RADACK, allows calculating the relative probability of damage of each nucleotide of DNA irradiated alone or in complexes with proteins. RADACK calculations require the knowledge of the three dimensional structure of DNA and its complexes (determined by X-ray crystallography, NMR spectroscopy or molecular modeling). The confrontation of the calculated values with the results of the radiolytic footprinting experiments together with molecular modeling calculations show that: (1) the extent and location of the lesions are strongly dependent on the structure of DNA, which in turns is modulated by the base sequence and by the binding of proteins and (2) the regions in contact with the protein can be protected against the attack by the hydroxyl radicals via masking of the binding site and by scavenging of the radicals. 2011 Elsevier B.V. All rights reserved.

Engineering of magnetic DNA nanoparticles for tumor-targeted therapy

International Nuclear Information System (INIS)

Hosseinkhani, Hossein; Chen Yiru; He Wenjie; Hong Poda; Yu, Dah-Shyong; Domb, Abraham J.

2013-01-01

This study aims to engineer novel targeted delivery system composed of magnetic DNA nanoparticles to be effective as an efficient targeted gene therapy vehicle for tumor therapy. A polysaccharide, dextran, was chosen as the vector of plasmid DNA-encoded NK4 that acts as an HGF-antagonist and anti-angiogenic regulator for inhibitions of tumor growth, invasion, and metastasis. Spermine (Sm) was chemically introduced to the hydroxyl groups of dextran to obtain dextran-Sm. When Fe 2+ solution was added to the mixture of dextran-Sm and a plasmid DNA, homogenous DNA nanoparticles were formed via chemical metal coordination bonding with average size of 230 nm. Characterization of DNA nanoparticles was performed via dynamic light scattering measurement, electrophoretic light scattering measurement, as well as transmission electron microscope. DNA nanoparticles effectively condensed plasmid DNA into nanoparticles and enhanced the stability of DNA, while significantly improved transfection efficiency in vitro and tumor accumulation in vivo. In addition, magnetic DNA nanoparticles exhibited high efficiency in antitumor therapy with regards to tumor growth as well as survival of animals evaluated in the presence of external magnetic field. We conclude that the magnetic properties of these DNA nanoparticles would enhance the tracking of non-viral gene delivery systems when administrated in vivo in a test model. These findings suggest that DNA nanoparticles effectively deliver DNA to tumor and thereby inhibiting tumor growth.

Engineering of magnetic DNA nanoparticles for tumor-targeted therapy

Energy Technology Data Exchange (ETDEWEB)

Hosseinkhani, Hossein, E-mail: hosseinkhani@yahoo.com [Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology (Taiwan Tech) (China); Chen Yiru [National Yang-Ming University, Department of Biomedical Engineering (China); He Wenjie; Hong Poda [Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology (Taiwan Tech) (China); Yu, Dah-Shyong [Nanomedicine Research Center, National Defense Medical Center (China); Domb, Abraham J. [Institute of Drug Research, The Center for Nanoscience and Nanotechnology, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem (Israel)

2013-01-15

This study aims to engineer novel targeted delivery system composed of magnetic DNA nanoparticles to be effective as an efficient targeted gene therapy vehicle for tumor therapy. A polysaccharide, dextran, was chosen as the vector of plasmid DNA-encoded NK4 that acts as an HGF-antagonist and anti-angiogenic regulator for inhibitions of tumor growth, invasion, and metastasis. Spermine (Sm) was chemically introduced to the hydroxyl groups of dextran to obtain dextran-Sm. When Fe{sup 2+} solution was added to the mixture of dextran-Sm and a plasmid DNA, homogenous DNA nanoparticles were formed via chemical metal coordination bonding with average size of 230 nm. Characterization of DNA nanoparticles was performed via dynamic light scattering measurement, electrophoretic light scattering measurement, as well as transmission electron microscope. DNA nanoparticles effectively condensed plasmid DNA into nanoparticles and enhanced the stability of DNA, while significantly improved transfection efficiency in vitro and tumor accumulation in vivo. In addition, magnetic DNA nanoparticles exhibited high efficiency in antitumor therapy with regards to tumor growth as well as survival of animals evaluated in the presence of external magnetic field. We conclude that the magnetic properties of these DNA nanoparticles would enhance the tracking of non-viral gene delivery systems when administrated in vivo in a test model. These findings suggest that DNA nanoparticles effectively deliver DNA to tumor and thereby inhibiting tumor growth.

Prototype Systems Containing Human Cytochrome P450 for High-Throughput Real-Time Detection of DNA Damage by Compounds That Form DNA-Reactive Metabolites.

Science.gov (United States)

Brito Palma, Bernardo; Fisher, Charles W; Rueff, José; Kranendonk, Michel

2016-05-16

The formation of reactive metabolites through biotransformation is the suspected cause of many adverse drug reactions. Testing for the propensity of a drug to form reactive metabolites has increasingly become an integral part of lead-optimization strategy in drug discovery. DNA reactivity is one undesirable facet of a drug or its metabolites and can lead to increased risk of cancer and reproductive toxicity. Many drugs are metabolized by cytochromes P450 in the liver and other tissues, and these reactions can generate hard electrophiles. These hard electrophilic reactive metabolites may react with DNA and may be detected in standard in vitro genotoxicity assays; however, the majority of these assays fall short due to the use of animal-derived organ extracts that inadequately represent human metabolism. The current study describes the development of bacterial systems that efficiently detect DNA-damaging electrophilic reactive metabolites generated by human P450 biotransformation. These assays use a GFP reporter system that detects DNA damage through induction of the SOS response and a GFP reporter to control for cytotoxicity. Two human CYP1A2-competent prototypes presented here have appropriate characteristics for the detection of DNA-damaging reactive metabolites in a high-throughput manner. The advantages of this approach include a short assay time (120-180 min) with real-time measurement, sensitivity to small amounts of compound, and adaptability to a microplate format. These systems are suitable for high-throughput assays and can serve as prototypes for the development of future enhanced versions.

Exploring the common molecular basis for the universal DNA mutation bias: Revival of Loewdin mutation model

International Nuclear Information System (INIS)

Fu, Liang-Yu; Wang, Guang-Zhong; Ma, Bin-Guang; Zhang, Hong-Yu

2011-01-01

Highlights: → There exists a universal G:C → A:T mutation bias in three domains of life. → This universal mutation bias has not been sufficiently explained. → A DNA mutation model proposed by Loewdin 40 years ago offers a common explanation. -- Abstract: Recently, numerous genome analyses revealed the existence of a universal G:C → A:T mutation bias in bacteria, fungi, plants and animals. To explore the molecular basis for this mutation bias, we examined the three well-known DNA mutation models, i.e., oxidative damage model, UV-radiation damage model and CpG hypermutation model. It was revealed that these models cannot provide a sufficient explanation to the universal mutation bias. Therefore, we resorted to a DNA mutation model proposed by Loewdin 40 years ago, which was based on inter-base double proton transfers (DPT). Since DPT is a fundamental and spontaneous chemical process and occurs much more frequently within GC pairs than AT pairs, Loewdin model offers a common explanation for the observed universal mutation bias and thus has broad biological implications.

Consequences of intramolecular dityrosine formation on a DNA-protein complex: a molecular modeling study

International Nuclear Information System (INIS)

Gras, Julien; Sy, Denise; Eon, Severine; Charlier, Michel; Spotheim-Maurizot, Melanie

2005-01-01

Irradiation of the free lac repressor with γ-rays abolishes protein's ability to specifically bind operator DNA. A possible radiation-induced protein damage is a dityrosine (DTyr) formed by two spatially close radiation-induced tyrosyl radicals. We performed the molecular modeling of complexes between operator DNA and DTyr-bearing parts (headpieces) of the repressor. The presence of DTyr affects the structure and the interactions between partners. A detailed analysis allows to conclude this damage can partially account for the loss of repressor ability to bind DNA

OPTSDNA: Performance evaluation of an efficient distributed bioinformatics system for DNA sequence analysis.

Science.gov (United States)

Khan, Mohammad Ibrahim; Sheel, Chotan

2013-01-01

Storage of sequence data is a big concern as the amount of data generated is exponential in nature at several locations. Therefore, there is a need to develop techniques to store data using compression algorithm. Here we describe optimal storage algorithm (OPTSDNA) for storing large amount of DNA sequences of varying length. This paper provides performance analysis of optimal storage algorithm (OPTSDNA) of a distributed bioinformatics computing system for analysis of DNA sequences. OPTSDNA algorithm is used for storing various sizes of DNA sequences into database. DNA sequences of different lengths were stored by using this algorithm. These input DNA sequences are varied in size from very small to very large. Storage size is calculated by this algorithm. Response time is also calculated in this work. The efficiency and performance of the algorithm is high (in size calculation with percentage) when compared with other known with sequential approach.

Isolation of proteins involved in the replication of adenoviral DNA in vitro

International Nuclear Information System (INIS)

Lichy, J.H.; Nagata, K.; Friefeld, B.R.; Enomoto, T.; Field, J.; Guggenheimer, R.A.; Ikeda, J.E.; Horwitz, M.S.; Hurwitz, J.

1983-01-01

The simple mechanism of replication of adenoviral DNA has made adenovirus an especially useful model system for studies of eukaryotic replication mechanisms. The availability of this in vitro system that replicates exogenously added Ad DNA-pro has made it possible to characterize the factors involved in replication. The results presented in this paper summarize our further fractionation of the in vitro system. First, the properties of two factors purified from the uninfected nuclear extract are described. Second, the separation of the pTP/Ad Pol complex into subunits and the properties of the isolated subunits are presented. The 140K protein is shown to possess the Ad DNA polymerase activity. The results suggest that the only DNA polymerase required for adenoviral DNA replication in vitro is the 140K Ad DNA polymerase and that this enzyme is probably a viral gene product. 50 references, 10 figures, 3 tables

Complex DNA structures and structures of DNA complexes

International Nuclear Information System (INIS)

Chazin, W.J.; Carlstroem, G.; Shiow-Meei Chen; Miick, S.; Gomez-Paloma, L.; Smith, J.; Rydzewski, J.

1994-01-01

Complex DNA structures (for example, triplexes, quadruplexes, junctions) and DNA-ligand complexes are more difficult to study by NMR than standard DNA duplexes are because they have high molecular weights, show nonstandard or distorted local conformations, and exhibit large resonance linewidths and severe 1 H spectral overlap. These systems also tend to have limited solubility and may require specialized solution conditions to maintain favorable spectral characteristics, which adds to the spectroscopic difficulties. Furthermore, with more atoms in the system, both assignment and structure calculation become more challenging. In this article, we focus on demonstrating the current status of NMR studies of such systems and the limitations to further progress; we also indicate in what ways isotopic enrichment can be useful

Complex DNA structures and structures of DNA complexes

Energy Technology Data Exchange (ETDEWEB)

Chazin, W.J.; Carlstroem, G.; Shiow-Meei Chen; Miick, S.; Gomez-Paloma, L.; Smith, J.; Rydzewski, J. [Scripps Research Institute, La Jolla, CA (United States)

1994-12-01

Complex DNA structures (for example, triplexes, quadruplexes, junctions) and DNA-ligand complexes are more difficult to study by NMR than standard DNA duplexes are because they have high molecular weights, show nonstandard or distorted local conformations, and exhibit large resonance linewidths and severe {sup 1}H spectral overlap. These systems also tend to have limited solubility and may require specialized solution conditions to maintain favorable spectral characteristics, which adds to the spectroscopic difficulties. Furthermore, with more atoms in the system, both assignment and structure calculation become more challenging. In this article, we focus on demonstrating the current status of NMR studies of such systems and the limitations to further progress; we also indicate in what ways isotopic enrichment can be useful.

Tumor transfection after systemic injection of DNA lipid nanocapsules.

Science.gov (United States)

Morille, Marie; Passirani, Catherine; Dufort, Sandrine; Bastiat, Guillaume; Pitard, Bruno; Coll, Jean-Luc; Benoit, Jean-Pierre

2011-03-01

With the goal of generating an efficient vector for systemic gene delivery, a new kind of nanocarrier consisting of lipid nanocapsules encapsulating DOTAP/DOPE lipoplexes (DNA LNCs) was pegylated by the post-insertion of amphiphilic and flexible polymers. The aim of this surface modification was to create a long-circulating vector, able to circulate in the blood stream and efficient in transfecting tumoral cells after passive targeting by enhanced permeability and retention effect (EPR effect). PEG conformation, electrostatic features, and hydrophylicity are known to be important factors able to influence the pharmacokinetic behaviour of vectors. In this context, the surface structure characteristics of the newly pegylated DNA LNCs were studied by measuring electrophoretic mobility as a function of ionic strength in order to establish a correlation between surface properties and in vivo performance of the vector. Finally, thanks to this PEGylation, gene expression was measured up to 84-fold higher in tumor compared to other tested organs after intravenous injection. The present results indicate that PEGylated DNA LNCs are promising carriers for an efficient cancer gene therapy. Copyright © 2010 Elsevier Ltd. All rights reserved.

Horizontal gene transfer of a chloroplast DnaJ-Fer protein to Thaumarchaeota and the evolutionary history of the DnaK chaperone system in Archaea.

Science.gov (United States)

Petitjean, Céline; Moreira, David; López-García, Purificación; Brochier-Armanet, Céline

2012-11-26

In 2004, we discovered an atypical protein in metagenomic data from marine thaumarchaeotal species. This protein, referred as DnaJ-Fer, is composed of a J domain fused to a Ferredoxin (Fer) domain. Surprisingly, the same protein was also found in Viridiplantae (green algae and land plants). Because J domain-containing proteins are known to interact with the major chaperone DnaK/Hsp70, this suggested that a DnaK protein was present in Thaumarchaeota. DnaK/Hsp70, its co-chaperone DnaJ and the nucleotide exchange factor GrpE are involved, among others, in heat shocks and heavy metal cellular stress responses. Using phylogenomic approaches we have investigated the evolutionary history of the DnaJ-Fer protein and of interacting proteins DnaK, DnaJ and GrpE in Thaumarchaeota. These proteins have very complex histories, involving several inter-domain horizontal gene transfers (HGTs) to explain the contemporary distribution of these proteins in archaea. These transfers include one from Cyanobacteria to Viridiplantae and one from Viridiplantae to Thaumarchaeota for the DnaJ-Fer protein, as well as independent HGTs from Bacteria to mesophilic archaea for the DnaK/DnaJ/GrpE system, followed by HGTs among mesophilic and thermophilic archaea. We highlight the chimerical origin of the set of proteins DnaK, DnaJ, GrpE and DnaJ-Fer in Thaumarchaeota and suggest that the HGT of these proteins has played an important role in the adaptation of several archaeal groups to mesophilic and thermophilic environments from hyperthermophilic ancestors. Finally, the evolutionary history of DnaJ-Fer provides information useful for the relative dating of the diversification of Archaeplastida and Thaumarchaeota.

DNA Binding by the Ribosomal DNA Transcription Factor Rrn3 Is Essential for Ribosomal DNA Transcription*

Science.gov (United States)

Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H.; Rothblum, Katrina; Schneider, David A.; Rothblum, Lawrence I.

2013-01-01

The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382–400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I. PMID:23393135

DNA binding by the ribosomal DNA transcription factor rrn3 is essential for ribosomal DNA transcription.

Science.gov (United States)

Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H; Rothblum, Katrina; Schneider, David A; Rothblum, Lawrence I

2013-03-29

The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382-400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I.

DNA Open states and DNA hydratation

International Nuclear Information System (INIS)

Lema-Larre, B. de; Martin-Landrove, M

1995-01-01

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 [es

High resolution optical DNA mapping

Science.gov (United States)

Baday, Murat

Many types of diseases including cancer and autism are associated with copy-number variations in the genome. Most of these variations could not be identified with existing sequencing and optical DNA mapping methods. We have developed Multi-color Super-resolution technique, with potential for high throughput and low cost, which can allow us to recognize more of these variations. Our technique has made 10--fold improvement in the resolution of optical DNA mapping. Using a 180 kb BAC clone as a model system, we resolved dense patterns from 108 fluorescent labels of two different colors representing two different sequence-motifs. Overall, a detailed DNA map with 100 bp resolution was achieved, which has the potential to reveal detailed information about genetic variance and to facilitate medical diagnosis of genetic disease.

Modeling early physical and chemical events for DNA damage induced by photons and tritium beta particles

Energy Technology Data Exchange (ETDEWEB)

Moiseenko, V [McMaster Univ., Dept. of Physics and Astronomy, Hamilton, Ontario (Canada); Waker, A J [Atomic Energy of Canada Limited, Chalk River, Ontario (Canada); Prestwich, W V [McMaster Univ., Dept. of Physics and Astronomy, Hamilton, Ontario (Canada)

1998-02-01

A method has been developed to model production of single-strand breaks (SSB) and double-strand breaks (DSB) in Deoxyribo Nucleic Acid (DNA) by ionizing radiations. Modeling is carried out by Monte Carlo means and includes consideration of direct energy depositions in DNA molecules, production of chemical species following water radiolysis, diffusion of chemical species, and their interactions with each other and DNA. Computer-generated electron tracks in liquid water are used to model energy deposition and to derive the initial localization of chemical species. Atomistic representation of the DNA with a first hydration shell is used to derive direct energy depositions in DNA molecules and the resulting consequences, and to derive coordinates of reactive sites for modeling of the chemical stage of radiation damage. Diffusion of chemical species is followed in time, and the reactions of species with each other and DNA are considered to occur in an encounter-controlled manner. Time of diffusion follow-up is restricted to 10{sup -12}- 10{sup -9} s, which yields a diffusion length of hydroxyl radicals comparable to that in the cellular environment. DNA SSB are assumed to result from any direct energy depositions in the sugar/phosphate moiety, ionizations in water molecules bound to sugar/phosphate and hydroxyl attacks on deoxyribose. DSB are assumed to result from two SSB on opposite strands separated by 10 or fewer base pairs. Photon radiations in the energy range 70 keV-1 MeV and tritium beta particles are considered. It is shown that for naked DNA in B-form (the configuration thought to be most biologically relevant) the effectiveness of tritium for SSB and DSB production is, within statistical uncertainties, comparable to photon radiation with energies in the range 70 keV-1 MeV, although a tendency for increased DSB production has been observed for 70 keV photons that represent orthovoltage X-rays and for tritium beta particles. It is predicted that hydroxyl

Different rates of DNA replication at early versus late S-phase sections: multiscale modeling of stochastic events related to DNA content/EdU (5-ethynyl-2'deoxyuridine) incorporation distributions.

Science.gov (United States)

Li, Biao; Zhao, Hong; Rybak, Paulina; Dobrucki, Jurek W; Darzynkiewicz, Zbigniew; Kimmel, Marek

2014-09-01

Mathematical modeling allows relating molecular events to single-cell characteristics assessed by multiparameter cytometry. In the present study we labeled newly synthesized DNA in A549 human lung carcinoma cells with 15-120 min pulses of EdU. All DNA was stained with DAPI and cellular fluorescence was measured by laser scanning cytometry. The frequency of cells in the ascending (left) side of the "horseshoe"-shaped EdU/DAPI bivariate distributions reports the rate of DNA replication at the time of entrance to S phase while their frequency in the descending (right) side is a marker of DNA replication rate at the time of transition from S to G2 phase. To understand the connection between molecular-scale events and scatterplot asymmetry, we developed a multiscale stochastic model, which simulates DNA replication and cell cycle progression of individual cells and produces in silico EdU/DAPI scatterplots. For each S-phase cell the time points at which replication origins are fired are modeled by a non-homogeneous Poisson Process (NHPP). Shifted gamma distributions are assumed for durations of cell cycle phases (G1, S and G2 M), Depending on the rate of DNA synthesis being an increasing or decreasing function, simulated EdU/DAPI bivariate graphs show predominance of cells in left (early-S) or right (late-S) side of the horseshoe distribution. Assuming NHPP rate estimated from independent experiments, simulated EdU/DAPI graphs are nearly indistinguishable from those experimentally observed. This finding proves consistency between the S-phase DNA-replication rate based on molecular-scale analyses, and cell population kinetics ascertained from EdU/DAPI scatterplots and demonstrates that DNA replication rate at entrance to S is relatively slow compared with its rather abrupt termination during S to G2 transition. Our approach opens a possibility of similar modeling to study the effect of anticancer drugs on DNA replication/cell cycle progression and also to quantify other

Electron transfer in DNA duplexes containing 2-methyl-1,4-naphthoquinone

OpenAIRE

Bergeron, François; Houde, Daniel; Hunting, Darel J.; Wagner, J. Richard

2004-01-01

2-Methyl-1,4-naphthoquinone (menadione, MQ) was linked to synthetic oligonucleotides and exposed to near-UV light to generate base radical cations in DNA. This model system of electron transfer induced alkali-labile breaks at GG doublets, similar to anthraquinone and metallointercalators systems. In sharp contrast to other systems, the photolysis of MQ–DNA duplexes gave interstrand cross-links and alkali-labile breaks at bases on the complementary strand opposite the MQ moiety. For sequences ...

Geant4-DNA coupling and validation in the GATE Monte Carlo platform for DNA molecules irradiation in a calculation grid environment

International Nuclear Information System (INIS)

Pham, Quang Trung

2014-01-01

The Monte Carlo simulation methods are successfully being used in various areas of medical physics but also at different scales, for example, from the radiation therapy treatment planning systems to the prediction of the effects of radiation in cancer cells. The Monte Carlo simulation platform GATE based on the Geant4 tool-kit offers features dedicated to simulations in medical physics (nuclear medicine and radiotherapy). For radiobiology applications, the Geant4-DNA physical models are implemented to track particles till very low energy (eV) and are adapted for estimation of micro-dosimetric quantities. In order to implement a multi-scale Monte Carlo platform, we first validated the physical models of Geant4-DNA, and integrated them into GATE. Finally, we validated this implementation in the context of radiation therapy and proton therapy. In order to validate the Geant4-DNA physical models, dose point kernels for monoenergetic electrons (10 keV to 100 keV) were simulated using the physical models of Geant4-DNA and were compared to those simulated with Geant4 Standard physical models and another Monte Carlo code EGSnrc. The range and the stopping powers of electrons (7.4 eV to 1 MeV) and protons (1 keV to 100 MeV) calculated with GATE/Geant4-DNA were then compared with literature. We proposed to simulate with the GATE platform the impact of clinical and preclinical beams on cellular DNA. We modeled a clinical proton beam of 193.1 MeV, 6 MeV clinical electron beam and a X-ray irradiator beam. The beams models were validated by comparing absorbed dose computed and measured in liquid water. Then, the beams were used to calculate the frequency of energy deposits in DNA represented by different geometries. First, the DNA molecule was represented by small cylinders: 2 nm x 2 nm (∼10 bp), 5 nm x 10 nm (nucleosome) and 25 nm x 25 nm (chromatin fiber). All these cylinders were placed randomly in a sphere of liquid water (500 nm radius). Then we reconstructed the DNA

DNA fragments assembly based on nicking enzyme system.

Directory of Open Access Journals (Sweden)

Rui-Yan Wang

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

Increased systemic oxidatively generated DNA and RNA damage in schizophrenia

DEFF Research Database (Denmark)

Jørgensen, Anders; Brødbæk, Kasper; Fink-Jensen, Anders

2013-01-01

such as cardiovascular disease, type 2 diabetes and dementia. We determined the urinary excretion of markers of systemic Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA) oxidation, 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydroguanosine, respectively, in 40 schizophrenia patients and 40 age- and sex...

Horizontal gene transfer of a chloroplast DnaJ-Fer protein to Thaumarchaeota and the evolutionary history of the DnaK chaperone system in Archaea

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Petitjean Céline

2012-11-01

Full Text Available Abstract Background In 2004, we discovered an atypical protein in metagenomic data from marine thaumarchaeotal species. This protein, referred as DnaJ-Fer, is composed of a J domain fused to a Ferredoxin (Fer domain. Surprisingly, the same protein was also found in Viridiplantae (green algae and land plants. Because J domain-containing proteins are known to interact with the major chaperone DnaK/Hsp70, this suggested that a DnaK protein was present in Thaumarchaeota. DnaK/Hsp70, its co-chaperone DnaJ and the nucleotide exchange factor GrpE are involved, among others, in heat shocks and heavy metal cellular stress responses. Results Using phylogenomic approaches we have investigated the evolutionary history of the DnaJ-Fer protein and of interacting proteins DnaK, DnaJ and GrpE in Thaumarchaeota. These proteins have very complex histories, involving several inter-domain horizontal gene transfers (HGTs to explain the contemporary distribution of these proteins in archaea. These transfers include one from Cyanobacteria to Viridiplantae and one from Viridiplantae to Thaumarchaeota for the DnaJ-Fer protein, as well as independent HGTs from Bacteria to mesophilic archaea for the DnaK/DnaJ/GrpE system, followed by HGTs among mesophilic and thermophilic archaea. Conclusions We highlight the chimerical origin of the set of proteins DnaK, DnaJ, GrpE and DnaJ-Fer in Thaumarchaeota and suggest that the HGT of these proteins has played an important role in the adaptation of several archaeal groups to mesophilic and thermophilic environments from hyperthermophilic ancestors. Finally, the evolutionary history of DnaJ-Fer provides information useful for the relative dating of the diversification of Archaeplastida and Thaumarchaeota.

DNA:DNA hybridization studies on the pink-pigmented facultative methylotrophs.

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Hood, D W; Dow, C S; Green, P N

1987-03-01

The genomic relatedness among 36 strains of pink-pigmented facultatively methylotrophic bacteria (PPFMs) was estimated by determination of DNA base composition and by DNA:DNA hybridization studies. A reproducible hybridization system was developed for the rapid analysis of multiple DNA samples. Results indicated that the PPFMs comprise four major and several minor homology groups, and that they should remain grouped in a single genus, Methylobacterium.

Clusters of DNA damage induced by ionizing radiation: Formation of short DNA fragments. I. Theoretical modeling

International Nuclear Information System (INIS)

Holley, W.R.; Chatterjee, A.

1996-01-01

We have developed a general theoretical model for the interaction of ionizing radiation with chromatin. Chromatin is modeled as a 30-nm-diameter solenoidal fiber composed of 20 turns of nucleosomes, 6 nucleosomes per turn. Charged-particle tracks are modeled by partitioning the energy deposition between primary track core, resulting from glancing collisions with 100 eV or less per event, and δ rays due to knock-on collisions involving energy transfers > 100 eV. A Monte Carlo simulation incorporates damages due to the following molecular mechanisms: (1) ionization of water molecules leading to the formation of circ OH, circ H, e aq , etc.; circ OH attack on sugar molecules leading to strand breaks; circ OH attack on bases; direct ionization of the sugar molecules leading to strand breaks; direct ionization of the bases. Our calculations predict significant clustering of damage both locally, over regions up to 40 hp and over regions extending to several kilobase pairs. A characteristic feature of the regional damage predicted by our model is the production of short fragments of DNA associated with multiple nearby strand breaks. Such fragments have subsequently been detected experimentally and are reported in an accompanying paper after exposure to both high- and low-LET radiation. The overall measured yields agree well quantitatively with the theoretical predictions. Our theoretical results predict the existence of a strong peak at about 85 bp, which represents the revolution period about the nucleosome. Other peaks at multiples of about 1,000 bp correspond to the periodicity of the particular solenoid model of chromatin used in these calculations. Theoretical results in combination with experimental data on fragmentation spectra may help determine the consensus or average structure of the chromatin fibers in mammalian DNA. 27 refs., 7 figs

Spectroscopic and molecular modeling study on the interaction of ctDNA with 3′-deoxy-3′-azido doxorubicin

International Nuclear Information System (INIS)

Geng, Shaoguang; Cui, Yanrui; Liu, Qingfeng; Cui, Fengling; Zhang, Guisheng; Chi, Yanwei; Peng, Hao

2013-01-01

The method of synthesizing 3′-deoxy-3′-azido doxorubicin (ADOX) directly from doxorubicin has been developed. This study presents the interaction between ADOX and calf thymus deoxyribonucleic acid (ctDNA) by using spectroscopic methods and molecular modeling techniques. Iodide quenching, fluorescence polarization, viscosity and molecular modeling studies of ADOX–ctDNA interactions indicated that ADOX was an intercalator of ctDNA and preferentially bound to C–G rich regions of ctDNA. Simultaneously, spectroscopic results indicated that the quenching mechanism of ADOX–ctDNA was a static quenching. According to thermodynamic parameters, electrostatic force played roles in the interaction of ADOX with ctDNA. -- Highlights: ●An approach to 3′-deoxy-3′-azido doxorubicin (ADOX) from doxorubicin was developed. ●The quenching mechanism of ADOX with ctDNA was a static quenching type. ●The binding mode between ADOX and ctDNA was intercalative binding. ●The results of molecular docking corroborated results of spectra investigations

DNA index determination with Automated Cellular Imaging System (ACIS in Barrett's esophagus: Comparison with CAS 200

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Klein Michael

2005-08-01

Full Text Available Abstract Background For solid tumors, image cytometry has been shown to be more sensitive for diagnosing DNA content abnormalities (aneuploidy than flow cytometry. Image cytometry has often been performed using the semi-automated CAS 200 system. Recently, an Automated Cellular Imaging System (ACIS was introduced to determine DNA content (DNA index, but it has not been validated. Methods Using the CAS 200 system and ACIS, we compared the DNA index (DI obtained from the same archived formalin-fixed and paraffin embedded tissue samples from Barrett's esophagus related lesions, including samples with specialized intestinal metaplasia without dysplasia, low-grade dysplasia, high-grade dysplasia and adenocarcinoma. Results Although there was a very good correlation between the DI values determined by ACIS and CAS 200, the former was 25% more sensitive in detecting aneuploidy. ACIS yielded a mean DI value 18% higher than that obtained by CAS 200 (p t test. In addition, the average time required to perform a DNA ploidy analysis was shorter with the ACIS (30–40 min than with the CAS 200 (40–70 min. Results obtained by ACIS gave excellent inter-and intra-observer variability (coefficient of correlation >0.9 for both, p Conclusion Compared with the CAS 200, the ACIS is a more sensitive and less time consuming technique for determining DNA ploidy. Results obtained by ACIS are also highly reproducible.

Heat degradation of eukaryotic and bacterial DNA: an experimental model for paleomicrobiology

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Nguyen-Hieu Tung

2012-09-01

Full Text Available Abstract Background Theoretical models suggest that DNA degradation would sharply limit the PCR-based detection of both eukaryotic and prokaryotic DNA within ancient specimens. However, the relative extent of decay of eukaryote and prokaryote DNA over time is a matter of debate. In this study, the murine macrophage cell line J774, alone or infected with Mycobacterium smegmatis bacteria, were killed after exposure to 90°C dry heat for intervals ranging from 1 to 48 h in order to compare eukaryotic cells, extracellular bacteria and intracellular bacteria. The sizes of the resulting mycobacterial rpoB and murine rpb2 homologous gene fragments were then determined by real-time PCR and fluorescent probing. Findings The cycle threshold (Ct values of PCR-amplified DNA fragments from J774 cells and the M. smegmatis negative controls (without heat exposure varied from 26–33 for the J774 rpb2 gene fragments and from 24–29 for M. smegmatis rpoB fragments. After 90°C dry heat incubation for up to 48 h, the Ct values of test samples increased relative to those of the controls for each amplicon size. For each dry heat exposure time, the Ct values of the 146-149-bp fragments were lower than those of 746-747-bp fragments. During the 4- to 24-h dry heat incubation, the non-infected J774 cell DNA was degraded into 597-bp rpb2 fragments. After 48 h, however, only 450-bp rpb2 fragments of both non-infected and infected J774 cells could be amplified. In contrast, the 746-bp rpoB fragments of M. smegmatis DNA could be amplified after the 48-h dry heat exposure in all experiments. Infected and non-infected J774 cell DNA was degraded more rapidly than M. smegmatis DNA after dry heat exposure (ANOVA test, p  Conclusion In this study, mycobacterial DNA was more resistant to dry-heat stress than eukaryotic DNA. Therefore, the detection of large, experimental, ancient mycobacterial DNA fragments is a suitable approach for paleomicrobiological studies.

Effects of tritium on DNA, 3

International Nuclear Information System (INIS)

Yamamoto, Osamu; Fuji, Izumi

1984-01-01

Lambda DNA solution was prepared at a concentration of 125μg/ml of 10mM Tris-HCl-5mM NaCl-1mM Na 2 EDTA (pH 7.4). Solutions were irradiated with 60 Co gamma-rays and 3 H beta-rays ( 3 HHO addition), respectively. After irradiation of DNA solutions, single-strand breaks and double-strand breaks were detected by a vertical gel electrophoretic system (BRL Model V16). In both strand breaks higher oxygen enhancement ratios were obtained with 60 Co erradiation. (J.P.N.)

Classification of human cancers based on DNA copy number amplification modeling

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Knuutila Sakari

2008-05-01

Full Text Available Abstract Background DNA amplifications alter gene dosage in cancer genomes by multiplying the gene copy number. Amplifications are quintessential in a considerable number of advanced cancers of various anatomical locations. The aims of this study were to classify human cancers based on their amplification patterns, explore the biological and clinical fundamentals behind their amplification-pattern based classification, and understand the characteristics in human genomic architecture that associate with amplification mechanisms. Methods We applied a machine learning approach to model DNA copy number amplifications using a data set of binary amplification records at chromosome sub-band resolution from 4400 cases that represent 82 cancer types. Amplification data was fused with background data: clinical, histological and biological classifications, and cytogenetic annotations. Statistical hypothesis testing was used to mine associations between the data sets. Results Probabilistic clustering of each chromosome identified 111 amplification models and divided the cancer cases into clusters. The distribution of classification terms in the amplification-model based clustering of cancer cases revealed cancer classes that were associated with specific DNA copy number amplification models. Amplification patterns – finite or bounded descriptions of the ranges of the amplifications in the chromosome – were extracted from the clustered data and expressed according to the original cytogenetic nomenclature. This was achieved by maximal frequent itemset mining using the cluster-specific data sets. The boundaries of amplification patterns were shown to be enriched with fragile sites, telomeres, centromeres, and light chromosome bands. Conclusions Our results demonstrate that amplifications are non-random chromosomal changes and specifically selected in tumor tissue microenvironment. Furthermore, statistical evidence showed that specific chromosomal features

Interleukin-6 promotes systemic lupus erythematosus progression with Treg suppression approach in a murine systemic lupus erythematosus model.

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Mao, Xiaoli; Wu, Yunyun; Diao, Huitian; Hao, Jianlei; Tian, Gaofei; Jia, Zhenghu; Li, Zheng; Xiong, Sidong; Wu, Zhenzhou; Wang, Puyue; Zhao, Liqing; Yin, Zhinan

2014-11-01

Our aim is to reveal the role of interleukin 6 (IL-6) in the pathogenesis of systemic lupus erythematosus (SLE) in a murine model of SLE. Normal female C57BL/6 mice were immunized with syngeneic-activated lymphocyte-derived DNA (ALD-DNA) to induce SLE. Non-immunized mice were used as control. SLE-associated markers, including anti-double-stranded DNA (anti-dsDNA) Abs, urine protein, and kidney histopathology, were assayed to ensure the induction of the disease. Compared with control mice, ALD-DNA immunized mice exhibited high levels of anti-dsDNA Abs, IL-6 expression in vivo and in vitro. We also found that IL-6 knockout (IL-6KO) mice were resistant to ALD-DNA-induced SLE. The activation of CD4(+) T cells in immunized IL-6KO mice was lower than in immunized wild-type (Wt) mice. Intracellular cytokine staining showed that Foxp3 expression in immunized IL-6KO mice was higher than in immunized Wt mice, which might be associated with the disease severity. We further discovered that ALD-DNA-stimulated dendritic cells supernatants could result in higher IL-6 and TNF-α expression and could suppress Foxp3 expression. In addition, blocking IL-6 could up-regulate Foxp3 expression. Therefore, our findings show that IL-6 promotes the progression of SLE via suppressing Treg differentiation.

Replicating animal mitochondrial DNA

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Emily A. McKinney

2013-01-01

Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

Postglacial species displacement in Triturus newts deduced from asymmetrically introgressed mitochondrial DNA and ecological niche models

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Wielstra Ben

2012-08-01

Full Text Available Abstract Background If the geographical displacement of one species by another is accompanied by hybridization, mitochondrial DNA can introgress asymmetrically, from the outcompeted species into the invading species, over a large area. We explore this phenomenon using the two parapatric crested newt species, Triturus macedonicus and T. karelinii, distributed on the Balkan Peninsula in south-eastern Europe, as a model. Results We first delimit a ca. 54,000 km2 area in which T. macedonicus contains T. karelinii mitochondrial DNA. This introgression zone bisects the range of T. karelinii, cutting off a T. karelinii enclave. The high similarity of introgressed mitochondrial DNA haplotypes with those found in T. karelinii suggests a recent transfer across the species boundary. We then use ecological niche modeling to explore habitat suitability of the location of the present day introgression zone under current, mid-Holocene and Last Glacial Maximum conditions. This area was inhospitable during the Last Glacial Maximum for both species, but would have been habitable at the mid-Holocene. Since the mid-Holocene, habitat suitability generally increased for T. macedonicus, whereas it decreased for T. karelinii. Conclusion The presence of a T. karelinii enclave suggests that T. karelinii was the first to colonize the area where the present day introgression zone is positioned after the Last Glacial Maximum. Subsequently, we propose T. karelinii was outcompeted by T. macedonicus, which captured T. karelinii mitochondrial DNA via introgressive hybridization in the process. Ecological niche modeling suggests that this replacement was likely facilitated by a shift in climate since the mid-Holocene. We suggest that the northwestern part of the current introgression zone was probably never inhabited by T. karelinii itself, and that T. karelinii mitochondrial DNA spread there through T. macedonicus exclusively. Considering the spatial distribution of the

An analytical framework for estimating aquatic species density from environmental DNA

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Chambert, Thierry; Pilliod, David S.; Goldberg, Caren S.; Doi, Hideyuki; Takahara, Teruhiko

2018-01-01

Environmental DNA (eDNA) analysis of water samples is on the brink of becoming a standard monitoring method for aquatic species. This method has improved detection rates over conventional survey methods and thus has demonstrated effectiveness for estimation of site occupancy and species distribution. The frontier of eDNA applications, however, is to infer species density. Building upon previous studies, we present and assess a modeling approach that aims at inferring animal density from eDNA. The modeling combines eDNA and animal count data from a subset of sites to estimate species density (and associated uncertainties) at other sites where only eDNA data are available. As a proof of concept, we first perform a cross-validation study using experimental data on carp in mesocosms. In these data, fish densities are known without error, which allows us to test the performance of the method with known data. We then evaluate the model using field data from a study on a stream salamander species to assess the potential of this method to work in natural settings, where density can never be known with absolute certainty. Two alternative distributions (Normal and Negative Binomial) to model variability in eDNA concentration data are assessed. Assessment based on the proof of concept data (carp) revealed that the Negative Binomial model provided much more accurate estimates than the model based on a Normal distribution, likely because eDNA data tend to be overdispersed. Greater imprecision was found when we applied the method to the field data, but the Negative Binomial model still provided useful density estimates. We call for further model development in this direction, as well as further research targeted at sampling design optimization. It will be important to assess these approaches on a broad range of study systems.

Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors.

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Miura, Hiromi; Quadros, Rolen M; Gurumurthy, Channabasavaiah B; Ohtsuka, Masato

2018-01-01

CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1-10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement. We call this method efficient additions with ssDNA inserts-CRISPR (Easi-CRISPR) because it is a highly efficient technology (efficiency is typically 30-60% and reaches as high as 100% in some cases). The protocol takes ∼2 months to generate the founder mice.

Molecular dynamics simulations of DNA-free and DNA-bound TAL effectors.

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Hua Wan

Full Text Available TAL (transcriptional activator-like effectors (TALEs are DNA-binding proteins, containing a modular central domain that recognizes specific DNA sequences. Recently, the crystallographic studies of TALEs revealed the structure of DNA-recognition domain. In this article, molecular dynamics (MD simulations are employed to study two crystal structures of an 11.5-repeat TALE, in the presence and absence of DNA, respectively. The simulated results indicate that the specific binding of RVDs (repeat-variable diresidues with DNA leads to the markedly reduced fluctuations of tandem repeats, especially at the two ends. In the DNA-bound TALE system, the base-specific interaction is formed mainly by the residue at position 13 within a TAL repeat. Tandem repeats with weak RVDs are unfavorable for the TALE-DNA binding. These observations are consistent with experimental studies. By using principal component analysis (PCA, the dominant motions are open-close movements between the two ends of the superhelical structure in both DNA-free and DNA-bound TALE systems. The open-close movements are found to be critical for the recognition and binding of TALE-DNA based on the analysis of free energy landscape (FEL. The conformational analysis of DNA indicates that the 5' end of DNA target sequence has more remarkable structural deformability than the other sites. Meanwhile, the conformational change of DNA is likely associated with the specific interaction of TALE-DNA. We further suggest that the arrangement of N-terminal repeats with strong RVDs may help in the design of efficient TALEs. This study provides some new insights into the understanding of the TALE-DNA recognition mechanism.

A Novel Image Encryption Algorithm Based on a Fractional-Order Hyperchaotic System and DNA Computing

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Taiyong Li

2017-01-01

Full Text Available In the era of the Internet, image encryption plays an important role in information security. Chaotic systems and DNA operations have been proven to be powerful for image encryption. To further enhance the security of image, in this paper, we propose a novel algorithm that combines the fractional-order hyperchaotic Lorenz system and DNA computing (FOHCLDNA for image encryption. Specifically, the algorithm consists of four parts: firstly, we use a fractional-order hyperchaotic Lorenz system to generate a pseudorandom sequence that will be utilized during the whole encryption process; secondly, a simple but effective diffusion scheme is performed to spread the little change in one pixel to all the other pixels; thirdly, the plain image is encoded by DNA rules and corresponding DNA operations are performed; finally, global permutation and 2D and 3D permutation are performed on pixels, bits, and acid bases. The extensive experimental results on eight publicly available testing images demonstrate that the encryption algorithm can achieve state-of-the-art performance in terms of security and robustness when compared with some existing methods, showing that the FOHCLDNA is promising for image encryption.

The assessment of cold atmospheric plasma treatment of DNA in synthetic models of tissue fluid, tissue and cells

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Szili, Endre J.; Gaur, Nishtha; Hong, Sung-Ha; Kurita, Hirofumi; Oh, Jun-Seok; Ito, Masafumi; Mizuno, Akira; Hatta, Akimitsu; Cowin, Allison J.; Graves, David B.; Short, Robert D.

2017-07-01

There is a growing literature database that demonstrates the therapeutic potential of cold atmospheric plasma (herein referred to as plasma). Given the breadth of proposed applications (e.g. from teeth whitening to cancer therapy) and vast gamut of plasma devices being researched, it is timely to consider plasma interactions with specific components of the cell in more detail. Plasma can produce highly reactive oxygen and nitrogen species (RONS) such as the hydroxyl radical (OH•), peroxynitrite (ONOO-) and superoxide (\\text{O}2- ) that would readily modify essential biomolecules such as DNA. These modifications could in principle drive a wide range of biological processes. Against this possibility, the reported therapeutic action of plasmas are not underpinned by a particularly deep knowledge of the potential plasma-tissue, -cell or -biomolecule interactions. In this study, we aim to partly address this issue by developing simple models to study plasma interactions with DNA, in the form of DNA-strand breaks. This is carried out using synthetic models of tissue fluid, tissue and cells. We argue that this approach makes experimentation simpler, more cost-effective and faster than compared to working with real biological materials and cells. Herein, a helium plasma jet source was utilised for these experiments. We show that the plasma jet readily induced DNA-strand breaks in the tissue fluid model and in the cell model, surprisingly without any significant poration or rupture of the phospholipid membrane. In the plasma jet treatment of the tissue model, DNA-strand breaks were detected in the tissue mass after pro-longed treatment (on the time-scale of minutes) with no DNA-strand breaks being detected in the tissue fluid model underneath the tissue model. These data are discussed in the context of the therapeutic potential of plasma.

Hands on Group Work Paper Model for Teaching DNA Structure, Central Dogma and Recombinant DNA

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Altiparmak, Melek; Nakiboglu Tezer, Mahmure

2009-01-01

Understanding life on a molecular level is greatly enhanced when students are given the opportunity to visualize the molecules. Especially understanding DNA structure and function is essential for understanding key concepts of molecular biology such as DNA, central dogma and the manipulation of DNA. Researches have shown that undergraduate…

DNA-binding, DNA cleavage and cytotoxicity studies of two anthraquinone derivatives.

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Gholivand, M B; Kashanian, S; Peyman, H

2012-02-15

The interaction of native calf thymus DNA (CT-DNA) with two anthraquinones including quinizarin (1,4-dihydroxy anthraquinone) and danthron (1,8-dihydroxy anthraquinone) in a mixture of 0.04M Brittone-Robinson buffer and 50% of ethanol were studied at physiological pH by spectrofluorometric and cyclic voltammetry techniques. The former technique was used to calculate the binding constants of anthraquinones-DNA complexes at different temperatures. Thermodynamic study indicated that the reactions of both anthraquinone-DNA systems are predominantly entropically driven. Furthermore, the binding mechanisms on the reaction of the two anthraquinones with DNA and the effect of ionic strength on the fluorescence property of the system have also been investigated. The results of the experiments indicated that the binding modes of quinizarin and danthron with DNA were evaluated to be groove binding. Moreover, the cytotoxic activity of both compounds against human chronic myelogenous leukemia K562 cell line and DNA cleavage were investigated. The results indicated that these compounds slightly cleavage pUC18 plasmid DNA and showed minor antitumor activity against K562 (human chronic myeloid leukemia) cell line. Copyright © 2011 Elsevier B.V. All rights reserved.

Theoretical approach of complex DNA lesions: from formation to repair

International Nuclear Information System (INIS)

Bignon, Emmanuelle

2017-01-01

This thesis work is focused on the theoretical modelling of DNA damages, from formation to repair. Several projects have been led in this framework, which can be sorted into three different parts. One on hand, we studied complex DNA reactivity. It included a study about 8-oxo-7,8-dihydro-guanine (8oxoG) mechanisms of formation, a project concerning the UV-induced pyrimidine 6-4 pyrimidone (6-4PP) endogenous photo-sensitizer features, and another one about DNA photo-sensitization by nonsteroidal anti-inflammatory drugs (i.e. ketoprofen and ibuprofen). On the other hand, we investigated mechanical properties of damaged DNA. The structural signature of a DNA lesion is of major importance for their repair, unfortunately only few NMR and X-ray structures of such systems are available. In order to gain insights into their dynamical structure, we investigated a series of complex damages: clustered abasic sites, interstrand cross-links, and the 6-4PP photo-lesion. Likewise, we studied the interaction modes DNA with several polyamines, which are well known to interact with the double helix, but also with the perspective to model DNA-protein cross-linking. The third part concerned the study of DNA interactions with repair enzymes. In line with the structural study about clustered abasic sites, we investigated the dynamics of the same system, but this time interacting with the APE1 endonuclease. We also studied interactions between the Fpg glycosylase with an oligonucleotides containing tandem 8-oxoG on one hand and 8-oxoG - abasic site as multiply damaged sites. Thus, we shed new lights on damaged DNA reactivity, structure and repair, which provides perspectives for biomedicine and life's mechanisms understanding as we begin to describe nucleosomal DNA. (author)

TEMPORAL MODELING OF DNA DEGRADATION IN BONE REMAINS

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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

Rescue of DNA-PK Signaling and T-Cell Differentiation by Targeted Genome Editing in a prkdc Deficient iPSC Disease Model.

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Shamim H Rahman

2015-05-01

Full Text Available In vitro disease modeling based on induced pluripotent stem cells (iPSCs provides a powerful system to study cellular pathophysiology, especially in combination with targeted genome editing and protocols to differentiate iPSCs into affected cell types. In this study, we established zinc-finger nuclease-mediated genome editing in primary fibroblasts and iPSCs generated from a mouse model for radiosensitive severe combined immunodeficiency (RS-SCID, a rare disorder characterized by cellular sensitivity to radiation and the absence of lymphocytes due to impaired DNA-dependent protein kinase (DNA-PK activity. Our results demonstrate that gene editing in RS-SCID fibroblasts rescued DNA-PK dependent signaling to overcome radiosensitivity. Furthermore, in vitro T-cell differentiation from iPSCs was employed to model the stage-specific T-cell maturation block induced by the disease causing mutation. Genetic correction of the RS-SCID iPSCs restored T-lymphocyte maturation, polyclonal V(DJ recombination of the T-cell receptor followed by successful beta-selection. In conclusion, we provide proof that iPSC-based in vitro T-cell differentiation is a valuable paradigm for SCID disease modeling, which can be utilized to investigate disorders of T-cell development and to validate gene therapy strategies for T-cell deficiencies. Moreover, this study emphasizes the significance of designer nucleases as a tool for generating isogenic disease models and their future role in producing autologous, genetically corrected transplants for various clinical applications.

Investigation of kinetics and thermodynamics of DNA hybridization by means of 2-D fluorescence spectroscopy and soft/hard modeling techniques